Oilseed Enzymes as Biological Indicators for Food Uses and

12 Oilseed Enzymes as Biological Indicators for Food Uses and Applications JOHN P. CHERRY

Downloaded by UNIV LAVAL on July 13, 2016 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0047.ch012

Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, New Orleans, La. 70179

Research u t i l i z i n g enzymes as indicators of physiological and biochemical changes in l i v i n g organisms is not new (25,38). Enzyme synthesis is genetically controlled, however, their specific task(s) and feedback control mechanisms are also influenced by other constituents in the cellular environment; i.e., cofactors, substrates and products associated with metabolic processes. Scientists have developed the technology to p a r t i a l l y imitate in vivo conditions i n order to study the active enzymes extracted from their natural environment. Nevertheless, enzymes do falter when abused, becoming excellent indicators of several types of cellular change. Gel electrophoresis is a method developed to qualitatively study enzymes. The background and theory of electrophoretic techniques were discussed by Ornstein (46), and applications of these procedures to analyze and compare proteins and enzymes have been presented by a number of investigators (6,8,24,29,48,55,57). Basically, the method applies an electric charge to separate aqueous extracts of proteins i n a gel matrix, such as polyacrylamide or starch. Protein mobility depends upon a combination of factors, including net charge, molecular size, and conformation. Histochemical staining procedures have been developed to detect the location of enzymes i n gels based on their catalytic activity (39). E l e c t r o p h o r e t i c a l l y detected enzyme changes have been used to c h a r a c t e r i z e Aspergillus-peanut i n t e r r e l a t i o n s h i p s ; in the fungus, development and d i f f e r e n t i a t i o n occurs w h i l e the peanuts undergo senescence (14,18). E l e c t r o p h o r e t i c zymograms of enzymes e x t r a c t e d from various plant and animal t i s s u e s were used in chemotaxonomic studies o f genetic s p e c i a t i o n and genera taxonomy (21,38). These s t u d i e s showed that a great amount of g e n e t i c a l l y c o n t r o l l e d molecular d i v e r s i t y , or enzyme multiplicity, e x i s t s in nature as isozymes; i . e . , g e n e t i c a l l y r e l a t e d enzymes with s i m i l a r substrate specificities but d i f f e r e n t e l e c t r o p h o r e t i c m o b i l i t i e s . Zymograms can a l s o vary because of t i s s u e ontogeny. These reports suggest that, in all types of e l e c t r o p h o r e t i c s t u d i e s , care should be taken to i n s u r e that standard known zymograms are developed f o r

209

Ory and St. Angelo; Enzymes in Food and Beverage Processing ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

E N Z Y M E S IN FOOD AND

210

the m a t e r i a l s effects.

B E V E R A G E PROCESSING

to be examined p r i o r to any e v a l u a t i o n of treatment


Complexity of I d e n t i f y i n g Isozymes as M u l t i p l e Enzyme Forms Hunter and Markert (32) revealed a vast m u l t i p l i c i t y of e l e c t r o p h o r e t i c a l l y d i s t i n c t bands w i t h enzyme a c t i v i t y . The term "isozyme" was proposed by Markert and M o l l e r (42) to r e f e r to enzymatically a c t i v e p r o t e i n s that are separated e l e c t r o p h o r e t i c a l l y and that c a t a l y z e the same biochemical r e a c t i o n . Markert (39) l a t e r proposed modifying the word isozyme with terms, such as a l l e l i c , n o n a l l e l i c , homopolymeric, conformational, h y b r i d , and conjugated (words which deal w i t h genetic and chemical terminology) . The p r i n c i p a l types of molecular m u l t i p l i c i t y that generate isozyme patterns were reviewed by Markert and Whitt (40). I t i s t h e o r i z e d that enzyme m u l t i p l i c i t y i s created at the molecu l a r l e v e l by various combinations of the f o l l o w i n g : (a) d i f f e r e n t polypeptides coded by a l l e l i c and n o n a l l e l i c genes; (b) polymers of v a r i o u s s i z e s ; (c) homo- and heteropolymers; (d) polypeptides s e c o n d a r i l y modified i n various ways; and (e) d i f f e r e n t conformations produced by permutations of polymer subunits, or a l t e r n a t e t e r t i a r y and quaternary c o n f i g u r a t i o n s of p r o t e i n s . Organisms may u t i l i z e both genetic and nongenetic mechanisms to form prope r t i e s of enzymes necessary to f i t s p e c i a l metabolic requirements. Shaw (54) c l a s s i f i e d isozymes i n t o primary types, which i n c l u d e d i s t i n c t molecular e n t i t i e s produced from d i f f e r e n t gen e t i c s i t e s , and secondary types, which r e s u l t from a s i g n i f i c a n t a l t e r a t i o n i n the s t r u c t u r e of a s i n g l e polypeptide. Scandalios (52) has employed the term isozyme to define the heterozygous s t a t e of g e n e t i c a l l y v a r i a n t enzymes. He showed a m u l t i p l i c i t y of a l l e l e s (genes) c a r r y i n g information f o r a s p e c i f i c type of enzyme a c t i v i t y . Crosses between s e v e r a l l i n e s of maize with d i f f e r e n t c a t a l a s e phenotypes revealed s i x a l l e l e s , whereas l e u c i n e aminopeptidase isozymes were c o n t r o l l e d by two separate genetic l o c i . I t was f u r t h e r noted that d i f f e r e n t isozymes were present i n d i f f e r e n t t i s s u e s and i n e x t r a c t s of t i s s u e s at d i f f e r ent stages of development. In 1971, the IUPAC-IUB Commission on Biochemical Nomenc l a t u r e (34) recommended that m u l t i p l e forms of an enzyme poss e s s i n g the same a c t i v i t y , separated and d i s t i n g u i s h e d by s u i t a b l e methods ( p r e f e r a b l y by e l e c t r o p h o r e t i c techniques but also by chromatography, k i n e t i c c r i t e r i a , chemical s t r u c t u r e , etc.) and o c c u r r i n g n a t u r a l l y i n a s i n g l e s p e c i e s , should be g e n e r a l l y defined as " m u l t i p l e forms of the enzyme...". M u l t i p l e forms of enzymes that have been s p e c i f i c a l l y c h a r a c t e r i z e d by genetic d i f f e r e n c e s i n primary s t r u c t u r e should be defined as isoenzyme or isozyme. These include g e n e t i c a l l y independent p r o t e i n s , heteropolymers (hybrids) of two or more noncovalently bound polypeptide chains, and genetic v a r i a n t s ( a l l e l i c ) . Enzymes not f a l l i n g i n t o t h i s l a t t e r category i n c l u d e p r o t e i n s conjugated with other groups,


12.

