Protein-Nitrogen Conservation in Fresh Stored Dioscorea Yams - ACS

21 Protein-Nitrogen Conservation in Fresh Stored Dioscorea Yams 1

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Godson O. Osuji , Robert L. Ory, and Elena E. Graves Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, New Orleans, L A 70179 Fresh and stored yams were examined for protein and selected nitrogen-metabolizing enzymes. Yams stored at room temperature lost weight (water), protein, and non-protein nitrogen. Sprouting depleted a l l of the protein. The major protein of yams is a 60-70% ethanol-soluble prolamine, virtually insoluble in NaCl or water. Purine-metabolizing enzymes in 7 Dioscorea yam cultivars were examined. Three of these (adenosine deaminase, uricase, allantoicase) give rise to ammonia; the others do not (xanthine oxidase, allantoinase). Allantoinase activity is low in yam tubers and may result in accumulation of allantoin. Yams also contain an alkaline proteinase that is active on yam prolamines, has a pH optimum at 9.5-10.0, and is stable to heat (60°C for 5 hours). It is not inhibited by thiol reagents, Ca, Mn, Mg, Zn or Cu but is strongly inhibited by Fe and Fe . Dehydrated yam flakes were prepared from fresh yams in over 60% yield, as a potential means of storing yams safely for food uses in the tropics. 3

2

The yam t u b e r ( D i o s c o r e a s p p . ) is a primary carbohydrate s t a p l e c r o p in West A f r i c a b u t i t s u f f e r s c o n s i d e r a b l e losses d u r i n g p o s t h a r v e s t storage due t o fungal decay and/or sprouting (1). S i n c e t h e yam is a l s o a minor source o f d i e t a r y n i t r o g e n f o r many people whose d i e t s c o n t a i n l i t t l e o r no animal p r o t e i n , l o s s of n i t r o g e n by t h e t u b e r s can be s e r i o u s s i n c e o t h e r d i e t a r y proteins are not a v a i l a b l e . The major sources of nonprotein n i t r o g e n in yams a r e t h e u r e i d e compounds o f t h e p u r i n e m e t a b o l i c pathway (2). Yams a r e e s s e n t i a l l y t r o p i c a l c r o p s t h a t cannot t o l e r a t e any f r o s t o r temperatures below 20°C (68°F) b u t t h e r a t e of growth i n c r e a s e s w i t h temperatures o f 25-30°C ( 7 7 - 8 6 F ) , which a r e normal in areas where yams a r e grown (_3> 4). The West A f r i c a n yam zone is t h e most i m p o r t a n t yam producing area o f t h e w o r l d . About h a l f o f t h e w o r l d crop appears t o be produced in N i g e r i a . Of 12.1 m i l l i o n m e t r i c tons produced in a l l o f West A f r i c a , about 9 m i l l i o n were produced in N i g e r i a U ) . There a r e many s p e c i e s o f D i o s c o r e a o f economic i n t e r e s t , o f which t e n a r e most i m p o r t a n t as e

1

Current address: Department of Agricultural Biochemistry, Anambra State University of Technology, Enugu, Nigeria. This chapter not subject to U.S. copyright. Published 1986, American Chemical Society

Ory; Plant Proteins: Applications, Biological Effects, and Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

