Biocatalysis in Agricultural Biotechnology 9780841215719, 9780841212411, 0-8412-1571-5

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ACS SYMPOSIUM SERIES

389

Biocatalysis in Agricultural Biotechnology JohnR.Whitaker,EDITOR Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.fw001

University of California

Philip E. Sonnet, EDITOR U.S. Department of Agriculture

Developed from a symposium sponsored by the Divisions of Agricultural and Food Chemistry and of Agrochemicals as part of the program of the Biotechnology Secretariat at the Third Chemical Congress of North America (195th National Meeting of the American Chemical Society), Toronto, Ontario, Canada, June 5-11, 1988

American Chemical Society, Washington, DC 1989

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Library of CongressCataloging-in-PublicalionData Biocatalysis in agricultural biotechnology / John R. Whitaker, editor Philip E . Sonnet, editor. p.

cm.—(ACS

symposium series, ISSN 0097-6156; 389)

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.fw001

Papers from the Symposium on Biocatalysis and Biomimetics held during the Third Chemical Congress of North America. Bibliography: p. Includes index. ISBN 0-8412-1571-5 1. Agricultural biotechnology—Congresses. 2. Enzymes—Biotechnology—Congresses. I. Whitaker, John R. II. Sonnet, Philip E., 1935III. Symposium on Biocatalysis and Biomimetics (1988: Toronto, Ont.) IV. Chemical Congress of North America (3rd: 1988: Toronto, Ont.) V. Series. S494.5.B563B5 668'.6—dcl9

1989 89-65 CIP

Copyright ©1989 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of the first page of each chapter in this volume indicates the copyright owner's consent that reprographic copies of the chapter may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per-copy fee through the Copyright Clearance Center, Inc., 27 Congress Street, Salem, M A 01970, for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, Tor creating a new collective work, for resale, or for information storage and retrieval systems. The copying fee for each chapter is indicated in the code at the bottom of the first page of the chapter. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected by law. PRINTED IN THE UNITED STATES OF AMERICA

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In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; Washington, D.C. Society: 20036Washington, DC, 1989. ACS Symposium Series; American Chemical

ACS Symposium Series M. JoanComstock,Series Editor

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.fw001

1989 ACS Books Advisory Board Paul S. Anderson

Mary A. Kaiser

Merck Sharp & Dohme Research Laboratories

E. I. du Pont de Nemours and Company

Alexis T. Bell

Michael R. Ladisch

University of California—Berkeley

Harvey W. Blanch University of California—Berkeley

Malcolm H. Chisholm

Purdue University

John L. Massingill Dow Chemical Company

Daniel M. Quinn University of Iowa

Indiana University

James C. Randall Alan Elzerman

Exxon Chemical Company

Clemson University

Elsa Reichmanis John W. Finley Nabisco Brands, Inc.

Natalie Foster Lehigh University

Marye Anne Fox The University of Texas—Austin

AT&T Bell Laboratories

C. M. Roland U.S. Naval Research Laboratory

Stephen A. Szabo Conoco Inc.

Wendy A. Warr Imperial Chemical Industries

G. Wayne Ivie U.S. Department of Agriculture, Agricultural Research Service

Robert A. Weiss University of Connecticut

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Foreword

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.fw001

The ACS S Y M P O S I U M S E R I E S was founded in 1974 to provide a

medium for publishing symposia quickly in book form. The format of the Series parallels that of the continuing A D V A N C E S IN C H E M I S T R Y SERIES except that, in order to save time, the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. Papers are reviewed under the supervision of the Editors with the assistance of the Series Advisory Board and are selected to maintain the integrity of the symposia; however, verbatim reproductions of previously published papers are not accepted. Both reviews and reports of research are acceptable, because symposia may embrace both types of presentation.

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Preface

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.pr001

THE GOLDEN ERA OF BIOLOGY,

including the agricultural sciences, is taking place in the 1980s, just as the early part of this century was considered the golden era for organic chemistry and the 1960s-1980s the golden era for electronics. Biology shares the present limelight with high-energy physics and computer sciences. The current focus on biology can be traced to several advances that are now affecting research concertedly. These developments include the solution of the structures of D N A and R N A by Watson and Crick, the remarkable progress in understanding protein structure and biological function, including the catalytic efficiency and specificity of enzymes, and the recognition of genomes as myriad genes and control mechanisms. Genetic engineering, recombinant DNA, transgenic animals and plants, chimeric macromolecules, gene deletion and addition, and a host of other terms are all "buzzwords" of biology in the 1980s. The nonscientific public is confused, and no wonder! Plant and animal breeding, fermentation-derived foods, processed and formulated foods, digestive aids, protease-containing detergents, rennin puddings, and cornstarch-derived sweeteners, although not really understood by the general public, have won their confidence. Yet we as biologists have not secured the trust and confidence of the public, and our modern buzzwords may be less a source of reassurance than of concern. Perhaps society's wariness stems from the fear that scientists will produce undesirable plants and animals through random, hit-and-miss genetic alterations. With detailed knowledge of the substrate and the catalyst, changes more precise than the surgeon's knife can be accomplished. This volume emphasizes the application of enzymes, the biological catalysts that can change plants and animals in precise and often remarkably dramatic fashion. In the near future, many of the chemicals that are now used to control pests and diseases will be eliminated, because the genetic composition of plants and animals will have been modified to make them resistant, or because the pests and plague organisms have been modified so that they cannot survive in the same environment with the desired plants and animals. Advances such as nitrogen fixation, tolerance to salinity, drought, and cold, and increased efficiency in fixing sunlight and nutrients into raw material for food uses

be

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.pr001

arc no longer 2 0 years in the future. Cultivation of plant and animal cells to produce needed flavors, colors, pharmaceuticals, and other desired products by methods now employed for culturing microbial cells is less than five to ten years away. All of this progress is possible because society, governments, educational institutions, research institutes, and scientists recognized that understanding the structure and function of biological molecules must precede the biotechnology and biology revolution that we are witnessing in the 1980s. We wish to express our appreciation to all the scientists who worked so diligently to describe their research. Initially these efforts were directed to the participants of the Symposium on Biocatalysis and Biomimetics: Aspects of Enzyme Chemistry for Agriculture. Subsequently, they sought to engage you, the reader, while meeting the exacting standards of the Books Department of the American Chemical Society and the idiosyncrasies of the co-editors. We also appreciate the support and confidence of the Division of Agricultural and Food Chemistry and the Division of Agrochemicals who encouraged us to undertake this project, under the auspices of the Biotechnology Secretariat. We especially appreciate the untiring efforts of Ms. Cheryl Shanks, who set high standards and strict guidelines for us, so that we never lost sight of the opportunities at hand. We trust that you, the reader and final judge, will agree with us that it was all worthwhile. JOHN R. WHITAKER

Department of Food Science and Technology University of California Davis, CA 95616 PHILIP E. SONNET

Eastern Regional Research Center Agricultural Research Service U.S. Department of Agriculture Wyndmoor, PA 19118 December 12, 1988

x In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 1

Interdependence of Enzymology and Agricultural Biotechnology

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch001

John R. Whitaker Department of Food Science and Technology, University of California, Davis, CA 95616 There is a strong dependence of advances in enzymology on agriculture, based on both historical and present data. Discovery of enzymes and elucidation of many of their properties were by agricultural chemists. The earliest enzymes studied were primarily from agriculturally important animals, plants and microorganisms, with substantial levels of enzymes and ready availability of raw material. The rapidly developing broad field of biochemical engineering/biotechnology, and its tremendous promise for the future, have brought basic and applied scientists together in private venture companies, at universities, and in government and industrial laboratories. Knowledge of enzymes and their utilization for the betterment of humans is literally exploding. This chapter provides a brief history of the contributions of enzymology to agricultural biotechnology, present state of applications of enzymology and a look at some future directions where enzymology can contribute. Enzymes have been a s s o c i a t e d w i t h humans and t h e i r food s i n c e creation. Many thousands o f y e a r s must have passed b e f o r e humans began t o wonder how the b e r r i e s they a t e and the meat they r e l i s h e d were c o n v e r t e d t o o t h e r s u b s t a n c e s i n the body and t o r e c o g n i z e t h a t t h e r e must be a c o n n e c t i o n between f o o d consumption and human growth and m o b i l i t y . They a l s o o b s e r v e d t h e p r o c e s s e s o f f e r m e n t a t i o n and t h e ease o f making b u t t e r m i l k , b e e r s , wines and cheeses, depending o n l y upon the m i c r o o r g a n i s m s u b i q u i t o u s l y present i n t h e i r surroundings. However, i t was n o t u n t i l 1833 t h a t Payen and P e r z o g (1) f o r t h e f i r s t time s p e c i f i c a l l y r e c o g n i z e d t h a t t h e r e was some compound p r e s e n t i n m a l t , used f o r brewing, w i t h t h e a b i l i t y t o s o l u b i l i z e t h e s t a r c h from the k e r n e l 0097-6156/89/0389-0001$06.00/0 • 1989 American Chemical Society

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch001

2

BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

o f g r a i n l e a v i n g b e h i n d the i n s o l u b l e e n v e l o p e s . They f u r t h e r n o t e d t h a t t h i s s u b s t a n c e c o u l d be p r e c i p i t a t e d from a m a l t e x t r a c t , and t h a t i t was d e n a t u r e d by h e a t . Payen and P e r z o g named t h i s s u b s t a n c e d i a s t a s e , because o f i t s s e p a r a t i n g a b i l i t y . We now know the enzyme as amylase. To t h i s day, the names o f enzymes g e n e r a l l y c o n t a i n the e n d i n g -ase. Remarkable c o n t r i b u t i o n on the p a r t o f Payen and P e r z o g ! Soon, t h e r e were r e p o r t s o f o t h e r d i s c r e t e a c t i v i t i e s i n p l a n t m a t e r i a l s caused by some endogenous compounds. In 1855, Schoenbein d e s c r i b e d a s u b s t a n c e ( p e r o x i d a s e ) i n p l a n t s t h a t caused a s o l u t i o n o f gum g u a i a c t o t u r n from brown t o b l u e i n the p r e s e n c e o f hydrogen p e r o x i d e (2). A y e a r l a t e r , Schoenbein i d e n t i f i e d a n o t h e r compound ( p o l y p h e n o l o x i d a s e ) i n mushrooms t h a t brought about the a e r o b i c o x i d a t i o n o f c e r t a i n compounds i n the p r e s e n c e o f m o l e c u l a r oxygen, t h e r e b y c a u s i n g browning i n the mushrooms (3), B e r t h e l o t , i n 1860 (4) d i s c o v e r e d a s u b s t a n c e i n y e a s t ( i n v e r t a s e ) t h a t caused a change i n the o p t i c a l r o t a t i o n o f s u c r o s e s o l u t i o n s . L i e b i g , i n e x p l o r i n g how food i s d i g e s t e d , o b s e r v e d t h a t the stomach o f v u l t u r e s c o n t a i n e d something ( p e p s i n ) t h a t d i s s o l v e d away meat t h a t had been p l a c e d i n a p e r f o r a t e d c a r t r i d g e and was swallowed by the b i r d . P a s t e u r r e c o g n i z e d t h a t f e r m e n t a t i o n i s caused by m i c r o o r g a n i s m s , and t h a t t h e r e was something i n the m i c r o b i a l c e l l s t h a t was a s s o c i a t e d w i t h t h i s fermentation (5). Because o f the c l o s e a s s o c i a t i o n between t h e s e s u b s t a n c e s and f e r m e n t a t i o n , they were named f e r m e n t s . Until 1879, when Kiihne proposed the word enzyme, (Greek, i n y e a s t ) ( 6 ) , t h e s e s u b s t a n c e s were c a l l e d oh^ayvLzoAfaeJunfLYVtA(thought t o r e q u i r e i n t a c t c e l l s f o r a c t i v i t y ) and unotiQCLVliztd ^tnmzwtii ( a c t i v i t y i n absence o f c e l l s ; p e p s i n f o r example). A g r i c u l t u r a l c h e m i s t s and a g r i c u l t u r a l p r o d u c t s c o n t i n u e d to c o n t r i b u t e t o the development o f enzymology i n major ways d u r i n g the 19th and 20th c e n t u r i e s . These c o n t r i b u t i o n s i n c l u d e d showing t h a t enzymes were s t i l l a c t i v e when s e p a r a t e d from l i v i n g c e l l s (7^), the c l o s e s t e r e o c h e m i c a l r e l a t i o n s h i p between an enzyme and i t s s u b s t r a t e ( 8 ) , and q u a l i t a t i v e methods f o r d e s c r i b i n g the a c t i o n o f enzymes (9^-12). P e p s i n , t r y p s i n and c h y m o t r y p s i n were p u r i f i e d , p a r t i c u l a r l y from cows and hogs because o f the a v a i l a b i l i t y o f t h e i r organs as b y p r o d u c t s (13, 14). The f i r s t enzyme t o be o b t a i n e d i n c r y s t a l l i n e form was from j a c k b e a n s , and i t was shown t o be a p r o t e i n ( 1 5 ) . The r e l a t i o n s h i p between enzyme a c t i v i t y and pH, u s i n g i n v e r t a s e , was s t u d i e d e x t e n s i v e l y by S^rensen ( 1 6 ) . The a p p l i c a t i o n o f endogenous and exogenous enzymes f o r q u a l i t y c o n t r o l and food m o d i f i c a t i o n , and f o r making s p e c i a l t y p r o d u c t s , became major a c t i v i t i e s . Enzymes a r e w i d e l y used i n the food i n d u s t r y t o m o d i f y p r o p e r t i e s o f raw p r o d u c t s i n t h e i r c o n v e r s i o n t o foods ( 1 7 ) . Most o f t h e s e a p p l i c a t i o n s use r a t h e r c r u d e enzyme systems, where a number o f r e a c t i o n s o c c u r . In r e c e n t y e a r s , t h e r e has been a tendency t o use more s p e c i f i c enzymes i n o r d e r to b r i n g about s e l e c t i v e and l i m i t e d changes, as w i l l be d e s c r i b e d below. Endogenous enzymes i n raw food m a t e r i a l s a l s o a r e o f major importance d u r i n g p o s t h a r v e s t s t o r a g e and i n s t o r a g e a f t e r processing (18). Enzymes have become v e r y i m p o r t a n t i n d e t e r -

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

1. WHITAKER

Interdependence of Enzymology and Biotechnology

m i n i n g and d e s c r i b i n g the g e n e t i c d i v e r s i t y o f p l a n t and a n i m a l genomes, i n d e v e l o p i n g new p l a n t s , and i n the c o n t r o l o f p e s t s and pathogens. The s e c t i o n below w i l l b r i e f l y summarize some o f t h e s e i m p o r t a n t uses o f enzymes i n a g r i c u l t u r e . Emphasis i s on u s i n g enzymes i n more s p e c i f i c ways, such as u n i q u e l y t a r g e t i n g compounds, o r s m a l l segments o f l a r g e polymers, f o r m o d i f i c a t i o n .

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch001

SOME NEW AND POTENTIAL APPLICATIONS OF ENZYMES IN AGRICULTURAL BIOTECHNOLOGY Heat Treatment f o r P r e s e r v a t i o n . Enzymes have pronounced e f f e c t s on the c o l o r , f l a v o r , aroma, t e x t u r e and n u t r i t i o n a l q u a l i t y o f foods d u r i n g growth and m a t u r a t i o n , d u r i n g h a r v e s t and p o s t h a r v e s t s t o r a g e and s t o r a g e a f t e r p r o c e s s i n g . The important enzymes v a r y from one food p r o d u c t t o a n o t h e r . In tomatoes, the s o f t e n i n g phenomenon i n r i p e n i n g i s caused by i t s p e c t i c enzymes. In peaches, a p p l e s , plums, grapes and avocados, the browning r e a c t i o n i s the r e s u l t o f p o l y p h e n o l o x i d a s e . In green l e a f y v e g e t a b l e s , l i p o x y g e n a s e and o t h e r enzymes cause f l a v o r and aroma d e t e r i o r a t i o n , but o t h e r enzymes may a l s o cause d i s c o l o r a t i o n ( l o s s o f green c o l o r and/or browning). Development o f the f r o z e n food i n d u s t r y was based on o b s e r v a t i o n s t h a t s u f f i c i e n t heat t r e a t m e n t o f v e g e t a b l e s and f r u i t s t o i n a c t i v a t e p e r o x i d a s e , f o l l o w e d by f r e e z i n g and f r o z e n s t o r a g e , c o u l d extend s h e l f l i f e from a few days o r a few weeks t o one t o two y e a r s (19-24). Other enzymes have been used l e s s f r e q u e n t l y t o m o n i t o r the adequacy o f heat t r e a t m e n t . These i n c l u d e c a t a l a s e (25) f o r E n g l i s h g r e e n peas and a few o t h e r vegetables, polyphenol oxidase i n f r u i t s , polygalacturonase f o r l o s s o f c o n s i s t e n c y o f tomatoes, p o t a t o e s and e g g p l a n t ; and l i p o x y g e n a s e and l i p a s e f o r o f f - f l a v o r development i n soybeans and c e r e a l products, r e s p e c t i v e l y . R e c e n t l y , W i l l i a m s e t a l . (18) made a s t r o n g case f o r u s i n g the s p e c i f i c enzyme r e s p o n s i b l e f o r q u a l i t y l o s s f o r m o n i t o r i n g the heat treatment by the food i n d u s t r y . These workers showed t h a t l i p o x y g e n a s e i s the major enzyme r e s p o n s i b l e f o r aroma d e t e r i o r a t i o n i n E n g l i s h g r e e n peas and g r e e n beans, w h i l e c y s t i n e l y a s e i s r e s p o n s i b l e f o r aroma d e t e r i o r a t i o n i n b r o c c o l i and c a u l i f l o w e r ( 1 8 ) . The f r o z e n food i n d u s t r y i s b e g i n n i n g t o adopt t h e s e s u g g e s t i o n s , because they use l e s s heat t o i n a c t i v a t e some o f t h e s e enzymes compared t o p e r o x i d a s e i n a c t i v a t i o n ; a t the same time they r e a l i z e c o n s i d e r a b l e energy s a v i n g s . To a s s i s t i n t h e s e changes, f a s t e r s e m i q u a n t i t a t i v e d e t e c t i o n methods a r e needed f o r the i n d i c a t o r enzymes. The consumers demand f o r more f r e s h - l i k e p r o d u c t s and f o r f o r m u l a t e d p r o d u c t s l e d t o o t h e r enzyme-caused problems. V e g e t a b l e s p a c k e t e d t o g e t h e r w i t h s t a r c h - b a s e d sauces s e r v e as an example. R e l a t i v e l y heat s t a b l e amylases from exogenous m i c r o o r g a n i s m s a r e o f t e n the problem, r e q u i r i n g m o d i f i e d washing c o n d i t i o n s and/or l o n g e r heat treatment o f the raw v e g e t a b l e s . T h i s was a problem i n canned a p r i c o t s a few y e a r s ago, where a r e l a t i v e l y h e a t - s t a b l e p o l y g a l a c t u r o n a s e from the brown r o t fungus was the c u l p r i t ( 2 6 ) . 1

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

E n z y m a t i c M o d i f i c a t i o n o f P r o t e i n s f o r Food Use. Proteolytic enzymes a r e used e x t e n s i v e l y f o r m o d i f y i n g p r o t e i n s i n v a r i o u s ways i n food p r o d u c t s and f o r waste management (17, 27). These a r e used i n baked and brewed p r o d u c t s , c e r e a l s , c h e e s e , c h o c o l a t e / c o c o a , egg and egg p r o d u c t s , f e e d s , f i s h , legumes, meats, m i l k , p r o t e i n h y d r o l y s a t e s and w i n e s . But t h e r e a r e many o t h e r u s e s , and p o t e n t i a l u s e s , o f enzymes t o m o d i f y p r o t e i n . W h i t a k e r (27, 28), and W h i t a k e r and P u i g s e r v e r (29) have d e s c r i b e d more than 100 enzymatic m o d i f i c a t i o n s o f p r o t e i n s i n v i v o , c h a l l e n g i n g s c i e n t i s t s t o l o o k a t the p o s s i b i l i t i e s o f m o d i f y i n g the amino a c i d s i d e c h a i n s by p r o t e o l y t i c and o t h e r methods. Feeney and W h i t a k e r (30, 31) have emphasized the importance o f the enzymes t r a n s g l u t a m i n a s e , l i p o x y g e n a s e , p o l y p h e n o l o x i d a s e and p e r o x i d a s e i n the c r o s s l i n k i n g o f p r o t e i n s . W i t h t r a n s g l u t a m i n a s e , c h i m e r i c p r o t e i n s can be made r e a d i l y by c r o s s l i n k i n g two p r o t e i n s , each w i t h q u i t e d i f f e r e n t p r o p e r t i e s (32, 33). P r o t e o l y t i c enzymes have l o n g been used t o produce p r o t e i n h y d r o l y s a t e s f o r use i n soups, b o u i l l o n , soy sauce, t a m a r i sauce, etc. Recent i n t e r e s t i n p r o d u c i n g l a r g e p o l y p e p t i d e s o f c o n t r o l l e d s i z e h a v i n g improved s o l u b i l i t y and f u n c t i o n a l p r o p e r t i e s f o r use i n the f o o d i n d u s t r y has l e d t o i n v e s t i g a t i o n o f h i g h l y s p e c i f i c p r o t e o l y t i c enzymes f o r t h a t purpose (34, 3 5 ) . Specialty Products. Enzymes, because o f t h e i r h i g h s u b s t r a t e s p e c i f i c i t y and s t e r e o s p e c i f i c i t y , a r e i d e a l f o r p r o d u c i n g s p e c i a l compounds r e q u i r e d by the f o o d and p h a r m a c e u t i c a l i n d u s t r i e s ( 3 6 ) . By a l l measures, the c o n v e r s i o n o f c o r n s t a r c h , produced by wet m i l l i n g , t o g l u c o s e and f r u c t o s e has been the most s u c c e s s f u l commercial o p e r a t i o n ( 3 7 ) . More than t e n b i l l i o n pounds o f f r u c t o s e a r e produced and used by the f o o d i n d u s t r y each y e a r . The i n d u s t r y u s e s r e l a t i v e l y heat s t a b l e a-amylase t o p a r t i a l l y h y d r o l y z e and s o l u b i l i z e s t a r c h , f o l l o w e d by g l u c o a m y l a s e t o produce g l u c o s e and i m m o b i l i z e d g l u c o s e isomerase t o c o n v e r t a p p r o x i m a t e l y 50% o f the g l u c o s e t o f r u c t o s e i n an e q u i l i b r i u m c o n t r o l l e d process. The f o l l o w i n g enzymatic h y d r o l y s e s a r e a l s o b e i n g used, but w i t h more l i m i t e d s u c c e s s : conversion of c e l l u l o s e t o f e e d s t o c k f o r s i n g l e c e l l p r o t e i n p r o d u c t i o n and o t h e r u s e s , l a c t o s e h y d r o l y s i s t o g l u c o s e and g a l a c t o s e ( t o remove l a c t o s e from m i l k o r f o r s w e e t e n e r s ) ; x y l a n h y d r o l y s i s t o x y l o s e and x y l i t o l ; DNA and RNA h y d r o l y s i s t o m o n o n u c l e o t i d e s as f l a v o r e n h a n c e r s , and l i g n i n and mannan d e g r a d a t i o n f o r waste t r e a t m e n t . L i p a s e s a r e now b e i n g s t u d i e d i n t e n s i v e l y t o a l t e r t r i g l y c e r i d e f a t t y acid composition. They a r e a l s o b e i n g e v a l u a t e d f o r s e l e c t i v e f o r m a t i o n o f mono- and d i g l y c e r i d e s and f o r p r o d u c i n g waxes. The d r i v i n g f o r c e f o r the r e s e a r c h i s r e l a t e d b o t h t o the c o n t i n u i n g abundance, e s p e c i a l l y i n the U.S., o f f a t s and o i l s , and t o the renewed i n t e r e s t i n a l l c l a s s e s o f enzymes t h a t can a c t on f a t t y a c i d s and t h e i r d e r i v a t i v e s . These workers p i n t h e i r hopes on improved u n d e r s t a n d i n g o f enzyme s t a b i l i t y and mechanism o f a c t i o n , and t o f i n d i n g new enzymes t h a t

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch001

1. WHITAKER

Interdependence of Enzymology and Biotechnology

have u n i q u e s e l e c t i v i t y w i t h r e s p e c t t o f a t t y a c i d c h a i n l e n g t h , degree o f u n s a t u r a t i o n , and a l c o h o l r e s i d u e ( o f the t r i g l y c e r i d e ) . Another l a r g e s u c c e s s f u l commercial a p p l i c a t i o n o f enzymes i s i n the amino a c i d i n d u s t r y . Amino a c i d s f o r food and f e e d f o r t i f i c a t i o n , n u t r i t i o n a l supplements, o r as f e e d s t o c k f o r downstream p r o d u c t s can be made by f e r m e n t a t i o n p r o c e s s e s , from p r o t e i n h y d r o l y s a t e s or by c h e m i c a l s y n t h e s i s . While chemical s y n t h e s i s i s cheaper f o r a number o f amino a c i d s , . i t o f t e n produces a r a c e m i c m i x t u r e . The r a c e m i c m i x t u r e i s s u c c e s s f u l l y r e s o l v e d on a commercial s c a l e by a c y l a t i n g the amino a c i d s , then u s i n g an a m i n o a c y l a s e t o remove the a c y l group from the L-amino a c i d and s e p a r a t i n g the f r e e L-amino a c i d from the s t i l l a c y l a t e d D-amino a c i d . A j i n a m o t o and o t h e r companies, e s p e c i a l l y i n Japan, make l a r g e amounts o f amino a c i d s by t h i s p r o c e s s . The p l a s t e i n r e a c t i o n i s b e i n g used s u c c e s s f u l l y i n Japan t o produce p h e n y l a l a n i n e - f r e e p e p t i d e p r o d u c t s f o r p a t i e n t s w i t h phenylketonuria, s u r f a c t a n t s f o r the c o s m e t i c and food i n d u s t r y and a n t i f r e e z e t y p e compounds t h a t have the p o t e n t i a l t o p r e v e n t "hard f r e e z i n g " o f f o o d s , b l o o d and sperm ( 3 8 ) . Aspartame ( L - a s p a r t y l - L - p h e n y l a l a n i n e m e t h y l e s t e r ) can be produced c h e m i c a l l y , o r more r e c e n t l y by m i c r o o r g a n i s m s , u s i n g recombinant DNA t e c h n o l o g y t o produce a l a r g e p o l y p e p t i d e o f r e p e a t i n g a s p a r t y l p h e n y l a l a n i n e u n i t s , then u s i n g a s p e c i f i c protease to h y d r o l y z e the p o l y p e p t i d e t o the d i p e p t i d e , f o l l o w e d by e s t e r i f i c a t i o n w i t h m e t h a n o l . Another a p p l i c a t i o n o f enzymes i s the use o f g l u c o s y l t r a n s f e r a s e s and g l y c o s y l a t i n g enzymes f o r the p r o d u c t i o n o f m o d i f i e d c a r b o h y d r a t e s t h a t r e t a i n t h e i r sweetness but a r e not m e t a b o l i z e d by the human. S t e v i o s i d e , a n a t u r a l sweetener from the p l a n t S t e v i a r e b a u d i a n a , has been m o d i f i e d by the use o f a - g l u c o s i d a s e i n o r d e r to b r i n g i t s t a s t e c l o s e r t o t h a t o f s u c r o s e (39, 4 0 ) . Enzymes a r e e s p e c i a l l y u s e f u l i n a d d i n g and removing r e s i d u e s from compounds, because o f t h e i r s u b s t r a t e and p r o d u c t stereospecificity. Only the b i o l o g i c a l l y a c t i v e isomer o f a compound i s formed by the enzyme because o f the s t r i c t s t e r e o c h e m i s t r y r e q u i r e d f o r b i n d i n g o f s u b s t r a t e t o the enzyme i n o r d e r t o have p r o p e r o r i e n t a t i o n t o the c a t a l y t i c s i t e o f the enzyme. Use o f more t r a d i t i o n a l c h e m i c a l methods l e a d s t o mixtures of isomers. O r g a n i c c h e m i s t s have found enzymes t o be i n v a l u a b l e i n p a r t i c u l a r steps of s y n t h e s i s . Enzymes a r e used q u i t e f r e q u e n t l y i n the s y n t h e s i s o f p h a r m a c e u t i c a l compounds, f o r example. Use o f a m i n o a c y l a s e s t o p e r m i t s e p a r a t i o n o f D- and Imm i x t u r e s o f amino a c i d s i s d e s c r i b e d above. The s t e r e o s p e c i f i c i t y o f enzymes i s a l s o i n v a l u a b l e i n a n a l y t i c a l uses f o r s e q u e n c i n g polymers, f o r c o n f i g u r a t i o n d e t e r m i n a t i o n o f monomeric u n i t s i n polymers ( f o r example c a r b o h y d r a t e s ) , as w e l l as f o r d e t e r m i n i n g how much o f each isomer i s p r e s e n t . Enzymes and Recombinant DNA T e c h n o l o g y . Recombinant DNA t e c h n o l o g y f o r whatever purpose depends a b s o l u t e l y upon enzymes. Keys t o the r a p i d advancement o f b i o c h e m i c a l e n g i n e e r i n g have been: 1) u n d e r s t a n d i n g o f the p r i m a r y and secondary s t r u c t u r e s o f DNA and RNA; 2) enzymes w i t h s t r i c t s p e c i f i c i t y t h a t p e r m i t the

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

5

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch001

6

BIOCATALYSIS IN AGRICULTURAL

BIOTECHNOLOGY

r e l a t i v e l y easy s e q u e n c i n g o f DNA and RNA ( w i t h the knowledge o f p r i m a r y s t r u c t u r e s o f p r o t e i n s encoded); 3) the a b i l i t y t o remove, v i a h y d r o l y s i s , s p e c i f i c n u c l e o t i d e segments from p l a s m i d s (by use o f r e s t r i c t i o n enzymes); and 4) a b i l i t y t o i n c o r p o r a t e n u c l e o t i d e sequences (by use o f l i g a s e s ) from o t h e r organisms o r by c h e m i c a l s y n t h e s i s i n t o the h o s t organism. T h i s r e l a t i v e l y easy c h e m i s t r y , mediated by the s p e c i f i c i t y o f enzymes, p e r m i t s enhanced p r o d u c t i o n o f a v a l u a b l e p r o t e i n , enzyme o r o t h e r p r o d u c t , e i t h e r i d e n t i c a l t o t h a t produced n a t u r a l l y by the donor organism, o r w i t h s e l e c t e d changes t h a t enhance s t a b i l i t y o r d i f f e r e n t functional properties. Examples o f t h i s i n c l u d e chymosin, i n s u l i n , b o v i n e growth hormone, and p r o t e o l y t i c enzymes f o r use i n detergents. Recombinant DNA t e c h n i q u e s p e r m i t b o t h an i n c r e a s e , as w e l l as d e c r e a s e , i n the l e v e l o f enzymes o r o t h e r p r o d u c t s i n an organism. Would i n c r e a s e d l e v e l s o f r i b u l o s e b i s p h o s p h a t e c a r b o x y l a s e e n g i n e e r e d i n t o a p l a n t i n c r e a s e the amount o f c a r b o n d i o x i d e f i x e d , o r would i t be b e t t e r t o d e c r e a s e the oxygenase a c t i v i t y o f t h i s enzyme, o r change the c o n c e n t r a t i o n o f hydrogenase? G l y p h o s a t e (N-phosphonomethylglycine; t r a d e name Roundup) i s an e f f e c t i v e g e n e r a l h e r b i c i d e , e f f e c t i v e a g a i n s t p l a n t s that c o n t a i n 5-enolpyruvylshikimate-3-phosphate synthase. T h i s enzyme has now been enhanced i n s e v e r a l a g r i c u l t u r a l l y i m p o r t a n t p l a n t s , p e r m i t t i n g the use o f Roundup t o c o n t r o l weeds and g r a s s e s i n t h e s e c r o p s . P o l y p h e n o l o x i d a s e causes up t o 50% l o s s o f t r o p i c a l f r u i t s because they brown when b r u i s e d , o t h e r w i s e damaged o r become too ripe. F r o z e n s t r a w b e r r i e s become mushy and brown when they a r e a l l o w e d t o warm t o room temperature f o l l o w i n g thawing. Y e t , they cannot be b l a n c h e d t o i n a c t i v a t e the p o l y g a l a c t u r o n a s e , because h e a t i n g i s d e t r i m e n t a l t o f l a v o r and t e x t u r e . L i p o x y g e n a s e causes o f f - f l a v o r and o f f - a r o m a i n soybeans (beany f l a v o r ) , E n g l i s h g r e e n peas, g r e e n beans, c o r n and p r o b a b l y o t h e r v e g e t a b l e s ( 1 8 ) . Can the l e v e l s o f t h e s e enzymes be r e d u c e d , o r e l i m i n a t e d e n t i r e l y , w i t h o u t an a d v e r s e e f f e c t on t h e p l a n t and p r o d u c t ? Traditional b r e e d i n g methods have been used t o d e c r e a s e the l e v e l s o f t h e s e enzymes, w i t h o u t apparent e f f e c t on p r o d u c t i v i t y and q u a l i t y . " L i p o x y g e n a s e - f r e e " soybeans have been b r e d , whereby the l e v e l o f the major l i p o x y g e n a s e isoenzyme has been g r e a t l y r e d u c e d . B i t t e r n e s s i n orange and g r a p e f r u i t j u i c e s i s a major economic problem. Orange j u i c e i s produced p r i m a r i l y from V a l e n c i a - t y p e oranges s i n c e t h e r e i s l e s s a s s o c i a t e d b i t t e r n e s s . L i m o n i n and n a r i n g i n a r e the major causes o f b i t t e r n e s s ( 4 1 ) . L i m o n i n c o n c e n t r a t i o n s as low as 5-6 ppm g i v e an u n a c c e p t a b l e l e v e l of b i t t e r n e s s . N a r i n g i n i s about 0.01 as b i t t e r as l i m o n i n but i s o f t e n produced i n h i g h e r amounts. B i t t e r n e s s due t o l i m o n i n can be e l i m i n a t e d by: a) p r e v e n t i n g i t s b i o s y n t h e s i s ( p r e h a r v e s t t r e a t m e n t w i t h 1-naphthalene a c e t i c a c i d ) ; b) removing the r a g and p u l p from f r e s h l y e x p r e s s e d j u i c e as soon as p o s s i b l e t o p r e v e n t the p r e c u r s o r , l i m o n i c a c i d A - r i n g (mono)lactone, from b e i n g c o n v e r t e d t o l i m o n i n (42, 43) o r , c ) enzymatic h y d r o l y s i s o f l i m o n i n t o n o n - b i t t e r p r o d u c t s by use o f i m m o b i l i z e d m i c r o b i a l c e l l s c o n t a i n i n g an NADP-dependent l i m o n i n dehydrogenase (44, 4 5 ) .

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

1. WHITAKER

Interdependence of Enzymology and Biotechnology

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch001

B i t t e r n e s s , caused by n a r i n g i n , i s a l s o removed by an enzyme, naringinase. Another p o s s i b i l i t y would be to e l i m i n a t e one o r more o f the enzymes i n the l i m o n i n b i o s y n t h e t i c pathway by u s i n g recombinant DNA t e c h n i q u e s . Limonin, a t r i t e r p e n o i d , i s probably s y n t h e s i z e d v i a the mevalonate pathway, as a r e the monoterpenoid f l a v o r compounds. I t appears t h a t n o m i l i n , a p r e c u r s o r o f l i m o n i n , i s s y n t h e s i z e d i n the stems and r o o t s o f c i t r u s and t h e n the p r e c u r s o r t r a n s p o r t e d t o the f r u i t where i t i s c o n v e r t e d by s e v e r a l enzymes t o l i m o n i n and o t h e r b i t t e r l i m o n o i d s ( 4 6 ) . I n t e g r a t e d C o n t r o l o f P e s t s and o f S y n t h e t i c P e s t i c i d e s . Much emphasis i s p l a c e d on r e d u c i n g the use o f s y n t h e t i c p e s t i c i d e s and i n d e s i g n i n g p e s t i c i d e s t h a t have a more r e s t r i c t e d spectrum o f a c t i v i t y w i t h s h o r t e r l i f e t i m e s i n the environment. There i s a l s o much emphasis on b e t t e r management o f t i m i n g a p p l i c a t i o n s and i n u s i n g b i o l o g i c a l c o n t r o l methods. T h i s f o c u s e s much a t t e n t i o n on enzymes o f the p l a n t s and o f the p e s t s . Enzyme and isoenzyme a c t i v i t i e s , d i s t i n g u i s h e d v i a s e p a r a t i o n s by p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s and a c t i v i t y s t a i n s , a r e used r o u t i n e l y i n d e t e r m i n i n g changes i n p l a n t s d u r i n g b r e e d i n g by t r a d i t i o n a l and n o n t r a d i t i o n a l methods. Enzyme p a t t e r n s e s t a b l i s h e d by the above t e c h n i q u e s a r e o f t e n r e l a t e d t o pathogen and i n s e c t r e s i s t a n c e , thus s a v i n g c o n s i d e r a b l e amount o f time i n a n a l y s e s . The c o r r e l a t i o n may be the r e s u l t o f compounds, such as p h e n o l i c s (47^), a l k a l o i d s ( 4 7 ) , p r o t e a s e i n h i b i t o r s (48, 49) and a-amylase i n h i b i t o r s (50; Ho and W h i t a k e r , U n i v e r s i t y o f C a l i f o r n i a , D a v i s , u n p u b l i s h e d d a t a ) produced by the p l a n t as a d e f e n s e mechanism a g a i n s t i n s e c t s and m i c r o o r g a n i s m s . I t i s a l s o important t o u n d e r s t a n d how p e s t i c i d e s a r e m e t a b o l i z e d by p l a n t s and o t h e r o r g a n i s m s . This w i l l aid i n d e t e r m i n i n g the r e s i d e n c e time o f the p e s t i c i d e s i n the environment, and c o u l d a l e r t us t o the p o s s i b i l i t y o f more t o x i c , and more p e r s i s t e n t p r o d u c t s t h a t may be formed. Removal o f Unwanted Compounds. The removal o f p h e n y l a l a n i n e from p r o t e i n s by the p l a s t e i n r e a c t i o n was mentioned above ( 3 8 ) . The p l a s t e i n r e a c t i o n has a l s o been used t o remove the b i t t e r p e p t i d e s from p r o t e i n h y d r o l y s a t e s by c a u s i n g r e s y n t h e s i s o f some p e p t i d e bonds c a t a l y z e d by p r o t e o l y t i c enzymes when the s u b s t r a t e c o n c e n t r a t i o n i s around 35% (w/w (5JL). A number o f food p l a n t s , i n c l u d i n g c a s s a v a and l i m a beans, c o n t a i n t o x i c l e v e l s o f cyanogenic g l y c o s i d e s (52). Soaking t h e s e v e g e t a b l e s o v e r n i g h t p e r m i t s h y d r o l y s i s o f the c y a n o g e n i c g l y c o s i d e s by s p e c i f i c g l y c o s i d a s e s ( l i n a m a r i n a s e f o r example when the s u b s t r a t e i s l i n a m a r i n ) t o g l u c o s e , HCN and a c e t o n e . The t o x i c compound, HCN, i s then removed by e v a p o r a t i o n from the food as i t i s c o o k i n g . When f r e s h l y squeezed orange j u i c e i s a l l o w e d t o s t a n d , the suspended p e c t i c compounds ("cloud") s e p a r a t e and s e t t l e from the juice. T h i s phenomenon can be p r e v e n t e d by h e a t i n g the f r e s h l y squeezed j u i c e t o i n a c t i v a t e the p e c t i n m e t h y l e s t e r a s e , producing a "cooked f l a v o r . " A l t e r n a t i v e l y , a d d i t i o n a l polygalacturonase can be added t o the j u i c e ( 5 3 ) . Cloud p r e c i p i t a t i o n i s caused by Ca2+ c h e l a t i n g the p e c t i c a c i d produced by p e c t i n m e t h y l e s t e r a s e .

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

7

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BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

The role of polygalacturonase i s to hydrolyze the pectic acid to fragments that do not precipitate when they chelate with Ca^ , but are s t i l l insoluble to give the cloud appearance of natural juice. +

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch001

CONCLUSION As b r i e f l y documented above, there has been a close association between the discovery of enzymes, and the development of a g r i c u l t u r a l biotechnology. Each period seems to bring more exciting advances i n use of enzymes. But the role of enzymes i n a g r i c u l t u r a l biotechnology has never been more exciting and important than i n the present period. New enzymes, and old enzymes, are being used more extensively to transform microorganisms, plants and animals, to make specialty food products and to carry out d i f f i c u l t stereochemical syntheses of needed b i o l o g i c a l compounds, with a purity and y i e l d that the organic chemist could only dream of i n the past.

LITERATURE CITED

1. 2.

Payen, A.; Perzog, J. F. Ann. Chim. (Phys.) 1833, 53, 73. Schoenbein, C. F. Verhandl. Naturforsch. Ges. Basel 1855, 1, 339. 3. Schoenbein, C. F. Phil. Mag. 1856, 11, 137. 4. Berthelot, M. Compt. Rend. Acad. Sci. 1860, 50, 980. 5. Pasteur, L. Compt. Rend. Acad. Sci. 1875, 80, 452. 6. Kühne, W. Unters. a.d. Physiol. Institut der Univ. Heidelberg 1878, 1, 291. 7. Büchner, E. Ber. 1897, 30, 117. 8. Fischer, E. Ber. 1894, 27, 2985. 9. Henri, V. Acad. Sci., Paris 1902, 135, 916; Lois générales de'action des diastases, Herman, Paris, 1903. 10. Brown, A. J. Chem. Soc. 1902, 81, 373. 11. Michaelis, L.; Menten, M. L. Biochem Z. 1913, 49, 333. 12. Lineweaver, H.; Burk, D. J. Amer. Chem. Soc. 1934, 56, 658. 13. Kunitz, M.; Northrop, J. H. J. Gen. Physiol. 1936, 19, 991. 14. Northrop, J. H.; Kunitz, M.; Herriott, R. M. Crystalline Enzymes, Columbia Univ. Press, New York, 1948. 15. Sumner, J. B. J. Biol. Chem. 1926, 69, 435. 16. Sørensen, S. P. L., Biochem. Z. 1909, 21, 131,201. 17. Whitaker, J. R. Principles of Enzymology for the Food Sciences, Marcel Dekker, Inc.: New York, 1972; pp 12-22. 18. Williams, D. C.; Lim, M. H.; Chen, A. O.; Pangborn, R. M.; Whitaker, J. R. Food Technol. 1986, 40, 130. 19. Kohman, E. F. Food Ind. 1936, 8, 287. 20. Diehl, H. C. Ind. Eng. Chem. 1932, 24, 661. 21. Diehl, H. C.; Dingle, J. H.; Berry, J. A. Food Ind. 1933, 5, 300. 22. Campbell, H. West. Canner Packer 1940, 32, 51. 23. Joslyn, M. S. Adv. Enzymol. 1949, 9, 613. 24. Enzyme inactivation tests (frozen vegetables). Technical inspection procedures for the use of USDA inspectors. U.S. Department of Agriculture, Agric. Mktg. Serv.: Washington, D.C., 1975. 25. Sapers, G. M.; Nickerson, J. T. R. J. Food Sci. 1962, 27, 277.

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

1. WHITAKER Interdependence of Enzymology and Biotechnology 26. 27. 28. 29. 30.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch001

31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53.

9

Luh, B. S.; Ozbilgin, S.; Liu, Y. K. J. Food Sci. 1978, 43, 713. Whitaker, J. R. Adv. Chem. Ser. 1977, 160, 95. Whitaker, J. R. In International Symposium on the Use of Enzymes in Food Technology, Edition Lavoisier, 1982, pp 329358. Whitaker, J. R.; Puigserver, A. J. Adv. Chem. Ser. 1982, 198, 57. Feeney, R. E.; Whitaker, J. R. In New Protein Foods; Vol. 5, Seed Storage Proteins; Wilcke, H. L.; Altschul, Α. Μ., Eds.; Academic Press: New York, 1985, pp 181-219. Feeney. R. E.; Whitaker, J. R. In Advances in Cereal Science and Technology, Vol. IX; Pomeranz, Y., Ed.; Amer. Assocn. Cereal Chem.: St. Paul, Minnesota, 1988, pp 21-46. Ikura, K.; Kometani, T.; Sasaki, R.; Chiba, H. Agric. Biol. Chem. 1980, 44, 2979. Ikura, K.; Kometani, T.; Yoshikawa, M.; Sasaki, R.; Chiba, H. Agric. Biol. Chem. 1980, 44, 1567. Chobert, J.-M; Sitohy, M.; Whitaker, J. R. J. Amer. Oil Chem. Soc. 1987, 64, 1704. Adler-Niseen, J. Enzymic Hydrolysis of Food Proteins, Elsevier Science Publishing Co.: New York, 1986, 427 pp. Feeney, R. E.; Whitaker, J. R., Eds. Protein Tailoring for Food and Medical Uses; Marcel Dekker: New York, 1985, 392 pp. Venkatasubramanian, K. In Enzymes: The Interface Between Technology and Economics; Danehy, J.P.; Wolnak, Β., Eds.; Marcel Dekker: New York, 1980, pp 35-52. Watanabe, M.; Arai, S. Adv. Chem. Ser. 1982, 198, 199. Miyaki, T. U.K. Patent 2 027 423-B, 1983. Nishihashi, H.; Mutsubayashi, T.; Matsuda, K. U.S. Patent 4 590 160, 1986. Chandler, Β. V.; Nicol, K.J. CSIRO Food Res. Q. 1975, 35, 79. Hasagawa, S. J. Agric. Food Chem. 1976, 24, 24.4 Brewster, L. C.; Hasagawa, S.; Maier, V. P. J. Agric. Food Chem. 1976, 24, 21. Vaks, B.; Lifshitz, A. Lebensm. Wiss. Technol. 1975, 8, 236. Maier, V. P.; Brewster, L. C.; Hsu, A. C. J. Agric. Food Chem. 1973, 21, 490. Herman, Z.; Hasagawa, S. Abst. No. 19, AGFD, 192nd Amer. Chem. Soc. Meeting, Anaheim, CA, Sept. 1986. Duffey, S. S.; Felton, G. W., this monograph. Ryan, C. A. Ann. Rev. Plant Physiol. 1973, 24, 173. Richardson, M. Phytochemistry 1977, 16, 159. Yetter, Μ. Α.; Saunders, R. M.; Boles, H. P. Cereal Chem. 1979, 56, 243. Fujimaki, M.; Arai, S.; Yamashita, M. Adv. Chem. Ser. 1977, 160, 156. Liener, I. E. Adv. Chem. Ser. 1977, 160, 283. Baker, R. Α.; Bruemmer, J. H. J. Agric. Food Chem. 1972, 20, 1169.

