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dissertation entitled

OL-GALACTOSIDASES 0F COWPEAS

(Vigna unguiculata)

 

presented by

Souod Rashid Alani

has been accepted towards fulfillment
of the requirements for

Ph.D. . Food Science
degree in

 

 

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.-

J.“

C: 9'
~
.

a-GALAC‘I'OSIDASES or commas
(219m immune)

BY

Souod Rashid Alani

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Food Science and Human Nutrition

1988

ABSTRACT

a-oanc'rosrnasrs 0" COM

(liens W)

By
Souod R. Alani

Cowpeas (11311: W) are the main source of

protein for many people in the world. However, like other
legume seeds cowpeas contain a-galactosides which cannot be
hydrolyzed and absorbed because of the lack of a-
galactosidase activity in the small intestine.

The objectives of this research were to (a) purify and
study the properties of cowpea a-galactosidases, (b) study
the activity of a-galactosidases in dormant and germinating
cowpeas and (c) determine the changes in oligosaccharides
during the germination of seeds and the incubation of a
paste from the seeds.

Three forms of a-galactosidases, I, II1 and 112 were
isolated from dormant cowpeas by means of gel filtration
and ion exchange chromatography. These forms exhibited
different values of optimum activity, pH 5.0, 5.9 and 5.3,
respectively. Their optimum activity temperature, 50' C,
was the same. The molecular weights (MW) obtained by gel
filtration chromatography were approximately 110,720 1
3780, 29,000 i 355 and 30,000 i 368, respectively. SOS-PAGE

of these enzymes yielded the following MW values: 30,916 i

Souod R. Alani

1,042, 29,875 i 413 and 30,350 i 550 daltons, respectively.
Based on the results obtained (Enzyme I and II2 were eluted
at the same pH and NaCl gradients from ion exchange
chromatography columns), enzyme I appears to be a tetramer
of enzyme 112. The Km and Vmax were determined using
artificial and natural substrates. The stability of enzymes
I, 111 and 112 during storage for 24 hrs at four different
temperatures and at various pH values were studied.
Germination of cowpeas resulted in a significant
increase in a-galactosidase activity. The oligosaccharide
changes during the germination of cowpeas and the
incubation of their paste were studied. Germination did not
have a significant effect on the oligosaccharide content
during 24 hr. Incubation of a cowpea paste made from
germinated seeds resulted in a considerable reduction of
the flatus-related oligosaccharides. It appears that the
most efficient way to decrease the flatus-related sugar of
cowpeas, especially stachyose, is to form a cowpea flour

paste and let it stand overnight in a warm place (30' C).

 

Dq1fliEbflflWElDFAJJJUH
1HIEthRIHFIH.I}flSCIHMPASSRJNAITZ

TO

My Father Rashid and My Mother Sabriah

ii

ACKNOWLEDGMENTS

All praise and thanks are due to Allah, Lord of the
universe, for His merciful divine direction throughout my
study. 3

The author is deeply indebted to his major professor
Pericles Markakis for his assistance, guidance and
encouragment in conducting this study. Sincere gratitude is
extended to Dr. Denise 14. Smith for her valuable advices
and constructive comments. The author also wishes to extend
his appreciation and gratitude to Dr. Won 0. Song for her
helpful discussions and crtitical evaluation of this
manuscript.

The author is most greatful to his freinds, T. Znati,
J. Dhahir, A. Salih and F. Fathy for their encouragements
throughout the course of his graduate studies.

Special thanks are extended to the members of my family

for their support, patience and understanding.

iii

TABLE OF CONTENTS
page

LIST OF TABLES.......................................vii
LIST OF FIGURES.......................................ix
INTRODUCTION...........................................l
LITERATURE REVIEW......................................4

OCCURRENCE AND LOCALIZATION.........................4
PROPERTIES OF a-GALACTOSIDASES......................5
MULTIMOLECULAR FORMS................................6
MOLECULAR WEIGHTS...................................7
SPECIFICITY.........................................7
A. Hydrolase Activity............................7
B. Transgalactosylation. .......................11
KINETIC PROPERTIES.................................12
A. Effect of Substrate Concentration............12
B. Effect of Temperature........................13
C. Effect of pH.................................13
D. Effect of Inhibitors.........................14
MECHANISM OF ACTION................................15
DETECTION AND METHODS OF ASSAY.....................19
PURIFICATION.......................................20
ROLE OF THE ENZYME.................................22
CHANGES IN a-GALACTOSIDASE DURING SEED MATURATION..23
OLIGOSACCHARIDES AND a-GALACTOSIDASE...............23
GERMINATING LEGUME SEEDS...........................25
CHANGES IN a-GALACTOSIDASES
DURING SEED GERMINATION.........................25
PHYSIOLOGICAL SIGNIFICANCE OF a-GALACTOSIDASES.....26
POSSIBLE APPLICATION..............................27
Sugar Factory...................................27
Soymilk ........................................27
Blood-group-B...................................28

EXPERInNTAL.PROCEDUREOOO00....O0....0.0.0.0000000000029

MATERIALS..........................................29
METHODS............................................3O
ENZYME ASSAY ....................................30
p-NITROPHENOL STANDARD CURVE......... ...........31
ENZYME ISOLATION AND PURIFICATION .... ..........31
MOLECULAR WEIGHT DETERMINATION......... .........33

Gel Filtration Chromatography
(Sephacryl 8-200)............................33
Disadvantages of gel chromatography .........35
1. Molecular weight markers (from Sigma)....35
2. Gel preparation...........................35
3. Column packing and equilibration......36
4. void volume determination.................36
5. Elution volume determination
for protein standards...................37
6. Standard curve...........................37
MW Estimation Using SDS-PAGE....................38
1. MW markers...............................38
2. Resolving gel-11% acrylamide..............38
3. Stacking gel-4% acrylamide................39
4. Sample preparation (Laemmli, 1970).....39
5. Standard curve............................40
POLXACRYLAMIDE GEL ELECTROPHORESIS (PAGE)..........40
Anionic PAGE (pH 8.3).........................40
Cationic PAGE ,pH 3.5
(Reisfeld et a1., 1962)....................40
Resolving gel, 7.5% acrylamide pH 4.3........41
Stacking gel, 4% acrylamide pH 6.8...........41
Detection of enzyme activity
on polyacrylamide gels (cationic).............41
ENZYME KINETICS....................................42
Initial Velocity................................42
Km and vmax Values for Various Substrates.......42
PNPG.........................................43
Raffinose....................................44
Stachyose....................................45
Enzyme I. ................................46
Enzyme II ...............................46
Effect of Substrate (Raffinose) Concentrations
on Enzyme Activity.............................47
STABILITY..........................................47
OPTIMUM ACTIVITY pH AT 30' C......................47
OPTIMUM ACTIVITY TEMPERATURE AT OPTIMUM pH......48
GERMINATION AND INCUBATION OF COWPEAS..............48
1- Experimental Design..........................48
2- Quantitation of 01igosaccharides............48
3- a-Galactosidase Activity and Oligosaccharides
Content of Germinating Cowpeas...............49
4- Incubation...................................49
5- Germination and Incubation ..................50
6- a-Galactosidase Activity During Sprouting....50
INVERTASE ACTIVITY.................................50
Extraction Method (Al-Baker and Whitaker, 1978).51
Invertase assay using HPLC..................51

“SULTS AND DIstSIONOOOOOOOOOO00.000.00.000.0.0.0.0053

ENZYME ASSAY.......................................53
ENZYME PURIFICATION................................53
POLYACRYLAMIDE GEL ELECTROPHORESIS.................7l
MOLECULAR WEIGHT DETERMINATION.....................74
Gel Filtration Chromatography
Using Sephacryl S-200...........................74
MW Estimation Using SOS-PAGE chromatography.....83
ENZYME KINETICS................1.......2...........87
and V x for Enzyme I, II and II
Using PNga as Substrate.......................187
Determiging Km and Vm x Values of Enzymes I, II
and II Using NaturaI Substrates...............87
Raffinose....................................91
Stachyose....................................95
Effect of a Natural Substrate Concentration
on Enzyme Activity............................98
STABILITY.........................................100
OPTIMUM pH AND OPTIMUM TEMPERATURE
FOR THE ENZYME ACTIVITY...........................104
GERMINATION EFFECT ON COWPEA a-GALACTOSIDASE......112
GERMINATION EFFECT ON COWPEA OLIGOSACCHARIDES.....114
SEED PASTE INCUBATION.............................116
GERMINATION PLUS INCUBATION OF THE SEED PASTE.....119
Safety Concerns................................122
INVERTASE ACTIVITY................................122

CONCLUSIONS AND DIRECTIONS FOR FUTURE RESEARCH.......124
APPENDICES O O O O O C O O O O O O O O O O O O O O O O O O O I O O O O O O O O O O O O O O O O O 128
Appendix 1 O O O O O O O O O O O O O O I O O O O C O O O O O O O O O O O O O O O O O O O O 128
Appendix 2 O O O O O O O O O O O O O O O O I O O O O O O I O I O O O O O O O O O O 0 O O O 129

BIBLIOGMPHY...O...00.0.0.0...OOOOOOOOOOOOOOO0.0.0.00130

vi

Table

. Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

9.

10.

11.

12.

LIST OF TABLES
Purification of cowpea (Vigna nnguignlata)

a-galactosidases, method I.....................54

Purification of cowpea (liens Mata)
a-galactosidases, method II...................55

Effect of acid precipitation on the
specific activity of the precipitate
(Stage 2)OOOOOOOOOOOOOO00.00.0000....0.0.0.0.0056

Effect of ammonium sulfate on the
specific activity of the precipitate
(Stage 3)OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO056

and Via values of a-galactosidases using
PPGas angstrate...OOOOCOOCOOOOOOOOOOO0.......87

and V x values of a-galactosidases
using rafIInose as substrate...................91

and Vb x values of a-galactosidases
using stacgyose as substrate...................95

Effect of substrate (raffinose) concentration
on a-galactosidase activity ...................99

Temperature and pH optima of a-galactosidases
from cowpeaSOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0.0105

Total a-galactosidase activity in sprout
and desprouted seeds of cowpeas at 30' C
(DB 505' PNm)OOCOOOCOOOOOOOOOOOOOOOOOOOOOO00.112

The statistical analysis of oligosaccharide
concentration changes during germination
for 24 hrs at 24.COOOOOOOOOOOOOOO0.0.0.0...114

The statistical analysis of oligosaccharide

concentration and pH changes during flour
paste incubation at different temperatures....117

vii

Table 13.

Table 14.

The statistical analysis of oligosaccharide
concentration and pH changes during 2 hrs of
soaking, 6 hrs of germination, followed by

12 hrs of incubation of a paste made from
germinated seeds at two different temperatures,
24' and 34‘ C.................................120

values of Enzymes I, II1 and 112 with
d fferent substrates..........................125

viii

Figure
Figure
Figure
Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

1.

2.

4.

10.

11.

12.

13.

14.

15.

LIST OF FIGURES

Structure of stachyose and related sugars......2
Glycosides hydrolyzed by a-galactosidase.......9
Mechanism of action of a-galactosidase........18
Enzymatic release of p-nitrophenyl

from PNPG (I). Color development by

increasing the pH of the p-nitrophenol
salution (II)OOOOOOOOOOOOOOOOOOI0.0.0.0000000021

Outline of the purification scheme for
a-galactosidases from cowpea..................34

Time-dependence of PNPG hydrolysis by
a-galactosidase...............................57

Absorption spectrum of p-nitrophenol freed
from PNPG by a-galactosidase..................58

p-Nitrophenol standard curve measured
at 405 MOIOOOOOOOOOOOOOOOOOOO00.0.0000000000059

Effect of extraction pH on a-galactosidase
activity measured at 405 nm ..................60

Specific activity of supernatant after
precipitation of in active protein at
various pH, measured at 405 nm (stage # 2)....61

Elution profiles of a-galactosidases
I and II from G-100 gel filtration column.....62

Elution profile of a-galactosidase I recycled
through a G-100 gel filtration column.........63

Elution profile of a-galactosidase II recycled
through a G-100 gel filtration column.........64

Elution profiles of a-galactosidases I
and II on an G-200 gel filtration column......66

Elution profile of a-galactosidase I on
an Accl-CM (Waters) column.............. ...... 67

ix

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

Elution profile of a-galactosidase II on
Accl-CM (Waters) column.......................68

Profile of a-galactosidase I on
an CM-cellulose column eluted with
a sodium chloride gradient....................69

Profile of a-galactosidase II on an
CM-cellulose column eluted with a sodium
chloride gradient.............................70

Anionic polyacrylamide disc gel
electfophores§s of a-galactosidases
, II and II at pH 8.3 (Coomassie stain).

Rf-0.26.00.00.0000000000......0.0.00.0...0.072

Polyacrylamide disc gel e1 crophorgsis
of a-galactosidases I, II and II at

pH 3.5. The same bands were visualized

by Coomasie stain and by enzyme activity
detection (Fast blue RR salt). Rf - 0.33......73

Elution profile of Blue Dextran
(MW 2,000,000, V0 - 54.5, VT - 144 ml)
from Sephacryl S-200 column...................75

Elution profiles of alcohol dehydrogenase

(MW 150,000, V - 62.0 ml) and carbonic
anhydrase (MW 39,000, Ve - 87.6 ml) from
Sephacryl S-200 column........................76

Elution profiles of bovine albumin

(MW 66,000, Ve - 69.0 ml) and cytochrome

C (MW 12,400, Ve s 97.0 ml) from Sephacryl
S-200 column..................................77

Elution profile of Enzyme I (at pH 5.0)
from Sephacryl S-200 column. Ve - 65.5 ml.....78

Elution profile of Enzyme II1 (at pH 7.0)
from Sephacryl S-200 column. Ve - 85.0 ml.....79

Elution profile of Enzyme 112 (at pH 7.0)
from Sephacryl S-200 column. Ve - 84.6 ml.....80

MW calibration curve using protein standards

on sephacnl 8-200, based on Ve /v0 0 o o o e e o e e 081

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

MW calibration curve using protein standards
on Sephacryl S-200, based on KaV’°'°'°"°""°82

SDS- {GE of cgwpea a-galactosidases

I, II and II . MW of markers:

1, bovine albumin, 66,000; 2, egg

albumin, 45,000: 3, glyceraldehyde-3-phosphate
dehydrogenase, 36,000: 4, carbonic anhydrase,
29,000; 5,trypsinogen, 24,000: 6,trypsin
inhibitor, soybean,20,000; 7, a-lactalbumin,

14,000.00....OOOOOOOOOOOOOOOOOOOOOO0.0.000000085

Molecular weight calibration curve
using protein standards and SDS-polyacrylamide
gel electrophoresis...........................86

Lineweaver-Burk plot of the hydrolysis
of PNPG by a-galactosidase I. n a 3..... ...... 88

Lineweaver-Burk plot of the hydrolysis
of PNPG by a-galactosidase II . n - 3... ...... 89

Lineweaver-Burk plot of the hydrolysis
of PNPG by a-galactosidase II . n - 3.........90

Lineweaver-Burk
of raffinose by

plot of the hydrolysis
a-galactosidase I. n a 3. ..... 92

Lineweaver-Burk plot of the hydrolysis

of raffinose by a-galactosidase II . n = 3....93
Lineweaver-Burk plot of the hydrolysis

of raffinose by a-galactosidase II . n - 3....94
Lineweaver-Burk plot of the hydrolysis

of stachyose by e-galactosidase I. n s 3 ...... 96
Lineweaver-Burk plot of the hydrolysis

of stachyose by a-galactosidase II . n = 3....97
Effect of pH and temperature on the

stability of Enzyme I after 24 hr

at storage.OOOOOOOOOOOOOOO0.0.0.0... ...... 00.101
Effect of pH and tempefature on the

stability of Enzyme II after 24 hr

at storage.IOOOOOOOOIOOOOOOOOOOOO0.000.0.00.0102

xi

Figure

Figure
Figure
Figure
Figure

Figure

Figure

Figure

Figure

Figure

Figure

41.

42.
43.
44.

45.

46.

47.

48.

49.

50.

51.

