Biocatalysis in Non-Conventional Media [1st Edition] 9781483298016

Table of contents :
Content:
Progress in BiotechnologyPage ii
Front MatterPage iii
Copyright pagePage iv
PrefacePages v-vii
AcknowledgementsPage viii
Medium and Biocatalyst EngineeringPages 3-9Ephraim Katchalski-Katzir
Physical-Chemical Nature of Low Water Systems for Biocatalysis: Especially Phase Behaviour, Water Activity and pHPages 13-21Peter J. Halling, Rao H. Valivety
Usefulness of NMR Methods for Assaying Cutinase Catalysed Synthesis of Ester in Organic MediaPages 23-29C. SARAZIN, G. GOETHALS, J.P. SEGUIN, M.D. LEGOY, J.N. BARBOTIN
Membrane Concentrations of Primary Alcohols Which Inhibit Progesterone 11α-Hydroxylase in Rhizopus NigricansPages 31-36S.J. Osborne, J. Leaver, M.K. Turner
A Comparison of Enzymatic Reactions in Aqueous, Organic and Multiphase SystemsPages 37-44T. Scheper, U. Bornscheuer, A. Capewell, A. Herar, H.G. Hundeck, H. Meyer, F. Schubert, F. Kolisis
Biocatalysis in Non-Conventional Media: Effect of Enzyme MicroenvironmentPages 45-52D. Combes
On the Importance of the Support Material for Enzymatic Synthesis in Organic Media. Support Effects at Controlled Water ActivityPages 55-61Patrick Adlercreutz
Enzyme Design for Nonaqueous MediaPages 63-66Jonathan S. Dordick, Zu-Feng Xu, Pramod Wangikar
Application of Q.S.A.R. Methodology to the Biocatalysis. I. Hydrolysis of EstersPages 67-74M.J. Cabezas, E.F. Llama, C. Campo, J.V. Sinisterra
Application of Q.S.A.R. Methodology to the Biocatalysis. II. Synthesis of PeptidesPages 75-82J.V. Sinisterra
Introduction to Gaseous and (Near-)Supercritical MediaPage 83Z. Knez
The Role of Water in Gaseous BiocatalysisPages 85-92H. Robert, S. Lamare, F. Parvaresh, M.D. Legoy
Pressure Control of Reactions in Supercritical Fluids: Thermodynamics and KineticsPages 93-100Theodore W. Randolph, Claude Carlier
Introduction to One-Liquid-Phase SystemsPage 101J.M.S. Cabral
Enzyme Mechanisms in Homogeneous Hydro-Organic Solutions. Solvents, Temperature and Pressure EffectsPages 103-110Claude Balny
Effect of Reaction Conditions on the Activity and Enantioselectivity of Lipases in Organic SolventsPages 111-119G. Carrea, G. Ottolina, S. Riva, F. Secundo
Correlations Between Enzyme Activity, Water Activity, and Log P in One-Liquid-Phase SystemsPages 121-128A. Manjón, J.M. Obón, M. Cánovas, J.L. Iborra
Microenvironmental Effects on Steroid δ1-Dehydrogenation in Organic Media Using Immobilised Whole CellsPages 129-136H.M. Pinheiro, J.M.S. Cabral
Enzyme Kinetics in Monophasic and Biphasic Aqueous-Organic SystemsPages 137-144A.J.J. Straathof, J.L.L. Rakels, J.J. Heijnen
Introduction to Two-Liquid-Phase Systems IPage 145R. Hilhorst
Process Engineering of Two-Liquid Phase BiocatalysisPages 147-154John M. Woodley, Malcolm D. Lilly
The Effect of Organic Solvents on Enzymatic Esterification of PolyolsPages 155-161Anja E.M. Janssen, Munasri Hadini, Nicolette Wessels Boer, Rob Walinga, Albert Van der Padt, Henk M. Van Sonsbeek, Klaas Van't Riet
Process Development for the Optical Resolution of Phenylalanine by Means of Chymotrypsin in a Liquid-Liquid-Solid Three-Phase Reaction SystemPages 163-170E. Flaschel, S. Crelier, K. Schulz, F.-U. Huneke, A. Renken
Introduction to Two-Liquid-Phase Systems IIPage 171M.D. Lilly
Understanding Protein Performance in Reversed Micelles: The Contribution of Transport Rate, Local Concentration and Water Content to Enzyme KineticsPages 173-180M.D. Raymond, Verhaert
Protein-Interface Interactions in Reverse MicellesPages 181-188A. Sánchez-Ferrer, M. Pérez-Gilabert, F. Garcií-Carmona
Kinetics of Enzyme-Catalysed Reactions in Water-in-Oil MicroemulsionsPages 189-198Christopher Oldfield, Cristina Otero, Maria L. Rua, Antonio Ballesteros
Concluding RemarksPages 201-206U. von Stockar
Enzyme Kinetics in a Self Evolving Microstructured MediumPages 211-212Joël Chopineau, Michel Ollivon, Marie-Dominique Legoy
Enzyme Deactivation Phenomena in Solid-State and Organic SolventsPages 213-220Guido Greco jr., Domenico Pirozzi, Giuseppe Toscano
Insolubilized Enzyme Derivatives in Organic Solvents: Mechanisms of Inactivation and Strategies for ReactivationPages 221-228J.M. Guisán, A. Bastida, R.M. Blanco, C. Cuesta, V. Rodriguez, R. Fernandez-Lafuente
Relation of Enzymatic Reaction Rate and Hydrophobicity of the SolventPages 229-235J. Bert, A. van Tol, Jan B. Odenthal, Jaap A. Jongejan, Johannis A. Duine
Kinetic Resolution of Racemic Glycidyl Esters with Porcine Pancreatic Lipase: A Major Effect of Ping-Pong KineticsPages 237-243J. Bert, A. van Tol, Jaap A. Jongejan, Johannis A. Duine
Biocatalysts Operating at High Substrate ConcentrationsPages 245-252P.W. Kühl
Regulation of Allosteric Enzymes in Water-Restricted MediaPages 253-260C. Lambert, V. Larreta-Garde
Quantitative Deuterium NMR of Protein Hydration in Air and Organic SolventsPages 261-266M.C. Parker, B.D. Moore, A.J. Blacker
Effects of Temperature on Stereochemistry of Alcohol Dehydrogenases from Thermoanaerobacter ethanolicusPages 267-273Robert S. Phillips, Van T. Pham, Changsheng Zheng, Francisco A.C. Andrade, Maria A.C. Andrade
Comparative Influence of Microenvironment on the Activity of Two Enzymes : Lipoxygenase and ThermolysinPages 275-282C. Pourplanche, P. Hertmanni, V. Larreta-Garde
On the Crucial Role of Water in the Lipase Catalysed Isomerisation of 1,2-(2,3)-Diglyceride Into 1,3-DiglyceridePages 283-290Claude Rabiller, Andreas Heisler, G. Hägele
Rapid Determination, Using Dielectric Spectroscopy, of the Toxicity of Organic Solvents to Intact CellsPages 291-297Gary J. Salter, Douglas B. Kell
Factors Affecting Protein Transfer from an Aqueous Phase Into a Reversed Micellar PhasePages 299-306R. Hilhorst, R. Wolbert, P. Fijneman, D. Heering, P. Rietveld, M. Dekker, K. van't Riet, B.H. Bijsterbosch
Cryo-Bioorganic Synthesis - Enzyme Catalysis at low Temperature and in Low Water Content EnvironmentsPages 307-312Ingibjörg Skúladóttir, Kurt Nilsson, Bo Mattiasson
Photoinduced Charge Separation in MicroemulsionsPages 313-320Raymond M.D. Verhaert, Fred Roeterdink, Riet Hilhorst, Tjeerd J. Schaafsma
Induced Stereo and Substrate Selectivity of Bio-Imprinted α-Chymotrypsin in Anhydrous Organic MediaPages 321-327Mats-Olle Månsson, Marianne Ståhl, Klaus Mosbach
The Effect of Attachment of Hydrophobic Modifiers on the Catalytic Activities of Lipase and TrypsinPages 331-338K. Ampon, M. Basri, C.N.A. Razak, W.M.Z. Yunus, A.B. Salleh
Inlfuence of the Solvent and the Solid Support on the Microenvironment of Immobilized α-ChymotrypsinPages 339-346A. Heras, M.T. Martin, N. Acosta, F. Debaillon-Vesque
Hydrophilic Gels as Immobilization Materials and Stabilizers for Enzyme-Catalysed Esterification in Organic MediaPages 347-354Sissel Hertzberg, Ole Martin Fuskevåg, Thorleif Anthonsen
Effect of Polyhydroxy Compounds on the Activity of Lipase from Rhizopus Arrhizus in Organic SolventPages 355-361Berit Nordvi, Holm Holmsen
Complex Formation Between Chymotrypsin and Polymers as a Means to Improve Exposure of the Enzyme to Organic SolventsPages 363-369Marina Otamiri, Patrick Adlercreutz, Bo Mattiasson
Synthesis of Enkephalins Using Modified Proteases in Organic MediaPages 371-376J.M. Sánchez-Montero, A. Ferjancic-Biagini, A. Puigserver, J.V. Sinisterra
Stabilization of Adsorbed Enzymes Used as Biocatalysts in Organic SolventsPages 377-382Ernst Wehtje, Patrick Adlercreutz, Bo Mattiasson
The use of Amylolytic and Proteolytic Enzymes in art RestorationPages 385-392P. Choisy, A. De La Chapelle, M.D. Legoy
Methyl Isobutyl and Methyl Ethyl Ketone Biodegradation in BiofiltersPages 393-399Marc A. Deshusses, Geoffrey Hamer
Lipase Catalysed Esterification in Supercritical Carbon DioxidePages 401-406Ž. Knez, M. Habulin
Effect of a Near- Critical and Supercritical Fluid on the Viability Ratio of Microbial CellsPages 407-416A. Isenschmid, I.W. Marison, U. von Stockar
Enzymatic Reaction in Organic Solvents and Supercritical GasesPages 417-423Xiao Mei Shen, Theo W. de Loos, Jakob de Swaan Arons
Fatty Acid Esterification in Supercritical Carbon DioxidePages 425-432Alain Marty, Didier Combes, Jean-Stéphane Condoret
Influence of Organic Solvents on the Specificity of α-Chymotrypsin and Subtilisin from B. Subtilis Strain 72 in Acyl Transfer ReactionsPages 435-442M.Yu. Gololobov, T.L. Voyushina, P. Adlercreutz
Peptide Synthesis in Organic-Aqueous Media Catalysed by α-Chymotrypsin Immobilised Over Different SupportsPages 443-450A.R. Alcántara, J.V. Sinisterra, C. Torres, J.M. Guisán, M.H. Gil, A. Williams
Control of Water Activity by Using Salt Hydrates in Enzyme Catalysed Esterifications in Organic MediaPages 451-457Birte Sjursnes, Lise Kvittingen, Thorleif Anthonsen, Peter Halling
Enzymatic Peptide Synthesis Using New Water-Soluble Amino Acid DerivativesPages 459-466D. Auriol, F. Paul, P. Monsan
Lipase-Catalyzed Resolution of 1,2-DiolsPages 467-474Aldo Bosetti, Daniele Bianchi, Pietro Cesti, Paolo Golini
Partitioning of Water During the Production of Terpene Esters Using Immobilized LipasePages 475-482H.F. de Castro, W.A. Anderson, M. Moo-Young, R.L. Legge
Thermoinactivation of Polyphenol Oxidase in Organic Solvents with Low Water ContentPages 483-489P. Estrada, W. Baroto, R. Sanchez-Muñiz, C. Acebal, M.P. Castillon, R. Arche
Continuous Enzymatic Transesterification of Rapeseed Oil and Lauric Acid in a Solvent-Free SystemPages 491-496P. Forssell, K. Poutanen
Effect of the Solvent on Enzyme EnantioselectivityPages 497-503László Gubicza
Modification of Waste Fats by Lipase-Catalyzed Reaction in Solvent-Free Substrate BlendsPages 505-512L. Haalck, H.C. Hedrich, J. Hassink, F. Spener
Thermolysin- and Chymotrypsin-Catalysed Peptide Synthesis in the Presence of Salt HydratesPages 513-518P. Kuhl, U. Eichhorn, H.-D. Jakubke
Lipase Catalyzed Triglyceride Synthesis. The Role of IsomerizationPages 519-524R. Lortie, M. Trani, F. Ergan
Resolution of 1-Benzamido-4-Carboxymethyl-Cyclopent-2-Ene Using Pig Liver EsterasePages 525-531M. Mahmoudian, B.S. Baines, M.J. Dawson, G.C. Lawrence
α-Substituted Primary Alcohols as Substrates for Enantioselective Lipase-Catalyzed Transesterification in Organic SolventsPages 533-540Enzo Santaniello, Patrizia Ferraboschi, Paride Grisenti, Ada Manzocchi
Soluble and Immobilized Saccharidases in Water-Miscible Organic SolventsPages 541-547R. Ulbrich-Hofmann, B. Selisko
Effect of Water Activity on Rate of Lipase Catalysed EsterificationPages 549-555Rao H. Valivety, Peter J. Halling, Alasdair R. Macrae
Synthesis of Triacylglycerols: The crucial role of water activity controlPages 557-562Albert Van der Padt, Jos J.W. Sewalt, Klaas Van't Riet
Chemo-Enzymatic Synthesis of Monosaccharide Fatty Acid Esters and their Preliminary CharacterizationPages 563-568Giuseppe Fregapane, Douglas B. Sarney, Sydney G. Greenberg, Dorothy J. Knight, Evgeny N. Vulfson
Reversing an α-Chymotrypsin Catalyzed Reaction, by Substituting a Water / 1,4-Butanediol Solvent Mixture for the Usual Aqueous Reaction MediumPages 569-576B. Deschrevel, M. Thellier, J.C. Vincent
Engineering Aspects of the Lipase-Catalyzed Production of (R)-1-Ferrocenylethylacetate in Organic MediaPages 577-584A. Wickli, E. Schmidt, J.R. Bourne
Variation of Tyrosinase Activity with Solvent at a Constant Water ActivityPages 585-592Zhen Yang, Donald A. Robb, Peter J. Halling
Hydrolase Activity of Pseudomonas Fluorescens Lipase in Organic MediaPages 593-600F. Ausseil, H. Biaudet, P. Masson
Factors Affecting Lipase Catalyzed n-Butyl Oleate SynthesisPages 601-608Y.-Y. Linko, O. Rantanen, H.-C. Yu, P. Linko
Behaviour of Soluble Aminoacylase in Water-Organic Solvent MixturesPages 609-614J. Kosáry, B. Szajáni, L. Boross
Effect of Organic Solvents on Growth and Anthraquinone Production in Morinda Citrifolia Cell CulturesPages 617-622L. Bassetti, J. Tramper
Functional Stability of Cytoplasmic Enzymes in Aqueous and Mixed-Phase SolventsPages 623-628Don A. Cowan, K.V. Ramana
Studies on Papain Catalyzed Synthesis of Gly-Phe in a Two-Liquid-Phase SystemPages 629-636J.A. Feliu, C. de Mas, J. López-Santín
The Effect of Alkanes on Viability, Enzyme Induction and Enzyme Activity in Flavobacterium DehydrogenansPages 637-643S. Boeren, C. Laane, R. Hilhorst
Kinetics and Engineering Studies of Lipase-Catalyzed Transesterification in Organic SolventPages 645-652M. Indlekofer, M. Hoeschele, M. Rizzi, M. Reuss
Kinetic Study of Enzymatic Reaction in Aqueous-Organic Two-Phase Systems - An Example of Enhanced Production of Aldehydes by Alcohol OxidasePages 653-658K. Kawakami, T. Yoshida
The Influence of Organic Cosolvents on the Lipase Catalyzed Hydrolysis of DecylchloroacetatePages 659-666J.G.T. Kierkels, E.T.F. Geladé, L.F.W. Vleugels, D.P. Vermeulen, C. Wandrey, W.J.J. van den Tweel, J. Kamphuis
Biotransformation of Benzaldehyde to Benzyl Alcohol by Whole Cells and Cell Extracts of Baker's Yeast in Two-Phase SystemsPages 667-673P. Nikolova, O.P. Ward
Production of Phenylacetyl Carbinol by Biotransformation Using Baker's Yeast in Two-Phase SystemsPages 675-680P. Nikolova, O.P. Ward
Stability and Activity of Cholesterol Oxidase in Supramolecular SystemsPages 683-690F. Alfani, S. Brandani, M. Cantarella, G. Savelli
Crown Ethers Can Enhance Enzyme Activity in Organic SolventsPages 691-695Jaap Broos, Willem Verboom, Johan F.J. Engbersen, David N. Reinhoudt
Dynamics, Structure and Stability of α-Chymotrypsin in Aqueous Solution and in Reverse Micelles as Studied by Fluorescence SpectroscopyPages 697-704V. Dorovska-Taran, C. Veeger, A.J.W.G. Visser
Comparison of Activity and Stability of Enzymes Suspended in Organic Solvents and Dissolved in Water-in-Oil MicroemulsionsPages 705-712Eleftheria Skrika-Alexopoulos, Jacqueline Muir, Robert B Freedman
Batch and Continuous Lipolysis/Product Separation in a Reversed Micellar Membrane BioreactorPages 713-718D.M.F. Prazeres, F.A.P. Garcia, J.M.S. Cabral

Citation preview

Progress in Biotechnology Volume 1

New Approaches to Research on Cereal Carbohydrates (Hill and Munck, Editors)

Volume 2

Biology of Anaerobic Bacteria (Dubourguier et al., Editors)

Volume 3

Modifications and Applications of Industrial Polysaccharides (Yalpani, Editor)

Volume 4

Interbiotech '87. Enzyme Technologies (Blazej and Zemek, Editors)

Volume 5

In Vitro Immunization in Hybridoma Technology (Borrebaeck, Editor)

Volume 6

Interbiotech '89. Mathematical Modelling in Biotechnology (Blazej and Ottova, Editors)

Volume 7

Xylans and Xylanases (Visser et al., Editors)

Volume 8

Biocatalysis in Non-Convential Media (Tramper et al., Editors)

Progress in Biotechnology 8

Biocatalysis in Non-Conventional Media Proceedings of an International Symposium Noordwijkerhout, 26-29 April 1992

Edited by J. Tramper M.H. Vermüe H.H. Beeftink Department of Process Engineering Agricultural University Wageningen, The Netherlands

U. von Stockar Institut de Génie Chimique Département de Chimie Lausanne, Switzerland

ELSEVIER Amsterdam — London — New York — Tokyo 1992

ELSEVIER SCIENCE PUBLISHERS B.V. Sara Burgerhartstraat 25 P.O. Box 211, 1000 AE Amsterdam, The Netherlands

L i b r a r y of Congress C a t a l o g 1 n g - 1 n - P u b l 1 c a t i o n

Data

B i o c a t a l y s i s i n n o n - c o n v e n t i o n a l m e d i a : p r o c e e d i n g s o f an i n t e r n a t i o n a l s y m p o s i u m , N o o r d w i j k e r h o u t , 2 6 - 2 9 A p r i l 1992 / e d i t e d by J . T r a m p e r . . . [ e t a l . ] . p. cm. — ( P r o g r e s s i n b i o t e c h n o l o g y ; 8 ) ISBN 0 - 4 4 4 - 8 9 0 4 6 - 7 (acid-free) 1. B i o r e a c t o r s — C o n g r e s s e s . 2. I m m o b i l i z e d enzymes—Congresses. I . T r a m p e r , J . , 1949. II. Series. T P 2 418 . 2 5 . B 5 5 B 5 4 1992 660 . 2 9 9 5 — d c 2 0 92-28341 CIP

ISBN 0-444-89046-7 © 1992 Elsevier Science Publishers B.V. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher, Elsevier Science Publishers B.V., Copyright & Permissions Department, P.O. Box 521, 1000 AM Amsterdam, The Netherlands. Special regulations for readers in the USA - This publication has been registered with the Copyright Clearance Center Inc. (CCC), Salem, Massachusetts. Information can be obtained from the CCC about conditions under which photocopies of parts of this publication may be made in the USA. All other copyright questions, including photocopying outside of the USA, should be referred to the copyright owner, Elsevier Science Publishers B.V., unless otherwise specified. No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. This book is printed on acid-free paper, pp. 525-532: copyright not transferred. Printed in The Netherlands

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PREFACE The idea to organize an international symposium on Fundamentals of Biocatalysis in Non-Conventional Media originated in the Working Party on Applied Biocatalysis of the European Federation of Biotechnology already quite a number of years ago as a result of earlier symposia and activities initiated by it. The main reason for this Working Party to organize symposia is to stimulate interest and activity in new areas of biocatalysis that could prove of importance to industry. This is usually pursued by initiating appropriate activities or international meetings, but it is not the intent or the politics of this Working Party to organize whole series of symposia on the same topic. Nevertheless, the one on Fundamentals of Biocatalysis in Non-Conventional Media can be understood as the last event in a succession of three symposia that were thematically related, although each one of them had a different topic and a different emphasis.

It began with the first symposium that our Working Party ever organized in its existence. It was devoted to Biocatalysis in Organic Syntheses and it was held almost exactly 7 years ago in the exact same facilities as the present one (réf. 1). Our aim in the Working Party at that time was to explore new possibilities for biocatalysis in an area traditionally reserved to organic synthesis. In organizing this first symposium the Working Party intended to identify those areas in which biocatalysis could be used as an alternative or complementary route to classical organic synthesis, and also to compare the respective merits and limitations of the two approaches.

It was at least partly as a result of the first symposium that the Working Party grew very sharply aware of one of the most important limitations on the side of biocatalysis: this approach tends to be restricted to aqueous media whereas many of the chemicals and molecules that are important to both chemical industry and to human society really require handling in organic solvents. It became obvious that if the application of biocatalysts could be extended beyond aqueous media to organic solvents, the potential contribution and usefulness of biocatalysis would immediately grow enormously and innumerable new and very promising applications would become accessible. The Working Party therefore decided quite quickly to collect the information then available and to stimulate work on Biocatalysis in Organic Media by organizing a symposium devoted to this topic. This second symposium was held only one and a half year later, that is in December 1986, in Wageningen (ref. 2).

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The Symposium on Biocatalysis in Organic Media was undoubtly a very stimulating event. A large number of novel applications have been presented for two phase organic-aqueous and aqueous two-phase systems. A considerable number of papers also demonstrated the usefulness of reverse micelles in order to maintain catalytically active enzyme molecules dispersed in an organic solvent. First hand information has been presented at this symposium on the then still novel work initiated by Prof. Klibanov and others concerning the use of dry enzymes directly in organic solvents. The now very well known log Ρ concept was for the first time discussed in great detail at a large meeting. Even at that time the crucial role of the water activity as a parameter for understanding the behaviour of enzymes in water poor systems was recognized and explained.

Despite all the excitement and the richness of results, the Working Party felt in analyzing the outcome of the symposium that much of the presented work had been obtained in a very empirical way. We concluded that there was probably no clear scientific base yet for a rational use of non-aqueous solvents in biocatalysis, due to an obvious lack in fundamental understanding of the phenomena associated with these systems. The questions we discussed in the Working Party at that time included, but were not restricted to the following examples: the activity as a parameter for water concentration had been pointed out clearly enough, but it seemed probable that a similar approach needed to be developed to characterize the effects of substrates, products and solvents. We might therefore need free energy relationships for all these chemicals. The role of pH in biocatalysts dispersed in organic liquids was discussed several times. It had been pointed out at the symposium that enzymes in organic solvents seemed to "remember" the pH of the last aqueous solution that they had seen before drying, and that this was the pH determining their activity in the organic solvents. This however appeared to be a rather fuzzy concept that clearly needed a better understanding. It was also felt that there was a great lack of data concerning mass transfer effects and thermodynamic phenomena in such non-aqueous solvent systems. Finally it was quite clear that the most important and central question about the influence of the solvents on both stability and activity of the biocatalyst itself had hardly been addressed in an appropriate way and was very far from being resolved.

As a result the Working Party decided that yet another symposium would have to be organized in due course, this time focussing specifically on Fundamentals of Biocatalysis in Non-Conventional Media. It is the results of this last symposium which are collected and presented in the present book.

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The fact that about 200 scientists from many parts of the world attended the symposium confirmed the relevance of its topic. We believe that the symposium did indeed improve our understanding of the Fundamentals of Biocatalysis in Non-Conventional Media, and we hope that the present book will contribute its share in helping interested scientists to gain insight into this fascinating topic of Applied Biocatalysis. The Editors, April 1992

REFERENCES 1) J. Tramper, H.C. van der Pias, P. Linko (Eds), Biocatalysts in Organic Synthesis, Elsevier, Amsterdam, 1985.

2) C. Laane, J. Tramper, M.D. Lilly (Eds), Biocatalysis in Organic Media, Elsevier, Amsterdam, 1987.

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ACKNOWLEDGEMENTS

The organizing committee of the international s y m p o s i u m "Fundamentals of Biocatalysis in Non-Conventional Media" acknowledges with gratitude the following organizations, that generously contributed to this s y m p o s i u m .

A n d e n o B V , Venlo, T h e Netherlands Applikon D e p e n d a b l e Instruments B V , S c h i e d a m , T h e Netherlands C o m m i s s i o n of the European C o m m u n i t i e s , Brussels, B e l g i u m D S M Research, Geleen, T h e Netherlands Q u e s t International B V , Naarden, The Netherlands Royal Gist-brocades N V , Delft, The Netherlands Unilever Research Laboratorium, Vlaardingen, T h e N e t h e r l a n d s W a g e n i n g e n Agricultural University, W a g e n i n g e n , T h e Netherlands

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

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Medium and biocatalyst engineering Ephraim Katchalski-Katzir Department of Membrane Research and Biophysics, The Weizmann Institute of Science, Rehovot 76100, Israel

1. INTRODUCTION The search for enzyme action in non-aqueous media seems a natural one, as many enzymes, or multienzyme complexes, including lipases, esterases, dehydrogenases, and oxido-reductive enzymes function within the cells in characteristic microenvironments which are often hydrophobic in nature. As a matter of fact when I started working on enzyme immobilization [1], I viewed these heterogeneous catalysts as potential models for enzymes embedded within the cell in organelles or lipid membranes. Rapid progress has been attained in the study of the stability and activity of enzymes in one-liquid-phase and two-liquid-phase systems. Indeed the range of lectures and variety of posters presented at this symposium, bear witness to the opening up of a new field of biocatalysis, both theoretical and applied in nature. Enzyme catalysis in monophasic organic solvent systems, occurs in the absence of a separate aqueous phase [2]. The enzyme, which itself contains an adequate amount of water, is usually suspended as a powder or in an immobilized form, adsorbed onto a suitable carrier. Both substrate and product are dissolved in the organic phase. A classification of biphasic systems was proposed some time ago by Lilly and Woodley [3]. They have indicated t h a t such reaction systems can be prepared and described as an emulsion in a continuous organic phase of soluble or immobilized biocatalysts dissolved or dispersed in a discrete aqueous phase [4]. The potential gains of employing enzymes in organic - as opposed to aqueous — media are rather obvious since such systems are characterized by: (1) an increased solubility of nonpolar reactants; (2) shifting of thermodynamic equilibria in favour of synthesis; (3) facilitating product recovery from low boiling temperature solvents and (4) improving thermal stability of the enzymes. Because of these advantages, enzymatic catalysis in monophasic organic solvents has been used in steroid oxidation, epoxidations and hydroxylations, phenolic polymerization, ester and peptide synthesis, and resolution of racemic mixtures [2].

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Similar progress has been recorded in the utilization of two-phase systems as is documented in the chapters dealing with two-liquid-phase systems. Also worth mentioning here is the remarkable progress attained in the understanding and application of enzymes embedded in reverse micelles suspended in organic solvent [5,6]. The lectures and discussions that follow will help us to gain a better understanding of the mode of action of enzymes in the nonconventional media. In particular, answers will be sought to the following questions: (1) How do enzymes remain catalytically active in organic solvents? (2) How do solvents and substrates affect enzyme structure and function? (3) What is the role of water in determining the activity of enzymes in organic solvents? (4) Can one predict the effect of a given solvent on the catalytic function of an enzyme?

2. THEORETICAL CONSIDERATIONS The above fundamental questions cannot be adequately answered without quantitative evaluation of the inter- and intramolecular forces prevailing in proteins upon their exposure to organic solvents. By now biophysicists have a good understanding of the thermodynamics of denaturation, and of the van der Waals, electrostatic, dipole-dipole and hydrogen bonding interactions in proteins dissolved in aqueous media (see for example [7]. Less well understood, however, is the nature of the alterations occurring in proteins as a result of their exposure to organic solvents. Since it is the enzyme's outer surface that is not only responsible for substrate recognition and modification, but also is exposed to the nonconventional environment, knowledge of the physical and chemical characteristics of protein surfaces may help us to understand enzyme behavior in the one- and two-liquid-phase systems. The molecular core of many enzymes and other globular proteins can roughly be represented by a tightly packed nucleus consisting mainly of hydrophobic amino acid residues. At their surface however these molecules seem to be relatively flexible and hydrophilic. The hydrophilic nature of the outer layer of the enzyme can account for the essential role of water in determining enzyme activity, whereas the flexibility of the surface amino acid residues might explain the molecular imprinting phenomenon [10,11] as well as the modification of enzyme enantioselectivity [12,13] in different organic solvents. The conformational memory' of enzymes in organic solvents and the conformational freezing of enzyme surfaces in the presence of excess ligand, are still not well understood. These phenomena disappear in water, a medium in which conformational fluctuations are restored.

3. THE ROLE OF WATER The role of enzyme-bound water in biocatalysis has still not been fully clarified. It is worth noting, however, that whereas some of the lipases act in organic solvent in the presence of minute amounts of water, other enzymes

5

reach optimal activity at a relatively high water.organic solvent ratio. It seems that all of the enzymes investigated so far require water for catalytic activity. In the organic solvent-water systems studied one has obviously to consider, as proposed by Hailing, the thermodynamic activity of water ( a w ) rather than the amount of water adsorbed by the protein [14]. This approach led to the important conclusion that at low water content ( a w less than 0.4) similar water adsorption isotherms are similar in each of the solvents studied and in the gas phase [15]. The presence of an organic solvent therefore seems to have little effect on the interaction between proteins and tightly bound water. It could further be demonstrated t h a t while nonpolar solvents increase the amount of water bound by the enzyme (at fixed a w ) , polar solvents (mainly EtOH) may reduce the amount of water bound by the enzyme, presumably by occupying part of the secondary hydration layers in place of water.

4. CHOICE OF ORGANIC SOLVENT It is not yet possible to issue reliable recommendations, based on theoretical considerations, with regard to the best organic solvent to be used in the one or two liquid phase enzyme systems for attaining the highest possible catalytic activity. One should therefore apply trial and error techniques whenever possible. Nevertheless, already at an early stage of the search for suitable organic media it became obvious that the hydrophobicity of the organic solvent plays a major role in determining catalytic activity. Various indicators representing hydrophobicity directly or indirectly, have been considered such as the δ-Hildebrand solubility parameter, the dielectric constant, the dipole moment, and the partition coefficient Ρ of the organic solvent defined as the ratio between the concentrations of the organic solvent in octanol and in water at equilibrium ρ _ [soluteJoctanol [S0lute] Water The best correlation between the hydrophobicity of the organic solvent and biocatalytic activity was obtained by the use of log Ρ [16]. In general, biocatalysis in organic solvents appears to be low in relatively hydrophilic solvents in which log Ρ < 2, is moderate in solvents where log Ρ is between 2 and 4, and high in hydrophobic solvents with log Ρ > 4. The specific action of the log Ρ > 4 solvents seems to result from the fact t h a t these solvents do not distort the essential water layer around the biocatalyst, thereby leaving the catalyst in an active state. Protection of the enzyme-bound water layer may be achieved by relatively hydrophobic supports or by soluble compounds such as polyalcohols stabilizing the structure of the bound layer. It thus seems reasonable to assume that in the presence of these compounds optimum enzyme activity is also attainable with log Ρ values lower than 4.

6

5. IMPORTANCE OF THE SUPPORT MATERIAL FOR ENZYMATIC ACTIVITY IN ORGANIC MEDIA Support m a t e r i a l s , or carriers, to which enzymes a r e adsorbed physically or bound chemically may effect the conformation, stability and activity of the bound enzymes. Carriers of various types have been shown to stabilize the bound enzyme, most probably by restriction of protein unfolding [17]. In addition to their direct effect on the conformation of the bound enzyme, however, the supporting materials are capable of creating around the enzyme a specific microenvironment that might either enhance or reduce its stability and activity. In aqueous systems, the effect of polyelectrolyte carriers on the mode of action of enzymes has been thoroughly investigated [18]. In organic media the amount and activity of the water retained by the support material are of major importance in determining enzyme activity. Indeed Adlercreutz [19] pointed out that the support material influences both the overall enzymic activity and the relative rates of the different catalytic steps. The choice of a suitable support material, characterized by a proper water-activity, is crucial for the successful employment of immobilized enzymes in organic solvents. At this stage the recent results of Wang et al. [20] should be mentioned. They immobilized their proteins on a carbohydrate polymer of aminoglucose units to form covalently bonded carbohydrate protein conjugates. In aqueous solution the conjugated enzymes showed about the same catalytic activity as the native enzymes. Moreover, conjugated proteases (e.g. chymotrypsin) were found catalytically active in organic solvents such as tetrahydrofuran, dioxane and acetonitrile, which denature and inactivate the native enzymes.

6. MOLECULAR IMPRINTING The flexibility of the amino acid side chains forming the protein surface is probably responsible for the induced conformational fit attained by proteins, under well-defined conditions, in the presence of a large excess of ligand. In the case of bovine serum albumin, for example, it was noted [10] that when the protein was dissolved in a concentrated solution of L-malic acid, the solution lyophilized and the solid residue thoroughly washed with tetrahydrofuran to extract all of the malic acid, the resulting (imprinted) protein was capable of preferentially binding to the ligand in anhydrous ethyl acetate. The imprinted protein lost this characteristic selectivity in water, a medium that probably facilitates flexibility of the binding region. Of particular note is the fact that imprinting could also be detected in a set of non-specific high molecular weight polymers, such as poly-L-aspartic acid. Induced stereo and substrate specificity were recently attained in chymotrypsin by bio-imprinting in anhydrous organic media [11]. The enzyme was precipitated with 1-propanol following molecular imprinting with Nacetyl-D-tryptophan. The precipitated enzyme was subsequently washed and dried under vacuum. Chymotrypsin prepared in this way could be used for the synthesis of N-acetyl-D-tryptophan ethyl ester. When small amounts of water were added to the enzyme in anhydrous solvent, the native stereospecificity was rapidly regained.

7

7. EFFECTS OF ORGANIC SOLVENTS ON THE ENANTIOSELECTWITY OF ENZYMES Recently it has been observed, to the surprise of the classical biochemists, t h a t different organic solvents can markedly affect the enantioselectivity of enzymes. Carrea et al. [12] for example, reports in this symposium t h a t water concentration, temperature and the nature of the organic solvent significantly influence not only the overall activity of lipases but also their enantioselectivity. Similarly, Fitzpatrick and Klibanov [13] found that the enantioselectivity of subtilisin Carlsberg in the transesterification between vinyl butyrate and homologous chiral alcohols in anhydrous solvents varies markedly with alteration of the medium in which the reaction is being carried out (dioxane, acetonitrile, tetrahydrofuran). The mechanism by which organic solvents affect the enantioselectivity of enzymes is not yet known, although it seems likely that the flexibility of the active site of the enzyme is involved. The use of chiral organic solvents may shed new light on this intriguing phenomenon.

8. ONE AND TWO LIQUID-PHASE SYSTEMS As the recent findings in connection with these systems have been thoroughly reviewed [2,4] I shall confine myself to a few comments on aspects of particular interest. With regard to the amount of water required for enzyme activity in a oneliquid-phase system, it is remarkable that chymotrypsin suspended in octane remains catalytically active even if only 50 molecules of water per enzyme molecule are present [21]. Other hydrolytic enzymes, such as subtilisin and various lipases may require even smaller amounts of water [2]. In many cases, however, much more water is needed. Horseradish peroxidase, for example, is more active by nearly two-orders of magnitude in toluene when 0.25% (v/v) aqueous buffer is added as compared with only 0.025% [2]. Organic solvents can also stabilize enzymes acting in a one-liquid-phase system. Porcine pancreatic lipase, for example, is completely inactivated within seconds at 100° C in an aqueous solution; but has a life time of 12 hours at 100° C in dry tributyrin containing heptanol [2]. In this connection it should be noted that when an enzyme is precipitated from aqueous solution at a given pH, its active site usually retains that which it possessed in the aqueous medium. Activity, and kinetics of an enzyme in an organic phase may thus be manipulated by the pH of the aqueous solution from which the desired enzyme preparation was obtained. Two liquid-phase biocatalytic systems were found to be particularly useful in alkene epoxidation [4]. Thus the addition of 20% (v/v) of cyclohexane to a fermentation broth of Pseudomonas oleovorans increased the conversion of 1,7 octadiene to epoxyacetone and diepoxyacetone from - 1 9 % to -90%. The two-liquid-phase bioreactor represents a most interesting model system for analysis of the mechanism and kinetics of the various steps involved. Several mass-transfer processes have to be taken into consideration, e.g. across the liquid/liquid interface, in the aqueous phase, across the

8

liquid/solid interface and in the biocatalyst phase. Recent progress in this direction promises a better quantitative evaluation of the processes involved in these bioreactor systems [4].

9. ENZYME DESIGN FOR NON-AQUEOUS MEDIA Relatively little theoretical information is available on the factors determining enzyme stability and activity in organic solvents so it is difficult to predict the proper manipulation which would enhance their stability and activity in non-conventional media. In general, the stability of enzymes in a non-aqueous media will be increased by tight internal packing, efficient hydrogen bonding, well located internal crosslinking, suitable salt bridges, and hydrophobic surfaces. Elucidation of the factors determining the conformational stability of thermostable enzymes at elevated temperatures, and of halophilic enzymes at high salt concentrations, is awaited with considerable interest. Experimentalists have meanwhile reported on some relevant findings concerning the possible design of enzymes for non-aqueous media. Matsumura et al. [22], for example, found that engineered disulphide mutants of phage T4 lysozyme, a disulphide-free enzyme, in which S-S crosslinking was carried out at amino acids 4-97, 9-164 and 21-142, are significantly more stable than the wild type protein. The recent work of Chen and Arnold [23] on random mutagenesis to enhance the activity of subtilisin Ε in polar organic media seems particularly promising, as it appears to offer a general technique for the preparation and selection of a desired enzyme mutant. As a matter of fact it was found that a triple mutant D60N + Q103R + N218S is 38 times more active than the wild-type subtilisin in 85% dimethyl formamide. The above authors concluded correctly that protein engineering is an effective approach to enhance enzyme activity in organic media.

10. CONCLUDING REMARKS A survey of the literature reveals a steady increasing body of information on the behavior of enzymes in nonconventional media. The number of enzymes examined so far is rather limited, but new important data can be expected as additional enzymes, particularly the highly stable and highly labile ones are tested. Some of the findings reported are rather surprising, and will have to be explained on a molecular level, in order to enable the design of new modified enzymes and the choice of novel media for enzyme catalysis. Among the surprising findings are: the stabilization of enzymes in hydrophobic media, the marked effects of different organic solvents on enzyme stability and activity; the solvent effect on enzyme enantioselectivity, and the molecular imprinting of proteins. Despite considerable progress in the evaluation of the activity of water bound to enzymes, no satisfactory theory is yet available to explain, on a molecular level, the role of water in determining enzyme activity.

9

Experimental and theoretical efforts should also be directed towards elucidation of the various effects of organic solvents on enzyme activity. As a first approximation one might assume that many of the enzymes so far investigated in liquid-phase systems, can be represented on a molecular level by a rigid, rather stable hydrophobic core, and a more flexible hydrophilic surface. It follows that in attempting to understand the behavior of enzymes in organic solvents one would need to elucidate the physical and chemical characteristics of the molecular surfaces of proteins. Recent attempts to acquire this information have been reviewed [8,9]. Finally, the recent success in the design of enzymes for non-aqueous media promises a supply of many new enzymes in the future. Studies of these enzymes will undoubtedly shed new light on the role of different amino acid residues and of the three dimensional s t r u c t u r e in determining the characteristic behaviour of enzymes in non-conventional media.

REFERENCES 1 LH. Silman and E . Katchalski, Ann. Rev. Biochem. 35 (1966) 873. 2 J . S . Dordick, Enzyme Microb. Technol. 11 (1989) 194. 3 M.D. Lilly and J.M. Woodley, in Biocatalysts in Organic Syntheses, pp. 179-192. ( J . Tramper, H.C. van der Pias, and P. Linko, eds.), Elsevier, Amsterdam, 1985. 4 L.E.S. Brink, J . Tramper, Κ. Ch. A.M. Luyben and K. Vant Riet, Enzyme Microb. Technol. 10 (1988) 736. 5 K. Martinek, A.V. Levashov, N. Klyachko, Y . L . Khmelnitski and I.V. Berezin, Eur. J . Biochem. 155 (1986) 453. 6 Y. L . Khmelnitsky, A.V. Levashov, N.L. Klyachko and K. Martinek, Enzyme Microb. Technol. 10 (1988) 710. 7 H. Scheraga, Chemical Scripta 29A (1989) 3. 8 M.L. Connoly, Biopolymers 25 (1986) 1229. 9 B.K. Shoichet and I.D. Kuntz, J . Mol. Biol. 221 (1991) 327. 10 K. Dabulis and A.M. Klibanov, Biotechnol. Bioeng. 39(2) (1992) 176. 11 M.-O. Mansson, M. Stahl and K. Mosbach, These Proceedings (1992). 12 G. Carrea, G. Ottolina, S. Riva and F. Seeundo, These Proceedings (1992). 13 P.A. Fitzpatrick and A.M. Klibanov. J . Am. Chem. Soc. 113 (1991) 3166. 14 P . J . Hailing and R.H. Valivety, These Proceedings (1992). 15 P.J. Hailing, Biochim. Biophys. Acta 1040 (1990) 225. 16 C. Laane, S. Borren, R. Hilhorst and C. Veeger, in Biocatalysis in Organic Media pp. 65-84 (C. Laane, J . Tramper and M.D. Lilly, eds.) Elsevier, Amsterdam 1987. 17 K. Mosbach (ed.), "Immobilized Enzymes", Methods Enzymol. Vol. 4 4 , Academic Press, New York (1976). 18 E . Katchalski, I. Silman and R. Goldman, Advan. Enzymol. 34 (1971) 445. 19 P. Adlercreutz, These Proceedings (1992). 20 P. Wang, T . J . Hill, C A . Wartchow, M.E. Huston, L.M. Oehler, M.B. Smith, M.D. Bednarski and M.R. Callstrom, J . Am. Chem. Soc. 114 (1992) 378. 21 A. Zaks, A.M. Klibanov, Proc. Natl. Acad. Sri. USA, 82 (1985) 3192. 22 M. Matsumura, G. Signor and B.W. Matthews, Nature 342 (1989) 291. 23 K. Chen and F.M. Arnold, Biotechnology 9 (1991) 1073.

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

13

Physical-chemical nature of low water systems for biocatalysis: especially phase behaviour, water activity and pH Peter J. Hailing and Rao H. Valivety Department of Bioscience & Biotechnology, University of Strathclyde, Glasgow G1 1XW, United Kingdom

Abstract

Thermodynamic analysis leads to useful predictions about the behaviour of biocatalysis in non-conventional media. Thermodynamic water activity is a good predictor of the optimal hydration conditions for catalytic activity, as other factors are changed, as well as of equilibrium positions. A number of methods are now available to set or control water activity in the reaction mixture. The pH of an inaccessible aqueous phase around the biocatalyst can be measured using special indicators in the organic phase, and is found sometimes to change in the presence of acidic reactants or products.

1. SOME GENERAL THERMODYNAMIC PRINCIPLES Most non-conventional media used for biocatalysis consist of more than 1 distinct phase. One phase is a relatively non-polar fluid, that acts as a reservoir and transport medium for at least some of the reactants. It is usually makes up the bulk of the reaction mixture, and is the continuous phase. Organic liquids, gases and supercritical fluids have all been used as the main component of this phase. The other phase is relatively polar, contains the biocatalyst, and is the site of the reaction. This catalyst phase is usually dispersed in the continuous phase, and may be of only microscopic dimensions (e.g. trapped within pores of a support particle). In some cases it can be a dilute aqueous solution, but it ranges all the way from this to solid enzymes with only traces of adsorbed water. Further phases may also be present, such as a gas headspace, or a solid support. Between the phases there are interfaces, which may be very important in the kinetic behaviour of the system, but normally do not significantly affect equilibria. Because of this multi-phase nature, the distribution of components between the phases is important. Assuming this reaches equilibrium (which may not always be true), then some useful predictions can be made from the basic thermodynamics of multi-phase systems. The composition of any one phase determines those of the others it is in equilibrium with, and hence may be used to predict them. The concentrations of individual components may of course be very different in the various phases; however thermodynamic activities will become equal, provided that the same standard state is used for each phase (i.e. avoiding those based on an activity coefficient of 1 for dilute solution in each solvent). Thus activities can be an attractive way to analyse and predict the observed behaviour. The relative volumes of the phases has no effect on the equilibrium distributions between them, or on equilibria within each; a very useful principle, not intuitively obvious, but which permits valuable predictions.

14

Most molecular processes are affected by the local concentrations in the phase in which they occur; the overall system composition does not influence them, and hence is not usually a very useful parameter for prediction. It might be said that most individual molecules are not aware of the presence and nature of other phases; only a small proportion are close to the interfaces at any one time. Thus much of the observed behaviour of the biocatalyst depends on the composition of the catalyst phase in which it is located. Similarly, the position of reaction equilibria in the non-polar phase (hence usually the bulk equilibria) are determined by solvation effects within that phase alone (independent of the presence and size of the catalyst phase).

2. EFFECTS OF WATER: GENERAL The precise level of remaining water is well known to be crucial to the performance of biocatalysts in most non-conventional media. Understanding and predicting the effects of water can be complicated, because it is typically distributed between all the phases present, so that total water content is a poor guide to behaviour. Some will be present in the bulk non-polar phase as a molecular solution, while some will be bound to the active enzyme molecules as hydration water. If a solid support is present, this too will bind some water, and a separate gas phase above an organic liquid can contain significant amounts of water vapour. In some cases water will also be present in a dilute aqueous solution. It is thus very useful to use the thermodynamic activity of water ( a w) in the analysis of the water relations of these systems. This is conventionally referred to a standard state of pure water at the same temperature, which thus has a w defined as 1. Hence, if water distribution reaches equilibrium, a w will become equal in all phases, and the entire system can be characterised by a single value. Some advantages of the use of a w are summarised in Table 1. The system a w is also linked to whether there is a second (polar) fluid phase, and its nature. If a w is close to 1, there must be a dilute aqueous solution phase somewhere in the system, and vice versa. If a w is significantly less than 1 , then no such phase can be present, only a concentrated aqueous solution, a semisolid dispersion or a solid with adsorbed water. These relationships may be predictively useful in either direction. The uses of a w in the analysis of these systems has been discussed in more detail [1-5]. Table 1. Advantages of using a

w

1. By definition, equal in all phases at equilibrium. 2. Measurable or controllable via relative humidity of equilibrated gas phase. 3. By definition, determines water mass action effects on hydrolytic equilibria.

3. WATER EFFECTS ON ACTIVITY Water has important effects on the activity of biocatalysts. Intuitively it seems reasonable to think that the water level in the immediate environment of the relevant enzyme molecules will be important, rather than that of the system as a whole. This was elegantly demonstrated experimentally by Zaks & Klibanov [6], who measured the amount of water actually bound to the solid enzyme particles. This would in turn be expected theoretically to be predicted, at least in part, by the a w of the system [3].

15

Studies have been reported of the extent of water adsorption by enzymes and other proteins in organic media, as a function of water concentration in the organic phase [6,7]. The relationship may be further explored by recalculating these data in terms of a w , [8] using activity coefficients from vapour-liquid equilibrium data. An example of the resulting isotherms is shown in Fig. 1. The isotherms for 4 proteins in solvents from a wide range of polarities were found to be rather similar to each other, and to those for most globular proteins in air, at least in the lower range of a w values [8]. Hence θ!ΐ 02 03 04 0.5 it seems that the presence of the < Water activity organic solvent does not greatly affect the higher affinity binding of water by Fig. 1. Water adsorption by proteins the protein. There are deviations at from air and from organic solvents. higher a w , with polar solvents reducing Lactoglobulin (open symbols), the amount of water bound (presumably bovine serum albumin (filled by replacement), while non-polar symbols); in air (O and line), ethanol solvents increase it. ( • ) , benzene (Δ) and ethyl acetate If a w determines water binding by the (V). Data from [1,2], recalculated [3] enzyme, and this in turn controls using activity coefficients estimated catalytic activity, then a w should from vapour-liquid equilibrium data. directly predict water effects on activity, at least when changing solvent. A direct experimental test with the lipase from Rhizomucor miehei confirmed this [5]. In solvents ranging in polarity from hexane to pentanone, optimal activity was found with a w about 0.55. The conventional presentation of these results, in terms of water concentration dissolved in the organic phase, showed the usual very different optimum levels, increasing with the solvent polarity. Different enzymes however differ in their profile of activity as a function of a w . Lipases from 7 different sources showed widely ranging behaviour, with some retaining high activity at a w 0.1 and less, while others needed up to 0.7 for good activity [9]. The lipase from Rh. miehei retains high activity even at the lowest a w values (obtained by drying over molecular sieve). The lower limit has been studied in more detail [10], using dry box techniques ( P 2 O 5 ) for the exclusion of environmental water (never before thought necessary during studies with enzymes, but essential at these low a w values). As well as freshly reactivated molecular sieve, we used two drying agents that act by interconversion of known chemical species, MgO and anhydrous C U S O 4 , for which a better defined a w could be estimated from literature data for water vapour pressures or free energies of formation. Table 2 shows that this lipase retains substantial activity even after exhaustive drying for over 90 days, with most rates not substantially different from those after initial drying. The significant fall in alcoholyis activity with molecular sieve may reflect batch differences with a drying agent acting by adsorption. All these rates are a significant fraction of those at higher a w values of 0.12 or 0.55 (the optimum for esterification [5]). This lower a w limit for good activity, of less than 0 . 0 0 0 1 , is orders of magnitude lower than those reported earlier for a range of different enzymes and conditions (Table 3).

16

Table 2. Initial reaction rates catalysed by exhaustively dried immobilised Rh. miehei lipase. [10] Pre-incubated with: Molecular sieve MgO/Mg(OH)2 CUSO4/CUSO4.H2O

(Initial drying) LiCI sat. Mg(N03>2 sat.

Water activity

1

1

Reaction rate (mmole s * kg" ) alcoholysis esterification a a a a AER PP AER PP

? 6 4-14 X 1 0 " 4 4-8 Χ Ι Ο "

0.10 0.39 0.32

2.1 2.8 2.8

0.69 0.76 0.69

3.7 3.5 3.7

< 0.01 0.12 0.55

0.10 0.32

2.8 3.1 -

0.76 0.83 1.77

3.9 4.9 10.3

-

a - The lipase was adsorbed to either anion exchange resin (AER) or macroporous polypropylene (PP). Catalyst and organic phase were pre-dried over molecular sieve, then further equilibrated with drying agents. The second drying stage could not be monitored by catalyst weight (no further detectable change) or organic phase water content (already zero by Karl Fischer), so the rate of subsequent reaction was used. Those shown are after at least 3 months; the rate was no greater after 7 days. Because of the noticeable acceleration in rate due to product water during esterification, activity was also determined in a transesterification reaction: alcoholysis (octanol) of dodecyl decanoate. Table 3. Minimum water level for enzymic activity System Powdered lysozyme. other enzymes Enzymes in foods Lipase in organic medium Chymotrypsin in organic media Alcohol oxidase in gas phase

Reported water level

Equivalent a w

Authors

0.07 g/g

0.2-0.3

Rupley et al [11]

a

w

0.1

0.1

Potthast [12]

a

w

0.1

0.1

Hailing & Macrae [13]

0.02 g/g

0.1

Zaks & Klibanov [14]

a

0.1

Barzana et al [15]

w

0.1

The retention of activity at extremely low a w may reflect: a) No requirement for water in the action of this lipase; b) Essential water is tightly bound (thermodynamically); or c) Water desorption rates are very slow. Hysteresis may make (b) and (c) hard to distinguish; our studies seem to relate to at least a metastable state. We attempted to determine residual water on the catalyst by Karl Fischer titration after extraction with dried dimethyl sulfoxide, finding 0.004 g/g (anionexchange resin) and 0.0025 g/g (polypropylene) [10]. However, the method is prone to error and interference, and these values may not be reliable.

17

4. WATER ACTIVITY CONTROL 100

As the determinant of water mass action, a w should affect the position of hydrolytic equilibria, and this has been shown to occur with organic biocatalysis [1, 13]. A comprehensive study of the effects on lipase-catalysed acylglycerol hydrolyses was reported by Muderwha et al 0 10 20 30 40 50 [16]. A recent demonstration time (h) from our laboratory [17] is shown in Fig. 2, for amino acid Fig. 2. Progress of Ac-Trp-OEt synthesis by agarose-chymotrypsin in ester synthesis, which also catalysed indicates the effect of a w on pentan-3-one at different a w . [17] At the start of the reaction water was reaction rate. (Note how the progress curves cross, as the removed via the headspace as necessary to lower initial rates at reduced a w bring measured a w to: close to 1 (I), 0.9 eventually proceed to higher ( · ) , 0.7 ( • ) , 0.6 (Δ), 0.4 (O), 0.25 (Δ). equilibrium yields.) Thus it may be seen that the optimal a w for many biocatalytic processes will be determined by the balance of two conflicting objectives. The equilibrium yield of a reverse hydrolysis, or the minimisation of an undesirable hydrolytic side reaction, will be favoured by use of the lowest possible a w . However, if a w is reduced too far, the biocatalyst activity will be greatly decreased (except for exceptional enzymes like Rh. miehei lipase). Hence there will usually be an optimum a w value, not close to either of those easily approached, 1 (with excess water) and 0 (with exhaustive drying). As a result, some means of controlling a w at the optimum intermediate value would be desirable. Some methods of setting or controlling a w in laboratory-scale organic reaction mixtures are listed in Table 4. Some further comments will be made on the last two, most recently investigated, methods. Kuhl et al [22] reported that chymotrypsin remained active in organic media in which water was supplied only in the form of the water of hydration in N a 2 C 0 3 . 1 0 H 2 0 . This was identified [1] as a system in which a w must be signifcantly less than 1. Later, we were able to collaborate in demonstrating that this salt hydrate acted to control the a w of the system [20]. Table 4. How to set or control water activity in organic media 1. Add pre-equilibrated silica gel to reaction mixture (Hailing & Macrae [13]) 2. Pre-equilibrate both phases with saturated salt solutions (Goderis et al [18]) 3. Use a w sensor and controlled drying of headspace (Khan et al [19]; Blanco et al [17]) 4. Recycle headspace gases through saturated salt solution (Patterson et al, unpublished) 5. Add solid salt hydrate(s) to reaction mixture (Kuhl & Hailing [20]) 6. Allow leak of wet air into evacuated headspace (Lacerda et al [21])

18

A salt hydrate will exhibit a certain partial pressure for water dissociation, converting to a lower hydrate or the anhydrous form. In theory, ideal behaviour is for the entire transformation to take place at a fixed water vapour pressure; provided a trace of each solid form remains, the equilibrium is not sensitive to their relative proportions. Many salt hydrate pairs are found experimentally to approach this ideal behaviour. A fixed water partial pressure corresponds to a fixed a w . For example, Na2CO3.10H2O dissociates to N a 2 C 0 3 . 7 H 2 0 with a pressure of 16.4 mbar at 20 ° C , giving an a w of 0.72. In a biocatalytic reaction mixture, therefore, salt hydrates can act to Water content (g/l) control or "buffer" a w at a fixed value. 50 This value is determined by the choice of the hydrate pair used. Fig. 3 illustrates how they act, showing the effective water adsorption isotherm for Na2HP04, which forms 3 different hydrates, and a typical smooth isotherm for the rest of the system (dissolved 30 water, enzyme-bound water etc). The combined isotherm for the mixed system has the 3 fixed a w sections from the 20 Salt salt hydrates. At these points, considerable changes in total water Tn»»l content (e.g. by exchange with the ^^-"Rest environment, or production or consumption in a reaction) do not affect the a w value, which is buffered by the —I 1 L salt hydrates so long as at least some of 0.2 0.4 0.6 0.8 each form remains. The theoretical and observed behaviour of salt hydrates, and Water activity data on salts giving a range of a w Fig. 3. Water activity buffering by values, is presented in more detail [23]. 1 The addition of salt hydrates is proving hydrated salts. to be a simple and attractive method for Na2HP04 (20 g I" ), and typical during laboratory-scale isotherm for rest of organic reaction fixing aw biocatalytic reactions. A number of salts mixture. have been found to be compatible with biocatalyst activity during protease-catalysed peptide synthesis [24], lipasecatalysed esterification [25], and catechol oxidation by polyphenol oxidase [26]. With lipases, the salt hydrates were shown to be able to maintain near optimal conditions, as expected, when changing the reactant or enzyme concentrations, or the identity of the solvent [25]. Partial evacuation of the reactor headspace has been used by several groups as an attractive means of removing product water during esterification in nonvolatile liquid reactant mixtures. Analysis of the temperature and pressure conditions reported to be successful indicate that simple removal of water into the vacuum line cannot account for the observed effects on equilibrium. One additional factor that may contribute is a significant leak of ambient air into the reactor, and the effectiveness of an intentional leak has been demonstrated experimentally [21]. Further analysis indicates that by (somewhat surprisingly) water-saturating the inlet gas, water activity can be conveniently controlled. For a sufficiently fast flow of gas through the leak and the vacuum line, and fast mass transfer between the reaction mixture and the headspace, the steady state a w is given by the following equation

I

19

Limiting a

w

=

Ph-Pi Po-Po

where P's are total pressures in the headspace (Ph) and atmosphere (P 0); and p's are water partial pressures, in the inlet leak before expansion (pj), and saturated at the reaction temperature ( p 0) . The term Pj/p 0 will usually be less than 1 (depending on reaction and water temperatures), so a w can be set at any desired lower value by control of PhAn initial demonstration of the use of such an a w control strategy is presented in Fig. 4. Evacuation to 0.2 bar causes some equilibrium shift in favour of synthesis, probably due to a small unintentional leak. A deliberate leak of air water-saturated at 2 0 ° C is expected to control a w at 0.0233 in the reaction mixture at 60 ° C . The result is a reduced initial rate but further increased equilibrium yield, as expected.

100

Conversion to ester (%) 0.2 bar + wet leak,

40

Reaction time (h)

Fig. 4. Effect of headspace evacuation and inlet leak on synthesis of solketal decanoate by immobilised Rh. miehei lipase. [21]. Leak 6.5 crri3 s - 1 1

5. MEASUREMENT AND EFFECTS OF pH it is well accepted that biocatalysts to be used in non-conventional media should preferably be adjusted to their optimal pH during preparation, when they are last in a bulk aqueous phase. However, there has been some speculation as to whether effective pH can change subsequently, for example as a result of acidic or basic reactants, and hence explain observed poor reaction rates. The limitation was the lack of a reliable method for the determination of the pH in the catalyst phase, which is inaccessible to direct measurement. Here at Strathclyde, some very hydrophobic indicators have been Organic developed that remain in the organic phase throughout, in both neutral and phase ionised forms, but which nevertheless respond to the pH of a microscopic In" aqueous phase around the enzyme molecules. The indicators are large branched alkyl esters of fluorescein, Hin specially synthesised [27] under the direction of Colin Suckling of our HH Chemistry Department. The principle of how they can respond to aqueous pH is illustrated in Fig. 5. The need for electroneutral partitioning between the phases means that estimation of pH requires knowledge of the concentration Fig. 5. Action of hydrophobic pH (or activity) of a monovalent cation indicators. (normally Na + ) in the aqueous phase; this is however usually better known

11

20

and less prone to change than pH. The 0.50. behaviour of the indicators was studied first in a system with a bulk aqueous phase whose pH could be independently monitored and adjusted, to show that the expected titration curves could be observed (e.g. Fig. 6) (see also [28, c 29]). Again as theoretically expected, ο the indicator response was found to be υ unchanged as the volume fraction of the ö aqueous phase was progressively reduced; thus we could be fairly 0.25. confident this would continue when the aqueous phase became too small for

Figure 1. Apparent enantioselectivity of hydrolysis or synthesis o f ethyl chloropropionate by Carboxyl Esterase NP. Experimental values were obtained by fitting curves o f enantiomeric excess vs. conversion. T h e line is equation 2 2 (AT* = 3.4 M , K? = 7.7 M ) .

8. R E F E R E N C E S Bell, R.P., J . E . Chrichlow and M.I. Page ( 1 9 7 4 ) / . Chem Soc., Perkin Trans. II, 2, 66-70. Chen, C.C., Y . Zhu and B . Evans (1989) Biotechnol Progr., 5, 111-118. Clement, G . E . , and M . L . Bender (1963) Biochemistry, 2, 836-843.

Dordick, J . S . (1989) Enzyme Microb. Technol, 11, 194-211. Fitzpatrick, P.A., and A M . Klibanov (1991) / . Am. Chem. Soc, 113, 3166-3171.

Kieboom, A.P.G. (1988) Reel Trav. Chim. Pays-Bas, 107, 347-348. Leo, A.L., C. Hansch and D. Elkins (1971) Chem. Rev., 7 1 , 525-616. Martinek, K., A.N. Semenov and I.V. Berezin (1981) Biochim. Biophys. Acta, 658, 76-89. Nakanishi, Κ , Y . Kimura and R . Matsuno (1986) Eur. J. Biochem. 1 6 1 , 541-549. Ramelmeier, R.A., and H.W. Blanch (1990) Biocatalysis, 4, 113-139. Straathof, A . J . J . , J . L . L . Rakels and J . J . Heijnen (1992) Biocatalysis, in press. Valivety, R.H., G.A. Johnson, C.J. Suckling and P.J. Hailing (1991) Biotechnol. Bioeng., 38, 1137-1143.

SESSION V TWO-LIQUID-PHASE SYSTEMS I Chairman: R. Hilhorst

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

147

Process engineering of two-liquid phase biocatalysis J o h n M . W o o d l e y and Malcolm D . Lilly Advanced Centre for Biochemical Engineering, Department o f Chemical and Biochemical Engineering, University College London, Torrington Place, L o n d o n W C 1 E 7 J E , United Kingdom. In the last decade there has been much interest in two-liquid p h a s e biocatalysis. Advances in the biochemical engineering o f two-liquid phase biocatalytic p r o c e s s e s have included the development o f a body of knowledge including established principles and rules, as well as experimental tools to collect the n e c e s s a r y data. E a c h o f these advances is discussed and illustrated with experimental results from the hydroxylation o f toluene by Pseudomonas putida to its corresponding dihydrodiol, Δ1 -dehydrogenation o f hydrocortisone by Arthrobacter simplex and hydrolysis o f benzyl a c e t a t e by pig liver esterase. Future research directions are addressed. 1. I N T R O D U C T I O N In 1 9 8 2 when w e first reviewed two-liquid phase biocatalysis [ 1 ] the subject w a s in its infancy and little was understood about the interactions between the biological catalyst, the organic phase and the process. Substantial progress has b e e n made during the last decade [ 2 , 3 ] and there have been important developments,

ESTABLISHED PRINCIPLES

SELECTION OF PROCESS ELEMENTS

EXPERIMENTAL TOOLS

• Location of reaction

· Organic phase

·

Solvent-substrate

• Substrate/product transfer

·

Biocatalyst

·

Biocatalyst-solvent

• Phase ratio/partitioning

·

Process operations

·

Biocatalyst-substrate

V PROCESS

UNDERSTANDING

1

V INTEGRATED

Figure 1. Overview o f advances in process engineering biocatalysis.

DESIGN

o f two-liquid

phase

148

including validation of the concept (for example, introduction of a second liquid phase to enhance reactor concentrations of poorly water-soluble organic components [ 4 ] and use of a two-phase system to reduce the inhibitory affects o f poorly water-soluble organic substrates and products [ 5 ] ) . In addition we have seen the first industrial exploitation of two-liquid phase biocatalytic processes [ 6 ] , in particular o f hydrolytic and oxidative biotransformations. In parallel the biochemical engineering framework has been advanced by a body o f knowledge including established principles and rules as well as experimental tools allowing rational process design. This framework is illustrated in Figure 1 and in this paper we give an overview o f each o f these advances. 2. ESTABLISHED PRINCIPLES A s knowledge in the area of two-liquid phase biocatalysis has grown, principles which embody the two-liquid biocatalytic concept have b e c o m e established. O n e key group o f principles relates to the understanding of the interaction between the substrate and the biocatalyst. Poorly water-soluble organic substrate may compose the organic phase itself or alternatively, b e dissolved in an added water-immiscible organic solvent. T h i s has implications for the required substrate transfer within the organic phase to the liquid-liquid interface [ 6 ] . Equally important is the transfer o f poorly water-soluble organic substrate from the liquid-liquid interface to the biocatalyst, m o s t likely within the bulk of the aqueous phase. Here a distinction can be drawn between those biotransformations where catalysis occurs at the liquid-liquid interface and those where catalysis takes place in the bulk of the aqueous phase. F o r both types of two-liquid phase biocatalysis, models have been developed to describe the mass transfer/biotransformation concept and in the latter case the model has been validated for poorly water-soluble organic substrate and product transfer in the presence o f both isolated enzyme and intact cell catalysts in a Lewis cell [ 7 ] and for substrate transfer in a stirred tank reactor [ 8 ] . Principles have also been established for the consequences o f alteration to the phase ratio (fraction of reaction volume occupied by organic phase) [ 6 ] . T h i s affects both reactor concentration of water-soluble and poorly water-soluble organic components and elimination or reduction of poorly water-soluble organic substrate or product inhibition respectively, all of which are dependent also upon component partitioning between the two liquid phases. 3. R U L E S In addition to these established principles, based upon the underlying biochemical engineering science, another body of knowledge has been developed based on observation, leading to general rules. This now provides the first basis for selection of biocatalyst, organic phase and process operations, including reactor and downstream processing. T h e use of rules for the selection of an organic solvent is described in more detail in the following section. T h e search for an indicator characteristic of a particular biocatalyst tolerance to

149

an organic solvent has led to various proposals. O n e of the most comprehensive early studies examined the relationship between solvent molecular weight and the Hildebrand solubility parameter with maintenance of biocatalytic activity in the presence o f that solvent [ 9 ] . More recently it was suggested that solvents with log Ρ (logarithm o f the partition coefficient o f an organic solvent in a standard octanol-water system) greater than 4 were most suitable while use o f those with a lower log Ρ resulted in considerable loss of activity [ 1 0 ] . However, further studies in our laboratory revealed that while the trend of increased activity with increased log Ρ appears universal, the solvent log Ρ - biocatalyst activity profile is specific to a particular biocatalyst and the extent o f its contact with the organic phase. For example differences have been observed between two liquid-phase biotransformations carried out in shaken flasks and stirred reactors [ 1 1 ] and between the use o f Gram-positive and Gram-negative bacteria as catalysts [ 1 2 ] . W h i l e immobilisation may protect Gram-positive bacteria from solvent damage this has not been conclusively observed for Gram-negative bacteria [ 1 3 ] . Such observations underline the empirical nature o f these rules and provide guidance for the further research required to understand the way in which different organic solvents damage different types o f biocatalyst. T h e s e studies will require examination o f the precise role o f the frequency, residence time and amount of the liquid/liquid interfacial contact with the biocatalyst. Guidance rules for selection of process parameters have also been established for phase ratio, agitation rate and catalyst concentration. Evaluation o f reactor and downstream operations is currently being addressed. 4. E X P E R I M E N T A L T O O L S W h i l e the preceding rules are not based on a complete understanding they nevertheless provide useful design guidelines for biotransformation processes. Consequently, these provide the starting point of a design by eliminating those options which are not worthy o f further study. Complementary to this knowledge is the development o f a set of experimental tools: protocols of experiments specifically designed to increase understanding and collect relevant process data (for design and operation). Such experiments are carried out in three distinct modes o f organic component (poorly water-soluble organic substrate or product) - biocatalyst contact: in an aqueous phase alone (with the organic component dissolved in the aqueous phase beneath the saturation concentration); in a biphasic aqueous-organic phase contactor using a flat liquid-liquid interface (with the organic component distributed between the phases in a Lewis cell) and; in a biphasic aqueous-organic dispersion o f one phase within the other (with the organic component distributed between the phases). In each case a series of measurements (kinetics, equilibria and, for microbial catalysts, suspension capacitance) may be made yielding data essential for process understanding and the successful design and scale-up from a laboratory biotransformation. Table 1 indicates which measurements are required in the three modes o f organic component - biocatalyst contactor in order to yield information characterising the organic solvent - substrate interaction (component partition, substrate saturation concentration and mass transfer kinetics with a particular

150

Table 1 Experimental tools

Measurements made

Aqueous

Aqueous-organic

Interaction Stirred tank

L e w i s cell

Stirred tank

Organic Solvent - substrate Partition

Equilibrium

Saturation

Concentration

M a s s transfer

M a s s transfer kinetics

Biocatalyst -solvent R e a c t i o n location

Biocatalyst kinetics

M a s s transfer kinetics Biocatalyst kinetics

Kinetics (with second phase)

Stability (range o f solvents)

Biocatalyst stability

Recovery

Biocatalyst kinetics

Biocatalyst* kinetics

Biocatalyst stability

Biocatalyst stability Phase separation

Biocatalyst - substrate Kinetics

Biocatalyst kinetics

W h e r e measurements to be made are boxed this implies data combination (dashed line) or comparison (full line). * Can also be used to indirectly determine the mass transfer kinetics in a stirred tank [ 8 ]

151

solvent), biocatalyst - organic solvent interaction (reaction location, biocatalyst kinetics, stability and phase separation in the presence o f a particular solvent) and biocatalyst - substrate interaction (biocatalyst kinetics). In s o m e c a s e s data collection requires direct routine measurements of component concentrations (as a function o f time) or indirect measurements (e.g. by comparison or by combination and comparison o f direct results) in standard laboratory reactors. In other c a s e s the use o f non-standard measurements (e.g. a dielectric permittivity probe to measure capacitance of microbial catalyst suspensions) and non-standard contactors (e.g. Lewis cell) is advocated. In the following section the use of a Lewis cell contactor and a dielectric permittivity probe, illustrative of the experimental tools now available, will be described in greater detail. 4 . 1 . Use of a Lewis Cell contactor A s previously indicated knowledge of the location of the reaction within the biocatalytic medium is o f key importance for process design. O n e method for elucidating this is a set o f experiments carried out in a defined flat liquid/liquid interface apparatus (Lewis cell) [ 1 4 ] . In such a device the rate o f m a s s transfer o f substrate from organic to aqueous phase under defined conditions can b e measured and thus a mass transfer coefficient obtained. Such information can b e combined with the aqueous phase biocatalyst kinetics (measured in an all aqueous phase solution with dissolved levels of poorly water-soluble organic substrate beneath saturation concentration) to predict substrate and product concentration - time profiles in a Lewis cell with biocatalyst present, which can then be compared with those measured experimentally. This technique is valid both for microbial and enzymically catalysed biotransformations [7, 1 4 ] . Figure 2 is a plot o f data for the hydrolysis o f benzyl acetate by pig liver esterase in a two-liquid phase system in a _ 2 4 . 2 m l (aqueous phase basis) specific interfacial area Lewis Cell. T h e data show substrate and product concentration - time profiles with 0 . 0 1 g 1"! pig liver esterase in 2 0 0 ml o f aqueous phase catalysing the hydrolysis o f 3 0 0 ml o f benzyl acetate (lower organic phase). Both phases were well mixed without disturbing the flat liquid-liquid interface using turbine impellers rotating at 1 2 0 rpm. T h e measured substrate m a s s transfer coefficient in the absence of biotransformation was 0 . 6 4 h"l. Close agreement was observed between the steady-state aqueous phase - 1 substrate concentration ( 0 . 6 m M ) and reaction rate ( 0 . 1 4 /zmol min-1 m l ) predicted by the aqueous phase bulk reaction model and the measured values. T h e Lewis cell may also be used to measure the partitioning of substrates and products between the phases. T h e generic use of the Lewis cell lies in the ability it gives to expose biocatalysts to defined amounts o f interface and consequently the L e w i s cell has a role in determining interfacial effects not only upon biocatalyst kinetics as illustrated here but also upon operational stability. 4 . 2 . Dielectric Permittivity Measurements T h e solvent tolerance of a microbial catalyst is a critical factor in its operational stability in a two liquid-phase reactor. This can be assessed by measuring the capacitance o f microbial suspensions when exposed to organic solvents using a novel dielectric permittivity probe [ 1 5 ] . T h e technique allows rapid evaluation o f

152

the biocatalyst compatibility of solvents. A s indicated earlier organic solvent damage is not j u s t a function of the solvent itself but the amount and time for which the biocatalyst is exposed to a solvent. This is illustrated by the results in Figure 3 for exposure of Saccharomyces cerevisiae to «-octanol [ 1 6 ] . Such measurements in the three types of reactor listed in Table 1 and other studies allow the role o f solvents in damaging microorganisms to be determined.

0

10

20

30

40

50

60

70

Time (min)

Figure 2 . Benzyl acetate hydrolysis by pig liver esterase in a Lewis cell with an aqueous phase saturated with benzyl acetate prior to enzyme addition. Aqueous phase substrate ( · ) and reactor product ( O ) concentrations.

0

10

20

30

40

50

60

Exposure time (min)

Figure 3 . Changes in the capacitance of a S.cerevisiae suspension, measured by dielectric permittivity, in a twoliquid phase reactor containing octanol added at 0.4 (A), 0.8 ( · ) and 1.2 ( • ) % by volume.

153 5. P R O C E S S APPLICATION In the preceding section we have described experimental tools for characterising two-liquid phase biotransformations. This has been illustrated using several enzymic and microbial reactions. T h e s e techniques have also been applied successfully in our laboratory to the complete process evaluation o f two biotransformations: phenol polymerisation by peroxidase and toluene hydroxylation to its cis-dihydrodiol by a strain of Pseudomonas punda. F o r the latter reaction, the tools have been used to determine the aqueous phase toluene concentration required for maximum reaction rate, the level at which toluene becomes toxic to the bacteria, the aqueous phase solubility o f toluene, its rate of transfer from the organic to aqueous p h a s e and the location of the reaction in the medium [ 7 , 1 7 ] . T h i s has allowed the various process options to be evaluated and best engineering solutions to be determined [ 1 8 ] . T h e emphasis has now shifted to optimising the properties of the biological catalyst. 6. F O R W A R D L O O K T h e application o f two-liquid phase biotransformations has grown dramatically in recent years as their potential for novel and environmentally acceptable chemistry has b e c o m e recognised. In addition, process development has been supported by significant advances in the biochemical engineering of two-liquid phase biotransformation processes as illustrated in this paper. T h e process engineering makes the best possible use of a given biocatalyst but the development o f bioprocess design and experimental tools is also identifying the key properties required o f the biocatalyst. G e n e t i c and protein engineering are now in the position to start delivering catalysts with properties tailored to the process [ 1 9 , 2 0 ] . F o r instance, r D N A technology will allow enzymes to be produced in alternative hosts which are easier to grow and have properties more amenable to the reaction process. Protein engineering can lead to changes in catalyst substrate specificity and improved kinetics and stability. Furthermore, changes to the properties of the catalyst m a y ease p h a s e separation and product recovery. All these potential improvements in biocatalysts and hence the processes will be accomplished through a collaborative approach by biologists, chemists and biochemical engineers. 7. R E F E R E N C E S 1 2 3

4 5

M . D . Lilly, J . Chem. Tech. Biotechnol., 3 2 ( 1 9 8 2 ) 1 6 2 - 1 6 9 . G . Carrea, Trends Biotechnol., 2 ( 1 9 8 4 ) 1 0 2 - 1 0 6 . M. D . Lilly, A . J . Brazier, M. D. Hocknull, A. C. Williams, and J . M. W o o d l e y , in Biocatalysis in Organic Media (C. Laane, J . Tramper, and M . D . Lilly, eds), Elsevier, Amsterdam, 1 9 8 7 , pp 3 - 1 7 . C. L a r r o c h e , C. Creuly and J - B . G r o s , Biocatalysis, 5 ( 1 9 9 2 ) 1 6 3 - 1 7 3 . K. Furuhashi, M . Shintani and M. Takagi, Appl. Microbiol. Biotechnol., 2 3 (1986) 218-223.

154

6

7 8 9 10 11 12

13 14 15 16 17 18 19 20

M. D . Lilly, G . A. Dervakos and J. M. Woodley, in Opportunties in Biotransformations ( L . G . Copping, R . E . Martin, J. E . Pickett, C. Bucke and A . W . Bunch, eds), Elsevier, London, 1 9 9 0 , pp 5 - 1 6 . J. M . Woodley, A. J. Brazier and M. D. Lilly, Biotechnol. Bioeng., 3 7 ( 1 9 9 1 ) 133-140. J. M . Woodley, P. J. Cunnah and M. D. Lilly, Biocatalysis, 3 ( 1 9 9 1 ) 1-12. L . E . S. Brink and J. Tramper, Biotechnol. Bioeng., 2 7 ( 1 9 8 5 ) 1 2 5 8 - 1 2 6 9 . C. L a a n e , S. Boeren and K . V o s , Trends Biotechnol., 3 ( 1 9 8 5 ) 2 5 1 - 2 5 2 . M . D . Hocknull and M. D. Lilly, Enzyme Microb. Technol., 3 3 ( 1 9 9 0 ) 148-153. J. M . Woodley, A. J. Harrop and M. D. Lilly, in E n z y m e Engineering 1 0 (H, Okada, A . Tanaka and H. W . Blanch, eds.) N. Y . Acad. Sei., New Y o r k , 1 9 9 1 , pp 1 9 1 - 2 0 0 . A . J. Harrop, J. M . Woodley and M. D. Lilly, E n z y m e Microb. Technol., in press. J. M . Woodley, in Biocatalysis (D. A. Abramowicz, ed.) V a n Nostrand Reinhold, New Y o r k , 1 9 9 0 , pp 3 3 7 - 3 5 5 . N. Stoicheva, C. L . Davey, G . H. Markx and D. B . Kell, Biocatalysis, 2 ( 1 9 8 9 ) 245-255. R . Eglin, J. M . W o o d l e y and M. D. Lilly, in preparation A . J. Brazier, M. D. Lilly and A. B . Herbert, E n z y m e Microb. Technol., 12 (1990) 90-94. C. J. Hack, J. M . W o o d l e y and M. D. Lilly, in preparation. T . G . Graycar, in Biocatalysts for Industry (J.S. Dordick, ed.) Plenum Press, New Y o r k , 1 9 9 0 , pp 2 5 7 - 2 8 3 . E . Falch, Biotech. Adv., 9 ( 1 9 9 1 ) 6 4 3 - 6 5 8 .

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

155

THE E F F E C T O F ORGANIC S O L V E N T S ON ENZYMATIC E S T E R I F I C A T I O N O F POLYOLS Anja E . M . Janssen, Munasri Hadini, Nicolette Wessels B o e r , R o b Walinga, Albert V a n der Padt, H e n k M. V a n Sonsbeek and Klaas V a n ' t R i e t Wageningen Agricultural University, Department o f F o o d Science, F o o d and Bioprocess Engineering Group, P.O. B o x 8129, 6 7 0 0 E V Wageningen, T h e Netherlands.

Abstract The lipase-catalyzed esterification of decanoic acid and several polyols (glycerol, 1,3-propanediol and sorbitol) has b e e n studied in aqueous-organic two-phase systems. The addition of an organic solvent is found to influence the ester mole fractions at equilibrium. T h e s e solvent effects can be predicted by the program T R E P (Two-phase R e a c t i o n Equilibrium Prediction), which is based on the U N I F A C group contribution method.

INTRODUCTION T h e use of organic solvents in enzymatic synthesis has been extensively studied. Attempts are made to establish rules for choosing a suitable solvent for each type o f reaction. Important parameters, such as the activity and stability of the enzyme in organic solvents, are investigated. In general, hydrophobic solvents are preferred, since the retention of enzyme activity is favourable in these solvents. L a a n e et al. [1] found a good correlation between biocatalytic activity and the log Ρ value o f the solvent, which is the partition coefficient in an octanol-water two-phase system. Besides an effect of solvents on the activity and stability of the enzyme, there is also a solvent effect on the equilibrium position of reactions. T h e s e effects are discussed by Martinek et al.[2], Eggers et al.[3], Halling[4] and recently by Valivety et al.[5]. T h e approaches used by these authors, are only valid for dilute systems a n d / o r systems with a water activity close to 1. However, in many esterification reactions polar substrates, such as glycerol, are used. T h e s e systems usually consist of two phases and to obtain high product concentrations at equilibrium, high glycerol and low water concentrations are necessary. This results in a low water activity and the approaches, mentioned in literature [2-5] cannot b e used therefore. In the present study, the effect of solvents on the esterification of decanoic acid and several polyols, such as glycerol, 1,3-propanediol and sorbitol is investigated. Furthermore, a thermodynamically based theory is developed for the prediction of the effect of solvents on esterification in aqueous-organic two-phase reaction systems with a low water activity.

156

THEORY F o r a reaction A

+

B

^

C

+

H2O

the reaction equilibrium constant Κ can be described by: a

c'°>H70

Κ

C '

=

-

=

X

H

2

7

0

X

Yc ' YHOO 2

-

2

(1)

where ah xx and y{ are the thermodynamic activity, the mole fraction and the activity coefficient o f component /, respectively. This equation shows that lowering the water activity will result in a higher product activity and subsequently a higher product concentration at equilibrium. T h e addition of an organic solvent will also affect the concentrations at equilibrium. T h e solvent will change the activity coefficients o f the reaction components and since the Κ value is constant for a given temperature this will lead to a change in the mole fractions. Furthermore, in a two-phase system at equilibrium, the activity of component / in phase 1 is equal to the activity of component / in phase 2. T h e phase equilibrium can be described by: aj

=

a?

*

x l - y l

=

xf-yf

(2)

M A T E R I A L S AND M E T H O D S Reaction T o study the effect o f solvents on the esterfication of decanoic acid and glycerol or 1,3-propanediol, 10 mmole decanoic acid, 2 0 mmole alcohol, 2 0 mmole water, 10 mmole solvent and 25 mg lipase from Chromobacterium viscosum were mixed in 10 ml stoppered glass bottles. T h e bottles were shaken by an end-over-end incubator ( 1 5 0 rpm) at 35 °C. After 200-300 hours samples were taken from the organic phase and analyzed by H P L C . F o r the esterification of decanoic acid and sorbitol the following amounts were mixed: 10 mmole decanoic acid, 2 0 mmole sorbitol, 71 mmole water, 10 mmole solvent and 5 0 mg lipase from Chromobacterium viscosum. T h e incubation is the same as described above and samples were taken from the organic phase after 1000 hours and analyzed by H P L C . Calculations F o r the estimation of activity coefficients the U N I F A C group contribution method was used [6], In this study the U N I F A C parameter table o f Magnussen et al. [7] for the prediction of liquid-liquid equilibria is used. T h e program T R E P , Two-phase Reaction Equilibrium Prediction, calculates the mole fractions of reactants in a two-phase system, in case both the reaction equilibrium as well as the phase equilibrium are achieved. T h e structure o f T R E P is schematically given in figure 1.

157

/

initial amounts

/

1

Φ—r calculate a\ UNIFAC J

adjust phases; mass balances |

> K m) but also relative to the droplet concentration (all reversed micelles need to contain substrate). This condition may be hard to realise experimentally due to e.g. partitioning into the interphase and extrapolation to an infinite concentration has to be performed. The question arises whether even this method yields the true apparent as defined and expressed in Table 1. The relationship between w 0 and this turnover number (Fig. 2 C ) demonstrates that at low w 0 values the maximum reaction rate is not reached due to transport factors and the presence of k_ 2 (Table 1). 5. STRUCTURAL STUDIES In section 3 it has been mentioned that one o f the basic assumptions o f the theory is that the strucural integrity o f the protein is maintained upon incorporation in reversed! micelles. This is important to prove since in calculating the rate equation, it is assumed that the enzyme behavior in the droplet is the same as in bulk water. In literature some indications for the validity o f this assumption were found, but we extended these studies for our systems. Different techniques were applied to investigate whether the structure is unaltered. In subsequent sections the results o f the study on three proteins are presented. Lipoamide dehydrogenase The structural stability o f lipoamide dehydrogenase was studied by circular dichroism (CD) and fluorescence techniques. C D is a sensitive technique to study changes in the conformation o f proteins. It was used to investigate both the protein backbone as well as the catalytic domain o f the enzyme [ 4 ] . The results demonstrated that changes in structure (if any) m reversed micelles composed o f the cationic surfactant CT AC (cetyltrimethylammonium chloride) as compared to aqueous solution are minor. The C D spectrum o f the flavin, which is part o f the active site, demonstrated that no changes occur in this reversed micellar medium. Addition o f small amounts o f ethanol (12 μΐ/ml^), which also caused a decreased activity o f the enzyme, induced considerable changes o f the 3 D structure [ 4 ] . Also fluorescence spectroscopy offers the fluorescence intensity (a.u.) opportunity to screen for minor changes in 6 • Λ * the active site. Furthermore it can be studied whether w 0-induced monomerisation proteins occurs. In the literature such monomerisation is reported to occur upon incorporating an enzyme in a reversed micellar medium consisting o f • f * droplets that are much smaller than the enzyme molecule [ 1 0 ] . According to this way o f reasoning, lipoamide dehydrogenase, a dimeric 100 kDa protein, with a size o f 6 . 4 x 8 . 4 x 1 9 . 2 nm ο [11] is bound to monomerize when 5 3 0 5 8 0 6 3 0 6 8 0 480 entrapped in a medium o f reversed emission wavelength (nm) micelles with a radius smaller than 3.5 nm Figure 3. Fluorescence spectra o f (the radius o f a empty C T A B reversed lipoamide dehydrogenase. micelle under the experimental conditions A. and B . : Reversed micelles used) [ 1 2 ] . This dissociation process can w o = 1 0 resp. 2 0 . C . Buffer. be studied easily by fluorescence spectroscopy, because the FAD-molecule is lost upon monomerization and its

178

fluorescence is quenched [ 1 3 j . The emission spectrum o f lipoamide dehydrogenase in CTAB-reversed micelles does not validate this assumption (Fig. 3 ) . Instead, its close similarity with the emission spectrum in aqueous buffered solution even indicates its structure is not changed at all. Enzyme activity measurements kinetic experiments on this enzyme in C T A B reversed micelles indicate that the kinetic behavior o f the enzyme is not changed and that the intramicellar substrate model is applicable (To be published elsewhere). Carbonic anhydrase Also measuring the kinetic performance o f an enzyme offers the possibility to probe whether the structure o f the enzyme is changed. Common kinetic parameters like K m and k^ cannot be used, since only apparent values are measured. However, deteriorations in the active site lead to an increased activation energy for the rate determining step. Therefore kinetics o f the hydrolysis o f /?ara-nitrophenylacetate by carbonic anhydrase were measured at different temperatures. Indeed preliminary results yield apparent values for and k ^ that are considerably different from aqueous solution. However an activation energy o f 5 0 + 5 kJ.mol is measured both in reversed micelles and in buffered aqueous solution. These data are in agreement with earlier results [ 1 4 , 1 5 ] . So a reversed micelle-induced conformational change for this enzyme is not likely. The same experimental data were also applied to investigate whether the kinetics o f this enzyme follow the model described above. Good agreement between experimental data and theory was observed despite complications caused by the preferential location o f the substrate in the interphase (In preparation). Cytochrome c. Absorbance 0.006 j

;

0.005;

!