RECEIVED

November 9, 1988

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 2

Design of Enzymatic Catalysts Donald Hilvert

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch002

Department of Molecular Biology, Research Institute of Scripps Clinic, Scripps Clinic and Research Foundation, LaJolla,CA92037 Two approaches to catalyst design are presented; these are site-directed mutagenesis of existing proteins and development of antibody-based catalysts with rationally designed immunogens. Specifically, semisynthetic selenoenzymes (proteases in which the active site nucleophile has been converted chemically into selenocysteine) and monoclonal antibodies with chorismate mutase activity are described. The complementary strategies yield tailored binding sites that couple novel chemical activity with high selectivity. The success achieved augurs well for the development of practical catalysts for general use in medicine and industry. Enzymes are remarkable c a t a l y s t s . Few chemical agents can match the rate accelerations or tremendous s p e c i f i c i t y that enzymes achieve under mild aqueous conditions. Consequently, i t i s no surprise that such molecules are being used increasingly i n medicine and industry to carry out important chemical transformations. The p o t e n t i a l usefulness of biocatalysts would be greatly increased, however, i f i t were possible to design and synthesize them de novo. But how i s t h i s to be accomplished? In the following a r t i c l e two viable approaches to catalyst design are i l l u s t r a t e d with examples from our laboratory. On the one hand, we are chemically mutating e x i s t i n g protein active s i t e s to develop a r t i f i c i a l selenoenzymes with p o t e n t i a l l y useful properties. On the other, we are employing r a t i o n a l l y designed immunogens to generate antibodies that catalyze concerted chemical transformations. Semisynthetic

Enzymes

As i t i s not yet possible to prepare t a i l o r e d protein binding pockets from their constituent amino acids, e x i s t i n g , well-characterized protein structures represent a t t r a c t i v e s t a r t i n g materials for the design of new enzymes. These can be used as s c a f f o l d i n g on which to mount c a t a l y t i c groups. For instance, Kaiser and co-workers have 0097-6156/89/0389-0014$06.00/0 © 1989 American Chemical Society

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch002

2.

HILVERT

15

Design of Enzymatic Catalysts

constructed successful semisynthetic flavoenzymes by incorporating reactive f l a v i n analogs into the active s i t e s of papain (1) and glyceraldehyde-3-phosphate dehydrogenase (2). The r e s u l t i n g hybrid enzymes couple the unique chemistry of the cofactor with the binding s p e c i f i c i t y of the protein template. A complementary approach u t i l i z e s chemical or recombinant DNA methodologies to a l t e r selected amino acid residues i n the protein binding s i t e . We are interested, for instance, i n the properties of selenium i n b i o l o g i c a l systems and are currently converting the active s i t e -OH and -SH groups of several serine and cysteine proteases into -SeH groups. Our reasons are threefold. F i r s t , selenium can serve as a molecular probe of enzyme mechanism. By comparing the properties of the analogous oxygen, s u l f u r and selenium variants i t may prove possible to sort out s t e r i c and electronic factors which are important for c a t a l y s i s . We also want to take advantage of some of the r i c h organic chemistry of selenium Q) to develop protein-based catalysts which may have p r a c t i c a l u t i l i t y . F i n a l l y , semisynthetic selenoenzymes are l i k e l y to be i n t e r e s t i n g models of n a t u r a l l y occurring selenoproteins, l i k e glutathione peroxidase. The l a t t e r enzyme protects mammalian c e l l s against oxidative damage and contains a c a t a l y t i c a l l y e s s e n t i a l selenocysteine residue (4). S e l e n o s u b t i l i s i n . Scheme 1 i l l u s t r a t e s our strategy for converting a serine residue i n a protein into a selenocysteine. The two step process i s analogous to that used by Bender (5) and Koshland (6) 20 years ago to convert a serine protease into a cysteine protease. It involves a c t i v a t i o n of the side chain alcohol group of a s e r y l residue by formation of a sulfonyl ester, followed by displacement of the sulfonate with an appropriate selenium nucleophile. We have successfully c a r r i e d out t h i s sequence on the b a c t e r i a l protease subt i l i s i n Carlsberg [EC 3.4.21.14] (Wu and H i l v e r t , unpublished results). Serine 221 i n the active s i t e of this enzyme was specif i c a l l y modified with S - l a b e l l e d phenylmethanesulfonyl f l u o r i d e (PMSF). The r e s u l t i n g PMS-subtilisin was treated with a large excess of hydrogen selenide at 40 °C and pH 6.8. After 36 hours, more than 95% of the r a d i o a c t i v i t y associated with the enzyme was l o s t . In the absence of hydrogen selenide less than 10% sulfonate was released from the enzyme. S e l e n o s u b t i l i s i n was separated from unreacted hydrogen selenide by gel f i l t r a t i o n on a Sephadex G-25 column and p u r i f i e d by ion exchange chromatography on CM-50 Sephadex. Greater than 0.9 equivalents of selenium were incorporated per mole of s u b t i l i s i n as judged by anaerobic t i t r a t i o n of the reduced enzyme with 5,5'-dithio-bis(2-nitrobenzoic acid) (7). 35

Scheme 1.

Preparation

of Semisynthetic

Selenoenzymes

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

16

BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

Serine and cysteine proteases cleave their substrates by a two step mechanism i n which the active s i t e nucleophile i s t r a n s i e n t l y acylated. In many cases i t i s possible to i s o l a t e the acyl enzyme intermediate. We have prepared an authentic acyl derivative of s e l e n o s u b t i l i s i n by treating the reduced protein (-SeH form) with excess cinnamoyl imidazole. Se-Cinnamoyl-selenosubtilisin is r e l a t i v e l y stable at pH 5 and can be separated from unreacted reagent by gel f i l t r a t i o n . A difference spectrum of the Se-cinnamoylated protein and unmodified s u b t i l i s i n showed an absorbance maximum at approximately 308 nm for the enzyme-bound chromophore; t h i s value i s red-shifted by 18 nm r e l a t i v e to the λ of the model compound Ncarbobenzoxy-Se-cinnamoyl-selenocysteine methyl ester (Wu andHilvert, unpublished r e s u l t s ) . For comparison, the absorbance maxima for the analogous cinnamoyl derivatives of native s u b t i l i s i n and thiosubt i l i s i n are 289 and 310 nm, respectively (5).

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch002

β β χ

Kinetics. The a v a i l a b i l i t y of a stable acyl enzyme intermediate o f f e r s the p o s s i b i l i t y of determining d i r e c t l y the deacylation rate for the modified protein (9). Selenoesters undergo non-enzymatic hydrolysis at roughly the same rate as s t r u c t u r a l l y analogous esters and t h i o l e s t e r s , while their aminolysis i s s i g n i f i c a n t l y faster (9). Consequently, comparison of the p a r t i t i o n i n g of the cinnamoyl-enzyme species between water and amine for the isologous oxygen, s u l f u r and selenium cases i s of p a r t i c u l a r i n t e r e s t . Rate constants were determined with the selenoenzyme by following decrease of absorbance at 310 nm i n aqueous buffer (pH 9.3) i n the presence and absence of glycinamide. Both the hydrolysis and aminolysis reactions were f i r s t order i n acyl enzyme. The second order rate constants for hydrolysis (k ) and aminolysis (k ) are given i n Table I, together with data for native Carlsberg s u b t i l i s i n and t h i o s u b t i l i s i n . x

2

Table I. Second Order Rate Constants for Hydrolysis (k ) and Aminolysis (k ) of Cinnamoylated S u b t i l i s i n s at 25.0 C and pH 9.3 x

2

e

Cinnamoyl derivatives of Subtilisin 1

s )

1

s )

k

x

(M"

k

2

(M"

k Ai 2

_1

_1

4.2

χ 10

-3

Thiosubtilisin 5

2.1 χ 10"

0.13

0.20

30

9,200

Selenosubtilisin 1.8 χ

5

10"

0.35 21,000

Although S-cinnamoyl-thiosubtilisin and Se-cinnamoyl-seleno­ s u b t i l i s i n are cleaved by water at comparable rates, t h e i r hydrolysis i s slower than that of native 0-cinnamoyl-subtililsin by more than two orders of magnitude. The decrease i n hydrolytic rate constant may r e f l e c t subtle s t r u c t u r a l changes within the relevant active s i t e geometries: the van der Waals r a d i i of s u l f u r and selenium are s i m i l a r (1.85 and 2.0 À, respectively) but that of oxygen i s much smaller

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

2.

HILVERT

17

Design of Enzymatic Catalysts

(1.45 Â) (10). What i s p a r t i c u l a r l y s t r i k i n g about the data summarized i n Table I, though, i s the fact that the r a t i o of rate constants for aminolysis and hydrolysis of the acyl enzyme species (k Ai) increases s u b s t a n t i a l l y as one proceeds from s u b t i l i s i n to s e l e n o s u b t i l i s i n . This r a t i o i s less than 30 for the native enzyme, while for s e l e n o s u b t i l i s i n aminolysis i s favored over hydrolysis by a factor of 21,000. Thus, by converting the active s i t e nucleophile of a serine protease into a selenocysteine the s e l e c t i v i t y of the enzyme can be altered i n a dramatic fashion. The enhanced s e l e c t i v i t y of deacylation suggests that semisynthetic s e l e n o s u b t i l i s i n , l i k e t h i o s u b t i l i s i n (11), may be a useful c a t a l y s t f o r peptide bond formation, for example i n fragment condensations. We are currently evaluating t h i s p o s s i b i l i t y , as well as studying the a b i l i t y of the modified protein to p a r t i c i p a t e i n other h y d r o l y t i c and redox processes.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch002

2

Catalytic

Antibodies

Ideally, one would l i k e to be able to generate a unique active s i t e for any chemical transformation. However, the number of s t r u c t u r a l l y well-characterized proteins for construction of semisynthetic enzymes i s l i m i t e d . An alternative approach exploits the mammalian immune system to produce binding pockets that are s p e c i f i c a l l y t a i l o r e d to the reaction of i n t e r e s t . The immune system i s a p r o l i f i c source of s p e c i f i c receptor molecules c a l l e d antibodies (12-14). They are large, dimeric proteins (Mr 160,000), consisting of two heavy and two l i g h t chains. Their normal function i n the body i s to recognize and t i g h t l y bind foreign materials, targeting them for eventual elimination. S i g n i f i c a n t l y , immunoglobulins can be e l i c i t e d against v i r t u a l l y any material, manmade or natural. The d i s s o c i a t i o n constants for t y p i c a l antigenantibody complexes are i n the range of 10"* to 10~ M (12-14), and binding apparently involves the same factors that are important for ligand binding to enzymes: hydrophobic interactions, ion p a i r i n g and dipolar interactions, and hydrogen bonding (13). Moreover, s t r u c t u r a l studies reveal that the size and shapes of the binding pockets of enzymes and antibodies are s i m i l a r (12). Nevertheless, immunoglobul i n s do not t y p i c a l l y catalyze reactions. Antibodies, according to Pauling (15), leave the molecules they bind chemically unchanged, because t h e i r combining s i t e s are complementary to the ground state of the antigen. An enzyme's active s i t e , on the other hand, must be complementary to the ephemeral, high energy t r a n s i t i o n state of the reaction i t catalyzes. In p r i n c i p l e , i t should be possible to prepare antibodies with c a t a l y t i c a c t i v i t y by challenging the immune system with compounds that resemble the high energy t r a n s i t i o n state of selected chemical reactions (16). This notion was recently reduced to practice. Phosphonates and phosphonamidates have been studied for some time as i n h i b i t o r s of hydrolytic enzymes (12). The tetrahedral phosphorus i s apparently an excellent mimic of the t r a n s i t i o n state geometry expected for ester and amide hydrolysis, and antibodies e l i c i t e d against a r y l phosphonate esters catalyze the cleavage of s t r u c t u r a l l y analogous esters (18.19) and carbonates (£0). Ester hydrolysis i s u n l i k e l y to be the optimal process for uncovering enzyme-like behavior i n 12

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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BIOCATALYSIS IN AGRICULTURAL

BIOTECHNOLOGY

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antibodies, however. This i s because there presumably exists a low p r o b a b i l i t y f o r generating an e f f e c t i v e c o n s t e l l a t i o n of c a t a l y t i c groups (eg. general acids, general bases and nucleophiles) i n the antibody combining s i t e during immunization. Consequently, we have targeted concerted reactions that do not require chemical c a t a l y s i s . Such reactions, including Claisen rearrangements and Diels-Alder c y c l i z a t i o n s , are expected to be especially sensitive to the p r i n c i p a l c a t a l y t i c e f f e c t s antibodies are l i k e l y to impart: induced s t r a i n and proximity (21). Moreover, these transformations are of enormous p r a c t i c a l and theoretical interest, especially f o r the synthesis of b i o l o g i c a l l y active molecules (£2)· Chorismate Mutase Antibodies. An example of a b i o l o g i c a l l y important Claisen rearrangement i s the conversion of (-)-chorismate 1 to prephenate 2, shown i n Scheme 2. This reaction occurs at a branch point i n the biosynthesis of aromatic amino acids i n bacteria, fungi and higher plants (23). The enzyme chorismate mutase [EC 5.4.99.5] catalyzes the rearrangement of chorismate to prephenate by a factor of two m i l l i o n over background (24) . Although the mechanism of action of the enzyme i s s t i l l controversial (25.26), i n h i b i t o r studies (27) and elegant stereochemical experiments (28) have implicated a d i a x i a l c h a i r - l i k e geometry f o r the t r a n s i t i o n state (Scheme 2). The oxabicyclic compound 3 was designed by Barlett and co-workers (29) to mimic the putative t r a n s i t i o n state structure. I t i s currently the best known i n h i b i t o r of chorismate mutase, binding 125 times more t i g h t l y to the enzyme than does chorismate i t s e l f . We have used t h i s material to prepare antibodies with chorismate mutase a c t i v i t y (30).

OH

Scheme 2.

OH

Rearrangement of Chorismate to Prephenate

We synthesized and i t s exo epimer 4, according to published procedures (29) . Since small molecules are generally not immunogenic, these compounds were linked i n d i v i d u a l l y to a c a r r i e r protein, keyhole limpet hemocyanin (KLH), with a g l u t a r i c acid l i n k e r . The r e s u l t i n g protein conjugates, containing approximately 15 to 20 molecules of 2 or 4 per KLH molecule, were used to immunize mice. Forty-five H

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d i f f e r e n t hybridomas which secreted monoclonal IgG antibodies s p e c i f i c for the t r a n s i t i o n state analog 3. were obtained using standard techniques (31.32). Antibodies were propagated i n ascites and p u r i f i e d by a f f i n i t y chromatography on immobilized Protein A (32) followed by FPLC ion-exchange chromatography on a Mono Q HR 10/10 column (33). The r e s u l t i n g antibodies were nearly homogeneous as judged by SDS PAGE (34) with Coomassie blue staining (Figure 1). Two of the p u r i f i e d monoclonals (1F7 and 27G5) were shown to catalyze the rearrangement of chorismate to prephenate to a s i g n i f i c a n t extent (30. H i l v e r t et a l . , unpublished r e s u l t s ) . An antibody with very high chorismate mutase a c t i v i t y has also been generated independently using immunogen 3 by B a r t l e t t , Schultz and t h e i r coworkers (35). Kinetics. Disappearance of chorismate was monitored spectroscopically. The catalyzed reaction was f i r s t order i n antibody, and formation of prephenate was v e r i f i e d by a standard assay (3j>). Furthermore, at high antibody concentrations the rate of chorismate disappearance equaled the rate of prephenate formation. At high substrate concentrations saturation k i n e t i c s were observed which suggests that c a t a l y s i s involves formation of a Michaelis-type complex. The k i n e t i c parameters determined f o r 1F7 at 14 °C were: cat " 0.025 min" and; ΐς, - 22 μΜ (30). Under these conditions the rate acceleration i n the presence of the antibody i s roughly 250-fold over background ( k a t / k u n c a t ) . The fact that the t r a n s i t i o n state analog 3 i s a competitive i n h i b i t o r (K i s ca. 0.6 μΜ) indicates that rearrangement i s occurring i n the induced binding pocket of the antibody and that binding interactions contribute to t r a n s i t i o n state stabilization. The k i n e t i c p r o f i l e of 27G5 was s i m i l a r to that of 1F7 (Hilvert et a l . , unpublished r e s u l t s ) . Hence, any s t r u c t u r a l differences between the two immunoglobulins are l i k e l y to be distant from the active s i t e . Values f o r the enthalpy and entropy of a c t i v a t i o n f o r the reaction catalyzed by 1F7 were determined from the temperature dependence of k . Apparently, the observed rate acceleration i s due e n t i r e l y to a lowering of the enthalpic b a r r i e r (15 kcal/mol versus 21 kcal/mol f o r the uncatalyzed reaction (24)), consistent with the notion that induced s t r a i n might be an important component of catalysis. The entropy of a c t i v a t i o n f o r the antibody-promoted reaction (-22 eu) i s actually less favorable than f o r the spontaneous reaction (-13 eu) (£3). This fact may r e f l e c t the need f o r some conformational change i n the antibody binding pocket during c a t a l y s i s . However, possible solvent effects make the interpretation of AS* difficult. The promoted rearrangement of chorismate i s notable as the f i r s t example of an antibody-catalyzed carbon-carbon bond forming reaction. The Claisen rearrangement i s , moreover, a prototype of a broad and important class of concerted chemical reactions. I t now seems a l l the more l i k e l y that t h i s strategy for catalyst design can be extended to related transformations, including additional sigmatropic rear­ rangements and bimolecular Diels-Alder cycloadditions. Bimolecular reactions are p a r t i c u l a r l y a t t r a c t i v e candidates f o r study, since the entropie advantage to be gained by binding two reactants together i n an active s i t e can approach ΙΟ M i n rate for 1 M standard states 1

k

C

A

cat

8

(21).

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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Figure 1. SDS-PAGE (34) of p u r i f i e d monoclonal antibody 1F7. Lane 1, protein r e l a t i v e molecular mass markers; lane 3, reduced 1F7; lane 5, non-reduced 1F7; lanes 2 and 4 are blank. The g e l was stained with Coomassie b r i l l i a n t blue.

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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S t e r e o s p e c i f i c i t y . Rate accelerations are only one aspect of enzyme catalyzed reactions. More important f o r p r a c t i c a l applications are the exacting regio- and s t e r e o s e l e c t i v i t y displayed by biocatalysts. Since antibodies are c h i r a l molecules, they might be expected to exert considerable control over reactions they promote. In fact, an antibody-catalyzed lactonization reaction was recently reported to be stereospecific (12). Not surprisingly, experiments with racemic chorismate establish that the antibodies with chorismate mutase a c t i v i t y also exhibit high enantioselectivity (17). Under conditions i n which a l l of (-)-chorismate rearranges, only h a l f of the racemic substrate i s converted to prephenate by 1F7 (37). The k value f o r (±)-chorismate i s the same as that measured f o r the pure (-)-isomer, but i t s apparent K,,, i s twice larger. Because the k value determined for the racemate i s unchanged r e l a t i v e to the o p t i c a l l y pure material, (+)-chorismate can be treated as a competit i v e i n h i b i t o r . From our data, the term 0.51^/^ must be much less than 1, i n d i c a t i n g that binding of the (+)-isomer to the antibody i s at least one or two orders of magnitude weaker than that of (-)chorismate. In order to provide a better estimate of the e n a n t i o s e l e c t i v i t y of the catalyst, we prepared an authentic sample of (+)-chorismate by k i n e t i c resolution of the racemate with 1F7 (37). Circular dichroism spectroscopy confirmed the i d e n t i t y and high o p t i c a l purity of the recovered, HPLC-purified compound. I n i t i a l rate measurements with the individual isomers show that (-)-chorismate i s favored over (+)-chorismate by the antibody by a factor of at l e a s t 90 to 1 at low substrate concentrations. The s l i g h t rate enhancements above background observed for the (+)-isomer may be due to general medium e f f e c t s rather than interaction with a s p e c i f i c locus on the antibody surface. To test this p o s s i b i l i t y we are currently examining the a b i l i t y of the t r a n s i t i o n state analog 3 to i n h i b i t rearrangement of t h i s o p t i c a l isomer. Since the immunizing antigen 2 i s racemic, c a t a l y t i c antibodies s p e c i f i c f o r rearrangement of both (-)- and (+)-chorismate might have been induced. Assays with racemic chorismate of the 45 monoclonals s p e c i f i c for 3, however, d i d not reveal any antibodies with reversed substrate preference. We are currently screening a second fusion for which the immunizing antigen was compound 4. More than 40 hybridomas that secrete antibody s p e c i f i c for t h i s compound have been i d e n t i f i e d (Hilvert, Carpenter and Auditor, unpublished r e s u l t s ) . Antibodies from t h i s fusion may also catalyze the rearrangement of chorismate to prephenate. Comparison of the s p e c i f i c a c t i v i t i e s , stereoselect i v i t i e s and structures of such molecules with the properties of 1F7 and 27G5 may lead to a better o v e r a l l understanding of the mutase reaction. c a t

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch002

c a t

Conclusions Binding interactions are essential f o r e f f i c i e n t b i o l o g i c a l catalys i s . The construction of enzyme-like molecules consequently requires the design of suitable binding s i t e s f o r organizing reactants and reducing the energy of the rate l i m i t i n g t r a n s i t i o n state. Although i t i s not yet possible to prepare protein binding pockets from their constituent amino acids, chemical p r i n c i p l e s can successfully inform

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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the redesign of e x i s t i n g structures as well as the manipulation of the immune system to provide t a i l o r e d active s i t e s . The success of semisynthetic s e l e n o s u b t i l i s i n and the chorismate mutase antibodies, described above, i l l u s t r a t e s the p o t e n t i a l of these complementary strategies to exploit binding interactions for highly s e l e c t i v e c a t a l y s i s . Continued study of these systems w i l l allow us to resolve important mechanistic issues and develop comprehensive structurefunction relationships. The tandem use of DNA-directed mutagenesis, random mutagenesis and genetic s e l e c t i o n may ultimately lead to the development of even more powerful c a t a l y t i c species. Exploration of t h i s e x c i t i n g f r o n t i e r i n molecular engineering w i l l bring us closer to our ultimate goal of designing enzymatic catalysts for v i r t u a l l y any chemical reaction. Acknowledgments This work was supported i n part by grants from the National Science Foundation (CHE-8615992), the National Institutes of Health (GM38273) and a Faculty Research Award from the American Cancer Society.

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1. Slama, J. T.; Radziejewski, C.; Oruganti, S. R.; Kaiser, Ε. T. J. Am. Chem. Soc. 1984, 106, 6778-6785. 2. Hilvert, D.; Hatanaka, Y.; Kaiser, E. T. J. Am. Chem. Soc. 1988, 110. 682-689. 3. Nicolaou, K. C.; Petasis, N. A. Selenium in Natural Products Synthesis: CIS, Inc.: Philadelphia, 1984. 4. Stadtman, T. C. Annual Rev. Biochem. 1980, 49, 93-110. 5. Polgár, L.; Bender, M. C., J. Am. Chem. Soc., 1966, 88, 31533154. 6. Neet, K. E.; Koshland, D. E. Proc. Natl. Acad. Sci. (USA) 1966, 56, 1606-1611. 7. Cavallini, D.; Graziani, M. T.; Dupre, S. Nature (London) 1966. 212. 294-295. 8. Polgár, L.; Bender, M. L. Biochemistry 1967, 6, 610-620. 9. Chu, S.-H.; Mautner, H. G. J. Org. Chem., 1966, 31, 308-312. 10. Pauling, L. The Nature of the Chemical Bond: Cornell University Press: Ithaca, New York, 1960; 3rd Ed., pp 246-60. 11. Nakatsuka, T.; Sasaki, T.; Kaiser, E. T. J. Am. Chem. Soc. 1987, 109, 3808-3809. 12. Kabat, E. A. Structural Concepts in Immunology and Immunochemistry: Holt, Reinhart and Winston: New York, 1976. 13. Pressman, D.; Grossberg, A. The Structural Basis of Antibody Specificity: Academic Press: New York, 1968. 14. Nisonoff, Α.; Hopper, J.; Spring, S. The Antibody Molecule: Academic Press: New York, 1975. 15. Pauling, L. Chem. Eng. News 1946, 24, 1375-1377. 16. Jencks, W. P. Catalysis in Chemistry and Enzymology: McGraw Hill: New York, 1969; p 288. 17. Bartlett, P. A. Bioact. Mol. 1987, 3, 429-440. 18. Tramontano, Α.; Janda, K. D.; Lerner, R. A. Science 1986, 234. 1566-1570.

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.

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Napper, A. D.; Benkovic, S. J.; Tramontane, Α.; Lerner, R. A. Science 1987, 237, 1041-1043. Jacobs, J. W.; Schultz, P. G.; Sugasawara, R.; Powell, M. J. Am. Chem. Soc. 1987, 109, 2174-2176. Jencks, W. P. Adv. Enzvmol. Relat. Areas Mol. Biol. 1975, 43, 219-410. March, J. AdvancedOrganicChemistry:Wiley: New York, 1985; pp 745-758, 1028-1032. Weiss, U.; Edwards, J. M. The Biosynthesis of Aromatic Amino Compounds; Wiley: New York, 1980; pp 134-184. Andrews, P. R.; Smith, G. D.; Young, I. G. Biochemistry 1973, 18, 3492-3498. Copley, S. D.; Knowles, J. R. J. Am. Chem. Soc. 1987, 109, 5008-5013. Gajewski, J. J.; Jurayj, J.; Kimbrough, D. R.; Gande, M. E.; Ganem, B.; Carpenter, Β. K. J. Am. Chem. Soc. 1987, 109. 11701186. Andrews, P. R.; Smith, G. D.; Young, I. G. Biochemistry 1977, 16, 4848-4852. Sogo, S. G.; Widlanski, T. S.; Hoare, J. H.; Grimshaw, C. E.; Berchthold, G. Α.; Knowles, J. R. J. Am. Chem. Soc. 1984, 106, 2701-2703. Bartlett, P. Α.; Johnson, C. R. J. Am. Chem. Soc. 1985, 107, 7792-7793. Hilvert, D.; Carpenter, S. H.; Nared, K. D.; Auditor, M.-T.M. Proc. Natl. Acad. Sci. (USA) 1988, 85, 4953-4955. Köhler, G.; Milstein, C. Nature (London) 1975, 256, 495-497. Goding, J. W. Monoclonal Antibodies: Principles and Practice: Academic Press: New York, 1983; pp 234-255. Clezardin, P.; McGregor, J. L.; Manach, M.; Boukerche, H.; Dechavanne, M. J. Chromatogr. 1985, 219, 67-77. Laemmli, V. Nature (London) 1970,227,680-685. Jackson, D. Y.; Jacobs, J. W.; Sugasawara, R.; Reich, S. H.; Bartlett, P. Α.; Schultz, P. G. J. Am. Chem. Soc. 1988, 110. 4841-4842. Görisch, H.; Lingens, F. Biochemistry 1974, 13, 3790-3794. Hilvert, D.; Nared, K. D. J. Am. Chem. Soc. 1988, 110, 55935594.

RECEIVED

October 26, 1988

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 3

Tailoring Enzyme Systems for Food Processing Joseph E. Spradlin

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch003

General Foods USA, White Plains,NY10625 Three illustrations are used to review the various approaches taken by the enzyme industry in tailoring enzyme preparations to meet the production and product quality needs of the food industry. Tailored enzyme preparations have been able to convert the corn syrup industry from an acid-based industry to an enzymebased industry, to overcome the problems created in the baking industry as grain technology improved and automation was introduced, and to rescue the cheese industry as the supply of bovine rennet decreased and the demand for cheese and cheese flavor increased. The use o f enzymes by t h e food i n d u s t r y as a p r o c e s s i n g t o o l i s i n c r e a s i n g r a p i d l y due t o t h e t a i l o r i n g o f enzyme p r e p a r a t i o n s t o f i t t h e p r o c e s s i n g needs o f the i n d u s t r y . The p o t e n t i a l o f u s i n g enzymes f o r c o n v e r t i n g a g r i c u l t u r a l p r o d u c t s i n t o food p r o d u c t s i s enormous, and has m i n i m a l l y been tapped as a method o f p r o d u c t i o n . To d a t e , the major modern a p p l i c a t i o n s coming a f t e r t h e a g e - o l d a p p l i c a t i o n s i n b a k i n g , brewing, and cheese have c e n t e r e d on the p r o d u c t i o n o f commodity i n g r e d i e n t s such as c o r n s y r u p s , whey u p g r a d i n g and h y d r o l y z e d p r o t e i n s . Today food p r o c e s s o r s a r e s t a r t i n g t o use enzymes as p r o c e s s i n g t o o l s t o improve the a t t r i b u t e s of t h e i r products. Appearance, t e x t u r e , f l a v o r , c o l o r , s t a b i l i t y , and n u t r i t i o n are a t t r i b u t e s b e i n g i n f l u e n c e d by a p p l y i n g t a i l o r e d enzyme p r e p a r a t i o n s d u r i n g t h e p r o c e s s o f c o n v e r t i n g a g r i c u l t u r a l products i n t o processed foods. Food p r o c e s s o r s a r e a l s o f i n d i n g s e v e r a l p r o c e s s i n g advantages i n u s i n g enzymes, such as i n c r e a s e d y i e l d s and improved p r o c e s s i n g parameters l i k e v i s c o s i t y , f i l t r a t i o n r a t e s , and ease o f h a n d l i n g ( 1 ) . Enzymes o f f e r s e v e r a l advantages t o t h e food p r o c e s s o r o v e r p h y s i c a l and c h e m i c a l methods o f p r o c e s s i n g . Enzymes work under v e r y m i l d c o n d i t i o n s o f pH and temperature w h i l e a c c o m p l i s h i n g the same r e a c t i o n t h a t o t h e r w i s e would r e q u i r e extreme pH and temperature c o n d i t i o n s . Consumers a r e becoming i n c r e a s i n g l y aware o f a r t i f i c i a l and p o t e n t i a l l y t o x i c c h e m i c a l s p r e s e n t i n f o o d s . The consumer, as w e l l as t h e government, i s a s k i n g f o r l e s s use o f chemical a d d i t i v e s t o c o n t r o l product a t t r i b u t e s . Enzymes a r e a l s o 0097-6156/89/0389-0024$06.00/0 © 1989 American Chemical Society

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v e r y s p e c i f i c and c a n s e l e c t o u t one s p e c i f i c r e a c t i o n , such as s t a r c h h y d r o l y s i s , i n a m i x t u r e o f s t a r c h , p r o t e i n , and f a t . Chemical and p h y s i c a l p r o c e s s i n g methods a r e not s e l e c t i v e and i n the s t a r c h , p r o t e i n , f a t m i x t u r e would h y d r o l y z e a l l t h r e e o f the polymers. Thus, by p e r f o r m i n g s p e c i f i c p r o c e s s i n g t a s k s under r e l a t i v e l y m i l d p r o c e s s i n g c o n d i t i o n s , enzymes c a n o f f e r s e l e c t e d chemical r e a c t i o n s i n a heterogeneous m i x t u r e , f r e e o f u n d e s i r a b l e side reactions. The f a c t o r s t o be c o n s i d e r e d i n t a i l o r i n g enzymes f o r the food i n d u s t r y can be d i v i d e d i n t o five areas: a) s e n s i t i v i t y t o p r o c e s s i n g c o n d i t i o n s ; b) c a t a l y t i c s p e c i f i c i t y o r a c t i o n p r o f i l e ; c) p u r i t y ; d) s o u r c e ; and e) a p p l i c a t i o n economics. S e n s i t i v i t y to Processing Conditions. I n most food a p p l i c a t i o n s , the environment i n which an enzyme must work has v e r y narrow limits. The c o m p o s i t i o n o f the raw m a t e r i a l s and the f i n a l p r o d u c t o f t e n d i c t a t e the pH, s a l t c o m p o s i t i o n , and i n h e r e n t enzyme inhibitor level. The temperature ranges a l l o w a b l e a r e o f t e n c o n t r o l l e d by the temperatures r e q u i r e d t o o b t a i n the d e s i r e d p r o d u c t a t t r i b u t e s from temperature-dependent p h y s i c a l and c h e m i c a l changes i n the raw m a t e r i a l s such as s t a r c h g e l a t i n i z a t i o n and heat induced p r o t e i n changes. T h i s temperature range r e s t r i c t i o n i s a l s o c o n t r o l l e d by temperature above o r below which u n d e s i r a b l e p r o d u c t a t t r i b u t e s develop. I t i s o f t e n d e s i r a b l e t o be a b l e t o c o n t r o l the a c t i v i t y o f an enzyme d u r i n g p r o c e s s i n g by a l t e r i n g t h e c o n d i t i o n s o f i t s environment such as pH o r temperature. Catalytic Specificity. The c a t a l y t i c s p e c i f i c i t y o r a c t i o n p r o f i l e o f an enzyme i s the t a i l o r i n g f a c t o r t h a t o f f e r s u n l i m i t e d potential. Being a b l e t o s e l e c t j u s t the e x a c t bond t o c l e a v e i n a polymer, o r t o c o n t r o l w i t h i n a v e r y narrow range the s i z e d i s t r i b u t i o n i n a polymer d i g e s t , o f f e r s enormous p r o d u c t q u a l i t y potential. Purity. The i s s u e o f o b t a i n i n g p u r i t y e c o n o m i c a l l y i s a challenging t a i l o r i n g factor. P u r i t y , i n an i n d u s t r i a l a p p l i c a t i o n sense, i s d e f i n e d by the absence o f s i d e r e a c t i o n s t h a t would cause n e g a t i v e impacts on one o r more p r o d u c t a t t r i b u t e s . U n l i k e most i n g r e d i e n t s used by the food p r o c e s s o r , enzymes a r e s o l d by t h e i r a c t i v i t y r a t h e r than t h e i r w e i g h t . T h e r e f o r e , the a c t i v i t y p e r u n i t weight o f the enzyme p r o d u c t i s o f prime importance. The enzymes used i n i n d u s t r y a r e r a r e l y c r y s t a l l i n e , c h e m i c a l l y pure, o r even single protein preparations. These i m p u r i t i e s may not i n t e r f e r e w i t h enzyme a c t i v i t y but enzyme i n p u r i t i e s c a n c a t a l y z e the formation of side products, present a t o x i c o l o g i c a l hazard, o r d e c r e a s e a d e s i r a b l e a t t r i b u t e o f t h e f i n a l food p r o d u c t such as c o l o r , t a s t e , o r t e x t u r e . What i s b e i n g added t o t h e food as p a r t of the enzyme p r e p a r a t i o n has t o be e v a l u a t e d as t o i t s impact on f i n a l product a t t r i b u t e s . In most a p p l i c a t i o n s the l e v e l o f enzyme used i s v e r y low and i s i n a c t i v a t e d d u r i n g the p r o c e s s i n g , a l l o w i n g the enzyme t o be regarded as a p r o c e s s i n g a i d . There a r e many p o t e n t i a l food a p p l i c a t i o n s f o r enzymes which r e q u i r e a c t i v i t y l e v e l s o r p r o c e s s i n g c o n d i t i o n s which would e i t h e r make the economics u n f a v o r a b l e o r would l e a v e u n d e s i r a b l e l e v e l s o f a c t i v e enzyme i n t h e f i n a l

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product. Immobilized enzymes are a way of overcoming these types of problems. Immobilized enzymes are usually completely recoverable from the reaction mixtures allowing them to be reused repeatedly without any contamination of the f i n a l product. Immobilized enzymes o f f e r the added advantage of eliminating the need to heat the product to inactivate the enzyme. In many applications heating cannot be done due to the temperature s e n s i t i v i t y of the product. Immobilization also allows the use of higher a c t i v i t y levels compared to using free enzyme, resulting i n a shortening of reaction times or reducing the size of the vessel needed to carry out the reaction. The absence of enzyme i n the f i n a l product i s often a major driving force i n using immobilized over soluble enzyme. Source. Enzymes for food applications come from a l l three kingdoms; plant, animal, and microbial. T r a d i t i o n a l l y used plant and animal enzymes are the plant proteases such as papain, f i c i n and bromelain, plant amylases from malt, and animal rennin which i s used i n cheese manufacture. Microbial c e l l s are the usual and most promising future source of i n d u s t r i a l enzymes. Estimates of the number of microorganisms i n the world tested as potential sources of enzymes f a l l around 2% with only about 25 organisms, including a dozen or so fungi, currently used for i n d u s t r i a l enzymes. Application Economics. The bottom line i s always the cost vs. benefit aspect of the process. The goal i s always to be the least cost producer of the highest quality product. Cost i s the most important decision-making aspect i n whether to put an enzyme-based process into production once the technical f e a s i b i l i t y has been demonstrated. The t a i l o r i n g of cost e f f e c t i v e enzyme preparations often becomes the challenge more than the t a i l o r i n g of c a t a l y t i c properties. Besides the direct cost of the enzyme product, enzymes need to be tailored to allow the processing of raw materials without adding long reaction times or costly unit operations such as material separation, cooling, heating, evaporation, or ion exchange, to the normal process flow. T a i l o r i n g Opportunities. There are many methods or approaches available to t a i l o r enzyme products. Early i n the history of enzyme companies, methods such as source selection, microbial s t r a i n selection, growth conditions, media, p u r i f i c a t i o n , and recovery systems, were primarily used to make each enzyme preparation unique. Later, immobilization, encapsulation, and chemical modification of the enzyme molecule i t s e l f were added as methods of t a i l o r i n g enzymes to better f i t i n d u s t r i a l applications. Today, a l l of these methods are s t i l l being used, and now we have added genetic engineering to our t a i l o r i n g expertises. Microbial enzymes o f f e r a much higher potential for being t a i l o r e d into cost e f f e c t i v e highly s p e c i f i c enzyme preparations for use i n food processing than do plant and animal enzymes. They o f f e r a great variety of c a t a l y t i c a c t i v i t i e s and ranges of environmental adaptability. They have the greatest potential for being produced in an inexpensive, regular and abundant supply. Microbial enzymes i n general are more stable than their corresponding plant and animal counterparts having similar a c t i v i t y . Microbial c e l l s also o f f e r an unlimited potential for genetic and environmental manipulation to

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increase y i e l d s , to produce altered enzymes, or to introduce new genes to produce t o t a l l y new enzymes. Several papers i n this symposium use site-directed mutagenesis as a means of t a i l o r i n g the properties of an enzyme. Today, recombinant DNA techniques are being used i n many laboratories to t a i l o r cost-effective enzyme products directed to the food industry. The technologies needed to synthesize a new gene for a new enzyme i n the laboratory, attach i t to an expression vector, and insert i t into a microorganism are available today. It i s only a matter of time before i t ' s done. Three food industries, corn syrup, baking, and dairy, w i l l be used to i l l u s t r a t e how enzyme preparations have been and are being tailored for use i n the food industry. Corn Syrup The corn syrup industry i s a good example of how enzymes took over and improved an industry after i t was established as a profitable and important industry. In the beginning, acid was used as the catalyst to hydrolyze starch into syrups i n order to impart new and desirable attributes t o food products. The process involved treating a 40%-45% solution of refined starch with hydrochloric acid at pH 1.5 and a temperature of 150°C for a few minutes. The acid was not selective and hydrolyzed the protein and fat present i n the starch preparation as well as the starch. This led to extensive undesired browning due to Maillard reactions. The syrup was neutralized, f i l t e r e d , carbon-treated, and ion-exchange treated to become a food product or an ingredient to be used by other food processors i n preparing products. The properties and subsequently the applications of the resulting corn syrup products, were dependent upon the extent of hydrolysis. The extent of hydrolysis i n corn syrup i s expressed i n dextrose equivalents (DE) which i s the percentage of glucosidic bonds hydrolyzed with native starch being 0 and pure glucose being 100. The DE of acid-catalyzed corn syrups depend only upon the time of reaction. A l l acid-catalyzed syrup at any single DE level w i l l always have the same dextrin p r o f i l e . Amylases. Acid hydrolyzed corn syrups were a great innovation f o r the food industry, but both the process and the product had their shortcomings. During the late 1950's, low DE corn syrups were gaining wide acceptance but had two major drawbacks. One was the development of a haze after concentration which limited i t s usefulness. The other was the high v i s c o s i t y of the hydrolyzates which made them d i f f i c u l t to process. The use of enzymes as a processing tool to improve the attributes of an existing food product was introduced to the industry. Up to that time enzymes were used only to enhance products that by their complex nature required enzyme reactions such as brewing and cheese production. In 1960, Corn Products Co. obtained a patent for using an alpha amylase to p a r t i a l l y hydrolyze the high molecular weight dextrins i n low DE acid hydrolyzed syrups without substantially increasing the DE (2). After concentration, the amylase-treated acid hydrolyzed syrups had a l l of the desirable c h a r a c t e r i s t i c s of low DE acid syrup but none of the undesirable features. During this same period of

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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time, h i g h DE a c i d h y d r o l y z e d syrups were b e i n g used as a d j u n c t s i n f e r m e n t a t i o n s but had a major problem of c r y s t a l l i z a t i o n due to the h i g h l e v e l of g l u c o s e . In 1962, Anheuser-Busch o b t a i n e d a p a t e n t d e s c r i b i n g the p r o d u c t i o n o f a h i g h l y f e r m e n t a b l e c o r n syrup i n which 80% to 90% o f the t o t a l c a r b o h y d r a t e i s f e r m e n t a b l e and t h e r e i s no u n d e s i r a b l e tendency toward c r y s t a l l i z a t i o n . T h e i r process i n v o l v e d a c i d c o n v e r s i o n o f s t a r c h to about DE 20 and then adding malt to i n c r e a s e the m a l t o s e l e v e l as h i g h as p o s s i b l e which r e s u l t e d i n a syrup o f about 55 DE. Next, a m i c r o b i a l amylase was added to f u r t h e r reduce the l o n g e r c h a i n d e x t r i n s to g l u c o s e t o g i v e a f i n a l DE i n the low 70's. T h i s was the b e g i n n i n g o f an i n d u s t r y t h a t now o f f e r s p r o d u c t s p r e p a r e d by c o n v e r t i n g s t a r c h i n a v a r i e t y o f ways: a c i d , acid-enzyme, enzyme, o r d u a l enzyme. Today t h e r e a r e two primary types o f enzyme produced h i g h m a l t o s e syrups a v a i l a b l e c o m m e r c i a l l y . One has a DE o f about 42 and c o n t a i n s about 40% m a l t o s e and o n l y about 8% g l u c o s e . This product i s w i d e l y used i n jams and c o n f e c t i o n e r i e s because i t i s r e s i s t a n t to c o l o r f o r m a t i o n , i s n o n - h y g r o s c o p i c and does not c r y s t a l l i z e as r e a d i l y as g l u c o s e s y r u p s . The o t h e r commercial h i g h m a l t o s e syrup has a DE o f about 62 and c o n t a i n s about 35% m a l t o s e and about 40% glucose. T h i s syrup i s h i g h l y f e r m e n t a b l e and s t a b l e to s t o r a g e ; t h e r e f o r e , i t i s used w i d e l y i n brewing and bread making. The p r o p e r t i e s o f low DE c o r n syrup and i t s e f f e c t when used i n a food system i s v e r y dependent upon i t s d e x t r i n p r o f i l e . By t a i l o r i n g the a c t i o n p a t t e r n o f the amylase, c o r n syrups o f unique and h i g h l y v a r y i n g d e x t r i n p r o f i l e s can be p r o d u c e d . Today t h i s i s an undeveloped a r e a . Knowledge i s c u r r e n t l y b e i n g g e n e r a t e d to understand how enzyme d i g e s t e d s t a r c h of c o n t r o l l e d d e x t r i n p r o f i l e can s e r v e as f a t m i m e t i c s , foam and g e l s t a b i l i z e r s and many o t h e r food a p p l i c a t i o n s p r e v i o u s l y f u l f i l l e d by c h e m i c a l a d d i t i v e s o r h i g h l e v e l s o f f a t . The t a i l o r i n g o f the a c t i o n p a t t e r n s of amylases w i l l be a major f i e l d i n the f u t u r e . The a c t i o n p a t t e r n of an amylase and the r e s u l t i n g c o m p o s i t i o n o f i t s d i g e s t are c o n t r o l l e d by the b i n d i n g e n e r g i e s o f the a c t i v e s i t e to the g l u c o s e r e s i d u e s i n the s t a r c h c h a i n . Each amylase has i t s own unique b i n d i n g energy p r o f i l e a c r o s s i t s a c t i v e s i t e . In a d d i t i o n , amylases d i f f e r i n the number o f g l u c o s e u n i t s t h a t w i l l f i t i n t o the a c t i v e s i t e . These two f a c t o r s w i l l c o n t r o l the d e x t r i n d i s t r i b u t i o n p r o f i l e of the r e s u l t i n g syrups. F o r example, the amylase from B a c i l l u s l i c h e n i f o r m i s produces m a l t o s e , m a l t o t r i o s e and m a l t o p e n t o s e as i t s main end p r o d u c t s w h i l e the amylase from B a c i l l u s a m y l o l i q u i f a c i e n s produces maltohexose as i t s major end p r o d u c t . The a c t i v e s i t e of the amylase from B a c i l l u s a m y l o l i q u i f a c i e n s has been e x t e n s i v e l y s t u d i e d t o the p o i n t t h a t i t i s known t h a t the a c t i v e s i t e i n t e r a c t s w i t h t e n g l u c o s e u n i t s a l o n g the s t a r c h c h a i n w i t h b i n d i n g o f the t e n t h g l u c o s e h a v i n g a l a r g e p o s i t i v e v a l u e t h a t h i n d e r s b i n d i n g o f s u b s t r a t e (_3). The c a t a l y t i c s i t e of the enzyme i s i n p r o x i m i t y o f the g l u c o s i d i c bond between g l u c o s e u n i t s s i x and seven, r e s u l t i n g i n the major p r o d u c t b e i n g maltohexose.Many amylases have the a b i l i t y to c l e a v e o f f m u l t i p l e p r o d u c t s from each s u c c e s s f u l enzyme/substrate e n c o u n t e r . This property allows f o r a c c u m u l a t i o n o f s m a l l o l i g o s a c c h a r i d e s i n the e a r l y phases o f amylase h y d r o l y s i s . The amylase has the a b i l i t y to c l e a v e a t the end o f a l a r g e s u b s t r a t e p r o d u c i n g s m a l l d e x t r i n s a f t e r the f i r s t random a t t a c k . A l t h o u g h the number o f r u p t u r e s per e f f e c t i v e