Effect of pH and tempesature on the
stability of Enzyme II after 24 hr
at storageOOOOOOOOOOOOOOOOOO0..0.0.0.0...0.0.103

Effect of pH on the activity of Enzyme I.....106

Effect of pH on the activity of Enzyme III...107
Effect of pH on the activity of Enzyme IIZ...108

Effect of
of Enzyme

temperature on the activity

IOOOOOCOOOOOOOOOOOOOC00.0.00...0.0.109

Effect of

teTperature on the activity
of Enzyme II

OOOOOOOOOOOOOOOOOOOOOOOCOO0.0.0.110

Effect of

tegperature on the activity
of Enzyme II

COOOOOOOOOOOOOO0.00.00.00.000.0.111

a-Galactosidase activity at different
germination temperatures (2 hrs soaking
prior to germination)........................113

Oligosaccharide changes during germination
for 24 hrs at 24' C (2 hrs soaking prior
to germination). n I3.0.00.000000000000000.115

Oligosaccharide and pH changes during
24 hrs of cowpea flour paste incubation
at different temperatures............ ........ 118

Oligosaccharide concentration and

pH changes during 2 hrs of soaking,

6 hrs of germination, followed

by 12 hrs of incubation of a paste made

from germinated seeds at two different
temperatures, 24' and 34' C............. ..... 121

xii

INTRODUCTION

Almost a hundred years ago, an enzyme was isolated from
yeast which hydrolyzed melibiose, 6-a-D-galactopyranosyl-D-
glucose. This enzyme was first called melibiase and later
renamed a-galactosidase (a-D-galactoside galactohydrolase,
E.C.3.2.1.22). The new name is due to the discovery that
the enzyme hydrolyzes a-galactosides in general. However,
it was also noticed that a-galactosidases catalyze trans-
a-D- galactosylation reactions.

These enzymes have multiple significance. They are used
in molecular structure studies in biochemistry. In plant,
animal and microbial physiology they serve to trace the
fate of the many a-galactosides present in nature. In
applied enzymology, they have been suggested as tools for:
a) Removing the flatus-generating galactosides, namely

raffinose, stachyose and verbascose present in legume

seeds (beans, peas, cowpeas, soybeans), Figure 1.

b) Facilitating the extraction of sucrose from molasses or
increasing the sucrose yield by eliminating raffinose
which interferes with the crystallization of sucrose.

c) Converting type-B human erythrocytes to type -o- by

removing the a-galactoside residues.

a-GALACTOSIDASE INVEHTASE

  

I
I
I
CH2 :
H H: Hoca' o H
OH H : H OH
i
OH .0 CH20H
H L0H OH H

. l , FRUC.
L MELIBIOSE

GALACTOBIOSE ——J L— sucnoss

L——wwmmormoss '
[ L RAFFINOSE

STACHYOSE ‘ I

 

 

 

 

 

 

 

Figure 1. Structure of stachyose and related sugars

Several factors motivated the choice of cowpeas in our
study. Cowpeas (yigna ungnignlata) are the main source of
protein for many people in Africa. The nutritional value
of cowpeas lies in their high protein content, which is 20-
253 and is double the protein value of most cereals
(Appendix 1).The practice of eating cowpea dishes in
combination with cereal foods enhance their protein value
(Dovlo et al., 1976) . Nevertheless, cowpeas like other
legume seeds contain a-galactosides which cannot be
hydrolyzed and absorbed in the gut for lack of a-
galactosidase activity in the small intestine. In the large
intestine,however, these galactosides are fermented by
microorganisms and may cause flatulence, distention, cramps
and diarrhea. These symptoms can be more severe in young
children.

The objectives of this research were to study:

a) The purification and properties of cowpea a-
galactosidases.

b) The activity of a- galactosidases in dormant and
germinating cowpeas.

c) The oligosaccharide changes during the germination of

seeds and the incubation of a seed paste.

LITERATURE REVIEW

OCCURRENCE AND LOCALIZATION

e-Galactosidases (a-D-galactoside galactohydrolase
,E.C.3.2.1.22) have been shown to be widely distributed in
plants, animals and microorganisms. As a rule, these
enzymes are present in all tissues containing a-D-
galactosides (Barham et al., 1971; French,1954: Wollenfels
and Malhotra,1961) . The galactosyl oligosaccharides rank
next to sucrose in their abundance in the plant kingdom
(Dey, 1980).

a-Galactosidases in most cases are found
intracellularly in various organelles (mitochondria,
chloroplasts, microsomes).In E;, 9911 (Burstein and
Kepes,1974; Gabriel and Bonner,1957; Budiger,1968) and
W meg, a-galactosidase is not constitutive
but can be induced by the introduction of a-D-
galactosidases in the culture media (Bailey and Howard,
1963).

Multiple forms of the enzyme have been found in various
parts of seeds (e.g., cotyledon, endosperm and embryo) and
root tips (Dey and Campillo, 1984). The a-galactosidase
activity is generally located in the water soluble fraction

of an extract. However, in several cases the activity was

cell-wall bound (Klis et al., 1974; Thomas and Webb,
1979).The bound enzymes can be extracted by salt solution .

The presence of a-galactosidase has been shown in the
protein bodies isolated from seeds (Plant and Meor, 1982;
Hankins and Shannon, 1978) . Protein bodies are generally
the sites of storage proteins and other hydrolytic enzymes

(Van der Wilden et al. 1980: Harris and Chrispeels, 1975).

PROPERTIES OF c-GALACTOSIDASES

a-Galactosidases catalyze the following reversible
reaction:

 

where R represents alkyl, aryl, monoglycosyl or
polyglycosyl groups.

Dey and Pridham (1969a) reported that most forms of the
enzyme are not absolutely specific for the glycone residue
and will hydrolyze the structurally related a-D-
fucopyranosides and fi-L-arabinopyranosides. Water can be

replaced by a number of organic galactose acceptors. In

this way simple galactosides and oligosaccharides have been
synthesized. De novo synthesis of oligosaccharides is also
possible when high concentrations of galactose are
incubated with the enzyme (Day and Pridham,1972) . Tanner
and Kandler (1968) observed that for an a-galactosidase
isolated from W W, the transgalactosylation
from galactinol to raffinose (with the formation of

stachyose) occurs more readily than hydrolysis.

NULTINOLECULAR FORMS

Petek and Dong (1961) were first to report multi-
molecular forms of a-galactosidases. Using alumina column
chromatography, they isolated tow separate forms of this
enzyme. Later Dey and Pridham (1969a) showed that dormant
seeds of L m have tow forms of a-galactosidase with
different molecular weights. In later work the tow forms of
the enzyme (I and II), obtained from Sephadex columns, were
re-examined by ion exchange (CM-cellulose) chromatography.
Enzyme II was eluted with tow active fractions (II1 and
112) (Dey and Pridham, 1972).

Some dormant seeds appear to contain a-galactosidase of
only one molecular size. The number of forms of a-
galactosidase may be related to the physiological state of
the seed (Barham et al.,1971). Forms of a-galactosidases

showed different pH optima and Km and Vmax values with

artificial and natural substrates, and also differed in
their thermal stabilities (Dey and Pridham, 1969b).

MOLECULAR WEIGHTS

Many dormant seeds contain two forms of the enzyme, one
form apparently having a substantially higher molecular
weight (MW) than the other. Dey and Pridham (1969b) showed
that a-galactosidase I, the high MW form from y. m, has
a subunit structure. This enzyme dissociated into six
inactive protein fractions when passed through a Sephadex
G-100 column in the presence of 6 M-urea. The a-
galactosidases isolated from various sources have different
molecular weight. Most of these were estimated by gel

filtration and SDS-PAGE and ranged between 25,000-200,000.

SPECIFICIT!

A. Hydrolase Activity

In general, change of configuration of hydrogen and
hydroxyl groups on any single carbon atom of a glycoside
substrate is sufficient to reduce the rate or completely
inhibit the hydrolytic action of the corresponding
hydrolase. Two main factors govern the rate of hydrolysis
of the substrate by a-galactosidases. First, the ring
structure must be pyranoid, and second, the configuration
of -H and -OH on carbon atoms 1,2,3, and 4 must be similar

to that of a-D-galactose. Changes at C-6 of the glycosyl

moiety of the substrate can be tolerated by a-
galactosidases as in the case of other glycosidases, such
as, p-galactosidase, fi-glycosidase and a-mannosidase (Adams
et al., 1943: Helferich and Appel, 1932). Hence the
glycosides indicated in Figure 2 can be hydrolyzed by the
a-galactosidases (Dey and Pridham, 1972). The affinity
of the enzyme (l/Km) for the substrate seems to depend
largely upon the structural changes in the sugar moiety and
follows the order :a-D-galactoside > a-D-fucoside > p-L-
arabinoside. This suggests that one of the specific points
of attachment of the substrate to the enzyme is through the
primary alcohol group of the galactose structure (Dey and
Pridham, 1972).

The aglycone group of a substrate may or may not have a
marked effect on hydrolysis by glycosidases. Normally, the
group does not completely inhibit hydrolysis. Various a-
galactosidases are known which hydrolyze the following
naturally occurring and synthetic a-D-galactosides :

1) Galactosides: methyl-, ethyl-, n-propyl-, phenyl, o-
nitrophenyl-, m-nitrophenyl-, p-nitrophenyl-, m-chloro-
phenyl-, and 6-bromo-2-naphthyl a-D-galactosides (Sheimin
and Crockar, 1961: Tsou and Su, 1964).

2) Oligosaccharides: melibiose, 0-a-D-gal-(1-> 4)-D-

gal, melibionic acid, raffinose, stachyose, verbascose, and

(:11on
MD A

OH 0—“

OH
cf-D-Galacteside

CH3

HO

OH

‘(‘D'Fucoside

HO
OH

OH

p—L-Arabinoside .

$H20H
HCOH

HO
OH

OH

U'Glyc ere-<-D-
galactohepteside

Figure 2. Glycosides hydrolyzed by a-galactosidase

10

higher homologues (Suzuki et al., 1970; Bailey and Howard,
1963: Bailey, 1962).

3) Polysaccharides: galactomannans are attacked by a-
galactosidases from various sources (Bahl and Agrawal,
1969: Courtois and Petek, 1966). These polysaccharides
normally have a basic structure cosisting of a backbone of
p-1,4-linked D-mannosyl residues to which D-galactosyl
residues are attached by a-1,6-linkages. The galactose
content varies depending on the plant source. Not all e-
galactosidases hydrolyze galactomannans: those that do,
appear to remove terminal galactose residues only. Some o-
galactosidase can also remove terminal a-D-galactosyl
residues from blood group B substance (Zarmitz and Rabat,
1960; Watkins, 1966). In general, aryl a-D-galactosides are
better substrates than alkyl derivatives or disaccharides
(Dey and Pridham, 1972: Wallenfels and Malhotra, 1961).

High affinity of the enzyme for the substrate does not
necessarily correlate with high V max values (Dey and
Pridham, 1972). Dey and Pridham (1969a) reported that the
Vmax of the enzymes from L Lab; is not influenced by the
nature of the substituent on the phenyl ring of aromatic
galactosides. The affinity (1\Km) shows some tendency to
increase when electron-attracting substituents are present
(Bailey, 1962: Bailey and Howard, 1963). The conversion of

melibiase to melibionic acid, oxidation of the reducing

11

group, does not appear to affect the rate of hydrolysis
(Dey and Pridham, 1972). The rate of hydrolysis decreased
with an increase in the chain length, in a homologous
series of a-D-galactosides, but in two microbial enzymes
the reverse was reported to occur (Petek et al.,1969:
Baily, 1963).

French et al. (1953) reported that almond a-galacto-
sidase was able to split the internal galactosidic linkage
of stachyose, forming galactobiose and sucrose. Coffee a-
galactosidase can only cleave stachyose in a stepwise
fashion starting from the nonreducing end. a-Galactosidase
of We b.9115 hydrolyzes verbascose in a similar
stepwise mode (Bailey and Howard, 1963).

B. Transgalactosylation

Blanchard and Albon (1950) were first to observe the
transgalactosylase activity of yeast a-galactosidase. They
showed that galactose could be transferred from melibiase
to a second melibiase acceptor molecule with the formation‘
of manninotriose. Transgalactosylation reactions to
aliphatic hydroxyl groups are normally accompanied by a
pronounced. hydrolytic activity. An a-galactosidase from
wheat bran with a high transfer/hydrolysis ratio has also
been described by Wohnlich (1963) . Li and Shetlar (1964)
have shown with pneumococcal a-galactosidase that

phosphorylation, reduction or oxidation of either C-1 or C-

12

6 of D-glucose or D-galactose destroyed the acceptor
properties of these hexoses. They have further reported
that the incubation of a-galactosidase from 9111:1211
Wis with 0- or p-nitrophenyl a-D-galactosides
gave rise to two oligosaccharides containing only D-
galactose.

In general, it appears that a-galactosidases
preferentially transfer galactosyl groups to the primary
alcoholic groups of the acceptor ( Dey and Pridham, 1972).
Galactosidases are known to synthesize oligosaccharides
when incubated with monosaccharides, and this procedure
has been used for the preparation of several glucose and
galactose derivatives (Helferich and Leete, 1955; Peat et
al., 1952: Stehen et al., 1962). Yeast a-galactosidase, for
example, in a solution of s 17% D-galactose polymerizes
7.5% of the sugar and about 60% of this appears as 6-0-a-D-
galactosyl-D-galactose (Clancy and Whelan, 1960; 1967a). By
allowing a mixture of D-galactose and N-acetyl-D-
glucosamine to react in the presence of yeast a-
galactosidase, Clancy and Whelan (1967b) have also produced

3-0- and 6-0-a-D-ga1actosyl-N-acetyl-D-glucosamine.

KINETIC PROPERTIES
A. Effect of Substrate Concentration
p-Nitrophenyl a-D-galactoside has been shown to be
inhibitory at high concentrations with both molecular forms

13

( I and II ) of a-galactosidase from L fab; (Dey and
Pridham, 1972). In contrast, galactose-containing
oligosaccharides, such as melibiase and raffinose, do not
show any inhibitory effect (Dey and Pridham, 1969b:
Malhotra and Dey, 1967) . Competitive inhibition of the
enzyme has generally been observed with D-galactose (Dey
and Pridham, 1972).
B. Effect of Temperature

a-Galactosidases display varying degrees of stability
depending on their origin (Dey and Campillo, 1984). Crude
enzyme from L 9311 is extremely unstable, whereas the
purified enzyme can be lyophilized and stored at 4' C for
up to 2 months without loss of activity (Jones and Kosman,
1980). The ionic strength of a-galactosidase solutions may
also be an important factor in stability (Carnie and
Porteons, 1963) . Most a-galactosidases behave normally in
the sense that the rate of the enzyme-catalyzed reaction
increases to an optimum value with increasing temperature
until inactivation of the enzyme occurs (Dey and Campillo,
1984).
c. Effect of pH

The pH optima of a-galactosidases vary. Most a-
galactosidases show single broad pH optima, but in several
cases the enzymes exhibit two peaks. The tetrameric enzyme

from mung bean was converted predominantly into the

14

monomeric form on storage in pH 7.0 buffer at -10° C for 2
years. However, on dialysis of the stored enzyme at pH 4.0
reconversion to the tetramer occurred (delCampillo et al.,

1981) .

D. Effect of Inhibitors

L m will; W a-Galactosidase from
W M can be inhibited by sulphydryl
reagents such as N-ethyl malcimide and iodoacetamide
(Flowers and. Sharon, 1979). On the other’ hand, a-
galactosidases from spinach leaves, sweet almond, and L
13b; are not specifically inhibited by such reagents. Thus
not all o-galactosidases require -SH groups for activity
(Somogyi, 1945; 1952; Fielding and Hough, 1971).
Photooxidation in the presence of methylene blue
inactivated a-galactosidases from sweet almond and y; {aha
(Somogyi, 1945: 1952; Dey and Pridham, 1972).

2‘ Metal ign§& The Ag+ inhibition of 2‘ fan; enzyme I
may be attributed to combination with carboxyl and /or
histidine residues in the active site (Dixon and Webb,
1964). Ag+ and Hg++ ions strongly inhibited o-
galactosidases. a-Galactosidase I from L Lana, which
inactivated by Ag+, regained most its activity on dialysis
against McIlvaine buffer (pH 4.0 or 6.0), particularly in

the presence of cysteine. The inhibitory effect of Ag+

15

decreased with low concentrations of galactose. Such
protection was observed only when galactose was added
before the addition of 119+. Thus, Ag+ is a competitive
inhibitor (Dey and Pridham, 1969a). Hg'” is a potent
inhibitor of several a-galactosidases, which suggests
reaction with thiol groups.