PLANT PROTEINS

262

sources of f o o d . Of t h e s e , D. a l a t a , D. cayenensis and D. r o t u n d a t a are the major s p e c i e s grown in N i g e r i a and are the species examined in these i n v e s t i g a t i o n s . Of these t h r e e , D. r o t u n d a t a is by f a r t h e major s p e c i e s grown as a food c r o p , f o l l o w e d by D. a l a t a and D. c a y e n e n s i s . In an e a r l i e r r e p o r t ΠΓΠ the decay of h e a l t h y yam t u b e r s d u r i n g s t o r a g e was shown to be a r e s u l t of c a t a b o l i s m of i t s p r o t e i n s by an a c t i v e α-glutamyl transpeptidase. There is a l s o some a l k a l i n e p r o t e o l y t i c a c t i v i t y in the yam t u b e r ( 6 h but little information is a v a i l a b l e on i n d i v i d u a l enzymes of the p u r i n e d e g r a d a t i v e pathway and on the p r o p e r t i e s of an a l k a l i n e p r o t e i n a s e t h a t may f u n c t i o n in yams d u r i n g s t o r a g e . This report d e s c r i b e s the i n t e r r e l a t i o n of f i v e enzymes of u r e i d e metabolism in f r e s h and s t o r e d yams, the r e l e a s e of ammonia in v i t r o by t h r e e of the enzymes t h a t may p r o v i d e an environment f o r a l k a l i n e p r o teinase a c t i v i t y in v i v o , and the in v i t r o p r o p e r t i e s of an a l k a l i n e p r o t e i n a s e i s o l a t e d from f r e s h yams. Using a method developed f o r the p r e p a r a t i o n of dehydrated f l a k e s from sweet p o t a t o e s (_7), dehydrated f l a k e s were prepared from f r e s h yams. These f l a k e s appear t o be a s u i t a b l e means f o r i n a c t i v a t i n g the yam enzymes and p r e s e r v i n g the d i e t a r y n i t r o g e n in yams f o r long term s t o r a g e under t r o p i c a l c o n d i t i o n s . M a t e r i a l s and Methods Yam t u b e r s of D i o s c o r e a a l a t a (Umudike c u l t i v a r ) , D. r o t u n d a t a (asukwu and o b i a t u r u g o c u l t i v a r s Π and D. c a y e n e n s i s (water yam and Nkokpu c u l t i v a r s ) were o b t a i n e d from the N a t i o n a l Root Crops Research I n s t i t u t e , Umudike, N i g e r i a . Some t u b e r s were s t o r e d 6 or 12 months a t room temperature (25-27°C), some in vacuum dessicators over a s u i t a b l e d e s s i c a n t , and some in paper bags p l a c e d in a dark c a b i n e t (absence of c i r c u l a t i n g a i r ) . Fresh t u b e r s were peeled by c a r e f u l l y s c r a p i n g away the cork l a y e r to m i n i m i z e l o s s of o u t e r t i s s u e s i n c e much of t h e p r o t e i n is c o n c e n t r a t e d here (8). They were then c u t i n t o 2 c u . cm. p i e c e s , q u i c k l y f r o z e n w i t h s o l i d CO2 in 50 g p o r t i o n s in p l a s t i c bags, and s t o r e d in a f r e e z e r u n t i l needed. Homogenization of Tuber T i s s u e . Frozen p i e c e s of t i s s u e (50g) were t r a n s f e r r e d to a Waring B l e n d o r l / and homogenized in 100 ml i c e - c o l d O.1 M K2HPO4 (4°C) and O.1 ml 3-mercaptoethanol a t low speed f o r 3 m i n . The homogenate was f i l t e r e d through two l a y e r s of c h e e s e c l o t h and the f i l t r a t e c e n t r i f u g e d a t 20,000 g. f o r 10 min. The p e l l e t was d i s c a r d e d and the s u p e r n a t a n t l i q u i d was d i a l y z e d a g a i n s t t h r e e changes of d e i o n i z e d water f o r 24 hr to remove low m o l e c u l a r weight sugars t h a t might i n t e r f e r e w i t h the phenyl h y d r a z i n e assays f o r a l l a n t o i n a s e and a l l a n t o i c a s e a c t i v i t y . Enzyme

Assays.

Adenosine

deaminase

(E.C.

3 . 5 . 4 . 4 ) was

assayed

by

2/ Trade names are g i v e n s o l e l y f o r t h e purpose of p r o v i d i n g specific information. T h e i r mention does not imply recommendation o r endorsement by t h e U. S. Department of A g r i c u l t u r e over o t h e r s not mentioned.


21.

Protein-Nitrogen Conservation in Yams

OSUJIETAL.

