Journal of Microbiology and Biotechnology Research
Scholars Research Library
J. Microbiol. Biotech. Res., 2011, 1 (3):91-99
(http://scholarsresearchlibrary.com/archive.html)
ISSN : 2231 –3168
CODEN (USA) : JMBRB4
A Maltotriose producing thermostable amylase from Bacillus sp KR11
Moumita Karmakar and Rina Rani Ray*
Microbiology Research Laboratory, Department of Zoology, Molecular Biology & Genetics,
Presidency University, Kolkata, India
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ABSRACT
A bacterial strain of Bacillus sp KR 11 was isolated from soil which was found to produce extra
cellular maltotriose producing amylase. The enzyme was partially purified from crude culture
filtrate by ammonium sulfate fractionation, followed by dialysis. The optimum pH and
temperature for the purified enzyme were 7.0 and 65 °C, respectively. The thermo inactivation
kinetics indicated that the half life of the enzyme at 65°C was 60 minutes. The enzyme activity
was completely inhibited by 10 mM Hg2+ and Cu2+ and thiol inhibitor like pCMB. Restoration of
enzyme activity in presence of external thiols like cysteine, reduced glutathione and dithiothreitol
indicated the presence of thiol groups at the active site. The starchy residues collected from
various wastes could be converted into sugars, of which starch extracted from tamarind kernel
showed the highest yield of sugar. Thin layer chromatographic analysis indicated the presence of
maltotriose as the major end product of amylase action of these starches. The enzyme
immobilized on 7% gelatin powder crosslinked with glutaraldehyde showed the best result and
could be used for judicious exploitation of this enzyme in future.
Key words: amylase, Bacillus sp, maltotriose, saccharification, immobilization.
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INTRODUCTION
Amylases are among the most important enzymes used in biotechnology, particularly in process
involving starch hydrolysis (1). Most amylases produce glucose and maltose from starch as their
main products, but only a few amylases produce oligosaccharides specifically from starch (2).
Among these oligosaccharides, at present, the maltotriose to maltotetraose, have a range of
potential uses in food, pharmaceutical, and fine-chemicals industries because of their unique
nature and special properties. Maltotriose possesses many excellent properties for the food
industry (3), including mild sweetness and the prevention of retrogradation of starch in
foodstuffs. They are all highly soluble and produce clear, viscous solutions which are tasty and
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J. Microbiol. Biotech. Res., 2011, 1 (3):91-99
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are superior nutrient foods for infants and aged persons (4).Although maltotrise forming
amylases have been discovered in some bacteria like Chloroflexus aurantiacus (2) Streptococcus
bovis (5) ,Thermobifida fusca (3) , Sclerotinia sclerotiorum (6), Nocardiopsis sp (7),
Natronococcus sp (8), Microbacterium impeniale (9), Bacillus subtilis (10) and Streptomyces
griseus (11)in comparison to other type of amylases, only a few reports are available on
maltotriose forming amylase.
This article deals with the partial purification and characterization of a thermo stable maltotrioseforming amylase from a bacterial strain, Bacillus sp. strain KR11.
MATERIALS AND METHODS
Collection of bacterial strain:
Soil samples were collected from starchy wastes of market place were dissolved in sterile water
and streaked on nutrient agar plates containing 0.5% starch. The plates were incubated at 37°C at
pH 7 and examined after 24 hours. Single colonies of different sizes were selected and the
diameters of colonies were measured.
Isolation of bacterial strain:
The starch plates were drained with iodine solution (I2 and KI) and the halo diameters of selected
single colonies were measured after 24 h of incubation to determine the halo diameter to colony
diameter ratio. Selected single colony was maintained on nutrient agar slants at 4°C.
Cultivation of bacterial strain:
The selected strain was grown in liquid state fermentation (LSF), in 100 ml Erlenmeyer flasks
each containing 20 ml Basal Medium (BM) composed of (gl-1): peptone 0.9; (NH4)2HPO4 0.4;
KCl 0.1;MgSO4.H2O 0.1 and starch 0.5. (pH: 7) at 37°C.