1‘ Sugar; and their deriyatiyes; Suzuki et al. (1966)
showed that D-galactose is a powerful and competitive
inhibitor’ of a-galactosidases. L-Arabinose and D-fucose
also inhibited the enzymes as structural analogues of D-
galactose, whereas their enantiomers were ineffective (Dey
and Pridham, 1969a). It has been established that 2-deoxy-
D-galactose, D-glucose, D-mannose, D-xylose, and D-ribose
do not inhabit a-galactosidases (Suzuki at el., 1970; Dey
and Pridham, 1969a). Dey and Pridham (1969a; 1969b) have
shown that various a-D-galactosides were competitive

inhibitors of a-galactosidases.

MECHANISM OF ACTION

Because of insufficient knowledge of the chemistry and
kinetics of the enzymes from most sources, few facts are
available regarding the mechanism of action of a-
galactosidases (Dey and Pridham, 1969a; 1969b; Dey, 1969).

So far there have been no studies on bond fission by a-
galactosidases, although by analogy with other glycosidases
it is likely that the galactose-oxygen bonds of substrates

16

are cleared. Nuclear magnetic resonance and polarimetry
studies have clearly shown that the liberated galactosyl
residues possess the same anomeric configuration as the
substrate in sweet almond a-galactosidase reactions.

Specificity studies on sweet almond a-galactosidase
with aryl a-D-galactosides show that the charges of the
hydroxyl groups of the aglycon has a noticeable influence
on the rate of enzymic hydrolysis. Thus, the hydrolytic
reaction may be attributed to the presence of basic and
acidic groups at the active site. These groups were
identified to be the carboxyl (deprotonated) and
imidazolium (protonated) groups, respectively (Dey and
Pridham, 1972). A similar behavior is also observed with
the 21 tab; enzyme I (Dey and Pridham, 1969a: 1969b: Nath
and Rydon, 1954) . It was further shown with the almond
enzyme that binding of p-nitrophenyl a-D-galactoside to the
active site lowers the pH of the aglycon dissociating on
the acidic side and raises the value of the aglycon
dissociating on the alkaline side of the pH optimum (Dey
and Pridham, 1969b).

A "two-step" mechanism has been postulated for the
action of sweet almond a-galactosidase, based on previous
results (Dey, 1969). The aglycon is cleaved by the
concerted action of carboxyl and imidazolium groups. This

is followed by a reaction with an acceptor molecule (R'OH),

17

which may be water or an aliphatic alcohol, resulting in
hydrolysis or transfer products. It is possible that the
electrophilic attack of the imidazolium group alone is
sufficient to cleave the glycosyl-oxygen bond with the
formation of a carbonium ion at C-1 of the galactose
moiety. In the complete two-step mechanism, two Walden
inversions probably occur, resulting in the retention of
the anomeric configuration in the final product (Dey and
Campillo, 1984).

A "one-step" mechanism of action also has been
suggested (Bahl and Agrawal, 1969). This involves the
formation of a ternary complex of the enzyme, substrate,
and acceptor with the carboxyl and the imidazolium groups
playing a similar role, as in the two-step mechanism.

Recently, Mathew and Balasubramaniam (1987) proposed
another mechanism of action of coconut a-galactosidase. The
presence of two carboxyl groups, a tryptophan and a
tyrosine at or near the active site of a-galactosidase was
indicated. Based on this a new mechanism of action for o-
galactosidase was suggested in which the ionizing group
with a pKa of 3.5 is a carboxyl group involved in
stabilizing a carbonium ion intermediate. The ionizing
group with a pKa of 6.5 is a carboxyl group perturbed due
to the presence of a hydrophobic residues in its vicinity

which donates a H+ ion in catalysis (Figure 3).

Figure 3. Mechanism of action of a-galactosidase

19

DETECTION AND METHODS OF ASSA!

The reducing disaccharide melibiose, or the non
reducing trisaccharide raffinose, are the natural
substrates of the plant enzyme and are often used for the
enzyme assay (Delente and Ladenburg,1972: Mital et al.,
1974). The liberated hexoses can be measured by monitoring
the increase in reducing power. Alternatively, the
liberated D-galactose can be measured using D-galactose
dehydrogenase (Dey and Kauss, 1981: Kurz and Wallenfels,
1970); in the case of melibiose, free glucose can be
measured using glucose oxidase (Lioyd, 1966).

Synthetic substrates, such as phenyl-, ortho-, or para-
substituted nitrophenyl a-D-galactosides are commonly used
for routine enzyme assays. The liberated phenol and
nitrophenols are estimated using the Folin reagent (Layne,
1957) and the absorbence of the yellow compound at 400-420
nm under alkaline condition, respectively. Plant a-
galactosidases do not have cofactor requirements for
activity (Dey and Campillo, 1984). However, in Escherichia
lei, the cell-free a-galactosidase extract requires Mn++
and NAD for activity (Schmitt and Rotman, 1966).

The enzyme can be located on gels by 4-
methylumbelliferyl a-D-galactoside. The liberated aglycone,
which is water-insoluble, fluoresces under UV light (Dey

and Wallenfels, 1974). Other substrates used for this

20

purpose are 1-naphthyl a-D-galactoside or 6-bromo-2-
naphthyl a-D-galactoside (Hadacov'a and Benes, 1977; Benes
and Hadacov'a, 1980: Williams et al., 1977: 1978; McCleary,
1979). The liberated water-insoluble aglycons are coupled
with Fast Blue BB, resulting in the formation of a colored
zone.

The enzymatic hydrolysis of PNPG by a-galactosidase
liberates p-nitrophenol, which has a pI<a of 7.2. p-
Nitrophenol in alkaline environment (1 to 2 pH units above
its pKa value) develops a deep yellow color suitable for
quantitative measurement of the enzyme activity. Advantages
of this method include simplicity, accuracy, sensitivity
and fast results (Hough and Jones, 1962) . The alkaline
compounds, used to develop the color of p-nitrophenol, also
stop the enzymatic reaction by raising the pH (5 ml of 0.1
M Na2C03 were used). Figure 4 shows the mechanism of the
chemical reactions occurring during the enzymatic
hydrolysis of PNPG by e-galactosidases and the color

development in the alkaline environment.

PURIFICATION
The separation of the multiple forms of plant a-
galactosidases have been achieved by applying methods that
resolve macromolecules according to their molecular weight
(gel filtration) and ionic characteristics (ion-exchange

chromatography, electrophoresis, and isoelectric focusing)

21

i:_@_mzn £91. 1%); o»—@—~°z

OH+NOZ + NaOH ——> 1)....»02-4- “4+ + H0“

Figure 4. Enzymatic release of p-nitrophenyl from PNPG (I).
Color development by increasing the pH of the p-nitrophenol
solution (II)

22

(Hadacov'a and Benes, 1977; Williams et al., 1977; 1978).
The procedure for extracting the enzyme from tissues
determines the level of the total enzyme as well as the
multiple-form composition.

The usual techniques of isolation include ammonium
sulfate and organic solvent fractionations, heat treatment,
acidification, ion exchange, gel chromatography and
isoelectric focusing. Affinity chromatography has been used
to separate several e-galactosidases. Melibiose-sepharose
(delCampillo and Shannon, 1982: Mapes and Sweeley, 1972)
and melibiose-Agarose affinity columns were used. Highly
purified and apparently homogeneous a-galactosidases have
only been isolated in a few cases (Dey and Pridham, 1969;

Lee and Wacek, 1970).

ROLE 01' THE ENZYME

The primary role of a-galactosidases in the storage
organs of plants is to mobilize the reserve a-galactosyl-
containing oligo- and polysaccharides (Dey and Pridham,
1972: Pridham and Dey, 1974: Dey, 1978; 1980). A low level
of the enzyme exists in the early stage of germination of
fenugreek seeds. At later stages, increase in the enzyme
level coincides with galactomannan degradation in the
endosperm. The embryo has a relatively high level of the
enzyme, which was found to remain constant during

germination and hydrolyze the raffinose family of sugars

23

(Reid and Meyer, 1973: McCleary and Matheson, 1974: Sciler,
1977).

The role of a-galactosidase in fruit is not clear: it
is probably involved in the degradation of
oligosaccharides. This enzyme participates in the control
of internal osmotic pressure in the unicellular alga

99hrgngna§ (Kreuzer and Kauss, 1980; Dey and Kauss, 1981).

CHANGES IN a-SALACTOSIDASE DURING SEED MATURATION

Pridham and Dey (1974) reported that examination of the
elution patterns from Sephadex gels and total activity of
a-galactosidases in immature seeds has shown that the
small, green beans of y; fab; contain only a low level of
enzyme II and that this increased with maturation.
Following this stage, a marked increase in the specific
activity occurred which can be related to the appearance of
enzyme I and a decrease in the activity of enzyme II.

OLIGOSACCNARIDES AND a-GALACTOSIDASE

It is well known that consumption of legume seeds by
humans may result in gastrointestinal distress and
flatulence. Nausea, cramps, and diarrhea may also occur in
varying degrees (Rackis et al., 1970a), depending upon the
legume consumed. Although the presence of raffinose and
stachyose in the active gas-producing fraction of beans has

been demonstrated, positive identification of all

24

flatulence-causing factors has not been made (Rackis et
al., 1970b: Murphy et al., 1972; Steggerda et al., 1966).

The c-galactoside sugars raffinose and stachyose are
not hydrolyzed or absorbed by the mammalian digestive
system (Taeufel et al., 1965). Mammalian digestive juices
lack the a-galactosidase (EC. 3.2.1.22) required to
hydrolyze these low molecular weight oligosaccharides. The
sugars pass into the lower bowel, ileum and colon, where
they may be fermented by various resident bacteria with the
production of gas. Major production of hydrogen, carbon
dioxide, and methane gases by normal intestinal microflora
takes place (Richards et al., 1968).

In attempt to reduce or eliminate the flatus activity of
legumes, several approaches have been taken. Induced seed
germination (Hsu et al., 1973) ethanolic and aqueous
extraction (Rackis et al., 1970a: Kim et al., 1973), and
adjustment of pH, temperature, and moisture content (Becker
et al., 1974) have been reported to decrease raffinose and
stachyose levels in beans. Antibiotics have been
successfully employed to suppress microbial activity in the
lower ileum and thereby reduce flatus (Murphy and Galloway,

1972; Richards and Steggerda, 1966).

25

GERMINATING LEGUME SEEDS

There is little doubt that seed a-galactosidases play
an important role in the early stages of germination by
hydrolyzing galactos-containing oligpsaccharides and,
hence, provide metabolites for the developing seedling
(Pridham and Dey, 1974: Courtois and Percheron, 1971: Dey
and Campillo, 1984; Dey et al., 1986a). It is not known how
the enzymes and their natural substrates are prevented from
interacting during the maturation of seeds. The
glycoprotien/lectin nature of the enzyme offers various
possibilities of in vivo compartmentation /inactivation of
the enzymes (Dey and Del, 1984: Dey and Pridham, 1982: Dey
et al., 1986b) and the carbohydrate chains may be involved
in the transport of these enzymes between compartments
(Chrispeels, 1983: Dey and Campillo, 1984).

McCleary and Matheson (1974) reported that germinating
seeds of lucerne, guar, carob and soybean initially
depleted raffinose. series oligosaccharides and then
galactomannan. This depletion was accompanied by a rapid

increase and then a decrease in a-galactosidases level.

CHANGES IN a-GALACTOSIDASES DURING SEED GERMINATION

Over the first 12 hr of L Lab; germination there is
rapid fall in total and specific a-galactosidase activities
followed by an increase to about 50% the original value.

After 24 hr, no marked change in activities occur up to 6

26

days (Pridham and Dey, 1974) . The Sephadex gel elution
patterns over this period show first a rapid decline in
enzyme I and then a slow increase in the level of enzyme II
(Pridham and Dey, 1974). The total activity after 6 days is
still much lower than the original activity of the mature
seeds in L fan. It is possible that at this time starch
takes over as the major reserve of the cotyledons (Pridham

and Dey, 1974).

PHYSIOLOGICAL SIGNIFICANCE OF a-GALACTOSIDASES

In higher plant tissues, galactose-containing oligo-
and poly-saccharides are common carbohydrate reserves,
particularly in seeds. There is new little doubt that e-
galactosidases function as hydrolytic agents in the
utilization of these compounds as sources of energy and
cell metabolites. (Pridham and Dey, 1974). The enzymes may
also play an important role in the metabolism of
galactolipids (Sastry and Rates, 1964) and in the function
of chloroplast membranes (Bomberger and Park, 1966: Gatt
and Baker, 1970). Few observations appear to have been made
on the role of a-galactosidase in the animal kingdom. The
enzyme does occur in brain tissues; in vivo it may be
involved in the hydrolysis of digalactosyl diglycerides
(Subba and Pieringer, 1970).

27

POSSIBLE APPLICATIONS
Sugar Factory

Crystallization of beet sugar may be further improved
and the yield of the sugar increased if raffinose in the
beet juice or molasses can be removed or decomposed by some
method. Many workers successfully removed raffinose from
molasses by incorporating crude preparations of a-
galactosidase isolated from various microorganisms.

Suzuki et al. (1969) studied the hydrolysis of
raffinose ‘ in beet molasses. They found that 80% of the
raffinose was hydrolyzed by the a-galactosidase obtained
from the mold W 2.1111933- The a-galactosidase
formation by the mold was induced by sugars such as
galactose, melibiose, raffinose and lactose.

Soymilk

Many attempts have been made to eliminate the
oligosaccharides from soybean and its products by soaking,
soaking and germination, fermentative processes, water
extraction and ratio and by ultrafiltration (Kim et al.,
1973: Mital et al., 1974). Enzyme treatment by microbial a-
galactosidase offers a promising solution for the
elimination of oligosaccharides from soymilk (Sugimoto and
Van, 1970: Smiley et al., 1976; McGhee et al., 1978).

Cruz and Park (1982) reported that the production of

extracellular’ a-galactosidase. by' Agpgzgillgg, erzag ‘was

28

induced by the addition of soybean carbohydrates to the
culture medium. Addition of only stachyose or raffinose
induced slight production of a-galactosidase. Treatment of
soymilk with e-galactosidase for 2 hr resulted in complete
despairing sucrose and a significance decrease in raffinose
and stachyose content. Sugimoto and Van (1970) removed all
the galactosaccharides from soymilk in three hours by
treating it with a-galactosidase from A; gaggi.
Blood-group-B

a-D-Galactosidases may be useful in studies of blood-
group substances or other glycoconjugated cell surface
(Harpaz et al., 1975). Hydrolysis of the terminal a-D-
galactopyranosyl moieties of blood-group-B substances have
been reported using a-galactosidases from different sources
(Watkins, 1956; Hakomori et al., 1971: Oishi and Aida,
1972). Harpaz et al. (1975) isolated a coffee bean a-D-
galactosidase which converted human type-B erythrocytes to
type 0. Pressey (1984) concluded that a-galactosidases
isolated from tomatoes may be employed to convert group B

erythrocytes to type 0.

EXPERIMENTAL PROCEDURE

MATERIALS

The materials used in the experiments and their sources
are listed below.

1. Cowpeas or Black-eye peas were purchased from
California.

2. p-Nitrophenyl-a-D-galactoside (PNPG) free of p-
nitrophenol (Sigma Chemical Co, St. Louis M0.).

3. Molecular weights standards used in SDS-PAGE and
Sephacryl S-200 (Sigma).

4. Sucrose, raffinose and stachyose (Sigma).

5. B-D-galactose dehydrogenase was obtained from
recombinant B. 991.1, using W W gene,
suspended in 3.2 M (NH4)2804, pH approximately 6.0, 58

units/mg of protein (Sigma).
6. Buffer solutions: MacIlvain (0.1 M citric acid and

0.2 M NazHPO4), potassium phosphate and Tris buffers were
prepared according to the Biochemists Handbook (1961) and
adjusted to the desired pH level electrometrically.

7. Sephadex G-100 and G-200 (Pharmacia, Uppsala,
Sweden).

8. CM-cellulose was purchased from Whatman (England),

Accl-CM-cation ( Waters Assoc. cationic resin).

29

30

9. SE 600 Series Vertical Slab Unit Electrophoresis
Apparatus (Hoeffer Scientific Instruments, San Francisco,
California).