CHERRY

Oilseed Enzymes as Biological Indicators

211

p r o t e i n s derived from one polypeptide chain, polymers of a s i n g l e subunit, and conformationally d i f f e r e n t forms.


Detecting M u l t i p l e Forms of Enzymes Esterases I n v e s t i g a t i o n s were completed to standardize techniques ( p r o t e i n and enzyme e x t r a c t i o n , p u r i f i c a t i o n , and g e l e l e c t r o p h o r e s i s ) used to examine enzymes (2,3,30,31,41). T h i s provided reproducible q u a l i t a t i v e data f o r comparative purposes; i . e . , u n i f o r m i t y i n i n t e n s i t y of g e l s t a i n i n g and repeatable s p a t i a l arrangement of enzyme bands between experiments. This a l s o e s t a b l i s h e d e l e c t r o p h o r e t i c patterns which serve as stand ards to which enzymes from damaged, processed, or mold-infected substances might be compared. A t y p i c a l example of on-gel e l e c t r o p h o r e t i c c h a r a c t e r i z a t i o n of enzyme a c t i v i t y was shown i n r a b b i t e r y t h r o c y t e s ( F i g . 1; 13), using polyacrylamide g e l e l e c t r o p h o r e s i s . This example revealed a complex array of n o n s p e c i f i c esterases i n samples of hypotonic e x t r a c t s and f r a c t i o n s . Although the r a b b i t s were considered to be i s o g e n i c , some genetic v a r i a b i l i t y i n esterase bands was noted ( c f . , region 1.5-2.5 cm of gels A and Β i n both membrane and cyto plasmic e x t r a c t s ) . N o n s p e c i f i c esterases were detected by i n c u b a t i n g gels i n a mixture of a- and β-naphthyl acetate. By using on-gel techniques, the esterases were c h a r a c t e r i z e d on the b a s i s of t h e i r s u b s t r a t e s p e c i f i c i t i e s and s u s c e p t i b i l i t i e s to i n h i b itors. Group I esterases were i n h i b i t e d by diisopropylphosphorof l u o r i d a t e (DFP), phenylmethanesulfonyl f l u o r i d a t e (PMSF) and e s e r i n e , but not by mercuric c h l o r i d e or p-chloromercuribenzenes u l f o n i c a c i d (CMBSA) ( F i g . 1). These esterases showed s p e c i i c i t y toward a c e t y l t h i o c h o l i n e i o d i d e and thiophenyl acetate and thus appeared to be composed of a c e t y l c h o l i n e s t e r a s e s . Group I I ( a c e t y l e s t e r a s e s ) c o n s i s t e d of enzymes that hydrolyzed e s t e r s of a c e t i c a c i d and β-naphthyl l a u r a t e and thiophenyl acetate, and was not i n h i b i t e d markedly by any of the i n h i b i t o r s t e s t e d . The enzymes i n Groups I I I a,b, and c, IV and VI, c o n s i s t i n g of slow, intermediate, and r a p i d migrating carboxylesterases, r e s p e c t i v e l y , were h i g h l y a c t i v e toward α-naphthyl butyrate, and were i n h i b i t e d p a r t i a l l y or completely by DFP and PMSF but not by mercurials or t r i - o - t o l y l phosphate. Only Groups I and VI were i n h i b i t e d by e s e r i n e . Groups V and VII showed s p e c i f i c i t y t y p i c a l of a r y l e s t e r a s e s , being p a r t i a l l y i n h i b i t e d by DFP, PMSF, and the m e r c u r i a l reagents. These data revealed that the n o n s p e c i f i c esterases i n hypo t o n i c cytoplasmic or membrane f r a c t i o n s from erythrocytes which could be detected using n o n s p e c i f i c substrates were r e a l l y heteromorphic ( s p e c i f i c e s t e r a s e s ) . Some of the d e f i n i t i o n s f o r d e s c r i b i n g the term isozyme may be a p p l i e d to these m u l t i p l e forms of esterases detected i n e r y t h r o c y t e s . However, u n t i l each of the e l e c t r o p h o r e t i c bands of esterase a c t i v i t y i s r e l a t e d to a s p e c i f i c gene(s) by breeding s t u d i e s , a true d e f i n i t i o n of isozyme cannot be used to describe them.



212

E N Z Y M E S IN FOOD A N D B E V E R A G E PROCESSING

Figure 1. Polyaery lamide gel electrophoretic patterns of nonspecific esterase activity in membrane and cytoplasmic fractions of rabbit erythrocytes. Methods for preparation of erythrocyte fractions are described by Cherry and Prescott (12). A composite shows the classes of esterase activity (I: acetylcholinesterases; II: acetylesterases; III a, b, and c, IV and VI: carboxylesterases and; V and VII: arylesterases. The bands in region 3.0S.5 cm of gels of the cytoplasmic fraction are hemoglobin.

Figure 2. Polyacrylamide gel electrophoretic patterns of peroxidases in peanut cotyledons at various developing and germinating stages. Gel patterns of the cultivar Spancross are presented as an example of typical standard gels.


12.

