Chemical Composition and Characteristics of
Alpha-amylase Inhibitor
By
A. Y. Gebriel;** A. A. Amin,* M. F. Madkour, ** H. A. El Banna * and K. F.
Mahmoud *
* Dept. of Food Sci. and Technol., National Research Center, Dokki, Giza, Egypt.
** Dept. of Food Sci., Faculty of Agric. Ain Shams University, Shoubra El-Kheima,
Cairo, Egypt.
ABSTRACT
In this study chemical constituents and characteristics of alpha amylase inhibitor extracted
from kidney bean Giza 133 and wheat Giza 164 were studied. Results indicated that the
purified alpha amylase inhibitors of kidney bean Giza 133 contained more total carbohydrate
content (14.4 %) than wheat Giza 164 (1.2 %), in addition to the differences in amino acid
content for both samples.
The alpha amylase inhibitor characteristics indicated that the molecular weight of kidney
bean Giza 133 (47.000 Da) was higher than both wheat Giza 164 (24.000 Da) and human
salivary alpha-amylase (32.000 Da) and lower than enzyme-inhibitor complex (61.000 Da).
The low activity of both human and pancreatic alpha amylase was achieved using low
concentration of kidney bean inhibitor (1.56 and 1.95 UI) compared with higher concentration
(4.69 and 5.47 UI) of wheat Giza 164.
The addition of salt (NaCl and bromide) in the purification step increased the inhibitor
activity up to 80, 91 %, and the inhibitor reaction rate reached maximum at ratio 1 : 1
(Inhibitor (I) : Enzyme (E)). Results also indicated that the low dissociation constant of
kidney bean inhibitor gave stability to the I-E complex at pH 6.9.
Increasing the pH values prevent further inhibition and the reaction is irreversible, while
raising the temperature up to 37 oC reverse the inhibition reaction to its primary rate.
Key Words: Alpha-amylase inhibitor; Wheat; Legumes.
INTRODUCTION
During the past 10 years considerable progress has been made in the
physicochemical properties, nutritional and physiological role of plant alphaamylase and its protein inhibitors. Most of these inhibitors are inactive toward plant
alpha-amylases references suggesting their physiological role is not to regulate
alpha-amylase levels in the plant, but controlling starch metabolism (Octavio and
Rigden, 2002). Purified alpha-amylase inhibitors are glycoproteins and active
against human pancreatic, salivary and insect alpha-amylase. They are inactive
against bacterial, mold and plant alpha-amylases (Frels and Rupnow, 1984 and
Santimone et al. 2004). Alpha amylase inhibitor have similar amino acid
compositions, molecular weight (49,000 Daltons) and contained two or more
subunits, with molecular weights ranging from 20,000 to 60,000 daltons and
emerging similarities in N-terminal amino acid residues and partial amino acid
sequences.
The amount of inhibitor extracted from whole wheat bread or barley was from 60
to 69 % lower than the corresponding meal.
The presence of carbohydrate as apart of the inhibitor molecule has been
demonstrated in beans but not in wheat (Saunders and Lang, 1973).
The carbohydrate content of alpha-amylase inhibitor and its nature play a specific
role in the inhibition mechanism of the enzyme. (Power and Whitaker 1977,
Yoshima et al., 1980 and Sharma and Pattabirman, 1982). However, the
complete absence of carbohydrate in wheat amylase inhibitors (Silano et al., 1973)
and the low carbohydrate content (10 %) of legume inhibitors (Power and
Whitaker 1977) decreases the possibility of inhibition process in cereals and
legumes.
The stoichiometry interaction between the alpha-amylase and wheat inhibitor
showed a binding complex of 1:1 enzyme : inhibitor. The purified alpha-amylase
inhibitor from wheat or legumes presents great potential for use in phaseulus genetic
improvement programs Octavio et al. (2005).