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e n c o u n t e r i s b a s i c a l l y a k i n e t i c phenomenon, the s i z e o f the p r o d u c t s r e l e a s e d w i l l p r o b a b l y be i n f l u e n c e d by monomer-subsite interactions. A f t e r the f i r s t c l e a v a g e , rearrangement o f the enzyme-substrate complex w i l l y i e l d a s e r i e s o f complexes whose r e l a t i v e c o n c e n t r a t i o n p a r t i a l l y depend upon the subsite-monomer i n t e r a c t i o n energies. The degree o f m u l t i p l e a t t a c k has been q u a n t i t a t e d f o r s e v e r a l alpha-amylases ( 4 ) . The h i g h e s t v a l u e i s f o r p o r c i n e p a n c r e a t i c a l p h a amylase w i t h s i x subsequent a t t a c k s p e r e f f e c t i v e e n c o u n t e r a t i t s o p t i m a l pH w h i l e A s p e r g i l l u s oryzae amylase undergoes two subsequent a t t a c k s p e r e f f e c t i v e e n c o u n t e r . I f p o r c i n e p a n c r e a t i c a l p h a amylase i s s t u d i e d a t pH 10, i t s subsequent number o f a t t a c k s drops t o 0.7. T h i s r e p e t i t i v e a t t a c k f e a t u r e i s one t h a t c o u l d be t a i l o r e d i n t o the a c t i o n p r o p e r t i e s o f l a b o r a t o r y d e s i g n e d amylases. In the f u t u r e , t a i l o r e d amylases w i l l be a b l e t o produce a range of p r o d u c t s o f f e r i n g a wide v a r i e t y o f u n i q u e p r o p e r t i e s t h a t c a n be used t o c o n t r o l the a t t r i b u t e s o f food p r o d u c t s . Heat S t a b l e A l p h a Amylase. One o f the key enzymes i n today's c o r n syrup i n d u s t r y i s a heat s t a b l e a l p h a amylase. I t i s now used i n the f i r s t s t e p i n p r o d u c i n g a l l v a r i e t i e s o f c o r n syrups such as low DE, h i g h m a l t o s e , h i g h g l u c o s e , and h i g h f r u c t o s e c o r n s y r u p s . In 1973, enzyme t a i l o r i n g i n t r o d u c e d the c o r n syrup b u s i n e s s t o an enzyme p r e p a r a t i o n t h a t was d e s i g n e d t o r e p l a c e the a c i d s t a r c h converters. The a l p h a amylase from B a c i l l u s l i c h e n i f o r m i s became a v a i l a b l e a t a c o s t e f f e c t i v e p r i c e . The enzyme had t o f u n c t i o n a t 105°C w i t h a h a l f - l i f e l o n g enough t o reduce the c h a i n l e n g t h o f the s t a r c h m o l e c u l e s t o a p o i n t t h a t would p r e v e n t p r e c i p i t a t i o n when the temperature dropped t o 95°C. One r e a s o n the a c i d c a t a l y z e d r e a c t i o n s were c a r r i e d out a t 150°C was due t o the need to open up the i n n e r s t r u c t u r e o f the s t a r c h g r a n u l e to make the bonds available for hydrolysis. A t lower t e m p e r a t u r e s , y i e l d was l o s t due to u n d i g e s t e d s t a r c h In the enzyme p r o c e s s u s i n g heat s t a b l e a l p h a amylase, the r e a c t i o n i s c a r r i e d out i n a j e t c o o k e r where m e c h a n i c a l s h e a r i s a p p l i e d as the temperature i s r a i s e d t o 105°C by i n j e c t i n g p r e s s u r i z e d steam. Under t h e s e c o n d i t i o n s , the enzyme c a t a l y z e s enough h y d r o l y s i s o f the i n n e r c o r e o f the s t a r c h g r a n u l e to p r e v e n t significant yield losses. I f the j e t c o o k i n g p r o c e s s i s r u n a t 95°C, where the enzyme i s much more s t a b l e , a s i g n i f i c a n t y i e l d l o s s i s obtained. A f t e r about f i v e minutes a t 105°C the s t a r c h s l u r r y i s f l a s h c o o l e d to 95°C and h e l d a t that temperature w h i l e the enzyme i n c r e a s e s the DE t o about t w e l v e . The temperature o f t h i s syrup c a n now be lowered t o 60°C o r l e s s and enzyme t r e a t e d i n a v a r i e t y o f ways t o produce a wide range o f c o r n syrup p r o d u c t s o f v a r y i n g p r o p e r t i e s and d e x t r i n d i s t r i b u t i o n s . Today, t h e r e a r e s e v e r a l heat s t a b l e a l p h a amylase p r o d u c t s on the market, many o f which were t a i l o r e d u s i n g m u t a t i o n and recombinant DNA t e c h n i q u e s . A major t a i l o r i n g t a r g e t has been improvements o f y i e l d d u r i n g enzyme p r o d u c t i o n i n o r d e r t o be more c o s t e f f e c t i v e i n the m a r k e t p l a c e . A l l o f the heat s t a b l e a l p h a amylases r e q u i r e c a l c i u m f o r s t a b i l i z a t i o n which must be added to the s t a r c h s l u r r y and then removed by i o n exchange l a t e r i n the process. The c a l c i u m r e q u i r e m e n t s v a r y w i d e l y . F o r example, the amylase from B. l i c h e n i f o r m i s r e q u i r e s o n l y 5 ppm c a l c i u m w h i l e the

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amylase from B. amyloliquifaciens requires 150 ppm calcium. A t a i l o r i n g need for the future would be a very stable non-calcium requiring amylase obtained i n high y i e l d during i t s production. Amyloglucosidases. Amyloglucosidases or glucoamylases as they are often c a l l e d , catalyze the stepwise hydrolysis of the alpha 1-4 links i n starch releasing beta glucose from the non-reducing end of the chain. Since glucoamylases can only bind at the end of each polymer chain i t s a c t i v i t y i s somewhat controlled by the size of the dextrin or starch molecules i n solution. Most glucoamylases w i l l also hydrolyze alpha 1-6 and alpha 1-3 bonds but do so at very slow rates. The major use of glucoamylases i n the corn starch industry today i s to digest to as near completion as possible the product resulting from the hydrolysis of starch with heat stable alpha amylase. The starting syrup i s usually at about 30% dry solids with a DE of about 12 to 15. Two major things must be done to the syrup as i t exits the heat stable amylase step. F i r s t , i t has to be cooled to 60°C because a heat stable glucoamylase has not yet been t a i l o r e d . Second, the pH must be adjusted to about pH 4.5. A major need of the industry i s a cost e f f e c t i v e heat stable glucoamylase that performs at about pH 6.5. This would eliminate the pH adjustment step as well as to allow both the alpha amylase and the glucoamylase reactions to proceed at the same time. Glucoamylases also require calcium ions for s t a b i l i t y but i n practice the calcium level i s adjusted before the alpha amylase step to s t a b i l i z e i t . The resulting product from glucoamylase treatment, a glucose syrup, usually has a DE of about 97 and a glucose content of 95% to 97% with 3-5% higher saccharides present. The glucose syrup i s then used i n several ways. I t can be used as a syrup or c r y s t a l l i z e d to give pure s o l i d glucose. Today a very large percentage of i t i s used as feed stock to produce high fructose corn syrup. In a l l cases, the higher the glucose level the better and each percent of carbohydrate not converted to glucose equates to m i l l i o n s of pounds of product lost annually for the industry. To push the reaction to as close to completion as possible, glucoamylase i s used batchwise for reaction periods of 48 to 92 hours. At pH 4.5 and 60°C the 30% syrups used for glucoamylase feed stocks are stable microbiologically since microorganisms w i l l not grow under these conditions. In practice, the maximum glucose level can be missed due to the thermodynamics of the system. In a 30% solution of glucose, there i s a small level of alpha 1-4, alpha 1-6, and alpha 1-3 disaccharides produced due to the systems thermodynamics. Since glucoamylase can catalyze a l l three of these reactions, we now have conditions that allow for the formation of alpha 1-6 and alpha 1-3 disaccharides. I f the reaction i s allowed to continue a glucose syrup reaching a level of 97% glucose would slowly decrease with time. Operating the glucoamylase reaction at a lower dry solids level w i l l greatly reduce the reversion reactions but unacceptable evaporation costs r e s u l t . Immobilization i s often suggested as a method of t a i l o r i n g enzymes for more e f f i c i e n t i n d u s t r i a l use. In the case of glucoamylase very superior properties would have to be obtained to warrant immobilizing the enzyme due to i t s r e l a t i v e l y low cost i n the soluble form. Some of the potential benefits would be l o g i s t i c

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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convenience, lower c a p i t a l and energy cost, reduction i n reaction time from 48 hours to less than one hour ,less color development during processing and lower in-process volume inventories. Glucoamylase has been successfully immobilized with h a l f - l i v e s of a month while operating under conditions that prevent microbial growth. Immobilized glucoamylase systems have a very high enzyme to substrate r a t i o . This causes a higher rate of formation of alpha 1,6 and 1,3 disaccharides leading to lower f i n a l glucose levels than in batch systems. A major problem during the 1970 s with commercial glucoamylase products was the presence of a contaminating enzyme which formed d i and trisaccharides with alpha 1-6 linkages. Thus, r e l a t i v e l y high levels of isomaltose and panose were observed which were much higher than predicted by recombination v i a glucoamylase reversion. T a i l o r i n g of preparations f i r s t by p u r i f i c a t i o n procedures and l a t e r by mutation and media control removed the contaminating transglucosidase from commercial products.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch003

1

Debranching Enzymes. One major source of y i e l d loss i n the production of high fructose corn syrup i s the loss due to incomplete conversion of starch to glucose. When a need exists with a s i g n i f i c a n t d o l l a r value associated with i t , someone w i l l develop a cost effective solution i f given enough time. There are enzymes whose primary reaction i s to catalyze the cleavage of the alpha 1-6 bonds of the amylopectin i n liquefied starch. These enzymes are referred to as debranching enzymes and include pullulanase and isoamylase. Enzyme companies are now t a i l o r i n g products for the primary purpose of obtaining another percent y i e l d of glucose during glucoamylase conversion of starch. One such product currently available commercially i s derived from Bacillus acidopullulyticus (5-6). The application conditions of this product are such that i t can be used at the same time as glucoamylase and results i n higher yields i n shorter time. Another company has taken a different approach to solving the glucose y i e l d problem (7-8). Its s c i e n t i s t s i d e n t i f i e d a new amylolytic enzyme from B a c i l l u s megaterium. The enzyme hydrolyzes alpha 1-4 bonds and does not d i r e c t l y hydrolyze alpha 1-6 bonds. The unique property of this enzyme i s i t s a b i l i t y , v i a a transfer reaction, to convert the glucoamylase resistant saccharides present in a syrup into forms easily hydrolyzed to glucose by the glucoamylase. This new amylolytic enzyme i s produced by B a c i l l u s megaterium as a trace contaminant i n a complex mixture of proteins which also include a large amount of beta amylase. By genetic engineering the B. megaterium gene for this enzyme was transferred into B. s u b t i l i s (9) resulting i n a 10,000-fold increase i n y i e l d and a product free of other amylases. Glucose Isomerase. The biggest success story i n the enzyme industry has to be glucose isomerase. The very f i r s t report of an enzyme that converts glucose to fructose was i n 1957 Q 0 ) . In less than 20 years the enzyme was studied, tailored into a cost e f f e c t i v e immobilized form and put into production to make a commodity corn syrup product. In less than 30 years from i t s f i r s t description, i t became the largest commercially used immobilized enzyme and responsible for making one of the world's major sweeteners. Since

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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glucose isomerase i s an i n t r a c e l l u l a r enzyme, stable above 60°C and a l l of i t s reactants are small molecules, glucose isomerase i s well suited for immobilization. Many approaches have been used to immobilize glucose isomerase using both whole c e l l s and c e l l free preparations. Commercial products of glucose isomerase are constantly being improved and today the enzyme cost per unit of fructose produced i s only a small f r a c t i o n of what i t was during the mid and late 1970's. In spite of the great commercial success of glucose isomerase, there i s s t i l l room for improvement. There are two major environmental problems that have been present since the beginning that s t i l l elude the researcher. A l l glucose isomerases require one or more heavy metal ions for a c t i v i t y and s t a b i l i t y . To make matters worse, calcium ions i n h i b i t the a c t i v i t y . In practice this means that calcium required for s t a b i l i z a t i o n of the alpha amylase and the glucoamylase must be removed by ion exchange before the magnesium ions, which are needed for glucose isomerase a c t i v i t y , can be added to the syrup. Of course, after isomerization the magnesium ions also have to be removed by ion exchange. Thus i n the production of high fructose corn syrup the syrup must be treated with ion exchangers twice. An enzyme not inhibited by calcium or not requiring a metal ion would find a ready market. The other big environmental problem i s pH. A l l currently available glucose isomerase products have to be used at a pH above 7.0 for cost e f f e c t i v e operation. Color formation and other alkaline catalyzed reactions increase refining cost and cause a loss of y i e l d . An enzyme capable of prolonged operation at a pH below 7 i s needed. Baking Enzymes play a very c r i t i c a l role in the baking industry. Traditional methods of baking are based on the presence of enzymes in the flour and i n the microorganisms used to ferment the dough. The endogenous alpha amylase hydrolyzes the low level of starch made available by the rupture of starch granules during the m i l l i n g process to dextrins. These dextrins become the substrate for the flour's endogenous beta amylase which produces maltose. The yeast i n the dough then uses i t s alpha glucosidase to convert the maltose to glucose, which the yeast can now use to produce carbon dioxide via i t s g l y c o l y t i c pathway. But this i s just the gas formation aspect of baking. Wheat flour also contains low levels of proteases, pentosanases, isoamylases, lipoxygenases, lipases, and esterases i n addition to the two types of amylases. A l l of these enzymes play a role i n the quality of the baked goods produced. Therefore, the enzyme content of flour i s of major importance i n the baking industry. Flour Amylases. Beta amylase i s produced i n wheat during the ripening stage and i s stored i n the aleurone layer u n t i l needed during germination and therefore i s present i n ungerminated grain. Plant alpha amylase, i n contrast, i s not present i n ungerminated grain to any s i g n i f i c a n t extent but i s synthesized de novo upon germination (11). Prior to 1920, the growing and harvesting of wheat were poorly controlled. F i e l d sprouting was routine and produced varying levels

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch003

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of a l p h a amylase i n the r e s u l t i n g f l o u r . With the i n t r o d u c t i o n o f m e c h a n i c a l h a r v e s t i n g of wheat i n the 1920's, f i e l d s p r o u t i n g became a rare occurrence. The l e v e l of a l p h a amylase i n f l o u r , produced from wheat t h a t has been c a r e f u l l y and e x p e r t l y grown, h a r v e s t e d , and m i l l e d , w i l l be too low t o produce a c c e p t a b l e b r e a d by the t r a d i t i o n a l system of dough p r e p a r a t i o n . The h i s t o r y of the use o f exogenous enzymes i n the food i n d u s t r y has n e a r l y p a r a l l e l e d the h i s t o r y o f the development of c e r e a l t e c h n o l o g y . The b e n e f i t s o f a d d i n g malt d i a s t a s e (amylase) t o bread to i n c r e a s e volume and q u a l i t y were known i n the 1800's. The p r a c t i c e o f adding malt to f l o u r became an e a r l y a r t i n the m i l l i n g and b a k i n g i n d u s t r y . I f the a l p h a amylase l e v e l was low, poor f e r m e n t a t i o n s were o b t a i n e d . I f the a l p h a amylase l e v e l was h i g h , the r e s u l t i n g bread had a s t i c k y , m o i s t , i n e l a s t i c crumb s t r u c t u r e . With p r o p e r a l p h a amylase s u p p l e m e n t a t i o n o f f l o u r , dough v i s c o s i t y i s reduced and the r a t e o f f e r m e n t a t i o n i n c r e a s e d r e s u l t i n g i n a p r o d u c t of improved volume and t e x t u r e . As bread making became more and more automated, r e p l a c i n g the bakers d e c i s i o n s on c r i t i c a l p r o c e s s i n g end p o i n t s w i t h p r e s e t p r o c e s s i n g parameters, the amylase l e v e l i n the f l o u r became a c r i t i c a l f a c t o r i n the i n d u s t r y . R e s p o n s i b i l i t y f o r the amylase l e v e l f e l l t o the m i l l e r t o s u p p l y f l o u r t h a t would perform t o the baker's s t a n d a r d s . The m i l l e r o r i g i n a l l y added the a l p h a amylase i n the form of m a l t . But i n 1884, Takamine d i s c o v e r e d t h a t the fungus A s p e r g i l l u s o r y z a e c o u l d be grown on wheat b r a n by s o l i d s t a t e f e r m e n t a t i o n t o produce a h i g h l e v e l o f a l p h a amylase. T h i s enzyme would prove to be an i d e a l amylase f o r supplementing f l o u r . Today i t i s the major method used by m i l l e r s to s t a n d a r d i z e f l o u r amylase activity. M a l t supplemented f l o u r i s d a r k e r than f u n g a l amylase supplemented f l o u r . T h i s d a r k e r c o l o r a d v e r s e l y a f f e c t s the c o l o r of the b r e a d . M a l t a l s o c o n t a i n s a r e l a t i v e l y h i g h l e v e l of p r o t e o l y t i c a c t i v i t y which can r e s u l t i n a r e d u c t i o n o f g l u t e n q u a l i t y i n low p r o t e i n f l o u r s . A. o r y z a e amylase p r o d u c t s c o n t a i n a n e g l i g i b l e protease content. Another major d i f f e r e n c e between malt a l p h a amylase and f u n g a l a l p h a amylase i s i n t h e i r t h e r m o s t a b i l i t y . M a l t amylase i s more s t a b l e and i f too much i s added can l e a d t o the formation of s t i c k y bread. The f u n g a l amylase i s i n a c t i v a t e d a t about 60°C b e f o r e a p p r e c i a b l e amounts o f s t a r c h a r e g e l a t i n i z e d , thus making the c o n t r o l o f the r e a c t i o n e a s i e r . A. o r y z a e amylase can be added to low a l p h a amylase f l o u r w i t h r e l a t i v e s a f e t y t o o b t a i n an i n c r e a s e d l o a f volume and l e s s f i r m crumb w i t h l i t t l e danger o f o v e r t r e a t m e n t . Flour Proteases. In the b a k i n g p r o c e s s , the q u a l i t y and q u a n t i t y of g l u t e n p r o t e i n determines the r h e o l o g i c a l c h a r a c t e r i s t i c s o f the dough. Wheat f l o u r c o n t a i n s 7% to 15% p r o t e i n w i t h about 80% b e i n g gluten. Ungerminated f l o u r c o n t a i n s low l e v e l s o f p r o t e a s e which p l a y a minor r o l e i n bread making. S i n c e the 1920's, p r o t e o l y t i c enzymes have been used as a d d i t i v e s i n b a k i n g t o improve dough h a n d l i n g p r o p e r t i e s . At t h a t time, the main s o u r c e was m a l t and i t was o f t e n d i f f i c u l t t o c o n t r o l the r a t i o o f a l p h a amylase and protease i n malt products. The a r t of t a i l o r i n g malt p r o d u c t s f o r the b a k i n g i n d u s t r y f o c u s e d p r i m a r i l y on the time and the c o n d i t i o n used f o r m a l t i n g . The use of p r o t e a s e s i n b a k i n g i n c r e a s e d s i g n i f i c a n t l y i n the 1950's a f t e r the i n t r o d u c t i o n o f f u n g a l

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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proteases from A. oryzae. Today the supplementation of flour made from hard, high protein wheat i s routinely practiced i n order to improve dough handling properties, to control mixing times, and to increase loaf volume. Without protease addition, high gluten doughs produce small loaves with b l i s t e r e d or cracked crusts. The level of protease a c t i v i t y i n a flour i s p a r t i c u l a r l y important today due to the use of automated baking operations that require s t r i c t control of mixing time. The controlled use of proteolytic enzymes can reduce the dough mixing time by 30% without adverse effect on dough properties and on bread quality. Excess proteolytic a c t i v i t y w i l l cause slack, sticky doughs and poor quality bread. Today the baking industry uses protease products tailored for their use from fungal proteases. Fungal proteases are less stable than malt proteases and are inactivated at a lower temperature. This feature allows for r e l a t i v e l y good tolerance to overusage. Fungal protease products contain both endopeptidase and exopeptidase a c t i v i t i e s . The endopeptidases affect the v i s c o e l a s t i c properties of gluten. The exopeptidases produce free amino acids which improve both bread flavor and crust color through increased Maillard reactions during baking. Bread Staling. Staling i n the baking industry i s a problem begging to be solved and the enzyme industry i s starting to t a i l o r products directed toward solving the problem. Staling of bakery products i s a general term used to cover a large number of changes that occur during the normal storage of bakery products. For the consumer, the staleness of a bakery product i s the sum of several perceptive judgements about the changes i n several product attributes. The major attributes used by a consumer to judge staleness are loss of perceived moisture and the firming or texture deterioration of the crumb. Up to very recently the dogma regarding the mechanism of s t a l i n g has focused on the rétrogradation of starch as the primary cause. The change i n the c r y s t a l l i n e structure of starch over time does play a role i n the s t a l i n g processes. Current theories on staling are focusing more on the level of free water i n the product with starch rétrogradation being one of the major mechanisms through which water becomes bound during s t a l i n g . It may not be the presence of retrograded starch that causes the firmness but the reduction of free water content below a c r i t i c a l l e v e l . Soon after the practice of supplementing flour with alpha amylase started, observations were made that the higher the amylase l e v e l , the longer the shelf l i f e . But, as discussed e a r l i e r , excess alpha amylase a c t i v i t y can lead to a gummy bread texture, d i f f i c u l t to s l i c e and unacceptable to the consumer. In 1953, a comprehensive study (12^) was reported i n which fungal, cereal and b a c t e r i a l alpha amylases were investigated at equivalent levels for their a b i l i t y to reduce firmness development. A l l three amylase products retarded firmness development and t h e i r effectiveness to do so was p o s i t i v e l y correlated to their heat s t a b i l i t i e s . The b a c t e r i a l amylase, having the highest heat s t a b i l i t y , produced the biggest effect while the fungal amylase showed the least having been inactivated before the starch was gelatinized. Theories were proposed that the amylase produced dextrins that interfered with the c r y s t a l l i z a t i o n of starch. However, i n 1959, based on X-ray studies of bread (13), i t was found that b a c t e r i a l alpha amylase treatment increased rather

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch003

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than decreased starch c r y s t a l l i z a t i o n . In 1980, another X-ray study (14) looked at the starch c r y s t a l l i z a t i o n pattern i n control bread and breads that had been treated with alpha amylases from malt, fungal and b a c t e r i a l sources. The basic findings agreed with the 1959 study i n that starch c r y s t a l l i n i t y and bread firming were not synonymous and that c r y s t a l l i z a t i o n increased upon amylase treatment. The news i n 1980 was that the type of crystals found i n amylase treated bread were different than the type of crystals found in the control bread. Previous to this 1980 study, i t was known that starches found i n nature are c r y s t a l l i n e and that they exist i n two forms, A and B. The A form contains less than 7% water i n i t s c r y s t a l l i n e structure while the Β form contains over 25% water i n i t s structure (L5). In the study, the control bread formed Β type crystals while the amylase treated bread formed A type c r y s t a l s . Due to the potential of producing gummy bread due to excess alpha amylase a c t i v i t y , the baking industry never adopted for regular use any of the commercially available amylase products for the primary purpose of freshness extension. Recently the enzyme industry set out to t a i l o r products for the baking industry that would decrease the rate of firming i n baked goods without the r i s k of producing a gummy texture. During the last two years, at least four enzyme companies have developed special products and are promoting them for this application. The two major challenges were to develop an enzyme product that would a) allow bread to r e t a i n i t s moisture as free water for as long as possible and b) would have a b u i l t - i n application tolerance l e v e l that would allow wide v a r i a t i o n i n use level without causing production or product problems. The approach one company i s employing i s to use a debranching enzyme along with a low level of b a c t e r i a l alpha amylase (16). They had developed a debranching enzyme from the bacterium Bacillus acidopullulyticus for use i n the corn syrup industry. By blending their debranching enzyme with an alpha amylase, an enzyme preparation was obtained with the a b i l i t y to greatly reduce the formation of Β starch crystals while maintaining a "forgiveness factor" before product or production problems are experienced. A second company also focused i n on changing the structure of the starch. Their approach i s to prevent Β type starch c r y s t a l l i z a t i o n by rearranging the branched portions of the starch chain v i a a transfer reaction. They developed a debranching system for the corn syrup business by transferring a unique amylase gene from Bacillus megaterium into Bacillus s u b t i l i s . This unique amylase has the a b i l i t y to transfer branched dextrin to glucose. In their a n t i - s t a l i n g enzyme preparation they have a blend of the B. megaterium amylase and a glucoamylase. Their l i t e r a t u r e claims that their new product w i l l improve moulding properties, crust color, height, flavor, and rate of s t a l i n g . Another component of flour, the pentosans, have a very high capacity to bind water. Pentosans are present i n wheat to about 4-5% and i n patent wheat flour to about 2.5-3%. Pentosans contribute to about one quarter of the adsorption of dough water which i s disproportionately more than the starch or protein fractions. Pentosans can absorb about 6.5 times their weight of water. A s i g n i f i c a n t a n t i - s t a l i n g effect has been claimed by the addition of pentosanase to bread (17-18).

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An enzyme company has now i n t r o d u c e d i n t o the market an enzyme p r e p a r a t i o n produced by T r i c h o d e r m a r e e s e i t h a t c o n t a i n s a m i x t u r e of c e l l u l a s e , b e t a - g l u c a n a s e and p e n t o s a n a s e . The c l a i m i s t h a t t h i s new p r o d u c t i s t a i l o r made f o r the b a k i n g i n d u s t r y t o extend the s o f t n e s s l i f e o f b r e a d . A f o u r t h enzyme company has added c e l l u l a s e , h e m i c e l l u l a s e and pentosanase t o f u n g a l a l p h a amylase as t h e i r e n t r y i n t o the a n t i - s t a l i n g market. They c l a i m t h a t the added enzymes work s y n e r g i s t i c a l l y w i t h the amylase t o produce a p r o d u c t w i t h reduced tendency t o become f i r m e r w i t h time.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch003

Dairy The d a i r y i n d u s t r y i s v e r y h e a v i l y dependent on the enzyme industry. Without enzymes t h e r e would be no cheese i n d u s t r y . The p r i n c i p a l enzymes used i n d a i r y t e c h n o l o g y a r e p r o t e a s e s , l i p a s e s / e s t e r a s e s , and l a c t a s e s . Cheese p r o d u c t i o n i s the l a r g e s t consumer o f p r o t e a s e s and has been the t a r g e t i n d u s t r y f o r t a i l o r e d enzyme p r e p a r a t i o n s f o r many y e a r s . Besides t a i l o r i n g protease p r e p a r a t i o n s , the enzyme i n d u s t r y has d e v e l o p e d many l i p a s e / e s t e r a s e p r e p a r a t i o n s s p e c i f i c a l l y f o r the r i p e n i n g and f l a v o r development o f cheese p r o d u c t s . The whole c o n c e p t o f r a p i d r i p e n e d cheeses and cheese f l a v o r i n g p r o d u c t s i s based on the use o f t a i l o r e d enzyme preparations. The f l a v o r l e v e l s o f enzyme m o d i f i e d cheese (EMC) p a s t e s and powders a r e 5-100 times g r e a t e r than cheeses aged by conventional procedures. The p o t e n t i a l uses o f l a c t a s e t o overcome n u t r i t i o n a l and p r o d u c t a t t r i b u t e problems have l e d t o the t a i l o r i n g o f many l a c t a s e p r o d u c t s , s e v e r a l i n an i m m o b i l i z e d state. Milk Coagulation. The f i r s t s t e p i n cheese manufacture i s t h e coagulation of milk. T r a d i t i o n a l l y , t h i s c o a g u l a t i o n step i s c a t a l y z e d by the enzyme r e n n e t . Rennet i s a s a l i n e e x t r a c t o f the 4th stomach o f c a l v e s , u s u a l l y s l a u g h t e r e d b e f o r e they a r e 30 days old. The p r i n c i p a l p r o t e a s e i n r e n n e t i s r e n n i n . In an attempt t o a v o i d c o n f u s i o n w i t h the hormone p e p t i d e r e n i n , t h e I n t e r n a t i o n a l Enzyme Nomenclature Committee has a s s i g n e d t h e name chymosin t o t h e protease i n c a l f rennet. D u r i n g the growth o f c a l v e s , chymosin i s r e p l a c e d by p e p s i n , the a c i d p r o t e a s e o f the mature stomach. Chymosin i n i t i a t e s t h e c l o t t i n g o f m i l k by s e l e c t i v e l y c l e a v i n g a s i n g l e p h e n y l a l a n i n e - m e t h i o n i n e bond i n k a p p a - c a s e i n . Cleavage o f t h i s one bond causes the l o s s o f a p e p t i d e from the m i l k ' s micelles. T h i s p e p t i d e i s t h e p a r t o f the n a t i v e m i c e l l e t h a t m a i n t a i n s a n e t n e g a t i v e c h a r g e on the m i c e l l e ' s s u r f a c e c a u s i n g the m i c e l l e s t o r e p e l one a n o t h e r . Once t h i s p e p t i d e i s l o s t the m i c e l l a r s u r f a c e c h a r g e s ( z e t a p o t e n t i a l ) a r e reduced t o about half. The m i c e l l e s c a n now a s s o c i a t e t o form a network s t r u c t u r e o f protein. On e l e c t r o n m i c r o s c o p y e x a m i n a t i o n the i n d i v i d u a l m i c e l l e s can s t i l l be c l e a r l y d i s t i n g u i s h e d i n d i c a t i n g t h a t chymosin does not d i s r u p t the m i c e l l e s t r u c t u r e . Chymosin i s h i g h l y s p e c i f i c i n i t s action. By s p l i t t i n g a s i n g l e p e p t i d e bond i n kappa c a s e i n , which i s o n l y about 10% o f the t o t a l c a s e i n i n m i l k and even l e s s than t h a t o f t h e t o t a l p r o t e i n , chymosin can produce d r a m a t i c changes i n the p h y s i c a l p r o p e r t i e s o f m i l k . Chymosin i s an e x c e l l e n t example of t h e marked e f f e c t enzymes c a n have on t h e f u n c t i o n a l p r o p e r t i e s of f o o d .

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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U n f o r t u n a t e l y , cheese consumption i s c o n s t a n t l y i n c r e a s i n g and v e a l consumption i s c o n s t a n t l y d e c r e a s i n g . F o r s e v e r a l y e a r s now, t h e r e has been a major s h o r t a g e i n c h y m o s i n . Chymosin i s an a c i d protease. More s p e c i f i c a l l y i t i s an a s p a r t a t e p r o t e a s e . I t i s so named because i t b e l o n g s to a c l a s s of a c i d p r o t e a s e s t h a t have two a s p a r t i c a c i d r e s i d u e s i n v o l v e d i n the a c t i v e s i t e . The t a s k f o r enzyme m a n u f a c t u r e r s i s to d e v e l o p m i c r o b i a l a c i d p r o t e a s e p r e p a r a t i o n s t h a t come as c l o s e as p o s s i b l e to the c a t a l y t i c and s t a b i l i t y p r o p e r t i e s of c h y m o s i n . Nearly a l l a c i d proteases w i l l c a t a l y z e the r a p i d h y d r o l y s i s o f the key p h e n y l a l a n i n e - m e t h i o n i n e bond i n kappa c a s e i n . The problem i s t h a t o t h e r bonds are c l e a v e d and the r e l a t i v e r a t e s o f t h e i r h y d r o l y s i s i s h i g h compared to the r e l a t i v e r a t e f o r chymosin. S u c c e s s f u l m i l k c o a g u l a n t s have been produced from m i c r o b i a l s o u r c e s f o r use i n cheesemaking. The major organisms used to produce m i c r o b i a l rennet p r o d u c t s a r e Mucor p u s i l l u s , Mucor m e i h e i , and E n d o t h i a p a r a s i t i c a w i t h Mucor p u s i l l u s b e i n g the most w i d e l y u s e d . Mucor p u s i l l u s was the f i r s t m i c r o b i a l rennet i d e n t i f i e d t h a t s u c c e s s f u l l y produced a good q u a l i t y c h e e s e . In the 20 y e a r s s i n c e t h e i r f i r s t d i s c o v e r y , m i c r o b i a l r e n n e t s have been t a i l o r e d to improve t h e i r f u n c t i o n a l i t y i n cheese making to more c l o s e l y match the f u n c t i o n a l i t y of c h y m o s i n . M i c r o b i a l rennets i n g e n e r a l have the d i s a d v a n t a g e of b e i n g too s t a b l e . The organisms have been mutated and the enzymes c h e m i c a l l y m o d i f i e d to i n c r e a s e t h e i r s p e c i f i c i t y and to d e c r e a s e t h e i r s t a b i l i t y . In the p r o c e s s o f t a i l o r i n g t h e r m a l l y d e s t a b i l i z e d m i c r o b i a l r e n n e t s , an unexpected b e n e f i t was o b t a i n e d as an i n c r e a s e i n the s p e c i f i c i t y of the m i c r o b i a l enzyme making i t s performance v i r t u a l l y i n d i s t i n g u i s h a b l e from c a l f r e n n e t (20) . Of c o u r s e , the u l t i m a t e t a i l o r i n g t a s k s t i l l needed to be done. U s i n g recombinant DNA t e c h n i q u e s , the b o v i n e chymosin gene needed to be e x p r e s s e d i n a m i c r o o r g a n i s m . Commercial p r o d u c t i o n o f chymosin u s i n g recombinant DNA t e c h n i q u e s became the t a r g e t o f a number of biotechnology firms. At f i r s t the gene was i n s e r t e d i n t o b a c t e r i a and y e a s t c e l l s m a i n l y because the s t a t e of the a r t i n e x p r e s s i o n v e c t o r s was b e s t u n d e r s t o o d i n these o r g a n i s m s . The recombinant DNA p r o c e d u r e s worked g r e a t but the chymosin t h a t was formed was i n an i n a c t i v e form and r e q u i r e d a c o s t l y r e n a t u r a t i o n p r o c e s s to o b t a i n an a c t i v e enzyme. The problem was one t h a t i s common to a number o f complex p r o t e i n s whose c o n f o r m a t i o n i s determined i n p a r t by f o r m a t i o n o f one o r more d i s u l f i d e bonds. When t h e s e p r o t e i n s are produced i n a m i c r o o r g a n i s m , the p r o p e r d i s u l f i d e bonds o f t e n do not form and the p r o t e i n assumes an i n a c t i v e c o n f o r m a t i o n . One way around t h i s problem i s to produce the p r o t e i n i n an o r g a n i s m t h a t w i l l s e c r e t e i t i n i t s a c t i v e form. M i c r o b i a l r e n n e t s , c u r r e n t l y produced and used i n cheese p r o d u c t i o n , are a s p a r t y l p r o t e a s e s l i k e c h y m o s i n . T h e r e f o r e , i f the b o v i n e gene f o r chymosin c o u l d be t r a n s f e r r e d to and e x p r e s s e d i n the same type o f organism t h a t c u r r e n t l y produces an a s p a r t y l p r o t e a s e , then the p r o b a b i l i t y of s u c c e s s i s much higher. S i n c e a l l m i c r o b i a l r e n n e t s a r e produced by f i l a m e n t o u s f u n g i , i n v e s t i g a t o r s t u r n e d to f i l a m e n t o u s f u n g i (19) i n o r d e r to s u c c e s s f u l l y o b t a i n e x p r e s s i o n and s e c r e t i o n of b o v i n e c h y m o s i n . Today, a c t i v e b o v i n e chymosin has been s u c c e s s f u l l y produced from s e v e r a l f i l a m e n t o u s f u n g i and they are i n d i s t i n g u i s h a b l e from the n a t u r a l enzyme o b t a i n e d from c a l v e s ' stomachs.

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch003

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Cheese R i p e n i n g . Rennet p l a y s a major r o l e i n the t e x t u r e and f l a v o r development o f cheese d u r i n g the r i p e n i n g p r o c e s s . Besides the r a p i d c l e a v a g e of the key p h e n y l a l a n i n e - m e t h i o n i n e bond to c o a g u l a t e m i l k , chymosin has been shown t o h y d r o l y z e a t l e a s t 22 o t h e r bonds i n the c a s e i n m o l e c u l e s . The f a v o r e d amino a c i d s a t the p o i n t of c l e a v a g e are l e u c i n e , i s o l e u c i n e and p h e n y l a l a n i n e . Chymosin produces cheese f r e e o f b i t t e r f l a v o r and o f e x c e l l e n t texture. The use o f o t h e r a c i d p r o t e a s e s such as p e p s i n and many o f the m i c r o b i a l r e n n e t s i n the c o a g u l a t i o n s t e p o f t e n l e a d s to b i t t e r f l a v o r and a s o f t t e x t u r e a f t e r r i p e n i n g . In t r a d i t i o n a l cheese making, w h i l e m i l k i s b e i n g c o a g u l a t e d u s i n g a rennet p r e p a r a t i o n , s t a r t e r c u l t u r e s of m i c r o o r g a n i s m s a r e added which a r e c a r e f u l l y s e l e c t e d f o r the type o f cheese b e i n g produced. Most v a r i e t i e s o f cheese undergo some degree of a g i n g which improves f l a v o r and t e x t u r e . C e r t a i n f r e e amino a c i d s appear w i t h i n a few hours a f t e r the c u r d i s h a r v e s t e d i n d i c a t i n g the presence of exopeptidases. In m o z z a r e l l a cheese d e s i r a b l e changes i n f l a v o r and t e x t u r e are e v i d e n t w i t h i n two weeks. Both p r o t e o l y s i s and l i p o l y s i s are i n v o l v e d i n the cheese ripening process. The r a t e and e x t e n t o f t h e i r i n t e r a c t i o n s a r e i n f l u e n c e d by the rennet p r e p a r a t i o n used, c h a r a c t e r i s t i c s of the s t a r t e r c u l t u r e , pH, m o i s t u r e range, s a l t i n g p r a c t i c e s , temperature, and the a c t i v i t y of a d v e n t i t i o u s m i c r o o r g a n i s m s p r e s e n t i n o r on the cheese. Rapid Ripened Cheese. Hard cheeses r e q u i r e p r o l o n g e d r i p e n i n g times thus the cheese m a n u f a c t u r e r must a c c e p t c e r t a i n time l a g s between the i n i t i a l c o n v e r s i o n o f m i l k and the r e a l i z a t i o n o f the v a l u e o f the r e s u l t i n g c h e e s e . The time l a g i s dependent upon the cheese variety. For example, mature cheddar r e q u i r e s up to one y e a r t o achieve f u l l maturity. The m a t u r a t i o n p e r i o d t i e s up l a r g e amounts o f c a p i t a l and i s a c o s t l y p r o c e s s . The c a p a c i t y of a cheese p r o d u c t i o n f a c i l i t y i s o f t e n l i m i t e d by the space a v a i l a b l e f o r m a t u r i n g the c h e e s e . Being a b l e to a c c e l e r a t e the a g i n g p r o c e s s would be a major c o s t e f f i c i e n c y improvement. S i n c e many v a r i e t i e s o f cheese a r e matured by the a c t i o n o f i n d i g e n o u s enzymes r a t h e r than by t h e i r v i a b l e m i c r o f l o r a , a g i n g o f these v a r i e t i e s has the p o t e n t i a l of b e i n g a c c e l e r a t e d by a r t i f i c i a l l y i n c r e a s i n g the c o n c e n t r a t i o n o f those enzymes t h a t have a d e f i n i t e r o l e i n the r i p e n i n g p r o c e s s . The t a i l o r i n g o f enzyme p r e p a r a t i o n s to a c c e l e r a t e the r i p e n i n g p r o c e s s , w h i l e g i v i n g e x c e l l e n t f l a v o r and t e x t u r e p r o p e r t i e s , i s a major c h a l l e n g e b e i n g t a c k l e d by the enzyme i n d u s t r y today. P r o t e a s e s f o r A c c e l e r a t i n g Cheese R i p e n i n g . P r o t e o l y s i s p l a y s such a major r o l e i n d e v e l o p i n g the f l a v o r and t e x t u r e o f hard cheese t h a t the a d d i t i o n o f exogenous m i c r o b i a l p r o t e a s e s has been attempted u s i n g every c o m m e r c i a l l y a v a i l a b l e p r o t e a s e p r e p a r a t i o n . By v e r y c a r e f u l l y c h o o s i n g the p r o t e a s e and i t s l e v e l , v e r y s t r o n g f l a v o r e d cheese can be produced i n a s h o r t time. However, i t i s v e r y d i f f i c u l t to produce the p r o p e r b a l a n c e of f l a v o r and t o a v o i d b i t t e r defects i n real applications. To v a r y i n g d e g r e e s , a l l p r o t e a s e treatments w i l l i n c r e a s e the s o f t n e s s o f the f i n a l p r o d u c t , thus a d d i n g a n o t h e r d e f e c t the cheese m a n u f a c t u r e r has to m i n i m i z e

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch003

3. SPRADLIN

Tailoring Enzyme Systems for Food Processing

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i n t h e r a p i d r i p e n e d cheese p r o d u c t s . The l i m i t o f the magnitude by which cheese f l a v o r s c a n be i n c r e a s e d w i t h p r o t e a s e treatment i s l i m i t e d by t h e o n s e t o f d e f e c t s . When f a c e d w i t h a problem t h a t c a r r i e s a b i g bottom l i n e p r o f i t p o t e n t i a l , t e c h n o l o g i e s w i l l be developed t o s o l v e t h a t problem. A new approach now b e i n g used t o t a i l o r enzyme p r e p a r a t i o n s f o r the cheese i n d u s t r y i s a s p i n - o f f o f the m e d i c a l / p h a r m a c e u t i c a l i n d u s t r i e s technology. Liposome t e c h n o l o g y i s t h e a r t o f p r o d u c i n g m u l t i l a y e r e d l i p i d p a r t i c l e s r a n g i n g i n s i z e from l e s s than one m i c r o n t o s e v e r a l microns w i t h n o n - l i p i d m a t e r i a l s t r a p p e d between layers. I n one c u r r e n t liposome enzyme p r e p a r a t i o n b e i n g t e s t e d now i n cheese making, p r o t e a s e s a r e e n c a p s u l a t e d i n a system o f p h o s p h o l i p i d b i l a y e r membranes ( 2 1 ) . T h i s liposome enzyme system i s a c o m b i n a t i o n o f an e n d o p e p t i d a s e and an e x o p e p t i d a s e . The enzymes a r e s l o w l y r e l e a s e d a f t e r the cheese i s put i n t o the a g i n g chamber. U s i n g these liposomes f o r cheddar cheese p r o d u c t i o n showed t h a t the liposomes were e v e n l y d i s t r i b u t e d and 90% o f the added enzyme was r e t a i n e d i n the cheese curd. The r e s u l t i n g cheese r i p e n e d i n h a l f the normal time w i t h e x c e l l e n t f l a v o r and t e x t u r a l p r o p e r t i e s . The t a i l o r i n g o f p r o t e a s e p r e p a r a t i o n s t o r a p i d l y produce h a r d cheeses i n a c o s t e f f e c t i v e manner and w i t h good f l a v o r and t e x t u r a l properties, i s progressing rapidly. I n t h e next few y e a r s s e v e r a l new and i n n o v a t i v e l y t a i l o r e d enzyme p r e p a r a t i o n s w i l l be a v a i l a b l e f o r a c c e l e r a t i n g the p r o d u c t i o n of hard cheeses. L i p a s e s f o r A c c e l e r a t i n g Cheese R i p e n i n g . Acceleration of l i p o l y s i s by the a d d i t i o n o f e i t h e r a n i m a l o r m i c r o b i a l l i p a s e s has been s u c c e s s f u l l y a p p l i e d to the r e l a t i v e l y s t r o n g f l a v o r e d cheeses. L i p a s e s d e r i v e d from the p r e g a s t r i c g l a n d o f k i d , c a l f and lamb a r e c u r r e n t l y b e i n g added t o a c c e l e r a t e r i p e n i n g i n I t a l i a n type cheeses where t h e c h a r a c t e r i s t i c r a n c i d f l a v o r o f low m o l e c u l a r weight f r e e f a t t y a c i d s , f o r example b u t y r i c a c i d , i s d e s i r a b l e . However, when these p r e g a s t r i c l i p a s e s a r e used t o a c c e l e r a t e m i l d f l a v o r e d cheese such as cheddar, t o o much o f the low m o l e c u l a r weight f a t t y a c i d s a r e produced and a r a n c i d f l a v o r d e v e l o p s . When the a n i m a l pancreas l i p a s e s a r e used i n a h i g h c o n c e n t r a t i o n , e x c e s s i v e amounts o f l a u r i c a c i d a r e produced g i v i n g the cheese a soapy t a s t e . Also, a major d i s a d v a n t a g e o f u s i n g any o f these l i p a s e p r e p a r a t i o n s t a i l o r e d from a n i m a l g l a n d s i s the p r o t e a s e t h a t i s always p r e s e n t . A l t h o u g h the p r o t e a s e i s p r e s e n t i n s m a l l amounts, cheese s o f t e n i n g o c c u r s and a t times b i t t e r o f f - f l a v o r s a r e produced. To d a t e , t h e use o f m i c r o b i a l l i p a s e has a l s o been p r i m a r i l y i n t h e v e r y s t r o n g f l a v o r e d cheeses, l i k e b l u e cheese where r a n c i d and soapy f l a v o r notes a r e l e s s n o t i c e a b l e . When t h e r e i s a need, someone w i l l s o l v e i t . A p a t e n t was i s s u e d i n 1987 (^2) d e s c r i b i n g a new and n o v e l l i p a s e produced by a mutant s t r a i n o f A s p e r g i l l u s t h a t has an a c c e l e r a t i n g e f f e c t on cheese f l a v o r development w i t h o u t l y p o l y t i c enzyme a s s o c i a t e d rancidity. The p a t e n t c l a i m s t h a t t h i s new l i p a s e w i l l be u s e f u l as a r i p e n i n g a c c e l e r a t o r i n t h e p r o d u c t i o n o f m i l d f l a v o r e d cheeses such as cheddar. Enzyme M o d i f i e d Cheese. I f the t e x t u r e i s s u e i s removed from the d e s i r a b l e a t t r i b u t e s o f cheese p r o d u c t i o n and the o n l y a t t r i b u t e

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch003

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d e s i r e d i s f l a v o r , then a whole new a r e a becomes a v a i l a b l e f o r t a i l o r e d enzyme p r e p a r a t i o n s . The importance of f r e e f a t t y a c i d s to the c h a r a c t e r i s t i c f l a v o r o f v a r i o u s t y p e s of cheese and to d a i r y p r o d u c t s i n g e n e r a l , becomes v e r y apparent as t e c h n o l o g i s t s e v a l u a t e and study the c h e m i s t r y o f food f l a v o r s . The mechanisms and c h e m i s t r y by which r e l a t i v e l y t a s t e l e s s cheese c u r d s become d i s t i n c t i v e l y f l a v o r e d cheeses have been under r e s e a r c h s c r u t i n y f o r many decades. L i p o l y s i s i s d e f i n e d as the enzyme-catalyzed h y d r o l y t i c c l e a v a g e o f t r i g l y c e r i d e s r e s u l t i n g i n the r e l e a s e o f f r e e f a t t y a c i d s . But l i p a s e s d i f f e r g r e a t l y i n t h e i r c a t a l y t i c s p e c i f i c i t y and a r e r e s p o n s i b l e f o r many o f the d i f f e r e n c e s among cheese f l a v o r s . The m i c r o b i a l l i p a s e s , produced by the s t a r t e r c u l t u r e m i c r o o r g a n i s m s , a r e o f t e n the d e c i d i n g f a c t o r on the type o f cheese b e i n g produced. The f l a v o r o f the I t a l i a n cheeses - romano, parmesan, and p r o v o l o n e - a r e e n t i r e l y dependent on f r e e f a t t y a c i d s f o r t h e i r d i s t i n c t i v e flavor. The c a t a l y t i c s p e c i f i c i t y of v a r i o u s l i p a s e s d i f f e r i n f o u r major a r e a s : f a t t y a c i d c h a i n l e n g t h ; p o s i t i o n o f the f a t t y a c i d i n the g l y c e r i d e s t r u c t u r e ; degree of e s t e r f i c a t i o n i n the g l y c e r i d e ( t r i - , d i - , o r mono-); and the p h y s i c a l s t a t e of the g l y c e r i d e . L i p o l y t i c enzymes r e s p o n s i b l e f o r v a r i o u s cheese f l a v o r s demonstrate p r e f e r e n t i a l r e l e a s e of s p e c i f i c f a t t y a c i d s from one p o s i t i o n i n the b u t t e r f a t when the b u t t e r f a t i s i n the c o r r e c t p h y s i c a l form. Enzyme p r e p a r a t i o n s a r e now b e i n g t a i l o r e d by p r e c u l t u r i n g the a p p r o p r i a t e organism under c o n d i t i o n s t h a t a l l o w the a c c u m u l a t i o n o f the d e s i r e d enzymes f r e e i n s o l u t i o n . Enzyme p r o d u c t s a r e now a v a i l a b l e t h a t a r e c a p a b l e o f p r o d u c i n g s p e c i f i c cheese f l a v o r s i n s l u r r i e s of b u t t e r f a t or i n s l u r r i e s of cheese c u r d . F o r example, a romano cheese f l a v o r was r e c e n t l y r e p o r t e d (^3) t h a t was produced from a v e r y s i m p l e b u t t e r f a t e m u l s i o n . An e m u l s i o n o f water washed b u t t e r and Tween 80 i n phosphate b u f f e r was t r e a t e d w i t h a c r u d e enzyme m i x t u r e i s o l a t e d from Candida r u g o s a . Today, cheese f l a v o r e d p a s t e and powders, c o n t a i n i n g from 5 to 100 times the f l a v o r l e v e l o f n a t i v e c h e e s e , a r e a v a i l a b l e i n the m a r k e t p l a c e f o r most cheese v a r i e t i e s . N e a r l y a l l o f these p r o d u c t s a r e made by u s i n g added enzymes w i t h most o f them u s i n g a b l e n d o f enzymes and m i c r o o r g a n i s m s . S e l e c t e d cheese f l a v o r s a r e produced i n a l i q u i d o r s l u r r y form i n a r e l a t i v e l y s h o r t time compared to making the t r u e cheese and a t a f l a v o r l e v e l many f o l d above the maximum f l a v o r o b t a i n a b l e i n the t r u e c h e e s e . The s y n e r g i s t i c use of enzymes and m i c r o o r g a n i s m s t o produce n a t u r a l f l a v o r s f o r foods i s an i n d u s t r y o f i t s own j u s t now b e i n g born. The t a i l o r i n g of enzyme p r o d u c t s w i t h and w i t h o u t m i c r o o r g a n i s m s , w i l l be a major market a r e a f o r the enzyme i n d u s t r y i n the f u t u r e . Lactase. L a c t o s e , p r e s e n t i n m i l k , i s a d i s a c c h a r i d e sugar composed o f g l u c o s e and g a l a c t o s e . L a c t o s e i s a major problem t o the d a i r y industry. I t i s non-sweet, v e r y low i n s o l u b i l i t y , cannot be absorbed d i r e c t l y from the i n t e s t i n e o f humans and i s m e t a b o l i z e d v e r y p o o r l y by m i c r o o r g a n i s m s . In c o n t r a s t , i t s components ( g l u c o s e and g a l a c t o s e ) have a sweetening power of about 0.8 r e l a t i v e to s u c r o s e , a r e 3 t o 4 times more s o l u b l e than l a c t o s e , a r e e a s i l y absorbed from the i n t e s t i n e and the g l u c o s e i s h i g h l y f e r m e n t a b l e by microorganisms.