CHERRY


213


A p p l i c a t i o n of Enzymes as B i o l o g i c a l I n d i c a t o r s Peroxidases Peroxidase i s probably the most widely used enzyme as a biochemical i n d i c a t o r of d i s e a s e , c e l l u l a r i n j u r y , trauma damage, i n f e c t i o n , e t c . , i n p l a n t s (4,7,16,23,36,37,47, 51,53). In peanuts, most peroxidase a c t i v i t y has been l o c a t e d i n the albumin f r a c t i o n of the p r o t e i n s (17). Peroxidase has been employed with peanuts as an index of b l a n c h i n g temperatures i n food q u a l i t y c o n t r o l and, with c e r t a i n other enzymes, has been i m p l i c a t e d i n the development of o f f - f l a v o r s during storage (1,28,59). Since most peroxidases seem to be l o c a l i z e d i n a s p e c i f i c storage p r o t e i n f r a c t i o n of peanuts, a n a l y s i s of t h i s f r a c t i o n may be a u s e f u l t o o l i n q u a l i t y c o n t r o l during prepara t i o n of p r o t e i n i s o l a t e s or concentrates. A l t e r n a t i v e l y , the removal of the albumin f r a c t i o n during i s o l a t e p r e p a r a t i o n may prevent or r e t a r d the production of o f f - f l a v o r s caused by peroxidase a c t i v i t y . Examination of many developing Spancross peanuts from i n d i v i d u a l p l a n t s grown i n Georgia showed a decrease i n peroxidase a c t i v i t y as seeds matured ( F i g . 2). Zymograms c o n t a i n i n g one band at the o r i g i n , one i n r e g i o n 0.7 cm and three i n r e g i o n 3.0-4.0 cm, were t y p i c a l of most p a t t e r n s of mature seeds. A peroxidase band i n r e g i o n 5.0 cm d i s t i n g u i s h e d immature and low intermediate seeds from high intermediate and mature peanuts. The presence of one major band i n r e g i o n 3.0-4.0 cm, the absence of isozymes at the o r i g i n and/or i n r e g i o n 0.7 cm, and the presence (or absence) of a c t i v i t y i n regions 1.0-2.7 and 4.0-5.0 cm d i s t i n g u i s h e d zymograms of mature seeds. Germinating peanuts showed i n c r e a s e d peroxidase a c t i v i t y i n e l e c t r o p h o r e t i c g e l s a f t e r 2 to 4 days ( F i g . 2) that became e s p e c i a l l y p r e v a l e n t i n r e g i o n 0-3.0 cm between 8 and 18 days germination. An i n c r e a s e i n peroxidase a c t i v i t y was noted i n r e g i o n 3.0-4.0 cm at days 6 and 8, decreasing slowly t h e r e a f t e r to 20 days germination. Esterases Esterases are popular i n e l e c t r o p h o r e t i c s t u d i e s of isoenzymes i n higher p l a n t s (8,10,15,19-21,23,43,44,52). Esterases from peanuts at immature and low intermediate stages of development showed much banding v a r i a t i o n that d i f f e r e d f o r Spancross and Florunner c u l t i v a r s ( F i g . 3). A f t e r the low intermediate stage of peanut development, esterase a c t i v i t y on e l e c t r o p h o r e t i c gels increased i n regions 3.0-4.0 and 5.0-7.5 cm. The two c u l t i v a r s could be d i s t i n g u i s h e d by e s t e r a s e bands i n region 4.0-5.0 cm. E x t r a c t s from overmature seeds of Florunner showed decreased esterase a c t i v i t y on e l e c t r o p h o r e t i c g e l s compared to those of mature seeds; no changes were observed i n overmature seeds of Spancross. I n t r a v a r i e t a l a n a l y s i s of these peanut c u l t i v a r s y i e l d e d both q u a l i t a t i v e and q u a n t i t a t i v e e s t e r a s e v a r i a t i o n s that were l i m i t e d p r i m a r i l y to regions 4.0-5.0 cm of the zymograms. V a r i a t i o n s w i t h i n and between various other



214

E N Z Y M E S IN FOOD A N D B E V E R A G E

PROCESSING

Figure 3. Typical polyacrylamide gel patterns of esterases in peanut cotyledons of Spancross and Florunner cultivars at various developing stages

Figure 4. Polyacryhmide gels of esterase activity in Florunner peanut cotyledons germinating for various times from 0-20 days



12.

CHERRY


215

peanut c u l t i v a r s were l i m i t e d to q u a n t i t a t i v e d i f f e r e n c e s i n region 4.4 cm. These s t u d i e s suggest that esterase a c t i v i t y i n peanuts may be u s e f u l i n i d e n t i f y i n g p a r t i c u l a r stages of peanut development to maturity, as w e l l as f o r d i s t i n g u i s h i n g Florunner and Spancross c u l t i v a r s . Both q u a l i t a t i v e and q u a n t i t a t i v e band v a r i a t i o n s appeared i n r e g i o n 2.0-7.0 cm s h o r t l y a f t e r Florunner seeds were germinated ( F i g . 4). These changes i n c r e a s e d markedly a f t e r 4 days, c l e a r l y d i s t i n g u i s h i n g e a r l y germinated cotyledons from those developing between 6 and 20 days. I n t e n s i t y of e s t e r a s e a c t i v i t y i n regions 2.0-2.5 and 4.5-5.0 cm of seeds germinated beyond 6 to 8 days increased q u a n t i t a t i v e l y . E s t e r a s e bands of mature seeds present i n other regions of the g e l , decreased between 2 and 8 days germination. Leucine Aminopeptidases Nearly a l l e l e c t r o p h o r e t i c d e t e c t i o n of v a r i a n t peptidases has i n v o l v e d the use of s y n t h e t i c s u b s t r a t e s such as aminoacylnaphthylamides, e s p e c i a l l y leucyl-3-naphthylamide. As w i t h the r e a c t i o n media used f o r e s t e r a s e s , t h i s s y n t h e t i c subs t r a t e i s not s p e c i f i c only f o r c e r t a i n peptidases. Despite the vagueness concerning s u b s t r a t e s p e c i f i c i t y , the l e u c i n e aminopeptidases are very u s e f u l enzymes i n e l e c t r o p h o r e t i c s t u d i e s of p r o t e i n polymorphism, p l a n t h y b r i d i z a t i o n , and polypeptide hydrol y s i s (19,21,22,38,58). Zymograms of l e u c i n e aminopeptidase a c t i v i t y d i s t i n g u i s h e d high intermediate, mature, and overmature peanuts of Spancross and Florunner c u l t i v a r s ( F i g . 5 ) . C u l t i v a r s and stages of seed development were s i m i l a r l y d i s t i n g u i s h e d by l e u c i n e aminopeptidases, as was shown with e s t e r a s e s . Changes i n l e u c i n e aminopeptidase a c t i v i t y i n Florunner cotyledonary e x t r a c t s were examined on gels during extended seed germination up to 20 days ( F i g . 6 ) . The peptidase a c t i v i t y i n these p r e p a r a t i o n s remained c o n s i s t e n t l y high d u r i n g e a r l y stages of germination to day 6, a f t e r which i t decreased. By day 12 most of i t could not be detected. T h i s o b s e r v a t i o n suggested that peptidase a c t i v i t y may be p a r t i a l l y r e s p o n s i b l e f o r the reserve p r o t e i n breakdown during e a r l y stages of germination. I n t e r e s t i n g l y , the fast-moving bands (region 5.0-6.0 cm) showed very l i t t l e a c t i v i t y up to 8 days of germination. However, a f t e r t h i s time, most of the remaining a c t i v i t y was a s s o c i a t e d with these bands, suggesting that they may have been a c t i v a t e d or synthesized de novo during t h i s i n t e r v a l . The breakdown of cotyledonary p r o t e i n s was i n v e s t i g a t e d q u a l i t a t i v e l y by g e l e l e c t r o p h o r e s i s ( F i g . 7). P r o t e i n e x t r a c t s from ungerminated seeds (0 day) c o n s i s t e d mainly of the monomeric (1.5 cm) and dimeric (1.0 cm) components of the major storage g l o b u l i n of peanuts, a r a c h i n . Except f o r i n t e n s i f i c a t i o n of two bands i n r e g i o n 0-0.5 cm, no major changes were observed during the f i r s t 2 days of germination. Major changes i n p r o t e i n comp o s i t i o n became evident at and a f t e r day 4. The a r a c h i n components and the bands i n r e g i o n 2.0-3.5 cm decreased q u a n t i t a t i v e l y