All characteristics indicated that the inhibitors act as highly specific substrates for
the enzyme, and inhibit at a unique peptide bond called the reactive site peptide
bond. Although the reaction inhibition mechanisms are not clearly understood, yet
reducing sugars, which are covalently bound to the inhibitor polypeptides chain,
may play a major role in it, or the inhibitor may induce conformational changes in
the enzyme molecule (Heidari, et al., 2005).
Therefore the present work was aimed to study the chemical composition of
alpha-amylase inhibitor extracted from wheat grains (Giza 164) and Kidney bean
(Giza 133), In addition the inhibitors characteristics and enzyme inhibitor
mechanism were also studied.
MATERIAL AND METHODS
Materials:
Alpha amylase inhibitors [extracted from wheat Giza 164 and kidney bean
Giza 133] and purified according to Mahmoud, 2006 were used.
Human pancreatic and salivary alpha-amylase were obtained from Sigma
Company, England.
Methods:
Chemical composition of the purified alpha-amylase inhibitor:
Carbohydrate composition:
The carbohydrate content of the amylase inhibitor was determined either in the
whole inhibitor by using the phenol-sulfuric acid reaction of Dubois et al. (1956)
or after separation of the carbohydrate moiety, as follows: 2 mg of the inhibitor
was hydrolyzed with 2 N HCl for 10 h at 100 oC in a closed Pyrex tube under
nitrogen. The solution was then passed through 2 ml Bio-Rad AG1-X-2 column,
and the free sugars were eluted with 70 % ethanol. To separate neutral sugars
from amino sugars, a Bio-Rad AG50W-X2 column was used and sugars obtained
were determined according to Krystal and Graham (1976). The identification of
individual carbohydrates was accomplished on whatman paper chromatography (1
MM) (Power and Whitaker, 1977).
Amino acids analysis of alpha-amylase inhibitor:
Amino acids analysis was performed in a Beckman 120C analyzer according to
Spackman et al. (1958). After hydrolysis with 6 N HCL in closed tubes, under
nitrogen, at 110 C, and for 72 h. Cysteine and methionine were determined
respectively as cysteic acid and methionine sulfone by oxidation with performic
acid (Moore, 1963). Tryptophan was determined according to Beaven and
Holiday (1952). For amino sugar determination, the inhibitor was hydrolyzed
with 4 N HCl at 110 oC for 4 and 6 hr (Spiro, 1973). Terminal amino acids were
identified basically as described by Gray, (1972).
Electrophoresis in polyacrylamide gel rods were run either in alkaline pH
according to Davis, (1964) or in acid media as proposed by Reisfield et al.
(1963), and in neutral solution by Weber and Osborn (1969).
Protein bands were visualized by staining with coomassie brilliant or by the
reaction with fucsine to demonstrate glycoproteins. The cross-linking reaction
with dimethyl suberimidate was performed as described by Davis and Stark
(1970). Dissociation of the inhibitor was studied by using different media: SDS
(1%), urea (6M), guanidine (6M), and β-mercaptoethanol (1%) in 0.1 M
phosphate buffer, pH 6.9, according to Tanford (1968). The elimination of the
reagents after the dissociation was done by dialysis against deionized water for 24
hr followed by dialysis against 0.1M phosphate buffer, pH 6.9; SDS was removed
from the media by using an ion-exchange (AGI-X8) resin (Lenard, 1971).
Determination of amylase activity:
Alpha-amylase activity was determined according to the method described by
Bernfeld (1955).
Alpha-amylase inhibitor assay:
The alpha-amylase inhibitor activity was determined using dinitrosalicylic
acid method described from Bernfeld, (1955) and modified by Ishimoto and
Chrispeels (1996).
One unit of inhibitor activity is defined as the amount which causes
approximately 50 % inhibition of α-amylase activity under the assay conditions.
2
Some characteristics of the purified alpha-amylase inhibitor:
Molecular weight of alpha-amylase inhibitor:
The molecular weight of the inhibitor was estimated by SDS-PAGE using the
method of Laemmli (1970).