0.004 %ν

0.003 h * 0.002 h

v

.-· .,,-vr 'A

X

0.001 ,/Λ

-.ν·) ' ^ ' ^ Λ ^

0 600

Figure 4.

650 700 750 800 Excitation wavelength (nm)

Absorption spectrum o f cytochrome c . T h e 6 9 5 nm absorption band, visible in buffer (upper l i n e ) , disappears in A O T microemulsion (lower curve).

In some cases studying the UV-Vis absorption enables elucidation o f small changes in protein structure. The 695 nm absorption band o f cytochrome c is known to disappear when the distance between the haem-group and the methionine-60 residue changes. In AOT reversed micelles this absorption band disappears, probably due to interaction between the positively charged protein and the negatively charged interface (Fig. 4 ) . Consequently alterations in the kinetic behavior o f the protein are expected. Therefore it is not surprising that the maximum protein reduction rate was decreased by a factor of 1000 in reversed micelles as derived from variation o f the reductant concentration. Simulation o f the data observed in these experiments succeed only if extreme low droplet 2exchange rate constants are used 1 ( k e x= 1 0 M ' s ) . Clearly the conditions to apply the intramicellar concentration are not met by this protein.

6. CONCLUSION We have shown that for enoate reductase [ 1 6 ] , hydroxy steroid dehydrogenase [17) and tyrosinase (To be published) the reactivity of proteins can be explained on the basis o f the intramicellar concentration model. Caution is important since, as demonstrated in the previous section, it is essential to perform structural studies before the kinetic theory can

179

be applied. Mere determination o f kinetic parameters is not sufficient: For carbonic anhydrase, lipoamide dehydrogenase and cytochrome c a decreased rate of the reaction and an altered substrate "affinity" is found. However, the changes in apparent kinetic parameters can be explained as arising from limitations in the rate o f substrate supply due to the micellar nature o f the reaction medium. The assumption that the enzymes themselves are unaltered is supported by spectrospic studies. The alterations in the cytochrome c structure prohibit the use o f the rate constants which were measured in aqueous solution whereas for the two other proteins the model seems applicable. The observation o f minor changes in a large number o f enzyme structures [ 18] indicates that kinetic studies have to be supplemented with structural studies. Furthermore it can be learned that pseudo-saturating kinetics can lead to deviant kinetic parameters. The fact that our kinetic theory is able to predict reaction rates, does not preclude the validity o f other kinetic theories, for in certain cases the enzymes can indeed be located in different environments in the reversed micellar medium [19] or the kinetic parameters o f the enzyme themselves can be influenced by the micellar environment. For example, for chymotrypsin it has been shown that the intrinsic rate constants are sensitive to charged groups in the immediate vicinity o f the enzyme [ 2 0 ] . ACKNOWLEDGEMENTS The author wishes to thank Y . Bruggeman, R. Hilhorst, J . W . Simons and J . P . W de Bruijn for their contributions. This work was supported by a fellowship o f the Royal Dutch Academy o f Arts and Sciences to R . V . REFERENCES

1.

2 3.

Hiromi, K . , Kinetics of Fast Enzyme Reactions. Theory and Practice, John Wiley &

Sons, New York, 1979. Verhaert, R . M . D . , Photoinduced charge separation and enzyme reactions in reversed micelles, PhD Thesis, Agricultural University, Wageningen, The Netherlands, 1989. Verhaert, R . M . D . , Hilhorst, R . , Vermuë, M . , Schaafsma, T . J . , and Veeger, C . , Description o f enzyme kinetics in reversed micelles. 1. Theory, Eur. J.

Biochem., 187, 59, 1990. 4. 5.

Verhaert R . M . D . , Hilhorst R . , Visser A . J . W . G . and Veeger C , The Optimization o f Enzyme Catalysis in Organic Media. In: Biomolecules in organic solvents A. Gomez Puyou (Ed.) Ch. 6, C R C Press Inc., Boca Raton, 133 1992. Bianucci, M . , Maestro, M . , and Walde, P . , Bell-shaped curves o f the enzyme activity in reverse micelles: a simplified model for hydrolytic reactions, Chem.

Phys., 141, 273, 1990.

6. 7. 8. 9. 10. 11. 12.

Ferreira, S . T . and Gratton, Ε . , Enzyme activity in reverse micelles: roles o f

diffusion and extent o f hydration, In: Biomolecules in organic solvents A. Gomez

Puyou (Ed.) Ch. 8, C R C Press Inc., Boca Raton, 189 1992. Verhaert, R . M . D . , and Hilhorst, R . , Enzymes in reversed micelles: 4 . Theoretical analysis o f a one-substrate one-product conversion and suggestions for an

efficient application, Receuil Trav. Chim. Pays-Bas, 110, 236, 1991.

Martinek, K . , Levashov, Α . V . , Klyachko, N . , Khmelnitski, Y . L . , and Berezin, L V . , Micellar enzymology. Eur. J. Biochem., 155, 4 5 3 , 1986. M a o , Q. and Walde, P . , Substrate effects on the enzymic activity o f

a-chymotrypsin in reverse micelles. Biochem. Biophys. Res. Commun. 178, 1105,

1991. Khmelnitski Y u . L . , Kabanov, A . V . , Klyachko, N . L . , Levashov, A . V . and Martinek K . , Enzyme catalysis in reversed micelles, in: Structure and reactivity in reverse micelles. Pileni, M . P . , Ed., Elsevier, Amsterdam, 1989, 2 3 0 . Schierbeek Β . , PhD Thesis State University, Groningen 1988. Vos, Κ . , Laane, C , Weijers, S . R . , Hoek, A . van, Veeger, C , and Visser, A . J . W . G . , Time-resolved fluorescence and circular dichroism o f porphyrin

180

cytochrome c and Zn-porphyrin cytochrome c incorporated in reversed micelles,

13.

Eur. J. Biochem., 169, 259, 1987.

van Berkel, W . J . H , Benen, J . A . E and Snoek, M . C . , On the FAD-induced dimerization o f apo-lipoamide dehydrogenase from Azotobacter vinelandii and

Pseudomonas fluorescens. Eur. J. Biochem. 197, 769 1991.

14.

a. J o b e , D. J . , Dunford, H. B . , Pickard, M . , and Holzwarth, J . F . , Kinetics o f azide binding to chloroperoxidase in water and reversed micelles o f S D S , hexanol

and water, in Reactions in Compartmentalized Liquids, Knoche, W., and

15.

Schumacher, R . , Eds., Springer Verlag, Berlin, 1989, 4 1 . b. Meier, P . , and Luisi, P . L . , Micellar solubilization o f biopolymers in hydrocarbon solvents. II The case o f horse liver alcohol dehydrogenase, J. Solid-Phase Biochem., 5, 2 6 9 , 1980. Fletcher, P . D. I . , Freed man, R . B . , Mead, J . , Oldfield, C , and Robinson, Β . H . , Reactivity o f a-chymotrypsin in water-in-oil microemulsions, Coll. and

Surf., 10, 193, 1984.

16.

Verhaert, R . M . D . , Tyrakowska, B . , Hilhorst, R . , Schaafsma, T . J . , and Veeger, C , Enzyme kinetics in reversed micelles. 2 . Behavior o f enoate reductase,

17.

Tyrakowska, B . , Verhaert, R . M. D . , Hilhorst, R . , and Veeger, C , Enzyme kinetics in reversed micelles. 3. Behavior o f 2 0 ß-hydroxysteroid dehydrogenase

Eur. J. Biochem., 187, 73, 1990. Eur. J. Biochem., 187, 81, 1990. 18. 19. 20.

Luisi, P . L . and Magid, L . J . , Solubilization o f enzymes and nucleic acids in hydrocarbon micellar solutions. CRC Cut. Rev. Biochem. 2 0 , 4 0 9 , 1986. B r u , R . , Sanchez-Ferrer, Α . , and Garcia-Carmona, F . , A theoretical study on the expression o f enzymic activity in reverse micelles, Biochem. J., 2 5 9 , 3 5 5 , 1989. Goldstein, L . , Kinetic behavior o f immobilized enzyme systems. Meth.Enz., 4 4 , 397, 1976.

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

181

PROTEIN-INTERFACE INTERACTIONS IN REVERSE MICELLES A. Sanchez-Ferrer, M . Pérez-Gilabert and F . Garcia-Carmona

Departamento de Bioquimica, Facultad de Biologia, Universidad de Murcia, E-30001 Murcia, Spain.

Reverse micelles are characterized by the presence o f different pseudophases (free water, bound water, surfactant tails and organic solvent) which are not only responsible for their physical-chemical properties but also affect the enzyme behaviour. This paper shows the experimental work carried out with different oxidases and with the kinetic multiphasic model described by our group, focussing on the possible explanation of controversial experimental data with respect to the ω 0 profile.

1. I N T R O D U C T I O N

Enzymology in amphiphilic self-organizing systems has attracted much attention over the last three decades since the majority o f the enzymes in vivo, function on the surface o f biological membranes or inside them in the so called "lipid particles". These associates o f lipid molecules consist o f reverse vesicles, reverse micelles and microheterogeneous aggregates (types o f lamellae, cylinders and/or ball-shaped micelles) [ 1 ] . In this article, we report comprehensively the experimental and theoretical results obtained with isotropic reverse micelles and show how the enzymatic activity changes as a result o f the different interfacial microenvironment in such aggregates.

2. R E V E R S E

MICELLES

Reverse micelles are microdroplets o f water dispersed in a hydrophobic medium from which they are shielded by a mono layer o f surfactant molecules. The hydrophilic part (the head) o f these molecules, which is often charged, is turned towards the water core o f the droplet, while its hydrophobic part (the tail) is exposed to the apolar medium. Their physical-chemical structure, when the amount o f water exceeds the hydratation requirements o f surfactant was outlined by El Seoud, [ 2 ] . This structure consist o f three different domains: - Free water, whose properties and structure become closer to those of bulk water

182

as ω 0 increases - Bound water, whose properties are qualitatively different to those o f bulk water, because o f the hydrophilic interactions with the polar headgroups o f the surfactant. - Surfactant tails, which penetrate into the apolar solvent. When the amount o f water is not enough to hydrate the surfactant molecules, there is no free water and reverse micelles have only two domains: bound water and surfactant. These reverse micelles are controlled by a dynamic equilibrium where amphiphilic molecules o f surfactant self-assemble spontaneously to form spherical or ellipsoidal aggregates. These interconvertible modes o f aggregation involve an exchange o f contents by fusion o f micelles to give a dimmer, which immediately splits into two monomelic micelles. The presence o f this dimmer explains why the real rate o f exchange experimentally measured 7 10 1 1 is about 1 0 instead o f the estimated ΙΟ M S if only the rate o f diffusion is involved. 7 Even taking into consideration this value, 1 0 , the rate o f enzymatic reaction is clearly several times lower. This permits us to consider reverse micelles as a pseudo-continuous phase [3]. Thus, from the enzymatic point o f view, a reverse micellar medium is equivalent to four phases in equilibrium: free water, bound water, surfactant tails and organic solvent. Enzyme and substrate are distributed among these phases (Figure 1).

^r^tC# — /

FREE

WATER

^ ' \\ BOUND WATER

\—\^

,, • •

FREE

ΒβΒβΙ «

•

W AB T E

BOUND SURFACT. ORGAN. W A RT E

SOLVENT

SURFACTANT

Figure 1. Reverse micelles can be considered as four phases in equilibrium since the rate of micelle exchange exceeds the enzymatic reaction rate. However, when the interacting species are two macromolecules (such as trypsin and soybean trypsin inhibitor) there are diffusional problems, since the exchange rate between these macromolecule-containing reverse micelles slows down by a thousand times and the 6 limiting-step in the exchange, the fusion, by 1 0 times [4J. As regards experimental conditions, the entrapment o f enzymes in reverse micelles is a very simple procedure and three different approaches have been used. In the first, the enzyme can be solubilized by spontaneous transfer o f the protein in a two phase system consisting o f approximately equal volumes o f the aqueous protein solution and the organic solvent containing the surfactant. A second procedure, consists o f the solubilization o f lyophilized enzyme in previously formed hydrated reverse micelles. The third, and nowadays the most widely used method, consists o f microinjection o f enzyme dissolved in a buffered aqueous medium into a stirred solution o f surfactant dissolved in organic solvent. This last method avoids the lengthy time required for the solubilization of

183

the proteins and the problem o f determining the actual amount o f water in the reverse micelles to determine their size, which is ruled by the molar ratio o f water to surfactant ( ω 0 ) . Also, the amount o f water with respect to the whole volume o f the reverse micellar medium, reflects the concentration of identically sized micelles and is designated as Θ. The above parameters ( ω 0 and Θ) affect the activity expressed by enzymes in reverse micelles. As for ω 0, three profiles are shown (Figure 2 ) : i) the saturation curve, interpreted by the need o f the enzyme for free water in order to reach its maximal activity; above this, the size o f the micelle has no effect on activity; ii) the bell-shaped curve, explained by the existence of an optimal inner cavity for catalytic activity, which, it is supposed, corresponds to the size o f the enzyme; and iii) where enzyme activity decreases continuously as ω 0 increases, as a result o f a decrease in the conformational flexibility o f the protein, which permits a greater catalytic efficiency o f the enzyme at low ω 0 .

ACTIVITY

1

2 3

Figure 2. Different ωα profiles expressed by enzymes in reverse micelles: (1) saturation curve; (2) bell-shaped curve and (3) curve expressing continuous decrease in enzyme activity as ω0 increases. In addition, there are some enzymes in reverse micelles that present a bigger than the one expressed in water, giving rise to the concept o f " superactivity ". This effect can be explained by a higher reactivity o f the structured water in the micelle and/or the relatively high rigidity o f the enzyme molecule caused by the surfactant layer. As regards Θ, two different responses in the activity o f the enzymes have been found (Figure 3 ) . The first is displayed by a group o f enzymes whose activity can change 10 or 100 fold when θ increases, while the activity in the second group does not depend on Θ. The difference between these two groups stems from the presence o f hydrophobic anchoring residues in the first group, which allow them to interact with the micellar monolayer.

LOG (ACTIVITY)

184

2

1

θ Figure 3. Different activity shown by enzyme as a function of water volume fraction (Θ): (1) enzyme whose activity does not depend on Θ; and (2) enzyme whose activity can change 10 or 100 fold when θ increases

Taking into account these basic concepts, some theoretical models have been developed [5-10]. They are classified in Table 1.

Table 1. Theoretical models described in reverse micelles

MODEL

BASED ON

DIFFUSIONAL

• ENTRAPMENT IN RM DOES NOT CHANGE ENZYME BEHAVIOUR • LIMITATIONS IN SUBSTRATE DIFFUSION

Ν 0 Ν D I F F U S I 0 Ν ;

GROUP VEEGER, 1990 (HOLLAND) OLDFIELD, 1990 (ENGLAND)

• NO DIFFUSIONAL PROBLEMS POLYDISPERSED

• ENZYME BEHAVIOR DEPENDS ON EXISTENCE OF DIFFERENT MICELLE RADII

KABANOV 1988 (OLD USSR)

• NO DIFFUSIONAL PROBLEMS MULTIPHASIC

• ENZYME BEHAVIOR DEPENDS ON ACTIVITY EXPRESSED. IN EACH PSEUDOCONTINUOUS PHASE

GARCIA CARMONA 1989 (SPAIN)

185

In this report we shall focus in the multiphasic model. It is based on the existence o f four different microenvironments in reverse micelles [ 8 - 1 0 ] . The mathematical expansion follows this order: a) The fusion o f micelles, which gives us the dimeric fraction o f micelles and the loss of surface area due to dimerization. b) These two parameters are inserted into the volume fraction formula o f each pseudophase (free water a, bound water ß, surfactant tails γ and organic solvent δ). c) Once the volume fractions are obtained we can distribute enzyme and substrate among the phases. In the case o f the enzyme, we assume that it is insoluble in organic solvent l 2 and so the enzyme partition coefficients K E and K E , can be calculated from enzyme concentration in free/bound water phases and from bound/surfactant phases. d) On the other hand, substrate can partition in all the phases, with its corresponding partition coefficients P, (free/bound phases), P 2 (bound/surfactant phases) and P 3 (surfactant/organic solvent phases). e) Taking into account all the above parameters, the enzymatic activity is expressed as a sum o f three Michaelis equations, which represent the activity expressed in free water, bound water and surfactant tails (Equation 1) where α,β,γ and δ represent the volume fraction o f free water, bound water, surfactant tails and organic solvent, respectively.

T

(·)

+

+

+

\Zl

+

izl

FREE WATER

BOUND WATER

SURFACTANT TAILS

(*)

= α + Ρ 1β + Ρ ]Ρ 2γ + Ρ 1Ρ 2Ρ 3 ( 1 - α - β - γ )

(·)

= α + κ\ β + Κ Ε Κ Ε γ

ι

2

Equation 1. Mathematical equations of the enzymatic activity in reverse micelles. The latter is expressed as a sum of three Michaelis equations, which represent the activity in free water, bound water and surfactant tails. Greek letters (α,β,γ and δ) represent the volume fraction of free water, bound water, surfactant tails and organic solvent, respectively.

186

As can be seen in each particular phase, the initial velocity includes a divided by enzyme partition coefficients and an apparent multiplied by a combination o f substrate partition coefficients. I f all the phases are taken into account, the equation predicts negative cooperativity, but, when enzyme is only active in one phase, the activity expressed is hyperbolic. This multiphasic model can simulate the different behaviour expressed by enzymes in reverse micelles. As regards ω 0, the saturation curve, the bell-shaped and the continuous decrease in activity are simulated when the enzyme is predominately active in free water, bound water and surfactant, respectively. However the shape o f the curve is modulated by enzyme distribution among the phases [ 8 , 1 0 ] . Furthermore, the model fits the experimental data o f superactivity [8] and predicts the not yet described superinhibition as a decrease in activity when bound water increases [ 8 ] . As for Θ, the model also simulates different behaviour depending on enzyme distribution. The model was used to fit experimental data obtained by our group with different oxidases [8,11-13], whose ω 0 profiles include both the saturation and the bell-shaped curves. The proportional relation between K . and θ found in polyphenol oxidase, also agrees with the model equations [9,11]. In the case o f an interfacial substrate, such as rm-butyl catechol, a new parameter called ρ can be defined as the ratio between substrate concentration and Θ. This ratio can be substituted in the general equation 1 to give:

Σ

i = f,b,

max

s

.

κ. Ill app. Ρ θ

The model also can be used to explain some controversial data about the real or artefactual bell-shapes (enzyme active mainly in free water and substrate equally distributed between free and bound water) found in the bibliography. Figure 4 shows how, under the same simulation parameters a bell-shaped curve and a saturation curve can be obtained. The only difference is the experimental procedure used to study ω 0 . In case Α , ω 0 was simulated by changing the water content and in B , the surfactant concentration at a fixed Θ. This simulation parameters were chosen to show the difference between the volume fraction o f bound water when ω 0 is studied changing water and when it is studied changing surfactant concentrations. In our opinion, to avoid this experimental effects, the following precautions must be taken into account: - ω 0 must studied starting at the highest ω 0 and then decreasing the ω 0 by adding surfactant. - the substrate must be kept under saturating conditions, when possible [ 1 4 ] . - the substrate effect on the micellar pH [ 1 5 ] .

187 - the θ used at a fixed ω 0 to entrap the enzyme, since not only is the size of the micelle important but also the amount o f micelles to reduce the exposition time o f the enzyme to the organic solvent.

00 ο

=

[

w a t e

r]/[surfactant]

Figure 4. ω0 profiles simulated (1) changing the water content and (2) changing the surfactant concentration. The simulation parameters used in both cases are: K\=0.1, K*B =0.1, kf=100, kb=l, k=l, P,=l, P2=0.1, P3= 0.1, Km/=0.1 KmJf= 0.1, Km,=0.1

3. CONCLUSIONS

The use o f optically transparent reverse micelles and reverse vesicles have great potential application in biotechnology [10] and analytical chemistry [16], since they provide the appropriate environment for bioconversion o f polar and apolar compounds. However, there is still much to learn before we fully understand enough how enzymes work in these ternary systems.

4. ACKNOWLEDGEMENTS

This work was partially supported by a grant from C I C Y T (BIO91-0790).A.S.F. is a holder o f a "Remcorporacion" grant ( M . E . C ) . M . P . G . is a holder o f a PFPI grant (M.E.C).

188

5. REFERENCES 1 N . L . Klyachko, A . V . Levashov, A . V . Pshezhetsky, N.G. Bogdanova, H.V. Berezin and K. Martinek, Eur. J . Biochem., 161 (1986) 149. 2 O.A. El Seoud, in Reverse Micelles, P . L . Luisi and B . E . Straub (Eds.) Plenum Press, New York, 1984. 3 P . L . Luisi, M . Giomini, M.P. Pileni and B . H . Robinson, Biochim. Biophis. Acta, 947 (1988) 2 0 9 . 4 R. Bru and F . Garcia Carmona, F E B S Lett., 282 (1991) 170. 5 A . V . Kabanov, A . V . Levashov, N . L . Klyachko, S.N. Namyotkin, A . V . Pshezhetskii and K. Martinek, J . Theor. Biol., 133 (1988) 327. 6 R . M . D . Verhaert, R. Hilhorst, M. Vermuë, T . J . Schaafsma & C.Veeger, Eur. J . Biochem., 187 (1990) 5 9 . 7 C. Oldfield, Biochem J . , 272 (1990) 15. 8 R . Bru, A . Sanchez-Ferrer and F . Garcia-Carmona, Biochem. J . , 2 5 9 (1989) 3 5 5 . 9 R . Bru, A. Sanchez-Ferrer and F . Garcia-Carmona, Biochem. / . , 2 6 8 (1990) 6 7 9 . 10 F . Garcia-Carmona, R. Bru and A. Sanchez-Ferrer, in Biomolecules in Organic Solvents, A. Gomez-Puyou (Ed), C R C Press, Boca Raton, Florida, U S A , 1992. 11 A. Sanchez-Ferrer, R. Bru and F . Garcia-Carmona, F E B S Lett., 233 (1988) 3 6 3 . 12 R . Bru, A. Sanchez-Ferrer and F . Garcia-Carmona, Biotechnol. Bioeng., 3 4 (1989) 3 0 4 . 13 R . Bru, A. Sanchez-Ferrer and F . Garcia-Carmona, Biotechnology Lett., 11 (1989) 2 3 7 . 14 R . Bru, and P. Walde, Eur.J. Biochem., 199 (1991) 9 5 . 15 P. Walde, M . Qingcheng, R. Bru, P . L . Luisi and R. Kuboi, Pure Applied Chem. (In press) 16 A. Sanchez-Ferrer, J . S . Santema, R . Hilhorst, and A . J . W . G . Visser, Anal. Biochem., 187 (1990) 129.

List of parameters Q 0:micelle size [H 20]/[surfactant] Θ: % o f water (v/v). If ω β is held constant this parameter represents the micelle concentration, p: [substrate]/Θ Subscripts: f, free water; fc, bound water;,, surfactant tails; m9 organic solvent. 2 K* E, K E : enzyme partition coefficients K ' E= [ E ] b / [ E ] f K \ = [ E ] s/ [ E ] b k j = catalytic constant in each domain. Pj 2 3: partition coefficients o f the substrate Pi = [ S y [ S ] f Pi=[SV[S] fc Pi = [SU[S] e

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

189

Kinetics of enzyme-catalysed reactions in water-in-oil microemulsions b

Christopher Oldfield**. Cristina Otero\ Maria L . Rua and Antonio Ballesteros

b

"Biological Laboratory, University o f Kent at Canterbury, Canterbury C T 2 7 N J , England b

C . S . I . C . Instituto de Catalisis, Campus Universidad Autonoma, Madrid 2 8 0 4 9 , Spain

Abstract In water-in-oil microemulsions the aqueous sub-phase is present as a monodispersion o f spherical droplets. A theoretical treatment (C. Oldfield (1990) Biochem. J 2 4 7 15-22 and (1991) J. Chem. Soc. Faraday Trans I 87 2607-2612) shows how the rates o f enzymecatalysed reactions involving water-soluble substrates can be expected to depend on the droplet size and concentration and on the rate o f exchange o f solutes between droplets. In this article the theory is briefly restated, with examples o f experimental systems which behave as predicted by the theory.

1. I N T R O D U C T I O N Water-in-oil microemulsions are optically transparent colloidal dispersions o f water and surfactant in an oil-continuous solvent medium. At low volume fractions ( < 0 . 2 ) , the dispersed phase is present as roughly equal sized, spherical water droplets coated by a monolayer o f close-packed surfactant molecules. The droplet size and concentration can be varied precisely and independently. T o a good first approximation, (i) there is a linear relationship between droplet radius and the molar water:surfactant ratio, R , and (ii) at constant R the droplet concentration is directly proportional to the (water+surfactant) volume fraction o f the microemulsion. The contents o f individual droplets are in communication with those in other droplets as a consequence o f the exchange process wherein droplet pairs transiently fuse, forming a dimer within which intermixing o f droplet contents occurs. W e are studying the ways in which the microdispersed nature o f the microemulsion aqueous domain influences the kinetics o f chemical and enzyme-catalysed reactions involving water-soluble reactants or substrates. In this paper we show how, for an enzyme-catalysed reaction which obeys Michaelis-Menten kinetics in aqueous solution, the steady-state rate o f substrate turnover, characterised by the first-order rate constant kcan and the affinity o f the enzyme for the substrate, characterised in aqueous solution by the Michaelis constant, Km, will depend on the size and concentration o f the microemulsion water droplets and on the rate of exchange o f solutes between droplets.

190

Κfus

k diss

ν

kdiss obs

obs

k1

k 1

CD Ψ

Θ

Scheme (I): Reaction between Λ and Β to yield product Ρ in a water-in-oil microemulsion^A and Β are located initially in separate droplets and the reaction is irreversible. The fused dimer species QJ^J) w i / / ultimately decompose to ΓΡ^Ι + , but this process will not influence the kinetics of the chemical reaction. 2. THEORY 2 . 1 . Bimolecular Reactions In microemulsions, water-soluble solutes are distributed over the total droplet population (following a normal distribution pattern [1]). A bimolecular reaction (e.g. between A and B ) can occur only in droplets which contain both A and Β and thus the steady-state rate o f the reaction must be a function o f the steady-state concentration o f droplets containing both A and B , generated by the exchange process. Scheme (I) shows how exchange permits the reaction between A and B , initially present in separate droplets, to give product, P , in an irreversible reaction. Reaction may take place either within the fused dimer or within the ( A ^ B ) daughter droplet, depending on whether the rate o f reaction is respectively fast or slow, relative to the rate o f dimer dissociation. Clearly, the reference frame for concentration units for a bimolecular reaction occurring within a droplet (Le the volume o f that droplet) is different from that for processes involving collisions between droplets, for which the reference frame is the total microemulsion volume. In our nomenclature the superscripts 'd' and ' m ' respectively are used to make clear the choice o f reference-frame for concentrations and for second-order rate constants and their units (first-order rate constants contain no concentration dimension and thus require no such distinction). In scheme (I) as written an immediate conflict o f reference frames is avoided by treating conversion o f

(^^B^)

^

0

T

C 3 C ^

^ ( P )

respectively as unimolecular processes characterised by the apparent first-order 005 (''observer's") rate constant, k+j . The expression relating the apparent first-order rate constants to their second-order counterparts is, for a droplet or dimer containing one molecule each o f A and Β [2,3], (1)

NAV* d

3d

l

1

where k+1 is the second-order rate constant for the reaction (units: dm mol' s" , reflecting 3d the fact that the concentration units for A and Β are mol d n r ) , V is the volume o f the

191

droplet and NA is Avogadro's Number. Substitution o f e q n . ( l ) for in scheme (I) gives a kinetic mechanism for which predicts a dependence o f reaction rate on droplet volume and concentration and on the rates o f the droplet fusion-dissociation process. Application o f the steady-state approximation ( d [ ^ A ^ ) ]/dt = d[ (A^j ] /dt = 0) gives the general-case rateequation for the steady-state rate o f product formation [ 2 ] . O f particular interest are its reductions to the limiting cases where reaction occurs only within the dimer (we call this type (i) kinetics) or only within the daughter droplet (type (ii) kinetics). Type (i) kinetics is characterised by a dependence o f rate on droplet volume only, whereas in type (ii) kinetics the reaction rate depends on both the volume and concentration o f the droplets. The dependence o f rate on droplet volume arises because, for fixed numbers o f reactant molecules per droplet, their concentration, and hence the reaction rate, is inversely proportional to the volume o f the droplet. The additional dependence o f type (ii) kinetics on droplet concentration arises because, from scheme (I), the reverse decomposition o f daughter droplet (AJB) , regenerating ^ A ^ and ^ B ^ ) ( v i a ( A ^ B ^ ) , is a function o f the droplet concentration 1

(the apparent first-order rate constant for this process is k^l Ç^)]c s' , where [ ( ^ ) ] c is the concentration o f 'empty' droplets (i.e. containing neither A nor B ) . Thus for example, increasing the droplet concentration leads to a decrease in the steady-state concentration o f (AJB^ , so lowering the steady-state rate o f Ρ formation. Since kdiss, the first-order rate constant for dissociation o f the transient dimer, has not yet been measured, it is not possible to predict a priori whether a system will obey type (i), type (ii) or 'mixed' ((i) + (ii)) kinetics. Thus a dependence o f rate on droplet concentration becomes an important criterion for distinguishing between type (i) and type (ii) kinetics. 2.2 Enzyme kinetics: The Michaelis Menten Mechanism In aqueous solution, under steady-state conditions, enzymes obey the kinetic mechanism: E + S * * E * S - » E + P i n which substrate, S, reversibly binds at the active site o f the enzyme, E , generating the enzyme-substrate, or Michaelis, complex, E - S . Chemical conversion o f enzyme-bound substrate to product, P, in a process characterised by the firstorder rate constant, kcan is followed by its release from the active site. Solution o f this mechanism using the steady-state approximation (setting d[E-S]/dt = 0 ) gives the Michaelis-

reg m

k

dur

=

ca.

dt

c

w

(2)

+ is]

w

w

where d[P] /dt is the steady-state rate o f product formation, [ E ] T is the total enzyme w concentration and Km is the Michaelis constant, where K

»

=

(*-i + KJ w

kK

*l

(3)

192

©

R

. o .

A

k_, k /N V d

+1

V

kcat k /NV d

- O2

A

d

®

A A

Scheme (11): An enzyme-catalysed reaction obeying type (ii) kinetics in water-in-oil microemulsions. The enzyme obeys Michaelis-Menten kinetics in aqueous solution in water-in-oil microemubions. The superscript 'w' is used to distinguish parameters containing a concentration dimension from their microemulsion counterparts. Equation (2) is valid under conditions where [S] > > [ E ] T, and under 'initial-rate' conditions where the concentration o f Ρ remains low, so that the rate o f the back-reaction (formation o f S from P) is negligible. 2.3 Microemulsion Equivalent of the Michaelis Menten Mechanism In this paper we shall concentrate on enzymic systems which obey type (ii) kinetics. W e have identified a number o f systems obeying this type o f kinetics, both in the literature and in our own work, as shown below. Type (i) systems are rather harder to identify, as discussed later. The analogous scheme for substrate conversion by an enzyme which, in aqueous solution obeys Michaelis-Menten kinetics is shown in scheme (II). Type (ii) kinetics are defined by excluding the possibility o f Michaelis complex formation in the fused dimer (done by setting k^X Ç) \ > k+fwy (c.f. scheme (I)). The transfer o f solutes between droplets during the fusion-dissociation process is thus condensed into a single step characterised by the apparent second-order rate constant ka (^-exchange) [ 2 ] . Equation (1) obs has been substituted for k+1 and k_*\ the observer's first-order rate constants for the formation o f the Michaelis complex from either Ε and S or Ε and P, respectively, within a droplet. Since the concentration o f Ρ in (E,PJ is very high the possibility o f the reverse reaction, i.e. resynthesis of S from P, cannot be ignored, and the reverse (E,P) -* fE-S step must be included in the scheme. The catalytic cycle is considered complete when Ρ exchanges out o f ( E , P ) (under initial-rate conditions the concentration o f ( Ρ ) droplets will be negligible, so that the back-reaction initiated by collision between ignored).

Ε

and Ρ can be

In principle the intermediate species ( E - P J should be included to balance the

193 scheme, but since steady-state kinetics are under consideration there is no need to do so. Application o f the steady-state approximation [3] yields the rate equation: m

a\F\

=

(4)

at constant 0

aq

(the units o f dfPJ/dt »α

are mol d n r

3m

1

s ) where,

ι

(5) 0

k^, is the maximum value o f kcalapp (see below), and (6) (excluding two numerically unimportant terms [3]) where,

d

K

=

(*-l + Ο K

(7)

*i

(cf. eqn 2 ) . In eqns (5) and (6) for a monodisperse system,

φ"* = ( NAV

) χ [ Ο 1,

(8)

a q

where # is the total aqueous volume-fraction and [ ( ^ ] T is the total droplet concentration. Thus rectangular-hyperbolic (Michaelis-Menten) kinetics are predicted for enzymecatalysed reactions in microemulsions o f constant composition (eqn.4), although both ^ ί α ρρ aq m (eqn.(5)) and Km (eqn.(6)) are dependent on A , is known as the enantiomeric ratio, E.

(D

238 The enantiomeric excess-value o f residual substrate, ees, in kinetic resolutions o f racemic substrates, can be related to the degree o f conversion, Ç, by Eqn. 2 :

1η[1-(1+Κ^.(ξ+«*.[1-ξ])]

(2)

ln[l-(l+K^).(i-ees.[l-Ç])]

2. PPL-CATALYZED RESOLUTION OF RACEMIC GLYCIDYL BUTYRATE Ladner and Whitesides [3] showed that chiral glycidyl esters with high enantiomeric excessvalues can be prepared by enantioselective hydrolysis o f racemic ester with commercial P P L at pH 7, Scheme 2 . A value o f E = 9 . 7 has been estimated [4] for commercial P P L on glycidyl butyrate based on Eqn. 2 (for ζ = 0 . 6 , ees = 0 . 8 6 ) , assuming non-equilibrium conditions. Ο

Ο Η

Ο Η

Lipase

CH2OH + HOCC 3H 7 Ο

λ

CH 2OÇC 3H 7 Ο

CH 2OCC 3H 7 Ο

(R) glycidyl butyrate

(R/S) glycidyl butyrate

(R) glycidol

butyric acid

Scheme 2 . Kinetic resolution o f racemic glycidyl butyrate. However, on raising ζ it appeared that ees did not improve according to Eqn. 2 (nonequilibrium). Measured values o f ees as a function o f ζ are shown in Figure 1, Panel A. A dramatic decrease o f the (apparent-) value o f Ε is evident from Figure 1, Panel Β for ζ > 0 . 6 1.00 0.80 h

3

UJ

0.60

I

0.40 0.20 O.OO

0.00

Figure 1.

0.20

0.40

0.60

0.80

1.00

0.00

0.20

0.40

0.60

0.80

1.00

Porcine pancreatic lipase catalyzed hydrolysis o f glycidyl butyrate at Τ = 298 and pH = 7 . 8 ; A. ees values versus ξ; Β . Actual apparent upvalues versus ξ.

239 3. CHEMICAL EQUILIBRIUM In order to determine whether the assumption o f non-equilibrium was valid (pH 7 . 8 , 293 K ) , was measured. Hydrolysis and condensation, conducted at various pH values, in the presence o f Pseudomonas cepacia lipase, taking advantage o f the lower pH-optimum o f this 3 lipase as compared to that o f P P L , afforded an estimate o f K«, = 1.4*10 at pH 7 . 8 . A 'best fit' o f experimental data for the enantioselective hydrolysis o f glycidyl butyrate at pH 7 . 8 by non-linear regression analysis showed substantial deviations from Eqn. 2 (K«, = \A*\(f).

4. H E T E R O G E N E I T Y O F P P L The presence o f hydrolytic activities with different values o f Ε in commercial samples o f P P L may effect the observed enantioselectivity. In particular, time-dependent inactivation o f hydrolytic species with high selectivities will result in lower apparent Ε-values as the conversion proceeds. Analysis o f commercial P P L (Sigma Chem. Co.) by chromatographic and electrophoretic techniques revealed the presence o f several species with hydrolytic activity for glycidyl esters. Fractions containing significant activities, however, showed similar E values in the hydrolysis o f racemic glycidyl esters. Time-dependent inactivation o f individual fractions had no influence on the measured E-value. Total protein recovered from the reaction mixture at various time intervals retained the initial enantioselectivity.

5. ESTEROLYTIC AND LIPOLYTIC ACTIVITY O F PPL The activity o f P P L as a function o f the concentration o f racemic glycidyl butyrate is shown in Figure 2 . The activity is seen to increase markedly at glycidyl butyrate concentrations that exceed the solubility in the aqueous phase. 20

15

• E=18

10

9 0.0

ι 0.1

0.2

0.3

0.4

[glybut] (l/l)

Figure 2 .

Activity and enantioselectivity o f P P L as a function o f the concentration o f racemic glycidyl butyrate.

240

Winkler and coworkers [5] suggested the occurrence o f a conformational change on adsorption o f H P L to an aqueous-lipid interface. A similar behaviour o f P P L might be accompanied by a change o f E. The enantioselective properties o f P P L acting in aqueous solution and at the water-ester interface were determined by the initial rate method developed by Jongejan and van Toi [ 6 , 7 ] . This method affords an estimate o f Ε at fixed concentrations of total substrate, all other conditions being equal. Results are shown in Figure 3. 20 ι

a: b: c:

Ε = 9(2) Ε = 14 (1) Ε = 18(2)

0.50 fraction S-glybut (-)

Figure 3.

E-values, determined with initial kinetics, o f P P L at different total concentrations o f glycidyl butyrate. Standard deviations are given between brackets.

The formation o f a second phase at [/?,5-glycidyl butyrate] > 25 g/1 has been ascertained by independent measurements. Although P P L appears to have a distinctly lower selectivity for dissolved glycidyl butyrate, the measured £-value is still substantially higher than the value (E=l) that is observed at ξ > 0 . 6 in kinetic resolutions (Figure 1).

6. PARTITIONING O F R E A C T I O N C O M P O N E N T S The equilibrium composition for the hydrolytic reaction in a single aqueous phase at pH 7 . 8 is at the product side (ζ > 0 . 9 8 ) . In the presence o f a second (organic) phase, partitioning of substrate and products over the two phases will occur. In bulk kinetic resolutions o f racemic ester, the volume o f the organic (ester) phase will diminish as a function o f ζ. To a first approximation, this change will affect the observed equilibrium constant according to Eqn. 3. ( W . P J app K

=

. ( W - P - ^ )

K

φ = phase ratio, Ρ = partition coefficient, K, = dissociation constant.

)

241

The concentrations o f substrate and products in both phases can then be evaluated from the equilibrium partitioning as a function o f ζ and the volume o f residual ester. Partition coefficients Ρ were experimentally determined, ρΚ,-values o f butyric acid in both aqueous and water-hexane mixtures were determined separately from titration experiments. i pp

Application o f K to Eqn. 2 resulted in minor depression o f ees as a function o f ζ for realistic values o f glycidyl butyrate concentrations. Simulated date are depicted in Figure 4 .

0.00

Figure 4 .

0.20

0.40

0.60

0.80

1.00

The effect o f partitioning on ees as a function o f ξ in the hydrolysis o f glycidyl butyrate for various values o f the pH.

7. PING-PONG K I N E T I C S O F P P L - C A T A L Y Z E D H Y D R O L Y S I S The minimal scheme, Scheme 1, that was used to derive Eqn. 1 (differential expression) and Eqn. 2 (integrated expression) considers the enantioselective properties o f enzymes to be the result o f competitive reactions o f both enantiomers o f a single substrate with generation o f single product enantiomers according to Michaelis Menten-type kinetics. In order to check the scope o f this approach, three alternative kinetic schemes were investigated: 1. reversible reactions; 2 . Rabin-type 'memory'-mechanisms [8]; and 3 . enantioselective product inhibition resulting from partitioning o f the P P L acyl-enzyme intermediate between water and product glycidol enantiomers. The effect o f the back-reaction has already been shown to be negligible under the conditions that are used (pH 7 . 8 ) . Moreover, curves that were simulated by applying Eqn. 2 and values of K«, that are appreciably smaller than the value that has been estimated, still show a bad fit to the experimental data. On the other hand, Rabin-type mechanisms, that require the existence o f at least two enzyme conformers with different enantioselectivities, could be modelled in such a way that convincing fits were obtained. However, unrealistic values o f the microscopic kinetic constants had to be introduced, while the effect o f added glycidol enantiomers could not be accommodated.

242

A convincing fit was also obtained for simulations o f the catalytic mechanism involving enantioselective product inhibition, Scheme 3.

Scheme 3.

Kinetic scheme (ping-pong BiBi) for the competition o f product glycidol enantiomers and H 20 for the P P L acyl-enzyme intermediate.

For this case, realistic values o f the microscopic kinetic constants were obtained. The validity of the model could be tested by adding enantiomerically enriched R- and 5-glycidol to the racemic ester and measuring ees as a function o f Ç, Figure 5.

0.00

Figure 5.

0.20

0.40

0.60

0.80

LOO

Enantiomeric excess-value o f remaining glycidyl butyrate as a function o f ξ in the presence o f excess R- and 5-glycidol.

Non-linear regression analysis o f experimental data according to the kinetic equations that were derived for the mechanism shown in Scheme 3, afforded consistent values for the kinetic constants.

243

8. C O N C L U D I N G R E M A R K S Investigations o f the kinetic resolution o f racemic glycidyl butyrate with P P L show that the relations derived by Chen and coworkers [ 1 , 2 ] have limited applicability. Mechanistic features o f PPL-catalyzed hydrolytic reactions, notably ping-pong kinetic schemes, are ubiquitous. Similar effects can be expected for e.g. NAD-dependent oxidoreductases as well as a number o f transferases and lyases that have been employed for enantioselective conversions. Partitioning effects as described here may be important for other hydrolytic enzymes acting in biphasic systems in which either substrates or products make up the second phase. Unambiguous evaluation o f the effect o f interfacial adsorption on the enantioselectivity was possible via the method o f initial kinetics on mixed enantiomers.

ACKNOWLEDGEMENTS We would like to thank Ms. Diana Kraayveld for the purification o f P P L from the crude pancreatin, and DSM-Andeno for financing o f the project.

REFERENCES 1. 2. 3. 4.

5. 6. 7. 8.

C . - S . Chen, Y . Fuyimoto, G. Girdaukas, and C.JH. Sih, J . Am. Chem. S o c . 1 0 9 (1987), 7194-7299. C . - S . Chen, S.-H. Wu, G. Girdaukas, and C . J . Sih, J . Am. Chem. Soc. 1 0 9 (1987), 2812-2817. W . T . Ladner and G. M . Whitesides, J . Am. Chem. S o c . 1 0 6 (1984), 7 2 5 0 - 7 2 5 1 . M . Philippi, J . A . Jongejan, and J . A . Duine (1987), 2 7 9 - 2 8 4 In Biocatalysis in organic media, C. Laane, J . Laane, and M . D . Lilly Eds., Elsevier Science Publishers, A'dam. F . K . Winkler, A. D'Arcy, and W . Hunziker (1990), Nature 343, 771-774. J . A . Jongejan, J . B . A . van T o i , A . Geerlof, and J . A . Duine, Reel. Trav. Chim. PaysBas 1 1 0 (1991), 247-254. J . B . A . van Toi, J . A . Jongejan, A. Geerlof, and J . A . Duine, Reel. Trav. Chim. PaysBas 1 1 0 (1991), 2 5 5 - 2 6 2 . B . R . Rabin (1967), Biochem. J . 1 0 2 , 22c-23c.

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

245

Biocatalysts operating at high substrate concentrations P. W. Kühl Institute of Theoretical Biology, Schaulistr. 2, CH-4142 Münchenstein BL, Switzerland Abstract At high substrate concentrations biocatalysts (enzymes, organelles, whole cells) often display substrate inhibition (SI) or substrate activation (SA). In this paper we confine ourselves to the treatment of SI and SA in enzymes. After briefly describing the complex phenomenology of SI and SA in conventional and nonconventional media and after summarizing the previously proposed mechanisms for explanation of SI and SA, we present our view of these phenomena. We pay particular attention to the temporal relationships between substrate arrival/departure events and the various phases which a working enzyme traverses during its operation cycle. Our "phasetheoretic" approach uses concepts of electrophysiology and queueing theory and leads to the conclusion that SI and SA are essentially a question of timing. 1. INTRODUCTION When biocatalysts (enzymes, organelles, whole cells) are exposed to high substrate concentrations, their catalytic efficiency may (i) decrease [substrate inhibition (SI)], (ii) increase [substrate activation (SA)] or (iii) remain unaffected. SI was already reported in the last century both for enzymes [1-3] and intact cells [4, 5] and has since been documented in thousands of publications both in enzymology and fermentation technology. SI was early recognized as an "almost universal phenomenon" [6] and there is perhaps no biocatalyst which does not display SI under certain reaction conditions with certain (non-natural or non-optimal) substrates. SA, defined by an attainable v m ax or u m ax greater than is consistent with classical Michaelis/Menten or Monod kinetics, is less frequently encountered in the literature. SA has been reported mainly for enzymes and occasionally also for organelles and transport processes across membranes, but it seems that SA kinetics has been only seldom explicitly referred to in studies concerning cell growth or product formation by intact cells. For this reason and because intact cells represent more complex and therefore less readily interprétable systems, in the following we focus on SI and SA in enzymes and do not treat SI and SA in higher biocatalytic systems such as metabolons, organelles or whole cells. 2. PHENOMENOLOGY OF SI AND SA Essential characteristics of typical SI are a descending limb in v(S) graphs (where reaction velocity ν is plotted versus substrate concentration [S]) and full reversibility of the inhibition; the attainable v m ax is often, but by no means always, lower than predicted by a Michaelian (i.e. hyperbolic) kinetics of the ascending limb in a v(S) graph. The

246

substrate concentration at which ν starts to decrease upon further increase of [S] can vary greatly: some enzymes display SI only at higher than 1M [S], others are already inhibited by submicromolar [S], but a major portion of enzymes shows SI in the millimolar range of [S]. SI occurs in fast and slow enzymes and in all enzyme classes (EC1 to EC6). SI can be observed in all kinds of kinetic mechanism (ping-pong, sequential, random, ordered) and with all possible sizes and kinds of substrate (gases [e.g. oxygen, acetylene], inorganic ions, sugars, aminoacids, nucleotides, oligo- and polysaccharides, proteins, nucleic acids etc.). SI can be induced, alleviated or modified by a great variety of means, e.g. by changing milieu parameters (pH, ionic strength, temperature, viscosity, organic cosolvents etc.) or by changing structural parameters of the substrate (number of hydrophobic groups, steric arrangement etc.) or the enzyme (modified or exchanged aminoacids, covalently or noncovalently attached molecules etc.). Furthermore, we can distinguish partial and total SI, fast and slow onsetting SI, competitive, noncompetitive and uncompetitive SI. A comprehensive description and treatment of these various manifestations of SI are however beyond the scope of this paper. SA is a similarly multifaceted phenomenon as SI. SA has also been reported in all enzyme classes (EC1 to EC6) and its presence, absence or degree can be manipulated essentially by the same means mentioned above for SI. Moreover, enzymes can be caused to switch from SI kinetics to SA kinetics and vice versa e.g. by varying pH [7-9] or temperature [10], by choosing a different substrate [11-13], by immobilizing the enzyme [14] or by mutationally or chemically changing the enzyme's structure [15-17]. In addition the mere increase of [S] from moderately high to very high levels can switch the kinetic pattern from SA to SI [18-21], but in some cases the opposite or stilJ more complex kinetic patterns have been reported giving rise to undulating v(S) graphs [22-24]. The bewildering phenomenological complexity and diversity of kinetic patterns at high [S] encountered already in conventional (i.e. aqueous) media are further augmented when enzymes are studied in nonconventional (e.g. water-poor, micellar or polyphasic) media. Microcompartmentalization of the medium and selective partitioning of substrates and other ligands between different compartments may drastically alter the overall [S] at which SI or SA sets in; for instance, microemulsions or reversed micellar media have been shown to shift up [25] or down [26] the inhibitory range of the overall [S] by one to two orders of magnitude. In spite of the quite complex phenomenology of enzyme kinetics at high [S] both in conventional and nonconventional media we suggest that the "aetiology" of genuine SI and SA is relatively simple, i.e. there are only a few causes and perhaps only one fundamental principle that give rise to genuine SI and SA (or their absence). In the subsequent section we first give a short summary of previously proposed mechanisms for explanation of SI and SA and then present our view of these phenomena. 3 . AETIOLOGY OF SI AND SA First one has to be aware that there are genuine and pseudo forms of SI and SA; the former are intrinsic properties of an enzyme, whereas the latter are consequences of impurities or of events taking place without participation of the enzyme in question. Pseudo SI and pseudo SA can be caused e.g. by complexation or micellation of the substrate, by contamination of the substrate with a noncompetitive inhibitor or activator, by physicochemical alteration of the solvent or by peculiarities of helper enzymes in coupled enzyme assays and other inadequacies of the analytical method. Since a mixture of two or more Michaelian isoenzymes with different kinetic parameters can

247

simulate SA (but not SI) behaviour, it is especially in cases of suspected genuine SA important to verify the chemical homogeneity of the studied enzyme. In the following we consider only the genuine forms of SI and SA.

3.1. Previously proposed mechanisms for explanation of SI Among the mechanisms proposed for explanation of genuine SI the following two have been most frequently11reported: (i) The "wrong-number mechanism which postulates that an increased number of substrate molecules are bound at the same time - either isosterically or allosterically [27-29 and numerous further references from 1930 up to now]. This mechanism, also called the (generalized) Haldane mechanism, has been advocated particularly in the case of hydrolytic enzymes (EC3). (ii) The "wrong-order " mechanism which postulates a changed order of substrate addition without an increased number of bound molecules of a given substrate. Examples: - Substrate Β unites with enzyme Ε before substrate A and thus gives rise to an abortive binary complex EB. - Substrate A unites with enzyme Ε before all products (P,Q,...) have been released and thus gives rise, for example, to an abortive ternary complex EQA. The wrong-order mechanism [30, 31 and numerous further references from 1956 up to now] is applicable to multiple-substrate and multiple-product enzymes (i.e. the great majority of enzymes) and is, for instance, very popular in the case of pyridine nucleotide dependent dehydrogenases (EC1). For some enzymes the following additional mechanisms have been proposed: (iii) The " wrong-aggregation " mechanism (applicable only to enzymes undergoing association/dissociation reactions) which postulates that high [S] favours a less active or inactive aggregation state of the enzyme; the less active state may be caused either by association [32, 33] or by dissociation [34, 35]. (iv) The " wrong-conformation " or " wrong-isomer " mechanism which postulates that high [S] favours a less active or inactive conformational (or other kind of ) isomer of the enzyme. Neither allosteric (or other kinds of ) interactions between multiple catalytic or noncatalytic substrate-binding sites nor an increased number of simultaneously bound substrate molecules are invoked. This mechanism does also not require a changed order of substrate addition or a changed aggregation state. Such a purely conformational or isomeric mechanism has been put forward as explanation of SI, for instance, in certain ribonucleases [36-38] and Bacillus subtilis L-alanine dehydrogenase [39]. Occasionally, mixed SI models which combine various elements of the mentioned mechanisms (i) to (iv) are encountered in the literature.

3.2. Previously proposed mechanisms for explanation of SA

All four types of mechanism listed above for explanation of SI have - with opposite sign - also been proposed for SA, but some of them are more popular or plausible for SA than for SI and vice versa. By far the most frequently advocated SA mechanism is the allosteric (or multiple catalytic sites) version of the increased-number mechanism (i) [40 and numerous further references from 1953 up to now], whereas the isosteric (single binding site) version of this mechanism has been rarely considered [41]. The changed - order mechanism (ii), e.g. the formation of EQA or EQB ternary complexes displaying enhanced product Q release [42-45], has been relatively often reported, whereas the changed-aggregation mechanism (iii) [46] and the changedconformation mechanism (iv) [47-49] as possible causes of SA are found relatively

248

seldom in the literature. Analogous to SI, some authors combined various elements of the mentioned mechanisms (i) to (iv) to interpret the experimental SA data. 3.3. The phase-theoretic approach The mechanisms enumerated above appear to cover all situations where genuine SI and SA have been observed and each mechanism may be ad hoc "correct", i.e. it can "explain" (at least approximately) the specific case(s) for which it was devised. Nevertheless, we think that most of the previously proposed mechanisms do not really touch the heart of the problem since they widely ignore basic temporal aspects. For an adequate interpretation of SI and SA it seems to us indispensable to pay due attention to the temporal relationships between internal isomerization processes of the enzyme and external ligand arrival/departure events. Our "phase-theoretic" interpretation of SI and SA is based on the following facts and ideas: 1. A working enzyme traverses during its operation cycle several temporal phases and various states of excitability and refractoriness [50]. The climax of the enzymic cycle is the formation or fission of a covalent bond which can be viewed as a quasi-point process dividing the operation cycle into two main phases: the prec/imax phase and the postclimax phase . (In enzymic mechanisms where two or more covalent bonds are sequentially formed or broken - e.g. in enzymes forming transitory covalent adducts with (a portion of) a substrate [51] - , we define the instant of the first chemical alteration of a substrate as end of the preclimax and start of the postclimax phase.) The start of the preclimax phase which coincides with the arrival of the first substrate molecule at the resting enzyme is set equal to phase 0. The end of the postclimax phase which is reached after restoration of the enzyme in the resting state and usually preceded or accompanied by release of the product(s) is set equal to phase 1. The position of the climax within the cycle can vary greatly from system to system and can adopt any phase value between 0 and 1. 2. At each phase of the operation cycle ligands (substrates, products, modifiers) may arrive at or depart from catalytic or noncatalytic sites in a more or less random fashion. We qualify these arrival or departure events as (i) euchronic, (ii) dyschronic, or (iii) time-indifferent (chrononeutra/) - depending on whether the time of arrival or departure during the operation cycle is of positve, negative or no influence on the reaction velocity v. 3. The substrate concentration is intrinsically a temporal quantity: the higher the concentration, the shorter the mean interarrivai time of substrate molecules at the binding site(s). We claim that application and elaboration of these ideas to situations where SI and SA are observed will show that most, if not all, previously proposed SI and SA mechanisms can be traced back to changed temporal relationships between substrate binding and enzymic isomerization events. (NB: Enzymic isomerizations are not necessarily identical with conformational rearrangements; the intramatrix migration of protons, substrate molecules, water or other ligands may as well give rise to a variety of isomeric forms without changing the enzyme's conformation.) Furthermore, we claim that the phase-theoretic approach is not only applicable to SI and SA but also to other types of non-Michaelian behaviour of enzymes, e.g. to sigmoidal or otherwise nonhyperbolic ascents in v(S) graphs. The basic tenet of this approach is that a non-Michaelian response of an enzyme is the consequence of a changed temporal pattern of substrate arrivals during the various phases and subphases of the operation cycle whereas for a Michaelian enzyme it is irrelevant whether, when and how often additional substrate molecules - besides the "successful" one arrive during the operation cycle. In other words, the Michaelian operation cycle is devoid of timing-sensitive phases, i.e. entirely chrononeutral, whereas a non-

249

Michaelian operation cycle may have a highly sophisticated time structure where refractory, excitable and superexcitable time segments (subphases) are dispersed over the preclimax and/or postclimax phase in a pattern specific for a given enzyme system. We have formulated our theory also in quantitative terms, but so far only for a simple case of SI (Kühl and Adams, to be published). Since the substrate arrival events during the various phases of the operation cycle are usually randomly distributed, only a stochastic approach appears adequate. The methods of queueing theory, recommended for the treatment of enzyme kinetic data already about thirty years ago [52], appear particularly useful for the quantitative elaboration of our theory.

3.4. The phase-theoretic interpretation of SI

According to our view SI is a consequence of temporal mismatch or "wrong" timing (dyschrontf of isomerization and association events. Specifically we postulate that SI arises when the association events of substrate molecules are not properly staggered in time but occur in too rapid a succession so that the enzyme does not have sufficient time between successive association events to complete an ongoing isomerization process; such untimely associations trap the enzyme in a refractory (= nonactivatable) state. Our SI model, termed RECOVERY MODEL (Kühl and Adams, to be published), does not require (but also does not exclude) that in substrate-inhibited enzymes substrate molecules bind with a wrong (= increased) multiplicity or in a wrong order; rather the mere fact that a substrate molecule binds untimely, i.e. before a critical time interval has elapsed after the preceding association or dissociation event, suffices to elicit SI. In other words, during the operation cycle of the enzyme there exist one or more critical time segments (vulnerable phases) during which the enzyme must not be reoccupied by a substrate molecule in order not to disturb (retard or revert) an ongoing isomerization process (postulate of obligatory nonoccupancy). A specific feature of a quantitatively elaborated version of our RECOVERY MODEL is that the mentioned isomerization is assumed to be a multistep process and - in accordance with earlier suggestions [53,54] - of quasi-fixed duration, i.e. with a negligibly small temporal variance, whereas the association reaction of an inhibitory substrate molecule is assumed to be a simple Poisson process and subject to statistical variation. We have shown by computer-aided calculations that in our model where two reactions (isomerization, association) with intrinsically different time structures compete with each other can lead to a very steep fall (a sudden switch off) of enzyme activity as soon as a critical substrate concentration is exceeded. Thus, steep descents in v(S) graphs, repeatedly reported experimentally, can be explained without invoking allostericity and multiple binding sites. Depending on whether dyschronic substrate binding occurs before or after the climax of the operation cycle, we distinguish preclimax and postclimax mechanisms of SI. The Haldane mechanism (see above under 3.1.) is a preclimax SI since the excess (inhibitory) molecule of e.g. substrate A arrives and exerts its inhibitory action before the other (noninhibitory) molecule of substrate A is converted to product(s). All other mechanisms (wrong-order, wrong-aggregation, wrong-isomer mechanisms; see above under 3.1.) can be preclimax or postclimax types of SI or both together. Experimentally, preclimax and postclimax SI can be readily distinguished: When excess substrate is added to a resting enzyme, preclimax SI suppresses enzyme activity already during the first catalytic cycle whereas postclimax SI usually gives rise to an initial (= first cycle) pulse of enzyme activity before SI sets in. If SI is total or almost total, postclimax SI thus provides an efficient mechanism for pulse or signal generation. (These differences between preclimax and postclimax SI hold only for nonhysteretic SI; hysteretic

250

SI where the inhibition develops only after multiple, e.g. hundred, catalytic cycles, is a special case and not considered here.) 3.5. The phase-theoretic interpretation of SA Before discussing SA a terminological remark appears appropriate here, since the term SA is used in the literature not with a uniform meaning. Many authors restrict the term to situations where v m ax is higher than predicted by Michaelis/Menten kinetics but others designate with SA all situations where with increasing [S] the reaction velocity ν increases more rapidly than is consistent with Michaelis/Menten kinetics. The latter definition includes cases where at low or intermediate [S] a steeply ascending segment in v(S) graphs is observed (e.g. in "negatively cooperative" or sigmoidal kinetics); in addition, it does not necessarily mean that v m ax is increased. In this paper (see INTRODUCTION) we apply the former definition and therefore focus the discussion on cases where v m ax (and not only a certain range of submaximal ν) is greater than expected for Michaelian behaviour. In the case of a Michaelian enzyme v m ax is attained when all enzyme molecules in the population are "busy" all the time, i.e. when the idle intervals between operation cycles have shrunk to virtually zero; the mean cycle duration and the 1:1 ratio of number of climaxes to number of cycles remain unchanged, i.e. they are independent on [S]. A sub- or supra-Michaelian v m ax can however be obtained when the mean cycle duration and/or the climax/cycle ratio are dependent on [S]. From our phase-theoretic point of view we suggest the following possible causes of SA. a) SA is a preclimax effect. Repetitive and properly timed substrate binding events during the preclimax phase may be necessary or facilitative to reach the climax [50]. Thus at elevated [S] the probability is increased to attain the climax within a shorter time and to shorten the total cycle duration; consequently also v m ax is increased and SA is obtained. b) SA is an early postclimax effect. Shortly after the catalytic act the enzyme may stay for a while in a superexcitable state before returning to the resting state. A substrate molecule arriving during this phase may be immediately converted to product(s) and thus bypass the timeconsuming preclimax phase. Repetitive arrivals of substrate molecules during the early postclimax phase may greatly prolong the superexcitable phase and give rise to a quasi-tetanic response, i.e. to a quick succession of catalytic acts without intervening relaxation periods. The result is a multiclimax operation cycle of (usually) prolonged duration. Since the increase of the catalytic efficiency by multiple climaxes usually outweighs the decrease of the catalytic efficiency by a prolonged cycle duration, SA is obtained. Another possibility, mentioned before under 3.2., is that substrate molecules arriving during the postclimax phase enhance product release - either directly by "pushing away" the still attached product molecule(s) or indirectly by conformational effects. The result is either a single-climax operation cycle of shortened duration due to a faster return to the resting state or, if the latter is not revisited, a multiclimax operation cycle of (usually) prolonged duration; in both cases SA is obtained. Enhancement of product release can be relevant also at relatively late times after the climax when a product is relatively sticky. c) SA is a late postclimax effect. In some enzymes one or more superexcitable phases may occur during or immediately after the recovery or refractory phase but before restoration of the stable resting state. (This situation bears some resemblance to the "supernormality" phase during the recovery process in nerve [55] and other excitable tissues.) Arrival

251

of substrate molecules at a late superexcitable phase may precociously terminate the recovery process, eliminate an idle intercycle interval and accelerate the preclimax processes of the next operation cycle. When in this way the mean cycle duration is shortened by high [S], v m ax increases and SA is obtained. 4 . CONCLUDING REMARKS Our phase-theoretic approach is equally well applicable to single-substrate, multiple-substrate, single-product and multiple-product enzymes and is capable of explaining SI and SA without postulating (but also without excluding) an increased number of bound substrate molecules, a changed order of substrate addition or a changed aggregation state of the enzyme. Decisive for the appearance of SI and SA is that arriving substrate molecules hit the enzyme during critical time segments in the preclimax or postclimax phase of the operation cycle. From this point of view SI is caused by "wrong" timing (dyschrony) due to temporal mismatch of external substrate arrival/departure events and internal rearrangement processes, whereas SA is caused by a particularly "fortunate" timing (euchrony) so that a more profitable use of the enzyme's catalytic potential is made. We do not, of course, deny that also spatial aspects ("eutopic" and "dystopic" binding modes [56,57], steric relationships between multiple binding sites etc.) are important for the functioning and the catalytic efficiency of enzymes and play a role in SI and SA. We assert, however, that SI and SA models which do not consider the basic temporal aspects addressed in this paper miss the root of the problem. 5. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

M. Barth, Ber. Dt. Chem. Ges., 11 (1878) 474. A. Medwedew, Arch. ges. Physiol., 65 (1896) 249. A. Medwedew, Arch. ges. Physiol., 74 (1899) 193. A. J. Brown, J. Chem. Soc. Trans., 61 (1892) 369. J. O'Sullivan, J. Chem. Soc. Trans., 61 (1892) 926. W. Langenbeck, Die organischen Katalysatoren und ihre Beziehungen zu den Fermenten, Verlag von Julius Springer, Berlin, 1935, p. 70. F. Fiedler and E. Werle, Eur. J. Biochem., 7 (1968) 27. C. M. McEwen Jr., G. Sasaki and W. R. Lenz Jr., J. Biol. Chem., 243 (1968) 5217. F. Schuber and P. Travo, Eur. J. Biochem., 65 (1976) 247. S. Pulvin, A. Fribouletand D. Thomas, Biochim. Biophys. Acta, 1041 (1990) 97. H. Nakata, Ν. Yoshida, Y. Narahashi and S. Ishii, J. Biochem., 71 (1972) 1085. Y. Hatanaka, H. Tsunematsu, K. Mizusaki and S.Makisumi, Biochim. Biophys. Acta, 832 (1985) 274. J. G. Bieth, S. Dirrig, M.- L. Jung, C. Boudier, E. Papamichael, C. Sakarellos and J.-L Dimicoli, Biochim. Biophys. Acta, 994 (1989) 64. Κ. C. O'Connor, H.- J. Schütz and J. E. Bailey, Biotechnol. Bioeng., 33 (1989) 896. F. Marcus and E. Hubert, J. Biol. Chem., 243 (1968) 4923. F. Messenguy, M. Penninckx and J.- M. Wiame, Eur. J. Biochem., 22 (1971) 277. T. J. Bazzone and B. L. Vallee, Biochemistry, 15 (1976) 868. R. A. Alberty, V. Massey, C. Frieden and A. R. Fuhlbrigge, J. Am. Chem. Soc, 76 (1954) 2485. R. C. Davies, J. F. Riordan, D. S. Auld and B. L. Vallee, Biochemistry, 7 (1968) 1090. J. S. Barton and J. R. Fisher, Biochemistry, 10 (1971) 577.

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B. Désiré, G. Blanchet and H. Philibert, Biochimie, 55 (1973) 643. N. F. Belyaeva and V. I. Telepneva, Doklady Akad. Nauk, Biochem. Sect., 209 (1973) 139. R. Müller and A. P. Sokolov, Z. Allg. Mikrobiol., 19 (1979) 261. P. V. Prasad and N. Appaji Rao, J. Biosci., 6 (1984) 613. J.-P. Samama, K. M. Lee and J.- F. Biellmann, Eur. J. Biochem., 163 (1987) 609. R. M. D. Verhaert, B. Tyrakowska, R. Hilhorst, T. J. Schaafsma and C. Veeger, Eur. J. Biochem., 187(1990) 73. A. Medwedew, Arch. ges. Physiol., 103 (1904) 403. J. B. S. Haldane, Enzymes, Longmans, Green and Co., London, 1930, p.84. D. R. P. Murray, Biochem. J., 24 (1930) 1890. Y. Takenaka and G. W. Schwert, J. Biol. Chem., 223 (1956) 157. I. B. Wilson and E. Cabib, J. Am. Chem. Soc, 78 (1956) 202. S. Kaufmann, J. Biol. Chem., 245 (1970) 4751. S. Yamamoto and Κ. B. Storey, Int. J. Biochem., 20 (1988) 1261. C. Frieden, J. Biol. Chem, 234 (1959) 809. G. Hathaway and R. S. Criddle, Proc. Nat. Acad. Sei. U.S.A., 56 (1966) 680. P. M. Kaiser, Doctoral dissertation, University or Marburg, FRG , 1972. H. Rübsamen, R. Khandker and H. Witzel, Hoppe-Seyler's Z. Physiol. Chem, 355 (1974) 687. R. Müller, Doctoral dissertation, University of Münster, FRG , 1975. C. E. Grimshaw and W. W. Cleland, Biochemistry, 20 (1981) 2650. R. A. Alberty and R. M. Bock, Proc. Nat. Acad. Sei. U.S.A., 39 (1953) 895. A. A. Klesov and B. L. Vallee, Sov. J. Bioorg. Chem, 3 (1977) 602. K. Dalziel and F. M. Dickinson, Biochem. J , 100 (1966) 491. G. H. Sheys and C. C. Doughty, Biochim. Biophys. Acta, 242 (1971) 523. C. C. Doughty and S. M. Conrad, Biochim. Biophys. Acta, 708 (1982) 358. F. M. Dickinson, Biochem. J , 225 (1985) 159. A. Tourian, Biochim. Biophys. Acta, 242 (1971) 345. W. P. Jencks in: N. O. Kaplan and E. P. Kennedy (eds.), Current Aspects of Biochemical Energetics, Academic Press, New York and London, 1966, p.273. U. Femfert and P. Cichocki, FEBS Lett, 27 (1972) 219. R. Jarabak and J. Westley, Biochemistry, 13 (1974) 3233. P. W. Kühl, Eur. J. Pharmacol, 183 (1990) 1547. L. B. Spector, Covalent Catalysis by Enzymes, Springer-Verlag, New York a.o, 1982. A. F. Bartholomay in: J. Gurland (ed.) Stochastic Models in Medicine and Biology, The University of Wisconsin Press, Madison, 1964, p. 101. V. I. Deshcherevskii, A. M. Zhabotinskii, Y. Y. Sel'kov, N. P. Sidorenko and S. E. Shnol', Biophysics, 15 (1970) 235. V. N. Morozov, Biophysics, 17 (1972) 974. H. T. Graham, Am. J. Physiol, 110 (1934) 225. E. A. Zeller, Ann. Ν. Y. Acad. Sei, 107 (1963) 811. Ε. Α. Zeller, Biochem. Ζ , 339 (1963) 13.

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

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Regulation of allosteric enzymes in water-restricted media C. Lambert and V. Larreta-Garde Laboratoire de Technologie Enzymatique, U.R.A. 1442 C.N.R.S., B P 649, 60206 Compiègne cédex, France.

Abstract

In presence of water soluble additives, the regulatory character of an allosteric enzyme, P F K , was modulated. Both sigmoidal behaviour with respect to F 6 P and inhibition by excess substrate with respect to ATP were modified. Depending on the medium conditions, either activation or inhition was measured, either allosteric regulation or michaelian behaviour was observed ; excess substrate inhibition increased or disappeared. These effects seemed independent of the nature of the additive used; enzyme activity and regulation appeared to be controlled by reaction medium properties such as viscosity and water activity. The diffusion role in the observed influence of soluble additives was discussed. The results may contribute to the understanding of in vivo enzyme behaviour.

1.INTRODUCTION All fundamental enzymology mechanisms have been obtained from experiments carried out in homogeneous conditions. However, enzyme must actually react in complex and heterogeneous media, either within the living cell or in industrial processes. The medium conditions encountered by enzymes may be very different depending on the purpose of the catalyzed reaction, but enzymes often have to react in water-restricted media. The various interactions between the biocatalyst and its substrate may be modulated, depending on the solvent properties, which results in modifications on activity , stability [1], nature of the reaction catalyzed [2], but also on specificity [3] of the concerned enzymes. The influence of solvents on enzyme behaviour has been largely studied, but very few studies dealing with the relation between enzymatic regulation and microenvironment are already available. To mimic biological media and try to understand the related enzymatic mechanisms, aqueous media with restricted water activity have been used. These media have been obtained by addition of water-binding agents such as polyols and sugars to ordinary buffer. Their physico-chemical properties ( a w , viscosity, solute concentrations,...) are comparable to cytoplasm ones and make them a good model for agrofood media.

254

A l l o s t e r i c e n z y m e s h a v e b e e n c h o s e n a s m o d e l b i o c a t a l y s t s for regulation studies. With these enzymes, a slight perturbation of the active m o l e c u l e s d u e to a c h a n g e i n t h e i r m i c r o e n v i r o n m e n t will b e e x p r e s s e d b y a n amplified m e s s a g e , which c o n s e q u e n t l y will be m e a s u r a b l e through apparent modifications in enzyme catalysis. T h e kinetic behaviour of rabbit m u s c l e p y r u v a t e k i n a s e h a s a l r e a d y b e e n d e s c r i b e d t o b e d e p e n d e n t on r e a c t i o n m e d i u m c o n d i t i o n s [ 4 ] , b u t t h i s e n z y m e is a l l o s t e r i c a l l y r e g u l a t e d o n l y i n t h e a b s e n c e o f a m o n o v a l e n t c a t i o n w h i c h i s n o r m a l l y r e q u i r e d for maximal activity [5]. R a b b i t muscle phospho-fructokinase ( P F K ) , was thus chosen as it displays regulatory kinetic behaviour in the form of sigmoidicity w i t h r e s p e c t to F 6 P , t h e s u b s t r a t e , and i n h i b i t i o n b y e x c e s s A T P , t h e c o s u b s t r a t e [ 6 ] . O n t h e o t h e r h a n d , t h i s e n z y m e r e g u l a t i o n h a s b e e n s h o w n to be d e p e n d e n t on m e d i u m conditions s u c h a s pH a n d t e m p e r a t u r e [ 7 , 8 ] . In t h e p r e s e n t paper, we a r e s t u d y i n g t h e r e l a t i o n b e t w e e n the regulation of P F K and the physico-chemical properties of the reaction medium.

2. EXPERIMENTAL Initial enzyme activity was measured spectrophotometrically at 3 4 0 n m . T h e r e a c t i o n w a s c a r r i e d o u t a t 2 5 ° C i n a 5 0 m M s o d i u m p h o s p h a t e buffer p H 6.9 containing 1 m M E D T A , 5 m M magnesium acetate, 2 5 m M potassium 1 chloride, 0.2 m M N A D H , 0.2 m M P E P , 12.5 I . U . m l - L D H , 1 2 . 5 I . U . m H P K 1 a n d 5 I . U . m l - P F K . A T P a n d F 6 P c o n c e n t r a t i o n s v a r i e d f r o m 0 . 1 to 2 . 5 m M . Glycerol, glucose, sorbitol and maltose were used a t concentrations r a n g i n g from 0 . 0 0 1 M to 1 1 . 5 M d e p e n d i n g on t h e i r s o l u b i l i t y i n w a t e r . All t h e u s e d a d d i t i v e s a n d e n z y m e s w e r e s a l t - f r e e . S u g a r a n d polyol s o l u t i o n s w e r e p r e p a r e d b y a d d i n g w a t e r to solid buffer c o m p o n e n t s a n d s u b s t r a t e s to o b t a i n c o n s t a n t s a l t c o n c e n t r a t i o n , a s a l l o s t e r i c e n z y m e s a r e v e r y s e n s i t i v e to i o n i c s t r e n g t h . V i s c o s i m e t r i c c o n s t a n t s [η] w e r e e i t h e r o b t a i n e d f r o m P h y s i c o c h e m i c a l H a n d b o o k s or m e a s u r e d in a s e m i - a u t o m a t i c S c h o t t A V S 4 0 0 viscosimeter.

3. RESULTS AND DISCUSSION 3.1. Activity measurements T h e e n z y m a t i c r e a c t i o n w a s first m e a s u r e d a s a f u n c t i o n o f F 6 P c o n c e n t r a t i o n i n p r e s e n c e o f s e v e r a l additives a t c o n c e n t r a t i o n s r a n g i n g from 0 . 0 0 1 M to 2 M . T h e A T P c o n c e n t r a t i o n w a s c h o s e n a c c o r d i n g to P e t t i g r e w a n d F r i e d e n [ 8 ] who showed t h a t t h e b e h a v i o u r w i t h r e s p e c t to one s u b s t r a t e d e p e n d e d on t h e c o n c e n t r a t i o n o f the other s u b s t r a t e . A n i l l u s t r a t i o n o f s o m e r e s u l t s o b t a i n e d w i t h m a l t o s e is p r e s e n t e d on figure 1. I n all t h e t e s t e d media, P F K exhibited sigmoidal k i n e t i c s . At high maltose concentrations (> 0.5 M), a sharp decrease in the enzyme activity was

255

observed (VM decreased by 4 5 % with 1 M maltose) while at low concentrations ( < 0 . 5 M ) an activation was obtained ( V M increased by 3 3 % with 0 . 0 1 M maltose).

Figure 1. P F K activity as a function of F6P concentration in reaction media containing 2.5 mM ATP and no (•), 0.01 M (•) or 1 M ( · ) maltose.

Figure 2.PFK activity as a function of ATP concentration in reaction media containing 0.5 mM F 6 P and no (•), 0.01 M (4) or 1 M ( · ) maltose

The apparent affinity modifications induced by a change in the enzyme microenvironment are negligible at low additive concentrations, then the affinity decreased. This emphasized additive influence : relative enzyme efficiency values varied from 154 to 31 % of the reference one. A similar profile was observed with the other additives (glycerol, glucose, sorbitol),when used in the same concentration range. The same measurements were realized in presence of 0.5 mM F6P as a function of ATP concentration. An illustration of the results obtained with maltose is presented on figure 2. The inhibition by high levels of ATP was also found sensitive to the reaction medium composition. While for the reference a 4 0 % inhibition was induced by 2 . 5 mM ATP, in presence of 0 . 0 1 M maltose the inhibition was reduced by 2 0 % and the measured activity in presence of high ATP concentrations was nearly equal to the maximal one observed in buffer. The 1 "positive' effect induced by low sugar concentrations and previously described with respect to F 6 P was also observed with respect to ATP. As for the other substrate, this positive effect decreased as the additive concentration increased and for high maltose concentrations (> 0 . 5 M) a negative influence was measured and the inhibition was found more pronounced ( 7 0 % inhibition for 2 . 5 mM maltose). A similar behaviour was observed with the other additives tested.

256 3.2.

Medium influence

The high solubility of glycerol made it possible to use this additive in a very large concentration range (from 0.01 to 11.5 M). At low glycerol concentrations, the behaviour of P F K was modified by glycerol as by the other sugars and polyols. For glycerol concentrations higher than 2 M, the activity drastically decreased : 75% decrease for 6 M glycerol, 90 % decrease for 8 M glycerol. The enzyme seemed to exhibit a kinetic behaviour following a Michaelis-Menten model which was confirmed by a Hill number close to 1. On the other hand, no more inhibition by excess ATP was observed. P F K activity was studied as a function of the thermodynamical and physico-chemical properties of the reaction medium induced by the presence of different additives at different concentrations. The results are presented on figures 3 and 4. JO

Ε c

Ο

ο Ε

>

I

ε

Ε >

0

100

200

Relative viscosity

Figure 3 P F K activity as a function of reaction medium water activity due to various water-soluble molecules : ( φ ) glucose, ( O ) sorbitol, ( · ) maltose, ( • ) glycerol.

Figure 4 . P F K activity as a function of r e a c t i o n m e d i u m relative viscosity due to various watersoluble molecules. Same symbols as on figure 3.

From the results on figure 3, it was obvious that P F K activity was directly controlled by reaction medium water activity. The influence of the thermodynamical parameter exerted independently of the nature of the additive used to depress water activity, as a single curve was obtained for either sugars and polyols, and with additives with 3 , 6 or 12 carbon atoms. For example, the same behaviour was observed with glucose, with its corresponding polyol and with the dimer. Such a result has already been described for egg-white lysozyme [9] ; but in this case, it was possible to distinguish between the effect of different additives inducing a different water molecules structuration (glycerol on the one hand and sorbitol and glucose on the other hand, [10]). The difference with the behaviour here observed could be due to the fact that in lysozyme reaction, water played the double role of reactant and solvent, while it did not

257

intervene in P F K reaction. In both cases, it was thermodynamical parameter may control enzyme activity.

shown

that

a

A similar general effect was obtained when activity was measured as a function of a physico-chemical parameter, viscosity. The results presented on figure 4 showed that P F K was very sensitive to the medium viscosity mainly when its value reached a threshold between 2 to 4 mPa.s. These values correspond to glucose, maltose or sorbitol concentrations higher than or equal to 0.5 M, the additive concentration over which the positive effect on activity previously described disappeared. Such an effect has also been described for lysozyme [11]. As for the influence of water activity on enzyme activity, no difference in the enzyme behaviour was observed whatever the viscosigen used. The physico-chemical parameter, viscosity, was found to control P F K activity when high. This last result made it possible to use a diffusion-reaction model in order to predict the reactivity of the enzyme in any medium only from the value of the diffusion coefficient of the substrate in this medium.

3.3. Role of diffusion

The bulk solution was considered to be an assembling of cubic identical elementary volumes, each of them containing one enzyme molecule. In this case, reaction and diffusion were separated in space, and a zero-dimensional diffusion-reaction model adopted. Under steady - state conditions, there was a balance between the reaction rate and the diffusion of the substrate into or out of the elementary volume. In this way, the "internal"concentration of the substrate S transformed in the enzyme vicinity was constant for a given concentration of So outside the enzyme microenvironment. The contribution of diffusion was approximated by Fick's law and it was possible to write the enzyme reaction rate as V

M

. «S).

in which : V m was a maximum enzyme activity f(S) was a dimensionless function (additive dependence) ranging from 0 to 1 In the present case, both the "positive" effect and the apparent inhibition were taken into account in the calculation of fl(S).

Jk'-SoJL e

e

vm«S)

s

(1)

with : e : the thickness of the diffusion layer (distance between the cube side and the outside surface of the active protein considered as a tiny cube)

258

Ds : the diffusion coefficient of S ( F 6 P ) measured using Stokes-Einstein's relation : Ds = kT / 6 π η R a with R a radius of F 6 P molecule considered as a sphere. We have verified the validity of this relation in our media by plotting In Ds = f (In [η]) [12], the slope was close to 1 (0.99). In a model system [13], a constant of the enzyme reaction is chosen as the unit of concentration, for example K M - A dimensionless parameter σ, proportional to η and similar to the Thiele modulus in chemical engineering [14], expresses the effect of the diffusion limitations, σ = V M e

2

/ KM DS .

In our case, the profile shown by the reaction when taking into account diffusion was unusual (figure 5).

0

10 20 Relative viscosity (mPa.s)

Figure 5. P F K reaction rate in additivecontaining media a s a function of reaction medium viscosity. Diffusion and additive effect on enzyme activity were taken into account. For low viscosities, no diffusion effect was observed, even for a viscosity double the buffer viscosity. Till a threshold value was attained, substrate diffusion was very fast and thus catalysis is reaction controlled. Above this value, the reaction was on the contrary directly diffusioncontrolled. A dimensionless parameter σ could not be used to express the effect of the diffusion limitations in these conditions, as apparent K M values were not constant, but declined simultaneously with k c at as viscosity increased. Such observations have already been reported by Hasinoff et al. [12], Pocker and Janjic [15] and Demchenko et al. [16] who studied respectively alcohol dehydrogenase, carbonic anhydrase and lactate dehydrogenase activities in high viscosity media. The particular behaviour shown by the two

259

oxidoreductases was attributed to a conformational modification which could modulate the enzyme-coenzyme association. The decrease of K M value is often considered to reveal a conformational change of the enzyme [17]. On the other hand, we have previously shown [4], with another allosteric enzyme, pyruvate kinase, that the presence of water soluble molecules in the enzyme reaction medium could induce slight conformational changes favourable for the enzyme activity. These structure modifications have been demonstrated through laser Raman spectroscopic observations. From the correlation between the data available in literature and our results presented here, we may postulate that the activation phenomenum observed with P F K is due to a slight change in the enzyme conformation. In media having a dielectric constant smaller than that of pure water, the active protein would adopt a structure more efficient to realize the phosphate transfer from ATP to the hydroxyl group of Ci on the F 6 P molecule .

4. CONCLUSION In presence of water soluble additives, both sigmoidal behaviour with respect to F 6 P and inhibition by excess substrate with respect to ATP of phosphofructokinase were modified. Depending on the medium conditions, either activation or inhition was observed. The regulatory properties of this allosteric enzyme were even lost when the reaction medium contained high additive concentrations : a michaelian behaviour was observed and no more inhibition by ATP was measured. These effects seemed independent of the nature of the additive used; enzyme activity and regulation appeared to be controlled by general reaction medium properties such as viscosity and water activity. Diffusion exerted an important role on enzyme catalysis when the viscosity of the medium exceeded a threshold value, below this value the reaction was the limiting step. This result confirmed informations already described in literature about enzyme catalysis in high viscosity media. A slight conformational change of the enzyme seemed to be responsible for the activation phenomenum observed at low additive concentrations. This hypothesis was well correlated to the steric exclusion principle [18]. O b s e r v a t i o n s with spectroscopic methods ( l a s e r R a m a n , F T I R , spectrofluorometry) have confirmed these structure rearrangements on related enzymes. These results are of importance as they were obtained in media well mimicking a living cell cytoplasm as far as physico-chemical and thermodynamical parameters are concerned. They may contribute to the better understanding of enzyme catalysis in vivo as they have shown that this kind of data may even influence the regulatory enzyme character. They also

260

confirm that the in vivo behaviour of enzymes could be different from the one observed in vitro and then partly controlled by the protein microenvironment.