10. Amicon pressure cell to concentrate the enzymes
(Amicon Division, W.R. Danvers, MA.).

11. Beckman DU single beam absorption
spectrophotometer.

12. HPLC system from Waters Assoc. Milford, MA.

METHODS
In order to achieve the objectives of the research,
several methods were used. All the plots in the results

were drawn using a computer program (Plotit).

ENZYME ASSAY

a-Galactosidase was assayed at 30' C by addition of
enzyme solution (0.1 ml) to a mixture of McIlvaine buffer,
pH 5.5 (0.25ml), and 12 mM p-nitrophenyl a-D-galactoside
(0.05ml) . After 15 min the reaction was stopped with 5 ml
of 0.1M Na2C03. The released p-nitrophenol was measured at
405 nm. The blank was prepared by adding either the enzyme
solution or the substrate solution to the mixture after
adding 5 ml 0.1 M Na2C03.

a-Galactosidase activity was expressed either as
A405/g dry wt. or katals (Kat). One katal is the amount of

enzyme activity that transforms 1 mole of substrate per

31

second. Specific activities were expressed as nkat/mg of

protein. Protein determinations were made by the method of

Lowry (1951) as modified by Cooper (1977) with crystalline

bovine serum albumin as standard.

p-NITROPHENOL STANDARD CURVE
A standard curve was prepared by measuring the

absorbence at 405 nm of p-nitrophenol concentrations

ranging from 0.005-0.5 uncles/ml. The following mixture was

used for each measurement: 0.35 ml MacIlvain buffer pH 5.5,
0.05 ml p-nitrophenol of correct dilution and 5.00 ml 0.1 M

N92C°3 for color development. All measurements were done in

triplicate. The buffer solution was used as blank.

ENZYME ISOLATION AND PURIFICATION

Cowpeas were ground in a Wiley-Mill and extracted with
McIlvaine buffer, pH 5.5, (1g seed powder / 2ml buffer) for
1 hr at 4' C. The slurry was centrifuged at 20,000 x g for
40min and the residue discarded. The pH of the supernatant
was lowered to 3.5 by gradual addition of 1 M citric acid.
The precipitated protein was discarded after centrifugation
at 16,000 x g for 40 min. The pH of the supernatant was
raised to 5.5 with NazHPO4 solution and brought to 25%
saturation with (NH4)ZSO4. The precipitated protein was
discarded and the supernatant was made 55% saturated with

(NH4)2804. The precipitated protein was collected by

32

centrifugation, resuspended (volumes; Tables 1 and 2) in
McIlvaine buffer, pH 5.5, and dialysed overnight against
the same buffer (2 l) . This dialysed fraction was applied
to a 1.9 x 70 cm Sephadex G-100 column and eluted with
McIlvaine buffer, pH 5.5 , containing 0.1 M KCl. The
fractions for each peak were pooled and concentrated
separately by ultrafiltration under N2 using an Amicon Ym10
membrane. Two different purification procedures were used
after this step.
W A

Enzyme I and II were dialysed against McIlvain buffer,
pH 4.0 and 3.0 respectively. The dialysed samples were
applied to a 0.9 x 10 cm Accel-CM-cation exchange column
and eluted with McIlvaine buffer containing 1 M NaCl at a
flow rate of 20 ml/hr. The pH gradient used to elute
Enzymes I and II ranged between 4.0 to 7.0, and 3.0 to
6.0, respectively. The elution resulted in resolving a-
galactosidase II in two enzyme fractions, II1 and II2.
21.929.91.113 3

Enzymes I and II from the gel filtration on G-100 were
dialysed against McIlvain buffer, pH 3.5 and applied to a
0.9 x 14 cm, CM-cellulose column equilibrated with the same
buffer. The flow rate was 25ml/hr. Following sample
application, the column was washed with the buffer and

elution of protein was carried out in a linear NaCl

33

gradient (0.05 to 0.4 M) in the same buffer. The elution
resulted in resolving a-galactosidase II in two enzyme
fractions, II1 and I12. All three enzymes I, II1 and 112
were recycled through the CM-cellulose column. The samples
were stored at -20' C in 1 ml fractions until used. An

outline of the purification scheme is shown in Figure 5.

MOLECULAR HEIGHT DETERMINATION

Gel-filtration and SOS-PAGE were used to determine the
molecular weight of the enzymes.

Gel Filtration Chromatography (Sephacryl 8-200)

The advantages of using gel chromatography to
determine the molecular weight of the enzymes (Freifelder,
1976) are:

1. Enzymes are not affected by chromatography, because
there is virtually no adsorption.

2. The zone spreading is less than other
chromatographic techniques.

3. The elution volume is related in a simple manner to
molecular weight.

4. The MW of an impure protein may be determined using

this procedure if a specific detection test is available.

34

Seed powder
V

Extraction with McIlvaine buffer, pH 5.5
(2.0 ml buffer/ 9 powder)

V

Citric acid precipitation of inactive protein
(pH 3.5)

V
(NH4)2804 precipitation (25-55%)
V

Sephadex G-100 gel filtration
(Enzyme I and II)

/ \

/ \
CM-cellulose chromatography Accl-CM-cation
(NaCl gradient) (pH gradient)

\ /

\ /

Enzymes I, II1 and II2

Figure 5. Outline of the purification scheme for a-

galactosidases from cowpea

35

Disadvantages of gel chromatography

1. Ve/Vo (V8 is the elution volume and V0 is the void
volume) may be temperature dependent.

2. Unreliable MW may be obtained if protein forms a
complex with the gel, contains a large amount of
carbohydrate, aggregates to larger complexes, or
dissociates into subunits (sigma, 1986).

3. In determining void volume, it is inadvisable to mix
Blue Dextran with standards or sample protein because many
proteins bind to Blue Dextran (Sigma, 1986).

The following steps were followed to determine the
molecular weight of the enzymes using gel filtration.

1. Molecular weight markers (from sigma)
The following protein MW standards were used:
. Cytochrome C, Horse Heart (MW 3 12,400 ).
. Carbonic Anhydrase, Bovine Erythrocytes (MW 3 29,000).
. Albumin, Bovine Serum (MW 3 66,000).
. Alcohol Dehydrogenase, Yeast (MW a 150,000).
. Blue Dextran was used as a tracking dye (MW s 2,000,000).
2. Gel preparation

MoIlvaine buffer, pH 7.0 (Enzymes II1 and II2) and pH
5.0 (Enzyme 1) containing 0.1M KCl was used as the
equilibration and elution buffer. Sephacryl S-200 was
prepared in the same buffer. The resin was suspended in

about 2 volumes of buffer, the gel allowed to settle for

36

15-30 min, and the smallest particles decanted. The last
step was repeated. The gel suspension was degassed before
use.
3. Column packing and equilibration

A slurry of approximately equal volumes of buffer and
gel was poured into a vertical column that was one-third
filled with buffer. The slurry was allowed to settle for
about 15 minutes without flow, then the excess buffer was
drained through the gel bed. Gel slurry was added
continuously until a bed height of 90 cm was obtained. The
inner diameter of the column was 1.45 cm. The buffer
reservoir was connected to the top of the column and 2-3
column volumes of buffer was allowed to pass through the
column. The final height of the column was 87 cm. This
column was built and run at 4' C. The column was
continuously eluted with buffer when not in use (Sigma,
1986).
4. void volume determination

Blue Dextran was dissolved in equilibration buffer. Two
ml of the Blue Dextran (2.0 mg/ ml) were carefully applied
to the column to determine V0 and to check column packing.
Fractions of 2.3 ml were collected. The flow rate was 23 ml
/hr. Ve for Blue Dextran and the V0 for the column were

determined spectrophotometrically at 280 nm.

37

5. Elution volume determination for protein standards

Individual protein standards were dissolved in
equilibration buffer at the following concentrations
(Cooper, 1977: Andrews, 1964): Cytochrome C (2 mg/ml),
Carbonic Anhydrase (3 mg/ml), Albumin (10 mg/ml) and
Alcohol Dehydrogenase (5 mg/ml).

The following proteins were mixed (1:1) and run
together (2.0 ml) on the column: Cytochrome C with Albumin
and Carbonic Anhydrase with Alcohol Dehydrogenase. Two ml
protein standards were applied to the column and fractions
were collected. The V3 for the protein standards were
determined spectrophotometrically (absorbence at 280 nm) by
measuring the volume of effluent collected from the point
of sample application to the center of effluent peak.
6.8tandard curve

Molecular weights versus relative elution volume (REV)
and distribution coefficient (Kav) for each protein
standard were plotted on semilog paper. Kav defined as Ve -
V0 /Vt - V0 (Vt is the total volume of the column) and REV
is defined as Ve / V0 (Andrews, 1964). MW determination of
unknown proteins were made by comparing the ratio of Ve/Vo
for the protein in question to the Ve/Vo of protein
standards. The V6 of the enzymes was determined by
measuring fractions at 405 nm after assaying for enzyme

activity using PNPG as substrate.

38

MW Estimation Using EDS-PAGE
The following steps were followed to obtain the MW of
the enzymes using the SOS-PAGE estimation method.
1. MW markers
The following protein MW standards were used.
. Albumin, Bovine ~ MW 66,000
. Albumin, Egg (Ovalbumin) s MW 45,000
. Glyceraldehyde-3-phosphate dehydrogenase, Rabbit muscle 3
MW 36,000
. Carbonic anhydrase, Bovine erythrocytes 3 MW 29,000
. Trypsinogeh, Bovine pancreas, PMSF treated 6 MW 24,000
. Trypsin inhibitor, Soybean s MW 20,100
. o-Lactalbumin, Bovine milk s MW 14,200
. Bromophenol Blue was used as a tracking dye
2. Resolving gel-11% acrylamide
The resolving gel was prepared according to the
following formula:
- Tris-SDS-stock solution (30 ml) pH 8.8, 0.75M Tris and
0.2% SDS.
- Acrylamide stock (22 ml) which contained 30% w/v
acrylamide and 0.8% w/v N,N -bis-methylene acrylamide.
- Water ( 8 ml).
- Tetramethylethylenediamine (TEMED) (25 ul)

- Freshly prepared 1% w/v ammonium persulphate (1.5 ml)

39

3. Stacking gel-4% acrylamide
The gel was prepared as follows:
- Tris-SDS-stock solution (10 ml) with pH 6.8, 0.2M Tris
and 0.2% SDS.
- Acrylamide stock (2.7 ml) which contained 30% w/v

acrylamide and 0.8% w/v N,N'-bis-methylene acrylamide.

Water ( 7.3 ml).

Freshly prepared 1% w/v ammonium persulphate (0.65 ml)
- TEMED (18 ul)
4. Sample preparation (Laemmli, 1970)

Samples were dialysed against buffer (pH 7.25)
containing 0.2% SDS and 0.025 M Tris. Then, 0.9 ml of each
sample was heated to 100' C with 0.1 ml 10% SDS. To each
sample 1-2 drops of 2-mercaptoethanol was added. The
samples , 5 to 50 ug protein, were subjected to
electrophoresis. The electrode buffer (pH 8.3) contained
0.025 M Tris, 0.192 M glycine and 0.1% SDS. The power was
set at lmA/channel (15 mA/slab gel) until the dye entered
the resolving gel then increased to 2 mA/channel.

Gels were stained with Coomassie brilliant blue-R 250
(0.4% w/v) in 40% acetic acid and 10% methanol for 4 hr at
room temperature. The gel was destained with a 7.5% (v/v)

acetic acid and 25% (v/v) methanol solution.

40

5. Standard curve
Molecular weights vs. relative mobility (Rf) for each

protein standard were plotted on semilog paper

POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE)
Anionic pass (pH 3.3)

All steps on this procedure were the same as for SDS-
PAGE, except that SDS and 2-mercabtoethanol were not
included in the preparations. Acrylamide , 7.5%, was used
in anionic PAGE instead of 11% in SDS-PAGE.

Cationic PAGE ,pE 3.5 (Reisfeld et al., 1962)
The following stock solutions were used to prepare the

resolving and stacking gel:

.A: (pH 4.3) C:

1. KOH (1N) 48 ml: 1. Acrylamide 30 gm:

2. Acetic acid 17.2 ml: 2. Methylene

3. TEMED 4 ml: bis-acrylamide 0.8 gm:
4. Water to make 100 ml. 3. Water to make 100 m1.
B: (pH 6.8) D:

1. ROM (1N) 48 ml; 1. Acrylamide 10 gm:

2. Acetic acid 2.87 ml: 2. Methylene
3. TEMED 0.46 ml: bis-acylamide 2.5 gm;
4. Water to make 100 ml. 3. Water to make 100 ml.

E:
1. Riboflavin 4.0 mg:

2. Water to make 100 ml.

41

Resolving gel, 7.5% acrylamide pH 4.3

A- 7 ml

C- 14 ml

Ammonium persulphate, fresh prepared (0.28 gm /100 ml)

28 ml.
Stacking gel, 4% acrylamide pH 6.8

B- 2.5 ml

D- 5 ml

E- 2.5 m1

A fluorescent lamp (15 W) was placed about 10 cm from
the gel. The stacking gel became opalescent and polymerized
within 15 min.

The sample buffer was prepared from stock A or B using
a 10-fold dilution. Methylene green was used as a tracking
dye (positively charged). The electrode buffer (pH 3.5)
contained 31.2 gm fi-alanine and enough acetic acid to
adjust the pH to 3.5. The electrode buffer was diluted 1:1
with H20 before use. The staining and destaining were the
same as in SDS-PAGE:
Detection of enzyme activity on polyacrylamide gels
(cationic)

Enzyme activity on the gels was detected using 0.3% 6-
bromo-2-naphthyl-o-D-galactoside in dimethylformamide as a
substrate (Hadacova and Benes, 1977). The medium consisted

of 20 ml MacIlvain buffer pH 5.5, 1 ml stock substrate

42

solution and 20 mg Fast blue RR diazonium salt. The gels
were incubated in this substrate solution for four hours at
room temperature. Bands containing active enzyme were

yellow.

ENZYME KINETICS
Initial Velocity
In an end point assay the initial velocity is the
amount of product formed in a reaction divided by the
elapsed time (Suelter, 1985; Allison and Purich, 1979). For

the determination of Rh and V’ initial velocities were

max'
used which corresponded to different reaction times for
different substrates. Initial velocities were based on 3
minute reaction times when PNPG was the substrate. With
raffinose as substrate, 45 min, 30 min and 90 min reaction
periods were used to calculate initial velocities for
Enzyme I, II1 and IIZ, respectively. With stachyose as
substrate, the initial velocities were based on 45 min and
30 min reactions for Enzyme I and Ill, respectively. Enzyme
II2 was not tested on stachyose since the reaction rate was
too slow and little enzyme was available.
E. and 7“: Values for Various Substrates

All the lines in the plots were drawn based upon the
method of least squares polynomials (first degree equation,

y - bx + a). The statistical manipulation of the

43

experimental data was achieved with the aid of a computer
and the estimated values of the coefficients, a and b,
along with the indexes of determinations, r, are indicated
with the plots.

The Km and Vmax values were calculated from the

following equation and formulas:

1 1 1
---- - --5L-- x ---------- (1)
v Vmax [S] vmax

where v 8 initial rate of reaction
8 - initial substrate concentration
Vmax = maximum reaction rate
Km - Michaelis constant
b 1

Km " """ ' vmax “ """
a a

The following substrates were used to determine Km and
Vmax using the Lineweaver-Burk equation:
1. p-Nitrophenyl-o-D-galactopyranoside (PNPG) as an

artificial substrate.
2. Raffinose and stachyose as natural substrates.
PNPG

The Km and Vmax values for each enzyme, with PNPG as

substrate, were determined using the Lineweaver-Burk double

reciprocal plot. Five or six PNPG concentrations were used.

44

Raffinose

A series of solutions, in MacIlvain buffer (pH 5.5),
with the following molarities of raffinose were prepared:
2, 3, 5, a, 13, and 20 mM. Solutions of enzyme 1 (0.2 ml),
enzyme II1 (0.1 ml) and enzyme II2 (0.15 ml) were added to
1 ml of raffinose solution. The mixtures were allowed to
react at 30‘ C for 45, 30 and 90 min for enzymes I, II 1
and 112, respectively (These times were based on
preliminary experiments which determined the linearity of
the enzymic reactions, after trying reaction periods
varying from 15 to 120 min, at 30' C with a 10 mM
raffinose substrate). At the end of the reaction time the
mixtures were placed in boiling water for three minutes.