Purified inhibitor, human salivary and pancreatic alpha-amylase activities:
The purified alpha amylase inhibitor and its effect on human salivary and
pancreatic alpha amylase activities was determined according to Hoover and
Sosulski (1984).
Alpha-amylase inhibitor activity at different salts:
The effect of salts i.e. (NaCl, KCl, Br- and I2 ) on alpha amylase inhibitor
incubated for 30 min at 30 oC with sodium phosphate buffer (0.02M, pH 6.9) was
determined according to the methods described by O'Donnell and McGeeney
(1976).
Starch hydrolysis by alpha-amylase in present of its inhibitor:
Human pancreatic alpha amylase was determined in three digests according to
Ceska et al. (1969).
Isoelectric point of alpha-amylase inhibitor:
Isoelectric point of the purified inhibitor was determined using polyacrylamide
gel electrophoresis (pH 4 to 6) according to the method described previously, by
Wrigley (1971).
Stoichiometry of inhibition:
Amylase inhibitor (4.5 unit inhibitor/mg protein) was incubated with human
alpha-amylase (4.1 unit/mg protein) in sodium phosphate buffer (0.02 M, pH
6.9) containing NaCl (0.05 M) at 30 oC. A control experiment containing only
alpha-amylase was incubated under identical condition. Aliquots containing 0.6
ml were removed at interval up to 10 h and transferred to tubes containing 0.35
ml of starch as a substrate (8 mg / ml). Alpha-amylase activity was determined
according to the usual assay procedure (Jane and John, 1985), and then the
procedure was repeated as follows:
1- Inhibitor + substrate. And added enzyme.
2- Inhibitor + enzyme. And added substrate.
3- Substrate + enzyme. And added inhibitor.
Dissociation constant:
Dissociation constant of alpha amylase and its inhibitor was measured by using
the method described by Bieth (1974).
Reversibility of inhibition:
The effect of high pH and low temperature on alpha amylase inhibitor was
determined according to John and Carmen (1975).
Specificity of alpha-amylase inhibitor:
The purified inhibitor was tested for its ability to inhibit alpha-amylase from a
number of sources i.e. plant, animal, fungal, and bacterial according to Franco
and Flavio (1985).
RESULTS AND DISCUSSION
Chemical analysis of alpha-amylase inhibitor from wheat flour Giza 164 and
kidney bean Giza 133.
1- Carbohydrate composition:
Carbohydrate composition of alpha-amylase inhibitor for both wheat flour and
kidney bean is shown in table (1).
3
Table (1): Carbohydrate composition of alpha-amylase inhibitor from both
wheat flour and kidney bean.
Sources of
alphaamylase
inhibitor
Total
carbohydrate
%
Sugars % after hydrolysis by phenolsulfuric acid reagent (H2SO4 0.1 N)
Neutral sugar %
Amino sugar %
Mannose
0.3
---1.2
Galactose
0.9
---Mannose
1
---Kidney bean
Xylose
2.4
---14.4
Giza 133
Galactose
8.0
----------Glucosamine
3
Results in table (1) showed that the glycoprotein nature of the purified kidney
bean Giza 133 inhibitor contained more total carbohydrate content than that in
wheat flour Giza 164 inhibitor being 14.4 and 1.2 % respectively, and that it
contained about 11.4 % neutral sugar (i.e. mannose, xylose and galactose) with 3
% amino sugar i.e. (glucosamine), while wheat flour inhibitor contained only 1.2
% neutral sugar mainly mannose and galactose and with no amino sugar content.
Wheat flour
Giza 164
2- Amino acid analysis of alpha-amylase inhibitor:
Amino acid composition of the wheat flour Giza 164 and kidney bean Giza 133
are shown in table (2). Values are given as mol of residue per mol of protein after
24 hr hydrolysis in 6 N HCl at 100 oC.
