Food Protein Deterioration - American Chemical Society

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Controlling Enzymic Degradation of Proteins T. RICHARDSON University of Wisconsin—Madison, Department of Food Science and the Walter V. Price Cheese Research Institute, Madison, WI 53706

Traditionally, the food industry has used a limited number of processes for controlling endogenous (in the raw material) and exogenous (added or adventitious) enzymic activities in foods including proteolysis. Control of enzymic activities in foods usually depends upon controlling the enzymic environment so as to maximize or prevent the action of enzymes on their substrates. All food scientists recognize that the activities of exogenous enzymes added to foods can be controlled by manipulating such factors as pH, temperature, presence of co-factors or inhibitors, etc. On the other hand exogenous, adventitious enzymes arising from spoilage microorganisms can often be controlled by the judicious use of sanitizers and pH controls which may be a function of product formation, water activity, osmolarity, etc. The control of endogenous food enzymes, however, is often very complex and requires a thorough knowledge of enzymic activities and how they are integrated into the post-harvest and post-mortem physiologies of foods. Control of these metabolic (usually catabolic) activities by the food scientist can have a profound influence on the quality and storage stability of various foods. Controlled atmosphere storage and packaging of various foods is an obvious example of environmental control of endogenous enzymic activities (1). Oxidative and fermentative metabolic reactions are of extreme importance i n the conversion of muscle to meat (2) and in the maintenance of quality in post-harvest fruits and vegetables (3). Endogenous enzymic reactions are of great importance in the maturation of dates and tea as well as other foods. Endogenous and exogenous proteolytic enzymes are well-documented as factors controlling the tenderization of meat (2). Blanching of vegetables and fruits is the time-honored method for inactivating endogenous enzymes to enhance stability of frozen foods (1). Within the intact tissues of foods, numerous enzymes are compartmentalized or constrained by intracellular membranes to physically prevent their interaction with substrates. This 0097-6156/82/0206-0031 $07.25/0 © 1982 American Chemical Society Cherry; Food Protein Deterioration ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

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FOOD P R O T E I N DETERIORATION

latency i s destroyed a f t e r harvest or slaughter as the membranes d e t e r i o r a t e , thereby a l l o w i n g admixture of enzymes with c e l l u l a r substrates (_2, _3» jO · T h i s r e s u l t s i n the degradation of c e l l u l a r s t r u c t u r a l elements which may be d e s i r a b l e f o r meat t e n d e r i z a t i o n or u n d e s i r a b l e i n the t e x t u r a l d e t e r i o r a t i o n of fruits. Thus, the c o n t r o l of enzymic l a t e n c y i n food p r o c e s s i n g can be extremely important. Recent research suggests some p o s s i b i l i t i e s f o r c o n t r o l l i n g the l a t e n c y of exogenous and endogenous enzymes i n food systems. I d e a l l y , the food s c i e n t i s t would l i k e t o c o n t r o l the v a r i o u s enzymic r e a c t i o n s a f f e c t i n g food q u a l i t y thereby l e a d i n g to products with b e t t e r t e x t u r e , c o l o r , f l a v o r and n u t r i t i o n a l v a l u e . At the present time, t h i s i d e a l i s unobtainable; however, f u r t h e r research w i l l undoubtedly lead to a d d i t i o n a l ways f o r c o n t r o l l i n g enzymic a c t i v i t i e s i n foods. The subsequent d i s c u s s i o n w i l l explore some p o s s i b i l i t i e s f o r manipulating enzymic a c t i v i t i e s which may serve as a b a s i s f o r a d d i t i o n a l research i n t h i s area. In many cases, i t i s n e c e s s a r i l y s p e c u l a t i v e but the s p e c u l a t i o n , I b e l i e v e , i s based on acceptable chemical, p h y s i c a l and b i o l o g i c a l p r i n c i p l e s . Since t h i s volume i s dedicated to the degradation of p r o t e i n s i n foods, the f o l l o w i n g text w i l l be r e s t r i c t e d p r i m a r i l y t o a d i s c u s s i o n of the c o n t r o l of p r o t e o l y t i c a c t i v i t i e s i n foods. Need to C o n t r o l P r o t e o l y s i s The c o n t r o l of p r o t e o l y t i c a c t i v i t y i n food m a t e r i a l s should be considered i n the broadest p o s s i b l e terms and not only i n i t s p r e v e n t i o n . The r e g u l a t i o n of the extent and timing of p r o t e o l y s i s by endogenous and exogenous enzymes could be of prime importance i n p r o c e s s i n g and storage of foods. I f one could s e l e c t i v e l y i n i t i a t e , enhance, r e t a r d or i n h i b i t v a r i o u s proteases, a g r e a t e r f l e x i b i l i t y i n food p r o c e s s i n g , i n product development and i n storage v a r i a b l e s might be p o s s i b l e . The r e q u i s i t e c o n t r o l might be achieved by manipulating enzymic l a t e n c y p h y s i c a l l y or c h e m i c a l l y , by j u d i c i o u s use of n a t u r a l or s y n t h e t i c , d i g e s t i b l e i n h i b i t o r s , by engineering exogenous proteases with recombinant DNA methods or by v a r i o u s other techniques discussed i n the succeeding s e c t i o n s . A few examples w i l l serve to i l l u s t r a t e why the c o n t r o l of p r o t e o l y t i c enzymes, or the l a c k of i t , i s important i n food systems. The s u b s t i t u t i o n of fungal rennets f o r c a l f - r e n n e t i n cheesemaking c r e a t e d an unexpected problem i n the c o n t r o l of p r o t e o l y t i c a c t i v i t y i n the cheese whey. Fungal rennets appearing i n the whey a f t e r curd formation a r e s u b s t a n t i a l l y more s t a b l e than c a l f rennet to thermal i n a c t i v a t i o n (5). Consequently, r e s i d u a l fungal p r o t e o l y t i c a c t i v i t y a f t e r c o n v e n t i o n a l thermal p r o c e s s i n g of the whey l e d to obvious problems when the d r i e d whey was subsequently used i n products c o n t a i n i n g c a s e i n . I d e a l l y , these proteases should be c o n t r o l l e d with a minimum heat

Cherry; Food Protein Deterioration ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