5. REFERENCES: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Gekko K. and Koga S., J . Biochem.94 (1983) 199. Zaks A. and Klibanov Α., J . Biol. Chem.263 (1988) 3194. Hertmanni P., Picque E . , Thomas D. and Larreta-Garde V., F E B S Lett.279(1991) 123. Xu Z.F., Thomas D. and Larreta-Garde V., Ann. Ν. Y. Acad. Sei. 6 1 3 , (1990)506. Muirhead H., Biological Macromolecules and Assemblies, vol 3, Frances A. and Mc Pherson A. (eds.) ,ppl44-186, John Wiley, New York, 1987. Bloxham D. and Lardy H., The enzymes, 3° ed., vol. 8, Boyer P. (ed.), pp 239-278, Acad. Press, New-York, London, 1973. Frieden C , Gilbert H. and Bock P., J . Biol. Chem. 251 (1976) 5644. Pettigrew D. and Frieden C , J . Biol. Chem. 254 (1979) 1896. Larreta Garde V., Xu Z.F., Lamy L . , Mathlouthi M. and Thomas D., Biophys. Biochem. Res. Commun. 155 (1988) 816. Mathlouhti M., Carbohyd. Res., 91 (1981) 113. Lamy L . , Portmann Μ.Ο., Mathlouthi M. and Larreta Garde V., Biophys. Chem. 36 (1990) 71. Hasinoff B., Dreher R. and Davey J . , Biochim. Biophys. Acta 911 (1987) 53. Thomas D., Barbotin J.N., David Α., Hervagault J . F . a n d Romette J . L . , Proc. Natl. Acad. Sci.USA 74 (1977) 5314. Levenspiel Ο., Chemical Reaction Engineering 1972, John Wiley, New York. Pocker Y. and Janjic N., Biochemistry 2 6 (1987) 2597. Demchenko Α., Rusyn O., and Saburova E . , Biochim. Biophys. Acta 998 (1989) 196. Engasser J.M. and Hisland P., Biochem. J . 173 (1978) 341. Gekko K. and Timasheff, Biochemistry 20 ( 1981) 4667.

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

261

Quantitative deuterium NMR of protein hydration in air and organic solvents 8

M.C Parker*, B.D. Moore and A.J. Blacker* department of Pure and Applied Chemistry, Thomas Graham Building, University of Strathclyde, 295 Cathedral Street, Glasgow, Gl 1XL, United Kingdom b

ICI Biological Products, PO Box 1, Billingham, Cleveland, TS23 1LB, United Kingdom

Abstract

2

We have developed a H NMR technique to measure the amount of water bound to a protein whilst in the presence of an organic solvent. This has been used to measure the water adsorption isotherms of subtilisin Carlsberg in the gas phase and while suspended in hexane. 1. INTRODUCTION It is well established that for almost all enzymes a layer of adsorbed 1 water is essential to promote catalytic activity in organic solvents . It is much less certain however what the nature and extent of this adsorbed water layer is and what interactions with the protein are important to sustain catalysis. Rehydration of enzymes has been shown to increase the conformational mobility of the protein matrix both in dry powders and in organic solvents and this process is often associated with the onset of 2 catalytic activity . It is interesting however that there appears to be a wide variation in the sensitivity of different enzymes to particular solvents and it is not yet clear what sort of protein-solvent and protein-water interactions are important in controlling this. This paper describes a method for determining water adsorption isotherms of proteins both as dry powders and whilst they are suspended in an organic solvent. Comparison of these isotherms gives an important insight into how the organic solvent perturbs 3 the protein hydration layer .

262

2. EXPERIMENTAL PROCEDURE 2.1 Equilibration in the gas phase. Subtilisin Carlsberg (Sigma), 5 mg/ml was dissolved in deuterium oxide (99.9 atom%, Aldrich) and after 5 minutes, lyophilysed and dried over molecular sieves. 10-15 mg samples of the enzyme powder were equilibrated in sealed vessels with D2O saturated salt solutions of known water activity for 2-3 days at 22°C. l-2ml of dry 1-propanol (HPLC grade, Aldrich) was added to the equilibrated enzyme and the mixture stirred for 15 minutes. A 0.5ml aliquot was removed following centrifugation and the NMR spectrum measured on a Bruker 400 MHz NMR spectrometer. The increase in intensity of the hyroxyl signal relative to the alkyl signals was used to determine the amount of water bound to the enzyme. The NMR technique is very sensitive and can measure D2O content to an accuracy of ± ^ g and thus the major experimental errors arise from weighing the protein and PrOH samples.

2.2 Equilibration in hexane A known volume of hexane was added to the dry enzyme prepared as above ensuring that a layer of solvent completely covered the sample. The enzyme-solvent samples were then equilibrated and treated in exactly the same way as the dry samples. The small contribution to the increase in hydroxyl signal intensity arising from free deuterated water in the hexane was allowed for in determining the amount of water adsorbed to the enzyme.

3. RESULTS AND DISCUSSION Figure 1 shows the natural abundance deuterium spectrum of npropanol. There are four peaks due to the four different chemical environments and they have relative intensities approaching 1:2:2:3 which is the statistical probability of deuteration at each site. The peak b which should have a relative intensity of two is found consistently to be depleted which is a consequence of the biological origins of the propanol. The deuterons/protons attached to the alkyl chain will be completely nonexchangeable under normal chemical conditions while the deuterons/protons on the hydroxyl group will exchange rapidly with protic solvents. This allows the possibility of selective enrichment at the propanol hydroxyl group on contact with a deuterated sample. The natural abundance of deuterium is 0.015 mole % and this provides a convenient and sensitive internal reference for quantifying the number of exchangeable deuterons in the sample.

263

CH,

*

Γ PPM

7

6

ι 5

4

J L 3

2

F i g u r e 1. 2H Natural abundance 400 MHz NMR spectrum of 1-propanol, 512 scans. Alkyl protons b, c, dart non-exchangeable, hydroxyl protons a, exchange rapidly with any protic source.

1

2

F i g u r e 2 . H NMR spectrum of 1-propanol after addition of 18mg of benzene saturated with D2O (665 scans). The intensity of the hydroxyl signal a, has increased due to complete exchange with the deuterons of the D2O.

Figure 2. shows the H NMR spectrum obtained on adding 18.0 mg of D 0 saturated benzene to the propanol sample. The PrOD peak has become enriched by exchange of deuterons from the D 0 with the large excess of PrOH in the sample as shown below. 2

2

2

PrOH (excess) + D2O

PrOH (excess) + 2 PrOD + H 0 2

The integrated area of the deuterium enriched hydroxyl signal can be ratioed against one of the non-exchangeable alkyl peaks to calculate the amount of water added to the system. The enrichment observed in Figure 2 corresponds to 0.571 μπιο^, 11.44 μg of D2O, within the benzene sample, which can be compared with the theoretical amount of deuterated water in the water saturated benzene sample of 0.599 pmoles, 11.99 μ& This result demonstrates that the method can measure total water content in the μg range and we were interested to try and apply it to the determination of protein hydration levels which are generally of the order of 1-800 μg/mg. In particular we were interested to see if the method could be applied to measurement of the hydration of enzymes suspended in organic solvents.

264

The enzyme subtilisin Carlsberg was chosen as a model system for the hydration measurements because it can be obtained commercially in a pure form and the data obtained could be conveniently compared with previous gravimetric measurements made on it and similar globular protein systems . The degree of hydration of lyophilised proteins is found to depend critically on the water vapour pressure in the surrounding air. By controlling the water vapour pressure, using saturated salt solutions of known thermodynamic water activity, a , it is straightforward to prepare a series of protein samples with a range of specific hydration levels. We required a fully deuterated hydration layer for the analysis and thus we lyphilised subtilisin from pure D 0 , dried it over molecular sieves and then equilibrated the samples with D 0 saturated salts in air for several 4

w

2

2

days. A second series was prepared by suspending lyophilised and dried subtilisin samples in hexane. The hexane/protein mixtures were then also equilibrated against D 0 saturated salt solutions. Following equilibration, known amounts of PrOH were added to the protein or protein/hexane samples and after stirring for 15 minutes an aliquot was removed and analysed by H NMR. The enrichment of the PrOD signal was used to determine the number of exchangeable deuterons in each of the samples. In general, the major contribution to this signal enrichment will be from deuterated water adsorbed on the protein, but at lower water contents, the contribution of deuterons from exchangeable groups on the protein may become significant. Our method cannot distinguish between these two sources of deuterons and therefore in our calculations we have assigned an adsorbed water molecule to every two deuterons observed. Figure 3 shows a plot of %(w/w) of adsorbed water on subtilisin versus water activity for the air equilibrated sample. It can be seen that the resultant water adsorption isotherm follows the familiar sigmoidal curve obtained in gravimetric measurements of globular proteins . There is an initial fast increase in water content up to a water activity of 0.1 , which is thought to be associated with hydration of the highly polar groups, after this the curve rises much more slowly due to the difficulty of hydrating the non-polar and hydrophobic regions. At a water activity of around 0.7 these areas finally become hydrated, a complete monolayer is formed, and rapid hydration can then take place leading eventually to dissolution . The form of the isotherms we obtain is completely reproducible although we have observed small (±l%w/w) variations in the hydration values obtained with different batches of lyophilised enzyme which may be due to small differences in the mOrphology of the solid enzyme particles. 2

2

4

2

265 80

60

40

20 1

•

•

ι1

0.0

• 1

1

0.2

1

'

0.4

1

«

0.6

1

'

0.8

1

1.0

water activity Figure 3. • Water adsorption isotherm for subtilisin equilibrated in air with saturated salt solutions of known water activity.

80

60

40

20 Η

Ο •

o° •

Q O 1

0.0

,

1

0.2

1

1

0.4

1

1

0.6

1

1

0.8

•

I

1.0

water activity Figure 4 . ο Water adsorption isotherm for subtilisin equilibrated in hexane with saturated salt solutions of known water activity. • Water adsorption isotherm as in Figure 4 .

266

The water adsorption isotherm obtained for subtilisin suspended in hexane is shown in Figure 4 superimposed over the isotherm measured in air. It can be seen that the general shapes are similar although there are small and reproducible differences in the two curves. At very low water activities the differences in measured water content are very small and the values lie on the same line within experimental error. Above a water activity of ~0.15 however, subtilisin appears to be more strongly hydrated in hexane than in air and over the range 0.2-0.7 aw the hexane isotherm deviates noticeably following a convex curve rather than the concave curve for air. The difference between the two isotherms at 0.4 a corresponds to an extra 0.5mg D2O adsorbed on the 10mg protein sample or in other words 80 more bound water molecules per subtilisin molecule. The NMR technique shows this difference clearly and we have obtained reproducible results in repeat experiments indicating that the difference cannot be simply explained by errors associated with weighing the protein sample. At ~0.7 aw the isotherms coincide again and at the highest water activity we have measured, 0.97 a there is less water bound to subtilisin in hexane than in air. These results show that the interaction between water and protein is definitely modified by the presence of the organic phase even using a hydrophobic solvent such as hexane. The isotherm indicates that at low to intermediate water activities, when a submonolayer of bound water is thought to be present and only the less polar and hydrophobic residues are left exposed, hexane promotes the adsorption of extra water molecules to subtilisin, while at higher water activities when mutiple monolayers are present, hexane appears to disrupt the binding of water. To the best of our knowledge this is the first adsorption isotherm carried out in such a hydrophobic solvent and more work is required to establish if the observed deviations from the air isotherm are general to these types of proteins or simply a reflection of the particular protein/solvent interface presented by subtilisin. We are currently applying the same methodology to the measurement of water adsorption isotherms in a number of other organic solvents in order to get a wider insight into the way solvents can effect the formation of the protein hydration layer. w

w

Acknowledgements W e would like to thank P.R. Dennison for his help in carrying out the N M R measurements.

4. REFERENCES 1. 2. 3. 4.

A. Zaks, A.M. Klibanov, J. Biol. Chem., 263 (1988) 8017. J.A. Rupley, G. Careri, Adv. Protein. Chem., 41 (1991) 37. PJ. Hailing, Biochim. Biophys. Acta, 1040 (1990) 225. H.B. Bull, K. Breese, Arch. Biochem. Biophys., 28 (1968) 488.

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

267

Effects of Temperature on Stereochemistry of Alcohol Dehydrogenases from Thermoanaerobacter ethanolicus a

8

Robert S. Phillips", Van T.3 Pham , Changsheng Zheng , Francisco A. C. Andrade and Maria A. C. Andrade'

b

"Departments of Chemistry and Biochemistry, University of Georgia, Athens, Georgia 30602 USA b

Instituto de Quimica, Universidade Federal da Bahia, Salvador, Bahia, Brasil 40210 Abstract

We have studied the effects of temperature on the stereochemistry of NADPdependent primary (PADH) and secondary (SADH) alcohol dehydrogenases from the thermophilic bacterium, Thermoanaerobacter ethanolicus. Reduction of 1% 2-butanone with SADH in 10% 2-propanol gives (R)-2-butanone, with increased stereoselectivity at high temperature and in the presence of NADP analogues. Reduction of 2-pentanone under the same conditions gives (S)-2-pentanol, with decreased stereoselectivity at high temperatures and in the presence of NADP analogues. Reduction of racemic 2-methylbutyraldehyde by PADH gives (S)-2methyl-1 -butanol, with the stereochemical purity increasing from 14% e.e. at 15° to 51% e.e. at 35°. These results show that optimal stereoselectivity in an enzymatic reaction may sometimes be obtained at the highest temperature compatible with the stability of the enzyme substrate and cofactor system. 1. INTRODUCTION It is widely believed that enzymes must exhibit maximal stereoselectivity at low temperatures. This belief is not without some experimental support. For example, the hydrolysis of some glutarate diesters catalyzed by pig liver esterase gives optimal enantioselectivity in 20% methanol at -10° [1]. Willaert et al. found that the diastereoselectivity of the reduction of 3-cyano-4,4dimethylcyclohexanone by horse liver alcohol dehydrogenase was greater at 5° than at 45° [2], In addition, Keinan et al. found that the reduction of 2pentanone by an alcohol dehydrogenase from Thermoanaerobium brockii gave (S)-2-pentanol with highest stereoselectivity at 5° [3]. However, these effects of temperature have been determined empirically, and a theoretical foundation was not developed. We have been studying the stereoselectivity of two thermostable alcohol dehydrogenases found in a thermophilic bacterium, Thermoanaerobacter ethanolicus, especially with regards to the effects of temperature and cofactor. One of these alcohol dehydrogenases (PADH), formed during the stationary phase of anaerobic growth on D-glucose, has high activity with primary alcohols and aldehydes [4]; in contrast, the other alcohol dehydrogenase (SADH) has low activity with ethanol, but very high activity with secondary alcohol and ketones [4]. We have found that the stereochemistry of these reactions are strongly influenced by the temperature of the reaction medium and by cofactor analogues.

268 2. E X P E R I M E N T A L 2.1 E n z y m e s C e l l s o f T. ethanolicus w e r e grown a n a e r o b i c a l l y o n 0 . 1 % D - g l u c o s e a s p r e v i o u s l y d e s c r i b e d [4]. T h e growth t e m p e r a t u r e h a s a s i g n i f i c a n t i n f l u e n c e o n t h e r e l a t i v e a m o u n t s of P A D H a n d S A D H . W h e n t h e cells w e r e g r o w n for 2 2 h o u r s a t 50°, t h e cells c o n t a i n e d p r e d o m i n a n t l y S A D H , w h e r e a s w h e n t h e cells w e r e g r o w n for 2 2 h o u r s a t 60°, t h e y c o n t a i n e d p r e d o m i n a n t l y P A D H . C e l l e x t r a c t s w e r e p r e p a r e d b y s o n i c a t i o n o f t h e cells s u s p e n d e d i n 0 . 0 5 M T r i s - H C l , p H 7 . 6 . F o r p r e p a r a t i v e purposes, t h e c r u d e cell e x t r a c t a f t e r c e n t r i f u g a t i o n w a s u s e d . F o r k i n e t i c e x p e r i m e n t s , t h e S A D H w a s purified a s d e s c r i b e d [ 5 ] . 2.2 P r e p a r a t i v e R e d u c t i o n s Typical S A D H reactions contained, in a total volume of 1 0 m L , 0 . 5 % - 1 . 0 % k e t o n e , 2 0 % 2-propanol (v/v), 0 . 0 5 m M N A D P (or a n a l o g u e ) , 3 m M 2 m e r c a p t o e t h a n o l , a n d 1 2 - 2 0 u n i t s of S A D H i n 0 . 0 5 M T r i s - H C l , p H 8 . T h e r e a c t i o n f l a s k s w e r e t i g h t l y stoppered a n d i n c u b a t e d a t t h e d e s i r e d t e m p e r a t u r e i n a b a t h . T h e r e a c t i o n s w e r e w o r k e d up b y addition of ( N H ^ S C X followed b y e t h e r extraction. Yields were measured by gas chromatography on a ChiralsilV a l 2 5 m c a p i l l a r y c o l u m n . O p t i c a l p u r i t i e s of t h e products w e r e d e t e r m i n e d b y G C of t h e N-trifluoroacetyl-L-prolyl e s t e r s on t h e C h i r a s i l - V a l c o l u m n [ 5 ] . F o r r e d u c t i o n of a l d e h y d e s w i t h P A D H , solutions c o n t a i n e d , in a t o t a l v o l u m e of 7.5 m L , 3 0 % e t h a n o l , 8 . 8 u n i t s P A D H , 5.3 m g N A D P a n d 9 0 m g 2 m e t h y l b u t a n a l . T h e r e a c t i o n s w e r e w o r k e d up a n d yields d e t e r m i n e d a s d e s c r i b e d for S A D H . O p t i c a l p u r i t i e s of t h e products w e r e d e t e r m i n e d b y Ή N M R of t h e L - v a l i n e e s t e r t o s y l a t e s [ 6 ] . 2.3 Kinetic M e a s u r e m e n t s T h e o x i d a t i o n of s e c o n d a r y alcohols c a t a l y s e d b y S A D H w a s followed s p e c t r o p h o t o m e t r i c a l l y i n order to o b t a i n q u a n t i t a t i v e d a t a on t h e r e a c t i v i t y of enantiomeric substrates. Cuvettes contained, in a total volume of 0.6 m L , 0.1 m M N A D P or a n a l o g u e , v a r i o u s a m o u n t s of t h e alcohols, a n d 0 . 1 M T r i s - H C l , p H 8 . 9 . D u e to t h e h i g h t e m p e r a t u r e coefficient of t h e p K a of T r i s buffer, t h e pH w a s a d j u s t e d to 8 . 9 a t e a c h t e m p e r a t u r e . T h e solutions w e r e p r e i n c u b a t e d i n t h e s p e c t r o p h o t o m e t e r for 5 m i n u t e s before t h e r e a c t i o n s w e r e i n i t i a t e d b y a d d i t i o n of 1 0 μί. o f e n z y m e solution. I n i t i a l r a t e s w e r e m e a s u r e d a t 3 4 0 n m , 3 6 3 n m o r 3 9 6 n m , r e s p e c t i v e l y , for N A D P H , A P A D P H , a n d S N A D P H f o r m a t i o n . K i n e t i c p a r a m e t e r s w e r e d e t e r m i n e d b y n o n l i n e a r fitting of t h e i n i t i a l r a t e d a t a t o t h e M i c h a e l i s - M e n t e n e q u a t i o n u s i n g E N Z F I T T E R , from E l s e v i e r Biosoft. 3. R E S U L T S AND D I S C U S S I O N 3.1 T h e o r y o f E f f e c t s o f T e m p e r a t u r e o n S t e r e o c h e m i s t r y T h e e n a n t i o m e r i c r a t i o , E , defines t h e s t e r e o s e l e c t i v i t y of a r e a c t i o n , a n d is e q u a l to t h e r a t i o of e n a n t i o m e r i c products or t h e r a t i o of t h e r a t e s of f o r m a t i o n (or r e a c t i o n ) of t h e e n a n t i o m e r s . T h e difference i n a c t i v a t i o n free e n e r g y , AAG*', for t h e two e n a n t i o m e r s is given b y E q u a t i o n 1. T h e t e m p e r a t u r e d e p e n d e n c e of 1 AAG is t h e n given b y E q u a t i o n 2 . I f t h e r e is no e n a n t i o s e l e c t i v i t y AAG* = - R T l n E AA& = ΔΔΗ^-ΤΔΔβ

(1) 1

(2)

269 i n a r e a c t i o n , E = l , a n d t h u s AAG*=0. E q u a t i o n 2 c a n t h e n be r e a r r a n g e d t o g i v e E q u a t i o n 3, w h e r e T r defines t h e "racemic t e m p e r a t u r e " for a g i v e n reaction, a t w h i c h t h e r e i s n o s t e r e o c h e m i c a l d i s c r i m i n a t i o n . H o w e v e r , a t t e m p e r a t u r e s less t h a n T r, t h e stereoselectivity is controlled b y t h e activation e n t h a l p y difference f o r t h e t w o e n a n t i o m e r s , a n d t h e e n a n t i o m e r i c r a t i o w i l l decrease as t h e t e m p e r a t u r e is i n c r e a s e d t o T r . I n c o n t r a s t , a t t e m p e r a t u r e s g r e a t e r t h a n T r , t h e reaction is controlled b y t h e activation entropy difference, a n d t h e enantiomeric r a t i o of t h e r e a c t i o n w i l l increase w i t h i n c r e a s i n g t e m p e r a t u r e . T h e m a j o r product observed a t Τ > T r is t h e antipode t o t h a t observed a t Τ < T r, a n d t h u s a temperature-dependent reversal of stereoselectivity is predicted. T r = AAHVAAS* ( 3 ) 3.2 R e a c t i o n of S e c o n d a r y Alcohol D e h y d r o g e n a s e T h e r e d u c t i o n o f 2 - b u t a n o n e c a t a l y s e d b y S A D H a t 37° gives ( R ) - 2 - b u t a n o l w i t h l o w s t e r e o s e l e c t i v i t y ( 2 8 % e.e.), b u t 2 - p e n t a n o n e gives ( S ) - 2 - p e n t a n o l ( 4 4 % e.e.) u n d e r t h e s a m e c o n d i t i o n s [ 5 ] . T h i s u n e x p e c t e d r e v e r s a l o f s t e r e o s e l e c t i v i t y w a s also r e p o r t e d b y K e i n a n e t a l . f o r t h e r e d u c t i o n o f t h e s e k e t o n e s b y T B A D H [3]. W e s t u d i e d t h e o x i d a t i o n of 2-butanol a n d 2-pentanol b y S A D H , i n order t o g a i n some i n s i g h t i n t o t h e m e c h a n i s t i c basis of t h i s u n u s u a l b e h a v i o r . T h e r a t i o of (kca(/Km)i/(kcat/Km)s w a s u s e d as t h e e n a n t i o m e r i c r a t i o , a n d t h e d a t a w e r e p l o t t e d u s i n g E q u a t i o n 2 , as s h o w n i n F i g u r e 1 . A t t e m p e r a t u r e s b e l o w 26°, (S)2 - b u t a n o l i s t h e p r e f e r r e d s u b s t r a t e , w h e r e a s above 26°, ( R ) - 2 - b u t a n o l r e a c t s f a s t e r . T h e r e a c t i o n of 2 - p e n t a n o l shows a g r a d u a l decrease i n e n a n t i o s p e c i f i c i t y w i t h t e m p e r a t u r e , w i t h (S)-2-pentanol being t h e preferred substrate a t t m e p e r a t u r e s b e l o w 70°. T h e s e d a t a a r e t h e f i r s t e x p e r i m e n t a l d e m o n s t r a t i o n o f a temperature-dependent reversal of stereospecificity i n a n e n z y m a t i c r e a c t i o n [5]. R e c e n t l y , w e f o u n d t h a t t h e use o f cofactor a n a l o g u e s h a s a s i g n i f i c a n t effect o n t h e e n a n t i o s e l e c t i v i t y of S A D H - c a t a l y s e d r e d u c t i o n s . F o r 2 - b u t a n o n e a t 37°, t h e Ε v a l u e o f t h e ( R ) - 2 - b u t a n o l p r o d u c t i n c r e a s e d f r o m 1.3 t o 3 . 7 , 2 . 9 , o r 4 . 6 , w h e n N A D P was replaced by acetylpyridineadenine dinucleotide phosphate (APADP), thionicotinamide adenine dinucleotide phosphate (SNADP), or N A D , r e s p e c t i v e l y [ 7 ] . A t 47°, t h e c o r r e s p o n d i n g Ε v a l u e s w e r e i n c r e a s e d from 1.9 t o 9.4, 6 . 6 , o r 7.0. A t 47°, w i t h A P A D P as t h e cofactor, w e o b t a i n e d ( R ) - 2 - b u t a n o l w i t h s t e r e o c h e m i c a l p u r i t y g r e a t e r t h a n 8 0 % e.e [ 7 ] . I n c o n t r a s t , w h e n r e d u c t i o n of 2 - p e n t a n o n e w a s p e r f o r m e d w i t h A P A D P , S N A D P , o r N A D , w e o b t a i n e d (S)-2p e n t a n o l of l o w e r p u r i t y t h a n o b t a i n e d u s i n g N A D P . T h e s e r e s u l t s s u g g e s t t h a t t h e cofactor a n a l o g u e s l o w e r t h e free e n e r g y of t h e t r a n s i t i o n s t a t e l e a d i n g t o t h e ( R ) - e n a n t i o m e r s . W e t h e n p e r f o r m e d s t e a d y - s t a t e k i n e t i c e x p e r i m e n t s of t h e o x i d a t i o n o f (R)- a n d ( S ) - 2 - b u t a n o l w i t h t h e cofactor a n a l o g u e s . T h e r e s u l t s o f t h e s e e x p e r i m e n t s a r e p r e s e n t e d i n F i g u r e 1 . W e f o u n d t h a t (kcat/Km)i/(kcat/Km)s for 2-butanol increased significantly w h e n N A D P was replaced b y A P A D P or S N A D P , as e x p e c t e d f r o m t h e r e s u l t s of t h e p r e p a r a t i v e r e d u c t i o n s t u d i e s . T h e s e d a t a s h o w a l i n e a r dependence of - R T l n E w i t h t e m p e r a t u r e s i m i l a r t o t h a t o b s e r v e d w i t h N A D P ( F i g u r e 1). T h e h i g h e r Ε v a l u e s a r e t h e r e s u l t o f a s h i f t i n t h e T r v a l u e f r o m a b o u t 17° f o r N A D P t o -11° f o r S N A D P a n d 9° f o r A P A D P . T h e use of t h e coenzyme a n a l o g u e s , i n c o m b i n a t i o n w i t h t e m p e r a t u r e , p r o v i d e s a novel strategy t o enhance t h e stereoselectivity of dehydrogenase reactions.

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F I G U R E 4 . S t e r e o c h e m i s t r y is c o n t r o l l e d b y b o t h b i n d i n g a n d a c t i v a t i o n e n e r g y differences.

272

3.3 R e a c t i o n o f P r i m a r y A l c o h o l D e h y d r o g e n a s e . P A D H o b t a i n e d from T. ethanolicus h a s h i g h a c t i v i t y f o r r e d u c t i o n o f a l d e h y d e s , b u t reduces k e t o n e s p o o r l y . W e h a v e e x a m i n e d t h e r e d u c t i o n o f racemic 2-methylbutyraldehyde b y P A D H a t several t e m p e r a t u r e s , i n order t o e v a l u a t e t h e effects of t e m p e r a t u r e o n t h e s t e r e o s p e c i f i c i t y . T h e s t e r e o c h e m i s t r y of t h e r e s u l t a n t 2 - m e t h y l - l - b u t a n o l w a s d e t e r m i n e d b y Ή N M R o f t h e N t r i f l u o r o a c e t y l ( T F A ) - L - v a l i n e esters [ 6 ] , as t h e N - T F A - L - p r o l y l o r v a l y l esters w e r e n o t s e p a r a t e d b y gas c h r o m a t o g r a p h y o n a C h i r a s i l - V a l c o l u m n . T h e r e s u l t s of t h e s e s t u d i e s a r e i l l u s t r a t e d i n F i g u r e 5. A t a l l t e m p e r a t u r e s investigated, (S)-2-methyl-l-butanol is t h e major product, b u t t h e s t e r e o s p e c i f i c i t y increases d r a m a t i c a l l y w h e n t h e r e a c t i o n t e m p e r a t u r e i s r a i s e d from 15° ( 1 4 % e.e.) t o 35° ( 5 1 % e.e.). T h u s , t h e s t e r e o s p e c i f i c i t y i s a p p a r e n t l y d e t e r m i n e d b y t h e TAAS* t e r m i n E q u a t i o n 2 , a n d c a n be s a i d t o b e " e n t r o p y d r i v e n " . A s w i t h S A D H , t h e stereochemical d i s c r i m i n a t i o n is b e t w e e n a m e t h y l a n d a n e t h y l g r o u p b i n d i n g t o t w o a l k y l b i n d i n g sites, d r i v e n b y h y d r o p h o b i c effects a n d v a n d e r W a a l s forces. 800

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F I G U R E 5. E f f e c t of t e m p e r a t u r e o n t h e s t e r e o s p e c i f i c i t y of 2 methylbutyraldehyde reduction by P A D H .

4. CONCLUSIONS I n contrast to the conventional wisdom, our data demonstrate t h a t m a x i m a l s t e r e o c h e m i c a l d i s c r i m i n a t i o n i n a n e n z y m a t i c r e a c t i o n m a y s o m e t i m e s be o b t a i n e d a t t h e h i g h e s t t e m p e r a t u r e c o m p a t i b l e w i t h t h e s t a b i l i t y of t h e e n z y m e , s u b s t r a t e a n d cofactor s y s t e m .

273 5.

REFERENCES

1 L a m , L . K . P., H u i , R. A . H . F., a n d J o n e s , J . B . J. Org. Chem. 1988, 51, 2 0 4 7 . 2 W i l l a e r t , J . J . , L e m i e r e , G . L., J o r i s , L . Α . , L e p o i v r e , J . A . a n d A l d e r w e i r e l d t , F. C. Bioorg. Chem. 1 9 8 8 , 1 6 , 2 2 3 . 3 K e i n a n , E., H a f e l i , F. V . , S e t h , Κ . Κ . a n d L a m e d , R. J. Am. Chem. Soc. 1 9 8 6 , 108, 1 6 2 . 4 B r y a n t , F. O., W i e g e l , J . a n d L j u n g d a h l , L . G . Appl. Environ. Microbiol. 1 9 8 8 , 45, 4 6 0 . 5 P h a m , V . T . a n d P h i l l i p s , R.S.J. Am. Chem. Soc. 1 9 9 0 , 1 1 2 , 3 6 2 9 . 6 A n d r a d e , F. A . C. a n d P h i l l i p s , R. S., i n p r e p a r a t i o n . 7 Z h e n g , C . a n d P h i l l i p s , R. S. J. Chem Soc, Perkin I, i n p r e s s . 8 A d o l p h , H . W . , M a u r e r , P., S c h n e i d e r - B e r n l o h r , H . , S a r t o r i u s , C. a n d Zeppezauer, M . Eur. J. Biochem. 1 9 9 1 , 201, 6 1 5 .

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

275

Comparative influence of microenvironment on the activity of two enzymes : lipoxygenase and thermolysin C. Pourplanche, P. Hertmanni and V. Larreta-Garde Laboratoire de Technologie Enzymatique, Centre de Recherches de Royallieu, BP 649, 60206 Compiègne, France

Abstract A comparative study of the influence of additives on an oxydoreductase lipoxygenase- and a metalloprotease -thermolysin- activity has been realized. The enzyme microenvironment has been modified by addition of water activity depressors and lipidoperoxidation and proteolysis have been evaluated in each medium. The enzyme catalysis was related to the n a t u r e of additive implicating the structuration of the reaction medium. 1. INTRODUCTION In industrial conditions or in vivo, enzymes function in heterogeneous media. To try to understand the catalytic behaviour of enzymes in such media, water activity depressors (polyols and sugars) were added. So, the physicochemical parameters (viscosity, hydrophobicity, water activity . . . ) of these media were modified. Two types of enzymes have been tested : an oxydoreductase (lipoxygenase) and an hydrolase (thermolysin) to study the effect of environment on two different action mechanisms : oxidation and hydrolysis respectively. Several water activity depressors (glycerol, maltose, mannitol, polyethylene glycol, sorbitol and sucrose) have been used to obtain different structurations of the reaction medium. 2. MATERIALS AND METHODS 2.1. Lipoxygenase Soybean lipoxygenase (type I-S) and linoleic acid (free acid) were purchased from Sigma. Additives (maltose, mannitol, sorbitol, sucrose) were obtained from Prolabo.

276

Lipoxygenase reaction was followed in 20ml of an air-saturated solution (pH9, 25°C) containing 300μΜ linoleic acid solubilized in the presence of Tween 20. The enzymatic reaction was initiated by addition of 50μ1 of a solution containing l,2mg lipoxygenase . ml'l. Polarographic assays were realized with a Clark oxygen electrode. 0% was represented by an anaerobic solution of dithionite and 100% by the substrate solution. Spectroscopic assays were realized according to Drapron [1] i.e. discontinously by solvent extraction. 2.2. H y d r o l y s i s b y t h e r m o l y s i n

Thermolysin and insulin (oxidized Β chain) were purchased from Sigma, additives, glycerol and P E G (polyethylene glycol), from Prolabo and Serva respectively. Insulin was purified by semi-preparative RP-HPLC using a C18 column and an acetonitrile/water gradient. Hydrolysis of insulin by thermolysin was performed in batch in 0.1M diethanolamine-HCl pH8 buffer, at 35°C. Various substrate/enzyme ratio were tested. Concentrations up to 920 mg/ml glycerol or polyethylene glycol were added to the reaction medium. Continuous agitation was applied. A RP-HPLC method (C18 column and acetonitrile/water gradient) was used to detect peptidic products and estimate the hydrolysis rate. The results were expressed in relative surface corresponding to hydrolysate peak area / total area ratio.

a RESULTS 3.1. L i p o x y g e n a s e

Lipoxygenases catalyze the oxygenation, by molecular oxygen, of fatty acids containing a cis,cis-l,4-pentadiene system into hydroperoxides with conjugated cis-trans double bonds. In mammalian, lipoxygenase acts in prostaglandins biosynthesis. In plants, there is not yet a unifying theme for the physiological role of lipoxygenase. To mimic biological media, various water activity depressors have been tested : monosaccharides, disaccharides (sucrose and maltose) and polyols (sorbitol and mannitol). The results with monosaccharides cannot be shown as they are protected by an industrial contract. The influence of additives on lipoxygenase activity was determined through oxygen consumption by a Polarographie method (figure D.The validity of this method in our media has been verified [2]. In all the studied media, the activity was first increased with the additive concentration and then decreased.

Additive Concentration (M)

Oxygen Consumption (μηΊθΙ/min)

Oxygen Consumption (μΓηοΙ/min)

277

Additive Concentration (M)

Figure 1. Influence of additive concentration on lipoxygenase activity. (A)DSorbitol; • Mannitol; (Β) • Sucrose; • Maltose. Lipoxygenase activity was also measured with a spectroscopic method which follows the appearance of the conjugated double bonds at 234nm. Without additive, the two methods were well correlated (initial rate = 19,5μΜ hydroperoxides produced /min and 19μΜ 0 2 consummed /min). The activity of lipoxygenase has been determined with a spectroscopic method at the additives concentrations inducing the most important activation effects (table 1). Table 1 Activation percentages of lipoxygenase activity by water activity depressor (%) Polarographic Method Spectroscopic Method Maltose 0,9M 51,5 21 Mannitol 0,7M 16 23 Sorbitol 2M 39,5 16 Sucrose 1,25M 61 23 In all cases, the enzyme activity was increased but according to the method, the activation was different. Secondary reactions of hydroperoxide decomposition can take place which leads to the formation of products absorbing a t 280nm. We have observed that the hydroperoxide stability was different according to the nature of the additive. This could explain the difference between the two methods. When additive concentration increases, water activity of the medium decreases. It could be interesting to represent lipoxygenase activity as a function of water activity (figure 2). In the range of concentrations used, mannitol did not induced significant water activity variations.

278

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F i g u r e 2. L i p o x y g e n a s e a c t i v i t y as a f u n c t i o n o f w a t e r a c t i v i t y . ( A ) • S o r b i t o l ; ( B ) D Sucrose; • M a l t o s e .

A m a x i m u m c a n be o b s e r v e d , w h i l e , u s u a l l y , t h e h i g h e r t h e w a t e r a c t i v i t y the higher the enzyme activity. This phenomenom has already been d e s c r i b e d f o r e n z y m e s s u c h as l i p a s e s [3] or f o r m i c r o o r g a n i s m s [ 4 ] . W i t h l i n o l e i c a c i d as s u b s t r a t e , t w o p o s i t i o n a l i s o m e r s ( 1 3 o r 9h y d r o p e r o x i d e ) c a n be o b t a i n e d w i t h l i p o x y g e n a s e . T h e s p e c i f i c i t y o f l i p o x y g e n a s e w a s s t u d i e d i n presence of a d d i t i v e s : w i t h a l l a d d i t i v e s , t h e r a t i o 13/9 w a s i n c r e a s e d [ 5 ] . T h e s e r e s u l t s h a v e s h o w n t h a t t h e m i c r o e n v i r o n m e n t was able to m o d u l a t e e n z y m a t i c catalysis (both a c t i v i t y a n d specificity). T h e effects seem t o be d i f f e r e n t a c c o r d i n g to t h e n a t u r e of t h e a d d i t i v e . Some p a r a m e t e r s have been m e a s u r e d to caracterize the most i n t e r e s t i n g m e d i a corresponding to a d d i t i v e concentrations i n d u c i n g t h e m o s t i m p o r t a n t a c t i v a t i o n effects ( t a b l e 2).

Table 2 Reaction media parameters corresponding m a x i m a l enzyme activity Additive Maltose Mannitol Sorbitol Sucrose Additive Concentration (M) 2 0,9 0,7 1,25 Water Activity 0,97 0,98 0,933 0,96 42 Water Concentration (M) 43,3 40,6 41,8 V i s c o s i t y (cp) 3,2 2,5 4,7 1,5

For t h e most a c t i v a t i n g additive concentrations, the best c o m m o n p o i n t is t h e w a t e r c o n c e n t r a t i o n . A n d t h i s v a l u e is close t o 4 0 M w h i c h i s t h e w a t e r concentration i n l i v i n g cell, whereas, i n a buffer, the w a t e r concentration is close t o 5 5 M .

1,0

279

A s c o m p a r e d t o b u f f e r w a t e r a c t i v i t y (0,99), t h e w a t e r a c t i v i t y v a r i a t i o n s i n o u r m e d i a are not i m p o r t a n t .

3.2. Thermolysin

/ H y d r o l y s i s of a m o d e l s u b s t r a t e The kinetics of i n s u l i n (oxidized Β chain) hydrolysis by t h e r m o l y s i n i n buffer was followed during 2 hours w i t h a R P - H P L C method. T h e hydrolysis p r o d u c t s were separated according to t h e i r h y d r o p h o b i c i t y . O x i d i z e d Β c h a i n r e v e a l e d 10 cleavage sites w h e n i t h a s b e e n c l e a v e d b y t h e r m o l y s i n i n b u f f e r conditions [6]. 16 d i f f e r e n t p e p t i d e s w e r e o b s e r v e d w h i c h h a v e b e e n d i v i d e d i n t o 2 classes depending on their evolution versus time: - class A : i n c l u d e d t h e 7 less h y d r o p h o b i c p e p t i d e s w h i c h c o n t i n u o u s l y appeared d u r i n g the reaction. They were presumably small peptides. - class B: 9 m o r e h y d r o p h o b i c a n d l a r g e p e p t i d e s w h i c h a p p e a r e d a t t h e b e g i n n i n g of the hydrolysis a n d were t h e n r a p i d l y hydrolyzed i n t o peptides i d e n t i c a l t o those o f class A . / H y d r o l y s i s i n presence of g l y c e r o l I n t h e same conditions, t h e k i n e t i c s was followed i n glycerol c o n t a i n i n g m e d i a ( u p t o 9 2 0 m g / m l ) [ 7 ] i n o r d e r to o b t a i n w a t e r a c t i v i t y r a n g i n g f r o m 0.5 t o 1. T h e v i s c o s i t y p a r a m e t e r w a s c o m p r i s e d b e t w e e n 1 a n d 60 cp. F o r g l y c e r o l , c o n c e n t r a t i o n s u p to 184 m g / m l a n d f o r S/E ( s u b s t r a t e / e n z y m e r a t i o ) = 4 0 0 0 , t h e i n i t i a l a c t i v i t y w a s o n l y s l i g h t l y decreased a n d 95 % h y d r o l y s i s w a s r e a c h e d w i t h i n 10 m i n u t e s . W h e n p r o t e o l y s i s w a s p e r f o r m e d i n m e d i u m c o n t a i n i n g 92 m g / m l g l y c e r o l , a n i n c r e a s e i n h y d r o l y s i s r a t e w a s o b s e r v e d ( f i g u r e 3). O n t h e c o n t r a r y , w i t h a glycerol concentration of 230 m g / m l , t h e proteolysis decreased; only i n s u l i n peak was present on H P L C profile [5].

l CD

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TIME (min) F i g u r e 3. H y d r o l y s i s p r o d u c t s o b t a i n e d f r o m l i m i t e d p r o t e o l y s i s o f i n s u l i n b y t h e r m o l y s i n a t 30 m i n u t e s i n b u f f e r (1), a n d i n r e a c t i o n m e d i u m c o n t a i n i n g 9 2 m g / m l g l y c e r o l (2).

280 I f t h e 2 classes o f p e p t i d e s w e r e c o n s i d e r e d , t h e e v o l u t i o n as a f u n c t i o n o f t i m e revealed t h a t glycerol increased the hydrolysis of the more hydrophobic p e p t i d e s (class B ) a n d so, s m a l l p e p t i d e s w e r e o b t a i n e d w i t h a g r e a t e r r a t i o ( f i g u r e 4). For a concentration of 460 m g / m l , the hydrolysis of hydrophobic peptides was decreased a n d t h u s , the hydrolysis products obtained were different. T h e h y d r o l y s i s r a t e a n d t h e s e l e c t i v i t y o f t h e protease w e r e m o d i f i e d .

a

TIME (min)

F i g u r e 4 . E v o l u t i o n as a f u n c t i o n o f t i m e o f class A (a) a n d class Β (b) p e p t i d e s obtained f r o m proteolysis of i n s u l i n by thermolysin i n buffer (•) a n d i n reaction m e d i a c o n t a i n i n g 9 2 m g / m l (O), 184 m g / m l (Δ) a n d 4 6 0 m g / m l ( · ) g l y c e r o l .

/ H y d r o l y s i s i n presence o f e t h y l e n e g l y c o l p o l y m e r s I n o r d e r to r e v e a l t h e possible i n f l u e n c e of a d d i t i v e size o n t h e m o d u l a t i o n of hydrolysis reaction, the kinetics were followed i n presence of v a r i o u s ethylene glycol polymers d u r i n g 4 hours. E t h y l e n e glycol 62 ( E G ) , p o l y e t h y l e n e g l y c o l 6 0 0 a n d 1550 w e r e t e s t e d a t5 t h e s a m e c o n c e n t r a t i o n s as g l y c e r o l . O n l y size w a s d i f f e r e n t . S/E r a t i o w a s 1 0 . F o r c o n c e n t r a t i o n s u p t o 184 m g / m l , E G i n d u c e d no e f f e c t o n i n i t i a l velocity. A slight increase was observed w i t h PEGs: for a 92 m g / m l c o n c e n t r a t i o n , t h e p e r c e n t a g e o f p e p t i d e s c o r r e s p o n d i n g t o class Β w a s h i g h e r than without PEG. F o r h i g h concentrations, E G s h o w n a more i m p o r t a n t i n h i b i t o r effect t h a n P E G . F o r e x a m p l e , a f t e r 2 h o u r s o f r e a c t i o n , w i t h 184 m g / m l a d d i t i v e , 4 6 % h y d r o l y s i s w i t h E G , 6 7 . 5 % a n d 8 7 % h y d r o l y s i s w i t h P E G 1550 a n d P E G 6 0 0 r e s p e c t i v e l y w e r e o b s e r v e d ( f i g u r e 5).

281

a

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100

b

200

0

100

200 TIME (min)

F i g u r e 5 . E v o l u t i o n as a f u n c t i o n o f t i m e o f class A (a) a n d class Β ( b ) p e p t i d e s obtained f r o m i n s u l i n proteolysis b y t h e r m o l y s i n i n buffer ( Δ ) a n d i n reaction m e d i a c o n t a i n i n g 184 m g / m l E G ( · ) , P E G 600 ( • ) a n d P E G 1550 (O).

To s u m u p , f o r small additive concentrations, glycerol i n d u c e d a p a r t i c u l a r enzyme behaviour: a n a c t i v a t i o n w a s observed w i t h a 92 m g / m l glycerol c o n c e n t r a t i o n . F o r h i g h e r a d d i t i v e c o n c e n t r a t i o n s , t h e size o f t h e a d d i t i v e s ( P E G s ) w a s i m p o r t a n t o n t h e decrease o f p r o t e o l y s i s : P E G 1550 ( h i g h P M ) seemed t o h a v e less effect t h a n E G ( l o w P M ) .

4. D I S C U S S I O N The physico-chemical properties of the enzyme microenvironment were m o d i f i e d b y t h e presence o f t h e a d d i t i v e s . A k n o w l e d g e of water activity in concentrated s o l u t i o n s i s i m p o r t a n t f o r a b e t t e r u n d e r s t a n d i n g of how components i n t e r a c t w i t h w a t e r . T h i s p a r a m e t e r c o n t r o l s t h e enzyme activity. For example, for w a t e r a c t i v i t y v a l u e s b e l o w 0 , 8 , l i p o x y g e n a s e exhibits no activity. In o u r c a s e , t h e w a t e r c o n c e n t r a t i o n a p p e a r s t o be i n t e r e s t i n g because, as c o m p a r e d t o a b u f f e r , t h e water concentration variations i n o u r media are more i m p o r t a n t t h a n water activity variations. I n t h e cell, t h e enzyme could be i n a m o r e active c o n f o r m a t i o n t h a n i n h o m o g e n e o u s b u f f e r m e d i a . So, w h e n t h e e n v i r o n m e n t becomes s i m i l a r t o t h e c e l l one d u e t o the presence of a d d i t i v e s , the e n z y m e c o u l d b e i n a m o r e a c t i v e c o n f o r m a t i o n , on the other hand, the viscosity is increased and the m o b i l i t y o f t h e m o l e c u l e s (therefore the rate of reaction) is limited. T h e w a t e r activity seems to be important but not sufficient to explain t h e r e s u l t s o b t a i n e d , i.e. d i f f e r e n c e s b e t w e e n effects o f each a d d i t i v e . T h e r e f o r e , a m o r e d e t a i l e d s t u d y o f t h e m e d i u m s t r u c t u r a t i o n w a s necessary. A t first, t h e r e s u l t s about p r o t e o l y s i s o f i n s u l i n i n presence o f P E G s , h a v e r e v e a l e d t h a t the size of additive i n f l u e n c e s catalysis. Arakawa a n d T i m a s h e f f [ 8 ] have showed that P E G 4 0 0 , 6 0 0 , 1 0 0 0 are preferentially excluded in this

282 order from the protein surface inducing a preferential h y d r a t a t i o n o f t h e molecule. Lee and T i m a s h e f f [9] have also indicated t h a t sucrose is p r e f e r e n t i a l l y e x c l u d e d from t h e p r o t e i n d o m a i n s t a b i l i z i n g i t . T h i s e x c l u s i o n p h e n o m e n u m m i g h t explain our results. I n t h e c a s e o f l i p o x y g e n a s e , t h e a d d i t i o n o f s u r f a c t a n t i s r e s p o n s i b l e for t h e m i c e l l a r o r g a n i z a t i o n o f t h e s u b s t r a t e . T h e a d d i t i o n o f s u g a r s a n d polyols i n f l u e n c e t h e m i c e l l e size a n d t h e o x y g e n c o n c e n t r a t i o n [ 2 ] . S o , t h e a c c e s s i b i l i t y o f t h e s u b s t r a t e w a s modified. F r o m t h e r e s u l t s obtained with t h e two e n z y m e s , i t a p p e a r s t h a t t h e m a c r o m o l e c u l a r s t r u c t u r a t i o n o f t h e r e a c t i o n m e d i u m i s o f i m p o r t a n c e for

b i o c a t a l y s i s . The modulations of enzyme behaviour induced by modifications in the microenvironment structuration of the protein are qualitative.

O n t h e o t h e r h a n d , a d d i t i v e s o l u b i l i t y m a y a l s o b e i n d i c a t i v e : for example, sorbitol solubility is g r e a t e r t h a n mannitol and they are isomers. However, the most important activation was not observed in the s a m e media c o n d i t i o n s ( w a t e r a c t i v i t y a n d additive c o n c e n t r a t i o n ) . A n o t h e r p a r a m e t e r a p p e a r e d to b e i m p o r t a n t : t h e c h e m i c a l n a t u r e o f t h e additive by itself. Indeed, e t h y l e n e glycol and glycerol w h i c h h a v e a p p r o x i m a t i v e l y the s a m e size, sucrose and m a l t o s e which a r e two d i s a c c h a r i d e s , r e v e a l e d d i f f e r e n t effects on p r o t e o l y s i s a n d l i p i d o p e r o x i d a t i o n respectively.

The modulations of enzyme behaviour induced chemical nature of the additive are quantitative.

by modifications

in the

F r o m o u r r e s u l t s , we m a y c o n c l u d e t h a t t h e a c t i v i t y o f t h e two e n z y m e s w a s affected b y a d d i t i v e s s u c h a s polyols or s u g a r s . T h e s e a d d i t i v e s i n f l u e n c e d the structuration of the enzyme microenvironment and thus its catalysis. T h e physico-chemical n a t u r e of the m e d i a is modified as c o m p a r e d with a buffer i n c l u d i n g v i s c o s i t y , h y d r o p h o b i c i t y , diffusion a n d s t r u c t u r a t i o n o f w a t e r . T h e s e m o d i f i c a t i o n s d e p e n d on a d d i t i v e c o n c e n t r a t i o n a n d n a t u r e .

PREFERENCES 1 2 3 4 5 6 7 8 9

R. Drapron and J . Nicolas, Riv. Ital. Sostanze G r a s s e 5 4 (1977) 2 1 8 . C. P o u r p l a n c h e , V . L a r r e t a G a r d e a n d D . T h o m a s , A n a l . B i o c h e m . 1 9 8 ( 1 9 9 1 ) 160. D . S i m a t o s a n d J . L . M u l t o n (eds.), P r o p e r t i e s o f W a t e r i n F o o d s , D o r d r e c h t , 1985. P . G e r v a i s , L e s C a h i e r s de l ' E N S B A N A 7 ( 1 9 9 0 ) 2 3 7 . P . H e r t m a n n i , C. P o u r p l a n c h e a n d V . L a r r e t a G a r d e , A n n . N . Y . A c a d . Sei., in press. P. D. Boyer, T h e Enzymes, Ed III, 3 (1971) 765. P. Hertmanni, E . Picque, D. T h o m a s and V. L a r r e t a Garde, F E B S Lett. 2 7 9 No. 1 ( 1 9 9 1 ) 1 2 3 . T. A r a k a w a and S. N. Timasheff, Biochemistry 24 (1985) 6 7 5 6 . J . C. L e e a n d S . N. T i m a s h e f f , J . B i o l . C h e m . 2 5 6 No. 1 4 ( 1 9 8 1 ) 7 1 9 3 .

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

283

ON T H E C R U C I A L R O L E O F W A T E R IN T H E L I P A S E C A T A L Y S E D ISOMERISATION O F l,2-(2,3)-DIGLYCERIDE INTO 1,3-DIGLYCERIDE Claude Rabiller*. A n d r e a s Heisler* and G. Hagele**

*Laboratoire de RMN et Réactivité Chimique -URA CNRS 472- Faculté des Sciences et des Techniques- 2, rue de la Houssinière - 44072 - Nantes cédex 03-(FRANCE) ** Institute of Inorganic Chemistry and Structural Chemistry, Heinrich-Heine-University Düsseldorf, Universitätsstr. 1, D-4000 Düsseldorf GFR.

Summary We have shown that the enzyme catalyzed isomerisation of1,2-dipalmitin into the 1,3isomer occurs with 1,3 regiospecific lipases. The mechanism of this transformation and the crucial role of the water still present in the enzyme preparation are elucidated. In particular, the percentage of the hydrolysis is found to be directly dependent to the amount of the enzyme used. Furthermore, an explanation of the poor enantioselectivity of the lipases in the transformation ofglycerides in organic media is given.

Introduction Glyceride synthesis from free fatty acids and glycerol or from transesterification and interesterification reactions is still a very tedious problem. Considering the great number of the possible equilibria which are likely to occur , it is never a trivial synthetic experiment to obtain the desired product with the right fatty acid chains. Furthermore , the reaction medium is often composed of a mixture of mono- , di- and triglycerides. T h e great potential o f lipases to catalyse regioselectively (Benzonana et al.,1971 ; Yokozeki et al.,1982 ; Macrae,1983 ; Jensen et al., 1983 ; Schuch et al., 1987 ; Bloomer et al., 1990) and stereoselectively (Akesson et al.,1983 ; Ransac et al.,1990 ; Rogalska et al.,1990 ; Rabiller et al.,1989) in the hydrolysis or the transesterification reactions of glyceride compounds has been recently demonstrated. A great deal of articles have already been published on this topic , but only a few were concerned with mechanistic problems , particularly when working with lipases in organic media. It is well known that the transesterification of a triglyceride with an alcohol ( e. g. ethanol) in the presence of different lipases affords a mixture of mono-, di- and of triglycerides. However, in such reactions several questions still remain : - Is the 13-diglyceride formed from a spontaneous isomerisation of the 1,2-diglyceride or not and what is the role of the enzyme in this transformation?

284

-Does the l-(3-)monoglyceride synthesized arise from a subsequent hydrolysis (or alcolysis) of the 1,3-diester and / or from the isomerisation of the 2-monoglyceride which could be formed from the 1,2-derivative ? A better knowledge about those mechanistic aspects should allow an improvement o f the univocal synthesis of the glycerides. In order to get more information about these questions we have undertaken the study o f the lipase catalyzed interesterification of l,2-(2,3-)dipalmitin into 1,3-dipalmitin and of the reverse reaction. A part of this study has been already reported (Rabiller et al, 1991).

Results and

discussion

The question of the isomerisation of di- or monoglycerides has been studied by several authors. For instance , it was shown recently that the isomerisation of 1,2-dipalmitoylglycerol occurred with a reasonable rate at 60°C in the presence of several catalysts : silica g e l , lecithin (Kodali et al.,1990). In the meantime , such a study has never been described in the presence of biological catalysts in organic solvents. Starting either from diluted solutions of the l , 2 - ( 2 3 ) dipalmitin , or from the 1,3-dipalmitin in the presence of lipases , we were very surprised to observe as the first chemical event catalysed by the lipase, the formation of noticeable amounts (15 to 2 0 % ) of the 2-monopalmitin and of the l-(3-)monopalmitin respectively. The figures 1 X

(a) and (b) show the H NMR spectra of such mixtures. At the same time the

spectra o f

13

the mixtures show an absorption at δ ( c ) = 179 ppm characteristic of the carboxylic acid carbon resonance. Two conclusions may be drawn from this observation : the water still present in the lyophil ized lipase powder can be used by the enzyme to hydrolyse the di glyceride and this reaction is the fastest one in the system ; the two enzymes used (Mucor miehei, Rhizopus arrhizus) have the same regioselectivity towards the l-(3-) position. A study of the evolution of the hydrolysis percentage versus the amount o f the lipase preparation used shows a linear dépendance (see Figure 2). The second event, clearly slower than the first one was, in the case o f 1,2-dipalmitin , the formation of tripalmitin and of l-(3-)monopalmitin. Furthermore , this last glyceride was synthesized from the non-catalysed isomerisation o f the 2-monoester as the concentration of the latter decreased when the concentration of the former increased [see Figure 3 (a) (c) (d) ] . The spontaneity of the latter isomerisation was clearly showed by means of NMR spectroscopy : after removal of the lipase the concentration of the 2-monoester still decreased.very quickly. It must be pointed out that the concentrations of each compound given in the figures do not take into account the glycerol. In fact, the concentration of glycerol is difficult to measure accurately

285

as it is not soluble in organic solvents and a part remains in suspension in the reaction solution or stuck to the enzyme. The third chemical event was the formation of 13-dipalmitin from the l-(3) monoester. Similarly, reversible reactions were observed starting from the 1,3-dipalmitin. The l - ( 3 - ) monopalmitin was very slowly converted to small amounts o f 2-monopalmitin which was converted further on into 1,2-dipalmitin and tripalmitin [see figure 3 (b)]. The last chemical event was the decrease of glycerol, mono- and triglyceride concentrations which occurred with an increase of either the 1,2- or 1,3-dipalmitin concentrations.

s e

*-S

3.s

va

vs

va

J

3.s

Figure 1 : (a) H NMR spectrum of the mixture obtained in the lipozyme catalysed isomerisation of the 1,2! dipalmitin in benzene after an incubation of one hour at 37°C. (b) HNMR spectrum of the mixture obtained in the Rhizopus catalyzed isomerisation of 1,3-dipalmitin in benzene after 2 hours at 37°C. Only the glyceryl protons are shown. (Solvent : CDClj/TMS, spectrometer : Bruker WM 250.; * : 1,2-dipalmitin ; : 1,3dipalmitin ; Δ : tripalmitin ; · : l-(3-)monopalmitin ; •: 2-monopalmitin.

HYDROLYSIS (%) 2 0

Figure

2

4

Ο

β

Κ)

12

Μ

Ιβ

Ιβ

20

2 2

2 : hydrolysis of tributyrin versus the amount of lipase used (Lipozyme)

LIPASE (mg )

286

Fieure 3 : Lipase catalyzed dipalmilin isomerisation from J,2-(2,3)-dipalmilin in the presence of lipozyme at 20°C (a) , at 37°C (c) and in the presence of Rhizopus arrhizus at 37°C (d) and from 1,3-dipalmitin at 20°C with lipozyme (b).; * : 1,2-dipalmilin monopalmilin.

1,3-dipalmilin ; A : tripalmitin ; · : l-(3-)monopalmitin ;

2-

287

The curves in figure 3 give an order of magnitude of the relative rate of each event and of the relative amounts o f each glyceride. The figures 3 ( c ) and 2(d) allow a comparison between the two kinds of lipases : Mucor miehei shows a faster reaction than Rhizopus arrhizus. A chemical description of all these events is given in the following scheme:

Ε Ε Ε

OCOR - OH - OCOR OCOR

P !J

Η 20

+

a se

RCOOH +

spontaneous

-OH M

OCOR - OH

E E

P -^T^

Η Ο

Γ fOCOR OH L i rOCOR Γ " OCOR

+

RCOOH H 20

+

L

OCOR

- OH

O

L i

a

- Ο COR

•

ι— OCOR U OH — OH

Lipase

Γ " OH

CR O

+

H 20

OH OH OCOR + OH OH

RCOOH

Lipase

OH — OH p 0C O R

«4 •

j — OH

+ 1

H +

RCOOH

2 °

LnmR

The interconversion between the l-(-3) and the 2-monopalmitin is a spontaneous reaction as we observed that the isomerisation of the 2-monoester was very fast in the abscence of the enzyme. S o , it was concluded that the glycerol formation was due to the hydrolysis o f the l-(-3)monoester. It should be pointed out that, in the conditions of our experiments the amount of water which is usually present at a percentage of about 5 % in the biological material , is sufficient to afford measurable quantities of the hydrolysis products (see experimental section, 0.1 mmole o f the diglyceride was used with 2 5 mg o f the lipase which contains about 1 mg, 0.06 mmole of water). However, only catalytic water to dipalmitin ratios are necessary to allow the turnover from a given dipalmitin to its corresponding positional isomer. S o it can be concluded that the lipase catalyzed interconversion o f 1,2-diglycerides into the 1,3-diesters and the reverse reaction mainly take their origin in a crucial initial step due to trace amounts of water. At this point we are not able to prove that no direct interconversion can occur , but in any case it is impossible to detect this reaction which is probably a very slow one. This result is important because in glyceride synthesis one has to consider that a trace o f water in a given catalyst (biological and chemical) or in the solvent may cause undesirable transformations. Furthermore , in certain circumstances , the presence o f water may be an advantage.

288

Considering the lipase catalyzed transesterification reaction of triglycerides with glycerol (Holmberg, 1988 ; Schlich, 1989 ; Holmberg, 1989 ; Rabiller 1992) it is very likely that the first step consists of a hydrolysis giving as a first product the l,2-(2,3-)diglyceride. A direct esterification o f the glycerol by the fatty acid then allows the formation o f the l - ( 3 - ) monoglyceride and the diglycerides. From figure 3, it can be seen that the reaction rate of the interconversion is faster when starting from the 1,2-dipalmitin than from the 1,3-isomer. An explanation of this phenomenon may be drawn from our mechanism : the hydrolysis of 1,2dipalmitin affords the 2-monoglyceride which is known to isomerise readily into the 1monoglyceride. The reverse reaction, considerably slower than the direct one , constitutes in that case the limitating step of the overall kinetic process. This result is also in complete accordance with those obtained by F. Ergan and al.(1991): the lipase catalyzed reaction of 1,2diolein with oleic acid is faster than with 1,3-triolein. The latter has first to be hydrolyzed , then the l-(3-) monoolein is isomerised before the transformation into l,2-(2,3-) diolein. In a similar way , the role of water is clearly demonstrated in Kodali's work (1990) : the rate of acyl migration increases when the organic solvent is replaced by a sodium phosphate buffer. A similar behaviour was observed as well in the transesterification of triglycerides in the presence of glycerol (Holmberg and al., 1989). Very recently, we obtained interesting results about the enantioselectivity of the lipases in organic media , which were in complete accordance with the mechanism proposed above. In effect, if the regioselectivity of the lipases and their enantioselectivity for the hydrolysis reaction are well known, their behaviour in the transesterification reactions is not so well documented. Furthemore, the few results reported in that field indicate a rather low enantioselectivity (see for instance C. Rabiller et al , 1989). Thus , in two separate experiments we compared the evolution at the same time of pure 1,2-dilaurine and of the racemate in presence of the same amount o f liposyme. After half an hour the mixture was composed o f the remaining diglyceride, 2-monoglyceride and of the triglyceride. The following percentages measured by l

means of H proton NMR spectra were respectively 7 1 , 2 0 , 9 in the case of 1,2-dilaurine and 7 1 , 13, 16 in the case of the racemate. From those results, it can be concluded that the snl position is clearly preferred and that the poor enantiomeric excess observed can be explained on the basis of the four concurrent reactions described hereafter.

fast

slow

RCOOH

289 OOOR RCOO.

.

' OH Γ-

OH

W

^ +

RCOOH

ROOO

fa

s t

s

r - OOOR RCOO— ]__ OCOR

+

l

o

H 20

OOOR

Experimental

Materials . Two kinds of lipases were used : Lipozyme (Mucor miehei) and Rhizopus arrhizus (ATCC 2 4 5 6 3 ) . All fatty materials were purchased from Sigma. The diglycerides used were 1,2-palmitoyl glycerol (racemic mixture) and 1,3-palmitoyl glycerol. The solvents (CDCI3 , CGDG from CEA Saclay) were used without further purification. Enzymatic reactions . A solution of 0.1 mmole of the dipalmitin in 2 ml of dry benzene was slowly stirred (magnetic stirrer) with 2 5 mg of the lipase. The experiments were run at room temperature and at 37°C during periods of time as long as several weeks.

NMR quantitative analysis of the different glycerides . Considering the relative unstability of certain glycerides , a non destructive analytical technique is required to measure accurately the percentages o f the different classes of these esters. Carbon 13 (Rabiller et al., 1989) and proton NMR spectroscopy are preferred techniques for this purpose. All the experiments were performed on a Bruker W M 2 5 0 spectrometer. Aliquots o f 1.5 ml o f the reaction media were filtered out in order to remove the non sedimented lipase.The solvent was removed under reduced vacuum and replaced by 1 ml of deuterochloroform. The composition o f the solution A

was then determined by means of the integration of the H NMR spectra. In particular , the region of the spectra containing the resonances of the glycerol moiety affords a lot o f information , because the CH-O and the C H 7 - O protons are well separated for the different classes of glycerides. The attribution of the resonances o f each compound was made by comparison with authentic standards.

Conclusion In this paper, we have shown that the lipase catalysed isomerisation in organic solvent of l,2-(2,3)-diglyceride into its 1,3-analogue does not occur directly. In fact the water still present around the enzyme induces an hydrolysis of the substrate as an initial first step of the transformation. Then the resulting 2-monoglyceride spontaneously isomerises into the l - ( 3 ) monoester. The acid liberated during the hydrolysis is able to acylate the latter monoester producing the 1,3-diglyceride. A side competitive reaction also occured forming noticeable amounts of the triglyceride. Similar but reverse reactions were observed when starting from the 1,3-diglyceride as a substrate. Our results clearly emphasize that lipozyme and Rhizopus

290

arrhizus lipases are strictly 1,3-regiospecific, the sn-1 position being largely preferred. The removal of the 2-acyl group is due to the non-catalysed and spontaneous equilibration between the two monoglycerides. Furthermore a study of the behaviour of the lipases in the presence of optically pure sn-l,2-diglyceride leads to the conclusion that the lack of enantioselectivity in organic solvent may be explained by the competition between the hydrolysis and the esterification reactions Aknowledgements Thanks are due to the European Community for an E R A S M U S support grant to A. H. This work conducted at Nantes University was a part of the Diplomarbeit of A. H. (Heinrich-Heine Universität Düsseldorf; March 1991 ). Elf Aquitaine (Groupe de Recherche de Lacq) and Amano are gratefully thanked for the generous gifts of the enzymes. We are equally grateful to Μ*. I. Jennings for improvements to the English text. References - Akesson B . , Gronowitz S. , Herslöf Β . , Michelsen P. and Olivecrona Τ. , Lipids , (1983) ,

18 , 3 1 3 . - Benzonana G. and Esposito S. Biochim. Biophys. Acta , (1971) , 231 , 15. - Bloomer S. , Adlercreutz P. , Mattiasson Β . , J. Am. Oil Chem. Soc. , (1990) , 67 , 5 1 9 . - Ergan F. and Trani Μ. , Biotechnology Letters , ( 1 9 9 1 ) , 13, 19. - Heisler Α., Rabiller C and Hublin L., Biotechnology Letters, (1991), 13(5), 327. - Holmberg Κ. and Osterberg E., J . Amer. Oil Chem. Soc., (1988), 65, 1544. - Holmberg Κ. , Lassen Β . and Stark Μ. Β . , J. Am. Oil Chem. Soc. , ( 1 9 8 9 ) , 66 , 1796. - Jensen R. G. , Dejong F.A. and Clark R.M. , Lipids , (1983) , 18 , 239.

- Kodali D. R. , Tercyak A. , Fahey D.A. and Small D. Μ. , Chemistry and Physics of Lipids (1990) , 52, ,163. - Macrae A.R. , J. Am.Oil Chem. Soc, (1983) , 60 , 243A.

- Rabiller C. and Maze F. , Magnetic Resonance in Chemistry , (1989) , 27 , 582. - Rabiller C. and Heisler A. , ( 1 9 9 1 ) , unpublished results. - Ransac S. , Rogalska Ε . , Gargouri Υ. , Deveer Α. M. T. J . , Paltauf F . , De Haas G. H. and - Verger R. , J. Biol. Chem. , (1990) , 265 , 20263. - Rogalska Ε. , Ransac S. and Verger R. , J. Biol. Chem. (1990) , 265 , 2 0 2 7 1 .

- Schuch R. and Mukherjee K. D. , J. Agric. Food Chem. , (1987) , 35 , 1005. - Schuch R. and Mukherjee K.D. , Appl. Microbiol. Biotechnol, (1989) , 30 , 3 3 2 . - Yokozeki K. , Yamanaka S. , Takinami Κ. , Hi rose Y . , Tanaka A. , Sonomoto K. and

Fukui S., European J. Appl. Microbiol. Biotechnol, (1982) , 14 , 1.

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

291

Rapid determination, using dielectric spectroscopy, of the toxicity of organic solvents to intact cells Gary J . Salter & Douglas B . Kell Department o f Biological Sciences, University College o f Wales, Aberystwyth, Dyfed S Y 2 3 3DA, U.K.

Abstract Dielectric spectroscopy utilising dual-frequency measurements has been used to study the toxic effects o f a number o f organic solvents to suspensions o f Saccharomyces cerevisie. Solvents o f a highly apoiar nature, such as hexadecane, were identified as being non-cytotoxic, and thus suitable for use with whole-cell systems. A novel approach to aid biotransformations, using mixed organic solvents, has also been studied. This has revealed that the cytotoxic nature of polar organic solvents, such as octan-l-ol, may be negated by dissolving them first in apolar, non-cytotoxic organic solvents, such as hexadecane, before exposure to the cell suspension. The use o f mixed organic solvents with immobilised cell systems was shown to be possible by the growth o f Lactobacillus brevis, (immobilised within hollow ceramic microspheres), in the presence o f 5 % (v/v) octan-l-ol dissolved within 5% (v/v) hexadecane, added to the media. Dispersion o f these immiscible solvents was improved by the addition o f 5% (v/v) ethanol and 0 . 0 5 % (v/v) tween 80. Cell growth occurred (as measured by dielectric spectroscopy) over a 7 0 - hour period. 1. I N T R O D U C T I O N There is much current interest in biotransformations using or aided by organic solvents (e.g. [1-3]). The organic solvent may itself be o f interest as a substrate, or it may be needed to shift the equilibrium composition in a favourable direction. In addition, many substrates are insoluble in aqueous media, and this will tend strongly to limit the rate o f the biotransformation. While the use o f organic solvents with enzymes is becoming more common, their use with intact, viable cells has received much less attention, and examples o f their use with immobilised cell reactors are few [4-6]. Many of the advantages conferred by the immobilisation o f cells for "normal" aqueous biotransformations also hold true for systems using organic solvents. It has been demonstrated that the passive electrical properties o f cellular suspensions at radio frequencies (described in considerable detail elsewhere [ 7 - 1 4 ] ) reflect the state (intactness) o f the cellular membrane. As the primary, cytotoxic site o f action by organic solvents is the cell membrane, one may expect that the passive electrical properties o f the cell suspension could be used to assess the level o f cell viability when challenged with organic solvents, in situ and in real-time [15]. Using this method to determine the suitability or otherwise o f organic solvents, and mixtures o f organic solvents, for use with intact viable cells, we show herein that it was possible to develop a system where organic solvents could be used within an immobilised cell reactor.

292 2. M A T E R I A L S AND iMETHODS Microorganisms All work involving the growth of cells immobilised within columns was carried out using Lactobacillus brevis NCIB 947. General studies using the Biomass Monitor to determine solvent toxicity were carried out using a strain of Saccharomyces cerevisiae in pressed form, obtained locally. Solvent toxicity All measurements were carried out using a Bugmeter™ Biomass Monitor (Aber Instruments, Aberystwyth Science Park, Cefn Llan, Aberystwyth, S Y 2 3 3AH, Dyfed, U K ) . Solvent toxicity, determined as the loss of integrity o f cellular membranes, was studied using a standard Biomass Monitor electrode inserted laterally into the base of a 100 ml polypropylene container and sealed with epoxy resin. A tight-fitting lid was placed on the container, onto which a large centrally positioned baffle was positioned. Agitation was achieved by the use of magnetic follower inserted into the container, the arrangement being placed on a stirrer. All sample volumes were 50 ml. Cells of 5. cerevisiae at a known concentration were suspended in 2 0 mM K H 2 P O 4 , to which one or more organic solvents were added. When two organic solvents were used the more polar of the two was first dissolved within the less polar solvent before addition to the cell suspension. The organic solvents were dispersed by the addition of 5% ethanol (Aldrich) and 0.05% Tween 80 (polyoxyethylene sorbitan mono-oleate, Sigma); the system was emulsified by agitation (see above). The effects of various solvents and solvent combinations were followed over time with the Biomass Monitor, using external control and two-frequency measurements (0.3 and 9.5 MHz). Method o f use o f the Biomass Monitor A "low" and a "high" frequency were selected (typically 0.3 and 9.5 MHz), and readings were taken at both frequencies throughout the experiment. The 'real' or delta capacitance was obtained by the subtraction o f the capacitance obtained at the high frequency, from that at the low frequency. External control of the Biomass Monitor was achieved by the use of an IBM-type personal computer and a Blackstar 2308 Interface (ADC). The relevant software was written by Dr.G.H. Markx. The data were stored in a file and analysed in a spreadsheet. Calibration [16] was achieved by the use of a dilution series of the relevant microorganism (L. brevis or S. cerevisiae) suspended in 2 0 mM K H 2 P O 4 or growth medium. Capacitance and/or conductance readings were taken over a range of known cell concentrations (determined by dry weight and/or wet weight and/or optical density; for immobilised biomass cells were lysed by boiling in 0.2 M NaOH for 10 min and assayed for protein by a modification of the Folin method). Through a calibration curve the capacitance was then directly related to biomass cocentration, resulting in the following calibrations: S. cerevisiae,1 pF = 1.75 g/1 dry weight o f cells; L brevis, 1 pF = 4.40 g/1 dry weight of cells. These values were used throughout.

293

Growth of immobilised cells Columns containing gold Biomass Monitor electrodes and packed with hollow ceramic microspheres as a cell support were prepared in a similar manner to that used previously [16]. Cells of L. brevis were then immobilised by pumping a cell suspension through the column from the top at a flow rate exceeding 3 column volumes/minute. The column was then washed by passing 10 column volumes of sterile, cell free buffer through the column, at a rate comparable to that above. Cells entrapped within a column were then grown by passing a suitable liquid medium (see below) through the column from the bottom. Cell growth was followed by the Biomass Monitor using two-frequency (0.3 and 9.5 MHz) measurements. A constant temperature o f 3 0 ° C was maintained by working within a drying oven with a temperature controller ( R S components) fitted. A commercially available medium, M R S (Merck), was used, and was autoclaved at 1 2 1 ° C for 3 0 min prior to inoculation. 3. R E S U L T S AND D I S C U S S I O N Preliminary experiments demonstrated a need to disperse the organic solvents through the aqueous phase as quickly as possible to attain the maximum cytotoxicity in a reasonable time. (It thus follows that the converse is also true, demonstrating that the expression of the toxic effects of many immiscible organic solvents is strongly diffusion-limited.) This would seem to be achieved most easily by the addition of a detergent, the detergent chosen being Tween 80 (polyoxyethylene sorbitan mono-oleate). We also added ethanol, in an attempt to make the dispersion o f the organic phase more stable. Indeed, overall mixing was improved (as observed visually), and the rate o f agitation could be reduced considerably (not shown). Comparison of data showing the toxicity of organic solvents used alone with those when Tween 80 and ethanol were added showed that the addition of Tween 80 and ethanol had little effect on the final toxicity for the organic solvents studied herein. The speed of action, however, was altered considerably, with a given extent o f cell death occurring after a shorter exposure to the test solvent (data not shown). Thus 5% ethanol and 0.05% Tween 80 (both v/v) were used in the experiments displayed. It is known that organic solvents with very low polarities or a high log Ρ value (log Ρ = octanol-water partition coefficient of the solvent) tend not to be cytotoxic [17-19], as the solvent is highly insoluble in water, and thus cannot gain access to the apolar regions o f enzymes and membranes via which they would be able to effect denaturation. Further, less polar solvents cannot disturb the water layer found around biomolecules, and which is essential to their activity. Toxicity studies on a range o f organic solvents confirmed this: as judged by dielectric measurements, polar organic solvents (as illustrated by octan-l-ol, Fig 1) are much more cytotoxic than are those that are relatively apolar (as illustrated by hexadecane, Fig 2 ) . This poses an interesting dilemma, however: highly apolar organic solvents that are most amenable for use with viable cells are not those capable of best solvating many compounds of interest in biotransformations [17-19]. In addition, the correlation between cytotoxicity and log Ρ is anyway less than perfect, and Schneider [20], for instance, when considering the choice o f individual organic solvents, has argued that other properties such as Η-bonding are o f importance. We consider that a better solution to this problem lies in the use of mixed organic solvents. It is well known from work with chromatographic methods such as T L C and HPLC that mixtures of solvents can have a much greater range of properties than can single solvents [21]. In the present case, it is to be expected that mixtures of a highly apolar solvent (such as hexadecane) with a more polar one which is itself poorly water-soluble will combine the desirable properties of each: the more polar, but still organic, solvent will allow the solvation o f (or be used as) the

294

100 ο

xi ^ S

CO

80 60 Η

ts timescale, leading to only a very low 5 7 conductivity (typically 10 -10" ^ S . c m *). Microstructures in which the communication between droplets is faster or in which the waterphase becomes continuous (e.g. connected

317 droplets or cylinders) will show an increased conductivity [ 2 2 ] . The conductivity o f the different microemulsions is presented in Table 2 . The results indicate a change from a droplet like structure (in which the different waterpools are not connected) to a bicontinuous structure. This change in structure will also affect the transport o f the photosystem components and, consequently, its efficiency. Indeed, also a relationship can be observed between the photoinduced charge separation efficiency and the microemulsion conductivity (Fig. 3 ) .

Yield (%)

Yield (%)

4.1

4.2 4.3 4.4 logP ol the o r g a n i c phase

2 3 (Alkanol) (M)

Figure 2 . Photoinduced charge separation yields in the various microemulsions in relation to A. the log Ρ o f the organic phase and B . the alkanol concentration in the microemulsion Table 2 Conductivity o f the various microemulsions Microemulsion

1

Conductivity (ptS.cm )

10% 20% 30% 40% 50%

pentanol pentanol pentanol pentanol pentanol

4.6 4.9 5.5 10.2 33.5

20% 20% 20% 20%

butanol pentanol hexanol octanol

7.6 4.9 4.3 4.1

3.2. Porphyrin structure P 16 used in the previous section contains both a charge (similar to the charge o f the interphase) and a hydrophobic tail (Fig. 1). Next we chose to study whether a relationship between porphyrin performance and its location in the medium. A porphyrin was used for this investigation which lacked the hexadecyl chain. The equivalence between P, and P 16 was tested using electrochemistry, UV-Vis and fluorescence spectroscopy. The oxidation potentials o f the porphyrins could not be

318

Y i e l d (%)

1 2

J

I ! ι ! 0 ,8 ' 0 -6

' I α

"

0 4

0

c

* 2!

i

0 Q 1

j

•

!

D

j i

D

n

""^"^"io

1

^

conductivity ( p s c m )

Figure 3. Dependence of the yield on the , . » . . microemulsion conductivity.

measured but those o f the Zn modified porphyrins were within expected error ( 0 . 8 6 and 0 . 8 8 V for Pi and P i 6 respectively), Therefore changes observed in the photoinduced methylviologen-reduction rate (vide infra) are not likely to be caused by changes in the redox properties o f the porphyrins. Finally also no differences exists in the absorption and the fluorescence spectra o f Pi and P 16 (not shown). First the location o f the two porphyrins was studied. The chemical shift o f fluorine modified Pj and P 16 porphyrins in a variety o f media was compared. The dependence o f the 19 F-shift on the alkanol used (Fig. 4A) demonstrates that an increased alkyl chain o f the alkanol results into a chemical shift to a r h yi a,g uh e e So fte c h e m i ,c as h j t f c an be

used to probe the changes in the porphyrin environment. The absence o f the alkyl tail induces a shift to a lower ppm value. In terms of polarity/hydrophobicity both variations imply that the environment o f the fluorine probe in the P 16 experiences a lower polarity than in Pj moreover also the decreased polarity by changing to higher alkanols can be probed. The resonance position was measured for both Pj and P 1 6, in reversed micelles with varying concentrations o f pentanol. The result, plotted in Fig. 4 B , demonstrates the dependence o f the probe on the composition. Remarkable however, is the scale o f the chemical shift: Not only is the range o f the variation much smaller, but also the values o f the shifts are more towards zero than the corresponding alcohols. This phenomenon was studied in more detail since it contains information on the composition o f the microemulsion at the location o f the probe. The resonance position o f Pj was studied in two other solutions: A C TA B solution in pentanol, comparable to the interphase, and a mixture o f pentanol and octane, a reference for the continuous phase. The chemical shifts were — 5 6 . 4 9 and — 5 4 . 9 3 ppm respectively. The position o f the resonances o f the Px in microemulsions is intermediate between those values. So most likely, this porphyrin is located in an interfacial layer consisting o f C TA B , pentanol and octane. This conclusion is also supported by the low solubility o f the porphyrin in the pentanol/octane mixture and its insolubility in buffer. Unfortunately the chemical shift o f Pie could not be measured since this porphyrine was insoluble in the pentanol/octane mixture. The close similarity in dependence o f the values o f P i 6 and Pi on the medium composition (CTAB/pentanol and microemulsions) suggests that also P 1 6 is located in the interphase. In Fig. 4C the dependence o f the charge separation on the nature o f the cosurfactant is presented. In contrast to the value o f Pj the resonance position o f P j 6 is barely dependent on the cosurfactant (cf. Fig. 4 B ) . This observation indicates that although both 19 porphyrins are located in the interphase, the F-P! environment is now gradually becoming more polar, while the P i 6 environment is unaltered. This implies that P i , but not Pi6, probes also part of the continuous phase, since in this series o f experiments the alkanol concentration in the interphase is constant but the concentration in the continuous phase increases with decreasing alkanol alkyl-chainlength [ 2 3 ] . This suggests either an average position o f the Pi towards the continuous phase or an increased mobility o f this porphyrin in comparison with P i 6 . Photoinduced charge separation measurements with P! as photosensitizer were carried out under the same conditions as using P 1 6. The difference in result between the series are not significant. Clearly the presence o f the alkyl chain and the altered position in the interphase do not affect its efficiency (Table 1). So despite the necessity to position the

319 photosensitizer in the interphase, allowing vectorial electron transport from the donor to the acceptor, the efficiency cannot be optimized by this slight modification o f the porphyrin. Our results, as presented in the first section, demonstrate that altering the microstructure o f the emulsion offers a better possibility to enhance this efficiency.

-55.0

C h e m i c a l shift ( p p m )

-54.5

-56.0

-55.0

-57.0

-55.5

-58.0

-56.0

-59.0

-54.5

-56.5

MeOH PentOH OctOH DecOH

C h e m i c a l shift ( p p m )

10

20

30

40

50

Chemical shift ( p p m )

-55.0

-55.5

1

-56.0

11

ButOH PentOH HexOH OctOH 19

Figure 4 . F Resonance in different media. A. Homologous series o f alkanols. B . Microemulsion; Varying pentanol concentration. C. Microemulsion; Different alkanols.

4. CONCLUSIONS We have demonstrated that the efficiency o f photoinduced charge separation using a porphyrin located in the interphase, an amphiphilic electron donor and methylviologen, a hydrophilic electron acceptor, is strongly depended on the microemulsion composition. A high cosurfactant concentration or a polar cosurfactant increased this yield. It was

320 established by conductivity measurements that the photoinduced charge separation efficiency is probably influenced by this microemulsion structure, indicating that the log Ρ dependence o f the reaction is most likely only a secundary effect. No relationship between the relative orientation o f the porphyrin relative to the other reactants o f the photosystem and the photoinduced charge separation could be observed since in all systems used, an altered location o f the porphyrin, induced by modification o f the porphyrin tail and probed I9 by F - N M R did not result into changes in the yield. The strong dependence o f photoinduced charge separation on the microemulsion structure indicates the microemulsion composition is the variable to study for optimization purposes. 5. A C K N O W L E D G E M E N T S This work is supported by a fellowship to R . M . D . Verhaert from the Royal Netherlands Academy o f Arts and Sciences. The authors wish to thank Dr. J . Vervoort for his valuable help in the NMR experiments. 6. REFERENCES 1. 2. 3. 4. 5. 6.

7. 8.

Hilhorst, R . , Laane, C . and Veeger, C . Proc. Natl. Acad. Sei. USA. 7 9 , 3927, 1982. Verhaert, R . M . D . , Schaafsma, T . J . , Laane, C , Hilhorst, R . and Veeger, C .

Photochem. Photobiol. 42, 209, 1989.

Pileni, M . P . , Brochette, P . and Lerebours Pigeonniere, B . Chemical Reactions

in Organic and Inorganic Constrained Systems, Setton, R . (Ed.), Reidel Publishing

Company, Dordrecht, 2 5 3 , 1986. Willner, I . , F o r d , W . E . , Otvos, J . W . and Calvin, M . Nature (London), 2 8 0 , 823, 1979. Mandler, D. and Willner, I . J. Am. Chem. Soc. 106, 5352, 1984. Infelta, P . P . , Fendler, J . H . and Grätzel, M . J. Am. Chem. Soc. 102, 1497, 1980.

Möbius, D. New. J. Chem. 11, 203, 1987. Brochette, P . and Pileni, M . P . Nouv. J. Chim. 9 , 5 5 1 , 1985.

11. 12.

Willner, L , Mandler, D . and Maidan, R . New. J. Chem. 1 1 , 109, 1987. Brochette, P . , Zemb, T . , Mathis, P . and Pileni, M.P. J. Phys. Chem. 9 1 , 1444, 1987. Verhaert, R.M.D. PhD Thesis, Agricultural University, Wageningen, 1990. Kano, K . , Takuma, K . , Ikeda, T . , Nakajima, D . , Tsutsui, Y . and Matsuo, T .

13.

Levstein, P . R . , Van Willigen, H . , Ebersole, M. and Pijpers, F . W . Mol

9. 10.

14.

15. 16. 17. 18. 19.

20.

21. 22. 23.

Photochem. Photobiol. 27, 695, 1978. Cryst. Liq. Cryst. 194, 123, 1991.

Grätzel, C . K . and Grätzel, M. J. Phys. Chem. 8 6 , 2710, 1982.

Schmehl, R H . and Whitten, D . G . J. Phys. Chem. 8 5 , 3 4 7 3 , 1 9 8 1 . Gunter, M . J . and Mander, L . N . J. Org. Chem. 4 6 , 4 7 9 2 , 1 9 8 1 . Y o u n g , R . and Chang, C . K . J. Am. Chem. Soc. 107, 8 9 8 , 1985. Gray, G . W . and Jones, B . J. Chem. Soc. 1467, 1954. Shaka, A . J . , Keeler, J . and Freeman, R . J. Magn. Res. 5 3 , 3 1 3 , 1983.

Mayhew, S.G. Eur. J. Biochem. 8 5 , 535, 1978.

Wegener, E . E . and Adamson, A . W . J. Am. Chem. Soc. 8 8 , 3 9 4 , 1966. Huruguen, J . P . , Authier, M . , Greffe, J . L . and Pileni, M.P. Langmuir. 7, 2 4 3 , 1991. Hilhorst, R . PhD Thesis, Agricultural University Wageningen, 1984.

