Enzymatic Modification of Soy Proteins To Improve Their Functional

Chapter 14

Downloaded by UNIV OF NORTH CAROLINA on November 4, 2014 | http://pubs.acs.org Publication Date: May 11, 1993 | doi: 10.1021/bk-1993-0528.ch014

Enzymatic Modification of Soy Proteins To Improve Their Functional Properties for Food Use F. F. Shih and N. F. Campbell Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 1100 Robert E. Lee Boulevard, New Orleans, LA 70124 Soy proteins modified by proteolysis, deamidation, and phosphorylation were characterized to determine the extent of modification and changes in molecular structure. Effects of modification on s o l u b i l i t y and emulsifying a c t i v i t y were investigated. Limited proteolysis in treatments with pronase Ε produced hydrolysates with up to 64 mmoles αamino groups per 100 g protein or 10.2% peptide bond hydrolysis. Also investigated were enzymatic deamidation which occurred with limited proteolysis during germination of soybean seeds. Soy proteins were also phosphorylated by a protein kinase from bovine cardiac muscle. Both deamidation and phosphorylation caused increase in negative charge of the protein and generally resulted in improved functional properties that are essential for use of the proteins in food products. For example, soy proteins with 5-10% peptide bond hydrolysis or 12% deamidation and less than 3% peptide bond hydrolysis could be used as a sodium caseinate substitute in coffee whiteners. Soy p r o t e i n i s a low-cost food p r o t e i n with good n u t r i t i o n a l value, but i t s uses i n foods a r e l i m i t e d because o f i n f e r i o r f u n c t i o n a l p r o p e r t i e s as compared t o those o f commonly used animal p r o t e i n s such as c a s e i n and albumin (1.2). Therefore, modifications are o f t e n r e q u i r e d t o make soy p r o t e i n more s u i t a b l e f o r food use. Improved f u n c t i o n a l p r o p e r t i e s , p a r t i c u l a r l y i n t h e pH range o f 3 t o 7 where most food systems belong, have been achieved by non-enzymatic methods, i n c l u d i n g s u c c i n y l a t i o n (3-5), deamidation (6.7), and phosphorylation (8.9). This chapter not subject to U.S. copyright Published 1993 American Chemical Society In Food Flavor and Safety; Spanier, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


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However, e f f e c t i v e enzymatic m o d i f i c a t i o n s , i f achievable, w i l l be a v i a b l e a l t e r n a t i v e because the methods are more s p e c i f i c and, most importantly, more l i k e l y t o c l e a r regulatory safety guidelines. Recently, functional changes or improvements i n soy p r o t e i n s have been reported by l i m i t e d p r o t e o l y s i s with v a r i o u s proteases (10-12). Hamada et al. (13) investigated the use of peptidoglutaminase, a deamidating enzyme, f o r soy p r o t e i n modification. They l a t e r reported t h a t deamidation was enhanced by p r i o r p r o t e o l y s i s or heating of the p r o t e i n s u b s t r a t e and the deamidated products showed improved f u n c t i o n a l p r o p e r t i e s (14). Soy p r o t e i n s have a l s o been modified by enzymatic phosphorylation using a protein kinase from bovine c a r d i a c muscle (15.16). The present study was conducted t o o b t a i n a d d i t i o n a l information on changes i n soy p r o t e i n subunits d u r i n g limited proteolysis. Enzymatic soy p r o t e i n deamidation t h a t occurred, i n a d d i t i o n t o l i m i t e d p r o t e o l y s i s , during germination of soybean seeds was investigated. The e f f e c t s of p r o t e o l y s i s and deamidation on s o l u b i l i t y and e m u l s i f y i n g a c t i v i t y were compared. Phosphorylation of soy p r o t e i n with a commercially a v a i l a b l e p r o t e i n kinase and i t s e f f e c t s on subsequent changes i n f u n c t i o n a l p r o p e r t i e s of the p r o t e i n were a l s o s t u d i e d . M a t e r i a l s and Methods Materials. Soy i s o l a t e , Mira Pro 111, was obtained from A.E. S t a l e y MFG. Co. (Decatur, IL) . BCA p r o t e i n assay reagent was purchased from P i e r c e Chemicals (Rockford, IL) . Reagents f o r ammonia a n a l y s i s which included glutamine dehydrogenase (GIDH), 2-oxoglutarate, and reduced nicotinamide adenine d i n u c l e o t i d e (NADH) were from Bowhinger Mannheim Biochemicals (Indianapolis, IN) . Pronase Ε and the p r o t e i n kinase from bovine heart muscle were from Sigma Chemical Co. (St. Louis, MO) . [γ- Ρ]ΑΤΡ was obtained from DuPont Chemical Co. (Boston, MA). 32