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treatment to save energy and minimize damage to whey p r o t e i n s . Recently, fungal rennets more l a b i l e to heat treatments have become commercially a v a i l a b l e . A c t i v i t i e s of h e a t - s t a b l e proteases and l i p a s e s from psychrotrophic microorganisms ( 6 , 7) p r o l i f e r a t i n g i n milk stored at low temperatures may adversely a f f e c t milk q u a l i t y and cheese yields. Inexpensive, p r a c t i c a l c o n t r o l of psychrotrophs and/or t h e i r h e a t - s t a b l e p r o t e o l y t i c and l i p o l y t i c enzymes would be highly desirable. Bovine plasmin i s secreted from the blood i n t o m i l k where i t a c t s p r i m a r i l y on B-casein to generate a s e r i e s of more hydrophobic γ-caseins and p o l a r peptides (8, 9 ) . Apparently, the plasmin can a t t a c k the milk p r o t e i n s while they are s t i l l i n the lumen of the mammary gland. In a d d i t i o n , p r o t e o l y s i s can continue, a f t e r m i l k i n g , i n the raw, stored product. The amount of plasmin i n milk seems to i n c r e a s e i n l a t e l a c t a t i o n m i l k and s u f f i c i e n t p l a s m i n o l y s i s of the milk p r o t e i n s may lead to d e f e c t s i n texture and l o s s of y i e l d i n the r e s u l t a n t cheese (10, 11). Since the plasmin has a s p e c i f i c i t y s i m i l a r t o t r y p s i n , i t s a c t i v i t y has been i n h i b i t e d with soybean t r y p s i n i n h i b i t o r . The c o n t r o l of endogenous plasmin a c t i v i t y i n milk would d e f i n i t e l y b e n e f i t the d a i r y i n d u s t r y . The foregoing three examples r e l a t i n g t o c o n t r o l o f added, m i c r o b i a l and endogenous enzymes serve to i l l u s t r a t e that the c o n t r o l of enzymic a c t i v i t y i s important i n a l l three general areas of food enzymology. In Table I are l i s t e d some major p o s s i b i l i t i e s f o r c o n t r o l l i n g p r o t e o l y t i c degradation o f food p r o t e i n s by enhancing, r e t a r d i n g , i n h i b i t i n g or d e l a y i n g enzymic a c t i v i t i e s . These are based i n p a r t on observations from the l i t e r a t u r e and w i l l be discussed i n the f o l l o w i n g s e c t i o n s . Enzymic Latency Latent a c t i v i t y of enzymes w i l l be defined as p o t e n t i a l a c t i v i t y which becomes expressed upon removal of p h y s i c a l and/or chemical c o n s t r a i n t s . C o n t r o l of enzymic l a t e n c y should allow r e l e a s e of enzyme a t d e f i n i t e p o i n t s i n a food process or during storage to e f f e c t d e s i r e d changes. Conversely, l a t e n t enzyme i n h i b i t o r s may a l s o be c o n t r o l l e d to i n h i b i t enzymic a c t i v i t y a f t e r a d e s i r e d i n t e r v a l . Various types of p o t e n t i a l enzymic latency are l i s t e d i n Table I I . Lysosomal l a t e n c y . A c l a s s i c a l example of enzymic l a t e n c y i s the v a r i o u s h y d r o l y t i c enzymes that are sequestered w i t h i n i n t r a c e l l u l a r lysosomal p a r t i c l e s (2, 4_, 12). Included among the h y d r o l y t i c enzymes r e t a i n e d w i t h i n the lysosomal membranes a r e the p r o t e o l y t i c cathepsins. Since these lysosomal proteases have pH optima i n the a c i d i c region around 5, there has been much d i s c u s s i o n concerning t h e i r relevance i n the economy or turnover


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FOOD PROTEIN DETERIORATION

of c e l l s with a presumed, nominal pH near n e u t r a l i t y . This has been e s p e c i a l l y true i n research on meat t e n d e r i z a t i o n whereby the post-mortem l i b e r a t i o n of l a t e n t c a t h e p t i c a c t i v i t y has been i m p l i c a t e d i n degradation of muscle p r o t e i n s to enhance the t e n d e r i z a t i o n of meat (2). However, the low pH optima of these enzymes coupled with the sparse numbers of lysosomal p a r t i c l e s and r e l a t i v e l y low c a t h e p t i c a c t i v i t y i n muscle t i s s u e have l e d some s c i e n t i s t s i n meat research to question t h e i r importance i n the t e n d e r i z a t i o n of meat (2, -4» 13, 14). Nevertheless, recent research by Dutson et a l . (15) on the increase i n meat tenderness r e s u l t i n g from high voltage e l e c t r i c s t i m u l a t i o n of ovine carcasses suggests that t h i s treatment r e l e a s e s l a t e n t c a t h e p t i c a c t i v i t y by r u p t u r i n g lysosomal p a r t i c l e s . An attendant decrease i n the pH of the muscle t i s s u e apparently favors the rupture of lysosomes and catheptic-mediated h y d r o l y s i s of muscle p r o t e i n s l e a d i n g to increased t e n d e r i z a t i o n of the meat. However, i t must be emphasized that the r e l e a s e of lysosomal enzymes r e s u l t ing from e l e c t r i c a l s t i m u l a t i o n i s only one of s e v e r a l p o s s i b l e mechanisms suggested f o r t h i s t e n d e r i z i n g e f f e c t . In a d d i t i o n , other endogenous proteases have a l s o been involved i n meat tenderization (2). Table I.

P o s s i b i l i t i e s f o r c o n t r o l l i n g exogenous and proteases.

endogenous

1)

Manipulation in vivo.

of enzymic latency i n s i t u , ex v i v o

2)

Chemical and b i o l o g i c a l engineering

3)

Use of n a t u r a l and

4)

C o n t r o l of e l e c t r o s t a t i c i n t e r a c t i o n s that may a f f e c t enzymic-substrate and post-enzymic product i n t e r a c t i o n s .

synthetic

and

of enzymes.

inhibitors.

Thus, the meat s c i e n t i s t may be able to c o n t r o l l a t e n c y of lysosomal enzymes i n muscle t i s s u e by e l e c t r i c a l s t i m u l a t i o n . In a d d i t i o n , other f a c t o r s of a biochemical nature may c o n t r i b u t e to the s t a b i l i t y or l a c k of i t i n c e l l u l a r membranes that d e l i m i t i n t r a c e l l u l a r enzymic r e a c t i o n s . For example, Lawrie (2) b r i e f l y discussed the l i b e r a t i o n of lysosomal enzymes as a r e s u l t of vitamin Ε d e f i c i e n c y or of excess vitamin A i n animal t i s s u e s . Polyunsaturated f a t t y a c i d s of membrane l i p i d s are known to enhance f l u i d i t y of c e l l u l a r membranes implying a decrease i n t h e i r p h y s i c a l s t a b i l i t y (16). This has obvious i m p l i c a t i o n s i n the post-mortem latency of lysosomal enzymes i n f i s h muscle. Mitochondria from f i s h l i v e r s w e l l much more r a p i d l y than those from r a t l i v e r (17) . A l s o , one might ask whether the latency of lysosomal enzymes i n the muscles of ruminants fed encapsulated polyunsaturated o i l s to y i e l d more unsaturated meat (and presumably membranes) (18, 19) would be d i f f e r e n t than the normal, more saturated counterpart.