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch003

3. SPRADLIN

Tailoring Enzyme Systems for Food Processing

41

L a c t a s e i s an enzyme t h a t s e l e c t i v e l y h y d r o l y z e s l a c t o s e i n t o g l u c o s e and g a l a c t o s e thus h a v i n g the p o t e n t i a l f o r the t a i l o r i n g of enzyme p r e p a r a t i o n s f o r wide use i n the d a i r y i n d u s t r y . Lactase treatment p r o v i d e s a method o f p r e v e n t i n g l a c t o s e c r y s t a l l i z a t i o n i n f r o z e n m i l k and whey based p r o d u c t s as w e l l as i n l i q u i d concentrated milk products. Humans i n t o l e r a n t of l a c t o s e can p r e v e n t s u f f e r i n g from m i l k i n d u c e d abdominal p a i n and o t h e r g a s t r o i n t e s t i n a l d i s t r e s s e s by u s i n g l a c t a s e - t r e a t e d m i l k . Today the l a r g e s t market f o r l a c t a s e i s by i n d i v i d u a l s s u f f e r i n g from lactose intolerance. S e v e r a l y e a r s ago the enzyme i n d u s t r y looked a t the cheese i n d u s t r y and saw l a c t a s e as a b i g o p p o r t u n i t y . B e s i d e s the l a c t o s e c r y s t a l l i z a t i o n problem i n f r o z e n and c o n c e n t r a t e d m i l k p r o d u c t s , the enzyme i n d u s t r y saw a n o t h e r p o t e n t i a l l y b i g p r o f i t a p p l i c a t i o n . Over h a l f o f the cheese whey produced i n making cheese i s d i v e r t e d to waste treatment o r waste d i s p o s a l systems r a t h e r than b e i n g p r o c e s s e d f o r food and feed u s e . U p g r a d i n g cheese whey became the hot development i t e m f o r the enzyme i n d u s t r y . The f i r s t problem f a c e d by the enzyme i n d u s t r y was to p r e p a r e p r e p a r a t i o n s h a v i n g the same c a t a l y t i c p r o p e r t i e s and y e t be a b l e to work a t e x t r e m e l y d i f f e r e n t pH v a l u e s . The pH o f m i l k i s 6.4 to 6.8 w h i l e the pH o f cheese whey i s c l o s e r to 4. A d j u s t i n g the pH o f e i t h e r feed stream i s not a f e a s i b l e answer to the p r o b l e m . Enzyme p r e p a r a t i o n s were d e v e l o p e d from d a i r y y e a s t t h a t a r e a c t i v e around pH 6.7 f o r use i n m i l k systems and o t h e r enzyme p r e p a r a t i o n s were d e v e l o p e d from f u n g a l organisms a c t i v e around pH 4 . 0 f o r use i n cheese whey. The use of s o l u b l e p r e p a r a t i o n s o f a c i d l a c t a s e was never d e v e l o p e d i n t o a c o s t e f f e c t i v e p r o c e s s f o r u p g r a d i n g whey. The low l e v e l of l a c t o s e i n the whey c r e a t e d a l a r g e expense i n c o n c e n t r a t i n g the p r o d u c t s . T h i s p l u s the h i g h c o s t o f food approved l a c t a s e made the p r o d u c t i o n o f sugar from whey uneconomical. The next t a i l o r i n g s t e p , i n an attempt to upgrade whey, was to i m m o b i l i z e the f u n g a l l a c t a s e and use the h y d r o l y z e d p r o d u c t as f e e d s t o c k to grow b a k e r ' s y e a s t . S e v e r a l companies spent y e a r s and a l o t o f money d e v e l o p i n g i m m o b i l i z e d l a c t a s e p r o d u c t s . The one system t h a t was put i n t o commercial p r o d u c t i o n i n the U n i t e d S t a t e s was t a i l o r e d by i m m o b i l i z i n g f u n g a l l a c t a s e onto m i c r o p o r o u s beads o f g l a s s - l i k e c a r r i e r . A f t e r s e v e r a l attempts to make the o p e r a t i o n a p r o f i t a b l e one, the f a c i l i t y was c l o s e d . Today, the l a c t a s e market i s p r i m a r i l y f o r s o l v i n g the l a c t o s e i n t o l e r a n c e problem i n human d i e t s . The t a i l o r i n g o f enzyme p r e p a r a t i o n s c o n t i n u e s and l a c t a s e may s t i l l be developed i n t o a b i g p r o f i t item. T a i l o r i n g Enzymes f o r F u t u r e Food A p p l i c a t i o n s The b i g t a i l o r i n g p r o j e c t s i n the f u t u r e w i l l be i n d e s i g n i n g enzymes u s i n g gene s y n t h e s i s , s i t e - d i r e c t e d mutagenesis and o t h e r genetic engineering technologies. The enzyme's r e s p o n s e s to i t s environment w i l l be the p r i m a r y t a r g e t s . Responses to t e m p e r a t u r e , pH, i o n s , and c h e m i c a l s w i l l be a d j u s t e d to b e s t t a i l o r an enzyme for i t s intended use. To i l l u s t r a t e the c h e m i c a l b a s i s f o r p o t e n t i a l changes, the t a i l o r i n g o f an enzyme's response to temperature w i l l be u s e d .

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch003

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BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

Enzymes respond to temperature i n two ways. F i r s t the enzyme's s t a b i l i t y i s a f f e c t e d by temperature. As the temperature i s i n c r e a s e d , i n a c t i v a t i o n o f the enzyme due t o thermal d e n a t u r a t i o n o f the p r o t e i n i n c r e a s e s r e s u l t i n g i n a d e c r e a s e i n the c o n c e n t r a t i o n of a c t i v e enzyme. The second e f f e c t of temperature i s on the reaction rate d i r e c t l y . F o r c h e m i c a l r e a c t i o n s , the A r r h e n i u s e q u a t i o n d e s c r i b e s the r e l a t i o n s h i p between the r a t e c o n s t a n t o f the r e a c t i o n and the a b s o l u t e temperature. T h i s e q u a t i o n , which i s e m p i r i c a l and has no t h e o r e t i c a l b a s i s , was developed i n 1889 by A r r h e n i u s to d e f i n e the a c t i v a t i o n energy o f a r e a c t i o n . The r o l e o f enzymes i n c h e m i c a l r e a c t i o n s i s to lower the a c t i v a t i o n energy. In f a c t , the a c t i v a t i o n energy o f e n z y m e - c a t a l y z e d r e a c t i o n s appear to be more c h a r a c t e r i s t i c o f the enzyme than o f the s u b s t r a t e s i n v o l v e d i n the reaction. For most e n z y m e - c a t a l y z e d r e a c t i o n s , the a c t i v a t i o n e n e r g i e s a r e between 6,000 and 15,000 c a l / m o l ( 2 4 ) . S i n c e the r a t e of r e a c t i o n i s dependent upon the a c t i v a t i o n energy, the g e n e t i c e n g i n e e r i n g t a s k i s to d e s i g n i n t o the enzyme a temperature s e n s i t i v e c o n f o r m a t i o n change. T h i s c o n f o r m a t i o n change needs to cause a major change i n the a c t i v a t i o n energy a t a d e s i r e d temperature. T h i s c o u l d be t o e i t h e r t u r n on o r t u r n o f f the a c t i v i t y depending upon the a p p l i c a t i o n . F o r example, i n cheese making i t may be d e s i r a b l e to t u r n o f f the a c t i v i t y as the temperature r i s e s , w h i l e i n bread b a k i n g i t may be d e s i r a b l e t o have enzyme a c t i v i t y o n l y a t h i g h temperatures. The s w i t c h can work i n either direction. The o t h e r t e m p e r a t u r e - r e l a t e d p r o p e r t y o f enzymes needing a t t e n t i o n i s thermal i n a c t i v a t i o n . T a i l o r i n g enzymes f o r the cheese i n d u s t r y has a l r e a d y produced l e s s s t a b l e p r o t e a s e s . T a i l o r i n g a l p h a amylase p r e p a r a t i o n s f o r the c o r n syrup i n d u s t r y has produced amylases w i t h long enough h a l f - l i v e s a t 105°C to a l l o w t h e i r use i n p r o d u c t i o n at t h a t temperature. The c u r r e n t p r o c e s s i n g t r e n d i n the food i n d u s t r y i s toward s h o r t e r p r o c e s s i n g t i m e s . One o f the major p r o c e s s i n g methods i n the food i n d u s t r y today i s r e f e r r e d t o as h i g h temperature and s h o r t time (HTST) p r o c e s s i n g . P r o d u c t s made under these c o n d i t i o n s o f t e n have p h y s i c a l p r o p e r t i e s v e r y s i m i l a r to p r o d u c t s made by much l o n g e r p r o c e s s i n g methods. However, most of the time the f l a v o r development i s not a c c e p t a b l e . Enzymes c a p a b l e o f working a t h i g h temperatures, even f o r s h o r t time p e r i o d s , are needed by the food i n d u s t r y . Microwave c o o k i n g i s a b l e to cook food r a p i d l y p r o d u c i n g the p h y s i c a l changes d e s i r e d but o f t e n f a l l s s h o r t i n f l a v o r development. Enzyme p r e p a r a t i o n s a r e needed to s o l v e the f l a v o r g e n e r a t i o n and t e x t u r a l problems a s s o c i a t e d w i t h microwave c o o k i n g and r e h e a t i n g . Conclusion The p a s t s u c c e s s o f t a i l o r i n g enzyme p r e p a r a t i o n s f o r a p p l i c a t i o n i n the food i n d u s t r y can be e x p r e s s e d by summarizing a few major a p p l i c a t i o n s o f enzymes i n the i n d u s t r y today. Almost a l l c l e a r f r u i t j u i c e s a r e enzyme t r e a t e d . A h i g h p r o p o r t i o n o f grape j u i c e , grape must and grape wine i s enzyme t r e a t e d . A l l p r o d u c t i o n o f g l u c o s e from s t a r c h i s done u s i n g the d u a l enzyme p r o c e s s . Corn syrups a r e produced by e i t h e r an acid-enzyme p r o c e s s o r by a d u a l enzyme p r o c e s s . A l l i s o m e r i z a t i o n s f o r the p r o d u c t i o n o f h i g h f r u c t o s e syrups a r e done e n z y m a t i c a l l y . Most bread i s p r o c e s s e d

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

3. SPRADLIN

Tailoring Enzyme Systems for Food Processing

w i t h enzyme a d d i t i v e s . Egg w h i t e s a r e g e n e r a l l y desugared enzymatically p r i o r to drying. Uses o f commercial enzymes i n the d a i r y and brewing i n d u s t r i e s a r e growing r a p i d l y . The a b i l i t y o f enzyme m a n u f a c t u r e r s t o d e s i g n enzyme p r e p a r a t i o n s and p r o c e s s e s f o r s p e c i f i c food a p p l i c a t i o n s i s l i m i t e d o n l y by t h e a b i l i t y o f t h e food p r o c e s s o r t o i d e n t i f y and communicate s p e c i f i c a p p l i c a t i o n needs t o the enzyme m a n u f a c t u r e r s .

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch003

Literature Cited

1. Cheetham, P. S. J. In Handbook of Enzyme Biotechnology; Wiseman, Α., Ed.; Ellis Horwood Limited: 1985, pp 323-361. 2. Snyder, E. C.; Kovi, E. R. U. S. Patent 2 965 520, 1960. 3. Thoma, J. Α.; Brothers, C.; Spradlin, J. Biochemistry 1970, 1768. 4. Robyt, J. F.; French, D. Arch. Biochem. Biophys. 1967, 122, 8. 5. Nielsen, G. C.; Dier, I. V.; Outtrup, N.; Norman, Β. E. U. K. Patent Appl. GB 2 097 405 A, 1982. 6. Jensen, B. F.; Norman, Β. E. Process Biochem. 1984, 19, 129. 7. David, M. H.; Gunther, H.; Roper, H. Starch/Starke 1970, 39, 436. 8. Hebeda, R. E.; Styslund, C. R.; Teague, M. Starch/Starke 1988, 40, 33. 9. Colson, C. Α.; Walon, C.; Lejeume, P.; Willinot, K. Eur. Patent Appl. 0 165 002, 1985. 10. Marshall, R. O.; Kooi, Ε. T. Science 1957, 125, 648. 11. Thoma, J. Α.; Spradlin, J. E.; Dygert, S. In The Enzymes; Boyer, P., Ed.; Academic Press: New York, 1971; Vol. 5, pp 115-189. 12. Miller, B. S.; Johnson, J. Α.; Palmer, D. L. Food Techno1 1953, 7, 38. 13. Zobel, H. F.; Senti, F. R. Cereal Chem. 1959, 36, 441. 14. Dragsdorf, R. D.; Varriano-Marston, E. Cereal Chem. 1980, 57, 310. 15. Sarko, Α.; Wu, H. C. H. Starch/Starke 1978, 30, 73. 16. Carroll, J. O.; Boyce, C. O. L.; Wong, T. M.; Starace, C. A. U. S. Patent 4 654 216, 1987. 17. Cook, Α.; Johannson, H. U. S. Patent 3 512 992, 1970. 18. Kulp, K.; Cereal Chem. 1968, 45, 339. 19. Cullen, D.; Gray, G. L.; Wilson, L. J.; Hayenga, K. J.; Lamso, M. H.; Rey, M. W.; Norton, S.; Berko, R. M. Biotech. 1987, 5, 369. 20. Branner-Jorgensen, S. U. S. Patent 4 386 160, 1983. 21. Kirby, C. J.; Brooker, Β. E.; Law, B. A. Int. J. Food Sci. Tech. 1987, 22, 355. 22. Arbige, M. V.; Newbeck, C. E. U. S. Patent 4 636 468, 1987. 23. Lee, D. -C. M.; Shi, H.; Huang, A. -S.; Carlin, J. T.; Ho, C. -T.; Chang, S. S. In Biogeneration of Aromas; Parliment, T. H.; Croteau, R., Eds.; ACS Symposium Series No. 317; American Chemical Society: Washington, DC, 1986; pp 370-378. 24. Sizer, I. W. Adv. Enzymol. 1943, 3, 35. RECEIVED

November 2, 1988

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 4

Characteristics of Some Enzymes Used in Genetic Engineering John R. Whitaker

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch004

Department of Food Science and Technology, University of California, Davis, CA 95616 Fundamental knowledge of the structure, function and mechanism of DNA-modifying enzymes has been important not only in understanding how these enzymes perform a myriad of chemical reactions in vivo but also for the development of the field of recombinant DNA technology. The functions of the major groups of enzymes in deoxyribonucleic acid synthesis, hydrolysis and modification are reviewed, as well as some structural and mechanistic aspects of the restriction endonucleases, ligases and polymerases. Two important commercial applications of enzymes are the use of carbohydrases i n making sweeteners from cornstarch and the r e s t r i c tion endonucleases/ligases/polymerases i n recombinant DNA technology and i n nucleic acid sequence studies. Both technologies developed rapidly as a result of substantial prior basic knowledge of the enzymes. Understanding the key roles of DNA and RNA i n microbes, plants and animals developed rapidly after enunciation of the Watson-Crick model, including the multitude of enzymes involved i n their biosynthesis, degradation and modification (Table I ) . Several enzymes are involved i n nucleic acid synthesis, especially when one considers the varied nature of enzymes i n each group. For example, with the polymerases, there are separate enzymes important i n biosynthesis of DNA and RNA, some with s p e c i f i c i t y f o r size of the chain length (gap) to be completed. The enzymes have different structural properties depending on whether they are from microorganisms, plants or animals. There are multiple forms within a single c e l l or organism. They vary from a single polypeptide enzyme of 40,000 daltons [mammalian 3-polymerase (1)], to a seven-subunit complex of about 500,000 daltons [E. coLL DNA polymerase III ( 2 ) ] . Unlike the polymerases, which add nucleotides sequentially to the 3'-0H terminus of single strands of pre-existing polynucleotides, the ligases catalyze formation of phosphodiester bonds 0097-6156/89/0389-0044$06.25/0 © 1989 American Chemical Society

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

4. WHITAKER Table I.

Enzymes Used in Genetic Engineering

45

Some Enzymes that Catalyze Reactions of Nucleic Acids

Enzyme

Reaction Catalyzed

Nucleic Acid Synthesis Polymerases

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch004

Ligases

RecA protein

Reverse transcriptases

Nucleotidyl transferases

Priming enzymes

Poly(A) adding enzymes Capping enzyme

Catalyze addition of nucleoside triphosphates to terminals of single strands of preexisting polynucleotides (primers) with release of pyrophosphate. Catalyze formation of phosphodiester linkages between DNA chain segments. Essential for homologous genetic recombination, DNA repair and other SOS functions as a result of DNA damage. Catalyze transcription of RNA nucleotide sequence into complementary DNA (cDNA). Catalyze attachment of mononucleotide triphosphates to the 3^-OH of the i n i t i a t o r polymer, with release of pyrophosphate. Provide a free 3^-OH at terminal end of i n i t i a t o r strand to serve as acceptor for transfer of the f i r s t nucleotide and formation of f i r s t phosphodiester bond i n DNA synthesis. Attach AMP residues, from ATP, to the 3', Y phosphodiester bond. Attaches 7-methylguanosine to the penultimate nucleoside of RNA v i a a Y 5^-triphosphate bridge. Replication of v i r a l RNA. y

Q3 replicase Nucleic Acid Hydrolysis Nucleases Restriction endonucleases

Catalyze hydrolysis of the phosphodiester bonds of polynucleotides. Catalyze hydrolysis of the phosphodiester bonds of polydeoxyribonucleotides. S t r i c t s p e c i f i c i t y for double stranded DNA and extended nucleotide sequences. Continued

on next page.

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

46

BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

Table I.

Continued.

Enzyme

Reaction Catalyzed

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch004

Nucleic Acid Modification

Kinases

Topoisomerases

Gyrase Unwinding enzymes Methylases

Insertases

Glycosylases

Alkaline phosphatase

Catalyze the transfer of the α-phos­ phate of a nucleoside 5'-triphosphate to the 5^-OH terminus of a deoxyribonucleic acid or ribonu­ c l e i c acid molecule. Catalyze changes i n the topological structure of DNA molecules by breakage and resynthesis of the same phosphodiester bond i n DNA strands. Type II topoisomerase that catalyzes conversion of relaxed DNA to a superhelical form; requires ATP. Catalyze separation of complementary strands of DNA; require ATP. Catalyzes transfer of methyl groups from donor j>-adenosyl-L-methionine (AdoMet) to s p e c i f i c bases of acceptor DNA molecules. Catalyze the reinsertion of a missing base into the appropriate apurinic or apyrimidinic AP sites of DNA. Catalyze hydrolysis of nucleoside bases-glycosidic bonds, resulting in production of an apurinic/ apyrimidinic (AP) s i t e i n DNA and a free nucleoside base. Catalyzes hydrolysis of monophosphate ester groups from compounds, including from the 3^-and Yterminal ends of nucleic acids and nucleotides.

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

4. WHITAKER

Enzymes Used in Genetic Engineering

47

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch004

between segments within double-stranded DNA chains. By use of r e s t r i c t i o n endonucleases, segments of double-stranded DNA can then be excised from a donor and the excised segments can then be covalently inserted into the host DNA by using ligases. Commercially available r e s t r i c t i o n endonucleases of w e l l defined s p e c i f i c i t y and purity (Table II) have made i t possible f o r recombinant DNA technology to be applied by b i o l o g i s t s and chemists with l i t t l e knowledge of the basic protein chemistry and enzymology that undergird this f i e l d . In fact, much of the basic knowledge of these endonucleases was developed i n the 1960-1981 period, making the modern era of molecular biotechnology possible. In the remainder of this paper, I s h a l l r e s t r i c t discussion to the r e s t r i c t i o n endonucleases, the ligases and the polymerases as being the most important i n genetic engineering. THE RESTRICTION ENDONUCLEASES Type II r e s t r i c t i o n endonucleases recognize s p e c i f i c base sequences i n double-stranded DNA, and cleave both strands of the duplex. More than 600 r e s t r i c t i o n endonucleases have been reported i n the literature. Some 121 r e s t r i c t i o n endonucleases are l i s t e d i n Current Protocols i n Molecular Biology as commercially available (_3). Table I I , adapted from the 1988 Sigma Chemical Company Catalog, l i s t s 35 of the endonucleases available from that source. Endonucleases are found i n many bacteria where t h e i r function i s to hydrolyze foreign DNA introduced by phage i n f e c t i o n , conjugation or transformation. The f i r s t r e s t r i c t i o n endonucleases were discovered and isolated i n 1968 (4,5). Restriction endonucleases are invaluable i n recombinant DNA applications and i n sequencing DNA. As shown i n Figure 1, s p e c i f i c r e s t r i c t i o n endonucleases hydrolyze the phosphodiester linkages at precise locations i n DNA. In a plasmid, such as pBR322, a portion of the chain can be excised by use of two r e s t r i c t i o n endonucleases, such as Bsm I and Pvu II i n Figure 1, removing a f r a g ment containing 714 base pairs. A fragment of s i m i l a r s i z e , with the right base sequence at the ends, can be excised from a porcine DNA molecule (for example, the region encoding porcine i n s u l i n ) with r e s t r i c t i o n endonucleases and, using DNA ligase to covalently attach the fragment into the vector (here pBR322), the gene f o r i n s u l i n can be inserted into the host DNA. Proper insertion of the modified plasmid into a bacterium ( E . doti for example) permits expression of porcine i n s u l i n by the E . cotl ( F i g . 2). SPECIFICITY. Restriction endonucleases are highly s p e c i f i c i n hydrolyzing doubled-stranded DNA. Not only do they recognize a s p e c i f i c sequence of four to s i x base pairs (up to 11 base pairs i n case of Bgl I; see Table II) but the base pairs must be paLindxomic i n the two strands of DNA. The cleavage s i t e s possess twofold rotational symmetry as shown i n Scheme I for r e s t r i c t i o n endonuclease Aat II from kcdtobacJiQA acttl (Table I I ; A, T, G, C indicate deoxyribonucleotides i n this paper). In this example, the T-C phosphodiester bond i n each strand two residues d i s t a l to the symmetry axis i s cleaved by r e s t r i c t i o n endonuclease Aat I I .

American Chemical Society Library 1155 18th St., M.W. Whitaker, J., el al.; In Biocatalysis in Agricultural Biotechnology; ACS Symposium Series; American Chemical Society: Washington, DC, 1989. Washington, O.C. 20036

48

BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

Table I I .

Some Commercially Available Restriction Endonucleases

Enzyme in

Specificity

No.

cleavage

a

sites

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch004

XDNA

Aat II Alu I Bal I Bam H-I Ban II Ban III Bel I Bgl I Bgl II Bst EII Cla I Eco RI Fsp I Hae II Hae III Hha I Hinc II Hind II Hind III Hinf I Hpa I Hpa II Kpn I Mbo I Mbo II Pst I Sac I Sal I Sau 3A I Sin I Sma I Sph I Taq I Xba I Xho I

5"-GACGT/C-3" 5'-AG/CT-3' 5'-TGG/CCA-3" 5'-G/GATCC-3' 5'-GPuGCPy/C-3' 5'-AT/CGAT-3" 5'-T/GATCA-3' 5'-GCCNNNN/NGGC-3" 5"-A/GATCT-3" 5"-G/GTNACC-3" 5'-AT/CGAT-3" 5"-G/AATTC-3" 5'-TGC/GCA-3" 5'-PuGCGC/Py-3' 5'-GG/CC-3' 5'-GCG/C-3" 5"-GTPy/PuAC-3" 5'-GTPy/PuAC-3' 5'-A/AGCTT-3' 5'-G/ANTC-3" 5"-GTT/AAC-3" 5'-C/CGG-5 5'-GGTAC/C-3' 5"-/GATC-3" 5'-GAAGA(N)g/-3' 5'-CTGCA/G-3' 5'-GAGCT/C-3' 5'-G/TCGAC-3' 5"-N/GATC-3" 5'-G/G(A,T)CC-3' 5'-CCC/GGG-3' 5'-GCATG/C-3' 5"-T/CGA-3' 5"-T/CTAGA-3" 5'-C/TCGAG-3"

10 >50 18 5 7 15 5 b 6 8 15 5 15 >30 >50 >50 34 34 6

b

1 2

b

> 5 0

κ 6 >50 2 8° >50 b

d

l 2 50 17 3 6 >50 4 1

L

Sigma Chemical Co. Catalog Feb. 1988; T, A, C, G specify deoxyribonucleotide residues; (N, nucleotide; Pu, purine; Py, pyrimidine; and /, point of cleavage. Using Adenovirus 2 DNA.

c

Using SV40 DNA.

d

Eco RI fragments of ADNA.

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch004

WHITAKER

F i g u r e 1. EAck&Uchia

Enzymes Used in Genetic Engineering

Some coti

r e s t r i c t i o n endonucleases p l a s m i d pBR322.

hydrolysis

sites

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

on

50

BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

Foreign DNA to be inserted

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch004

^ Joining

Recombinant DNA molecule I Introduction into host cells by I transformation or viral infection

I

^

Selection for cells containing a recombinant ONA molecule

Cloning

Figure 2. Schematic of insertion of foreign DNA fragment into plasmid vector and cloning of DNA molecules i n host c e l l s .

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

4. WHITAKER

51

Enzymes Used in Genetic Engineering cleavage s i t e

5'-G-A-C-G-T-C-3' .**!#!!!

Scheme I

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch004

3'-C-T-G$C-A-G-5' t V cleavage^symmetry site axis The s p e c i f i c i t i e s of some of the r e s t r i c t i o n endonucleases l i s t e d i n Table II are included i n Figure 3. The cleavage s i t e s may be staggered or even. The high s p e c i f i c i t y of r e s t r i c t i o n endonucleases permits precise and reproducible cleavage of phosphodiester bonds i n DNA substrates. They are indispensable tools for sequencing very long strands of DNA, i s o l a t i n g genes, analyzing chromosome structures and creating new DNA molecules that can be cloned (recombinant DNA technology). Obviously the enzymes cleave variable numbers of bonds, depending on a given DNA s sequence. For example, as shown i n Table I I , r e s t r i c t i o n endonucleases Xho I, Sal I, Hind I I I and Sau 3A I hydrolyze 1, 2, 6 and 50 sites i n XDNA, respectively. 1

ECO RI ENDONUCLEASE. Eco RI endonuclease i s one of the better studied endonucleases. It i s a small protein of 276 amino acid residues (31,065 daltons) of known sequence (6,7). The active enzyme contains two subunits (8,9) and hydrolyzes the phosphodiester bond between the deoxyguanylic and deoxyadenylic acid residues of duplex 5'-GAATTC-3'. On hydrolysis, there i s an inversion of configuration at the reactive phosphorus (10) i n d i cating there i s an odd number of chemical steps i n the o v e r a l l reaction. This could mean that the endonuclease does not form a coyalent intermediate with the DNA. Hydrolysis of DNA requires Mg , but binding of duplex 5'-GAATTC-3' occurs i n the absence of Mg with a dissociation constant of 1 0 " M (11,12). Eco RI endonuclease does not cleave duplex 5^-GAATTC-3' when the central adenine moiety of either one or both chains i s methylated ( 13). DNA of the host organism i s protected from hydrolysis by i t s own r e s t r i c t i o n endonucleases by such s p e c i f i c methylation, which i s catalyzed by a s p e c i f i c methylase that recognizes the same nucleotide sequence as the r e s t r i c t i o n endonuclease. 2+

11

Crystalline enzyme-substrate complex. Recently, Eco RI endonuclease has been c o - c r y s t a l l i z e d with the dodeca- and tridecanucleotides 5'-CGCGAATTCGCG-3' and 5"-TCGCGAATTCGCG-3' (Eco RI endonuclease recognition s i t e s underlined) ( 13) and the c r y s t a l structure of the complex solved at 3 Â resolution. The enzyme consists of two globular subunits forming a crevice on one side of the molecule. Each subunit forms a single domain consisting of a five-stranded 3 sheet surrounded on both sides by α-helices. One subunit of the enzyme i s shown i n F i g . 4, depicting the crevice at the upper center part. The complex i s formed by binding of DNA into the crevice (Fig. 4) with the major groove of the polynucleotide i n contact

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

5'-GGATCC-3'

: : »: :

BamH I

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch004

3'-CCTAGG-5'

5'-GGTACC-3

y

Kpn I S'-CCATGG-S'

5'-GGCC-3' Hae

III

3'-CCGG-5'

5'-GCGC-3'

:*:

Hha I

3'-CGCG-5'

5'-GCCNNNNNGGC-3' Bgi ι 3'-CGGNNNNNCCG-5' F i g u r e 3. R e c o g n i t i o n and c l e a v a g e (arrows) s i t e s of double stranded nucleotide sequences by some restriction endo­ nucleases. The c l o s e d circle indicates axis of twofold r o t a t i o n a l symmetry of the two s t r a n d s .

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch004

4.

WHITAKER

Enzymes Used in Genetic Engineering

Figure 4· α-Carbon backbone of one polypeptide subunit of Eco RI endonuclease. (Reproduced with permission from Ref. 13. Copyright 1986 American Association for the Advancement of Science.)

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

53

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch004

54

BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

with the enzyme and the minor groove exposed to the solvent. The complex has twofold symmetry as shown i n Figure 5. The N-terminal end of each subunit of the enzyme wraps around the double stranded polynucleotide, l i k e arms. More details of the complex are given i n Figure 6, showing the backbone schematic drawing of one subunit of the dimeric endonuclease and both strands of the polynucleotide. The endonuclease-polynucleotide complex i s s t a b i l i z e d by 12 hydrogen bonds that determine the substrate s p e c i f i c i t y of the enzyme ( F i g . 7). As shown i n Figure 7, six hydrogen bonds between the amino acid side chains of Glul44, Argl45 and Arg200 of the enzyme with the purine and pyrimidine bases of the hexanucleotide s p e c i f i c i t y region of the substrate are contributed by each subunit. This explains the basis of the twofold rotation symmetry i l l u s t r a t e d i n Figure 3. In solution, these interactions are more l i k e l y ion pairs (9). When Mg i s infused into the complex, there i s an i s o merization of the complex to give the active conformation. McClarin et a l . (13) suggested that "the isomerization plays the important functional role of enhancing the s p e c i f i c i t y of Eco RI endonucleases by a l l o s t e r i c activation." Halford and Johnson (14), i n an elegant series of single turnover experiments, have shown also that a conformational change i n the enzyme, after DNA i s bound, i s required before i t can cleave DNA. Depending on whether such a conformational change occurs i n one or both of the enzyme subunits, one or both strands of DNA are cleaved at the s c i s s i l e bonds during the lifetime of the enzyme-DNA complex. Under conditions used i n steady state experiments, the bonds i n both strands are cleaved. 2+

Catalysis by enzyme. The s p e c i f i c events resulting i n catalysis of hydrolysis of the phosphodiester bonds are not determinable d i r e c t l y from the X-ray crystallographic structure. Determinable are the physical shape changes (13). When double-stranded polynucleotides (including DNA) with the correct sequence of base pairs bind into the active s i t e of the enzyme, d i s t o r t i o n s of the double strands occur. These d i s t o r t i o n s are concentrated into separate features that are localized disruptions of the double h e l i c a l symmetry. Unwinding of the double helix results i n widening of the major groove of the polynucleotide. The phosphate-phosphate distances of the backbone of the polynucleotide are increased by approximately 3.5 Â, while there i s no change i n the distance between the purine and pyrimidine bases of the two strands. These physical changes result i n neokinks i n the chains. When Mg^ i s added to the complex, conformational changes (isomerization) occur resulting i n a c t i v a t i o n of the enzyme and cleavage of the s c i s s i l e bonds. Hydrolysis of the phosphodiester bond of the nucleotide occurs at the 3^-phosphate position ( F i g . 8). Most l i k e l y the mechanism i s a general acid-general base catalyzed reaction, such as i s known to occur with ribonuclease A where His12 serves as a general base and H i s l l 9 serves as the general acid ( F i g . 9). No covalent intermediate would be formed, which i s consistent with data on the mechanism of Eco RI endonuclease (10).

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch004

WHITAKER

Enzymes Used in Genetic Engineering

F i g u r e 5. α-Carbon structure of Eco RI endonuclease-DNA complex. The l e f t p a r t s h o w s a f r o n t v i e w w i t h d o u b l e - s t r a n d e d DNA (heavier white lines running v e r t i c a l l y through center) setting i n the c r e v i c e formed by t h e two s u b u n i t s of the enzyme. The r i g h t p a r t i s a p r o j e c t i o n f r o m t h e " t o p " w i t h a x i s 90° f r o m t h a t on t h e l e f t . ( T h e d o u b l e - s t r a n d e d DNA i s the c i r c u l a r s t r u c t u r e at bottom c e n t e r . ) (Reproduced with p e r m i s s i o n f r o m R e f . 13. C o p y r i g h t 1986 A m e r i c a n A s s o c i a t i o n f o r the Advancement of S c i e n c e . )

F i g u r e 6. S c h e m a t i c d r a w i n g o f t h e α-carbon b a c k b o n e o f one s u b u n i t o f E c o R I e n d o n u c l e a s e a n d b o t h s t r a n d s o f DNA i n t h e complex. ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m R e f . 13. Copyright 1986 A m e r i c a n A s s o c i a t i o n f o r t h e A d v a n c e m e n t o f S c i e n c e . )

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

56

BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

Arg 200?

ι I I I

N7

06 L

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch004

J

6

Arg 200a

Figure 7. Schematic representation of the hydrogen bond i n t e r ­ action of the deoxynucleotide sequence C-T-T-A-A-G with Argl45, Arg200 and Glul44 residues of the α and 3 subunits of Eco RI endonuclease. (Reproduced with permission from Ref. 13. Copy­ right 1986 American Association f o r the Advancement of Science.)

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Enzymes Used in Genetic Engineering

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch004

WHITAKER

Figure 9. Proposed transition state i n the ribonuclease Α-catalyzed hydrolysis of cytidine 2^-3^-phosphate. Two h i s t i d i n e residues of the enzyme participate i n c a t a l y s i s .

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

58

BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

McLaughlin et a l , ( 15) have recently examined effects of changes i n the functional group pattern of a series of polynucleotides for binding and catalysis by Eco RI. They reported that only the exocyclic amino group of the outer adenine residue of the substrate i s necessary for e f f i c i e n t catalysis although both are involved i n complex formation (13). They found that the endonuclease could discriminate between the inner and outer A-T base pairs. Furthermore, addition of an amino group to the minor groove of the nucleotide at the outer A-T base pair did not prevent binding to the enzyme active s i t e but the s c i s s i l e bond was not cleaved.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch004

DNA

LIGASES

DNA ligases catalyze the joining of two DNA chain segments or the closure of a single DNA chain, by condensation of the 5^-phosphoryl group with the adjacent 3^-hydroxyl group, coupled with hydrolysis of the pyrophosphate group of NAD (E. coLL enzyme) or ATP (T4 phage or eukaryotic enzymes). E. doti DNA ligase i s a single-chain protein of 74,000 daltons, thought to be somewhat elongated i n shape (16). The T4 DNA ligase i s also a single-chain protein of approximately 68,000 daltons, also elongated i n shape (17). Bacteriophage T7 DNA ligase i s 41,133 daltons (18), while mammalian c e l l s contain at least two DNA ligases of about 200,000 and 85,000 daltons (19). The mechanism of DNA ligase has been extensively studied as summarized by Lehman (20) and Engler and Richardson (21). The overall reaction catalyzed by E. colÂ, DNA ligase consists of three steps as shown i n Figure 10. In the f i r s t step, the ε-amino group of a l y s y l residue of the ligase, i n a nucleophilic attack on the adenylyl phosphorus of NAD , i s phosphorylated to give the adenylated ligase with elimination of nicotinamide mononucleotide (NMN). In the second step, the adenylyl group i s transferred from the enzyme to the 5^-phosphoryl terminus of the nicked DNA to form a pyrophosphate bond. In the third step the 3^-0H group, i n a nucleophilic attack on the 5^-phosphoryl group, forms a phospho­ diester bond, with elimination of AMP, thereby joining the two ends of the nicked chain. There are two covalent intermediates formed i n the reaction, both by nucleophilic attack mechanisms. T4 DNA ligase catalyzes a similar condensation reaction except that i n the f i r s t step there i s a nucleophilic attack by the ε-amino group of a l y s y l residue of the enzyme on the adenylyl phosphorus of ATP, with elimination of pyrophosphate (PP^. T4 DNA ligase ligates blunt ends of DNA where both strands have been nicked (22). Presumably, i t does this by duplicating the reaction ( F i g . 10) for both strands. Both ligases condense strands with cohesive (sticky) ends. +

THE POLYMERASES Both DNA and RNA polymerases have been isolated and studied. I s h a l l concentrate on DNA polymerase and s p e c i f i c a l l y DNA polymerase I from E. coti i n this paper.

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

3

+

Η

u

ο

ι ι

1

Ρ

I I

Ο-

>

1

1

0Η

θ Ν

1 1 \*

of

the

"

w

reaction

\

AMP J

Ρ Ο-

Ο

Ρ °"

J

Μ

θΝ

·Ε

1

I I I I

1111

catalyzed

by

I I I I I I 1^ο-?- | ι ι ι ι ι ι

ι ι ι ι ι ι

AR

Ο

π

1 ι ι ι ι ^ ιι ι ι ι ι ' " «' " Ι ^ ^ μ^"ί-

ο-

» I 1 I ι ι 1 I-

Μ

Ο-

1 11

Η

E-N-P-O-RA

Figure 10. Proposed mechanism E. Q.oti DNA ligase.

AR

ο' ο-

Ν

Ρ

'ΦΟΓΓ^Ο^

1 1

ΝΜΝ

ι ι ιι ι ι «' " '^ΟΗ ο, ,ο^'

•ι ι ι ιII

! I I ! I

E-N-P-0-RA+ ό-

+Η

ο- ο-

Ε-ΝΗ, + A R - 0 - P - O - P - O - R N

Ε - ΝΗ

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch004

Γ

60

BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

DNA polymerase I, obtained from wild-type E. coLi i n which there are about 400 molecules per bacterium, or by molecular cloning of the po£A gene i n a lambda transducing bacterium, i s a single-chain protein of 103,000 daltons. The protein i s approximately spherical, folded into three domains ( F i g . 11), and contains a substantial amount of α-helical structure ( F i g . 12)(23). The enzyme contains one sulfhydryl group which can be modified without loss of a c t i v i t y . The active center of the enzyme contains three d i s t i n c t but closely juxtaposed binding sites f o r the DNA template, the nucleotide primer and the deoxynucleoside triphos­ phates (24). The four deoxynucleoside triphosphates appear to be accommodated equally well i n the binding s i t e . There i s also a Mg -binding s i t e . DNA polymerase I can be cleaved by limited proteolysis into a 35,000-dalton fragment containing a l l the o r i g i n a l 5'-»· 3' exonuclease a c t i v i t y , and a 68,000-dalton fragment (Klenow enzyme) with a l l the polymerase and 3'+5' exonuclease a c t i v i t i e s . The exonuclease a c t i v i t i e s play a role i n editing out mismatches of nucleoside units and i n removing modified pyrimidine bases (damaged by UV l i g h t f o r example; 5'*3' exonuclease a c t i v i t y only). A schematic drawing of the three domains of DNA polymerase I, each containing one of the three a c t i v i t i e s , i s shown i n Figure 11. The portion of structural gene f o r E. (loti DNA polymerase I Klenow fragment has been cloned into an expression vector (25), permitting production of large amounts of this segment of the poly­ merase. X-ray d i f f r a c t i o n data indicate that the Klenow fragment has the t e r t i a r y structure shown i n Figure 12 (23). The N-terminal residues 1-323 (not shown) of DNA polymerase I are thought to con­ t a i n the 5'-^'-exonuclease a c t i v i t y ( F i g . 11). Residues 324-517, which provide the 3'+5'-exonuclease a c t i v i t y , consist of s i x α-helical regions (A-F) and f i v e 3-sheets (1-5), which are mostly parallel. The domain has a central core of β-pleated sheets (1-4) with α-helices on both sides. In c r y s t a l form, the enzyme binds one molecule of dTMP and a divalent metal ion i n this domain. The divalent metal ion i s bound to the protein by the carboxyl groups of Asp335, Asp501 and Glu357 and the 5"-phosphate of dTMP. The remainder of the molecule (residues 518-928) i s a domain with a very deep c l e f t , about 20-24 A wide and 25-35 Â deep (23). The bottom of the c l e f t i s formed from s i x a n t i p a r a l l e l 3-sheets and the walls of α-helices. Steitz and Joyce (23) compared the c l e f t to a structure similar i n shape to a right hand grasping a rod. "Thus, one side of the c l e f t forms a wall, 50 Â long, that can be compared with the curled-over fingers of a right hand. The other side of the c l e f t i s formed primarily by two long α-helices, I and H, projecting from the protein l i k e a thumb. At the t i p of the thumb-like protrusion and hanging over the crevice are 50 amino acid residues (not shown i n F i g . 12) that appear to be p a r t i a l l y disordered i n the c r y s t a l . " Evidence that this i s the binding s i t e for double-stranded DNA comes from model building based on the X-ray data (23) and from two mutations, pol5 and po£6, of DNA polymerase I that change amino acids i n the c l e f t region and change binding of DNA to the enzyme (26).

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch004

2+

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

WHITAKER

Enzymes Used in Genetic Engineering

polymerase

ϊ-5'exo

5-ïexo

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch004

KLENOW FRAGMENT

Figure 11. Schematic drawing of the three-domain structure of E. cotl DNA polymerase I. (See text for details.) (Reproduced with permission from Ref. 23. Copyright 1987 Alan R. L i s s , Inc.)

Figure 12. Tertiary structure of the Klenow fragment of E. cotl DNA polymerase I, based on X-ray crystallographic data. The α-helices are represented by tubes (lettered) and 3-sheets by arrows (numbered). (Reproduced with permission from Ref. 23. Copyright 1987 Alan R. L i s s , Inc.)

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

61

62

BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

E. cotl DNA polymerase shown i n Equation 1. Mg (Deoxynucleotide)

n

+ dNTP

I catalyzes

the elongation

reaction

2+

(Deoxynucleotide)

n+1

+ ??

±

(1)

As indicated above, the enzyme also possesses 3'+5" and 5'-»·3" exonuclease a c t i v i t i e s (24); the 5"+3* exonuclease a c t i v i t y i s not present i n the large proteolytic (Klenow) fragment (25). For DNA synthesis, a l l four deoxyribonucleoside 5'-triphosphates (dATP, dGTP, dCTP and dTTP) must be present, as well as Mg , a primer molecule ((deoxynucleotide) shown i n Equation 1) and a DNA template. A detailed mechanism of the reaction must consider that the enzyme binds at least four compounds simultaneously, these being a deoxynucleoside 5'-triphosphate, Mg , a deoxynucleotide primer molecule and a DNA template. The enzyme i s thus able to convert an impossible f i v e component reactant system into a unimolecular reaction, contributing substantially to the e f f i c i e n c y of the catalyzed reaction. More d e t a i l of the elongation reaction catalyzed by DNA polymerase I is shown i n Equation 2.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch004

n

OH 5'-C-C-G^ . · · 3'-G-G-C-T-A-T-C-G-A...

„ _ „ 5'-C-C-G-A-T-A-G-C-T... 2dATP 2+ dCTP î * — > : : : : : : : : : (2) dGTP , * 3'-G-G-C-T-A-T-C-G-A... 2dTTP i J 4

M

+

b

The substrate i s a DNA template (bottom strand) annealed to a DNA primer possessing a 3"-OH terminus. The enzyme adds the nucleoside triphosphates to the 3'-OH terminus of the DNA primer i n the order specified by the DNA template. The chemistry of the elongation reaction catalyzed by DNA polymerase I i s shown i n Figure 13. The enzyme catalyzes the nucleophilic attack of the 3"-0H terminus of the primer molecule on the α-phosphorus of the deoxyribonucleoside triphosphate to form a new phosphodiester bond with release of pyrophosphate. Elongation of the DNA chain proceeds i n the 5*+3' d i r e c t i o n at a rate of approximately ten nucleotides per second per molecule of DNA polymerase I. It i s thought that the reaction i s processive, i n that many nucleotide units are added without release of the enzyme from the template. Other DNA polymerases are known. DNA polymerase II and I I I are produced by the polB and dnaA genes of E. cotl. They are similar to DNA polymerase I i n most properties. They d i f f e r however, i n template preferences. While DNA polymerase I acts best to f i l l i n extended single-stranded regions near double-helical regions, DNA polymerases II and I I I act optimally on double­ nt randed DNA templates that have short gaps.

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch004

WHITAKER

Enzymes Used in Genetic Engineering

I Primer strand I

| Primer strand |

Ο

Ο

HO

Figure 13. Proposed DNA polymerase I.