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

321

INDUCED STEREO AND SUBSTRATE SELECTIVITY OF BIOIMPRINTED α-CHYMOTRYPSBSr IN ANHYDROUS ORGANIC MEDIA Mats-Olle Mânsson, Marianne Stahl and Klaus Mosbach Pure and Applied Biochemistry, Chemical Center, University of Lund P.O.B. 124 S-221 00 Lund, Sweden

1. INTRODUCTION During recent years much interest has been given to attempts to change or manipulate the properties of proteins and enzymes. A major feature of enzymes and proteins in water-poor media is the rigidity of the conformation which forms the basis for new properties, e.g. a new catalytic property [1,2], new binding properties [3,4], enhanced thermostability [5] and inhibitor-induced enzyme activation [6]. We have showed that precipitation of a-chymotrypsin with 1-propanol in the presence of N-acetyl-D-tryptophan, an inhibitor of a-chymotrypsin, followed by drying of the precipitate, induced a new conformation to the active site. α-Chymotrypsin, in an anhydrous solvent, with this new conformation could catalyze the synthesis of the D-form of N-acetyl-tryptophan ethyl ester, which is not possible using a-chymotrypsin precipitated in the absence of the D-inhibitor.

2. MATERIALS AND METHODS Crystalline bovine pancreatic a-chymotrypsin type II, (EC 3.4.21.1), was purchased from Sigma (St.Louis Mo.) as a lyophilized powder (specific activity 46 U/mg with N-benzoyl-L-tyrosine ethyl ester (BTEE) as substrate, pH 7.8 and 2 5 C). N-acetyl-D-tryptophan, N-acetyl-L-tryptophan and Nacetyl-L-tryptophane ethyl ester were also obtained from Sigma. All the solvents used were purchased from Merck (Darmstadt, FRG) and were of highest purity commercially available. They were dried by 3 Â molecular sieves before use.

Precipitation of a-chymotrypsin and ester synthesis. The method typically used for precipitation and ester synthesis is described below. 30 mg of a-chymotrypsin was dissolved in 1 ml 10 mM sodium phosphate buffer at pH 7.8 containing 20 mM of either N-acetyl-Dtryptophan, the corresponding L-isomer or no ligand and the enzyme solution was cooled to 0 °C. 1-propanol (4 ml) was added at a temperature of 20 °C. The precipitate was recovered by centrifugation after 30 minutes on ice. After several washes with pure 1-propanol the precipitate was dried, under vacuum, for 4 hours. The chymotrypsin precipitate was then suspended in cyclohexane (4 mg/ml) and sonicated to reduce the particle size.

322 The synthesis was started by adding the substrate dissolved in 99.5 % ethanol. The final solution contained 80% cyclohexane, 20 mM substrate (Nacetyl-D-tryptophane or the L-isomer), 3.4 M ethanol and 8 mg of enzyme in a total volume of 2.5 ml.

Analysis of ester synthesis The rate of esterification was measured by determining the amount of N-acetyl-D-tryptophan ethyl ester formed in the mixture at certain intervals. A 20 μΐ aliquot of the reaction mixture, from which the enzyme had been removed by centrifugation, was injected directly into an HPLC RP-18 column (Nucleosil C-18). It was then eluted with water:acetonitrile:acetic acid (60:35:5, v/v). The ester was detected at 275 nm.

3. RESULTS AND DISCUSSION The synthesis of N-acetyl-D-tryptophan ethyl ester was measured using three different enzyme preparations. In the first the enzyme was precipitated in the presence of N-acetyl-D-tryptophan, in the second the enzyme was precipitated in the presence of N-acetyl-L-tryptophan and in the third no ligand at all was present during precipitation. The result of ester synthesis can be seen in figure 1 and it is only when the enzyme was precipitated in the presence of the D-form of N-acetyl tryptophan that the D-ester was formed.

3η

0

20

40

60

80

100

120

Time (hours) Figure 1. The synthesis of N-acetyl-D-tryptophane ethyl ester in cyclohexane with chymotrypsin previously precipitated in 1-propanol in the presence of ( • ) N-acetyl-L-tryptophan, ( A ) N-acetyl-D-tryptophane or ( • ) no ligand

323

O f t h e six s o l v e n t s t e s t e d ( f i g u r e 2) 1 - p r o p a n o l g a v e t h e b e s t r e s u l t s a n d w a s t h e r e f o r e u s e d as t h e p r e c i p i t a t i n g s o l v e n t i n a l l t h e s u b s e q u e n t e x p e r i m e n t s . C y c l o h e x a n e w a s c o n s i d e r e d s u i t a b l e as a s o l v e n t f o r t h e s y n t h e s i s since i t p r e v i o u s l y h a d b e e n s h o w n t h a t i t w a s p o s s i b l e t o efficiently synthesize N-acetyl-L-tyrosine e t h y l ester w i t h c h y m o t r y p s i n i n this solvent [7].

5n

Time (hours) F i g u r e 2. T h e s y n t h e s i s o f N - a c e t y l - L - t r y p t o p h a n e e t h y l e s t e r i n c y c l o h e x a n e w i t h c h y m o t r y p s i n p r e v i o u s l y p r e c i p i t a t e d i n t h e presence o f 2 0 m M N a c e t y l - L - t r y p t o p h a n e i n ( A ) 1 - p r o p a n o l , ( • ) 2 - p r o p a n o l , ( • ) acetone. E t h y l a c e t a t e , d i e t h y l e t h e r a n d a c e t o n i t r i l e as p r e c i p i t a t i n g a g e n t s g a v e i n a c t i v e enzyme preparations.

W h e n t h e p r e c i p i t a t i o n w a s p e r f o r m e d i n t h e presence o f t h e D - f o r m s o f N a c e t y l - t y r o s i n e or N - a c e t y l - p h e n y l a l a n i n e t h e D - e s t e r s o f t h o s e a m i n o a c i d s c o u l d also be s y n t h e s i z e d . W e h a v e c o i n e d t h i s m e t h o d , l e a d i n g t o a c h a n g e of the c o n f o r m a t i o n of t h e active site, " b i o - i m p r i n t i n g " i n analogy to molecular i m p r i n t i n g i n synthetic polymers [8]. T h e synthesis of N - a c e t y l - D - t r y p t o p h a n e t h y l ester a n d N-acetyl-Lt r y p t o p h a n e t h y l ester w i t h a - c h y m o t r y p s i n b i o - i m p r i n t e d w i t h N-acetyl-Dt r y p t o p h a n was investigated w i t h small additions of w a t e r to the reaction solution. The activity of the enzyme, i n the synthesis of N-acetyl-Lt r y p t o p h a n e t h y l ester, i s r a p i d l y i n c r e a s i n g as t h e w a t e r c o n c e n t r a t i o n i s i n c r e a s e d ( f i g u r e 3) w h e r e a s t h e o p p o s i t e i s t h e case i n t h e s y n t h e s i s o f N a c e t y l - D - t r y p t o p h a n e t h y l e s t e r ( f i g u r e 4). T h e i n i t i a l w a t e r c o n t e n t o f t h e s o l v e n t , p r i o r t o a d d i t i o n s , w a s 10 m M (0.02%) a n d 1.5 h a f t e r a d d i t i o n i t h a d i n c r e a s e d t o 20.5 m M , i n d i c a t i n g t h a t w a t e r h a d b e e n r e m o v e d f r o m t h e enzyme a n d i n t o the solvent. The w a t e r content of the d r i e d enzyme was

324

determined to be 12.2% (w/w), the water released into the solvent thus representing only a minor part of the enzyme bound water (3%). After equilibration there were still around 140 water molecules surrounding each enzyme molecule. This value is below the one calculated to give a monolayer around the enzyme (500 water molecules / enzyme molecule) and above the minimun amount of water necessary for activity (50 water molecules / enzyme molecule) [5]. 120

-ι

0

100 Water

200

300

(mM)

Figure 3. Synthesis of N-acetyl-L-tryptophan at different water additions

1.0-1

Water

(mM)

Figure 4. The syntesis of N-acetyl-D-tryptophan at different water additions Similar effects of water on the catalysis, as described in figure 3, has been observed earlier both for a-chymotrypsin [9] and other enzymes [10] and has

325

been interpreted as an effect of the increasing mobility of the enzyme polypeptide chains as the possible interactions with surrounding water molecules are increased. A certain amount of water is needed for the most efficient catalysis. The results described in figure 4 must be interpreted in another fashion since the enzymatic activity is dramatically decreased even in the presence of very minor amounts of water. A possible explanation can be that the conformation of a-chymotrypsin obtained upon bio-imprinting in the presence of N-acetyl-D-tryptophan is stable only below a certain water concentration. When the water concentration is increased above this level the interactions between the enzyme and the surrounding water molecules are such that the original native enzyme conformation, active only towards the L-form, is regained. The maximum water addition increase the total water concentration only from 20 to 24 mM and yet the activity is totally lost. To what extent the added water partitions to the enzyme is not known and merits further studies as does the possible effect of ethanol and 1-propanol acting as water mimics [11 ] in the interactions between solvent and enzyme. The decreasing activity of a-chymotrypsin towards N-acetyl-D-tryptophan as the water concentration increased could be interpreted as a function of the mobility of the polypeptide chains in a-chymotrypsin. In order to investigate whether the same increased movements could be obtained by increasing the temperature in the reaction media, synthesis of N-acetyl-D-tryptophan ethyl ester at different temperatures was investigated. The enzymatic activity reached a maximun at about 35 °C followed by a decline. The enzyme was however still active at 70 °C. The same pattern however, was obtained both when N-acetyl-L-tryptophan and N-acetyl-D-trypophan were used as a substrates showing that the decrease in activity is an inherent property of a-chymotrypsin dispersed in cyclohexane /ethanol and not due to the disruption of the conformation active also towards N-acetyl-D-tryptophan.

Initial rates (nmol/h, mg enzyme) during synthesis of ethyl esters of L- and D- forms of N-acetyl-tryptophan, N-acetyl-phenylalanine and N-acetyltyrosine with bio-imprinted ai

ty

The method to test the a-chymotrypsin hydrolytic and syntethic activity were decribed in previous papers (18, 1 9 ) . R E S U L T S AND

DISCUSSION

α-Chymotrypsin immobilized on tresyl and tosyl activated sepharose 4B and 6B respectively, and chitin, were used to catalyse the hydrolysis of BTEE and the synthesis of R-Tyr-Leu-NH2 peptide from BTEE and Leu-amide. The reactions were carried out in different aqueous-organic media. The aim of the experiments was to study the relationship between the activity of a-chymotrypsin and the structure of the protein layer on the support. The activation of sepharose by reaction with Tosyl chloride (TsCl) and Tresyl chloride involved the primary alcohol groups of galactose, which are less strongly hindered and thus react more readily than the other hydroxyl groups with TsCl and Tresyl Cl by esterification (20) under these mild conditions. The activation of chitin by glutaraldehyde affects the NH2 groups of this compound.

342 The i n s o l u b i l i z a t i o n o f s u p p o r t s was c a r r i e d out elsewhere (18,19). F i g u r e 1 shows enzyme support used sepharose (a)

60 -

*J

rt

OX)

a 'e

ethanol

A

ethanol and Brij 35 0,1%

40 -

Ο •

"«3

ε

Δ

20 -

2-methoxyethanol 2-methoxyethanol and Brij 35 0,1%

0 20

40

Concentration

60 of

alcohol

80

100

(V/V)

fig 5 : remaining activity of α-amylase in alcoholic solutions with and without Brij 35

T h e effect o f B r i j 35 o n r e m o v a l

We checked that, without the enzyme, the surfactant had only a limited effect on removal (less than 5% of D.E.). With the addition of 0,1% of Brij 35 in the enzymatic solution, the D.E. of the treatment dramatically increased at low temperature (25°C). The D.E. doubles with α-amylase for 30 minutes of treatment on the starch linings and, with collagenase, there is an increase by a factor of seven on rabbit skin glue (fig. 6). Even if the surfactant may enhance the solubilisation of some pigments and some media, the reduction in treatment duration could reduce this risk. However, the presence of Brij 35 could be satisfactory for most of old master engravings.

391

time

(min)

fig 6 : influence of Brij 35 on removal at 25°C

Removal with alcoholic solutions and surfactant We previously discussed the problem of fugitive pigments and the possibility for using alcoholic solutions with a non-ionic surfactant to protect the enzyme from denaturation. We used a ternary solution of 2-methoxyethanol, Brij 35 and amylase in water to reach a compromise between the duration of the treatment, the efficiency and a possible reduction of pigment unstability (fig. 7).

O

2-methoxyethanol 5 0 %

Δ

2-methoxyethanol 20%

Brij 3 5 0,1%

Brij 3 5 0,1% Δ

2-methoxyethanol 10% Brij 3 5 0,1%

•

Time

α -amylase only

(min)

fig 7 : the use of ternary solutions (alcohols, Brij 35 and water) for paper removal

392

Conclusion The medium that has been studied is highly complex and depends on many parameters which are not completely understood. Under the conditions of our work we have found evidence that efficient enzymatic treatments were obtained at low temperatures and with reduced enzyme concentration. The papers studied were hydrophobic because of their surface layer. Under these conditions the diffusion of enzymes through paper and paste mainly constrained the efficiency of removal. The diffusion is significantly improved when using a non ionic surfactant even below CMC concentration, but surfactants tend to increase pigment solubilisation whereas lower primary alcohols tend to stabilise them. The use of ternary solutions of surfactant, alcohol and water could maintain a good enzymatic activity, a good diffusion rate and a good pigment stability. Further studies concerning hydrolysis in low water environments to preserve pigment stability will be carried out. To this end we intend to use entrapped enzymes in reversed micelles in organic solvents [15], [16].

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

M. Keghel, les encres, les cires, les colles et leurs préparations, Paris, Baillière J . E . et fils (eds.),Paris, 1922. F. Duprat, D. Gallant, A. Guilbot, C Mercier, J . P . Robin, les polymères végétaux, B.Monties, (ed.), Gauthier-Villars, Paris, 1980 A. Guilbot and C. Mercier, The polysaccharides, vol 3, G.O. Aspinal (ed.), Academic Press, New York, 1985 F. Hermann., Encyclopedia of polymer science and technology, vol. 4, 1981. G. Perreault, L'estampille, No. 222, (1989), 28. A. Karpowicz, Studies in conservation, 26, (1981), 153. R.W. Perkins, R.E. Mark, Preprints of the seventh fundamental research symposium, Cambridge, Paper, n°5/3, 1983. U.B. Möhlin, Svensk Papperstidn, 77,(1974), 131 P.G. de Gennes, Journal de Physique, 40, (1979),783. D. Coopper, C. King and J . Segal, preprints of the institute of paper conservation Cambridge conference, the conservation of library and archive materials and the graphic arts, 1980, 25 K. Martinek, A.V. Levashov, N. Klyachko, Y . L . Khmelnitski, I.V. Berezin, European Journal of Biochemistry, 155, (1986),453. C. J . Drummond, S. Albers, D. N. Furlong, Colloids and surfaces, 62, (1992), 75. R. Collison, Starch and its derivatives, fourth edition, Chapman, Londres, 1968. J.W. Park, Y. Takahata, T. Kajiuchi and T. Akehata, Biotechnology and Bioengineering, 39, (1992), 117. A.V. Kabanov, Α. V. Levashov, N. Klyachko, S.N. Namyotkin and A.V. Pshezhetsky, Journal of theoretical Biology, 133, (1988), 327. P.L. Luisi, C. Laane, TIBTECH., June 1986, 153.

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

393

Methyl Isobutyl and Methyl Ethyl Ketone Biodegradation in Biofilters Marc A. Deshusses and Geoffrey Hamer Institute of Aquatic Sciences and Water Pollution Control, Swiss Federa! Institute of Technology Zürich, Ueberlandstr. 133, CH-8600 Dübendorf, Switzerland

Abstract

Microbiological waste gas treatment is a most promising new development in environmental biotechnology. Herein effective methyl ethyl ketone (MEK) vapour removal and effective methyl isobutyl ketone (MIBK) vapour removal from polluted air streams are demonstrated and various aspects of such treatment processes identified for subsequent investigation. Biofilters of the type employed provide an example of microbes functioning effectively in a non-conventional medium.

1. INTRODUCTION Microbiological waste gas treatment represents a major development in environmental protection. Until some 30 years ago the concept of using microbes for pollutant removal from waste gases had not been subject to serious consideration [1], However, increasingly stringent environmental legislation, together with its more effective enforcement during the past 15 years, has forced technologists to examine hitherto neglected possibilities for efficient and economic waste gas treatment. The prospective biotreatment of waste gases has resulted in the development of two distinct types of bioreactor systems: biofilters and bioscrubbers. Biofilters are those systems where the resting or growing process culture is attached to a stationary solid support material such that direct contact occurs between the attached microbial film and the humid pollutant containing waste gas stream undergoing treatment. On the other hand, bioscrubbers are those systems where the pollutant œntaining waste gas stream is contacted with an aqueous solution in which either simultaneous or subsequent biodégradation of absorbed pollutants occurs as a result of the actions of growing microbes in or contacted

394

with the aqueous scrubbing solution. However, in the case of biofilters, filtration, as strictly defined, is not a pollutant separation mechanism. Pollutants present in waste gas streams can be either permanent gases or vapours. It has been shown that in biofilters pollutant biodégradation is directly associated with the aqueous solubility of the pollutant under consideration. Microbially mediated biodégradation on surfaces in contact with a humidified pollutant containing gas phase clearly represents an important example of biocatalysis in a nonconventional medium. In the present communication the aerobic biodégradation of both methyl isobutyl ketone (ΜΓΒΚ) vapour as a single gaseous phase pollutant and when mixed with methyl ethyl ketone (MEK) vapour as a pollutant mixture in humid air in packed bed biofilters is considered. Both MIBK and MEK are widely used industrial chemicals with annual productions exceeding 250,000 and 600,000 tonnes, respectively. The nature of use of the two chemicals, i.e., in coatings and as solvents for adhesives, inks and paints, and in the case of MIBK, in the extraction of metal salts from solutions, results in their widespread presence in both concentrated and dilute waste streams as either single or multiple pollutants. In general, concentrated waste streams can be subjected to economic recovery processes, recycling or where a diverse spectrum of pollutants are present, treatment and disposal by incineration. Dilute waste streams frequently present problems with respect to their economic and efficient treatment. Gaseous waste streams, particularly those containing pollutant mixtures, are remarkably problematical in this latter context and, therefore, offer major challenges as far as waste gas biotreatment is concerned. Fundamental factors relevant to the successful scaling-up and optimization of industrial scale biofilters include the definition of pollutant transfer mechanisms, questions of substrate and nutrient limitation, the role of the solid support material and effects resulting from its physical and chemical properties and, by no means least, the needs of the microbial process culture for specific nutrients. In this last matter, the question of the relative effectiveness of resting microbial cells and growing cells for efficient biodégradation requires resolution, but also effects resulting from death by lysis and subsequent "cryptic" growth [2] must be considered.

395

2. MATERIALS AND METHODS A schematic diagram of the experimental system used is shown in Fig. 1.

7K

Compressed air ^prp^

MEK Inlet air

Water

Effluent air

Fig. 1. Schematic diagram of the experimental system

Pollutant containing humid air stream. Compressed

oil-free air is saturated with water vapour by sparging the air into a 50 L bottle containing deionized water and thermostated at 28°C. Two smaller compressed air streams were sparged into 0.5 L bottles containing either MIBK or M E K as required and subsequently mixed with the major humidified air stream. A metered flow of pollutant containing humid air stream was passed downwards through each of five vertical packed column biofilters operating under a range of selected parameters.

Biofilters and packing material. The biofilters were constructed from plexiglas tubing and were 1 m in length and 80 mm in internal diameter. The upper and lower 60 mm of each column was packed with expanded clay spheres and the remaining 880 mm with a commercially available biofilter packing (Bioton: ClairTech, Utrecht, NL), comprising an equivolume mixture of compost and polystyrene spheres. Acid neutralizing components are also present in the biofilter material. Before use the packing material was inoculated with a concentrated enrichment culture. Sample ports were located at 0, 2 5 , 52, 78 and 100 percent of the active packing height in each column. Column temperatures were maintained between 21° and 25°C.

396

Inoculum. Solvent degrading enrichment cultures were grown on M E K and MIBK in shake flasks, with regular transfers, over a period of six months. The inoculum was prepared by concentrating 3 L of enrichment culture to 15 m L by centrifugation. This was sufficient to coat 1 kg of packing material prior to introduction into the columns. Operating conditions. The

active bed volume was 4.4 L and the mass of damp support material, comprising 60 percent water, was 950 g per filter. The void space in the bed was 50 percent. Gas flow rates of 200 to 300 L h"l were used. This gave a surface loading of 40 to 60 m h~l and a volumetric loading of 4 5 to 70 h~l. The gas had a relative humidity greater than 95 percent and contained between 300 and 1200 mg m"3 solvent carbon. The pressure drop over the filter was less than 10 mm water column.

Analysis.

The concentrations of MEK, MIBK oxygen and carbon dioxide in gas phase samples were determined by gas chromatography. In the case of MEK and MIBK, 1 m L gas samples were introduced into a Carlo Erba (Milan, I) type HRGC 5160 gas Chromatograph fitted with a SE 54 column and operated 1 isothermally at 70°C. The carrier gas used was 1.83 L h" hydrogen and detection was with a flame ionization detector. The detection limit was ca. 15 mg solvent carbon per mß of gas. Oxygen and carbon dioxide were determined by injecting 1 m L gas samples into a Shimadzu (Kyoto, J ) type GC-8A gas Chromatograph fitted with a combined 5Â molecular sieve / Porapak Q column and operated isothermally at 80°C. The carrier gas used was 2.64 L h'l helium and detection was with a thermal conductivity detector. The detection limit was ca. 30 mg carbon dioxide carbon per m^ of gas.

3. R E S U L T S Results for the performance of a biofilter treating a mixed MEK/MIBK vapour in air are shown in Pig. 2, where the reduction in MEK/MIBK vapour concentration and carbon dioxide production and temperature relative to biofilter height are reported. Whereas an extremely high level of removal (ca. 98 percent) of the pollutant vapours is indicated, the measured production of carbon dioxide only corresponds to ca. 42 percent recovery of the carbon entering the system. The temperature profile in the biofilter showed a relatively slight gradient in the direction of air flow (ca. 1 C°). Several explanations exists for the low carbon recovery. Probably the most plausible is that the biofilter had only been started up 7 days prior to the experiment and the active biofilm of microbes was still being built-up. In similar biofilters where more prolonged operation had occurred,

397

up to 75 percent carbon recovery was observed. Neither volatile nor gaseous products, other than carbon dioxide or remaining traces of MIBK, could be found in the effluent gas stream. However, a fraction of the carbon dioxide produced was probably absorbed in water films within the biofilter and because of the neutralizing capacity of the packing, carbonate would be expected to be retained within the packing, thus accounting for a part of the missing carbon.

0.00

0.20

0.40

0.60

0.80

1.00

relative height [-] Fig. 2. Solvent, carbon dioxide and temperature profiles as a function of the relative height in the biofilter bed for MEK/MIBK removal from air. (inlet concentrations: MIBK=385 mg m"3; MEK=383 mg m"3; gas flow=284Lh-!).

In Fig. 3, results are shown for a biofilter operating on an air stream containing MIBK alone, in one case, and a MEK/MIBK mixture, in the other case. In the former case, virtually the whole concentration of MIBK vapour is removed in the upper 20 percent of the biofilter. However, in the latter case, MIBK removal occurs throughout the upper 80 percent of the biofilter, but M E K removal only in the upper 60 percent of the biofilter. Clearly the presence of M E K in the air stream has a marked effect on MIBK removal. Both MEK and MIBK

398

removal were virtually complete and neither vapour could be detected in the effluent stream. Removal occurred simultaneously and in a parallel manner in the upper 60 percent of the biofilter. In shake-flask mixed cultures of enriched biofilter inocula, MEK was utilized as carbon and energy substrate by the cultures grown on MIBK and vice versa. With respect to MIBK as a single pollutant in the air stream, the hourly biodégradation potential was 67 g MIBK per m^ biofilter volume, whereas when MIBK was mixed with MEK, the hourly biodégradation potential was reduced to 21 g MIBK per mß biofilter volume. This suggests that the rate determining step in the overall process is most probably biological in nature, rather than a transfer limitation. This proposal will be confirmed by recirculating a fraction of the gaseous effluent stream, thereby estabhshing a differential reactor [3].

Fig. 3. Biodegradation profile of MIBK alone and of a MEK/MIBK mixture as a function of the relative height in the biofilter bed. (inlet concentrations: ΜΠ3Κ=385 mg m"3; MEK=383 mg m~3; gas 1 flow=284Lh" ).

399

4. CONCLUDING REMARKS The present investigation demonstrates that biofilters of the type that have been constructed and operated here exhibit high efficiencies for the removal of both MEK and MDLBK vapours from polluted air streams. However, the additional presence of MEK in an air stream polluted with MIBK markedly reduced performance with respect to MIBK removal potential. The poor carbon recoveries reported emphasize a need for further studies to elucidate the fate of the removed pollutants, although recoveries suggest that biodégradation is coupled with growth in the systems investigated.

5. REFERENCES [1] [2] [3]

H. Brauer, Chem. Ing. Tech., 56 (1984) 279. CA. Mason, G. Hamer and J.D. Bryers, FEMS Microbiol. Revs., 39 (1986)373. D. Greiner, M. Kolb, J . Endler and R. Faust, Staub-Reinhalten der Luft, 50(1990)289.

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

401

Lipase catalysed esterification in supercritical carbon dioxide Z.Knez, M . Habulin Faculty o f Technical Sciences, Dept. Chemical Engineering, University o f Maribor, S L O - 6 2 0 0 0 M A R I B O R , S L O V E N I A

Abstract Oleic acid esterification has been performed in a stirred batch reactor by lipase from Mucor miehei in solvent free system and in supercritical carbon dioxide. T h e reaction rate was influenced by temperature and reaction media. Future research will b e concentrated on the influence of the process parameters on final conversion, initial velocity, enzyme stability and on fractionation of the product from the substrates and scaling up of the production process.

1. I N T R O D U C T I O N Supercritical fluids have been used as solvents for a wide variety of extractive applications (also on a commercial scale) in the last few years.Recently supercritical fluids have been applied as a solvent for non extractive applications in high pressure micronisation, in chromatography, and as chemical and biochemical reactions media. U s e o f supercritical fluids decrease mass transfer limitations because of the high diffusivity o f reactants in supercritical medium, low surface tension and because o f the relatively low viscosity of the mixture. An additional benefit of using enzymatic reactions in supercritical fluids is that it provides a very convenient way for the recovery of products or recovery of non reacted components. T h e advantages expected from using supercritical carbon dioxide as a medium for enzymatically catalyzed reactions, compared with aqueous and organic liquid reaction media, is well documented in [1]. T h e most useful temperature range of supercritical carbon dioxide and the typical, low operation temperatures of enzymes ideally overlap each other. T h e limitation of this process may arise only from the non-polarity of carbon dioxide and therefore mainly hydrophobic compounds are suitable as substrates or products. Since the work of Nakamura et al. in 1985 [2] on interesterification of triglycerides by a lipase, several other studies are now available and have proved the feasibility of enzymatic reactions in supercritical fluids [3-12]. In our previous research work the influence of process parameters on: (i) final conversion, (ii) initial velocity, (iii) enzyme activation and deactivation in the ezymatic synthesis of η-butyl oleate [13-16] and l,2-isopropylidene-3-oleyl glycerol [17] in a solvent free system was studied. T h e main objectives of our present research programme are: (i) the comparison of enzymatic reactions in supercritical fluids and in a solvent free system in a batch stirred tank reactor, (ii) the development of an integrated production and product recovery process based on enzymatic catalysis and product fractionation in supercritical carbon dioxide, (iii) the development of a small scale multi-purpose enzyme reactor.

402 The model system used for our research was esterification of oleic acid with oleyl alcohol catalysed by lipase from Mucor miehei.

2. M A T E R I A L S AND M E T H O D S 2.1. Materials 2.1.1. Enzyme preparation For the synthesis of oleyl oleate Mucor miehei lipase was used as a catalyst. T h e lipase Lipozyme - immobilized on a macroporous anion exchange resin, was kindly donated from Novo Nordisk A/S (Copenhagen, Denmark). T h e enzyme beads contained 10 w/w% of water. In some experiments Palatase 1000 L was used, which is a lipase of Mucor miehei in water soluble form. 2.1.2. Lipase activity The lipase activity was measured according to Novo Nordisk A / S . T h e activity of the Lipozyme preparation used for our synthesis was 23 B l U / g . O n e Batch Interesterification Unit ( B I U ) corresponds to 1 micromole of palmitic acid incorporated in triolein per min at standard conditions [ 1 8 ] . The activity of Palatase preparation was 1000 LU/g. O n e Lipase Unit ( L U ) is the amount of enzyme which liberates one micromole butyric acid per minute from a tributyrin substrate at standard conditions. 2.1.3. Chemicals The oleic acid was purchased from Merck (Darmstadt, Germany) and the oleyl alcohol was supplied by Aldrich ( U S A ) . All other chemicals, including n-butanol as substrate, were from Kemika (Zagreb, Croatia). Carbon dioxide was 99,97 % volume pure and was supplied by Rogaska (Rogaska Slatina, Slovenia). 2.2. Methods 2.2.1. Analytical Method The amount of oleic acid in the reaction mixture was determined by the H P L C method. The Milton Roy H P L C model was equiped with a variable wavelenght U V detector. Separation was achieved on a stainless steel column (4,6 χ 250 mm) packed with Spherisorb O D S 2 , at a wavelength 223 nm. T h e mobile phase was pure acetonitrile and the flow rate was 3.50 ml/min. 2.2.2. Synthesis o f oleyl oleate in a batch stirred tank reactor - Solvent free system The reaction mixture contained 47 mmol of oleic acid, 47 mmol of oleyl alcohol, 1,04 g of enzyme preparation. T h e mixture was stirred in a 250 mL, round bottom flask with a magnetic stirrer and heated up to the desired temperature in a water bath. At set times samples were taken from the reaction mixture and the level of free fatty acids was determined. F r o m these values the concentrations of oleyl oleate were calculated.

403

2.2.3. Synthesis o f oleyl oleate in a batch stirred tank reactor - Supercritical system T h e design of the apparatus is shown in Figure 1. T h e volume of the reactor is 150 ml and is designed to operate to a pressure of 500 bar. T h e reaction mixture was mixed via an oscillating device. T h e whole system was placed in a constant temperature bath. First the reaction mixture, which contained 25 mmol of oleic acid and 25 mmol ofoleyl alcohol, was pumped into the reactor. Then 0,5 g of enzyme preparation (Lypozyme or Palatase 1000 L ) was added. Finally dry CO2 was pumped into the reactor up to the desired pressure. During the reaction samples were taken out of the reactor and the amount o f free oleic acid was determined by the H P L C method.

1....REACTOR 2....SEPARATOR P r. . . H I G H P R E S S U R E PUMP

Τ =

konst^,

2

CO

2

Figure 1. Design of experimental apparatus.

3. R E S U L T S AND D I S C U S I O N 3.1.Solvent free system A synthesis o f oleyl oleate with immobilized Mucor miehei lipase in the solvent free B S T R was done at various temperatures. Figure 2 shows the variation of ester concentration vs. time at a pressure 1 bar and water activity 1% by weight. As observed in our previous research work on esterification of oleic acid with n-butanol [13-17] the increase o f the temperature increases the equilibrium conversion. Initial reaction rates for enzymatic synthesis of oleyl oleate in a solvent free B S T R was determined. T h e highest value 1,429 mmol/g/h per g of enzyme preparation was found for the temperature 50 C. With the decreasing of the temperature the initial reaction rates decreased and were 0,625 mmol/g/h per g o f enzyme preparation at 4 0 ° C and 0,465 mmol/g/h per g of enzyme preparation at 30 C.

404

&

-S? "5 Ε c

8 Ö 8

•

•

20oC 50

+

30oC

*

40oC

π

50oC

200 time (min)

Figure 2. Concentration of oleyl oleate vs. time for various temperatures in a solvent free system (pressure 1 bar, water activity 1 % by weight). 3.2. Supercritical fluid system A synthesis of oleyl oleate with immobilized Mucor miehei lipase (Lipozyme ) and water soluble lipase Palatase was performed at various pressures and temperatures. T h e first results are presented in Figure 3 and the systematic investigation on the influence o f process parameters on the equilibrium conversion is still under investigation.

_ 3.5-

1 υ W> Î5 2.5ο. -S? "δ ε 2υ 8

ϋ ö

1.5-

• (Lipozyme) J 1 ° C ,

8 4 , 5 bar

-f- (Lipozyme) 40°C,

167 bar

*

141 bar

(Palatase) 40°C,

1-

0.5-

25 time (h)

Figure 3. Concentration of oleyl oleate vs. time at various procès parameters.

405

i i V 1

F o r enzymatic synthesis of oleyl oleate catalyzed with Lipozyme in B S T R operated at supercritical conditions the initial reaction rates were higher. T h e highest value (1,428 mmol/g/h per g of enzyme preparation) was found for 30 C and pressure 84,5 bar. At a higher temperature and a higher pressure (40°C, 167 bar) the initial reaction rate was lower and was 0,615 mmol/g/h per g of enzyme preparation. F o r the reaction catalyzed with Palatase 1000 L the initial reaction rate was the lowest (0,454 mmol/g/h per g enzyme preparation) probably due to high water activity in the system. It is evident that water activity has a strong influence on the reaction rates.

4. P R O C E S O U T L I N E A hypothetical, idealized flowsheet of continuous processes for enzymetic synthesis of esters in supercritical fluids is presented in figure 4. T h e reaction mixture is fractionated in separators S i and S2. T h e product is continuously being drawn from the reaction system, unreacted substances are recycled back to the saturation column and the enzymatic reactor.

(PIRCV- — — — —;

1....SATURATION COLUMN 2....ENZYMATIC REACTOR 3....SEPARATOR 1 4....SEPARATOR 2 Ρ ..HIGH PRESSURE PUMP

Figure 4. Hypothetical flowsheet of an enzymatic supercritical esterification production process.

406 5. C O N C L U S I O N T h e s e are only preliminary results and the detailed research on the influence of process parameters on the initial reaction rate is still under investigation. F o r the design of the separation process of oleyl oleate from the reaction mixture the solubility of oleyl oleate, oleyl alcohol and oleic acid in supercritical CO2 in temperature ranges of 30 to 5 0 ° C and at pressures of 80 to 300 bar are being investigated.

6. R E F E R E N C E S 1 2 3 4 5 6

7

8 9 10 11 12 13 14 15 16 17 18

n d

Aaltonen and M . Rantakyla, Proceedings 2 International Symposium on Supercritical Fluids, Ed. M.A. McHugh Boston Mass. (1991) 146. K . Nakamura, Y . M . Chi, Y . Yamada, Chem. Eng. Commun., 45 (1985) 207. Κ. Nakamura, T . Hoshino, Int. Workshop on »Role of Food Engineering« Research in the development of Indonesian Food Industry, Jakarta, Sept. 2-6 1991. K . Nakamura, T I B T E C H , 8 (1990) 288. A.M.M. van Eijs, J . P . J , de Jong, H.J. Doddema, D . R . Lindeboom, Proceedings International Symposium on Supercritical Fluids, E d . M . Perrut, Nice-F, 2 (1988) 933. H . J . Doddema, R . J . J . Janssens, J . P . J , de Jong, J . P . van der Ingt, H.H.M. Oostrom, 1 Proceedings 5 European Congress on Biotechnology, Ed.C.Christiansen et al., Munksgaard., Int. Publisher, Copenhagen, 1 (1990) 239. n International Symposium T . Dumont, D . Barth, M. Perrut, Abstract Handbook 2 High Pressure Chemical Engineering, E d . G. Vetter, D E C H E M A , G V C - V D I , Erlangen, ( 1 9 9 0 ) 6 5 . A. Marty, W. Chulalaksanamukul, J . S . Condoret, R . M . Willermont, G.Durand, B i o technology Letters, 12 (1990) 11. A. Marty, W.Chulalaksanamukul,, R.M.Willermont, J . S . Condoret, Biotechnology and Bioengineering (in press). D.A. Miller,H.W. Blanch,J.M. Prausnitz, Ind.Eng.Chem. Res., 30 (1991) 9 3 9 . J . C . Erickson, P. Schyns, C L . Cooney, A I C h E Journal, 36 (1990) 299. n T . Dumont, D . Barth, M. Perrut, Proceedings 2 International Symposium on Supercritical Fluids, E d . M . A. McHugh, Boston Mass. (1991) 150. M . Leitgeb, Z. Knez, J A O C S , 67 (1990) 775. M . Habulin, È . Knez, J . M e m b . Sei., 61 (1991) 315. È . Knez, M. Habulin, S. Pecnik, Proceedings 5 European Congress on Biotechnology, Ed. C.Christiansen et al., Munksgaard Int.Publisher,Copenhagen, 1 (1990) 437. £ . Knez, M. Leitgeb, D . Zavrsnik, B . Lavrià, Fat. Sei. Technol., 92 (1990) 169. S. Pecnik, Z. Knez, J A O C S , 69 (1992) 2 6 1 . Preliminary Product Informations Lypozyme and Palatase Novo Nordisk A/S Copenhagen - D K , 1985.

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

407

Effect of a near- critical and supercritical fluid on the viability ratio of microbial cells A. Isenschmid, L W . Marison and U von Stockar I n s t i t u t e of Chemical E n g i n e e r i n g , Swiss Federal I n s t i t u t e of Technology, C H 1015 L a u s a n n e , S w i t z e r l a n d

Abstract T h e f o l l o w i n g p a p e r d e s c r i b e s h o w p r e s s u r i z e d CO2 u n d e r n e a r - c r i t i c a l a n d s u p e r c r i t i c a l c o n d i t i o n s affects t h e s u r v i v a l o f a r a n g e o f y e a s t a n d b a c t e r i a l cells. Kluyveromyces fragilis w a s f o u n d t o b e t h e m o s t r e s i s t a n t o f t h e t e s t o r g a n i s m s studied w i t h relative viabilities of > 70% u n d e r near- critical conditions. T h e g r o w t h h i s t o r y o f t h e cells b e f o r e t r e a t m e n t w a s t h e m a j o r f a c t o r a f f e c t i n g s u r v i v a b i l i t y w i t h u p t o 100- f o l d h i g h e r v i a b i l i t i e s f o r cells t r e a t e d f r o m t h e s t a t i o n a r y p h a s e o f b a t c h g r o w t h c o m p a r e d w i t h those f r o m a c t i v e l y g r o w i n g cells i n t h e e a r l y e x p o n e n t i a l phase.

1. INTRODUCTION T h e e x t r a c t i o n o f f r e q u e n t l y d i l u t e b i o p r o d u c t s f r o m bioprocess m e d i a r e p r e s e n t s one o f t h e m a j o r l i m i t a t i o n s associated w i t h b i o t e c h n o l o g i c a l processes. F r e q u e n t l y such bioproducts are heat sensitive or r e q u i r e the a d d i t i o n o f organic solvents, t h e residues o f w h i c h m u s t be completely removed f r o m t h e f i n a l product. O n e e x t r a c t i o n m e t h o d w h i c h m a y be r e a d i l y i n t e g r a t e d w i t h bioprocesses, a n d l e a v e s n o r e s i d u e s , i n v o l v e s t h e u s e o f c o m p o u n d s , s u c h as c a r b o n d i o x i d e , n e a r t h e i r vapour- l i q u i d critical points. Such compounds e x h i b i t a range of properties w h i c h can be used to advantage i n e x t r a c t i o n processes. T h u s (1) t h e d i s s o l v i n g p o w e r i s s t r o n g l y d e p e n d e n t o n t h e d e n s i t y o f t h e e x t r a c t a n t a n d t h i s m a y be c a r e f u l l y c o n t r o l l e d b y r e g u l a t i o n o f t h e t e m p e r a t u r e a n d p r e s s u r e ( F i g u r e 1); (2) d i f f u s i o n r a t e s i n a s u p e r c r i t i c a l fluid m a y be u p t o 100 t i m e s h i g h e r t h a n i n l i q u i d s ( T a b l e 1) w i t h a r e s u l t i n g i n c r e a s e i n e x t r a c t i o n r a t e s ( S t e y l e r e t a l . , 1 9 9 1 ; M a r t y e t a l . , 1 9 9 0 ; P a s t a e t a l . , 1 9 8 9 ) ; (3) t h e s u r f a c e t e n s i o n a p p r o a c h e s zero f o r s u p e r c r i t i c a l gases, w h i c h r e s u l t s i n a r a p i d w e t t i n g o f t h e c a t a l y s t b y t h e s o l v e n t ; (4) t h e v i s c o s i t y o f s u p e r c r i t i c a l gases i s a b o u t 1 0 0 t i m e s l o w e r t h a n i n l i q u i d s ( T a b l e 1) e v e n t h o u g h t h e r e i s a l a r g e increase a r o u n d t h e c r i t i c a l p o i n t ; (5) a d d i t i o n o f co- s o l v e n t s o r e n t r a i n e r s , s u c h as methanol or ethanol, can significantly improve t h e solvent characteristics (Wong et a l . , 1986; P e t e r e t a l . , 1978).

408 Table 1 Typical properties o f a g a s , liquid a n d supercritical fluid property

gas

density [kg/m3]

0.6 - 2.0

(0.2 - 0.9) xl03

(0.6 3 - 1.6) xlO

diffusion coeff. [m2/s] viscosity [ k g / m s]

(1 3 - 4 ) χ 10

2-7

(0.2 1- 2.0) xlO"

(1 5- 3) χ 10'

(1 - 9) χ 10-5

(0.2 - 3.0) 3 χ ΙΟ"

-0

7,3 (water)

60Ό00 (carbon dioxide)

10-75

surface tension [N/m2] heat capacity [ J / m o l K]

supercrit. fluid

0

liquid

(From: H o y e r , 1985)

F i g u r e 1. D e n s i t y o f p u r e c a r b o n dioxide a s a f u n c t i o n o f t e m p e r a t u r e a n d p r e s s u r e . T h e c r i t i c a l point o f CO2 is 3 1 ° C a n d 7 3 , 8 3 b a r ( B u s e , 1 9 8 5 ) . I

I gas or liquid

Η

near-critical liquid

||j

supercritical fluid

409

As a result of these properties near- or super- critical fluids, particularly CO2, have been used for the extraction of propanic, acetic and butyric acids from cultures of Clostridium thermoaceticum (Shimshick, 1981), acetone, butanol and ethanol from cultures of C. acetobutylicum (van Eijs et al., 1988) and ethanol from cultures of Saccharomyces cerevisiae and S. rouxi (l'Italien et al., 1988). Conversely, it has also been reported that near- critical CO2 may be used to destroy microbial cells (Kamihira et al., 1987; Ho Mu Lin, 1991). Thus if near- and super- critical fluids are to be used successfully for the extraction of bioproducts from bioprocesses it is essential that data is obtained which quantifies the damage to living microbial cells by such techniques. This is the aim of the present paper. 2. M A T E R I A L S AND METHODS

LEGEND 1 pressurized carbon dioxide 2 valve J thermostat: J°C 4 precooling coil 5 HPLC - pump 6 preheating coil 7 thermostat: JJ°C 8 pressure vessel (stirred, 80.9 ml) 9 magnetic stirrer 10a temperature controller J°C 10b temperature controller 11 pH indicator 12 pressure controller (computer directed) 1J pressure reduction valve 14 decompression valves (computer directed) 15 outlet

Figure 2. Schematic representation of the pressurizable reactor configuration. A pressurizable reactor system has been constructed (Figure 2) in which the pressure within the reactor is continuously measured by quartz pressure transducer coupled to a Mac Ilci computer operating with LabView acquisition/ control software. A control alorithm enabled careful control of the compression, holding and decompression time as well as ensuring reproducibility between experiments. Carbon dioxide was chosen as the test extractant. The test organisms included the yeasts, Candida utilis NCYC 9950, Kluyveromyces fragilis NRRL 1109 and

410 Saccharomyces cerevisiae CBS 426, as well as the bacterium Escherichia coli W3110. Each microbial strain was grown in a 3 litre bioreactor under batch conditions as previously described (Birou, 1986). Cells were harvested after different periods of growth corresponding to 27%, 60% and 85% of the final cell density, divided into aliquots, and incubated at -70°C for 24 h followed by storage in liquid nitrogen. Aliquots were thawed rapidly at room temperature, diluted to give a range of cell concentrations and exposed to CO2 in the pressure reactor over a range of pressures for different periods of time. The total cell count, viable cell count and cell size distribution were determined before and after CO2 treatment. The former and the latter were determined using a Coulter counter (Coultronics Model ZM) whilst viable cell number was determined by counting the number of colony forming units (CFU) obtained after plating of suitabely diluted culture samples on a medium composed of yeast extract (1% w/v), peptone (2% w/v), glucose (2% w/v) and agar (1,6% w/v) followed by incubation at 30°C for 24- 48 h. Relative viable count is defined as the ratio of the CFU ml-1 of cell containing samples exposed to CO2 compared with identical samples not subjected to CO2 treatment. Since the pressure reactor contains a cell suspension in contact with CO2, the pH of the liquid phase varies with the pressure applied. Consequently the pH in the reactor vessel was continuously measured using a pH probe, modified in our laboratory, to withstand high pressures. The results are shown in Figure 3.

7

Î >

χ

6.5» 6» 5.5» 5 » 4.5» 4 » 3.5 —

-+-

20

h40

60

pressure [bar]

Figure 3. Effect of CO2 pressure on the pH of the liquid phase. 3. R E S U L T S A N D D I S C U S S I O N 3.1 E f f e c t o f p r e s s u r e o n c e l l s u r v i v a l

Samples (2 ml) of the test organisms were incubated in the pressure vessel, with and without stirring, and subjected to a range of CO2 pressures at a temperature of 27,5°C, corresponding to near- critical conditions. The results for C.utilis (Figure 4) show that the relative viability remains high (approximately 40-

411

100%) over the range 0- 60 bar CO2 and then falls dramatically at pressures above 70 bar. Similar results were obtained for all of the test organisms. It is noticable that the decrease in viability correlates well with the large increase in density of the CO2 between 60- 70 bar pressure (Figure 4). The agitation rate had no appreciable effect under these conditions due to the small liquid phase volume (2 ml). It was also shown t h a t , whilst the pH falls to a value of 3- 3,5 at 60 bar, this did not account for the decrease in viability (Wallhäuser, 1988).

Figure 4. The effect of pressure on the viability of C. utilis in near- critical CO2. Conditions: 27,5°C, 0- 1500 rpm, 2ml cell sample volume, harvested a t 70% completion of growth. When the temperature was increased to 33°C the relative viabilities for all of the test organisms decreased by a factor of between 20- 30 fold when the CO2 pressure was increased from 65 to 85 bar.The order of sensitivity of the test organisms was shown to be: K. fragilis < E. coli < S.cerevisiae! C. utilis

3.2. Effect of cell age on survival The phase of batch growth from which the cells were harvested was found to play an important role in the survivability of cells to treatment with CO2. Thus cells harvested during the early exponential phase exhibited greater sensitivity to CO2 under near- (65 bar) or super- critical (85 bar) conditions. Figure 5 shows the results for C. utilis. Clearly cells harvested in the stationary phase (after approximately 800 h of growth) were the most resistant, with 70- 80% relative viability even after 7 χ 10^ minutes in the stationary phase. Similar results were obtained for the other test organisms. Once again it can be observed (Figure 5)

412 that at 85 bar the survivability was significantly lower than obtained in the nearcritical region (65 bar).

·.··

• • •

ο

ο

ÇS

5

0

•

2000

4000

fermentation

•

ο

ο

6000

8000

time [min.]

Figure 5. The effect of the period of harvesting from batch cultures of C. utilis on survivability.

80

j

70 6 0 --

ο υ

o" ο

50

φ

η

.2 '> φ > φ

40 30

•

2 0 ·-

ο

1 0 » 0

OP Q Q Ο

0%

20%

60%

40%

100%

80%

% completion of growth ° C.utilis

•

K.fragilis

D

S.cerevisiae

•

E.coli

Figure 6. Comparison of the effect of the period of harvesting of different test organisms on survivability. Conditions: 65 bar C02,33°C, 250 rpm, 5 ml culture sample and 5 min exposure to CO2.

413

Due to these results, the time of harvesting of the cells was expressed as a percentage of the time before entry into the stationary phase of batch growth. In the case of C. utilis 100% completion of growth represents 8 0 0 min after inoculation. This method allows comparison of results from different strains in which the growth kinetics are different for each. Figures 5 and 6 show how, regardless of strain, the survivability was a direct function of the period of harvesting. These results clearly show that actively growing cells are much more sensitive to CO2, particularly at pressures above 65 bar.

3.3. Effect of cell density on survival

coun

When cells were harvested after 70% completion of growth, diluted to give between 4 1 0 and 10^ cells ml'*, and incubated in the reactor vessel at 62 bar CO2 it was found that the survivability increased with decreasing cell concentration. Thus for C. utilis (Figure 7) the relative viability falls from approximately 70% a t a cell concentration of 2 χ 10^ cells ml'l to less than 10% at a cell concentration of 3 χ 10? cells ml~l. Although the reason for this remains unclear it is possible that when the cell concentration is high the accumulation of hydrolytic enzymes from damaged cells reaches high levels which may result in increased damage to remaining viable cells. This effect must be examined in more detail before any definite conclusions may be drawn.

80 j 60--

viabl

Φ

elat

Φ >

0^

40-20Ο

0 -1.E+03

1.E+05 cell density

1.E+07

1.E+09

[cells/ml]

Figure 7. Effect of cell density on the survivability of C. utilis to treatment with CO2. Conditions: 62 bar, 27,5°C, 2 ml sample volume, 0 rpm and 5 min exposure to C 0 2 .

3.4. Effect of sample volume and agitation on cell survival A series of experiments demonstrated that as the volume of the cell suspension, which was incubated in the pressure reactor, was increased the higher

414 was t h e level o f cell s u r v i v a l . F u r t h e r m o r e , i f t h i s l i q u i d phase was a g i t a t e d b y m e a n s o f a m a g n e t i c s t i r r e r r o d , t h e s u r v i v a b i l i t y decreased ( F i g u r e 8). T h i s effect i s p r o b a b l y r e l a t e d t o t h e i n t e r n a l g e o m e t r y o f t h e c y l i n d r i c a l p r e s s u r e reactor w h i c h h a d a t o t a l v o l u m e o f 80,9 m l . T h e l a r g e r t h e l i q u i d phase cell s u s p e n s i o n v o l u m e i n t r o d u c e d i n t o t h e r e a c t o r , t h e l o w e r i s t h e a m o u n t o f CO2 w h i c h can be added to fill t h e reactor a n d thus a lower level of extractant. F u r t h e r m o r e , t h e r a t i o o f t h e i n t e r f a c i a l a r e a f o r CO2 t r a n s f e r t o l i q u i d v o l u m e decreases w i t h i n c r e a s i n g l i q u i d v o l u m e , r e s u l t i n g i n less CO2 c o m i n g i n t o c o n t a c t w i t h t h e m i c r o b i a l cells. I f t h e l i q u i d i s a g i t a t e d a t a h i g h r a t e t h e i n t e r f a c i a l a r e a i n c r e a s e s r e s u l t i n g i n g r e a t e r CO2 c o n t a c t w i t h t h e c e l l s a n d , as a r e s u l t , decreased v i a b i l i t y .

F i g u r e 8. E f f e c t o f a g i t a t i o n speed a n d s a m p l e v o l u m e o n t h e s u r v i v a l o f C. utilis. Conditions: Cells harvested a t 7 0 % completion of g r o w t h , 2 m l sample volume, 80 b a r CO2 p r e s s u r e , 2 7 , 5 ° C , 5 m i n e x p o s u r e t o CO2. r.v.c, r e l a t i v e v i a b l e c o u n t [%]; v, v o l u m e o f l i q u i d phase [ m l ] ; s, s t i r r e r speed [ r . p . m . ]

A f u r t h e r i n t e r e s t i n g f e a t u r e i s t h a t w h e r e a s v i a b i l i t y decreases w i t h d e c r e a s i n g l i q u i d phase v o l u m e a n d h i g h a g i t a t i o n r a t e a t n e a r - c r i t i c a l c o n d i t i o n s , e x a c t l y t h e r e v e r s e i s t r u e a t s u p e r c r i t i c a l c o n d i t i o n s ( t e m p e r a t u r e above 3 1 ° C a n d p r e s s u r e above 74 b a r s ) . T h u s w h e n cells o f C. utilis w e r e h a r v e s t e d a f t e r 9 5 % c o m p l e t i o n o f g r o w t h a n d 2 ml o f cell s u s p e n s i o n i n c u b a t e d w i t h s t i r r i n g (1500 r p m ) a t 85 b a r for 5 m i n , t h e v i a b i l i t y w a s 3- f o l d higher t h a n w h e n t h e c o r r e s p o n d i n g v o l u m e w a s 10 ml. I f h o w e v e r i d e n t i c a l c o n d i t i o n s w e r e u s e d b u t w i t h a p r e s s u r e o f 65 b a r

415

(near- critical) the viability was 2,5- fold lower with a volume of 2 ml compared with a volume of 10 ml. It should be added however that the viability of the cells was generally much higher under near critical conditions as compared with supercritical conditions. A series of further experiments demonstrated that either the time during which the pressure was hold constant and the time needed for decompression had a significant effect on the viability of the test strains. Both of these parameters were kept constant during all the experiments. These effects are the base of future work. However addition of culture media, buffers or salts to the liquid phase in the pressure reactor had no significant effect on cell survival. The results of the present work indicate that of all of the factors studied, the period of batch growth from which the cells were harvested played a major role in the ability of the test organisms to survive pressures of C O 2 upto 8 5 bar. Furthermore cell survival was significantly higher under near- critical conditions as compared with supercritical conditions, particularly if a large liquid phase volume and low agitation rate were used. Under these conditions K. fragilis was found to be the most resistent organisms with relative viabilities greater than 70%.

4. CONCLUSIONS Near- critical CO 2 has little effect on the viability of a range of microbial cells providing the conditions for cultivation and harvesting are carefully determined. This offers the possibility of using CO 2 for the extraction of important bioproducts from culture media, particularly products which are formed during the lateexponential and stationary phases of batch growth, since under these conditions the cells are most resistent. An important development of this work will be to determine the effect of more 'neutral' compounds such as ethane as extractants, since these should not effect cell metabolism and should not cause important changes in the physico- chemical environment, such as pH.

5. R E F E R E N C E S BIROU B. (1986) Etude de al chaleur dégagée par des cultures microbiennes dans un fermenteur de laboratoire. Thèse No. 6 1 2 , E P F Lausanne HO-MU LIN, ERR-CHENG CHAN, CHEESHAN C H E N and L I - F U CHEN, (1991) Disintegration of Yeast Cells by Pressurized Carbon Dioxide, Biotechnol. Prog., 7, 201 - 204 HOYER G.G., (1985) Extraction with supercritical fluids: Why, how, and so what, Chemtech, 15, 440448

416 KAMIHIRA M., TANIGUCHI M. and KOBAYASHI T., (1987) Sterilization of Microorganisms with Supercritical Carbon Dioxide, Acric. Biol. Chem., 5 1 , 407 - 412 MARTY Α., CHULALAKSNANUKUL W., CONDORET J . S . , WILLEMOT R.M. and DURAND G., (1990) Comparison of Lipase-Catalysed Esterification in Supercritical Carbon Dioxide and in n-Hexanef Biotechnol. letters, 1 2 (1), 11 - 16 L'ITALIEN Yves, THIBAULT Jules and LEDUY Anh , (1989) Improvement of Ethanol Fermentation under Hyperbaric Conditions, Bioengin. 3 3 (1), 471-476

Biotechnol.

PASTA P., G. MAZZOLA, G. CARREA and S. RIVA, (1989) Subtilisin-catalysed transesterification in Supercritical Carbon Dioxide, Biotechnol. letters, 1 1 (9), 643-648 P E T E R S., BRUNNER G. and RIHA R., (1978) Economic Aspects of the Separation of Substances by Means of Compressed Gases in Countercurrent Processes Ger. Chem. Eng. 1, 26-30 Shimshick E . J . , (1981) Removal of organic acids from dilute aqueous solutios of salts of organic acids by supercritical fluids, U.S. Pat. No. 4,250,331. STEYLER D.C., P.S. MOULSON and J . REYNOLDS, (1991) Biotransformations in near-critical carbon dioxide. Enzyme Microb. Technol., 1 3 (3), 221-226 van E U S A.M.M., WOKKE J.M.P., TEN BRINK B . and DEKKER K.A., (1988) Downstream Processing of Fermentation broths with supercritical carbon dioxide International Symposium on Supercritical Fluids, October 17.- 19.1988, Mice, France. van E U S A.M.M., WOKKE J.M.P., TEN BRINK B. (1988) Supercritical extraction of fermentation products. Preconcentration and Drying of Food Materials, pp. 135-143, edited by S. Bruin Elsevier Science Publishers B.V., Amsterdam. WALLHAEUSSER K.H. (1988) Praxis der Sterilisation, Desinfektion, Konservierung, Elsevier Science Publishers B.V., Amsterdam.

p. 29, edited by S. Bruin

WONG J.M. and JOHNSTON K.P., (1986) Solubilisation of Biomolecules in Carbon Dioxide Based Supercritical Biotechnol. Progress, 2 (1), 29-39

Fluids

HANDBOOK OF PHYSICS AND CHEMISTRY, 6 4 ^ edition (1983-1984) Robert C. WEAST, Melvin J . ASTLE, William H. B E Y E R , CRC-Press, Inc. Boca Raton, Florida.

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

417

E N Z Y M A T I C R E A C T I O N IN O R G A N I C S O L V E N T S AND S U P E R C R I T I C A L G A S E S Xiao Mei Shen, Theo W . de Loos and Jakob de Swaan Arons Laboratory o f Applied Thermodynamics and Phase Equilibria, Department o f Chemical Technology and Materials Science, Delft University o f Technology, Julianalaan 136, 2628 B L Delft, The Netherlands. The enzymatic esterification o f glycidol and butyric acid, catalysed by porcine pancreatic lipase (ppl), has been performed in different organic solvents and compared with equilibrium conversions estimated from theory. Preliminary results show that the predicted equilibrium conversions were in good agreement with experiments for most o f the organic solvents tested. In different organic solvents the enzyme exhibited enantioselectivity . It appears that the more polar the organic solvent, the higher was the selectivity towards the R-isomer. Conversions were also predicted for supercritical solvents. The conversion in supercritical carbon dioxide was predicted to be greater than that in the organic solvents. Moreover, the conversion in carbon dioxide was predicted to be greater than that in supercritical ethane. In the phase behavior o f the glycidol and carbon dioxide binary two liquid phases coexisted near the critical point o f pure carbon dioxide. Taking into account the three phase (liquid-liquid-vapor) lines o f glycidol-carbon dioxide, the gas phase reaction conditions should be selected at temperatures above 3 1 0 Κ and pressures above 80 bar.

1.

Introduction.

Supercritical fluids (SCFs) can be used as reaction media. Chemical reactions in S C F s have been investigated for some years(l). The reason for this is the interesting physical properties o f S C F s . The transport properties o f S C F s are intermediate between those o f gases and liquids. The viscosities are about one order o f magnitude less than those o f liquids whilst the diffusion coefficients are typically one to two orders o f magnitude larger(l). Solubilities of solutes in the region o f the critical point are highly sensitive to changes in pressure and temperature due to large density changes. Thus, in the critical region , solubility changes o f orders o f magnitude can be obtained from relatively small changes in pressure or temperature(2). Biochemical reactions in supercritical fluids were demonstrated first in 1985(3). In the pioneering work o f Randolph, et. al. it was found that the enzyme, alkaline phosphatase, was active in supercritical carbon dioxide. Supercritical fluids offer several advantages for enzymatic reactions in addition to improved mass-transport and simplified separations. Because most of the more interesting supercritical solvents are non-polar, and do not dissolve enzymes, separation and recovery o f the catalyst after reaction is possible. The activity o f water, and thermodynamic equilibrium o f some enzymatic reactions, can be altered by changing the water content. The reduced water activity in S C F s can cause the reversal o f

418 hydrolytic reactions(4). Recently, two papers were presented concerning lipase catalyzed reactions of chiral compounds in supercritical carbon dioxide. Both papers demonstrated that in supercritical carbon dioxide the S(-)enantiomer was favoured over the R( + )isomer. No explanation for these observations was offered(5,6). Our purpose was to test whether supercritical fluids were good solvents for the production o f pure enantiomers. In this paper, our preliminary results o f experiments and calculations are presented. Different enantio-selectivities o f ppl in various solvent media were observed.

2.

Experimental

2.1

Experimental method for organic solvents The esterification was performed in each o f the organic solvents saturated with water at room temperature . Then 0.1 mol. glycidol, 0.1 mol. butyric acid and 5 g.crude ppl ( 2 0 % purity ) were added. The stirring speed o f the magnetic stirrer was the same in all experiments. Samples of the product were analyzed by gas chromatography using a capillary column. The ester was washed out o f the reaction mixture with water. The concentrations o f the enantiomers were determined with HPLC. 2.2

Experimental method for measuring phase behaviour For phase behaviour measurements a conventional cailletet-apparatus was used(7,8).

2.3

Procedure for supercrical esterification A high pressure flow reactor has been designed and built. It has capabilities for controlling water activities o f the reactants. The enzyme is immobilized on a glass substrate.

3.

Results o f preliminary experiments and calculations

3.1

Equilibrium esterification in different organic solvents The model reaction chosen was the esterification o f racemic pure glycidol (2,3-epoxyl1-propanol) with butyric acid, catalysed by a crude porcine pancreatic lipase purchased from Sigma. The reaction was carried out in dichloromethane, chloroform, tetrachloromethane and hexane. The liquid phase equilibrium constant was calculated from classical thermodynamics as follows:

RT In K th l

= - Δ Gf

AG f was estimated by the method o f Joback(9), from which l

K th

« 4.06

( 25° C, 1 atm.)

For the esterification, the thermodynamic equilibrium constant is given by:

419 V. ΊαΙ

X

X

w

V\v

e

X

Ί ac

al

X

a

Where y ' s are activity coefficients, and x ' s are mole fractions. If a is the equilibrium conversion for the model reaction, then the quotient: 1 ( 1 - α

f

( or

1

- 1

Ϋ

from which α can be calculated.

[

Ν

+1 r

1

Activity coefficients were estimated using UNIFAC. Calculated conversions were compared with the experimental results. Except for chloroform, the predicted equilibrium conversions were in good agreement with experiments ( F i g . l ) . The discrepancy in the case o f chloroform is unexplained.

Ο

20

40

60

80

100

Predicted Conversion (%)

Fig.l 3.2

Predicted conversions compared with experimental results-organic solvents.

Enantioselectivity in different organic solvents. Using a reverse phase HPLC column enantioselectivities were also measured. The results, shown in Fig.2, suggest that the more polar the solvent, the higher the initial reaction rate. The glycidyl butyrate was separated and enantiomeric excess (ee) was analyzed by HPLC.

420

ee The ee value was over 9 0 % in water saturated dichloromethane, 65 % in chloroform and a little less in tetrochloromethane. The same tendency is shown in Fig.3: the more polar the solvent, the higher the selectivity towards the R-isomer.

Fig.2 Initial reaction rate in different organic solvents

Fig.3 Enantiomeric excesses in different organic solvents

421 3.3

Prediction o f conversion in supercritical fluids. Chemical conversion was estimated from the equilibrium relationship given previously

and rearranged to give: Οφ

*

Qy * Qp Qe-

Where:

(for the model reaction) =

Op*

6.7

( 298.15 Κ )

Q y was determined from the phase equilibrium relationship applied to each component Yi

Q

*

t * ί*

x

0

= Φι * y * ?

ρ

β

-

Ρ Χ

t

(

}

RTL

The second virial coefficients were estimated by the use o f a four-parameter intermolecular potential function for polar fluids ( 1 0 ) . At 50°C and 10 MPa, the estimated equilibrium conversion was 9 2 % in supercritical carbon dioxide. Under the same conditions in supercritical ethane, the estimated conversion was only 57 % . It seems that it may be preferable to perform this reaction in carbon dioxide. 3.4

Selection o f gas-phase reaction conditions.

9 Vapor curve of C O e

3 Φ ω OL

θ

6 Critical point of glycidol

5 290

300

310

T e m p e r a t u r e

540

550

(K)

Fig.4 Three phase line o f glycidol- C 0 2 (x = 0.0764)

422

In order to select appropriate gas phase reaction conditions, the solubilities o f glycidol in supercritical carbon dioxide were measured for selected concentrations. The three phase line (liquid-liquid-vapor) o f glycidol-carbon dioxide was measured and is given in Fig. 4 . It can be seen mat the L L V critical end point was above the critical point o f pure carbon dioxide. Therefore, if we wish to work in a homogenous phase, the reaction conditions should be chosen at temperatures above 310 K, and pressures above 80 bar.

4.

Conclusions.

4.1 Prediction and estimation of enzymatic reaction equilibrium conversion in different organic solvents can be very useful. It can be used to choose optimum solvent environment and reduce the number of experiments to be performed. 4.2 Calculations indicate that to perform this model reaction supercritical carbon dioxide might be preferable over ethane. 4.3 Based on the phase behavior o f glycidol-carbon dioxide system, preliminary gas phase reaction conditions were established.

Acknowledgments This work has been supported by a special financial grant o f Delft University o f Technology. We thank Mr. H. Gaebel and Dr. G . T . Wilkinson in assisting us with phase behaviour measurements. We also thank Drs. J . B . A . van Toi, J . A . Jongejan and J . A . Duine for their co-operation and helpful discussions.

Nomenclature By Er ES Gf Κ Ρ Q Q P8 QP * QX QY QY Q* R Τ χ y Ζ

second virial coefficient peak area o f HPLC o f R-form ester peak area o f HPLC o f S-form ester Gibbs energy o f formation (Kcal mol ') chemical reaction equilibrium constant pressure (MPa) quotient quotient o f partial pressures quotient o f saturated pressures quotient o f mole fractions in liquid phase quotient o f mole fractions in gas phase quotient o f activity coefficients quotient o f fugacity coefficients 2 1 1 gas constant (cm MPa m o l Κ ) temperature (Κ) mole fraction in liquid phase mole fraction in gas phase compressibility factor

423

Greek Letters α γ Δ Φ

equilibrium conversion for model reaction activity coefficient change in a property fugacity coefficient

Superscripts and Subscripts a al e i

acid (butyric acid) alcohol (glycidol) ester (glycidyl butyrate) substrates and products o f model reaction

j

co

1 s sat th w

liquid solvent saturation thermodynamic water

2

References 1. 2. 3. 4 5.

6. 7. 8. 9. 10.

B . Subramanlam and M . A. McHugh, Ind. Eng. Chem. Process Des. Dev., 25 ( 1986) T . W . Randolph, H.W. Blanch, J . M . Prausnitz and C R . Wilke, Biotechnology Letters, 7-5 (1985) 3 2 5 . D.A. Miller, H.W. Blanch and T . M . Prausnitz, Ind. Eng. Chem.Res., 3 0 (1991) 9 3 9 . O. Aaltonen and M. Rantakylä, ChemTech, April (1991) 2 4 0 . J . F . Martins, S . F . Barreiros, E . G . Azevedo and M.N. da Ponte, Proc. o f 2nd Int. Sym. on Supercritical Fluids, pp. 406-407, 2 0 - 2 2 , May, 1991, Boston, Johns Hopkins Univ. O. Aaltonen and M . Rantakylä, Proc. o f 2nd Int. Sym. on Supercritical Fluids,pp. 146-149, 2 0 - 2 2 , May, 1 9 9 1 , Boston, Johns Hopkins Univ. H.J. van der Kooi, Ph.D. Thesis, 1 9 8 1 , Delft. T h . W . de Loos, H . J . van der Kooi, W. Poot and P . L . Ott, Delft, Progr. Rep., 8 (1983) 2 0 0 . R . C . Reid, J . M . Pransnitz and B . E . Poling, The Properties o f Gases and Liquids, pp. 154-156, 4 Edn (1986), McGraw-Hill Book Company. N . J . Marls and L . I . Stiel, Ind. Eng. Chem. Process Des. Dev., 2 4 (1985) 183.

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

425

FATTY ACID ESTERIFICATION IN SUPERCRITICAL CARBON DIOXIDE

Alain Marty, Didier Combes, Jean-Stéphane Condoret Institut National des Sciences Appliquées, Département de Génie Biochimique et Alimentaire, Complexe Scientifique de Rangueil, 31077 Toulouse FRANCE.

Abstract Enzymatic synthesis of ethyloleate, from oleic acid and ethanol, catalyzed by an immobilized lipase from Mucor miehei, has been performed in supercritical carbon dioxide (SCCO2) and in n-hexane. Batch studies of initial velocities have allowed the investigation of protein stability, influence of water content and the reactional mechanism. A continuous packed bed reactor has been modelized, after generalization of the initial velocity equation, using a plug-flow reactor model. Finally, the complete reaction-separation process with recycling of the solvent has been examined in a small pilot plant with four post reactional separators.

1. INTRODUCTION Numerous reports (1-3) have already shown the advantages of using organic solvents or low water content media to operate enzymatic reactions. Indeed, these media allow good stability of enzymes, increased solubility of hydrophobic compounds, and recovery of enzymes when they are, as is generally the case, insoluble in these media. Furthermore, in the case of hydrolytic enzymes, the reaction may be reversed, because thermodynamic equilibrium is shifted, due to the poor water content. Another kind of non aqueous solvent has been considered for several years : the supercritical fluid. These media were initially used for liquid-fluid and solidfluid extraction processes and exhibited good efficiency, due to favourable transport properties. Their main advantage lies however in the great variability of their solving power as a function of temperature and pressure, leading to easy recovery of extracted products. Supercritical carbon dioxide (SCCO2) has been intensively used because of its relatively low critical pressure (7.3 MPa), its non dangerous character and its low cost. Its low critical temperature (31°C) prevents thermal denaturation when processing biological compounds, and furthermore its non toxic nature is greatly appreciated in the food and health industry. All these features make it suitable

426

for operating enzymatic catalysis. Besides the already quoted advantages for recovery of extracted products, the fact that CO2 is in gaseous state at atmospheric pressure avoids problems of dilution in a solvent. Since the first works of Nakamura et al. (4) in 1985, feasibility of enzymatic reactions in SCCO2 has been demonstrated for various synthesis reactions such as interesterifications (Nakamura et αΖ.(1985) (4), Chi et αΖ.(1988) (5), and Erickson et α/.(1990) (6)), transesterifications (van Eijs et αΖ.(1988) (7), Pasta et αΖ.(1989) (8), and Kamat et α/.(1992) (9)), esterifications (Marty et αΖ.(1991-1992) (10-11)) and oxidations (Randolph et αΖ.(1988) (12) and Hammond et al. (1985) (13)). Two well documented reviews (14-15) have been published on the subject. In these studies, stability of enzymes in SCCO2 has been proved to be good. In a specific study, Taniguchi et al. (1987) (16) have examined the stability of 9 commercial enzymes in SCCO2. As for organic solvents, water content has a negative influence on enzyme stability. The influence of the depressurization step has also been considered, and particularly in the case of enzymes lacking the S-S bridges (which contibute to protein stability), for which some negative effects have been detected (Kasche et al. (1988) (17)). In addition to denaturation effects, water content also plays an important role in the catalytic activity of the enzyme, because it is quite closely related to the hydration of the protein. Water content is also involved in the global kinetics through the reverse hydrolytic reaction. The direct influence of pressure on the kinetics has not yet been looked at. Nevertheless, solubility of reactants, which improves as the pressure increases (5) is a factor which improves the productivity. Comparisons between SCCO2 and organic solvents (5,8,9,13) seem to indicate that the reaction rates are faster in SCCO2, but all conditions were not clearly enough defined to validate these conclusions. A mechanistic determination, as described in this paper will provide a better basis for comparison. Our work has dealt with feasibility, kinetic study and extension to continuous reaction-separation process and has employed a model reaction for the esterification of a fatty acid. The synthesis of ethyloleate, from oleic acid and ethanol, catalyzed by an immobilized lipase from Mucor miehei (Lipozyme™ from Novo Industri) has been chosen. Batch studies of initial velocities in SCCO2 and n-hexane have allowed protein stability, influence of water content and reactional mechanism to be investigated. A generalized equation has been derived and used to modelize a continuous packed bed reactor. Finally, the complete reaction-separation process with recycling of the solvent has been undertaken on a small pilot plant with four post reactional separators.

427

2. MATERIALS AND METHODS 2.1. Experimental apparatus T h e e x p e r i m e n t a l a p p a r a t u s for t h e b a t c h k i n e t i c s t u d i e s h a s b e e n d e s c r i b e d in o u r p r e v i o u s w o r k s ( 1 0 - 1 1 ) . T h e pilot p l a n t for c o n t i n u o u s r e a c t i o n h a s b e e n built by S e p a r e x Company, Nancy, F r a n c e (Figure 1). I n t h i s s y s t e m c a r b o n dioxide is pumped a t -5 °C a n d p r e s s u r i z e d t o a b o u t 1 5 M P a and 4 0 ° C . It percolates the packed bed of enzyme. At the output of the r e a c t o r , a s a m p l e loop o f 0 . 6 3 m L allows a s s a y i n g t h e effluent m i x t u r e . A c a s c a d e of four s e p a r a t o r s i s following, w h e r e t h e p r e s s u r e t u n i n g in e a c h v e s s e l i s obtained by needle valves.

-1-Liquid C 0 2 tank. - 2- Heater. -3- High pressure pump. -4- HPLC pump (substrates input). -5- Cooler. -6- Reaction vessel. -7- Separators. -8- Needle valves -9- Heater. -10- Mass flow meter. -X- Temperature and pressure measurements and security valves.

F i g u r e 1 : P i l o t p l a n t for c o n t i n u o u s r e a c t i o n - s e p a r a t i o n p r o c e s s .

3. RESULTS OF THE BATCH STUDY IN AGITATED VESSEL 3.1. Stability I t h a s b e e n e s t a b l i s h e d t h a t t h e i m m o b i l i z e d l i p a s e e x h i b i t e d good s t a b i l i t y , s i m i l a r in b o t h s o l v e n t s ( 1 0 - 1 1 ) . A s m a l l decay o f activity, o f a b o u t 1 0 % , h a s b e e n d e t e c t e d a f t e r 6 d a y s o f i n c u b a t i o n a t p r e s s u r e from 1 3 t o 1 8 M P a a n d a t 4 0 ° C . I n b o t h s o l v e n t s , w a t e r proved t o b e a d e n a t u r i n g factor. A d d i t i o n a l i n f o r m a t i o n h a s b e e n d r a w n from c o n t i n u o u s e x p e r i m e n t s i n p a c k e d b e d s w h e r e s t a b i l i t y i n SCCO2 h a s b e e n confirmed w h e r e a s , in n - h e x a n e , a m e c h a n i c a l d e g r a d a t i o n o f t h e s u p p o r t , l e a d i n g t o a l e s s a c t i v e powder, h a s b e e n o b s e r v e d after two or t h r e e hours of percolation.

428

3.2. Influence of water content Two effects on reaction rates have been demonstrated: an influence on kinetic constants and hydration of the protein, the latter probably being the most important. Water solubility is greater in S C C O 2 (a few g/L, depending on pressure and temperature) than in n-hexane (a few tenths of g/L). The effective parameter to take into account is therefore not the water added to the heterogeneous system but the water content of the solid resulting from the partition of water between solvent and solid. This partition may be described by isotherms of adsorption of water (11), that in the case of S C C O 2 are strongly dependent on pressure, temperature and the ethanol content that retains water. It has been shown (11) that the optimum water content, referred to the solid water content, was the same in both solvents, with a value of about 10% g/g of dry solid support.

3.3. Reactional mechanism and kinetic constants It has been demonstrated (11-18) from initial velocity studies, operated at optimum water content in both media, that the mechanism was a Ping Pong Bi Bi pattern with inhibition by ethanol, whose equation is : v

=

V"UOlJ[Eth] Km,ai [Eth](l + [Eth]/Ki) + Km,Bh, [Ol] + [OI][Eth]

)

with [01] and [Eth] the initial oleic and ethanol concentration, Km( 0 i) and Km( e th) their affinity constants, Ki the inhibition constant of ethanol, V and Virigynt the initial and the initial and maximum velocities of the reaction. Kinetic constants were numerically determined by parametric identification and, although their values differed in S C C O 2 and hexane, kinetics were globally in the same range and no clear advantage may be found. Numerical values and some attempts for explaining their differences may be found in reference 11.

4. STUDY OF THE CONTINUOUS REACTION 4.1. Determination of a general equation The equation (1) is only valid for initial velocities, when reaction products do not interfere. To describe a continuous reactor, a more complete equation is needed, involving the affinity and inhibition constants of products of the reaction m a na (Km(ethol) ^ (H20))» the maximum velocity of the reverse hydrolysis a n ( (Vm(hyd)) * the equilibrium constant (Keq). This general equation is derived from King and Altman method (19) and is written as :

( 1

429

Vmsynl([OI][Eth]-[H20][Ethol]/Keq)

v

D

(2)

with,

D = Km,a, [Eth](l + [Bh]/W + [Ethol]/KJiB,dl ) + Km.., [Ol](l + [H20]/W, H 2 0, ) + [Ol][Eth] + Vm.Km,H20, [H20] (1 + [Eth]/Ki)/(Vm,h)a,Keq) + VmKm,H20, [Ethol]/(Vm,hidlKeq) + VmjH20][Ethol]/(Vm, h > d,Keq)

S o m e specific e x p e r i m e n t s h a v e allowed u s t o a s s e r t t h a t e t h y l o l e a t e h a s no i n h i b i t i n g effects. T h e hydrolysis o f e t h y l o l e a t e h a s b e e n o p e r a t e d i n S C C O 2 a n d a v a l u e o f K e q o f a b o u t 7.2 h a s b e e n o b t a i n e d . T h e w a t e r c o n t e n t a c t e d s i m u l t a n e o u s l y on t h e k i n e t i c c o n s t a n t s a n d b y t h e h y d r a t i o n effect. W e h a v e simplified t h e m o d e l b y i g n o r i n g i t s k i n e t i c s a n d c o n s i d e r i n g t h a t t h e h y d r a t i o n effect w a s p r e d o m i n a n t . A simplified e q u a t i o n m a y t h u s b e w r i t t e n a s :

_

v

Vrn^, ([Q][Bh] -[H20][Bhol]/[Keq3) Km^lEthJO+tEth^+Km^^+talEth]

)

B a t c h e x p e r i m e n t s u p to e q u i l i b r i u m h a v e b e e n p e r f o r m e d a n d h a v e b e e n m o d e l i z e d b y a s e t o f differential e q u a t i o n s . T h e n u m e r i c a l r e s o l u t i o n b y R u n g e K u t t a m e t h o d h a s led t o t h e c o n c e n t r a t i o n o f e t h y l o l e a t e v e r s u s t i m e . F i g u r e 2 s h o w s t h e s e r e s u l t s a n d t h e corresponding e x p e r i m e n t a l d a t a i n t h e c a s e o f S C C O 2 . A good a g r e e m e n t h a s b e e n obtained.

I

12

I

I

I

I

I

C02SC

-

•

EXPERIMENT MODELIZATION

• ~

ENZYME : 44.4 MG — OLEIC ACID :12.17 MM ETHANOL : 150 MM ~ CO

I 50

I 100

1 150

1 200

1 250

Time ( m n )

1 300

350

EXPERIMENT MODELIZATION

ENZYME : 10.2 MG OLEIC ACID : 15.3 MMETHANOL : 93.9 MM I I 100

150

200

Time ( m n )

F i g u r e s 2 a n d 3: E s t e r s y n t h e s i s v e r s u s t i m e in S C C O 2 ( 1 3 M P a , 4 0 ° C ) a n d i n n-hexane (40°C): experiments and modelization

( 3

430

Figure 3 is related to n-hexane experiments and its modelization. In this case, because no hydrolysis was observed, the general equation reduces to the initial velocity equation.

4.2. Continuous reaction in fixed bed A tubular fixed bed of immobilized enzyme has been operated with S C C O 2 (40°C, 15 MPa) and with n-hexane (40°C). In each case the amount of water introduced with the feed has been calculated in order to maintain the solid at its optimum water content of 10%. Ethanol concentration has also been taken in each case at its optimum value, according to the kinetics. The flow rate was varied to determine the effect of residence time on the conversion rate. The modeling of the reactor was performed using a very simple plug-flow reactor model written as : dX _ V

M

~Q.[Ol]0

)

with X=([01]o-[01])/[01]o, the conversion, M the mass of catalyst, Q the flow rate and [01]Q the initial oleic acid concentration. The integration has been numerically done by Simpson's method. In the case of S C C O 2 , this model gave good results (Figure 4) but appears to slightly overestimate results in hexane (Figure 5), due to eventual diffusional limitations. This very simple model is an efficient tool to model this fixed bed reactor, provided adequate kinetic information is used

Figure 4 and 5 : Conversion of ethyl oleate versus residence time in the reactor in S C C 0 2 (15 MPa, 40°C) and in n-hexane (40°C).

431

5. POST REACTIONAL SEPARATION The post reactional separation was obtained by operating a cascade of depressurizations, (possibly at different temperatures) in order to recover the products and the non-reacted substrates with the greatest selectivity and yield. At the output of the last separator C O 2 was recycled. The operating conditions (flow rates and quantity of enzyme) were fixed so as to obtain a 60% conversion (a nearly 100% conversion would have eliminated the problem of oleic acid-ester separation). The tuning of the separation is not straightforward, and results vary very sharply with small changes in the set of pressures and temperatures. Nevertheless, an empirical approach has allowed us to optimize our system and an example of "good" separation is given in table 1 Table 1: Selective post reactional separation of oleic acid, ethyl oleate, ethanol and water. Output of the reactor : oleic acid: 2.4g/L; ethyl oleate: 3.7g/L of C O 2 (P: 15 MPa, T: 40°C, flow rate: 40mL/min, duration: 185 min). SEP1 10.2 MPa 18.5 mL Oleic acid (g/L) Ethyl oleate (g/L) Ethanol (g/L) Water (g/L) Ester purity (% Mol.) Ester purity (% Mass.) Ester recovery (%)

605 138 2.1 2.8 15.8 18.4 10.2

SEP2 8.1 MPa 9.9 mL 148 741 11 5.9 68.7 81.8 29.3

SEP3 7.2 MPa 17.5 m L 99 790 15.5 4.4 73.1 86.9 55.1

SEP4 5.2 MPa 2.8 m L 56 489 91 327 7.2 50.4 5.4

The global mass balance on the separator cascade was incorrect but this can be attributed to incomplete recovery of the liquid phase in the separators (i.e, the retention in the separator had not reached steady state). For these reasons, the yields were calculated in respect to the total quantity effectively recovered. These results present a good selectivity in the separation. Indeed, 77% of oleic acid is recovered in Sep 1, and by mixing effluents of Sep.2 and Sep.3, 84.6% of the ester is recovered. Its purity is about 85% in mass (with oleic acid: 13%; ethanol: 1.5% and water: 0.5%). As a comparison, when operating the same reaction with the same conversion in n-hexane, an ester concentration in the effluent of 3.7 g/L would be obtained, leading after complete elimination of the solvent to an ester concentration of only 378 g/L with a purity of 43 % in mass (or 14% molaire). These results emphasize the interest of using the fractionation potential of the S C C O 2 . The preceding results have been obtained with four separators, but if

432

the product separation was a crucial point of the process, an increase of the number of separators would improve greatly the results. Since no clear advantage of S C C O 2 as a reaction medium has been demonstrated, this technology applied to enzymatic conversion, will be of interest only when considering the coupled reaction-separation processes.

References : 1 A.Zaks and A.M.Klibanov, The Journal of Biological Chemistry, 263, 1988, 8017 2 C.Laane, S.Boeren, R.Hilhorst and C.Veeger, Biocatalysis in Organic Media, Proceedings of Wageningen Symposium (Elsevier Publishers), 1987, 65 3 B.Hahn-Hägerdal, Enzyme Microb. Technol, 8, 1986, 322 4 K.Nakamura, Y.M.Chi and Y. Yamada, Chem. Eng. Commun., 45, 1985, 207 5 Y.M.Chi and K.Nakamura, Agric. Eng. Chem., 52, 1988, 1541 6 J.C.Erickson, P.Schyns and C L . Cooney, AICHE, 36, 1990, 299 7 A.M.M.van Eijs, J.P.L.de Jong, H.J.Doddema and D.R. Lindeboom, Proceedings of the International Symposium on Supercritical Fluids (Nice, France), 1988, 933 8 P.Pasta, G.Mazzola, G.Carrea and S.Riva, Biotechnol. Lett., 2, 1989, 643 9 S.Kamat, J.Burrera, E.J.Beckman and A.J.Russell, Biotechnol. Bioeng., 1992, in press 10 A.Marty, W.Chulalaksananukul, J.S.Condoret, R.M. Willemot and G.Durand, Biotechnol. Lett.,12, 1990, 11 11 A.Marty, W.Chulalaksananukul, R.M. Willemot and J.S.Condoret, Biotechnol. Bioeng.,39, 1992, 273 12 T.W.Randolph, D.S.Clark, H.W.Blanch and J.N.Prausnitz, Science, 1988, 387 13 D.A.Hammond, M.Karel and V.J.Krukonis, Appl. Biochem. Biotechnol., 1985, 11, 393 14 K. Nakamura, Tibtech, 8, 1990, 288 15 Ο .Aaltonen, Chemtech, april 1991, 240 16 M.Taniguchi, M.Kamihira and T.Kobayashi, Agric. Biol. Chem, 51, 1987, 593 17 V.Kasche, R.Schlothauer and G.Brunner, Biotechnol. Lett., 10, 1988, 569 18 W.Chulalaksananukul, J.S.Condoret, P.Delorme and R.M.Willemot, F E B S Letters, 276, 1990, 181 19 E.L.King and C.Altman, J . Phys. Chem., 60, 1956, 1375

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

435

Influence of organic solvents on the specificity of a chymotrypsin and subtilisin from B. subtilis strain 7 2 in acyl transfer reactions M.Yu. Gololobova, T.L. Voyushinab and P. Adlercreutz* a

D e p a r t m e n t of Biotechnology, Chemical Center, University of Lund, P.O. B o x 1 2 4 , 2 2 1 0 0 Lund, Sweden

^Institute for Genetics and Selection of Industrial Microorganisms, 1-st Dorozhny 1, Moscow 1 1 3 5 4 5 , Russia

Abstract

Nucleophilic properties of amino acid amides were studied systematically in acyl-transfer reactions catalyzed by a-chymotrypsin and subtilisin from Bacillus subtilis strain 7 2 (subtilisin 72) using Mal-L-Ala-LAla-L-PheOMe and Bz-L-TyrOMe a s the acyl g r o u p d o n o r s . In a chymotrypsin-catalyzed reactions in water the reactivity of the amino acid amides increases with hydrophobicity of the nucleophiles. Hydrophobic interactions in this case are responsible for the differences between the reactivity of the nucleophiles for amides of all the amino acids tested with the exception of D-AlaNH2 and L-ArgNH2. In a low water system (4% of H 2 O , 4 7 . 5 % of acetonitrile, 4 7 . 5 % of dimethyl formamide, 1% of ( C 2 H 5 ) 3 N ) t h e specificity of a - c h y m o t r y p s i n towards the amino acid amides in acyl transfer reactions decreases and does not depend on the amino acid side c h a i n hydrophobicity. The specificity in this c a s e correlates with the bulk c h a r a c t e r i s t i c s of the amino acid side chains (normalized van der Waals volume, polarizability, molecular weight). The bulky amino acid amides are less efficient nucleophiles. In reactions catalyzed by subtilisin 7 2 , amino acid side chain characteristics do not correlate with the nucleophile reactivities. The data obtained show that different factors m a y be responsible for the specificity of enzymes in water and in low water s y s t e m s and in general, specificity p a t t e r n s obtained in water can not be used for low water systems.