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Some meat s c i e n t i s t s and muscle b i o l o g i s t s have suggested that lysosomal enzymes r e l e a s e d from phagocytic c e l l s w i t h i n muscle t i s s u e may p l a y a r o l e i n the t e n d e r i z a t i o n o f meat. Catheptic a c t i v i t y i n phagocytic c e l l s i s very high (4). I f the m i g r a t i o n of the phagocytic c e l l s i n t o muscle t i s s u e could be c o n t r o l l e d , i t may be p o s s i b l e t o r e g u l a t e , i n p a r t , the tenderi z a t i o n process. In t h i s regard, chemotactic peptides (e.g., M-formyl-methionyl-leucyl-phenylalanine) that a t t r a c t phagocytic c e l l s have been i s o l a t e d and c h a r a c t e r i z e d (20). I f these peptides c o u l d be s e l e c t i v e l y d e l i v e r e d to s k e l e t a l muscles, they might prove u s e f u l i n manipulating the m i g r a t i o n of phagocytic c e l l s i n t o muscle t i s s u e thereby p a r t i a l l y c o n t r o l l i n g l e v e l s of lysosomal c a t h e p s i n s . Thus, p o s s i b i l i t i e s e x i s t , a l b e i t remote a t t h i s p o i n t , f o r c o n t r o l l i n g post-mortem lysosomal enzymic l a t e n c y i n muscle t i s s u e . Latent enzymic a c t i v i t i e s are a l s o very important i n the post-harvest physiology of p l a n t t i s s u e (3). I t i s l e s s c l e a r , however, how these a c t i v i t i e s might be b e t t e r c o n t r o l l e d . Table I I .

Enzymic l a t e n c y .

1)

C o n t r o l of lysosomal proteases i n v i v o and ex v i v o .

2)

M i c r o e n c a p s u l a t i o n of proteases ( c o n t r o l l e d r e l e a s e ) .

3)

C o n t r o l of zymogen a c t i v a t i o n .

4)

Latency o f chemical d e r i v a t i v e s o f proteases latency).

5)

Latency o f p o l y c a t i o n i c p r o t e i n s - protease complexes.

6)

Genetic engineering of food microorganisms. P r o t e o l y t i c a c t i v i t y a f t e r a u t o l y s i s or s e c r e t i o n of enzymes.

(chemical

Chemical l a t e n c y . An e x c e l l e n t example o f chemical l a t e n c y a r i s e s out o f the p r e - s l a u g h t e r , intravenous i n j e c t i o n of the s u l f h y d r y l protease papain i n t o animals, again to enhance tenderi z a t i o n of meat v i a post-mortem p r o t e o l y t i c a c t i v i t y . In t h i s case, the enzyme i n j e c t e d before slaughter of the animal i s disseminated by the c i r c u l a t i o n o f the animal throughout i t s musculature. The uniform d i s t r i b u t i o n of enzyme w i t h i n the t i s s u e s r e s u l t s i n post-mortem t e n d e r i z a t i o n o f the r e s u l t a n t meat. I n j e c t i o n of the f r e e , a c t i v e enzyme r e s u l t s i n s t r e s s to the animal accompanied by i n t e r n a l hemorrhaging l e a d i n g to r e j e c t i o n o f the c a r c a s s . However, i f the e s s e n t i a l t h i o l group i n the a c t i v e s i t e of papain i s r e v e r s i b l y blocked by t h i o l d i s u l f i d e interchange, t h i s i n a c t i v e enzyme can be administered to the animal without i l l e f f e c t s . The l a t e n t papain a c t i v i t y i s subsequently regenerated i n s i t u by reducing agents i n the meat (perhaps by g l u t a t h i o n e while cooking) (21) . I f the


36


e s s e n t i a l f u n c t i o n a l g r o u p s f o r e n z y m i c a c t i v i t y a r e known, i t s h o u l d be p o s s i b l e t o i n t r o d u c e c h e m i c a l l a t e n c y i n t o v i r t u a l l y any e x o g e n o u s enzyme u s e d i n f o o d p r o c e s s i n g . The a c i d p r o t e a s e s u s e d i n t h e m a n u f a c t u r e o f c h e e s e may offer opportunities f o r l a t e n t p r o t e o l y s i s i n the ripening of cheese. Conversion of e s s e n t i a l c a r b o x y l a t e r e s i d u e s i n the a c i d p r o t e a s e s t o l a b i l e a n h y d r i d e s o r e s t e r s may a l l o w s l o w b u t sustained r e l e a s e of p r o t e o l y t i c a c t i v i t y w i t h i n the cheese. In t h i s r e g a r d , i t s h o u l d be u s e f u l t o d i s c u s s t h e i n h i b i t i o n o f c h y m o s i n a c t i v i t y upon t r e a t i n g t h e enzyme w i t h d a n s y l c h l o r i d e (22, 23, 2 4 ) . Since l y s i n e r e s i d u e s i n p r o t e i n s a r e r e a d i l y d a n s y l a t e d , an e s s e n t i a l l y s i n e was p r o p o s e d f o r c h y m o s i n a c t i v i t y ( 2 2 , 2 3 ) . However, t h e s l o w r e g e n e r a t i o n o f l a t e n t p r o t e o l y t i c a c t i v i t y upon s t o r a g e o f t h e d a n s y l a t e d c h y m o s i n ( 2 3 ) and r a p i d r e a c t i v a t i o n upon t r e a t m e n t w i t h NH^OH ( 2 4 ) w e r e n o t c o n s i s t e n t w i t h p r o p o s e d d a n s y l a t i o n o f an e s s e n t i a l l y s i n e residue, which y i e l d s a very s t a b l e covalent d e r i v a t i v e . Subsequently, formation of a l a b i l e , dansylated h i s t i d i n e residue i n c h y m o s i n was s u g g e s t e d ( 2 4 ) t o e x p l a i n t h e r e g e n e r a t i o n o f activity. However, i n v i e w o f t h e e s s e n t i a l i t y o f t h e γ-carboxyl g r o u p s o f two a s p a r t a t e r e s i d u e s i n a c i d p r o t e a s e s ( 2 5 ) , i t i s p l a u s i b l e t h a t t h e d a n s y l c h l o r i d e r e a c t e d w i t h an e s s e n t i a l c a r b o x y l a t e a n i o n a s shown i n F i g u r e 1 t o y i e l d a m i x e d a n h y d r i d e thereby i n a c t i v a t i n g the chymosin. T h i s c o u l d subsequently r e a c t w i t h ammonium h y d r o x i d e o r a l y s i n e r e s i d u e t o r e g e n e r a t e p r o t e o l y t i c a c t i v i t y and y i e l d , i n t h e l a t t e r case a d a n s y l a t e d lysine. T h i s s u g g e s t i o n i s c o n s i s t e n t w i t h t h e mechanism f o r h y d r o l y s i s o f s u l f i t e e s t e r s b y p e p s i n p r o p o s e d b y K a i s e r and Nakagawa ( 2 6 ) who w e r e a b l e t o i n f e r a n h y d r i d e i n t e r m e d i a t e s b y u s i n g strong n u c l e o p h i l e s as t r a p p i n g agents (Figure 2 ) . Microencapsulation. Enzymes h a v e b e e n e n t r a p p e d w i t h i n m i c r o c a p s u l e s u s i n g a v a r i e t y o f methods ( 2 7 , 28) t o y i e l d s o c a l l e d m i c r o c e l l s and i m m o b i l i z e d enzymes f o r b i o m e d i c a l o r f o o d p r o c e s s i n g p u r p o s e s . The f e a s i b i l i t y o f e n c a p s u l a t i n g enzymes o r c e l l - f r e e e x t r a c t s c o n t a i n i n g m i x t u r e s o f enzymes i n t o e d i b l e m i c r o c a p s u l e s o f m i l k f a t h a s b e e n e s t a b l i s h e d b y O l s o n and c o w o r k e r s ( 2 9 , 30, 3 1 ) . These w o r k e r s a r e d e v e l o p i n g a cheese r i p e n i n g system whereby c e l l - f r e e e x t r a c t s o f m i c r o o r g a n i s m s can be e n c a p s u l a t e d w i t h s u b s t r a t e s and c o - f a c t o r s f o r g e n e r a t i o n o f f l a v o r s s u c h a s d i a c e t y l and a c e t o i n . The m i l k - f a t c o a t e d m i c r o c a p s u l e s c o n t a i n i n g t h e a p p r o p r i a t e enzyme s y s t e m s a r e a d d e d t o m i l k and r e t a i n e d i n t h e r e s u l t i n g c h e e s e . The j u x t a p o s i t i o n o f enzyme, s u b s t r a t e s and c o - f a c t o r s a l l o w s t h e enhancement and r e g u l a t i o n o f f l a v o r development i n the cheese. Proteolytic enzymes c o u l d b e e n c a p s u l a t e d a n d t h e l a t e n t a c t i v i t y r e l e a s e d by t h e r m a l o r l i p o l y t i c treatment o f t h e p r o d u c t a f t e r an a p p r o p r i a t e period. Physical latency.

