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JILLEZJFT A

LIBRARY
Michigan: State
l University

 

 

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’_~—

This is to certify that the
thesis entitled

ENZYMATIC CHARACTERISTICS OF OL-GALACTOSIDASES
FROM ASPERGILLUS NIGER, COFFEE BEANS
AND ESCHERICHIA COLI

 

 

presented by

Penelope Gavriel

has been accepted towards fulfillment
of the requirements for

M.S. degree in Food Science

 

 

fizctaa ”gm

Major professor

Pericles Markakis

 

Date 1/ Z 9/] 9 85

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ENZYMATIC CHARACTERISTICS OF a-GALACTOSIDASES
FROM ASPERGILLUS NIGER, COFFEE BEANS
AND ESCHERICHIA COLI

 

 

By

Penelope Gavriel

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1984

 

ABSTRACT

ENZYMATIC CHARACTERISTICS OF a-GALACTOSIDASES
FROM ASPERGILLUS NIGER, COFFEE BEANS
AND ESCHERICHIA COLI

 

By

Penelope Gavriel

A crude laboratory preparation of a-galactosidase from
A. 11933 and two commercial preparations of the enzyme, one
from g. coll and another from coffee beans, were compared
as follows: their optimum reaction pH and temperature and
their storage stability were determined with p-nitro-
phenyl-a-D-galactoside (PNPG) as substrate. Their Km and
Vmax values were assessed with PNPG, melibiose, raffinose
and stachyose as substrates. The A. gigs: and coffee bean
enzymes had similar pH and temperature optima, 5.0 and about
52°C, respectively. The E. coll enzyme had a pH optimum
near 38°C. The latter enzyme was much more unstable in
storage than the other two. The E. coll enzyme hydrolyzed
melibiose faster and stachyose more slowly than the other

two enzymes. Ag+ and Hg2+

inhibited all three enzymes, but
while KI lifted the inhibition of the A. 1133: and coffee
bean enzymes, it did not affect the inhibition of the

E. coil enzyme. There were considerable differences among

the Km and Vmax values of the three enzymes.

 

ACKNOWLEDGMENTS

The author wishes to express her sincere gratitude to
Professor Pericles Markakis, her academic advisor, for his
encouragement and able guidance throughout the course of
this work and during the writing of this manuscript. She
also appreciates the assistance of Professors Robert Herner
and C.M. Stine in preparing this thesis. Special thanks
are extended to Dr. Basil Makris, research associate at the
Greek Atomic Energy Commission, for the generous donation
of an enzymic preparation from A. niggg. She also wishes
to thank Dr. John Partridge for the loan of a water bath.
Dr. Wanda Chenoweth was particularly helpful by donating
stachyose for the completion of the experiments during the
last stages of this work. Many thanks particularly to my
family members for their assistance, patience and under-

standing throughout the course of my graduate studies.

ii

 

TABLE OF CONTENTS

LIST OF TABLES.
LIST OF FIGURES
INTRODUCTION.

Occurrence . . .

Detection and Methods .of Assay .

Isolation. . .

Physical Properties.

Hydrolase Activity

Inhibitors

Mechanism of Action.

Physiological Significance and Possible
Applications of a- -galactosidases

LITERATURE REVIEW .

The a- galactosidases of A. niger, coffee beans
and E. coli. . . . . . . . . . . . . . . .

MATERIALS AND METHODS

D-Galactose Standard Curve. .

p- Nitro- phenol Standard Curve . .

a-Galactosidase from Aspergillus niger.
Isolation Procedures. . .
Assay of the Enzymatic Activity .
Optimum Reaction Temperature.
Optimum Reaction pH .
Determination of the Hydrolysis Rates
Determination of the Km and Vmax Values
Stability Tests
Other Enzymes
Inhibitors. . .

a-Galactosidase from. Coffee Beans
Dialysis. . . .
Assay of the Enzymatic Activity
Optimum Reaction Temperature.
Optimum Reaction pH
Reaction Rates.

 

 

Determination of the Km and Vmax Values.
Inhibitors . . .
a- Galactosidase from E. cLli. . .
Optimum Reaction Temperature .
Optimum Reaction pH.
Reaction Rates .
Determination of the Km and Vmax Values.
Inhibitors . . . . . . . . .

RESULTS AND DISCUSSION

Standard Curve of Galactose . .
Standard Curve of p-nitrophenol
a-Galactosidase from A. niger .
Optimum Reaction Temperature
Optimum Reaction pH.
Reaction Rates
Km and Vmax Values
Stability.
Other Enzymes.
Inhibition . .
a- -Galactosidase from Coffee Beans

Optimum Reaction Temperature and Stability :

Optimum Reaction pH.
Reaction Rates
Km and Vmax Values
Inhibition
a- Galactosidase from E. cLIi. .
Stability, Optimum Reaction Temperature,
Reaction Rates .
Optimum Reaction pH.
Km and Vmax Values
Inhibition

CONCLUSIONS.
BIBLIOGRAPHY

iv

Page

Table

10
ll

LIST OF TABLES

Page
Substrate specificity of a-galactosidase from
V. faba. . . . . . . . . . . . . . . . . . . . . l0
Absorbance of galactose solutions in acetate and
phosphate buffers at 340 nm. . . . . . . . . . . 54

Thermal stability of a-galactosidase from
A. nige . . . . . . . . . . . . . . . . . . . . 6l

Kinetic constants of a-galactosidase from
A. niger for PNPG, melibiose, raffinose and
stachyose, determined by the Lineweaver-Burk

(L-B) and Eadie-Hofstee (E-H) methods. . . . . . 75
Inhibitors of a-galactosidase from A. niger. . . 77

The regenerating effect of KI upon a- alactosi-
dase from A. niger, inactivated by Ag and

Hg++ ions. . . . . . . . . . . . . . . 79
Activity restoration in Ag+ or Hg++ inactivated
a-galactosidase from A. niger by KI, EOTA,
citrate and cysteine . . . . . . . . . 80
Thermal stability of a-galactosidase from coffee

beans. . . . . . . . . . . . . . . . . . . . . . 84

Kinetic constants of a-galactosidase from coffee

beans for PNPG, melibiose, raffinose and

stachyose, determined by the Lineweaver-Burk

(L-B) method and Eadie-Hofstee (E-H) method. . . 94

Inhibitors of a-galactosidase from coffee beans. 94

Kinetic constants of a-galactosidase from

E. coli, for PNPG, melibiose, raffinose and

stachyose by the Lineweaver-Burk (L-B) method

and Eadie-Hofstee (E-H) method . . . . . . . . . 102

 

Table Page

l2 Inhibitors of a-galactosidase from E. coli. . . . lO8

l3 Summary of the Optima temperatures and pH of the
a-galactosidases from A. niger, coffee beans and

E. coli . lll

l4 Summary of the Km values of a-galactosidases from

A- £1321. coffee beans and E. coli. 111

15 Summary of the Vmax values of a-galactosidases
from A. niger, coffee beans and E. coli . . . . . 112

vi

Figure

l

2
3
4

10
ll

12

l3

14

LIST OF FIGURES

a-Galactosidase hydrolysis.
Transferase activity of a-galactosidase
Glycosides hydrolyzed by a-galactosidase.

Hydrolysis of stachyose by a-galactosidase
from almonds. . . . . . . .

"Two step" mechanism of action of a-galactosi-
dase from sweet almonds . . . . . . .

Reaction of galactose oxidase

Standard curve for determining galactose.
Reactions occurring during the hydrolysis of
PNPG by a galactosidase and color development
of the liberated p-nitrophenol. . . . . .

Formation of intramolecular hydrogen bond by
o-nitrophenol . . . . . . . . . . .
Standard curve for determining p-nitrophenol.
Temperature activity relationship of a-galacto-
sidase from A. niger with PNPG as substrate at
pH 4.5. . . . . . . . . . . . . . . . . . . . .

pH- activity relationship of a- galactosidase
from A. niger with PNPG as substrate at 55°C.

Hydrolysis of PNPG by a-galactosidase from
19er, at optimua conditions of temperature

andn pH. . . . . . . . . . . . . .

Hydrolysis of melibiose, raffinose, stachyose

by a- -galactosidase from A. n1 ger at 45° C and
pH 5.0.. . . . . . . . .

vii

ll

13
50
53

56

56
58

59

62

64

65

 

Hi i

Figure Page

15 Optimum substrate concentration during the
hydrolysis of PNPG by a- o-galactosidase from

niger. . . . . . . . . . . . 67
16 Lineweaver—Burk double reciprocal plot of
a-galactosidase from A. niger for PNPG. . . . . 70

l7 Eadie-Hofstee reciprocal plot of a-galactosi-
dase of A. niger for PNPG . . . . . . . . . . . 70

l8 Lineweaver-Burk double reciprocal plot of
a-galactosidase of A. niger for melibiose . . . 7]

l9 Eadie-Hofstee reciprocal plot of a-galactosi-
dase of A. niger for melibiose. . . . . . . . . 71

20 Lineweaver-Burk double reciprocal plot of
a-galactosidase of A. niger for raffinose . . . 72

2l Eadie-Hofstee reciprocal plot of a-galactosi-
dase of A. niger for raffinose. . . . . . . . . 72

22 Lineweaver-Burk double reciprocal plot of
a-galactosidase from A. niger for stachyose . . 73

23 Eadie-Hofstee reciprocal plot of a-galactosi~
dase of A. niger for stachyose. . . . . . . . . 73

24 Temperature-activity relationship of the coffee
bean, a-galactosidase, with PNPG as substrate,
pH45 82

25 pH activity relationship of the coffee bean
a-galactosidase with PNPG as substrate,
temperature 50°C.. . . . . . . . . . . . . . . 85

26 Hydrolysis of PNPG by coffee bean a-galactosi-

dase at optimum conditions of temperature and 87
pH. . . . . . . . . . . . . . . . . . . . . . .

27 Hydrolysis of melibiose, raffinose, stachyose
by coffee bean a-galactosidase at 45° C and
optimum pH. . . . . . . . . . . . . . . . . . . 88

28 Lineweaver-Burk double reciprocal plot of
coffee bean a-galactosidase for PNPG. . . . . . 89

29 Eadie-Hofstee reciprocal plot of coffee bean
a-galactosidase for PNPG. . . . . . . . . . . . 89

viii

 

Figure Page

30 Lineweaver-Burk double reciprocal plot of coffee
bean a-galactosidase for melibiose. . . . . . . . 90

31 Eadie-Hofstee reciprocal plot of coffee bean
a-galactosidase for melibiose . . . . . . . . . . 90

32 Lineweaver-Burk double reciprocal plot of coffee
bean a-galactosidase for raffinose. . . . . . . . 91

33 Eadie-Hofstee reciprocal plot of coffee bean
a-galactosidase for raffinose . . . . . . . . . . 91

34 Lineweaver-Burk double reciprocal plots of coffee
bean a-galactosidase for stachyose. . . . . . . . 92

35 Eadie-Hofstee reciprocal plot of coffee bean
a-galactosidase for stachyose . . . . . . . . . . 92

36 Temperature-activity relationship of the a-galac-
tosidase from E. coli, pH 6.5 . . . . . . . . . . 95

37 pH-activity relationship of the a-galactosidase
from E. coli at 45°C. . . . . . . . . . . . . . . 98

38 Hydrolysis of PNPG by a-galactosidase from

E. coli at optimum conditions of temperature and 100
pH. . . . . . . . . . . . . . . . . . . . . . . .

39 Hydrolysis of melibiose, raffinose and stachyose
by a-galactosidase from E. coli at optimum
conditions of temperature and pH. . . . . . . . . 101

 

40 Lineweaver-Burk double reciprocal plot of
a-galactosidase from E. coli for PNPG . . . . . . 103

4l Eadie-Hofstee reciprocal plot of a-galactosidase
from E. coli for PNPG . . . . . . . . . . . . . . 103

42 Lineweaver-Burk double reciprocal plot of
a-galactosidase from E. coli for melibiose. . . . 104

43 Eadie-Hofstee double reciprocal plot of a-galac-
tosidase from E. coli for melibiose . . . . . . . 104

44 Lineweaver-Burk double reciprocal plot of
a-galactosidase from E. coli for raffinose. . . . 105

45 Eadie-Hofstee reciprocal plot of a-galactosidase
from E. coli for raffinose. . . . . . . . . . . . 105

ix

Figure Page

46 Lineweaver-Burk double reciprocal plot of
a-galactosidase from E. coli for stachyose. . . 106

47 Eadie-Hofstee reciprocal plot of a-galactosi-
dase from E. coli for stachyose . . . . . . . . 106

INTRODUCTION

In 1895, Bau, Fisher and Lindner (21) isolated certain
enzymes from bottom yeast, which hydrolyzed the disaccharide
melibiose,and they named them melibiases. This name was
later changed to a-galactosidases by Heidenhagen, who
studied the specificity of action of the enzyme using a
variety of carbohydrates possessing non-reducing, terminal
a-D-galactosyl residues.

a-Galactosidases or a-D-galactoside galactohydrolases
(E.C. 3.2.1.22) catalyze the following reaction (Figure 1).

The enzyme may hydrolyze a variety of simple a-D-
galactosides as well as more complex molecules, such as
oligo- and polysaccharides. The ease of hydrolysis of the
a-galactopyranosyl residues decreases progressively with
increasing size of the substrate molecule.

In addition to their hydrolytic ability a-galactosidases
from certain sources (bacteria, plants) can catalyze trans-
a-D-galactosylation reactions from aryl a-D-galactopyranosides
to alcohols, mono- and oligosaccharides (21).

Interest has centered around the mode of action and
physiological significance of these enzymes and their use as
tools for structural studies of biological molecules as well

as practical applications (40).

CHZOH - CHon

OH H H OH . H

*4 OH H O—R +-HOH‘;:: H OH '4- R0“
H OH H 0

Figure l. a-Galactosidase hydrolysis.

R: Aromatic or aliphatic residue.

Occurrence

 

a-Galactosidases have been reported to occur widely
in microorganisms, plants and animals. In most cases they
are found intracellularly in various organelles (mitochondria,
chloroplasts, microsomes). In E. coli (8, 26, 51, 52, 53)

and Aerobacter aerogenes a-galactosidase is not constitutive

 

but can be induced by the introduction of several a-D-

galactosides in the culture media (6).

Detection and Methods of Assay

 

Melibiose and raffinose, presumed to be natural
substrates for plant a-galactosidase, are commonly used to
determine enzyme activity (14, 42, 44, 58, 60). Following
incubation the extent of hydrolysis is measured in terms of
liberated molecules of galactose (product) or non-hydrolyzed
molecules of substrate. The galactose determination can be
achieved by various methods, such as colorimetry (23, 45),
measurement of the increased reducing power of the hydro-
lyzate, chromatography (33) or enzymatically (22, 30), while
the determination of the remaining substrate can be estimated
with sufficient accuracy by GLC or HPLC (l3, 14, 31). Often
substituted phenyl-a-D-galactosides can be used as convenient
assay substrates (35). In this case the enzymatic activity
can be estimated spectrophotometrically, by the appearance

of the phenyl compound, which is usually a chromogen.