1· INTRODUCTION Acyl transfer reactions catalyzed by chymotrypsin and different subtilisins are widely used in mechanistic studies and in preparative synthesis (see [1-4] and references therein). However, there is a n evident lack of systematic d a t a related to the specificity of nucleophiles in these

436 reactions. The medium effects have to our knowledge not been studied in this case. In this paper we report systematic d a t a on the reactivity of different amino acid amides in acyl transfer r e a c t i o n s catalyzed by chymotrypsin (α-form of bovine chymotrypsin, E C 3 . 4 . 2 1 . 1 was used) and subtilisin 7 2 . F o r in-depth understanding of the factors influencing nucleophile reactivities we have attempted to correlate our d a t a with different c h a r a c t e r i s t i c s of amino acid side c h a i n s available in the literature [5].

2. RESULTS AND DISCUSSION 2.1. Choice of the kinetic model and the acyl group donors

Under the experimental condition, hydrolysis of t h e peptide p r o d u c t s w a s not detected. Therefore, the simplest s c h e m e of the reactions discussed is as follows:

"

ΕΑΝ

"4

•

E + P

where Ε is the enzyme, S is the donor (BzTyrOMe or MalAlaAlaPheOMe) of the acyl moiety (BzTyr or MalAlaAlaPhe) to be transferred to the added nucleophile Ν (amino acid amide); E S is the complex of the enzyme with S; Ρ d e n o t e s t h e peptide s y n t h e t i c p r o d u c t ( B z T y r X a a N H 2 or MalAlaAlaPheXaaNH2), EA represents the acylenzyme intermediate, ΕΑΝ is the complex of EA with the added nucleophile, Pi is methanol and P2 is BzTyrOH or MalAlaAlaPheOH. The meaning of the constants follows from the scheme. If not otherwise stated, amino acid residues are of the reconfiguration. In organic solvents, the synthesis from MalAlaAlaPheOMe results in the formation of MalAlaAlaPheXaaNH2 and a number of other unknown compounds. The analysis of these products is now in progress. In water, the acyl transfer of the BzTyr-moiety to some amino acid amides proceeds in a more complex manner than that described above. B e c a u s e of that, MalAlaAlaPheOMe was used for the analysis of the reactivity of the

437

amino acid amides in water, and d a t a for low w a t e r s y s t e m s were obtained using BzTyrOMe. The height of the energy b a r r i e r between t h e a c y l e n z y m e i n t e r m e d i a t e and the s y n t h e t i c p r o d u c t (P) is a m e a s u r e of the nucleophile specificity of the enzyme. This height is proportional to 1^4/K n. To calculate this quantity from the experimental d a t a a t different water content, the activity of water m u s t be m e a s u r e d . To avoid this problem, all d a t a were normalized to the reactivity of GlyNH^.

2.2. Water systems

Plotting the data obtained shows that good correlation exists in the case of the chymotrypsin-catalyzed reaction. The logarithm of the relative nucleophile reactivities correlates with the hydrophobicity of amino acid

water

2

L

0 μ

hydrophobicity

The numbers denote the following amino acid amides: 1. Arginine amide 2. D-alanine amide 3 . Glycine amide 4. Alanine amide 5. Histidine amide 6. Threonine amide 7. Tyrosine amide 8. Tryptophane amide 9. Valine amide 10. Leucine amide 11. Methionine amide 12. Norvaline amide 13. Isoleucine amide 14. Phenylalanine amide

Figure 1. Correlation between hydrophobicity [5] and the n a t u r a l logarithm of normalized nucleophile reactivity for acyl-transfer reactions in water ( R w at e r ) catalyzed by chymotrypsin. Conditions: pH 9 . 0 (0.1 M veronal), 30°C, 1% (v/v) of DMSO. side chains [5] for all c a s e s except acyl-transfer to ArgNH2 and DA l a N Ü 2 (Fig. 1). Moreover, experimental points mainly lay around the straight line with a unit slope. The points for TyrNÜ2 and TrpNH 2 lay a little apart from this line but deviations are not significant. Therefore, the data obtained shows that hydrophobic interactions are mainly responsible for the differences between the reactivity of the different nucleophiles in chymotrypsin-catalyzed reactions for practically all amino acid amides. The higher reactivity of ArgNH 2 is to all appearance the result of ionic interactions between two aspartic acid residues (Asp-64 and Asp-35) and the positively c h a r g e d side c h a i n of the arginine nucleophile [2].

438 Incorrect orientation of a methyl group in the binding of MalAlaAlaPhechymotrypsin with D-AlaNH2 is probably the reason for the low reactivity of the latter compound. No correlation between amino acid side c h a i n p a r a m e t e r s and nucleophile reactivities were found for acyl-transfer reactions catalyzed by subtilisin 7 2 . We only b e c a m e firmly convinced of the negative influence of the size of the substituent of the nucleophile molecule on transfer reactions (Fig. 2 ) . The high reactivity of D-AlaNÜ2 (even higher than AlaNÜ2) affirms the low selectivity of the S'i-subsite [6] of subtilisin 72.

2.3. Some comments on amino acid side chain parameters

Numerous amino acid side chain p a r a m e t e r s are described in the literature [5], E a c h of them can be used in QSAR studies. Some comments on the parameters used in this work are given below. The hydrophobicity of the side chain (π) reflects the ability of the enzyme active site to extract the given amino acid residue from water.

>

3

water 2

-

:·

15

4

9

11

n

12· 13

•

6

8

•

•

1

•

• 10 I

1

The numbers denote the following amino acid amides: 1. Arginine amide 2. D-Alanine amide 3. Glycine amide 4. Alanine amide 6. Threonine amide 8. Tryptophane amide 9. Valine amide 10. Leucine amide 11. Methionine amide 12. Norvaline amide 13. Isoleucine amide 15. Serine amide

normalized van der Waals volume Figure 2. Correlation between the normalized van der Waals volume and the n a t u r a l logarithm of the normalized nucleophile reactivity for reactions catalyzed by subtilisin 7 2 in water (R Water)- Conditions: pH 9 . 0 (0.1 M veronal), 30°C, 1% (v/v) of DMSO. The values used in this work were determined [5] according to the following equation: π = logP(Ac-amino acid amide) - logP(Ac-glycine amide), where Ρ is the partition coefficient of the compound in the wateroctanol two-phase system. Therefore, Fig. 1 shows t h a t (i) the properties of the S'i-subsite of chymotrypsin in many respects are similar to those of octanol and, (ii) the differences of the nucleophile reactivity in

439

chymotrypsin-catalyzed reactions in water are mainly the result of the extraction of the nucleophile molecule by this subsite. The normalized van der Waals volume (iv) is a bulk parameter of the amino acid side chain and it is calculated according to the following equation iv = [V(side chain) - V(H)]/V(CH 2 ), where V is the van der Waals volume. F r o m this, iv = 1 for the side chain of alanine and it increases by one unit for each CH2-group. The polarizability (a) (and m o l a r refractivity, which is closely related to a) is another popular bulk parameter [7,8]. However, for amino acid amides it depends mainly on the molecular weight of the substituent b e c a u s e other quantities used in the calculation (index of refraction, density) c h a n g e for these c o m p o u n d s to a smaller e x t e n t t h a n the molecular weight. This fact results in a high correlation between iv. α and molecular weight of amino acid side chains (Fig. 3 and 4 ) . Therefore, the choice of the bulk parameter for a correlation study is mainly a matter of convenience in this c a s e . We used the normalized van der Waals volume b e c a u s e in o u r opinion it reflects better the size of the amino acid residues.

Figure 3 and 4. Correlation between different amino acid p a r a m e t e r s . The straight lines were drawn using the l e a s t - s q u a r e s method. D a t a were taken from the literature [5].

2.4. Low water systems The specificity patterns in low water systems (4% of H2O, 4 7 . 5 % of acetonitrile, 4 7 . 5 % of dimethyl formamide, 1% of (C2Hs)N) the enzyme

440

adsorbed on Celite) and water are completely different (Fig. 5). In water,

D-Ala Gly Ala Thr His Val Leu Met Nva Phe He Trp Tyr Arg Figure 5. Influence of the replacement of organic solvent for water on specificity p a t t e r s of the enzyme with r e s p e c t to the nucleophiles (amides of the listed amino acids) in chymotrypsin-catalyzed acyl-transfer reactions. amides of all the L-amino acids tested show higher reactivity t h a n GlyNH2. In organic solvent, GlyNÜ2 is one of the best nucleophiles. The difference between the reactivity of the amino acid amides (except TrpNH2) in low water system decreases a s compared to water. There is a poor correlation between the nucleophile reactivity of the amino acid amides in low water systems with both its reactivity in water (Fig. 6) as well a s the amino acid side chain hydrophobicity (Fig. 7) but there is a good correlation between the reactivity of different nucleophiles and the normalized van der Waals volume (Fig. 8). D-Alanine amide and arginine amide a r e the exceptions both in w a t e r and organic s y s t e m s . The probable reasons were considered above.

3· CONCLUSIONS This work is a n a t t e m p t toward s y s t e m a t i c study of QSAR of nucleophiles p a r t i c i p a t i n g in acyl t r a n s f e r r e a c t i o n c a t a l y z e d by proteolytic enzymes in water and low water systems. The results show the striking difference between factors influencing the nucleophile specificity in water and in the organic solvent. In water, the specificity of the S'i-subsite of chymotrypsin is mainly the result of hydrophobic interactions between amino acid side chain and this subsite. The size of the nucleophile molecule is not very important for reactivity. In organic solvent, the size of the amino acid residue is the m o s t important parameter. In reactions catalyzed by subtilisin 7 2 , no definite correlation between the reactivity of the nucleophiles and amino acid side chain parameters was found.

441

'org

• 4

0.0

3 · 6

-1.0 L

9 • 12 11 · » · 1

•

•

5

#

3 •

9

β·

•

•7

2

Ο

>

· 12

11 · » • 13 • 10 5 • 14 7

3

10 • 14

2

-2.0

• 4

1 Ο

1°

#

Ο

ÇFlg.6 ) 8

8

-3.0

ι

-0.5

I

•

1.0

•

-0.5

2.5

4.0

1.0

hydrophobicity

water

0.0

•

8.0

The numbers in Figs. 6 - 8 denote the following amino acid amides: 1. Arginine amide 2. D-Alanine amide 3. Glycine amide 4. Alanine amide 5. Histidine amide 6. Threonine amide 7. Tyrosine amide 8. Tryptophane amide 9. Valine amide 10. Leucine amide 11. Methionine amide 12. Norvaline amide 13. Isoleucine amide 14. Phenylalanine amide

normalized van der Waals volume Figures 6, 7, 8. Correlation between the natural logarithm of normalized nucleophile reactivity of amino acid amides in low water system ( R o rg ) and the n a t u r a l logarithm of normalized nucleophile reactivity of amino acid amides in water ( R Wa t e r ) (Fig. 6 ) , hydrophobicity (Fig. 7) and the normalized van der W a a l s volume (Fig. 8) for chymotrypsin-catalyzed reactions.

442

4. ACKNOWLEDGEMENTS W e w i s h to t h a n k Prof. V . M . S t e p a n o v f o r h i s e n c o u r a g e m e n t , L P . M o r o z o v a for p u r i f i c a t i o n of s u b t i l i s i n 7 2 , E.Yu. Terent'eva for h e r help i n s y n t h e t i c w o r k a n d the S w e d i s h I n s t i t u t e for the f i n a n c i a l s u p p o r t of M.Yu. Gololobov.

5· REFERENCES 1. M . Y u . G o l o l o b o v , L P . M o r o z o v a , T . L . V o y u s h i n a , E.A. T i m o k h i n a a n d V . M . Stepanov, B i o c h i m . B i o p h y s . A c t a , 1118 (1991) 2 6 7 . 2. V. Schellenberger, U. Schellenberger, Yu.V. M i t i n a n d H.-D. J a k u b k e , E u r . J . B i o c h e m . , 187 (1990) 163. 3. T.L. V o y u s h i n a , E.Yu. Terent'eva, V . F . Pozdnev, A . V . G a i d a , M . Y u . G o l o l o b o v , L.A. L y u b l i n s k a y a a n d V . M . S t e p a n o v , B i o o r g . K h i m . , 17 ( 1 9 9 1 ) 1066. 4 . M . Y u . G o l o l o b o v , T . L . V o y u s h i n a , V . M . S t e p a n o v a n d P. A d l e r c r e u t z , B i o c h i m . B i o p h y s . A c t a (1992) s u b m i t t e d . 5. J . - L . F a u c h e r e , M . C h a r t o n , L.B. Kier, A . V e r l o o p a n d V . P l i s k a , I n t . J . Peptide a n d P r o t e i n Res., 3 2 (1988) 2 6 9 . 6 . I. S c h e c h t e r a n d A . B e r g e r , 2 7 ( 1 9 6 7 ) 1 5 7 . 7 . C. G r i e c o , C . H a n s c h , C. S i l i p o , R . N . S m i t h , A . V i t t o r i a a n d K. Y a m a d a , A r c h . B i o c h e m . B i o p h y s . , 194 (1979) 5 4 2 . 8 . P. C l a p e s a n d P. A d l e r c r e u t z , B i o c h i m . B i o p h y s . A c t a , 1 1 1 8 ( 1 9 9 1 ) 7 0 .

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

443

Peptide synthesis in organic-aqueous media catalysed by a-chymotrypsin immobilised over different supports b

A. R. Alcantara*, J. V. Sinisterra", C. Torres', J, M. Guisân , M. H. G i T and A. Williams* "Organic & Pharmaceutical Chemistry Department. Pharmacy Faculty. Complutense University 28040 MADRID (SPAIN) b

Instituto de Catâlisis. C.S.I.C. Cantoblanco 28049 MADRID (SPAIN)

department (PORTUGAL)

of Chemistry,

d

University

Chemistry Labs. University CANTERBURY (UNITED KINGDOM)

of

Kent

Universidad of Coimbra, at

Autonoma,

COIMBRA

Canterbury.

CT 2

3000 7NH,

Abstract Peptide synthesis has been carried out using a-chymotrypsin (aCT) immobilised on different supports (agarose, two polyethyleneg-co-2-hydroxyethyl methacrilate (PE/HEMA) copolymers, and two soluble acrilic microgels), using different organic-aqueous media. Fairly good yields were obtained with all of them, and their reutilisation at least two times was excellent. The influence of the physicochemical properties of supports and solvents in the yield in peptide synthesis is discussed.

l.INTRODUCTION The Merrifield synthesis of peptides [1] made a very important contribution to the progress in peptide research. The drawbacks of this methodology (sequential synthesis, with a decrease of the yield in every step, heterogeneity of the synthetic peptide, secondary reactions,...) can be avoided using the enzymecatalysed approach. Furthermore, with this methodology it is possible to use much milder reaction conditions, with less contamination risks. The main dissadvantage of the enzymatic synthesis, the high cost of the biocatalyst, can be easily overcome by simply immobilising on an appropiate carrier. In this way, the enzyme can be reused and the overall cost of the process will be decreased. The enzymatic synthesis of active peptides, such as Leu- and Met-enkephalins [2] is a brilliant example of the synthetic applicability of the enzymatic approach.

444 In this paper we present the results obtained in the synthesis of the Bz-L-Tyr-L-Leu-NH 2 dipeptide, using a-chymotrypsin (a-CT) immobilised by different covalent coupling methods on different supports, in different organic-aqueous media. The influence of the physicochemical properties of the support and solvent in the yield in peptide synthesis are discussed.

2. EXPERIMENTAL 2.1. Covalent immobilisation of α-CT on different supports The supports used for immobilising the α-CT were two polyethylene-g-co-2-hydroxyethyl methacrylate (PE/HEMA) copolymers, agarose and two synthetic soluble acrylic microgels. 2.1.a. Immobilisation on PE/HEMA copolymer The polyethylene-gco-2-hydroxyethyl methacrylate copolymer was synthetised using a previously described methodology [3]: in that way, two copolymers were obtained, CI and C2, with 61.25 and 47.66 % of grafting, respectively, possesing 1.21 and 2.2 0 mmol of -COOH groups per gram of copolymer. The enzyme coupling to these copolymers was made following a previously described method [3]: 2 g of copolymer were mixed with 400 mg of l-cyclohexyl-3 (2-morpholinoethyl) carbodiimide metho-ptoluene sulphonate (CMC) in 20 mL of an enzyme solution (4 mg/g copolymer) in 0.1 M acetate buffer, pH=5.00. The mixture was stirred for 20 h at 4°C, and the amount of enzyme immobilised was determined by the Lowry assay procedure [4]. The enzymatic activity was tested using hemoglobin as substrate [5]. The loading results were 87.2 and 33.3 mg of active enzyme per gram of CI and C2, respectively. 2.1.b. Immobilisation on agarose The activation of the agarose gel was carried out following the previously described glycidol methodology [6]: in that way, an activated agarose gel was obtained, with 2.4 μπιοί aldehyde/mL gel, which corresponds to a surface density of 0.5 aldehyde residues/1000 À gel surface. The enzyme coupling to these gels was carried out following a previously described method [6]: 2 mg of α-CT dissolved in 40 mL of 0.1 M borate buffer, pH=10.00 were mixed with 40 mL of an aqueous suspension of activated agarose containing 2 0 mL packed gel. This reaction mixture was stirred very gently at 25°C, and aliquots of supernatant and whole suspension were withdrawn at different times. The catalytic activity was determined spectrophotometrically using benzoyl-L-tyrosine ethyl ester (BTEE) as substrate [6]. After 3 h the enzymatic derivative was reduced with borohydride following the same experimental procedure previously described for trypsine-agarose derivatives [7]. The loading obtained was 27.14 mg α-CT/mg gel. 2.I.e. Immobilisation different microgels (Ml

on and

acrylic soluble microcrels Two M2) were synthetised following.

445

respectively, a previously described method [8] for Ml and a modification of that method [9] for M2. Microgel Ml solution was analysed for particle size (89 nm diameter) with a Malvern Automesure instrument 4700V4. The enzyme coupling to Ml microgel containg -COOH and -COOEt groups was carried out following a previously described method [8]: the enzyme (150 mg) was mixed with polymer (30 mL, 50/50 v/v in 0.1 M Tris/HCl buffer, pH=7.00). The pH was adjusted to 6.80, and 200 mg of l-ethyl-3-(3'-dimethylaminopropyl) carbodiimide (EDC) were added. The mixture was stirred 1 h at 25°C. No precipitation occured when the enzyme and polymer were mixed. During the time period the carboxyl groups reacted completely with the polymer, and any isourea or anhydride intermediate coupled with the enzyme or hydrolysed [10]. The urea by-products and unbound enzyme were removed by ultrafiltration on Amicon filters with 300.000 m. wt. cut-off. The integrity of the enzyme-polymer conjugate was demonstrated by chromatography on an analytical Fractogel^TSK HW65(F) column. To test the covalent binding, the enzyme-polymer conjugate was subjected to ultrafiltration with an Amicon Centricon filter (300.000 m. wt. cut-off) and washed three times with 30 mL of Tris pH=7.00 buffer and three times with 0.1 M borate buffer pH=9.00. Control experiments were carried out to demonstrate that the filters passed the native enzyme but not the polymer and the enzyme concentration in both the effluent and the supernatant were determined using the colorimetric Lowry method [4] . The enzymatic activity was determined spectrophotometrically using benzoyl-L-tyrosine p-nitro anilide (BTpNA) [11]. The loading obtained was 0.6 mg α-CT/mL microgel. For the M2 synthesis, 3-vinyl benzyl chloride was used at 5%* as a part of the monomer feed, just to have 3-chlorobenzyl groups in its structure, and the amount of chloride was determined by the titrimetic Möhr method [12]. The enzyme coupling to M2 was carried out in the following way: the enzyme (401.2 mg) was mixed with polymer (50 mL, borate buffer, 0.05 M, 0.05 M in NaCl, pH=9.00, 10 % (v/v) Ν,Ν-dimethyl formamide). The mixture stirred overnight at 2 5 ° C No precipitation occured when the enzyme and polymer were mixed. The urea by-products and unbound enzyme were removed by ultrafiltration on Amicon filters with 300.000 m. wt. cut-off. The integrity of the enzyme-polymer conjugate was demonstrated by chromatography on an analytical Fractogel^TSK HW65(F) column. The enzyme-polymer conjugate was subjected to ultrafiltration with an Amicon Centricon filter (300.000 m. wt. cut-off) and washed three times with 50 mL of immobilisation buffer without any sodium chloride ( borate buffer, 0.05 M, pH=9.00, 10 % (v/v) Ν,Ν-dimethyl formamide). Enzyme concentration (2.83 mg α-CT/mL gel) and activity were determined as for Ml. 2.2. Synthesis of the Bz-L-Tyr-L-Leu-NH 2 dipeptide The enzymatic synthesis of the model dipeptide were performed using 0.1 M Tris buffer, pH=9.00 as aqueous medium, with different amounts of ethyl acetate or 1,4-butanediol, which were the organic cosolvents employed in most of the experiments, considering a 1/2 (M/M) acyl donor/nucleophile ratio. The reaction mixture was introduced in a thermostatted water bath at

446

25°C and stirred magnetically for 5 min. Then, an amount of immobilised enzyme (corresponding to 4 mg of native enzyme) was added. To analyse the increase in the reaction yield, samples of 0.05 mL were extracted at different reaction times, mixed with 0.1 mL of ethanol and 0.85 mL of the internal standard (naphtalene in acetonitrile, 0.47 mM) and stored at -15°C. The reaction was monitored by analytical HPLC on a LDC Analytical CM 4000 multiple solvent delivery system fitted with a 4 χ 200 mm Nucleosil 128 (10 μ) column. Adequate resolution of samples was accomplished by isocratic dilution with a solvent system composed by an helium-degassed mixture of acetonitrile and deionised water in a proportion of 50/50. The flow rate was always maintained at 0.8 mL/min and analysis lasted 10 min. The elution was spectrophotometrically monitored at 270 nm. The amount of eluted substances was calculated by the internal standard method.

3· RESULTS AND DISCUSSION 3.1 Synthesis of the model dipetide by α-CT immobilised on agarose The results obtained for the synthesis of Bz-L-Tyr-L-Leu-NH 2 catalysed by α-CT immobilised on agarose in different organicaqueous media are shown in Table 1. Table 1 Synthesis of Bz-L-Tyr-L-Leu-NH2 catalysed by α-CT immobilised on agarose.* Experimental error = ± 5 % medium 1, 4-butanediol/pH=9, 1 / 2 ( v / v ) 1, 4-butanediο1/pH=9, l / K v / v ) 1, 4-butanediol/pH=9, 2 / l ( v / v ) 1, 4-butanediol/pH=9, 4 / l ( v / v ) 1, 4-butanediol/pH=9, 9 9 / l ( v / v ) ethyl acetate/pH=9, 2/l(v/v) ethyl acetate/pH=9, 4/l(v/v) ethyl acetate/pH=9, 9 9 / l ( v / v ) a b c d

% peptide 12.8 16.7 22.0 44.6 50.0 60.0 100.0

d

0

%acid

c

26.0 39.0 26.2 24.0 45.0 39.5 0.0

time (min) 10 30 30 90

— 300 1440 2820

Reaction conditions as described in Experimental Maximum yield in Bz-L-Tyr-L-Leu-NH 2 Yield obtained in Bz-Tyr-COOH as secondary reaction. No reaction was observed

From this data we can observe that the higher the proportion of organic solvent, the greater the maximum yield in peptide (as a

447 result of a thermodynamical shift of the equilibrium composition towards the desired product [13]) and the longer the time to reach that yield. This is true except for 1,4-butanediol at 99% (v/v), where no synthesis was observed. This fact could be explained if we take into consideration the distribution of water in the medium between the organic solvent and the system enzymesupport; in fact, 1,4-butanediol can retain by hydrogen bonding the small amount of water molecules present in the medium, avoiding their access to the system enzyme-support, thereby hindering the creation of the hydration sphere of the enzyme, which is essential for the catalytic activity. When ethyl acetate is used, due to the fact that this solvent presents much weaker hydrogen bonding with water, and because of the high tendency of agarose to retain water (we could use the term aquaphilicity described by Reslow et ai. [14] and defined as the support abbility to absorb water from water-saturated diisopryl ether), the accès of water molecules to the enzyme-support system is allowed, and the greatest yields are obtained at high percentages of organic solvent and large reaction times (thermodynamically controlled synthesis). 3.2 Synthesis of the model dipeptide by α-CT immobilised on PE/HEMA copolymers The results obtained for the synthesis of Bz-L-Tyr-L-Leu-NH 2 catalysed by α-CT immobilised on the two PE/HEMA copolymers CI and C2 in different organic-aqueous media are shown in Tables 2 and 3. Table 2 Synthesis of Bz-L-Tyr-L-Leu-NH 2 catalysed by α-CT immobilised on a Cl . Experimental error = ± 5 % medium 1, 4-butanediol/pH=9, 2/l(v/v) 1,4-butanediol/pH=9, 4/l(v/v) 1,4-butanediol/pH=9, 99/l(v/v) ethyl acetate/pH=9, 2/l(v/v) ethyl acetate/pH=9, 4/l(v/v) ethyl acetate/pH=9, 99/l(v/v) dimethylformamide/pH=9 , 1/4(v/v) a b c d

% peptide

0

%acid

c

time(min)

30. 7 31. 0

67. 3 21. 5

1320 1380

100. 0 50. 0 15. 0 2. 9

50. 0 8. 0 32. 0

—

2440 4440 5550 60

Reaction conditions as described in Experimental Maximum yield in Bz-L-Tyr-L-Leu-NH 2 Yield obtained in Bz-Tyr-COOH as secondary reaction. No reaction was observed

448 Table 3 Synthesis of Bz-L-Tyr-L-Leu-NH 2 catalysed by α-CT immobilised on a C2 . Experimental error = ± 5 % medium

% peptide" 42. 7 43. 2 d 82. 0 50. 0 15. 0 20. 1

2/l(v/v) 1,4-butanediol/pH=9, 1,4-butanediol/pH=9, 4/l(v/v) 1,4-butanediol/pH=9, 99/l(v/v) ethyl acetate/pH=9, 2/l(v/v) ethyl acetate/pH=9, 4/l(v/v) ethyl acetate/pH=9, 99/l(v/v) d imethy1formamide/pH=9 , 1/4(v/v) a b c

%acid

c

time(min)

43. 6 25. 4

120 1320

16. 0 45. 0 20. 0 71. 6

2440 2880 5550 60

Reaction conditions as described in Experimental Maximum yield in Bz-L-Tyr-L-Leu-NH 2 Yield obtained in Bz-Tyr-COOH as secondary reaction. No reaction was observed

For α-CT immobilised on PE/HEMA copolymers CI and C2 (Table 2 and 3 ) , when 1,4-butanediol is used at 99% (v/v) no synthesis was observed, and this fact could be explained by the same reasons exposed for the agarose immobilisation. As a result of the much smaller aquaphilicity of this support compared to agarose, the higher yields should be expected for solvent systems with a relatively high percentage of water, as in fact is observed both for CI and C2 in Tables 2 and 3. Comparing the results obtained with 1,4-butanediol and ethyl acetate at the same proportions, the higher yields are obtained for the later, as expected from the differences in the hydrogen-bonding ability of both solvents. Then, the ratio 2/1 (v/v) for ethyl acetate is the most adequate for the synthesis, because excellent yields are obtained (100 and 82 % for CI and C2 respectively) , although it is neccesary to allow for large reaction times. 3.3 Synthesis of the model dipetide by α-CT soluble acrilic microaels The first results obtained for the synthesis of Leu-NH 2 dipeptide using α-CT immobilised on the synthetised soluble acrilic microgels Ml and M2 Table 4.

immobilised

on

the Bz-L-Tyr-Ltwo previously are reported in

Table 4 Synthesis of Bz-L-Tyr-L-Leu-NH 2 catalysed by α-CT immobilised on microgels Ml and M2. Experimental error = ± 5 % medium

microgel a

l,4-butanediol/pH=9, 2/l(v/v) Ml b l,4-butanediol/pH=9, 2/l(v/v) M 2

% peptide 67.9 60.7

%acid 26.2 19.6

t(min) 25 120

449

The two derivatives of α-CT immobilised on the soluble acrylic supports Ml and M2 has been tested so far only in one solvent system, 1,4-butanediol/pH=9 2/1 (v/v). The results are shown in Table 4. For both of them, relatively high yields are obtained (67.9 and 60.7, respectively), and, what is more interesting, this yields were reached at very short reaction times (25 and 120 minutes). We can observe that both enzymatic derivatives present a similar synthetase activity, which therefore let us think that both immobilisation methods (through -C00H groups with EDC for Ml, or directly through Ph-CH 2-Cl groups for M2) do not alterate the structure of the enzyme, due to the fact that the coupling reaction takes place by the e-NH 2 of the lysine residues. This immobilisation method has been reported previously as a causing a moderate stabilisation versus Τ and little disturbance in the catalytic activity of α-CT [8] and some other proteins [10, 1 5 ] , allowing the enzyme to work very efficiently and quickly in organic-aqueous solvents, because the fact that the enzyme is immobilised on a liquid support decrease the steric hindrance or diffusional problems during the reaction of the soluble enzymepolymer conjugate with the substrate [15, 1 6 ] . So that, the results obtained with this derivative could be considered as fairly good. 3.4 Reutilisation of the enzymatic derivatives The different α-CT immobilised derivatives were tested for their reutilisation after one synthetic cycle. In that way, the derivatives were separated from the reaction mixture by vacuum filtering through sintered glass (Cl, C2 and agarose) or by ultrafiltration through a 300,000 Dalton cut-off membrane (Ml and M2) , washed three times with the immobilisation buffer, and reused. Some of the results obtained are shown in Table 5. Table 5 Reutilisation of some enzymatic derivatives. Experimental error = ± 5 % Deriv. Medium CI C2 C2 Ml M2

1 1 2 2 2

Cycle 0 %peptide %acid 50.0 50.0 42.7 67.9 60.7

50.0 45.5 43.6 26.2 33.2

Cycle 1 %peptide %acid 68.2 26.6 51.2 70.4 66.6

12.0 14.8 8.5 12.9 30.5

Cycle 2 %peptide %acid 40.0 16.0 34.4 48.9 47.3

4.0 4.2 58.8 10.7 19.6

1 ethyl acetate/pH=9.00, 4/1 (v/v) 2 l,4-butanediol/pH=9.00, 2/1 (v/v)

The agarose support presented the worst mechanical properties due to its gel structure, being very difficult to be recovered

450 after the reaction process. From Table 5 we can observe that the other enzymatic derivatives can be reused at least three times without excessive deactivation. Normally a higher peptide synthesis and a lower extension in the secondary acid synthesis is obtained after one reaction cycle. This finding could be explained if we consider that the excess of water in the enzyme microenvirontment can be removed for the organic solvent in the first cycle. In the third reutilisation of the derivative (Cycle 2 ) , this effect is not that pronunciated, although we can still retain a important percentage of activity, normally around 80 % of the obtained in Cycle 0. Therefore, these enzymatic derivatives could be very useful in peptide synthesis. In this way, we are using this methodology in the synthesis of some other peptides using both natural and unnatural aminoacids, which results will be published. This work has been possible thanks to a Spanish-British Accion Integrada (no. 112 A, 1991) and a Spanish-Portuguese Accion Integrada (no. 30 B, 1992)

REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

B. Merrifield, Chem. Script., 25 (1985) 121 W. Kullman, Enzymatic Peptide Synthesis, CRC Press, Florida, 1987 C. G. Beddows, M. H. Gil and J. T. Guthrie, J. Appl. Polym. Science, 35 (1988) 135 O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, J. Biol. Chem., 193 (1951) 265 M. L. Anson and A. E. Mirsky, J. Gen. Physiol., 17 (1933) 151 J. M. Guisân, A. Bastida, C. Cuesta, R. Fernandez-Lafuente and C. M. Rosell, Biotech, and Bioeng., 38 (1991) 144 R. M. Blanco and J. M. Guisân, Enz. Microb. Technol., 11 (1989), 360 A. K. Luthra, R. J. Pryce and A. Williams, J. C. S. Perkin Trans. II (1987) 1575 A. R. Alcantara, J. V. Sinisterra, C. Torres, P. Romanelli and A. Williams -Umpublished data J. P. Davey, R. J. Pryce and A. Williams, Enzyme Microbiol. Technol., 11 (1989), 657 H. F. Gaertner and A. J. Puigserver, Proteins : Structure, Function and Genetics, 3 (1988) 13 0 F. Möhr, Angewandte, 97 (1856) 335 A. M. Klibanov, Chemtech., 16 (1986) 354 M. Reslow, P. Adlercreutz and B. Mattiasson, Eur. J. Biochem., 172 (1988) 573 A. R. Alcantara, J. V. Sinisterra, G. Guanti, S. Thea and A. Williams, J. Mol. Catal., 70 (1991) 381 G. Manecke and S. Heise, Reactive Polymers, 3 (1985) 251

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. 451

© 1992 Elsevier Science Publishers B.V. All rights reserved.

C O N T R O L OF WATER ACTIVITY B Y USING SALT HYDRATES IN ENZYME CATALYSED ESTERIFICATIONS IN ORGANIC MEDIA

Birte Sjursnes,* Lise Kvittingen, and Thorleif Anthonsen Chemistry Department, The University of Trondheim, N-7055 Trondheim, Norway

Peter Hailing Department of Bioscience and Biotechnology, University of Strathclyde, Glasgow G l 1XW, Scotland, U K

A b s t r a c t . E n z y m e s u s e d as catalysts in o r g a n i c m e d i a are k n o w n to n e e d s o m e w a t e r to function. W e h a v e u s e d salt h y d r a t e s to control t h e w a t e r c o n t e n t in t h e r e a c t i o n m i x t u r e a n d s t u d i e d t h e effect w h e n t h e c o m p o s i t i o n in a m o d e l r e a c t i o n w a s c h a n g e d . A s a m o d e l r e a c t i o n w a s c h o s e n the esterification o f b u t a n o i c a c i d w i t h b u t a n o l c a t a l y s e d b y l i p a s e from Candida rugosa. S o m e o t h e r lipases w e r e tested to d e t e r m i n e the w a t e r c o n t e n t for o p t i m a l a c t i v i t y .

INTRODUCTION It h a s b e e n s h o w n

that

the activity of enzymes

used

in o r g a n i c m e d i a

is

d e p e n d e n t o n t h e w a t e r c o n t e n t , a n d that o n l y a n a r r o w r a n g e g i v e s o p t i m a l a c t i v i t y . T h e w a t e r c o n t e n t in t h e r e a c t i o n m i x t u r e is b e t t e r e x p r e s s e d b y t h e t h e r m o d y n a m i c activity o f w a t e r ( a w ) than b y concentration. T h e w a t e r activity o f the reaction m i x t u r e can b e m e a s u r e d in the g a s p h a s e [1,2].

C o m m o n w a y s o f adjusting o p t i m a l w a t e r activity a r e e i t h e r b y a d d i n g v a r i o u s a m o u n t s o f w a t e r a n d m e a s u r i n g the initial v e l o c i t y in o r d e r to o b t a i n o p t i m u m condition

or b y p r e - e q u i l i b r a t i n g

the solvent and

saturated salt solutions o f k n o w n w a t e r activity.

enzyme

separately

over

452 It is also p o s s i b l e to a d d a p a i r o f solid salt h y d r a t e s d i r e c t l y to t h e r e a c t i o n m i x t u r e , c o r r e s p o n d i n g to a k n o w n water activity [3]. Ideally this will g i v e a fixed w a t e r a c t i v i t y , w h i c h w i l l b e m a i n t a i n e d b e c a u s e t h e h i g h e r salt h y d r a t e c a n release w a t e r w h i l s t the l o w e r salt h y d r a t e can t a k e u p w a t e r . In c o n t r a s t to the use o f saturated salt solution, w h e r e o n l y the initial a

w

o f a r e a c t i o n m i x t u r e can

b e adjusted b y pre-equlibrating through t h e gas p h a s e , the solid salt h y d r a t e pair will buffer a

w

at a constant v a l u e t h r o u g h o u t the reaction. T h e a

w

corresponding

to a salt h y d r a t e pair will differ from that o f a saturated salt s o l u t i o n o f the s a m e salt, e.g. a

w

c o r r e s p o n d i n g to a saturated salt solution o f s o d i u m c a r b o n a t e is 0.9,

w h i l s t the c o r r e s p o n d i n g a

w

o f a m i x t u r e o f solid d e c a - a n d h e p t a h y d r a t e is 0.72.

M a n y salt h y d r a t e pairs s h o w nearly ideal b e h a v i o u r . Different salt h y d r a t e pairs release water at different water activity a n d b y c h o o s i n g an appropriate pair o f salt h y d r a t e s a particular w a t e r activity in the reaction m i x t u r e can b e o b t a i n e d . It is n e c e s s a r y that b o t h salt h y d r a t e f o r m s are p r e s e n t , b u t t h e r a t i o o f t h e m is u n i m p o r t a n t . A t h e o r e t i c a l b a c k g r o u n d o f w a t e r a c t i v i t y , t h e f u n c t i o n o f salt h y d r a t e pairs as well as tables o f a

w

h a v e b e e n g i v e n p r e v i o u s l y [4,5].

In the f o l l o w i n g the state o f h y d r a t i o n will b e i n d i c a t e d in p a r e n t h e s i s after the salt h y d r a t e , e.g. s o d i u m s u l p h a t e d e c a h y d r a t e as N a 2 S 0 4 (10) a n d a m i x t u r e o f d e c a h y d r a t e a n d a n h y d r o u s s o d i u m sulphate as Na2SC>4 ( 1 0 / 0 ) .

RESULTS AND DISCUSSION A s a m o d e l reaction w a s c h o s e n the esterification o f b u t a n o i c acid w i t h b u t a n o l in h e x a n e catalysed b y l i p a s e from Candida rugosa. W i t h o u t w a t e r a d d e d a s i g m o i d c u r v e w a s o b t a i n e d , reflecting the v a r y i n g w a t e r activity d u r i n g the r e a c t i o n ( 1 , figure 1). A d d i t i o n o f w a t e r g a v e a linear initial rate, b u t a d e c r e a s i n g rate t o w a r d s t h e e n d o f t h e reaction. T h e r e w a s n o influence o n t h e c h e m i c a l e q u i l i b r i u m as the reaction proceeded

to 9 9 . 9 % c o n v e r s i o n . H o w e v e r , as t h e p o s i t i o n o f

e q u i l i b r i u m is d e t e r m i n e d b y the l a w of m a s s action, o n e m i g h t e x p e c t a greater influence of w a t e r in other reactions. In the p r e s e n t c a s e o p t i m u m w a t e r addition w a s 3 μΐ. With the addition of N a 2 S 0 4 ( 1 0 / 0 )

with a

w

0.76 a s m o o t h c u r v e w i t h linear

initial rate w a s o b t a i n e d (2, fig. 1). T h e salt h y d r a t e pair buffers the w a t e r activity as the d e c a h y d r a t e can release water whilst the a n h y d r o u s salt can take up water,

453 thus maintaining

constant water

activity. A comparison of the

maximum

g r a d i e n t s for the t w o r e a c t i o n s i n d i c a t e s that this salt h y d r a t e p a i r g i v e s nearo p t i m a l conditions for the e n z y m e . A s e n z y m e activity is d e p e n d e n t o n a w ^ other salt h y d r a t e s w i t h v a r i o u s a lipase from Candida

rugosa,

w

w e r e i n v e s t i g a t e d . F o r this p a r t i c u l a r e n z y m e ,

the initial activity d r o p p e d w i t h d e c r e a s i n g a w . T h i s

is s h o w n for N a 2 H P 0 4 ( 7 / 2 ) , a

w

0.57 (3, fig. 1).

Time, min Figure

1. M o d e l r e a c t i o n ( r e a c t i o n n o t

shown

to 1 0 0 %

c o n v e r s i o n for 1 a n d 3 ) . N o t h i n g a d d e d ( 1 ) , in the p r e s e n c e o f 1 g Na2S0

4

( 1 0 / 0 ) a w 0.76 (2) a n d in t h e p r e s e n c e o f l g

N a 2 H P 0 4 ( 7 / 2 ) a w 0.57(3). S o m e o f t h e investigated salts s h o w e d non-ideal b e h a v i o u r (not s h o w n ) . N a 2 C 0 3 ( 1 0 / 7 ) , w h i c h w a s successfully u s e d in p e p t i d e synthesis [ 6 ] , r e a c t e d in this c a s e w i t h b u t a n o i c a c i d to g i v e its s o d i u m salt, r e m o v i n g it f r o m s o l u t i o n , t h u s n o esterification occurred. With N a 2 P 2 0

7

(10), C a S 0 4 (2/0,5), Z n S 0 4 ( 7 / 1 ) and

K 4 F e ( C N ) 6 (3) the p r o g r e s s c u r v e s s h o w e d a lag o f a b o u t 1 0 0 0 m i n , a n d then a c c e l e r a t e d to a r a t e s i m i l a r to that at h i g h e r w a t e r a c t i v i t y . T h i s s i g m o i d b e h a v i o u r is n o t n e c e s s a r i l y r e l a t e d to t h e rates o f w a t e r e q u i l i b r a t i o n , as for C a S 0 4 ( 2 / 0 , 5 ) it persisted to a certain e x t e n t e v e n with pre-equilibration. W a t e r dissociation from this h y d r a t e is k n o w n to b e particularly s l o w [7]. H y d r a t e forms o f Z n S 0 4 also s h o w e d a n o m a l o u s b e h a v i o r a n d h e n c e w e c o n s i d e r t h e s e salt hydrates unsuitable for this application.

454

W h e n Na2SC>4 ( 1 0 / 0 ) w a s a d d e d , n e a r - o p t i m a l c o n d i t i o n s w e r e o b t a i n e d e v e n if the c o m p o s i t i o n o f the reaction m i x t u r e w a s c h a n g e d : • A n i n c r e a s e o f the a m o u n t o f e n z y m e from 5 m g to 3 0 m g d i d n o t alter the initial rate per m g . e n z y m e w h e n salt hydrates w e r e present. If salt hydrates w e r e n o t p r e s e n t , a n d w a t e r w a s to b e a d d e d , a p r o p o r t i o n a l i n c r e a s e in t h e total a m o u n t o f w a t e r in the s y s t e n c o m p a r e d to the i n c r e a s e in w e i g h t o f e n z y m e w o u l d result in a surplus o f water. T h i s is b e c a u s e there will b e n o c h a n g e in the r e q u i r e d a m o u n t o f w a t e r dissolved in the o r g a n i c p h a s e . • W h e n the substrate concentration is i n c r e a s e d , the s o l u b i l i t y o f w a t e r in the o r g a n i c p h a s e will i n c r e a s e , h o w e v e r the a m o u n t o f w a t e r a s s o c i a t e d w i t h the e n z y m e will not c h a n g e , t h u s a p r o p o r t i o n a l i n c r e a s e o f w a t e r c o m p a r e d to the substrate concentration will not b e correct. F i g u r e 2 s h o w s t h e effect o f a threefold increase o f substrate concentration with (1) a n d w i t h o u t (2) salt h y d r a t e s present.

Time, min

Figure and

2. M o d e l reaction with (3) a n d w i t h o u t (4) salt h y d r a t e s three times concentration of substrates with

(1) a n d

without (2) salt hydrates. • C h a n g i n g the solvent will alter the solubility o f w a t e r in the o r g a n i c phase. F i g u r e 3 s h o w s t h e m o d e l reaction in h e x a n e , t r i c h l o r o e t h y l e n e a n d toluene.

455

Figure 3. M o d e l r e a c t i o n in h e x a n e w i t h ( 1 ) , in t r i c h l o r o e t h y l e n e w i t h (2) a n d w i t h o u t (3) a n d in toluene with (4) a n d w i t h o u t (5) salt hydrates. W h e n c h a n g i n g the s u b s t r a t e c o n c e n t r a t i o n o r t h e s o l v e n t t h e h y d r a t i o n o f the e n z y m e m i g h t b e i n f l u e n c e d d i r e c t l y . H o w e v e r , at l e a s t o n e l i p a s e h a s b e e n reported to s h o w the s a m e d e p e n d e n c e o f the a w in different solvents [1].

Time, min Figure 4. M o d e l reaction catalysed b y lipase Ρ A m a n o in the p r e s e n c e o f N a 2 S 0 4 ( 1 0 / 0 ) a w 0.76 (1), N a 2 H P 0 4 ( 1 2 / 7 ) a w 0.74 (2), N a 2 H P 0 4 ( 7 / 2 ) a w 0.57 (3) and N a 2 H P 0 4 ( 2 / 0 ) a w 0.15 (4).

456 O n t h e b a s i s o f t h e results w i t h l i p a s e from Candida

a series o f salt

rugosa,

h y d r a t e s w h i c h s h o w e d near-ideal b e h a v i o u r w e r e c h o s e n to i n v e s t i g a t e t h e u s e o f salt h y d r a t e s a n d w a t e r activity o f t h r e e o t h e r l i p a s e s . T h e f o l l o w i n g salt hydrates were used: N a 2 S 0 4 ( 1 0 / 0 ) a (7/2) a

w

0.57 a n d N a 2 H P 0 4 a

w

w

0.76, N a 2 H P 0 4 ( 1 2 / 7 ) a

a n d l i p a s e Ρ ( A m a n o ) from Pseudomonas the s a m e o p t i m u m as Candida

w

0.74, N a 2 H P 0 4

0.15. L i p a s e from p o r c i n e p a n c r e a s (not s h o w n )

rugosa

fluorescens

(fig. 4 ) a p p e a r e d to h a v e

lipase ( N a 2 S 0 4 ( 1 0 / 0 ) , a

w

0.76) a n d s h o w e d

declining activity with l o w e r water activity. H o w e v e r , L i p o z y m e I M 2 0 a p p e a r e d to h a v e the best activity for N a 2 H P 0 4 ( 7 / 2 ) , a

w

0.57 (not s h o w n ) .

CONCLUSION A d d i t i o n o f salt h y d r a t e s is a s i m p l e a n d c o n v e n i e n t m e t h o d for c o n t r o l l i n g water activity in e n z y m e catalysed reactions in o r g a n i c m e d i a . In the p r e s e n c e o f a g i v e n pair o f salt h y d r a t e s the fixed w a t e r activity v a l u e m a i n t a i n s near-optimal c o n d i t i o n s e v e n w h e n c h a n g e s are m a d e in e n z y m e c o n c e n t r a t i o n , s o l v e n t u s e d or reactant concentrations. T h e h y d r a t e pair is also able to take u p or release water (at c o n s t a n t w a t e r a c t i v i t y ) to m a i n t a i n o p t i m a l c o n d i t i o n s a s t h e r e a c t i o n proceeds.

ACKNOWLEDGEMENTS W e are grateful to A m a n o P h a r m a c e u t i c a l C o for a gift o f lipase Ρ a n d to N o v o N o r d i s k , Bagsvaerd, D e n m a r k for a gift o f L i p o z y m e I M 2 0 . A g r a n t from T h e Norwegian

R e s e a r c h C o u n c i l for S c i e n c e a n d

Humanities,

NAVF

is

also

gratefully a c k n o w l e d g e d .

EXPERIMENTAL Materials.

L i p a s e from Candida

rugosa

(formerly C. cylindracea,

E C 3.1.1.3), 9 0 0

units pr. m g w a s p u r c h a s e d from S i g m a C h e m i c a l C o . All c h e m i c a l s i n c l u d i n g organic solvents w e r e o f analytical grade.

457

Analytical

Methods.

G L C was performed

using a Varian 3400

instrument

e q u i p p e d w i t h a u t o s a m p l e r 8 1 0 0 a n d a V i s t a 4 0 2 d a t a s y s t e m for integrations. A c a p i l l a r y c o l u m n , J & W Scientific, D B - 1 7 0 1 , 3 0 m 0.255 m m , film t h i c k n e s s 0.25 μ ι η , w a s u s e d w i t h a t e m p e r a t u r e p r o g r a m , 6 0 - 1 2 0 ° C , 10 ° C pr. m i n . T h e i n c r e a s e o f p r o d u c t s w a s calculated from the internal s t a n d a r d . C a l i b r a t i o n c u r v e s w e r e e s t a b l i s h e d from 7 i n d e p e n d e n t concentrations.

Determination of water in the organic phase. T h e concentration o f w a t e r in t h e standard organic phase of the reaction mixture (1.0 m m o l B u O H , 0.50 m m o l b u t a n o i c acid, 8 m L h e x a n e ) after equilibration at 2 0 ° C with N a 2 S O 4 1 0 H 2 0 ( a w 0.76) w a s d e t e r m i n e d b y c o u l o m e t r i c K a r l F i s c h e r t i t r a t i o n u s i n g a M e t r o h m 1

1

C o u l o m e t e r 6 8 4 K F as 100 m g L " (5.7 m m o l L " ) .

General experimental procedure for enzymatic esterification. A typical reaction m i x t u r e c o n s i s t e d o f lipase (5.0 m g , w h e n not o t h e r w i s e stated), n - h e x a n e (8 m L ) , n - b u t a n o l (1.0 m m o l ) , b u t a n o i c a c i d (0.50 m m o l ) a n d , as i n t e r n a l s t a n d a r d , nd e c a n e (0.31 m m o l ) . Salt hydrates in pairs, (0.5 g - 1 . 5 g) w e r e a d d e d to the reaction m i x t u r e s , w h e n n o t o t h e r w i s e s t a t e d . T h e r e a c t i o n s w e r e c a r r i e d o u t at r o o m t e m p , in c l o s e d vials ( 1 0 m L , i.d. 2 0 m m ) w i t h s h a k i n g 1 3 0 s t r o k e s / m i n . All e x p e r i m e n t s w e r e c o n d u c t e d at least t w i c e a n d t h e m a x i m u m d e v i a t i o n s w e r e less than 1 0 % .

REFERENCES 1.

Valivety, R.H.; Hailing, P.J. a n d M a c r a e , A.R. Biochim. Biophys. Acta (1991) In press.

2.

H a i l i n g , P.J. Trends Biotechnol 7 (1989) 50-52.

3.

K v i t t i n g e n , L., S j u r s n e s , B . , A n t h o n s e n , T. a n d Hailing, P. J.

Tetrahedron 48(1992) 2793 - 2802.

4.

Eisenberg, D . a n d Crothers, D . Physical Chemisty with Applications to the Life Sciences, T h e B e n j a m i n / C u m m i n s P u b l i s h i n g C o . , Inc. 1 9 7 9 , p. 2 9 7 - 3 0 0 .

5.

Hailing, P.J. Biotechnology Letters / Techniques 14 (1992) In press

6.

K u h l , P. a n d Hailing, P. J. Biochim. Biophys Acta (1991) 1078, 326-328

7.

Gmelins Handbuch der Anorganischen Chemie, 8th Ed., Vol . 28, Part B 3 , p. 7 2 1 - 7 2 2 . 1 9 6 1

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

459

E N Z Y M A T I C PEPTIDE SYNTHESIS USING NEW WATER-SOLUBLE A M I N O A C I D DERIVATIVES D. Auriol, F . Paul and P. Monsan

BioEurope S.A., BP 4 1 9 6 , 4 Impasse Didier-Daurat, 31031 Toulouse Cedex, France

Abstract

N-L-malyl-L-tyrosine ethyl ester (MTEE), produced at a large scale by a Staphylococcus chromogenes aminopeptidase, was used as a highly water-soluble N-protected form of L-tyrosine in the kinetically controlled synthesis of N-Lmalyl-L-tyrosylglycylglycine ethyl ester (MTGGEE) and N-L-malyl-Ltyrosylarginine ethyl ester (MTAEE) by a-chymotrypsin. The S. chromogenes aminopeptidase was used for product deprotection, allowing the proposal of a selective and mild complete enzymatic peptide synthesis process.

1. I N T R O D U C T I O N Protease-catalyzed synthesis of short peptides usually requires the protection of the α-amino group of the acyl donor amino acid. The commonly used α-amino protecting groups (Boc, Fmoc, F o r , and Z) yield amino acid derivatives poorly soluble in aqueous media, especially those containing an aromatic side chain such as tyrosine, phenylalanine and tryptophan. An increasing attention is presently devoted to the use of solubilizing Nterminal protecting groups: highly water-soluble aromatic amino acid derivatives containing the maleyl and citraconyl moitiés were synthesized and used in enzyme-catalyzed peptide synthesis (1, 2). Nevertheless, N-terminal protecting group removal is achieved through chemical procedures usually requiring drastic reaction conditions with the risk of side reactions and sometimes difficult purification operations (3). Keeping in mind the solubility problem of N-protected aromatic acids and the drawbacks of chemical procedures in product deprotection, we looked for a selective and mild enzymatic protection and deprotection process involving the use of a solubilizing N-terminal protecting group. Several approaches to the use of enzymes as reagents for a-amino group protection and deprotection have already been described (4). Nevertheless, protease-catalyzed protection and deprotection may involve unwanted proteolysis of the product, and non-pro tease enzymes are particularly suitable for this purpose.

460

We have discovered an aminopeptidase produced by a non-pathogenic S. chromogenes strain isolated from dairy environment, able to catalyze the equilibrium controlled synthesis of Aspartame from unprotected L-aspartic acid and L-phenylalanine methyl ester (5). In search of stable (unable to produce diketopiperazine compounds), highly water-soluble aromatic amino acid derivatives, we succeeded in the enzymatic synthesis of α-hydroxy acyl-amino acids among which L-malyl, L-lactyl and L-a-hydroxyglutaryl-amino acids are particularly a t t r a c t i v e for cosmetic and nutritive applications (6). Though initial studies were performed with a partially purified aminopeptidase preparation, the obtainment of an electrophoretically pure enzyme enabled us to demonstrate definitively that the same enzyme was able to use L amino acids as well as α-hydroxy acids as the acyl donor substrate (7). Here, we report on the production of N-L-malyl-L-tyrosine ethyl ester and its use in the kinetically controlled synthesis of L-tyrosylglycylglycine (the N-terminal moiety of enkephalin) and L-tyrosyl-L-arginine (opioid dipeptide kyotorphin) by achymotrypsin. 2. MATERIALS AND METHODS Chemicals: a-chymotrypsin (50 BTEE-U/mg), glycylglycine ethyl ester-HCl, L-arginine ethyl ester-2HCl, N-acetyl L-tyrosine ethyl ester were from Sigma. Synthesis reaction conditions: Substrates: the nucleophile was prepared a t a 2.0 M concentration in 1.0 Ν NaOH; the acyl donor was dissolved in the concentrated nucleophile solution. The final nucleophile concentration in reaction synthesis mixtures was 0,4 M. During the incubation, the pH was maintained a t 7.5 with 2.0 Ν NaOH; the temperature was 30°C. Aliquots (incubation time: 0, 5, 10, 15, 20, 30, 4 5 , 60, 90, 120, 150, 180, 240, 300 min) of reaction media were diluted 50 times with 0.05 Ν HCl for αchymotrypsin inactivation and the concentration of r e a c t a n t s was determined by HPLC. HPLC conditions: a μ-Bondapack C18 column (Millipore-Waters) is coupled with a UV spectrophotometer set at a wavelength of 274 nm. The concentration of the products was determined by the external standard method (specific absorbance of L-tyrosine). The elution was carried out with 12.5 mM N a H 2 P 0 4 at pH 3.5, and an increasing concentration of acetonitrile from 4 % to 25 % in 30 min. The flow r a t e was 1.5 ml/min. 3. RESULTS AND DISCUSSION 3.1 Production of N-L-malyl-L-tyrosine ethyl ester MTEE synthesis from L-malic acid and L-tyrosine ethyl ester was carried out with an S. chromogenes aminopeptidase preparation as previously described (7). The de-esterified form of MTEE, N-L-malyl-L-tyrosine (MT), is currently produced by BioEurope on a large scale (several tons per year) using the S. chromogenes aminopeptidase original technology. So far, this enzymatic process is the second largest enzyme-catalyzed peptide bond synthesis process after the Aspartame process. MT is a highly water-soluble tyrosine derivative commercialized as a melanin precursor for sun-tan formulas in Cosmetics.

461 3.2 L-malyl as a solubilizing N-terminal protecting group The water-solubility of MTEE is 0.20 M, pH 7 . 5 , 3 0 ° C . The L-malyl residue appears consequently as an N-protecting group allowing the obtainment of a highly water-soluble N-protected L-tyrosine ethyl e s t e r . A description o f the synthesis of N-L-malyl-L-tyrosyl peptides in reaction mixtures containing one solvent, water, or a mixture of water and ethanol (ethanol 40 % v/v) is given below. Nucleophile: glvcvlglvcine ethyl e s t e r The synthesis reaction was first carried out in water at pH 7.5 with an initial MTEE concentration of 170 mM. The a-chymotrypsin concentration was 0.02 g/1. The mixture was heterogeneous at the s t a r t of the reaction (solubility of MTEE in the presence of 400 mM glycylglycine ethyl e s t e r = 158 mM), and b e c a m e totally c l e a r after about 30 min of incubation. The variation of the concentration of r e a c t a n t s during the incubation is described in Figure 1. Though a slight hydrolysis of the MTEE-ethyl e s t e r bond occured when carrying out the initial pH adjustment (initial MT concentration: 34 mM), MTEE was chemically stable in the conditions of incubation. The final concentrations of MTGGEE and N-L-malyl-L-tyrosylglycylglycine (MTGG) remained stable after 2.5 h of incubation. The achieved MTEE conversion yield, corresponding to the proportion of MTEE converted in N-L-malyl-L-tyrosyl peptides, was 83 %, with a major proportion of esterified form (90 % ) . The specific initial rates for substrate consumption and product appearance are listed in Table 1. The a-chymotrypsin specific activity measured as the initial MTEE consumption r a t e per mg a-chymotrypsin, is 5 times higher than that measured without the nucleophile glycylglycine ethyl ester (35 μιηοΐ/min/mg). This suggests that the de-acylation of the acyl-enzyme complex is the rate-determining step for the substrate transformation. mM

150

100

50

0

1

2

3 hours

Figure 1. N-L-malyl-L-tyrosyl peptides synthesis water using glycylglycine ethyl e s t e r as the nucleophile.

by

a-chymotrypsin

in

462 Table 1 Specific initial reaction rates for the α-chymotrypsin-catalyzed synthesis of N - L malyl-L-tyrosyl peptides using glycylglycine ethyl e s t e r as the nucleophile. Specific Reaction

initial (jumol/min/mg) Water

reaction

rate

Ethanol 40 % (v/v)

MTEE consumption

172

1.59

MTGGEE appearance

149

1.50

MTGG appearance

12

0.06

MT appearance

12

0.03

MTGGEE synthesis was investigated in the presence of 40 % (v/v) ethanol to decrease the substrate de-esterification. Due to a-chymotrypsin denaturation by ethanol, the a-chymotrypsin concentration was increased to 5.0 g/1 to obtain the total MTEE transformation. The reaction mixture contained 180 mM MTEE, 6 6 % being soluble a t the s t a r t of the reaction. After 2 h of incubation, the final concentration of N - L malyl-L-tyrosyl peptides was 160 mM, corresponding to a conversion yield of 89 % (Figure 2). The MT appearance specific r a t e was 400-fold lower than that measured in water, whereas the MTEE consumption and MTGGEE appearance specific rates were 100-fold lower than those obtained in water (Table 1), indicating a protective e f f e c t of ethanol toward the substrate de-esterification reaction. N-acetyl-L-tyrosine ethyl e s t e r (ATEE), a recognized substrate of achymotrypsin, was finally used as the acyl donor for the synthesis o f N - a c e t y l - L tyrosylglycylglycine ethyl e s t e r (ATGGEE) in the presence of 0.01 g/1 achymotrypsin. A T E E (200 mM) was almost completely insoluble a t the beginning o f the reaction. Nevertheless, it was completely transformed after 30 min o f incubation. N-acetyl-L-tyrosyl peptides were obtained with an A T E E conversion yield of 72 %, 28 % of the substrate having been hydrolyzed. Though the a-chymotrypsin specific activity when A T E E is the substrate is about 20-fold higher than when MTEE is the substrate, A T E E has the drawback o f being more sensitive to deesterification than MTEE. Indeed, the ratio of the initial rates of transacylation/hydrolysis was 6.2 (Table 2), whereas it was 12.4 in t h e c a s e of MTEE.

463

Figure 2. N-L-malyl-L-tyrosyl peptides synthesis by α-chymotrypsin in 40 % (v/v) ethanol using glycylglycine ethyl e s t e r as the nucleophile. Table 2 Specific initial reaction rates for the or-chymotrypsin-catalyzed synthesis of Nacetyl-L-tyrosyl peptides in water using glycylglycine ethyl e s t e r as the nucleophile. Specific Reaction

initial reaction (//mol/min/mg)

A T E E consumption

3330

ATGGEE appearance

2600

ATGG appearance

310

AT appearance

420

rate

464 Nucleophile: L-arginine ethyl e s t e r A homogeneous reaction medium was obtained when incubating MTEE (163 mM) in the presence of 0.40 M L-arginine ethyl e s t e r (solubility limit: 296 mM). The concentration of a-chymotrypsin was 0.02 g/1. The variation of the concentration of the reactants is described in Figure 3. The specific initial reaction r a t e s for substrate consumption and product appearance (Table 3) were similar to those obtained with glycylglycine ethyl e s t e r as the nucleophile. Maximum concentration of N-L-malyl-L-tyrosyl peptides was achieved after 1 h of incubation (114 mM, corresponding to a MTEE conversion yield o f 7 0 %) and remained constant for up to 5 h of incubation. Nevertheless, MTAEE was, after 2 h of incubation, gradually de-esterified (0.20 mM/min) to produce N-L-malyl-L-tyrosylarginine (MTA). MTGGEE remained stable under the same conditions. A by-product, attributed to N-L-malyl-Ltyrosyl-arginylarginine ethyl ester, was also observed (final concentration of 10 mM).

Figure 3. N-L-malyl-L-tyrosyl peptides synthesis water using L-arginine ethyl ester as the nucleophile.

by

a-chymotrypsin

in

465

Table 3 Specific initial reaction r a t e s for the α-chymotrypsin-catalyzed synthesis of N - L malyl-L-tyrosyl peptides using L-arginine ethyl e s t e r as the nucleophile. Specific Reaction

initial reaction (μιηοΐ/min/mg)

MTEE consumption

170

MTAEE appearance

155

MTA appearance

rate

8

MT appearance

15

MTAEE synthesis differs from MTGGEE synthesis by the higher amount of N-L-malyl-L-tyrosine using L-arginine ethyl e s t e r as the nucleophile, the difference resulting from higher peptide hydrolysis. Finally, the N-L-malyl-L-tyrosylarginine peptides conversion yield is very close to that of N-L-maleyl-L-tyrosylargine peptides reported elsewhere (1). 3.3 5 . chromogenes

aminopeptidase-catalyzed product deprotection

Product deprotection was carried out using a highly purified S. chromogenes aminopeptidase preparation (specific activity: 1400 //moles Aspartame hydrolyzed per min and mg aminopeptidase, pH 6 . 4 , 30°C) under the following conditions:

S. chromogenes aminopeptidase

:

0.24 mg/1

Substrate Tris-HCl pH 8.5 Temperature

: : :

30 mM 50 mM 30°C

The L-malyl residue was liberated at a specific r a t e o f 7 0 #moles/min/mg from MTGGEE and at 25 //moles/min/mg from MTAEE. By comparison, the specific activity of the S. chromogenes aminopeptidase-catalyzed MTEE hydrolysis was 260 //moles/min/mg, suggesting a decrease in the aminopeptidase activity when the C-terminal chain is extended.

466 4.

CONCLUSION

The present paper describes the use solubilizing N-terminal protecting group in synthesis process:

L-malic acid + L-tyrosine ethyl ester

of the L-malyl residue as a a complete enzymatic peptide

S. chromogenes aminopeptidase

N-L-malyl L-tyrosine ethyl ester Nucleophile + a-chymotrypsin

L-malic acid + L-tyrosyl peptides

S. chromogenes ^ninopeptidase

N-L-malylL-tyrosyl peptides

MTEE is a highly water-soluble N-protected form of L-tyrosine, efficiently used by a-chymotrypsin in the kinetically controlled synthesis of peptides. The 5. chromogenes aminopeptidase, unable to hydrolyse L-tyrosyl peptides, is a quite promising catalyst for selective and mild substrate protection and product deprotection. REFERENCES 1 2 3 4 5 6 7

A. Fischer, A. Schwarz, C. Wandrey, A.S. Bommarius, G. Knaup and K. Drauz, Biomed. Biochem. A c t a , 50 (1991) 10/11, S 169. Α. Schwarz, A. Fischer and C. Wandrey, Biomed. Biochem. A c t a , 50 (1991) 10/11, S 193. P. Hermann, Biomed. Biochem. A c t a , 50 (1991) 10/11, S 19. W. Kullman, Enzymatic Peptide Synthesis, C R C Press, Boca Raton, 1987. F . Paul, D. Auriol and P. Monsan, Ann. N.Y. Acad. Sei., 542 (1988) 351. D. Auriol, F . Paul and P. Monsan, Ann. N.Y. Acad. Sei., 613 (1990) 201. D. Auriol, F . Paul, I. Yoshpe, J . C . Gripon and P. Monsan, Biomed. Biochem. A c t a , 50 (1991) 10/11, S 163.

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

467

Lipase-catalyzed resolution of 1,2-diols Aldo Bosetti. Daniele Bianchi, Pietro Cesti and Paolo Golini. Istituto G. Donegani, Via G. Fauser 4, 1-28100 Novara, ITALY.

Abstract: A new, practical procedure for the enzymatic resolution of racemic 1,2-diols la,f has been developed. Lipase-catalyzed hydrolysis and lipase-catalyzed alcoholysis of the corresponding diesters additionally to the enzymatic esterification of the diols, were compared using lipase PS from Pseudomonas cepacia. In hydrolytic reactions lipase PS showed either a low regioselectivity and a poor stereoselectivity. Conversely, esterification reactions, using anhydrides as acylating agents and alcoholysis reactions, using n-propanol as nucleophile in several organic solvents, displayed a higher degree of both chemo- and enantioselectivity.

INTRODUCTION Optically active enantiomers of 1,2-diols are important intermediates in asymmetric synthesis, being valuable chiral building blocks in the industrial preparation of pharmaceuticals, agrochemicals, pheromones and liquid crystals. They are usually prepared via classical resolution or via chiral pool synthesis, starting from naturally occurring 1 products, such as L-aminoacids, (S)-malic acid or D-mannitol . However, these methods are hampered by several disadvantages, such as the need of expensive chemical reagents and sometimes laborious procedures. Looking for a method of general applicability, we focused our attention on the use of enzymes because they can offer significant advantages over classical chemical methods in the 2 synthesis of optically active compounds . A number of reports have been recently published on lipase-catalyzed resolutions of racemic alcohols, either via enantioselective hydrolysis of the 3 corresponding esters in aqueous solutions , or via enantioselective esterification and transesterification in 3 4 organic s o l v e n t s · . The latter method was extended to the

468

resolution of 1,2-diols by using porcine pancreatic lipase (PPL) and aliphatic esters or anhydrides as acylating 5 6 agents » . In these conditions PPL showed a remarkable regioselectivity in the esterification of the primary alcohols 7 but a very poor stereoselectivity . In the present work, we wish to present a new, efficient method to prepare optically active 1,2-diols (la,f) in both enantiomeric forms by lipase-catalyzed resolution using a different enzyme: the lipase PS from Pseudomonas cepacia. We studied the selectivity of this enzyme comparing its behavior in water and in organic media. OH

la,f

la

lb

lc

Id

le

If

MATERIALS AND METHODS

Materials Lipase Amano PS from Pseudomonas cepacia was purchased from Amano Chemical Co. All the organic chemicals used were purchased from Fluka Chemie. The optical rotation was measured with a Perkin Elmer 241 Polarimeter. All hydrolytic reactions were performed with a Metrohm pH-stat. Enzymatic hydrolysis of (R,SÏ-2a A mixture of 3 g (12 mmol) of racemic 2 a and 0.27 g of lipase PS in 50 ml of 0.01 M phosphate buffer, pH 7, was shaken at 30 °C. The pH was kept constant by addition of aqueous sodium hydroxide and the reaction was followed by HPLC. The reaction was stopped extracting the products with ethyl acetate. The products were purified by silica gel chromatography. Adsorption of enzyme on Celite Celite 577 (2g) was washed with water and 0.1 Ν phosphate buffer and then added to a solution of lipase Amano PS (0.5 g, 15000 units) in 10 ml of a 0.1 Ν phosphate buffer. The mixture e was spread on a watch glass and left to dry at 2 5 C with occasional mixing until visible dry. The water content was

469

about 1 % as determined by the Karl Fischer method. Enzymatic esterification of (R,S)-la (R,S)-la (11 mmol) was dissolved in 40 ml of 2-methyl-2-butanol. Acetic anhydrides ( 23.8 mmol ) and lipase PS ( 0.3 g ) supported on Celite 577 ( 1 g ) were added to the e solution, stirring the reaction mixture at 25 C . At the end of the reaction, the solid enzyme preparation was filtered off and the products purified by silica gel chromatography. Enzymatic alcoholysis of (R,S)-2a The reaction was carried out by suspending 0.1 g of Lipase PS supported on Celite 577 in a solution of 1.54 mmol of (R,S)-2a and 12.3 mmol of n-propanol in 10 ml of anhydrous 2-methyl-2-butanol. The suspension was shaken on an orbital shaker at 200 rpm at 30 °C, following the reaction by HPLC. After filtration of the immobilized enzyme, the products were separated using the above procedure. Determination of optical purity The optical purity of compounds la and 2f was determined by HPLC analysis, performed on chiral columns Chiralcel OC and OD (Daicel Chemical Industries, LTD) respectively, using hexane/2-propanol 9:1 (V/V) as eluent, flow rate 0.8 ml/min. The optical purities of compounds lb,c were determined after chemical conversion to the diol la, following literature methods. The absolute configurations were assigned comparing 8 9 the sign of the optical rotation with the reported o n e s · . The optical purity of Id was determined by comparison with the 1 0 literature d a t a .

RESULTS AND DISCUSSION 1,2-Phenylethandiol was chosen as a model compound to study the enzymatic resolution of 1,2-diols. The presence in the molecule of primary and secondary hydroxyl groups allowed us to investigate both the chemoselectivity and the stereoselectivity of lipase PS. It should be noted that the chemical reactivity of both hydroxyl groups is comparable. In a first approach, the resolution of the corresponding diacetate (R,S)-2a was attempted via enzymatic hydrolysis using lipase Amano PS (Scheme 1 ) . The reaction was carried out in phosphate buffer at pH 7, kept constant by pH-stat addition of sodium hydroxide. The hydrolysis was stopped after addition of a molar ratio NaOH/2a = 0.89.

470

Scheme 1 OH

(S)-4a At this step the amounts of diol la, primary monoester 3a, secondary monoester 4a and diester 2a were quantitatively determined to be 20/22/27/31 (see table 1 ) . The products were separated by column chromatography on silica gel and the optical purities were determined by HPLC after alkaline 1 1 hydrolysis to l a . Kinetic studies showed that the rate of hydrolysis of the primary ester moiety was 2.6 times faster than that of the secondary one. When the reaction was allowed to proceed, both monoesters 3a and 4a underwent further hydrolysis to give the racemic diol la, demonstrating the low regioselectivity of lipase PS in these reaction conditions. As shown in table 1, the S isomer of 2a was preferentially hydrolyzed, nevertheless the low optical purities and the heterogeneity of the products were not satisfying for a practical use. In a second approach we investigated the alcoholysis reaction of (R,S)-2a in organic solvent using n-propanol as nucleophile (Scheme 2 ) . The reaction was carried out by suspending lipase PS, supported on Celite, in a solution of (R,S)-2a and n-propanol (molar ratio 1:10) in anhydrous 2-methyl-2-butanol at 30°C. The suspension was shaken at 200 rpm. Scheme 2

(R.S)-2a

(S)"

4a

W-2a

The reaction, stopped after 40 % of conversion, afforded as only products the unreacted (R)-2a and the secondary monoester (S)-4a. As shown in table 1, both compounds were obtained with moderate optical purity. These findings demonstrated that

471

alcoholysis reaction proceeded with much more chemoselectivity than the hydrolysis, although maintaining the poor stereoselectivity displayed in water. In spite of the chemoselectivity of the enzyme, we couldn't recover chemically pure monoester 4a due to an intramolecular non-enzymatic transesterification giving the primary monoester 3a. In a different strategy we studied the lipase-catalyzed esterification of (R,S)-la using acetic anhydride as acylating 11 agent (scheme 3 ) . Scheme 3 OAc

(^^T^OAc (S)-2a

The esterification reaction was performed by adding the lipase, supported on Celite, to a solution of la and acetic e anhydride in 2-methyl-2-butanol at 2 5 C . The suspension was shaken at 200 rpm. Surprisingly the behavior of lipase PS in these reaction conditions was dramatically different than in water. As shown in table 1, the diol la was initially quantitatively transformed into the primary monoester 3a, which was subsequently, stereoselectively acylated to give the diester 2a in the S form and 3a in the R form. No traces of the secondary ester 4a were detected, indicating a higher regioselectivity compared to that displayed by the same enzyme in water. The optical purities of the products, isolated at different degrees of conversions showed that lipase PS was specific for the S enantiomer in both steps of esterification of la, with the enantiospecificity of the esterification of the secondary hydroxyl group being considerably larger than that of the primary one. On the basis of the marked selectivity expressed by lipase PS in the esterification of la in 2-methyl-2-butanol, we extended the above method to the preparative resolution of several racemic 1,2-diols lb,e. The results are summarized in table 1. Substrates bearing an aromatic group l b f d confirmed the regioselectivity and stereoselectivity observed for la. To prove the synthetic potential of these compounds, the diol Id, obtained from the corresponding enzymatically-prepared esters by chemical hydrolysis, was converted into optically pure 3-tosyloxy-l,2-propanediol acetonide, an extremely versatile 10 chiral intermediate in synthetic organic chemistry . Conversely, lipase PS-catalyzed acylation of aliphatic

472

substrates, e.g. le, afforded as intermediate products a mixture of secondary monoester 4e, primary monoester 3e, diester 2e and unreacted diol le with very low optical purities. These findings confirmed the literature data reporting that lipase PS requires the presence of bulky groups on the substrate, such as aromatic or tert-butyl moiety, in order to 12 increase the degree of Stereodifferentiation . Table 1 Lipase PS-catalyzed resolution of 1,2-diols MONOESTER 3

DIOL 1

SUBSTRATE REACTION

YIELOX

CONF.

YIELD*

CONF.

e.ejc

MONOESTER 4 YIELD

CONF.

β.β,Χ

DIESTER 2 YIELD*

CONF.

·.·.*

2a

a

20

S

75

22

S

75

27

S

13

31

R

96

la

b

-

-

-

53

R

77

-

-

-

47

S

93

2a

c

-

-

-

-

-

-

40

S

78

60

R

36

lb

b

-

-

-

55

R

75

-

-

-

45

S

95

lc

b

-

-

-

52

R

85

-

-

-

48

S

92

Id

b

-

-

-

48

S

98

-

-

-

52

R

86

c

-

-

-

100

-

80

-

60

-

40

-

20

-

0

10

20

30

40

50

60

70

80

90

temperature [°C]

Figure 4 . pH-dependence o f lipolytic activi-

Figure 5. Temperature stability o f native

ty o f native and immobilized Rhizopus arrhi-

and immobilized Rhizopus arrhizus lipases.

zus lipases,

ν native lipase I;

Δ native lipase II;

• Duolite ES 762-immobilisate; pH-stat method.

ο native lipases;

·

Duolite ES 762-immobili-

sate; pH-stat method; •

interesterification activ-

ity in a solvent-free substrate blend (after 12h).

511 For the interesterification o f beef tallow and castor oil the immobilisate o f Rhizopus arrhizus lipase on Duolite E S 762 was chosen due to its high lipolytic activity. T o minimize downstream processing and to enhance the product homogeneity, we later chose a solventfree column reactor. Blends o f beef tallow/castor oil (1:1 to 1:3, w/w) were still solid at ambient temperature. T o overcome this problem, the reaction was carried out at 5 0 - 6 0 ° C with a thermostatically controlled substrate feed. The same content o f hydroxylated triglycerides was obtained using the Duolite E S 7 6 2 immobilisate as well as Lipozyme or the native Chromobacterium viscosum lipase [ 1 2 ] . Moreover, the Duolite support allowed to rise the substrate flow to 4 0 ml/h without any loss o f activity (Fig. 6 ) . Under optimized conditions the column was run 1 month with a flow o f 2 0 ml/h, yielding 7 0 0 ml interesterification product/g immobilisate in total.

flow [ m l / h ]

Figure 6 . Dependence o f product formation from Substrate flow. · Duolite ES 762 -immobilisate;

Δ Lipozyme.

weight [ g ]

Figure 7. Spreading properties o f transesterification products. - - - subcutaneous calf fat; - - triglycerides after acidolysis; - — beef tallow/castor oil (1:2, w/w); —

interesterification

product.

In contrast to the initial blend o f beef tallow and castor oil, the interesterified triglyceride mixture is characterized by a significantly enhanced spreading (Fig. 7 ) , leading to liquefaction at room temperature. The high amount o f hydroxylated fatty acids enabled a sulfonation with sulphuric acid for production o f 'turkish red o i l ' , a helpful additive for leather tanning.

512 4. C O N C L U S I O N S

The lipase-catalyzed modification o f beef tallow or subcutaneous calf fat with both, hydroxylated- or branched fatty acids, afforded a liquefied product o f newly structured triglycerides. As a result the processing o f industrial fats is simplified. Moreover, the incorporation o f hydroxylated fatty acids opens the way for further derivatisations. With the goal o f minimized downstream processing in mind, the catalysts were immobilized and the transesterification reactions were carried out at 5 0 - 6 0 ° C in a solvent-free substrate blend.

5. A C K N O W L E D G E M E N T S

This work was supported by a grant from the German Ministry o f Research and Technology ( B M F T Project Nr. 0318877 AS) and from the European Economic Community (BIOT-CT91-0258).

6. R E F E R E N C E S

1 2 3 4 5 6 7 8 9 10 11 12

T . Yamane, in 'Proceedings World Conference on Biotechnology for Fats and Oils Industry', T . H . Applewhite, (ed.), J . Am. Oil Chem. S o c . , (1988) pp 17-22 R . G . Jensen, D . R . Galluzzo and V . J . Bush, Biocatalysis, 3 (1990) 307 F . Ergan, M . Trani and G. Andre, Biotechnol. Lett., 10 (1988) 6 2 9 C. Miller, H. Austin, L . Posorske and J . Gonzlez, J . Am. Oil Chem. S o c . , 65 (1988) 9 2 7 K . D . Haase, A . J . Heynen and N . L . M . Laane, Fat Sei. Technol., 9 (1989) 3 5 0 H . C . Hedrich, F . Spener, U . Menge, H . J . Hecht and R . D . Schmid, Microb. Technol., 13 (1991) 8 4 0 H.H. Weetall, Methods Enzymol., 41 (1976) 134 R . Ruyssen and A. Lauwers, in 'Pharmaceutical Enzymes', E . Story-Scientia, Gent, (1978) pp. 210-213 K . Munzel, J . Büchi and O. Schulz, in 'Galenisches Praktikum', Wissenschaftliche Verlagsgesellschaft, Stuttgart, (1959) pp. 534 M . W . Baillargeon, R . G . Bistline and P . E . Sonnet, Appl. Microbiol. Biotechnol., 3 0 (1989) 9 2 L . Haalck and F . Spener, in 'Downstream Processing in Biotechnology', R . N . Mukherjea, (ed.), Tata-McGraw Hill Publ., Bombay, (1992), in press L . Haalck and F . Spener, Dechema Biotechnol. Conf., 3 (1989) 113

Biocatalysis in Non-Conventional Media, edited by J . Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

513

THERMOLYSIN- AND CHYMOTRYPSIΝ-CATALYSED PEPTIDE SYNTHESIS IN THE PRESENCE OF SALT HYDRATES P. Kuhl, U. Eichhorn and H.-D. Jakubke Leipzig University, Biosciences Division, Department of Biochemistry, Talstraße 33, D-O-7010 Leipzig, Germany

Abstract Thermolysin (EC 3.4.24.4.) suspended in hexane in the presence of N a ^ S O ^ l O H ^ O catalyses synthesis of N-protected dipeptide- and tripeptide amides in good yields. The influence of different mixing conditions including sonification has been investigated. Besides further results of the chymotrypsincatalysed peptide coupling of Y-Ala-Phe-OMe (Y = Z, Boc) with Leu-NH 2 under modified reaction conditions are reported.

Introduction The promising approach of biocatalytic conversions of substrates in non-conventional media invariably attracts the attention of chemists interested in the synthesis of organic compounds. For more than a dozen of years some methodical contributions in this line have been developed in the field of enzymatic peptide synthesis. This is easily understandable, because one of its important aims has been to reverse a protease-catalysed reaction, the equilibrium of which for thermodynamic reasons lies far on the side of what we call the "starting compounds". Medium engineering has proved to be a valuable means of shifting the equilibrium to the peptide product side. Recently we have shown that chymotrypsin suspended in hexane in the presence of Na 2CO3'10H 2O catalyses peptide synthesis from Y-Ala-Phe-OMe (Y = Z, Boc) and Leu-NH 2 in high yields (1,2). In our opinion this non-conventional reaction system, in which all starting compounds are present mainly in the form of undissolved particles, is of special interest. The possibility of using such reaction mixtures should considerably broaden the scope of protease-catalysed peptide

514

synthesis, because it is solvent for the reactants. or peptide derivatives has the selection of conditions

not necessary to find a suitable Poor solubility of some amino acid often been seen as a restriction in for further coupling.

Another problem very often involved in enzymatic peptide synthesis are side reactions due to a relative high molar water content of the reaction medium. However, it is not possible to omit water completely, since enzymes are not active without it. Thus the best should be a balanced situation with a reaction system allowing a diminished and controlled water activity. When a salt hydrate as N a 2 C 0 3 * 10H 2O is added to a dry reaction mixture, it may lose water forming the lower hydrate N a 2C 0 3* 7 H 20 . The released water can be taken up by the enzyme, thus ensuring its catalytic activity. Because ideally the activity of a solid is independent of the amount present, a characteristic water vapour pressure, corresponding to a certain water activity, will be found above the mixture of the two salt hydrate forms of Na 2CO^. Thus, at constant temperature, the water activity will remain fixed, or "buffered", as long as a changing water amount corresponds to the coexistance of the salt hydrate pair (3).

Results and Discussions Continuing our previous studies (2) on the chymotrypsincatalysed reaction of Z-Ala-Phe-OMe with Leu-NH 2 giving Z-Ala-Phe-Leu-NH 2, we were interested in further methodical improvements by varying some reaction conditions. Because the reactants are mainly undissolved in the medium used, sufficient mixing is an important prerequisite for effective coupling. In order to accomplish peptide synthesis at shorter reaction time, we applied ultrasonic treatment using an ultrasonic cleaner (33 kHz, 50 W) . In this way we were able to obtain the tripeptide derivative within 30 minutes in 90 % yield, whereas otherwise a reaction time of 2 hour had been necessary. The hydrolysis product Z-Ala-Phe-OH was found with 3-4 %. A further reduction of the reaction time was not possible without a drastic decrease of the desired coupling product. A reaction volume of 2 ml proved to be optimal. Using 0.3 and 0.2 ml hexane resulted in yields of 69 and 39 % Z-Ala-Phe-Leu-NH 2, resp. Confirming previous results (2) , substitution of N a 2 B 4 0 7 · 1 0 H 2 O for N a 2C O 3" 1 0 H 2O was not successful, even under ultrasonic conditions, giving coupling yields less than 10 %.

515

The sonically assisted reaction of 0.1 mmol Boc-Ala-Phe-OMe with 0.1 mmol Leu-NH 2 to Boc-Ala-Phe-Leu-NH^ in 2 ml hexane in # the presence of 5 mg chymotrypsin and 0.2 mmol N a 2C 0 3 1 0 H 20 gave yields of 59 and 77 % after 30 and 60 minutes, resp. We were not succesful to couple the N-protected dipeptide acid Z-Ala-Phe-OH with Leu-NH 2 in appreciable amounts under the same conditions. In another series of experiments we also applied a hexane/salt hydrate medium for thermolysin-catalysed peptide bond formation. First we studied the model reaction of Z-Phe-OH with Leu-NH^ to Z-Phe-Leu-NHp, but in the presence of we obtained no satisfying results, possibly N a 2C O 3' 1 0 H 2 O because of the alkaline reaction of soda. Therefore we successfully replaced it with the neutral N a 2S 0 4· 1 0 H 2O . Figure 1 demonstrates the time course of this synthesis using two different mixing methods, i. e., magnetic stirring and sonif ication, which in this case gave very similar effects.