263

the method of Coddington (9) and absorbance was read a t 265 nm in a spectrophotometer over the f i r s t 5 min of r e a c t i o n t i m e . The change in absorbance was used to c a l c u l a t e enzyme a c t i v i t y . X a n t h i n e o x i d a s e (E.C. 1.2.3.2) was measured a c c o r d i n g to Bray (10) with xanthine as substrate and oxygen as the e l e c t r o n acceptor. The change in absorbance a t 293 nm between 5 and 10 min of r e a c t i o n was used t o calculate activity. Uricase (E.C. 1.7.3.3) was assayed a c c o r d i n g t o Mahler (11) w i t h oxygen as electron acceptor. The change in absorbance was measured a t 293 nm in a spectrophotometer over t h e f i r s t 5 min and used t o calculate activity. A l l a n t o i n a s e (E.C. 3.5.2.5) was measured w i t h a l l a n t o i n as s u b s t r a t e by a m o d i f i c a t i o n of t h e methods of Singh e t a l . (12) and T r i j b e l s and Yogels U 3 ) . To 1 ml of a l l a n t o i n s o l u t i o n T 3 mM in O.1 M t r i s b u f f e r , pH 7 . 4 ) , O.5 ml of t u b e r e x t r a c t was added and t h e m i x t u r e i n c u b a t e d a t 40°C in a water bath f o r 1 h r . The r e a c t i o n was stopped by adding 1 ml each of cone. HC1 and phenyl h y d r a z i n e (100mg/30ml d e i o n i z e d water) which r e a c t s w i t h the g l y o x y l i c a c i d formed from a l l a n t o i n . Reaction tubes were placed in b o i l i n g water f o r 5 m i n , then c o o l e d r a p i d l y by immersing in an i c e / w a t e r b a t h . A f t e r r e t u r n i n g the tubes t o room t e m p e r a t u r e , 1 m l . of potassium f e r r i c y a n i d e (500 mg/30 m l . d e i o n i z e d water) was added, mixed w e l l , then c e n t r i f u g e d a t 10,000 g f o r 5 min to remove p r e c i p i t a t e d p r o t e i n s . Absorbance was measured a t 525 nm in a s p e c t r o p h o t o m e t e r . Controls contained a l l a n t o i n but no t u b e r e x t r a c t and were t r e a t e d the same as t e s t samples. Absorbance of c o n t r o l s was s u b t r a c t e d from t e s t samples and c a l c u l a t e d from a s t a n d a r d curve prepared w i t h g l y o x y l i c a c i d . A l l a n t o i c a s e (E.C. 3 . 5 . 3 . 4 ) was assayed w i t h a l l a n t o i c a c i d as s u b s t r a t e by a m o d i f i c a t i o n of the methods of T r i j b e l s and Yogels (13) and Ory, e t a l . ( 1 4 ) . To O.5 ml a l l a n t o i c a c i d s o l u t i o n T 3 . 5 nM in O.1 M t r i s ~ ï ï u f f e r , pH 7 . 4 ) , O.5 ml of the t u b e r e x t r a c t was added and i n c u b a t e d a t 40°C in a water bath f o r 1 hr. T e s t tubes were p l a c e d in i c e , then 1 ml each of cone. HC1 and phenyl h y d r a z i n e (100 mg/30 ml d e i o n i z e d water) were added, f o l l o w e d by 1 ml of the potassium f e r r i c y a n i d e s o l u t i o n and m i x i n g to promote r e a c t i o n w i t h g l y o x y l i c a c i d . The p r e c i p i t a t e was removed by c e n t r i f u g a t i o n (as above) and absorbance read a t 525 nm in the spectrophotometer. Controls containing allantoic acid substrate but no tuber extract were t r e a t e d as above and absorbance of controls was subtracted from test samples to calculate activity. Ureidoglycine and u r e i d o g l y c o l a t e produced from a l l a n t o i c a c i d by t u b e r e x t r a c t s were determined by the d i f f e r e n t i a l g l y o x y l a t e method of T r i j b e l s and Yogels (JL3) a f t e r the t u b e r e x t r a c t s and a l l a n t o i c a c i d had been i n c u b a t e d as described for a l l a n t o i c a s e . P r o t e i n s and P Isolation/Assay. P i e c e s of t u b e r were homogenized U00g/150 ml a b s o l u t e e t h a n o l ) in a Waring Blendor f o r 3 min (as b e f o r e ) , f i l t e r e d through c h e e s e c l o t h , and the f i l t r a t e c e n t r i f u g e d a t 10,000 g f o r 15 m i n . The supernatant was made to 10% w i t h t r i c h l o r o a c e t i c a c i d to p r e c i p i t a t e t h e p r o t e i n and s t o r e d a t 0°C f o r s e v e r a l hours t o s e p a r a t e the yam p r o l a m i n e . A f t e r c e n t r i f u g i n g a t 10,000 g f o r 15 m i n , t h e p r e c i p i t a t e was suspended in a minimum volume o f water and d i a l y z e d against r

o

t

e

i

n

a

s

e


264

PLANT PROTEINS

d e i o n i z e d water o v e r n i g h t , then f r e e z e - d r i e d and s t o r e d a t 5°C u n t i l needed. For the p r o t e i n a s e , 100 g of yam cubes was homogenized in 150 ml i c e c o l d (4°C) d e i o n i z e d water c o n t a i n i n g 2 g p o l y v i n y l p y r r o l i done (PYP) and 100 mg e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d (EDTA) in a Waring Blendor at low speed f o r 3 min, f i l t e r e d through two l a y e r s of c h e e s e c l o t h , then c e n t r i f u g e d as before. The supernatant l i q u i d was made up to 85% s a t u r a t i o n w i t h s o l i d ( N H ^ SO4; t h e p r e c i p i t a t e was removed by c e n t r i f u g i n g a t 10,000 g f o r 15 m i n , then d i s s o l v e d in 2-5 ml of d e i o n i z e d water f o r d i a l y s i s a g a i n s t d e i o n i z e d water o v e r n i g h t . The d i a l y z e d enzyme was f r e e z e - d r i e d f o r s t o r a g e in the f r e e z e r u n t i l needed. In some c a s e s , the o r i g i n a l homogenate was made up to 35% w i t h ( N H ^ p SO4 t o remove small amounts of p r o t e i n ; then the f i l t r a t e was r a i s e d from 35% t o 85% w i t h (NH4)? SO4, p r e c i p i t a t e d , c e n t r i f u g e d , d i a l y z e d , and f r e e z e - d r i e d as b e f o r e , to e f f e c t a h i g h e r p u r i f i c a t i o n . P r o t e i n c o n t e n t s of t u b e r e x t r a c t s were determined by the method of Lowry e t a l . (^5) using b o v i n e serum albumin as t h e standard. For a c t i v i t y a s s a y s , p r o t e i n a s e s o l u t i o n s were made f r e s h d a i l y (10 mg f r e e z e - d r i e d s o l i d s in 1 ml pH 10.0 phosphate b u f f e r , O.1 M). Two ml of t h e p r o t e i n a s e , O.5 ml of s u b s t r a t e ( a z o c a s e i n or o t h e r p r o t e i n s in pH 10 b u f f e r ) , O.3 ml of O.1% EDTA, and d e i o n i z e d water were made up to a volume of 3.5 m l . Reaction tubes were i n c u b a t e d in a 4 0 C water bath f o r 1 h r , then the r e a c t i o n was stopped by a d d i t i o n of 1 ml 5% TCA. A f t e r removal of the p r e c i p i t a t e d p r o t e i n s by c e n t r i f u g a t i o n , absorbance was read a t 366 nm ( f o r a z o c a s e i n ) or by the Lowry method (15) f o r o t h e r s u b s t r a t e s , t o c a l c u l a t e p r o t e i n a s e a c t i v i t y w i t h TRe d i f f e r e n t substrates. For e l e c t r o p h o r e s i s of the p r o t e i n a s e , s a t u r a t e d s o l u t i o n s of crude and p u r i f i e d p r e p a r a t i o n s were made in O.1 M T r i s - g l y c i n e b u f f e r , pH 10.5, and e l e c t r o p h o r e s e d in 7% p o l y a c r y l a m i d e gel w i t h and w i t h o u t sodium dodecyl s u l f a t e (SDS). e