Often cofactors such as NAD+ or metal ions such as
Mn++, as in the case of cell free enzymic extracts of A. £311
(8), or activators, such as K+ ions for the a-galactosidase
isolated from 1. :gba seeds (17), may be necessary in the
assay medium.

Galactosyl transferase activity, as opposed to hydro-
lysis, is normally assayed by carrying out the enzymic
reaction in the presence of a galactose donor, such as PNPG
and an acceptor, such as raffinose (Figure 2). The resulting
mixture is fractionated and the products are determined

usually by chromatography (l6).

Isolation

 

a-Galactosidases from various sources can be isolated
by conventional methods of extraction. Often NaCl solutions
are used to extract the enzyme from the cells of the micro-
organisms that produce it. At times,suspensions of cultures
are marketed as sources of a-galactosidase (8). Commonly
a-galactosidases occur in the cells in association with
other glycosidases and often have been difficult to fraction-
ate these activities.

The techniques of isolation include ammonium sulfate
or cold acetone, fractionation, ion exchange, molecular
exclusion and isoelectric focusing (8, 23, 28, 40, 57, 59).
All or some of these techniques are employed, according to

the desired purity of the enzymic preparation or the

 

C"'20” H2014. CHZOH

ow
0H 0
@2+ 0” ° . H02” 014
o O ow O CHZOH
H

p-nitro—phenyl- Raffinose
a-D-galactoside

CH20H
0 HO C
H OH OH CHZOH

0H

p-nitrophenol Stachyose

Figure 2. Transferase activity of a-galactosidase.

difficulty during fractionation. Highly purified and
apparently homogeneous a-galactosidases have only been

isolated in a few cases (19, 40).

Physical Properties

 

The existence of multi-molecular forms of a-galactosi-
dases was first reported by Petek and his collaborators
(48). They isolated two separate forms of this enzyme from
coffee beans by chromatography on alumina columns. Dey and
Pridham (20) later showed that dormant seeds of 1. £323_
possessed two a-galactosidases, which had different molecular
weights. The separation was achieved by Sephadex gel filtra-
tion. Multimolecular forms of a-galactosidases may have
very similar properties and may be difficult to resolve.
For example, the isolated enzyme from A, gigs; was homo-
geneous, as judged by gel filtration but, when passed through
an ion exchange column, it was resolved into three active
forms (23, 40). The a-galactosidases isolated from various
sources have different molecular weights. Most of these
were estimated by gel filtration and ranged between 25,000-

200,000.

Hydrolase Activity

 

In general, change of configuration of hydrogen and/or
hydroxyl groups or any single carbon atom of a galactoside

substrate is sufficient to reduce or completely inhibit the

hydrolytic action of the enzyme. Two main factors govern

the rate of hydrolysis of the substrate: the ring structure
must be a pyranoid, and the configuration of -H and -OH on
carbon atoms 1,2,3 and 4 must be similar to that on galactose.
Changes at C-6 are tolerated by a-galactosidases. Hence the
glycosides indicated in Figure 3 can be hydrolyzed by this
enzyme (21).

Kinetic studies of the hydrolytic activity of a-galacto-
sidases on various galactosides or glycosides, which have
similar molecular conformation have shown that the Km and
Vmax values for different substrates vary greatly and that
the hydrolyzability (velocity of the reaction) is not
related to the affinity (l/Km) of the enzyme for the
substrate(20, 21). The affinity of the enzyme 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<h-B-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 (21).

During the hydrolysis of various galactosides, the
velocity of the reaction was higher for those galactosides
in which the aglycon had aromatic structure than those with
alkyl aglycon. In addition it was found that the affinity
of a-galactosidases isolated from 1. £363 seeds and sweet

almonds tends to increase, when electron attracting

CHéfii
0H 0

0H 0—2

OH

a-D-Galactoside

H

0“ O—R

OH

B-L-Arabinoside

 

“'3
OH
0“ O--R

OH

a-D-Fucoside
jHZOH
CHOH
OH
OH 0—12

OH

D-Glycero-a-D-galacto-
heptoside

Figure 3. Glycosides hydrolyzed by a-galactosidase.

 

substituents are present in the phenyl ring of a phenyl-a-
0-galactosides,which are used as substrates (Table 1).

Some a-galactosidases can attack galactomannans (12).
Almond a-galactosidase can Split the internal galactosidic
bond of stachyose forming galactobiose and sucrose (21),
as indicated in Figure 4.

0n the other hand coffee bean a-galactosidase can only
cleave stachyose in a stepwise fashion, starting from the

non-reducing end (12).

Inhibitors

 

Most a-galactosidases are inhibited by "sulfhydryl
reagents", such as p-chloromercuribenzoate, iodoacetamide
and N-ethyl-maleimide. But not all a-galactosidases are

-SH enzymes (e.g. the sweet almond a-galactosidase, 1. faba).

 

Ag+ and Hg++ ions exhibited strong inhibition upon
a-galactosidases. In all cases a-D-galactose at concentra-
tion higher than 10 mM exhibited competitive inhibition.
Also some substrates, such as PNPG, above certain concentra-
tion can cause decrease of the velocity of the enzymic
reaction, which becomes more pronounced as the substrate

concentration increases (20).

 

10

Table l. Substrate specificity of a-galactosidase from

 

 

1. faba (20 .
Substrate Km
n-propyl-a-D-galactoside 6.13
ethyl-a-D-galactoside 6.93
phenyl-a-D-galactoside 1.11
m-chloro-phenyl~a-D-galactoside 0.83

p-nitro-phenyl-a-D-ga1actoside 0.38

 

ll

14 Hog«:o
0 o
OWHZOH
0 OH

 

Stachyose
CH 20H CHZOH
GHQ; 0 OHH +0®HOZHC o
0H0H 0H 0 HZOH
Galactobiose Sucrose

Figure 4. Hydrolysis of stachyose by a-galactosidase from
almonds.

12

Mechanism of Action

Few concrete facts are available regarding the mechanism
of action of a-galactosidases. So far there have been no
studies of the bond fission by a-galactosidases, although
by analogy with other glycosidases it is likely that the
galactose-oxygen bonds of substrates are cleaved (21).
Nuclear magnetic resonance and polarimetry studies have
clearly shown with sweet almond a-galactosidase that the
liberated galactosyl residues possess the same anomeric
configuration as the substrate (16).

The substrate specificity studies on sweet almond and
1. faba a-galactosidases (20, 42) showed that the electronic
nature of the aglycon has a noticeable influence on the rate
of enzymic hydrolysis. Thus the hydrolytic reaction could
be a result of the presence of basic and acidic groups at
the active site. These groups were identified by kinetic
studies as carboxyl (deprotonated) and imidazolium (protonated),
respectively. On the basis of these results a "two step"
mechanism has been postulated for the action of sweet almond
a-galactosidase (16). The aglycon is cleaved off by the
combined action of carboxyl and imidazolium groups (Figure 5).
This is followed by reaction with an acceptor molecule, which
may be water or another substrate molecule, resulting in

hydrolysis or transfer products.

 

 

Figure 5. "Two step" mechanism of action of a-galactosidase
from sweet almonds.

14

It is possible that the imidazolium group alone is
sufficient to cleave the glycosyl-oxygen bond, with the
formation of a carbonium ion at the C-1 of the liberated
galactose moiety. The free carbonium ion though could lead
to mutarotation products (a- and a-anomeric forms of galac-
tose) in the mixture. For this reason it is believed that
the carbonium ion is stabilized somewhere on the active site
(probably COO“) retaining the stereochemical configuration

of the liberated galactose.

Physiological Significance and Possible

 

Applications of a-galactosidases

 

In the plant kingdom galactose containing oligo- and
polysaccharides and lipids are ubiquitous (3, 9, 12, 15, 39,
58, 60). Among the oligosaccharides known are the galactosyl
sucrose derivatives of raffinose and stachyose, which are
found in substantial amounts in the legume seeds. In many
seeds there is a concomitant synthesis of these galactosyl-
sucrose compounds and an apparent high content of a-galacto-
sidase. It is apparent that, in order for those galactosyl
derivatives to accumulate in the seed during maturation,
there must be some mechanism, that prevents the interaction
between enzyme and substrate; perhaps this is achieved by
compartmentalization or an endogenous inhibitor (21). During
germination,the a-galactosidase must be involved in the

hydrolysis of these oligosaccharides, which serve as soluble

IS

and readily metabolizable energy reserves.

It appears that 02 is somehow involved in the hydrolytic
reaction of the sugars in vivo, as this is inhibited if
moistened seeds are kept in a reduced oxygen atmosphere
(54).

The disappearance of raffinose and stachyose during
germination is not accompanied by the concomitant presence
of galactose in the tissues, but only a marked increase in
the sucrose content has been observed (21, 29, 39). What
could happen in this case is that galactose is immediately
phosphorylated, converted into glucose and metabolized.
Besides the biochemical and biophysical aspects of a-galacto-
sidase there is another important aspect of this enzyme.
This is the potential of this enzyme for industrial or
medical applications.

Raffinose is found in variable amounts in sugar beets,
which usually increase during storage. The amount of
raffinose in sugar beets could come up to 0.15% of raw
material (58). In the sugar industry raffinose is known as
an obstacle substance for the normal crystallization of the
beet sugar. When the raffinose content in the beet molasses
is gradually increased to an amount of 6% to 10%, the
crystallization of the sugar becomes unprofitable and it is
abandoned (41, 58).

If raffinose in the beet juice or beet molasses can be

removed or decomposed by some method, it is possible that

 

16

the crystallization of the beet sugar will be further
improved and consequently the yield of the sugar increased.
Many workers attempted successfully the removal of raffinose
by incorporating into the molasses crude preparations of
a-galactosidase, isolated from various microorganisms. Of
all the available sources of a-galactosidase, members of the
fungi have been used successfully (9, 32, 38, 46, 59).

In Japan, Suzuki et al. have employed the mycellial

a—galactosidase from a fungus, Mortierella vinaceae, in the

 

form of a pellet and incorporated it into the beet molasses
to accomplish the hydrolysis of raffinose (58). The micro-
scopical crystal examination of the recovered sugar from the
hydrolyzate revealed normal crystal formation, due to the
absence of the inhibitory action of raffinose. A similar
experiment was attempted in Poland with the same successful
results (46).

An economic evaluation allowing for investment and
operational costs shows that the a-galactosidase process is
profitable, when 1) there is a considerable price difference
between sugar and molasses, 2) the factory is isolated,

3) the molasses is of little value, 4) the beet raffinose
content is high, 5) the campaign is protracted.

Soybeans are used in increasing amounts, as low cost
high quality protein supplement. The potential of soy milk,
as a substitute for cow's or human milk has been emphasized

during the years. It can be used successfully, in cases

 

l7

where cow's milk is not available or there is some kind of
physical disorder in humans (13, 60).

Soy milk is deficient in the sulfur containing amino
acids (36). It also contains considerable amounts of
galactosyl sucrose oligosaccharides, which have been impli-
cated as factors responsible for flatulence (56). Various
methods have been employed for the removal of these trouble-
some compounds from the soybeans (soaking, soaking and
germination, fermentative process (44), water extraction,
ultrafiltration). All these methods are tedious and time
consuming.

Enzyme treatment by microbial a-galactosidase offers a
promising solution to this problem (13, 43, 55, 60).

Sugimoto et al. succeeded in removing all the oligosaccharides
from soy milk in three hours, by treating it with 16x10“3
units of a-galactosidase per gram of solid material.

Commercially prepared crude enzyme preparation from
A. 11221 was used (57). Microbial a-galactosidases have been
used for the removal of the oligosaccharides from soy milk
commercially in a hollow fiber reactor (55). Entrapment of
a-galactosidase from a fungus in polyacrylamide gel has also
been carried out (60). These methods allowed continuous and
multiple use of the enzyme.

(Finally there is an additional interest in some plant
a-galactosidases (coffee beans, tomatoes). Experimental work

proved that they can modify the B specificity of intact human

18

erythrocytes by removing a-l,3-linked galactose residues

from cell surfaces (49, 61). A similar reaction was
demonstrated by a-galactosidases isolated from figs and
soybeans. Pressey working with a-galactosidase isolated from
tomatoes concluded that his enzyme may be employed in the
future to convert group B erythrocytes to type 0 (49).

This process has drawn considerable interest recently as a

way of utilizing unused type B blood (49).

LITERATURE REVIEW

0

The a—galactosidases of A. n1ger,

Coffee Beans and E. coli

 

Although oligosaccharides containing a-D-galactosyl
groups in their structure, such as raffinose and stachyose,
and galactosyl glycerides in lipid fractions, are widely
distributed in the plant kingdom, a-galactosidases have not
been studied as extensively as s-galactosidases. Microbial
galactosidases have been reported in brewers yeast and some
strains of bacteria and molds, but detailed information on
their characteristics have not been given until recently.

In preliminary studies Sugimoto et al. (57) found that
a considerable amount of fungal strains belonging to the
genus Aspergillus exhibited powerful abilities to produce
galacto-oligosaccharide decomposing enzymes, such as
a-galactosidase and invertase.

Some commercial enzyme preparations were derived from
the same fungi containing considerable activities of both
enzymes and were distributed under various names, such as
Molsin (Seishin Pharmacetuical Co., Ltd., Tokyo) from A. 53391

and Rhozyme HP-lSO (Rhom and Haas Co., Philadelphia) from

A. niger.

 

20

Some of the research which has been conducted around
enzymic preparations from the various species of Aspergillus
included mainly various purification and isolation procedures
and a few practical applications, mainly limited to experi-
mental levels, employing the use of a-galactosidase for the
removal of oligosaccharides from soy milk. Specifically
Sugimoto et al. (57) working with a crude commercial prepara—
tion from A. 53:31 determined an optimum reaction temperature
for this enzyme at 55°C, an optimum pH between 5.0 and 5.5
and the Km value of this enzyme for melibiose, as substrate,
was found to be 3.1x10‘3 M. Furthermore, investigations by
means of thin-layer chromatography indicated that addition of
small amounts of this enzyme preparation to soy milk resulted
in complete hydrolysis of the galacto-oligosaccharides.
McGhee et al. (43) also investigated the removal of flatu-
lence factors from soy milk by employing a crude enzyme
preparation from A. awamori, isolated in their laboratory
from cultures of this fungi. Gel filtration techniques
estimated the molecular weight of this enzyme at 290,000.
Their enzyme exhibited maximum activity at 50°C and pH 5.0.
The Km values of this a-galactosidase with melibiose were

2 M and 3.6x10‘2 M for raffinose. The molecular

3.0x10-
weight of this enzyme was estimated to be about 130,000,
on the basis of Sephadex and sodium dodecyl sulfate—poly—
acrylamide gel electrophoresis. This enzyme has the

advantage of not being inhibited by the presence of

21

galactose.