Enzyme extraction and assay:
The bacterial culture broth was centrifuged at 10,000 rpm for 10 min and the supernatant was
used as the crude enzyme. To measure the activity of extracellular amylase, the assay mixture
(1ml) containing an equal volume of properly diluted enzyme and 1 %( w/v) starch in 50mM
phosphate buffer (pH-6) was incubated at 65°C for 10 min. The reducing sugar produced was
measured by the DNSA method (12) taking glucose as standard. One unit of enzyme activity was
defined as the amount of enzyme releasing 1 µmole of reducing sugar per minute per ml. under
standard assay conditions.
Partial purification of the enzyme:
The supernatant was subjected to fractionated ammonium sulfate precipitation for partial
purification of the enzyme. Ammonium sulfate (60-80% w/v) was gradually added to the
supernatant followed by centrifugation (10,000 rpm for 10mins at 4°C). The pellet was
resuspended in 50 mM phosphate buffer (pH 6) and the suspension was dialyzed against the
same buffer for desalting (13).
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Characterization of the enzyme:
The temperature and pH optima were detected by incubating the assay mixture at different
temperatures (30°-80°C) at constant pH and at various pH ranges (4-9) at 65°C for 10 minutes
respectively. Thermostability of the enzyme was determined by exposing the enzyme at 65°C for
various time periods, up to 120 minutes followed by the measurement of their residual activities.
The pH stability was determined by keeping the enzyme in presence of various buffers 0.1M
acetate buffer (pH 4-6), 0.1M phosphate buffer (pH 5-8) and 0.1m Tris glycine buffer (pH 8-9)
at 25°C for 120 minutes followed by the estimation of their residual activities. Effect of divalent
ions and additives on enzyme activity was measured by adding 10mM of various metallic salts or
additives followed by the measurement of the respective residual activities. The effect of
substrate concentration was determined by varying the concentration of starch (0.5-2.0% w/v) in
the assay mixture.
Saccharification of enzyme:
A suspension of substrate (10 mg/ml) in 0.1(M) phosphate buffer (pH: 6) was incubated with
equal amount of amylase (238 U/ml) in a screw capped tube for 30 minutes at 65°C [14]. The
resultant supernatant following centrifugation at 2000 g for 2 minutes was analyzed by DNSA
method (12) using glucose as standard.
Determination of end product of saccharification : The end products of saccharification of
agro wastes by amylase was analysed by TLC on a pre coated TLC plate (Merck) using a solvent
system of butanol: acetic acid: water (5:3:2 v/v), developing it with 2% methanolic orcinol in
10% H2SO4. (15).
Immobilization of enzyme: The enzyme was immobilized in 5-10% (w/v) gelatin powder
crosslinked with gluteraldehyde (16). The enzyme activity after immobilization was measured by
incubating the enzyme with 1% starch solution in 50mM phosphate buffer (pH- 6) at 65°C for 10
min with vigorous shaking.
Each experiment was carried out in triplicate and their values were averaged.
RESULTS AND DISCUSSION
Isolation and identification of working strain:
Out of 24 isolated bacterial strains, strain KR 11 showed the highest production and was selected
for further work. The strain was identified by the basic morphology and biochemical properties
according to Bergey’s Manual of Systematic Bacteriology (17) as Bacillus sp.
Effect of pH
The pH profile study indicated that the optimum pH for enzyme activity was 6.0, higher than
Sclerotinia sclerotiorum (6) but lower than that of Natronococcus sp (8).
About 70% activity of the enzyme was restored at a pH range of 4-9 (Fig. 1) This broad range of
pH tolerance would increase the applicability of the enzyme at industrial level.
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120
Relative Activity (%)
100
80
60
40
20
0
4
5
6
7
8
9
pH
Activity
Stability
Fig. 1.Effect of pH on activity and stability of the enzyme
Relative activity (%)
120
100
80
60
40
20
0
30
40
50
60
65
70
80
Temperature ('C)
Fig. 2.Effect of temperature on the activity of the enzyme.