Although

enzymic c o m p a r t m e n t a l i z a t i o n


in

RICHARDSON

Enzymic Degradation

Ο 1» Chym-C-O

+

DanSO^l

of

-—•

Proteins

Ο Il Chym-C-O-SO^Dan (mixed

II Chym-C-O-SC^-Dan

Ν» OH

HC1

anhydride)

fj _ Chym-C-0

•

+

+

Dan-S0

3

Figure 1. Suggested mechanism for inhibition of chymosin by dansyl chloride (top) and subsequent reactivation (bottom). Although aromatic sulfonates are good leaving groups (56), it is possible that an incoming amine could attack the sulfur moiety in such an asymmetric anhydride, yielding the appropriate sulfonamide (61).

ο

r-c-OH O R Ε

r

r

S=0

f HO-S-OR' ^e-o"~^

OR'

c-o~

II

t o H ç )

C-0"

Ε OH ^-C-O-S-OR

II

II

0

r

C - O H

Ε

r

^

Ε

^~Ç-OH

r

Ο

Ο II

VI

Ο R'

C-0"

Ε

—

^C-O-S-OR'

Ο VII

\

I

Ο π ι

II

ο V + ROH

L f Μ ΣII- 0 Ο IV

O H ι ι S-ÇR OR'

Figure 2. Proposed mechanism for the pepsin-catalyzed hydrolysis of sulfite esters. (Reproduced, with permission, from Ref. 26. Copyright 1977, Plenum Publishing Corp.)


38


lysosomes and microencapsulation of enzymes are forms of p h y s i c a l l a t e n c y , t h i s s e c t i o n w i l l be r e s t r i c t e d to l a t e n c y v i a formation of d i g e s t i b l e , macromolecular aggregates. G e n e r a l l y , most p r o t e i n s and enzymes c a r r y a net negative charge a t n e u t r a l pH values ( 3 2 ) . I t f o l l o w s that there are few p r o t e i n s and enzymes with net p o s i t i v e charges i n food systems. However, p o s i t i v e l y charged p r o t e i n s ( p i = 7 to 10) can be prepared by simply e s t e r i f y i n g or amidating f r e e carboxyl groups (33, 3 4 , 3 5 ) . These p o s i t i v e l y charged p r o t e i n s a v i d l y i n t e r a c t with n e g a t i v e l y charged c a s e i n s , f o r example, to y i e l d a p r e c i p i t a t e d complex (33, 34, 35). Thus, i t seems p o s s i b l e to form i n s o l u b l e , i n a c t i v e complexes between p o s i t i v e l y charged p r o t e i n s and n e g a t i v e l y charged proteases such as porcine pepsin ( p i = 2.2) and bovine chymosin ( p i = 4 . 6 ) . P r o t e o l y s i s of the complexed p o l y c a t i o n by the entrapped protease should y i e l d the l a t e n t activity. The p u t a t i v e l a t e n c y might be u s e f u l i n c o n t r o l l i n g r i p e n i n g of cheese. D i g e s t i b l e , macromolecular complexes with other f o o d - r e l a t e d enzymes such as the amylases should a l s o be p o s s i b l e to prepare. Zymogen a c t i v a t i o n . As i s w e l l known, many p r o t e o l y t i c enzymes are secreted as i n a c t i v e zymogens. Subsequent cleavage of an a c t i v a t i o n peptide from the N-terminus, f o r example, of pepsinogen ( 3 6 ) or prochymosin ( a u t o c a t a l y t i c a l l y or by other proteases) r e s u l t s i n r e a l i z a t i o n of p r o t e o l y t i c a c t i v i t y (Figure 3 ) . The r a t e s of a c t i v a t i o n of the f o r e g o i n g zymogens are a f f e c t e d by a number of environmental f a c t o r s such as pH, i o n i c s t r e n g t h , temperature, e t c . (25, 3 7 ) . Apparently, e l e c t r o s t a t i c i n t e r a c t i o n s between the c o v a l e n t l y bound a c t i v a t i o n peptide sequence and the a c t i v e s i t e of the protease s t e r i c a l l y prevents enzyme-substrate i n t e r a c t i o n s (25). With pepsinogen, at l e a s t , the r e l e a s e d a c t i v a t i o n peptide can, at r e l a t i v e l y high molar r a t i o s , s t i l l e l e c t r o s t a t i c a l l y bind to the enzyme as an i n h i b i t o r (Figure 4 ) . Guanidation of the a c t i v a t i o n peptide enhanced the i n h i b i t i o n ( 3 8 ) suggesting that chemical s y n t h e s i s and m o d i f i c a t i o n of peptides may y i e l d u s e f u l protease i n h i b i t o r s . The c o n t r o l of zymogen a c t i v a t i o n by other than environmenta l f a c t o r s might be accomplished by v a r y i n g the sequence of the a c t i v a t i o n peptide with recombinant DNA techniques. For example, N i s h i m o r i et a l . ( 3 9 ) r e c e n t l y cloned the gene f o r c a l f p r o chymosin i n t o E s c h e r i c h i a c o l i . I t i s w e l l known that many of the a c i d proteases are g e n e t i c a l l y r e l a t e d w i t h long amino a c i d sequences conserved (40) (Table I I I ) . However, there are a l s o a number of non-homologous regions that apparently are not essential for proteolytic a c t i v i t y . Therefore, these nonhomologous r e g i o n s w i t h i n the v a r i o u s a c i d proteases or t h e i r a c t i v a t i o n peptides might be a l t e r e d u s i n g p o i n t mutations and recombinant DNA techniques (41, 42, 43) to engineer the r a t e of zymogen a c t i v a t i o n and perhaps the a c t i v i t y of the p r o t e o l y t i c


RICHARDSON

Enzymic

Degradation

Ρ

of

p

Proteins

Ρ

Ρ

Figure 3. Activation of pepsinogen. In the conversion of pepsinogen to pepsin, hydrolysis occurs at several points (P) releasing peptides (A), a pepsin inhibitor (B), and pepsin (C). (Reproduced, with permission, from Ref. 36. Copyright 1960, Academic Press.)

too

0 < LU

OC

50

È >

0

~

Ίθ

*"

30

~

P E P T I D E i P E P S I N M O L A R RATIO Figure 4. Inhibition of pepsin by varying amounts of its activation. Key: | , pep tide 1-16; and Φ, its guanidinated derivative. (Reproduced, with permission, from Ref. 38. Copyright 1977, Plenum Publishing Corp.)