216

ENZYMES

IN FOOD

AND BEVERAGE

PROCESSING

Figure 5. Polyacrylamide gel patterns of leucine aminopeptidase activity in developing peanut cotyledons of Spancross and Florunner cultivars

Figure 6. Polyacrylamide gels of leucine aminopeptidases in germinating Florunner peanut cotyledons


12.

CHERRY


217

i n the g e l p a t t e r n s , and simultaneously there appeared numerous p r o t e i n s i n region 2.5-7.0 cm. The appearance of small p r o t e i n components i n the g e l patterns i n d i c a t e s h y d r o l y s i s of the major storage p r o t e i n s of peanuts by proteases and peptidases to polypeptides of various s i z e s and to f r e e amino a c i d s , p r i o r to t h e i r t r a n s p o r t to the embryo. A f t e r day 14, no f u r t h e r major changes were noted i n the few remaining p r o t e i n s detectable by g e l e l e c t r o phoresis. This c o i n c i d e d w i t h the observation that a c o n s i s t e n t l y low quantity of p r o t e i n and peptidase a c t i v i t y remained i n the cotyledons between days 12 and 20 a f t e r germination began.


Fungal-Caused Seed D e t e r i o r a t i o n Studies have shown that standard enzyme e l e c t r o p h o r e t i c patterns of peanuts are d i s t i n c t l y modified by i n f e c t i o n with various A s p e r g i l l u s species (14,18). Biochemical transformations i n c l u d e d e l e t i o n of some enzymes, i n t e n s i f i c a t i o n of others, and/or production of new bands. These changes i n g e l patterns of peanut enzymes i n d i c a t e that the biochemical mechanisms operative i n the saphrophyte-seed i n t e r r e l a t i o n s h i p f u n c t i o n very e f f i c i e n t l y and s y s t e m a t i c a l l y f o r growth of the fungus at the expense of the seed. This i s based on the observation that i n a d d i t i o n to enzyme changes i n the i n t e r r e l a t i o n s h i p , decomposition of storage g l o b u l i n s of l a r g e molecular weight to small p r o t e i n components and f r e e amino acids occurs i n i n f e c t e d peanuts (9,11,14). The type of enzyme systems included i n studies suggested a degradation of storage p r o t e i n peptides by l e u c i n e aminopeptidases, h y d r o l y s i s of many e s t e r linkages by e s t e r a s e s , hormonal i n t e r a c t i o n , and/or o x i d a t i o n of organic substances w i t h peroxides by peroxidases and oxidases, and decomposition of p o t e n t i a l l y t o x i c hydrogen peroxide by c a t a l a s e s . Many of the peanut enzymes remained a c t i v e during A s p e r g i l l u s invasion. In a number of cases, i n t e n s i f i c a t i o n of a c t i v i t i e s of some enzymes and the production of new bands were c o r r e l a t e d w i t h enzyme bands i n m y c e l i a l t i s s u e of the fungus c o l l e c t e d from the seed s u r f a c e . Q u a n t i t a t i v e and q u a l i t a t i v e v a r i a t i o n s i n g e l patterns of c e r t a i n enzymes a l s o d i s t i n g u i s h e d between fungal t i s s u e from the e x t e r i o r surface of peanuts and that grown on a s y n t h e t i c medium, Czapek's s o l u t i o n (14,18). These v a r i a t i o n s i n banding patterns of t i s s u e s c o l l e c t e d from two separate sources may p o s s i b l y be r e l a t e d to d i f f e r e n c e s i n n u t r i t i o n a l needs of the fungus grown under the two c o n d i t i o n s . Phosphogluconate Dehydrogenase, A l c o h o l Dehydrogenase and A l k a l i n e Phosphatase Examples of enzyme changes i n peanuts i n f e c t e d f o r 4 days with various s t r a i n s of A s p e r g i l l u s f l a v u s capable of producing no, intermediate, and high l e v e l s of a f l a t o x i n are shown i n Figure 8. In general, phosphogluconate and a l c o h o l dehydrogenase banding a c t i v i t i e s detected on s t a r c h e l e c t r o p h o r e t i c gels become d i f f i c u l t to d i s c e r n i n e x t r a c t s of



218

ENZYMES IN FOOD AND BEVERAGE PROCESSING

Figure 7. Polyacrylamide gel electrophoretic patterns of proteins in extracts from germinated Florunner peanut cotyledons

Figure 8. Starch gel electrophoretic patterns of phosphogluconate dehydrogenase, alcohol dehydrogenase, and alkaline phosphatase activity of noninfected and A. flavusinfected peanuts after four days. Controls 1 and 2 represent peanuts at day zero and after incubation for four days at 29°C in a high humidity chamber, respectively. A. flavus strains from the Northern Regional Research Center, USDA-ARS, Peoria, III, were labeled as follows: 1. NRRL 5520, 2. NRRL 5518, 3. A-14152, 4. NRRL 8517, 5. A-62462, 6. NRRL 3353, 7. A-4018b, 8. NRRL 3251, and 9. A-13838.