Bahl et a1. (5) conducted an extensive study centered
around the purification and isolation of the a-galactosidase
(among the other glycosidases) from a crude commercial
preparation (Rhozyme HP-lSO). Their work included ammonium
sulfate precipitation, pressure dialysis, chromatography and
study of the hydrolytic action of the isolated enzyme upon
oligo— and polysaccharides, such as melibiose, raffinose,
stachyose and galactomannans from guar and locust beans.
During the hydrolysis of the latter substrate the enzyme
liberated 35% to 40% of the total D-galactose residues.

The Km and Vmax values of this enzyme for PNPG was estimated
to be 3.5x10‘4 M and 58.8 umoles of p-nitro-phenol/min/mg of
crude enzyme preparation, respectively.

Lee et al. (40) worked on the isolation of the a-galacto-
sidase from Rhozyme HP-150. Their work included precipitation
and isolation procedures by using gel chromatography with
Sephadex, ion exchange chromatography on DEAE, electro-
focusing and rechromatography of the isolated a-galactosidase
on CM-cellulose. The additional procedure of electrofocusing
suggested that there were more than one component (isozymes)
of a-galactosidase. The observation that different optimum
pH values existed agreed with the above suggestion.

The determination of the Km constant for PNPG gave a
value of 4.6x10'3 M and a distinct substrate inhibition has

been reported.

 

22

Courtois et al. (12, 17) and Petek et al. (48) included
in their carbohydrate studies some experiments for the isola-
tion and investigation of the enzymatic characteristics of
a-galactosidases from coffee beans. Their work is the most
prominent in the literature review, related to the study of
a-galactosidases from this source. Petek and ToDong (48)
obtained their enzymatic preparations from ground coffee
beans by the method of acetone powder. During the purifica-
tion and isolation procedures on alumina column, they succeeded
in separating two a-galactosidases, which were defined as
a-galactosidase I, with optimum pH 5.3 and Km for PNPG
2.2x10'3 M, and a-galactosidase II, with optimum pH 6.0 and
Km for PNPG l.1xlO'3 M. 0n the contrary the optimum pH
during the hydrolysis of melibiose by a-galactosidase I was
3.8 (51a).

Studying the transferase activity of these enzymes the
same workers used PNPG as a donor, while D-mannose, D-
galactose, sucrdse, cellobiose and maltose were used as
acceptors. The appearance of compounds such as raffinose
and stachyose in the reaction mixture, determined chromato-
graphically, was used as indication of transferase activity.

Courtois et al. studied the mode of hydrolysis of
stachyose by a-galactosidase from coffee beans. His conclu-
sion was that this enzyme hydrolyzes stachyose in two stages:
first one molecule of galactose is removed and raffinose is

formed; the latter is then split into galactose and sucrose

23

(12).
Studying the action of a-galactosidase from coffee
beans upon various galactomannans, isolated from Trifolium

repens, Gleditschia triacanthos and some other species,

 

 

Courtois et al. concluded that this hydrolase splits at
similar initial rates a-D-galactosyl units from all galacto-
mannans. At pH 4.6 and at 37°C the Km value was estimated
for raffinose to be 5.3x10'3 M and 2.5x10‘3

(12).

M for stachyose

a-Galactosidase from coffee beans was also employed to
demonstrate the hydrolyzation of galactose molecules from B
blood group substance (61). The study of a-galactosidase
from g. 9211 was limited only in the area of biochemical
research, as subject of the overall process of induced enzyme
formation by certain microorganisms, such as E. 3311.

It has been known for some time that the a-galactoside
melibiose can induce in E. coli simultaneously the synthesis
of a-galactosidase (53). Growth of A. ggli on melibiose
requires the induced synthesis of a-galactoside permease
and a-galactosidase (8). This is achieved by a "melibiose
operon" controlling the synthesis of the above proteins
(51). It was observed that raffinose utilizing E. coli
strains contained B-galactosidase. When these strains were
tested for the presence of a-galactosidase, only those grown
on melibiose and raffinose showed high activity (26). This

indicates that the formation of a-galactosidase in E. coli is

 

24

induced, while Bgalactosidase has a constitutive character.
Cell free extracts from induced E. 3911 cultures were
assayed for a-galactosidase activity with PNPG (8). Enzyme
activity was observed only at high protein concentrations
and the activity decreased exponentially with dilution. The
reason for this is that this hydrolase, unlike other known
similar enzymes, requires NAD+ as a cofactor.

Burstein et a1. studying the a-galactosidase from
E. ggli K12 determined that for optimal activity the enzyme
requires Mn++ and a high concentration of mercaptoethanol
with an optimum pH of 8.1. The Km values of this a-galacto-
sidase for PNPG is 3.0x10'3 M and for melibiose 1.0x10‘2 M.
In an older paper the behavior of a-galactosidase was
studied in intact induced cells of E. 3911 B12 (52).
Successful extraction of the a-galactosidase in active form
from the E. £911 812 cells was not possible. The Km value
of this enzyme at all levels of activity was the same,
5.5x10'5M for PNPG. When induced E. 3911 B12 cultures were
subsequently incubated in a growth medium without an inducer,
loss of the ability to produce a-galactosidase was observed
(no induction). This was indicated by a constant decrease of
a-galactosidase activity.

The present study was aimed at obtaining data upon the
various physical and kinetic parameters of three a-galacto-
sidases derived from three different sources, A. £1121,

coffee beans, and E. coli.

 

25

Although the previously reviewed communications included
excellent experimental procedures, they did not provide much
information upon the kinetic constants, Km and Vmax, of these
enzymes for various substrates. Four compounds were used as
substrates, PNPG, melibiose, raffinose and stachyose. The
experiments were centered around the determination of the
optimum reaction temperature and pH during the hydrolysis of
PNPG and estimation of the Km, Vmax values for all four

substrates.

MATERIALS AND METHODS

p-Nitro-phenyl-a-D-galactoside (PNPG) free of p-nitro-
phenol (Sigma).

o-Nitro-phenyl-a-D-galactoside (ONPG) free of p-nitro-
phenol (Sigma).

Melibiose, raffinose, stachyose (Sigma).

Crude a-galactosidase preparation from A. £1122.
a-Galactosidase from coffee beans (25 units) in 3.2 M
(NH4)ZSO4 solution, pH 6.0, 10 units/mg of protein
(Sigma).

a-Galactosidase from E, £211 (10 units). Lyophilized
powder, 20-40 units/mg of protein (Sigma).
B-D-galactose dehydrogenase (25 units) from recombinant

E. coli, using Pseudomonas fluorescent gene, in suspension

 

in 3.2 M (NH4)SO4, pH approximately 6.0, 58 units/mg of
protein (Sigma).

Buffer solutions: acetate, phosphate, Tris-HCl, prepared
according to reference #51 and adjusted to the desired
pH level electrometrically.

Beckman DU single beam spectrophotometer.

26

27

D-Galactose Standard Curve

 

Two 0.6 mM D-galactose stock solutions were prepared,
one in 0.05 M acetate buffer, pH 5.0 and the other in 0.01 M
phosphate buffer, pH 7.0. Serial dilutions were prepared
from the stock solutions with the following molarities:

0.30 mM, 0.24 mM, 0.18 mM, 0.12 mM, 0.06 mM and 0.03 mM
galactose. A 0.6 mM NAD+ solution was prepared, in 0.1 M
Tris-H01 buffer, pH 8.6. Twenty five units of B-D-galactose
dehydrogenase were disolved into 2.5 mL of Tris-HCl buffer.
This enzyme solution was kept in the freezer at ~10°C and
was stable for five weeks.

Two mL of each of the previously prepared serial solutions
were transferred into stoppered glass tubes, 2.0 mL of the
NAD+ solution were added, mixed well and the absorbance of
these mixtures was measured spectrophotometrically against
blanks, at 340 nm. Next 10.0 uL (0.1 units of enzyme) of
B-D-galactose dehydrogenase solution were added into each
tube, the contents were mixed well again and the stoppered
tubes were set aside at room temperature (25°C) for 12 hrs.
Two series of experiments were conducted, in triplicates,
one for the galactose solutions in the acetate and the other
for the phosphate buffer solutions. Conditions and proce-
dures were identical during both experiments.

The final pH in the tubes was measured after the addition

of NAD+ solution and was 8.4 for the acetate and 8.5 for the

28

phosphate tubes. The optimum pH level for the irreversible
lactonization of galactose, catalyzed by a-D-galactose
dehydrogenase was about 8.6 (28).

The blanks were prepared by mixing 2.0 mL of the
corresponding buffer with 2.0 mL of NAD+ solution and 10.0 uL
of deactivated B-D-galactose dehydrogenase. The blanks were
prepared immediately after the galactose solutions.

After 12 hours of reaction the absorbance of the NADH in
each tube was measured again at 340 nm. The final absorbance
was estimated by subtracting the first reading from the last
one. The absorbance readings were plotted vs. the galactose
concentrations and the line was drawn by the method of least
squares polynomials. The coefficients (a,b) and the index

of determination (r) were estimated by computer.

p-Nitrophenol Standard Curve

 

Twenty five mg of p-nitrophenol were diluted into
100.0 mL of 0.05 M acetate buffer, pH 5.0. One hundred and
fifty mL of 0.3 N NaOH were added to the previous solution
(1.5 1.0 v/v) and the pH of the system was elevated to about
11.5. The above mixture was used as stock solution, contain-
ing 0.1 mg p-nitrophenol/mL.

For the preparation of the serial dilutions 5.0 mL,
4.0 mL, 3.0 mL, 2.0 mL and 0.5 mL of stock solution were
transferred into 100.0 mL volumetric flasks and made to

volume with a mixture of 0.3 N NaOH and acetate buffer

29

(1.5:1.0 v/v). The absorbance of these solutions was
measured at 420 nm. As a blank during the measurements
was used the NaOHzacetate (l.5:l.0) mixture.

All measurements were done in triplicate and the
extinction coefficient of p-nitrophenol was estimated to be

28.9 units of absorbance per umole of p-nitrophenol per mL.

a-Galactosidase from Aspergillus niger

 

Isolation Procedures

 

A crude enzymic preparation in the form of powder was
derived from A. niger Au 37 grown as follows:
Growth Medium (g/100 mL Hzoh

(NH4)3P04 1.00
(NH4)2504 0.15
NaNO3 0.50
KZHPO4 0.01
MgSO4-7H20 0.08
wheat bran 4.00

Growth conditions (shake flasks):

pH 4.0 - 4.5
Temperature 30°C
Time 5 to 5 days

Harvesting:
Centrifugation for 10 min at 2,000 g, to separate the
biomass from the culture medium. Filtration through a

membrane filter (1.2 upore size). The filtrate containing

 

30

the enzyme was lyophilized.

Assay of the Enzymatic Activity

 

An enzyme solution was prepared, containing 1.0 mg of
the crude preparation per mL, in 0.05 M acetate buffer, pH
5.0. The substrate used was a 0.05 mM PNPG solution in the
above buffer. The assay procedure included mixing 0.5 mL of
enzyme solution with 2.0 mL of substrate, let react for 5 min
at 55°C. The enzymic reaction was stopped by adding into the
tube 9.0 mL of 0.3 N NaOH and the absorbance of the hydro—
lyzed p-nitrophenol was measured against a blank at 420 nm.
The blank contained 2.0 mL of acetate buffer, 0.5 mL of
enzyme solution and 9.0 mL of 0.3 N NaOH. One unit of the
enzyme activity was defined as the amount of enzyme which
hydrolyzed 1.0 umole of p-nitrophenol per min, under the
previously described reaction conditions. The specific
activity of an enzyme expresses the number of units per mg
of protein. The nitrogen content of this crude enzymic
preparation, estimated by the microkjeldahl method (3),
was 13.6%. This corresponds to about 84.3% protein content
(N% x 6.25). The specific activity of this enzyme was
estimated to be 0.06 units/mg of protein.

Optimum Reaction Temperature

 

For the determination of the optimum reaction tempera-

ture the activity of this a—galactosidase was tested at

31

various temperatures, ranging from 25°C to 75°C. Half mL of
the enzyme solution, containing 1.0 mg of crude enzyme crude
enzyme preparation/mL of solution, in acetate buffer (pH

4.5), was transferred into stoppered tubes (in triplicate)

and incubated for 15 minutes under the following temperatures:

°, 60°, 63°, 67.5°, 70°, 75°C.

25°, 30°, 36°, 40°, 45°, 50°, 58
At the end of the incubation time 2.0 mL of substrate

(0.1 mM of PNPG solution, in 0.05M acetate buffer, pH 4.5)

were added into each tube containing the enzyme, mixed well
and allowed to react for 5 minutes. The enzymatic reaction
was stopped by adding 9.0 mL of 0.3 N NaOH. The enzymatic

activity at each temperature was estimated by the hydrolyzed

nmoles of p-nitrophenol/min.

Optimum Reaction pH

 

The effect of pH on the rate of the hydrolysis, when
PNPG was used as substrate, was studied with a seriesof
acetate buffers, ranging between pH 3.0 and 6.5. Substrate
solutions were prepared in each buffer separately at the
tested pH level (substrate concentration was 0.1 mM of
PNPG). The enzyme solution was prepared in a lower molarity
buffer (0.01 M acetate, pH 4.5), so that the addition of the
enzyme solution into the reaction system will not change
the final pH. The reaction systems were prepared as
previously and the enzymatic reaction was run at 55°C, for

5 min.

 

32

Determination of the Hydrolysis Rates

 

When the hydrolysis rate of PNPG was studied, 42.0 mL
of PNPG solution (0.2 mM) in acetate buffer, 0.05 M, pH
5.0, were transferred into a stoppered vessel, 10.5 mL of
enzyme solution (1.0 mg/mL) were added, mixed well and
allowed to react in a water bath at 55°C. At various time
intervals (1, 3, 5, ...30 min) aliquots of 1.25 mL each were
taken and transferred into tubes, containing 4.5 mL NaOH
each. Before each aliquot was taken the main vessel was
shaken, in order to achieve thorough mixing of the contents
within it. The absorbance of the hydrolyzates was measured
as previously. The hydrolysis rate of PNPG by this enzyme
was expressed as nmoles of hydrolyzed p-nitrophenol/mole of
PNPG.

When melibiose, raffinose and stachyose were the
substrates the reaction rate was determined as follows: 10.0
mM solution for each substrate in acetate buffer (0.05 M,
pH 5.0) was prepared. Fifteen mL of each substrate were
transferred into a vessel and 15.0 mL of enzyme solution in
buffer at a concentration of 5.0 mg of enzyme preparation/mL
of solution, were added. The vessels were stoppered, shaken
adequately and placed in a water bath at 45°C. All substrate
and enzyme solutions were freshly prepared and the vessels
were stoppered tightly, to avoid any losses due to evaporation,

during the prolonged incubation time.