P r e p a r a t i o n of Protease-Treated P r o t e i n s . The p r o t e o l y s i s of soy i s o l a t e was c a r r i e d out by i n t r o d u c i n g 6 mL pronase Ε (mg/mL) t o a w e l l d i s p e r s e d mixture of 12 g Mira Pro 111 i n 760 mL water. D i f f e r e n t l e v e l s of p r o t e o l y s i s were achieved by r e a c t i o n a t 50 °C f o r v a r i o u s p e r i o d s of time. The r e a c t i o n was stopped by heating a t 100 °C f o r 3 min, and the modified protein was then recovered by lyophilization. P r e p a r a t i o n of M o d i f i e d Soy P r o t e i n s from Germinating Soybeans. Soybean seeds (12 g dry weight) were germinated on wet V e r m i c u l i t e i n a metal pan covered w i t h aluminum foil. Water was added t o the t r a y as needed during the course of germination. To collect products, the germinated seeds were washed and then ground with mortar

In Food Flavor and Safety; Spanier, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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and p e s t l e . E x t r a c t i o n o f ground seeds was c a r r i e d out w i t h 200 mL 0.2 M sodium bicarbonate b u f f e r (pH 8.0) . The e x t r a c t was c o l l e c t e d by f i l t r a t i o n through 5 l a y e r s o f cheesecloth. Enzymatic r e a c t i o n s i n t h e e x t r a c t were stopped by p r o t e i n p r e c i p i t a t i o n a t pH 4.7 with t h e a d d i t i o n o f HC1. The residues were r e d i s s o l v e d i n water a t pH 7 and f i l t e r e d , and t h e p r o t e i n s i n t h e f i l t r a t e were recovered by l y o p h i l i z a t i o n . P r o t e i n phosphorylation. Soy p r o t e i n s were phosphorylated a t 37 °C i n a r e a c t i o n mixture c o n t a i n i n g 0.07% Miro Pro 111, 60 μΜ ATP (100-250 cpm/pmole) , 2 mM MgCl , and 2.5 U of p r o t e i n kinase i n a 100 r e a c t i o n volume. The amount of P incorporation was determined by liquid s c i n t i l l a t i o n counting as described p r e v i o u s l y (15) . When l a r g e r amounts (up t o 350 mg) o f phosphorylated p r o t e i n were needed f o r f u n c t i o n a l i t y t e s t s , a l l r e a c t a n t s were increased by a l i n e a r scale-up, with n o n r a d i o a c t i v e ATP s u b s t i t u t i n g f o r r a d i o a c t i v e ATP. 2