The following procedure was used to determine the
amount of liberated galactose (Kurz and Wallenfels, 1981):

The absorbence (A'334) of 3.0 ml 0.1 M Tris buffer, pH
8.6, containing 0.1 ml 16.5 mM nicotinamide-adenine
dinucleotide (NAD) and 0.2 ml of the D-galactose-containing
solution was measured.D-Galactose dehydrogenase, 0.02 ml,
(25 U/ml) was added and the mixture incubated at 37' C for
20 min or until the absorbence became constant, A"334.
Delta A x A' - A" was used for the calculations.

Under the above conditions the reaction proceeded

stoichiometrically and the following formulla was used to

45

determine the concentration of galactose (Bergmeyer et al.,
1991):

delta A x V

C = ----------------- [umole/ml]

a x d x v
e : extinction coefficient [ cm2 /umole]
delta A: extinction change
V: assay volume [ml]
v: volume of sample used in assay [ml]
d: light path [cm]
C: concentration [umole/ml], [ug/ml]

For a total assay volume of 3.32 ml and a sample volume

of 0.20 ml the D-galactose concentration of the sample was

obtained using the following equations:

C = delta A x 2.72 [uncle/ml] or

C a delta A x 0.490 [mg/ml].

Stachyose

The Km and Vmax values for enzyme I and II:L were
determined using stachyose as substrate. The disappearance
of stachyose was measured by HPLC: An aliquot of the
solution was passed through a 0.22 um memberane filter and
a Sep-pak C18 cartridge. A 20 ul sample was injected into
an HPLC system (Waters Assoc.,Milford, MA), composed of an

M-45 pump, a guard-pack precolumn, an 1r-

46

Bondapak/carbohydrate column and an RI-401 refractometric
detector. A Kontes model-100 recorder was used. The solvent
(acetonitrile/water, 65/35 v/v) flow rate was 2.0 ml/min.
Enzyme I

The following concentrations of stachyose in MacIlvain
buffer, pH 5.5, were prepared: 5, 8, 13, 20 and 30 mM.
Enzyme I solution, 0.1 ml, was added to 1.0 ml of each
stachyose concentration. The reaction was carried out at
30' C for 45 min. After stopping the reaction by boiling
for 3 min, each mixture was injected in to the HPLC. The
area of the stachyose peak was measured and compared to the
peak area of standard stachyose solutions subjected to the
same HPLC separation. The linearity of the enzymic
reactions (0.1 ml enzyme and 1.0 ml stachyose) was
confirmed after trying reaction times of 0, 30, 45 and 60
min at 30' C with a 5 mM stachyose substrate.
Enzyme 111

A 0.1 ml of the enzyme was used with 1.0 ml of each of
the following concentrations of stachyose in MacIlvain
buffer (pH 5.5): 3, 5, 8, l3 and 20 mM. The reaction was
carried out at 30' C for 30 min. The 30-min reaction was
used as the initial velocity was linear by a mixture of 0.1
ml enzyme and 1.0 ml stachyose solution 3 mM for 30, 45 and
60 min. The calculation of the disappearing of stachyose

was as with enzyme I above.

47

Effect of Substrate (Raffinose) Concentrations on Enzyme
Activity

The following raffinose concentrations were used for
enzymes I, 111 and 112: 10, 20, 40, so and 160 mM. At 30'
C, enzymes I, II1 and II2 were incubated for 45, 30 and 90
minutes, respectively. Solutions of I, II1 and II2 0.1, 0.1
and 0.4 ml respectively were added to 1.0 ml raffinose.
Enzyme quantities were chosen to give readable results. For
galactose quantitation , the galactose dehydrogenase test

was used (Kurz and Wallenfels, 1981).

STABILITY

The stability of enzymes I, II1 and II2 during 24 hrs
storage at four different temperatures, 4, 22, 37 and 45'
C, and at various pH values from 2.0 to 8.0 was studied.
MacIlvain buffer and PNPG as substrate were used. Enzyme
activity was assayed at time zero and after 24 hr. Activity
at time zero was considered 100% in order to calculate the
relative activity. The MacIlvain buffer (0.5 M citric acid
and 1 M NazHPO4) was used to keep the pH of the assay

around 5.5.

OPTIMUM ACTIVITY pH AT 30' C
The effect of pH on the rate of PNPG hydrolysis at 30°
C was studied with a series of MacIlvain buffers ranging

between 2.0 and 8.0 pH. The pH of the reaction mixture was

48
measured before the initiation of the reaction.

OPTIMUM ACTIVITY TEMPERATURE AT OPTIMUM pH
The optimum temperature for activity was determined
between 0' and 70' C at the optimum pH for each enzyme.

PNPG, was used as substrate to assay the enzyme.

GERMINATION AND INCUBATION OF COWPEAS

1- Experimental Design

Experiments were designed to follow the activity of a-
galactosidase and changes in oligosaccharides during the
germination, incubation and germination plus incubation of
cowpeas. All experiments were performed in duplicates. The
results were statistically analyzed (ANOVA) using a
computer program (Microstat). The statistical analysis was
used to show the significant differences within and between
the treatments.
2- Quantitation of Oligosaccharides

Samples from the germinated and incubated cowpeas, 1-3
9 dry wt., were ground and extracted twice with 25 ml 80%
ethanol in an 80' C bath for 4 hr with occasional shaking.
The extract was centrifuged at 2500 x g for 15 min. The
supernatants were saved and 1 ml 10% lead acetate was added
twice. After 10 min at 60' C, the solutions were
centrifuged at 2500 x g for 15 min. Five drops of 10%

oxalic acid were added to the supernatants which were then

49

centrifuged at 2500 xg for 15 min. The purified extracts
were concentrated by vacuum evaporation below 50' C to a
final volume of 25 ml. An aliquot of the extract was passed
through a 0.22 um membrane filter and a Sep-pak C18
cartridge. A 20 ul sample was injected into an HPLC system
as previously described. The concentrations of the sucrose,
raffinose and stachyose standard solutions ranged from 0.2-
0.6%, 0.02-0.06% and 0.1-0.3%, respectively. Results were
expressed as g sugar/ 100 g dry wt.

The A.O.A.C. procedure No. 14.003, was used for
moisture determination.
3- o-Galactosidase Activity and Oligosaccharides Content of
Germinating Cowpeas

Cowpea seeds, 2-3 9 dry wt, were soaked in distilled
water for 2 hr and germinated at 24' C for 24 hr for
oligosaccharides measurment. The grmination time for a-
galactosidase activity measurment was 30 hr at 24' and 30‘
C. The seeds were germinated in darkin Petri dishes lined
with wet filter paper. At intervals of 6 hrs, samples were
removed from the incubators and frozen at -20' C. The
frozen seeds were ground and analyzed for oligosaccharide
content and o-galactosidase activity.
4- Incubation

Flour was made from cowpeas by the dry method (Dovlo et

al., 1976). The cowpea seeds were ground in a Wiley mill

50

and passed through a 20-mesh seive after blowing off the
seed coats. A paste was made from cowpea flour by mixing
100 g flour with 140 ml distilled water. Samples, 2-3 gm
dry wt., were incubated at 24' C and 34' C for 0, 3, 6, 12
and 24 hr. After each period of time, the samples were
removed from the incubator and their pH was measured.
Samples were kept in the freezer at -20' C until analyzed
for oligosaccharide content using HPLC as previously
described.
5- Germination and Incubation
Samples ' were soaked for 2 hr, germinated for 6 hr,

ground, incubated for 0, 3, 6 and 12 hrs at 24 and 34' C
and analyzed for oligosaccharides using HPLC. The pH of the
suspension was also determined.
6- o-Galactosidase Activity During Sprouting

The seeds started to sprout after 24 hr of
germination. The a-galactosidase activity was measured at
42 and 72 hr of germination at 30' C in the sprouts and

the remainder of the seeds.-

INVERTASE ACTIVITY
Samples from zero time, 12 hr incubation and 12 hr
germination were analyzed for invertase activity. The
analysis were performed on samples obtained by the

following extraction method:

51

Extraction Method (Al-Baker and Nhitaker, 1978)

Five gram samples were ground with 20 ml distilled
water and centrifuged at 15,000 xg for 30 min. The pellets
were extracted three times with 0.25 M potassium phosphate
buffer (1:1,w/v), pH 7.5, in a Vortex shaker for 2 min. The
three extracts were combined after centrifugation at 15,000
xg for 30 min. The pH of the supernatant was adjusted to
4.0 with 30% acetic acid. Ammonium sulfate was added to 30%
saturation. The supernatant was 95% saturated with ammonium
sulfate and the precipitate formed was removed by
centrifugation at 20,000 xg for 25 min. The pellet was
dissolved in 0.01 M sodium acetate buffer, pH 4.0, dialyzed
against the same buffer for 40 hr with several changes in
buffer and tested for invertase activity.

Invertase assay using HPLC

Enzyme solution, 0.3 ml, was added to 5 ml of 0.1 M
sucrose-0.1 M sodium acetate buffer, pH 4.0, at 30' C for
10 min. The reaction was stopped by boiling for 2 min. The
reaction solution was diluted four-fold with distilled
water before being injected in to the HPLC. Sucrose
solution (25 mM) was used as a standard. Substracting the
sucrose peak height of the enzyme reaction mixture from the
peak height of the standard represented the mM of sucrose

disappeared.

52

One unit of invertase activity was that amount of
invertase which hydrolyzed 1 uncle of sucrose per min at pH

4.0, 30‘ C at an initial sucrose concentration of 100 mM.

RESULTS AND DISCUSSION

ENZYME ASSAY

The validity of the PNPG assay for a-galactosidase
activity was established.

The stopped assay was linear at a reaction time of 15
min or less (Figures 6). The wavelength of maximum
absorbence for p-nitrophenol was 405 nm (Figure 7). Figure
8 shows the standard curve for determining p-nitrophenol

concentration.

ENZYME PURIFICATION
The purification of cowpea a-galactosidases is
summarized in Table 1 and 2. The difference between method
I (Table 1) and method II (Table 2) was in using different
ion exchange columns. The elution time, using CM-cellulose
column was longer than the elution time when Accl-CM column
was used. Method I resulted in better yields.

In stage 1, the extraction pH which resulted in the
maximum enzyme activity and the clearest extract was 5.5
(Figure 9). In stage 2, acid precipitation (citric acid)
almost tripled the specific activity of the extract with
very little loss of total activity. The addition of citric
acid also increased the specific activity obtained after

ammonium sulfate precipitation (Table 3) . The optimum pH

53

54

for acid precipitation of the inactive protein was 3.5
(Figure 10).

Table 1. Purification of cowpea (ll-SEA W) o-
galactosidases, method I.

 

Volume Activity Protein Sp. activity Recovery1

 

Stage (ml) (nKat/ml) (mg/ml) (nKat/mg) (%)
1.Cnfle
Extract 1575 63 174 0.4 ---
2. citric Acid
Precipitation 2060 42 37 1.1 87
25-55% 154 324 220 1.5 50

4. Sephadex G-100

a-galactosidase I 152 180 4 45.0 28
o-galactosidase II 113 77 40 1.9 8.8

5. CM-cellulose chromatography

I 18.5 395 0.1 3950 7.4
111 15.5 368 10.0 37 5.8
112 13.5 5.3 0.2 27 0.1

 

1% recovery was calculated relative to the first stage, i.e. crude
extract. Seed powder wt. taken - 1 kg.

In stage 3, ammonium sulfate fractionation was used
rather than acetone precipitation as suggested by Dey and
Pridham (1969), because ..10- C acetone inactivated the
enzyme. The cut point in ammonium sulfate precipitation for

maximizing the activity of the precipitation was found to

55

be 25% to 55% ammonium sulfate (Table 4). The specific

activity after this stage increased considerably.

Table 2. Purification of cowpea (11311: W) a-
galactosidases, method II.

 

velume Activity Protein Sp. activity Recovery1

 

Stage (ml) (nKat/ml) (mg/m1) (nKat/mg) (%)
1.Cnme
extract 500 77 210 0.4 ---
2. Citric Acid
Precipitation 870 44 45 1.0 99
3. (NH4) SO4
25-55% 55 299 160 1.9 43

4. Sephadex G-100

a-galactosidase I 126 119 3 40.0 39
a-galactosidase II 110 65 39 1.7 19

5. Sephadex G-100, recycle

a-galactosidase I 44 159 3 53 18
a-galactosidase II 57 67 16 4 10

6. Accl-CM-cation

I 10 250 0.14 1786 6.5
111 3 45 0.7 64 0.4
112 8 13 0.3 43 0.3

 

1% recovery was calculated relative to the first stage, i.e. crude
extract. Seed powder wt. taken - 0.5 kg.

In stage 4, the enzyme solution was dialyzed against
McIlvain buffer, pH 5.5, and then passed through Sephadex
G-100 column. Two protein fractions with a-galactosidase

activity were resolved. Figure 11 shows the elution

56

profiles of enzyme I and II from this column. Recycling the
fractions I and II removed some inactive protein and raised
the specific activity as shown in Figures 12 and 13 and
Tables 1 and 2.

Table 3. Effect of acid precipitation on the specific
activity of the precipitate (stage 2)

 

 

Specific 25% (NH ) $04 60% (NH4)ZSO4
activity Sp.act.(nRa /mg) Sp.act.(nKat/mg)
(nKat/mg) supernatant suprnt. ppt.
1.Extract
at pH 5.5 0.30 0.30 0.01 0.50
2.Precipitate
at pH 3.5 0.40 0.40 0.03 0.80

 

Table 4. Effect of ammonium sulfate concentration on the
specific activity of the precipitate (stage 3)

 

Specific activity Specific activity

 

(nKat/mg) (nKat/mg)
supernatant precipitate
1.Extract at pH 5.5 0.43
2.Acid precipitation
at pH 3.5 0.63

3.(NH4)ZSO4 precipitation

a. 25% 0.65
b. 35% 0.26

4.Supernatant (from a):

b. 60% (111102504 0.0

H)-
. 0
ht”

 

57

1.4-1

ABSORBANCE (405 nm)

 

 

 

TIME (MINUTES)

Figure 6. Time-dependence of PNPG hydrolysis by a-

galactosidase

I I I Mlil-UMNMMI‘I‘M‘MIM Iii IIIM‘I‘M'I'M‘I'NMil}.{NMOU‘I-‘IM‘N‘I‘IIf!“MiiiflfiiMOOIMQII‘N'M’IMCII’Q‘IMUNIQ

58

n4 1 P-E LRNBDR 48: ID Bab C‘a’C/Tlfl 081/ 0.th
4.. . -. Nl1 SCAN (R)= 3LT l= R3? 3= 3P0 129: C'I'Ci 081

”1.893 a

'7—
.p.
III)
N-
I

 

 

 

;
I
— I/ .
l \
/ \
”LENA 9 if \K. ..1.
Mea.e 498.9 , 698.3 883.8 933.8

Figure 7. Absorption spectrum of p-nitrophenol freed from

PNPG by a-galactosidase

ABSORBANCE (405 nm)

1.00
T

0.90-

0.80-

0.70—

d

0.60—

d

0.50-

0.40-

d

0.30—

0.20-

0.10-

d

 

0.00

59

 

j” r T I r I I

0.00 0.04

0.08

TTIIT‘IF TTTrIIIIIII TIrIVIIITIT

l
0.12 0.16 0.20 0.24 0.28 0.32 0.36 0.40
0 moles p—nitrophenol/ml

Figure 8. p-Nitrophenol standard curve measured at 405 nm

60

0.40-

0.35-

0.30-

0.25-

0.20-

0.15-

Absorbonce, 405 nm

0.10-

0.05-
e-—e White Cowpeas

0.00 T 4_—r-4 Block Cowtflm

3 I: 5 6 7
Extraction pH

 

Figure 9. Effect of extraction pH on a-galactosidase

activity measured at 405 nm

1.00-
0.90-
a
E 0.80-
> 070
.345 ° 7
53 0.50-J
3‘0
55 0.50-
450
<g 0.40-4
m
o
E 0.30—
8
Q 0.20-
m
0.10-
0.00
Figure

 

61

 

10.