33

The hydrolysis of the oligosaccharides was observed
for 24 hours. Aliquots of 1 mL each were taken from the
reaction mixture at predetermined time intervals (1 1/2,

3, 5, ...20, 24 hours), transferred into st0ppered tubes and
placed in boiling water for 3 minutes to inactivate the
enzyme (3 minutes of boiling was previously determined to be
enough for full enzyme inactivation).

The quantitation of the liberated galactose, in the
aliquots, was performed as follows. After boiling the
aliquots were allowed to cool at room temperature, 4.0 mL
Tris-HCl buffer (0.1 M, pH about 8.6) were added into each
aliquot and mixed well. Two mL of the previous mixtures
were transferred into other tubes containing 2.0 mL of
0.6 mM NAD+ solution in Tris-HCl buffer, mixed well and their
absorbance was measured against blanks at 340 nm. Following
that, 10.0 uL of B-D-galactose dehydrogenase solution were
added into each tube. The tubes were stoppered, vortexed
and let aside, at room temperature to react for 12 hours.
Blanks were treated similarly. Finally the absorbance of
the contents in each tube was measured again and the amount
of galactose was estimated by the difference of the second
minus the first absorbance reading, multiplied by a dilution

factor of 20.

Determination of the Km and Vmax Values

 

The determination of the kinetic constants of this

enzyme was achieved both by the method of Lineweaver-Burk

34

double reciprocal and the Eadie—Hofstee reciprocal plots.

When PNPG was the substrate, solutions of this compound
in acetate buffer with the following molarities were
prepared: 0.1 mM, 0.125 mM, 0.15 mM, 0.2 mM, 0.25 mM and
0.5 mM. Two mL of each substrate solution were transferred
into a tube (triplicates), 0.5 mL of enzyme solution were
added, mixed well and allowed to react for 3 minutes at 55°C.
The reaction was stopped by the addition of 9.0 mL 0.3N NaOH.

The velocity of the reaction was estimated after the
quantitation of the hydrolyzed p-nitrophenol and plots were
drawn. The method of least squares polynomials was also
employed. The a, b and r coefficients are indicated with
the Figures, in the Discussion section.

The Km and Vmax values for the oligosaccharides were
determined as follows. For each substrate a series of
solutions, in acetate buffer, with the following molarities
were prepared: 2.5 mM, 5.0 mM, 10.0 mM, 20.0 mM, 40.0 mM
and 80.0 mM of each oligosaccharide. One mL solution from
each substrate concentration was pipetted into each of three
tubes. One mL of enzyme solution was added, mixed well and
allowed to react at 45°C for 1 1/2 hour for melibiose and
raffinose and for 2 1/2 hour for stachyose. At the end of
the reaction time the tubes were placed in boiling water for
three minutes. For the galactose quantitation the same
procedures were followed, as previously. The reaction

velocities were used along with the corresponding substrate

 

35

concentrations for the derivation of the Km and Vmax values,

as with PNPG

Stability Tests

Studying how changes of the ionic strength in the
solution medium (buffers) affect the enzymic activity, two
solutions of the enzyme were prepared, one in 0.05 M acetate
buffer, pH 5.0, and the other in 0.5 M, same pH, containing
1.0 mg of enzyme preparation/mL of solution. The enzymatic
activity of each solution was tested during the hydrolysis
of two mL of substrate (0.75 mM PNPG solution in the corre-
sponding buffer). Similar reaction conditions were used as
previously (10 min at 55°C). In order to study the stability
of the enzyme solutions during longer periods of time, two
identical solutions in 0.05 M acetate buffer were kept, one
under refrigeration (2°-3°C) and the other under freezing
conditions (-100 to -12°C). Every two weeks the solutions
were taken out and their activity was assayed with PNPG.
Studying the thermal stability of this enzyme, two identical
reaction mixtures for each oligosaccharide were prepared.
One of them was incubated at 45°C and the other at 25°C.
After 0, 6, 12, 18 and 24 hrs aliquots were taken from each
solution and the enzymatic activity was determined, based

upon the estimated amount of hydrolyzed galactose.

36

Other Enzymes Found in the Enzymic Preparation from A. niger

 

The presence of some other enzymes, which are known to
coexist with a—galactosidase in crude preparations, such as
this one, was also examined. Among the most commonly found
enzymes is B-galactosidase and particularly invertase.

The activity of B-galactosidase was assayed with o-nitro-
phenyl-B-galactoside (ONPG). A 0.5 mM solution of this compound,
in 0.05 M acetate buffer, pH 4.0 was prepared and 2.0 mL of
this substrate solution were mixed with 0.5 mL of enzyme.

solution. The reaction was run at 37°C and the hydrolyzed

 

o-nitrophenol was determined qualitatively with NaOH.

The presence of invertase was confirmed by its hydro-
lytic action upon sucrose. A 20.0 mM sucrose solution was
prepared, in 0.05 M acetate buffer, pH 5.5 (at lower pH
levels sucrose is subjected to acid hydrolysis) and 2.0 mL
of this solution were mixed with 2.0 mL of enzyme solution
(5.0 mg/mL of solution). The reaction was run at 37°C, for
24 hours. The presence of glucose and fructose in the
hydrolyzate was determined qualitatively by descending paper
chromatography. As solvent was used, a mixture of n-butanol,
ethanol, water (5:3:2 v/v). The elution lasted 24 hours.

For the identification of the sugars the guide strip method
was used. To develop the spots, the chromatograms were
sprayed with 3% p—anisidine (some hydrochloric acid was

added into the p-anisidine solution) and heated for 5 minutes

at 105°C.

 

37

The presence of invertase was also detected by HPLC
but not very successfully. Two mL of a 20.0 mM sucrose
solution in distilled water were subjected to hydrolysis
by 2.0 mL of enzyme solution in water (5 mg/mL of solution),
for 24 hours at 37°C.

Another mixture of sucrose solution with heat inacti-
vated enzyme was prepared and injected into the pump of an
HPLC analyzer (by Waters). As carrier solvent was used a
mixture of acetonitrile, water and ethanol (85:12:3 v/v),
with a flow rate of 2.0 mL/min.

Twenty 0L of the sucrose hydrolyzate were injected and
the component separation was recorded. The separation was
not very successful but the sucrose in the mixture with the
deactivated enzyme gave a distinct peak, which was not
observed during the elution of the components in the hydro-
lyzate.

Due to the high amount of protein in the hydrolyzates,
before the initiation of both chromatographic techniques
described above, protein precipitation and removal was
performed by the addition of 10% (w/v) lead acetate and
centrifugation at 5,000 xg. The excess of lead acetate was

precipitated by 10% (w/v) oxalate and centrifuged out.

Inhibitors
Metal ions, sugars and other inhibitory substances were

added to reaction mixtures and their effect upon the enzyme

 

38

activity was tested, using a 0.75 mM PNPG solution as sub-
strate. Table 6 in the Discussion section indicates the
inhibitors, their final concentration in the reaction
mixtures and the relative activity of the enzyme in each
case. The reaction mixtures consisted of 1.0 mL substrate
solution, 0.25 mL of enzyme solution and the indicated
concentration of the inhibitor. The inhibition was expressed

as % of the activity demonstrated by control samples.

a-Galactosidase from Coffee Bean

 

Dialysis

A 0.5 mL suspension of a-galactosidase from coffee
beans (5 units) in 3.2 M (NH4)ZSO4, pH 6.5 was purchased from
Sigma. This enzyme preparation was subjected to dialysis
against 0.01 M acetate buffer, pH 5.4. The enzyme suspension
was transferred quantitatively into a cellulose dialysis
tube (2 inch width), which was previously prepared as
follows. The tubing was simmered in 1.0 L of 50% ethanol,
for 1 hour. Immersion of the tubing, after boiling, again
in 50% ethanol for 1 hour followed. Another immersion in
10.0 mM NaHC03 solution, which was repeated with a change of
solution. Immersion in 1.0 mM EDTA for one hour followed.
Finally the tubing was rinsed in distilled water for 1 hour.
One end was tied into a double knot and so was the other,

after the enzyme was transferred into the tube.

 

39

The enzyme was transferred with some acetate buffer and
the total volume of its solution in the tubing was about
10.0 mL. The dialysis lasted for 20 hours and was performed
under continuous stirring in cold room at 2°C. Following
the dialysis the enzyme solution was transferred into a
10 mL volumetric flask and was made to volume. This enzyme

solution contained about 0.5 units/mL, a total of 5 units.

Assay of the Enzymatic Activity

 

The reaction mixture consisted of 10.0 mL acetate
buffer, 0.2 mL of substrate solution (10 mM PNPG in the same
buffer) and 0.5 mL of the dialyzed enzyme solution. The
reaction lasted for 10 minutes at 45°C. At the end of the
reaction time 10.0 mL of 0.3 N NaOH were added. Blanks were
prepared by mixing 10.2 mL of acetate buffer, 0.01 M,
pH 5.4, with a 0.5 mL of enzyme solution and 10.0 mL NaOH.
Samples were prepared in triplicate. The activity of this
enzyme was estimated to be 62.5 units/mL of solution, under

the previously described experimental conditions.

Optimum Reaction Temperature

 

For the determination of the optimum temperature similar
procedures were followed as in the case of the a-galactosi-
dase from A. Aiggg. Ten mL of buffer (0.01 M, pH 5.4) were
mixed with 0.5 mL of enzyme solution in steppered tubes and
incubated for 15 minutes at various temperatures ranging from

25° to 70°C. The rest of the reaction conditions were kept

 

40

the same as during the enzyme assay.

Optimum Reaction pH

 

The rate of hydrolysis of PNPG by this a-galactosidase
was studied with a series of acetate buffers, 0.1 M and pH
ranging from 3.3 to 5.75. Similar volumes of substrate,
enzyme and NaOH solutions as during the assay experiment
were used and the reaction was run at optimum temperature
level (50°C). The reaction rates were determined based upon

the amount of hydrolyzed p-nitrophenol.

Reaction Rates

 

During the next series of experiments another enzyme
solution was used, which after the dialysis contained 1 unit/
mL of solution. The reason for diluting this enzyme less
was the fact that enzymes maintain their activity longer at
higher concentrations.

The procedures for the determination of the reaction
rates, during the hydrolysis of the four substrates by this
enzyme, are similar to those followed during the reaction
rate experiments for a-galactosidase from A. 2133;, Only
the volumes and the concentrations in the reaction mixtures
vary.

Thirty three mL of substrate solution (1 mM of PNPG,
in 0.01 acetate buffer, pH 5.0) were mixed with 330.0 L of
enzyme solution in the above buffer and allowed to react for

35 minutes at 50°C. One mL aliquots were taken at various

 

41

reaction times and the hydrolyzed p-nitrophenol was quanti-
tated spectrophotometrically, after the addition of 3.6 mL

of NaOH. The rate of hydrolysis of the oligosaccharides

was studied during a 24 hour observation. Three main vessels
were prepared, containing the reaction mixture for each
oligosaccharide, each having a molarity of 10.0 mM. Thirty
mL of each substrate solution were mixed with 600.0 L of
enzyme solution. The reaction lasted for 24 hours at 45°C.
Aliquots of 1.0 mL each were taken into screw-capped test
tubes and boiled for three minutes.

The galactose quantitation was done in a similar way
as before. The dilution factor was in this case twice as
large (1/10.02), because in the aliquots of a-galactosidase
from A. giggg the volume of the substrate was 1/2 of the

total volume.

Determination of the Km and Vmax Values

 

The values of the kinetic constants of this enzyme

were estimated as in the case of the a-galactosidase from

A. Aiggg, for the initial rates of the reaction. The estima-
tion of Km and Vmax values was achieved as previously, by

the Lineweaver-Burk and Eadie—Hofstee, after the statistical
manipulation of the experimental data. For the determina-
tion of the Km and Vmax values for PNPG a series of solutions
with the following molarities were prepared: 0.25 mM, 0.5 mM,

0.75 mM, 1.0 mM, 1.5 mM and 2.0 mM.

42

One mL of substrate solution was hydrolyzed by 5.0 L
of enzyme solution for 3 minutes at 50°C. The reaction was
stopped by the addition of 3.6 mL of 0.3 N NaOH solution.

For the determination of the kinetic parameters of the
oligosaccharides, solutions of melibiose and raffinose were
prepared in acetate buffer (0.01 M, pH 5.0), with the
following molarities: 2.5 mM, 5.0 mM, 10.0 mM, 20.0 mM,

40.0 mM and 80.0 mM. For stachyose solutions with the
following molarities were prepared: 1.875 mM, 3.75 mM,

7.5 mM, 15.0 mM, 30.0 mM and 60.0 mM. An additional
solution was prepared for melibiose, with molarity of

1.25 mM, due to substrate inhibition phenomena observed at
concentrations of about 70.0 mM of melibiose and above.

The reaction mixtures contained 2.0 mL of each substrate and
40.0 0L of enzyme solution (1 unit/mL of solution). The
reaction mixtures of melibiose and raffinose were incubated
for 1 1/2 hours and those with stachyose for 3 hours. At
the end of the reaction time the tubes were boiled for three
minutes.

For the galactose quantitation 2.0 mL of Tris-HCl
buffer were added into each tube, which contained the meli-
biose and stachyose solutions and 5.0 mL of the same buffer
into each tube containing the raffinose solutions. The rest
of the procedures, for the galactose determination, were the
same as previously (a-galactoseidase from A. Aiggg). The

dilution factors were 5, for melibiose and stachyose and 7,

 

43

for raffinose aliquots.

Inhibitors

The inhibitory effect of several compounds upon the
enzymatic activity of the a-galactosidase from coffee beans
was studied, using PNPG as substrate. To study the inhibi-
tory action of Ag+ ions, 2.5x10'7 moles of AgN03 were added
to 5.0 mL of active enzyme solution, containing 1.0 unit of
enzyme per mL of solution. Next 5 0L of this enzyme solu-
tion were incubated with 1 mL of substrate solution (0.75 mM
PNPG, in acetate buffer) for 5 minutes at 45°C. Control
samples were also tested, containing active enzyme solutions.

The enzyme inactivation was determined from the ratio:

1 umol of hydrolyzed p-nitrophenol by enz w/ inhibitor
00 X umol of hydrolyzed p-n1trophenol by enz w/o 1nh1b1tor

 

When Hg++ ion was the inhibitor, 1.0x10'7 moles of HgClZ
were added into 5 mL of active enzyme solution. Experiments
for the Hg++ inhibition were also performed as previously.
As in the case of the a-galactosidase from A. fligg_,
the effect of KI was tested upon enzyme solutions, inacti—
vated by the addition of heavy metal ions, such as Ag+ and

Hg++.

44

a-Galactosidase from E. coli

 

The a-galactosidase from E. ggli was purchased in the
form of lyophilized powder, containing 20-40 units/mg of
protein. An enzyme solution containing approximately
1 unit/mL was prepared in 0.01 M phosphate buffer, pH 6.5.
This enzyme solution was constantly kept in iced water, to
prevent rapid loss of its activity. For the assay of this
enzyme 1.0 mL of the above phosphate buffer was mixed in a
tube with 100 uL of a PNPG solution, with a molarity of
5.0 mM, also prepared in phosphate buffer and 5.0 0L of the

enzyme solution.