40


enzyme i t s e l f . Other p o s s i b i l i t i e s f o r g e n e t i c e n g i n e e r i n g of p r o t e a s e s w i l l be d i s c u s s e d b e l o w . The f o r e g o i n g e x a m p l e s s e r v e t o i l l u s t r a t e how t h e r e g u l a t i o n o f t h e l a t e n c y o f enzymes, e s p e c i a l l y p r o t e a s e s , may be a t t a i n a b l e t o f a c i l i t a t e f o o d p r o c e s s i n g and t o c o n t r o l s t o r a g e . Table

I I I . Homology i n some a c i d p r o t e a s e s

Homology i n B o v i n e C h y m o s i n and P e p s i n

Homology i n B o v i n e and P o r c i n e P e p s i n s % Homology 67

% Homology

Sequences Activation

(40).

Peptide

Sequences

40

1-47

83

48-111

67

48-111

100

259-265

86

259-265

79

355-373

57

355-373

Chemical

Engineering

The c h e m i c a l m o d i f i c a t i o n o f enzymes i s p r o b a b l y o f l i m i t e d p r a c t i c a l v a l u e . However, i t may h e l p t o u n d e r s t a n d s u c h f a c t o r s a s how p r o t e a s e - p r o t e i n i n t e r a c t i o n s may be a f f e c t e d by t h e i r relative p i values. In a d d i t i o n , chemical m o d i f i c a t i o n s of p r o t e a s e s may p r o v i d e w o r t h w h i l e i n f o r m a t i o n f o r e n g i n e e r i n g p r o t e a s e s u s i n g r e c o m b i n a n t DNA m e t h o d s . I t i s l i k e l y t h a t any a t t e m p t s t o s u b s t a n t i a l l y m o d i f y t h e s u r f a c e c h a r g e o f enzymes by e s t e r i f i c a t i o n o f c a r b o x y l g r o u p s o r by a c y l a t i o n o f amino g r o u p s , f o r e x a m p l e , may l e a d t o d i s r u p t i o n o f t h e i r t h r e e d i m e n s i o n a l s t r u c t u r e s and t h e r e b y i n a c t i v a t e them. F o r e x a m p l e , e x t e n s i v e p h o s p h o r y l a t i o n , a m i d a t i o n or e s t e r i f i c a t i o n of 3 - l a c t o g l o b u l i n tends to d i s r u p t i t s g l o b u l a r s t r u c t u r e (35, 44). N e v e r t h e l e s s , J o h a n s e n e t a l . (45) f o u n d t h a t s u c c i n y l a t e d , n i t r a t e d and i o d i n a t e d p r e p a r a t i o n s o f s u b t i l i s i n C a r l s b e r g w e r e more a c t i v e t o w a r d s c l u p e i n and g e l a t i n but not c a s e i n or fi-benzoyltyrosine e t h y l e s t e r s as compared t o t h e n a t i v e enzyme. By t h e same t o k e n , M i t z and Summaria (46) r e p o r t e d i n c r e a s e d a c t i v i t y o f s o l u b l e c h y m o t r y p s i n c e l l u l o s e d e r i v a t i v e s i n r e l a t i o n t o t h e n a t i v e enzyme. T h e r e a r e a number o f e x a m p l e s o f c h e m i c a l m o d i f i c a t i o n s o f enzymes t h a t s u g g e s t p o s s i b i l i t i e s f o r e n g i n e e r i n g enzymes by r e c o m b i n a n t DNA t e c h n i q u e s . F o r e x a m p l e , p o l y p e p t i d y l enzymes h a v e b e e n s t u d i e d i n some d e t a i l ( 4 7 ) . I n g e n e r a l , t h e enzyme i s r e a c t e d w i t h an a p p r o p r i a t e amino a c i d ϋ-carboxyanhydride t o y i e l d a p r o d u c t whereby p o l y a m i n o a c i d c h a i n s a r e c o v a l e n t l y bound t o t h e enzyme t o r e n d e r i t more c a t i o n i c , a n i o n i c o r hydrophobic, e t c .


2,

RICHARDSON

Enzymic Degradation

of

41

Proteins

Soluble, p o l y p e p t i d y l proteases are g e n e r a l l y more r e s i s t a n t to i n a c t i v a t i o n by a u t o l y s i s or by p r o t e o l y s i s r e s u l t i n g from other enzymes. A l s o , p o l y p e p t i d y l proteases may have much d i f f e r ent a c t i v i t i e s on the same substrates compared t o the n a t i v e enzymes. P o l y - D L - t - l e u c y l chymotrypsin had 64% of the esterase a c t i v i t y of chymotrypsin a c t i n g on L - p h e n y l a l a n y l - e t h y l e s t e r . On the other hand, i t was 9 0 % more a c t i v e than chymotrypsin as an amidase a c t i n g on benzoyl-L-phenylalanylhydroxamide (48). P o l y - L - l y s y l - r i b o n u c l e a s e (P-L-RNase) has been prepared as i n d i c a t e d i n Figure 5 to y i e l d a p o s i t i v e l y charged enzyme (49) which, of course, i n t e r a c t s with p o l y a n i o n i c r i b o n u c l e i c a c i d . As shown i n Table I V , the P-L-RNase a c t s d i f f e r e n t l y on v a r i o u s substrates as the pH i s changed compared t o the unmodified enzyme i n d i c a t i n g a marked a l t e r a t i o n i n i n t e r a c t i o n s with s u b s t r a t e . Although v a r i o u s P-L-RNase preparations possessed only 4 to 25% of the n a t i v e a c t i v i t y on RNA, P-L-RNase d i s p l a y s maximal a c t i v i t y at i o n i c strengths 2 to 6 times greater than f o r the n a t i v e enzyme at pH 5 or 8 (49). Optimum pH a t low i o n i c strengths was s h i f t e d i n the same d i r e c t i o n as that o f the i s o e l e c t r i c p o i n t of the modified enzyme f o r RNA. Removal o f l y s y l peptides by t r y p t i c treatment of P-L-RNase tended to reverse the changes i n enzymic p r o p e r t i e s . I n t e r e s t i n g l y , d i s u l f i d e bridges i n the P-L-RNase (as with the c o n t r o l RNase) could be reduced and subsequently reformed by o x i d a t i o n to completely recover a c t i v i t y . Table

IV.