12.

CHERRY


219

peanuts i n f e c t e d w i t h f l a v u s . Small amounts of a c t i v i t y were observed only i n c e r t a i n n o n a f l a t o x i n producing s t r a i n s of A. f l a v u s . A phosphogluconate dehydrogenase band i n region 3.5 cm s p e c i f i c a l l y d i s t i n g u i s h e d peanuts i n f e c t e d with n o n a f l a t o x i n producing A. f l a v u s s t r a i n 1 from a l l other contaminants. In c o n t r a s t , a l k a l i n e phosphatase was detected only i n A. f l a v u s - i n f e c t e d peanuts. Two bands of s i m i l a r a c t i v i t y were noted i n a l l contaminated peanuts, regardless of a f l a t o x i n l e v e l s . Since a l k a l i n e phosphatase i s not normally present i n peanuts, t h i s enzyme could be used as an i n d i c a t o r of A^ f l a v u s contami n a t i o n i n peanut p r o t e i n p r e p a r a t i o n s . T h i s would be e s p e c i a l l y suggestive of compositional changes i n p r o t e i n s or other storage c o n s t i t u e n t s and p o s s i b l e a f l a t o x i n contamination of peanut products that could a f f e c t t h e i r f u n c t i o n a l and n u t r i t i o n a l p r o p e r t i e s and t h e i r use i n food products. Preliminary tests for a l k a l i n e phosphatase a c t i v i t y i n peanut products p r i o r to t h e i r u t i l i z a t i o n as food i n g r e d i e n t s could be i n c l u d e d as p a r t of q u a l i t y c o n t r o l measures normally used by food processors. Esterases Region 3.0-7.0 cm contained d e t e c t a b l e changes i n esterase patterns a f t e r A. f l a v u s i n f e c t i o n of peanuts, compared with uninoculated or c o n t r o l seeds ( F i g . 9 ) . Four days a f t e r i n f e c t i o n , esterase a c t i v i t y increased both q u a n t i t a t i v e l y and q u a l i t a t i v e l y i n regions 4.0-5.0 and 6.5-7.5 cm. Enzyme bands of most i n f e c t e d seeds i n regions 3.0-4.0 and 5.0-6.0 cm decreased or were not r e a d i l y detected i n the g e l p a t t e r n s . E a r l i e r s t u d i e s with A^ p a r a s i t i c u s showed s i m i l a r i n t e n s i f i c a t i o n of esterase a c t i v i t y (14,18). These changes with A. f l a v u s were s i m i l a r to those of fungal t i s s u e c o l l e c t e d from the e x t e r i o r s u r f a c e of peanuts i n the e a r l i e r work. Some of the esterase p a t t e r n s d i s t i n g u i s h e d A. f l a v u s s t r a i n s producing no, intermediate, or high l e v e l s of a f l a t o x i n s . Leucine aminopeptidase A n a l y s i s f o r l e u c i n e aminopeptidase a c t i v i t y i n polyacrylamide gels c o n t a i n i n g e x t r a c t s from uninoculated peanut t i s s u e s y i e l d e d f i v e bands i n r e g i o n 4.0-7.5 cm ( F i g . 10). Four days a f t e r i n o c u l a t i o n , each of the 9 A. flavus s t r a i n s produced a zymogram that d i f f e r e d from that of the c o n t r o l s and was d i s t i n c t from a l l other f u n g i . The number of bands i n gels of i n f e c t e d peanuts ranged between 6 and 8, compared to 4 and 5 bands i n c o n t r o l g e l s . In a d d i t i o n , band a c t i v i t y was more intense i n many gels of i n f e c t e d seeds than i n those of the c o n t r o l s . As suggested e a r l i e r i n s t u d i e s with developing and germinating peanuts ( F i g s . 5,6,7), changes i n peptidase a c t i v i t y may be i n d i c a t i v e of p r o t e i n h y d r o l y s i s . Gel e l e c t r o p h o r e t i c patterns of p r o t e i n s i n s o l u b l e f r a c t i o n s showed t h a t each A^ f l a v u s s t r a i n caused changes i n these components during the i n f e c t i o n p e r i o d that were d i f f e r e n t from those of the c o n t r o l ( F i g . 11); no p r o t e i n changes were noted i n g e l patterns of noninfected seeds. In general, p r o t e i n patterns of seeds i n f e c t e d by the various f u n g i showed new p r o t e i n components



220

E N Z Y M E S IN FOOD A N D B E V E R A G E

PROCESSING

Figure 9. Polyacryhmide gel electrophoretic patterns of esterase activity in noninfected and A. fiavus-infected peanuts. Gel descriptions are given in Figure 8.

Figure 10. Polyacrylamide gel electrophoretic patterns of leucine aminopeptidase activity in noninfected and A. Ravus-infected peanuts. Gel descriptions are given in Figure 8.


12.

CHERRY


221

i n region 0-1,0 cm p l u s increased m o b i l i t y and poor r e s o l u t i o n of the bands i n region 1.0-2.5 cm as the i n f e c t i o n progressed to day 4. At the same time, bands normally l o c a t e d i n region 2.0-3.5 cm disappeared, and a new group of polypeptides appeared i n r e g i o n 5.5-7.0 cm.