32

A n a l y s i s o f P r o t e o l y s i s and Deamidation. Degree o f p r o t e o l y s i s f o r the modified p r o t e i n s was measured i n terms o f peptide bonds hydrolyzed o r i n c r e a s e o f amino groups. Total amino groups was analyzed by t h e t r i n i t r o b e z e n e s u l f o n i c a c i d (TNBS) method (17). Alphaamino and e-amino groups were d i s t i n g u i s h e d according t o Kakade and L i e n e r (18). Complete peptide bond h y d r o l y s i s (100%) was achieved by h y d r o l y s i s with 6N HC1 a t 110 °C f o r 24 h r . Degree o f deamidation was analyzed by measuring the ammonia generated u s i n g the enzymatic method of Bergmeyer and B e u t l e r (19). Complete protein deamidation (100%) was achieved by h y d r o l y s i s with 2N HC1 a t 100 °C f o r 3 h r . Electrophoresis. SDS polyacrylamide g e l e l e c t r o p h o r e s i s (4-20% gradient) was performed using the method o f Laemmli (20) . Autoradiographic a n a l y s i s was performed on g e l s s t a i n e d with Coomassie B r i l l i a n t Blue (R-250), d r i e d , and s t o r e d a t -70 °C with a sheet o f X-ray f i l m (Kodak X-OMAT RP) i n d i r e c t contact with t h e g e l . Functionality Analysis. S o l u b i l i t y was determined on 1% (w/v) d i s p e r s i o n o f p r o t e i n i n 0.2 M phosphate b u f f e r s a t a pH o f 3.0 t o 8.0. A f t e r s t i r r i n g f o r 0.5 h r , the d i s p e r s i o n was f i l t e r e d (0.45 μπι. M i l l i p o r e ) , and t h e f i l t r a t e was analyzed f o r p r o t e i n by the BCA method (21) . E m u l s i f y i n g a c t i v i t y index (EAI) , expressed as i n t e r f a c i a l a r e a / u n i t weight p r o t e i n (m /g)/ was assessed by t h e t u r b i d o m e t r i c method o f Pearce and K i n s e l l a (22.) . 2

Feathering Test. Modified soy p r o t e i n s were i n v e s t i g a t e d as a s u b s t i t u t e f o r sodium caseinate i n c o f f e e whitener. A 15 mg/mL p r o t e i n s o l u t i o n was heated a t 70 °C f o r one hour with i n t e r m i t t e n t s t i r r i n g . To 10 mL o f t h e s o l u t i o n , 1.5 g o f Louana vegetable o i l was added. The


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mixture, heated a t 70 °C f o r 15 min, was then homogenized f o r 2 min. A f t e r c o o l i n g t o room temperature, 3 mL of t h i s l i q u i d c o f f e e whitener was added t o 30 mL 2% c o f f e e s o l u t i o n a t 90-95 °C. The whitening c a p a c i t y and the extent of f e a t h e r i n g (coagulation) were recorded. A c o n t r o l was conducted u s i n g sodium c a s e i n a t e .