R e l a t i v e r a t e s of h y d r o l y s i s of nucleoside 2*-3* c y c l o phosphates by p o l y p e p t i d y l RNase. Native RNase = 100% (49).

Preparation (Number of Lysines/RNase)

pH 5.0 C--c-P

pH 8 .0

pH 7.0

;U-c-P

2

C-c-P ;U-c-P 1

2

C-c-P ;U-c-P 1

p-Lys-RNase (21 lysines/RNase)

53

125

60

65

107

100

p-Lys-RNase (26 lysines/RNase)

88

250

112

95

120

108

p-DL-Ala-RNase

84

42

177

47

210

25

f

Cytidine 2 -3 !

f

c y c l i c phosphate

f

U r i d i n e 2 - 3 c y c l i c phosphate The foregoing data suggest m o d i f i c a t i o n of proteases using genetic engineering techniques to a l t e r t h e i r p i , i n t e r a c t i o n s with s u b s t r a t e s , k i n e t i c parameters, hydrophobicity, pH optimum,


2

42

FOOD PROTEIN

1. DioxanePhosphate ε-Ν-TFA-a-N-carboxy-L-lysine · _ + Buffer RNase pH 7, 0°C J

J

c

c

DETERIORATION

__ ' . RNase L

1

J

J

2. 1,0 M P i p e r i d i n e Figure 5.

Synthesis of polylysyl ribonuclease (49J.


2.

RICHARDSON

Enzymic Degradation

of

Proteins

43

thermal s t a b i l i t y , e t c . T h i s o f f e r s numerous o p p o r t u n i t i e s f o r designing novel enzymes to be used i n food p r o c e s s i n g . I t may e v e n t u a l l y be p o s s i b l e t o manipulate secondary and t e r t i a r y s t r u c t u r e s of enzymes using genetic and computer techniques which could d e f i n e the e f f e c t s of changes i n primary amino a c i d sequences on three dimensional s t r u c t u r e (50). B i o l o g i c a l Engineering In a d d i t i o n to modifying p u t a t i v e non-homologous or other regions of the primary sequence of zymogens and proteases w i t h p o i n t mutation-recombinant DNA techniques, other p o s s i b i l i t i e s are suggested by the previous d i s c u s s i o n of p o l y p e p t i d y l enzymes. For example, i n s e r t i o n i n t o a c l o n i n g v e c t o r of the a p p r o p r i a t e o l i g o n u c l e o t i d e sequence coding f o r p o l y l y s i n e , p o l y c y s t e i n e , p o l y l e u c i n e , polyglutamic a c i d , e t c . c o v a l e n t l y bound to the C-terminus of the protease might allow the p r o d u c t i o n of foodr e l a t e d enzymes with v a s t l y a l t e r e d p r o p e r t i e s which could be e x p l o i t e d . The r e g e n e r a t i o n of a c t i v i t y a f t e r o x i d a t i o n of the i n a c t i v e , reduced P-L-RNase d i s c u s s e d p r e v i o u s l y suggests that polyamino a c i d s added t o proteases by genetic manipulation should not a f f e c t f o l d i n g of the modified proteases and o x i d a t i o n of t h i o l groups to y i e l d an a c t i v e three-dimensional s t r u c t u r e . Recombinant DNA technology i s c u r r e n t l y a t a stage where the aforementioned m o d i f i c a t i o n s of enzymes are c l e a r l y possible. N a t u r a l and Synthetic Protease

Inhibitors

There i s a wide range of proteases that are i n h i b i t e d by n a t u r a l l y o c c u r r i n g p r o t e i n i n h i b i t o r s i n p l a n t s (51) . I n h i b i t o r s are known t o occur i n c e r t a i n animal t i s s u e s and f l u i d s as w e l l (52). E v i d e n t l y there are numerous i n h i b i t o r s s p e c i f i c f o r proteases with d i f f e r e n t a c t i v e s i t e s such as the s e r i n e proteases and the a c i d proteases. There i s a s u b s t a n t i a l amount of i n f o r m a t i o n on the s t r u c t u r e s and a c t i v e s i t e s of these n a t u r a l i n h i b i t o r s a v a i l a b l e with which t o design p o t e n t i a l s y n t h e t i c peptide i n h i b i t o r s ( 5 1 , 5 3 ) . Although the use of these n a t u r a l i n h i b i t o r s i n foods may not meet with the approval o f the U.S. Food and Drug A d m i n i s t r a t i o n , a d e t a i l e d knowledge of t h e i r s t r u c t u r e s and mechanisms of a c t i o n may e v e n t u a l l y allow the s y n t h e s i s of r e v e r s i b l e and d i g e s t i b l e peptide i n h i b i t o r s that c o u l d prove u s e f u l . For example, p e p s t a t i n analogs, p e p s t a t i n - l i k e p e p t i d e s , or modified a c t i v a t i o n peptides from zymogens may be used t o c o n t r o l a c i d proteases i n the production of cheese. Or peptides patterned a f t e r the a c t i v e s i t e i n t r y p s i n i n h i b i t o r s could i n h i b i t unwanted plasmin a c t i v i t y i n m i l k mentioned p r e v i o u s l y . P e p s t a t i n i s a pentapeptide, i s o l a t e d from v a r i o u s species of actinomycetes, which s t r o n g l y i n h i b i t s s e v e r a l a c i d


44


proteases; the K f o r i n h i b i t i o n of pepsin i s Ι Ο " M (25, 54). P e p s t a t i n , which c o n t a i n s a unique, c e n t r a l s t a t y l r e s i d u e i n v o l v e d i n i n t e r a c t i o n s with the a c t i v e s i t e s o f a c i d proteases has the f o l l o w i n g s t r u c t u r e (25, 54): 1 0