Optimum Reaction Temperature

 

The determination of the optimum reaction temperature
was carried out by incubating 100 0L of substrate solution
(5.0 mM of PNPG in 0.01 M phosphate buffer, pH 6.5), with
1.0 mL phosphate buffer and 5.0 0L of the previously prepared
enzyme solution. The reaction lasted for 10 minutes and was
run at various temperatures ranging from 25° to 60°C.

Prior to the addition of the substrate, the enzyme was
incubated along with the buffer at the tested temperature
for 10 minutes. The reaction was stopped by the addition of
2 mL of 0.3 N NaOH solution. The blanks used during the
spectrophotometric determination of the hydrolyzed p-nitro-
phenol were prepared by mixing 1.1 mL of phosphate buffer
with 5 0L of enzyme solution and 2 mL of 0.3 N NaOH.

 

45

Optimum Reaction pH

 

The effect of pH upon the enzymatic activity of
a-galactosidase from E. ggli was studied again during the
hydrolysis of PNPG. Two different series of buffers were
prepared each with a molarity of 0.1 M. For the pH levels
between 4.0 and 5.75 a series of acetate buffers was
prepared. For the pH range 6.0 through 8.75 a series of
phosphate buffers were prepared. The reaction mixtures
were prepared as previously and the reaction was run at

45°C.

Reaction Rates

 

The hydrolysis of PNPG by this a-galactosidase was
followed for 36 minutes. Aliquots were taken from the
reaction mixture in the same way as before, at various
times and the enzymatic activity was plotted vs. the reaction
time. Optimum temperature (45°C) and pH (7.0) conditions
were kept. The hydrolysis of melibiose and raffinose were
followed for 24 hours and that of stachyose for 38 hours.
The reaction mixtures were prepared as for the other two
enzymes, in three main vessels, one for each substrate. All
substrate solutions were prepared with the same molarity,

10 mM, in phosphate buffer (0.01 M, pH 7.0). The volume
ratio of the substrate and enzyme solution was 1 mL of
substrate/25 0L enzyme solution (1 unit/mL of solution).

Aliquots of 1 mL each were taken at various times, depending

46

upon how fast the reaction was progressing. For example,
during the hydrolysis of stachyose the aliquots were taken
every 3 hours in the beginning of the reaction and every

5 hours later, in order to yield measurable differences in
the amount of hydrolyzed galactose.

Initially a temperature of 45°C was selected for the
reaction. The galactose quantitation data though, particu-
1arly those from the hydrolysis of stachyose, gave very low
values, even after many hours of hydrolysis. For this reason
a second experiment was run, only for stachyose, at 25°C and
for the same reaction time, which gave better results. For
the determination of the galactose concentration in the
aliquots, the absorbance readings were multiplied by 20, for

melibiose and raffinose, and by 4 for stachyose.

Determination of the Km and Vmax Values

 

During the determination of the Km and Vmax values for
PNPG a series of substrate solutions with the following
molarities was prepared: 0.0625 mM, 0.125 mM, 0.250 mM,
0.50 mM, 0.75 and 1.0 mM, in phosphate buffer (0.01 M and
pH 7.0). One mL of each substrate solution was mixed with
5 0L of enzyme solution and the reaction was run for 10
minutes at 45°C. The reaction was stopped by the addition
of 2 mL of 0.3 N NaOH. Quantitation of the hydrolyzed
p-nitrophenol followed.

For the determination of the enzyme kinetic constants

for the oligosaccharides, solutions with the following

1 '

47

concentrations were prepared, in the above phosphate buffer:
2.5 mM, 5.0 mM, 10.0 mM, 20.0 mM, 40.0 mM, and 80.0 mM for
melibiose and raffinose and for stachyose 1.25 mM, 2.5 mM,
5.0 mM, 10.0 mM, 20.0 mM and 40.0 mM.

The reaction mixtures contained 2 mL of each substrate
solution and 50 pL of enzyme solution. The tubes were
incubated at 25°C (all three substrates), for 2 hours, for
melibiose and raffinose and for 3 hours for stachyose. At
the end of the reaction time the tubes were placed in boiling
water for three minutes. The galactose determination
followed and the absorbance readings were multiplied by

4, for raffinose and stachyose and by 20 for melibiose.

Inhibitors

A few inhibition experiments were conducted, with
several inhibitory agents, such as galactose, Ag+ and Hg++.
Also the effect of KI upon inactivated enzyme solutions was
studied. The reaction mixtures consisted of 1 mL phosphate
buffer, 50 0L of substrate solution (5.0 mM PNPG in buffer)
and 0.25 mL of either active (controls) or inactivated
enzyme solution, after the addition of various amounts of
inhibitors, as indicated in Table 12 in the Discussion section

(1 unit of enzyme/mL of solution).

RESULTS AND DISCUSSION

Standard Curve of Galactose

 

The enzymatic activity of all three enzymes was deter-
mined by a dual approach. When PNPG was the substrate, the
amount of liberated p-nitro-phenol was determined. When

melibiose, raffinose and stachyose were the substrates, the

' 'LAL

enzymatic activity was measured by the amount of liberated
galactose.

Several methods were tried for the galactose determi-
nation: paper chromatography (PC), thin layer chromatography
(TLC), high pressure liquid chromatography (HPLC) and an
enzymatic method employing the use of B-D-galactose dehydro-
genase. The chromatographic methods did not give satisfactory
results. The major reason for this was the microquantities
of galactose, which had to be determined.

The enzymic methods are more specific, precise and
rather simpler, in cases where specificity and accurate
determination of microquantities are crucial factors.

For the enzymic determination of galactose one can
choose between two methods, depending upon the conditions
and the components in the reactivation system. Factors such

as pH, temperature and reactants affecting the rate of the

48

 

49

enzymic reaction can greatly influence the results. One of
the methods is galactose oxidase, which catalyzes the oxida-
tion of galactose at the sixth carbon atom (49). This is
indicated by Figure 6. During this reaction the properties
of H202, produced stoichiometrically are exploited in a
manner analogous to the determination of glucose, with
glucose oxidation (33). This method, though, was considered
improper in this case, because other sugars, such as galac-
tose derivatives (melibiose, raffinose, stachyose) can react
appreciably with their terminal galactose moieties, leading
to erroneous results (49).

An alternative method is the a-D-galactose dehydro-
genase or D-galactose NAD-l-oxydoreductase (E.C. 1.1.1.48),
which catalyzes the conversion of B-D-galactose into 0-
galactono-lactone in the presence of coenzymes NAD+ or DPN,

as indicated below:
B-D-galactose+NAD+——).D-galactono-a-lactone+NADH+H+
B-D-galactose+DPN —91D-galactono-y-lactone+DPNH

This enzyme is selective only for s-D-galactose, a-L-
arabinose, and B—D-fucose. The optimum pH for the reduction
of NAD+ or DPN is between 8.0 and 9.0. In this range the
galactonolactone undergoes spontaneous hydrolysis and the
reaction is virtually irreversible (21).

The galactose concentration is a function of the

absorbance of the formed NADH at 340 nm. The extinction

 

Figure 6.

SO

CHO

OH

OH

Reaction of galactose oxidase.

OH

OH

Hé%1

 

51

coefficient of NADH is 4.25 absorbance units/“mole/mL.
Since the formation of NADH occurs stoichiometrically with
the galactose lactonization, the amount of galactose is
equivalent to the formed NADH (29).

In aqueous solutions D-galactose exists as an equili-
brated mixture of various isomers. For this reason it takes
a long time for the entire amount of galactose to be
lactonized. Mutarotase or aldose-l-epimerase (E.C. 5.1.3.3)
can be added to convert all the galactose into the 8-0-
configuration for rapid enzymatic reaction (6). The addi-
tion of mutarotase though can be omitted from the system, if
the reaction is allowed to run long enough.

Several researchers who employed the use of this enzyme
in their work (26, 27) decided that 1 hour at 37°C would be
enough time for the completion of the reaction, without
mutarotase. Another factor influencing the reaction time
is the number of B~D-galactose dehydrogenase units used in
the system. The determination of an adequate incubation
time for the B-D—galactose dehydrogenase reaction was
achieved after repeated measurements of the NADH absorbance,
formed in tubes of samples containing D-galactose, NAD+ and
enzyme. It was found that at room temperature (25°C) an
incubation time of 12 hours was sufficient, when 5 0L of a
B-D-galactose dehydrogenase solution, containing 0.1 units/mL,

were added into the reaction system.

 

52

The determination of the galactose standard curve was
accomplished by measuring the absorbance of samples with
various galactose concentrations ranging from 0.0 to 0.3
umoles/mL of solution (Figure 7). The absorbance of 0.3
nmoles of galactose/mL was 1.25 A, and was considered to be
the highest galactose concentration that should be found in
a reaction mixture for accurate readings. The galactose
concentration in the experimental samples was adjusted up to
this level by various dilutions with Tris—HCl buffer, 0.1 M
and pH 8.6.

Two kinds of buffers were used during our experiments,
acetate and phosphate. The presence of two different ionic
species in the reaction mixtures of galactose dehydrogenase
could affect differently the activity of this enzyme. Two
series of samples were prepared (in triplicate), one with
various galactose solutions in acetate buffer (0.05 M, pH
5.0) and the other in phosphate (0.01 M, pH 7.0). The rest
of the reaction conditions were identical. The galactose
absorbance readings, corresponding to equal galactose con-

centrations, were very close (Table 2).

Standard Curve of p-nitro-phenol

 

The p-nitro-phenol derivatives are very popular com-
pounds, used in many cases, as chromogenic substrates for
the colorimetric assays of various enzymes. Pure solutions

of PNPG are colorless. During the enzymatic hydrolysis of

53

1.5 -

1.0 L

A420

 

I l

.12 .24
fbmoles of galactose/mL

Figure 7. Standard curve for determining galactose.

54

Table 2. Absorbance of galactose in acetate and phosphate
buffers at 340 nm.

 

 

Galactose Acetate Phosphate

(”moles/mL) buffer buffer
0.300 1.230 1.205
0.150 0.585 0.600
0.120 0.473 0.450
0.090 0.345 0.320
0.060 0.235 0.250
0.030 0.130 0.145
0.015 0.073 0.080

 

55

this compound by a-galactosidases, p-nitro-phenol is liberated,
which has a pKa of 7.2. This compound in alkaline environ-
ment (1 to 2 pH units above its pKa value) develops a deep
yellow color suitable for the quantitative measurement of
the enzyme activity. Advantages of this method include
simplicity, accuracy, sensitivity and fast results (34).

The alkaline compounds, used to develop the color of p-
nitro-phenol, also stop the enzymic reaction by raising the
pH (when 0.3 N NaOH is added, the pH of the reaction mixture
reaches 12.8). The mechanism of the chemical reactions
occurring during the enzymatic hydrolysis of PNPG by
a-galactosidases and the color development in the alkaline
environment are shown in Figure 8.

In many cases o-nitro-phenol derivatives are used
instead. It seems though that the p—derivative is the
compound of preference, because the o-nitro-phenol is steam
distilled, due to the formation of intramolecular hydrogen
bonds (Figure 9).

The blanks, used during the various spectrophotometric
measurements, initially were prepared by mixing equal volumes
of PNPG solutions, to those used in the experimental samples,
heat inactivated enzyme solution and NaOH. Immediately after
mixing the blanks were colorless but with time they developed
a deep yellow color. This was due to the gradual liberation
of p-nitro-phenol, after the addition of NaOH. For this

reason the PNPG solution in the blanks was replaced by the

 

56

CH H

CHZOH
1:; o
HOH
0H O_J<C)—No2 ——9 OH + 0H—-<C:>>——~o2
0

OH

OH——<©>—wo2 + NaOH H ‘o—Q—NOZ-l- MT 4» HOH
NOz-4<::2;:O

5102019172..) Moz£©:§ e—a ::>°-<:>=o< -

5‘ 1.

2,0,.“

\

Figure 8. Reactions occurring dUring the hydrolysis of
PNPG by a-galactosidase and color development
of the liberated p-nitro-phenol.

 

Figure 9. Formation of intramolecular hydrogen bond by
o-nitro-phenol.

57

corresponding buffer. Pure PNPG solutions do not absorb
any light at 420 nm,so do the buffers (Figure 10).
Other workers used water, as a reference, in similar

experiments.

a—Galactosidase from A. niger

 

Optimum Reaction Temperature

 

Figure 11 indicates that the optimum temperature for
the hydrolysis of PNPG, by a-galactosidase from A. nigg_,
is 55°C. Between 50° and 60°C the enzyme shows 94 to 100%
of its maximum activity. At 65°C the activity drops to
86%. Beyond 65°C an abrupt decrease of the enzymic
activity is demonstrated, due to heat denaturation of the
enzyme molecule. At 70°C the enzyme loses 65% of its
maximum activity in 15 minutes. Holding the enzyme solu-
tion for several hours at room temperature also resulted
in some loss of activity. For this reason the enzyme was
always kept in iced water.

The thermal stability of other a—galactosidases,
isolated from various species of the genus Aspergillus,
such as A. ggtgi (57) demonstrated a similar optimum
temperature, about 55°C. Another a-galactosidase, from
A. awamori (43), was found to be less thermostable, with an
optimum temperature at 50°C. This a-galactosidase was
inactivated at 55°C in 15 minutes. The enzyme did not

lose activity during prolonged incubation at 45°C.

58

A420

 

I l

0 .008 .016
famoles of p-nitrophenol/mL

Figure 10. Standard curve for determining p—nitrophenol.

nmoles of p-nitrophenol/mIn/mL of substrate solution

 

Figure 11.

59

l l

30 o 60
Temperature(c )

Temperature—activity relationship of q-galacto-
sidase from A. niger with PNPG as substrate

(pH 4.5). Reaction mixture: 1 mL substrate
solution (0.1 mM PNPG) 0.25 mL enzyme solution
(1 mg/mL).

V

60

The data in Table 3 makes a comparison of the absorb-
ance readings of two identical samples of the oligo-
saccharides, incubated at 25°C and 45°C. The reaction
mixtures contained 1 mL of substrate solution (10 mM of
each oligosaccharide in acetate buffer) and 1.0 mL of
enzyme solution (5.0 mg of enzyme preparation/mL of acetate

buffer).

Optimum Reaction pH

 

Figure 12 indicates the relation between the enzyme
activity and the pH in the reaction system. The optimum
reaction pH is about 5.0. It is apparent that this enzyme
favors acidic environments, showing increased activity at
the pH range between 3.75 and 5.0. Actually at pH 3.75,
where many enzymes show very low activity and some of them
are totally inactivated, this enzyme maintains 96% of its
apparent activity. The increased activity of this enzyme
in acidic environment can be explained by the fact that it
is derived from a mold. These microorganisms can tolerate
acidic environments well. As a result of this their enzymes
also remain active in acidic environments.