Table (2): Amino acid analysis of alpha-amylase inhibitor from
both wheat flour and kidney bean (per mol of protein).
Amino acid
Asparagines
Thereonine
Serine
Glutamine
Proline
Glycine
Alanine
Cysteine
Half-Cystine
Valine
Methionine
Inhibitor Analysis
(mol %)
Wheat
Kidney
flour
bean
Giza 164 Giza 133
15.5
77.1
5.7
32.4
14.7
52.2
23.6
36.1
15.3
13.5
22.4
20.3
29.1
23.6
16.8
1
8.8
--19.1
36.0
4.6
2.3
Amino acid
Iso-Leucine
Leucine
Tyrosine
Phenylalanine
Histidine
Lysine
Arginine
Trptophane
Glucosamine
C.M.Cysteine
Inhibitor Analysis
(mol %)
Wheat
Kidney
flour
bean
Giza 164
Giza 133
4.9
18.2
19.4
20.1
9.1
16.4
3.8
23.1
2.2
15.0
7.1
2.0
12.0
13.3
3.1
2.0
--2.1
18.5
---
Results in table (2) showed that there were differences in amino acid content
for both wheat flour Giza 164 and kidney bean Giza 133 inhibitors. Kidney bean
had higher contents of asparatic acid, serine, glutamic acid and thereonine being
77.1, 52.2, 36.1 and 32.4 mol %, respectively. Wheat flour had higher content of
Alanine (29.1 %), glutamic acid (23.6 %) and glycine (22.4 %). Results also
indicated that wheat flour Giza 164 inhibitor had 8.8 % half-cystine which is
slightly less than that found in malted barley inhibitor being 9 % (Randall et al.,
1983).
The amino acids liberated with time as C-terminal i.e. leucine and tyrosine and
third C-terminal amino acid i.e. serine, alanine and asparatic acid is given in figure
(1).
4
Moles Librated
25
20
15
10
5
0
0
10
20
30
40
50
60
70
80
90
100
Min
Leu
Tyr
Ala
Ser
Asp
Val
Figure (1): Identification of liberated amino acid.
2- Characterization of the purified alpha-amylase inhibitor
Molecular weight estimation of alpha-amylase inhibitor
Molecular weight of the two purified wheat flour Giza 164 and kidney bean
Giza 133 inhibitors were estimated and the results are shown in figures (2 and 3).
Ribo-Nuclease
0.5
Alpha-Chymo Trypsinogen
0.4
Inhibitor
24.000 Da
Bovine Serum Albumin
4.
5
4.
4
4.
3
4.
2
0
4
Ov-Albumin
5
0.1
Beta-Lactoglobulin
4.
9
0.2
Complex
41.000 Da
Alpha-amylase
32.000 Da
4.
8
0.3
4.
7
0.6
4.
6
Kda (Kilo Dalton)
0.7
Fraction num ber
Com plex
Alpha-am ylase
Inhibitor
Figure (2): Molecular weight of purified wheat flour inhibitor, human
salivary alpha-amylase and alpha-amylase-inhibitor complex.
Results in figure (2) indicated that the molecular weight of wheat flour Giza 164
inhibitor was approximately 24,000 Da, which is in agreement with that found by
Randall et al. (1983) suggesting that the inhibitor is not a dimer. Results also
showed that the molecular weight of human salivary alpha-amylase was 32,000 Da
which is low compared to the value reported by Greenwood, (1968) being 45,000
Da.
Results also showed that gel filtration of the inhibitor with human salivary alphaamylase at pH 8.0 yielded a new peak having a molecular weight of 41,000 Da,
presumably, due to the formation of enzyme-inhibitor complex. These results are in
agreement with that demonstrated by Weselake et al. (1983).