S p e c i a t i o n and Genetic

Crossability

S u b s t a n t i a l evidence has been accumulated suggesting that the genetic base, or gene p o o l , of c u l t i v a t e d p l a n t s does not have the reserve germ plasm needed to r e s i s t many of the new problems brought on by p o l l u t i o n , dwindling water s u p p l i e s , i n s e c t s , and p l a n t pathogens. F o r t u n a t e l y , there are w i l d species of most c u l t i v a t e d p l a n t s c o n t a i n i n g untapped genetic resources f o r f u t u r e breeding programs. Wild species contain o l d sources of germ plasm which can be used to decrease "genetic e r o s i o n " of commercial crops by broadening the genetic base i n c u l t i v a t e d v a r i e t i e s (45). Once the c r o s s a b i l i t y of species w i t h i n a genus i s understood, s t u d i e s can begin to determine "bridge s p e c i e s " f o r the i n t r o d u c t i o n of new genetic information i n t o c u l t i v a t e d v a r i e t i e s (56). Gel e l e c t r o p h o r e s i s of enzymes o f f e r s a biochemical approach to the e v o l u t i o n a r y aspects of p l a n t s p e c i a t i o n . Amino a c i d changes w i t h i n a p r o t e i n caused by genetic mutational changes can r e s u l t i n a l t e r e d migration r a t e s when the p r o t e i n s are compared i n a matrix system of polyacrylamide or s t a r c h g e l . Since numerous e l e c t r o p h o r e t i c analyses have shown that species d i f f e r from one another i n band frequencies, the i n d i v i d u a l i t y of each p l a n t species can u s u a l l y be expressed according to i t s enzyme banding p a t t e r n s (9,10,19-22,52). The genus Gossypium i s comprised of about 30 d i p l o i d and three n a t u r a l a l l o t e t r a p l o i d species (5,27,33,50). The d i p l o i d (2n) p l a n t s were d i v i d e d i n t o s i x groups or genomes l a b e l e d A to F; t e t r a p l o i d (4n) species were described 2(AD)η s i n c e they were hypothesized to have o r i g i n a t e d from a cross between p l a n t s from the A and D genomes. A n a l y s i s of species r e l a t i o n s h i p s w i t h i n Gossypium by polyacrylamide g e l e l e c t r o p h o r e s i s of p r o t e i n s and enzymes i n e x t r a c t s of dormant seeds was reported by Cherry et a l . (19-22). The chemotaxonomic comparisons of p r o t e i n and enzyme patterns f o r species w i t h i n and between genomes supported the present c l a s s i f i c a t i o n of Gossypium and the o r i g i n of the natural allotetraploids. In a d d i t i o n , the esterase s t u d i e s showed that much more v a r i a t i o n i n banding patterns e x i s t e d w i t h i n the w i l d species than i n new c u l t i v a r s . Figure 12 presents an example of species w i t h i n the D genome which have esterase banding patterns that are more s i m i l a r to each other than to members of other genome groups (A-C, E-F). E n d r i z z i (26) showed c y t o l o g i c a l l y that G. lobaturn and £ . aridum were c l o s e l y r e l a t e d . Gossypium h a r k n e s s i i was shown to cross f r e e l y with G. armourianum(35). The highest chiasma frequency



222

E N Z Y M E S IN FOOD A N D B E V E R A G E PROCESSING

Figure 11. Polyacrylamide gel electrophoretic patterns of proteins in noninfected and A. Rawis-infected peanuts. Gel descriptions are given in Figure 8.


12.

CHERRY


223


among the D genome species was produced i n crosses between G. klotzschianum and G^ d a v i d s o n i i (49). These comparisons were f u r t h e r supported by the isozyme patterns of the e s t e r a s e s ; i . e . , the more c l o s e l y r e l a t e d the species were g e n e t i c a l l y , the more s i m i l a r were t h e i r banding p a t t e r n s . On the other hand, G. r a i m o n d i i appeared to produce esterase patterns uniquely d i f f e r e n t from other species of the D genome. Much esterase v a r i a b i l i t y i s observed w i t h i n and between species of the D genome. For example, s i x d i f f e r e n t zymograms (A-F), were observed f o r the G^ t h u r b e r i seeds (268 as compared to 24 f o r a l l other s p e c i e s ) . The presence of s i x esterase g e l patterns i n d i c a t e s that notable genetic v a r i a b i l i t y e x i s t s w i t h i n species at the molecular l e v e l , and i n t r a s p e c i f i c v a r i a b i l i t y increases when many seeds are examined. Of major i n t e r e s t i n t h i s study with G. t h u r b e r i are the esterases l o c a t e d i n a r e l a t i v e l y narrow region of the g e l s , 6.5-9.2 cm. Within t h i s region, d i s t i n c t v a r i a t i o n s (both q u a l i t a t i v e and q u a n t i t a t i v e ) occur i n the bands. A comparison of the r e l a t i v e frequencies of seed expressing a s p e c i f i c p a t t e r n , from each of the four populations analyzed, i s shown i n Table I . In general, zymograms A-D occur much more f r e q u e n t l y i n n a t u r a l populations than do zymograms Ε and F. These data suggest that banding v a r i a t i o n may be expressed both w i t h i n and between popu lations. The o r i g i n of t h i s banding v a r i a t i o n noted f o r G^ t h u r b e r i (and other s p e c i e s ; F i g . 12) may be due to one or more genetic p o s s i b i l i t i e s i n the p l a n t s : (a) isozymic v a r i a n t s of a s p e c i f i c esterase gene i n homozygous or heterozygous p a i r i n g w i t h i t s p a i r e d gene; (b) a number of d i f f e r e n t and s i m i l a r polypeptide subunits i n various combinations derived from c l o s e l y r e l a t e d n o n a l l e l i c genes; (c) enzymes completely unrelated and not from a common source; and (d) a s e l e c t i v e combination i n v o l v i n g a l l of these p o s s i b i l i t i e s . As to which of these a l t e r n a t i v e s i s most l i k e l y , i t can be noted from the data f o r G. t h u r b e r i that the band i n region 8.0 cm i s the only one present i n a l l of the zymograms and i t i s a l s o present i n greater amounts than any of the other esterases of t h i s s p e c i e s . Thus, a l l of the l a t t e r bands may have a r i s e n from t h i s main esterase through mutation w i t h i n a s i n g l e gene ( i n t r a g e n i c v a r i a t i o n ) or through d u p l i c a t i o n of a s i n g l e gene that then underwent mutation ( i n t e r g e n i c v a r i a t i o n ) . H y b r i d i z a t i o n s t u d i e s of pure p l a n t s that produce each of these esterase bands, however, would be needed to t e s t t h i s hypothesis. Much v a r i a b i l i t y of the esterases can a l s o be noted between species of the D genomes. In a number of cases, s p e c i f i c banding patterns occurred with a low frequency i n one species and a h i g h frequency i n another, whereas the reverse s i t u a t i o n could be obtained with other g e l p a t t e r n s . For example, i n G. h a r k n e s s i i , a zymogram with three esterase bands occurs with a h i g h frequency of 67%. Within t h i s s p e c i e s , a zymogram with four esterase bands


224

E N Z Y M E S IN FOOD AND

TABLE I.