Lee et al. (40) determined the optimum reaction pH
for the a-galactosidase from A. flifléfi at 4.6, while Bahl
estimated it at 4.9 (5). Note that there is a wide
optimum pH level, in which the enzyme demonstrated increased

activity. This could indicate the presence of more

61

Table 3. Thermal stability of a— —galactosidase from ni er,
during incubation for 24 hours at 25°C and A450 0.
A34o* of a system containing 1 mL of substrate
solution (10 mM melibiose, raffinose or stachyose)
and 1 mL of enzyme solution (5.0 mg of enzyme per
mL of solution) after 6, 12, 18, 24 hours of

 

reaction.
Incubation at 25°C Incubation at 45°C
Substrate Reaction time (hours) Reaction time (hours)

 

6 12 18 24 6 12 18 24

 

Melibiose 9.6 11.6 12.6 13.4 9.7 11.8 12.8 13.4
Raffinose 10.2 13.1 13.5 13.6 10.6 12.8 13.4 13.8
Stachyose 5.2 7.6 8.6 10.0 5.0 7.7 9.0 9.8

 

*A3 of NADH is a measure of galactose determined enzymati-
a1°y. The A340 values represent the original absorbance
readings multiplied by a dilution factor of 20.

62

 

 

 

A
6..
C
O

4"

a
5
H
O
U)

Q

‘3
(I!

f-

p

B
= A —
m

c.

O
.J
E

\

1:

d
E

\

r-G
O
C!

Q)

.2
8‘24
L
U
I“

C
I
Q.

s.
O
(D
0

H
O
E
I:

O I l ‘>
3.0 4.0 6.0

pH

Figure 12. pH-activity relationship for a-galactosidase from
A. niger, with PNPG as substrate at 550C.
Reaction mixture: 1 mL substrate solution (0.1
mM PNPG) 1 mL enzyme solution (1 mg/mL).

 

63

a-galactosidases in this crude enzyme preparation (40).

Reaction Rates

 

During the study of an enzyme for the determination
of its kinetic constants, Km and Vmax, it is important
that the data of the velocity are taken during the steady
state of the reaction, when the disappearance rate of the
substrate, S (equation 1) is equal to the appearance rate
of the product, P. In this way the concentration of the
complex ES is constant (by definition) in the steady state
(Briggs-Haldane hypothesis, ref. 24).

k1 k2
E+s——-*.__.1=.s..__-——-:E+P (l)
k-1 k-z
where: E = enzyme concentration
S = substrate concentration
P = product concentration

The steady state is achieved very rapidly after mixing

the enzyme with the substrate. Fast reaction techniques
have shown that in most cases it is achieved within a few
miliseconds and before any finite product has been formed.
This means that, when one measures the initial reaction
rate of an enzyme by the usual experimental methods, one
is actually measuring the steady state of the reaction (24).
Figures 13 and 14 show the course of the reaction of

a-galactosidase from A. niger, during the hydrolysis of

 

64

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A5505;
on ON CH C
A a q _

 

 

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uotsntos enemasqns JO qm/Ioueqdoaatu-d JO sstowu

65

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Amuwgmgoucmommpo mo 45 opp =o_u:_om mumcumnzm be 45 P ”mczuwa comuommm .mmmv
iwmouoepmmia .mevc .< 54 mmoxsumum new mmocwwwac .wmownwpos $0 mwmxpocvz: .¢_ mczmvu

 

 

'ctzn os aqeaqsons '0 1m 950309 e? 0 ea o"u
J

4m5454525
cm cm 0. o
A q q 4
fl
3

1.oo~

ouomsoanm o
G

occanuaoz 1
ouocduumm 4

1 co:

 

 

66

PNPG and melibiose, raffinose and stachyose. For PNPG the
reaction rate plateaus within 12 minutes, while for the
oligosaccharides this is not observed until several hours
are gone. Among the three oligosaccharides stachyose is
hydrolyzed most slowly, while raffinose seemed to be the
substrate of preference. Melibiose was initially hydro-
lyzed at a slower rate than raffinose and reached later

the hydrolysis rate of raffinose.

Km and Vmax Values

 

The determination of the Km and Vmax values was esti-
mated for the initial reaction rates, about 3 minutes for
PNPG, 1 1/2 hours for melibiose and raffinose, 2 1/2 hours
for stachyose. When PNPG was the substrate, a decrease of
the reaction rate was observed, when the substrate concen-
tration in the reaction mixture was higher than 0.75 nmoles
of PNPG/mL of reaction mixture. As Figure 15 shOws,among
the various PNPG concentrations, the reaction rate was
higher, when the substrate solution had a molarity of about
0.75 mM. The same observation was made by Dey and Pridham,
when they were collecting data during kinetic studies upon

the a-galactosidases I and II from Vicia faba (20). These

 

researchers observed that, when PNPG was the substrate, at
concentrations above 0.75 mM the simple Michaelis-Menten
law was not obeyed. They characterized this phenomenon as

”substrate inhibition". One possible explanation for this

nmoles of p-nitrophenol/mL of substrate solution

 

67

 

A
200 —
100 —
1 L,
O 1.0 2.0
Substrate solution concentration(mM)
Figure 15. Optimum substrate concentration during the

hydrolysis of PNPG by a-galactosidase of

A. niger. Reaction mixture: 1 mL of substrate
solution, 1 mL of enzyme solution (1 mg enzyme
preparation/mL).

 

68

is that for the formation of an effective enzyme-substrate
complex a single substrate molecule may associate with a
binding site on the enzyme. At higher substrate concen-
trations though a second molecule may associate with the

enzyme to form an inactive complex (20), i.e.

E + SHESHE + P

1

E85 (inactive complex)

During the hydrolysis of the oligosaccharides,inhibition
phenomena were observed with melibiose at concentrations
above 0.07 mmoles per mL of reaction mixture. The presence
of stachyose at the same concentration in the reaction
mixture did not cause any inhibition. If Haldane's theory
about the substrate inhibition is correct, then the forma-
tion of the inactive compound ESS is not possible, when
stachyose is used as substrate. Probably stereochemical
limitations at the enzyme's active site do not allow more
than one large molecule, such as stachyose, to bind. This
substrate inhibition phenomenon is not observed in the
various biological systems, due to the small concentrations
of substrate and continuous removal of the product, which
renders available more enzyme sites.

The determination of the kinetic constants of
a-galactosidase from A. 31333 was based upon the Michaelis-

Menten equation. Two modifications of this equation were

4 4.._..

69

used to obtain the Km and Vmax values of the four sub-
strates. One was the well known Lineweaver-Burk equation

(2) and the other was the Eadie-Hofstee equation (3).

 

l _ Km 1 l

V - Vmax X S x Vmax (2)

v = Vmax - % x Km (3)
where: v = initial rate of reaction

S initial substrate concentration
Vmax = maximum reaction rate

Km = Michaelis constant

Figures 16 through 23 indicate the derivation of the Km and
Vmax values of the a-galactosidase from A. giggg, for the
four studied substrates, by both methods. All the lines
were drawn based upon the method of least squares poly—
nomials (first degree equation, y = bx+a). The statistical
manipulation of the experimental data was achieved with the
aid of a computer and the estimated values of the coeffi-
cients, a and 8, along with the indexes of determinations,
r, are indicated with the plots. The Km and Vmax values

were calculated from the formulas:

mIU

Lineweaver-Burk plots

m|—‘

Vmax

Km

II
D)

- b Vmax Eadie-Hofstee plots

 

7O

 

 

 

 

 

A
?§0— a=33.05
13321.1“
1/' r=0.918
150. xm-6.7x10'°Mz 1
Vmax=3.0x10 min-
/ 1 1\
o 6 m ’
1/s
Figure 16. Lineweaver-Burk double reciprocal plot of
a-galactosidase of A. ni er for PNPG. Reaction
mixture: 1 mL substrate so ution,0.25 mL of
enzyme solution (1 mg enzyme preparation/mL).
33:
ES.
e—‘E.
E2
5'22
3.5,.
‘65-.
52;;
Figure 17. Eadie-Hofstee reciprocal plot of a-galactosi-

dase of A. niger for PNPG. Reaction mixture:
1 mL substrate solution, 0.25 mL enzyme
solution (1 mg/mL).

5. -4

3“.

71

1\ 3263.1“
b’389.07

20° T r¢O.99“

  
 
 

Km-6.16x10'3M
Vmax 1.6x10 min

1/v

 

1/

l/S

Figure 18. Lineweaver-Burk double reciprocal plot of
a-galactosidase of A. niger for melibiose.
Reaction mixture: 1 mL of substrate solution,
1 mL of enzyme solution (5 mg of enzyme
preparation/mL).

  
 
 
  
  

 

 

O 016 s-0.0164
? . ‘ b:-6.72
E r'0.97“
e 5
§ 3 Kn'6.7x10’3n
3 .3 Vux"°°’”° mm
0 O
I
'2 3 0.008 _
U U
L
h a
O D
D
to 3
0 O
H
g:
‘:
J4 >
o 0.001 0.002

'/s

Figure 19. Eadie-Hofstee reciprocal plot of a-galactosidase
from A. niger for melibiose. Reaction mixture:
1 mL substrate solution, 1 mL enzyme solution
(5 mg/mL).

72

0'19.28
0'739.83
r80.969

   
 

200 -
Kn-3.83110 2n
1/' lex-5.2110'2n1n'1

o 0.3 0.6 ’

Figure 20. Lineweaver-Burk double reciprocal plot of
a-galactosidase of A. niger for raffinose.
Reaction mixture: 1 mL of substrate solution,
1 mL of enzyme solution (5 mg of enzyme
preparation/mL).

  
  
 
  

 

 

E

E A
-1 1 “0.0304
5 2 0.030
} 4 bI-17.59
a 3
g 3 r=0.843
0 In
3 o x :1 7x10'2M
° ° VII ;3 1x10‘2nm‘1
c. 5 an: ‘
o 2 0.015
I'D 3
0 VI
F1
0 S.
E O
1.
I’

l l
o .0008 .0016
V/s

Figure 21. Eadie-Hofstee reciprocal plot of a-galactosidase
of A. niger for raffinose. Reaction mixture:
1 mL of substrate solution, 1 mL of enzyme
solution (5 mg enzyme preparation/mL).

 

73

 

1000
0:70.“)
I/v b‘2,017.h
r-0.999
5°C Km=2.9x10-2M
Vu.x-1.ux10'zaln'1
l 1 Q
0 .2 .

Figure 22. Lineweaver-Burk double reciprocal plot of
a-galactosidase of A. niger for stachyose.
Reaction mixture: 1 mL of substrate solution,
1 mL enzyme solution (5 mg enzyme preparation/mL).

  

 

 

\

T 330.0145“
_. T ba-29.11
{r E .012 ~ ’=°-993
% ;
-' = K =2.9x10'2M
5 T m -2 -1
i E anx"'““° min
L— C
L. 5
c : mos—
”: V)
we
3 5
E L-
3,5
>

I l
o .0002 .0004

Figure 23. Eadie-Hofstee reciprocal plot of a-galactosidase
from A. niger for stachyose. Reaction mixture:
1 mL substrate solution, 1 mL enzyme solution
(5 mg/mL).

 

74

The above formulas were derived from equations Q) and B).
Table 4 gives the Km and Vmax values of a-galactosi-
dase from A. 31331 for PNPG, melibiose, raffinose and
stachyose, as they were estimated by both methods. As one
can see the Km and Vmax values change independently, among
the various substrates. Comparing the Km and Vmax values
obtained for each substrate by the two methods, one may
conclude that, when the indexes of determination (r) are
closer to 1.0 the estimated values of the kinetic constants
estimated by both methods are almost the same. This enzyme

had higher affinity (l/Km) for PNPG.

Stability

Stability tests indicated that this a-galactosidase
is more stable than the enzymes from coffee beans and
E. 2211. Dissolving the enzyme in 0.5 M acetate buffer
caused a 10% increase of the enzyme activity, compared to
that in 0.05 M acetate buffer. The enzyme solutions kept
under refrigeration and freezing both demonstrated 99% of
their original activity during the first month. After
1 1/2 months the refrigerated solution indicated only 65%
of each original activity, while the frozen one retained
90% of it. In two months the refrigerated solution had
lost all its activity, while the frozen one still retained

75% of it.

an.

 

75

 

 

 

Table 4. Kinetic constants of a-galactosidase from A. niger
for 4 substrates determined by the Lineweaver-Burk
(L-B) and Eadie-Hofstee (E-H) methods.
Substrate Km (moles) Vmax (nmoles/mL)
substrate/min)
L-B E-H L-B E-H
PNPN 5.7x10'4 5.1x1o‘4 3.0x10"2 2.5x10'2
Melibiose 6.2x10'3 6.7x10‘3 1.6x10-2 1.5x10’2
Raffinose 3.8x10'2 1.8x10'2 5.2x10"2 3.0x1o‘2
Stachyose 2.9x10“2 2.9x1o-2 1.4x1o‘2 1.4x1o”2

 

 

76

Other Enzymes

 

The experimental procedures for the determination of
the presence of other enzymes, in this crude a-galactosi-
dase were not very detailed and accurate. Yet they
revealed some information about enzymes that can be expected
to be found in this crude a-galactosidase preparation. The
experiments with NPG indicated the presence of B—galacto-
sidase. The chromatographic analysis of the sucrose
hydrolyzates, by TLC and HPLC, revealed the presence of

invertase.

Inhibition

 

Table 5 presents data taken during the inhibition
experiments. EDTA and citrate, known as chelating agents,
did not cause significant inhibition. This led to the
assumption that there are not any activating metals at the
active side of this enzyme. On the other hand this enzyme
seemed to be sensitive to heavy metals, such as Ag+ and

2+ Although this sensitivity indicates participation

H9
of thiol groups (-SH) during the enzymic catalysis (20),
typical thiol specific reagents, such as iodoacetamide
and chloro-mercury-benzoate, were not tested to confirm
the presence of this thiol group related inhibition. The
addition of Ag+ and Hg2+ caused a considerable amount of

inhibition, even without incubation, and within 2 hours the

enzyme solutions were completely inactivated.

 

77

Table 5. Inhibitors of a-galactosidase from A. niger.a

 

 

Inhibitor Amount of inhibitor % Inhibition
(moles) in reaction
mixture
-4
EDTA 2.5x10 15
-5
Citrate 2.5x10 3O
-5
HgClZ 2.5x10 100 (without incubation)
-5
AgN03 2.45x10 100 (after 2 hrs incub)
-8
AgNO3 4.9x10 50 (without incubation)
Galactose 3.0x10.6 100
-5
Galactose 3.0x10 26

 

aReaction mixture: 1 mL of substrate solution (0.5 mM
PNPG), 0.25 mL enzyme solution (1 mg of crude a-galacto-
sidase preparation per mL of solution), 0.5 mL of
inhibitor solution.