Η

mechanism

of

the reaction

catalyzed

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

by

64

BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

LITERATURE CITED

1. 2. 3.

4. 5. 6.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch004

7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

Korn, D.; Fisher, P.A.; Battey, J.; Wong, T.S.-F. Cold Spring Harb. Symp. Quant. Biol. 1978, 43, 613. McHenry, C; Romberg, A. J. Biol. Chem. 1977, 252, 6478. Ausubel, F.M.; Brent, R.; Kingston, R.E.; Moore, D.D.; Smith, J.A.; Seidman, J.G.; Struhl, K. Current Protocols in Molecular Biology; Greene Publishing Associates and Wiley-Interscience: New York, 1987; p. 1.5.3. Meselson, M.; Yuan, R. Nature (London) 1968, 217, 1110. Linn, S.; Arber, W. Proc. Nat. Acad. Sci. USA 1968, J59, 1300. Greene, P.J.; Gupta, M.; Boyer, H.W.; Brown, W.E.; Rosenberg, J.M. J. Biol. Chem. 1981, 256, 2143. Newman, A.K.; Rubin, R.A.; Kim, S.-H.; Modrich, P. J. Biol. Chem. 1981, 256, 2131. Modrich, P.; Zabel, D. J. Biol. Chem. 1976, 251, 5866. Jen-Jacobsen, L.; Kurpiewski, M.; Lesser, D.; Grable, J.; Boyer, H.W.; Rosenberg, J.M.; Greene, P.J. J. Biol. Chem. 1983, 258, 14638. Connolly, B.A.; Eckstein, F.; Pingoud, A. J. Biol. Chem. 1984, 259, 10760. Modrich, P. Q. Rev. Biophys. 1979, 12, 315. Halford, S.E.; Johnson, N.P. Biochem. J. 1980, 191, 593. McClarin, J.Α.; Frederick, C.A.; Wang, B.-C.; Greene, P.; Boyer, H.W.; Grable, J.; Rosenberg, J.M. Science 1986, 234, 1526. Halford, S.E.; Johnson, N.P. Biochem. J. 1983, 211, 405. McLaughlin, L.W.; Benseler, F.; Graeser, E.; Piel, N.; Scholtissek, S. Biochemistry 1987, 26, 7238. Modrich, P.; Anraku, Y.; Lehman, I.P. J. Biol. Chem. 1973, 248, 7495. Panet, Α.; van de Sande, J.H.; Loewen, P.C.; Khorana, H.G.; Raae, A.J.; Lillehaug, J.R.; Kleppe, K. Biochemistry 1973, 12, 5045. Dunn, J.J.; Studier, F.W. J. Mol. Biol. 1981, 148, 303. Soderhall, S.; Lindahl, T. FEBS Lett. 1976, 67, 1. Lehman, I.R. In The Enzymes, Third Edition; Boyer, P.D., Ed.; Academic: New York, 1974; Vol. 10, p 237. Engler, M.J.; Richardson, C.C. In The Enzymes, Third Edition; Boyer, P.D., Ed.; Academic: New York, 1982; Vol. 15B, p 3. Sgaramella, V.; van de Sande, J.H.; Khorana, H.G. Proc. Nat. Acad. Sci. USA 1970, 67, 1468. Steitz, T.A.; Joyce, C.M. In Protein Engineering; Oxender, D.L.; Fox, C.F., Eds.; Alan R. Liss, Inc.: New York, 1987; p 227. Kornberg, A. DNA Replication; Freeman: San Francisco, CA, 1980. Joyce, C.M.; Grindley, N.D.F. Proc. Nat. Acad. Sci. USA 1983, 80, 1830. Joyce, C.M.; Fujii, D.N.; Laks, H.F.; Hughes, C.M.; Grindley, N.D.F. J. Mol. Biol. 1985, 186, 283.

RECEIVED

December 6, 1988

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 5

Plant Cells for Secondary Metabolite Production

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch005

Dietrich Knorr Department of Food Technology, Berlin University of Technology, D—1000 Berlin 33, Federal Republic of Germany, and Biotechnology Group, Department of Food Science, University of Delaware, Newark, DE 19716 Plant cell cultures represent a potentially rich source of secondary metabolites of commercial importance and have been shown to produce them in higher concentrations than the related intact plants. However, plant cell cultures often produce metabolites in lower concentrations than desired and commonly store them intracellularly. These limitations can be overcome by product yield enhancement procedures, including immobilization of cultured cells, and permeabilization, or ideally using a combined immobilization/ permeabilization process with retained plant cell viability. Complex coacervate capsules consisting of chitosan and alginate or carrageenan proved to be effective biomaterials for entrapment, controlled permeabilization of cells and to allow control of capsule membrane diffusivity. Using immediate precursors of desired food aroma compounds also increased metabolite yields. For example, by applying ferulic acid to cultured Vanilla planifolia cells, vanillin concentration could be enhanced as compared to untreated cells. Vanilla concentration was also increased in green vanilla bean extracts when the cells were treated with β-glucosidase. M i c r o b i a l p o l y s a c c h a r i d e s have been shown t o s t r e s s p l a n t c e l l s , r e s u l t i n g i n ' e l i c i t a t i o n ' ( i n d u c t i o n ) and increased metabolite synthesis. Induction of various enzymes has been r e p o r t e d . Chitosan s u c c e s s f u l l y e l i c i t e d c h i t i n a s e p r o d u c t i o n i n c a r r o t (Daucus c a r o t a ) c e l l c u l t u r e s and e l i c i t a t i o n o f d e s i r e d food i n g r e d i e n t s and p r o c e s s i n g a i d s v i a c h i t o s a n has been a t t e m p t e d .

β

0097-6156/89/0389-0065$06.00/0 1989 American Chemical Society

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch005

66

BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

The b a s i c concept o f t o t i p o t e n c y l e a d i n g t o t h e r e c e n t advances o f p l a n t c e l l c u l t u r e was f o r m u l a t e d i n 1838 (1) and 1839 ( 2 ) . I n 1902 H a b e r l a n d t ( 3 ) r e p o r t e d h i s p i o n e e r i n g e x p e r i m e n t s on m a i n t a i n i n g p l a n t c e l l s i n a v i a b l e s t a t e away from t h e p a r e n t body by b a t h i n g them i n s i m p l e n u t r i e n t s o l u t i o n s . S i n c e t h e n , t h e s y n t h e t i c p o t e n t i a l o f p l a n t c e l l c u l t u r e s was e s t a b l i s h e d ( 4 ) . Large s c a l e p r o d u c t i o n o f p l a n t biomass from c u l t u r e d c e l l s was suggested as e a r l y a s 1962 ( 5 ) . P l a n t c e l l c u l t u r e s can now be e s t a b l i s h e d f o r a wide range o f h i g h e r p l a n t s s p e c i e s ( 6 ) and r e p r e s e n t a p o t e n t i a l l y r i c h s o u r c e of c o m m e r c i a l l y i m p o r t a n t secondary m e t a b o l i t e s (7_,8). P l a n t c e l l and organ c u l t u r e s c a n produce h i g h e r m e t a b o l i t e c o n c e n t r a t i o n s t h a n found i n t h e c o r r e s p o n d i n g i n t a c t p l a n t organs ( 6 , 9 ) . However, p l a n t c e l l s grown i n c u l t u r e may a l s o produce lower q u a n t i t i e s o f t h e d e s i r e d secondary m e t a b o l i t e s w h i c h a r e commonly stored i n t r a c e l l u l a r l y . The c h a l l e n g e s t o i n c r e a s e p r o d u c t y i e l d and t o enhance t h e r e l e a s e o f secondary m e t a b o l i t e s c a n be met i n v a r i o u s ways (7_). These i n c l u d e i m m o b i l i z a t i o n ( 9 ) , p e r m e a b i l i z a t i o n (10,11), t h e use o f p r e c u r s o r s ( 1 2 , 1 3 ) , and t h e i n d u c t i o n o f secondary m e t a b o l i t e p r o d u c t i o n v i a e l i c i t o r s ( 1 4 ) . Immobilization

of Cultured P l a n t

Cells

Nunez and Lema (15) viewed t h e main o b j e c t i v e i n i m m o b i l i z i n g a b i o l o g i c a l l y a c t i v e agent as t h e c o n c e n t r a t i o n o f i t s a c t i v i t y i n as s m a l l a volume as p o s s i b l e . T h i s r e q u i r e s a p r o c e d u r e ( 1 6 ) w h i c h c o n f i n e s a c a t a l y t i c a l l y a c t i v e enzyme o r c e l l i n s i d e a r e a c t o r system t h a t p r e v e n t s i t s e n t e r i n g t h e m o b i l e phase c a r r y i n g t h e s u b s t r a t e and t h e p r o d u c t . Some o f t h e advantages o f p l a n t c e l l i m m o b i l i z a t i o n a r e l i s t e d i n T a b l e I . The advantages o f c e l l i m m o b i l i z a t i o n have l o n g been r e c o g n i z e d i n c l u d i n g i t s importance i n n i t r o g e n f i x a t i o n i n legumes, t h e adherence o f f l u v i a l m i c r o o r g a n i s m s t o sand and s t o n e o r t h e p r o d u c t i o n o f v i n e g a r u s i n g woodshavings as s u p p o r t medium ( 1 5 ) . T a b l e I . Some advantages o f p l a n t c e l l Advantage

immobilization

a

Reason/Consequence

Reuse o f b i o c a t a l y s t

entrapment

Maintenance o f s t a b l e and active biocatalyst

e l i m i n a t i o n of non-productive growth phase; reduced r i s k o f d e c l i n i n g p r o d u c t i v i t y from a selected c e l l l i n e

P r o t e c t i o n o f c e l l s by m a t r i x

s i m p l i f i c a t i o n o f s c a l e up; p r o t e c t i o n from p h y s i c a l damage

Employment o f c o n t i n u o u s p r o c e s s

c o n t i n u o u s removal o f m e t a b o l i c i n h i b i t o r s ; improved p r o c e s s c o n t r o l and p r o d u c t r e c o v e r y

Increased

increased c e l l d e n s i t i e s ; e l i m i n a t i o n o f growth phase

a

productivity

Adapted from ( 9 , 17)

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch005

5.

KNORR

Plant Cells for Secondary Metabolite Production

67

Polymers commonly used f o r p l a n t c e l l i m m o b i l i z a t i o n i n c l u d e agar, agarose, a l g i n a t e , carrageenan, c h i t o s a n , g e l a t i n , polya c r y l a m i d e , p r e p o l y m e r i z e d p o l y a c r y l a m i d e (Γ7,18) and p o l y u r e t h a n e foam ( 1 9 ) . Complex c o a c e r v a t e g e l c a p s u l e s have a l s o been developed (8,10,20-22) c o n s i s t i n g of c o u p l e d c h i t o s a n - a l g i n a t e or c h i t o s a n carrageenan networks ( 2 3 ) . Entrapment methods have been used almost e x c l u s i v e l y t o i m m o b i l i z e p l a n t c e l l s ( 1 7 ) . They can be c l a s s i f i e d i n t o t h r e e g e n e r a l groups (24,25): a. g e l f o r m a t i o n by i o n i c c r o s s l i n k i n g of a charged polymer ( i o n o t r o p i c ) ; b. g e l f o r m a t i o n by c o o l i n g of a heated polymer ( t h e r m a l ) , and c. g e l f o r m a t i o n by c h e m i c a l r e a c t i o n (cross-linking, r a d i c a l polymerization). I o n i c c r o s s l i n k i n g i n v o l v i n g marine p o l y s a c c h a r i d e s , such as a l g i n a t e , c a r r a g e e n a n , or c h i t o s a n , as charged p o l y e l e c t r o l y t e s w i t h c a l c i u m , p o t a s s i u m or polyphosphate i o n s as low m o l e c u l a r weight c o u n t e r i o n s , have r e c e i v e d much a t t e n t i o n ( 9 ) . The p o l y c a t i o n i c n a t u r e of c h i t o s a n , as opposed t o the p o l y a n i o n i c p r o p e r t i e s of most o t h e r marine polymers, p r o v i d e s v a r i o u s unique p r o p e r t i e s and o f f e r s a wide range of a p p l i c a t i o n s i n food b i o t e c h n o l o g y (26,27). The p o l y c a t i o n i c p r o p e r t i e s o f c h i t o s a n p e r m i t i t s use as a h i g h m o l e c u l a r weight c o u n t e r i o n i n p o l y e l e c t r o l y t e complex f o r m a t i o n r e s u l t i n g i n a c o u p l e d network c o n s i s t i n g of c h i t o s a n and one or more p o l y a n i o n i c polymers ( F i g u r e 1). M e c h a n i c a l s t a b i l i t y of the complex c o a c e r v a t e c a p s u l e s ( w i t h a l i q u i d c o r e ) was improved by a c o m b i n a t i o n o f h i g h and low m o l e c u l a r weight c o u n t e r i o n s , as w e l l as by the a d d i t i o n of g l u c o s e (Table I I ) . The use o f g l u c o s e has a d d i t i o n a l p r a c t i c a l relevance a l l o w i n g shorter r e a c t i o n times f o r bead f o r m a t i o n w h i c h i s e s s e n t i a l f o r the v i a b i l i t y of the entrapped c e l l s , and lower a l g i n a t e c o n c e n t r a t i o n s , which - due t o the d e c r e a s e d v i s c o s i t y of the a l g i n a t e s o l u t i o n - p r o v i d e s b e t t e r c o n d i t i o n s f o r capsule formation. The m e c h a n i c a l s t a b i l i t y of the c h i t o s a n / a l g i n a t e g e l capsules decreases w i t h i n c r e a s i n g e s t e r i f i c a t i o n of a l g i n a t e but g e l s t a b i l i t y was improved by a u t o c l a v i n g the c a p s u l e s i n T r i z m a ( t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e ) b u f f e r s o l u t i o n (Table I I I ) . T h i s i s of i m p o r t a n c e f o r food b i o t e c h n o l o g y a p p l i c a t i o n s of the complex c o a c e r v a t e c a p s u l e s (e.g. m i c r o e n c a p s u l a t i o n ) where p r o d u c t s r e q u i r e a u t o c l a v i n g . S i n c e the g e l m a t r i x p r o v i d e s a mass t r a n s p o r t b a r r i e r , i t can a f f e c t v i a b i l i t y of entrapped p l a n t c e l l s as w e l l as the r e l e a s e of desired plant metabolites. C o n s e q u e n t l y , adequate d i f f u s i v i t y of the polymer network i s e s s e n t i a l f o r development of i m m o b i l i z e d p l a n t c e l l r e a c t o r s . C a p s u l e w a l l p r o p e r t i e s of c h i t o s a n - a l g i n a t e c o a c e r v a t e g e l c a p s u l e s a r e a f f e c t e d by pH, b u f f e r t r e a t m e n t i n c l u d i n g p h t h a l a t e , borax and t r i s ( h y d r o x y m e t h y l ) amino- methane b u f f e r , as w e l l as by v a r i o u s t y p e s of polymers (20, 21_, Pandya, Y. and K n o r r , D., U n i v . of Delaware, u n p u b l i s h e d d a t a ) . F o r example, degree o f e s t e r i f i c a t i o n of a l g i n a t e s used ( 2 0 ) , c h i t o s a n c o n c e n t r a t i o n and degree o f d e a c e t y l a t i o n of the c h i t o s a n ( r e s u l t i n g i n c h i t o s a n p r o d u c t s of d i f f e r e n t m o l e c u l a r weight and v i s c o s i t y ) were shown t o a l t e r g e l d i f f u s i v i t y . The e f f e c t s of c h i t o s a n c o n c e n t r a t i o n and v i s c o s i t y of a l g i n a t e - c h i t o s a n c o a c e r v a t e c a p s u l e s on the r e l e a s e o f low m o l e c u l a r w e i g h t food dyes from such c a p s u l e s , i s shown i n F i g u r e 2. A l s o , s i g n i f i c a n t d i f f e r e n c e s i n g e l p e r m e a b i l i t y o c c u r r e d when the a l g i n a t e o f the complex c o a c e r v a t e g e l

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch005

Plant cells (20 - 30 percent loading density)

(2.5 - 3 percent chitosan in 260 mM citric acid plus 450 mM CaCI ) or 2

Polymer solution

(2 - 3 percent Na alginate plus 3 percent sodium hexametaphosphate) droplet formation via syringe or peristaltic pump

counterion solution

0.75 percent alginate plus 2.25 percent glucose or 1 percent chitosan in 3 percent ascorbic acid

stir for 30 minutes

wash with sterile, distilled water •

transfer into nutrient medium ,

immobilized cell reactor Figure 1. S i m p l i f i e d protocol of chitosan/alginate or a l g i n a t e / chitosan complex coacervate capsule formation (adapted from 10.20.22)

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

5.

KNORR

Plant Cells for Secondary Metabolite Production

69

T a b l e I I . E f f e c t of C a l c i u m C h l o r i d e and G l u c o s e on M e c h a n i c a l S t a b i l i t y o f C h i t o s a n - a l g i n a t e Coacervate G e l C a p s u l e s 3

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch005

Polyelectrolyte (capsule core)

Setting Time (hrs)

Force t o Burst (Newtons)^

Counterion ( o u t e r membrane)

Reaction Time (min)

3.0% c h i t o s a n

2.0% N a - a l g i n a t e

20

3.0

0.73+0.30

3.0% c h i t o s a n + 460 mM C a C l

2.0% N a - a l g i n a t e

5

3.0

0.10+ 0.05

3.0% c h i t o s a n + 460 mM C a C l

2.0% N a - a l g i n a t e

15

3.0

0.97+0.61

3.0% c h i t o s a n + 460 mM C a C l

2.0% N a - a l g i n a t e

20

3.0

10.8 + 5.70

3.0% c h i t o s a n + 460 mM C a C l

2.0% N a - a l g i n a t e + 6.0% g l u c o s e

10

0.5

9.8 + 6 . 1 0

3.0% c h i t o s a n + 460 mM C a C l

2.0% N a - a l g i n a t e + 6.0% g l u c o s e

15

0.5

16.5 + 3.00

3.0% c h i t o s a n + 460 mM C a C l

2.0% N a - a l g i n a t e + 6.0% g l u c o s e

20

0.5

11.6 + 5.70

3.0% c h i t o s a n + 460 mM C a C l

1.0% N a - a l g i n a t e + 3.0% g l u c o s e

15

0.5

24.5 + 2.40

3.0% c h i t o s a n + 460 mM C a C l

1.0% N a - a l g i n a t e + 3.0% g l u c o s e

15

3.0

24.8 +10.50

3.0% c h i t o s a n + 460 mM C a C l

0.5% N a - a l g i n a t e + 2.25% g l u c o s e

15

0.5

19.4 + 7.10

3.0% c h i t o s a n + 460 mM C a C l

0.5% N a - a l g i n a t e + 2.25% g l u c o s e

15

24.5

17.2 +10.20

2

2

2

2

2

2

2

2

2

2

Average c a p s u l e diameter 5.7 mm; adapted from ( 2 0 ) Mean ±_ s t a n d a r d d e v i a t i o n (N > 5)

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

70

BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

c a p s u l e s was r e p l a c e d by kappa-carrageenan (Pandya, Y. and K n o r r , U n i v . of Delaware, u n p u b l i s h e d d a t a ) .

D.,

T a b l e I I I . E f f e c t of b u f f e r t r e a t m e n t and e s t e r i f i c a t i o n of a l g i n a t e on the m e c h a n i c a l s t a b i l i t y of c h i t o s a n / a l g i n a t e c o a c e r v a t e c a p s u l e s 3

Setting s o l u t i o n P e r c e n t

of a l g i n a t e c a r b o x y l groups e s t e r i f i e d ^ 0

5

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch005

Pressure

D i s t i l l e d water 100 mM

CaCl

2

100 mM T r i z m a + 100 mM C a C l

d

to

12.5 burst

0

_

23.6

6.0

172.5

119.6

37.2

149.9

108.9

37.8

189.3

176.6

100.1

2

d

100 mM T r i z m a + 100 mM C a C l

e 2

Adapted from (21) b E s t e r i f i c a t i o n a c h i e v e d by s u b s t i t u t i n g 50% e s t e r i f i e d p r o p y l e n e g l y c o l a l g i n a t e f o r 0, 10 and 25% of sodium a l g i n a t e c χ 102 P a s c a l ; Per i n i t i a l c r o s s - s e c t i o n a l a r e a of c a p s u l e T r i s (hydroxymethyl)aminomethane C a p s u l e s were a u t o c l a v e d a

d

e

P e r m e a b i l i z a t i o n of P l a n t C e l l s Secondary m e t a b o l i t e s produced by p l a n t c e l l c u l t u r e a r e commonly accumulated i n the c e l l s . W i t h few e x c e p t i o n s such as Capsicum f r u t e s c e n s , T h a l i c t r u m minus (9) and V a n i l l a p l a n i f o l i a ( K n o r r , D. and Romagnoli, L., U n i v . of Delaware, u n p u b l i s h e d d a t a ) c u l t u r e s , which r e l e a s e v a l u a b l e compounds such as c a p s a i c i n , b e r b e r i n e and v a n i l l i n , r e s p e c t i v e l y , i n t o the medium, p r o c e d u r e s t o i n d u c e p r o d u c t r e l e a s e are r e q u i r e d to develop continuous production processes. R e p o r t e d p e r m e a b i l i z a t i o n methods i n c l u d e t r e a t m e n t w i t h d i m e t h y l s u l f o x i d e (DMS0), i s o p r o p a n o l , t o l u e n e , p h e n e t h y l a l c o h o l or c h l o r o f o r m (9_, 28). But as F o n t a n e l and Tabata (9) p o i n t e d o u t , such t r e a t m e n t s w i t h o r g a n i c s o l v e n t s are s e v e r e and o t h e r methods of p e r m e a b i l i z a t i o n need t o be developed. Based on the p i o n e e r i n g work on c h i t o s a n and i n d u c e d p l a n t membrane p e r m e a b i l i t y i n c u l t u r e d G l y c i n e max c e l l s conducted by Young et a l . ( 2 9 ) , the r e l e a s e of secondary m e t a b o l i t e s from v a r i o u s c e l l c u l t u r e s i n t o the r e l a t e d n u t r i e n t medium was made p o s s i b l e i n the presence of c h i t o s a n (18, 22, 30, 31^, Beaumont, M. and K n o r r , D., U n i v . of Delaware, u n p u b l i s h e d d a t a ) . Leuba and S t o s s e l (32) suggested t h a t the r e l e a s e of C a and subsequent e f f l u x of + +

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


( E q n . 1) CH COCOH(COOH)CH 3

5

+ *0

2

-> CH3COCOCH3 + C 0

2

+ H-,0

(1)

T y p i c a l l y the l e v e l of d i a c e t y l might be 0 . 1 0 - 0 . 1 5 m g / L , w e l l above the t a s t e t h r e s h h o l d l e v e l of 0.0T> mg/L. The purpose of the s e c o n d a r y f e r m e n t a t i o n i s to a l l o w t h e e x c e s s a c e t o l a c t a t e that d i f f u s e d out of the y e a s t c e l l s to d i f f u s e back i n and be reduced to a c e t o i n , before it is spontaneously c o n v e r t e d to d i a c e t y l ; that secondary f e r m e n t a t i o n can take s e v e r a l weeks ( J . Power, J . E . S i e b e l & Sons, P e r s o n a l Communication). I f a c e t o l a c t a t e d e c a r b o x y l a s e i s used i t then decarboxylat.es the a c e t o l a c t a t e to produce C 0 and a c e t o i n i n 9

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

178

BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

days. The a c e t o i n may be f u r t h e r reduced by the a p p r o p r i a t e dehydroge­ nase t o 2,3-butylene g l y c o l , which doesn't a f f e c t t h e f l a v o r o f beer i n the c o n c e n t r a t i o n s encountered (Eqn. 2 ) . CH COH(C0 "")COCH

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch012

3

2

3

+

H

+

->

CH3-CHOH-CO-CH3

+

C0

2

(2)

T h i s c a n be done i n a day or two. A unique t w i s t t o t h i s has been added by The T e c h n i c a l R e s e a r c h C e n t e r o f F i n l a n d ( J . K n o w l e s , p e r s o n a l communication g i v i n g d e t a i l s o f F i n n i s h P a t e n t A p p l i c a t i o n ) . The gene r e s p o n s i b l e f o r t h e a c e t o l a c t a t e d e c a r b o x y l a s e production was removed from Aerobacter aerogenes and s p l i c e d i n t o a s t r a i n o f Saccharomyces used f o r brewing; t h e r e s u l t i s c o n v e r s i o n o f the excess a c e t o l a c t a t e t o a c e t o i n i n t h e p r i m a r y f e r m e n t a t i o n . With t h e e n g i ­ neered y e a s t , d i a c e t y l l e v e l s of about 0.01 mg/L o r l e s s a r e achieved w i t h o u t a secondary f e r m e n t a t i o n . By making the secondary f e r m e n t a t i o n unnecessary, t h i s engineered y e a s t reduces the o v e r a l l beer p r o d u c t i o n time from about 5 weeks t o 2 weeks, w i t h no o t h e r brewing p r o p e r t i e s affected. T h i s same l a b has a l s o c o n s t r u c t e d two g l u c a n o l y t i c b r e w i n g y e a s t s by t r a n s f e r i n g a s i n g l e β-glucanase gene i n t o t h e y e a s t ; the r e s u l t a n t beer i s v i r t u a l l y d e v o i d of β-glucans and f i l t e r s much more e a s i l y ( 3 ) . ENZYMES USED IN FLAVOR MODIFICATION Cheese/butter f l a v o r . P r e g a s t r i c l i p a s e s , have, been used f o r years t o i n t e n s i f y f l a v o r i n "enzyme-modified cheese", and f o r an i n t e n s i f i e d butter f l a v o r i n l i p o l y z e d b u t t e r . G e n e r a l l y the f a t t y acid residues t h a t need t o be s p l i t o f f ( t o generate t h e r i g h t f l a v o r ) a r e the s h o r t c h a i n f a t t y a c i d s , e s p e c i a l l y the C ^ t o C-JQ a c i d s t y p i c a l o f I t a l i a n cheeses. The b u t y r i c a c i d s a r e produced from b u t t e r f a t more s p e c i f i ­ c a l l y by newly developed l i p a s e s ( r e a l l y e s t e r a s e s ) from Mucor meihei and a v e r y new one, from Aspergillus oryzae, e s p e c i a l l y f o r cheddar cheese f l a v o r development. The l a t t e r enzyme i s marketed under t h e name " F l a v o r Age" ( 4 ) . F l a v o r s produced i n t h i s manner a r e used w i d e l y i n c h e e s e - f l a v o r e d snack foods; t h e v a l u e o f t h e i n t e n s i f i e d cheese f l a v o r s i s on the order o f $50 m i l l i o n , b u t t h e v a l u e o f the enzymes employed i s o n l y about $2-3 m i l l i o n . Cheese f l a v o r s a r e not o n l y developed by f a t m o d i f i c a t i o n , or f r e e f a t t y a c i d s , b u t a l s o by the m o d i f i c a t i o n o f the m i l k p r o t e i n s by endopeptidases. An aminopeptidase from Lactobacillus lactis i s being used t o h a l v e the r i p e n i n g time o f hard cheeses, l i k e cheddar ( 5 ) . F l a v o r E s t e r s . Some o f t h e newest a p p l i c a t i o n s o f l i p a s e s / e s t e r a s e s i n v o l v e the production of f l a v o r e s t e r s , such as e t h y l b u t y r a t e ( p i n e a p p l e f l a v o r ) , which c a n be s y n t h e s i z e d by l i p a s e s suspended i n o r g a n i c s o l v e n t s such as heptane or l o n g e r a l k a n e s ( 6 ) . The water o f h y d r a t i o n i n the enzyme p r e p a r a t i o n i s s u f f i c i e n t f o r t h e r e a c t i o n t o proceed, i n most cases. The r e a c t i o n i s a r e v e r s a l o f t h e h y d r o l y s i s r e a c t i o n l i p a s e s / e s t e r a s e s are t r a d i t i o n a l l y used f o r ; the r e a c t i o n i s an enzymatic c o n d e n s a t i o n combining an o r g a n i c a c i d w i t h an a l c o h o l . A p r e f e r r e d l i p a s e i s d e r i v e d from Candida cylindracea (7). Y e a s t - d e r i v e d meaty f l a v o r . Y e a s t e x t r a c t i s a n i m p o r t a n t food i n g r e d i e n t used p r i m a r i l y f o r i t s meaty f l a v o r . The c o n v e n t i o n a l method f o r p r o d u c t i o n from the yeast Saccharomyces cerevisiae i s done by p l a s m o l y s i s , f o l l o w e d by a u t o l y s i s by t h e y e a s t ' s own enzymes, p a s t e u r i z a t i o n , f i l t r a t i o n , c l a r i f i c a t i o n , arid d r y i n g . The t y p i c a l y i e l d i s about 70% o f the dry weight o f the y e ^ t . The enzymes thought t o be a c t i v e i n t h e a u t o l y s i s process a r e p r o t e a s e s ; i t has been

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch012

12.

SCOTT

Specialty Enzymes ami Products for the Fori

179

common t o supplement the n a t i v e enzyme w i t h p a p a i n , t o a c c e l e r a t e t h e p r o c e s s . Other d e p o l y m e r i z i n g enzymes from t h e y e a s t a r e a l s o i n v o l v ­ ed. An Arthrobacter luteus p r e p a r a t i o n known as "Zymolase" ( 8 ) i s r e p o r t e d l y e f f e c t i v e i n y e a s t l y s i n g because i t c o n t a i n s a β-glucan l a m i n a r i p e n t a o h y d r o l a s e t h a t has a h i g h a f f i n i t y f o r i n s o l u b l e g l u c a n . We know we have enzyme p r e p a r a t i o n s t h a t work, but we a r e n o t y e t c e r t a i n we know a l l o f the enzymes present o r i n v o l v e d . A d d i t i o n o f a p r e p a r a t i o n w i t h h i g h endo- and exo β-glucanase a c t i v i t i e s , with p r o t e a s e a c t i v i t y t o o , from t h e p l a n t pathogen Rhizoctonium solani (Basidiomycete aphyllophorales) r a i s e d t h e y i e l d t o 9 5 % ( 9 - 1 0 ) . The added y i e l d p r o b a b l y comes from t h e y e a s t c e l l w a l l ( 1 5 % o f t h e d r y weight o f the y e a s t ) and s o l u b i l i z a t i o n o f o t h e r c e l l components. Of course t h e important t h i n g about yeast e x t r a c t i s t h e f l a v o r as w e l l as t h e y i e l d . D i f f e r e n t p r o c e s s e s , e s p e c i a l l y t h e p a s t e u r i z a t i o n and drying that r e s u l t i nM a i l l a r d browning, g i v e t h e v a r i o u s yeast e x t r a c t s t h e i r i n d i v i d u a l i t i e s f o r food a p p l i c a t i o n s . CYCLODEXTRINS Cyclodextrins a r e non-reducing c y c l i c m o l e c u l e s w i t h 6-8 g l u c o s e r e s i d u e s arranged i n a doughnut shape. They e f f e c t i v e l y t r a p many d i f f e r e n t k i n d s o f molecules i n t h e h o l e o f t h e doughnut, a p r o c e s s now c a l l e d " i n c l u s i o n c o m p l e x a t i o n " . C y c l o d e x t r i n s a r e made from starch u s i n g c y c l o m a l t o d e x t r i n g l u c a n o t r a n s f e r a s e (CGTase) o b t a i n e d from

Bacillus

macerans

and o t h e r

Bacillus

sp.

(11).

The p r o c e s s used by American Maize begins w i t h p o t a t o o r waxy maize s t a r c h c o n v e r s i o n t o m a l t o d e x t r i n s and then s u b j e c t s these t o the a c t i o n s o f CGTase. T h i s enzyme h y d r o l y z e s o f f t h e 6-, 7-, o r 8g l u c o s y l r e s i d u e s and then c o v a l e n t l y a t t a c h e s the r e d u c i n g end t o t h e non-reducing end. The t h r e e d i f f e r e n t c y c l o d e x t r i n s can be s e p a r a t e d by e i t h e r s o l v e n t f r a c t i o n a t i o n o r u l t r a f i l t r a t i o n . Whereas s t a r c h c o n t a i n s c h a i n s o f between one and t h r e e m i l l i o n g l u c o s y l r e s i d u e s , the m a l t o d e x t r i n s u b j e c t e d t o CGTase a c t i o n h a s o n l y a b o u t 14-70 g l u c o s y l r e s i d u e s (G. A. Reed, A m e r i c a n M a i z e , p e r s o n a l communi­ c a t i o n ) , and the c y c l o d e x t r i n has 6, 7, o r 8 g l u c o s y l groups. These compounds o f f e r s u b s t a n t i a l promise because o f t h e hydrop h i 1 i c - h y d r o p h o b i c n a t u r e , and t h e a b i l i t y t o p r o t e c t many d i f f e r e n t k i n d s o f molecules a g a i n s t d e t e r i o r a t i o n o r e v a p o r a t i o n . LOW SWEETNESS SWEETENERS Low sweetness sweeteners a r e o f i n t e r e s t because they are g e n e r a l l y m e t a b o l i z e d i n t h e body y e t do not c o n t r i b u t e t o d e n t a l c a r i e s , and tend t o have a lower e f f e c t on b l o o d g l u c o s e i n d i a b e t i c s . Four e x a m p l e s a r e d i s c u s s e d h e r e . Where s w e e t n e s s e q u a l t o s u c r o s e i s d e s i r e d , a h i g h i n t e n s i t y sweetener c a n be mixed w i t h i t i n most c o u n t r i e s . However low sweetness c a l o r i c sweeteners are o f p a r t i c u l a r i n t e r e s t f o r persons w i t h k i d n e y d i s e a s e , where i t i s o f t e n d i f f i c u l t t o get them t o take enough c a l o r i e s . Use o f g l u c o s e syrups t o " s t u f f c a l o r i e s " w i t h l i t t l e water i s o f t e n u n a c c e p t a b l e t o t h e p a t i e n t because o f the e x c e s s i v e l y sweet t a s t e . Perhaps someone w i l l develop a low sweetness m e t a b o l i z a b l e sweetener f o r these p e o p l e . Non-reducing f r u c t o s e d e r i v a t i v e sweeteners. A Hyashibara process (12) s u b j e c t s a m i x t u r e o f s t a r c h and s u c r o s e t o c y c l o d e x t r i n g l u c o s y l t r a n s f e r a s e and a m y l o p e c t i n - a - 1 , 6 - g l u c o s i d a s e (such as p u l l u l a n a s e o r i s o a m y l a s e ) . C y c l o d e x t r i n s a r e formed as i n t e r m e d i a t e s , but then a c t as g l u c o s y l d o n o r s f o r a t t a c h m e n t t o t h e g l u c o s e p o r t i o n o f t h e

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch012

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s u c r o s e . T h i s r e s u l t s i n sweet n o n - r e d u c i n g oligomers with a fructose end. T h e s w e e t n e s s a n d t h e a v e r a g e number o f g l u c o s y l residues attached t o the fructose will vary with the r a t i o o f s t a r c h t o s u c r o s e , t h e enzyme l e v e l , a n d r e a c t i o n t i m e a n d c o n d i t i o n s . Isomaltulose. Isomaltulose i s a reducing disaccharide comprised o f glucose and f r u c t o s e . I t occurs n a t u r a l l y , b u t i t i s p r i m a r i l y obtained by t h e rearrangement o f sucrose by cr-glucosyl t r a n s f e r a s e d e r i v e d f r o m Protaminobacter rubrum. C o n v e r s i o n y i e l d s o f 8 5 % o r more are n o t unusual. I t i s made i n J a p a n b y M i t s u i S u g a r C o . ( 1 3 ) . I s o m a l t u l o s e i s 4 2 % as sweet a s s u c r o s e , b u t f u l l y a s c a l o r i c . I t o f f e r s advantages as a non-cariogenic n u t r i t i v e sweetener, (and p o s s i b l y a n t i c a r i o g e n i c as, l i k e x y l i t o l , i t i n h i b i t s i n s o l u b l e glucan f o r m a t i o n b y Streptococcus /nutans f r o m s u c r o s e when c o n s u m e d with sucrose. I t i s a c i d - s t a b l e , a n d a p p e a r s t o be u s e f u l i n t h e m a n u f a c t u r e o f h a r d c a n d i e s (1_4). I s o m a l t u l o s e i s a r e d u c i n g d i s a c c h a r i d e , w i t h a f r e e aldehyde group. Consequently, o n hydrogénation i t y i e l d s Isomalt. an equimolar mixture o f a-D-glucopyranosyl-1,6-mannitol and a - D - g l u c o p y r a n o s y l - 1 , 6 - s o r b i t o l . T h i s i s r e p o r t e d t o be h a l f a s sweet as s u c r o s e w i t h 2 c a l o r i e s p e r gram, m a k i n g i t u s e f u l a s a s u c r o s e s u b s t i t u t e i n r e d u c e d c a l o r i e f o r m u l a t i o n s where i t i s used w i t h a h i g h - i n t e n s i t y n u t r i t i v e or n o n - n u t r i t i v e sweetener. I t i s r e p o r t e d l y a p p r o v e d f o r f o o d u s e i n t h e E E C s i n c e 1985 (_1_5). L e u c i o s e i s 5 0 - 6 0 % a s s w e e t a s s u c r o s e a n d i s made b y t h e a c t i o n o f a n a - 1 , 6 - g l y c o s y l t r a n s f e r a s e on sucrose i n t h e presence o f f r u c t o s e , whereby t h e glucose p o r t i o n o f t h e s u c r o s e i s t r a n s f e r r e d t o t h e f r u c t o s e , and f r u c t o s e regenerated (16).

NORMAL & ENHANCED SWEETNESS SWEETENERS Immobilized i n v e r t a s e . H y d r o l y s i s o f sucrose by immobilized i n v e r t a s e now i s c h e a p e r a n d b e t t e r t h a n h y d r o l y s i s b y a c i d i c r e s i n s . I n v e r t a s e i m m o b i l i z e d i o n i c a l l y o n a reusable carrier, diethylaminoethylc e l l u l o s e i na weighted p o l y s t y r e n e (17-18), i s being used i n c o u n t r i e s w h e r e b e e t o r c a n e s u g a r i n t e r e s t s o r g o v e r n m e n t a l c o n s t r a i n t s make c o r n s u g a r p r o h i b i t i v e l y e x p e n s i v e a s a s t a r t i n g m a t e r i a l ( H . Lommi, F i n n i s h Sugar Co., p e r s o n a l c o m m u n i c a t i o n ) . The i m m o b i l i z e d i n v e r t a s e g i v e s g o o d c o n v e r s i o n ; a t f l o w r a t e s o f 2-4 b e d v o l u m e s p e r h o u r 99.5% c o n v e r s i o n i s a c h i e v e d , w h i l e even a t 40 b e d volumes p e r hour 5 0 % hydrolysis i s achieved. The c o s t o f enzymatic h y d r o l y s i s , a t about $US 0 . 1 0 - 0 . 1 5 / c w t d r y b a s i s , a p p e a r s h i g h e r when o n l y t h e h y d r o l y s i s s t e p i s c o n s i d e r e d b u t n o t when t h e o v e r a l l p r o c e s s i s considered. O v e r a l l , immobilized invertase is cheaper; strong cat ionic hydrolysis costs more than immobilized invertase because t h e a c i d method results i n significant f r u c t o s e p o l y m e r i z a t i o n forming t r i o s e s and t e t r o s e s , lowering the y i e l d . The a c i d r e s i n method r e q u i r e s a d e c o l o r i z a t i o n s t e p b e c a u s e o f t h e c o l o r f o r m a t i o n ; t h e a c i d method forms s i g n i f i c a n t amounts o f h y d r o x y m e t h y 1 f u r f u r a l . The mineral content resulting from t h e a c i d i f i c a t i o n - n e u t r a l i z a t i o n requires a demineralization step. A second i m m o b i l i z e d i n v e r t a s e , w h e r e i n t h e c a r r i e r must be d i s c a r d e d w i t h t h e s p e n t e n z y m e , w i t h o u t p o s s i b i l i t y o f in situ r e g e n e r a t i o n , has a l s o been d e s c r i b e d ( 1 9 ) . G l u c o s e i s o m e r a s e . G l u c o s e i s o m e r a s e f o r HFCS i n t h e USA i s a s u c c e s s s t o r y p r i m a r i l y because o f t h e a r t i f i c i a l d i f f e r e n c e i n p r i c e between corn s t a r c h and t h e supported p r i c e o f sucrose. This p r i c e d i f f e r e n c e

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does not e x i s t i n Canada, where w o r l d market s u c r o s e p r i c e s p r e v a i l , and, f o r t h a t r e a s o n , t h e i n c e n t i v e t o s w i t c h t o HFCS does n o t e x i s t i n Canada. Enhancing the sweetness o f n a t u r a l i n v e r t sugar i s another i n t e r e s t i n g i m m o b i l i z e d g l u c o s e isomerase ( I G I ) a p p l i c a t i o n which s t a r t s w i t h i n v e r t sugar. T h i s i s based on l a b e l i n g laws. C o n c e n t r a t e d w h i t e grape j u i c e i s used as a sweetener, b u t appears on the l a b e l as " j u i c e " , not sugar. J e l l i e s made w i t h c o n c e n t r a t e d grape j u i c e a r e o f t e n promoted as "no sugar added". I t i s now p o s s i b l e t o make supersweet c o n c e n t r a t e d grape j u i c e by s e p a r a t i n g t h e g l u c o s e from t h e f r u c t o s e , i s o m e r i z i n g t h e g l u c o s e a n d a d d i n g i t b a c k f o r a 60:40 f r u c t o s e : g l u c o s e o r even an 80:20 f r uc t o s e : g l u c o s e ( F . Hammer, Finnsugar Biochemicals Inc., personal communication). This gives h i g h e r sweetness per u n i t weight. The 80:20 would have a sweetness o f as much as 25% more than t h e 50:50 p r o d u c t , a l l o w i n g t h e use o f 20% less concentrated j u i c e . L a b e l d e c l a r a t i o n r e q u i r e m e n t s , and consumer p e r c e p t i o n o f these l a b e l e d i n g r e d i e n t s , may make t h i s a t t r a c t i v e f o r s p e c i f i c uses. HIGH INTENSITY SWEETENERS High i n t e n s i t y sweeteners i n c l u d e both n a t u r a l and s y n t h e t i c compounds. S a c c h a r i n and aspartame are s y n t h e t i c . On t h e o t h e r hand both s t e v i o s i d e and r i b a u d i o s i d e - A a t e n a t u r a l h i g h i n t e n s i t y sweeteners d e r i v e d from t h e Stevia plant. Aspartame and g l y c o s y l a t e d s t e v i o s i d e s are c o m m e r c i a l l y produced w i t h enzymes. Aspartame. The success o f L - a s p a r t y l - L - p h e n y l a l a n i n e methyl ester (aspartame) as a h i g h q u a l i t y low c a l o r i e sweetener has r e s u l t e d i n c o n s i d e r a b l e r e s e a r c h e f f o r t d i r e c t e d a t a more e f f i c i e n t d i r e c t enzymatic c o u p l i n g o f t h e components. The o r i g i n a l c h e m i c a l process was done a t v e r y low temperatures a t h i g h c o s t . The f i r s t enzymatic process commercialized used i m m o b i l i z e d t h e r m o l y s i n t o c o u p l e Np r o t e c t e d a s p a r t i c a c i d w i t h t h e methyl e s t e r o f p h e n y l a l a n i n e , then removing t h e p r o t e c t i v e group by hydrogénation. Newer methods have been developed by s e v e r a l companies. The phenyl s e r i n e e s t e r has been s y n t h e s i z e d (20) u s i n g s e r i n e h y d r o x y m e t h y l t r a n s f e r a s e , f o l l o w e d by coupling with blocked a s p a r t i c acid using thermolysin; t h i s i s then hydrogenated t o g i v e hydroxyaspartame o r aspartame. L - A s p a r t i c a c i d has been d i r e c t l y coupled w i t h L - p h e n y l a l a n i n e (H)» u s i n g u n s t a t e d enzymes produced by E. coli or o t h e r mixed organisms i n t h e fermentat i o n b r o t h , t o which t h e r e a c t a n t s a r e added. The y i e l d s i n t h i s p r o c e s s a r e v e r y low, but i t i s obvious t h a t the d i r e c t process i s b e i n g r a p i d l y a p p r o a c h e d as t h e b a s i c p a t e n t s o n a s p a r t a m e h a v e a l r e a d y , o r w i l l soon e x p i r e . A new e n t r y i n t h e aspartame f i e l d i s s a i d t o employ i m m o b i l i z e d enzymes ( i n i m m o b i l i z e d c e l l s ) t o make t h e p h e n y l a l a n i n e from c i n n a m i c a c i d a n d ammonia, p r i o r t o c o u p l i n g d i r e c t l y to a s p a r t i c acid (22). The l i m i t a t i o n s o f aspartame as a s w e e t e n e r have s p u r r e d work on o t h e r c l e a n - s w e e t h i g h i n t e n s i t y sweeteners as w e l l . S t e v i o s i d e s and ιibaudioside-A. S t e v i o s i d e and r i b a u d i o s i d e - A (βg l u c o s y l s t e v i o s i d e ) both occur n a t u r a l l y i n t h e p l a n t Stevia rebaudiana, w i t h the s t e v i o s i d e s c o m p r i s i n g 10-20% o f the d r i e d weight of the l e a v e s . R i b a u d i o s i d e - A may c o n s t i t u t e as much as 60-70% of t h e m i x t u r e f o r some s t r a i n s o f the p l a n t ( 2 3 ) . While s t e v i o s i d e i s about 150-300 times as sweet as s u c r o s e , t h e f l a v o r p r o f i l e has a b i t t e r