Frequency of occurrence


Location (Tucson, A r i z o n a area)


of n o n s p e c i f i c e s t e r a s e p a t t e r n s .

Total Seeds Analyzed

A

Β

Santa C a t a l i n a Mountains

78

0.72

0.00

0.18

0.09

0.03

0.00

Rincon Mountains

72

0.13

0.26

0.08

0.49

0.04

0.00

Santa R i t a Mountains

72

0.81

0.08

0.00

0.11

0.00

0.00

Baboquivari Mountains

45

0.18

0.07

0.36

0.22

0.11

0.07

268

0.49

0.10

0.13

0.22

0.04

0.01

TOTALS

Zymograms

*Zymograms A-F of the n a t u r a l populations of G. t h u r b e r i ( F i g . 12). occurs i n low frequency of 21%. In G. armourianum, the frequencies of these two g e l patterns are reversed (25% versus 50%). Similar comparisons can be made among g e l p a t t e r n s of G^ aridurn, G. lobaturn, G. h a r k n e s s i i and G^ gossypioides. T h i s i n d i c a t e s that d i f f e r e n t combinations of genes coding f o r esterases are p o s s i b l y present w i t h i n the species of t h i s group. The v a r y i n g zymograms may be a r e f l e c t i o n of n a t u r a l s e l e c t i o n pressures operating to produce the most s t a b l e genetic composition f o r each s p e c i e s . These isozyme patterns may a l s o be due to d i f f e r e n t i a l genetic c o n t r o l of seed esterase production and composition i n the p l a n t caused by environmental pressures during a p a r t i c u l a r growing season. A l l o p o l y p l o i d s as Bridge

Species

Examination of enzyme patterns of seeds from a l l o t e t r a p l o i d s formed by genetic breeding s t u d i e s showed that these gels compared c l o s e l y to the a d d i t i v e zymograms of the parent species when the l a t t e r were combined i n a s y n t h e t i c mixture (19,21). T h i s technique can be used to determine p a r e n t a l species of n a t u r a l l y o c c u r r i n g h y b r i d s . Where w i l d h y b r i d species cross with c u l t i v a t e d v a r i e t i e s and form v i a b l e seed, the p a r e n t a l s or bridge species could be determined f o r i n t r o d u c t i o n of v a l u a b l e genetic information i n t o new c u l t i v a r s . An example of such a study i s shown i n Figure 13. Esterases from seeds of a man-made v i a b l e a l l o t e t r a p l o i d 2^20.^), species (AZ 239) were examined by g e l e l e c t r o p h o r e s i s . T h i s a l l o t e t r a -



CHERRY


Figure 12. Polyacrylamide gel electrophoretic patterns of seed esterases from nine species wtihin the D genome of the genus Gossypium. Included are the frequencies of zymograms formed from seeds of each species.

Figure IS. Polyacrylamide gel electrophoretic patterns of seed esterases in a comparison between parentals, their synthetic mixture, and the allotetraploid. Comparisons include G. arboreum var G24 (A genome), G. thurberi (Ό genome), their syn thetic mixture, and their synthetic allotetraploid: 2(A D ) , AZ 239. 2

2

2

1


i

226

E N Z Y M E S I N FOOD AND


p l o i d was formed from a cross between a v a r i e t y o f arboreurn (A^) and t h u r b e r i (D^). The esterases of a s y n t h e t i c mixture of seed e x t r a c t s formed from arboreum and G^_ t h u r b e r i and the i n d i v i d u a l e x t r a c t s o f these p a r e n t a l s were compared with that of the a l l o t e t r a p l o i d AZ 239. I n the s y n t h e t i c mixture, an a d d i t i v e esterase zymogram was formed. The a d d i t i v e zymogram compared c l o s e l y t o the g e l p a t t e r n of AZ 239. This a l l o t e t r a p l o i d has the p o t e n t i a l of s e r v i n g as a bridge species t o cross with other w i l d p l a n t s shown by c l a s s i c a l genetic techniques and g e l e l e c t r o p h o r e s i s to be c l o s e l y r e l a t e d to G^_ arboreum and G. t h u r b e r i .


Conclusions Technology has enabled s c i e n t i s t s to take enzymes out of t h e i r i n v i v o environments to study them i n t e s t tubes. Enzyme a c t i v i t y during i s o l a t i o n , c h a r a c t e r i z a t i o n , and i d e n t i f i c a t i o n procedures i s a b i o l o g i c a l i n d i c a t o r o f these c a t a l y t i c p r o t e i n s . Nevertheless, enzymes do change i n a c t i v i t y and/or are a l t e r e d s t r u c t u r a l l y when abused. Because o f t h i s they can be e x c e l l e n t i n d i c a t o r s of changes r e l a t e d to food p r o c e s s i n g or p h y s i o l o g i c a l mechanisms. S p e c i f i c task(s) performed by enzymes are genet i c a l l y c o n t r o l l e d and can be used to evaluate genetic s p e c i a t i o n and taxonomy to determine bridge species i n breeding s t u d i e s f o r i n t r o d u c i n g genes o f w i l d species i n t o c u l t i v a t e d v a r i e t i e s . Fungi-host i n t e r r e l a t i o n s h i p s can be determined and evaluated, using enzymes to f o l l o w changes i n development and d i f f e r e n t i a t i o n of the former and a r a p i d form of senescence i n the l a t t e r . F u n c t i o n a l molecules such as enzymes should be s t u d i e d with an understanding o f g e n e t i c and ontogenetic v a r i a b i l i t y . A s u f f i c i e n t number of d i f f e r e n t enzymes should be examined to i n s u r e the development of proper standards f o r reaching appropriate conclusions.

LITERATURE CITED 1. 2. 3. 4. 5. 6. 7. 8.