78

Experiments with KI indicated that the addition of this
compound into enzyme solutions, which were inactivated by
Ag+ and Hg++ ions, regenerated their activity, to a degree
relative to the amount of KI in the reaction mixtures.

This compound added into enzyme solutions before the intro-
duction of the inhibitory metals protected the enzyme.
Ag+-inactivated enzyme solutions were not regenerated by

‘4 M.

the addition of KI at concentrations higher than 10
Table 6 presents data upon the regenerating effect of KI
at various concentrations.

Table 7 shows that adding 5.5xlO'5 moles of K1 into
the reaction mixtures resulted in 90—100% restoration of
the enzyme activity. Adding 5x10'5 moles of EDTA into
5 mL of enzyme solution inactivated by Ag+ or Hg++ resulted
in 75% regeneration of the enzyme. Treatment with citrate
was not effective at all. When cysteine was added to
similarly inactivated enzyme, testing the enzyme activity
immediately after the addition of cysteine did not indi-
cate any activity restoration. Fourty minutes 1ater,though,
90% of the original activity was restored, when Ag+ was
the inhibitor, and 80% in the case of Hg°+. The same
observation was made by Dey and Pridham (20).

Galactose caused considerable inhibition in amounts

above 10'6 moles. Adding 3.5x10~° moles of galactose in
the reaction system caused 100% inhibition. At concentra-

tions 3.0x10‘6 M galactose caused 86% inhibition. Addition

 

79

Table 6. The regenerating effect of KI upon a-galactoai-
dase from A. niger inactivated by Ag+ and Hg +.
% inhibition and relative amounts of K1 (moles)
in the reaction mixture.

 

 

KI (moles) None 2.5x1o‘5 5.0x10'5 7.5x1o‘5
Control 0 0 2 5
ch12 88 34 o 1
AgNO3 15 32 0 1

 

Reaction mixtures. Control: 1 mL substrate solution
0.5 mM PNPG), 0.25 mL enzyme solu-
tion (1 mg of crude a—galactosidase
preparation per mL of solution), 1 mL
of distilled water, or 1 mL KI
solution.

with HgCLZ: 1 mL substrate solution, 0.25
mL enzyme solution, 1 mL of HgC12
solution (2.5x10'7 moles of H9012),
1 mL KI solution.

with AgN03: 1 mL substrate solution, 0.25
mL enzyme solution, 1 mL AgNO3
solution (2.5x10-7 moles of AgNO3),
1 mL KI solution.

80

Table 7. Regeneration of a-galactoEidase from A. niger
inactivated by Ag+ and Hg + expressed as % of
control. Effect of KI, EDTA, citrate and

 

 

cysteine.
Inhibitor KI 5 EDTA Citrate Cysteine
2.5x10-7 5.5x10' 5.0x10‘5 5.0x1o-5 5.5x10-3
(moles) (moles) (moles) (moles) (moles)
AgN03 100 75 0 90
ch12 90 - - 80

 

Reaction mixture: 1 mL substrate (0.5 mM PNPG), 0.25 mL
solution, 1 mL inhibitor solution, 1 mL
KI, EDTA, citrate, or cysteine solution.

81

KI into the reaction system prior to the addition of
galactose did not prevent the inhibitory effects of
galactose. Dey and Pridham (20) reported that the addition
of small amounts of galactose into their system protected
the enzyme (a-galactosidase from Vicia £323) from inactiva-
tion by heavy metals. Similar experiments with a-galac-
tosidase from A. 31331 did not have the same results. This
might lead to the assumption that the action center of KI
is not located at the active site of the enzyme.

The regenerating effect of KI needs further study to
elucidate its action upon this and other enzymes. For
example KI restored 80% of the activity of B-galactosidase
(A. £1333) after a 100% inactivation by HgC12, when PNPG
was the substrate. It is also worth repeating this
experiment with oligosaccharides.

The action of K+ and I“ions separately upon the
enzyme molecule is worth investigating. K+ have been
previously reported to act as activators of a-galactosi-

dases (21).

a-Galactosidase from Coffee Beans

 

Optimum Reaction Temperature and Stability

 

Figure 24 shows that the a-galactosidase from coffee
beans demonstrated maximum activity at a temperature range
between 45°C and 50°C. At 60°C the enzyme retained only

80% of its maximum activity and at 70°C only 26% of it.

nmoles of p-nitrophenol/min/mL of substrate solution

 

82

 

IN
20 _
10 H
b L I >
25 40 o 60
Temperature (C )
Figure 24. Temperature-activity relationship of a-galacto-

sidase from coffee beans with PNPG as substrate,
pH 4.5. Reaction mixture: 10 mL acetate buffer,
0.5 mL enzyme solution (0.5 units/mL) 0.2 mL
substrate solution (10 mM PNPG).

 

83

The above data indicate that the a-galactosidase from

A. giggg is more thermostable than that from coffee beans.

0n the other hand though during a long incubation period at
45°C this enzyme showed increased activity. Table 8 presents
data of the enzyme activity, during incubation for 24 hours,
at 25°C and at 45°C.

The a-galactosidase from coffee beans demonstrated
higher storage stability than that from A, £1333, Solutions
of this enzyme in 0.01 M acetate buffer, pH 5.0, containing
1 unit/mL of solution demonstrated 95% of their original
activity, after two months in the freezer. This increased
stability of the enzyme from coffee beans could be contri-
buted to a higher degree of purity and concentration. The
solutions of the enzyme from A. niger had a concentration

of 0.06 units/mL.

Optimum Reaction pH

 

Figure 25 shows that the optimum pH for this enzyme is
at 5.0. Petek and ToDong isolated two a-galactosidases
from coffee beans, I and II, and determined their optima
pH at 5.3 and 6.0 respectively, using phenyl-a-D-galacto-
side as substrate. The pH-activity curve of the a-galacto-
sidase from coffee beans had a sharp peak at the optimum pH

level, in contrast to that from A. niger (Figure 12).

 

84

Table 8. Thermal stability of a-galactosidase from coffee
beans during incubation for 24 hours at 25°C and
45°C. A340 of a system containing 1 mL of
substrate solution (10 mM melibiose or raffinose
or stachyose) and 10 uL of enzyme solution (1
unit per mL of solution), after 6, 12, 18, 24
hours of reaction.

 

 

Incubation at 25°C Incubation at 45°C
Substrate Reaction time (hours) Reaction time (hours)
6 12 18 24 6 12 18 24

 

Melibiose 3.8 6.9 7.0 7.5 4.2 7.6 8.1 8.4
Raffinose 6.2 7.5 8.0 9.0 6.9 8.3 9.0 9.9
Stachyose 2.9 4.1 5.0 5.8 3.3 5.0 5.6 6.0

 

*A340 of NADH is a measure of galactose determined
enzymatically. The A3§0 values represent the original
absorbance readings mu tiplied by a dilultion factor of
10.02.

20_.

10

nmoles of p-nitrophenol/min/mL of substrate solution

 

2.5

Figure 25.

85

pH

pH-activity relationship for a-galactosidase
from coffee beans, with PNPG as substrate at
50°C. Reaction mixture: 10 mL acetate buffer,
0.5 mL enzyme solution (0.5 units/mL), 0.2 mL
substrate solution (10 mM PNPG).

 

86

Reaction Rates

 

Figures 26 and 27 represent graphically the hydrolysis
on PNPG and the oligosaccharides (melibiose, raffinose,
stachyose). When PNPG is the substrate, the hydrolysis
reaction plateaus after approximately 30 minutes of incu-
bation, under optimum conditions of temperature and pH.
When the oligosaccharides are the substrates, the reaction
of their hydrolysis levels off after approximately 24 hours
of incubation. Raffinose seems to be the substrate of
preference, while stachyose was hydrolyzed at the slowest
rate.

Phenomena of substrate inhibition were also observed
with this enzyme when PNPG at a concentration above 2.0 mM
was used in a reaction mixture containing 0.005 enzyme
units/mL. The inhibition was manifested in terms of slower
reaction rates. Similar inhibition phenomena were observed,
when melibiose was the substrate, at concentrations above

70 mM (0.02 enzyme units/mL).

Km and Vmax Values

 

Figures 28 through 35 show the Km and Vmax values
along with the Lineweaver-Burk double reciprocal and
Eadie-Hofstee reciprocal plots. The values of the kinetic
constants were determined as those for the o-galactosidase
from A. Aiggg. The substrate preference of this enzyme for

raffinose was indicated by its Vmax value for this substrate,

 

 

87

45

P

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”st covaommm .mcmwn wwmmoo 50;» ommc_mouom_mmuo xn enz; eo m_mxpocux:

.uaevoEAE
on em ca

 

J H —

 

.om oa=m_a

UOTQEIOS aseuzsqns jo qw/Iouaqdoaqtu-d jo seiowu

88

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a
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89

   
 

 

 

I
150 L
a-Sk.33
0 $20.61
KmtlbdxlOJ°H
5 _ Vu‘x81.8x10‘2c1n"
I J l \
0 1 3 5

Figure 28. Lineweaver-Burk double reciprocal plot of

Figure 29.

a-galactosidase from coffee beans for PNPG.
Reaction mixture: 1 mL substrate solution,
5 0L enzyme solution (1 unit/mL).

 
    
   

 

 

5 c

\ v:

—4 b

o \.

E: C?

o :—

g .-

Q. a

o r

L p-

a o

E If.

a 3 2° ’ a=1.85

G. g b=-09u60

° 2

a ,6 r'00995

£5 3 1o —

8 45 xm=4.6x10'°r.

B -2 -1

;?\ Vmax 1.8110 min

1 L \
0 .015 .030

v/s

Eadie-Hofstee reciprocal plot of coffee bean
o-galactosidase for PNPG. Reaction mixture:
1 mL substrate solution, 5 0L enzyme solution
(1 unit/mL).

 

90

A _2

    

 

 

33179.8 Km'1.1x10 H
b-206u.7 vmx-s.6x10'3n1n'1

2000-
P'0.97O

l/v
1000
/ l I L 1.
0 03 .6 09
1/8

Figure 30. Lineweaver-Burk double reciprocal plot of coffee
bean a-galactosidase for melibiose. Reaction
[mixture: 1 mL substrate solution, 5 0L enzyme
solution (1 unit/mL).

a-0.0oh
bI-6.6h
r-0.86O

.00“

   
  

Km-6.6310'3M
-3 -1
Vmax-“.0110 min

.002 -

of subrtate solution/min)

v(74moles of gnlactose/mL

 

 

l \
O .0003 .0006
v 8

Figure 31. Eadie-Hofstee reciprocal plot of coffee bean
a-galactosidase for melibiose. Reaction
mixture: 1 mL substrate solution, 5 0L enzyme
solution (1 unit/mL).

 

 

91

   
 

a-55.lu

MOO b'931.°00
l/v r'0.987
a -2

20° Km 1.7x10 H

-2 -l
Vmax=l.flxlo min

Figure 32. Lineweaver-Burk double reciprocal plot of
o-galactosidase of coffee beans for raffinose.
Reaction mixture: 1 mL substrate solution,
5 0L enzyme solution (1 unit/mL).

a-0.01567
b'-13.0#u
r-0.9“0

Km=1.3x10'zfl

-2 -l
Vmax'1'6xlo min

v(1zmoles of galactose/mL of
substrate solution/min)

 

 

 

0 .000 .0012

Figure 33. Eadie-Hofstee reciprocal plot of coffee bean
o-galactosidase for raffinose. Reaction
mixture: 1 mL substrate solution, 5 0L enzyme
solution (1 unit/mL).

 

 

 

92

6000 _
0-217.26
4000 b!9037.31
rco.972

l/v
xm-u.2x10‘zu

2000 -3 _,
I 1
Vmax 4.61.0 min

 

V

 

Figure 34. Lineweaver-Burk double reciprocal plot of coffee
-bean o-galactosidase for stachyose. Reaction
mixture: 1 mL substrate solution, 5 uL enzyme
solution (1 unit/mL).

 

  
  
    

. 380.00218
b=-1A,733
r=0.810

.0020

xm-1.5x10'2n
g -2 -1
Vmax 2.2110 min

 

T

' .0010

v(,.moles of galactose/mL of
substrate solution/min)

 

l

0 6 12 18
10-52V/S

Figure 35. Eadie-Hofstee reciprocal plot of coffee bean
a-galactosidase for stachyose. Reaction mixture:
1 mL substrate solution, 5 0L enzyme solution
(1 unit/mL).

 

93

which had the highest value among those for the three
oligosaccharides. The enzyme also demonstrated a substrate
preference for the synthetic compound PNPG. The Vmax
values for PNPG and raffinose were similar. The enzyme

affinities (l/Km) for these substrates varied (Table 9).

Inhibition

 

2+

Ag+ and Hg caused inhibition, when they were added

at low concentrations into solutions of a-galactosidase

from coffee beans. HgZ+

caused stronger inhibition than
Ag+ and the addition of K1 was less effective in restoring
the activity than in the case of o-galactosidase from

A. Aiggg. Addition of KI restored the coffee bean enzyme
activity by 90% when Ag+ was used as inhibitor, and to

85% when Hg+ was the inhibitor. Table 10 indicates some

inhibitors of the coffee bean o-galactosidase, their final

concentration and % inhibition.

a-Galactosidase from E. coli

 

Stability, Optimum Reaction Temperature, Reaction Rates

The o-galactosidase from E. 3911 was a very unstable
enzyme. Solutions in 0.01 M phosphate buffer, pH 7.0
containing 1 unit of enzyme per mL, kept under refrigeration
at 2-3°C lost in 24 hours 45% of the original activity. In
48 hours 70% of the activity was gone and in 72 hours 80%.

Due to this instability the absorbance readings during the

 

 

94

Table 9. Kinetic constants of a—galactosidase from coffee
beans for 4 substrates determined by the
Lineweaver-Burk (L-B) and Eadie-Hofstee (E-H)

 

 

 

methods.

Km (moles) Vmax (moles/mL of

Substrate substrate/min)
L-B E-H L-B E-H

PNPG 4.5x10'4 4.6x1o’4 1.8x10'2 1.8x10‘2
Melibiose 1.1x1o'2 6.6x10‘3 5.5x10'3 4.0x10‘3
Raffinose 1.7x10‘2 1.3x10"2 1.8x10"2 1.5x10‘2
Stachyose 4.2x10'2 1.5x1o‘2 4.5.110"3 2.2x10'3

 

Table 10. Inhibitors of a-galactosidase from coffee beans.

 

Amount of inhibitor in

 

Inhibitor the reaction mixture % Inhibition
(moles)
-8 .
AgNO3 5.0x10 85 (15 min)
AgNO3 5.0x10‘8 100 (60 min)
ch12 2.0x10‘8 95 (15 min)
EDTA 2.0x10'3 0
Galactose 1.2x10“2 95

 

Reaction mixture: 1 mL substrate solution (0.75 mM PNPG),
5 0L enzyme solution, inhibitor.

