Interdependence of Enzymology and Agricultural Biotechnology

Chapter 1

Interdependence of Enzymology and Agricultural Biotechnology

Downloaded by 64.184.2.5 on January 24, 2016 | http://pubs.acs.org 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, John R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


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 -


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 .


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


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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



1. WHITAKER


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


5


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 ) .


1. WHITAKER



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 .


7

8


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. +


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

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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.


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


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.

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Interdependence of Enzymology and Agricultural Biotechnology

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