R e s u l t s and D i s c u s s i o n Protease-Treated Soy Proteins. P r o t e o l y s i s of soy p r o t e i n s with pronase Ε proceeded a t a c o n s i s t e n t r a t e d u r i n g the time of i n v e s t i g a t i o n (Figure 1). Alpha-amino groups i n c r e a s e d from 3.0 mmoles/100 g p r o t e i n a t 0 h r t o about 64 mmoles/100 g a t 3 h r . The extent o f p e p t i d e bonds hydrolyzed was c a l c u l a t e d t o be from 0 t o 10.2% i n the same p e r i o d of time. E l e c t r o p h o r e s i s a n a l y s i s showed more d e t a i l s about the p r o t e o l y s i s of soy p r o t e i n s (Figure 2). The 7S subunits disappeared from the p r o f i l e f a s t e r than the l i s subunits as p r o t e o l y s i s proceeded. At the end of 3 hr, 7S subunits were completely converted t o s m a l l e r p o l y p e p t i d e s , whereas both a c i d i c and b a s i c l i s subunits remained mostly unchanged. Similar f i n d i n g s t h a t the smaller 7S c o n g l y c i n i n of soy p r o t e i n s , more than the l a r g e r l i s g l y c i n i n , was s u s c e p t i b l e t o r e a c t i o n with p r o t e o l y t i c enzymes such as t r y p s i n and chymotrypsin have been r e p o r t e d before (10). As soy p r o t e i n s c o n s i s t of about one t h i r d each of 7S c o n g l y c i n i n and l i s g l y c i n i n , the e f f e c t of 7S p r o t e o l y s i s on the f u n c t i o n a l p r o p e r t i e s of soy p r o t e i n , such as s o l u b i l i t y , may be l i m i t e d . P r o t e i n s M o d i f i e d by Deamidation. The presence of proteases i n germinating seeds i s w e l l known, but these enzymes may or may not be a c t i v a t e d depending on the germinating c o n d i t i o n s . According t o α-amino group a n a l y s i s , r e l a t i v e l y low l e v e l s of p r o t e o l y s i s were observed during germination. Instead, we found s i g n i f i c a n t amount of ammonia, i n d i c a t i n g deamidation. As can be seen i n F i g u r e 3, both deamidation and p e p t i d e bond h y d r o l y s i s of soybean storage p r o t e i n i n c r e a s e d with i n c r e a s e d germination. However, a t the end of 4 days, about 12% of the amide bonds were hydrolyzed, with o n l y 2.6% peptide bond h y d r o l y s i s . The l i m i t e d p e p t i d e bond h y d r o l y s i s was confirmed by both HPLC a n a l y s i s (not shown) and e l e c t r o p h o r e s i s . The l a t t e r showed t h a t , f o r the h y d r o l y s a t e with 12% deamidation, 7S subunits were p a r t i a l l y hydrolyzed whereas the 11S subunits remained p r a c t i c a l l y unchanged and very l i t t l e small p e p t i d e s were generated (Figure 2). For t h i s m o d i f i e d protein, t h e r e f o r e , amide bond h y d r o l y s i s may be r e s p o n s i b l e , more so than peptide bond h y d r o l y s i s , f o r the subsequent changes i n i t s f u n c t i o n a l i t y . P r o t e i n s M o d i f i e d by Phosphorylation. The r e a c t i o n of p r o t e i n kinase with soy p r o t e i n was very s p e c i f i c . Only



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TIME (hours) Figure 1. Proteolysis of soy protein with pronase Ε in termas of α-amino groups generated as a function of time.

Figure 2. Electrophoretic profiles for intact and pronase Ε-modified soy proteins. Intact soy protein (a); soy protein proteolyzed for 0.5 h (b), 1.0 h (c), 1.5 h (d), 2.0 h (e), and 3.0 h (f); deamidated soy isolate (g).


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13 μπιοΙβΒ o f phosphorus per g p r o t e i n was incorporated i n soy p r o t e i n a f t e r r e a c t i o n with the enzyme f o r 4 h r a t 37 °C. Autoradiography o f the g e l s showed P incorporated i n t o t h e g l y c i n i n a c i d i c polypeptides f i r s t , then t h e g l y c i n i n b a s i c polypeptides, and f i n a l l y t h e β and a/a' subunits o f /9-conglycinin (Figure 4). The order i n which the subunits were phosphorylated corresponded t o t h e number o f p o t e n t i a l phosphorylation s i t e s i n the known p r o t e i n primary s t r u c t u r e s (23-26). 32