95

various spectrophotometric measurements from experiments
performed under identical conditions were different.

The addition of NADT and Mn+ and/or mercaptoethanol
neither improved the enzyme stability nor optimized its
activity. Enzyme solutions kept in the freezer for 2 1/2
months retained 90% of their initial activity. Figure 36
represents the relation between the temperature and the
enzyme activity, expressed as nmoles of liberated p-nitr04
phenol/min/mL reaction mixture. This a-galactosidase
demonstrated maximum activity at 45°C, during the hydrolysis
of PNPG. Between 45°C and 50°C the enzyme exhibited 90% to
100% of its maximum activity. At 60°C only 10% of its
activity remained, while the other two a-galactosidases,
from A. 11921 and coffee beans, retained 94% and 80% of
their activity, respectively.

Although incubation of PNPG with this enzyme at 45°C
for 10 min indicated a very high enzymatic activity, the
enzyme lost a substantial amount of its activity when it was
incubated at that temperature for longer periods. This
sensitivity of the enzyme was demonstrated by data taken
during prolonged incubation periods for the hydrolysis of
the oligosaccharides. Thermal stability experiments with
two identical mixtures, one with melibiose and another
with raffinose as substrates, were conducted at two
different temperatures, 45°C and 30°C. The results showed

that for melibiose the ratio of the reaction rates at 45°C

 

 

 

96

nmoles of p-nitrophenol/mL of reaction mixture/min

 

o L L 1 a
25 45 60

Temperature(C°)

Figure 36. Temperature-activity relationship of a—galactosi-
dase from E. coli, with PNPG as substrate, at
pH 6.5. Reaction mixture: 1 mL phosphate buffer,
100 0L substrate solution (5 mM PNPG), 5 0L
enzyme solution (1 unit of enzyme/mL solution).

 

97

to that at 25°C was 1:2 and for raffinose 1:4. Raffinose
was hydrolyzed more slowly than melibiose. The latter was
the substrate of preference for this enzyme. Stachyose

was hydrolyzed much more slowly, compared to the other

two oligosaccharides. This is clearly indicated by

Figure 39, where the hydrolysis rates of the three oligo-
saccharides were plotted vs. the reaction times. Raffinose
at concentrations above 80 mM demonstrated the same inhi-

bition phenomena, as melibiose did with the coffee beans

IL.

o-galactosidase (the enzyme concentration in the reaction
mixture was 0.005 units).

Substrate inhibition phenomena were observed with
PNPG as substrate at concentrations higher than 2 mM. The
rate of hydrolysis of PNPG plateaued after 30 minutes
(Figure 38) while that of melibiose after about 12 hours,
of raffinose after 18 hours, and of stachyose after about

10 hours (Figure 39).

Optimum Reaction pH

 

Figure 37 shows how the enzyme activity changes at
various pH levels. It is apparent, that at those pH levels,
where the previously examined enzymes demonstrated maximum
activity (pH between 4.5 and 5.0) this enzyme does not have
any activity at all.

Since phosphate buffers do not have good buffering

capacity at pH levels below 6.0, a series of acetate buffers

nmoles of p-nitrophenol/mL of reaction mixture/min

 

98

 

1 J J

 

Figure 37.

6.5 7.5 8.5
pH

pH-activity relationship of the a-galactosidase
from E. coli, under optimum temperature (45°C).
Reaction mixture: 1 mL phosphate buffer,

100 uL substrate solution (5 mM PNPG), 5 0L
enzyme solution (1 unit/mL).

99

was prepared to cover the pH range from 5.00 through 5.75
during the pH experiments.

The difference of ionic species in the buffers did not
affect the enzyme activity. At pH of 6.75 through 7.75 the
enzyme activity shows a sharp increase and then it decreases
slowly, as the pH of the reaction mixture becomes more
alkaline.

It is obvious that this enzyme favors slightly alkaline
media. The optimum pH is 7.0. At pH 8.5 the enzyme has
almost the same activity as at pH of 6.5. A change of the
pH by 0.5, in the acidic range, below the optimum level,
decreased the enzyme activity as much as a change of the pH
by 1.5 above the optimum level, in the alkaline range.

The behavior of the enzyme at the various pH levels is
compatible with the fact that it was derived from bacteria.
Most bacteria can not tolerate acidic environments. In

fact the optimum pH for the growth of E. coli is 7.0.

Km and Vmax Values

 

Table 11 shows that the kinetic constants of this
enzyme also change independently, between the four sub-
strates. The Vmax for melibiose had the highest value and
that of stachyose had the lowest. The Km values for these
substrates were similar. Figures 40 to 47 show the Line-
weaver-Burk and Eadie-Hofstee plots pertaining to the E. 3911

a-galactosidase.

 

.—-.--. ——.—-.

 

100

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euecumezm 4: cop .gewwae awesomegel45 p "ecsuwa :ewueeem .eczueceQEmu new :e
we mcewuwecee Ezswuee we .wpee .m segw mmeewmeueepemio we aaza we mwmx_ecexz

 

4:4350249
n: on we

.mm oezmwe

 

sansxtm 00130594 30 qu/[ouaququu-d jo setomu

 

 

 

 

 

101

.A45\uw=: pv cewuzpem e5x~ce 4: mm .Aeewceseeememer :5 owv cewuzpem
euegamebm 45 . "ogzuxw5 cowaoeem .ze ecu ocaaecee5mu we mcewuwecee 5:5wuae an
wwee .m 5ecw emeewmeueepemia 54 mmexcoeam .mmecwwwec .mmew4__e5 we mwmxpegex: .mm eezmwe

 

«oesvoeae _ .
e: on ea c. e

¢ d — 1 d

 

ihT11111311111511111115T .0

oceanonum

a Low”

sunqxtm notions; 30 qw/esoioeze? so sstowu

.1

oeondwwem . o o
ouodnaaoz.ll o.

 

 

 

102

 

 

 

Table 11. Kinetic constants of a-galactosidase from E. col'
determined by the Lineweaver-Burk (L-B) and
Eadie-Hofstee (E-H) methods.

Substrate Vmax (moles/mL of

substrate/min)
L-B E-H L-B E-H

PNPG 2.8x10'4 .6x10'4 3.3x10'3 3.2x10‘3

Melibiose 2.1x10‘2 .9x10'2 3.1x10‘2 3.0x10’2

Raffinose 1.2x1o‘2 .2x10"2 9.5x10‘3 9.5x10"3

Stachyose 2.1x10'2 .3x1o‘2 2.6x1o'3 1.9x10’3

 

 

 

 

103

 

 

a=2?°.9 A
*‘83'7 2000_
-34
Km52.8x10 H
-3 -l 1000
Vmax'3."x10 min -
/ J a l >
0 5 10 15

Figure 40.

.0032

v(f4moles of p-nitrophenol/mL
of substrate solution/min)

 

    
   

i/S

Lineweaver-Burk double reciprocal plot of
o-galactosidase from E. coli for PNPG. Reaction
mixture: 1 mL substrate solution, 25 at enzyme
solution (1 unit/mL).

e-0.00323
r80.99l

K.t2.6x10'°n

Vn‘x'3.2810'3lin'1

 

.0016 -
O . 00“ . 008 .012 >
v/s
Figure 41. Eadie-Hofstee reciprocal plot of a-galactosidase

from E. coli for PNPG. Reaction mix: 1 mL
substrate solution, 25 0L enzyme solution
(1 unit/mL).

 

 

Figure 42.

v(/4molee of galactose/mL

Figure 43.

of substrate solution/mln)

104

7

 

uoo _ a-31.93
1/1 b8667.1
r-0.996
2°° ’ Km~2.1x10'zx
Vm‘x‘3.1x10'°min'1

l 1 $
0 0.2 0.4
1/5

Lineweaver-Burk double reciprocal plot of
o-galactosidase from E. coli for melibiose.
Reaction mixture: 1 mL substrate solution,
25 0L enzyme solution (1 unit/mL).

e20.0298
b3-18.9
r'o.984

0.030

   
  

Km=1.9x10'2M

Vinax33.0110-2511:.1
0.015 -

 

0 .0005 .0010 .0020
v/S

Eadie-Hofstee reciprocal plot of o-galactosidase
from E. coli for melibiose. Reaction mixture:

1 mL substrate solution, 25 uL enzyme solution
(1 unit/mL).

1.

105

 

 

»\

60° - .=105.67

b.1213.92

1" r-0.999

-2
V3.189.5110 -nin

o . .4 6"
1/s

Figure 44. Lineweaver-Burk double reciproca] plot of
a-gaiactosidase from E. coli, for raffinose.
Reaction mixture: 1 mL substrate soiution,
25 uL enzyme soiution (i unit/mL).

 

   
 
 

 

 

?
-2 '5
s \t
\ I:
o o
a an
O u
3 £3 A
'3 3 380.009“?
:, u «015: bn-anz
0.. 3 P30 98?
o x. ‘
fl ‘0'; 2
2 3 .010 . “fl-”1°- ”3
a Vm‘3'9.5110 min
:\3
>
o .0004 .0008 ;,

v/s

Figure 45. Eadie-Hofstee reciprocal piot of o-gaiactosidase
from E. coii for raffinose. Reaction mixture:

1 mL Eubstrate soiution, 25 uL enzyme soiution
(1 unit/mL).

 

6000 »

l/V

3000

 

106

02386.15
b-7751.8

r-0.995

x -2.1x1o'2n

VII -2 6x10'3nin'1
an: ‘

   
 

 

1A 1 1‘
0 h .8 "
us
Figure 46. Lineweaver-Burk doub1e reciproca1 p1ot of

.002

.001

moles of galactose/nL
of substrate solution/min)

V(

Figure 47.

 

 

a-ga1actosidase from g. coli for stachyose.
Reaction mixture: 1 mL 0? substrate so1ution,
25 uL enzyme so1ution (1 unit/mL).

310.00186
bs-12.508
r-0.878

. -2
Km 1.3x10 H

Vmax-1.9x10'3m1n-1

\
I

Eadie-Hofstee reciproca1 p1ot of a-ga1actosidase
from g. co1i for stachyose. Reaction mixture:

1 mL substrate so1ution, 25 uL enzyme so1ution
(1 unit/mL).

 

 

107

On some Eadie-Hofstee p1ots, for the three enzymes,

particu1ar1y those of stachyose, a curvature can be noticed

between the points. This cou1d be due to the presence of

more than one a-ga1actosidases, in the enzymic preparations.

Inhibition

 

Few experiments were conducted, to study the inhibitory
action of various compounds on the activity of the
a—ga1actosidase from E. coli, using PNPG as substrate
(Tab1e 12). The addition of K1 into enzyme so1utions,
inactivated by Ag+ and Hg++ did not restore the activity.

On the contrary, KI added to active enzyme soiutions caused
inhibition (Tab1e 12).

The behavior of the a-ga1actosidase from g. 3311 during
the addition of K1, a1ong with its Optimum pH represent some
of the major differences between this enzyme and those from

A. niger and coffee beans.

 

 

 

108

Tab1e 12. Inhibitors of a-ga1actosidase from g. co1i, their
amounts in the reaction mixtures (mo1esi and
caused inhibition, expressed as % of contr01

 

 

activity.
Inhibitors Amount (mo1es) % Inhibition
KI 2.5x10"6 98
ch12 1.0x10‘7 100
AgNO3 2.5x10'6 100
EDTA 2.5x10‘5 0
Ga1actose 1.5x10'6 95

 

Reaction mix:

1 mL phosphate buffer, 50 uL substrate
so1ution (5 mM PNPG), 0.25 mL enzyme
so1ution (1 unit/mL), inhibitor.

CONCLUSIONS

The a-ga1actosidases from A, giggr_and coffee beans
demonstrated a resemb1ance in their behavior, when their
activity was tested at various pH 1eve1s and temperatures.

They both seemed to be quite thermostabTe and t01erant
to acidic environments. Their kinetic constants indicated
that both hydronzed raffinose at a faster rate than the
other two o1igosaccharides, under optima1 conditions of
temperature and pH. Stachyose was hydronzed faster by the
a-ga1actosidase from coffee beans than the enzyme of the
other sources.

Stabi1ity tests showed that so1utions of both enzymes
in buffers, with proper pH, maintained a substantia1 amount
of their activity, when they were kept under refrigeration
and even more under freezing.

Inhibition studies indicated that both enzymes are
inhibited by heavy meta1s. Their activity was restored by
the addition of proper amounts of KI into the reaction
mixtures. Due to their stabi1ity and hydro1ytic characteris-
tics, these a-ga1actosidases cou1d be successfu11y emp1oyed
in various commercia1 app1ications.

The a-ga1actosidase from g. 3311 showed different

behavior from that of the other two enzymes. This enzyme

109

 

 

110

was very unstab1e (particu1ar1y in so1utions). It was more
active in s1ight1y a1ka1ine environments. During the
kinetic studies of this a-ga1actosidase a strong substrate
preference for me1ibiose was observed. ‘The hydro1ysis rate
of stachyose was very s1ow, compared to the other two
a-ga1actosidases. When this enzyme was inactivated by heavy
metaIS, KI did not restore its activity. The curvature
noticed in some of the Eadie-Hofstee p1ots may indicate the

simu1taneous action of two enzymes on the substrate.

The enzymatic characteristics of the a-ga1actosidases
from A. niger, coffee beans and g. c01i are summarized on
Tab1es 13, 14 and 15. The indicated Km and Vmax va1ues are

those determined by the Lineweaver-Burk method.

 

111

Tab1e 13. Summary of the optima temperatures, pH of
a-ga1actosidases from A. niger, coffee beans
and E. c01i.

 

 

Enzyme Opt. Temp. (00) Opt. pH
A. niger 52 5.0
Coffee beans 50 5.0
E. c01i 45 7.0

 

 

Tab1e 14. Summary of the Km va1ues of a-ga1actosidases
from A. niger, coffee beans and E. c01i (M).

 

 

Enzyme PNPG Me1ibiose Raffinose Stachyose
. -4 —3 -2 -2
A. niger 6.7x10 6.2x1O 3.8x10 2.9x1O
Coffee beans 4.5xio‘4 1.1xio'2 1.7xio‘2 4.2xio‘2
g. c01i 2.8xio‘4 2.1xio'2 1.2xio'2 2.1xio'2

 

 

 

 

112

Tab1e 15. Summary of the Vmax va1ues of a-ga1actosidases
from A. niger, coffee beans and E. c01i, in

 

 

min-1.
Enzyme PNPG Me1ibiose Raffinose Stachyose
A. giggg 3.1x1O'2 1.6x1O‘2 5.2x10"2 1.4x10'2
Coffee beans 1.8xio‘2 5.6x10'3 1.8x10'2 4.6x10'3
g. c01i 3.3xio‘3 3.1xio’2 9.5xio‘3 2.6xio‘3

 

 

 

 

 

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10.

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