Acker, L., and H. O. Beutler, Fette Seifen Anstrichm., 67:430 (1965). Augustinsson, Κ. B., B. Axenfors, I . Andersson, and H. Ericksson, Biochim. Biophys. Acta 293:424 (1973). Augustinsson, Κ. Β., Ann. N.Y. Acad. S c i . 94:844 (1961). Baptist, J. N., C. R. Shaw, and M. Mandel, J. Bacteriol. 99:180 (1969). Beasley, J. O., Genet. 27:25 (1942). Berry, J. Α . , and R. G. Franke, Amer. J. Bot. 60:976 (1973). Bhatia, C. R . , and J. P. Nilson, Biochem. Genet. 3:207 (1969). Brewbaker, J. L., M. D. Upadhya, Y. Makinen and T. MacDonald. Physiol. Plant. 21:930 (1968).


12.

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. 35. 36.

CHERRY


227

Cherry, J. P., and L. R. Beuchat, Cereal Chem. 53:750 (1976). Cherry, J. P., Peanut S c i . 2:57 (1975). Cherry, J. P., C. T. Young, and L. R. Beuchat, Can. J. Bot. 53:2639 (1975). Cherry, J. P., and J. M. Prescott, Proc. Exp. B i o l . & Med. 147:418 (1974). Cherry, J. P., and J. M. Prescott, (Unpublished data, 1974). Cherry, J. P., R. Y. Mayne, and R. L. Ory, Physiol. P1. Path. 4:425 (1974). Cherry, J. P., and R. L. Ory, Phytochem. 12:283 (1973). Cherry, J. P., and R. L. Ory, Phytochem. 12:1581 (1973). Cherry, J. P., J. M. Dechary, and R. L. Ory, J. Agric. Food Chem. 21:652 (1973). Cherry, J. P., R. L. Ory, and R. Y. Mayne, J. Amer. Peanut Res. Ed. Assoc. 4:32 (1972). Cherry, J. P., F.R.H. Katterman, and J. E . Endrizzi, Theoret. & Appl. Genet. 42:218 (1972). Cherry, J. P., and F. R. H. Katterman, Phytochem. 10:141 (1971). Cherry, J. P., "Comparative Studies of Seed Enzymes of Species of Gossypium by Polyacrylamide and Starch Gel Electrophoresis," Ph.D. Dissertation, University of Arizona, Tucson (1971). Cherry, J. P., F. R. H. Katterman, and J. E . Endrizzi, Evolution 24:431 (1970). Cubadda, R . , A. Bozzini and E . Quattrucci, Theoret. & Appl. Genet. 45:290 (1975). Davis, B. J., Ann. N.Y. Acad. S c i . 121:404 (1964). Dixon, M., and E . C. Webb, "Enzymes," Academic Press Inc., New York (1964). Endrizzi, J. E., J. Hered. 48:221 (1957). F r y x e l l , P. Α . , Adv. Front. Plant S c i . 10:31 (1965). Gardner, H. W., G. E . Inglett, and R. A. Anderson, Cereal Chem. 46:626 (1969). Gottlieb, L. D . , BioSci. 21:939 (1971). Holmes, R. S., and C. J. Masters, Biochim. Biophys. Acta 151:147 (1968). Holmes, R. S., and C. J. Masters, Biochim. Biophys. Acta 132:379 (1967). Hunter, R. L., and C. L. Markert, Science 125:1294 (1957). Hutchinson, J. Β., R. A. Silow, and S. G. Stephens, "The Evolution of Gossypium and the Differentiation of the Cultivated Cottons," Oxford University Press, London (1947). IUPAC-IUB Commission on Biochemical Nomenclature, J. B i o l . Chem. 246:6127 (1971). Kearny, Τ. Η . , Leaflets Western Bot. 8:103 (1957). Kruger, J. E., and D. E . LaBerge, Cereal Chem. 51:578 (1974).


228

37. 38.

39. 40. 41. 42.


43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59.

E N Z Y M E S I N FOOD A N D B E V E R A G E PROCESSING

LaBerge, D. E., J. E . Kruger, and W.O.S. Meredith, Can. J. Plant S c i . 53:705 (1973). Manwell, C . , and C. M. A. Baker, "Molecular Biology and the Origin of Species: Heterosis, Protein Polymorphism and Animal Breeding, University of Washington Press, Seattle, Washington (1970). Markert, C. L., Ann. N.Y. Acad. S c i . 151:14 (1968). Markert, C. L., and G. S. Whitt, Experientia 24:977 (1968). Markert, C. L., and R. L . Hunter, Histochem. Cytochem. 7:42 (1959). Markert, C. L., and F. Moller, Proc. Natl. Acad. S c i . 45:753 (1959). Marshall, D. R . , and R. W. A l l a r d , J. Hered. 60:17 (1969). Menke, J. F., R. S. Singh, C. O. Qualset, and S. K. Jain, C a l i f . Agric. 27:3 (1973). M i l l e r , J., Science 182:1231 (1973). Ornstein, L., Ann. Ν. Y. Acad. S c i . 121:321 (1964). Pawar, V. S., and V. K. Gupta, Ann. Bot. 39:777 (1975). Payne, W. J., J. W. Fitzgerald, and K. S. Dodgson, Appl. Microbiol. 27:154 (1974). P h i l l i p s , L . L., Amer. J. Bot. 53:328 (1966). P h i l l i p s , L . L., Evolution 17:460 (1963). Reddy, Μ. Μ., and S. F. H. Threlkeld, Can. J. Genet. Cytol. 13:298 (1971). Scandalios, J. G . , Biochem. Genet. 3:37 (1969). Schipper, A. L., Phytopath. 65:440 (1975). Shaw, C. R . , Intern. Rev. Cytol. 25:297 (1969). Shaw, C. R . , Science 149:936 (1965). Simpson, C. E., and R. L . Haney, Texas Agric. Prog. 19:10 (1973). Smithies, O., Adv. Prot. Chem. 14:65 (1959). Sopanen, T . , and J. Mikola, Plant Physiol. 55:809 (1975). Wagenknecht, A. C., Food Res. 24:539 (1959).


Oilseed Enzymes as Biological Indicators for Food Uses and

Recommend Documents