f

E f f e c t s o f P r o t e i n M o d i f i c a t i o n on F u n c t i o n a l i t y . F i g u r e 5 shows the s o l u b i l i t y - p H p r o f i l e s o f soy p r o t e i n s modified by pronase E. S o l u b i l i t y i n the pH range o f 3 t o 8 increased i n i t i a l l y with increased p r o t e o l y s i s . The s o l u b i l i t y increase was caused mostly by t h e decrease i n s i z e o f the 7S subunits. A f t e r 2 h r , almost a l l the down-sized 7S subunits became s o l u b l e and t h e i n s o l u b l e r e s i d u e s were the remains of s l i g h t l y hydrolyzed l i s subunits. The s o l u b i l i t y ceased t o i n c r e a s e and even decreased s l i g h t l y f o r hydrolysates with more than 2 h r proteolysis indicating crosslinking among the hydrolysates. Minimum s o l u b i l i t y occurred a t pH ranging form 4.5 t o 5.0, depending on p r o t e o l y s i s . The e f f e c t s o f pH on the s o l u b i l i t y became i n s i g n i f i c a n t a f t e r 2 h r . For comparison, the s o l u b i l i t y - p H p r o f i l e o f t h e deamidated p r o t e i n was added t o t h e p l o t c o n t a i n i n g t h e p r o f i l e s f o r the pronase Ε-treated p r o t e i n s (Figure 5 ) . The deamidated p r o t e i n , with 2.6% peptide bond h y d r o l y s i s , showed improved minimum s o l u b i l i t y , comparable t o the protein with 5.7% peptide bond h y d r o l y s i s and no deamidation. The shape o f s o l u b i l i t y - p H p r o f i l e f o r t h e deamidated sample resembled t h a t o f the i n t a c t p r o t e i n more than those o f the pronase Ε-treated samples. F o r t h e deamidated sample, both the increase i n s o l u b i l i t y and t h e s l i g h t s h i f t o f minimum s o l u b i l i t y t o the a c i d s i d e were the r e s u l t o f the increase i n negative charges from deamidation. Obviously, deamidation was more capable o f maintaining the original protein structure than p r o t e o l y s i s , which i s e s s e n t i a l f o r the development o f desirable functional properties. E m u l s i f y i n g a c t i v i t y index (EAI) i s a measure o f t h e a b i l i t y o f p r o t e i n t o emulsify o i l , which depends on s o l u b i l i t y , s i z e , charge, and s u r f a c e a c t i v i t y o f t h e p r o t e i n molecules. The e f f e c t o f p r o t e o l y s i s with pronase Ε on EAI o f the modified protein was relatively i n s i g n i f i c a n t (Figure 6 ) . However, deamidation appeared t o enhance EAI, e s p e c i a l l y a t pH values more b a s i c than the i s o e l e c t r i c p o i n t (pH 4.7). The e f f e c t o f phosphorylation a t 13 Mmoles/g p r o t e i n on f u n c t i o n a l p r o p e r t i e s was minimal. Both s o l u b i l i t y and EAI o f the phosphorylated p r o t e i n were s l i g h t l y higher, compared t o those o f i n t a c t p r o t e i n (not shown). Phosphorylation with p r o t e i n kinase from bovine c a r d i a c muscle i s r e s t r i c t e d by the l i m i t e d number o f p o t e n t i a l phosphorylation s i t e s i n soy p r o t e i n s . Experiments a r e


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20

0

2

4

6

TIME (days)

F i g u r e 3. Deamidation and p e p t i d e bond h y d r o l y s i s o f soy p r o t e i n during germination of soybean seeds.

F i g u r e 4. Autoradiogram o f SDS-PAGE phosphorylation o f soy p r o t e i n s .

gel

f o r the



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0.0

1

0

" 2

1

ι 6

4

ι 8

I 10

pH Figure 5. Solubility of soy proteins as a function of pH. Intact soy protein (O); soy protein proteolyzed with pronase Ε for 1 h (•), 2 h (V), and 3 h (Δ); deamidated soy protein (#).

pH Figure 6. Emulsifying activity index of soy proteins as a function of pH. Intact soy protein (O); soy protein proteolyzed with pronase Ε for 1 h (V) and 3 h (•); deamidated soy protein (#).