±

CH CH. CH CH CH(CH-). \3 ^3 ^ 3 j 3 2 CH

CH

I I I

CH

0

0

0

CH

0

I

2

( C H „ ) CHCH CONHCHCONHCHCONHCH 3 2 2 j HO — CH —

CH(CH ) j 32

3

0

CH

0

I

2

CONHCHCONHCH j j CH HO — CH — CH — 2

2

CO^

There are a number of n o v e l f e a t u r e s i n the s t r u c t u r e o f p e p s t a t i n that a r e important i n the i n h i b i t i o n of pepsin (Figure 6). I t i s a very hydrophobic peptide that has poor s o l u b i l i t y i n water. The c e n t r a l s t a t y l r e s i d u e (Figure 6) i s thought t o combine with the a c t i v e s i t e of pepsin t o mimic the t r a n s i t i o n s t a t e during normal p e p t i c p r o t e o l y s i s . P e p s t a t i n has thus been r e f e r r e d t o as a " t r a n s i t i o n s t a t e " i n h i b i t o r (25). The c e n t r a l hydroxy1 group i s e s s e n t i a l f o r assuming a p s e u d o - t r a n s i t i o n s t a t e and i s , t h e r e f o r e , necessary f o r i n h i b i t i o n of pepsin (25, 5 4 , 55). The t e r m i n a l c a r b o x y l group i s not r e q u i r e d f o r inhibitory activity. Under comparable c o n d i t i o n s , p e p s t a t i n i n h i b i t s chymosin a t 48% of the i n h i b i t i o n f o r p e p s i n , suggesting a c e r t a i n s t r u c t u r a l s p e c i f i c i t y f o r i n h i b i t i o n that might be e x p l o i t e d (55). Although there a r e a number of unique f e a t u r e s about the s t r u c t u r e of p e p s t a t i n , the j u d i c i o u s use o f modelb u i l d i n g combined with a p p r o p r i a t e peptide chemistry might a l l o w the s y n t h e s i s of r e v e r s i b l e , d i g e s t i b l e i n h i b i t o r s o f a c i d proteases f o r use i n foods. Chemical m o d i f i c a t i o n s of p e p s t a t i n such as those suggested i n F i g u r e 7 may help d e f i n e s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s as w e l l as i n d e s i g n i n g some novel i n h i b i t o r s of a c i d proteases. Removal o f the e s s e n t i a l hydroxy1 group (dideoxypepstatin) i n c r e a s e s the f o r p o r c i n e pepsin 2,000 f o l d (54). In F i g u r e 7, r e a c t i o n s a r e proposed whereby a t h i o l , amino o r other n u c l e o p h i l i c group might be s u b s t i t u t e d f o r the e s s e n t i a l hydroxy1 f u n c t i o n . These m o d i f i c a t i o n s r e l y on the good l e a v i n g p r o p e r t i e s of aromatic s u l f o n a t e s (56). Such s t r u c t u r a l a l t e r a t i o n s should l e a d t o a b e t t e r understanding o f molecular requirements f o r producing p e p s t a t i n analogs. Various types of e s t e r s i n v o l v i n g the p e p s t a t i n hydroxy1 group should be e a s i l y o b t a i n a b l e t o impart l a t e n t i n h i b i t o r s or other f e a t u r e s to these d e r i v a t i v e s . For example, phosphoryla t i o n of the hydroxyl group (Figure 7) p o t e n t i a l l y r e s u l t s i n a more w a t e r - s o l u b l e , i n a c t i v e but l a t e n t i n h i b i t o r . Slow h y d r o l y s i s of the phosphate e s t e r i n a c i d i c foods (optimum pH f o r h y d r o l y s i s of phosphate e s t e r s -4-6) (57) should r e l e a s e a c t i v e inhibitor. The n o n - e s s e n t i a l c a r b o x y l group of p e p s t a t i n can be


RICHARDSON

Enzymic Degradation

of

Proteins

Pepsin Asp 2 ! 5

~, .

P^3

L

I H

9 2

I - N H —

/

/

6

H

I

!

C H — Ç

Ô Pepsin - A s p

'

CH

CH

-7

I C

i Ν

Η

H

-2

ι 2

Ο

l — NH—CH —

Η

l C

C

Η

Η

CO —

JC—Ο

P R O P O S E D TRANSITION STATE OF P E P T I C CATALYSIS Figure 6.

1

Δ STATYL R E S I D U E IN PEPSTATIN

Inhibition of pepsin by pepstatin. (Reproduced, with permission, ft Ref. 55. Copyright 1977, Plenum Publishing Corp.)


46


1)

M o d i f i c a t i o n of e s s e n t i a l -OH on s t a t y l r e s i d u e :

+ C H CH SO F _

P e p s t a t i n -OH

—Pepstatin-O-SO^H^^

R p

+ AcSH -C H CH S0 6

5

2

or

3

+ NH. Pepstatin-S-Ac

Pepstatin-SH

or Pepstatin-NH

2)

A c t i v e e s t e r s of e s s e n t i a l -OH group:

A.

0 + OH" II Pepstatin-OH + P0C1 — P e p s t a t i n - O - P - 0 + 3C1 3

8B.

3)

2

Pepstatin-O-P-0 0"

+

+

9 -

+ H

-> P e p s t a t i n OH + HO-j>-0 Ο slow? R

Q

2

D e r i v a t i v e s of n o n - e s s e n t i a l c a r b o x y l group : Pepstatin-COO

+ X

•

Pepstatin-COX ( a c t i v a t e d c a r b o x y l group)

* Pepstatin-COX Figure 7.

+ Z: *

•

Pepstatin-COZ

Possible chemical modifications of pepstatin.


2.

RICHARDSON

Enzymic

Degradation

of

Proteins

47

a c t i v a t e d or otherwise modified to immobilize the i n h i b i t o r or t o couple i t to u n d i g e s t i b l e macromolecules such as carboxymethyl c e l l u l o s e f o r use i n foods. T h i s l a t t e r compound would impart water s o l u b i l i t y to the hydrophobic i n h i b i t o r . Immobilized p e p s t a t i n has been used to i s o l a t e a c i d proteases (58). Pepsin i s known to be i n h i b i t e d by high molar r a t i o s of i t s a c t i v a t i o n peptide (38). When the a c t i v a t i o n peptide i s guanidated, i t s i n h i b i t o r y a c t i v i t y can be more than doubled (Figure 4 ) . The sequence o f t h i s peptide and r e l e v a n t chemical m o d i f i c a t i o n s suggest routes f o r p r e p a r a t i o n of e f f e c t i v e , digestible synthetic inhibitors. T r y p s i n i s i n h i b i t e d by oligomers of homoarginine (n = 10) with a K i approaching 10"" M (53) (Figure 8 ) . T h i s o l i g o m e r i c s u b s t r a t e analog f o r t r y p s i n apparently binds t o the a c t i v e s i t e of t r y p s i n where the geometry f o r r a p i d h y d r o l y s i s i s not adequate. Synthetic peptide i n h i b i t o r s with high a f f i n i t i e s f o r the a c t i v e s i t e of proteases are a l s o p o s s i b l e based on a f f i n i t y l a b e l i n g techniques ( 5 9 ) . There are thus numerous p o s s i b i l i t i e s f o r designing s y n t h e t i c peptide (60) protease i n h i b i t o r s that c o u l d prove u s e f u l i n food systems. Again based on an understanding o f the peptide chemistry o f i n h i b i t o r s , recombinant DNA technology could probably be employed to e v e n t u a l l y produce d e s i r a b l e peptide i n h i b i t o r s u s i n g conventional fermentation technology. Solidphase o l i g o n u c l e o t i d e s y n t h e t i c procedures are p r o g r e s s i n g q u i c k l y and i t i s r a p i d l y becoming p o s s i b l e t o synthesize o l i g o n u c l e o t i d e s , coding f o r a p a r t i c u l a r amino a c i d sequence, f o r i n s e r t i o n i n t o a c l o n i n g v e c t o r (61, 62). 5