100 80 60 40 20

~0

5

10

15 20 Time ( min)

25

30

Figure 1. Time course of the thermolysin-catalysed synthesis of Z-Phe-Leu-NH 2 in hexane in the presence of N a 2S 0 4' 1 0 H 20 at different mixing conditions. Z-Phe-OH and Leu-NH 2 0.1 mmol each; 0.5 mmol N a 2S 0 4* 1 0 H 20 ; 5 mg thermolysin; 2 ml hexane; 40 °C; * magnetic stirring (800 r p m ) ; ο sonification with USG 50 ( 33 kHz, 50 W) Variation of the amount of salt hydrate 1 mmol had only little influence, but N a 2S 0 4' 1 0 H 20 the yield markedly decreased.

between 0.1 and below 0.1 mmol

516 We used the reactants in equivalent amounts and obtain better results with an excess of nucleophile.

did

Increasing the sonic energy resulted in remarkable reaction rates and good dipeptide yields (Fig. 2 ) .

not high

100 80 60 40 20

0

10

20 T i m e ( min)

30

40

Figure 2. Thermolysin-catalysed synthesis of Z-Phe-Leu-NH 2 using ultrasound of different energy. Ultrasound devices: ο USG 50 ( 33 kHz, 50 W ) ; • Labsonic U (20 kHz, ca. 350 W ) ; for other reaction conditions see Fig. 1. Further model reactions under the same conditions comprising other C- and N-components are compiled in Table 1. When Z-Ala-Phe-OH was used as substrate we observed besides the mainly synthesized tripeptide derivative formation of about 3 % of a byproduct, which was identified as Z-Ala-Leu-NH 2. In principle, N-components can also be used in form of its hydrochloride salts. In these cases we employed the basic N a 2 P 0 4 · 1 2 H 2 0 as salt hydrate in order to get the free base of the nucleophile. Table 2 summarizes the results we obtained when D-isomers of C- and/or N-component had been used as reactants in the described reaction medium. Except of Z-L-Phe-D-Leu-NH 2 which, however, was synthesized in a very low yield, only couplings with reactants having both L-configuration were succesful.

517

Table 1 Thermolysin-catalysed synthesis of N-protected dipeptide- and tripeptide amides in sonificated hexane/sait hydrate media. C-component

N-component

Z-Phe-OH

Leu-NHo Phe-NH 2 Met-NH 2 Ile-NH 2

Z-Phe-Leu-NH 2 Z-Phe-Phe-NH 2 Z-Phe-Met-NH 2 Z-Phe-Ile-NH 2

86, 8 4 87 45 a 41

Leu-NH 9

Z-Ala-Leu-NH 2 Z-Pro-Leu-NH 2 Z-Ala-Phe-Leu-NH 2

92 33 78

Ac-Ala-Trp-Leu-NH 2 Ac-Gly-Trp-Leu-NH 2

84 77

II II

II

Z-Ala-OH Z-Pro-OH Z-Ala-Phe-OH Ac-Ala-Trp-OH Ac-Gly-Trp-OH

Il

^

Leu-NH 2 Leu-NHo Il

*

Product

Yield

(%) a

Reaction conditions: C- and N-component o T l mmol each; 0.5 mmol N a 2S 0 4* 1 0 H 20 ; 5 mg thermolysin; 2 ml hexane; 40 °C; a 10 min sonificated, Labsonic U ( 20 kHz, 350 W ) ; using Ncomponent as hydrochloride salt; 0.2 mmol N a 2H P 0 4· 1 2 H 20 ; 20 min sonificated. Table 2 Thermolysin-catalysed synthesis of Z-Xaa-Leu-NH^ (Xaa = Phe, Ala) using L- eand D-isomers as reactants in sonificated hexane/ N a 2 S O 4 1 0 H 2 O suspensions. C-component

N-component

Product

Yield

(%)

Z-L-Phe-OH Z-L-Phe-OH

L-Leu-NH 2 D-Leu-NH 2

Z-L-Phe-L-Leu-NH 2 Z-L-Phe-D-Leu-NH 2

90 3

Z-D-Phe-OH Z-D-Phe-OH

L-Leu-NH 2 D-Leu-NH 2

Z-D-Phe-L-Leu-NH 2 Z-D-Phe-D-Leu-NH 2

0 0

Z-L-Ala-OH Z-L-Ala-OH

L-Leu-NH 2 D-Leu-NH 2

Z-L-Ala-L-Leu-NH 2 Z-L-Ala-D-Leu-NH 2

92 0

Z-D-Ala-OH Z-D-Ala-OH

L-Leu-NH 2 D-Leu-NH 2

Z-D-Ala-L-Leu-NH 2 Z-D-Ala-D-Leu-NH 2

0 0

0.2 mmol N a 2S 0 4· 1 0 H 2O ; other reaction conditions as given in Table 1

518

References 1. 2. 3.

P. Kuhl, P.J. Hailing, H.-D. Jakubke, Tetrahedron Lett. 31, (1990) 5213 P. Kuhl, U. Eichhorn, H.-D. Jakubke, Pharmazie 46, (1991) 53 P. Kuhl, P. J. Hailing, Biochim. Biophys. Acta 1078, (1991) 326

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

519

Lipase Catalyzed Triglyceride Synthesis. The Role of Isomerization R. Lortie, M . Trani and F . Ergan Biotechnology Research Institute, National Research Council Canada, 6 1 0 0 Royalmount Av. Montréal (Qc), H4P 2 R 2 , Canada.

Abstract The synthesis o f triolein from oleic acid and glycerol catalyzed by an immobilized Mucor miehei lipase has been studied. The equilibrium constants for the synthesis o f mono-, diand triglycerides were determined. The rate o f isomerization o f 1,3-diolein and its dependence on oleic acid concentration has been measured. A kinetic study o f the synthesis of triolein from 1,3-diolein and oleic acid showed that the isomerization to 1,2-diolein is the rate limiting step.

1. INTRODUCTION It is known that lipases considered as 1,2-specific can catalyze the synthesis o f triglycerides in concentrated media [ 1 ] . The possible explanations for this are a change in the specificity o f the enzyme caused by the unusual environment or the isomerization o f the 1,3-diglyceride to 1,2-diglyceride which can then undergo a lipase catalyzed esterification. The objective o f the work reported here is to verify the possibility o f isomerization in the case o f triolein synthesis catalyzed by an immobilized Mucor miehei lipase.

2 . MATERIAL AND METHODS 2 . 1 . Equilibrium Composition Different ratios o f oleic acid and glycerol were mixed with 0 . 0 5 g Lipozyme I M - 2 0 (Mucor miehei lipase immobilized on weak anion exchange resins, a gift from Novo Industri) in Eppendorf tubes. The reaction took place in open tubes that were placed in an Eppendorf shaker inside an incubator, thermostated at 6 0 ° C , and the course o f the reaction was followed by HPLC to ensure that equilibrium was reached.

2 . 2 . Time Course of the Triolein Synthesis Stoichiometric amounts o f the substrates - 0 . 1 8 3 2 g (.295 mmol) o f 1,3-diolein (NuCheck-Prep, Elysian, MN) and 0 . 0 8 3 3 g oleic acid (Sigma Co. St. Louis, M O ) - were mixed in an Eppendorf tube, followed by addition o f the 0 . 0 2 5 g o f the immobilized lipase. The

520 three phase system was then stirred vigorously in a chamber thermostated at 6 0 ° C . samples were taken, diluted in 8 0 0 ,uL acetone and analyzed by H P L C .

5

2 . 3 . Time Course o f Diolein Isomerization 0 . 1 8 3 2 g o f 1,3-diolein were weighed in Eppendorf tubes and amounts corresponding to 0.25, 0 . 5 , 0 . 7 5 and 1 times the stoichiometric quantity o f oleic acid were added. The tubes were kept open and agitated in a controlled temperature chamber (T = 60 °C). The isomerization was followed by HPLC.

3. M O D E L L I N G 3 . 1 . Equilibrium I f all the possible equilibria are considered, the following scheme is obtained: κ·2

OA •+ G q

V I

By considering that the activity o f water is constant because o f the equilibrium with the surrounding atmosphere, apparent constants may be written : K,1

y

_

/_

3

[MQJ [G\[OA]

k

h_ 1

[TO]

, _K 2

[G1[CM]

„ _ K 3

[DOl2][OA]

[PQiJ [ 3 / 0 2] [ O 4 ]

[TO] [DOl3][OA]

/ y/ _

[PQi,J [MO,][04]

2

_WK+K» K K

521

[G\o = [ G l [MO\ + [DO] + [ Γ 0 ]

(1)

[ΟΛ] 0 = [OA] + [MO] + 2 [DO] + 3 [ΓΟ]

(2)

+

which can be rewritten in terms o f oleic acid and glycerol only:

[ G ] 0 = [G] ^K,[G][OA] +KlK2[Gi[OAY+KlK&[Gl[OA]'

(3)

2

[OA]0 = [OA] + K, [G][OA] +2KXK2 [G][OA] + 3 J ^ J ^ [G][OA]

3

(4)

This non-linear set o f equations was solved numerically for [G] and [OA] by a GaussNewton method. The estimation o f the equilibrium constants was performed with a multiresponse regression algorithm [2] based on the minimization o f the B o x and Draper criterion [ 3 ] . 3 . 2 . Kinetics For the synthesis o f triolein from 1,2-diolein or 1,3-diolein, very little monoolein formation was observed during the period o f the experiments, so the following triangular reaction scheme was considered for the analysis o f the kinetics o f the reaction. DO,

^

3

A

The isomerisation step was studied separately, as described in the materials and methods section. The variation o f the concentrations o f the two isomers may be written as follows: d[D l 2]

°-

= kJD013] = ki[D012]

-

- kffX), 2]

(5)

kjpo^

(6)

This set o f differential equations was solved with an adaptive step-size Runge-Kutta method. The subroutine written to solve this set was used in the programme performing the multi-response parameter estimation and the rate constants were determined. Since the synthesis o f triolein is slow, it was assumed that the reaction mixture is at equilibrium with the surrounding atmosphere, in which the water activity is constant. It was also assumed that the activity coefficient o f water is constant so that the concentration o f water in the mixture is approximately constant. As a consequence, the rate equations may be written as follows.

522

= [ki[DOlt2] d[DOl2]

= * _ ' 3[ Γ 0 ]

dt d l D l

° *

]

= kjLDOia]

+ k?[DOl3])[OA]

+ M^i, ] 3

- (ki3 + k!!3)[TO]

(7)

(*, + * 3 ' [ O 4 ] ) [ D 0 1 2]

(8)

+ ^[ro] - (t., + #'[α4])[ζ>ο ]

(9)

1ι3

7

= ( * ' 3 + *-3)[7Ό] - (*3 [0Ο1>2] + ^ [ ^

(10)

])[(9A]

1 3

This set o f differential equations was solved with an adaptive step-size Runge-Kutta method. The subroutine written to solve this set was used in the programme performing the multi-response parameter estimation. The equilibrium constants determined earlier were used to link the forward and reverse rate constants and the isomerization rate constants, function o f the oleic acid concentration, determined earlier were used.

4. R E S U L T S 4 . 1 . Equilibrium The equilibrium compositions for different oleic acid/glycerol initial ratios are shown on Figure 1. The maximum concentration o f triolein is obtained for the stoichiometric 1 1 quantities. The curves calculated with the identified parameters ( ^ = 2 . 1 M , K2=5l M ' , 1 ΛΓ3 = 12 M" ) represent the experimental data quite well. 2.5

^

0.0

0.2

0.4

0.6

1.5

0

5

[ G ] 0/ [ O A ] 0 ( - ) Figure 1. Equilibrium concentration as a function o f the initial [G]/[0/4] ratio, ο [OA], ν [MO], Δ [DO], • [ 7 0 ]

10 15 TIME ( h )

20

Figure 2 . Isomerization o f 1,3-diolein.

ο [ D 0 1 3] , · [DOl2].

[OA]/[DO]=0J5

523

The isomerization o f 1,3-diolein is easily measurable, as seen on Fig. 2 , and this isomerization is a function o f the oleic acid concentration, as shown on Fig. 3. The relationships between the isomerization rate constants and the oleic acid concentration are: 1 1 k. = 0.050+0.090[OA] h" and k_x = 0 . 0 3 0 + 0 . 0 4 6 [ O 4 ] h" .

OLEIC ACID

(mmol/mL)

Figure 3. Variation o f the isomerization rate constants as a function o f the oleic acid concentration.

Figure 4 . Time course o f lipase catalyzed synthesis o f triolein from 1,3-diolein and oleic acid, ο oleic acid, Δ 1,3-diolein, • 1,2-diolein, ο triolein.

The results o f the time course o f triolein synthesis from 1,3-diolein are shown on Fig. 4 . The concentration o f oleic acid increases at the beginning o f the reaction, probably because of a rapid hydrolysis o f diolein giving oleic acid and glycerol. T o take this hydrolysis into account, it should be possible to measure the concentrations o f 1-monoolein and 2 monoolein, which is difficult because o f their very low concentrations. Despite this fact, the curves calculated with the simple model used describe quite well the variation o f the different 1 concentrations. The values o f the two identified rate constants are : k3 = 7 . 4 x l O " 2 mL/mmole·h and k'3' = 5 . 2 x l O ' mL/mmole-h. By comparing the constants and the resulting reaction rates with the rate o f isomerization, it appears that, for the amount o f enzyme used, the isomerization is the limiting step for the synthesis o f triolein from 1,3diolein. The fact that simple rate equations may be used to describe enzyme catalyzed reactions probably means that there is a limiting step that can be represented by such equations. It does not give information on the mechanism o f the reaction, only on the general behaviour o f the system.

5. CONCLUSIONS The results presented here show that isomerization o f 1,3-diolein and the enzyme catalyzed esterification o f the freed primary hydroxyl is responsible for the largest part o f the triolein synthesis catalyzed by the lipase from Mucor miehei. They also show that, when looking at the action o f lipases on glycerides, in esterification as well as in hydrolysis, the

524

chemistry o f the glycerides should be taken into account. selectivity o f lipases is investigated.

This is especially true when the

6. R E F E R E N C E S 1 F . Ergan, M . Trani and G. André, Biotechnol. Bioeng., 35 (1990) 195. 2 D . M . Bates and D . G . Watts, Commun. Statis.-Simula. Computa., 13 (1984) 7 0 5 . 3 G . E . P . Box and N . R . Draper, Biometrika, 4 6 (1965) 7 7 .

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. 1992 Elsevier Science Publishers B.V.

525

Resolution of l-benzamido-4-carboxymethyl-cyclopent-2-ene using pig liver esterase M. Mahmoudian, B . S . Baines, M . J . Dawson and G.C. Lawrence Department o f Natural Products Discovery, Glaxo Group Research, Greenford, Middlesex U B 6 0 H E , UK

Abstract l-/?-benzamido-4S-carboxymethyl-cyclopent-2-ene (Ia) is a useful intermediate in the production o f carbocyclic nucleosides. Resolution o f racemic I (ie Ia + lb) has been attempted using pig liver esterase (PLE). The methyl ester was readily hydrolysedrhydrolysis o f the 15, 4/?-ester (lb) being 3-4 times faster than the 1R, 45-ester (Ia) as judged by chiral HPLC. This is in contrast to the work o f Sicsic et al (1) who found the 1R, 4 5 enantiomer o f the closely related substrate l - a c e t a m i d o - 4 - c a r b o x y m e t h y l - c y c l o p e n t - 2 - e n e to be preferentially hydrolysed. Solvents are well known to affect the stereochemical course o f esterase reactions ( 2 , 3 ) . Eleven solvents with a range o f log Ρ values were tested for their effect on the rate and enantioselectivity o f the PLE-catalysed hydrolysis o f racemic I. There appeared to be no systematic relationship between log Ρ and the effect o f the solvent on the reaction. The most interesting effects were found with dimethylsulphoxide ( D M S O ) and methanol. D M S O was found to decrease the relative rate o f hydrolysis o f Ib/Ia in a concentration dependent manner. Indeed, at 5 0 % (v/v) D M S O , Ia was preferentially hydrolysed. Methanol, on the other hand, significantly increased the enantioselectivity o f the reaction. At 1 0 % (v/v) methanol, the ratio of the hydrolysis rates o f Ib/Ia was approximately doubled. Although absolute enantiospecificity was not obtained under any o f the reaction conditions tested here, the residual ester (Ia) o f correct conformation for synthesis o f 'natural' carbocyclic nucleosides, could be obtained in high optical purity. For example, Ia could be obtained in 9 0 % ee ( 6 5 % yield) by allowing the reaction to proceed to 6 5 % conversion, or 9 5 % ee ( 5 8 % yield) at 6 9 % conversion. Higher optical purities, albeit with lower yield, could be obtained by allowing the reaction to proceed further.

526 1.

INTRODUCTION E n z y m e s are widely used in synthetic organic chemistry for the enantioselective hydrolysis o f esters (4). Hydrolytic enzymes often display low substrate specificity, are robust, and do not require coenzymes. However, these enzymes often display only a limited degree o f enantioselectivity with non-physiological substrates. Here we describe the use o f pig liver esterase to resolve the carbocyclic nucleoside intermediate, l-benzamido-4-carboxymethyl-cyclopent-2-ene (I), (Fig 1). The effect of solvents has been studied and methanol was found to improve the enantioselectivity o f the enzyme.

(la)

(lb) Fig. 1 2.

Structure o f enantiomers o f l-benzamido-4-carboxymethyl-cyclopent-2-ene

M A T E R I A L S AND M E T H O D S Pig liver esterase ( 2 0 0 units/mg protein) was obtained from Sigma Chemical Co Ltd. Enzyme reactions were carried out at 2 0 ° C in 5-10 ml volumes containing substrate (1 mg/ml), enzyme (5-10 U/ml) and C a C ^ (2 mM). The pH was maintained at 8.0 using a pH-stat (Metrohm Herisau, Switzerland), or by inclusion o f Tris/HCl buffer (100 mM). Reaction mixtures were deproteinated by ultrafiltration and analysed by chiral HPLC on a chiral-AGP column (10 cm χ 4 mm, Technicol, Stockport, Cheshire) using 5% (v/v) propanol, 25 mM N a H 2 P 0 4 (pH 5.0) as the mobile phase. The flow rate and detection wavelength were 0.5 ml/min and 2 3 0 nm, respectively.

527

3.

R E S U L T S AND D I S C U S S I O N T i m e course o f Hydrolysis Figure 2 shows the time course o f hydrolysis o f I using P L E . The 1.S, 4/?-ester (lb) was hydrolysed approximately 3 times more rapidly than the IR, 45-ester (Ia). Although the degree o f enantioselectivity was modest, the enantiomeric excess o f the residual ester (Ia) increased during the reaction and 'optically pure' product could be obtained by allowing the reaction to proceed further.

Fig. 2

Resolution o f I using P L E . The reaction was carried out in a pH-stat with 5 U PLE/ml.

Effect o f Solvents In an attempt to improve the enantioselectivity o f the reaction, the effect o f the addition o f solvents ( 1 0 % v/v) with a range o f log Ρ values was investigated. The effect on the reaction rate and degree o f enantioselectivity is shown in Table 1. There appeared to be no systematic relationship between log Ρ and the effect o f the solvent on the reaction. D M S O and methanol were chosen for further study on the basis o f good relative enantioselectivity and specific activity.

528 Table 1

+

Solvent 1 0 % (v/v)

Log P

None

-

1666

4.0

52

Acetonitrile

-0.33

1494

4.1

50

Methanol

-0.76

544

7.5

56

1,4-Dioxan

-1.1

1129

2.4

40

DMSO

-1.3

1598

3.4

50

n-Propanol

+0.28

129

4.5

28

Tetrahydrofuran

+0.49

306

4.4

44

Toluene*

+2.5

2720

2.7

27

+3.5

1938

3.5

50

* «-Hexane

Specific activity (nmole/min/mg)

Ratio of initial rates of hydrolysis of Ib/Ia

% ee Ia at 5 0 % conversion

+ Partition coefficient in a standard octanol-water biphasic system. Indicates biphasic systems Reactions were carried out in stirred vials (5-7 ml volume) buffered with 100 mM/Tris/HCl (pH 8) with 1 mg/ml substrate at 20°C. The enzyme concentration was 10 U/ml. Analyses were carried out by chiral HPLC. There was no reaction with n-butanol, phenol or chloroform.

529

Effect of D M S O Increasing concentrations o f D M S O were found to decrease progressively the degree o f enantioselectivity o f the reaction (Fig. 3 ) . Interestingly, at 5 0 % (v/v) D M S O the stereochemical preference o f the reaction was reversed and the IR, 45-ester (Ia) was preferentially hydrolysed.

lb

% conversion Fig. 3

Effect o f D M S O concentration on the enantioselectivity o f PLE-catalysed hydrolysis of I. The reactions were carried out in a pH stat with 5 U PLE/ml. DMSO concentrations (% v/v) as shown.

Effect of Methanol I n c r e a s i n g c o n c e n t r a t i o n s o f m e t h a n o l p r o g r e s s i v e l y i n c r e a s e d the degree o f enantioselectivity o f the reaction (Table 2 ) . The improvement in enantioselectivity was balanced by a decrease in the specific activity o f the enzyme. The time-course o f the reaction at 5 % (v/v) methanol is shown in Figure 4. The addition o f methanol allowed la to be obtained at 9 0 % ee after approximately 6 5 % conversion ( 6 5 % yield) compared with 7 5 % conversion ( 4 8 % yield) in its absence (cf Fig. 2 ) .

530

Table 2

[Methanol] % v/v

Specific activity (nmole/min/mg)

Ratio of initial rates of hydrolysis of Ib/Ia

%ee l a at 5 0 % conversion

0

1211

3.0

50

1

597

4.9

58

5

364

5.5

58

10

297

6.2

n.d.

The reactions were carried out in a pH-stat with 5-10 U PLE/ml. (n.d. = not determined)

Fig. 4

Resolution o f I using PLE in the presence o f 5% (v/v) methanol. The reaction was carried out in a pH-stat with 10 U PLE/ml and 5% (v/v) methanol.

531

4.

CONCLUSIONS P i g l i v e r esterase c a t a l y s e s the e n a n t i o s e l e c t i v e hydrolysis o f l - b e n z a m i d o - 4 carboxymethyl-cyclopent-2-ene (I). The degree o f enantioselectivity can be increased by the addition o f methanol to the reaction mixture. Although enantiospecificity was not achieved, this method can be used to prepare the chiral intermediate (Ia) in good optical purity. Acknowledgements W e would l i k e to thank D r C. M e e r h o l z and c o l l e a g u e s in P r o c e s s R e s e a r c h Department, G G R for supply o f I and Ia, and Mrs J . Cooper for development o f the chiral HPLC method. References

1.

Sicsic, S., Ikbal, M. and L e Goffic, F . Tetrahedron Lett. 2 8 , (1987) 1887-1888.

2.

Lam, L.K.P., Hui, R.A.H.F. and Jones, J . B . J . Org. Chem. 5 1 , (1986) 2047-2050.

3.

Boutelje, J . , Hjalmarsson, M., Huit, Κ., Lindback, M. and Norm, T. Bioorg. Chem. 16, (1988) 364-375.

4.

Jones, J . Enzymes in Organic Synthesis: 3rd International Conference on Chemistry and Biotechnology o f Bilogically Active Natural Products 1, (1987) 18-39.

5.

L a a n e , C , B o e v e n , S., Hilhorst, R . and V e e g e r , C. Biocatalysis in Organic Media (Laane, C , Tramper, J . and Lilly, M.M. (ed.) pp65-84 (1987). Elsevier, Amsterdam.

Biocatalysis in Non-Conventional Media, edited by J . Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

533

α-SUBSTITUTED PRIMARY ALCOHOLS AS SUBSTRATES FOR ENANTIOSELECTIVE LIPASE-CATALYZED TRANSESTERIFICATION

IN

ORGANIC SOLVENTS Enzo Santaniello, Patrizia Ferraboschi, Paride Grisenti, Ada Manzocchi Dipartimento di Chimica e Biochimica Medica, Universita' di Milano, Italy Summary Primary alcohols containing at the α-carbon small groups such as methyl or an oxirane ring are efficiently and with high enantioselectivity resolved by an irreversible transesterification reaction with vinyl acetate in chloroform or dichloromethane, catalyzed by a lipase from Ρ seudomonas fluores cens. Introduction Enantiomerically configurations

pure

can

transesterification

be of

alcohols prepared

racemic

and

esters

by

an

alcohols

of

opposite

enzyme-catalyzed [1]·

One

of

the

available procedures uses hydrolytic enzymes such as lipases as the biocatalysts method

is

quite

irreversible,

and vinyl

esters

effective

since

because

rapidly tautomerizes therefore

one

of

the

the

to acetaldehyde

prevented.

[2,3].

as acyl donors

Additionally,

This

transesterification

products,

vinyl

is

alcohol,

and the back reaction the

reaction

can

is be

efficiently carried out in organic solvents (Eq. 1 ) .

Enzyme R-CH 2OH

+

CH 2=CH-OCOCH 3

•

R-CH 2OCOCH 3

+

CH 3CH0

(1)

solvent

We

have

alcohols the

used

the

above method

which bear

general

a chiral

structure

for

carbon -

the

resolution

of

primary

at the alpha-position

R-(CH2)n CH(CH3)CH2OH.

The

lipase

of

used

534 was

Pseudomonas

from

were

fluorescens

dichloromethane

acylating

or

(PFL),

the

and

vinyl

chloroform

organic

solvents

acetate

was

the

reagent.

Materials and Methods Ρseudomonas fluorescens lipase (PFL) and all the c h e m i c a l s w e r e p u r c h a s e d from Fluka (Buchs, S w i t z e r l a n d ) . The o p t i c a l p u r i t i e s w e r e d e t e r m i n e d by m e a s u r i n g the o p t i c a l r o t a t i o n s on a P e r k i n - E l m e r M o d e l 241 P o l a r i m e t e r and by ^H-NMR a n a l y s i s of the esters w i t h (-)-MTPA c h l o r i d e [ 4 ] . The •'-H-NMR s p e c t r a w e r e r e c o r d e d in CDCI3, u s i n g SiMe4 as i n t e r n a l s t a n d a r d . The 200 and 500 M H z -'-H-NMR spectra w e r e r e c o r d e d on V a r i a n XL 200 and AM 500 B r u k e r s p e c t r o m e t e r s .

PFL-Catalyzed Transesterification: General Procedure. The e x p e r i m e n t a l p r o t o c o l w a s e s s e n t i a l l y i d e n t i c a l to the method d e s c r i b e d by W a n g et al. [ 3 ] . To a solution of the (R, S)-alcohol (0.01 m o l ) in m e t h y l e n e c h l o r i d e (20 m L ) , v i n y l a c e t a t e (3.6 m l , 0.044 m o l ) and PFL (Fluka, 0.14 g, 31.5 U / m g ) w e r e added. The s u s p e n s i o n w a s kept at 30 °C for the t i m e necessary to reach 40% or 60% conversion to acetate, r e s p e c t i v e l y . The enzyme w a s r e m o v e d by f i l t r a t i o n and the m i x t u r e of the o p t i c a l l y a c t i v e a l c o h o l and the c o r r e s p o n d i n g acetate was obtained after evaporation of the solvent. P u r i f i c a t i o n on silica gel column c h r o m a t o g r a p h y a f f o r d e d the a c e t a t e and the a l c o h o l (hexane/ethyl a c e t a t e , as e l u a n t s ) w i t h a ratio d e p e n d i n g on the i n c u b a t i o n t i m e .

Results and Discussion The high

PFL-catalyzed

enantioselectivity

4-phenylselenyl,

and

w e r e the s u b s t r a t e s f#)-alcohol In

transesterification

order

and

to

enantiomeric

brought

about

(>98%

to

with

S)-4-phenylthio,

(R,

4-phenylsulphonyl-2-methylbutanol

a

preparation

of

the

60%

conversion

(R)-alcohol is d e s i r e d

to

the

(40% y i e l d )

(Eq. 2, alcohol

for

acetate.

is n e a r l y

In

Table 1 ) . with

be

this

way,

optically

pure

H - N M R a n a l y s i s of its M T P A (sJ-acetate,

the

has to

1

the

la-c

corresponding

e x c e s s , the t r a n s e s t e r i f i c a t i o n

e e ) , as e s t a b l i s h e d by >98% ee

when

f s ) - a c e t a t e could be p r e p a r e d

highest

If a

ee)

achieved

[ 5 , 6 ] . By this p r o c e d u r e , the

achieve

the u n r e a c t e d

(>98%

was

the

ester.

reaction

has

to be stopped at 4 0 % c o n v e r s i o n and the r e c o v e r y of the p r o d u c t is e s s e n t i a l l y q u a n t i t a t i v e The since

above

the

synthons.

examples

chiral In

(38-40%).

are

of

special

compounds

so

far

fact,

synthetic

obtained

enantiomerically

are pure

significance, useful

chiral

(R)-

and

535

PFL,

r

CH 9=CH-0Ac

OH

OH

• OAc

(2)

CHC1, (R,S)-1

(S)-2

(R)-l a.

R=CH 2SPh

d.

R=CH 20CH 2Ph

b.

R=CH 2SePh

e . R=Ph

c.

R = C H 2S 0 2P h

f.

R=CH=CH-CH Q

Table 1 ACETATE (CONFIGURATION)

SUBSTRATE

ALCOHOL (CONFIGURATION)

a

R

98

s

98

b

R

>98

S

98

c

R

98

s

98

d

R

98

S

85

e

R

>98

S

90

f

R

>98

S

>98

( •S y)-4-phenylthiole

are

C-5

moiety

in

chiral

building

Due

to

molecule,

the we

presence

have

namely,

and

alcohols

with

resolved

the

compounds

fSj-acetate

by

la-c, 2d

[7].

was

(>98%

We

and have

(25S)-26-hydroxy

the

phenylselenyl (R)-

transformed

into

la

two

useful

and

and chiral

2-methyl-l,3-

of high ee [ 6 ] . same

structural

the

4-Benzyloxy-2-methylbutanol of

lb

2-methyl-3-butenol

propanediol derivatives Other

of

easily

C5 /)-4-phenylselenyl-2-methylbutanol synthons,

blocks

( S ) - i s o m e r for a s y n t h e s i s of

[8]. the

ee%

4-phenylsulphonyl-2-methylbutanol

isoprenoid

a l r e a d y used the cholesterol

or

ee%

same Id,

nicely ee).

The

o b t a i n e d also from the r a c e m i c

same

alcohols

were

enzymatic

strictly

resolved

feature

related

into

prepared reaction.

to

the

(RJ-alcohol

positive le and

If.

results

series Id

and were

536

Optically

pure

derivatives

of

2-methyl-l,3-propanediol

3a-e

could also be prepared by the same biocatalytic approach. Thus, the

enantioselective

acetylation

of

the

racemic

silyl

ethers

of

2-methyl-l, 3-propanediol, compounds 3b and 3c, afford >98%

ee

(Sj-alcohols

applied

3b

and

also to the

3c.

The

3-benzyl

same

ether

stereochemical

3d and the

outcome

3-benzoate

When the racemic monoacetate of 2-methyl-l,3-propanediol substrate, the unreacted >

98%

ee)

when

the

can be isolated

(S)-monoacetate

reaction

is

brought

about

to

3e.

is the (38%,

60%

of

diacetate [9] (Eq. 3, Table 2 ) .

PFL, RO

OH

CH=CH 2-OAc

I

CHCI3

I

RO

+

I

OH

(R,S)-3

RO

3

OAc 4

a.

R=C0CH 3

b.

R = S i P h 2t B u

c.

R = S i ( C H 3) 2t B u

d.

R=CH 2Ph

e.

(3)

I

R=C0Ph

Table 2

SUBSTRATE

ALCOHOL

ee%

(CONFIGURATION)

ACETATE (CONFIGURATION)

ee%

a

s

>98

-

-

b

s

>98

R

98

c

s

98

R

98

d

S

90

s

85

e

s

85

R

90

From the above results, it appeared that the structural feature common

to the alcohols

1 and

3 is that

they are all

primary

alcohols bearing a methyl group at the α-position. Therefore, we

decided

to

extend

our

observation

to

other

similar

537 substrates. (2R)-

Thus,

enantiomerically [10].

Also

virtually the

data

from

by

no

same

useful

chiral

could

on

the

the

PFL

other

formed.

From

substrates

in

an

la-e

organic

are a v a i l a b l e

lipases,

this

serine

for a n o t h e r

and

an be

on

the

enzyme

hydrolytic

large

apolar

hydrophobic R,

and,

a methyl

be

order at

the

pig

promote

recently

liver

shown.

This

integrated to

t h e PFL a c t i v e

define

carbon

( H L)

if

chirality

more

atom

this

can

will

are

a

of the

proposed

esterase

body

further

of

(PLE),

( H s)

and

the

the

pattern

in

other of

group,

as

such

however,

a

size

in way

will needs

substrates,

the

Hs

long-chain

formed a c e t a t e

informations,

hydroxy

a

contains,

oriented

on

organic

i n d i c a t e d as H L and

center

be

studies

properly

bearing

i n an

accomodate

of the e n z y m a t i c a l l y

initial by

the

site

pockets

pocket

group

that the c o n f i g u r a t i o n

to

i.e.,

be

derivative

two h y d r o p h o b i c p o c k e t s of small

Large and s m a l l h y d r o p h o b i c

group

case,

b e as

enzyme,

can

irreversible

should

1 ) . As

catalytic

also

acetyl

nearby

(Figure

1. A p o s s i b l e m o d e l o f

solvent.

our

the

of

( H L) size [ 1 2 ] .

Figure

The

base

process

our m o d e l m a y p r e s e n t large

a

afford

the

bulk 3a-e,

procedure

can

le,

the

could

transacetylation catalytic

procedure

and

[ 1 1 ] . T h u s , the acyl d o n o r in the

the

prepared

solvent

serine h y d r o l a s e

transesterification

synthons

be

transacetylation

was

informations

for

If

2-methyl-4-phenylbutanol (R)-le

us

for

as

the

pure by

model

PFL,

synthetically

racemic

collected

drawn. Although of

pure

optically

active-site

site

the

4£-2-methyl-4-hexenol

(2S)-

and

in

substitution and

number

538

of

substituents

structural

at

d e s i g n of the m o s t

of

the

of

and

any

relevance

for

the

2-benzyl-

and

3)

2-Substituted

2-nonyl

PFL,

PFL-catalyzed

oxiranemethanols

other

for

the

site.

of the a b o v e m o d e l , w e s y n t h e t i z e d

substrates

[13].

ß-carbons,

and

substrates

s u i t a b l e m o d e l of a c t i v e

As an a p p l i c a t i o n as

a-

the

feature

and

tested

transesterification

5a

and

oxiranemethanols

5

5b

(Eq.4,

can

be

Table

considered

CH 2=CH-OAc

•

(4)

CHCI3

I

(S)-5

(R,S)-5 a.

(S)-6

R=PhCH 2

b.

R = n C gH 11

Table 3 ALCOHOL (CONFIGURATION)

SUBSTRATE

S

98

S

98

b

S

98

S

98

carbon at

useful

constituted 60%

building

by

an

conversion

(sj-(-)-alcohol corresponding

acetate, (R)-(

group

r i n g could

methyl fit

of

procedure

oxiranemethanol synthesis

to

us,

applies

of

At

the

the

a

its

a p o l a r p o c k e t H L,

long

chain

but

is

an

>98% indeed

same

to the p r e p a r a t i o n chiral

placed

of

intermediate

malyngolide

be

that,

oxirane

of our m o d e l . The

and

5b w a s e s p e c i a l l y could

the

showed

3,

the

to

obtained,

5a,

1 and

valuable

antibiotic

epoxyalcohol obtained

This result

compounds Hs

quaternary

the we

was

successfully 5b,

a

conversion

(s)-(+)-6a

[ 1 4 ] , This e p o x y a l c o h o l

because

6a,

40%

small p o c k e t

(S)-nonyl the

From

is S as the a l c o h o l

enzymatic

compounds

ring.

+ )-alcohol 5a.

into the

bearing

acetate

compound 6a

as

the

the

>98%ee.

the a c e t a t e of the for

blocks

oxirane

to

5a,

ee. H e r e , the a c e t a t e

for

ee%

a

synthetically

5a,

ACETATE (CONFIGURATION)

ee%

in

thus a d d i n g some o t h e r s t r u c t u r a l

related

interesting the

large

information

539

about

our

model

of

active

site.

We

were

able

to

prepare

and (S)-(+)-6b in 3 5 - 3 8 % y i e l d s and 9 8 % e e .

(S)-(-)-5b

Conclusions The organic of

irreversible solvents

α-methyl

which

optically

pure

stereochemical is

seems

especially

substituted

compounds

model

PFL-catalyzed primary

for

outcome

proposed

oxiranemethanols

5

for

the

of

been

found

to

1 and 3. T h e are

examined is

by

fit

A

action in

the

nearly

us, the

constant.

catalytic

in

resolution

procedure

substrates

enzyme

for t h e

of type

by this

the r e a c t i o n

the

have

suitable

alcohols

can be p r e p a r e d and,

transesterification

simple

and

also

postulated

a c t i v e site, so that e n a n t i o m e r i c a l l y p u r e 5a-b and 6a-b can b e prepared.

Acknowledgements.We t h a n k M i n i s t e r o Scientifica e Tecnologica

dell'Universita'

e

Ricerca

(MURST) for f i n a n c i a l help, M s . E l i s a

V e r z a and M r . C a r l o C a v a r r e t t a for t e c h n i c a l

assistance.

REFERENCES 1.

Cambou,

B.; K l i b a n o v ,

2.

Degueil-Castaing,

A . M . J. Am. Chem.

Soc. 1984, 106,

2687-2692. M.;

De

M a i l l a r d , Β . Tetrahedron 3.

Wang, D.E.;

Y. F.; L a l o n d e , Wong,

C.

H.

Jeso,

J. J.;

Drouillard,

S.;

1987, 28, 9 5 3 - 9 5 4 .

Lett.

J.

B.;

Momongan,

Am.

Chem.

M.;

Bergbreiter, 1988,

110,

Org.

Chem.

Manzocchi,

Α.;

Soc.

7200-7205. 4.

Dale,

J.

Α.;

Dull,

D . L.; M o s h e r ,

H.

S.

J.

1969, 3Λ, 5 2 4 3 - 5 2 4 9 . 5.

Ferraboschi,

6.

Ferraboschi,

7.

Fuganti,

P.;

Santaniello, 1990, Chem.

Grisenti,

E . J. Org. Chem. P.;

Grisenti,

P.;

1990, 55, 6 2 1 4 - 6 2 1 6 .

P.;

Santaniello,

E.

Synlett

545-546. C ;

Grasselli,

Soc., Perkin

Trans.

P.;

Servi,

S.; H o g b e r g ,

1 1988, 3 0 6 1 - 3 0 6 5 .

H. E .

J.

540

8.

Ferraboschi, P.; Fiecchi, A. ; Grisenti, P.; E. J. Chem.

9.

Soc,

Santaniello, Tetrahedron

Perkin

E.; Lett.

1, 1987,

Trans.

Ferraboschi, 1990,

31,

P.;

5657-5660;

Santaniello,

1749-1752.

Grisenti, ibid.

1991,

P. 32,

430. 10.

Ferraboschi,

P.;

Brembilla,

11.

Maraganore, J. M.; Heinrikson, R. L. T r e n d s

Santaniello, E. Synlett 1986, 12. 13.

P.;

1991, 310-312. Biochem.

Sei.

E. J.;

Werth,

M. J. ; Jones, J. B. J. Am.

Chem.

1990, 2 2 2 , 4946-4952.

Ferraboschi,

P.;

Brembilla,

Santaniello, E., J. Org. Chem., 14.

Grisenti,

497-498.

Toone, Soc.

D.;

Giese, B.; Rupaner, R. Liebigs

D.;

Grisenti,

P.;

1991, 56, 5478-5480. Ann. Chem.

1987, 231-233.

Biocatalysis in Non-Conventional Media, edited by J . Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

541

SOLUBLE AND IMMOBILIZED SACCHARIDASES IN WATER-MISCIBLE ORGANIC SOLVENTS R. Ulbrich-Hofmann

a

and B. Selisko

a

Martin-Luther university, Department of Biochemistry/ Biotechnology, Weinbergweg 16a, D-0-4050 Halle, Germany D

University of London, Royal Free Hospital School of Medicine, Department of Molecular Cell Pathology, Rowland Hill Street, London NW3 2PF, UK

SUMMARY For application of enzymes in organic solvents, two different effects of solvents must be differentiated: denaturing influences and changes of the catalytic parameters. Studies on glucoamyläse and invertase in mixtures of buffer and aliphatic alcohols or other water-miscible solvents demonstrate that both the effects are influenced by solvents in a non-parallel way so that they have to be analyzed separately. Irreversible denaturation is mostly small at solvent concentrations lower than 30%, whereas it drastically increases above 50%. In most systems studied, immobilization of the enzymes to polystyrene or silica matrices does not improve their stability towards solvent denaturation but prevents protein precipitation and allows the usage of enzymes at solvent concentrations higher than 50%. The kinetic characterization of the enzymes in the buffer-solvent mixtures shows above all increases of the values. In the case of invertase, substrate inhibition is not observed in the presence of alcohols, dioxane or dimethylformamide.

INTRODUCTION The catalytic efficiency of enzymes in organic solvents is mostly reduced in comparison with their efficiency in aqueous solutions. Reasons for this can be changes in the catalytic parameters K M and V or the reduction of the concentration of active enzyme molecules by reversible or irreversible denaturation. For application of enzymes in non-aqueous media.

542

a differentiation of these effects is important. Irreversible enzyme denaturation is especially disadvantageous, because it destroys enzyme activity irreparably, whereas partial reversible denaturation and reduction of the catalytic power can be compensated for by long reaction times. So far, the interplay of these effects has been rarely considered. With regard to non-conventional enzymatic reactions such as the synthesis of unusual oligosaccharides [1] or the alcoholysis of saccharides to obtain saccharide derivatives [2], the reactivity and stability of invertase and glucoamylase were studied in mixtures of buffer and aliphatic alcohols or other water-miscible solvents. The influence of immobilizing the enzymes to macroporous carriers such as functionalized polystyrene-divinylbenzene copolymers or silica on the solvent effects was tested. Differences in the behaviour of these enzymes , especially in stability, were expected because of the different carbohydrate content in their protein structure (48% in invertase, S. cerevisiae, 10.2% in glucoamylase, A. nigrer) [3] .

RESULTS AND DISCUSSION

Contribution of irreversible inactivation substrate conversion in organic solvents

to

the

reduced

The conversion of starch or maltose by glucoamylase and of sucrose by invertase was determined in a number of 50 or 30 % (v/v) water-miscible solvents before and after immobilization of the enzymes. In order to estimate the contribution of enzyme denaturation to reduced conversion, compared with the reaction in buffer, irreversible inactivation of the samples occurring during the time of assay was determined separately. In dependence on the individual solvents, great differences in the conversion rates and the extent of irreversible inactivation were observed. The tendencies, however, were the same for invertase and glucoamylase, for soluble as well as for immobilized enzymes. In Table 1 they are demonstrated with immobilized glucoamylase as an example. The contribution of irreversible denaturation to observed activity losses is zero in the case of glycerol and ethylene glycol, which are known as enzyme stabilizers [7], whereas it is greatest in acetonitrile and dimethylformamide. In all cases, however, the activity was much more reduced than caused by irreversible denaturation. Reversible denaturation and changes of the catalytic parameters might be the reason for this additional reduction of activity. The great differences between the conversion rates measured by different substrates such as soluble starch or maltose (Table 1) as well as the results of Table 2, however, suggest that changes in the catalytic properties are more responsible for reduced activities than reversible denaturation.

543

Table 1 Contribution of irreversible of immobilized glucoamylase Solvent

inactivation to observed activity

Activity (%) in the presence of 50% solvent to soluble starch (or maltose)

none glycerol ethylene glycol methanol ethanol acetonitrile dimethylsulfoxide dioxane dimethylformamide

100 21,.1 28,.4 54..9 46,.7 29,.2 20,.4 19..0 5,.9

Remaining activity (%) after solvent contact (irreversible denaturation) 100 100.9 104.0 86.6 93.8 77.2 95.1 90.0 84.5

(100) (53.3) (58.4) (76.9) (76.3) (53.3) (35.2) (69.2) (34.2)

Glucoamylase (A.niger) was bound to aminomethyl polystyrenedivinylbenzene copolymer by glutaraldehyde [4] . The enzyme activity in 50% (v/v) solvent/25 mM sodium acetate buffer, pH 4.5, was determined by incubation with 1% soluble starch (Zulkowsky) or 11.6 mM maltose at 30°C for 20 min and measuring the increase of reducing groups [5] or released glucose [6] . For measuring irreversible inactivation, enzyme samples were incubated with 50% (v/v) solvent/25 mM sodium acetate buffer , pH 4.5, at 30°C for 20 min, then filtered, washed with water and assayed with 1% starch as described above but in pure buffer. Irreversible concentrât ion

inactivation

in

dependence

on

solvent

The resistance of glucoamylase and invertase to irreversible damage of protein structure by different organic solvents was studied in dependence on solvent concentration. Furthermore, the influence of different immobilization methods (aminopropyl silica or aminomethylpolystyrene-divinylbenzene copolymer as carrier matrices, glutaraldehyde or trichlorotriazine as coupling methods) on the solvent stability of the enzymes was invest i gated. The results reveal the individual character of denaturation dependent on the special solvent and the special enzyme. Nevertheless, there were some common features, which are demonstrated for the denaturation in aliphatic alcohols in the Figures 1 and 2. In all cases, small alcohol concentrations (

diglyceride + water

(2)

— t r i g l y c e r i d e + water

(3)

^

558

Compared to the other reaction components, an excess of triglycerides can be obtained only when the water produced is removed continuously. This is found in literature for batch experiments [2]. In this system the reaction medium was dried using free evaporation or vacuum, among others. The system changes from a two-phase system into a one phase system. When a mixture of fatty acids, mono-, di- and triglycerides is chosen as substrate for the enzymatic esterification, there is no need to add glycerol and a one-phase system is obtained (reactions 2 and 3). However, if the enzyme is immobilized onto a resin, often water accumulates in the resin which results in the hydrolysis of the ester-bonds. This results in an equilibrium state in which mono- and diglycerides are present [4], The aim of this study is to produce an excess of triglycerides relative to the other reaction components. Therefore, a new membrane reactor concept is developed (figure 1). In the upper two-phase membrane device, a mixture of mono-, di- and triglycerides is produced [3]. A second reactor is placed in series. At one side of the membrane, the non-polar phase is brought into contact with the enzyme. At the other side of the membrane an extraction phase, air, is pumped through to remove the water produced, thus avoiding water accumulation in the immobilization carrier resulting in a reaction that should proceed to an equilibrium state of pure triglycerides (figure 1). This paper shows preliminary results of the triester synthesis in a batch membrane system at various water activity conditions. Emphasis will be laid to the equilibrium concentration which can be achieved as a function of the water activity. Also the stability of lipase at low water activity conditions will be discussed.

559

glycerol/water

ι lipase }

membrane reactor

fatty acids

glycerides/fatty acid

membrane reactor ψ excess triglycerides condenser lipase

air

Figure 1.

A membrane reactor design for the production of a surplus of tnacylglycerols.

EXPERIMENTAL

Experiments were performed in batch systems only. The membrane selection and mass transfer characteristics will be discussed elsewhere [ 5 ] . A cellulose hollow fibre device (ENKA) is chosen as reactor unit, Candida rugosa lipase is immobilized onto the membrane ( 2 5 ° C ) . The non-polar phase is produced in an emulsion system starting with a 0 . 7 4 mole.mole'l decanoic acid in hexadecane non-polar phase, a 0 . 4 mole fraction glycerol water phase ( a w = 0 . 5 ) and Candida rugosa lipase [ 3 ] , Only small amounts of triester are formed. The equilibrium solution is brought in the pervaporation reactor at various water activities. The water activity is varied as follows: In the condenser, the air stream is saturated with water at a reduced temperature. Before the air stream enters the reactor, the temperature of the air phase is adjusted to 2 5 ° C , hence the water activity decreases. By varying the temperature in the condenser, any a w can be obtained in the reactor. The water activity of the inlet air stream is determined by measuring the dew-point using a silicon sensor (Mitchell Instruments).

560 R E S U L T S AND D I S C U S S I O N Equilibrium Measurements T h e equilibrium mole fractions are measured as a function of the water activity (figure 2 ) . An excess o f triacylglycerols is obtained only at low water activities ( a w < 0.1). This is in agreement with model calculation using the program T R E P [6]. Calculations can be made without water production during synthesis, thus simulating that the water produced is removed instantaneously. T h e s e calculations show that only at extremely low water activity conditions a surplus of triesters is formed [7].

(-) Figure 2.

Equilibrium mono- (n), di- (o) and triester (Δ) mole fraction in the non-polar phase as a function of the water activity. The curves only connect the datum points.

561 Enzyme activity and stability T h e enzyme activity is measured as a function o f time at different water activities. = /c · ( C - C e < ) 7 ), at 150 hours after the T h e reaction constant (d(C - Ceq)/dt immobilization o f the enzyme, is given in figure 3 ( • ) . T h e enzyme activity is more or less constant at different water activities. This is in contrast with the findings of Valivety et al. [8]. They found an increase of the enzyme activity upon an increase o f the water activity (0 < a w < 0 . 5 ) . However, at 6 5 0 hours after the immobilization o f the enzyme, the system presented in this study shows an increase o f the enzymatic activity upon increasing water activity too. Apparently, at low water activity conditions the enzyme has a higher inactivation rate compared to the system at a w = 0.5. T h e half-life time at a w = 0.1 approximates 100 hours and at a w = 0.5 the half-life time exceeds 5 0 0 hours.

k(10" 20

6

1/s)

•

I

—•

15 10

A

5H

ο

θ' ,

ι

0.25

0

0.5

0

aw ("> Figure 3.

Reaction constant k of an oil batch at 150 h (π) and 625 h (ο) as a function of the water activity.

CONCLUSIONS This paper shows, that an excess of triacylglycerols can be produced at low water conditions in a membrane system. T h e immobilized lipase has a half-life time o f 100 hours at low water activity conditions and the half-life time exceeds 5 0 0 hours at a w = 0.45. T h e enzyme activity is about constant at different water activities.

562

LITERATURE 1. 2.

3.

4.

5.

6.

7. 8.

Oberkobusch, D . (1990) Polymere aus fettchemische Rohstoffen durch polymeranaloge Umsetzung. Fat Sei. Technol., 1 0 , 3 9 7 - 4 0 0 Ergan, F . , M. Trani and G . André (1990) Production o f glycerides from glycerol and fatty acid by immobilized lipases in non-aqueous media. Biotechnol. Bioeng., 3 5 , 195-200 V a n der Padt, Α., M . J . Edema, J . J . W . Sewalt and K. V a n 't Riet, Κ. (1990) Enzymatic acylglycerol synthesis in a membrane b i o r e a c t o r . / . Am. Oil Chem. Soc, 67,347-352 Goldberg, M., F . Parvaresh, D . Thomas and M.-D. Legoy ( 1 9 8 8 ) Enzymatic ester synthesis with continuous measurement o f water activity. Biochim. Biophys. Acta 9 5 7 359-362 V a n der Padt, J . J . W . Sewalt and K. V a n ' t R i e t ( 1 9 9 2 ) Specific by-product removal in a membrane bioreactor. Presentation to be held at 'Congress on engineering of membrane processes', May 13-15, 1992, Garmisch-Partenkirchen, Germany Janssen, A.E.M., M. Hadini, Ν. Wessels Boer, R . Wallinga, A. V a n der Padt, H.M. Van Sonsbeek and K. V a n ' t Riet (1992) T h e effect of organic solvents on enzymatic esterification of polyols. Presented at 'Fundamentals of biocatalysis in non-convential media' April 26-29, 1992 Noordwijkerhout, T h e Netherlands V a n der Padt, Α., A . E . M . Janssen, J . J . W . Sewalt and K. V a n ' t R i e t Acylglycerol equilibrium in a two-phase system. Submitted for publication Valivety, R.H., P . J . Hailing and A . R . Macrae (1992) R e a c t i o n rate with suspended lipase catalyst shows similar dependence on water activity in different organic

solvents. Biochim. Biophys. Acta 1 1 1 8 218-222

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

CHEMO-ENZYMATIC SYNTHESIS O F MONOSACCHARIDE F A T T Y E S T E R S AND T H E I R P R E L I M I N A R Y C H A R A C T E R I Z A T I O N

563

ACID

Giuseppe Fregapane, Douglas B . Sarney, Sydney G. Greenberg, Dorothy J . Knight and Evgeny N.Vulfson Department of Biotechnology & Enzymology, A F R C Institute of Food Research, Earley Gate, Whiteknights Road, Reading RG6 2 E F , U K

Abstract A range of galactose and xylose monoesters were prepared by lipase-catalysed esterification of the corresponding sugar acetals with commercial (crude) fatty acid preparations followed by acid-catalysed cleavage of the isopropylidene group(s). Overall yields of 60-85% were obtained. GC and GC-MS analysis snowed that the "crude" products contained only small amounts of unreacted fatty acids and monosaccharides. This compared favourably to chemically synthesized sorbitan monoesters, prepared from the same fatty acid mixtures. GC separation of sorbitan monoesters revealed the presence of a large number of individual compounds, many of which were identified by MS as various isomers of sorbitan, isosorbide and their mono- and diesters.

Introduction Application of enzymatic methods to the production of functional food ingredients has attracted much attention in recent years (see reviews [ 1 , 2 ] ) . This is mainly due to the significantly increased demand for higher quality products as well as to the tightening of regulations with regard to the safety of artificial food additives. The latter proved to be especially important for additives resulting from high temperature processes, such as emulsifiers, since the formation of potentially health hazardous side-products is practically unavoidable under these conditions. Consequently numerous attempts have been made to develop a relatively mild biotechnological approach to the synthesis of food acceptable emulsifiers [3-6]. However, very low productivity obtained in these studies hampered their practical application. Recently it has been shown that various sugar acetals could be regioselectively esterified by long-chain fatty acids using lipases in an equimolar mixture of substrates, i.e. in a solventfree process [ 7 ] . It has also been suggested that this approach could provide a viable addition/alternative to current manufacturing methods. This paper describes preliminary results concerning the preparation of monosaccharide esters from commercial (crude) fatty acid mixtures and compares the quality of products obtainable via this route, with the conventional, chemically manufactured food-grade emulsifiers.

564

Materials and Methods Chemicals: Lipase from Mucor miehei (Lipozyme IM-60) was supplied by Novo-Nordisk A/S. 1,2:3,4-di-O-isopropy lidene-D-galactopyranose and 1, 2-O-isopropylidene-Dxylofuranose were obtained from Aldrich Chemical Co. Ltd. Sorbitan monoesters were kindly gifted by ICI (Spans), Grinstead (Famodans) and Croda Ltd (Crills). Fatty acid mixtures used for manufacturing of the corresponding sorbitan esters were obtained from the same manufacturers. Synthesis of monosaccharide fatty acid esters: Enzymatic esterification was carried out in a 1:1 equimolar mixture o f sugar acetal and fatty acid as previously described [ 7 ] . Acidcatalyzed cleavage of the isopropylidene group(s) was achieved using aqueous acids according to [ 8 , 9 ] . The products were recovered by evaporation and extensively washed to remove traces of the acid. Yields of 60-85% were typically obtained. GC and GC-MS analysis: GC analysis was performed on a Hewlett-Packard series 5890A gas Chromatograph equipped with FID detector. 1 μ\ of trimethylsilyl derivatives, prepared according to [ 1 0 ] , were applied to a Hewlett-Packard Ultra 2 , capillary (25M* 0 . 2 2 ID) fused silica, wall coated open tubular column with 5% phenylmethyl silicone phase, 0 . 3 3 μ film thickness. A 6 0 cm length of deactivated fused silica, 0.32mm ID was attached, as a retention gap, between the injector and the analytical column. The carrier gas was helium flown at a linear rate of 25cm/sec with a head pressure of 2 0 psi. The temperature of both the injector and the detector was 285°C. The temperature programme was: 100°C for 0 . 5 min, then increased to 240°C at 8°C/min, held for 3 min, increased to 300°C at 10°C/min and held for 3 0 min. GC-MS was carried out in the CI mode using a Hewlett-Packard series 5 9 8 8 mass-spectrometer with isobutane as the reagent gas (source temp, 20CPC; source pressure, ltorr; scan rate, 0.5scan/sec.).

Results Comprehensive GC analysis of currently used food-grade sorbitan esters was undertaken in order to establish a reference point. Very similar results were obtained with the products from all three manufacturers. A typical chromatogram is presented in Figure 1. GC separation revealed the presence of a large number of individual compounds (up to 6 5 ) , many of which were identified by MS as various isomers of sorbitan, isosorbide and their monoand di-esters. For instance, more than 10 isomers of sorbitan have been identified on the basis o f their molecular ions and some possible structures are depicted in Figure 2. This clearly demonstrates that there is a strong reason to suspect sorbitan esters for causing adverse allergic effects and to re-open the question of their food acceptability. In contrast to chemically produced emulsifiers the esters resulting from low-temperature enzymatic esterification contained practically no undesirable side-products. The composition of crude stearic acid (the same preparation as used for synthesis of sorbitan monostearate), the product of lipase-catalysed esterification and the resultant galactose-6-stearate are shown in Figure 3 ( A , B and C respectively). It is evident that only very minor amounts of unidentified contaminants were present.

565 300

SORBITAN MONOLAURATE

200 Silylation reagent • other

100 C180

M

aJluul ι

0 1 0:0

In

Β SORBITAN MONOSTEARATE

300

SORBITAN MONO-OLEATE

200

C 1 1 8Silylation , reagent • + other

100 012:0 . « « Λ

C16:0 |

20

30

40

F i g . l . G L C separation o f food-grade sorbitan esters.

50

(Time)

566

Fïg. 2. Possible dehydration products o f sorbitol identified by G C - M S . Discussion Two main approaches have been pursued so far to develop an efficient method for the enzymatic synthesis o f sugar fatty acid esters: the first was based on the use o f organic solvents, suitable for solubilization o f both substrates [3-6], while the second relied on prior "hydrophobization" o f sugars and their subsequent solvent-free esterification in molten fatty acids [ 7 , 1 1 ] . Although the former appears simpler, it suffers from poor kinetics and low overall productivity. Due to these reasons the second method is technologically more attractive. Thus, the synthesis o f 6-O-acyl-glucopyranosides from simple alkyl-glucosides [11] has recently entered pilot scale trials; the products are expected to find applications as industrial and household detergents [ 1 2 ] .

567 1000 ρ

A

Silylatio n reagen t

'STEARIC ACID' 600 r

C14:0 Silylatio n reagen t

200 γ

600 ρ

U-

Silylatio n reagen t

Β DI-ISOPROPYLIDENE GALACTOSE FATTY ACID ESTERS 400 h

D-IPGALACTOS E

D-IPGAL-C16: 0 D IPGAL-C18: 0

> Ε C18:0

200 r C16:0

I I

Silylatio n reagen t

D-IPGAL-C14: 0

C14:0

JL. 600

- Silylatio n reagen t

r

c GALACTOSE FATTY ACID ESTERS

400 h

200 h C16:0

C18:0 Silylatio n ι reagen t

I

X

Hi 10

20

30

40

(Time)

Fig.3. G L C separation o f crude stearic acid (A) and galactose fatty acid esters before (B) and after (C) cleavage o f the isopropylidene groups

568 The large-scale acetalisation and subsequent deprotection, required in our approach [7], should not present any additional technological difficulties since these reactions are currently run on a pilot plant scale as a part of the vitamin C manufacturing cycle. It remains to be seen, however, whether these steps can be justified economically. At the same time the use of isopropylidene-sugars is probably a more versatile approach as compared to alkylglycosides: it provides an efficient route to the synthesis of monosaccharide fatty acid esters and can be readily extended to oligosaccharides (Sarney et al> in preparation) which may not be amenable to the latter approach. Finally, this communication reinforces the advantages associated with the application of enzymes to the synthesis of food ingredients and also illustrates that the rational combination of chemistry and enzymology can provide the required productivity and quality of the final product.

Acknowledgements W e would like to thank Mr. S. Elmor for the use of GC-MS facilities and some technical assistance. The helpful discussions and suggestions of Professor B . A. Law and Mr. I. Gill are appreciated. W e are also grateful to Mr. R. Young and Miss. J . Wilcock for the graphics and to Mrs. J . Hodgkinson for the preparation of the manuscript.

References 1. J.R.Whitaker, Food Biotech. 1990, 4 , 699-725 2. E.N.Vulfson and B.A.Law, Food Technology International Europe. 1991, pp 171-174 Sterling Publications Ltd. 3. M.Therisod and A.M.Klibanov, J.Am.Chem.Soc. 1986, 108, 5 6 3 8 - 5 6 4 0 4. J.Chopineau, F.D.McCafferty, M.Therisod and A.M.Klibanov, Biotechnol. Bioeng. 1988, 3 1 , 208-214 5. A.E.M.Janssen, A.G.Lefferts and K.van't Riet, Biotech.Lett. 1990, 12, 7 1 1 - 7 1 6 6. A.E.MJanssen, C.Klabbers, M . C . R . Franssen and K.van't Riet, Enzyme Microb. Technol. 1991, 13, 565-572 7. G.Fregapane, D.B.Sarney and E.N.Vulfson, Enzyme Microb.Technol. 1 9 9 1 , 1 3 , 7 9 6 - 8 0 0 8. J.A.Mills, Advan. Carbohydr. Chem. 1955, 10, 1 9. A.N. De Beider, Advan. Carbohydr. Chem. 1965, 2 0 , 2 1 9 10. C.C.Sweeley, R.Bentley, M.Marita and W.W.Wells, J.Am.Chem.Soc. 1963, 8 5 , 2497 11. K.Adlehorst, F.Bjorkling, S.E.Godtfresen and O.Kirk, Synthesis. 1990, 112-115 12. F.Bjorkling, S.E.Godtfredsen and O.Kirk, Trends Biotech. 1991, 9 , 360-363

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. 569

© 1992 Elsevier Science Publishers B.V. All rights reserved.

Reversing an OC-chymotrypsin catalyzed reaction, by substituting a water / 1,4-butanediol solvent mixture for the usual aqueous reaction medium a

b

B. Deschrevel , M. Thellier and J.C. Vincent

3

l a b o r a t o i r e "Polymères, Biopolymères, Membranes" URA 5 0 0 CNRS Laboratoire "Echanges Cellulaires" URA 203 CNRS Université de Rouen, B P 118, F-76134 Mont Saint Aignan Cedex, France

b

Abstract The hydrolysis and synthesis reactions o f N-Cbz-L-tryptophanyl-glycineamide catalyzed by Oi-chymotrypsin have been examined as a function o f the relative concentrations o f water and 1,4-butanediol in the reaction medium. An aqueous medium and a ( 2 0 % water/80% 1,4-butanediol) mixture have been selected to kinetically study the hydrolysis and synthesis reactions respectively. They have been studied with respect to the enzyme and substrate concentrations, the pH and the calcium ion concentration.

1. INTRODUCTION Artificial models o f active transport systems have been based on reversible enzyme reactions occurring in gel slabs in which the asymmetrical distribution o f enzyme activities was ensured by maintaining a difference in pH between the two faces o f the barrier [1-3]. The same kind o f functional asymmetry may be induced by a difference in the water concentration between the two faces o f the barrier i f water participates in the reversible reaction. A good candidate would be a reversible synthesis-hydrolysis reaction for which the concentration o f water could monitor the direction o f the reaction. The design o f such a model requires partly aqueous/partly organic media. Different types of systems have been worked out in order to maintain the activity o f enzyme in the presence of organic solvents [4-9]. Monophasic systems are the most widely used. The first class o f such systems uses water-miscible organic solvents in which the proportion o f organic solvent does not usually exceed 5 0 % in order to avoid a loss o f enzyme activity. More recently, a second class o f monophasic systems has appeared which uses enzyme suspensions in nearly anhydrous organic media. Among the various hydrolytic systems described in the littérature, we selected the synthesis-hydrolysis reaction o f the peptide bond involved in N-Cbz-L-tryptophanylglycineamide. This reaction is catalyzed by α-chymotrypsin which is probably one o f the best studied enzymes, and whose reaction mechanism is now well understood. The reaction:

N-Cbz-L-tryptophanyl-glycineamide + water

chymotrypsin ^ *

N-Cbz-L-tryptophan + glycineamide

(1)

570

has been tested in the presence o f different miscible organic solvents by Homandberg et al [10]. From these data, we selected a water/1,4-butanediol medium in order to study this reaction more extensively. The presence o f 1,4-butanediol in the reaction medium leads to a decrease in the water concentration and also to a reduction o f the ionization o f the substrates [10]. These two effects encourage the synthesis reaction. This monophasic system has been chosen for the simplicity o f its conception and its possible use even at low water concentration without significant loss o f ος-chymotrypsin activity. In addition, the reaction seemed to be a good candidate for a model system o f active transport in which a hydrophilic species would be carried across a hydrophobic barrier. We deal here with the effects o f various parameters on the kinetics o f the hydrolysis and synthesis reactions.

2. MATERIALS AND METHODS 2.1. Reagents As the peptide was not commercially available, it was prepared by enzymatic synthesis and purified by preparative chromatography [11]. All the other reagents were purchased: glycineamide (SIGMA, G 7378), N-Cbz-L-tryptophan (SIGMA, C 5377), CX-chymotrypsin (EC 3.4.21.1) from bovine pancreas (SIGMA, C 4129), calcium chloride (PROLABO, 22 317.297), 1,4-butanediol ( 9 9 % ) (FLUKA, 18960), methanol (99.9%) (CARLO E R B A , 414 818) and Tris Base (PROLABO, 28 812.232).

2.2. Measurements of N-Cbz-L-tryptophan and peptide concentrations All the reactions were performed in stirred solution under standardized conditions. The reaction kinetics were followed by high performance liquid chromatography (HPLC). The device was composed o f an isocratic pump (Spectra-Physics, SP 8810), a C18 inverse phase column (Beckman, Ultrasphere ODS, 3 μηι, 0.46 χ 7.5 cm) and a spectrophotometric detector (Merck, L 3 0 0 0 ) connected to a micro-computer (Kenitec, 3 8 6 S X - 2 0 ) . Home-made software was used for data treatment. Throughout each reaction, samples were withdrawn, rapidly diluted with a Tris Base solution (50 mM, pH 6.7), filtered on filter units (Millipore, SJHV004NS, 0.45 μηι) and finally injected into the HPLC loop (20 ul). The elution was performed with a 5 0 % Tris Base (10 mM) / 5 0 % methanol (v/v) mixture adjusted to pH 6.7 1 with HCl ( I M ) . The pressure remained close to 130 bar with a flow rate o f 1 ml m i n . Under such conditions, chromatograms at 2 1 0 nm showed three peaks characterized by reproducible retention times, and corresponding to glycineamide plus 1,4-butanediol plus chymotrypsin (0.9 min), N-Cbz-L-tryptophan (2.7 min) and peptide (7.3 min) respectively. Concentrations o f N-Cbz-L-tryptophan and peptide were then determined with previously established calibration curves for peak areas.

2.3. Measurements of pH Measurements o f pH were performed with a conventional combined glass electrode (Tacussel X C 111) for most o f the hydrolysis experiments in the aqueous medium. In the water/1,4-butanediol medium, the apparent pH values, p H a p p, were measured by using a two-electrode device (Tacussel) specially designed for etiianolic media and composed o f a measuring electrode ( X G 920) and a reference electrode ( X R 920). The reference electrode was plunged into a (20% water/80% 1,4-butanediol) mixture saturated with KCl, thus

571 forming an electrolytic junction (AL 100). The two devices were calibrated using aqueous pH buffers.

3. RESULTS AND DISCUSSION For each reaction, the initial rate, noted V h for hydrolysis o f the peptide bond and V s for its synthesis, was calculated from the kinetic curves. 3 . 1 . Influence o f the water content o f the reaction medium The initial rates o f reaction, V h and V s , were plotted as a function o f the water content o f the reaction medium (Fig. 1). Under the experimental conditions, both V h and V s increased significantly when the water content o f the reaction medium was increased. The more concentrated in water the reaction medium, the larger the ratio Vj/Vg, with V h being practically equal to V s when the water content o f the medium was close to 5%.

Figure 1. Initial rates of hydrolysis, V h, (curve a) and synthesis, V s, (curve b) plotted against the water content of 1 the reaction medium. In each reaction medium: chymotrypsin 0.1 mg m l , Tris Base 200 mM and p H a pp 8.10. Initial concentrations: peptide 1 mM and glycineamide 19 mM, for hydrolysis; glycineamide 20 mM and N-Cbz-L-tryptophan 1 mM, for synthesis.

As shown below, the hydrolysis catalyzed by chymotrypsin is o f the Michaelis-Menten type [12,13]. As long as the water content o f the medium is above 2 % , the concentration o f water may be assumed to be saturating for the hydrolysis o f the peptide bond. This allows V h to be expressed as:

v =

V«* C 3o (2)

h

K m 3 + C 3'

572

where is the apparent maximum rate, K m 3 the Michaelis constant and C ° 3 the initial concentration o f peptide. Since the initial concentration o f peptide was the same in all the hydrolysis experiments, the variations o f V h with the water content o f the reaction medium are clearly due to the variations o f the enzyme parameters, V , ^ and/or K m 3. The K m usually characterizes the interactions o f the substrate with the active site o f the enzyme; hence it depends on the partitioning o f the substrate between the active sites and the bulk o f the solvent. In the case o f chymotrypsin, the interactions o f the substrate with the enzyme are mainly hydrophobic, and the peptide exhibits a rather high hydrophobicity. Increasing the water content o f the reaction medium thus diminishes its hydrophobicity. This tends to weaken the interactions o f the substrate with the solvent in favour o f those o f the substrate with the active site o f the enzyme. As a consequence, the K m is decreased [6,14], which causes the initial rate of hydrolysis to increase. This theoretical expectation is consistent with our experimental results and with other experimental results. For instance, the K m values for the hydrolysis o f a peptide bond catalyzed by chymotrypsin in a 2 6 water/dioxane mixture ranged from 10~ to Ι Ο M when the relative water content o f the medium ranged from 10% to 100% [15]. Similarly, the papain-catalyzed hydrolysis o f a peptide bond in a mixture o f water with 7/3 (v/v) Ν,Ν-dimethylformamide and dimethylsulfoxide exhibited K m values ranging from 52 to 3 mM when the water content o f the medium ranged from 5 0 % to 100% [16]. Variations o f V ^ under partial aqueous conditions may be due to conformational changes o f the enzyme, induced either by the nature o f the water-miscible organic solvent used [4], or by the decrease in water concentration causing an insufficient hydratation o f the enzyme molecules [6]. Both these effects tend to decrease the V ^ value, via enzyme inactivation. In our study, the enzyme activity still remained in a reaction medium containing as little as 2 % water. In pure 1,4-butanediol ( 0 % water) no synthesis at all was observed in 10 days, thus revealing a total inactivation o f the enzyme. However the inactivation was reversible since the addition o f an aqueous buffer up to a relative water concentration o f 2 0 % caused the enzyme to start working. Preliminary experiments concerning the synthesis substrate-dependencies have shown us that the synthesis reaction catalyzed by chymotrypsin is also o f the Michaelis-Menten type. B y assuming that the synthesis reaction mechanism is the opposite o f the hydrolysis reaction mechanism [12], V s may be expressed as: V ms

(3)

V s= 1 +

+ [RîCOOH]

0

[ R 2N H 2] °

where V m s is the maximum rate, K m l and K m 2 are the Michaelis constants with respect to 0 0 the N-Cbz-L-tryptophan and glycineamide respectively, and [ R j C O O H ] and [R2NH2] are the initial concentrations of the non-ionic forms o f N-Cbz-L-tryptophan and glycineamide, respectively. These concentrations were different from one experiment to another because o f the variations o f the ionization constants o f the two amino acids with the water content o f the medium. Although these variations contributed to the observed variations o f V s with the water content o f the reaction medium, it could be that these V s variations could also be due to V m s, K m l and K m 2 variations, as was the case for hydrolysis.

573 From a strictly kinetic standpoint, a purely aqueous medium is the most suitable one in which to perform the hydrolysis and synthesis reactions. However, as expected, increasing the concentration o f organic solvent thermodynamically favours the synthesis o f the peptide bond. Thus an aqueous medium and a ( 2 0 % water/80% 1,4-butanediol) mixture represent a good compromise for the study o f the hydrolysis and synthesis reactions, respectively.

3.2. Effect of the chymotrypsin concentration The variations o f the initial hydrolysis rate as a function o f the chymotrypsin concentration are presented in Figure 2. As expected, V h was proportional to the enzyme concentration. For the synthesis reaction, Figure 3 reveals that V s was also proportional to 1 the chymotrypsin concentration up to at least 0.1 mg ml" . For larger enzyme concentrations, V s still greatly increased, although not linearly.

τ

1

1

1

Enzyme cone, (mg ml

Figure 2. Initial rates of hydrolysis, V h, plotted against the chymotrypsin concentration in the aqueous reaction medium (Tris Base 50 mM, pH 6.7). Initial peptide concentration ImM.

Γ

)

Figure 3. Initial rates of synthesis, V s , plotted against chymotrypsin concentration in the (20% water/80% 1,4-butanediol) medium (Tris Base 50 mM, p H a pp 7.02). Initial concentrations: glycineamide 100 mM and N-Cbz-L-tryptophan 1 mM.

Whereas the enzyme was completely dissolved in the aqueous medium, whatever the enzyme concentration used, the ( 2 0 % water/80% 1,4-butanediol) medium progressively became turbid, with the aspect o f a colloidal dispersion, for enzyme concentrations higher 1 than 0.1 mg m l . In other words, the non-solubility o f the enzyme in the reaction medium did not imply its inactivation. It has been shown that enzymes such as chymotrypsin, suspended in nearly anhydrous media, were still active because enzyme molecules remained sufficiently hydrated [6,17]. The more hydrophobic die organic solvent used, the smaller the water content required for the enzyme to be active. The 1,4-butanediol is not a highly hydrophobic solvent. Thus, in a ( 2 0 % water/80% 1,4-butanediol) medium, some o f the enzyme molecules may be insufficiently hydrated and thus inactive. This percentage

574 increases with an increase in the total enzyme concentration. Another possible explanation o f the observed V s variations is that enzyme molecules form aggregates whose size increases with the enzyme concentration. Therefore, the substrate diffusion becomes more and more difficult. Both situations will lead to V s variations similar to those observed in our experiments. 3 . 3 . pH-dependencies The initial hydrolysis rates were plotted as a function o f pH o f the reaction medium (Fig. 4). The curve is similar to the pH-dependence curves classically obtained for chymotrypsin-catalyzed reactions [12,13]. The optimum pH for the hydrolysis o f the peptide bond catalyzed by chymotrypsin was found to be close to 7.8. It is worth noting that the N-Cbz-L-tryptophanyl-glycineamide is not an ionizable compound and thus the pHdependence curve only results from the enzyme activity variations as a function o f pH. For the synthesis, as shown in Figure 5, V s varied with the apparent pH o f the reaction medium and reached a maximum for p H a pp 6.10. Because both die substrates are ionizable and their initial concentrations were not saturating (refering to preliminary experiments o f synthesis substrate-dependencies), the curve does not correspond to a true pH-dependence curve. The curve obtained results from the variations o f both the enzyme activity and the initial concentrations o f the non-ionic forms o f the substrates as a function o f p H a p p. Once again, i f we assume that the synthesis reaction mechanism is the opposite o f the hydrolysis reaction mechanism, we may expect that, in a given medium, the enzyme synthesis activity will vary in the same way as the enzyme hydrolysis activity as a function o f p H a p p. However, by taking account o f the effect of the organic solvent content on the ionization constants o f the enzyme, the optimum p H a pp would slightly increase with a decrease in the water content o f the reaction medium.

Figure 4. pH-dependence of hydrolysis. Aqueous medium: Tris Base 50 mM. Chymotrypsin concentra1 tion 0.1 mg m l and initial peptide concentration 1 mM.

Figure 5. pH-dependence (apparent pH) of synthesis. (20% water/80% 1,4-butanediol) medium with Tris Base 50 mM. Chymotrypsin concentration 0.1 mg 1 m l . Initial concentrations: glycineamide 100 mM and N-Cbz-L-tryptophan 1 mM.

575

3.4. Hydrolysis substrate-dependence The maximum concentration o f peptide soluble in the aqueous medium was determined and was equal to 0.59 mM. Therefore, the substrate-dependence was performed with initial peptide concentrations ranging from 0 to 0.3 mM. The observed variations o f V h as a function o f the initial peptide concentration corresponded to a Michaelian behaviour o f the enzyme (Fig. 6). Then, by using a Lineweaver and Burk plot, a K m 3 value o f 0.72 mM and a value o f 3.95 umole per hour and per mg o f enzyme were deduced. In fact, since the peptide solubility in water is close to K m 3, the V j ^ value is purely theoretical. The maximum hydrolysis rate which could be experimentally obtained is thus calculated by using the maximum soluble peptide concentration, and is equal to 1.78 umole per hour and per mg o f enzyme.

Peptide c o n c e n t r a t i o n (mM) Figure 6. Substrate-dependence of hydrolysis. Aqueous medium: Tris Base 200 mM. pH 8.10. 1 Chymotrypsin concentration 0.1 mg m l .

3 . 5 . Effect o f the c a l c i u m ion concentration Calcium ions are known to improve the chymotrypsin stability and its hydrolysis activity [ 1 8 ] . The synthesis activity o f chymotrypsin was thus examined as a function o f the concentration o f calcium ions and the results are presented in Figure 7. It shows that the enzyme synthesis ++ activity depends on the C a concentration and is maximum for a calcium ion concentration close to 5 mM. For calcium ion concentrations smaller than 5 mM, the increasing enzyme activity exhibited corresponded to the favourable effect expected. The decrease in the enzyme activity observed with larger calcium ion concentrations, was probably due to an + + unfavourable effect o f C a . Considering the overall process o f the peptide bond formation [ 1 1 ] , it appears that calcium ions may interact with the ionization-neutralization reactions o f N-Cbz-L-tryptophan. At ++ high C a concentrations, the concentration of the non-ionic form o f N-Cbz-L-trypto-

0.04

1

0.00 0

1

20

1

40

1

60

1

80

1

100

Ca** c o n c e n t r a t i o n (mM) Figure 7. Initial rates of synthesis, V s , plotted against the calcium ion concentration. (20% water/ 80% 1,4-butanediol) medium with Tris Base 50 mM, p H a pp 6.10. Chymotrypsin concentration 0.1 1 mg m l . Initial concentrations: glycineamide 100 mM and N-Cbz-L-tryptophan 1 mM.

576

phan would thus be decreased and, V s , as expressed by equation ( 3 ) , would therefore tend to decrease. In such a case, the experimental curve obtained would result from the combination of the two opposite effects o f the calcium ions on the synthesis o f the peptide bond catalyzed by chymotrypsin.

4. CONCLUSION Although the presence o f the organic solvent in the reaction medium is useful to shift the synthesis-hydrolysis equilibrium toward synthesis, it tends to decrease both the synthesis and hydrolysis reaction rates. An aqueous medium is thermodynamically and kinetically favourable for the hydrolysis reaction while a ( 2 0 % water/80% 1,4-butanediol) mixture is much more thermodynamically than kinetically favourable for the synthesis reaction. In such a reaction medium, the synthesis reaction rate may be increased by using: (i) high amino acid concentrations, (ii) an appropriate calcium ion concentration and (iii) a high enzyme concentration. As suggested, the decrease in the reaction rates with increasing organic solvent contents in the reaction medium may be due to K m variations, related to the partitioning o f the substrates between the medium and the enzyme, but also to V m variations. A better knowledge o f the enzyme/organic solvent relationships might help us to understand these possible V m variations and the V s variations observed for high enzyme concentrations. More generally, the knowledge o f the effects o f the organic solvent on the catalyst would possibly allow the solvent system to be modified in order to preserve a better enzyme activity. Finally, the present study has provided information about the kinetic behaviour o f the synthesis and hydrolysis reactions catalyzed by chymotrypsin in water/1,4-butanediol media. Nevertheless, a complete kinetic optimization o f the system would require many more experiments.

REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

S. Alexandre, Dr. Univ. Thesis Rouen (1988). J.C. Vincent, S. Alexandre and Thellier, Arch. Biochem. Biophys., 261 (1988) 405-408. J.C. Vincent, S. Alexandre and M. Thellier, Bioelectrochem. Bioenerg. 20 (1988) 215-222. L.G. Butler, Enz Microb. Technol., 1 (1979) 253-259. K. Morihara, T. Oka and H. Tsuzuki, Nature, 280 (1979) 412-413. A.M. Klibanov, Trends Biochem. Sei., 14 (1989) 141-144. Ε. Antonini, G. Carrea and P. Cremonesi, Enz. Microb. Technol., 3 (1981) 291-296. K. Martinek, A.N. Semenov and I.V. Berezin, Biochim. Biophys. Acta, 658 (1981) 76-89. P. Douzou, Cryoconservation Cell. Norm. Neoplasiques, C.-R. Conf. Int., (1973) 11-16. G.A. Homandberg, J.A. Mattis and M. Laskowski, Biochemistry., 17 (1978) 5220-5227. B . Deschrevel, J . Y . Dugast and J.C. Vincent, Compt. Rend. Acad. Sei., submitted. C. Walsh, in "Enzymatic Reaction Mechanisms", H.W. Freeman and Co., San Francisco, (1979) 56-97. G.P. Hess, in "The Enzymes", 3rd ed., P.D. Boyer, Ed., Acad. Press, New-York, 3 (1971) 213-248. P. Maurel, J . Biol. Chem., 253 (1978) 1677-1683. K. Tanizawa and M X . Bender, J . Biol. Chem., 249 (1974) 2130-2134. M.M. Fernandez, D.S. Clark and H.W. Blanch, Biotechnol. Bioeng., 37 (1991) 967-972. J.S. Dordick, in "Applied Biocatalysis", H.W. Blanch and D.S. Clark, Ed., New-York, 1 (1991) 1-51. W. Rick, in "Method of Enzymatic Analysis", 2nd ed., H.U. Bergmeyer, Ed., Acad. Press, 2 ,1006.

Biocatalysis in Non-Conventional Media, edited by J . Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

577

Engineering aspects of the lipase-catalyzed production of (R)1-Ferrocenylethylacetate in organic media A. Wickli , E . Schmidt and J . R. B o u r n e a

b

a

a

Swiss Federal Institute of Technology, Chemical Engineering Department, Ε Τ Η Zentrum, CH-8092 Zurich b

Ciba-Geigy AG, Pharmaceutical Division, CH-4002 Basle

Abstract

In this study we investigated aspects of the lipase-catalyzed optical resolution of 1-ferrocenylethanol by esterification. Lipase P S (Amano Pharmaceutical Co.) is the most effective enzyme, toluene the best solvent and vinyl acetate should be used as acylating agent. The water content and the temperature of the reaction system have a significant influence on the enantioselectivity and on the activity of the enzyme. In an attempt to develop a continuous reaction system, different immobilization methods and several carriers have been examined. It was found that adsorption is more favourable than covalent binding. The kinetics of the native and the immobilized enzyme have been determined. Due to strong attrition effects found in a stirred tank reactor, mechanical mixing must be avoided. Fixed bed reactors seem to be suitable for a large scale process. In a fixed bed reactor with recirculation, engineering aspects such as mass transfer have been investigated. On the basis of promising results, a continuous fixed bed reactor has been developed. For a reactor operating at a conversion of 40% and T= 30°C a space-time yield of 1.28 kg (R)-FEA/(1 d) was obtained.

1. Introduction Water is for many applications in chemistry a poor solvent. Chemists realised the limitations of aqueous-based catalysis and have long ago replaced water with more suitable organic solvents. Unlike chemical processes, conventional biocatalysis has been performed in aqueous solutions. This is mainly due to the preconceived notion that nature intended enzymes to be catalytically active in water and that organic solvents serve only to destroy the catalytic power of enzymes. Today one knows that this notion was wrong. From the biotechnological perspective there are numerous potential advantages in employing enzymes in organic as opposed to aqueous medial. Because such an approach is attractive, enzymatic catalysis in non aqueous media has undergone a rapid expansion, particularly in the last decade. Today one is trying to combine enzymatic processes with conventional chemistry. An important class of compounds of interest in this context are the optically active organic esters. Specifically, this

578

paper describes various aspects of the enantiospecific esterification of 1ferrocenylethanol ( F C E ) with vinyl a c e t a t e (VAC) to (R)-lferrocenylethylacetate2>3 (FEA), which is an important intermediate in the production of ligands for optically active hydrogénation catalysts (Fig. 1).

racemic 1-ferrocenylethanol

Lipase PS

vinylacetate

Toluene

CH 3

(R)-l-ferrocenylethylacetate

(S)-1 -ferrocenylethanol

CHO

acetaldehyde

Figure 1. Reaction system 2. Influence of t h e w a t e r c o n t e n t on e n z y m e a c t i v i t y The overall water content and the partition of water between the enzyme, the solvent and the support material are both very important in enzymatic organic reaction systems . On the one hand an extremely low water content results in a low enzyme activity, on the other, especially in esterification reactions, too high a water content supports the back reaction with water.

579

1.0-ο (Λ 0)

υ χ

îantio»rneric

0)

0)

0.8-

m

•

0.6•

0.40.20.0-

• 10000

20000 ppm Η20

30000

40000

F i g u r e 2 . E n a n t i o m e r i c excess as a f u n c t i o n o f t h e w a t e r c o n t e n t i n t h e r e a c t i o n system

A s s h o w n i n F i g u r e 2 a n d F i g u r e 3, t h e r e a c t i o n r a t e a n d t h e e n a n t i o m e r i c excess (ee) o f t h e ( R ) - l - f e r r o c e n y l e t h y l a c e t a t e d e p e n d s i g n i f i c a n t l y o n t h e w a t e r c o n t e n t i n t h e r e a c t i o n s y s t e m . I t c a n be s h o w n t h a t l o w e r i n g t h e w a t e r c o n t e n t of the system i n a certain range results i n a n increase of the reaction rate a n d of t h e e n a n t i o m e r i c excess o f t h e p r o d u c t u p t o a m a x i m u m v a l u e ( a t a p p r o x i m a t e l y 1 0 0 p p m H2O). T h e r e f o r e , i n o r d e r t o r e d u c e t h e w a t e r c o n t e n t i n t h e r e a c t i o n s y s t e m , a l l s o l v e n t s w h i c h w e r e u s e d h a v e b e e n d r i e d o v e r m o l e c u l a r sieves.