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being conducted i n our l a b o r a t o r y t o prepare new p r o t e i n kinases with d i f f e r e n t and l e s s s p e c i f i c phosphorylation s i t e requirements. For instance, p r o t e i n kinase from germinating soybeans, i f a v a i l a b l e , w i l l most l i k e l y be more e f f e c t i v e i n soy p r o t e i n phosphorylation, because both enzyme and s u b s t r a t e are from the same source. P o t e n t i a l Uses o f M o d i f i e d P r o t e i n s . Deamidation and l i m i t e d p r o t e o l y s i s are e f f e c t i v e i n the development o f d e s i r a b l e f u n c t i o n a l p r o p e r t i e s , because they cause increase i n negative charges without significantly changing the b a s i c p r o t e i n s t r u c t u r e . T h i s can be demonstrated by using the modified soy p r o t e i n s as c a s e i n s u b s t i t u t e i n c o f f e e whiteners. The c o f f e e whitener was formulated u s i n g only the key i n g r e d i e n t s , p r o t e i n and o i l , without other e m u l s i f y i n g a d d i t i v e s t h a t a r e normally presence i n commercial c o f f e e whiteners. The method was chosen t o provide the most severe t e s t f o r the p r o t e i n i n g r e d i e n t o f i t s whitening c a p a c i t y and the a b i l i t y t o r e s i s t c o a g u l a t i o n ( f e a t h e r i n g ) . The r e s u l t s showed t h a t proteolyzed soy p r o t e i n s , a t 10-60 mmoles α-amino groups/100 g p r o t e i n , and the deamidated p r o t e i n , a t 12% deamidation, were most e f f e c t i v e as sodium c a s e i n a t e s u b s t i t u t e i n c o f f e e whiteners (Table 1).

Table 1. R e l a t i v e E f f e c t i v e n e s s of P r o t e i n s as an Ingredient i n Coffee Whitener Proteins

Whitening

Sodium Caseinate I n t a c t Soy P r o t e i n Soy P r o t e i n Proteolyzed 0.5 h r Proteolyzed 1.0 hr Proteolyzed 2.0 h r P r o t e o l y z e d 3.0 h r Deamidated

+ + + + + +

non-feathering almost i n s t a n t l y

+ + + + +

e x t e n s i v e l y w i t h i n 1 min e x t e n s i v e l y w i t h i n 1 min s l i g h t l y a f t e r 1 min s l i g h t l y a f t e r 1 min s l i g h t l y a f t e r 1 min

+ + + + +

+ + + + + + +

Feathering

Conclusions In l i m i t e d p r o t e o l y s i s , proteases such as pronase Ε hydrolyzed the 7S subunits of soy p r o t e i n s more than the 11S subunits, r e s u l t i n g i n enhanced p r o t e i n s o l u b i l i t y . Deamidation with r e l a t i v e l y i n s i g n i f i c a n t peptide bond h y d r o l y s i s t h a t occurred during the germination o f soybeans imparted t o the storage p r o t e i n improved s o l u b i l i t y and e m u l s i f y i n g a c t i v i t y . On the other hand, the i n c o r p o r a t i o n of phosphorus i n soy p r o t e i n s by the p r o t e i n kinase cAMPdPK was too low t o e f f e c t s i g n i f i c a n t


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improvements i n the f u n c t i o n a l p r o p e r t i e s f o r the m o d i f i e d proteins. Consequently, soy p r o t e i n s m o d i f i e d by deamidation and/or l i m i t e d p r o t e o l y s i s were u s e f u l as a sodium c a s e i n a t e s u b s t i t u t e i n food systems such as c o f f e e whiteners. However, phosphorylating enzymes more e f f e c t i v e than cAMPdPK a r e needed t o r a i s e the l e v e l o f phosphorylation before i t can be considered f o r food use.


Acknowledgment. We thank Kim D a i g l e f o r a s s i s t a n c e i n conducting experiments f o r the i n v e s t i g a t i o n and i n the p r e p a r a t i o n o f the manuscript.

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Received October 28, 1992


Enzymatic Modification of Soy Proteins To Improve Their Functional

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