F l I W “-1

1 2 :5 4 5.. 6 i 8, 9 10
pH Precipitation

Specific activity of supernatant after

precipitation of inactive protein at various pH, measured

at 405 nm (stage # 2)

62

 

 

1.0-
Enz.l
0 L0 0.84 Protein
CD 0
N <I'
< < l
'E _..__.>_~ 0.6—
3 -.-.>-
8 o ‘
O < Enz."
a) 0.4“
.E E
.3 RT «
8 c:
O. LLJ 0.24
' l
0.01....e...‘.,..l
O 10 20 30

Fraction # (4.5 ml/fraction)

Figure 11. Elution profiles of a-galactosidases I and II

from G-100 gel filtration column

.35

 

63

 

 

1.0-
U3 C3 C18—
0 (I)
si' (\I
<1 <1 ‘
- * Enz.|
g ‘E 0.6— .
.2 .93
‘5 g ‘
< o
a) 0.4-
E C
>. .93 -
E 8
UJ 0. CL2-
OsO $1 TL 7 T r I I I I r I I I l I Y—Y r ; : ; ; fl I r I I
O 10 20 3O
Fraction # (3.0 ml/fraction)
Figure 12. Elution profile of o-galactosidase I recycled

through a G-100 gel filtration column

Enzyme Activity, A405

64

 

 

0:4‘
53
CV 043—
<{
*5 Bah
c:
B .
C 0.2- Protein
0
C)
.E
(D
.H
g 0.1-—
CL
0.04;?;?;;;?—r??rfrrrli[Irrtfirv—Fifif
O 10 20 30
Fraction # (2.5 ml/fraction)
Figure 13. Elution profile of a-galactosidase II recycled

through a G-100 gel filtration column

65

Similar results were observed on Sephadex G-200 but the
elution was slower and the peaks were broader compared with
Sephadex G-100. Figure 14 shows the elution profiles of
enzyme I and II from Sephadex G-200.

Whatman CM-cellulose resin (Table 1) and a newly
introduced cation Accell-CM by Waters Association (Table 2)
were compared in stage 5. When enzyme I was passed through
Accel-CM with the pH gradient of 4.0 to 7.0, one active
peak was observed with higher specific activity (Figure
15). Two active fractions, enzymes II1 and 112
respectively, were obtained from enzyme II when passed
through Accel-CM (Figure 16). Enzyme I and II were also
fractionated on CM-cellulose using a NaCl gradient. Again,
Enzyme I eluted as one peak, and enzyme II eluted as two
peaks (Figures 17, 18 and Table 1).

The apparent specific activity of enzyme II1 was higher
than that of II2 and the proportions of the two total
activities varied with different batches of seeds. Enzymes
I and II2 were eluted from CM-cellulose and Accel-CM
columns by similar sodium chloride concentrations and pH
values, respectively, which indicated that they are charged

in a similar way.

Enzyme Activity, A405

0.61

C15—

0.4-J

0&3-

(12-

Protein Content, A280

0.1—

 

011

66

Ean Protein

.,. .r....r...
10 3O 4O
Fraction # (2.5 mI/fraction)

1il

50.

Figure 14. Elution profiles of o-galactosidases I and II on

an G-200 gel filtration column

Enzyme Activity, A405

Protein Content, A280

1.0—

CL8—

Ou4‘

0.2-i

 

013

O

L

67

pH

\

‘Frfrfirrrrrrrfr’w
10 20

Fraction # (3.5 mI/fraction)

Figure 15. Elution profile of a-galactosidase I on an Accl-

CM (Waters) column

no $> of cm

 

3O

68

(16-
Protein

pH
>12

5an11 _.

(O

 

 

0.2-

Enzyme Activity, A405
Protein Content, A280

 

 

 

 

OI) rr~.. .. .. r, ......
O 10 20 30

Fraction #

Figure 16. Elution profile of a-galactosidase II on Accl-CM

(Waters) column

Enzyme Activity, A405

SLOW
CD 1.6-
00
(\l
< .
*5 1.2—
_2
C d
O
U 0.8-
.E
8 .
O
L.
Q_ 094-

 

‘10

   
 
 

l I i I I I I '

" 20 30 ...40.. 50 60 70 80

69

Ean T

C15

(14

[NaCl], M

(12

Protein

 

 

 

   

Fraction # (3 ml/fraction)

Figure 17. Profile of a-galactosidase I on an CM-cellulose

column eluted with a sodium chloride gradient

Enzyme Activity, A405

Protein Content, A280

SLOW

1.6-

1.2-

(18-

Ou4—

 

 

CLOd

70

 

 

Bufi‘ _
Protein .0.5 2.
J34 E
E.
NaCl ‘01)
Buflz _Cl2
.01
i' 'i""i°"'I"'-:i:? ‘50
10 20 30 4O

Fraction # (3 rni/froction)

Figure 18. Profile of a-galactosidase II on an CM-cellulose

column eluted with a sodium chloride gradient

71

POLYACRYWIDB OBI. ELECTROPHORBBIB

The purpose of this experiment was to test the purity
of the enzymes and their mobility in different ionic
environments. PAGE was carried out under cationic, pH 3.5,
and anionic conditions, pH 8.3. A slight mobility for all
three enzymes was observed on anionic PAGE. One protein
band was observed for Enzyme I and II1 while enzyme 112
migrated in more than one band. Enzymes I,II1 and the main
band in enzyme 112 migrated the same distance towards the
anode (Figure 19). The R: value for all enzymes was 0.26.
At pH 3.5 the enzymes migrated towards the cathode as
discrete bands (Figure 20) when detected with a-
galactosidase-specific reagents and by Coomasie blue stain.
The Rf value was 0.33.

Dey et a1 (1982) reported that the mobility of the
three forms of the enzyme of yigia {aha on PAGE at pH 8.3
was zero. however, they all migrated at pH 3.5. The
mobilities of I and II1 appeared to be identical and these
proteins were apparently less basic than 111. In view of
these results, they suggested that the order of elution of
the enzymes from Chi-cellulose columns by a NaCl gradient
was not expected. They suggested that only a proportion of
the charged regions of the enzymes interact with the ion
exchange material and the elution is not a function of the

total charges on the proteins. In our study the mobility of

72

 

 

l
O
4:. on N

 

 

I
0
U1

 

 

 

 

 

 

 

 

 

Figure 19. Anionic polyacrylamide disc gel electrophoresis
of a-galactosidases I, II1 and II2 at pH 8.3 (Coomassie
stain). Rf = 0.26

'73

 

[Hi
I
i

 

 

 

 

 

 

 

 

Figure 20. Polyacrylamide disc gel elecrophoresis of a-
galactosidases I, II1 and II2 at pH 3.5. The same bands
were visualized by Coomasie stain and by enzyme activity

detection (Fast blue RR salt). Rf = 0.33

74

the Enzymes I, III and 112 appeared to be identical and

these proteins may have the same charges.

MOLECULAR HEIGHT DBTBRKINITION

Two methods, gel filtration and. SDS-PAGE
chromatography, were used to determine the molecular weight
of the enzymes.

Gel Filtration Chromatography Using Sephacryl 8-200

Figures 21, 22 and 23 show the elution profiles of MW
standards and Blue Dextran on the Sephacryl gel filtration
column. Figures 24, 25 and 26 show the elution volume
profiles of enzyme 1,111 and II2 respectively. The Ve (half
maximum peak height of the leading edge of a protein peak
after the extrapolation of the leading side to the base
line) for all proteins, including the enzymes, were
measured. This is more reliable than the peak crest because
the position of that crest depends on the sample volume
(Cooper, 1977).

Figures 27 and 28 show calibration curves for the MW
determination of the enzymes by plotting REV and Kav vs. MW
of the known proteins,respectively. Enzyme I, Enzyme III
and Enzyme II2 have approximate MW of 110,720 1 3780,
29,000 i 355 and 30,000 i 368, respectively. The most
likely causes of error in protein MW estimated by gel-

filtration are inaccurate estimation of elution volumes,

ABSORBANCE (280 nm)

75

 

 

2.0-—
0.0- . . r , . . . l 1 1
2o 22 24 26 28 so

FRACTION NUMBER (2.3 ml each)

Figure 21. Elution profile of Blue Dextran (MW 2,000,000,

Vo = 54.5, VT a 144 ml) from Sephacryl S-200 column

76

i .0 j
A
g 08— Alcohol Dehydrogenase
O - Carbonic Anhydrase
00
N 0.6—
v
DJ .
0
Z
< 0.4—
CD
0:
O 7
E}
< 0.2—
0.0

 

 

I ' l r l ' I ' I . I I I I I ' r I I ‘ I I '
2O 22 24- 26 28 30 32 34 36 38 4O 42 44
FRACTION NUMBER (2.3 ml each)

Figure 22. Elution profiles of alcohol dehydrogenase (MW
150,000, Ve a 62.0 ml) and carbonic anhydrase (MW 29,000,

Ve - 87.6 ml) from Sephacryl S-200 column‘

77

1.0fi

Albumine,Bovine Serum

0.8—

0 5- Cytochrome C

0.4-

ABSORBANCE (280 nm)

0.2-

 

 

CLO . I ‘* I ' III*I I*I ‘r r*? I‘F‘W . l I I ' I ' r“
25 27 29 31 33 35 37 39 41 43 45 47 49
FRACTION NUMBER (2.3 ml each)

Figure 23. Elution profiles of bovine albumin (MW 66,000,

Ve =- 69.0 ml) and cytochrome C (MW 12,400, V = 97.0 ml)

e
from Sephacryl S-200 column

ABSORBANCE (405 nm)

78

(105-

(104—

(103—

(102—

(101—

 

 

(100 I If I I I I I T I I
25 26 27 28 29 3O 31 32 33 34 35

FRACTION NUMBER (2.3 ml each)

Figure 24. Elution profile of Enzyme I (at pH 5.0)

from Sephacryl S-200 column. Ve = 65.5 ml

ABSORBANCE (405 nm)

79

0.2-

0.0

 

 

I I l I I l I I I I T
34 35 36 37 3a 39 4o 41 42 43 44 45

FRACTION NUMBER (2.3 ml each)

Figure 25. Elution profile of Enzyme II1 (at pH 7.0)

from Sephacryl S-200 column. Ve = 85.0 ml

80

0.03-1

0.02-

0.01-

ABSORBANCE (405 nm)

 

 

4| I
0.0031 32 3'3 3‘4 3'5 3'6 3‘7 3'8 39 4'0 41 4:2 43
FRACTION NUMBER (2.3 ml each)

Figure 26. Elution profile of Enzyme II2 (at pH 7.0)

from Sephacryl 8-200 column. Ve = 84.6 ml

LOG MW

4.25—

4.00

81

Alcohol Dehydrogenase

  
   
  

Albumine, Bovine

310'!»

o Carbonic

Anhydrase

o Cytochrome

 

 

1.00

Ve/Vo

Figure 27. MW calibration curve using protein standards on

Sephacryl 8-200, based on Ve /Vo

Kav

82

     
  

 

 

(LS—
Cytochrome
(l4—
, Carbonic Anhydrase

0.3_' a = 2 o 0

b = -0.38

r = -0.98

_ n = 3
(124
Albumine. Bovine
0'1— Alcohol Deh drogenose
a Y
03 4 “I I r* I I I I I I 2‘ I . I . I
4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00
LOG MW

Figure 28. MW calibration curve using protein standards on

Sephacryl S-200, based on Kav

83

differences in shape between one protein molecule and
another, and density differences between solvated protein
molecules, resulting in different ratios between sizes and
MW (Andrews, 1964).

Dey and Pridham (1969) located two forms, I and II, of
the enzyme in resting L Lab; seeds. Using Sephadex gel
filtration G-200, they found that the MW of I and II were
209,000 and 38,000, respectively. Later work (Dey et al.,
1971) showed that form II was a mixture of two enzymes, II1
and II2 with MW (sedimentation equilibrium method) of
45,730 1 3,037 and 43,390 1 1,409, respectively.
Examination of the MW of I by the sedimentation equilibrium
gave a value of 160,400 1 2,850 (Pridham and Dey, 1974).

Ml Estimation Using ans-PAGE chromatography

The mobility of a protein in acrylamide gels is a
function of both its net charge and size. Hypothetically,
two proteins of different MW may migrate toward the anode
at the same rate if their size differences are balanced by
compensating charge difference (Shapiro et al., 1967; Weber
and Osborn, 1969). For this reason acrylamide gel
electrophoresis using gels of only one pore size may not be
used to gain information about the MW of a molecules. In an
effort to surmount these problems Shapiro et al. (1967)
attempted to separate a mixture of proteins in the presence

of SDS.

84

Figure 29 shows the SDS-PAGE mobilities of a-
galactosidases I, II1 and 112. These mobilities were
sufficient to allow an estimation of their MW by plotting
log MW vs. relative mobility (Rf) (Figure 30). Enzyme I,
Enzyme 111 and Enzyme II2 have approximate MWs equal to
30,916 :I: 1,042, 29,875 :I: 413 and 30,350 i: 550 daltons with
R: values of 0.63, 0.65 and 0.64, respectively. The weak
bands appearing with the main enzyme bands in the SDS-PAGE
picture (Figure 29) may indicate the presence of minor
polypeptides or contaminating proteins. Various We have
been reported in the literature for a-galactosidases from
different plant sources (Appendix 2).

The following observations suggest that Enzyme I is a
tetramer of Enzyme 112:

a) Gel filtration indicated that the MW of Enzyme I is
110,720 :I: 3780 vs. 30,000 i 368 for Enzyme I12.

b) SDS-PAGE which depolymerized Enzyme I resulted in almost
equal MW estimates of the two enzymes; 30,916 1 1,042 vs.
30,350 i 550 for Enzymes I and 112, respectively.

c) The two enzymes are eluted at the same pH value (4.4-
4.8) in Accel-CM chromatography and at the same NaCl
concentration value (a 0 . 24M) in CM-cellulose
chromatography. These methods separate compounds on the

basis of charge only.

 

 

 

 

 

Rf
-0.0
-0.1
-0.2
‘
1
III. ..0,3
-0.4
2,1III
-0.
3- 5
112 111 I
‘1‘, «1!? '-0 6
4-- ~—
- -' '07
5 —-
-0.8
6"IIP - 0.9
l
741...: . ' "‘-’ “J _ 1.0

 

Figure 29. SDS-PAGE of cowpea a-galactosidases I, II1 and
112. MW of markers: 1, bovine albumin, 66,000: 2, egg
albumin, 45,000; 3, glyceraldehyde-3-phosphate
dehydrogenase, 36,000; 4, carbonic anhydrase, 29,000; 5,
trypsinogen, 24,000: 6, trypsin inhibitor, soybean,20,000;

7, a-lactalbumin, 14,000.

LOG MW

5.00-I

4.75 -

4.50-

4.25-

 

4.00

86

(Jr!
(:2)

, AlbumIn. Bovme _ ' . 88
0 . 99

2:! "S (3" III
II II II II

(.31 I

 
   
   
  
   
  

Albumin, Egg
Glyceroldehyde—3—phosphate Dehydrogenase

Corbonic Anhydrase
Trypsinogen

.Trypsin Inhibitor, Soybean

. olpho-Lactalbumin

 

I

I I I I I I I I I I I I
0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40

RELATIVE MOBILITY (Rf)

Figure 30. Molecular weight calibration curve using protein

standards and SDS-polyacrylamide gel electrophoresis

87

882!!! I183T168

x. and Vmax for Ensyme I, 111 and 112 Using me as
Substrate

Lineweaver-Burk plots (equation 1) were used to
calculate Km and Vmax values (Figures 31, 32, 33). Table 5
summarizes the Xi and Vmax values for the enzymes. The Km
value of enzyme II2 with PNPG (5.3 mM) was more than twice
as high as the value obtained for Enzyme II1 (2.2 mM) and
approximately four times as high as the Km of Enzyme I (1.5
mM). These results are not very different from other
researchers (Appendix 2).
Table 5. Km and Vmax values of a-galactosidases using PNPG

as substrate

 

 

Enzyme I - 111 112
Km (mM) 1.5 2.2 5.3
Vmax (umole/min/mg) 0.10 0.22 0.01

 

Determining x. and vh‘: Values of Enzymes I, 111 and 112
Using Natural Substrates

Raffinose and stachyose as natural enzyme substrates
were used to determine Km and VmaX' Stachyose is hydrolyzed
more slowly, while raffinose seemed to be the substrate of
preference because a-galactosidase affinity towards
raffinose was higher than towards stachyose (Gavriel,

1985).