6.00e-5 5.00e-5 -É a)

4.00e-5 -

•M

ra

c 3.00e-5 1οra 2.00e-5 1.00e-5 0.00e+0

A GYA

10000

5 20000 ppm H20

30000

40000

F i g u r e 3. I n i t i a l r a t e as a f u n c t i o n o f t h e w a t e r c o n t e n t i n t h e r e a c t i o n s y s t e m

580

3. Kinetics In recent years, a lot has been done in the field of using enzymes in organic 4 media but kinetic constants have hardly been published . We investigated the kinetics of the considered esterification with the native Lipase P S (LPS) in a suspension batch system and determined the kinetic constants. The reaction follows a two-step mechanism (ping-pong mechanism).

P

_J

B

L i

Ε

EA

«

A = VAC Β = F C E

*

E'P

Ε'

ΕΉ

^

* EQ

Ε

Ε = Enzym Ε' = Acyl-Enzym Ρ = Acetaldehyd Q = F E A

Figure 4. Scheme of the reaction mechanism Neither substrate inhibitions nor mass transfer limitations have been observed. A small product inhibition was found at a high product concentration (> 0.1 M). Experimental data were compared with various mathematical reaction rate attempts by using the software package SIMUSOLV (Dow Chemical Inc.) in order to obtain a reliable model for the kinetics. The following attempt fitted the experimental data best and is therefore proposed as the model for the kinetics ν m a x„ [FCE] [VAC] K

m,FCE

[VAC] + K m




VC A[FCE] +

^ Y

m m

K

Ffl?

AC ) P

V[FEA] A

+ [FCE] [VAC]

Kinetic constants (T = 25°C): -Km >C F E = 1.402 ± 0 . 1 4 M - Km^vAC = 0.22 ± 0.02 M - vmax = 0.024 ± 0.006 Units/mg LPS - K i = 0.112 ± 0 . 0 0 5 M

4. Enzyme Immobilization In order to develop a continuously operated reactor for the production of (R)-lferrocenylethylacetate, the L P S had to be immobilized on a convenient support. Various immobilization methods and different support materials have been tested (Table 1).

581

Table 1 A c t i v i t y o f different i m m o b i l i z e d L P S on v a r i o u s s u p p o r t m a t e r i a l s FEA-Production Immol/Cml ç Protein h Π Immobilization ££ support 0.99

12.1

Alox neutral

precipitated

0.95

29.4

Celite 560

dried

1.00

6.3

Celite 560

precipitated

0.99

9.4

BioFix C2

dried

0.99

21.1

native

BioFix C2

precipitated

0.98

52.4

Biosynth VA

covalent

0.95

7.9

Eupergit C

covalent

0.96

8.1

BioFix E2

covalent

0.12

10

While the covalent immobilization reduced significantly the activity and enantiospecifity o f the enzyme, adsorbed lipase was still highly active and retained i t s selectivity. Since proteins are not soluble in organic solvents t h e r e is no a d v a n t a g e to u s e c o m p l i c a t e d c o v a l e n t a t t a c h m e n t s . T h e r e f o r e , a d s o r p t i o n was chosen as the immobilization method T h e h i g h e s t r e a c t i o n r a t e s w e r e o b t a i n e d w h e n l i p a s e w a s a d s o r b e d onto hydrophobic c a r r i e r s s u c h a s t h e c e r a m i c B i o F i x C 2 . T h i s effect c a n b e i n t e r p r e t e d a s e v i d e n c e o f t h e e n z y m e a n d t h e s u p p o r t m a t e r i a l b o t h c o m p e t i n g for t h e w a t e r i n t h e s y s t e m . T h e p r o t e i n l o a d i n g o f t h e s u p p o r t m a t e r i a l s w a s d e p e n d e n t on t h e adsorption method used. T h e b e s t enzyme-carrier-complexes have been obtained by precipitating t h e e n z y m on the c a r r i e r . T h e protein loading, as shown in F i g u r e 5 , c o u l d b e r e p r o d u c e d . D r y i n g w a s found to b e n o t s u i t a b l e for h i g h e r protein loading (> 0 . 0 1 g protein/ g support material) because of the lack of reproduction. U n d e r due consideration o f all results, B i o F i x C 2 ( E C C International Ltd.) was chosen as the support material and precipitation as the immobilization method.

c ο CO

φ > c ο Ü I

LU

Ο

F i g u r e 5 . L P S l o a d i n g on B i o F i x C 2 b y p r e c i p i t a t i o n

582

5. Continuous reaction system An immobilized enzyme packed bed reactor (IEPBR) has been developed for the continuous production of (R)-l-ferrocenylethylacetate. Due to the particular reactor requirements selected, such as - high productivity - good long-term characteristics - simple equipment the following reactor concept was applied

immobilized enzyme

product solution filter

pressure intrument pump

substrate solution

Figure 6. Continuous immobilized enzyme packed bed reactor The loading of the immobilized enzyme used in the experiments was 2 g LPS/ g BioFix C2. The concentration of the substrate solution was 0.35 molar in 1ferrocenylethanol and 3.5 molar in vinyl acetate. The flow rate of the substrate solution was varied between 10 and 30 millilitres per hour. As Figure 6 shows there was no recycle and the substrate solution passed just once through the reactor. The composition of the product solution was analysed off-line by HPLC. The results of two long-term experiments at different temperatures are shown in Figure 7 and 8.

583

Time [h]

Figure 7. Long-term experiment at Τ = 25°C with 2 g of immobilized enzyme ( • = FCE-Conversion · = ee of FEA)

0

20

40

60

80

Time [h]

Figure 8. Long-term experiment at Τ = 30°C with 10 g of immobilized enzyme ( • = FCE-Conversion · = ee of FEA)

584

It can be shown that the enantioselectivity of the lipase is highly dependent on the conversion of 1-ferrocenylethanol and on the reaction temperature. At room temperature, the activity and the enantiospecifity (at a given conversion) of L P S adsorbed on BioFix C2 remained constant over a long period of time. Increasing the temperature caused a very small enzyme deactivation and a decrease in the enzyme's enantiospecifity. The space-time yield for the reactor operating at a conversion of 40% and T= 30°C was 1.28 kg (R)-FEA/(1 d). The productivity of the reactor operating at T= 25 °C was 6.5 g (R)-FEA/(g immobilized enzyme d).

6. Conclusions In this study the kinetics of the LPS-catalyzed optical resolution of 1-ferrocenylethanol to (R)-l-ferrocenylethylacetate have been determined. It was found that the overall water content of the reaction system had to be minimised to a certain extent in order to maximise the activity of the enzyme and to get the highest possible enantiomeric excess of the product. Various methods for enzyme immobilization and different supporting materials have been tested. The best enzyme activity and enantioselectivity have been obtained with precipitated LPS on an hydrophobic carrier. Finally, a continuous immobilized enzyme packed bed reactor was developed. The productivity and the space-time yield of this reactor are very high. At room temperature and at a 1-ferrocenylethanol conversion of 30 % , the activity and enantiospecifity (ee = 0.96) of the LPS-BioFix-complex were retained over a long period of time (210 hours). An increase of the reaction temperature to 30°C and of the conversion to 40 % caused a small but steady decrease of the enzyme's enantioselectivity while the activity remained almost constant.

7. References 1 Dordick, J . S., (1989) Enzyme Microb. Technol., U 194 2 Boaz, N. W, (1989) Tetrahedron Lett., 3 0 , 2061 3 Wang, Y.- F . , Lalonde, J . J . , Momongan, M., Bergbreiter, D., Wong, C - H., (1988) J . Am. Chem. Soc, HQ 7200 4 Miller, D. P., Prausnitz, J . M., Blanch, H. W., (1991) Enzyme Microb. Technol., 13, 98

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

585

VARIATION OF TYROSINASE ACTIVITY WITH SOLVENT AT A CONSTANT WATER ACTIVITY Zhen Yang, Donald A. Robb and Peter J . Hailing Department of Bioscience & Biotechnology, The Todd Centre, 31 Taylor Street, University of strathclyde, Glasgow G 4 ONR, U.K. Mushroom tyrosinase (EC 1.14.18.1) adsorbed on celite was very active in organic media containing a low amount of water. In the absence of water the enzyme was inactive but activity increased dramatically as phosphate buffer was added, reaching a maximum at a water activity (a^J of 0.96 (corresponding to a water content of 0 . 4 5 % v/v for chloroform). Enzyme activities in different organic solvents were compared at the same level of water activity using two methods: 1) Pre-equilibration of substrate solutions and enzyme preparation separately under constant humidity; 2) direct addition of salt hydrates to the reaction mixtures to act as water buffers to achieve a constant ν The latter was much simpler, and since it provided comparable reaction rates it was the preferred method. Solvents were chosen to give a variety of log Ρ values but no obvious relationship was found between enzyme activity and log Ρ at constant a ^ Also the optimal solvent seemed to depend on the substrate used. The half-life of the enzyme in chloroform was about 10-fold higher than in aqueous solutions, whereas the optimal reaction temperature was lower.

INTRODUCTION Research extensively conducted over the last several years has demonstrated that enzymes can competently function in organic media containing a low amount of water [1,2]. Solvent selection is one of the key factors to influence enzyme activity in such a system. A number of solvent properties have been considered for correlation with the enzyme activity, but the most popular one is log Ρ [3] which reflects the hydrophobicity of the solvent; usually enzymes are found to be more active in an organic solvent with a higher log P. Studies on the effect of solvent are often conducted by adding the same amount of water to the whole system. But Zaks and Klibanov [4] have revealed that the enzyme activity depends only on the amount of water adsorbed to the enzyme rather than on the water content of the whole system; and depending on the hydrophobicity of the solvent, the same water content results in entirely different amounts of water associated with the enzyme. So it is important to compare the enzyme activity in different organic solvents when the hydration of the enzyme is kept constant. Since there is a proportional relationship between water activity (a^) and water content of the enzyme, which is little affected by changing the solvent [ 5 ] , water activity provides a convenient means of controlling the amount of water adsorbed on the enzyme. There are two ways of obtaining constant water activity: The generally used method is pre-

586

equilibration of substrate solution and enzyme preparation separately with a saturated aqueous solution of a salt to obtain a required ^ [6]; the other one is direct addition of a salt hydrate to the reaction mixture to act as a water buffer. The latter method was discussed by Hailing [7] who considered that the equilibrium of a salt hydrate pair can provide a constant vapour pressure of water at a fixed temperature; the effectiveness of this method in buffering water activity in a low-water enzymic reaction was demonstrated recently [ 8 ] . In this study, mushroom tyrosinase (polyphenoloxidase, E C 1.14.18.1) was employed as a model enzyme. It was adsorbed on celite and used in the oxidation of o-diphenols with oxygen to ö-quinones in various organic solvents. The dependence of the enzyme activity on water activity was examined, the effect of solvent on the enzyme activity at a constant ^ was studied, and the thermostability of the enzyme in organic and aqueous media was compared.

EXPERIMENTAL Enzyme: Mushroom tyrosinase was extracted from fresh mushroom into 50mM phosphate buffer (pH6.0), precipitated with ammonium sulphate and then adsorbed onto celite and dried in a vacuum oven until a constant weight was obtained. Substrates: 4-methylcatechol (MC), 4-tert-butylcatechol ( B C ) . Solvents: All solvents were washed twice with distilled water and then dried over molecular sieves (4A) before use. Salt hydrate: ^ Η Ρ 0 4· 1 2 Η 20 Reactor: All reactions in organic solvents were carried out in a 250ml stoppered roundbottom flask incubated in a water bath at 20°C and stirred with an overhead stirrer (lOOrpm) for one hour. The progress of the reaction was followed by sampling at regular intervals and measuring the absorbance of oquinone at 390nm. Since the extinction coefficient of ö-quinone is the same in all the solvents used, the reaction rate was determined from progress curves and simply expressed as Δ Α per hour. The thermodynamic water activity ( a j was determined with the aid of a Philips LiCl sensor (Philips Instruments, Cambridge). Thermostability: 1. Aqueous system Six test tubes containing 1ml of enzyme in phosphate buffer (50mM, pH6.0) were incubated at each temperature for various specified periods of time. They were then transferred to a water bath at 20°C for 5 minutes, and 1ml aqueous solution of 2.5mM M C (already incubated at 20°C) was added to start the reaction. The change in absorbance at 400nm was recorded and the half-life of thermal inactivation calculated. 2. Organic system 0.25g enzyme/celite was suspended in the reactor used above containing 50ml chloroform (saturated with water at ~20°C) incubated in a water bath at a certain temperature for a specified period of time; ice was then immediately added to bring

587

the temperature of the water bath back to 20°C, and after stirring for another 5 minutes to aid equilibration of the temperature of the solution inside the reactor, 0.1241 g M C (20mM) was added to start the reaction. Water-saturated chloroform was set as blank and the absorbance at 390nm after 10 minutes was used as the initial reaction rate. Partition coefficient of substrate (P c) and product ( P r ) : After mixing 1ml of aqueous substrate solution (0.02% w/v for M C and 0 . 4 % w/v for B C ) and 1ml organic solvent with a Vortex mixer, the substrate concentration in the aqueous phase ( [ S ] H 2)0 was determined spectrophotometrically [ 9 ] . The substrate determined as the difference between concentration in the organic phase ( [ S ] s o l v )e nwas t the original concentration and [ S ] H 2. 0 P s was then calculated using the equation P s = [ ^ s o l v e n t / [S]H20*

P p was determined in a similar fashion where the 0-quinone in the aqueous phase w as was produced by oxidation of substrate with N a I 0 4 , and [P] H 2o determined from the absorbance at 400nm and calculated by using the extinction coefficient of the quinone.

RESULTS AND DISCUSSION 1.Dependence of enzyme activity on water activity

100

Ί

80 Η

60 Η

20 Η

0 Η 0.70

1

1

1

0.75

0.80

0.85

— ι 0.90

1

1

0.95

1.00

aw Fig.l Effect of water activity ( a j on enzyme activity ( V ) . The activity of tyrosinase was found to be greatly affected by the water activity of the whole system (Fig.l). When chloroform was used as a solvent, the enzyme had no activity

588

with an a^, lower than 0.7, but its activity then increased dramatically as phosphate buffer (50mM, pH6.0) was added to exceed the water content of 0 . 1 % v/v (approximately the solubility of water in chloroform). After reaching a maximum at a water activity of 0.96 (corresponding to a water content of 0.45% v/v for chloroform), the reaction rate declined. This experimental result was in good agreement with the earlier work [10] and revealed that tyrosinase required more extensive hydration ( 2 ^ = 0 . 7 - 1 ) than certain lipases ^ = 0 . 1 3 - 0 . 8 9 ) [11] and thermolysin ( ^ = 0 . 3 - 1 ) [12]. A discrete aqueous phase on the top of the solvent could be easily seen after the water activity reached 0.9, and this supported the result obtained by Zaks and Klibanov [4] that mushroom tyrosinase was catalytically active in organic solvents when several hundred water molecules were bound per enzyme molecule, enough to form a monolayer of water on the surface of the enzyme. The decline of the enzyme activity after reaching the maximum was due to the catalyst particles becoming clumped together, reducing interfacial area and limiting mass transfer. 2. Enzyme activity in various solvents at a fixed 3^, As was mentioned in the Introduction, the enzyme activity in different organic solvents should be compared at a constant water activity. In the first series of experiments an ^ of 0.83 was established by separate pre-equilibration of solvent containing substrate and enzyme with a saturated solution of KCl at room temperature (~20°C). The enzyme activities obtained on subsequent analysis are given in Table 1, which shows that chloroform was the optimal solvent for oxidation of MC but carbon tetrachloride was best for B C oxidation.

Table 1. Relative enzyme activities obtained with pre-equilibration ( ^ = 0 . 8 3 ) Solvent

log Ρ

V (MC)

V (BC)

ethyl acetate

0.68

2.8

0.6

butyl acetate

1.7

2.1

0.8

diisopropyl ether

1.9

18.1

1.6

chloroform

2.0

100

47.5

benzene

2.0

47.7

52.5

amyl acetate

2.2

4.9

2.8

toluene

2.5

46.7

51.6

chlorobenzene

2.8

35.8

37.8

carbon tetrachloride

3.0

5.8

100 19.9

pentane

3.0

ethylbenzene

3.1

cyclohexane

3.2

13.8

hexane

3.5

21.8

heptane

4.0

11.0

isooctane

4.5

9.9

29.3

53.0

589 It has recently been shown that salt hydrates are effective in buffering water activity in lowwater enzyme systems [8]. N a 2 H P 0 4 - 1 2 H 2 0 can be directly added to an anhydrous mixture of solvent containing substrate and tyrosinase to promote enzyme activity at an ^ of 0.74 at 20°C. Various chlorinated solvents of similar densities were chosen because the relative density of the salt to solvent could affect the efficiency of water transfer to the enzyme. Results shown in Table 2 confirm that chloroform is the most effective solvent for MC, but for B C tetrachloroethylene is superior to carbon tetrachloride. Table 2. Relative enzyme activities obtained by direct addition of N a 2 H P 0 4 - 1 2 H 2 0 (^=0.74) Solvent

Methylcatechol

Butylcatechol

chloroform

100

36.0

1,2-dichloroethane

93.3

30.9

1,1,2,2-tetrachloroethane

88.5

34.4

dichloromethane

72.0

22.0

1,1,1 -trichloroethane

66.7

52.1

carbon tetrachloride

5.3

75.5

trichloroethylene

4

4.7

tetrachloroethylene

4

100

Examination of the data in Table 1 indicates that there is no relation between enzyme activity and log P. Several solvents with similar log Ρ values (benzene, chloroform, diisopropyl ether and amyl acetate, log Ρ 1.9-2.2) gave widely divergent activities and very little activity was observed with high log Ρ solvents (hexane, heptane, isooctane). Therefore it is important to consider other parameters which might aid solvent selection. Tyrosinase, like alcohol oxidase and alcohol dehydrogenase, possesses a monolayer o f water when fully active[4]. In this case the partition coefficients of the substrate (P s) and product ( P p) should be considered in place of log P. The substrate must partition out of the solvent into the aqueous environment of the enzyme before reaction can occur. Equally, enzyme activity is likely to remain high when there is efficient removal of the product from the vicinity o f the enzyme back to the solvent and product inhibition is minimised. It has been suggested previously [10] that poor activity in solvents like hexane is due to product inhibition because of the low solubility of the product in these solvents. This seems a reasonable explanation and can be used to reject all solvents with a low P p — these are solvents with a log Ρ greater than 3.1. But the substrate partition coefficient should be considered also. It is expected that there is an optimum substrate partition coefficient, for at low P s values so much substrate may enter the hydration layer that substrate inhibition results and at high P s values so little may enter that V m xa is not attained. Such behaviour is observed as Fig.2 demonstrates (based on rates listed in Table 1). There is an increase in enzyme activity with P s until an abrupt maximum is reached and then activity declines at higher P s values. Each substrate has its own optimum P s: 0.67 (chloroform) for MC and 1.28 (carbon tetrachloride) for B C . The results obtained by direct addition of salt hydrate confirm this relationship between enzyme activity and P s (data not shown).

590

Methylcatechol

Butylcatechol

log Ps

log Ps

Fig.2 The relation between enzyme activity and log P s. It should be noted that there are several solvents in which the product is freely soluble but which do not promote enzyme activity. These are the ones with the highest P s and this leads us to propose that the Pp/Ps ratio is a better criterion than consideration of just one parameter. Use of this ratio leads to a reasonable correlation with enzyme activity (Fig.3) and aids the choice of solvent for either substrate.

Fig.3 The relation between enzyme activity and the ratio of partition coefficients: MCO),

BC(a).

591

However, Reslow et al [13] found no correlation between P s, P p and the reaction rates of esterification catalyzed by a-chymotrypsin in different solvents, but this can be explained by the fact that the amount of water bound to the enzyme (no more than 5 0 water molecules per enzyme molecule in octane) is about 10 times less than needed to form a monolayer on the enzyme surface [2] so that most of the enzyme molecule is exposed to the bulk organic solvent and not surrounded by a layer of water.

3.Thermostability of the enzyme Although the optimal reaction temperature of the tyrosinase-catalysed reaction in watersaturated chloroform (20°C) was lower than 45°C for the aqueous environment [14], the halflife of the enzyme (t 1 / 2) in water-saturated chloroform was about 10-fold higher (Fig.4). This fact agreed with the enhanced thermal stability of a-chymotrypsin [13] and lipase [15], and supported the hypothesis that dehydration drastically hindered the thermoinactivation of the enzyme and protected its conformational rigidity [16]. The half-life of tyrosinase in dry chloroform should therefore be expected to be even higher than in water-saturated chloroform. 4 π

20

30

40

50

60

70

Incubation temperature ( *C) Fig.4

Half-life of the enzyme in water-saturated chloroform (*) and in aqueous solution ( · ) at different incubation temperature.

REFERENCES 1. 2. 3.

Dordick, J . S . (1989) Enzyme Microb. Technol. 11, 194-211. Klibanov, A.M. (1989) T I B S 14, 141-144. Laane, C , Boeren, S., Vos, Κ. and Veeger, C. (1987) Biotechnol. Bioeng. 3 0 , 81-87.

592

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Zaks, A. and Klibanov, A.M. (1988) J . Biol. Chem. 2 6 3 , 8017-8021. Hailing, P.J. (1989) Ή B T E C H 7, 50-52. Valivety, R.H., Hailing, P.J. and Macrae, A.R. (1992) Biochim. Biophys. Acta 1118, 218-222. Hailing, P.J. (1992) Biotechnology Techniques (in press). Yang, Z. and Robb, D.A. (unpublished observations) Waite, J.H. and Tanzer, M.L. (1981) Anal. Biochem. I l l , 131-136. Kazandjian, R.Z. and Klibanov, A.M. (1985) J . Am. Chem. Soc. 107, 5448-5450. Goldberg, M., Thomas, D. and Legoy, M.-D. (1990) Enzyme Microb. Technol. 12, 976-981. Cassells, J.M. and Hailing, P.J. (1988) Enzyme Microb. Technol. 10, 4 8 6 - 4 9 1 . Reslow, M., Adlercreutz, P. and Mattiasson, B . (1987) Appl. Microbiol. Biotechnol. 26, 1-8. Yang, Z. and Robb, D.A. (1991) Biochem. Soc. Trans. 2 0 , 13S. Zaks, A. and Klibanov, A.M. (1984) Science 224, 1249-1251. Volkin, D . B . , Staubli, Α., Langer, R. and Klibanov, A.M. (1991) Biotechnol. Bioeng. 37, 843-853.

ACKNOWLEDGEMENT The S B F S S award (Sino-British Friendship Scholarship Scheme) to Z . Y . is very much appreciated.

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

Hydrolase activity of Pseudomonas fluorescens organic media

593

lipase in

F.Ausseil, H.Biaudet and P.Masson Centre de Recherches du Service de Santé des Armées. Unité de Biochimie. B.P. 87 - 3 8 7 0 2 L a Tronche Cedex, France.

Abstract The hydrolytic activity of a model esterase : lipase ( E C . 3 . 1 . 1 . 3 ) from Pseudomonas fluorescens in suspension in water restricted organic media was studied. The role of water, regarded as the reaction cosubstrate was investigated. Kinetics of indoxyl acetate hydrolysis in dioxane and in toluene were studied as a function of water concentration. Apparent affinity (Km H 2 0 ) of the enzyme for water was found to be about 16 M in dioxane (log P = - l . l ) and about 120 mM in toluene (log P = 2 . 5 ) . The catalytic activity of the enzyme was found to be identical in both solvents. Thermal stability of the enzyme in dioxane was compared to the s t a b i l i t y in a q u e o u s 0 . 1 M p h o s p h a t e b u f f e r , p H = 7 . 0 . In both c a s e s the e n z y m e thermoinactivation is a complex process that can be described by a double exponential decay model. Stability in dioxane at 90°C was found to be 20 times higher than in phosphate buffer.

1. I N T R O D U C T I O N Lipases are certainly the most studied enzymes in organic media. They can be used for transesterifications, synthesis and hydrolysis r e a c t i o n s [ 1 , 2 ] . Furthermore their high thermostability allows catalysis at high temperatures [3]. Most of these reactions are becoming of great industrial importance. According to the protein hydratation shell theory [4], the catalytic activity of enzymes in organic media depends on the water content of the solvent [ 5 , 6 ] . Since thermodenaturation o f proteins involves free water [ 4 ] , the enzyme thermostability greatly depends on the water content of organic media [ 7 ] . Moreover, water is the cosubstrate of lipase-catalyzed ester hydrolysis reactions. Thus, in the lipase hydrolytic system, water is important in the acquisition and the stabilization of the enzyme structure and in the reaction. The hydrolysis of esters ( R - X ) by lipases ( E ) can be depicted by the following minimum reaction scheme :

594

,

^

k

Κ

k

1

Ε + R-X "

2

3

E R-X

• E-R X-

k-i

• 2 °

Ε + ROH + H+ H

(i;

where Ε R - X stands for the Michaelian enzyme substrate complex and E - R for the acyl enzyme intermediate; the reaction products X " and ROH are respectively the alcohol and acid moieties of the substrate. Since water is the reaction cosubstrate the hydrolytic step may be considered to proceed through the intermediacy of a water-acyl enzyme complex. Thus a K m for water can be determined.

E-R + H 2 0

-

E-R H 2 0

•

Ε + R-OH + H+

kM

)

W e studied the effect of water on the hydrolase activity o f l i p a s e (triacylglycerol acyl hydrolase, E C . 3 . 1 . 1 . 3 ) from Pseudomonas

fluorescens

in

organic media. Kinetics were carried out in a water-miscible solvent (dioxane) and in a water-immiscible solvent (toluene). The hydrolytic activity of the enzyme in suspension in o r g a n i c solvents was m e a s u r e d as a function o f water concentration in the solvent, using indoxyl acetate (IA) as the substrate [ 8 ] . L i p a s e thermostability in

a n h y d r o u s d i o x a n e w a s a l s o c o m p a r e d to

thermostability in water.

2. M A T E R I A L S

AND

METHODS

2.1 M a t e r i a l s Lyophilized lipase from Pseudomonas

fluorescens

was purchased from

Fluka ; its specific activity was 3 1 . 5 μηιοί of oleic acid liberated / min / mg of enzyme preparation, with triolein as the substrate at pH 8.0 and 40°C. Dioxane (pure for anhydrous analysis) and tetrahydrofurane ( T H F ) were from SDS (Solvants Documentation Synthèses - Peypin, France ) . Titration of water in dioxane was determined by the coulometric Karl Fisher method and was found to be 0.1 M ( 0 . 1 5 % w:v). Toluene was dehydrated by distillation on a P2O5

(

2

595

column. Both toluene and dioxane were stored dessicated. Deuterium oxide (atom % D > 99.8) was from Fluka and indoxyl acetate was from Sigma.

2.2 Activity assay Enzyme hydrolysis measurements in organic solvents were performed after diluting 200 μΐ of reaction mixture in 2.8 ml T H F . The amount o f the reaction product (oxidized 3-hydroxy indole) was determined by a spectrophotometric assay at 385 nm.

2.3 Kinetic measurements as a function of water concentration Lipase-catalyzed hydrolysis of IA in organic solvents was measured as follows: a suspension of the lyophilized enzyme preparation (0.5 mg/ml) was diluted in 1 ml of organic solvent containing the substrate. The final concentration of enzyme suspension was 20 μg/ml. The indoxyl acetate concentrations were 100, 200, 300, 400 and 480 mM in dioxane and 20, 30, 45 and 70 m M in t o l u e n e . B u f f e r e d w a t e r ( a q u e o u s 0.1 M p h o s p h a t e b u f f e r , pH=7.0) concentrations were 3.3, 3.9, 4.4 and 5.5 M in dioxane and 11.0, 13.9, 16.7, 22.2 and 33.3 mM in toluene. In parallel, the solvent isotope effect of deuterium oxide upon the kinetics in dioxane was investigated using heavy water in place of water.Reaction mixtures were placed in 3 ml watertight vials. Sealed vials were shaken on a 30°C thermostated orbit shaker at 210 rpm in the dark. At a given time, 200 μΐ aliquots were withdrawn and a s s a y e d

for a c t i v i t y as above

mentioned.

2.4 Thermal inactivation The residual activity of the lipase after irreversible thermal inactivation was measured as follows: a suspension of lipase (0.5 mg/ml) in dioxane was placed in 2 ml screw-cap vials. Vials were then heated at 90°C in a dry bath. After a certain period of time, each vial was removed from the bath. A 200 μΐ aliquot was witdrawn and diluted in 1.8 ml dioxane containing 250 mM indoxyl acetate and 0.55 M water. The mixture was incubated at 30°C in the dark for 25 hours on an orbit shaker at 210 rpm and assayed for activity as described above.

596

3. RESULTS AND DISCUSSION 3.1 Apparent kinetic parameters T h e r e a c t i o n rate o f i n d o x y l acetate hydrolysis in d i o x a n e a n d t o l u e n e w a s d e t e r m i n e d as a function o f water c o n c e n t r a t i o n with f i x e d i n d o x y l acetate c o n c e n t r a t i o n s . T h e d o u b l e r e c i p r o c a l plot o f the h y d r o l y s i s r e a c t i o n rate against the c o n c e n t r a t i o n o f water at f i x e d i n d o x y l a c e t a t e c o n c e n t r a t i o n s s h o w e d straight l i n e s . T h e l i n e s w e r e found to b e parallel to e a c h o t h e r , ( F i g 1 ) s u g g e s t i n g that h y d r o l y s i s t o o k p l a c e in a P i n g P o n g bi bi s y s t e m [ 9 ] T h e equation o f e a c h line is: 1 =K m H 2 Q v

Vmax

1

_J

+

[ h 20 ]

L

Vmax \

|

KmIA \ [ h 2o ] ]

(3)

Figure 1: Double-reciprocal plots of the hydrolysis reaction rate, V, against water concentration [ H 2 0 ] at given indoxyl acetate concentrations [IA]: A in dioxane, Β in toluene. In dioxane [IA] were: •

100 mM, Ο 200 mM, ·

300 mM, • 4 0 0 mM, •

4 8 0 mM.

In toluene [IA] were: • 20 mM, Ο 30 mM, · 45 mM, • 7 0 mM. R e p l o t o f the 1 / [ H 20 ] axis intercepts against 1/[IA] w a s u s e d to o b t a i n K m a P p H 2 0 ( F i g . 2 ) . T h e equation o f the line is: 1 1 / H 20-axis intercept

KmlA 1 = Km H 2 0 [IA]

+

1 Km H 2 0

(4)

597

The K m a p p H2O values were 120 mM in toluene (log Ρ = 2.5) and 16 M in d i o x a n e ( l o g Ρ = - 1 . 1 ) . These values were c o r r e l a t e d with the solvent hydrophobicity, i.e, the ability of solvent to keep water in the enzyme hydratation shell.

Figure 2: Replot o f the 1 / [ H 2 0 ] axis intercepts against the 1/[IA]: A in dioxane and Β in toluene. The intercept on the 1/[H 20] axis intercept represent s 1/Km

a

pp

H 20 .

The slopes of the F i g . 2 lines correspond to K m a p p I A / K m a p p H 2 0 . Apparent KmlA in dioxane and in toluene were calculated from slopes ; their values were 5 9 0 mM in dioxane and 4 3 0 mM in toluene. T h e s e values are roughly similar, suggesting that Km does not depend on the solvent nature. These Km values are about 3 times higher than the value found for the P E G modified lipase in benzene (Km = 1 6 0 mM) [ 8 ] . Such a discrepancy may be attributed to the P E G modification. Indeed, it is known that P E G modification alters the microenvironment of enzymes and improves their catalytic efficiency [ 1 0 , 1 1 ] . Calculation of Vmax from slopes of figure 1 are: 4 5 . 5 μmol/min/mg in toluene and 4 7 . 1 μηιοΐ/min/mg in dioxane. The c o r r e s p o n d i n g apparent 1

specificity constants (Vmax/Km) are 0.106 mM-ï.min-ï.mg" in toluene and 0 . 0 8 1

mM-ï.min-ï.mg" in dioxane.

598

3.2 Dependence of the hydrolase activity on added water and heavy water The water concentration in toluene was varied up to 3 5 m M which is the saturation concentration of water in toluene. Increasing water concentration beyond 35 mM would have led to a biphasic system in which hydrolysis kinetics would have been different [12]. On the other hand, dioxane is a completely water miscible solvent. Studies have shown that enzyme activity in dioxane displays a bell-shaped dependence on the water content [ 1 3 ] . Above a critical water concentration, reaction rate decreases. Such a behaviour was observed for indoxyl acetate hydrolysis by lipase in dioxane (Fig.3).For water concentrations higher than 8 M, the reaction rate decreased. This was due to the solvent-induced enzyme denaturation which is promoted by high water contents[14]. Since dioxane strips off the essential water from the enzyme hydration shell [ 1 5 ] , the apparent K m for water was paradoxically higher than the optimum water concentration. Another nucleophilic cosubstrate, i.e. deuterium oxide, was used instead of water. The activity profile was shifted toward higher D 2 0 concentrations, suggesting a solvent isotope effect of deuterium oxide.

trichloroethylene > carbon tetrachloride. Enzyme activity and stability in 1.40 h presence of organic solvents is usually correlated with log Ρ ( 7 ) . T h e rule of log 1.20 k Ρ predicts better stability in solvents with 1.00 higher log Ρ which implies lower polarity. Thus, this rule would have predicted toluene (log 0.40 Ρ = 2.5) > trichloroethylene (log Ρ = 1.9). 0.20 0.00

Interfacial deactivation 25 30 In all the experiments performed in TIME (Hours) dipeptide synthesis or enzyme stability, an early white precipitate appeared at the Figure 5. Comparative solvents effect water/solvent interphase. Other researchers ( 3 ) have attributed this to reactant precipitation. In our research, the appearance o f such a precipitate was closely related with the presence o f papain in the aqueous phase. Analysis o f the precipitate confirmed that there was a high protein concentration in the interphase. T h e protein concentration remained nearly constant in the aqueous phase. Loss o f protein in the aqueous phase was less than 1 0 % . O n e explanation could be that papain which goes to the interphase is deactivated by the interfacial tension. A dynamic equilibrium is then established between papain at the aqueous phase and deactivated papain at the interphase. Finally, papain on both sides is completely deactivated. Thus, enzyme stability will depend on the organic surface available to papain (inverselly proportional to droplet size) and on the interfacial tension between aqueous/organic phases. So, for similar droplet size, a higher interfacial tension will result in a faster enzyme deactivation; and for similar interfacial tension, a smaller diameter (implying greater surface area per unit volume) will lead to faster deactivation. For agitated vessels, as well as for pipes, the Sauter mean drop size is the term used in the literature (8,9). This is related to the W e b e r number ( W e ) , a dimensionless group relating inertial forces and surface-tension forces ( 1 0 ) . 32.

—

= 0 . 0 8 1 ( 1 + 4 . 4 7 0)

0 6

We' '

(1)

634 2

We =

3

N DT Q

, 2

σ

In the above equations, d 32 is the Sauter mean drop size, φ is the volume ratio ( V o r /gV a q) , Ν is the impeller rotational speed, D , is the impeller diameter, ρ is the density and σ is the interfacial tension. As all the experiments were carried out under similar conditions (constant 0 , N, and D j ) , equations 1 and 2 can be simplified to

d3

2 =c

. ( i ) -0.6

(3)

σ where c is a constant dependent on 0 , N, and D,. Table 1

Carbon tetrachloride Trichloroethylene Cyclopentanone Toluene Benzene n-Heptane T C E + Tween 8 0 ( 0 . 4 % (v/v))

d 3 2/ c

Interf. Tension 1 (mNm )

LogP

1.68 1.63 N.D. 2.28 2.18 2.92 2.66

37.93 32.94 N.D. 34.07 32.24 40.62 7.50

3.0 1.9 0.4 2.5 2.0 4.0 -

Complete deactivation (hours)

5 8 15 20 20 > 25 > > 50

N.D: Not determined

T a b l e 1 summarizes the values o f d 3 2/ c , interfacial tensions and log Ρ for the solvents of figure 5, and for other solvents also tested. Values o f log Ρ were taken from the literature ( 7 ) . Values of density and interfacial tension were measured in our research as explained in Materials and Methods. It can be seen that for toluene and benzene, which have similar d 3 2/ c and interfacial tension, the enzyme stability is the same. Papain is more stable in a two-liquid system with trichloroethylene than with carbon tetrachloride, the latter having a similar d 3 2/ c , but a higher interfacial tension. T h e enzyme is also more stable with toluene or benzene than with trichloroethylene, which has a similar interfacial tension, but a lower

95% as determined by SDS-PAGE). All other chemicals were of the higest purity available. Decylchloroacetate was prepared by slowly adding chloroacetic anhydride in dichloroethane to a solution of decanol in dichloroethane at 4° C. The reaction mixture was stirred at 20° C for 6 h and the dichloroethane was evaporated under reduced pressure. Destillation of the crude ester (140° C, 6 mm Hg) resulted in > 99.5% pure (GC) decylchloroacetate. In all experiments the interfacial area of the emulsions was measured simultaneously with the hydrolytic activity using the experimental setup as decribed in our previous paper [11]. Experiments were carried out in a 3 0 0 ml thermostated (25° C) reaction vessel with baffles and a bottom drain. For all experiments 4% (v/v) organic phase was emulsified in 50 mM Tri^HCl pH 8.0 (total volume 2 0 0 ml). After stirring for 5 minutes, to allow equilibration of the emulsion, the reaction was started by addition of the lipase. The liberated acid was neutralized by adding 0.1 Ν NaOH using a pH-stat. Interfacial tension measurements were performed with a Kruss K-12 tensiometer using a DuNouy ring. All data were corrected for the density difference according to Zuidema and Waters [12].

661

RESULTS AND DISCUSSION Adsorption of lipase In enzyme catalysis one of the parameters that determine the reaction rate is the enzyme concentration. In case of enzymes that are active at interfaces (heterogeneous systems) also the available interfacial area should be taken into account as this can become rate limiting at high enzyme amounts. To assess the optimal enzyme loading a series of experiments was performed during which the initial hydrolytic activity of a 4% (v/v) emulsion upon varying amounts of enzyme was measured. The organic phase contained 20% (w/w) decylchloroacetate in toluene. Figure 1 illustrates that there is a linear dependence of the initial activity as a function of the amount of enzyme up to approximately 0.6 mg, indicating a progressive saturation of the liquid-liquid interface. A fully saturated interface is reached at about 2-3 mg of lipase resulting in a maximum activity of about 2 6 9 0 μmoKmin*m ). 800 CM

ε

600

X

ο ε

400

Ι

200

2

4

6

8

10

lipase(mg)/200 ml emulsion

Figure 1. Initial activity as a function of the amount of Pseudomonas fluorescens lipase using a 4% (v/v) emulsion in 50 mM Tri^HCl at pH 8.0 and 25° C. The organic phase contained 20% (w/w) decylchloroacetate in toluene. Assuming that only a monolayer of enzyme is active at the interface, the intersection of the dashed lines at 1.0 mg lipase corresponds to a fully covered interface. As the total initial interfacial area of the emulsion reactor under these conditions was about 2 2450 + 100 cm (independently of the amount of lipase added), approximately 4 mg 2 of lipase per m is needed to form a monolayer. This is in close agreement with values reported by colloid chemists [13] and other investigators [14]. From this value an 2 average molecular area of 1500 + 100 Â per lipase molecule was calculated. This value is within the range reported by others [15].

662

Comparing the average molecular area found under these conditions with the 2 theoretical value of 2000 Â (based on a closed packed enzyme layer a molecular mass 3 of 31000 Dalton and a partial specific volume of 0.73 g/cm ), suggests that only a slight conformational change has occurred upon adsorption to the interface. The molecular area for proteins unfolding upon adsorption to an interface were reported to be orders of magnitude larger [14].

Cosolvent addition To analyze the effects of organic solvents on the hydrolysis of decylchloroacetate initial rates of hydrolysis were measured in three organic solvent-aqueous buffer emulsions. The experiments were carried out using 4% (v/v) organic phase containing varying amounts of substrate. To be sure that saturation of the interface was not rate limiting only 0.2 mg of lipase was used. Using toluene, dibutylether and isooctane as a cosolvent Michaelis-Menten like curves were obtained as shown in Figure 2. 800

0 « 0

•

' 20

« 40

•

i 60

•

. 80

1 100

mol % decylchloroacetate

Figure 2. Effects of toluene ( • ) , dibutylether ( · ) and isooctane ( A ) on the lipase catalyzed hydrolysis of decylchloroacetate. Both toluene and dibutylether show a slight but significant increase in activity between 45-70 mol% decylchloroacetate. Similar results were reported by Mukataka [16] for the hydrolysis of beef tallow and palm oil. In their experiments, however, a maximum activity was obtained for isooctane at about 20% (v/v) organic solvent. For all three organic solvents the interfacial area for the various mixtures was 2 between 3900 and 6800 cm . It is thus very unlikely that the interfacial area is rate limiting.

663

From Figure 2 the amount of substrate needed to reach half of the maximum velocity, (So)^, was calculated (see Table 1). Table 1. Amount of substrate, (So)^, corresponding to half of V, cosolvent

V

(So)^ (mol %)

log Ρ

toluene

22.7

2.4

(μηιοΐ/mïn *mg) 625

dibutylether

10.0

2.9

675

isooctane

2.8

4.5

575

max

In the presence of isooctane the activity is relatively high at low substrate amounts. This was also reported by other investigators [9,16]. An explanation given by Mukataka [9] that may account for this effect is an enhanced affinity of the lipase for the substrate. According to this assumption one should expect the lowest affinity of the lipase for the substrate using the toluene system. We have found that Pseudomonas fluorescens lipase shows a very high affinity to the interface irrespective the presence of cosolvent. Using pure decylchloroacetate as the organic phase and 0.2 mg of lipase no differences in activity could be observed at various stirrer speeds and organic/aqueous phase ratios [to be published]. This indicates that the lipase almost quantitatively adsorbs to the interface. This is also consistent with the observation that biomolecules in general absorb very strongly to surfaces [13]. Furthermore, using the data from Figure 1 the Gibbs free energy of adsorption was calculated according to the following equation:

=

(1-Θ)

χ exp

KI

55.5

were Θ is the fraction of surface covered with lipase. Θ is equal to V/V m a x, the measured velocity divided by the maximum obtained velocity. The enzyme concentration expressed in mol/1 is represented by C. The Gibbs energy calculated for these experiments was about - 5 5 kJ/mol which also indicates that this lipase has a very high affinity to the interface. Evidently there must be other factors that account for the observed differences (Figure 2). In general activities correlate rather well with log Ρ values of the cosolvents [17,18]. Our results also indicate a correlation between the log Ρ of the organic solvent and the amount of substrate needed to reach half of the maximum velocity (Figure 3). This indicates that physicochemical properties of the reaction components should not be neglected when studying kinetics in two-phase systems.

664

Figure 3. Relation between the log (So)^ and the log Ρ of the organic solvent.

Figure 4. Interfacial tension of various amounts of decylchloroacetate in toluene ( • ) , dibutylether ( · ) and isooctane ( A ) .

To investigate the physicochemical aspects in more detail interfacial tension measurements of the organic solvent-substrate mixtures against 5 0 mM Tri^HCl buffer at pH 8.0 and 25° C were performed. Figure 4 shows that in case of toluene and dibutylether, within experimental error, no changes in interfacial tension were observed as a function of the amount of decylchloroacetate. Although a rather small difference in interfacial tension between toluene and dibutylether was observed, this probably does not explain the differences presented in Figure 2 and Table 1. The interfacial tension of pure dibutylether and pure toluene is almost identical to the interfacial tension of pure decylchloroacetate (Figure 4). This implies that no specific molecular interaction exists. Consequently no interfacial concentrations can be calculated based on these interfacial tension data. A quite different effect was observed in case of isooctane. Already at low substrate amounts (< 1 mol%) the interfacial tension dropped from a value of 55.0 mN/m for pure isooctane to 32.8 mN/m, the value for decylchloroacetate (see inset of Figure 4). This observation strongly suggests that decylchloroacetate is predominantly located at the interface. Consequently, it is very likely that the low value for (So)^ using isooctane is due to large differences in physicochemical properties of the substrate and the organic solvent.

665

CONCLUDING R E M A R K S Since lipases act at interfaces the interfacial substrate concentration is an important parameter determining the kinetics. As shown by our results the interfacial concentration is largely determined by the physicochemical properties of the various reaction components. Although various data have been gathered it is very complicated to calculate the interfacial substrate concentration. Currently we are, together with the Department of Physical and Colloid Chemistry of the Agricultural University Wageningen, trying to calculate interfacial substrate concentrations by modelling the specific interactions of the different reaction components.

ACKNOWLEDGEMENT The authors would like to thank Dr. W. Norde and Dr. F . Leermakers of the Agricultural University Wageningen for interesting and valuable discussions.

REFERENCES 1 2 3 4 5

6

7 8

9 10 11 12 '

A.M. Klibanov, Chemtech., 16 (1986) 354. A.M. Klibanov, Acc. Chem. Res., 23 (1990) 114. Z.H. Xie, Tetrahedron Asymm., 23 (1991) 733. C.J. Sih, Q.M. Gu, X. Holdgrun and K. Harris, Chirality, 4 (1992) 9 1 . E.M. Meijer, J . Kamphuis, J.A.M. van Balken, H.F.M. Hermes, W . J . J . van den Tweel, M. Kloosterman, W.H.J. Boesten and H.E. Schoemaker, Trends in Drug Research Vol. 13 (V. Claasen, Ed.), Elsevier, Amsterdam, 1990, pp. 363. J . Kamphuis, M. Kloosterman, H.E. Schoemaker, W.H.J. Boesten and E.M. Meijer, Proceedings of the 4th European Congress on Biotechnology Vol. 4 ( O . Neijsel, R. van der Meer and Κ. Luyben, Eds.), Elsevier, Amsterdam, 1987, pp. 331. M. Kloosterman, V.H.M. Elferink, J . van Iersel, J . H . Roskam, E.M. Meijer, A.H. Hulshof and R.A. Sheldon, Tibtech., 6 (1988) 251. M. Kloosterman, J.G.T. Kierkels, R.P.M. Guit, L.F.W. Vleugels, E.T.F. Geladé, W . J . J . van den Tweel, V.H.M. Elferink, L.A. Hulshof and J.Kamphuis, Mechanism and Genetic Engineering, CEC-GBF International Workshop 1990 Braunschweig (L. Alberghina, R.D. Schmid and R. Verger, Eds.), VCH, Weinheim, 1990, pp. 187. S. Mukataka, T. Kobayashi and J . Takahashi, J . Ferment. Technol., 6 3 (1985) 4 6 1 . Y . J . Wang, J . Y . Sheu, F . F . Wang and J . F . Shaw, Biotechnol. Bioeng., 31 (1988) 628. J.G.T. Kierkels, L.F.W. Vleugels, J.H.A. Kern, E.M. Meijer and M. Kloosterman, Enzyme Microb. Technol., 12 (1990) 760. H.H. Zuidema and G.W. Waters, Ind. Eng. Chem., Analytical Edition, 13 (1941) 312.

666

13 14 15 16 17

18

W. Norde, Advances in Colloid and Interface Science, 2 5 (1986) 267. F . MacRitchie, Advances in Protein Chemistry Vol. 32, Academic Press, New York, 1987, pp. 283. G.A. Roberts and M.P. Tombs, Biochim. Biophys. Acta., 902 (1987) 327 S. Mukataka, T. Kobayashi, S. Sato and T. Takahashi, J . Ferment. Technol., 6 5 (1987) 23. C. Laane, S. Boeren, R. Hilhorst and C. Veeger, Biocatalysis in Organic Media (C. Laane, J . Tramper and M.D. Lilly, Eds.), Elsevier, Amsterdam 1987, pp. 6 5 . R.H. Valivety, G.A. Johnston, C.J. Suckling and P . J . Hailing, Biotechnol. Bioeng., 38 (1991) 1137.

Biocatalysis in Non-Conventional Media, edited by J . Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

667

Biotransformation of benzaldehyde to benzyl alcohol by whole cells and cell extracts of baker's yeast in two-phase systems P. Nikolova and O.P. Ward Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N 2 L 3 G 1 .

Abstract W h o l e cells o f baker's yeast were found to catalyse conversion of benzaldehyde to benzyl alcohol in aqueous-organic two-phase systems. F r o m five organic solvents tested, namely chloroform, toluene, carbon tetrachloride, hexane and decane, product formation was maximum in whole cell biotransformations with hexane. Increase in activity paralleled increasing values of Log Ρ up to 3.5. Although decane has a higher Log Ρ than hexane (5.6 compared to 3.5), reductive biotransformation activity was lower with decane. Biotransformation o f benzaldehyde to benzyl alcohol was also observed with crude cell-free extracts of baker's yeast in biphasic systems containing the above solvents. N A D H was incorporated as cofactor in this system and cofactor regeneration was promoted by addition of ethanol into the medium. However, rates of benzyl alcohol formation catalyzed by cell-free crude extracts were lower than those observed with whole cells. Reductive biotransformation reactions were also investigated in aqueous and two-phase systems using mutants of Saccharomyces cerevisiae lacking some or all of the A D H isoenzymes, A D H I, II and III. Biotransformation rates for conversion of benzaldehyde to benzyl alcohol using these mutants were similar to the rates observed with the wild type strain. R a t e s of conversion of benzaldehyde to benzyl alcohol were generally higher in aqueous media as compared with biphasic systems. INTRODUCTION T h e vast majority of studies on the biotransformation capacity of baker's yeast relate to oxidation reduction reactions and, in most cases, whole yeast cells rather than isolated enzymes were used as biocatalyst [1]. Traditionally, yeast cells have b e e n used in conventional aqueous biotransformation media [2]. However, because many organic substrates are poorly water soluble, implementation o f biotransformation reactions in organic solvent containing media offers potential for utilisation o f these substrates in biocatalytic systems [3,4]. While some investigations on oxido-reductase biotransformations using isolated enzymes have b e e n carried out in two-phase systems and micro-aqueous organic solvent systems, little emphasis has b e e n placed on whole cell systems [5,6]. T h e s e systems are advantageous in oxidation-reduction biotransformation reactions in that conditions for promotion o f cofactor regeneration can often be established [7,8]. Baker's yeast manifests the capacity to reduce benzaldehyde and the range of substituted aromatic aldehydes to corresponding aromatic alcohols and the

668 same biotransformations have b e e n accomplished with purified commercial yeast cell alcohol dehydrogenase [9]. However, when the biotransformation o f benzaldehyde to benzyl alcohol was investigated using wild type and A D H isoenzyme mutant strains of S. cerevisiae in aqueous media, no correlation was observed between A D H activity in the cells and their capacity to produce benzyl alcohol [10]. In this study, conversion of benzaldehyde to benzyl alcohol by wild type and mutant strains o f baker's yeast has been investigated in aqueous-organic biphasic systems using whole cells and cell-free crude extracts. MATERIALS AND M E T H O D S Microbial cultures Details o f Saccharomyces mutants have previously b e e n described [10]. T h e s e strains were maintained on agar containing glycerol/lactate medium (glycerol, 3 0 g/1; lactate, 3 0 g/1; yeast extract, 10 g/1; peptone, 2 0 g/1; p H 5.0). Cells for use as biocatalysts were incubated in the glycerol/lactate medium in shake flasks for 2 4 h on an orbital incubator set at 175 rpm and 3 0 °C. Commercial baker's yeast cake ( 3 0 % dry weight, Fleischman's, Canada) was activated by incubation in glycerol/lactate medium on an orbital shaker at 175 rpm at 3 0 ° C for 1 h. Centrifuged pellets o f yeast cells were used in the whole cell biotransformation experiments and for preparation o f yeast cell-free extract. Preparation of biocatalyst Cell pellets, resuspended in 2 0 m M T R I S - H C 1 , p H 7 (buffer A ) , were lyophilised. Celite (30-80 mesh, British Drug House, England) 1 g, was mixed with 3 0 0 mg o f lyophilised cells and resuspended in 15 ml of buffer A and relyophilised. F o r biotransformation experiments using cell-free extracts, yeast pellet (5 g), suspended in 15 ml 5 0 m M T R I S - H C 1 , p H 7.2, containing 0.6 M sorbitol and 0.25 m M E D T A , was homogenised with 2 0 g glass beads (0.45-0.5 m m dia., British Drug House, England) in a bead-beater (Biospec Products, Bartlesville, O K ) for 5 χ 3 0 s cycles with chamber surrounded by ice. T h e cell-free supernatant was then recovered, lyophilised and 2 0 0 mg o f lyophilised material was suspended in 15 ml buffer A, containing 1 /ig N A D H and mixed with 1 g glass beads (140-270 mesh, Sigma, St. Louis, M O ) , and relyophilised. Biotransformation conditions F o r whole cell biotransformations, solvents were presaturated with buffer A and then the moisture content adjusted to 0.5, 2 and 1 0 % v / v with buffer A above the saturation point. Lyophilised whole cell biocatalysts, 1.3 g, were suspended in 10-20 ml o f biotransformation medium. F o r cell-free extract biotransformations, 1.2 g o f lyophilised biocatalyst was suspended in 10 ml of biotransformation medium, supplemented with 10 m M ethanol for cofactor regeneration (Figure 1).

669

Figure 1 Schematic diagram o f the cofactor regeneration system Analytical methods Benzaldehyde and benzyl alcohol concentrations chromatography as previously described [10].

were

determined

by

gas

RESULTS T h e effect o f moisture content on conversion o f benzaldehyde to benzyl alcohol by whole cells o f commercial baker's yeast was investigated in two-phase systems containing hexane. A comparison o f initial biotransformation rates is provided in T a b l e 1. Biotransformation activity increased with moisture content in the range 0 - 1 0 % . Using different moisture contents, studies were carried out on the effect o f solvent on the production o f benzyl alcohol by commercial baker's yeast over a 2 h incubation. T h e results are presented in T a b l e 2. U n d e r these conditions, highest production o f benzyl alcohol was observed with hexane as solvent, having a Log Ρ o f 3.5. Table 1 Effect o f moisture content on catalytic activity in whole cell biotransformation with wild

type S. cerevisiae Hexane containing various % moisture 0.5 2.0 10.0

1

Activities (mmoLh'^mg" dry cells) 1.0 20.3 22.2

670

Table 2 Effect o f organic solvent and % moisture on product formation with whole cells o f wild

type S. cerevisiae Benzyl alcohol ( m M ) Solvent

LogP

Butylacetate Chloroform Toluene Carbon tetrachloride Hexane Decane

1.7 2.0 2.5 3.0 3.5 5.6

0.5%

2%

10%

0.18 0.18 0.18 0.19 0.20 0.19

0.29 0.20 0.36 0.70 0.89 0.77

0.41 0.27 0.66 0.98 1.21 1.10

A similar experiment was carried out using crude cell extract rather than whole cells, except in this case it was necessary to incorporate a system for cofactor recycling (from N A D to N A D H ) mediated by conversion of ethanol to acetaldehyde. In the case of cellfree extracts, much lower rates of product formation were observed and, consequently, the incubation was extended to 24 h. T h e results, indicated in T a b l e 3, illustrate that highest bioconversions were observed with hexane and decane. Table 3 Effect o f organic solvent and % moisture on product formation with crude extracts o f wild type S. cerevisiae Benzyl alcohol ( m M ) Solvent

LogP

Butylacetate Chloroform Toluene Carbon tetrachloride Hexane Decane

1.7 2.0 2.5 3.0 3.5 5.6

0.5%

2%

10%

0.19 0.18 0.16 0.22 0.37 0.32

0.19 0.18 0.18 0.19 0.28 0.28

0.20 0.18 0.16 0.20 1.25 1.23

Studies were also carried out on the conversion o f benzaldehyde to benzyl alcohol by whole cell mutant strains of S. cerevisiae which contained only one of the three A D H isoenzymes, I, II or III, and also with a mutant strain lacking A D H I, II and III. Biotransformations were carried out in aqueous-organic systems containing 2 % moisture over a 6 h incubation period, with regular sampling. T i m e courses for production of benzyl alcohol from each mutant strain are presented in Figure 2. E a c h o f the mutant strains manifested highest activity in hexane. Patterns o f conversion o f benzaldehyde to

671

benzyl alcohol were not substantially different with each mutant and, in hexane media, product concentrations after a 6 h incubation ranged from 0.8-1.1 m M . A comparison o f the kinetics o f production o f benzyl alcohol by whole cells of S. cerevisiae containing different isoenzymes in hexane containing 2 % moisture and an aqueous media is illustrated in Figure 3. In general, biotransformation rates observed in aqueous media were substantially higher than the corresponding values in hexane containing 2% moisture.

a, 73 Ο

T i m e (h) Figure 2

T i m e courses for production o f benzyl alcohol by whole cells o f different S.

cerevisiae strains Strain:

#3 #4 #5 #7

contains A D H I I I ; lacks A D H I and I I contains A D H I ; lacks A D H I I and ΠΙ contains A D H I I ; lacks A D H I and I I I lacks A D H I , I I and ΙΠ

Solvents: o, butyl acetate; · , chloroform; • , toluene; • , carbon tetrachloride; A , hexane. ( E a c h solvent contained 2 % moisture.)

672

4

Strain no. Figure 3

Catalytic activities o f whole cells o f different 5. cerevisiae strains in aqueous media and hexane containing 2 % moisture

DISCUSSION In the case o f whole cell biphasic systems, biotransformation activity increased substantially as moisture content was increased from 0 - 1 0 % , with highest activity expressed in media containing 1 0 % moisture. In contrast, highest biotransformation activity with crude cell extract was observed at a moisture content o f 0 . 5 % . Enzymatic activity was likewise found to increase with increasing water content in organic solvents for three unrelated enzymes, alcohol oxidase from yeast, polyphenol oxidase from mushroom, and alcohol dehydrogenase from horse liver [4,5]. Changes in the ratios of the aqueous/organic phases were found to have little effect on the rate o f steroid transformation reactions using Nocardia species, provided the aqueous phase was sufficient to completely swell the cells [11,12]. T h e capacity o f mutant strains, lacking A D H I, I I and III, to convert benzaldehyde to benzyl alcohol in aqueous media is consistent with earlier findings that these mutant strains act as biocatalysts for benzyl alcohol in aqueous media [10]. While another alcohol dehydrogenase, A D H I V , has recently b e e n purified and characterised [13], its role in reduction o f aromatic aldehydes has yet to b e established. This work is being extended to identify the enzymes responsible for reduction o f benzaldehyde in A D H negative mutants with a view to exploring the potential applications o f these enzymes in biotransformations involving conventional and nonconventional media.

673 Acknowledgement Support for this research by the Natural Sciences and Engineering R e s e a r c h Council of Canada is gratefully acknowledged. O.P. Ward is holder o f an N S E R C Industrial Research Chair, co-sponsored by Allelix Biopharmaceuticals Inc., Canada. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

O.P. Ward and C S . Young, Enzyme Microb. Technol. 12 (1990) 4 8 2 . S. Servi, Synthesis (1990) 1. A . M . Klibanov, Chem. Technol. 16 (1986) 3 5 4 . A . Zaks and A . M . Klibanov. J . Biol. Chem. 263 (1988) 8017. J . S . D e e t z and J . D . Rozzell, T I B T E C H , 6 (1988) 15. J . Grunwald, B . Wirz, M. Scollar and A . M . Klibanov. J . Am. Chem. Soc. 108 (1986) 6732. O.P. Ward, Bioprocessing. V a n Nostrand Reinhold, New Y o r k , 1 9 9 1 . A . Long and O.P. Ward, J . Indust. Microbiol. 4 (1989) 4 9 . A . Long, P. J a m e s and O.P. Ward, Biotechnol. Bioeng. 33 ( 1 9 8 9 ) 657. P. Nikolova and O.P. Ward, Biotechnol. Bioeng. 3 8 (1991) 4 9 3 . L . E . S . Brink and J . Tramper, Biotechnol. Bioeng. 27 ( 1 9 8 5 ) 1258. T . Y a m a n e , H. Nakatani, E . Sada, T . Omata, A . T a n a k a and S. Fukiu, Biotechnol. Bioeng. 2 1 ( 1 9 7 9 ) 2 1 3 3 . C. Drewke and M . Ciriacy, 1988. Biochim. Biophys. A c t a 9 5 1 (1988) 5 4 .

Biocatalysis in Non-Conventional Media, edited by J . Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

675

Production of phenylacetyl carbinol by biotransformation using baker's yeast in two-phase systems P. Nikolova and O.P. Ward Department o f Biology, University o f Waterloo, Waterloo, Ontario, Canada N 2 L 3 G 1 . Abstract Production o f L-phenylacetyl carbinol, a precursor o f the pharmaceutical L ephedrine, catalyzed by baker's yeast, was one of the first microbial biotransformation processes to b e commercialised. W e have studied this biotransformation with whole cells in two-phase systems. Highest biotransformation rates were observed with hexane and hexadecane, having Log Ρ values of 3.5 and 8.8 respectively. Lowest bioconversion rates were observed with toluene and chloroform. T h e effect o f moisture content on biotransformation rate was investigated using hexane as organic phase and a moisture content of 1 0 % was found to b e optimal. T h e effect o f organic solvent type on yeast cell structure was investigated. Examination of cells incubated in the two-phase medium for up to 2 6 h by electron scanning microscopy was performed. Ethylacetate, butylacetate, and chloroform, solvents with relatively low Log Ρ values, exhibited lower biotransformation activities, and appeared to puncture the cells, as shown in electron micrographs. In contrast, cells incubated in media containing hexane and decane appeared quite intact and not distorted. D a t a are presented on the biotransformation activities observed in the range of aqueous-organic two-phase systems and the effects of solvents on cell structure are illustrated using scanning electron micrographs. INTRODUCTION W h o l e yeast cells have b e e n used widely as a biocatalyst for organic synthesis in aqueous media, and, indeed, production o f L-phenylacetyl carbinol ( P A C ) was one o f the first classical microbial biotransformation processes to b e commercialised [1], An interesting feature of the production o f acyloin compounds by biotransformation is the capacity o f the system to convert a range o f substituted aromatic aldehydes to the corresponding acyloin compounds [2,3]. Since many organic chemicals manifest low solubility in water, the implementation o f biotransformations in non-conventional media offers potential to extend the range o f substrates which can b e used in bio-organic synthesis [4]. While isolated yeast enzymes have b e e n investigated in non-conventional media, little emphasis has b e e n directed towards examining the performance of whole cells biocatalytic systems in these media [5-7].

676 MATERIALS AND M E T H O D S Preparation of biocatalyst Fresh pressed baker's yeast ( 3 0 % dry weight) was suspended, 5 0 g in 5 0 ml o f 0.05M sodium citrate buffer, p H 6, (buffer A ) and lyophilised. Celite (30-80 mesh, British Drug House, England) 1 g, was mixed with 3 0 0 mg o f lyophilised cells, resuspended in the same buffer and relyophilised. Biotransformation conditions Lyophilised biocatalyst, 1.3 g, was suspended in 3 0 ml o f biotransformation medium. Prior to use, organic solvents were saturated with buffer A. T h e moisture content was then adjusted to 0 . 5 - 2 0 % v/v above the saturation point with buffer A. Sodium pyruvate ( 5 0 g/1) was added as substrate and the reaction initiated by addition o f benzaldehyde (6 g/1). Biotransformations were conducted on an orbital shaker set at 2 8 ° C and 3 0 0 rpm. Analytical methods P A C was determined by gas chromatography ( G C ) , as previously described [8]. F o r protein and lipid determination, 2 0 ml o f the medium, after removal o f the biocatalyst, was evaporated to dryness under vacuum. T h e fatty acid content in this residue was analyzed by G C following methylation, as previously described [9]. T o determine protein content, the residue was dissolved in 100 μ\ o f 2 % S D S and analyzed by scanning densitometry according to the procedure described by Ghosh et al [10]. F o r scanning electron microscopy, the yeast cells were recovered from the organic solvents, air dried and then gold coated (200-300 Â ) with S E M coating unit P S 3 . Accelerating voltage of 15 kv was used in a Hitachi S-570 scanning electron microscope.

RESULTS Production o f P A C from benzaldehyde and pyruvate by whole yeast cells adsorbed on celite was evaluated in different organic solvents containing 1 0 % moisture and the effect o f the biphasic medium on the cell structure was investigated by monitoring the release o f cellular fatty acids and proteins due to damage o f the cells. A time course study on the production o f P A C in hexane containing different moisture contents ( 0 . 5 - 2 0 % v / v ) was investigated (Figure 1). Highest P A C production was observed when the moisture content was 1 0 % . T h e pattern o f production o f P A C in different organic solvents containing 1 0 % moisture is illustrated in Figure 2. Lower biocatalytic activity was observed with chloroform and toluene, intermediate values were observed with ethylacetate and butylacetate and highest P A C production was observed with hexane.

677

0.5

Figure 1 T i m e course o f production o f P A C by whole yeast cells in hexane containing different moisture levels

0.5

EtOAc BuOAc

Chloroform Toluene Hexane Dodecane Hexadecane

Time [h] Figure 2

Pattern of production o f P A C by whole yeast cells in different organic media containing 1 0 % v / v moisture

678

T h e effect o f the different biphasic media on cell structure and fatty acid and protein release was then investigated. Biocatalyst, recovered from the biotransformation medium after various time periods, was examined using scanning electron microscopy. No apparent damage was observed for yeast cells recovered from toluene, hexane and decane containing media. Biocatalyst, recovered from the more hydrophilic solvents (solvents with a lower Log P ) , exhibited damage appearing in the form o f puncturing of the cells in scanning electron micrographs. A comparison o f the scanning electron micrographs obtained for biocatalyst recovered from hexane and chloroform biphasic media after biotransformation periods of 2 6 h and 2 h, respectively, is illustrated in Figure 3.

Figure 3

Scanning electron micrographs of yeast biocatalyst recovered from (a) hexane and ( b ) chloroform

In order to test for release of cellular material into the biotransformation medium, total fatty acids and total protein were determined in the biphasic medium after 2 6 h (Table 1). T h e results indicate that, in the case o f all biphasic media, fatty acids and proteins are released. However, it should b e noted that lowest fatty acid and protein contents were observed in the hexane containing medium which exhibited highest biocatalytic activity. T h e spectrum of fatty acids observed in each biphasic medium is presented in T a b l e 2.

679

Table 1 Comparison o f yeast fatty acids and proteins released into the biotransformation medium with observed biocatalytic activity Solvent*

T o t a l fatty acids (Mg/ml) 140 164 240 170 178

Hexane Butylacetate Ethylacetate Chloroform Toluene

T o t a l protein (/ig/ml)

Activity (mmole.h .mg dry cells) 60.0 31.0 28.4 10.0 7.0

45 50 60 115 80

•containing 1 0 % v/v aqueous phase

Table 2 Spectrum o f yeast fatty acids released into the biotransformation media Solvent Concentration o f fatty acids (/ig/ml)

14:0 16:0 16:1 16:2 16:3 16:4 18:0 18:1 18:2 18:3-6 18:3-3 >18C Total

EtOAc

BuOAc

3.6 26.7 44.5 3.3 6.4 2.2 9.9 93.0 28.8 0.0 12.5 9.3

1.3 15.6 19.2 2.0 3.4 0.7 7.4 77.3 25.0 1.8 10.7 0.0

240.2

164.4

Chloroform

Toluene

Hexane

1.2 16.0 17.0 1.6 3.4 0.0 8.5 83.5 26.0 1.7 11.4 0.0

1.9 14.7 15.3 1.2 2.7 0.0 7.5 89.0 30.9 1.7 13.0 0.0

0.8 12.0 12.4 1.1 2.6 0.0 5.8 70.0 24.0 1.4 10.0 0.0

170.3

177.9

140.1

680 DISCUSSION As was noted above for L-phenylacetyl carbinol synthesis, production o f benzyl alcohol from benzaldehyde in two-phase systems increased with moisture content and highest biotransformation activity was observed with hexane. It has b e e n suggested that, when solvent concentrations exceed a critical level in the cell membrane, the consequent increase in membrane fluidity can result in loss o f biocatalytic activity [12,13]. Rhizopus migricans retained full activity of 11-a-hydroxalase at saturating aqueous phase concentrations o f hexane because o f the inability o f this solvent to attain a critical concentration in the cell membrane [13]. T h e observation that hexane caused less apparent cell damage and released less lipid and protein material from yeast cell biocatalyst than the other solvents tested correlated well with the higher catalytic activity observed in this biotransformation medium. Acknowledgement Support for this research by the Natural Sciences and Engineering R e s e a r c h Council of Canada is gratefully acknowledged. O.P. Ward is holder o f an N S E R C Industrial R e s e a r c h Chair, co-sponsored by Allelix Biopharmaceuticals Inc., Canada. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

K . Kieslich (ed), Biotechnology 6a: Biotransformations, Verlag Chemie, Weinheim, 1984. A . Long and O.P. Ward, J . Indust. Microbiol. 4 ( 1 9 8 9 ) 4 9 . A. Long, P. J a m e s and O.P. Ward, Biotechnol. Bioeng. ( 1 9 8 9 ) 3 3 . O.P. Ward, Bioprocessing, V a n Nostrand Reinhold, New Y o r k , 1 9 9 1 . J . S . D e e t z and J . D . Rozzell, T I B T E C H , 6 ( 1 9 8 8 ) 15. J . Grunwald, B . Wirz, M . Scollar and A . M . Klibanov, J . A m . Chem. Soc. 108 (1986) 6732. A . Zaks and A . M . Klibanov, J . Biol. Chem. 2 6 3 ( 1 9 8 8 ) 8 0 1 7 . P. Nikolova and O.P. Ward, 1991. Biotechnol. Bioeng. 3 8 ( 1 9 9 1 ) 4 9 3 . W . Yongmanitchai and O.P. Ward, O.P. Appl. Environ. Microbiol. 57 (1991) 4 1 9 . S. Ghosh, S. Gepstein, J . J . Heikkila and E . B . Dumbroff. Anal. Chem. 169 (1988) 227. P. Nikolova and O.P. Ward - this volume S . J . Osborne, J . Leaver, M.K. Turner and P. Dunnill, Enzyme Microb. Technol. 12 (1990) 281. L . J . B r u c e and A . J . Daugulis, Biotechnol. Prog. 7 ( 1 9 9 1 ) 116.

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al.

683

© 1992 Elsevier Science Publishers B.V. All rights reserved.

Stability and activity of supramolecular systems 3

a

cholesterol

3

F. A l f a n i , S. B r a n d a n i , M . C a n t a r e l l a , G. S a v e l l i a

oxidase

in

b

D e p a r t m e n t o f C h e m i s t r y , C h e m i c a l E n g i n e e r i n g and M a t e r i a l s , U n i v e r s i t y o f

L'Aquila, M o n t e l u c o di R o i o , 6 7 0 4 0 L'Aquila, Italy b

D e p a r t m e n t o f C h e m i s t r y , U n i v e r s i t y of Perugia, via Elce di Sotto 8, 0 6 1 0 0 P e r u g i a ,

Italy

Abstract O x i d a t i o n of c h o l e s t e r o l to cholest-4-ene-3-one was carried out w i t h a commercial p r e p a r a t i o n of c h o l e s t e r o l o x i d a s e from S t r e p t o m y c e s sp.. Reaction medium was prepared with ethyl acetate p l u s 0.05 M d i d o d e c y l d i m e t h y l a m m o n i u m c h l o r i d e and 2% v/v of 50 mM p h o s p h a t e b u f f e r at pH 7. A two s u b s t r a t e e n z y m e kinetic model holds and the following set of apparent parameters was d e t e r m i n e d : V m Äc = μηιοίβε/π^ m i n , α = 1 . 1 2 1 , 3 59.88 K ( O a ) = 0 . 1 8 0 b a r , Κ ( c h o l e s t e r o l ) = 6 9 . 6 1 m g / m l . Enzyme a c t i v i t y is higher than in pure solvent without surfactant. Activation e n e r g y is equal to 13.07 KJ/mol and indicates that o v e r a l l r a t e of c h o l e s t e r o l o x i d a t i o n is not controlled by enzyme k i n e t i c s u n d e r the e x p e r i m e n t a l c o n d i t i o n s adopted.

1.

INTRODUCTION

S t u d i e s on the n e g a t i v e effects of cholesterol on human health became m o r e f r e q u e n t since 1960 and proved that several cholesterol oxides lead to d e s e a s e s such as a r t e r i o s c l e r o s i s m o r e than c h o l e s t e r o l itself [ 1 ] . These c h o l e s t e r o l d e r i v a t i v e s in m a n y c i r c u m s t a n c e s are formed d u r i n g storage and p r o c e s s i n g of foods [ 2 ] . O n c e t o x i c i t y of these c h e m i c a l s w a s a s c e r t a i n e d new processes were developed which allow to minimize a u t o o x i d a t i o n of c h o l e s t e r o l but m e t h o d s w e r e also i n v e s t i g a t e d to r e d u c e the a m o u n t of c h o l e s t e r o l in plant f e e d s t o c k s . Extraction with supercritical fluids and the use of cholesterol reductases (categories of enzymes w h i c h catalyze c o n v e r s i o n of c h o l e s t e r o l into not d a n g e r o u s d e r i v a t i v e s ) w e r e suggested. The l i t e r a t u r e also indicates that c h o l e s t - 4 - e n e - 3 one is p r o b a b l y the m o s t i n t e r e s t i n g end p r o d u c t of c h o l e s t e r o l o x i d a t i o n and can be p r o d u c e d through a b i o c o n v e r s i o n c a t a l y z e d by c h o l e s t e r o l o x i d a s e (EC 1 . 1 . 3 . 6 ) . Attempts have been made in non aqueous solvents [3,4] and in

684 s u p e r c r i t i c a l fluids w i t h free and immobilized e n z y m e s [ 5 ] , This study deals with an e x p e r i m e n t a l i n v e s t i g a t i o n c a r r i e d out w i t h commercially available cholesterol oxidase in m e d i a prepared with ethyl or butyl a c e t a t e and the ionic s u r f a c t a n t , didodecyldimethylammonium chloride (DDDAC1). C h o l e s t e r o l is indeed very poorly water soluble and its b i o c o n v e r s i o n must be carried out in organic s o l v e n t s . These are selected in order to r e t a i n the a c t i v i t y and s t a b i l i t y of biocatalyst but also to allow their easy utilization in industrial bioprocesses. Ethyl a c e t a t e is already largely used in the food i n d u s t r y for the e x t r a c t i o n of fatty s t u f f s . On the other hand, p r e v i o u s studies [6] showed that a p p r e c i a b l e a m o u n t s of w a t e r , n e c e s s a r y to a s s u r e enzyme a c t i v i t y , can be d i s s o l v e d in b u t y l a c e t a t e and DDDAC1 mixtures. The three components present a supramolecular organization (reverse micelles) which, as o b s e r v e d in p h y s i c a l , chemical and b i o l o g i c a l s y s t e m s , can be used to d e v e l o p new f u n c t i o n s [ 7 ] .

2. EXPERIMENTAL 2.1. M a t e r i a l s The c h o l e s t e r o l o x i d a s e from S t r e p t o m y c e s sp. used in this study w a s from SIGMA ( U S A ) . Ethyl a c e t a t e and η-butyl a c e t a t e w e r e supplied by Baker C h e m i c a l s (Holland) and used without further purification. C h o l e s t e r o l and c h o l e s t - 4 - e n e - 3 - o n e w e r e b o u g h t from SIGMA ( U S A ) . All other c h e m i c a l s w e r e pure g r a d e reagents commercially available. The p r e p a r a t i o n and p u r i f i c a t i o n of the twin chain s u r f a c t a n t (DDDAC1) w a s performed according to the methods previously d e s c r i b e d in [ 8 ] .

2.2 Assay of cholesterol oxidase activity

e

The enzyme w a s incubated 15 m i n at 30 C w i t h c h o l e s t e r o l (2 m g / m l ) and the liquid phase was c o n t i n u o u s l y saturated with p u r e oxygen a t m o s p h e r e at 1.1 b a r , u n l e s s o t h e r w i s e s p e c i f i e d . The following composition of reaction medium was used as s t a n d a r d : 50 μΐ of enzyme solution in 50 mM p h o s p h a t e b u f f e r p H 7 and 2.45 ml of a c e t a t e plus DDDAC1 (0.05 M ) . Final e n z y m e c o n c e n t r a t i o n was 0.022 m g / m l . The r e a c t i o n w a s carried out in stirred r e a c t o r s (100 r e v / m i n ) . The formed p r o d u c t w a s m e a s u r e d s p e c t r o - p h o t o m e t r i c a l l y at 254 nm. A c a l i b r a t i o n curve w a s used to turn m o l a r a b s o r b a n c e into product c o n c e n t r a t i o n .

2.3 S t a b i l i t y of c h o l e s t e r o l o x i d a s e C h o lee s t e r o l o x i d a s e w a s stored at d i f f e r e n t t e m p e r a t u r e s (20 40 C ) under stirred conditions (100 rev/min); 0.6 ml of e n z y m a t i c solution (1.1 m g / m l in 50 mM p h o s p h a t e b u f f e r pH 7) w e r e added to 14.7 ml of acetate and DDDAC1. Surfactant c o n c e n t r a t i o n in the organic phase w a s 0.1 M. At r e g u l a r time

685

intervals samples (1.275 ml) were withdrawn from the storage vessel and the assay of residual activity was carried out at the above mentioned conditions. For the sake of comparison cholesterol oxidase was also stored in purely aqueous media (enzyme concentration equal to 1.1 mg/ml and 50 mM phosphate buffer pH 7 ) . The system was unstirred. At the end of the storage period, 50 μΐ were sucked up from the glass tube and added to 2.45 ml of organic solvent, DDDAC1 and cholesterol in order to test residual activity at the standard assay conditions. 3. RESULTS AND DISCUSSION 3.1 Enzyme storage stability The experiments discussed in this study were always carried out with cholesterol oxidase from Streptomyces sp. since the results of a previous investigation [9] showed that the enzyme from Nocardia erythropolis is less active (41.2 % of relative activity) and exhibits a higher rate of deactivation while that from Pseudomonas fluorescens is less active (44.1 % of relative activity) and shows a comparable deactivation rate. Since it can be assumed that the enzyme in the supramolecular system is totally partitioned in the water pools of the micelles its concentration in the microenvironment should be equal to that in the aqueous solutions used to prepare the storage mixture. Furthermore, it can be also assumed that inside these water pools the enzyme is enough protected from the shear stress caused by stirring devices in the bulk. Therefore, stability of cholesterol oxidase in stirred supramolecular media was compared with that in unstirred purely aqueous buffer at the same enzyme concentration (1.1 mg/ml) in the water pools. The stability in buffer was measured at 30 *C while that in supramolecular media was studied between 20 and e 40 C . The experimental results are reported in Figure 1 and show that initial rate of enzyme deactivation is higher in supramolecular systems than in buffer. This finding suggests that the hypothesis of enzymes protected by shear in the micelles is unsound or that enzyme deactivation is also induced by some of the chemical species present in the enzyme microenvironment (DDDAC1 and ethyl acetate are partially soluble in water). The possibility of a chemical deactivation of the enzyme in the micelles being more important than the thermal one seems confirmed by these preliminar results which indicate that the rate of deactivation is independent of temperature in spite of the large activation energy usually observed for the thermal deactivation phenomenon [10]. The enzyme behaviour observed in this study is also consistent with the stability of hydrolytic enzymes in biphasic media prepared with buffer and several organic solvents [11]. The data of Figure 1 also show that cholesterol oxidase activity rapidly decreases during the first two hours (almost

686

QeJ

i

Ο

ι

ι

200

ι

ι

400

1

ι

1

600 Time,

1

Θ00

1

1

1000

1

ι

1200

1

ι

1400

min

Figure 1. Enzyme storage stability. Pure b u f f e r : ^ 30 *C supramolecular system: φ 20*C, g| 40"C. 65% of initial activity is lost) and then attains roughly a constant value. The curves of residual activity versus storage time can be representative of two different rates of deactivation for first order mechanisms in the active enzyme concentration. The kinetic constant of the second deactivation 4 1 regime in water k 2 w= 2.36 1 0 ~ min- is higher than those for 4 1 10~ minstorage in supramolecular systems k 2.(20*C)= 0.79 1 and k2«(40*C)= 1.55 10-* m i n - . Furthermore, the rate of the second deactivation regime in supramolecular systems is slightly dependent on temperature. 3.2 Cholesterol oxidase activity in pure solvent and in supramolecular systems. Experiments were carried at 30"C during 15 min of incubation with 2 mg/ml of cholesterol and in different media saturated with oxygen at 1.1 bar. Total reaction volume was 10 ml and 200 μΐ of buffered cholesterol oxidase solution (1.1 mg/ml) were always present. The following organic phases were tested: pure ethyl and η-butyl acetate, ethyl and η-butyl acetate with 0.05M DDDAC1. The reactor operated at differential conditions since the highest cholesterol conversion reached under the adopted experimental conditions was 10.2%. The initial rate of cholesterol oxidation is reported in Table 1. Enzyme activity is significantly higher in supramolecular systems than in pure solvents. Media rich in η-butyl acetate allow better enzyme performances than those prepared with ethyl acetate. This

687

b e h a v i o u r can be assumed again as a consequence of the partial solubility of ethyl a c e t a t e in the w a t e r p h a s e w h e r e the enzyme is confined. A chemical c u r t a i l m e n t of the e n z y m e a c t i v i t y can be caused by this solvent w h i c h on the c o n t r a r y is a b s e n t or m u c h less important when η-butyl a c e t a t e is used since this solvent is p o o r l y soluble in w a t e r . In the p r e s e n c e of s u r f a c t a n t the s u p r a m o l e c u l a r o r g a n i z a t i o n can f u r t h e r m o r e p r o t e c t the enzyme from the solvent.

Table 1 Initial rate of c h o l e s t e r o l

Reaction

medium

Ethyl a c e t a t e Ethyl a c e t a t e + D D D A C l η-butyl a c e t a t e η-butyl a c e t a t e + D D D A C l

oxidation

R e a c t i o n rate μπιοίβε/π^ m i n

0.042 0.245 0.561 1. 603

Conversion %

0. 30 1. 60 3.60 10.20

H o w e v e r , in spite of these r e s u l t s on the initial r e a c t i o n rate which w o u l d suggest to w o r k in η-butyl a c e t a t e m e d i a , the f o l l o w i n g part of this e x p l o r a t o r y i n v e s t i g a t i o n w a s c o n t i n u e d in ethyl acetate because of the w e l l e s t a b l i s h e d use of t h i s solvent in the food i n d u s t r y .

3.3 Evaluation of kinetic parameters It has been already p o i n t e d out that c h o l e s t e r o l o x i d a t i o n cannot be p e r f o r m e d in aqueous media since the s u b s t r a t e is a l m o s t t o t a l l y u n s o l u b l e . A n o t h e r a d v a n t a g e w h i c h a r i s e s by the u t i l i z a t i o n of organic solvents is the higher solubility of o x y g e n (second substrate of the r e a c t i o n ) in these c h e m i c a l s with respect to the one in w a t e r . In fact, a c c o r d i n g to the v a l u e s of Henry's constant d3 e t e r m i n e d in [ 1 0 ] , the s a t u r a t i o n molar concentration ( m o l e s / m ) of o x y g e n at 25*C and 1 atm gas p r e s s u r e is 1.38 in w a t e r , 11.78 in ethyl a c e t a t e and 10.83 in η-butyl a c e t a t e . T h e r e f o r e , if m a s s t r a n s f e r is s u f f i c i e n t fast the r e a c t i o n rate should be a c c e l e r a t e d in these organic m e d i a . This is specially e v i d e n t if h i g h c o n c e n t r a t i o n of o x y g e n are n e e d e d to reach substrate s a t u r a t i o n c o n d i t i o n s (zeroth o r d e r kinetics). Furthermore, the stoichiometry of cholesterol o x i d a t i o n is e q u i m o l a r and in the 3 tested solvents up to 10 of cholesterol can be mg/ml equivalent to 25.86 moles/m solubilized. Therefore, the above levels of dissolved oxygen a s s u r e that if the liquid is c o n t i n u o u s l y saturated w i t h gas at r o u g h l y 2.2 bar all c h o l e s t e r o l can be c o n v e r t e d and c o s t l y o p e r a t i o n s at h i g h p r e s s u r e avoided.

688 The reaction network of cholesterol schematically represented as follows:

Ε + Β

±

+

A

A

Jî

EA + Β

can

be

EB

+

J!

oxidation

QRa

• EAB

Ε +

Pi

+

Ps

where Ε is the enzyme, A and Β are oxygen and cholesterol respectively, P i and P 2 are cholest-4-ene-3-one and hydrogen peroxide. According to the classic enzyme theory the following rate equation holds:

V

[B] αΚ Β {1

+ W[A]}

+ [B]{1 + a W [ A ] }

Experiments were carried out at 30"C with 4, 6, 10 mg/ml of cholesterol and oxygen pressures equal to 0.315, 0.400 and 1.100 bar. Enzyme concentration was 0.0022 mg/ml, incubation time was 15 min and the reaction medium was prepared with ethyl acetate plus 0.05M DDDAC1. Water was 2% of total volume. The results are plotted in Figure 2 and seem to confirm the correctness of the kinetic model.

0.05

-0-05

-0-2 Figure

0.15

0.25

l/[cholesterol], ml/mg

2 : L i n e w e a v e r - B u r k p l o t . T = 3 0 * C . Oxygen p r e s s u r e # 0.315; • 0.400; # 1.100.