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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component and a d e l a y e d sweetness onset t h a t makes i t not f u l l y a s u c r o s e - t a s t e s u b s t i t u t e . R i b a u d i o s i d e - A , as w e l l as a - g l u c o s y l s t e v i o s i d e , do n o t have t h e b i t t e r t a s t e , a n d more c l o s e l y m i m i c t h e d e s i r a b l e t a s t e o f s u c r o s e . The best sweeteners a r e made by c o n v e r t ­ i n g t h e s t e v i o s i d e i n t o the g l y c o s y l a t e d d e r i v a t i v e (24-26). The Hayashibara process (2J>) c o n s i s t s o f making t h e a - g l u c o s y l d e r i v a t i v e o f s t e v i o s i d e by t h e use o f an α-glucosyltransferase such as a - g l u c o s i d a s e , α-amylase, c y c l o d e x t r i n g l y c a n o t r a n s f e r a s e , o r a-1,6g l u c o s y l t r a n s f e r a s e , depending on t h e α-glycosyl donor. U s u a l l y a m i x t u r e o f α-ηιοηο-, α-di-, and α-tri-glucosyl s t e v i o s i d e s r e s u l t , as w e l l as α-glycosylribaudiosides, i f r i b a u d i o s i d e was p r e s e n t . The ag l y c o s y l donor c a n be g e l a t i n i z e d s t a r c h , d e x t r i n s , m a l t o s e , o r even s u c r o s e , f o r example. The commercial donor used i s d e x t r i n . I n t h e Dainippon Ink & Chemical ("DIC") process ( 2 6 ) , s t e v i o s i d e i s r e a c t e d w i t h a β-1,3- o r 0 - 1 , 4 - g l y c o s y I s u g a r compound i n t h e presence o f t h e a p p r o p r i a t e β-glycosyl t r a n s f e r a s e t o make t h e βg L y c o s y l s t e v i o s i d e ( r i b a u d i o s i d e - A . ) Donors o f t h e β-glycosyl group can be g l u c a n from y e a s t c e l l w a l l , laminarin, lactose, cellobiose, or c u r d l a n f o r example. C u r d l a n , a β-1,3-glucan, i s t h e p r e f e r r e d donor. Any β-1,3- or β-1,4-glycosyl t r a n s f e r a s e can be used, i n c l u d i n g c e l l u l a s e s , e m u l s i n ( t h e β-glucosidase from almonds), depending on the β-glucosyl donor, but t h e p r e f e r r e d g l u c o s y l t r a n s f e r a s e f o r use w i t h c u r d l a n i s a Streptomyces t r a n s g l u c o s i d a s e . T h i s Streptomyces c u l t u r e i s a l s o t h e s o u r c e o f t h e l a m i n a r i p e n t a o h y d r o l a s e (2_7) u s e d t o h y d r o l y z e the c u r d l a n i n t o 5-unit fragments (M. Yamada, DIC, p e r s o n a l communication). I n t h i s process t h e endo- and exo- β-glueanases w i t h t r a n s f e r a s e a c t i v i t y a r e s e p a r a t e d and i m m o b i l i z e d ; the l a m i n a r i p e n t a o h y d r o l a s e i s a l s o removed and added t o t h e c u r d l a n f e r m e n t a t i o n b r o t h , c u t t i n g the curdlan i n t o laminaripentose (5-glucose r e s i d u e s ) . The c e l l s a r e removed and t h e e n t i r e m i x t u r e i s added t o t h e s t e v i o ­ s i d e and passed over t h e i m m o b i l i z e d β-glucanases t o produce t h e βg l y c o s y l a t e d s t e v i o s i d e , r i b a u d i o s i d e - A . DIC, however, i s working w i t h p l a n t improvement t h r u c o n v e n t i o n a l and p r o b a b l y g e n e t i c e n g i n e e r i n g and c l o n i n g t o produce a p l a n t t h a t makes p r i m a r i l y r i b a u d i o s i d e - A . ENZYMES IN CORN WET MILLING Wet m i l l i n g o f c o r n begins w i t h a s t e e p i n g s t e p . Corn k e r n e l s a r e s o f t e n e d by absorbed water w h i l e soluble s o l i d s d i f f u s e into the steep water. S t e e p i n g makes i t easy t o s e p a r a t e t h e s t a r c h g r a n u l e s , p r o t e i n , f i b e r and germ. The c o r n k e r n e l i s steeped i n water, w i t h s u l f u r d i o x i d e added, then ground s l i g h t l y t o break down t h e s t r u c ­ t u r e . Much o f t h e s t a r c h i s embedded i n f i b r o u s m a t e r i a l , and bound t o the g l u t e n ; as much as 5-10% o f the s t a r c h may remain thus bound. The j u d i c i o u s a d d i t i o n o f Trichoderma reesii cellulases rich i n side a c t i v i t i e s c a n s i g n i f i c a n t l y improve the s t a r c h y i e l d by f r e e i n g bound s t a r c h g r a n u l e s , which a r e then e a s i l y s e p a r a t e d from t h e p r o t e i n and o t h e r components based on d e n s i t y . (T. Vaara, A l k o L t d . , p e r s o n a l communication, and J . Z e l k o , Genencor, p e r s o n a l communication). One v e r y important a d d i t i o n a l c o n c u r r e n t enzyme p r o c e s s has been developed by A l k o and D o r r - O l i v e r ( 2 8 ) . T h i s i s t h e s t e e p i n g o f c o r n w i t h phytase i n c o m b i n a t i o n w i t h a f . reesii c e l l u l a s e r i c h i n side a c t i v i t i e s . ( T h i s enzyme combination was o r i g i n a l l y developed under the Econase 434 name f o r use i n p i g f e e d s ; t h e breakdown o f p h y t a t e by phytase r e l e a s e d t h e phosphorus bound by t h e p h y t a t e . T h i s amounts t o 50% o f t h e phosphate i n c o r n , f o r example.) Anyone working w i t h Corn

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch012

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Steep L i q u o r as a m i c r o b i o l o g i c a l n u t r i e n t o r i n animal f e e d has r u n i n t o problems w i t h t h e i n s o l u b l e c a l c i u m phytate-phosphate sludge. The A l k o / D o r r O l i v e r p r o c e s s i n v o l v e s a two-stage s t e e p i n g , which increases the e f f i c i e n c y o f the e x t r a c t i o n , s u b s t a n t i a l l y shortens s t e e p i n g time t o 12 hours, and g i v e s a c o n c e n t r a t e d c o r n s t e e p l i q u o r reduced i n p h y t i c a c i d and f r e e o f p r e c i p i t a t e , making the c a l c i u m and phosphorus a v a i l a b l e t o t h e organism o r animal b e i n g so f e d . Making the phosphate i n t h e c o r n a v a i l a b l e t o a n i m a l s e a t i n g i t a v o i d s t h e need f o r a d d i t i o n o f p h o s p h a t e t o t h e a n i m a l f e e d ; i n t u r n t h i s r e s u l t s i n r e d u c i n g the amount o f phosphorus e x c r e t e d by t h e a n i m a l . In some c o u n t r i e s farmers a r e p e n a l i z e d f o r adding phosphorus compounds t o s u r f a c e waters (when d i s p o s i n g o f manure r i c h i n p h y t a t e phosphorus m a t e r i a l s , f o r example) because o f t h e e u t r i f i c a t i o n o f ponds and streams. I t i s f o r t h i s same reason t h a t phosphates have been removed from household d e t e r g e n t s i n many a r e a s . OXIDASES

There a r e many o x i d a s e s used i n c l i n i c a l and a n a l y t i c a l c h e m i s t r y f o r the s p e c i f i c d e t e r m i n a t i o n o f c o n s t i t u e n t s o f complex samples, based on t h e g e n e r a t i o n o f hydrogen p e r o x i d e by t h e a c t i o n o f an a e r o b i c o x i d a s e on i t s s u b s t r a t e . C h o l e s t e r o l , g l u c o s e , g a l a c t o s e and many o t h e r a n a l y t e s a r e d e t e r m i n e d t h a t way. T h e s e a n a l y s e s a r e a l s o i m p o r t a n t i n f o o d a n a l y s i s . But the volume o f o x i d a s e s consumed i n f o o d p r o c e s s i n g i s p r i m a r i l y based on t h e p r o t e c t i o n a f f o r d e d t h e f o o d by t h e removal o f e i t h e r oxygen o r t h e o t h e r s u b s t r a t e a c t e d upon. Glucose o x i d a s e . Glucose o x i d a s e has been used f o r f o r t y y e a r s f o r t h e s t a b i l i z a t i o n o f egg p r o d u c t s by c o n v e r s i o n o f g l u c o s e t o g l u c o n i c a c i d , preventing the M a i l l a r d r e a c t i o n t h a t i n t h i s i n s t a n c e i s u n d e s i r a b l e . Glucose o x i d a s e has a l s o been used f o r t h e p r o t e c t i o n o f foods a g a i n s t growth o f a e r o b i c o r g a n i s m s and a g a i n s t v a r i o u s o x i d a t i v e r e a c t i o n s t h a t cannot take p l a c e i n t h e absence o f oxygen. I t now appears t h a t some o f the deoxygenation uses a n t i c i p a t e d decades ago w i l l be o f commercial importance due t o improvements i n packaging m a t e r i a l s , o r changes i n the form o f p r o d u c t s b e i n g marketed. F o r e x a m p l e , OVAZYME was p r o m o t e d a s a s u r f a c e c o a t i n g f o r f i l m f o r p a c k a g i n g cheese, t o s e r v e as an oxygen b a r r i e r , over 30 y e a r s ago, but never r e a l l y used because vacuum p a c k i n g t e c h n i q u e s were, g e n e r a l l y , a d e q u a t e . Now, 30 y e a r s l a t e r , c h e e s e i s b e i n g m a r k e t e d i n shredded form, a form t h a t does not l e n d i t s e l f t o vacuum p a c k i n g . I n s t e a d 50% o f t h e a n t i c a k i n g m i x t u r e d u s t e d onto t h e shredded cheese has been r e p l a c e d by a m i x t u r e o f g l u c o s e o r g l u c o g e n i c m a t e r i a l , w i t h added p u l v e r a n t g l u c o s e o x i d a s e and c a t a l a s e , which i s a c t i v a t e d by the m o i s t u r e i n t h e cheese and c o m p l e t e l y deoxygenates t h e s e a l e d p a c k a g e , r e t a r d i n g m o l d i n g (D. S c o t t a n d T. S z a l k u c k i , F i n n s u g a r Biochemicals Inc, unpublished data). O x a l a t e o x i d a s e . I n some i n s t a n c e s , as i n d r y wine, t h e r e i s n o t s u f f i c i e n t g l u c o s e t o e f f e c t deoxygenation. However t h e r e appears t o be s u f f i c i e n t o x a l i c a c i d i n many wines t o a c t as t h e s u b s t r a t e f o r c o m b i n a t i o n w i t h oxygen by o x a l a t e o x i d a s e , d e r i v e d from the d i s c a r d e d l e a f y tops o f t h e sugar beet p l a n t ( 2 9 ) . The a p p l i c a t i o n may s t i l l not work w e l l i f i t r e l i e s on c a t a l a s e t o decompose t h e H2O2 formed by the o x i d a s e , as c a t a l a s e i s s t r o n g l y i n h i b i t e d by e t h a n o l a t the pH o f wine.

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A l c o h o l o x i d a s e . Another p u r i f i e d o x i d a s e o f p a r t i c u l a r interest i s P t o v e s t a ' s a l c o h o l o x i d a s e , f r o m t h e y e a s t Pichia pastoris. Described as " e q u a l l y a c t i v e on m e t h a n o l a n d c t h a n o l " ( D. R a n a s i a k a n d T. H o p k i n s , P r o v e s t a, p e i s o n a l c o m m u n i c a t i o n ) i t i s "one-third as a c t i v e u n c t h a n o ! t h a n o n m e t h a n o l . . " o n a n enzyme e l e c t r o d e ( H o p k i n s and Mul l e i (3ÇP). The commercial p r o d u c t i s v i r t u a l l y d e v o i d o f catalase a n d l i a s -Acetates

I

I

Aldehydes Figure 3. Reduction pathways for pheromone biosynthesis.

The reactions mentioned above account f o r many, but by no means a l l , pheromones whose b i o s y n t h e s i s i s r e l a t e d to f a t t y acids. For example, the silkworm moth, Bombyx mori, has as i t s main pheromone component (Ε, Z) -10,12-hexadecadien-l-ol (1). The corresponding diunsaturated f a t t y a c i d a l s o i s present i n the gland (14.) as i s a large quantity of (Z)-11-hexadecenoic acid (lu) . Recent studies have demonstrated that the monounsaturated a c i d i s formed f i r s t , followed by what appears to be a 1,4desaturation y i e l d i n g the diunsaturated a c i d (16). Other desaturases also may play a part i n these pathways. The greenheaded l e a f r o l l e r moth, Planotortrix excessana, a New Zealand species, uses (Z)-8-tetradecenyl acetate as i t s main pheromone component. In vivo studies using deuterated precursors showed t h a t the path given i n F i g 4 was o p e r a t i v e , i n d i c a t i n g a desaturase that introduced a double bond at the 10-11 p o s i t i o n was operative (12). Another New Zealand insect, the brownheaded l e a f r

16:Acid

•

(Z)-10-16:Acid

•

(Z)-8-14:Acid

Figure 4. Desaturase and chain-shortening activity in Planotortrix excessana.

r o l l e r moth, Ctenopseustis obliquana, uses the same Z8 compound as well as (Z)-5-tetradecenyl acetate as major components. Studies with deuterated precursors showed the path indicated i n F i g . 5 was

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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o p e r a t i v e , i m p l i c a t i n g o l e i c a c i d , (Z)-9-octadecenoic a c i d , and t h e common 9-10 d e s a t u r a s e i n t h e b i o s y n t h e s i s o f t h e s e pheromones

(lfi.) . 18:Acid-**

(Z)-9-18:Acid

(Z)-7-16:Acid-*>

(Z)-5-14:Acid

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Figure 5. Desaturase and chain-shortening activity in Ctenopseustis obliquana.

An u n u s u a l 9-10 d e s a t u r a s e i s p r e s e n t i n t h e c o d l i n g moth, Cydia pomunella. I t forms (E)-9-dodecenoic a c i d , which then undergoes 1,4-desaturation a n d r e d u c t i o n t o g i v e t h e pheromone (E,E)-B,10-dodecadien-l-ol (11). Some i n s e c t s use h y d r o c a r b o n s as s e x pheromones. F o r example, t h e h o u s e f l y , Musca domestica, uses a m i x t u r e i n c l u d i n g (Z)-9tricosene, the corresponding e p o x i d e and ketone, and s e v e r a l m e t h y l a l k a n e s ( 2 0 ) . The t r i c o s e n e i s d e r i v e d by c h a i n e l o n g a t i o n o f o l e i c a c i d , and t h e e p o x i d e and k e t o n e a r e made f r o m i t . The m e t h y l a l k a n e s a r e made de novo f r o m a c e t a t e and p r o p i o n a t e , with one p r o p i o n a t e u n i t per molecule supplying the branch carbon. Propionate can a r i s e from t h e degradation of valine or i s o l e u c i n e , b u t n o t from s u c c i n a t e , a l t h o u g h s u c c i n a t e may s e r v e as an a c e t a t e p r e c u r s o r . S e v e r a l s p e c i e s o f t i g e r moths, Holomelina s p p . , u s e 2methylheptadecane as a m a i n pheromone component. Studies i n v o l v i n g t h e i n j e c t i o n o f r a d i o l a b e l e d compounds i n t o whole i n s e c t s have d e m o n s t r a t e d t h a t t h e p a t h g i v e n i n F i g 6 i s most likely functional (2-1) . T h i s i s s i m i l a r t o a known p a t h i n c r i c k e t s (22). (CH ) CHCH(NH )COOH 3

2

2

(CH ) CH (CH ) COOH 3

2

2

15

—

( C H 3 ) CHCH COOH leucine

•

—

2

•

2

( C H 3 ) CH (CH ) 2

2

1 4

CH

3

Figure 6. Biosynthetic pathway for Holomelina spp.

A n o t h e r s t e p i n t h e b i o s y n t h e s i s worthy o f m e n t i o n i s t h a t o f the synthesis of the f a t t y a c i d s themselves. A l t h o u g h we o f t e n c o n s i d e r t h e pathways t o s t a r t w i t h t h e a c i d s ( p a l m i t i c , o l e i c ) , f a t t y a c i d s y n t h e t a s e c o u l d be c o n s i d e r e d p a r t o f them, though, o f course, not a unique p a r t (23). Enzvmatic

Properties

The s t u d y o f t h e enzymes i n v o l v e d i n t h e s e pathways i s s t i l l i n i t s e a r l y stages. However, enough h a s been done t o g i v e some insight i n t o t h e c h a r a c t e r i s t i c s and o r g a n i z a t i o n of these systems. a) Chain Shortening. The f i r s t s t e p i n t h e s e x pheromone b i o s y n t h e s i s i n t h e redbanded l e a f r o l l e r i n v o l v e s c h a i n s h o r t e n i n g f r o m h e x a d e c a n o i c a c i d t o t e t r a d e c a n o i c a c i d (3.) . Crude c e l l - f r e e preparations f r o m pheromone g l a n d s e x h i b i t t h i s r e a c t i o n when

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p r o v i d e d with u n i f o r m l y C l a b e l e d hexadecanoic a c i d NADH, NADPH, Coenzyme A, ATP, magnesium c h l o r i d e and BSA (ZA) . Assumedly, t h e Co A, ATP and M g C l 2 a l l o w t h e c r u d e p r e p a r a t i o n t o s y n t h e s i z e t h e Co A d e r i v a t i v e o f t h e a c i d . O m i s s i o n o f t h e Coenzyme A c a u s e s t h e r e a c t i o n t o s t o p . A l s o , f a i l u r e t o p r o v i d e NADP c a u s e s c h a i n s h o r t e n i n g t o no l o n g e r be e x h i b i t e d . A s m a l l amount o f dodecanoic a c i d and no s h o r t e r - c h a i n a c i d s were o b s e r v e d , showing t h e r e a c t i o n t o be l i m i t e d , u n l i k e t h e normal m i t o c h o n d r i a l d e g r a d a t i o n sequence. b) Δ 1 1 d e s a t u r a s e . The d e s a t u r a s e from t h e cabbage l o o p e r moth has b e e n p a r t i a l l y p u r i f i e d a n d i t s p r o p e r t i e s d e t e r m i n e d (2JL). The pheromone g l a n d s were e x c i s e d , g r o u n d i n p h o s p h a t e b u f f e r c o n t a i n i n g s u c r o s e and d i t h i o t h r e i t o l and t h e m i c r o s o m a l f r a c t i o n i s o l a t e d by c e n t r i f u g a t i o n a t 100,000 g f o l l o w e d b y resuspension of the p e l l e t . The p r o d u c t was d e t e r m i n e d t o be solely (Z)-11-hexadecenoic a c i d , u s i n g 1- C h e x a d e c a n o y l Co A as substrate. The pH optimum was b r o a d a n d c e n t e r e d a r o u n d 7.8. P r o d u c t i o n o f p r o d u c t was l i n e a r f o r one hour, and t h e n i t q u i c k l y ceased. A c t i v i t y was maximum i n t w o - d a y - o l d i n s e c t s a n d was a l m o s t gone by f i v e d a y s .

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f

14

The normal a s s a y i n c l u d e d , i n a d d i t i o n t o enzyme s o l u t i o n and s u b s t r a t e , BSA, NADH and NADPH. O m i t t i n g t h e BSA o r NADH r e s u l t e d i n marked r e d u c t i o n i n a c t i v i t y , whereas o m i s s i o n o f NADPH gave only s l i g h t reduction i n a c t i v i t y . The enzyme a c t i v i t y was h i g h e s t when o c t a d e c a n o y l Co A was u s e d a s s u b s t r a t e , a l m o s t as h i g h when h e x a d e c a n o y l Co A was u s e d , a n d v e r y s m a l l when t e t r a d e c a n o y l Co A was u s e d as s u b s t r a t e . The most common d e s a t u r a s e i n most o r g a n i s m s , including i n s e c t s , i s s t e a r o y l Co A d e s a t u r a s e , which i n t r o d u c e s a d o u b l e b o n d i n t h e 9-10 p o s i t i o n o f l o n g - c h a i n f a t t y a c i d s (2JL) . S i m i l a r i t i e s between t h i s enzyme a n d t h e Δ 1 1 d e s a t u r a s e f r o m cabbage l o o p e r i n c l u d e : l o c a t i o n i n the microsomal f r a c t i o n , l a c k o f s e n s i t i v i t y t o c a r b o n monoxide, i n h i b i t i o n b y c y a n i d e , use o f a r e d u c e d n i c o t i n e - a d e n i n e n u c l e o t i d e c o f a c t o r as an e l e c t r o n s o u r c e and use o f 16 and 18 c a r b o n a c i d s as p r e f e r r e d s u b s t r a t e s . T h e r e a l s o a r e s i g n i f i c a n t d i f f e r e n c e s between t h e enzymes. F i r s t , t h e r e i s the d i f f e r e n t s i t e of i n t r o d u c t i o n o f the double bond. Then t h e r e i s t h e p r e f e r r e d c o f a c t o r ; b o t h w i l l a c c e p t NADPH o r NADH, b u t t h e Δ9 enzyme p r e f e r s t h e f o r m e r and t h e Δ 1 1 enzyme t h e l a t t e r . Both a r e s e n s i t i v e t o c y a n i d e , b u t t h e Δ 1 1 i s about 50 t i m e s l e s s s e n s i t i v e . The pH optimum o f t h e Δ9 enzyme i s somewhat more a c i d i c (7.8-7.2). F i n a l l y , there i s the t i s s u e location. The Δ9 enzyme i s g e n e r a l l y p r e s e n t t h r o u g h o u t t h e i n s e c t , w h i l e t h e Δ 1 1 enzyme i s l o c a l i z e d i n t h e pheromone g l a n d The s m a l l amounts o f t h e Δ 1 1 d e s a t u r a s e a v a i l a b l e make s o l u b i l i z a t i o n and s t a b i l i z a t i o n n e c e s s a r y b e f o r e p u r i f i c a t i o n c a n be a t t e m p t e d . A series of detergents previously reported useful i n d e s a t u r a s e p u r i f i c a t i o n s p r o v e d n o t t o be u s e f u l i n t h i s c a s e i n d e e d , most o f them a c t u a l l y l o w e r e d t h e a c t i v i t y . However, o c t a n o y l - N - m e t h y l g l u c a m i d e (21) d i d a p p e a r t o s t a b i l i z e and a t l e a s t p a r t i a l l y s o l u b i l i z e t h e enzyme. Treatment o f enzyme solutions with Blue Sepharose, an a f f i n i t y chromatography

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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substrate, gave a degree of p u r i f i c a t i o n , but the a c t i v i t y was so low that further studies have proved d i f f i c u l t (24.) · c) Reduction. The l a s t step i n the proposed b i o s y n t h e t i c paths involves reduction of the acids formed by chain shortening and desaturation. This can give product d i r e c t l y or can produce an intermediate that then gives the f i n a l product. For example, i n the corn ear worm, Heliothis zea, which uses aldehydes as pheromone components, the immediate product of the reduction of 11-hexadecenoic a c i d could well be the corresponding a l c o h o l . There e x i s t s i n the insect c u t i c l e a primary alcohol oxidase that converts alcohols to aldehydes (13) . This oxidase shows l i t t l e s p e c i f i c i t y for geometry or number of double bonds, has a broad pH p r o f i l e (5-9) and was f u n c t i o n a l i n both dichloromethane and hexane, an unusual and p o t e n t i a l l y quite i n t e r e s t i n g property. More extensive studies have been done on the spruce budworm, Choristoneura fumiferana, (2iL) which uses (E) - and (Z)-lltetradecenal as pheromone components. Once again, the f i r s t step appears to be reduction of the a c i d to an alcohol. Then an a c e t y l Co A: f a t t y alcohol acetyltransferase converts the alcohol to the acetate e s t e r (12.) . The enzyme i s s p e c i f i c f o r alcohols with chain lengths of 12-15 carbons, and p r e f e r s monounsaturated alcohols to saturated ones. It i s located s p e c i f i c a l l y i n the pheromone gland, and appears to be microsomal. Two other enzymes are involved i n aldehyde production. The f i r s t i s an acetate esterase, which i s soluble, located i n the gland, and hydrolyzes tetradecanyl acetate and i t s Δ11 unsaturated d e r i v a t i v e s (Z or E) at the same rate. The second i s an alcohol oxidase, which also i s soluble and located i n the gland. The oxidase requires oxygen, but does not require reducing cofactors (12) . The saturated and unsaturated 14-carbon acids a l l react at s i m i l a r rates i n the reaction catalyzed by t h i s enzyme. The redbanded l e a f r o l l e r moth uses a mixture of (E) and (Z) 11-tetradecenyl acetates as pheromone components. The pheromone gland of t h i s insect also contains an a c e t y l Co A: f a t t y alcohol a c y l transferase (25.) . This enzyme w i l l acetylate 12, 14 and 16 carbon a l c o h o l s , and p r e f e r s the saturated a l c o h o l to the Ζ monounsaturated derivative, and the Ζ to the E, i n the approximate r a t i o s of 5:3:1 f o r saturated, Ζ and E, respectively. d) Fatty Acid Synthetase (FAS) . FAS i s a commonly occurring enzyme complex whose occurrence and properties i n insects has been r e c e n t l y reviewed (23) . FAS a c t i v i t y i n the pheromone gland of spruce budworm has been observed both in vivo and in vitro (12., 20.) . In both cases, the r a t i o of incorporation of malonate to acetate was 8:1 though in vitro t h i s r a t i o decreased to 7:1 a f t e r two hours. Isolated acids ranged i n chain length from 12 to 18 carbons and appeared to be p r i m a r i l y saturated. In the redbanded l e a f r o l l e r moth, treatment of i n t a c t glands with DMSO containing radioactive acetate l e d to the formation of radiolabeled 14, 16 and 18 carbon saturated acids as well as Δ 1 1 tetradecenoic acids and pheromone components (2JL) . Cell-free preparations from redbanded l e a f r o l l e r pheromone glands a l s o showed FAS a c t i v i t y when r a d i o l a b e l e d a c e t y l Co A, NADPH and malonyl Co A are supplied (21). The major products are the 16 and 18 carbon saturated acids, with the 18 carbon a c i d predominating.

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The a c t i v i t y does not sediment i n a 100,000 g f r a c t i o n , i n d i c a t i n g i t i s soluble. A s i m i l a r a c t i v i t y may be observed i n preparations from the pheromone gland of the cabbage looper moth (21)·

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch022

Areas f o r further study The study of the enzymes involved i n sex pheromone biosynthesis i n i n s e c t s has b a r e l y begun. There are f a r too many f r u i t f u l research projects i n t h i s f i e l d to even l i s t them here. We w i l l mention a few just to introduce the v a r i e t y possible. F i r s t , the use of two s p e c i f i c reactions — Δ11 desaturation and c o n t r o l l e d 2 carbon chain shortening of f a t t y a c i d precursors to account for the biosynthesis of a large number of pheromones — has been an extremely f r u i t f u l approach. Even i n a case where i t seemed uncertain i f t h i s approach was appropriate (22.) , i t turned out that i t was (22.) . Other reactions should now be added t o increase the range of products accounted f o r . Examples already mentioned include the Δ10 desaturase and the chain elongation of branched-chain s t a r t i n g materials. Other f u n c t i o n a l groups that appear i n sex pheromones should also be accounted f o r , such as epoxides. Another i n t e r e s t i n g problem i n v o l v e s the c o n t r o l of the synthesis. Hormonal c o n t r o l of the b i o s y n t h e t i c pathway i s c u r r e n t l y being i n v e s t i g a t e d (22) , and the existence of a brain hormone has been demonstrated. Other aspects of control, such as the r a t i o of compounds and when they are a c t u a l l y released, also require extensive further study. F i n a l l y , although the Δ11 desaturase from cabbage looper i s probably the most studied enzyme involved i n these pathways, much more work should be done with i t and r e l a t e d enzymes. For example, i t only produces Ζ products and does not f u n c t i o n e f f i c i e n t l y with 14 carbon substrates, i n d i c a t i n g a fundamental d i f f e r e n c e with the enzyme from redbanded l e a f r o l l e r moths, which desaturates 14-carbon acids to give a mixture of Ζ and Ε product (22). Projects such as t h i s may well prove t e c h n i c a l l y d i f f i c u l t , but w i l l provide great insight into the enzymatic mechanisms.

Literature Cited 1. 2. 3.

4. 5. 6. 7. 8.

Butenandt, Α.; Beckman, R.; Stamm, D.; Hecker, E. Z. Naturforsch 1959, 14, 283-84. Roelofs, W. L.; Bjostad, L. B. Biorg. Chem. 1984, 12, 279-98. Bjostad, L. B.; Wolf, W. Α.; Roelofs, W. L. In Pheromone Biochemistry: Prestwich, G. D.; Blomquist, G. L., Eds; Academic Press: New York, 1987; Chapter 3. Wolf, W. Α.; Bjostad, L. B.; Roelofs, W. L. Environ. Entomol. 1981, 10, 943-46. Hill, Α.; Carde, R.; Comeau, Α.; Bede, W., Roelofs, W. L. Environ. Entomol. 1974, 3, 249-52. Bjostad, L. B.; Wolf, W. Α.; Roelofs, W. L. Insect Biochem. 1981, 11, 73-9. Roelofs, W. L.; Brown, R. L. Ann. Rev. Ecol. Syst. 1982, 13, 395-422. Wolf, W. Α.; Roelofs, W. L. Insect Biochem. 1983, 13, 37579.

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

22. WOLF AND ROELOFS Biosynthesis of Sex Pheromones in Moths331 9. 10. 11. 12. 13. 14. 15.

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16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.

Dillwith, J. W.; Blomquist, G. J.; Nelson, D. R. Insect Biochem. 1981, 11, 247-53. Bjostad, L. B.; Roelofs, W. L. J. Biol. Chem. 1981, 256, 7936-40. Bjostad, L. B.; Roelofs, W. L. Science 1983, 220, 1387-89. Morse, D; Meighen, E. In Pheromone Biochemistry: Prestwich, G. D.; Blomquist, G. L., Eds; Academic Press: New York, 1987; Chapter 4. Teal, P. Ε. Α.; Tumlinson, J. H. This volume. Bjostad, L. B.; Roelofs, W. L. Insect Biochem. 1984, 14, 275-78. Yamaoka, R.; Hayashiya, K. Jpn. J. Appl. Entomol. Zool. 1982, 26, 125-30. Ando, T; Hase, T.; Arima, R.; Uchiyama, M. Agr. Biol. Chem. 1988, 52, 473-78. Foster, S. P; Roelofs, W. L. Arch. Insect Biochem. Physiol., In Press. Foster, S. P; Roelofs, W. L. Experientia In Press. Löfstedt, C.; Bengtsson, M. J. Chem. Ecol. 1988, 14, 903-15. Blomquist, G. L.; Vaz, A. H.; Jurenka, R. Α.; Reitz, R. C. This volume. Charlton, R.; Roelofs, W. L. Unpublished results. Blailock, T. T.; Blomquist, G. L.; Jackson, L. L. Biochem. Biophys. Res. Commun. 1976, 68, 841-49. Stanley-Samuelson, D. W.; Jurenka, R. Α.; Cripps, C.; Blomquist, G. J.; de Renobales, M. Arch. Insect Biochem Physiol. In Press. Roelofs, W. L.; Wolf, W. A. J. Chem. Ecol., In Press. Wolf, W. Α.; Roelofs, W. L. Arch. Insect Biochem. Physiol 1986, 3, 45-52. Wang, D. L.; Dillwith, J. W.; Ryan, R. O.; Blomquist, G. J.; Reitz, R. C. Insect Biochem. 1982, 13, 375-79. Hildreth, J. Ε. Κ. Biochem J. 1982, 207, 363-66. Silk, P. J.; Kuenen, L. P. S. Ann. Rev. Entomol. 1988, 33, 83-101. Jurenka, R. A. Unpublished results. Morse, D; Meighen, E. J. Chem. Ecol. 1986, 12, 335-51. Wolf, W. Α.; Roelofs, W. L. Unpublished results. Morse, D; Meighen, E. Science 1984, 226, 1434-36. Wolf, W. Α.; Roelofs, W. L. J. Chem. Ecol. 1987, 13, 101927.

34. Bird, T. G.; Klun, J. Α.; Raina, Α. Κ. RECEIVED

This volume.

September 20, 1988

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 23

Enzyme-Catalyzed Pheromone Synthesis by Heliothis Moths Peter E. A. Teal, J. H. Tumlinson, andA.Oostendorp

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch023

Insect Attractants, Behavior, and Basic Biology Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Gainesville, FL 32604 The specificity of blends of compounds used for pheromone communication by Lepidoptera species is the result of essentially two distinct sets of biosynthetic enzymes which regulate the production of specific olefinic bonds and synthesis of the oxygenated functional moiety, respectively. In Heliothis moths the regulatory systems that are responsible for production of the functional group during the final stages of pheromone biosynthesis consist of cellular acetate esterases and extracellular alcohol oxidases. Evidence indicates that the relative activities of these enzymes differ for each species of Heliothis. Thus, pheromone mediated reproductive isolation between closely related species of Heliothis is probably the result, in large measure, of the fact that some species require only aldehydes for communication while others use acetates, alcohols and aldehydes.

Knowledge of the chemistry, associated behavior and physiology of chemical communication systems of Lepidoptera has increased dramatically over the past 3 decades. However, studies on the biosynthesis of pheromone components are r e l a t i v e l y new. Biochemical investigations are c r i t i c a l f o r accurately defining pheromone communication systems because biosynthesis determines the types of compounds that w i l l be emitted as pheromones and establishes the r a t i o of components released. The importance of elucidation of pathways of biosynthesis which regulate pheromone production i s exemplified by the i d e n t i f i c a t i o n of additional pheromone components f o r the cabbage looper moth. U n t i l the above research had been conducted, (Z)-7-dodecenyl acetate was the sole i d e n t i f i e d pheromone component. This compound was an e f f e c t i v e attractant f o r males, but insects d i d not perform the complete range of behaviors that were exhibited i n response to females. The additional components i d e n t i f i e d by Bjostad et a l . ( 1 ) were found only after studies on biochemical precursors were This chapter not subject to U.S. copyright Published 1989 American Chemical Society

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch023

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333

conducted and these compounds were subsequently found to be c r i t i c a l for maximizing the pheromone mediated response of males (2). Similarly, knowledge of the biochemical mechanisms which regulate the release of actual pheromone components i s of value i n explaining why analogues of behaviorally relevant pheromone components are present within the pheromone gland but are either b i o l o g i c a l l y inactive, or i n h i b i t the response of conspecific insects. For example, ethyl ether extracts of the pheromone glands obtained from females of Heliothis zea contain large amounts of (Z) 11-hexadecen-l-ol, the alcohol analogue of the aldehydic pheromone component present i n greatest amount (3). However, the addition of as l i t t l e as 0.1% of this alcohol to the four aldehydes that are released i n the v o l a t i l e pheromone blend (4) resulted i n a s i g n i f i c a n t reduction i n the number of males captured i n traps i n f i e l d studies (3). The presence of the alcohol i n the pheromone gland was subsequently explained (5-7) by studies on the terminal step of pheromone biosynthesis which showed that a primary alcohol oxidase present i n the c u t i c l e overlying the pheromone gland converts the alcohol to the behaviorally active aldehyde very rapidly, and apparently completely, as the alcohol passes through the c u t i c l e . Biosynthesis of the Hydrocarbon Skeleton. I n i t i a l studies on biosynthesis of pheromones of Lepidoptera were begun i n the early 1970's when Inoue and Hamamura (8) showed that r a d i o l a b e l e d (Ε,Z)-10,12-hexadecadien-l-ol was produced when the pheromone glands of Bombyx mori were incubated with radiolabelled palmitic acid, and Kasang and Schneider (9) induced female gypsy moths to synthesize t r i t i a t e d disparlure by i n j e c t i n g the H l a b e l l e d hydrocarbon analog. Subsequently, Jones and Berger (10) succeeded i n inducing female cabbage loopers to produce C l a b e l l e d (Z)-7-dodecenyl acetate by i n j e c t i n g (1- C) acetate. These i n i t i a l studies were performed on isolated species from families which have d i s t i n c t types of components. Systematic studies on biosynthesis by phylogenically related insects were not undertaken u n t i l the works by Roelofs and Brown (11) and Steck et a l . (12) outlined the analogies i n pheromones used by Tortricidae and Noctuidae respectively. The s i g n i f i c a n t feature of these pheromones i s that most of the Tortricinae use 14-carbon compounds with a double bond at the 11 position whereas the majority of i d e n t i f i e d compounds from noctuid moths have a double bond between the 5th and 6th carbons from the terminal methyl group ( i e . ±7dodecenyl, A9-tetradecenyl, All-hexadecenyl). Studies by Roelofs, Bjostad and Wolf have been instrumental i n elucidating the mechanisms by which these compounds are synthesized. Their studies on the cabbage looper (2, 13, 14), orange t o r t r i x (15) and red banded l e a f r o l l e r moth (16) have demonstrated that a great number of insects u t i l i z e common saturated f a t t y acyl compounds as precursors of unsaturated pheromone components. In this biosynthetic scheme the common acid, palmitate, i s the i n i t i a l precursive compound. Palmitate can be acted upon d i r e c t l y by a ±11desaturase to give (Z)-11-hexadecenoate, which can then be subjected to p a r t i a l β-oxidation to y i e l d (Z)-9-tetradecenoate and (Z)-7dodecenoate, or can be chain elongated to provide 3

14

14

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(Z)-13-octadecenoate. A l t e r n a t i v e l y , palmitate can undergo chain shortening to y i e l d myristate which, when acted upon by the A11desaturase, r e s u l t s i n the production of (E)- or ( Z ) - l l tetradecenoate (17). The A-11-desaturase also appears to explain the production of a number of d i f f e r e n t types of compounds when associated with other enzymatic reactions. For example, the 2,13or 3,13-octadecadienyl compounds found i n moths of the family Aegeriidae can be explained by the action of the A-11 desaturase on palmitate, followed by chain elongation coupled with the action of a β-ketoacyl reductase. Subsequent desaturation would y i e l d either the A2, A13- or A3, Al3-octadecadienoate (17). The biosynthesis of the conjugated diene, (Ε,Z)-10,12-hexadecadien-l-ol (Bombykol) also appears to r e s u l t from incorporation of a double bond at the 11 p o s i t i o n . In this case the (Z)-11-hexadecenoate formed i s apparently acted upon by enzymes capable of isomerizing the double bond to y i e l d the 10,12 conjugated system (17, 18, 19). A more complete discussion of the biosynthesis of the hydrocarbon portion of pheromone molecules i s given by Wolf and Roelofs (this volume). Biosynthesis of Oxygenated Functional Groups. While studies on biosynthesis of the hydrocarbon portion of pheromone molecules are of considerable importance, studies on the functional group are also c r i t i c a l . Often species use blends of compounds having the same functional group. However, numerous cases e x i s t i n which pheromone blends are comprised of compounds which have the same basic structure but d i f f e r i n t h e i r functional group. Also many instances have been documented i n which extracts of pheromone glands contain compounds that have d i f f e r e n t functional groups from the behaviorally s i g n i f i c a n t compounds (3, 20, 21, 22, 23, 24, 25). In many of the above cases clear biosynthetic relationships e x i s t between the classes of compounds. The obvious f i r s t step i n biosynthesis of a functional group i s the reduction of the fatty acid or f a t t y acyl-CoA analogues. Among most organisms this i s accomplished by microsomal acyl-CoA reductases which r e s u l t i n the production of the f a t t y aldehyde (26, 27, 28). This simple enzymatic system would seem a l o g i c a l method of production of aldehydic pheromone components. However, as indicated by Morse and Meighen (29) and Teal and Tumlinson (5, 6, 7), aldehydes are highly toxic and reactive compounds. Consequently, as occurs i n the pheromone gland of the spruce budworm moth the aldehydes are almost instantaneously converted to the alcohol analogues v i a the action of an aldehyde reductase. Evidence that acid reductases function i n pheromone biosynthesis comes from i n v i t r o studies on the orange t o r t r i x moth (15) as well as i n vivo studies on the redbanded l e a f r o l l e r (30). Aldehyde reductase a c t i v i t y occurs i n the pheromone gland of the spruce budworm moth (29). Alcohols formed i n the above reactions can be released as pheromone components d i r e c t l y , subjected to further enzymatic reactions, or both. Two enzyme systems that act on alcohols formed i n the above fashion have been documented to date. By far the most common i s the action of an acetyl transferase which y i e l d s acetates. Acetyl transferase a c t i v i t y can be invoked to explain the production of acetate pheromone components by most t o r t r i c i d moths (17) , and we

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch023

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TEAL ET AL

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have demonstrated that this enzyme converts alcohols to the acetate components i n a noctuid moth species (25). Acetyl transferases also produce acetate intermediates which are further metabolized to alcohol and aldehyde pheromone components. This occurs i n the spruce budworm moth (31) and has been implicated i n pheromone biosynthesis by Heliothis subflexa (25) as well as H. virescens (6, 25). The action of this enzyme, i n these cases, appears to be somewhat of an anomaly inasmuch as the acetates are further metabolized back to the alcohols p r i o r to oxidation to the aldehyde analogues (31-33). However, as discussed by Morse and Meighen (33) the acetates are stable high energy compounds which are r e l a t i v e l y non toxic and thus make good storage precursors. As indicated above, acetates formed i n the pheromone gland of the above species are subsequently converted back to the alcohol v i a the action of an acetate esterase. Evidence to date suggests that the esterase resides i n the soluble f r a c t i o n of pheromone gland homogenates while the acetyl transferase i s found i n the microsomal f r a c t i o n of homogenates (33, 34). Aldehydes are produced v i a the action of alcohol oxidizing enzymes. In those species studied, a primary alcohol oxidase which appears to require only molecular oxygen and the alcohol substrate i s involved (5, 29, 33, 34). However, the action of alcohol dehydrogenases which do not require molecular oxygen to function can not be discounted where aldehydes or ketones are produced as pheromones by other insects. We have recently shown that the oxidase employed to convert the alcohols to aldehydes by three species of Heliothis moths i s present i n the c u t i c l e which covers the c e l l s of the pheromone gland (37), and evidence suggests that the oxidase used by the spruce budworm also resides i n the c u t i c l e (29). An important feature of the enzymatic system i n Heliothis moths i s that the c u t i c l e oxidase converts a l l primary alcohols with equal e f f i c i e n c y . Consequently there i s an excellent c o r r e l a t i o n between the ratios of alcohols produced i n the gland and the ratios of aldehydes released (3, 22, 24,). Comparison of Oxidase and Esterase A c t i v i t i e s i n H e l i o t h i s Members of the Heliothis genus of noctuid moths have been the subjects of considerable study i n our laboratory over the past 10 years not only because of their economic importance, but also because of the a b i l i t y to hybridize two of the species, H. subflexa and H. virescens. Since members of this genus use variations of the same types of compounds for pheromone communication a commonality i n the biosynthetic c a p a b i l i t y of these insects seems indicated (Table I ) . As discussed e a r l i e r , the sixteen carbon aldehydes, used as pheromone components by a l l Heliothis species studied to date, are formed by the action of a primary alcohol oxidase i n H. virescens. H. zea and H. subflexa. Interestingly, although acetate analogues of these aldehydes have only been found i n extracts of the pheromone glands of H. subflexa (22, 35, 36), a l l three species possess esterases i n the pheromone gland that are capable of converting the acetates to primary alcohols (25). Females of H. zea and H. virescens are capable of converting s u b s t a n t i a l l y more t o p i c a l l y

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Table I:

Compounds I d e n t i f i e d from E x t r a c t s o f Pheromone Glands o f North American H e l i o t h i s S p e c i e s * Species

Compound

H, Sea

H. v i r e s c e n s

H. s u b f l e x a

H. phlaxiphaga

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Tetradecanal (Z)-9-Tetradecenal Hexadecanal (Z)-7-Hexadecenal (Z)-9-Hexadecenal (Ζ)-11-Hexadecenal Tetradecan-1-ol (Z)-9-Tetradecen-l-ol Hexadecan-1-ol (Z)-9-Hexadecen-l-ol (Z)-11-Hexadecen-l-ol Hexadecan-l-ol acetate (Z)-9-Hexadecen-l-ol acetate (Z)-11-Hexadecen-l-ol acetate

*The presence o f "+" i n d i c a t e s that a compound has been i d e n t i f i e d as being present i n e x t r a c t s o f pheromone glands, a -" i n d i c a t e s the absence o f the compound. Data f o r H. phlaxiphaga are from Raina e t a l (47). Other data are from r e f e r e n c e s 3, 5, 22, 35 and 36. 11

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch023

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337

applied acetate to alcohol than are females of H. subflexa (Figure 1), indicating that the r e l a t i v e a c t i v i t y of the esterase i n glands i s higher f o r the species which do not use acetates f o r pheromone communication. Consequently, acetates produced i n the pheromone glands of H. virescens and H. zea are probably transitory intermediates i n pheromone biosynthesis, and are converted to the corresponding alcohols as rapidly as they are produced. However, i n H. subflexa this i s not the case and acetates are released as pheromone components (22,35,46). Studies of esterase a c t i v i t y i n c e l l - f r e e c u t i c l e preparations and homogenates of pheromone gland c e l l s have yielded interesting r e s u l t s . Over 96% of the esterase a c t i v i t y was associated with the c e l l u l a r f r a c t i o n of glands i n H. zea and H. subflexa. However, the c e l l u l a r f r a c t i o n of the glands of H. virescens had less a c t i v i t y (85%) (figure 2). The small amount of esterase a c t i v i t y associated with the c u t i c l e of both H. subflexa and H. zea may be an a r t i f a c t of our experimental approach since m i c r o v i l l a r extensions of the a p i c a l c e l l membranes go into the c u t i c l e overlying the gland (37). These membranes would remain associated with the c u t i c l e fragments unless the c u t i c l e was f i n e l y ground thus s o l u b i l i z i n g the enzyme. Therefore esterases bound to the apical c e l l membrane would be present i n the c u t i c l e used i n our experiments. This i s supported, i n part, by preliminary studies conducted i n our laboratory i n which f i n e l y ground c u t i c l e of the glands of H. zea had high oxidase a c t i v i t y but no esterase a c t i v i t y . Esterase a c t i v i t y i n the c u t i c l e of H. virescens i s twice that found i n the c u t i c u l a r fractions of H. subflexa and H. zea. This suggests that females of this species maintain esterases i n the c u t i c l e , as has been hypothesized f o r the spruce budworm moth (33), and esterase a c t i v i t y has been found i n c u t i c u l a r pore canals of other insects (38). H. virescens and H. subflexa are very closely related species that are capable of hybridizing (39). However, i n nature there i s no cross a t t r a c t i o n between the two species (40) and studies have shown that the sixteen carbon acetates are required f o r communication by H. subflexa (46). Consequently, the presence of the esterase i n the c u t i c l e of H. virescens may act as a f a i l - s a f e mechanism which converts any acetate which might enter the c u t i c l e to the alcohol analogue p r i o r to conversion to the aldehyde, thus maintaining the species s p e c i f i c i t y of the pheromone blend of H. virescens. The oxidase that subsequently converts alcohol metabolites to aldehydic pheromone components i s e s s e n t i a l l y limited to the c u t i c l e which covers the c e l l s of the pheromone gland (7). This enzyme becomes active after the adult c u t i c l e has become f u l l y developed j u s t p r i o r to eclosion from the pupal stage i n H. zea and i s active when females of H. virescens and H. subflexa emerge from the pupal case (Figure 3). This stage of development correlates well with s t r u c t u r a l changes i n the pheromone gland that include the development of numerous l i p o p h i l i c v e s i c l e s i n the c e l l s (41). In other species, notably the spruce budworm moth (42) which also appears to use a cuticular alcohol oxidase i n pheromone biosynthesis, c e l l u l a r changes that occur at this time include a switch from protein to l i p i d synthesis, as i s indicated by the degradation of rough endoplasmic reticulum and evolution of numerous Golgi complexes, microbodies and extensive smooth tubular endoplasmic reticulum. Pore canals have been implicated as

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch023

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BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

Κ SUBFLEXA KVIRESCENS Figure 1. Percentage of (Z)-11-tetradecen-l-ol acetate (500 ng) converted to alcohol by the action of acetate esterase ( s o l i d bars) and subsequently to aldehyde (checked bars) v i a the oxidase i n the intact pheromone glands of H. subflexa. H. virescens and H. zea (n - 10, each species). 120 ι

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In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

24. MORI

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OH f.c.d)

~40V.' Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch024

(S).J8 Figure 5. Synthesis of ( S ) - s u l c a t o l and (2R,5S)-pityol. Reagents: a) DHP, TsOH (quant.); b) LAH/ether (89%); c) TSCI/C5H5N (quant.); d) Me2C=CHMgBr Cul/THF (quant.); e) AcOH-THF-H 0, Δ (82%); f ) Tl(OAc) /HBF -An-H20 (99%; 12% after MPLC purification). f

2

OH A/ 2 (SH5 C 0

3

OH

OTHP E t

4

d.e)

a.b.c), OTs

f)

Λ Λ Λ (S)-16 Sulcatol

^0' " f OH (2R,5S)-17 Pityol Figure 6. Preparation of the enantiomers of methyl 3 hydroxypentanoate. Reagents: a) Candida rugosa; b) MeOHH2SO4 (80%); c) 3 5-(02N)9CeH3C02H DMAP, DCC/CH Cl2; r e c r y s t a l l i z a t i o n ; d) KOH/THF-MeOH-H20; e) Saccharomyces cerevisiae (70%); f) I^CX^/MeOH. f

f

2

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

OH ^A^C02Me

a,b,c,d,e )

f) v

0CH2Ph

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(R)-18 e

( 93 / e.e.) e

QSif

0

m)

I) 20 (1007.e.e.) OH 1

0

OH

1

(1

: 2.8)

OH

0

OH

Γ

(4S,6S,7S)-19

(4R,6S,7S)-19

Figure 7. Synthesis of (4S,6S,7S)-serricornin. Reagents: a) LDA, Mel; b) DHP, PPTS (61% from 18); c) LAH (99%); d) NaH, P h C H C l (94%); e) TsOH, MeOH (quant.); f ) , 3,5(02N)2C6H C02H, PI13P, Et02CN=NC02Et, r e c r y s t a l l i z a t i o n (40%); g) KOH (95%); h) t-BuMe2SiCl, imidazole (quant.); i ) H /Pd-C (97%), j ) T s C l 7 c H N ; k) Nal (99.6%, 2 steps); 1) Et200, LDA (80%); m) AcOH-THF-H 0 (43%). 2

3

2

5

5

2

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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24. MORI

355

Preparation of Chiral Building Blocks

Ο

Ο

OSi-|-

(+)-23 Sporogen-AO 1

Figure 8. Synthesis o f (+)-sporogen-AO 1. Reagents: a) baker's yeast (74%); b) DHP, PPTS (quant.); c) LAH/ether (98%); d) TsCl/C5H5N (95%); e) PPTS/MeOH; f ) aq HC104~ether (85%); g) t-BuOK/t-BuOH (78.5%); h) Ph P, Et0 CN=NC02Et, PhC02H/THF; LiOH/MeOH (80%); i ) t-BuMe SiCl, imidazole-DMF (quant.); j ) Li/NH -t-BuOH, M e l (83%); k) M e S i I (Me Si)2NH; CH2=CHCOMe B F OEt2/i-PrOH-MeN0 (68%); 1) p y r r o l i d i n e / C H (78.6%); m) LDA, MeCHO (92%); n) (COCl)2, DMSO E t N (78.5%); o) DDQ/ether (83%); p) t-BuOOH-Triton Β (70%); q) Me SiCH MgCl; H2SO4-THF; HF-MeCN (88%). 3

2

2

3

3

3

f

6

f

3

2

6

3

3

2

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

r

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BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

Figure 9. Synthesis of the enantiomers of p o l y g o d i a l . Reagents: a) baker's yeast (63-79%); b) t-BuMe SiCl (81%); c) LDA/THF-HMPA, Mel (89%); d) NaC=CH/liq NH (99%); e) CuSO^xylene, Δ (51%); f ) H /Pd-CaC03, q u i n o l i n e i n npentane (quant.); g) Me0 CC=CC0 Me, 110°C, 30 h (97%); h) aq HF-MeCN (27% of 27 and 27% of 28 after MPLC); i ) DBU/THF, Δ; H /Pd-C (80% from 27); j ) TfC1-DMAP/CH C1 (86%); k) H /Pd (89%); 1) LAH/ether (82%); m) (C0C1) , DMSO Et3N/CH Cl (63%); n) DBU/THF 2

3

2

2

2

2

2

2

2

2

f

Figure 10. Synthesis of juvenile hormones I and II.