R e s u l t s and D i s c u s s i o n A summary of the p r o x i m a t e a n a l y s e s of these yam tubers is shown in T a b l e 1. On a f r e s h weight b a s i s , p r o t e i n is q u i t e low because of the high water and s t a r c h c o n t e n t s of yams (dry w e i g h t p r o t e i n c o n t e n t is 4 - 8 % ) . Yams are r a r e l y consumed alone b u t , depending upon t h e economic s t a t u s and t a s t e s of t h e consumer, they are accompanied by meat or f i s h , green v e g e t a b l e s , or s p i c e s . Where animal p r o t e i n is not i n c l u d e d , c o n s e r v a t i o n of t h e l i m i t e d amounts o f crude p r o t e i n in yams is e s s e n t i a l f o r areas t h a t depend upon yams as t h e i r primary food s o u r c e . Because yams are poor sources of dietary nitrogen, a knowledge of n o n p r o t e i n n i t r o g e n metabolism in yams would p r o v i d e useful i n f o r m a t i o n f o r r e t a i n i n g the amount p r e s e n t in fresh t u b e r s and p r e v e n t i n g the usual l o s s e s s u f f e r e d d u r i n g s t o r a g e in the t r o p i c s . Yams accumulate l a r g e q u a n t i t i e s of nonprotein n i t r o g e n as a l l a n t o i n (16) but the u r e i d e compounds ( a l l a n t o i n , a l l a n t o i c a c i d , e t c . ) are more i m p o r t a n t i n t e r m e d i a t e s in the n i t r o g e n - r i c h plant species (17-19) than in n i t r o g e n - d e f i c i e n t


21.

OSUJIETAL.

Table I.

265


Summary o f P r o x i m a t e Analyses of Fresh Yam Tubers

Component

D. a l a t a

t

Species D. c a y e n e n s i s %

U.

D.rotundata %

Moisture

65-73

83

58-73

Carbohydrate

22-29

15

23

O.03-O.27

O.05

O.12

1.1-2.8

1.0

1-2

Crude F i b e r

O.6-1.4

O.4

O.3-O.8

Ash

O.7-2.1

O.5

O.7-2.6

Fat Crude P r o t e i n (Ν χ

U

6.25)

Data taken from Coursey

U),

pp. 154-157.