   

1/V (umoles p.NitrophenoI/mn/mg)—1

 

88

    

"H
001

= mM
x .1 uMoIe/min/mg

g;

 

Figure 31.

r T r
0T4 018 1.2 _ 1.6 2.0

1/[81 (mMr‘1

Lineweaver-Burk plot of the hydrolysis of PNPG

by a-galactosidase I. n a 3

89

2C}-

—A
CD
I

l/v (“moles p.Nitrophenol/mn/mg)"1

 

2
0.22 uMole/min/mg

 

 

I" I I I I I I I I T j r I I l
-0.5 -0.4-0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

I/IS] ImM)“1

Figure 32. Idneweaver-Burk plot of the hydrolysis of PNPG
by a-galactosidase III. n = 3

90

O
O
J

   
  

lLlllllllllle

 
     
 

L4 80

CD

E

\ .

C ..

E ..

2> I

o _.

C 604

q; .

.2 .I

o- .1

o u

b -I

'2. :

a. 40_

a) I

53 .

o a:

E I

1 " =
V d

> .I =
\ 20 . =

.3
0.01 uMoIe/min/mg

 

 

Fri |' I I I I I I I I I II
-Ou4-Oq3-O.2—CLIIJJ3 0.1 052 On3 0.4 035 CL6 CL7 CLB CL9 1.0

I/[S] (myr-1

Figure 33. Idneweaver-Burk plot of the hydrolysis of PNPG

by a-galactosidase IIZ. n - 3

91

Raffinose

When raffinose was the substrate, the enzymatic
activity of Enzymes I, II:1 and 112 was measured by the
amount of liberated galactose. Originally, it was attempted
to determine the freed galactose using HPLC but the
method was not sensitive enough to measure the small
quantities of galactose present. An enzymic method of
determining galactose was used as microquantities of
galactose had to be determined,

Km and V for Enzymes I, II1 and II2 with raffinose

max
as substrate were determined using Lineweaver-Burk plots
(Figures 34, 35, 36). Table 6 summarizes the Km and Vmax
values for the different enzymes. The Km value of enzymes I
(4.6 mM) and II1 (5.0 mM) were close to each other, and
were approximately three times as high as the Km of Enzyme
II2 (1.6 mM). Thus, Enzyme II2 has higher affinity (1/Km)

for raffinose than Enzyme I and III.

Table 6. Km and Vmax values of a-galactosidases using

raf f inose as substrate

 

Enzyme I 111 112

 

Km (mM) 4.6 5.0 1.6

Vmax (umole/min/mg) 0 . 0034 0 . 043 0 . 001

 

92

 

 

 

1000— .
800-4
600-—
>
\
400—4
a = 295
b = 1350
200-— r = 0.98
Km = 4 6 IIIM .
Vmax = 0.0034 uMoIe/mIn/mg
F ' f I r I L I I I r I r I I I f I
—.25 —.1 5 -.05 0.05 0.15 0.25 0.35 0.45 0.55

I/[S]

Figure 34. Lineweaver-Burk plot of the hydrolysis of

raffinose by a-galactosidase I. n = 3

 

    

93

 

100-
80— .
60—
>
\
404
a = 23
b = 116
_, r - .99
Km = 5.0 mM
Vmax = 0.043 uMoIe/min/mg
I I I I I I I I I I I I I I I I
—.25 -.15 -.05 0.05 0.15 0.25 0.35 0.45 0.55

1/[S]

Figure 35. Lineweaver-Burk plot of the hydrolysis of

raffinose by a-galactosidase 111. n =

3

 

94

2000 -

1800-

1600-

1400-

 

1200-—

>
\ 10 0(—
BOO-I

b =
500- a = 1076
r =

 

r

fi I I

I In I FI I I I If I I
-.55 -.45 -.35 -.25 -.15 --.05 0.05 0.15

I/IS]

r ' r j r T- I F I
—.65 0.25 0.35 0.45 0.55

Figure 36. Lineweaver-Burk plot of the hydrolysis of

raffinose by a-galactosidase IIZ. n = 3

95

stachyose

It was difficult to determine the Ru and Vmax for a-
galactosidases using stachyose as substrate by measuring
the amount of liberated galactose. Theoretically, two moles
of galactose will be produced from hydrolysis of one mole
of stachyose by a-galactosidases. While the reaction is in
progress, it is impossible to assume a definite ratio of
freed galactose to hydrolyzed stachyose. Therefore, Km and

V values were determined using HPLC to measure the

max ’
disappearance of stachyose.

The Lineweaver-Burk plots of Enzymes I, and II]- are
shown in Figures 37, 38. Table 7 shows the Km and Vmax
values of enzymes I and Ill. The Km value for enzyme II1
was higher than the value of Enzyme I, which means the
affinity of Enzyme I towards stachyose was higher than the
affinity of Enzyme III. The Km values for Enzymes I and II1

were 11.0 and 15.0 mM, respectively, using Lineweaver-Burk

plot.

Table 7. Km and Vmax values of a-galactosidases using

stachyose as substrate

 

Enzyme I II1

 

Km (mM) 11 15

Vmax (umole/min/mg) 0.05 0.1

 

96

70-

60—

50—

4o-

I/v

30~

 

20
224
0. 98

=11mM

20—

g "30"”

10— Vmax 0.05 uMoIe/mIn/mg

 

 

I ‘ I I I
—.15 —.10 -.05 0.00 0.05 0.10 0.15 0.20 0.25

I/[S]

Figure 37. Lineweaver-Burk plot of the hydrolysis of

stachyose by a-galactosidase I. n = 3

97

   
 

 

 

I f I I
—.15 -.05 0.05 0.15 0.25 0.35 0.45

l/[S]

Figure 38. Lineweaver-Burk plot of the hydrolysis of

stachyose by a-galactosidase III. n = 3

98

Different values have been reported in the literature
for the Km of a—galactosidases of different sources using
PNPG as substrate (Appendix 2). The Km for c-galactosidases
I and II from y. m, using raffinose and stachyose as
substrates were also reported (Dey and Pridham, 1972). The
Km values for Enzyme I were 4.0 and 7.5 mM and for Enzyme
II were 5.0 and 5.26 mM using raffinose and stachyose,
respectively. The method of measuring the Km using

stachyose as substrate was not mentioned.

Effect of a Natural Substrate Concentration on Enzyme
Activity

Table 8 shows the effect of increasing raffinose
concentrations on Enzymes I, II1 and 112. The activity of
Enzyme I started declining at concentrations higher than 40
mM of raffinose. The activity of Enzyme IIl started
diminishing at 160 mM raffinose and that of Enzyme 112
showed a similar decline at-80 mM raffinose. With different
Km and enzyme amount, the comparison between the enzymes

was not possible.

99

Table 8. Effect of substrate (raffinose) concentration on

c-galactosidase activity

 

Enzyme activity Kat/ml (at 30' C)

 

 

Raffinose Enzyme I Enzyme II1 Enzyme II2
conc.(mM) Rh 4.6 mM Km 5.0 mM Km 1.6 mM
10 217 552 98
20 233 665 . 108
40 . 227 756 133
80 207 816 118

160 200 718 93

 

The observations that, above a certain substrate
concentration, for each enzyme, the reaction rate started
decreasing indicates that substrate itself act as an
inhibitor of the reaction, probably by forming inactive
complexes with the enzyme as shown in the following scheme

(Dey and Pridham, 1972).

E + S <---=-> ES ------ > E + product

V
E82 (inactive complex)

100

STABILITY

The stability of Enzymes I, II1 and II2 during storage
for 24 hr at four different temperatures and at various pH
values was studied. Figure 39 shows the effect of pH and
temperature on the stability of Enzyme I. The enzyme
maintained its full activity in the pn range of 4.5 - 5.0
during 24 hr at 4' C. The enzyme lost almost 35% of its
original activity in the pH range of 4.5 - 6.0 at 22' C.
The enzyme was very unstable at 37' C, in general. The
minimum loss of activity occurred at pH 5.8.

Figure 40 shows the effect of pH and temperature on
the stability of Enzyme III. At 4' C and 22' C, Enzyme II1
was more stable than Enzyme I and 112; it maintained 100%
activity in the pH range of 3.5 to 7.0 for 24 hrs. At 37' C
almost 90% of original activity was preserved for 24 hr, at
pH 4.5 - 5.5.

Figure 41 shows the effect of pH and temperature on
the stability of Enzyme 112. At 4° c, it maintained 75-80%
of its original activity for 24 hrs in a large pH range
(3.5 to 7.5). At 22' C the stability of the enzyme was
somewhat lower at pH 4.0 but it did keep 75% of its

original activity in the pH range of 5.0 to 6.5.

101

 

 

1001
: k—A 4°C
3 o—o 22°C
3 e—e 37°C
801
>\ 2
ti :
.2 :
“(3 j
<( 60:
m E
.2 1
2 4.0-5
m _
O: 2
m a
20—2
1
0‘ I FT :T I II I I II
0 1 2 3 4 5 6 7 8
pH

Figure 39. Effect of pH and temperature on the stability of

Enzyme I after 24 hr of storage

102

 

 

 

100: r 5 ‘ I 7%
: m—x 45°C
5 e—e 37°C
5 k—i 22°C
80? e—e 4°C
)5 1
ti 1
._>_ :
‘8 2
<1 60?
a) 3
.2 :
+4 4
2 40-5
0) .
O: I
L\° 3
20%
3
i
O T l" :l I I I I ‘1
0 2 4 6 8

Figure 40. Effect of pH and temperature on the stability of

Enzyme II1 after 24 hr of storage

7. Relative Activity

 

 

100
a—a 4°C
H 22°C
e—e 37°C
80
60
4O
2O
0 I I FI Ll / I I I
0 I 2 3 4 5 6 7 8

pH

Figure 41. Effect of pH and temperature on the stability of

Enzyme II2 after 24 hr of storage

104

In general, all three enzymes showed their greatest
stability at 4' C and at intermediate pH values (Table 9).
Enzyme II1 was more stable than Enzyme II2 3 Enzyme I was
the least stable under the conditions of this experiment.
Enzyme III, the only one showed some activity at 45' C.

The thermal stability of other a-galactosidases,
isolated from various species of the genus W,
such as A. my; demonstrated an optimum temperature at 55'

C (Sugimoto and Van, 1970).

OPTIMUM p8 AND OPTIMUM TEMPERATURE
FOR m ENZYME ACTIVITY

The pH optimum for the c-galactosidases I, II1 and 112
were tested at 30’ C and found to be 5.0, 5.9 and 5.3
respectively (Figures 42, 43, 44) . The activity decreased
rather rapidly on either side of the optimum pH. various
figures have been reported in the literature as the optimum
pH of a-galactosidase activity (Appendix 2).

The optimum temperature for the activity of each of .
the three enzymes was tested at the optimum pH. For all
three enzymes the optimum activity temperature was found to
be 50' C. The activity decreased rapidly at temperature
higher than the optimum temperature indicating heat
denaturation (Figures 45 , 46, 47) . Most a-galactosidases
behave normally as rate of the enzyme-catalyzed reaction

increases to an optimum value with increasing temperature

105

until inactivation of the enzyme occurs (Dey and Pridham,
1972) . a-Galactosidase from W W has an
optimum temperature of 50' C (McGhee et al., 1978). Optimum
temperature of a-galactosidase from L m for
hydrolysis of PNPG was 50' C (Cruz and Park, 1982).

Table 9 summarized the results of the pH and
temperature of optimum activity along with the pH of
optimum stability at 22' C .

Table 9. Temperature and pH optima of a-galactosidases from

 

 

cowpeas
Enzyme I 111 112
Opt. activity pH (30' C) 5.0 5.9 5.3
Opt. activity temp.(at opt. pH) 50'C 50'C 50’C

Opt. stability pH (22'C) 4.5-6.0 3.5-7.0 4.5-6.5

 

106

 

 

1001
80—
3‘
22>:
*5
< 60"
(D
.2
4..)
2 40—
o
[I
N
20‘
O T I ' I r r l r 1
O 1 2 3 4 5 6 7 8

Figure 42. Effect of pH on the activity of Enzyme I

107

 

 

1001
ao—i
3‘ E
1.5. E
“5 ;
<1 60:
<12 3
.2 :
£2 40%
m _
CK a
N E
205

0‘ I I I I I I I

O 1 2 3 4 5 6 7'

Figure 43. Effect of pH on the activity of Enzyme II1

108

 

 

100m
80—1
k?
>.
:2:
.2 50..
5 \
< /
o
.2 40—
4.;
.9
Q)
o:
201
O r T: I I I I I
O 1 2 3 4 5 6 7
pH

Figure 44. Effect of pH on the activity of Enzyme II2

109

 

 

100:
530-:
.J
3‘ 1
.2 :
44 -
U I.
<1 60:
a) E
> d
'5 :
.9 40:
(1) ..
0: :
B\° :
20—3
.
.
.4
i
I

O rrfiTFIIIIrIIIIFrIIrTTIIKrillliilrrTYIFI

O 20 40 . 60 80

Temperature (CO)

Figure 45. Effect of temperature on the activity of Enzyme
I

110

   

100

lllllllllLl

CI)
0

60

4O

73 Relative Activity

 

llllllllllllllllllllllllllllllllljllll

 

Or—rlIllIilIlTlIU'UUI'lililirjrilirlIIIIFI‘

o 20 40 so 80

Temperature (CO)

Figure 46. Effect of temperature on the activity of Enzyme
1
II

111

 

 

100:
I
80{
>‘ 2
ii I
.> :
*8 :
<1 60:
m 2
.2 -
4.1 3
2 401
m -
D: 2
R I
20%
1
0‘IUIIVUrIIrfIITIITFIIIIIFIIjrrlIIIIYIIIII
O 20 4O 60 80

Temperature (C 0)

Figure 47. Effect of temperature on the activity of Enzyme
I12

112

GBRIINITION EFFECT ON COWPEA a-GALACTOBIDABB

The a-galactosidase activity increased rapidly during
the first 6 hrs of germination at 24' C and leveled off
during the subsequent 24 hrs (Figure 48). Germination at

30' C showed a similar trend in a-galactosidase activity.
In a second experiment, the a-galactosidase activity
was assayed for up to 72 hrs of germination at 30' C in the
sprouts and the remaining seeds. As shown in Table 10, this
activity increased less rapidly in the sprouts during the
first 42 hrs, than during the following 30 hrs, while the
activity in the desprouted seeds did not change very much

during the entire 72 hrs period.

Table 10. Total a-galactosidase activity in sprout and

desprouted seeds of cowpeas at 30' C (pH 5.5, PNPG)

 

 

Time of sprouting Part Enzyme activity
(hr) (A405 /9 dry Wto)
0 whole seed 32
42 sprout 39
72 sprout 68
42 seeds w/o sprout 51

72 seeds w/o sprout 58

 

113

 

ACTIVITY (A 405/ g dry wt)

 

 

60-
24 C
50- ._
30 C
40-
i
30..
20-
10-4
O I” I I I I I’ II I
O 5 10 15 20 25 30 35 40
TIME (HOURS)
Figure 48. a-Galactosidase activity at different

germination temperatures (2 hrs soaking prior to

germination)

114

GIRIINIIION 3,386! O! COWPEA OLIGOBLCCEARIDBB

The oligosaccharide changes during germination for 24
hours at 24' C (room temp.) are shown in Figure 49.
Sucrose, raffinose and stachyose concentration did not
change significantly during the 24 hr germination period (P
< 0.05). The correlation coefficients between
concentrations of each sugar and time were around 0.8
(sucrose 0.78, raffinose -0.78 and stachyose -0.8). The
statistical analysis of oligosaccharide changes during

germination for 24 hours at 24‘ C shows in Table 11.

Table 11. The statistical analysis of odigosaccharide

concentration changes during germination for 24 hrs at 24'

 

 

Sucrose Raffinose Stachyose
Slope (arithmetic)* 0.029 -o.oos -o.042
Slope (logarithmic) 0.0065 -0.0182 -0.007
Intercept (arithmetic) 1.61 0.213 3.15
Intercept (logarithmic) 0.21 —0.64 0.49
r value (arithmetic) 0.78 -0.78 -0.80
r value (logarithmic) 0.74 -0.81 -0.77
probability (arith) 0.12 0.12 0.11
probability (logarith) 0.16 0.09 0.13
Standard error (arith) 0.013 0.0024 0.019
Standard error (log) 0.0034 0.0076 0.003

 

* arithmetic and logarithmic in the Table were for
linearity. n - 2

115

 

Concentration

* rofl'nou

 

(9 Sugar/ 100 9 Sample, Dry wt.)