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

2

2

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24. MORI

357

Preparation of Chiral Building Blocks

B a e y e r - V i l l i g e r o x i d a t i o n r e a c t i o n t o cleave the cyclohexane r i n g (31). ( 2S, 3S ) - 2 - E t h y 1 - 3 - h y d r o x y - 2 - m e t h y l c y c l o h e x a n o n e a s a B u i l d i n g B l o c k f o r J u v e n i l e Hormones I a n d I I . R e d u c t i o n o f a p r o c h i r a l 1,3d i k e t o n e 30 w i t h P i c h i a t e r r i c o l a KI 0117 f u r n i s h e s (2S,3S)-31 o f 99% e.e. (32). As shown i n F i g u r e 10, 31 was e m p l o y e d a s t h e s t a r t i n g m a t e r i a l f o r the s y n t h e s i s o f e n a n t i o m e r i c a l l y pure (+)-juvenile h o r m o n e s I a n d I I (32). P u r i f i c a t i o n o f 3 1 was a c h i e v e d by r e c r y s t a l l i z a t i o n o f 3 2 , whose B a e y e r - V i l l i g e r o x i d a t i o n g a v e a c r y s t a l l i n e l a c t o n e 33. M e t h a n o l y s i s o f t h e l a c t o n e 33 was f o l l w e d by p r o t e c t i o n o f the g l y c o l system t o g i v e 34. T h i s was c o n v e r t e d t o j u v e n i l e hormones I (35) and I I (36). ( I S , 4 R , 5 S ) - 5 - H y d r o x y - l - m e t h y l b i c y c l o [ 2 . 2 . 1 ]heptan-3-one as a B u i l d i n g B l o c k f o r G l y c i n o e c l e p i n A. R e d u c t i o n o f l - m e t h y l b i c y c l o [ 2.2.1 ] heptane-3,5-dione (37) w i t h baker's y e a s t g i v e s (lS,4R,5S)-5-hydroxyl - m e t h y l b i c y c l o [ 2 . 2 . 1 ] h e p t a n - 3 - o n e (38) o f 8 0 - 8 7 % e.e. a s shown i n F i g u r e 11 ( M o r i , K.; W a t a n a b e , H., T h e U n i v e r s i t y o f T o k y o , unpublished data). By e m p l o y i n g two β-hydroxy ketones 25 and 38 a s t h e b u i l d i n g b l o c k s , g l y c i n o e c l e p i n A (40) was s y n t h e s i z e d v i a 3 9 ( M o r i , K.j Watanabe, H., t h e U n i v e r s i t y o f T o k y o , u n p u b l i s h e d d a t a ) . G l y c i n o e c l e p i n A i s a degraded t r i t e r p e n o i d w i t h s i g n i f i c a n t h a t c h s t i m u l a t i n g a c t i v i t y f o r t h e soybean c y s t nematode (33, 34).

CÛ2H Glycinoeclepin A 40

Figure 11.

Synthesis of glycinoeclepin A.

Acknowledgments I thank my co-workers whose names appear i n t h e r e f e r e n c e s f o r t h e i r e n t h u s i a s m i n c a r r y i n g o u t t h e b i o c h e m i c a l and s y n t h e t i c works. F i n a n c i a l s u p p o r t o f t h i s p r o j e c t by Japanese M i n i s t r y o f E d u c a t i o n , S c i e n c e and C u l t u r e i s acknowledged w i t h thanks.

Literature Cited

1. 2.

Morrison, J.D., Ed. Asymmetric Synthesis Vols. Academic Press: Orlando, 1983-1985. Jones, J.B.; Sih, C.J.; Perlman, D., Eds. Application Biochemical Systems in Organic Chemistry Part 1; Wiley: New York, 1976.

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

1-5; of John

358 3. 4. 5. 6. 7.

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8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.

BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

Mori, K. In Studies in Natural Products Chemistry Vol. 1; Rahman, A.-u., Ed.; Elsevier: Amsterdam, 1988. Okazaki, T.; Noguchi, T. Igarashi, K.; Sakagami. Y.; Seto, H.; Mori, K.; Naito, H.; Masumura, T.; Sugahara, M. Agric. Biol. Chem. 1983, 47, 2949. Mori,K.; Okazaki, T.; Noguchi, T.; Naito, H. Agric.Biol.Chem. 1983, 47, 2131. Mori, K.; Sugai, T.; Maeda, Y.; Okazaki, T.; Noguchi, T.; Naito, H. Tetrahedron 1985, 41, 5307. Baker, B.J.; Okuda, R.K.; Yu, P.T.K.; Scheuer, P.J. J.Am. Chem. Soc. 1985, 107, 2976. Mori, K.; Takeuchi, T. Tetrahedron 1988, 44, 333. Kleiner, E.M.; Pliner, S.A.; Soifer, V.S.; Onoprienko, V.V.; Blasheva, T.A.; Rozynov, B.V.; Khokhlov, A.S. Bioorg. Khim. 1976, 2, 1142. Mori, K.; Yamane, K. Tetrahedron 1982, 38, 2919. Mori, K. Tetrahedron 1983, 39, 3107. Deol, B.S.; Ridley, D.D.; Simpson, G.W. Aust. J. Chem. 1976, 29, 2459. Mori, K. Tetrahedron 1981, 37, 1341. Hungerbuhler, E; Seebach, D.; Wasmuth, D.S.; Helv. Chim. Acta 1981, 64, 1467. Sugai, T. Fujita, M.; Mori, K. Nippon Kagaku Kaishi 1983, 1315. Seebach, D.; Zuger, M.F. Helv. Chim. Acta 1982, 65, 495. Mori, K.; Watanabe, H. Tetrahedron 1984, 40, 299. Mori, K.; Puapoomchareon, P. Liebigs Ann. Chem 1987, 271. Hasegawa, J.; Hamaguchi,S.;Ogura, M.; Watanabe, K. J. Ferment. Technol. 1981, 59, 257. Mori, K.; Watanabe, H. Tetrahedron 1985, 41, 3423. Mori, K.; Mori, H.; Sugai, T. Tetrahedron 1985, 41, 919. Chuman, T.; Kohno, M.; Kato, K; Noguchi, M. Tetrahedron Lett. 1979, 2361. Kitahara, T.; Mori, K. Tetrahedron Lett. 1985, 26, 451. Tanaka, S.; Wada, K.; Marumo, S.: Hattori, H. Tetrahedron Lett 1984, 25, 5907. Mori, K.; Tamura, H. Liebigs Ann. Chem. 1988, 97. Kitahara, T.; Kurata, H.; Mori, K. Tetrahedron 1988, 43, 4339. Mori, K.; Mori, H. Tetrahedron 1985, 41, 5487. Mori, K.; Watanabe, H. Tetrahedron 1986, 42, 273. Barnes, C.S.; Loder, J.W. Aust. J. Chem. 1962, 15, 322. Kubo, I.; Lee, Y.-W.; Pettei, M.; Pilkiewicz, F.; Nakanishi, K. J. Chem. Soc., Chem. Commun. 1976, 1013. Mori, K.; Mori, H. Tetrahedron 1987, 43, 4097. Mori, K.; Fujiwhara, M. Tetrahedron 1988, 44, 343. Masamune,T.;Anetani, M; Fukuzawa, A.; Takasugi, M.; Matsue, H.; Kobayashi, K.; Ueno, S.; Katsui, N. Bull. Chem. Soc. Jpn. 1987, 60, 981. Masamune,T.;Fukuzawa, A.; Furusaki, A.; Ikura, M.; Matsue, H.; Kaneko, T.; Abiko, A.; Sakamoto, N.; Tanimoto, N.; Murai, A. Bull. Chem. Soc. Jpn. 1987, 60. 1001.

RECEIVED

September 19, 1988

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 25

Baker's Yeast-Mediated Synthesis of Natural Products Claudio Fuganti and Piero Grasselli

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch025

Dipartimento di Chimica del Politecnico, Centro del Consiglio Nazionale delle Ricerche per la Chimica delle Sostanze Organiche Naturali, 20133 Milan, Italy

Baker's yeast, in a manner that is dependent upon the fermentation conditions and the nature of the αand γsubstituents, converts α,βunsaturated aldehydes into three sets of chiral adducts. These material have been used for the synthesis of optically active forms of several insect pheromones. The a b i l i t y o f b a k e r ' s y e a s t t o t r a n s f o r m unconventional s u b s t r a t e s s t e r e o s e l e c t i v e l y i s w e l l known, as one can judge from the c o n s i d e r a b l e amount o f work i n t h i s a r e a r e p o r t e d i n the e a r l y literature (_1). However, o r g a n i c c h e m i s t s o n l y r e c e n t l y r e c o g n i z e d the s y n t h e ­ t i c p o t e n t i a l o f b a k e r ' s y e a s t , i n t h e more g e n e r a l c o n t e x t o f a s y n t h e t i c approach t o e n a n t i o m e r i c a l l y pure forms o f b i o l o g i c a l l y a c t i v e n a t u r a l p r o d u c t s such as i n s e c t pheromones.These m a t e r i a l s a r e o f c o n s i d e r a b l e i m p o r t a n c e i n a g r i c u l t u r e , and have been s y n ­ t h e s i z e d i n some c a s e s u s i n g r e a d i l y a v a i l a b l e o p t i c a l l y a c t i v e n a t u r a l p r o d u c t s (2), (_3) .The n a t u r a l m a t e r i a l s employed, however, u s u a l l y a r e a v a i l a b l e i n o n l y one enantiomeric form, a circumstance w h i c h r e p r e s e n t s a m a j o r d r a w b a c k when both enantiomers o f t h e t a r g e t m o l e c u l e a r e n e e d e d , o r when t h e a b s o l u t e c o n f i g u r a t i o n o f t h e f i n a l p r o d u c t c a n n o t be o b t a i n e d from t h e s t a r t i n g m a t e r i a l . F u r t h e r m o r e , t h e t y p e s o f c h i r a l i t y p r e s e n t amongst t h i s s e t o f compounds are r a t h e r l i m i t e d ; they a r e p a r t i c u l a r l y a b u n d a n t i n t h e R,R'CHX s t r u c t u r e (X=oxygen o r n i t r o g e n f u n c t i o n s ) and s p a r s e i n t h e R,R',R"CH and R, R', R"C (OR"') moieties. These o b s e r v a t i o n s s t i m u l a t e d a s e a r c h f o r new, relatively small, h i g h l y f u n c t i o n a l i z e d c h i r a l m a t e r i a l s complementary to those a l r e a d y p r o d u c e d by n a t u r e , and i t was t h o u g h t t h a t t h i s need c o u l d be s a t i s f i e d p a r t i a l l y by t h e compounds a v a i l a b l e by enzymatic t r a n s f o r m a t i o n s o f u n c o n v e n t i o n a l substrates using e i t h e r i s o l a t e d enzymes or m i c r o o r g a n i s m s (4). Organic c h e m i s t s , who a r e n o t g e n e r a l l y k e e n on g r o w i n g c u l t u r e s , began u s i n g baker's y e a s t which i s r e a d i l y a v a i l a b l e and cheap, u s i n g i t l i k e a common s h e l f r e a g e n t i n r e a c t i o n s v e r y s i m i l a r to the n a t u r a l t r a n s f o r m a t i o n s t h a t i t c a t a l y z e s . M o s t o f the c u r r e n t a p p l i c a t i o n s o f b a k e r ' s y e a s t i n v o l v e s t e r e o s p e c i f i c

c

0097-6156/89/0389-0359$06.00/0 1989 American Chemical Society

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r e d u c t i o n o f compounds c o n t a i n i n g c a r b o n y l groups and/or ( c a r b o n y l a c t i v a t e d ) d o u b l e b o n d s , and w i d e s u b s t r a t e a c c e p t a n c e i s shown. However, i n some i n s t a n c e s , t h e s y n t h e t i c s i g n i f i c a n c e o f t h e s e transformations is diminished by the limited reaction s t e r e o s e l e c t i v i t y perhaps, because s e v e r a l e n z y m a t i c a l l y a c t i v e p r i n c i p l e s a r e a c t i n g on the same s u b s t r a t e w i t h o p p o s i n g s t e r e o b i a s e s ( 5 ) , (6) .We became i n t e r e s t e d i n the baker's yeast transformations of u n c o n v e n t i o n a l s u b s t r a t e s i n a study of the s t e r i c c o u r s e o f the known (7) c o n v e r s i o n o f cinnamaldehyde i n t o 3 - p h e n y l p r o p a n o l . By means o f d e u t e r i a t e d s u b s t r a t e s i t has b e e n shown t h a t s a t u r a t i o n of the double bond o c c u r s w i t h f o r m a l t r a n s 2 a d d i t i o n of hydrogen; [2- H ] cinnamaldehyde b e i n g t r a n s f o r m e d i n t o (2S) [ 2 - H ] 3 - p h e n y l p r o p a n o l (8). The latter m a t e r i a l has been u s e d i n the s y n t h e s i s of a s y m m e t r i c a l l y l a b e l l e d L-homoserine i n a study on the mechanism o f t h e e n z y m a t i c f o r m a t i o n o f L - t h r e o n i n e (9).However, we s o o n r e a l i z e d t h a t under the e x p e r i m e n t a l c o n d i t i o n s u s e d , the r e d u c t i o n o f the c a r b o n y l carbon and the s a t u r a t i o n o f the double bond a r e o n l y two o f t h e s y n t h e t i c m a n i f e s t a t i o n s t h a t a r e p o s s i b l e u s i n g an a - B - u n s a t u r a t e d a l d e h y d e (10). S i n c e t h e n , we have b e e n e x p l o r i n g t h i s a r e a and i t now appears t h a t α - βu n s a t u r a t e d aldehydes can be reduced by baker's y e a s t t o y i e l d the s y n t h e t i c a l l y u s e f u l c h i r a l p r o d u c t s i n d i c a t e d i n Scheme 1 i n a manner t h a t d e p e n d s upon t h e f e r m e n t a t i o n c o n d i t i o n s and t h e n a t u r e o f the α and Ύ s u b s t i t u e n t s . The f o r m a t i o n from cinnamaldehyde (1) o f the (2S,3R) d i o l (3) (Scheme 2) t h a t c o n t a i n e d two a d d i t i o n a l carbon atoms t o those o f cinnamaldehyde and two a d j a c e n t c h i r a l c e n t r e s was new. They o c c u r r e d as t h e c o n s e q u e n c e o f two d i s t i n c t c h e m i c a l operations. A d d i t i o n of a C e q u i v a l e n t of a c e t a l d e h y d e onto the s i f a c e of the c a r b o n y l c a r b o n f o r m e d t h e (R) α - h y d r o x y ketone (2), t h a t was s u b s e q u e n t l y reduced on the r e f a c e t o g i v e the (2S, 3R) d i o l (3) i l l ) . The C-C bond f o r m i n g p r o c e s s i s i d e n t i c a l t o the one r e p o r t e d by N e u b e r g (12) w h e r e i n benzaldehyde was t r a n s f o r m e d t o ( R ) - a c e t y l p h e n y l c a r b i n o l , a key i n t e r m e d i a t e i n the manufacture o f (-) e p h e d r i n e , i n f e r m e n t i n g baker's y e a s t . The i d e n t i t y o f the enzyme(s) i n v o l v e d i n the l a t t e r r e a c t i o n has been debated {13). However, the f o r m a t i o n o f the above hydroxyketone, i n a n a l o g y w i t h a c e t o i n , has been c o n c e p t u a l i z e d as the consequence o f the c o n d e n s a t i o n o f the " a c t i v e " form o f a c e t a l d e ­ hyde, t h a t i s f o r m e d by d e c a r b o x y l a t i v e a d d i t i o n o f p y r u v a t e t o t h i a m i n e pyrophospate, w i t h benzaldehyde.The r o l e o f p y r u v a t e , i n f a c t has been e s t a b l i s h e d . The same mechanism can be invoked f o r the r e a c t i o n o f c i n n a m a l d e h y d e . l t i s known t h a t t h e p y r u v a t e d e c a r b o x y l a s e (E.C. 4.1.1.1) a c c e p t s as s u b s t r a t e s a - o x o a c i d s h i g h e r t h a n p y r u v a t e . When we i n c u b a t e d c i n n a m a l d e h y d e (1) w i t h baker's y e a s t t h a t had b e e n w a s h e d w i t h w a t e r a t pH 5.5-6, and w i t h α - o x o b u t a n o a t e , we o b t a i n e d (4R) 5, t h a t was subsequently reduced by y e a s t i n t h e p r e s e n c e o f D - g l u c o s e t o t h e (3S,4R) d i o l 6 (14). I t s h o u l d be n o t e d t h a t t h e f i n a l p r o d u c t 6 i s t h e r e s u l t o f c o u p l i n g two r e a g e n t s ( a l d e h y d e and α - o x o a c i d ) t h a t had b e e n added t o the r e a c t i o n mixture. T h i s r e a c t i o n , w h i c h w o u l d be o f t e n d e s i r a b l e , i s s e l d o m observed i n the e n z y m a t i c c a t a l y s i s (15). There i s some t o l e r a n c e by the enzyme (s) i n v o l v e d i n the above

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2

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25. FUGANTI & GRASSELLI

Yeast-Mediated Synthesis ofNatural Products

Scheme 1

Ο 1

R= H,Me

H +

OH 2

R= H,Me

OH 3

R= Η

4

R= Me

Scheme 2

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p r o c e s s w i t h r e s p e c t t o the s t r u c t u r e o f the a,β-unsaturated a l d e ­ hyde. I n d e e d , α - m e t h y l and o t - b r o m o c i n n a m a l d e h y d e s a f f o r d e d t h e c o r r e s p o n d i n g d i o l s 7 and 8 (Scheme 3), but α-ethylcinnamaldehyde is not a s u b s t r a t e f o r t h i s reaction. Furthermore, α m e t h y l c r o t o n a l d e h y d e was not a c c e p t e d , but when the p o s i t i o n was o x i d i z e d , as i n e t h y l 3-methyl-4-oxo c r o t o n a t e , up t o 35% y i e l d o f the d i o l 9 was o b t a i n e d i n b a k e r ' s y e a s t f e r m e n t i n g on Dg l u c o s e . U s i n g washed y e a s t , the same a l d e h y d o e s t e r i n the presence o f e t h y l α - o x o b u t a n o a t e a f f o r d e d the c o r r e s p o n d i n g (R) α-hydroxy ketone, t h a t was s u b s e q u e n t l y r e d u c e d t o t h e d i o l (10) , a l t h o u g h i n lower o v e r a l l y i e l d . The d i o l s 4 and 6-10 a r e p a r t i c u l a r l y s u i t e d f o r the p r e p a r a ­ t i o n of s m a l l m o l e c u l e s , l i k e i n s e c t pheromones, t h a t c o n t a i n r e l a t i v e l y few c h i r a l carbon atoms i n t h e i r framework and whose c h i r a l i t y i s due t o oxygen s u b s t i t u t i o n . F r o m r e l a t e d i s o p r o p y l i dene s t r u c t u r e s i t i s p o s s i b l e t o s y n t h e s i z e the c h i r a l C^,C^ and C c a r b o n y l compounds (11)-(14) by o z o n o l y s i s , t h a t a r e c o n v e r t i b l e i n t o t h e α - e p i m e r s ( 1 5 ) - ( 1 8 ) by b a s e treatment. Furthermore, the number o f synthons t h a t a r e p o t e n t i a l l y a v a i l a b l e i s i n c r e a s e d by the adducts a c c e s s i b l e from (11)-(18) by r e a c t i o n with s u i t a b l e carbon n u c l e o p h i l e s under c o n d i t i o n s a l l o w i n g c o n t r o l o f the s t e r e o c h e m i s t r y by the two oxygen f u n c t i o n s . 6

S y n t h e s i s o f Pheromones from Condensation

the P r o d u c t s

of

Acyloin-Type

Thus, ( - ) - f r o n t a l i n 19 (Scheme 4), p h e r o m o n e o f Dentroctonus F r o n t a l i s b a r k b e e t l e , was o b t a i n e d f r o m t h e m e t h y l k e t o n e 12 through t h e i n t e r m e d i a c y o f t h e a d d u c t 20, t h a t was o b t a i n e d as the s o l e t r a n s f o r m a t i o n p r o d u c t by r e a c t i o n w i t h 4-methyl-pent-4enylmagnesium bromide i n THF. The l a t t e r m a t e r i a l , once 0-benzyl a t e d , w i t h a c i d h y d r o l y s i s y i e l d e d 22, and was t h e n c o n v e r t e d i n t o 23 by p e r i o d a t e o x i d a t i o n , f o l l o w e d by NaBH^ r e d u c t i o n . T h i s s e q u e n c e d e s t r o y e d t h e two c h i r a l c e n t e r s o f 12 t h a t had a s s i s t e d the f o r m a t i o n o f the q u a t e r n a r y c h i r a l c e n t e r r e q u i r e d i n 26. The c o n v e r s i o n o f 23 i n t o 26 s i m p l y r e q u i r e d o z o n o l y s i s t o 24 and h y d r o g e n o l y s i s t o the d i h y d r o x y k e t o n e 25, which s p o n t a n e o u s l y c y c l i z e s t o 26 ( 27% o v e r a l l y i e l d f r o m 20) (16). The aldehyde 11 and i t s α -epimer 15, on r e a c t i o n w i t h s a t u r a ­ ted Grignard reagents, afforded separable mixtures of d i a s t e r e o i s o m e r s i n w h i c h the syn adducts p r e v a i l e d . The two s e t s o f a d d u c t s 2 6 - 3 1 so o b t a i n e d (Scheme 5) w e r e 0b e n z y l a t e d , h y d r o l y z e d under a c i d i c c o n d i t i o n s , and oxidized with p e r i o d a t e , t o y i e l d the e n a n t i o m e r i c aldehydes 32-37. The aldehyde 32 was t r e a t e d w i t h a s u i t a b l e Grignard rea­ gent t o p r o d u c e t h e a d d u c t s 38 and 39, (Scheme 6) t h a t w e r e t h e n c o n v e r t e d i n u n e x c e p t i o n a l s t e p s , i n t o ( + ) - e x o b r e v i c o m i n 40 and i n t o t h e ( ) e n d o d i a s t e r e o i s o m e r 41, t h a t w e r e s e p a r a t e d by p r e p a r a t i v e gas chromatography. S i m i l a r l y , from 35 the (-) and (+)- e n a n t i o m e r s , respectively, were p r e p a r e d (17). Compound 33, was a l l o w e d t o r e a c t w i t h BrMg (CH ) CH=CH , and the s y n and a n t i d i a s t e r e o i s o m e r s so o b t a i n e d w e r e s e p a r a t e d t o u l t i m a t e l y y i e l d , (5S,6R) 6 - a c e t o x y - 5 - h e x a d e c a n o l i d e 42 and t h e -

_

2

3

2

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

25. FUGAOTI & GRASSELLI

Yeast-Mediated Synthesis ofNatural Products

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch025

OH

OH

oVr 6

R= Me; R = H

7

R= H; R = Me

8

R

1

X

R= H; R = Br

OH

1

Ο

„ > > o

OH

9 10

R= Η

11 R=

R= Me

12

R= H; R = Me

13

R= Me; R = Η

14

R= R = Me

1

1

ft 15

R= R = Η

16

R= H; R = Me

17

R= Me; R = Η

18

R= R = Me

1

1

1

Scheme 3

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

363

BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

364

0

x

0

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch025

HQ

20

R= Η

21

R=CH C H

pH

22 2

6

5

23

R= CH C H ; X= CH

24

R= CH C H ; X= Ο

25

R= Η; X= Ο

2

6

2

5

6

R= C H C H 26 5

19

2

5

Scheme 4

Λ

qXp

R v

n

OH

26

R= Me

29

R= Me

27

R= C H n

30

R= CgH^n

28

R= (CH ) CH=CH

31

R= (CH^CIfeCH,,

9

l g

2

2

2

0

H ^ Y ^ R OR'

OR'

32

R= Me; R*= CHgCgt^

35

R= Me; R*= CHgCgHg

33

R= C H n ; R = CH^H,.

36

R= CgH^n; R =

34

R= (CH ) CH=CH

37

R= ( C H ^ C f e C H g

X

9

ig

2

2

2

X

CHgC^

Scheme 5

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

25. FUGANTI & GRASSELLI

Yeast-Mediated Synthesis of Natural Products

(5R,6R) d i a s t e r e o i s o m e r (Scheme 7). A g a i n , f r o m 34 t h e (5R,6R) p r o d u c t 43 was o b t a i n e d . P r o d u c t s 42 and 43 r e p r e s e n t t h e two enantiomers o f the proposed major component of a mosquito o v i p o s i t i o n a t t r a c t a n t p h e r o m o n e (18). The e n a n t i o m e r i c f o r m s o f 5h e x a d e c a n o l i d e , 44 and 45, the pheromone o f Vespa o r i e n t a l i s , were o b t a i n e d f r o m t h e e n a n t i o m e r i c a l d e h y d e s 34 and 37 (12)· H ^ compounds o b t a i n e d up t o t h i s p o i n t h a v e b e e n p r e p a r e d i n b o t h e n a n t i o m e r i c forms, s t a r t i n g from a s i n g l e c h i r a l m a t e r i a l . Contrastingly, the c h i r a l framework of the t h r e o aldehyde 15 was i n c o r p o r a t e d i n t o (2S, 3S) o c t a n e - 2 , 3 - d i o l 46, and i n t o the d e r i v e d k e t o n e 47, by a W i t t i g c o n d e n s a t i o n w i t h a C reagent, f o l l o w e d by hydrogénation and h y d r o l y s i s . These m a t e r i a l s a r e pheromones o f X y l o t r e c h u s p y r r h o r e c u s . From the same i n t e r m e d i a t e , by u n e x c e p t i o n a l s t e p s , 47 was o b t a i n e d (20). I n c o r p o r a t i o n o f t h e carbon framework o f the a l d e h y d e 11 i n t o (3S,4S) 4-methyl-3-heptan o l 49 (Scheme 7) was a c c o m p l i s h e d v i a the epoxide 48, but w i t h extended m a n i p u l a t i o n o f t h e c h i r a l i t y p r e s e n t i n t h e p r e c u r s o r (2_1) . P r o d u c t 49 i s a p h e r o m o n e o f S c o l y t u s Eliiî:iËÎ:£i£Î:H:£* study o f the mechanism o f the a c y l o i n - t y p e c o n d e n s a t i o n r e p o r t e d i n Scheme 2, we o b t a i n e d the (3S,4R) d i o l 50 by y e a s t r e d u c t i o n o f the c o r r e s p o n d i n g h y d r o x y k e t o n e . T h i s s e r v e d as t h e s t a r t i n g m a t e r i a l f o r the s y n t h e s i s o f (4S,5R) s i t o p h i l u r e 53 (Scheme 8), a pheromone o f t h e g e n u s S i t o p h i l u s . The key i n t e r m e d i a t e was t h e a l l y l i c epoxide 51, t h a t y i e l d e d the c h i r a l h o m o a l l y l i c a l c o h o l 52 on A l H ^ o p e n i n g , and c o n t a i n e d t h e c h i r a l i t y and t h e masked f u n c t i o n a l i t y f o r c o n v e r s i o n i n t o 53. I n d e e d , O - p r o t e c t i o n by silylation, o z o n o l y s i s , a d d i t i o n of ethylmagnesium bromide, o x i d a t i o n o f t h e c a r b i n o l and d e p r o t e c t i o n y i e l d t h e r e q u i r e d p r o d u c t 5 3 (22). A

t

i e

n

a

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch025

4

I

Synthesis of pheromones Double Bond.

from

the

Products

of

S a t u r a t i o n of

the

With t h e e x c e p t i o n o f t h e d i o l 9, t h a t was o b t a i n e d f r o m t h e c o r r e s p o n d i n g a l d e h y d e i n up t o 35% y i e l d , m o s t o f t h e c h i r a l d i o l s mentioned above were i s o l a t e d i n y i e l d s o f o n l y 20-25%. The formation o f t h e a c y l o i n - t y p e c o n d e n s a t i o n products i s in c o m p e t i t i o n w i t h the much more e f f i c i e n t r e d u c t i o n o f the c a r b o n y l carbon and s a t u r a t i o n o f the double bond o f the u n s a t u r a t e d a l d e hydes t h a t w e r e u s e d as s u b s t r a t e s . We became i n t e r e s t e d i n t h e mode o f r e d u c t i o n o f p a r t i c u l a r aldehydes such as 54-56 (Scheme 8) i n a study o f the t o t a l s y n t h e s i s o f n a t u r a l α-tocopherol ( v i t a m i n E) (2J3) . We e x p e c t e d t o o b t a i n c h i r a l a l c o h o l s t h a t w o u l d be u s e f u l f o r c o n v e r s i o n i n t o n a t u r a l i s o p r e n o i d s from the r e d u c t i o n o f t h e α - d o u b l e bond o f t h e a b o v e a l d e h y d e s . Indeed, 54-56 a f f o r d e d up t o 75% y i e l d o f t h e s a t u r a t e d c a r b i n o l s 5 7 - 5 9 by treatment w i t h y e a s t . Whereas t h e ee o f 57 and 58 was c a 85%-90%, t h a t o f 59 i s 99%, as shown by NMR ex­ periments on the (-)-MTPA d e r i v a t i v e (24). The s y n t h e t i c s i g n i f i ­ cance o f c a r b i n o l 59 was b a s e d on t h e s t r u c t u r a l unit present i n n a t u r a l i s o p r e n o i d s (see b r a c k e t s i n s t r u c t u r a l formulas) .This p r o t e c t e d s y n t h o n c a n be u n m a s k e d by o z o n o l y s i s , as i n d i c a t e d by the h i g h y i e l d c o n v e r s i o n o f 59 i n t o (S)-(-) - 3-methy1-γb u t y r o l a c t o n e 60 (Scheme 9). P r o d u c t 59 i s a b i f u n c t i o n a l c h i r a l i n t e r m e d i a t e which does not need p r o t e c t i v e m a n i p u l a t i o n i n t h a t

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

365

366

BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

OH

R= 0(CH ) 0; R*= l

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch025

38

2

2

OH

CH^^

39

R= 0(CH ) 0; R*= CI^CgHg l

2

40

2

41

Scheme 6 OAc

42

OAc

R= C H n g

43 R= C H n

i g

g

i g

44 R= C_H ,n 45 R= C ^ H n 10 21 10 21 0

OH

OH

OH

Ο

46

47

OR

48

R=CH C H 2

6

49

5

OH

Scheme 7

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

0 1

25. FUGANTI & GRASSELLI

Yeast-Mediated Synthesis of Natural Products

PH

OH 51

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch025

50

Ο

OH 53

52

54

R= H; R = Me

55

R= Me; R = Η

56

X

OH

J

57

R= H; R = Me

58

R= Me; R = Η

Cr: 59

1

Scheme 8

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

367

BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch025

368

Scheme 9.

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

25. FUGANTI & GRASSELLI

Yeast-Mediated Synthesis ofNatural Products

the f u r a n r i n g can be seen as a c a r b o n y l p r o t e c t i n g group and can be s e l e c t i v e l y removed p e r m i t t i n g e l o n g a t i o n a t b o t h ends o f t h e molecule.The f u n c t i o n a l i t i e s o f 59 c a n be u s e d i n a l t e r n a t i n g manner. The d i r e c t o z o n i z a t i o n w i t h o x i d a t i v e w o r k up l e d t o 6 0 . A l t e r n a t i v e l y , t h e a l c o h o l f u n c t i o n a l g r o u p c o u l d be u s e d t o modify t h e s i d e c h a i n and t h e f u r a n r i n g o z o n i z e d a t a subsequent stage. T a k i n g advantage o f t h e above mentioned p r o p e r t i e s , p r o d u c t 59 has been used f o r t h e p r e p a r a t i o n o f (4R,8R)-4,8-dimethyldecan a l 64, a p h e r o m o n e c o m p o n e n t o f T r i b o l i u m c a s t a n eu m (2^4). The s y n t h e s i s o f 64 i n v o l v e d t h e u s e o f two e q u i v a l e n t s o f 59 a n d proceeded t h r o u g h t h e i n t e r m e d i a t e s 6 1 - 6 3 t h a t w e r e a s s e m b l e d according t o the sequences: + C-^ — C ; C + n'> n C-^ — C a d d i t i o n t o t h e two s e t s o f c h i r a l products mentioned a b o v e , a n o t h e r t y p e o f o p t i c a l l y a c t i v e m a t e r i a l i s available i n fermenting baker's y e a s t from α , 3 -unsaturated a l d e h y d e s . D u r i n g s t u d i e s on t h e s u b s t r a t e s p e c i f i c i t y o f t h e a c y l o i n - t y p e c o n d e n s a t i o n i n d i c a t e d i n Scheme 2 we s u b m i t t e d Ύ-oxygen substituted crotonaldehydes t o t h e a c t i o n o f f e r m e n t i n g baker's y e a s t a t pH 5.Whereas 6 5 a f f o r d e d 35 % y i e l d o f t h e d i o l 9, 66 gave r i s e t o t h e d i o l 6 1 i n c a 2 0 % y i e l d a s s o l e chiral t r a n s f o r m a t i o n product. Compound 67 i s b e l i e v e d t o a r i s e by f o r m a l 1 , 4 - a d d i t i o n o f water a c r o s s t h e d o u b l e bond o f t h e α ,3 - u n s a t u r a ­ t e d a l d e h y d e 6 6 , f o l l o w e d by r e d u c t i o n o f t h e i n t e r m e d i a t e 3hydroxy aldehyde. The s y n t h e t i c s i g n i f i c a n c e o f 67 i s t h a t i t has the (R) c o n f i g u r a t i o n . T h i s i s o p p o s i t e t o t h e i d e n t i c a l m a t e r i a l f o r m a l l y a c c e s i b l e f r o m n a t u r a l (S) m a l i c a c i d , w h i c h h a s b e e n a l r e a d y u s e d a s a s t a r t i n g m a t e r i a l i n p h e r o m o n e s y n t h e s i s . We e x p e c t e d t o o b s e r v e an e x t e n s i o n o f t h e l a t t e r mode o f a c t i o n o f f e r m e n t i n g baker's y e a s t t o more f u n c t i o n a l i z e d γ-oxygen s u b s t i t u ­ t e d a, 3 - u n s a t u r a t e d aldehydes, h o p e f u l l y t o o b t a i n s t e r e o s p e c i f i c h y d r a t i o n / r e d u c t i o n and k i n e t i c r e s o l u t i o n w i t h s u b s t r a t e s l i k e 68. I n s u c h an e v e n t , we s h o u l d h a v e a c c e s s t o new chiral i n t e r m e d i a t e s t h a t c o u l d be u s e f u l i n pheromone s y n t h e s i s . Many o t h e r s u b s t a n c e s b e l o n g i n g t o q u i t e d i f f e r e n t s t r u c t u r a l c l a s s e s , i n a d d i t i o n t o t h o s e m e n t i o n e d above, have been s y n t h e t i s e d from t h e c h i r a l m a t e r i a l s o b t a i n e d i n f e r m e n t i n g baker's y e a s t and α, 3 - u n s a t u r a t e d a l d e h y d e s , thus l e n d i n g f u r t h e r s u p p o r t to the s i g n i f i c a n c e f o r organic synthesis o f the products obtained by b i o t r a n s f o r m a t i o n s . c

6

I

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch025

1

c

+

6

n

2

Literature cited

1. Neuberg, C. Adv. Carbohydr. Chem. 1949, 4, 75 2. Seebach, D.; Kalinowski, H. O.,; Nachr. Chem. Techn. 1976, 24, 415 3. Hanessian, S. Total Synthesis of Natural Products: The Chiron Approach; Pergamon Press, London, 1983 4. Fischli, A. in Modern Synthetic Methods 1980; Scheffold, R., Ed.,; Salle + Sauerlander; Aarau, 1980; p 269 5. Zhou, B.; Gopalan, A.S.; Van Middles worth, F.; Shieh, W.; Sih, C. J. Am. Chem. Soc. 1983, 105, 5925 6. Sih, C.; Chen, C.S. Angew. Chem. Int. Ed. Engl. 1984, 23 570 7. Fisher, F.G.; Wiedemann,O.Annalen 1934, 513, 265 8. Fuganti, C; Ghiringhelli, D; Grasselli, P. J.Chem. Soc. Chem. Commun. 1975, 846

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

369

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch025

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BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

9. Fuganti, C. J. Chem. Soc. Chem. Commun. 1979, 337 10. Fuganti, C.; Grasselli, P. Chem. & Ind. (London) 1977, 983 11. Fuganti, C.; Grasselli, P.; Servi, S.; Spreafico, F.; Zirotti, C.; Casati, P. J. Org. Chem. 1984, 49, 4087 12. Neuberg, C.; Hirsch, P. J. Biochem. Z. 1921, 115, 281 13. Smith, P. F.; Hendlin, D. J. Bacteriol. 1953, 65, 440; Hanc, O.; Karac, B. Naturwiss. 1956, 43, 498 14. Fuganti, C.; Grasselli, P.; Ploi, G.; Servi, S.; Zorzella, A. J. Chem. Soc. Chem. Commun. in press 15. Whitesides, G. M.; Wang, C. H. Angew. Chem. Int. Ed. Engl. 1985, 24, 617 16. Fuganti, C.; Grasselli, P.; Servi, S. J. Chem. Soc. Perkin I 1983, 241 17. Bernardi, R.; Fuganti, C.; Grasselli, P. Tetrahedron Lett. 1981, 22, 4021 18. Fuganti, C.; Grasselli, P.; Servi, S. J. Chem. Soc. Chem. Commun. 1982, 1285 19. Servi, S. Tetrahedron Lett. 1983, 24, 2023 20. Pedrocchi-Fantoni, G.; Servi, S. J. Chem. Res. (S) 1986, 199 (M) 1986, 1834 21. Fuganti, C.; Grasselli, P.; Pedrocchi-Fantoni, G.; Servi, S. Zirotti, C. Tetrahedron Lett. 1983, 24, 3753 22. Fronza, G.; Fuganti, C.; Hogberg, H. E.; Pedrocchi-Fantoni, G.; Servi, S. Chem. Lett. 1988, 385 23. Fuganti, C.; Grasselli, P. J. Chem. Soc. Chem. Commun. 1979, 995; ibidem 1982, 205 24. Fuganti, C.; Grasselli, P.; Hogberg, H. E.; Servi, S. J. Chem. Soc. Perkin I in press RECEIVED

December 29, 1988

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 26

Preparation of Optically Active Pyrethroids via Enzymatic Resolution Takeaki Umemura and Hideo Hirohara

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch026

Takarazuka Research Center, Sumitomo Chemical Company, 4-2-1 Takatsukasa, Takarazuka 665, Japan Efficient biochemical processes were developed for the preparation of the two optically active pyrethroid insecticides by a combination of enzyme-catalyzed reactions and chemical transformations. These are based on the findings that a lipase from Arthrobacter species hydrolyzes the acetates of the two important secondary alcohols of synthetic pyrethroids with high enantioselectivity and reaction rate. The two alcohols are 4-hydroxy-3-methy1-2-(2'-propynyl)-2-cyclopentenone (HMPC) andα-cyano-3-phenoxybenzy1alcohol (CPBA). The enzyme gave optically pure (R)-HMPC or (S)-CPBA and the unhydrolyzed esters of their respective antipodes. Kinetic studies suggested that the high enantioselectivity resulted from very selective binding of the substrates to the enzyme. S y n t h e t i c p y r e t h r o i d s are a group o f e s t e r compounds h a v i n g e x c e l l e n t insecticidal activities. A f t e r the d i s c o v e r y o f a l l e t h r i n (1), a variety o f u s e f u l s y n t h e t i c p y r e t h r o i d s have been produced m a i n l y by structural m o d i f i c a t i o n o f an a l c o h o l h a v i n g an asymmetric center. The insecticidal a c t i v i t i e s g r e a t l y depend upon the stereoisomers. Therefore, much e f f o r t has been expended to develop t e c h n o l o g i e s f o r obtaining o p t i c a l l y active isomers. However, c o n t r a r y to the case o f chrysanthemic a c i d , c h e m i c a l methods o f o p t i c a l r e s o l u t i o n were not very e f f e c t i v e f o r these a l c o h o l s . Because of the s p e c i f i c i t y and the e n a n t i o s e l e c t i v i t y of some enzyme-catalyzed reactions, the application of enzymes is i n c r e a s i n g l y i m p o r t a n t i n asymmetric i n d u c t i o n and k i n e t i c r e s o l u t i o n in organic synthesis. A l a r g e number o f p u b l i c a t i o n s were recently reviewed, focusing on u t i l i z a t i o n of enzymes and m i c r o o r g a n i s m s to stereospecific hydrolysis and o t h e r reactions to produce pure stereoisomers (2,3). However, the use o f an enzyme as a c a t a l y s t has u s u a l l y been l i m i t e d to s m a l l - s c a l e experiments i n the l a b o r a t o r y . Lipases are a c l a s s of enzymes h a v i n g h i g h s t a b i l i t y in the presence of l a r g e amounts o f w a t e r - i n s o l u b l e o i l y substrates, and catalyze hydrolysis at a water-oil interface. Such enzymes, 0097-6156/89/0389-0371$06.00/0 ο 1989 American Chemical Society

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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requiring no cofactors, are now commercially available from various sources, and might be one of the most promising enzymes for largescale application i n organic synthesis. This a r t i c l e describes our results on lipase-catalyzed enantioselective hydrolysis of carboxylic acid esters of two i n d u s t r i a l l y important alcohols related to synthetic pyrethroids i n two-liquid phase systems. The two alcohols are 4-hydroxy-3-methyl-2-(2 propynyl)-2-cyclopentenone (HMPC) and the cyanohydrin, a-cyano-3phenoxybenzyl alcohol (CPBA). The configurations, and 2, are given for the liberated stereoisomers of the two alcohols i n our lipasecatalyzed reactions.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch026

f

It i s known that the (S)-forms are the essential stereoisomers f o r the i n s e c t i c i d a l a c t i v i t i e s of both alcohols (4,5). Chemicoenzymatic processes are also reported i n this a r t i c l e on the preparation of the o p t i c a l l y active pyrethroid i n s e c t i c i d e s having the (S)-isomers of the two alcohols. Processes were developed that use enantioselective hydrolysis with a lipase. 1

4-Hydroxy-3-methy1-2-(2 -propyny1)-2-cyclopentenone (HMPC) HMPC i s an alcohol moiety of a new promising synthetic pyrethroid for household use, p r a l l e t h r i n . This ester has several times greater knockdown and k i l l i n g effects than a l l e t h r i n against various insect pests (4). Hydrolysis by Lipase. The screening of our stock cultures revealed that a large number of microorganisms including bacteria, actinomycetes, yeasts and molds enantioselectively hydrolyzed the acetate of racemic HMPC to give (R)-HMPC p r e f e r e n t i a l l y i n most cases (6). A study on the addition of an o i l to the culture media suggested that the resposible enzyme might be a lipase rather than a carboxylic esterase. Thus, commercially available lipases were tested for the hydrolysis of the racemic ester. Ten out of twelve lipases tested were found to hydrolyze the ester with various rates of hydrolysis, y i e l d i n g varying o p t i c a l p u r i t i e s of the alcohol. Examples of microbial enzymes that gave the (R)-alcohol and the (S)ester with r e l a t i v e l y high o p t i c a l p u r i t i e s are shown i n Table I. The experiments were conducted at a substrate concentration of 8.8w/v%, pH 7.0 and 40°C. A lipase from Arthrobacter species yielded pure (R)-HMPC at 50% hydrolysis with the smallest amount of the enzyme. Lipases from Pseudomonas fluorescens, Chromobacterium viscosum and Alcaligenes species were of less interest to us than the Arthrobacter lipase among others, judging from the o p t i c a l purity of the product, degree

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

26. UMEMURA AND HIROHARA

Preparation ofOptically Active Pyrethroids 373

of hydrolysis, and amount of the enzyme used. The three enzymes also gave high o p t i c a l p u r i t i e s at around 50% hydrolysis.