root tubers. However, u r e i d e s are known t o be m e t a b o l i z e d by plant roots (20-22). F i v e enzymes of the p u r i n e d e g r a d a t i v e pathway in two s p e c i e s of yams were i n v e s t i g a t e d t o o b t a i n a b e t t e r u n d e r s t a n d i n g of n i t r o g e n metabolism in f r e s h and s t o r e d t u b e r s . The results ( T a b l e 2) show t h a t 12 months s t o r a g e had v a r i e d but s t r i k i n g e f f e c t s on a c t i v i t y of adenosine deaminase, x a n t h i n e oxidase, u r i c a s e , a l l a n t o i n a s e , and a l l a n t o i c a s e in both D. a l a t a and D. rotundata. In the general scheme of p u r i n e d e g r a d a t i o n , adenosine is deaminated t o y i e l d x a n t h i n e , which in t u r n is o x i d i z e d to u r i c acid, the substrate for uricase. Subsequent deamination by uricase produces allantoin, the substrate for allantoinase. A l l a n t o i n d e g r a d a t i o n t o a l l a n t o i c a c i d p r o v i d e s the s u b s t r a t e f o r a l l a n t o i c a s e , which c o n v e r t s t h i s t o urea and g l o x y l i c acid. Thus, low a c t i v i t y ( o r i n h i b i t i o n ) of t h e l a s t two enzymes of t h i s c h a i n c o u l d be r e s p o n s i b l e f o r the a c c u m u l a t i o n of a l l a n t o i n in certain plants. Whereas t h e water yam c u l t i v a r shows a general i n c r e a s e in a c t i v i t y f o r a l l f i v e enzymes d u r i n g s t o r a g e , the o t h e r c u l t i v a r s show mixed e f f e c t s ; some l o s e a c t i v i t y and some increase activity upon storage. Comparing allantoinase/ a l l a n t o i c a s e a c t i v i t i e s , these enzymes in t h e D. a l a t a c u l t i v a r s increased during storage whereas those in t h e ÏÏ7" r o t u n d a t a c u l t i v a r s decreased. Such decreased a c t i v i t i e s d u r i n g s t o r a g e may provide a mechanism f o r increased a c c u m u l a t i o n of a l l a n t o i n / a l l a n t o i c a c i d and subsequent r e t e n t i o n of n o n p r o t e i n n i t r o g e n in the t u b e r s . This also provides useful information f o r breeding yam t u b e r s w i t h h i g h e r n i t r o g e n ; t u b e r s w i t h lower a c t i v i t i e s of these enzymes would be more d e s i r a b l e . I f the s p e c i f i c a c t i v i t i e s of these two enzymes in f r e s h yams are compared ( T a b l e 3 ) , i t appears t h a t a l l a n t o i c a s e a c t i v i t y is h i g h e r in a l l c u l t i v a r s . T h i s suggests t h a t a l l a n t o i n a s e may be the l i m i t i n g enzyme in the p u r i n e d e g r a d a t i v e pathway.


Ory; Plant Proteins: Applications, Biological Effects, and Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1986. 225

100

159

100

D. r o t u n d a t a (asukwu)

100

100

100

•

89

100

D. r o t u n d a t a ( o b i a t u r u g o )

100

210

100

155

100

(water yam)

D. c a y e n e n s i s

100

%

100

48

116

100

45

97

118

% 100

%

Fresh Stored

Allantoinase

100

of

109

152

120

%

Fresh Stored

16

100

46

(umudike) 100

D. a l a t a

%

Uricase

Relative Activities

Xanthine Oxidase Fresh Stored

on

%

%

Adenosine Deaminase Fresh Stored

Storage

%

Species/Cultivar

Table 2 . E f f e c t s of 12 Months U r e i d e Enzymes in Yam T u b e r s .

82 37

100 100

112

100

% 100

100

%

Fresh Stored

Allantoicase

21. OSUJIETAL.


T a b l e 3. Specific Activities A l l a n t o i c a s e in F r e s h Yams. Species/Cultivar

D. a l a t a

(umudike)

D. c a y e n e n s i s

(water yam)

(μΜ/min/mg)

Allantoinase

267

of

Allantoinase

and

Allantoicase

1.1

1.5

O.8

2.5

D.rotundata

(obiaturugo

1.0

1.7

D.rotundata

(asukwu)