(bar):

689

The following set of kinetic parameters was determined: Vmax

=

Κα

=

K b

= 6 9 . 6 1

α

=

5 9 . 8 8 0 . 1 8 0

μηιοίβε/π^ min bar mg/ml

1 . 1 2 1

These values indicate that the reaction can not occur at a saturating cholesterol concentration while at an oxygen partial pressure above 1 bar the reaction rate is independent of dissolved oxygen concentration. Finally, experiments were # carried out at different in order to determine the temperatures from 1 5 to 4 0 C activation energy of the enzymatic reaction. Oxidation was carried out in the same medium with the composition above reported but with the exception of a constant cholesterol concentration ( 1 0 mg/ml) and a saturating oxygen partial pressure. This latter condition is achieved at 1 . 1 bar for e temperatures below or equal to 3 0 * C and 1 . 4 bar at 3 5 and 4 0 C . Enzyme concentration was also constant and equal to 0 . 0 2 2 mg/ml. Results were reported in the form of an Arrhenius plot and showed that the rate of oxidation is controlled by a single mechanism in the whole range explored (unique slope). The value of the activation energy ( 1 3 . 0 7 KJ/mol) was calculated by a linear regression of data and indicates that overall rate of cholesterol oxidation is not controlled by enzyme kinetics. Besides, experiments carried out at different oxygen flow rates gave the same value of activation energy and therefore, the conclusion was reached that interphase mass transfer of oxygen from gas to liquid did not control overall reaction rate. On the other hand, since both oxygen and cholesterol preferentially partition in the organic phase it seems reasonable to assume that either cholesterol or oxygen transfer from outside to inside the assemblies where the enzyme is confined may act as rate limiting step. This implies that the above values of kinetic parameters are apparent. 4. ACKNOWLEDGEMENTS The support of the Science Research Council (CNR, grants 8803616-14 and 89-04592-14) is acknowledged. 5. REFERENCES 1 2 3

C.B. Taylor, S.K. Peng, N.T. Werthessen, Ρ.Tham and K.T. Lee, Amer. J. Clinical Nutrition, 32 (1979) 40 J. Nourooz-Zedeh and L.A. Appelqvist, J. Food Science, 53 (1988) 74 B.C. Buckland, P. Dunnill and M.D. Lilly, Biotechnol. Bioeng., 17 (1975) 815

690

4

T. O m a k a , A. Tanaka and S. F u k u i , J. F e r m e n t . T e c h n o l . , 5 8 (1980) 339 5 T.W. R a n d o p h , H.W. Blanch and J.M. P r a u s n i t z , A I C h E J., 34 (1988) 1354 6 F. A l f a n i , M. C a n t a r e l l a , N. C u t a r e l l a , N. S p r e t i , R. G e r m a n i , G. S a v e l l i , J. B i o t e c h n o l . , (1992) in p r e s s 7 H. R i n g s d o r f , B. S c h l a r b and J. V e n z m e r , A n g e w . C h e m . I n t . E d . E n g l . , 27 (1988) 113 8 A. C i p i c i a n i , R. G e r m a n i , G. S a v e l l i , C.A. B u n t o n , M. M h a l a and J.R. M o f f a t , J . C h e . S o c . P e r k i n I I , (1987) 541 9 S. B r a n d a n i , Thesis in C h e m i c a l E n g i n e e r i n g , U n i v e r s i t y of L ' A q u i l a , (1991) 10 F. A l f a n i , L. C a n t a r e l l a , M. C a n t a r e l l a , A. G a l l i f u o c o and V. S c a r d i , L a t . A m . A p p l . R e s . , 20 (1990) 47 11 M. C a n t a r e l l a , L. C a n t a r e l l a and F. A l f a n i , Enzyme M i c r o b . T e c h n o l , 13 (1991) 547 12 P. L u h r i n g and A. Schumper, J . C h e m . E n g . D a t a , 34 (1989) 250

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

691

C R O W N E T H E R S CAN E N H A N C E E N Z Y M E A C T I V I T Y IN O R G A N I C S O L V E N T S

Jaap Broos, Willem Verboom, Johan F. J. Engbersen and David N. Reinhoudt* Laboratory of Organic Chemistry, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands The effect of various crown ethers on the transesterification reaction of N-acetyl(L)phenylalanine ethyl ester with propanol catalyzed by a-chymotrypsin was investigated. Crown ethers can strongly enhance this reaction in different organic solvents. A macrocyclic effect is responsible for the increased enzymatic activity. Several possible crown ether enzyme interactions are discussed.

1. I N T R O D U C T I O N

Nowadays it is well established that enzymes can be catalytically active in dry organic solvents . Compared to aqueous solutions, the use of an organic reaction medium can have some interesting advantages, like a different substrate and stereospecificity, a higher thermal stability of the enzyme, and the absence of interference of water as nucleophile in 1 the catalyzed reaction . Best activity of the enzyme in organic solvent is obtained when the enzyme is first lyophilized or precipitated from an aqueous solution of the pH where the enzyme has its optimal activity and, if necessary, in the presence of high concentrations of 2 an inhibitor . An important drawback of the use of organic solvents as the reaction medium is that the activity of the enzyme is generally up to several orders of magnitude lower than in aqueous solution. The nature of this lower activity is still unknown, although some reasons have been proposed to explain this phenomenon. In comparing the activity of 3 horseradish peroxidase in aqueous and organic media, Ryu and Dordick concluded that the K m value is strongly increased in the presence of organic solvents, while the k c at value is less influenced, resulting in a much lower k c a l/ K m value. Activity studies in relation to the hydration of dry enzyme powders revealed that the enzyme started to become catalytically 4 active when the hydration has reached a certain minimal level . Concomitant with the increase in hydration, the flexibility of the enzyme is increased and it is believed that some flexibility of the peptide backbone is necessary for enzymatic activity. The reason for the larger flexibility of the hydrated enzyme is explained by the weakening of salt bridges and hydrogen bonds upon the hydration of the charged groups on the enzyme. Moreover, the strong electric field of the charged groups on their environment which might be deleterious for good enzymatic activity, becomes reduced by the high dielectric constant of water. It has been found that the activity can be markedly enhanced when the amount of water on 5 the enzyme, suspended in an organic solvent, is increased . However, increment of the amount of water on the enzyme also results in a more "water-like" enzyme behaviour, thus reducing the advantages which are typical for the use of organic solvents. Therefore, adding water to the organic reaction medium is generally not a good method to enhance enzyme 5,6 activity. As an alternative hydrogen-bond formers like glycol or formamide can be added . However, this method is not generally applicable since these additives are not miscible with the more apolar solvents, which is the most attractive class of solvents to be used for 7 enzymatic reactions . Another approach is to make the enzyme more soluble in organic solvents by linking the

692 enzymes covalently with amphiphiles which renders the enzyme surface more 8 hydrophobic . It has9 ,been reported that enzymes modified in this way show a higher activity 10 in organic solvents . We have investigated the influence of crown ethers on the enzymatic activity of enzymes 11,12 suspended in organic s o l v e n t s . Depending on their ring size and structure, crown ethers are known to form complexes with several types of guests like metal ions, ammonium 1 3 , 1 4 , 11 5 , groups, water, and the guanidinium i 0 n . All these guests are present in enzymatic reactions. Furthermore, the strongest interaction between crown ethers and guest species occurs in aprotic organic solvents. Several influences of crown ethers might be proposed as there are: increasing the accessibility of the active site for the product by complexation of the buffer cations and/or water; reduction of the high electrostatic influence of charged residues, by complexation with the crown ether, and dissolution of the enzyme by making 17 the surface more hydrophobic .

2. R E S U L T S AND D I S C U S S I O N

The influence of crown ethers has been explored on the α-chymotrypsin catalyzed transesterification of N-acetyl(L)phenylalanine ethyl ester with propanol (reaction 1). ο α-chymotrypsin

OCH 2CH 3

ΗΝ

Y"

^

γ

"

ο

Y^

+HOCH 9CH 0CH 3

°

^

·

^

^

HN

+ C H 3C H 2O H

0

1

reaction 1 2

This reaction was chosen since it has been well studied in various organic solvents . The 18 effect of addition of the well studied crown ether 18-crown-6 (18-C-6) on this model reaction in toluene is depicted in Fig. 1.

Vo [M/min] 5 Ί0

0

4

1

•

,

.

.

•

•

1

2 4 • cone. 18-C-6 [mM] Fig. 1: Influence of the 18-C-6 concentration on the initial a velocity of reaction l . 0

a

Conditions: 2.5 mM ester, 1 M PrOH in toluene, 0.5 mg enzyme/ml (pretreated with potassium phosphate buffer), 25°C.

693 Table 1: Influence of 18-C-6 on reaction 1 in various solvents. 18-C-6 [mM]

solvent octane

0

cyclohexane

2 0 2

dibutyl ether

0 2 0 2 0 2 0 2

toluene t-amyl alcohol THF + 1 % H 20

Vo [ M / m i n - ^ l O

7

Vo(18-C-6)/Vo(blank)

54 ± 6 1545 ± 8 0

29

42±8 805 ± 95

19

1.7 ± 0 . 1 53 ± 4 5.0 ± 0 . 2

20

98 ± 13 0.20 ± 0 . 0 1

2

0.40 ± 0.02 1.8 ± 0 . 0 14.5 ± 0 . 5

3

31

8

a Conditions: 2.5 mM ester, 1 M PrOH, 0.5 mg enzyme/ml ( pretreated with potassium phosphate buffer), 25°C.

Initial additions of 18-C-6 do not exhibit a large effect on the enzyme activity but above ca. 0.2 mM 18-C-6 a sharp increase in the initial rate is observed until a plateau value is reached at concentrations above 1 mM. In the concentration range 1-4 mM the initial rate is 20 times higher than in the absence of 18-C-6. The effect of 18-C-6 in different organic solvents has been studied at a fixed concentration of 2 mM 18-C-6. Results are presented in Table 1. In all solvents tested, 18-C-6 has a significant influence on the enzyme activity. The largest effects have been found in dibutyl ether and octane where the initial velocity increases 30 times. Notably, in octane, in which the enzyme shows also its highest activity in the absence of crown ether, also the largest rate increase is observed.

Table 2: Influence of various crown ethers on reaction 1 in toluene.

0

crown ether 18-C-6 pentaglyme monoaza-18-C-6 decyl-18-C-6 dicyclohexyl-18-C-6 dibenzo-18-C-6 dibenzo-24-C-8 15-C-5 kryptofix 22 didecyl

Vo(crown ether)/Vo (blank) 19.6 1.4 11.3 1.9 4.0 6.0 1.8 1.8 2.6

a Conditions: 2.5 mM ester, 1 M PrOH, 0.5 mg enzyme/ml (pretreated with potassium phosphate buffer), 25 °C.

694

Next to 18-C-6 also the effect of other crown ethers has been studied on the model reaction in toluene. As shown in Table 2 the best results are observed with the crown ether 18-C-6, although also other crown ethers like monoaza-18-C-6 and dibenzo-18-C-6 strongly enhance the reaction. The effect of a crown ether is decreased when the ring size becomes smaller or larger than 18 atoms. Most remarkably, the open chain analog of 18-C-6, pentaglyme, shows hardly any effect on the activity of the enzyme. This clearly points out that a macrocyclic effect is responsible for the observed activation of the enzyme. Species which form complexes with 18-membered crown ethers are alkali metal ions, ammonium groups, and water. Alkali metal ions are present as the result of the pretreatment of the enzyme with 0.1 M K H 2 P 0 4 buffer at pH= 7.8 and subsequent lyophilization. Instead of the potassium ion buffer, we also tested pretreatment with sodium and lithium phosphate buffer and found for all these enzyme preparations an enhancement of the reaction (6-29 12 times). However, the largest effects where obtained with potassium as the counterion . Potassium ions form more stable complexes with 18-C-6 than do sodium or lithium ions. This might be used as an indication that the enhancing effect of crown ethers is due to complexation of the buffer cation on the surface of the enzyme, which could possibly lead to a better accessibility of the active site. However, some caution with such an interpretation is necessary since upon changing the buffer solution not only the cation is changed but also the amount of water present on the enzyme after lyophilization. The possible role of the ammonium group in the enzyme activation caused by crown ethers has been elucidated comparing the activities of the protease trypsin in its native form (from Bovine pancreas, Sigma) and trypsin, in which the ammonium residues of the enzyme are acylated. The effect of 18-C-6 on the activity of both enzymes was investigated with the model reaction, in toluene, as described for α-chymotrypsin. It was found that 2 mM 18-C-6 enhanced the activity of native and acylated trypsin 29 and 48 times respectively. From these results it can be concluded that complexation of the ammonium groups by 18-C-6 is not responsible for the observed increase in enzymatic activity. Apart from complexing alkali metal ions and ammonium groups, crown ethers are also 15,19 known to form complexes with water . The water complexing ability of several of the 19 tested crown ethers in CDC13 has been reported . Interestingly, the ability of the crown ethers to enhance the reaction (in toluene) corresponds well with its affinity for water (in CDC13). Thus the better the crown ether complexes water, the better it is able to enhance the enzymatic activity. This might be an indication that dehydration of the active site and/or of the substrate by crown ethers play a role in the activation process. This view is supported by the fact that more hydrophobic crown ethers like decyl-18-C-6 and dicyclohexyl-18-C-6 show a lower enhancing effect than other 18-membered crown ethers (Table 2). The reason may then be that these former compounds are not as good able to enter the polar active site. The proposed dehydration mechanism is quite difficult to verify by variation of the experimental conditions, since any alteration of the amount of water on the enzyme concomitantly changes other characteristics of the catalyst like its flexibility, while water also can act as a reactant. An experimental approach to this problem might be to test the crown ether effect on a series of N-acetyl amino acids with side chains of different size. If dehydration of the active site by the crown ethers is responsible for the catalytic effect, an enhanced influence of crown ethers can be expected when the substrate size is increased. Indeed we have found that the effect of 2 mM 18-C-6 on the transesterification of N-acetyl(L)phenylalanine ethyl ester is larger than that on the alanine ester analogue. However, more research is needed to elucidate the importance of the dehydration mechanism. Research is in progress to study the influence of crown ethers on the stereochemical outcome of enzymatic reactions. Moreover, the rate-limiting step in the described transesterification reactions is studied by variation of the leaving alcoholate groups in the N-acetyl alanine esters.

695

Preliminary results show that for substituted ethyl esters of N-acetylalanine, acylation of the enzyme is rate-limiting. In conclusion, we present here a simple method to strongly activate enzyme activity in organic solvents by adding low concentrations of crown ethers. It is shown that a macrocyclic effect is responsible for the observed enzyme activation, although the exact mechanism has still to be elucidated.

3. ACKNOWLEDGEMENTS This investigation was supported by the Netherlands Technology Foundation [ S T W , Future Technology Science Branch of the Netherlands Organisation for the Advancement of Pure Research (NWO)] and DSM Research Geleen.

4. R E F E R E N C E S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

A. M. Klibanov, Trends Biochem. Sei. 14, 141 (1989). A. Zaks and A. M. Klibanov, J . Biol. Chem. 2 6 3 , 3194 (1988). K. Ryu and J . S. Dordick, J. Am. Chem. Soc. I l l , 8026 (1989). J . A. Rupley, E. Gratton and G. Cared, Trends Biochem. Sei. 8 , 18, (1983). A. Zaks and A. M. Klibanov, J . Biol. Chem. 2 6 3 , 8017 (1988). H. Kitaguchi and A. M. Klibanov, J . Am. Chem. Soc. I l l , 9272 (1989). C. Laane, S. Boeren, R. Hilhorst and C. Veeger in " Biocatalysis in Organic Media", C. Laane, J . Tramper and M. D. Lilly, eds., Elsevier Science Publishers B.V., Amsterdam, 1987, ρ 65. Y. Inada, Κ. Takahashi, T. Yoshimoto, A. Ajima, A. Matsushima and Y. Saito, Trends Biotechnol. 4, 190 (1986). M. T. Babonneau, R. Jacquier, R. Lazaro and P. Viallefont, Tetrahedron Lett. 3 0 , 2787 (1989). Y. Okahata and K. Ljiro, J . Chem. S o c , Chem. Commun. 1392 (1988). D.N. Reinhoudt, A. M. Eendebak, W. F. Nijenhuis, W. Verboom, M. Kloosterman and H. E. Schoemaker, J . Chem. S o c , Chem. Commun. 399 (1989). J . Broos, M-N. Martin, I. Rouwenhorst, W. Verboom and D. N. Reinhoudt, Reel. Trav. Chim. Pays-Bas 110, 222 (1991). J.W. H. M. Uiterwijk, C. J . van Staveren, D. N. Reinhoudt, H. J . den Hertog Jr., L. Kruise and S. Harkema, J . Org. Chem. 5 1 , 1575 (1986). J . A. A. de Boer and D. N. Reinhoudt, J . Am. Chem. S o c 107, 5347 (1985). F. de Jong, D. N. Reinhoudt and C. J . Smit, Tetrahedron Lett. 1371 (1976). W. L. Dom, A. Knöchel, J . Oehler and G. Rudolph, Z. Naturforsch., Teil Β 3 2 , 7 7 6 (1977). B. Odell and G. Earlam, J . Chem. S o c , Chem. Commun. 359 (1985). "Crown Ethers and Analogs", E. Weber, S. Fatal and Z. Rappoport, eds. J . Wiley & Sons., Chichester, 1989. L.P. Golovkova, A. I. Telyatnik, V. A. Bidzilya, Ν. E. Akhmetova and V. I. Konovalova, Theor. Exp. Chem. (Engl. Transi.) 2, 248 (1985).

Biocatalysis in Non-Conventional Media, edited by J . Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

697

Dynamics, structure and stability of α-chymotrypsin in aqueous solution and in reverse micelles as studied by fluorescence spectroscopy

V.

Dorovska-Taran

a , b

,

C.Veeger

department of Biochemistry, W a g e n i n g e n , The N e t h e r l a n d s b

3

and

A.J.W.G.Visser

Agricultural

Permanent address: I n s t i t u t e of Organic S c i e n c e s , 1113 S o f i a , B u l g a r i a

3

University,

Chemistry,

Dreyenlaan 3,

6703 HA

B u l g a r i a n Academy

of

ABSTRACT I n t h e l a s t few y e a r s d i f f e r e n t e x p e r i m e n t a l approaches have been made t o a n s w e r t h e q u e s t i o n : how many w a t e r m o l e c u l e s d o e s a n enzyme r e q u i r e t o d i s p l a y i t s e n z y m a t i c a c t i v i t y and t o w h i c h e x t e n t has t h e p r o t e i n g l o b u l e t o be h y d r a t e d t o m a i n t a i n t h e maximum c o n f o r m a t i o n a l s t a b i l i t y ? The q u e s t i o n u n d e r s t u d y r e q u i r e s t o u s e a s y s t e m i n w h i c h t h e a m o u n t o f w a t e r m o l e c u l e s c a n be v e r y p r e c i s e l y c o n t r o l l e d w h i l e t h e enzyme c o n f o r m a t i o n - a c t i v i t y r e l a t i o n s h i p c a n be f o l l o w e d b y some v e r y s e n s i t i v e t e c h n i q u e l i k e f l u o r e s c e n c e . Some a m p h i p h i l i c s u r f a c t a n t s l i k e a e r o z o l - 0 T (AOT) i n o r g a n i c s o l v e n t s f o r m t h e r m o d y n a m i c a l l y s t a b l e , o p t i c a l l y t r a n s p a r e n t r e v e r s e m i c e l l e s i n w h i c h a q u e o u s s o l u t i o n s o f a n enzyme c a n be s o l u b i l i z e d . The h y d r a t i o n d e g r e e , w 0, d e f i n e d a s [ H 20 ] / [ A 0 T ] i s d e t e r m i n e d v e r y p r e c i s e l y . As a m o d e l p r o t e i n we h a v e c h o s e n α - c h y m o t r y p s i n ( C T ) a s one o f t h e b e s t s t u d i e d e n z y m e s . I n a d d i t i o n t o n a t u r a l f l u o r e s c e n c e o f α-chymotrypsin a d e t a i l e d fluorescence study of anthraniloyl-a-chymotrypsin ( A n t - C T ) , i n w h i c h a h i g h l y f l u o r e s c e n t a n t h r a n i l o y l g r o u p was c o v a l e n t l y a t t a c h e d t o t h e a c t i v e s i t e , was c a r r i e d o u t t o i n v e s t i g a t e i t s p h y s i c a l p r o p e r t i e s i n aqueous s o l u t i o n and i n a r e s t r i c t e d w a t e r s y s t e m . R e s u l t s p r e s e n t e d i n t h i s s t u d y i n d i c a t e a c h a r a c t e r i s t i c dependence f o r t h e s t a b i l i t y a n d f l u o r e s c e n c e p r o p e r t i e s o f CT o n t h e a m o u n t o f w a t e r i n t h e r a n g e o f wD a r o u n d 5 . T h i s f e a t u r e i s q u a l i t a t i v e l y d i s c u s s e d i n t e r m s o f a " t h r e e p h a s e m o d e l " o f t h e s t a t e o f w a t e r i n r e v e r s e d m i c e l l e s ( A . G o t o et al., T h e r m o c h i m . A c t a 163 ( 1 9 9 0 ) 1 3 9 ) . A t v e r y l o w w 0 t h e c o n f o r m a t i o n o f CT c h a n g e s t o v e r y r i g i d i n c o m p a r i s o n t o w a t e r s o l u t i o n . The f o u r p e a k s i n t h e f l u o r e s c e n c e l i f e t i m e d i s t r i b u t i o n i n aqueous s o l u t i o n a r e c o n v e r t e d i n t o a s i n g l e p e a k . The o v e r a l l c e n t e r o f g r a v i t y o f t h e t r y p t o p h a n f l u o r e s c e n c e s p e c t r u m o f t h e enzyme a t w D = 0 . 6 5 i s b l u e s h i f t e d i n comparison t o t h e s i t u a t i o n i n w a t e r . I n t h e absence o f a h y d r a t i o n s h e l l , the p r o t e i n i s e s s e n t i a l l y f r o z e n and i n a c t i v e . Small i n c r e a s e s i n w a t e r c o n t e n t t r a n s f e r r e d new w a t e r m o l e c u l e s t o t h e n e g a t i v e l y c h a r g e d s u r f a c t a n t s h e a d s a n d t h e enzyme m o l e c u l e becomes e v e n m o r e r i g i d t h a n i t i s a t w 0 = 0 . 6 5 . The s h o r t c o r r e l a t i o n t i m e p e a k o f t h e a n t h r a n i l o y l g r o u p , w h i c h r e p r e s e n t s t h e i n t e r n a l a c t i v e s i t e m o t i o n o f CT shows a n i n c r e a s e u p t o w 0 e q u a l t o a b o u t 5 w h e r e t h e enzyme d i s p l a y s a m i n i m u m o f e n z y m a t i c a c t i v i t y . A n i n c r e a s e i n e n z y m a t i c a c t i v i t y w i t h i n a r e l a t i v e l y s m a l l wD range o f 5 t o 8 i n d i c a t e s a c o o p e r a t i v e h y d r a t i o n - d e p e n d e n t a c t i v a t i o n of

698

c a t a l y s i s which accounts f o r cooperative b i n d i n g of water molecules to the p r o t e i n and c o v e r s a l i m i t e d h y d r a t i o n range (< 1 g o f w a t e r per gram o f p r o t e i n ) . Above wD = 1 0 , h o w e v e r , t h e e f f e c t o f h y d r a t i o n on t h e a c t i v a t i o n o f c a t a l y s i s i s c o m p l e t e w h i c h shows t h a t t h e enzyme a c t i v i t y d e p e n d s o n t h e a m o u n t o f w a t e r i n c o n t a c t w i t h t h e enzyme a n d n o t o n t h e t o t a l a m o u n t of water i n the system. I n other words, bulk water i s not necessary f o r a c t i v i t y . The r e s u l t s a r e c o n s i s t e n t w i t h t h e p i c t u r e d e s c r i b e d b y R u p l e y et al. ( A d v . C h e m . , S e r . 80 ( 1 9 8 0 ) 1 1 1 ) .

1.INTRODUCTION I t i s w e l l known t h a t p r o t e i n s c a n e x i s t i n a number o f d i f f e r e n t main c o n f o r m a t i o n s and s u b c o n f o r m a t i o n s d e p e n d i n g on t h e c h e m i c o - p h y s i c a l parameters of the exact environmental c o n d i t i o n s [ 1 - 3 ] . I t i s o f t e n s u g g e s t e d t h a t t h e i n t e r c o n v e r s i o n t o t h e a c t i v e c o n f o r m a t i o n may o c c u r i n t h e course o f t h e enzymatic r e a c t i o n ( " i n d u c e d f i t mechanism" [ 4 , 5 ] ) or by f i x i n g t h e enzyme m o l e c u l e i n a p r o p e r l y c h o s e n m a t r i x [ 6 ] . The p r o t e i n h y d r a t i o n p l a y s an i m p o r t a n t r o l e i n c a t a l y t i c a c t i v i t y and c o n f o r m a t i o n a l s t a b i l i t y o f enzymes [ 7 , 8 ] . I n t h e l a s t f e w y e a r s d i f f e r e n t e x p e r i m e n t a l a p p r o a c h e s h a v e b e e n made t o a n s w e r t h e q u e s t i o n : how many w a t e r m o l e c u l e s d o e s a n enzyme r e q u i r e t o e x p r e s s i t s a c t i v i t y a n d t o w h i c h e x t e n t h a s t h e p r o t e i n g l o b u l e t o be h y d r a t e d t o m a i n t a i n t h e maximum c o n f o r m a t i o n a l s t a b i l i t y ? [ 7 , 9 ] . The q u e s t i o n u n d e r s t u d y r e q u i r e s a s y s t e m i n w h i c h t h e a m o u n t o f w a t e r m o l e c u l e s c a n be v e r y p r e c i s e l y c o n t r o l l e d a n d t h e enzyme c o n f o r m a t i o n - a c t i v i t y r e l a t i o n s h i p c a n be f o l l o w e d b y some v e r y s e n s i t i v e t e c h n i q u e . Some a m p h i p h i l i c s u r f a c t a n t s i n o r g a n i c s o l v e n t s f o r m thermodynamically stable o p t i c a l l y transparent reverse micelles i n which a q u e o u s s o l u t i o n s o f a n enzyme c a n b e s o l u b i l i z e d . The h y d r a t i o n d e g r e e , w 0, d e f i n e d a s [ H ^ 0 ] / [ A O T ] i s a v e r y p r e c i s e l y d e t e r m i n e d q u a n t i t y . The m i c e l l a r m a t r i x may p l a y t h e r o l e o f a n " e n z y m a t i c t r a p " f o r t h e s o l u b i l i z e d enzyme c o n v e r t i n g a n d f i x i n g i t i n a c e r t a i n c o n f o r m a t i o n a l s t a t e [ 6 ] . As a m o d e l p r o t e i n we h a v e c h o s e n α - c h y m o t r y p s i n ( C T ) a s one o f t h e b e s t s t u d i e d e n z y m e s . I t i s k n o w n t h a t CT a l o n e a s w e l l a s some l i g a n d - C T complexes e x i s t i n a number o f d i f f e r e n t c o n f o r m a t i o n a l s t a t e s a n d s u b s t a t e s [ 1 ] . R e c e n t l y L e v a s h o v et al. [ 6 ] p o s t u l a t e d t w o i n t e r c o n v e r t i b l e f o r m s o f CT d i f f e r i n g i n c o n f o r m a t i o n a l m o b i l i t y , i . e . a r e l a x e d one p r e d o m i n a n t l y e x i s t i n g i n a q u e o u s s o l u t i o n a n d a t e n s e one whose p r o p o r t i o n r i s e s w i t h a i n c r e a s e i n t h e c o n c e n t r a t i o n o f t h e water-miscible organic solvent i n the reverse m i c e l l a r system. S i n c e enzymes i n r e v e r s e d m i c e l l e s a r e o p t i c a l l y t r a n s p a r e n t s o l u t i o n s , f l u o r e s c e n c e s p e c t r o s c o p y h a s p r o v e n t o be a h i g h l y s e n s i t i v e t e c h n i q u e t o i n v e s t i g a t e t h e dynamics, s t a b i l i t y and s t r u c t u r e o f t h e p r o t e i n . T h i s method can p r o v i d e d e t a i l e d i n f o r m a t i o n on t h e a c t i v e s i t e i f a s u i t a b l e f l u o r e s c e n t s u b s t r a t e a n a l o g u e c a n be bound t o t h e enzyme. A l s o n a t u r a l f l u o r o p h o r e s l i k e t r y p t o p h a n c a n be m o n i t o r e d a l t h o u g h d e t a i l e d i n f o r m a t i o n c a n n o t a l w a y s be o b t a i n e d i n case o f m u l t i - t r y p t o p h a n c o n t a i n i n g p r o t e i n s l i k e CT. A n a d v a n t a g e o f t h e f l u o r e s c e n c e m e t h o d i s i t s h i g h s e n s i t i v i t y a l l o w i n g t h e u s e o f v e r y l o w enzyme c o n c e n t r a t i o n s a t w h i c h t h e enzyme o n l y e x i s t s i n a m o n o m e r i c f o r m . The a i m o f t h i s s t u d y was t o f i n d t h e r e l a t i o n s h i p b e t w e e n c a t a l y t i c a c t i v i t y o f t h e enzyme a n d t h e s t r u c t u r e a n d a m o u n t o f w a t e r n e c e s s a r y f o r a c o n f o r m a t i o n a l l y a c t i v e e n z y m a t i c f o r m . The r e v e r s e d m i c e l l a r s y s t e m h a s t h e p o t e n t i a l t o s t a b i l i z e t h e a c t i v e c o n f o r m a t i o n a l s t a t e o f t h e enzyme induced by s u b s t r a t e b i n d i n g . I n a d d i t i o n t o n a t u r a l f l u o r e s c e n c e o f α-chymotrypsin a d e t a i l e d fluorescence study of anthraniloyl-a-chymotrypsin ( A n t - C T ) a s a m o d e l p r o t e i n was c a r r i e d o u t t o i n v e s t i g a t e i t s p h y s i c a l p r o p e r t i e s i n aqueous s o l u t i o n and i n a r e s t r i c t e d w a t e r s y s t e m .

699

2. MATERIALS AND METHODS Chemicals. B o v i n e p a n c r e a t i c α - c h y m o t r y p s i n ( C T ) ( 3 χ r e c r y s t a l l i z e d ) was o b t a i n e d f r o m S i g m a . The n o r m a l i t y o f t h e enzyme s t o c k s o l u t i o n was d e t e r m i n e d by a c t i v e s i t e t i t r a t i o n w i t h Ν - t r a n s e i n n a m o y l i m i d a z o l e [10]. A n t h r a n i l o y l - α - c h y m o t r y p s i n ( A n t - C T ) was p r e p a r e d f o l l o w i n g t h e p r o c e d u r e o f H a u g l a n d a n d S t r y e r ( 1 1 ) . The f i n a l s o l u t i o n was l y o p h i l i z e d a n d s t o r e d i n s m a l l p o r t i o n s a t -20 ° C I n a l l e x p e r i m e n t s the c o n c e n t r a t i o n o f Ant-CT was 90 n M . S o d i u m b i s ( 2 - e t h y l h e x y l ) s u l f o s u c c i n a t e (AOT) was o b t a i n e d f r o m S i g m a . R e s o r u f i n b u t y r a t e ( R B ) was o b t a i n e d f r o m Lambda P r o b e s & D i a g n o s t i c s a n d was d i s s o l v e d i n d i m e t h y l s u l f o x i d e . A l l o t h e r r e a g e n t s were a n a l y t i c a l grade and p r o p e r l y p u r i f i e d a d d i t i o n a l l y .

Preparation of reversed micelles The m i c e l l a r s o l u t i o n s w e r e p r e p a r e d b y a d d i t i o n o f m e a s u r e d v o l u m e s o f n - o c t a n e a n d w a t e r t o d r y , p r e w e i g h e d a m o u n t s o f AOT. 1 μ ΐ o f A n t - C T ( o r α - c h y m o t r y p s i n ) s o l u t i o n i n 0 . 1 M t r i s - H C l b u f f e r , pH 8 . 2 , was i n j e c t e d i n t o 3 m l r e v e r s e d m i c e l l a r o r g a n i c s o l u t i o n . T h e m i x t u r e was g e n t l y s h a k e n u n t i l a c l e a r s o l u t i o n was o b t a i n e d . The d e s i r e d w D was r e a c h e d b y a d d i t i o n o f a p r e d e t e r m i n e d v o l u m e o f 0 . 1 M t r i s - H C l b u f f e r , pH 8 . 2 . The h i g h e s t w D was a b o u t 6 1 a t 2 5 ° C A b o v e t h i s w D v a l u e t h e s o l u t i o n became t u r b i d .

Fluorescence measurements A b s o r p t i o n and s t e a d y - s t a t e f l u o r e s c e n c e s p e c t r a were r e c o r d e d on a C a r y - 1 4 s p e c t r o p h o t o m e t e r a n d a S L M - A m i n c o SPF-500C f l u o r o m e t e r , r e s p e c t i v e l y . The r a t e s o f C T - c a t a l y z e d h y d r o l y s i s o f r e s o r u f i n b u t y r a t e ( n o n f l u o r e s c e n t ) was f o l l o w e d b y means o f p r o d u c t r e s o r u f i n f l u o r e s c e n c e e m i s s i o n a t 590 nm ( e x c i t a t i o n a t 540 n m ) . The t r y p t o p h a n y l f l u o r e s c e n c e s p e c t r a o f CT w e r e m e a s u r e d a t e x c i t a t i o n w a v e l e n g t h 300 n m . A n t - C T f l u o r e s c e n c e was o b t a i n e d a t e x c i t a t i o n w a v e l e n g t h 340 n m . T i m e - r e s o l v e d f l u o r e s c e n c e e x p e r i m e n t s w i l l be d e t a i l e d e l s e w h e r e . A l l e x p e r i m e n t s w e r e c a r r i e d o u t a t 25 ° C

3. RESULTS Conformational change of α-chymotrypsin induced upon solubilization in reverse micelles at low wQ

I n s p i t e o f many r e p o r t s o n t h e w D d e p e n d e n c e o f t h e c a t a l y t i c a c t i v i t y o f CT s o l u b i l i z e d i n r e v e r s e d m i c e l l e s n o d a t a a r e p r e s e n t e d a t v e r y l o w d e g r e e o f h y d r a t i o n , w Q < 5 . The t i m e c o u r s e o f t h e r e a c t i o n o f RB w i t h CT i n 0 . 1 M A O T / n - o c t a n e r e v e r s e m i c e l l e s i s i n d i c a t i v e f o r a v e r y s m a l l r e a c t i o n r a t e w h e n CT i s a d d e d t o RB i n r e v e r s e m i c e l l e s w i t h w 0 = 0 . 6 5 . I n c r e a s i n g t h e a m o u n t o f w a t e r i n t h e same s a m p l e t o w D = 2 . 3 a n d t h e n t o 3 . 8 i s n o t l e a d i n g t o some v i s i b l e a c c e l e r a t i o n o f t h e r e a c t i o n . H o w e v e r , i f we s t a r t t h e r e a c t i o n w i t h a d d i t i o n o f t h e enzyme t o s u b s t r a t e i n r e v e r s e m i c e l l e s w i t h w D = 3 . 8 a c o n s i d e r a b l e r e a c t i o n o f RB w i t h CT o c c u r s .

Irreversible change in the conformation of α-chymotrypsin upon solubilization in reversed micelles. The s o l u b i l i z a t i o n o f CT i n 0 . 1 M A O T / n - o c t a n e r e v e r s e d m i c e l l e s i s accompanied by r a p i d i n a c t i v a t i o n . A f t e r two m i n u t e s o f i n c u b a t i o n t h e i n a c t i v a t i o n i s c o m p l e t e d . When CT i s s o l u b i l i z e d i n r e v e r s e m i c e l l e s w i t h d i f f e r e n t wD and a f t e r a s h o r t i n c u b a t i o n p e r i o d , each sample i s b r o u g h t t o

700 c o n s t a n t h i g h w Q, t h e enzyme " r e m e m b e r s " t h e w a t e r p o o l i n w h i c h i t h a s b e e n i n c u b a t e d a n d i t shows d i f f e r e n t c a t a l y t i c a c t i v i t y . T h i s i s s h o w n i n F i g . l A . E a c h p o i n t r e p r e s e n t s t h e c a t a l y t i c a c t i v i t y o f enzyme a t w Q = 2 8 . 6 a f t e r 10 m i n u t e s o f i n c u b a t i o n a t t h e p a r t i c u l a r w D s h o w n o n t h e a b s c i s s a . A t v e r y l o w w 0 t h e c a t a l y t i c a c t i v i t y o f CT d e c r e a s e s w i t h i n c r e a s i n g w 0 p a s s i n g a minimum v a l u e a t wQ = 5 . 0 4 a f t e r w h i c h i t v e r y r a p i d l y i n c r e a s e s a n d r e a c h e s a c o n s t a n t v a l u e i n t h e r a n g e o f w Q - 1 0 - 3 0 . H o w e v e r t h e enzyme c a n n o t r e a c h t h e " n o r m a l " a c t i v i t y w h i c h i t h a s when a d d i n g t o r e v e r s e m i c e l l e s c o n s i s t i n g o f s u b s t r a t e and p e r f o r m i n g t h e r e a c t i o n w i t h o u t any p r e l i m i n a r y i n c u b a t i o n (dashed l i n e i n F i g . l A ) .

Steady-state

fluorescence

measurements

of

anthraniloyl-a-chymotrypsin

The f l u o r e s c e n c e e m i s s i o n s p e c t r u m o f A n t - C T was r e c o r d e d i n m i c e l l a r s o l u t i o n s o f d i f f e r e n t wD a n d c o m p a r e d t o i t s s p e c t r u m i n a q u e o u s s o l u t i o n . I n w a t e r , t h e c e n t e r o f g r a v i t y o f t h e e m i s s i o n s p e c t r u m was 4 1 7 . 3 n m . U p o n i n c o r p o r a t i o n of Ant-CT i n t o a reversed m i c e l l a r s o l u t i o n a blue s h i f t of t h e e m i s s i o n maximum was o b s e r v e d . W i t h i n c r e a s i n g w a t e r c o n t e n t t h e c e n t e r o f g r a v i t y o f t h e f l u o r e s c e n c e s p e c t r u m o f A n t - C T as a f u n c t i o n o f wQ showed a g r a d u a l r e d s h i f t w h i c h a t v e r y h i g h wD a p p r o a c h e s t h a t i n b u l k water ( F i g . I B ) .

Steady-state

tryptophanyl

fluorescence

of

a-chymotrypsin

The f l u o r e s c e n c e p r o p e r t i e s o f α - c h y m o t r y p s i n a s a f u n c t i o n o f h y d r a t i o n d e g r e e , w 0, a n d t h e i n c u b a t i o n t i m e i n r e v e r s e m i c e l l e s a r e s h o w n i n F i g . l C . The c e n t e r o f g r a v i t y o f t r y p t o p h a n y l f l u o r e s c e n c e o f α - c h y m o t r y p s i n a t v e r y l o w wQ = 0.65 ( F i g . l C , a ) i s b l u e s h i f t e d i n c o m p a r i s o n t o t h a t i n aqueous s o l u t i o n ( F i g . l C , dashed l i n e ) . W i t h i n c r e a s i n g the wD v a l u e t o 5 the c e n t e r o f g r a v i t y o f t h e e m i s s i o n spectrum increases to a s l i g h t l y h i g h e r v a l u e t h a n i t has i n aqueous s o l u t i o n and w i t h f u r t h e r i n c r e a s e o f w 0 u p t o 2 8 . 6 i t shows n o c o n s i d e r a b l e d i f f e r e n c e f r o m t h e v a l u e i n t h e a q u e o u s s y s t e m . A f t e r 10 m i n u t e s i n c u b a t i o n o f CT i n r e v e r s e m i c e l l e s a t d i f f e r e n t wD t h e c e n t e r o f g r a v i t y shows a r e d s h i f t i n c o m p a r i s o n t o n o n i n c u b a t e d s a m p l e s w h i c h r e a c h e s t h e maximum v a l u e a t w0 - 5 ( F i g . l C , b ) .

aqueous

Fluorescence solution

lifetime measurements of anthraniloyl-a-chymotrypsin and in reversed micelles

in

The f l u o r e s c e n c e l i f e t i m e d i s t r i b u t i o n o f A n t - C T i n a q u e o u s s o l u t i o n c o n s i s t s o f f o u r p e a k s : 9 . 8 n s ( 4 1 % ) , 5 . 2 n s ( 3 0 % ) , 2 . 2 n s (15%) a n d 0 . 1 n s (14%). I n reversed m i c e l l a r s o l u t i o n w i t h a v e r y low w a t e r c o n t e n t ( w 0 - 0 . 6 5 ) t h e image i s d o m i n a t e d by a s i n g l e peak w i t h a b a r y c e n t e r a t 8.7 ns ( 9 9 . 6 % ) . A t i n c r e a s i n g w a t e r c o n t e n t i n r e v e r s e d m i c e l l e s up t o w0 = 28.6 the r e s o l u t i o n i n t o f o u r peaks i s r e s t o r e d , a p p r o a c h i n g t h e s i t u a t i o n i n bulk water.

Correlation

time

distribution.

The c o r r e l a t i o n - t i m e i m a g e o f t h e f l u o r e s c e n c e a n i s o t r o p y d e c a y o f α-CT i n b u f f e r d i s p l a y s one m a i n p e a k w i t h a b a r y c e n t e r a t 1 2 . 8 η ( 9 7 . 8 % o f t h e d e c a y ) and one m i n o r p e a k w i t h s h o r t c o r r e l a t i o n t i m e ( 0 . 3 n s ) w i t h a v e r y s m a l l c o n t r i b u t i o n ( 2 . 2 % ) . I n r e v e r s e d m i c e l l e s a t t h e l o w e s t w0 ( 0 . 6 5 ) b o t h p e a k s a r e s h i f t e d t o l o n g e r c o r r e l a t i o n t i m e s . When t h e w a t e r c o n t e n t o f r e v e r s e m i c e l l e s i s i n c r e a s e d u p t o wD = 2 . 5 t h e s h o r t c o r r e l a t i o n peak i n c r e a s e s t o 4 ns ( 1 4 . 5 % ) , f o l l o w e d by a d e c r e a s e t o 1.2 n s ( 1 2 . 5 % ) a t w Q - 2 8 . 6 ( F i g . I D , a ) . The l o n g e r c o r r e l a t i o n t i m e s h i f t s u p t o 4 5 . 4 n s ( 7 8 . 6 % ) a t w D= 9 . 6 a n d r e m a i n s m o s t l y i n d e p e n d e n t o n w D u p t o 2 8 . 6 (Fig.ID,b).

701

Fig.lA.

C a t a l y t i c a c t i v i t y of α-CT a t w 0 - 2 8 . 6 a f t e r 10 minutes o£ i n c u b a t i o n a t the p a r t i c u l a r w Q shown on t h e a b s c i s s a . The dashed l i n e r e p r e s e n t s the c a t a l y t i c a c t i v i t y of α-CT a t wQ - 2 8 . 6 .

Fig.IB.

Center of g r a v i t y of t h e f l u o r e s c e n c e s p e c t r a o f Ant-CT as f u n c t i o n o f w Q. The dashed l i n e i n d i c a t e s the v a l u e i n aqueous s o l u t i o n .

Fig.lC.

Center of g r a v i t y of the tryptophanyl fluorescence o f α-CT as f u n c t i o n o f w Q. a) without incubation b ) a f t e r 10 minutes i n c u b a t i o n o f o-CT. The dashed l i n e i n d i c a t e s the v a l u e i n aqueous s o l u t i o n .

702

FIG.ID.

10

15

20

25

THE W0-DEPENDENCE OF BARYCENTERS OBTAINED FROM THE SHORT (A) AND LONG (B) CORRELATIONTIME DISTRIBUTION OF ANT-CT IN AOT REVERSED MICELLES. THE DASHED LINE INDICATES THE VALUE (B) IN AQUEOUS SOLUTION.

30

FIG.IE. THE ENTHALPY OF WATER IN AOT/CHLOROFORM SOLUTION AS A FUNCTION OF W 0. DATA ARE TAKEN FROM [12].

703 4. DISCUSSION C a l o r i m e t r i c s t u d i e s o n t h e s t a t e o f w a t e r i n AOT r e v e r s e m i c e l l e s y i e l d e d a c h a r a c t e r i s t i c p a t t e r n f o r t h e averaged molar e n t h a l p y dependence o f w a t e r as a f u n c t i o n o f wD ( F i g . I E ) , [ 1 2 ] . T h i s d e p e n d e n c e h a s b e e n o b t a i n e d b y m i x i n g AOT s o l u t i o n s o f v a r i o u s w 0 w i t h t h o s e w i t h o u t w a t e r a d d e d . The a u t h o r s e x p l a i n e d t h e s e r e s u l t s a s s u m i n g a " t h r e e p h a s e m o d e l " . A t s m a l l wQ t h e w a t e r h a s a s t r o n g i n t e r a c t i o n w i t h t h e h e a d g r o u p s o f m i c e l l e s ( p h a s e S x ) . W i t h i n c r e a s i n g w 0 a new w a t e r s t r u c t u r e i s f o r m e d ( p h a s e S 2) b e t w e e n t h i s s t r o n g l y b o u n d w a t e r a n d t h e b u l k w a t e r ( p h a s e B ) . The i n t e r m e d i a t e p h a s e S 2 i s e n t h a l p i c a l l y u n s t a b l e b e c a u s e i t i s d e s t r o y e d b y t h e s t r o n g p h a s e S l t As a r e s u l t , a maximum i s o b s e r v e d i n t h e e n t h a l p y curve. Results presented i n t h i s study i n d i c a t e a s i m i l a r p a t t e r n f o r the w Q- d e p e n d e n c e o f enzyme k i n e t i c s a n d f l u o r e s c e n c e p r o p e r t i e s o f CT i n r e v e r s e m i c e l l e s o n t h e a m o u n t o f w a t e r i n t h e r a n g e o f wQ a r o u n d 5 . A t v e r y l o w w 0 t h e c o n f o r m a t i o n o f CT c h a n g e s t o v e r y r i g i d i n c o m p a r i s o n t o w a t e r s o l u t i o n . The f o u r p e a k s i n t h e f l u o r e s c e n c e l i f e t i m e d i s t r i b u t i o n i n a q u e o u s s o l u t i o n a r e c o n v e r t e d i n t o a s i n g l e p e a k . The o v e r a l l c e n t e r o f g r a v i t y o f t h e t r y p t o p h a n f l u o r e s c e n c e s p e c t r u m o f t h e enzyme a t w 0 = 0 . 6 5 i s b l u e s h i f t e d i n c o m p a r i s o n t o s i t u a t i o n i n w a t e r . T h i s means t h a t t h e CT m o l e c u l e when e n t r a p p e d i n d r y r e v e r s e m i c e l l e s i s r e s t r i c t e d f r o m t h e s o l v e n t making the molecule v e r y r i g i d ( t h e d i p o l a r r e l a x a t i o n time o f s o l v e n t a n d a m i n o a c i d d i p o l e s w i l l be l o n g o n a n a n o s e c o n d t i m e s c a l e ) . I n t h e absence o f a h y d r a t i o n s h e l l , t h e p r o t e i n i s e s s e n t i a l l y f r o z e n and i n a c t i v e . A s m a l l i n c r e a s e i n w a t e r c o n t e n t w i l l t r a n s f e r new w a t e r m o l e c u l e s t o t h e n e g a t i v e l y c h a r g e d s u r f a c t a n t s h e a d s a n d t h e enzyme m o l e c u l e becomes e v e n more r i g i d t h a n i t i s a t w Q = 0 . 6 5 . The s h o r t c o r r e l a t i o n t i m e peak o f t h e a n t h r a n i l o y l group c o v a l e n t l y a t t a c h e d t o S e r - 1 9 5 , w h i c h r e p r e s e n t s t h e i n t e r n a l a c t i v e s i t e m o t i o n o f CT shows a n i n c r e a s e u p t o w Q a r o u n d 5 ( F i g . l D a ) . A t t h e same t i m e t h e c e n t e r o f g r a v i t y o f t h e t r y p t o p h a n f l u o r e s c e n c e s p e c t r u m shows a r e d s h i f t w h i c h i s h i g h e r t h a n t h e c e n t e r o f g r a v i t y o f t h a t i n aqueous s o l u t i o n ( F i g . l C ) . T h i s means t h a t t h e a v e r a g e p o l a r i t y a r o u n d t h e enzyme m o l e c u l e i s h i g h e r t h a n i n w a t e r . T h i s s i t u a t i o n may o c c u r w h e n t h e p r o t e i n g l o b u l e i s s u r r o u n d e d b y p h a s e S 2 w a t e r m o l e c u l e s i n r e v e r s e m i c e l l e s . The l o n g c o r r e l a t i o n t i m e w h i c h r e p r e s e n t s t h e s i z e o f t h e w h o l e p a r t i c l e becomes l o n g e r ( F i g . l D b ) w h e n w 0 i n c r e a s e s f r o m 0 . 6 5 t o 5 . We assume t h a t a t w 0 around 5 t h e phase S2 o f r e v e r s e m i c e l l a r w a t e r i s c o m p l e t e d and u n d e r t h e s e c o n d i t i o n s t h e enzyme d i s p l a y s a m i n i m u m o f e n z y m a t i c a c t i v i t y ( F i g . l A ) . A sharp increase i n enzymatic a c t i v i t y w i t h i n a r e l a t i v e l y small w0 range v a l u e f r o m 5 t o 8 i n d i c a t e s a c o o p e r a t i v e b i n d i n g o f w a t e r molecules t o t h e p r o t e i n . H y d r a t i o n d a t a o f lysozyme powders [13] were f i t t e d i n terms o f a c o o p e r a t i v e model o f h y d r a t i o n - d e p e n d e n t a c t i v a t i o n o f c a t a l y s i s which accounts f o r binding of water molecules to the p r o t e i n . T h i s f i t y i e l d e d a degree o f h y d r a t i o n o f 0.35 g o f w a t e r p e r gram o f p r o t e i n f o r p r o m o t i n g t h e h a l f - m a x i m a l a c t i v a t i o n o f c a t a l y s i s and a h i g h p o s i i v e H i l l c o e f f i c i e n t o f 4 . 9 5 . The c o n v e r s i o n f r o m w 0 i n t o h y d r a t i o n b y making use o f p r o c e d u r e s g i v e n i n [ 1 2 ] and [ 1 3 ] c o v e r s a l i m i t e d h y d r a t i o n range ( < 1 g o f w a t e r p e r gram o f p r o t e i n ) . Above w0 = 1 0 , h o w e v e r , t h e e f f e c t o f h y d r a t i o n o n t h e a c t i v a t i o n o f c a t a l y s i s i s c o m p l e t e w h i c h shows t h a t t h e enzyme a c t i v i t y d e p e n d s o n t h e a m o u n t o f w a t e r i n c o n t a c t w i t h t h e enzyme a n d n o t o n t h e t o t a l a m o u n t o f w a t e r i n t h e s y s t e m . I n o t h e r w o r d s , bulk water i s not necessary f o r a c t i v i t y . The w a t e r c o n t r o l i n a r e v e r s e m i c e l l a r s y s t e m h a s t h e a d v a n t a g e t o show how h y d r a t i o n o f p r o t e i n a f f e c t s t h e d y n a m i c a l s t r u c t u r e o f t h e

704

p r o t e i n m a t r i x w h i c h i s e s s e n t i a l t o t h e e n z y m a t i c a c t i v i t y . The o b s e r v e d r e s u l t s a r e c o n s i s t e n t w i t h t h e p i c t u r e d e s c r i b e d b y R u p l e y et al. [ 7 ] : The f i r s t molecules o f w a t e r added i n t e r a c t p r e d o m i n a n t l y w i t h i o n i z a b l e g r o u p s . T h i s s t r o n g l y bound w a t e r i s d i s p e r s e d over t h e p r o t e i n s u r f a c e . It c o n s t i t u t e s 25% o f t h e w a t e r o f h y d r a t i o n , w h i c h i s e q u a l t o t h e p e r c e n t a g e of the s u r f a c e c o n t r i b u t e d by i o n i z a b l e groups. At the c o n c e n t r a t i o n of n e a r 0 . 1 g w a t e r / g p r o t e i n , w a t e r c l u s t e r s d e v e l o p . When t h e h y d r a t i o n l e v e l i s between 0.2 and 0 . 3 , h y d r a t i o n o f t h e h y d r o g e n - b o n d i n g s i t e s i s c o m p l e t e . At a h y d r a t i o n l e v e l o f 0 . 4 t h e n o n p o l a r r e g i o n s a r e c o v e r e d by a w a t e r m o n o l a y e r . I t i s a t t h i s p o i n t w h e r e t h e e n z y m a t i c a c t i v i t y shows t h e l a r g e s t c h a n g e . The p r o t e i n a t t h i s h y d r a t i o n l e v e l meshes w e l l w i t h b u l k w a t e r . I n t h i s r e g i o n t h e r e a r e no changes i n t h e t h e r m a l p r o p e r t i e s a t a h y d r a t i o n l e v e l above 0 . 4 . Dynamic p r o p e r t i e s change as t h e h y d r a t i o n i s i n c r e a s e d a b o v e 0 . 4 , h o w e v e r , t h e s e c h a n g e s a p p e a r t o be s m a l l c o m p a r e d t o t h o s e o c c u r r i n g i n t h e r a n g e 0 . 2 5 - 0 . 4 . The e n z y m a t i c a c t i v i t y i s o b s e r v e d before completing the monolayer. By c o m b i n i n g t h e r e s u l t s o f F i g . l D b ( l a r g e c h a n g e i n c o r r e l a t i o n t i m e w i t h r e s p e c t t o w 0) , F i g . I E ( l a r g e i n c r e a s e i n e n t h a l p y b e t w e e n w 0 0 . 6 5 a n d 5 ) a n d F i g . l C ( b l u e s h i f t e d f l u o r e s c e n c e s p e c t r a a t w Q, f o l l o w e d b y a r e d s h i f t u p t o w 0 = 5 ) , i t c a n be c o n c l u d e d t h a t e n z y m e - c o n t a i n i n g r e v e r s e m i c e l l e s change d r a s t i c a l l y i n shape and s i z e u n d e r t h e s e c o n d i t i o n s , w h i c h i s accompanied by a c o n s i d e r a b l e change i n a c t i v e s i t e c o n f o r m a t i o n .

5. ACKNOWLEDGEMENT T h i s work has been s u p p o r t e d by a F e l l o w s h i p o f t h e Wageningen A g r i c u l t u r a l U n i v e r s i t y t o V . D-T a n d i n p a r t b y t h e B u l g a r i a n M i n i s t r y Science and C u l t u r e .

6. 1.

of

REFERENCES

R. L u m r y a n d R. B i l t o n e n , S t r u c t u r e a n d S t a b i l i t y o f B i o l o g i c a l M a c r o m o l e c u l e s , M a r c e l D e k k e r , New Yowk ( 1 9 6 9 ) 6 8 . 2. M. K a r p l u s a n d J . A . McCammon, A n n . R e v . B i o c h e m . 53 ( 1 9 8 3 ) 2 6 3 . 3. E. G r a t t o n , J . R . A l c a l a a n d F . G . P r e n d e r g a s t , i n F l u o r e s c e n t B i o m o l e c u l e s , ( D . M . J a m e s o n a n d G . D . R e i n h a r t , E d s . ) P l e n u m P r e s s , New York (1989) 17. 4. D . E . J r . K o s h l a n d a n d K . E . N e e t , A n n . R e v . B i o c h e m . 37 ( 1 9 6 8 ) 3 5 9 . 5. D. H a r s c h l a g , B i o o r g . Chem. 16 ( 1 9 8 8 ) 6 2 . 6. A . V . L e v a s h o v , N . L . K l y a c h k o , N . G . B o g d a n o v a a n d K. M a r t i n e k , FEBS L e t t . 268 ( 1 9 9 0 ) 2 3 8 . 7. J . A . R u p l e y , P . - H . Y a n g a n d G. T o l l i n , A d v . C h e m . , S e r . 80 ( 1 9 8 0 ) 1 1 1 . 8. M. L u s c h e r a n d M. R u e g g , B i o c h i m . B i o p h y s . A c t a 533 ( 1 9 7 8 ) 4 2 8 . 9. A . Z a k s a n d A . M . K l i b a n o v , P r o c . N a t l . A c a d . S e i . USA 82 ( 1 9 8 5 ) 3 1 9 2 . 1 0 . G . R . S c h o n b a u m , Β. Z e r n e r a n d M . L . B e n d e r , J . B i o l . Chem. 236 ( 1 9 6 1 ) 2930. 1 1 . R.P. H a u g l a n d and L. S t r y e r , i n C o n f o r m a t i o n o f B i o p o l y m e r s , ( G . H . R a m a c h a n d r a n , E d . A c a d e m i c P r e s s , New Y o r k , 1 ( 1 9 6 7 ) 3 2 1 . 1 2 . A . G o t o , H. Y o s h i o k a , H. K i s h i m o t o a n d T . F u j u t a , T h e r m o c h i m . A c t a 163 ( 1 9 9 0 ) 1 3 9 . 1 3 . S . T . F e r r e i r a a n d E. G r a t t o n , i n B i o m o l e c u l e s i n O r g a n i c S o l v e n t s , ( A . G o m e z - P u y o u , E d . ) CRC P r e s s ( 1 9 9 2 ) 1 8 9 .

Biocatalysis in Non-Conventional Media, edited by J. Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

705

Comparison of activity and stability of enzymes suspended in organic solvents and dissolved in water-in-oil microemulsions Eleftheria Skrika-Alexopoulos, Jacqueline Muir and Robert Β Freedman The Biological Laboratory, The University, Canterbury, Kent, C T 2 7 N J , United Kingdom. In order to focus on the effects o f the reaction system on the properties o f enzymes in low water systems, we have studied two representative enzymes, bilirubin oxidase (Myrothecium

verrucaria) and triglyceride lipase (Chromobacterium viscosum) in two commonly used lowwater reaction systems, namely suspensions o f lyophilised enzymes directly in organic solvents, and solutions o f enzymes in the aqueous droplet phase o f oil-in-water microemulsions. For each enzyme, the same organic solvents have been studied in both reaction systems. For both enzymes we observe that the activity as suspensions in organic solvent is highly dependent on the nature and quantity o f co-lyophilised buffer; the effects suggest a role for the buffer materials additional to the maintenance o f the appropriate protonation state o f the enzyme. For both enzymes in microemulsions we observe that the process o f inactivation is best described by a double-exponential biphasic decay, whereas inactivation in the suspensions is characterised by single-exponential decay. F o r bilirubin oxidase in C T A B microemulsions, stability is independent o f droplet concentration and o f limited changes in log Ρ o f the oil phase (2.5 - 3 . 5 ) ; by contrast, stability o f lipase in A O T microemulsions is dependent on the identity o f the oil phase.

1. I N T R O D U C T I O N Enzymatic catalysis in organic media has received much attention during the past decade. Many systems for presenting enzymes to predominantly organic media have been devised and used successfully. These include the direct suspension o f enzyme particles in the organic phase ( 1 ) , solubilisation in water-in-oil (w/o) microemulsions ( 2 ) , adsorption onto solid supports ( 3 ) , use o f covalent modifications to render enzyme molecules soluble in organic solvents ( 4 ) , as well as solubilisation in water/organic solvent biphasic media, or in water/water-miscible organic solvents. Each system presents both advantages and disadvantages; so far, there has been a lack o f any systematic comparison o f enzymatic properties, activity and stability, between these systems. The purpose o f this study has been to compare in a systematic way various aspects o f enzymatic activity and stability o f two enzymes, namely bilirubin oxidase from Myrothecium verrucaria, and lipase from Chromobacterium viscosum, in two well characterised low-water systems, namely liquid-solid two phase suspensions o f enzyme powder in organic solvents, and w/o microemulsions, where the enzyme is solubilised in the aqueous core o f the microemulsion droplets. The two enzymes are representative o f two distinctly different groups o f enzymes; bilirubin oxidase represents a class o f oxidases which use molecular oxygen and do not require

706

extrinsic cofactors for activity, whereas lipases catalyse the hydrolysis o f fatty acyl esters and are known to act at water/oil interfaces. Moreover, the comparative study described here could draw on previous work in this laboratory on the activities o f both enzymes in w/o microemulsions stabilised by charged surfactants ( 5 , 6 ) . Throughout the study, for both enzymes as many experimental parameters as possible were kept similar between the two low-water systems, so as to enable their valid comparison. This has allowed attention to be focused on intrinsic differences between the reaction systems. Our work has highlighted differences in the kinetics o f enzyme inactivation between the systems and identified factors influencing activity in the suspension systems.

2. A C T I V I T Y S T U D I E S ON E N Z Y M E S U S P E N S I O N S IN O R G A N I C S O L V E N T S Whereas the activity o f both bilirubin oxidase and lipase had been well characterised in w/o microemulsions, there was a need for optimisation o f their activity in liquid-solid two phase suspensions. One o f the most interesting aspects o f this study turned out to be the effect on enzymatic activity o f buffer salts, co-lyophilised with the enzyme from an aqueous buffered solution prior to suspension in the organic phase. All experiments were carried out at 2 5 ° C , and at atmospheric pressure.

Fig. 1 Dependence of activity of BO on pH of buffer from which enzyme was lyophilised prior to its suspension in liquid-solid two phase system. Solvent chloroform: nheptane 5 0 : 5 0 v/v; BO lmg/ml; added water 0 . 5 % v/v.

bilirubin 4 . 5 μΜ;

buffer 0.24mmoles/mg B O ;

707 2.1. Bilirubin oxidase Bilirubin Oxidase (BO) (EC 1.3.3.5) from Myrothecium verrucaria (Genzyme Diagnostics, Kent U K ) was suspended in anhydrous chloroform:n-heptane 50:50(v/v), to which known amounts o f water ( 0 - 0 . 5 % v/v) had been previously added and dispersed. A simple spectrophotometric assay was used to measure the oxidation o f the substrate bilirubin at 442nm ( 7 ) . Suspensions o f the commercial enzyme powder were found to be completely inactive, thus lyophilisation o f the enzyme from a buffered aqueous solution was attempted. The pH optimisation is described in Fig 1 (activities expressed in terms o f second order rate constant, k 2 ) ; optimal pH was found to be 8.4, very close to that previously found for this enzyme in a w/o microemulsion system ( 5 ) . There were, though, a number o f unexpected observations that led us to elaborate further on the pH-dependency. B O lyophilised from a volatile buffer solution (ammonium acetate), at optimal pH, was completely inactive (Table 1). By contrast, enzymatic activity in C T AB w/o microemulsions buffered with ammonium acetate at optimal pH was optimal (results not shown). Secondly, activity varied widely when B O was lyophilised from three different buffer solutions all at pH 8.4 (EPPS, T A P S , AMPSO) (Table 1). No similar specific buffer effect was observed in microemulsions ( 5 ) . Nevertheless, there is a genuine pH-optimum distinct from the specific buffer effect. Thus, samples lyophilised from AMPSO at pH 9 . 0 and from EPPS at pH 7.5 have k 2 values o f 4 4 . 6 and 1.1 M " V (cf data in Table 1).

Table 1 Dependence of activity of BO on buffer species in liquid-solid two phase system. Solvent chloroform : n-heptane 5 0 : 5 0 v/v; B O 1 mg/ml (EPPS, T A P S , ammonium acetate) 0.1 mg/ml (AMPSO); bilirubin 4.5μΜ; buffer pH 8.4, 0.24 mmoles buffer/mg B O ; added water 0 . 5 % v/v (EPPS, T A P S ) , 0 - 0 . 5 % (ammonium acetate), 0 . 1 % (AMPSO). 1

1

Buffer

k 2 ( M s" )

AMPSO EPPS TAPS Ammonium Acetate (volatile buffer)

208.0 12.6 3.9 0.0

Thirdly, activity increased with increasing amount o f buffer salts co-lyophilised with B O (not shown). No such effect was observed at the same range o f buffer concentrations in microemulsions. Although a number o f possible explanations could be given for the phenomena observed, such as difference in the ability o f various buffer species to act as buffers o f water activity and pH ( 8 ) , or more efficient dispersion o f enzyme at higher buffer salt concentrations, it is evident that considerable work is yet required before the molecular basis o f these effects is established. Nevertheless, it is obvious that the nature and quantity o f buffer species may play an important role in enzymatic catalysis in these highly anhydrous systems, an area largely unexplored.

708 2 . 2 . Lipase A transesterification reaction between heptanol and paranitrophenyl acetate was successfully catalysed by C. viscosum lipase (EC 3 . 1 . 1 . 3 ) (Genzyme Diagnostics, Kent, U K ) in anhydrous organic solvents. This enzyme, in contrast to B O , showed some activity when the commercial enzyme, as supplied, was suspended in solvent. However, lyophilisation from M E S buffer at optimal pH 6.7 (6) increased its activity in anhydrous cyclohexane, heptane, and dodecane by two orders o f magnitude (Table 2 ) ; interestingly, activity varied linearly with M E S content, being increased more than three-fold when M E S was at 0 . 2 4 mmoles/mg enzyme powder in the lyophilised preparation, compared with the enzyme preparation containing no buffer at all, lyophilised from ammonium acetate solution at pH 6.7 (not shown). Table 2

Comparison of activity of C viscosum lipase, in liquid-solid two phase system, between (a) enzyme "straight from the bottle" (b) lyophilised preparation (buffer M E S pH 6.7, 0 . 2 4 mmoles MES/mg lipase) Solvent

Log P*

Cyclohexane 3.2 Heptane 4.0 Dodecane 6.6

k2

3

1

1 (g" dm s ) χ KT*

(a)

(b)

2.5 1.4 3.2

150.0 116.0 118.0

* Log Ρ values o f individual solvents are based on calculations following the method o f the hydrophobic fragmental constants ( 1 0 ) . It is clear in this case how the dependency o f activity on quantity o f buffer in the lyophilised preparation is superimposed on the pH-effect. More important, similar effects are observed with two enzymes very different in nature indicating the generality o f these observations on the importance o f buffering species, in addition to pH adjustment.

3 . I N A C T I V A T I O N IN W / O M I C R O E M U L S I O N S 3 . 1 . Bilirubin oxidase The kinetics o f inactivation o f BO solubilised in C T A B w/o microemulsions have been studied in detail ( 9 ) . The inactivation process is best described by a double exponential decay equation as follows: y = A! exp-Wjt + A 2 exp-W 2t where Αλ and A 2 represent the proportion o f activity, expressed as percentage o f activity at time 0 , decaying at rates W, and W 2 respectively. A typical decay pattern is illustrated in Fig 2 .

709

36

72

108

144

180

216

252

2

Time (seconds * Ί 0 )

Fig. 2 Time-dependent inactivation of BO stored in CT AB w/o microemulsion system, in chloroformm-heptane; 50:50 v/v. R 10; C T AB 0. I M ; B O 0.04mg/ml; bilirubin 12μΜ (added for assaying the activity at various time points o f storage period); EPPS buffer pH 8.0, 1.8mM.

Kinetic parameters were measured for C T A B w/o microemulsions o f varying solvent phase (Table 3 ) , and various C TA B contents (ie different droplet concentrations) at constant R (Table 4 ) . No significant effect was observed on any o f the parameters Au A 2 , W b W 2 ; in all cases, inactivation follows a similar pattern which consists o f a "fast" phase o f decay o f activity which occurs during the first 60-80 minutes o f storage period and affects 4 0 - 6 0 % o f the enzymatic activity, and o f a "slow" phase which affects the remaining activity. 3 . 2 . Lipase A double exponential decay equation best described the inactivation process o f lipase in AOT w/o microemulsions. Again, the fast phase and the slow phase account for loss o f about 4 0 % and 6 0 % o f the activity respectively, but the rates o f inactivation were found to be considerably slower for lipase compared with B O . Preliminary results with cyclohexane have indicated that, although a double exponential decay is followed, kinetic parameters deviate from values obtained with heptane (results not shown). When lipase activity was studied in nonane, decane and dodecane as the organic phase, no inactivation was observed at all over at least six days.

710

Table 3 Double exponential decay constants obtainedfor the time-dependent inactivation of BO stored in CTAB w/o microemulsions (in the absence of bilirubin) varying the hydrophobicity (log P) of the organic phase by (a) varying the ratio of chloroform (log Ρ 2) to heptane (log Ρ 4), (b) varying the co-solvent keeping chloroform at 50% v/v (other conditions as for legend to Figure 2 ) . (a) Log P *

2.46 2.54 2.71 2.81

Chloroform (% v/v)

Heptane (%v/v)

A, (%)

w, 4 (s'xlO- )

A2 (%)

w2 6 (s-'xlO- )

35 40 50 55

61.5 50.8 60.7 50.6

2.98 2.93 3.05 3.70

38.5 40.1 48.1 48.9

7.55 10.40 4.63 10.70

65 60 50 45

(b) LogP

Chloroform (% v/v)

2.51 2.71 3.06 3.21 3.47

50 50 50 50 50

Cosolvent (50% v/v)

Ai (%)

W, 4 (s'xlO- )

(%)

(s'xlO )

Cyclohexane Heptane Decane Dodecane Hexadecane

34.8 60.7 54.4 47.0 59.2

2.66 3.05 3.66 4.12 3.97

65.3 40.1 46.1 52.4 41.3

19.17 4.63 14.23 15.67 13.18

6

* Log Ρ values o f each organic mixture were calculated based on the semi empirical formula logP = X ^ o g P ! + X 2l o g P 2 where X „ X 2 are the mole fractions o f constituents 1, 2 o f the mixture ( 1 1 ) . Table 4 Double exponential decay constants obtainedfor the time-dependent inactivation of BO stored in CTAB w/o microemulsions R 10 at various CTAB concentrations. Chloroform to heptane ratio was 5 0 : 5 0 v/v. The rest o f the experimental parameters were as described in legend of Table 3.

w

LogP Cosolvent

CTAB conc(mM)

A, (%)

(s'xlO )

2.71 2.71 2.71

100 50 25

60.7 56.6 57.9

3.05 3.32 4.27

Heptane Heptane Heptane

1

4

A2

(*)

W2 6 (s^xlO )

k2 at time 0 1 1 (M" s")

40.1 42.4 41.3

4.63 12.92 5.45

475 895 1490

711

4. INACTIVATION IN LIQUID-SOLID TWO PHASE SUSPENSIONS The decay o f activity o f B O suspended in chloroform:n-heptane mixture ( 5 0 : 5 0 , v/v), under optimal activity conditions, is best described by a single exponential curve (y = A exp-Wt), ie all o f the enzymatic activity is decaying at the same rate (Fig 3 ) . Interestingly, the half-life o f inactivation is ca. 5 0 minutes, ie it is comparable to the half-life o f the "fast" phase o f inactivation in C T A B w/o microemulsions (ca. 5 0 min). Operational stability o f B O under the same experimental conditions, is very good for at least two hours, indicating that the presence o f substrate improves stability. The same phenomenon had been previously observed in w/o microemulsions ( 5 ) . Inactivation o f lipase in anhydrous heptane, cyclohexane and dodecane is currently under study. Preliminary results indicate that decay o f activity is slower compared with B O . Nevertheless, it seems that the process o f inactivation is best described by a single exponential curve. Presence in the storage medium o f heptanol which is one o f the two substrates o f the transesterification reaction studied greatly enhances stability. Current work is focused on developing physical models o f the inactivation processes which can account for the differences in inactivation kinetics between the two reaction systems.

Fig. 3 Time-dependent single phase inactivation of BO stored in liquid-solid two phase suspension. Solvent: chloroform:n-heptane 5 0 : 5 0 v/v; B O lmg/ml; buffer EPPS pH 8.3, 0 . 2 4 mmoles EPPS/mg B O ; added water 0 . 3 7 5 % v/v; bilirubin, 4 . 5 μ Μ was added at fixed time points o f storage.

712

Acknowledgements This work was carried out as part o f the Inter-University Biotransformation Centre supported by the Science and Engineering Research Council, the Department o f Trade and Industry and 11 industrial partners under the auspices o f the LINK programme in Biotransformations. Abbreviations CTAB cetyltrimethyl ammonium bromide. AOT sodium bis-2-ethylhexyl sulphosuccinate. BES N, N-bis [2-hydroxyethyl]-2-aminoethane sulfonic acid. , EPPS N-[2-hydroxyethyl]-piperazine-N -[3-propane-sulfonic acid]. AMPSO 3 - [ ( l , l-dimethyl-2-hydroxyethyl) amino]-2-hydroxypropane sulfonic acid. TAPS N-tris[hydroxylmethyl] methyl-3-aminopropane sulfonic acid. MES 2-[N-morpholino] ethane sulfonic acid. CAPS 3-[cyclohexylamino]-l-propane sulfonic acid. R molar ratio o f water to surfactant. LogP Logarithm to the base 10 o f the partition coefficient o f the solvent in an octane-water two phase system.

REFERENCES 1 2 3 4 5 6 7 8 9 10 11

Klibanov, A M , 1989. T I B S 14: 141-144. Martinek, K, Levashov, A V , Klyachko, N, Khmelnitski, Y L , Berezin, I V , 1986. Eur J Biochem 1 5 5 : 4 5 3 - 4 6 8 . Reslow, M , Adlercreutz, P, Mattiasson, B , 1988. Eur J Biochem 1 7 2 : 5 7 3 - 5 7 8 . Inada, Y , Takahashi, K, Yochimoto, T , Ajima, A, Matsushima, A, Saito, Y , July 1986. Tibtech: 190-194. Oldfïeld, C, Freedman, R B , 1989. Eur J Biochem 183: 3 4 7 - 3 5 5 . Fletcher, P D I, Robinson, Β Η, Freedman, R B , Oldfïeld, C, 1985. J Chem Soc Faraday Trans 1: 2667-2679. Skrika-Alexopoulos, E , Freedman, R B . Submitted for publication. Adlercreutz, P, 1 9 9 1 . Eur J Biochem 199: 609-614. Skrika-Alexopoulos, E , Muir, J , Freedman, R B . Submitted for publication. Rekker, R F , de Kort, H M , 1979. Eur J Med Chim Therapeutica 1 4 : 4 7 9 - 4 8 8 . Hilhorst, R, Spruiyt, R, Laane, C, Veeger, C, 1984. Eur J Biochem 114: 459-466.

Biocatalysis in Non-Conventional Media, edited by J . Tramper et al. © 1992 Elsevier Science Publishers B.V. All rights reserved.

713

Batch and continuous lipolysis/product reversed micellar membrane bioreactor a

b

D.M.F. Prazeres , F.A.P. Garcia and J.M.S. Cabral

separation

in a

a

a

Laboratorio de Engenharia Bioquimica, Instituto Superior Técnico 1000 Lisboa, Portugal

b Departamento de Engenharia Quimica, Universidade 3 0 0 0 Coimbra, Portugal

de Coimbra

Abstract The enzymatic hydrolysis of olive oil using Chromobacterium viscosum lipase Β encapsulated in reversed micelles of dioctyl sodium sulfosuccinate ( A O T ) in isooctane was carried out in batch and continuous mode in a reversed micellar membrane bioreactor. The influence of some process parameters (substrate concentration, residence time) in the performance of the system was investigated.

1.

INTRODUCTION

Reversed micellar systems present several advantages over conventional reaction media, such as enhancement of catalytic activity, increased stability and greater interfacial area. Despite of this potencial, biocatalysis in reversed micellar systems has been mostly performed in batch type reactors with few references on other types of reversed micelle reactors [1-3]. The development of reactor design that enables reactions in semibatch or continuous mode, allowing for the separation of substrates, products

714

a n d e n z y m e s is t h e r e f o r e o n e o f t h e c r i t i c a l d e m a n d s in r e v e r s e d micelles technology [ 4 ] . T h e p r e s e n t w o r k r e p o r t s the d e v e l o p m e n t o f a m e m b r a n e b i o r e a c t o r f o r t h e b a t c h and c o n t i n u o u s l i p o l y s i s / b i o p r o d u c t s s e p a r a t i o n i n a reversed micelle media. Following previous studies [5], Chromobacterium viscosum l i p a s e Β e n c a p s u l a t e d in A O T / i s o o c t a n e r e v e r s e d m i c e l l e s w a s used with o l i v e o i l as the m o d e l s u b s t r a t e .

2. MATERIALS AND METHODS 2 . 1 . SYSTEM A

thermostatized

ceramic

membrane

was

used

both

The

reaction

reactor

coupled

with

an

ultrafiltration

( C A R B O S E P ® - R h o n e P o u l e n c ) - M W cutoff

in b a t c h and

mixture

was

continuous

recirculated

mode with

(Figure a flow

1 and

rate

of

tubular

10000 DaFigure 2 ) . 1080 cm

3

min'l.

2.2. REACTION EXPERIMENTS M i c e l l a r solutions o f Lipase Β were prepared by a mass transfer p r o c e s s i n v o l v i n g the l i q u i d - l i q u i d e x t r a c t i o n o f t h e e n z y m e f r o m an a q u e o u s p h a s e to the r e v e r s e d m i c e l l e s s y s t e m , as d e s c r i b e d e a r l i e r [ 5 ] . L i p a s e c o n c e n t r a t i o n s o b t a i n e d w e r e in the r a n g e 0 . 0 1 - 0 . 0 4 m g m l " l . The amount of solubilized water in the micellar system ( W o = [ H 2 0 ] / [ A O T ] ) was determined using a K a r l F i s c h e r titrator (Mettler D L 1 8 ) . E n z y m a t i c solutions o f different A O T concentrations ( 5 0 - 2 5 0 m M ) and W 0 v a l u e s ( 2 - 1 0 ) w e r e o b t a i n e d b y d i l u t i o n s o f the f o r m e r s o l u t i o n s with i s o o c t a n e and/or i s o o c t a n e / A O T . T h e e x t e n t o f h y d r o l y s i s o f o l i v e o i l was e s t i m a t e d by d e t e r m i n a t i o n o f o l e i c a c i d t h r o u g h a l k a l i m e t r i c titration o f the s a m p l e s ( 1 0 0 μ ΐ ) with K O H in e t h a n o l / a c e t o n e m e d i a ( 1 0 m l , 1:1 v / v ) , u s i n g p h e n o l p h t a l e i n as the i n d i c a t o r . B a t c h o p e r a t i o n s w e r e c a r r i e d out by r e c y c l i n g the p e r m e a t e s t r e a m as s h o w n in F i g u r e 1. C o n t i n u o u s o p e r a t i o n s w e r e c a r r i e d out by f e e d i n g t h e r e a c t o r with a reversed m i c e l l a r solution ( [ A O T ] = 2 5 0 m M , W o = 7 ) containing the s u b s t r a t e at t h e d e s i r e d c o n c e n t r a t i o n and r e c o v e r i n g t h e p e r m e a t e stream (Figure 2 ) .

715

I-Β

•μ a

Figure module,

1. B a t c h

operation

(Α-Reactor, Β-Ultrafiltration

C-Ceramic membrane,

D-Pump)

Figure 2 . Continuous operation ( Α - R e a c t o r , Β-Ultrafiltration module, CC e r a m i c m e m b r a n e , D , H - P u m p , Ε - S u b s t r a t e and F - P r o d u c t r e s e r v o i r s )

Inlet flow rate ( Q ) was kept equal to

maintain

changed

by

a

constant

varing

the

level inlet

in and

to the p e r m e a t e

the

reactor.

outlet

flow

The rates

f l o w r a t e in residence whenever

order

time a

state w a s a c h i e v e d . R e a c t i o n s w e r e c a r r i e d out at 3 0 ° C and p H 7.

was

steady

716

3. RESULTS 3.1. BATCH R E A C T O R Higher

equilibrium

concentrations inhibition

conversions

probably

due

(Xe

to

x )p

water

w e r e o b t a i n e d for l o w e r o l i v e oil limitation

and

perhaps

product

(Figure 3 ) .

100 [OIL] 25 mM 49 mM 134 mM 171 mM 237 mM

ο Δ

9

12

Time (hour) Figure 3 Effect

An

inhibition

200

mM.

substrate The

phenomena

This

can

be

was

detected

caused

either

for

by

oil

the

parameters

determined

for

of

the

substrate

Michaelis

influence kinetic

under

the

the

equation inhibition

z o n e ( < 1 7 1 m M ) . M a x i m u m v e l o c i t y ( V m xa , a p p ) was 6 0 3 2 μ ι η ο ΐ

min" 1

and the M i c h a e l i s c o n s t a n t , K M , a p p » 7 0 3 m M .

mg~l The

above of

structure.

Menten

concentrations

W 0= 7 )

concentrations

direct

on the l i p a s e or by a c h a n g e in the m i c e l l a r

apparent

were

o f substrate c o n c e n t r a t i o n ( A O T 2 5 0 m M ,

separation

process

was

ways.

Although

fluxes,

a d e c r e a s e in the

rejection

lower

coefficient

oil

affected

separation

varied

by

the

concentrations from

efficiency

1%

for

( 2 3 7 m M ) to 2 7 % for the l o w e s t ( 2 5 m M ) .

the

substrate led

to

was

in

two

opposite

better

permeation

observed:

oleic acid

higher

oil

concentrations

717

3.2. CONTINUOUS R E A C T O R -Effect

o f Substrate

Concentration

and

Residence Time

S t e a d y s t a t e c o n v e r s i o n ( Χ β χ )ρ w a s found to b e d e p e n d e n t on r e s i d e n c e t i m e o f t h e r e a c t i o n m i x t u r e in t h e r e a c t o r , e s p e c i a l l y f o r l o w e r s u b s t r a t e c o n c e n t r a t i o n s ( F i g u r e 4 ) . F o r o i l c o n c e n t r a t i o n s o f 1 7 1 and 2 3 7 m M , t h e i n c r e a s e in c o n v e r s i o n d e g r e e w i t h r e s i d e n c e t i m e is c o n s i d e r a b l y l o w e r . T h i s is p r o b a b l y d u e t o a s u b s t r a t e inhibition phenomena that b a t c h studies indicated to o c c u r in the higher concentrations.

[OIL] 25 mM 94 mM 134 mM 171 mM 237 mM

120 Normalized Residence Time E/Q (mg min/ml) F i g u r e 4 E f f e c t o f r e s i d e n c e t i m e and substrate c o n c e n t r a t i o n on s t e a d y state c o n v e r s i o n ( A O T 2 5 0 m M , W 0 = 7 , [ L i p a s e ] = 0 . 0 1 8 ml"l)

- Ε - t o t a l a m o u n t o f lipase ( m g ) , Q-inlet flow rate ( c m

3

mg

min"l)

T h e p r o d u c t i v i t y o f o l e i c a c i d d e c l i n e d sharply as with an i n c r e a s e in the r e s i d e n c e time. A limit o f productivity for e a c h r e s i d e n c e time w h i c h i s i n d e p e n d e n t o f s u b s t r a t e c o n c e n t r a t i o n s h i g h e r than 9 4 m M w a s f o u n d , and i s p r o b a b l y a c o n s e q u e n c e o f p r o d u c t ( f a t t y a c i d ) inhibition. Whithin the operation conditions tested, a maximum oleic acid p r o d u c t i v i t y o f 2 5 μ m o l m g ' l m i n ! or 7 . 1 m g a c i d m g " l m i n ~ l w a s a c h i e v e d , w h i c h in t h e c a s e o f an o i l c o n c e n t r a t i o n o f 1 3 4 m M c o r r e s p o n d s to a 6 0 % c o n v e r s i o n d e g r e e .

718 -Stability A

long

term

stability

of

operation the

Qin=Qout=0-025 over

a

week

conversion system

carried

out

([OIL]=94

in

mM,

order

degree), the

significant

confirming complete

membrane.

Total

oleic

continuous

operation

was

acid

loss

the

to test

continuously

o f activity

high

rejection

stability o f the

production

the

[Lipase]=0.018

ml m i n ' l ) . T h e r e a c t o r was

without

[ 6 ] , and

was

reactor

in

operational 1

mg

ml" ,

operated

(approximately o f the

enzyme the

lipase by

whole

the

for

60 in

% this

ceramic

170

hour

19.4 g o f acid.

4 . CONCLUSIONS A membrane bioreactor for the hydrolysis of olive oil with microencapsulated lipase in reversed micelles was successfully o p e r a t e d in b a t c h and c o n t i n u o u s m o d e . Long term continuous operation with product recovery in this b i o r e a c t o r is f e a s i b l e p r o v i d e d that w a t e r f i l l e d m i c e l l e s a r e s u p p l i e d to t h e r e a c t o r t o g e t h e r with the s u b s t r a t e o r g a n i c s o l v e n t s o l u t i o n , to c o m p e n s a t e for the p e r m e a t i o n o f e m p t y r e v e r s e d m i c e l l e s . T h e h i g h o p e r a t i o n a l s t a b i l i t y o f this l i p a s e / r e a c t i o n media/bioreactor s y s t e m c o u p l e d with the g o o d s p e c i f i c p r o d u c t i v i t i e s and c o n v e r s i o n d e g r e e s o b t a i n e d i n d i c a t e s that the a s s o c i a t i o n o f r e v e r s e d m i c e l l e s e n z y m o l o g y with the m e m b r a n e r e a c t o r c o n c e p t has r e a l p o t e n t i a l in the biotransformations area.

5. R E F E R E N C E S 1 2 3 4 5

P. Luthi and P . L . L u i s i , J . A m . C h e m . S o c , 1 0 6 ( 1 9 8 4 ) 7 2 8 5 . D . K . E g g e r s and H . W . B l o c h , B i o p r o c e s s E n g i n e e r i n g , 3 ( 1 9 8 8 ) 8 3 . P . L u t h i and T . A . Hatton, B i o s e p a r a t i o n , 2 ( 1 9 9 1 ) 5 . J . W . S h i e l d , H . D . F e r g u s o n , A . S . B o m m a r i u s and T . A . Hatton, Ind. E n g . Chem. Fundam., 2 5 ( 4 ) ( 1 9 8 6 ) 6 0 3 . D . M . F . Prazeres, F . A . P . G a r c i a and J . M . S . Cabrai, J . C h e m . T e c h n o l . Biotechn., 53 (1992) 159.

ACKNOWLEDGEMENTS F i n a n c i a l support from Junta N a c i o n a l de I n v e s t i g a ç â o C i e n t i f i c a e T e c n o l o g i c a - P o r t u g a l ( P r o j e c t 9 6 2 . 9 0 - B I O ) and a f e l l o w s h i p to D . M . P . P r a z e r e s from P r o g r a m a C I E N C I A ( B D / 5 1 / 9 0 - I S ) are a c k n o w l e d g e d .