Table I. Enantioselective Hydrolysis of Acetate of HMPC with Microbial Lipases

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch026

Origin of Lipase

Amount of Lipase Used (mg)

Arthrobacter sp. Pseudomonas fluorescens Chromobacterium viscosum Alcaligenes sp. Achromobacter sp. Humicola sp. Candida cylindracea

3 6 6 20 100 100 100

Degree of Hydrolysis (%)

49.9 47.1 58.6 47.4 32.9 36.8 10.8

Isomer Ratio Enantiomeric of Liberated HMPC Ratio*) Ε (S)/(R)

0.4/99.6 2.6/97.4 4.5/95.5 2.2/97.8 2.4/97.6 9.3/90.7 8.7/91.3

1280 101

-

124 64 15 12

*) see Literature Cited (7). A l l of the lipases used here are crude preparations, hydrolyzed the acetate much slower than o l i v e o i l (Table I I ) .

Table I I .

and

A c t i v i t y of Microbial Lipase on Acetate of HMPC and Olive O i l

A c t i v i t y on *) Origin of lipase

Arthrobacter sp. Chromobacterium viscosum Pseudomonas fluorescens Alcaligenes sp. Achromobacter sp. Humicola sp. Candida cylindracea

olive o i l acetate of HMPC o l i v e o i l (ymol/min(umol/min(ymol/min» mg-protein) mg-powder) mg-protein)

17.4 6.7 3.9 2.0 0.8 0.2 0.1.

230 790 133 59 82 400 110

190 330 70 22 10 120 21

*) pH 7.0 and 40°C

It should be noted that i n the lipase-catalyzed hydrolysis the reaction mixture i s biphasic consisting of water and water-insoluble o i l y substrate, and that the reaction i s easily controlled by

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch026

starting and t e r m i n a t i n g v i g o r o u s stirring. Furthermore, product r e c o v e r y i s s i m p l i f i e d i n the absence o f o r g a n i c s o l v e n t s . T h u s , the use o f l i p a s e s t h a t can be u t i l i z e d as "an o r g a n i c reagent" i s much more advantageous than that of microorganisms for the enantios e l e c t i v e h y d r o l y s i s o f a l k y l e s t e r s o f secondary a l c o h o l s . This is e s p e c i a l l y t r u e f o r the i n d u s t r i a l p r e p a r a t i o n of c h i r a l a l c o h o l s w i t h biocatalysts (8). Enantioselective Hydrolysis with Arthrobacter Lipase. Reaction performance w i t h the A r t h r o b a c t e r l i p a s e was s t u d i e d i n detail. The pH p r o f i l e c u r v e o f the z e r o - o r d e r r e a c t i o n e x h i b i t e d a pH-optimum around 7.0, and spontaneous h y d r o l y s i s was not s i g n i f i c a n t at pH lower than 8 . 0 ( F i g u r e 1 ) . The enzyme was s u f f i c i e n t l y s t a b l e as long as the r e a c t i o n was c a r r i e d out at 4 0 ° to 50°C. The optical purity of the (R)-HMPC l i b e r a t e d was not i n f l u e n c e d by the chain length from a c e t y l to capryl moiety at all ((S)/(R)=0.4/99.60.1/99.9). Time-course o f the h y d r o l y s i s c l e a r l y showed i n F i g u r e 2 that the reaction proceeded without difficulty even at a substrate concentration as high as 80%, and stopped spontaneously at 50% hydrolysis at w h i c h p o i n t the a c e t a t e o f (R)-HMPC was completely hydrolyzed. The HMPC l i b e r a t e d was optically pure at each experimental p o i n t regardless of percent h y d r o l y s i s . Therefore, the Arthrobacter lipase d i s t i n g u i s h e s the ( R ) - i s o m e r from i t s antipode quite completely. Kinetic Study o f H y d r o l y s i s . I t i s o f g r e a t i n t e r e s t to know the nature of the h i g h e n a n t i o s e l e c t i v i t y o f the A r t h r o b a c t e r lipase. The i n i t i a l v e l o c i t y measurements were conducted f o r the purpose of knowing w h i c h is the main f a c t o r o f the enantioselectivity, the apparent M i c h a e l i s c o n s t a n t K m o r the c a t a l y t i c c o n s t a n t k c a t . In a lipase-catalyzed reaction of a water-insoluble oily substrate, the enzyme reversibly adsorbs at the substrate-water interface, and the enzymatic r e a c t i o n takes p l a c e at t h a t i n t e r f a c e . The i n t e r f a c i a l a r e a s t r o n g l y depends upon some p h y s i c a l parameters of e m u l s i o n , such as the s i z e o f the e m u l s i o n d r o p l e t s as w e l l as the amount o f the s u b s t r a t e p r e s e n t . However, the importance of the interfacial a r e a s h o u l d not be o v e r e s t i m a t e d i n some cases ( 9 ) . If the experiments were c a r r i e d out whereby one makes v e r y f i n e d r o p l e t s of the e m u l s i o n and has a s u f f i c i e n t l y h i g h concentration of the substrate, a l l o f the enzyme m o l e c u l e s are adsorbed at the i n t e r f a c e and a "maximal v e l o c i t y " i s o b s e r v e d . In such a c a s e , the usual kinetic treatment o f an aqueous homogeneous r e a c t i o n can be applied for the e s t i m a t i o n o f K m and k c a t i n a t w o - l i q u i d phase reaction system. Two experiments were conducted to confirm that the conditions mentioned above are fulfilled when the substrate c o n c e n t r a t i o n i s much g r e a t e r than t h a t o f the enzyme. A study o f the i n f l u e n c e o f s t i r r i n g c y c l e s on the specific activity of the Arthrobacter lipase revealed that the activity i n c r e a s e d w i t h the s t i r r i n g c y c l e s and l e v e l e d o f f at 800 rpm. This might imply t h a t the maximal i n t e r f a c i a l a r e a was o b t a i n e d a t more than 800 rpm o f the a g i t a t i o n . T h i s was s u p p o r t e d by the e f f e c t of detergents on the activity at 1 ,000 rpm. Addition of various d e t e r g e n t s to the r e a c t i o n m i x t u r e d e c r e a s e d the a c t i v i t y . This is attributed to the d e c r e a s e of the i n t e r f a c i a l a r e a , a p a r t of which f

f

?

f

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

26. UMEMURA AND HIROHARA

Preparation ofOptically Active Pyrethroids 375

•H

ω ο u α oo e c •H

e

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch026

ι

4-» (J

F i g u r e 1. pH P r o f i l e f o r A r t h r o b a c t e r L i p a s e - c a t a l y z e d of A c e t a t e of Racemic HMPC.

O,

20W/V% o f

Δ,

40W/V%

•

Subst.

Hydrolysis

Cone.

, 80W/V%

3 Time

4

12

(hr)

F i g u r e 2. R e a c t i o n Performance o f A c e t a t e o f Racemic HMPC w i t h Arthrobacter Lipase.

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

is occupied by the detergent added. Thus, a l l of the experiments were performed under the conditions of 1,000 rpm and the substrate concentration greater than that of the enzyme. According to the kinetic treatment of Lavayre et a l (10) on two insoluble enantiomeric isomers, the r e l a t i v e i n i t i a l v e l o c i t i e s , V/VR were plotted against various ratios i n the (S)-enantiomer. A l l of the i n i t i a l v e l o c i t y measurements were made at 50°C instead of 40°C to avoid c r y s t a l l i z a t i o n of the acetate of pure (R)-HMPC, and at pH 6.0 to prevent spontaneous hydrolysis of the substrate at 50°C. The result gives a concave type of curve as i l l u s t r a t e d i n Figure 3. This implies that K mS < K'mR and that the (S)-enantiomer i s a strong competitive inhibitor. Thus, i t i s concluded that a very high o p t i c a l purity of (R)-HMPC liberated with Arthrobacter lipase i s entirely the result of the great c a t a l y t i c constant of the (R)-HMPC ester. The Lineweaver-Burk plots of the (R)-ester, i n the presence and absence of the (S)-enantiomer, exhibited a t y p i c a l pattern of a competitive i n h i b i t o r with two straight lines crossing on the v e r t i c a l axis. Apparent k i n e t i c constants were evaluated from the plots and summarized i n Table I I I .

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch026

f

Table I I I .

Apparent K i n e t i c Constants of Hydrolysis of Acetate of HMPC with Arthrobacter Lipase

Enantiomeric Isomer

k'cat (ymole/min-mg-protein)

K» (mM)

100 52

(R)-HMPC (S)-HMPC

f

(K m) (K»i)

66 0

From the r e s u l t s , the enantioselectivity may be explained by the binding s i t u a t i o n of the two enantiomers to the Arthrobacter lipase as i l l u s t r a t e d schematically (Figure 4). This p a r t i c u l a r enzyme may have the a b i l i t y to recognize the s t e r i c bulk of the two substituents on the asymmetric carbon of the alcohol so that the larger hydrophobic substituent L, i s t i g h t l y bound to the larger hydrophobic subsite P i , within the active center and the smaller substituents S, to the smaller subsite p . The carbonyl group of the (R)-ester i s positioned very near to the c a t a l y t i c s i t e of the enzyme so that the (S)-enantiomer i s hydrolyzed with the high velocity whereas that of the (S)-ester i s placed too far from the c a t a l y t i c site to be attached. 2

Preparation of Optically Active (S)-Prallethrin To obtain the i n s e c t i c i d a l l y active (S)-HMPC J5, the enzymatically hydrolyzed (S)-acetate ji must be separated from inactive (R)-HMPC ^ and hydrolyzed chemically to the desired (S)-HMPC £ . This might not be very attractive from the i n d u s t r i a l viewpoints, even i f the

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch026

UMEMURA AND HIROHARA

Preparation of Optically Active Pyrethroids

% of (S) i n Ester Figure 3. I n i t i a l Velocity of Hydrolysis of Mixtures of (R)- and (S)-HMPC Acetate with Arthrobacter Lipase.

Catalytic site (R)-HMPC

Catalytic site (S)-HMPC

Figure 4. Binding Mode for High Enantioselectivity of Arthrobacter Lipase.

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

377

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BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

enzymatic optical r e s o l u t i o n was q u i t e e f f i c i e n t as was mentioned above. However, this p r o b l e m was u s e f u l l y s o l v e d by means o f the c h e m i c a l t r a n s f o r m a t i o n o f the (R)-HMPC 1 to the d e s i r e d (S)-HMPC 6, as shown i n F i g u r e 5. The mixture o f the (R)-HMPC ^ and the ( S ) - a c e t a t e 4, from the enzymatic h y d r o l y s i s was e s t e r i f i e d w i t h m e t h a n e s u l f o n y l choride and triethylamine to a f f o r d a m i x t u r e of the corresponding (R)s u l f o n a t e 5a and the ( S ) - a c e t a t e 4 . The ( S ) - a c e t a t e 4 was u n a f f e c t e d by the sulfonation conditions. The r e s u l t a n t mixture of two esters, 4, and 5 a , was h y d r o l y z e d i n the p r e s e n c e o f a s m a l l amount of c a l c i u m c a r b o n a t e . The ( R ) - s u l f o n a t e 5a was converted into the (S)-HMPC 6 as a r e s u l t o f i n v e r s i o n o f c o n f i g u r a t i o n . On the other hand, the (S)-acetate 4, was hydrolyzed with retention of configuration. Consequently, all of the r a c e m i c a c e t a t e J3, was converted w i t h maximum e f f i c i e n c y to the d e s i r e d (S)-HMPC 6, by the sequence: enzymatic h y d r o l y s i s and s u l f o n a t i o n f o l l o w e d by i n v e r s i o n of the c h i r a l c e n t e r i n (R)-HMPC ^ w i t h o u t s e p a r a t i o n o f the (S)a c e t a t e j4. S i m i l a r t r a n s f o r m a t i o n s c o u l d a l s o be c a r r i e d out via nitrate e s t e r i n t e r m e d i a t e 5b o b t a i n e d from the r e a c t i o n o f the ( R ) HMPC w i t h n i t r i c a c i d and a c e t i c a n h y d r i d e . Finally, the derived (S)-HMPC 6, was esterified with (1R)chrysanthemoy1 c h l o r i d e 7 to g i v e the most p o t e n t optically active form 8 of a new synthetic pyrethroid, prallethrin, which has significant knock-down and k i l l i n g e f f e c t s a g a i n s t v a r i o u s h o u s e h o l d insect pests. The i n s e c t i c i d a l a c t i v i t i e s o f some s t e r e o i s o m e r s of prallethin are summarized i n T a b l e IV. Optically active (S)p r a l l e t h r i n showed s e v e r a l times the l e t h a l a c t i v i t i e s o f the r a c e m i c m i x t u r e a g a i n s t h o u s e f l y and c o c k r o a c h .

Table IV. I n s e c t i c i d a l A c t i v i t i e s of Stereoisomers of P r a l l e t h r i n a g a i n s t H o u s e f l y and Cockroach (Topical Application)

Stereoisomers of P r a l l e t h r i n Acid

Housefly (M. domestica)

(1R)-trans (1R)-cis (1R)-trans (1R)-cis (1RS)-cis,trans

419 164 72 57 100

Alcohol

S S R R RS

Relative

a-Cyano-3-phenoxybenzyl A l c o h o l

Potencies

(B.

Cockroach germanica)

611 185 56 9 100

(CPBA)

CPBA i s an a l c o h o l moiety o f h i g h l y p o t e n t s y n t h e t i c p y r e t h r o i d s such as f e n v a l e r a t e ( a - c y a n o - 3 - p h e n o x y b e n z y l 2 - ( 4 - c h l o r o p h e n y l ) - 3 - m e t h y 1 butyrate) or c y p h e n o t h r i n (a-cyano-3-phenoxybenzy1 chrysanthmate).

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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UMEMURA AND HIROHARA

Preparation of Optically Active Pyrethroids

Figure 5. Chemico-enzymatic Preparation of Optically Active (S)-Prallethrin from Racemic HMPC.

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The former i s a photostable and e f f i c i e n t a g r i c u l t u r a l having no cyclopropane ring that i s employed for cotton, vegetables.

insecticide f r u i t s and

Enantioselective Hydrolysis with Arthrobacter Lipase. The results of the enantioselective hydrolysis of the acetate of racemic CPBA are summarized i n Table V f o r several commercial lipases that liberate very o p t i c a l l y pure CPBA. The experimental conditions were chosen to give approximately 50% hydrolysis for each enzyme. I t i s noticed that a l l of the lipases i n Table V hydrolyzed the ester of (S)-CPBA p r e f e r e n t i a l l y to give the i n s e c t i c i d a l l y active (S)-isomer. This i s apparently d i f f e r e n t from the case of HMPC. The highest a c t i v i t y and optical purity were again given by the Arthrobacter lipase. Spontaneous termination of the reaction at 50% hydrolysis was observed with this enzyme as was the case of HMPC. The reaction rate of the CPBA ester was the same order of magnitude with that of the HMPC ester. This i s astonishing considering the presence of two bulky substituents, 3-phenoxybenzyl and cyano groups, on the carbon atom i n the α-position of the ester bond. Brockerhoff s generalization (11) predicts strong i n h i b i t i o n in such a case. The pH p r o f i l e of the hydrolysis of the acetate of CPBA showed that the a c t i v i t y of the enzyme was not greatly influenced i n the range of pH 4 to pH 7. An undesirable conversion from the liberated (S)-CPBA to 3-phenoxybenzaldehyde was sharply increased when the pH was higher than pH 5. The reaction was therefore carried out at pH 4 to pH 5 as shown i n Table V. Other reaction behavior was similar to that f o r the HMPC ester. 1

Table V. Enantioselective Hydrolysis of Acetate of CPBA with Microbial Lipases

Temp. Origin of Lipase

Arthrobacter sp. Pseudomonas fluoresens Chromobacterium viscosum Alcaligenes sp. Achromob acter sp.

(°C)

Cone, of Substrate (w/v%)

Degree of Hydrolysis (%)

Isomer Ratio of CPBA (S)/(R)

4.0 5.0

35 30

20 10

49.0 49.8

99.9/0.1 96.4/3.6

4.0

40

20

49.6

97.8/2.2

5.0 5.0

30 30

10 10

49.8 47.5

96.7/3.3 96.7/3.3

PH

Discussion on Binding Mode. The i n i t i a l velocity measurements of the acetate of CPBA with various ratios of the (R)-enantiomer were observed to give almost the same l i n e having a concave curve as i l l u s t r a t e d f o r the acetate of HMPC i n Figure 3. This r e s u l t

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

26. UMEMURA AND HIROHARA

Preparation ofOptically Active Pyrethroids 381

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indicates that K'mR < K'mS i n the case of the CPBA ester and that the (R)-enantiomer i s a strong competitive i n h i b i t o r . The high o p t i c a l purity of the liberated (S)-CPBA we conclude i s due to the great c a t a l y t i c constant of the (S)-enantiomer. Why was the (S)-CPBA ester enantioselectively hydrolyzed when the same enzyme catalyzes the hydrolysis of the (R)-HMPC ester ? The answer to this question may be given by the comparison of the configuration of (R)-HMPC and (S)-CPBA and 2) as well as the R and S d e f i n i t i o n s by the conventions of the sequence rule (12). The r e l a t i v e sizes of the groups attached to the asymmetric carbon atom i n HMPC and CPBA are, respectively:

C K

>

- C = C— C h L C E C H

-

C H

2

— C = 0

The stereochemistry of the (S)-CPBA i s i n good accordance with that of the (R)-HMPC as i l l u s t r a t e d i n Z* However, the d e f i n i t i o n of (R) and (S) i n the two compounds are the results of nomenclature. Thus, the binding situation of the L and S substituents to the subsites p and p i n the active center of the enzyme and the positioning of the carbonyl group of the (S)- and (R)-CPBA esters to the c a t a l y t i c s i t e are considered to be very similar to those i n the case of the (R)- and (S)-HMPC esters. a

x

n

d

2

Preparation of Esfenvalerate ((S)-(S)-Fenvalerate) In contrast to the case of HMPC, most lipases hydrolyze the racemic acetate of CPBA 9 to give a mixture of the i n s e c t i c i d a l l y active (S)CPBA 2 and the (R)-acetate 1£. Thus, the desired (S)-CPBA ,2 could be separated from the (R)-acetate 10 by means of a continuous countercurrent extraction using η-heptane solvent at 80°C. However, i t i s important to u t i l i z e the recovered (R)-acetate 10, for an e f f i c i e n t process. Fortunately, since the proton of the asymmetric carbon of the cyanohydrin acetate i s l a b i l e , the antipodal (R)-acetate is e a s i l y racemized by treatment with weak organic base such as triethylamine without any side reactions. The racemized acetate £ thus obtained was recycled as shown i n Figure 6. Therefore, a l l of the racemic acetate 9 was converted to the desired (S)-CPBA ^2 i n this recycling process. The (S)-CPBA 2 obtained was e s t e r i f i e d with (S)2-(4-chlorophenyl)-3-methylbutyry1 chloride 11 to produce the most insecticidally active stereoisomer JT2 of f envalerate, namely esfenvalerate. The relative biocidal activities between

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch026

382

BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

Figure 6.

Preparation of Esfenvalerate from Racemic CPBA.

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

26. UMEMURA AND HIROHARA

Preparation of Optically Active Pyrethroids 383

esfenvalerate 12 and fenvalerate are shown in Table VI. Esfenvalerate has 3.5-4.0 times the insecticidal activities of f e n v a l e r a t e and i s not at a l l p h y t o t o x i c .

Table V I .

R e l a t i v e B i o c i d a l A c t i v i t i e s between and F e n v a l e r a t e

absolute configuration

compound

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alcohol

esfenvalerate fenvalerate *) **)

S RS

acid

S RS

biocidal

i n s e c t i c i d a l *)

3.5-4.0 1

Esfenvalerate

activities

chlorotic

**)

1/16 1

r e l a t i v e p o t e n c y to h o u s e f l y and cabbage army worm r e l a t i v e c h l o r o t i c e f f i c a c y i n tomato or C h i n e s e cabbage

In conclusion, the combination of an enzymatic optical resolution and subsequent c h e m i c a l t r a n s f o r m a t i o n s of epimerization or r a c e m i z a t i o n o f the asymmetric c e n t e r o f the unwanted antipodes have l e d to the s u c c e s s f u l development o f p r o c e s s e s f o r p r e p a r a t i o n o f the two o p t i c a l l y a c t i v e p y r e t h r o i d i n s e c t i c i d e s . T h i s work w i l l provide a n o v e l f e a t u r e i n the a p p l i c a t i o n o f enzymes, especially l i p a s e s f o r the i n d u s t r i a l p r o d u c t i o n o f c h i r a l compounds.

Acknowledgment The a u t h o r s a r e i n d e b t e d to t h e i r c o l l e a g u e s who c a r r i e d out some o f the experiments i n t h i s work. They a l s o e x p r e s s t h e i r thanks to one o f e d i t o r s f o r s c i e n t i f i c d i s c u s s i o n on t h i s work.

Literature Cited

1. 2. 3. 4. 5. 6. 7.

Schechter, M. S.; Green, N.; LaForge, F. B. J. Am. Chem. Soc. 1949, 71, 1517. Poter, R.; Clark, S., Ed. Enzymes in Organic Synthesis; Pitman: London, 1985. Whitesides, G. M.; Wong, C. H. Angew. Chem. 1985, 24, 617. Matsuo, T.; Nishioka, T.; Hirano, M.; Suzuki, Y.; Tsushima, K.; Itaya, N; Yoshioka. H. Pestic. Sci. 1980, 11, 202. Aketa, K.; Ohno, N.; Yoshioka, H. Agric. Biol. Chem. 1978, 42, 895. Mitsuda, S.; Umemura, T.; Hirohara, H. Appl. Microbiol. Biotech. in press. Chen, C-S.; Fujimoto, Y.; Girdaukas, G.; Sih, C.J. J. Am. Chem. Soc. 1982, 104, 7294.

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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8.

Hirohara, H.; Mitsuda, S.; Ando, E.; Komaki, R. In Biocatalysts in Organic Syntheses; Tramper, J.; van der Plas, H. C.; Linko, P., Ed.; Elsevier: Amsterdam, 1985; p119. 9. Bonsen, P. P. M.; de Haas, G. H.; Pieterson, W. Α.; van Deenen, L. L. M. Biochim. Biophys. Acta 1972, 270, 364. 10. Lavayre, J.; Verrier, J.; Baretti, J. Biotechnol. Bioeng. 1982, 24, 2175. 11. Brockerhoff, H.; Jenson, R. G. Lipolytic Enzymes; Academic: New York, 1974; p 58. 12. For example ; Hendrickson, J. B.; Cram, D. J.; Hammond, G. S. Organic Chemistry; 3rd Ed.; McGraw-Hill: New York, 1970, p 203. November 18, 1988

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ch026

RECEIVED

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ix001

Author Index Anderson, Joseph, 84 Blomquist, Gary J., 314 Bloom, Mark, 84 Chin, C. K., 193 Cress, Douglas, 84 Cséke, Csaba, 265 Dangler, Charles A , 230 Dordick, Jonathan S., 141 Duffey, Sean S., 289 Eigtved, Peter, 158 Felton, Gary W., 289 Fuganti, Claudio, 359 Gerwick, B. Clifford, 277 Gold, Michael H., 127 Grasselli, Piero, 359 Hansen, Tomas T., 158 Hardy, Daniel M., 215 Hedrick, Jerry L., 215 Heldt-Hansen, Hans Peter, 158 Hilvert, Donald, 14 Hirohara, Hideo, 371 Horton, H . Robert, 242 Hutny, Jan, 84 Ishii, Michiyo, 158 Jurenka, Russell A , 314 Knorr, Dietrich, 65 Morell, Matthew, 84 Mori, Kenji, 348 Okita, Thomas, 84 Oostendorp, A , 332

Osburn, Bennie I., 230 Owen, W. John, 265 Patkar, Shamkant A , 158 Pedersen, H., 193 Pilnik, W., 93 Preiss, Jack, 84 Reitz, Ronald C , 314 Rodriguez, Raymond L., 202 Roelofs, Wendell L., 323 Ryu, Keungarp, 141 Scott, Don, 176 Secor, Jacob, 265 Simmons, Carl R., 202 Sonnet, Philip E., 264,346 Spradlin, Joseph E., 24 Stafford, Douglas R., 141 Subramanian, Mani V., 277 Swaisgood, Harold E., 242 Teal, Peter E . A , 332 Tumlinson, J. R , 332 Umemura, Takeaki, 371 Urch, Umbert A , 215 Valli, Khadar, 127 Vaz, Apollo H., 314 Venkatasubramanian, K., 193 Voragen, A G. J., 93 Wariishi, Hiroyuki, 127 Whitaker, John R., 1,12,44,82,174 Wolf, Walter A , 323 Zikakis, John P., 116

Affiliation Index Agricultural University, 93 Berlin University of Technology, 65 Centro del Consiglio Nazionale delle Ricerche per la Chimica delle Sostanze Organiche Naturali, 359 Cornell University, 323 Dow Chemical Company, 265,277 General Foods USA, 24 H. J. Heinz Company, 193 Michigan State University, 84 North Carolina State University, 242 Novo Industri AyS, 158 Novo Industri (Japan) Ltd., 158 Oregon Graduate Center, 127

Royal Holloway and Bedford New College, 265 Rutgers University, 193 Scott Biotechnology, Inc., 176 Scripps Clinic and Research Foundation, 14 Sumitomo Chemical Company, 371 University of California, 1,12,44,82,174, 202,215,230,289 University of Delaware, 65,116 University of Iowa, 141 University of Nevada, 314 University of Tokyo, 348 U.S. Department of Agriculture, 264,332,346 Washington State University, 84

385 In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

386

BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

Subject Index

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A

Α-factor, synthesis, 349,351/ Acetate esterase, use in sex pheromone biosynthesis, 329 Acetolactate decarboxylase, use in brewing, 177-178 Acetolactate synthase activities from various plant sources, 282/ inhibition by triazolopyrimidines, 277-286 interaction with triazolo­ pyrimidines, 283,284/ kinetics, 283 purification from barley seedlings, 282/,283 (5S,6/?)-6-Acetoxy-5-hexacanolide, synthesis, 362,365-366 (/?)-2-(Acetoxymethy)l-3-phenyl-1 -propanol, chiral building block, 349,351/ Acetyl coenzyme A carboxylase effect of desalting on activity, 272,273/ inhibition of activity, 269,270/271/,274/ Acetyl coenzyme A carboxylation reaction rates, 273/ effect of herbicides on reaction rates, 273,275/ Acidolysis of tricaprylin, activity with different fatty acids, 165/,166 Acrosin cellular function as a sperm lysin, 222-223 description, 216 discovery, 216 functional domains, 220-223 future research, 225,227 purification, 216-217 structural properties, 217-220 structure-function models, 223-226 Acyl derivation of selenosubtilisin, preparation, 16 Adenosine 5'-diphosphate glucose synthetase activators, 85-86 starch synthesis, 85-91 structure, 85 5'-Adenylic deaminase, application, 187 Aggregated cell cultures, examples, 198-199 Agonists definition, 290 direct removal, 296,297/ indirect removal, 297-302 Agricultural biotechnology, relationship to enzymology, 1-8

Agrochemicals, biocatalytic synthesis, 346-347 Air lift fermentors, use in growing plant cell cultures, 196-197 Alcohol, chiral building blocks, 349,350-351/ Alcohol oxidase applications, 184 use in sex pheromone biosynthesis, 329 Aldehydes, a,0-unsaturated formation of chiral adducts, 359,361 reaction products, 362 Aldehydic pheromone components, production, 334-335 Alkaline phosphatase, use as detection enzyme, 237-238 Alkenes, biosynthesis by houseflies, 318,319/320 Alko/Dorr-Oliver process, description, 183 α-Amino acids chiral building blocks, 348 production, 5 synthesis of gizzerosine, 348-349,350/ Aminoacylase, commercial application, 252 Ammonia, release by ureases, 294 Amylase(s) active site, 28-29 degree of multiple attack, 29 tailoring of action patterns, 28 use in brewing, 177 use in corn syrup, 27-29 α-Amylase discovery, 1-2 a(l-4)-endoglycolytic cleavage of amylose and amylopectin, 203 features of rice gene enzyme system, 202-203 See also Heat-stable α-amylase α-Amylase in rice callus advantages of callus system, 212 comparison of expression in seed and shoot callus, 205,209/ effect of gibberellin, 205,206/209,210/ effect of gibberellins on expression, 211-212 effect of tissue source on expression, 209,211 electrophoretic profile, 205,206/ levels in germinating seedlings, 204 Western blot, 205,207/ Amyloglucosidases function, 30 tailoring by immobilization, 30-31 use in brewing, 177 use in corn starch industry, 30

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ix002

INDEX

387

Analytical bioreactors determination of protein digestibility, 257,258/ preparation of enzymic hydrolysates of protein for amino acid analysis, 256,257/ Analytical detection systems, enzymc-probc conjugates, 230-240 Animals, distribution of chitinolytic enzymes, 118 Antagonists definition, 290 direct production, 291-294 indirect production, 294-296 Antibodies catalytic, See Catalytic antibodies description, 17 preparation, 17-18 Antibody probes use in enzymatic detection systems, 231 use in genetic sequence identification, 231 Antienzyme enzymes, examples, 189 Antigens, detection, 236-237 Antimicrobial enzymes, examples, 187-189 Apple juice amounts and composition of high molecular weight fragments, 106,107/ tentative structure of pectin fragments released by enzyme liquefaction, 106,108/ Apple pectin, structure, 94,96/ Applied enzymology, advances, 176 Arthrobacter lipase catalyzed hydrolysis of acetate of 4-hydroxy-3-mcthyl-2(2-propynyl)-2-cyclopentenonc apparent kinetic constants, 376/ binding mode, 376,377/ effect of stirring cycles, 374,376 initial velocity, 376,377/ pH profile, 374,375/ reaction performance, 374,375/ Arthrobacter lipase hydrolysis of a-cyano-3-phenoxybenzyl alcohol binding mode, 380-381 enantioselectivity, 380/ pH profile, 380/ Aryloxyphenoxypropanoate(s) mode of action, 266 structures, 265,268/ Aryloxyphenoxypropanoatc herbicides, effect on maize acetyl coenzyme A carboxylase activity, 271/ Aspartame production, 5 use of enzymes in production, 181 Aspartame synthesis, commercial application, 254

Aspartase, commercial application, 253 8-Azido nucleotide substrates electrophoresis of inactivated ADPGlc synthetase, 89,90/ photoinactivation of ADPGlc synthetase, 86,88/89 saturation curves of ADPGlc synthetase, 86,87/

Β Baker's yeast applications, 359-360 synthesis of natural products, 359-369 Baking, role of enzymes, 32-36 Biocatalytic synthesis, agrochemicals, 346-347 Biochemical systems in organic chemistry, application, 348 Bioreactors, factors influencing economics, 251 Biosynthesis hydrocarbon skeleton of sex pheromones, 333-334 oxygenated functional groups of sex pheromones, 334-335 Biosynthesis of polymers, maximization of process, 82-83 Biotin-containing proteins, Western blot, 275/ Biotransformation, description, 194 Bread staling mechanism, 34 use of amylases, 34-36 Brewing, use of enzymes, 177-178 (lS,4/?)-4-(/er/-Butyldimcthylsilyloxy)3-chloro-2-cyclopenten-l-ol, chiral building block, 349,350/

C Cabbage looper moth, pheromone components, 332—333 Candida cylindracea lipase, description, 165 Capsaicin, yield enhancement, 74-75 Carrot cell cultures effect of aggregated states on productivity and morphogenic capacity, 199 effect of treatment on chitinolytic activity, 76/ Casein, effect of alkylation on protein digestibility and insect growth, 299-300,301/302

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

Catalase, applications, 3 Catalytic antibodies kinetics, 19 preparation, 18-19,20/ properties, 17-18 stereospecificity, 21 Cell stability, control of cell growth, 197-198 Cheese-butter flavor, use of enzymes, 178 Cheeseflavoring,use of enzymes, 40 Cheese production, use of enzymes, 36 Cheese ripening, role of rennet, 38 Chiral building blocks, preparation by biochemical methods, 348-357 Chitin applications, 116-117 bioprocessing of shellfish and whey waste, 122-123 degradation, 118-119 description, 116 Chitinases comparison of production of costs, 119,120/ defense against pathogenic pests, 120-121 degradation of chitin, 118-119 description, 189 distribution in animals, 118 distribution in microorganisms, 117 distribution in plants, 117-118 insect resistance, 302 plants, 295-2% sources, 119 Chitobiase, degradation of chitin, 118-119 Chitosan effect on carrot cells, 74/ effect on cell viability, 72 effect on permeabilization of plant cells, 71,72/,73/ Chitosan-alginate coaccrvate capsules effect of buffer treatment and esterification of alginate on mechanical stability, 67,71/ effect of viscosity and concentration on dye releases, 67,70/ Chitosan-alginate coacervate gel capsules effect of calcium chloride and glucose on mechanical stability, 67,69/ protocol, 67,68/ Chorismatc kinetics of disappearance, 19 rearrangement to prephenate, 18 Chorismatc mutasc, rearrangement of chorismate to prephenate, 18 Chorismatc mutase antibodies, preparation, 18-19,20/ Chymosin role in milk clotting, 36-37 synthetic production, 12-13

Cinnamaldehyde conversion to 3-phenylpropanol, 360 formation of (2S,3/?)-diol, 360-361 Cinnamoylated subtilisins, second-order rate constants for hydrolysis and aminolysis, 16/, 17 Clasien rearrangement, example, 18-21 Coffee cell cultures, effects of aggregated states on productivity and morphogenic capacity, 198-199 Corn starch, conversion to glucose and fructose, 4 Corn syrup, use of enzymes, 27-32 Corn wet milling, use of enzymes, 182 Covalent enzyme immobilization methods applications, 244 preparation of supports, 246,248-251 Crude enzyme preparation from Candida antarctica

activity assays, 160 activity on olive oil, 160,162/ batch interesterification assay, 160,162/ immobilization, 160 performance in continuous acidolysis, 163,167/ properties, 160-162 temperature optimum, 160,164/ thermostability, 160,161/163,164/ Cultured plant cells, immobilization, 66-71 Cuticular lipids components, 314 role in insects, 314 Cyanidase, application, 187 a-Çyano-3-phenoxybenzyl alcohol binding mode, 380-381 description, 378,380 enantioselective hydrolysis with Arthrobacter lipase, 380 structure, 372 Cyanogenesis, resistance against insects, 291,293 Cyanogenic glycosides, function in plants, 291 Cyclic ^-hydroxy ester, chiral building block, 352,355/ Cyclodextrin description, 179 preparation, 179 Cystine lyase, application, 3

D Dainippon ink and chemical process, preparation of ribaudioside A, 182 Dairy industry, use of enzymes, 36-42 Debranching enzymes applications, 31

In Biocatalysis in Agricultural Biotechnology; Whitaker, J., el al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

INDEX

389

Debranching enzymes—Continued definition, 31 use to counteract bread staling, 35 Dehydrogenases, potential industrial applications, 256 Δ -Desaturase, biosynthesis of sex pheromone, 328-329 Dextrin distribution profile, influencing factors, 28 Dextrose equivalents, definition, 27 Diastase, discovery, 1-2 2,4-Dihydroxy-7-methoxy-1,4-benzoxazi η -3 -one glucoside, role in insect resistance, 293 Dioxane, purification, 144 Direct-indirect detection systems comparison of direct to indirect detection, 233-234 target-probe-enzyme interactions, 233,234/ DNA ligases description, 58 function, 58 mechanism, 58,59/ DNA polymerase I catalysis of elongation reaction, 60,62 comparison to DNA polymerases II and III, 62 description, 60 mechanism of elongation reaction catalysis, 60,62,63/ schematic drawing of three-domain structure, 60,61/ tertiary structure of Klenow fragment, 60,61/ DNA polymerases II and III, comparison to DNA polymerase I, 62 Downstream processing, influencing factors, 198

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11

EcoRl endonuclease crystalline enzyme-substrate complex, 51,54,55-56/ description, 51 subunit, 51,53/54 Egg envelopes, nomenclature, 215 Elongation reactions, hydrocarbon biosynthesis, 317-320 Embryogénie callus, definition, 204 Enantiotopicity, definition, 347 Endonuclease-polynucleotide complex backbone schematic drawing, 54,55/ α-carbon structure, 54,55/ isomerization, 54 schematic representation of hydrogen-bond interaction, 54,56/

Endopectin lyases, activity on pectins, 97/,98 Endopeptidases, potential industrial applications, 255-256 Endopolygalacturonases, kinetics and degradation limits, 95,97/98 Enzymatic catalysts, design, 14-21 Enzymatic detection systems future applications, 239—240 selection of probes, 231 types, 231-232,233/ Enzymatic hydrolyses, production of specialty products, 4 Enzyme(s) addition and removal of residues from compounds, 5 advantages, 174-175 applications in food industry, 2-3 commercial applications, 44 de novo synthesis, 13 discovery of function in food, 1 immobilization methods, 244-251 lowering of activation energy, 42 plant, See Plant enzymes preparation of cyclodextrins, 179 response to temperature, 42 role in plant resistance to insects, 289-307 specialty uses, 174-175 tailoring for industrial uses, 12-13 use brewing, 177-178 corn wet milling, 182 feed, 184-185 flavor modification, 178-179 high-intensity sweeteners, 181-182 low-sweetness sweeteners, 179-180 normal and enhanced-swectness sweeteners, 180 stereospecific hydrolysis, 371 Enzyme activity, definition of one unit of activity, 204 Enzyme applications, factors influencing diversity, 176-177 Enzyme-catalyzed pheromone synthesis, Heliothis moths, 332-342 Enzyme kinetics, comparison in organic media to that in aqueous solutions, 143 Enzyme-modified cheese,flavoring,39-40 Enzymes in agricultural biotechnology heat treatment for preservation, 3 modification of proteins for food use, 4 production of specialty products, 4-5 recombinant DNA technology, 5-7 removal of unwanted compounds, 7-8 use in pesticides, 7 Enzymes in food processing advantages, 24-25

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BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY

Enzymes in food processing—Continued application economics, 26 applications, 24 catalytic specificity, 25 purity, 25-26 sensitivity to processing conditions, 25 source, 26 tailoring for future food applications, 41-42 tailoring opportunities, 26-27 use in baking industry, 32-36 in corn syrup industry, 27-32 in dairy industry, 36-41 Enzymes of plant cell cultures, effect of elicitors on induction, 76,77/ Enzymic hydrolysates of protein, preparation for amino acid analysis, 256,257/ Enzymology, relationship to agricultural biotechnology, 1-8 Epoxide, chiral building blocks, 349 Esfenvalerate biocidal activities, 383/ preparation, 381,382/,383 Ester synthesis, lipase components, 171/ Ethyl 3-hydroxybutanoate enanliomcrs chiral building block, 349,353/ preparation, 349,351/ (2S,3K)-2-Ethyl-3-hydroxy-2-methylcyclohexanone, chiral building block, 356/357

F Fatty acid specificity, acidolysis of tricaprylin, 165/,166 Fatty acid synthetase, use in sex pheromone biosynthesis, 329-330 Feed, use of enzymes, 184 Fenvalerate, biocidal activities, 383/ Ferments, description, 2 Flavor esters, use of enzymes, 178 Flavor modification, use of enzymes, 178-179 Flour amylases, critical processing parameter in baking, 32-33 Flour proteases, role in bread making, 33-34 Food processing, tailoring enzyme systems, 24-42 (-)-Frontalin synthesis, 362-363 Fumarase, commercial application, 253 Functional domains of acrosin hydrophobic binding, 221-222 proteolytic activity, 220-221 Zona pellucida binding, 221

G 0-Galactosidase application, 186 commercial application, 252-253 Genetic engineering, characteristics of enzymes, 44-63 Gibberellins, effect on α-amylase expression, 205,206/209,210/ Gizzerosine, synthesis, 348-349,350/ Glass surfaces, adsorption of glutamate dehydrogenase, 248,250/,251 0-Glucanases applications, 184-185 role in defense against pathogenic pests, 120-121 Glucoamylase commercial application, 253 See also Amyloglucosidases Glucose isomerase commercial application, 252 commercial success, 31-32 environmental problems, 32 use in sweeteners, 180-181 Glucose oxidase, applications, 183 Glucosinolates, role in insect resistance, 293 Glucosyl transferases, application, 5 Glutathione-decomposing enzymes, application, 186 Glycinoeclepin A synthesis, 357/ Glycoamylase hydrolysis rate maximization, 12 Glycosylating enzymes, application, 5 Glyphosate, applications, 6 Green vanilla bean extracts, effect of 0-glucosidase on flavor components, 75,76/

H Haloxyfop effect of desalting on inhibition of enzyme activity, 272,273/ effect on fatty acid biosynthesis, 267,269/ effect on lipid metabolism, 266,267/,26S/ effect on whole-plant growth and acetyl coenzyme A carboxylase inhibition, 272/ inhibition of acetyl coenzyme A carboxylase, 269,270/271/,274/ resistant and susceptible species, 272/ structure, 265,268/ Hayashibara process, preparation of stevioside, 182

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391

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INDEX Heat-stable α-amylase tailoring target, 29-30 use in production of corn syrup, 29 Heliothis genus comparison of oxidase and esterase activities, 335-342 compounds from extracts of pheromone glands, 335,336/ dependence of oxidase activity on insect development, 337,339/,340 distribution of esterase activity in pheromone glands, 335,337,338/ effect of duration of incubation on oxidase activity, 340,341/ effect on oxidase activity of soaking cuticle in organic solvents, 340,341/ Hemicellulases, degradation of cell walls of fruit pulps, 106 (Z,Z)-6,9-Heptacosadiene, biosynthesis, 315,317,319/ Herbicide-resistant mutants, applications, 285-286 High-intensity sweetness, use of enzymes, 181-182 Horseradish peroxidase critical enzyme loading vs. bead size, 149,150/ effect of aqueous component pH on peroxidase catalysis, 147,148/ effect of hydrogen peroxide concentration on catalysis, 150 electronic adsorption spectral maxima, 129,130/,131 elimination of intraparticle diffusional limitations, 147,148/149 oxidation of phenols, 142 properties, 143 stoichiometry of catalysis of p-crcsol oxidation, 143-144 surface area coverage on glass bead surface, 149 Horseradish peroxidase-catalyzed polymerization of p-cresol adsorption of peroxidase onto glass beads, 145 cresol oxidation products in organic media, 146 determination of initial catalysis rates in organic media, 145-146 experimental procedure, 144-145 materials, 144 product identification, 145 reaction optimization, 146-150 Hydrocarbon skeleton of sex pheromones, biosynthesis, 333-334 Hydrocarbons in insects biosynthesis of methyl-branched hydrocarbons, 315,316/ components, 314-315

Hydrocarbons in insects—Continued conversion of very long chain fatty acids, 318 elongation reaction, 317 formation, 318 regulation of chain length, 318,319/320 Hydrogen peroxidase catalyzed oxidation-polymerization of p-cresol effect of methanol and acetone on kinetics, 153,154-155/ effect of organic solvents, 150-155 K , 151,152f kinetics in aqueous buffer, 151 Michaelis-Menton kinetics, 151-156 saturation kinetics experiments, 150-151,152/ Hydroperoxides, production, 297 Hydrophobic binding, acrosin, 221-222 α-Hydroxy acids, chiral building blocks, 349 (S)-3-Hydroxy-2,2-dimethylcyclohexanonc, chiral building block, 352,356/357 ^-Hydroxy esters, chiral building blocks, 349,351-354 ^-Hydroxy ketones, chiral building blocks, 352,356-357/ 4-Hydroxy-3-methyl-2-(2-propynyl)-2-cyclopentenone enantioselective hydrolysis with Arthrobacter lipase, 372 hydrolysis by lipase, 372,373/,374 kinetic study of lipase hydrolysis, 374,376/ structure, 372 (lS,4/?,15)-5-Hydroxy-l-mcthylbicyclo[2.2.1|heptan-3-one, chiral building block, 357/ m

Imidazolinones binding sites, 285 herbicidal activity, 277-278 herbicidal activity through acetolactatc synthase inhibition, 277 structure, 277,280/ Immobilization, tailoring enzymes, 30 Immobilization of enzymes characteristics of bioreactors, 251 methods, 244-251 Immobilization of plant cells, advantage, 194 Immobilized enzyme(s), potential partitioning of ions and substrates, 248,249/ Immobilized enzyme technology advantages, 243,244/ analytical applications, 256—258 commercial applications, 252-254 factors influencing growth rate, 242

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Publication Date: January 1, 1989 | doi: 10.1021/bk-1989-0389.ix002

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Immobilized enzyme technology—Continued lipid modification, 243 potential applications, 254-256 protein modification, 243 sugar conversions, 243 synthesis of organic flavor compounds, 243-244 Immobilized invertase, use in sweeteners, 180 Immobilized lipase(s) activities in ester synthesis, 166,167/ nonspccificity, 163/,165/ reactions under low water activity, 158,159/ Immobilized lipase components acidolytic activity, 168,169/ fatty acid specificity, 170/ positional specificity, 169/,170 Immobilized plant cell preparations, effect of cell aggregation on secondary metabolite formation and cell metabolism, 194 Inclusion complexation, definition, 179 Insect enzymology, development, 264 Insect hydrocarbons, See Hydrocarbons in insects Invertase, function, 2 Ionic cross-linking, description, 67 Isomalt, description, 180 Isomaltulose, description, 180 Isomaltulose production, commercial application, 254 Isothiocyanatcs, production, 298

J Juvenile hormones I and II, synthesis, 356/,357

Κ Killer enzymes, examples, 189 Kinetics, hydrolysis and aminolysis of cinnamoylated subtilisins, 16/,17

L Lactase applications, 41 description, 41 upgrading whey, 41 Lactoperoxidasc, applications, 187-188 Lactose, description, 40 Lepidoptera, biosynthesis of pheromone components, 332

Lepidopteran species, fatty acid content, 324 Leucrose, description, 180 Lignin degradation, 127,129 description, 127 mechanism of oxidation, 129 schematic representation, 127,128/ Lignin peroxidase catalytic cycle, 132,133/ catalyzed reactions, 132,134/136 electronic adsorption spectral maxima, 129,130/,131 general properties, 129 kinetics, 131-132 mechanism of C - C « cleavage, 132,135/136 one-electron oxidation, 136 oxidized intermediates, 131/ side-chain cleavage, 136 spectroscopic properties, 129,130/,131 Lipase application for ester synthesis and fat modification, 158 applications, 3,187 catalytic specificity, 40 description, 371-372 hydrolysis of 4-hydroxy-3-methyI-2(2-propynyl)-2-cyclopentenonc, 372,373/,374 potential industrial applications, 254-255 production of specialty products, 4-5 See also Immobilized lipases Lipase components actions, 171,172/ ester synthesis, 171/ Lipase components of Candida antarctica characterization, 168/ immobilization, 168,169/ purification, 168 Lipases for accelerating cheese ripening, tailoring, 39 Lipolysis definition, 40 role in cheese ripening, 38 Liposome technology, definition, 39 Lipoxygenases applications, 3 production of hydroperoxides, 297 reduction in plants, 6 role in insect resistance, 296-298 Low dextrose equivalent corn syrup drawbacks, 27 effect of dextrin content on properties, 28 use of