3.6

11.8

R e l e a s e o f Ammonia. Of the f i v e enzymes, t h r e e lead to r e l e a s e of ami de/ami no nitrogen as ammonia during ureide metabolism: adenosine deaminase, uricase, and allantoicase. This could p r o v i d e a mechanism f o r the o v e r a l l net l o s s of n i t r o g e n by the t u b e r s or f o r a c c u m u l a t i o n in t h e t i s s u e s . A c c u m u l a t i o n of NHo is b e l i e v e d t o r e s u l t in the b u i l d up of u r e i d e s in p l a n t s ( 2 3 ) . S i n c e yams are t r a d i t i o n a l l y s t o r e d in open barns in West A f r i c a f o r s e v e r a l months, both p r o t e i n and n o n p r o t e i n n i t r o g e n can be l o s t . Yams are t h e primary source of d i e t a r y c a r b o h y d r a t e but are a l s o a minor source of n i t r o g e n . K j e l d a h l a n a l y s i s of yam t i s s u e b e f o r e and a f t e r storage showed t h a t D. a l a t a Umudike c u l t i v a r l o s t 31% of i t s t o t a l nitrogen a f t e r 12 month's storage and 65% of the n o n p r o t e i n n i t r o g e n . D. r o t u n d a t a asukwu c u l t i v a r l o s t 15% of t h e t o t a l n i t r o g e n and 5"U% oF tHë "nonprotein n i t r o g e n a f t e r s t o r a g e . Loss of ammonia by purine degradation c o u l d be s i g n i f i c a n t since the tubers are a l r e a d y low in n i t r o g e n . A l k a l i n e P r o t e i n a s e A c t i v i t y in Yams. The r e l e a s e of ammonia a t s e v e r a l stages d u r i n g u r e i d e metabolism suggested a p o t e n t i a l f o r a l k a l i n e c o n d i t i o n s in yam t u b e r s , r a t h e r than the usual n e u t r a l or a c i d c o n d i t i o n s g e n e r a l l y found in seeds and p l a n t s . Because yams are s t o r e d in open systems a t ambient temperatures ( u s u a l l y warm), t u b e r t i s s u e was examined f o r p r o t e i n a s e a c t i v i t y a t 40°C. Some t u b e r s had high apparent polyphenol o x i d a s e a c t i v i t y upon p e e l i n g of the t u b e r s ( t i s s u e t u r n e d deep p u r p l e a t the peeled s u r f a c e ) so t h a t PYP was added to e x t r a c t s to combine w i t h p o l y p h e n o l i c compounds and p r o t e c t the p r o t e i n a s e from r e a c t i n g w i t h these compounds. E a r l i e r s t u d i e s had shown some i n h i b i t i o n o f a l k a l i n e p r o t e i n a s e a c t i v i t y by f e r r i c i o n (24) so t h a t EDTA was a l s o added t o the e x t r a c t s t o c h e l a t e any f r e e i r o n . Two a l k a l i n e pH optima were f o u n d , a t 9.0 and 10.5. The a l k a l i n e p r o t e i n a s e s of w h i t e potatoes (Solanum tuberosum) have pH optima between 8.6 and 9 (25) and those of C a r i l l a c h o c o l a t u b e r s have pH optima between 8.0 and 9.5 U6,2771 s u g g e s t i n g t h a t a l k a l i n e p r o t e i n a s e s of D i o s c o r e a t u b e r s mayHiave t h e h i g h e s t pH optima y e t r e p o r t e d in p l a n t s . The temperature optimum f o r t h i s p r o t e i n a s e may a l s o be a result of evolutionary adaptation to the hot temperatures e x p e r i e n c e d d u r i n g growing and p o s t h a r v e s t storage in West A f r i c a .


268

PLANT PROTEINS

Maximum a c t i v i t y was found a t 6 0 ° ( 2 4 ) . As seen in F i g . 1, t h e enzyme l o s t o n l y 10-15% o f i t s a c t i v i t y a t 60°C a f t e r 5 hr a t t h a t temperature, suggesting a remarkable stability toward heat. However, a l l t e s t s were conducted a t 40°C t o a v o i d d e n a t u r a t i o n e f f e c t s on p r o t e i n a s e s u b s t r a t e s and d i f f i c u l t i e s i n v o l v e d w i t h maintaining the higher temperature. Optimal temperature o f the t h i o l p r o t e i n a s e of w h i t e potato is 40°C (25) and a membrane-bound p r o t e i n a s e in g e r m i n a t i n g pea seeds is s t a B l e a t 60°C f o r 1 h r . (28). The yam tuber proteinase does not seem to be membrane-bound, s i n c e i t is e a s i l y e x t r a c t e d from t h e t i s s u e without detergents. To study s u b s t r a t e s p e c i f i c i t y , two assays were used. In i n i t i a l t e s t s (pH optimun, heat s t a b i l i t y / o p t i m u m ) , a z o c a s e i n was employed as a chromogenic s u b s t r a t e because of i t s sensitivity. Because the yam p r o t e i n a s e had o n l y h a l f or l e s s o f the a c t i v i t y of t r y p s i n and p e p s i n on a z o c a s e i n , a d d i t i o n a l t e s t s on s u b s t r a t e s employed t h e Lowry method (15) t o measure h y d r o l y s i s of s u b s t r a t e s by yam p r o t e i n a s e . R e s u l t s o f these t e s t s a r e summarized in Table 4. Lowest activity was measured with a z o c a s e i n , peanut g l o b u l i n ( a r a c h i n ) , egg a l b u m i n , bovine serum albumin and hemoglobin, and 6 - l a c t o g l o b u l i n , a l l r e a d i l y s o l u b l e proteins. Highest activity was exhibited towards t h e two p r o l a m i n e s , wheat g l i a d i n and yam p r o t e i n ; both p r o t e i n s e x t r a c t e d with ethanol. The yam p r o t e i n a s e showed as much a c t i v i t y toward the yam p r o l a m i n e as i t d i d on wheat g l i a d i n . T h i s suggests t h a t t h e yam p r o t e i n a s e may f u n c t i o n in v i v o by h y d r o l y z i n g t h e d i f f i c u l t l y - s o l u b l e p r o l a m i n e s d u r i n g l o n g term s t o r a g e o f the t u b e r s under t r o p i c a l c o n d i t i o n s . R e s u l t s in T a b l e 4 a l s o suggest t h a t the p r o t e i n a s e is not a s e r i n e p r o t e i n a s e . T e s t s w i t h the two e f f e c t o r s , l e u p e p t i n and p e p s t a t i n - A , showed no i n h i b i t i o n of a c t i v i t y w i t h a z o c a s e i n as s u b s t r a t e . The observed i n h i b i t i o n of a c t i v i t y by f e r r i c ions suggests t h a t i t is not a metal loenzyme. Thus, i t appears to be d i f f e r e n t from the known p l a n t p r o t e i n a s e s . Attempts t o p u r i f y the enzyme by Sephadex chromatography were not s u c c e s s f u l because o f e x c e s s i v e losses of a c t i v i t y a f t e r passage over t h e columns. Such losses after chromatography suggested t h a t the p r o t e i n a s e may be d i s s o c i a t i n g i n t o s u b u n i t s ( o r changing c o n f o r m a t i o n ) t h a t have very l i t t l e a c t i v i t y . The f i n a l bands o f a c t i v i t y were so f a i n t t h a t they c o u l d not be photographed. The p u r i f i e d enzyme gave a s i n g l e , very l i g h t band upon e l e c t r o p h o r e s i s and attempts t o improve sharpness of the band in the g e l s were not s u c c e s s f u l . Electrophoresis with molecular weight markers p r o v i d e d a crude e s t i m a t e o f t h e s i z e . The crude enzyme ( u n d i s s o c i a t e d ) d i d not e n t e r t h e gel as r e a d i l y as d i d r a b b i t muscle myosin ( 2 0 5 , 0 0 0 ) , but w i t h sodium dodecyl s u l f a t e (SDS), the subunits migrated faster than d i d the carbonic anhydrase marker ( 2 9 , 0 0 0 ) . Protein in Stored Tubers. The heat t o l e r a n c e , a l k a l i n e pH optimum, and s u b s t r a t e s p e c i f i c i t y of the yam p r o t e i n a s e suggested t h i s enzyme as t h e primary cause of p r o t e i n l o s s d u r i n g s t o r a g e . F i g . 2 shows f r e s h yams l e s s than a month a f t e r h a r v e s t and a similar t u b e r a f t e r 4 months storage a t ambient temperature (24-27°C) in open paper bags in a c l o s e d l a b o r a t o r y c a b i n e t . The t e m p e r a t u r e , h u m i d i t y , and l i m i t e d a i r f l o w promoted s p r o u t i n g of the t u b e r s . The new s p r o u t was over 25 cm. in l e n g t h and attempts