5
RiboNuclease
0.6
0.5
Alpha-Chymo Trypsinogen
0.4
Complex
61.0000
Da
0.3
Alpha-amylase
32.000 Da
Inhibitor
47.000 Da
0.2
Beta-Lactoglobulin
5
4.
9
4.
8
4.
5
4.
4
4.
3
4
4.
2
0
4.
7
Ov-Albumin
Bovine Serum Albumin
0.1
4.
6
Kda (Kilo Dalton)
0.7
Fraction num ber
Com plex
Alpha-am ylase
Inhibitor
Figure (3): Molecular weight of purified kidney bean inhibitor, human salivary
alpha-amylase and alpha-amylase-inhibitor complex.
Results in figure (3) indicated that the molecular weight of the purified kidney
bean Giza 133 inhibitor, human salivary alpha-amylase and enzyme-inhibitor
complex was 47,000, 32,000 and 61,000 Da, respectively.
Alpha-amylase Activity %
Effect of the purified inhibitor on human salivary and pancreatic alphaamylase activity.
The effect of the purified wheat Giza 164 inhibitor on human salivary (HSA)
and pancreatic alpha-amylase (HPA) is shown in figure (4). The results indicated
that the relative ratio of inhibition was 1.0 : 0.5, and its magnitude was linear
against inhibitor concentration. Beyond this point the inhibition was progressive
but non linear, reaching nearly 100 % inhibition at high concentrations. No
activity of both human salivary and pancreatic alpha-amylase was achieved when
inhibitor concentration was 6 and 7 μg of protein, respectively.
100
90
80
70
60
50
40
30
20
10
0
0
1
2
3
HSA
4
5
6
7
8
9
10
Inhibitor Concentration (μg)
HPA
Figure (4): The purified wheat Giza 164 inhibitor and the activity of HSA
& HPA.
HSA: Human Salivary Amylase -- HPA: Human pancreatic Amylase
HSA
10
9
8
7
6
5
4
3
2
1
100
90
80
70
60
50
40
30
20
10
0
0
Alpha-amylase Activity %
The same experiment was repeated using the purified kidney bean Giza 133
inhibitor on both enzymes figure (5).
Inhibitor Concentration (μg)
HPA
Figure (5): The purified kidney bean Giza 133 inhibitor and the activity
of HSA & HPA.
6
The results showed that the low activity of both human salivary and pancreatic
alpha-amylase was achieved when inhibitor concentration was 2.0 and 2.5 μg of
protein, respectively.
So, the purified inhibitor from kidney bean Giza 133 was chosen to study its
characteristics and properties because: Firstly, the amount of the inhibitor used is
small and capable to inhibit the activity of both human salivary and pancreatic
alpha-amylase comparing with the amount used from the purified wheat Giza 164.
Secondly, wheat flour Giza 164 inhibitor was not heat stable and heat treatment at
50 and 70 oC for 10 min caused 50 and 100 % loss in its activity, respectively.
While, storage at 4 oC for 2 weeks caused also 50 % loss in activity, and also
below pH 4.0 the loss reached 60 %. The inhibitor was maximally stable at pH 6.9
(Mahmoud, 2006).
100
90
80
70
60
50
40
30
20
10
0
HSAI
HPAI
% Inhibition
c
50
40
30
20
10
0
100
90
HPAI
d
80
70
60
50
40
30
20
Br¯ (mg)
HSAI
0.9
0.7
0.5
0.1
0.9
0.7
0.5
0.3
0.1
10
0
0.3
% Inhibition
100
90
80
70
60
0.9
KCl (mg)
NaCl (mg)
HSAI
0.7
0.5
b
0.1
0.9
0.7
0.5
0.3
a
0.3
% Inhibition
100
90
80
70
60
50
40
30
20
10
0
0.1
% Inhibition
Effect of salts on alpha-amylase inhibitor activity:
It was found that the purified alpha-amylase inhibitor lost its activity after
dialysis by the early purification steps. Such activity could be restored by adding
small amounts of NaCl to the media.