.J

T j' I i T l’ _ j I—

13 18 24
GERMINATION. HOURS

Figure 49. Oligosaccharide changes during germination for

24 hours at 24' C (2 hrs soaking prior to germination). n=3

116

8330 EAST! 130083110”

Figure 50 shows the changes in oligosaccharide
contents during 24 hrs of cowpea flour paste incubation at
two different temperatures 24' C and 34’ C. The 24 and 34'
C were chosen to cover the temperature range of the places
in which the cowpeas are consumed. Sucrose content
significantly decreased during incubation at 24' C and 34'
C (P < 0.05). The rates of sucrose decrease (slope) at 24'
and 34; C were -0.057 and -0.061 % sugar/hr, respectively.
Raffinose content did not change significantly at 24' C (P
< 0.05) although a significant decrease was observed at 34'
C (P < 0.05). Raffinose decreased twice as fast at 34' C
than at 24' C (-0.0074% / hr, P < 0.05). The rate (slope)
of stachyose depletion at 24' C and 34' C were -0.064 and -
0.1 % sugar/ hr, respectively, and there was a significant
difference between the two (P < 0.05). Stachyose and
sucrose concentrations approached zero at 34' C after 24 hr
incubation. The pH of the paste was 6.35 and did not change
significantly (P < 0.05) at 24' C. At 34' C the pH changed
significantly (P < 0.05) at a rate (slope) of -0.016. The
pH decreased from 6.35 to 6.00. The rate of oligosaccharide
content of the paste decreased in the following order
during incubation: stachyose> sucrose> raffinose.

Since the arithmetic linear correlation is better than

the logarithmic linear correlation for raffinose and

117

stachyose, the rate comparisons (slopes) between the two

temperatures refer to the arithmetic linearities. The
arithmetic linear correlations were easier to plot and to
interpret although the logarithmic correlations were higher
within the same temperature. Table 12 shows the statistical

analysis of oligosaccharide and pH changes during flour

paste incubation at different temperatures.

Table 12. The statistical analysis of oligosaccharide
concentration and pH changes during flour paste incubation

at different temperatures

 

 

 

Sucrose Raffinose Stachyose pH

24°C 34°C 24°C 34°C 24°C 34°C 24°C 34°C
slope1 —0.057 -0.061 -0.004 -0.0074 -0.064 -0.10 -0.021 -0.016
slope2 -0.034 -0.041 -0.003 0.018 -0.015 -0.05
Incpt.1 1.54 1.46 0.27 0.28 2.66 2.50 6.46 6.34
Incpt.2 0.24 0.22 -0.57 0.53 0.43 0.50
r value1 -0.94 -0.93 -0.75 -0.98 -0.90 -0.96 -O.85 -0.88
r value -0.98 -0.99 -0.78 -0.99 -0.94 -0.99
Prob.1 0.017 0.023 0.14 0.004 0.036 0.003 0.07 0.047
Prob.2 0.004 0.001 0.123 0.002 0.02 0.001
Std. 2.1 0.012 0.014 0.002 0.001 0.018 0.016 0.01 0.005
Std. 2.2 0.004 0.003 0.004 0.002 0.003 0.004

 

1- arithmetic linearity, 2- logarithmic linearity, n=

118

 

 

 

 

 

 

 

 

 

 

 

 

7
”T
4.;
g 6
E;
. 5
c .2
O 0.
2.: E
8 ° 4
.H In
S 01
0
O
5 O 3
O ‘—
E
0 2
0‘
3
(I)
0‘ 1
v
_._-'_"_‘!‘°_°.°£4¢L g
'"Mnna4m “fi\‘fi‘
0'0 fl r I 7 T T fi— 1 i I F I T 1 l T T i i. I I 13"
0 3 6 9 12 Is 18 21 . 24

INCUBATION TIME (HOURS)

Figure 50. Oligosaccharide and pH changes during 24 hrs of

cowpea flour paste incubation at different temperatures

119

GBRIIIMTIO)! PLUS 111008113108 01' m SEED P1813!

Figure 51 contains plots of stachyose, raffinose and
sucrose concentrations during two hours of soaking, six
hours of germination, followed by 12 hours of incubation of
a paste made from germinated seeds at two different
temperatures, 24' C and 34‘ C. Two hours soaking was used
prior to germination in order to speed up the germination
process. The a-galactosidase activity in the germinating
seeds peaked at about 6 hours (Figure 48). The incubation
of the paste was stopped at 12 hrs because considerable
oligosaccharide breakdown was achieved by then without
apparent microbial spoilage.

Stachyose content significantly decreased (P < 0.01) at
both 24' C and 34' C. Raffinose was significantly decreased
at both 24' C and 34' C (P < 0.05). The rates of decrease
(slope) at both temperatures were rather low (-0.004 % /hr
at 24' .C: -0.003 8 /hr at 34' C). Sucrose increased
significantly at 24' C (slope - 0.02) at P < 0.01, but it
did not change significantly at 34' C (slope = -0.011). The
pH increased significantly from 6.35 to 6.6, (P < 0.01) at
24' C (slope - 0.014) but did not change significantly (pH
6.35) at 34' C (P < 0.05). Raffinose content was not
significantly altered with increasing temperature (P <
0.05). Stachyose, sucrose and pH changed significantly with

increasing temperature (P < 0.05) . Stachyose decrease was

120

significantly greater at 34° C than at 24' C. The rate

comparisons (slopes) between the two temperatures (P <
0.05) refer to the arithmetic linearities which is better
than logarithmic linear correlation for sucrose, stachyose
and pH. Table 13 illustrates the statistical analysis of
oligosaccharide and pH changes during the situation

described in Figure 51.

Table 13. The statistical analysis of oligosaccharide
concentration and pH changes during 2 hrs of soaking, 6 hrs
of germination, followed by 12 hrs of incubation of a paste

made from germinated seeds at two different temperatures,

 

 

 

24 and 34‘C
Sucrose Raffinose Stachyose pH

24°C 34°C 24°C 34°C 24°C 34°C 24°C 34°C
slope; 0.02 -0.011 -0.004 -0.003 -0.072 -0.12 0.014 0.00
slope 0.004 -0.003 -0.008 -0.006 -0.015 -0.04
Incpt.; 1.68 1.78 0.28 0.27 2.92 2.98 6.30 6.37
Incpt. 0.23 0.25 -0.54 -0.57 0.48 0.56
r value; 0.96 -0.83 -0.95 -0.90 -0.99 -0.99 0.94 0.01
r value 0.94 -0.89 -0.91 -0.88 -0.98 -0.94
Prob.; 0.01 0.084 0.014 0.04 0.0001 0.001 0.005 0.99
Prob. 0.018 0.044 0.03 0.046 0.002 0.02
Std. 8.; 0.003 0.005 0.001 0.001 0.003 0.007 0.002 0.01
Std. E. 0.001 0.001 0.002 0.002 0.002 0.008

 

1- arithmetic linearity, 2-

logarithmic linearity, n= 3

121

 

 

 

 

 

 

 

 

 

3.5— #4046) '1 7
I - #4 -I
" fl ‘ a
’7 ' ' we”)
4" 3.0"J "‘ 6
3
E‘ ..
D - 5
c 35 .
.9 g
“2’ o - 4
4_, (n
C '1
8 01
O ‘4 3
S o
o *- 4 -
\
L- —-I —J
O 1.0 2
g‘ - 3
(D
005— 4 d1
3 tm(240)
' r W6340) 4
0.0 r T fTfi I T I 1 l I I T I 1 T I l T I
0 2 4 6 8 10 12 14 16 18 20

TIME (HOURS)

Figure 51. Oligosaccharide concentration and pH changes
during 2 hrs of soaking, 6 hrs of germination, followed by
12 hrs of incubation of a paste made from germinated seeds

at two different temperatures, 24' and 34' C

122

These three experiments show that it is possible to
decrease the flatus-related sugar content of cowpeas,
especially stachyose which is present in much larger
concentration than raffinose. The most efficient way
appears to be forming a paste with cowpea flour and let it
stand in a warm place (around 30' C) overnight.

Safety Concerns

The relatively high pH of the paste makes it
susceptible to spoilage by microorganisms, including
W 1193111111111!- Although microbiological studies
were not conducted, the paste did not show signs of
spoilage (smell, gas). The possibility of an outgrowth of
9“ Wm may be eliminated, if Dovlo's et al. (1976)
procedure is followed which includes aerating of the paste

and subsequent frying.

IMRTBBB ACTIVITY

The invertase activity of dormant seeds was 5.8
nkat/mg sample, that of seeds germinated for 12 hours at
24° C was 10.3 nkat/mg sample, and that of seed paste
incubation for 12 hours at 24' C was 20.0 nkat/mg sample.
The high invertase activity in the paste explains the quick
disappearance of sucrose in that material. Invertase
activity was lower in dormant and germinated seeds than in

the paste made from cowpea flour. Invertase activity

123

increased in the paste probably as a result of the
decompartmentation of the enzyme and its substrate.
Schwimmer et al. (1961) suggested that the low
invertase activity in potato tubers may be due to the
presence of an inhibitor. Enzyme action can be accelerated
by decompartmentation of enzyme and substrate via cell and

organella breakage (Schwimmer, 1981).

CONCLUSIONS AID DIRICTIONS FOR FUTURE 33883303

The objectives of this research focused on
investigating the properties of cowpea a-galactosidases,
activity, in dormant and germinating cowpeas, and
determining the oligosaccharide changes during the
germination of seeds and the incubation of a paste from
these seeds. In the following sections, the main research
contributions of this research are summarized and avenues
for future research development are described.

Two forms of a-galactosidases, I and II, were isolated
using gel filtration on Sephadex G-100 and G-200. Form II
of a-galactosidase was further resolved into two fractions,
II1 and 112, using Accl-CM and CM-cellulose cation exchange
chromatography. The three enzymes exhibited different Rh ,
V

ma
for enzymes I, II1 and II2 with different substrates. The

x and optimum activity pH. Table 14 shows the Km values

values of the optimum activity pH obtained for enzymes I,
II1 and 112 were 5.0, 5.9 and 5.3, respectively. All three
enzymes displayed maximum stability at 4’ C and at
intermediate pH levels (4.5-6.5). The optimum activity
temperature was the same for all three enzymes, 50' C.

The effect of increasing raffinose concentration on
Enzyme I, II1 and II2 was studied. Above a certain level of

substrate concentration, the reaction rate started

124

125

decreasing indicating that the substrate itself started

acting as an inhibitor of the reaction.

Table 14. Km values of Enzymes I, II1 and II2 with

different substrates

 

 

 

substrate
Enzyme PNPG Raffinose Stachyose
I 1.5 4.6 11.0
111 2.2 5.0 15.0
112 5.3 '1.6 ---

 

The enzymes were purified to homogeneity as determined
by disc gel electrophoresis. Enzymes I, II1 and II2 moved
slightly in alkaline pH (8.3) and acidic pH (3.5) PAGE. The
protein bands on the gel at pH 3.5 were confirmed as the a-
galactosidases I, II1 and 112.

Using gel filtration, the NW of Enzymes I, II1 and II2
were 110,720 :1: 3,780, 29,000 :I: 355 and 30,000 1: 368
daltons. Using SDS-PAGE polyacrylamide gel electrophoresis,
the MW values obtained were 30,916 1 1,042, 29,875 :t 413
and 30,350 1 550 for the three enzymes, respectively. Based
on the SDS-PAGE and two types of ion exchange
chromatography, Enzyme I appears to be a tetramer of Enzyme
II2 rather than Enzyme IIz.

126

Stachyose decreased at a rapid rate while raffinose
declined slowly during incubation and germination plus
incubation of cowpeas for 24 hrs at 24' and 34' C. The
sucrose concentration slightly increased during germination
plus incubation of cowpeas for 24 hrs at 24° C. The
activity of a-galactosidase increased during germination
for 24 hrs, especially during the first 6 hrs. The change
in oligosaccharide content during 24 hr of cowpea flour
paste incubation was higher at 34' C than at 24' C. The
rate of stachyose decrease was higher than sucrose which in
turn was higher than raffinose. Germination did not have
significant effect on the oligosaccharide changes during 24
hr. Incubation of a paste made from germinated seeds or
flour of the seeds resulted in considerable reduction of
the flatus-related oligosaccharide of the cowpea. As a
result of these investigations, it appears that the most
efficient way to decrease flatus-related sugar of cowpeas
especially stachyose, is to form a cowpea flour paste and
let it stand overnight in a warm place (30' C).
Microbiological safety concerns of this procedure need to
be evaluated.

Future research can be continued at the enzyme level
and the nutritional level.

At the enzyme level, the following aspects of a-

galactosidases may be studied.

127

- Purification of the cowpea a-galactosidases using
affinity chromatography.

- Amino acid composition and sequence of cowpea a-
galactosidases

- The effect of using invertase with a-galactosidase on
the oligosaccharides in cowpeas.

- The influence of germination on the ratio of Enzyme I
and II in cowpeas.

From a nutritional point of iview, it has been shown
that incubation of a cowpea paste resulted in
oligosaccharide decrease. It will be of interest to study
the effect of such pastes in reducing flatulence. Another
issue of interest which requires further investigations,
concerns the use of immobilized a-galactosidase to remove

the oligosaccharides from cowpea slurries.

APPEN DI CES

 

APPENDICES
Appendix 1
Table. Nutritional value of cowpeas and certain other foods

(Dovlo et al., 1976)

 

 

 

 

 

 

 

 

 

 

Cal/ Protein Fat Carbo- Vit- Thi- Ribo- Nia- Ascorb
1009 hydrate amin A amin flavin cin acid
% % % 10/ mg/ mg/ m9/ m9/
_1009 _100g _1009 _1009 _1009_
Cowpea 340 22 1.5 60 20 0.9 0.15 2 0
Soybean 382 35 18 20 0 1.1 0.3 2 0
Rice,
slightly
milled 354 8 1.5 77 0 0.25 0.05 2 O
Sorghum
flour 353 10 2.5 73 0 0.4 0.1 3 0
Fish,
lean,
fillet 73 17 0.5 0 0 0.05 0.1 2.5 0
Beef,
lean 202 19 14 0 0 0.1 0.2 5 O
3993:
hen 158 13 12 0.5 1000 0.12 0.35 0.1 0

 

 

Cereal proteins are relatively deficient in the amino acid
lysine, which is prsent in a relatively high amount in
legumes.Legumes are a poor source of the sulfur-containing
amino acids, methionine and cystine, which are adequate in

cereals.

128

129

Appendix 2
Table. Molecular forms and properties of some plant seed a-

galactosidases (Dey and Pridham, 1972)

 

 

Source Form Method MW pH Km (mM)
of separation Optimum PNPG
substrate
gaigngg indigus I Gel filtration High 5.0 6.3
II Low 4.5 1.2
ggngggnig fiiligua I DEAE-Cellulose 37,000 3 5.0 0.42
II 37,000 a 5.0 0.42
III 23,000 ~ 5.0 0.45
glycine max I Gel filtration 160,000 6.8 0.15
II 40,000 5.6 0.39
Medigagg flatly; I DEAE-Cellulose 34,000 s 5.0 0.49
II 23,000 3 5.0 2.36
2191; {aha ' I Gel filtration 160,000 2.0 a 5.5 0.44
II1 SDS-PAGE 43,000 2.0 & 5.2 0.97
112 41,000 2.0 a 5.5 0.33
yigng Indies; I Gel filtration 160,000 5.6 8 7.2 0.2
.11 40,000 5.6 0.1
yignagngnigulgtg I Gel filtration 110,000 5.0 1.2
111 SDS-PAGE 30,000 5.9 2.2
112 29,000 5.3 5.3

 

BIBLIOGRAPHY

BIBLIOGRAPHY

Adams, H., Richtmyer, N. R., and Hudson, C. 8.1943. Amer.
Chem. Soc., 65:1369.

Al-Bakir, A.,and Whitaker, J.R. 1978. Purification and
characterization of invertase from dates (Phoenix

DagtxlifgzaILL42aszgngi).J. Food Biochem. 2:132-160.

Allison, R.D., and Purich, D.L. 1979. Practicle
consieration in the design of initial velocity enzyme
rate assays. Methods Enzymol., 63:3-22.

A.O.A.C. 1980. "Official methods of analysis". 13th, Ed.
Association of Official Agricultural Chemists.
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