21.

OSUJI ET AL.


2

r

1

ol F i g u r e 1.

F i g u r e 2.

U

U

1

2

U

U

I I

3 4 5 HOURS Heat S t a b i l i t y o f Yam Tuber A l k a l i n e P r o t e i n a s e a t 60°(with a z o c a s e i n as s u b s t r a t e ) .

Photograph o f Yam T u b e r s , Fresh (A) and a f t e r 4 Months Storage (B) Showing a T y p i c a l Sprout t h a t Occurs in S t o r e d T u b e r s .


269


Peanut Arachin

O.26Ù

Substrate

Activity ( a t 750nm)

O.018

Azocasein

+

Ô.âdÔ

Egg Albumin

Bovine Hemoglobin O.3έ5

Azocasein + P e p s t a t i n-A

Bovine Serum Albumin Ô.3Ô5

O.018 O.016

Azocasein Leupepti η

3-Lactoglobulin

S u b s t r a t e S p e c i f i c i t y of Yam Tuber A l k a l i n e P r o t e i n a s e (Activity: mg p r o t e i n h y d r o l y z e d ) / 1 0 mg enzyme/2hr).

Activity ( a t 366 nm)

Substrate

Table 4.

Wheat Gliadin O.771O.805

Yam Protein Ô.Ô6Ô

21.

OSUJIETAL.