The ionic strength of the pre-incubation buffer had a profound effect on the
inhibitory activities of alpha-amylase inhibitor (figure 6).
I¯ (mg)
HPAI
HSAI
HPAI
Figure (6): Salts ionic strength, inhibitor of salivary and pancreatic
alpha-amylase activity.
Results in figure (6) showed that the activities of inhibitor at 0.9 ionic strength
were 25 % more than 0.1 and were in agreement with the activities reported by
Jane and John (1985). The same results were also observed with Bromide.
These results suggested that an increase in salt ionic strength might facilitate
inhibition by shielding charge groups on the enzyme or inhibitor, which could
cause a conformational change or an alkaline shift at the optimum pH. Although
7
O`Donnell and McGreeney (1976) reported that increasing phosphate buffer
concentrations from (0.1 to 50 mM) did not have any effect on the degree of
inhibition of salivary alpha-amylase by inhibitor isolated from wheat, while the
addition of NaCl (1 mg) to phosphate buffer (0.1 mM), increased the inhibitor
activity from 68 to 85 %.
The results also showed that addition of KCl and iodine up to 1.0 mg have no
effect on the inhibition of human salivary and pancreatic alpha-amylase by the
inhibitor.
iodine stain
(% of original value)
Starch hydrolysis by alpha amylase in presence of its inhibitor:
The effect of starch on the interaction between kidney bean Giza 133 inhibitor
and human pancreatic alpha-amylase was determined. Figure (7) showed that
whether inhibitor can interact with alpha-amylase in the presence of substrate
(starch), or substrate can dissociate the enzyme-inhibitor complex.
Results in figure (7) showed that there is a noticeable decrease in iodine
staining power (digest b) after 10min incubation indicating that starch can interact
with inhibitor–enzyme complex causing reduction in its amount.
120
100
a
80
60
b
40
c
20
0
0
10
20
30
40
50
60
Time of Incubation (min)
Figure (7): Degradation of starch by human pancreatic alpha-amylase.
Digest (a): starch solution added after incubation at 37 oC for 30 min.
Digest (b): addition of starch to enzyme-inhibitor mixture.
Digest (c): Addition of inhibitor to enzyme-substrate mixture.
Above results showed that either formation of Enzyme-inhibitor or enzymesubstrate and addition of inhibitor caused a reduction in the hydrolysis action of
enzyme.
Such effect could be attributed to the formation of enzyme-inhibitor-substrate and
enzyme-substrate-inhibitor by the some rate and gave the same results.
Isoelectric point of alpha-amylase inhibitor:
The isoelectric point of kidney bean alpha-amylase inhibitor obtained by using
polyacrylamide gel electrophoresis is shown in figure (8).
Solubility %
100
80
60
40
20
0
3.5
4
4.5
5
5.5
6
pH value
Figure (8): Isoelectric point of alpha-amylase inhibitor
Results showed that the isoelectric point of kidney bean alpha-amylase
inhibitor was at 4.5, similar to that obtained by Power and Whitaker (1977)
being 4.65. The inhibitor isolated from wheat had higher isoelectric points (around
6.7) and curiously also had higher pH optimum, reaching values in the alkaline
range which is in agreement with that reported by Saunders and Lang (1973).
8
Stoichiometry of inhibition:
The stoichiometry of inhibition alpha-amylase interaction was investigated
during a different period of pre-incubation i.e. 0 to 60 min, and the maximum
extent of inhibition was given in figure (9).
INHIBITION (%)
100
80
60
40
20
0
0
5
10
15
20
25
30
35
40
45
50
55
60
Duration of Preincubation (m in)
Figure (9): The determination of the stoichiometry of inhibitor-human
salivary alpha-amylase.