Control of Interactions A few, b r i e f examples from the l i t e r a t u r e should be s u f f i c i e n t to i l l u s t r a t e the p o t e n t i a l of d i r e c t l y managing enzyme-substrate i n t e r a c t i o n s and i n d i r e c t l y c o n t r o l l i n g subsequent product i n t e r a c t i o n s . Since v i r t u a l l y a l l food p r o t e i n s have a net negative charge a t the pH o f most foods, enzymes engineered to be more p o s i t i v e l y or n e g a t i v e l y charged or more hydrophobic may be r e t a i n e d more or l e s s i n a food as d e s i r e d . For example, the amount o f m i l k - c l o t t i n g or l i p o l y t i c enzymes r e t a i n e d i n cheese curd could be governed by charge or f u n c t i o n a l groups on a modified enzyme. I n t e r a c t i o n s of t h i s type might be important i n subsequent r i p e n i n g or aging of the cheese. Holmes e t a l . ( 6 3 ) have demonstrated the d i f f e r e n t i a l r e t e n t i o n of v a r i o u s , p r o t e o l y t i c m i l k - c l o t t i n g enzymes i n cheese curd. M a r s h a l l and Green ( 6 4 ) have s t u d i e d i n some d e t a i l the e f f e c t s of c a t i o n i c substances i n c l u d i n g p r o t e i n s on the rennet c l o t t i n g time o f m i l k . Normal renneting of m i l k i s thought to r e s u l t i n a decreased charge r e p u l s i o n between c a s e i n a t e

American Chemical Society Library 1155 16th t ft W. Cherry; FoodS Protein Deterioration Washington, 0.Chemical C. 20039 ACS Symposium Series; American Society: Washington, DC, 1982.

48

FOOD PROTEIN

Figure 8. Inhibition of trypsin by polyhomoarginine. (Reproduced, with per mission, from Ref. 53. Copyright 1974, Springer-Verlag New York, Inc.)

Π,

DETERIORATION

Residues per molecule


2.

RICHARDSON

Enzymic Degradation

of

Proteins

49

p a r t i c l e s from the enzymic-mediated r e l e a s e of a h y d r o p h i l i c , n e g a t i v e l y charged p e p t i d e . The decreased charge r e p u l s i o n favors c o a g u l a t i o n of the modified caseinate p a r t i c l e s to y i e l d curd. M a r s h a l l and Green (64) observed that as i n c r e a s i n g amounts of c a t i o n i c m a t e r i a l s were added to the milk to pro g r e s s i v e l y n e u t r a l i z e the net negative charge on the c a s e i n m i c e l l e s , the r a t e of enzymic-mediated c o a g u l a t i o n of the c a s e i n increased with the reduced net negative charge i n the system. Many o f these observations have been v e r i f i e d and extended by DiGregorio and S i s t o (33, 34) and by M a t t a r e l l a (35), who s t u d i e d the i n t e r a c t i o n s of p o s i t i v e l y charged β-lactoglobu l i n d e r i v a t i v e s with c a s e i n a t e systems. This has r e s u l t e d i n the use of p o s i t i v e l y charged p r o t e i n s as e l e c t r o s t a t i c coagulants of milk which d r a m a t i c a l l y increased the y i e l d of p r o t e i n s i n cheesemaking ( 3 3 , 34). However, i t i s not known what the e f f e c t s would be on the q u a l i t y of r e s u l t a n t cheese. Kang and Kepplinger (65) showed that a d d i t i o n of a c a t i o n i c peptide, p a l m i t o y l - L - l y s y l - L - l y s i n e e t h y l e s t e r , to a pepsin s o l u t i o n before a d d i t i o n t o milk i n h i b i t e d the c l o t t i n g a c t i v i t y of pepsin. On the other hand, when the peptide was added f i r s t to the milk, an enhanced r a t e o f c l o t t i n g was observed. This i s an extreme example of how p r o t e o l y s i s and subsequent product i n t e r a c t i o n s might be r e g u l a t e d e l e c t r o s t a t i c a l l y . C o n t r o l l i n g the p a t t e r n of p r o t e o l y s i s i n p r o t e i n hydrolysates by r e l y i n g on s t e r i c or charge e f f e c t s between p r o t e i n and an immobilized protease i s a d i s t i n c t p o s s i b i l i t y . By analogy, the p a t t e r n s of s t a r c h h y d r o l y s i s are s u b s t a n t i a l l y d i f f e r e n t depending upon whether a s o l u b l e o r immobilized aamylase i s used (66). Conclusions C o n t r o l of endogenous and exogenous enzymic a c t i v i t y i n foods must be extended beyond c u r r e n t , conventional methods t o r e a l i z e advances i n food p r o c e s s i n g and storage. Suggestions i n t h i s review f o r manipulating food enzymes are based on accepted p h y s i c a l , chemical and b i o l o g i c a l p r i n c i p l e s . Among p o s s i b i l i t i e s discussed are: (1) management of enzymic l a t e n c y i n s i t u , ex v i v o and i n v i v o ; ( 2 ) engineering of enzymes using chemical and b i o l o g i c a l techniques; (3) c o n t r o l of enzymes using n a t u r a l and s y n t h e t i c i n h i b i t o r s : and (4) a l t e r a t i o n of e l e c t r o s t a t i c and hydrophobic f o r c e s on exogenous enzymes and w i t h i n foods that may r e g u l a t e enzymic-substrate and post-enzymic product i n t e r a c t i o n s . Although p r o t e o l y t i c enzymes are s p e c i f i c a l l y d i s c u s s e d , many of the concepts can be g e n e r a l i z e d to other f o o d - r e l a t e d enzymes. Future food s c i e n t i s t s may thus be able to exert b e t t e r c o n t r o l over exogenous and a d v e n t i t i o u s enzymes i n foods by designing and engineering the enzymes as w e l l as by using acceptable enzymic i n h i b i t o r s . Powerful techniques such as


50

FOOD PROTEIN

DETERIORATION

those used f o r chemical m o d i f i c a t i o n of enzymes and i n h i b i t o r s , and s o l i d phase s y n t h e s i s of polypeptides and o l i g o n u c l e o t i d e s coupled w i t h emerging recombinant DNA technology can be brought t o b e a r t o e v e n t u a l l y r e a l i z e b e t t e r c o n t r o l of enzymic r e a c t i o n s i n f o o d p r o c e s s i n g a n d i n food d e t e r i o r a t i o n * Regulation o f endogenous enzymes i n foods i s o b v i o u s l y more d i f f i c u l t a n d must a w a i t a b e t t e r understanding of the physiology o f p o s t - h a r v e s t a n d post-mortem processes. Acknowledgments T h i s c o n t r i b u t i o n was made p o s s i b l e b y support from the Walter ?. P r i c e Cheese Research I n s t i t u t e a n d f r o m the College of A g r i c u l t u r a l a n d L i f e Sciences, U n i v e r s i t y o f Wisconsin-Madison, Madison, WI 53706.

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RECEIVED August 30, 1982.


Food Protein Deterioration - American Chemical Society

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