271

to extract protein from the residual t u b e r were fruitless, i n d i c a t i n g a r a p i d and severe l o s s of p r o t e i n n i t r o g e n . S i n c e the tubers a l s o l o s e n i t r o g e n v i a u r e i d e metabolism d u r i n g storage, consumption of yams a f t e r extended s t o r a g e under adverse c o n d i t i o n s could introduce nutritional disorders where o t h e r sources of p r o t e i n are l a c k i n g . E i t h e r the t u b e r s would have t o be consumed soon a f t e r h a r v e s t or a method must be found to improve the t r a d i t i o n a l ways o f s t o r i n g yams in the t r o p i c s . An attempt was made t o p r e p a r e dehydrated f l a k e s from yam t u b e r s by the SRRC-developed method f o r p r e p a r i n g sweet p o t a t o flakes (]). T h i s would i n h i b i t a l l enzyme a c t i v i t i e s , remove the l a r g e amounts of water ( T a b l e 1 ) , and r e t a i n the i n i t i a l p r o t e i n and nitrogen of the tubers. Since the SRRC process is e n e r g y - i n t e n s i v e and a p p l i c a t i o n s in West A f r i c a would have to depend p r i m a r i l y on a v a i l a b l e manpower, 12.5 kg of yam tubers were p e e l e d by hand, d i c e d i n t o 2 χ 2 cm. p i e c e s , b o i l e d in water and s t i r r e d w i t h a wooden paddle in a l a r g e k e t t l e , then poured i n t o the mechanical f i l t e r f o r removing f i b r o u s m a t e r i a l and mashing. The puree was manually t r a n s f e r r e d t o a hopper and drum-dried by p a s s i n g steam through the s l o w l y r e v o l v i n g drum (the only step t h a t w i l l r e q u i r e f o s s i l energy in A f r i c a ) . The f l a k e s o b t a i n e d ( F i g . 3) were o f f - w h i t e in c o l o r , had good t e x t u r e and r e p r e s e n t e d over 60% r e c o v e r y on a dry weight b a s i s . Over 95% of the o r i g i n a l n i t r o g e n of f r e s h t u b e r s was r e c o v e r e d , s u g g e s t i n g a p o t e n t i a l means of p r e s e r v i n g yams w i t h o u t f e a r of l o s s e s due t o s p r o u t i n g , i n s e c t s , fungal d e g r a d a t i o n , e t c . , in the t r o p i c s . Yam f l a k e s s h o u l d be more s t a b l e f o r extended s t o r a g e than f r e s h yams and, as f l a k e s , they would r e q u i r e l e s s storage s p a c e .

F i g u r e 3.

Dehydrated F l a k e s Prepared from F r e s h P e e l e d Yam Tubers by t h e SRRC-Sweet P o t a t o F l a k i n g Process.


272

PLANT PROTEINS

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

Coursey, D. G.: "Yams"; Longmans, Green: London, 1967; p. 172; 135. Osuji, G.O.; Ory, R. L. J. Agric. Food Chem., In Journal review 1965. Copeland, Ε. B. Philipp. J . Sci. 1916, 11, 277. Prain, Sir D.; Burkill, I. H. Ann. R. Bot. Gdn., Calcutta 1936, 14, 1. Osuji, G. O. Acta Biol. Med. Germ., 1981, 40, 1497. Osuji, G. O.; Umezurike, G. "The Biochemistry and Technology of the Yam Tuber"; ASUTECH Press, Enugu, Nigeria, 1985. Wadsworth, J. I.; Koltun, S. P.; Gallo, A. S.; Ziegler, G. M.; Spadaro, J. J. Food Technol. 1966, 20 (6), 111. Walter, W. M.; Collins, W.; Purcell, A. E. J. Agric. Fd. Chem. 1984, 34, 695. Coddington, A. Biochim. Biophys. Acta 1965, 99, 442. Bray, R. C. "The Enzymes." Eds. : Boyer, P. D.; Lardy, H.; Myrbäch,K., Academic Press, Inc., New York, 1963; no. 7, 533. Mahler, H. R. "The Enzymes." Eds. : Boyer, P. D.; Lardy, Η.: Myrbäch, K., Academic Press, Inc., New York, 1963; vol. 8, 285. Singh, R.; St. Angelο, A. J.; Neucere, N. J. Phytochemistry 1970, 9, 1535. Trijbels, F.; Vogels, G. D. Biochim. Biophys. Acta 1966, 113, 292. Ory, R. L.; Gordon, C. V.; and Singh, R. Phytochemistry 1969, 8, 401. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265. Ueda, H.; Sasaki, T. J. Pharm. Soc. Japan 1956, 76, 745. Fosse, R., C. R. Acad. Sci. 1926, 182, 869. Mothes, K.; Engelbrecht, L. Flora 1952, 139, 586. Krupk, R. M.; Towers, G. H. N. Canad. J. Bot. 1959, 37, 539. Brunel, Α.; Capelle, G. Bull. Soc. Chim. 1947, 29, 427. Kushizaki, M.; Ishiguka"; J.; Alkamatsu, F J. Sci. Soil Manure, Japan 1964, 35, 232. Ishizuka, J.; Okino, F.; and Hoshi, S. J. Sci. Soil Manure, Japan 1970, 44, 78. Thomas, R. J.; Feller, U.; Erismann, Κ. H. Plant Physiol. 1979, 63, Suppl. 50. Osuji, G. O.; Ory, R. L.; Graves, Ε. E. J. Agric. Food Chem., in journal review 1985. Santarius, K., and Belitz, H. D. Planta 1978, 141, 145. Ryan, C. A. Ann. Rev. Plant Physiol. 1973, 24, 173. Tookey, H. L.; Gentry, H. S. Phytochemistry 1969, 8, 989. Ashton, F. M. Ann. Rev. Plant Physiol. 1976, 27, 95.

RECEIVED January 24, 1986


Protein-Nitrogen Conservation in Fresh Stored Dioscorea Yams - ACS

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