Results showed that the maximum extent of inhibition 93 % was observed
when the 0.40 μg of inhibitor (MW 47.000 daltons) and at 60 min pre-incubation
was added to 0.34 μg alpha-amylase (MW 32.000 daltons), suggesting that the
inhibition reaction was approximately at ratio 1 : 1. These results are in agreement
with that reported by John and Carmen (1975) and Makoto et al. (1987).
Dissociation constant:
The dissociation constant for kidney bean alpha-amylase inhibitor against
pancreatic alpha-amylase is shown in figure (10).
60
(1-a) (10-9)
I0
-----------------------
50
40
30
20
10
0
0
2
4
6
8
10
12
14
1
----a
16
Figure (10): Dissociation constant of the complex of human pancreatic
alpha-amylase and the inhibitor from kidney bean.
Where: (I) the initial inhibitor concentration
(a) the activity of free enzyme without inhibitor
Results in figure (10) showed that the dissociation constant of complex kidney
bean Giza 133 inhibitor with pancreatic alpha-amylase was 2.4 X 10-9 M, higher
from that reported by Power and Whitaker (1977) being 3.5 X 10-11 M for red
kidney bean indicating that the lower the dissociation constant is the stable the
inhibitor-enzyme complex forming is at pH 6.9.
Reversibility of inhibition:
Attempts were made to determine whether the conditions found to be
unfavorable for inhibition, namely high pH and low temperature, will reverse
inhibition, which has already taken place.
Effect of pH
The activities of alpha-amylase comparing with those in appropriate control
digests from which the alpha-amylase inhibitor was omitted.
Results indicated that pre-incubation at pH 5.5 followed by pre-incubation at
pH 8.2 gave alpha-amylase activity identical to each other, but lower than that
after pre-incubation at pH 6.9. Thus, elevation the pH prevents further
inhibition, and doesn't reverse inhibition, which has already occurred.
9
Inhibition %
Effect of low temperature
The effect of low temperature on alpha-amylase inhibitor activity at pH 6.9 and
after pre-incubation of an identical digest under the same conditions for the same
length of time, followed by 45 min at 0 oC, is shown in figure (11).
100
80
60
40
20
0
0
15
30
45
60
75
90
Time (min)
Reversible
Figure (11): Effect of low temperature at (0 oC) on reversibility of
human salivary alpha-amylase inhibition.
Results showed that at low temperature (0oC) the inhibition reduced with time
up to 45 min (being 51 %), followed by stability stage up to 90 min. Raising the
temperature up to 37oC reversed the inhibition reaction to its primary rate.
Specificity of kidney bean alpha-amylase inhibitor
The purified kidney bean inhibitor was tested for its ability to inhibit alpha
amylase from different sources i.e. plant, animal, fungal and bacteria, and the
results are given in table (3).
Table (3): Specificity of kidney bean Giza 133 alpha-amylase inhibitor
against different sources of alpha-amylase.
Sources of alpha-amylase
Extent of
inhibition %
Animal
95
 Human salivary
100
 Human pancreatic
97
 Hog pancreatic
Bacteria
5
 Bacillus subtilis
0
 Bacillus licheniformis
Fungus
3
 Aspergillus oryzae
Plant
0
 Wheat
0
 Kidney bean
0
 Barley
Results in table (3) showed a complete inhibition of human pancreatic alphaamylase and reduction to 95 and 97 % for human salivary and hog pancreatic
alpha-amylase, respectively. The inhibitor was also active on bacteria and fungus
i.e. Bacillus subtilis (5 %) and Aspergillus oryzae (3 %) but not on plant i.e.
Wheat, Kidney bean and Barley. These results are in agreement with that reported
by John and Carmen (1975), Fronco and Flavio (1985) and Ramanaoppa and
Thillaisthanam (1985).
Conclusion:
It could be concluded that kidney bean inhibitor reaction rate reached
maximum at 93 % when 0.4 µg inhibitor at 60 min pre-incubation was added to
0.34 µg alpha-amylase indicating that the inhibitor reaction was at ratio 1 :1.
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