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Relative activity (%)
100
80
60
40
20
0
0
5
15
30
45
60
80 100 120
Time exposed (mins)
Fig. 3.Effect of temperature on the stability of the enzyme
120
Relative activity(%)
100
80
60
40
20
0
0.5
1
1.5
2
2.5
Substrate conc.(%)
Fig. 4.Effect of substrate concentration on the activity of the enzyme
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Relative activity (%)
140
120
100
80
60
40
20
g2
+
H
u2
+
C
a2
+
C
Fe
2+
M
g2
+
M
n2
+
K
+
N
a+
C
on
tr
ol
0
Metal ions (10mM)
Fig. 5.Effect of metal ions on the activity of the enzyme
Relative activity (%)
140
120
100
80
60
40
20
C
on
C trol
ys
te
in
e
C
ys
t in
e
pC
M
B
D
TT
G
SH
ED
TA
SD
Tw
S
ee
Tw n 8
ee 0
Tw n 4
e 0
Ph e n
yt 20
ic
ac
id
0
Additives
Fig. 6.Effect of various additives on the activity of the enzyme
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Fig. 7.Effect of various additives on the activity of the enzyme
G: Glucose (standard) M:Maltose, Mt: Malto triose
1: end product of amylase action of potato starch
2: end product of amylase action of tamarind kernel starch
Table. 1- Saccharification of various waste starches
Starch (10 mg)
Sugar present (mg)
Rice powder
2.06
Arrowroot powder
2.24
Tapioca
1.89
Tamarind kernel
4.82
Oat
1.98
Corn flour
2.15
Millets
2.06
Arum powder
2.15
Corn
3.62
Potato starch (Merck)
3.87
Shredded potato
2.06
Pulse powder
1.72
Enzyme used: 238 U/ml
Table 2-Effect of immobilization on relative activity of the enzyme
Enzyme type
Relative Activity (%)
Free enzyme
100
Immobilized enzyme in 5% gelatin
58.33
Immobilized enzyme in 7.5% gelatin
75
Immobilized enzyme in 10% gelatin
42
100% activity: 200U/ml
Effect of temperature
Maximum activity was found at 65°C (Fig. 2.), lower than that reported to be produced by
Chloroflexus aurantiacus (2) but higher those secreted by Thermobifida fusca (19) and
Sclerotinia sclerotiorum (6). The temperature optima of the maltotriose presenting amylase of
the present strain and from the alkaliphilic actinomycete, Nocardiopsis sp. strain TOA-1(7) was
found to be identical.
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J. Microbiol. Biotech. Res., 2011, 1 (3):91-99
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The thermo inactivation kinetics of the enzyme indicated that the half life of the partially purified
enzyme at 65°C was 60 minutes and about 80% activity was retained even after 15 minutes of
exposure at 65°C (Fig. 3.).
Effect of substrate concentration
The enzyme showed highest hydrolytic activity in presence of 1.5% (w/v) starch. Further
increase in starch concentration brought about a remarkable decrease in enzyme activity (Fig. 5.),
which might be due to enzyme limitation (18).
Effect of metal ions and additives
The enzyme was strongly inhibited in presence of various metallic ions like Hg2+, Mg2+, Cu2+
and Mn2+ probably due to the destruction of the active site of the enzyme, but activated in
presence of Fe2+ and Ca2+ an observation more or less similar to the chemostability of
maltotriose forming amylases from Chloroflexus aurantiacus (2) and Sclerotinia sclerotiorum
(6).
The activity of malto triose producing amylase from Bacillus sp KR-11 was remarkably
enhanced (Fig. 6.) in presence of thiols like Cysteine HCl, dithiothreitol (DTT) and reduced
glutathione (GSH).This result indicated the presence of thiol group at the active site of the
enzyme which was confirmed by the deactivation of enzyme in presence of p-chloromercuri
benzoate (pCMB) a potent thiol inhibitor. The enzyme was significantly deactivated in presence
of detergents and surfactants and chelating agent (EDTA).
Saccharification of various raw starches
The enzyme was found to digest various waste starches (Table 1) collected from market
effluents. The enzyme showed highest potency in bioconversion of starch from tamarind kernel
seed followed by potato starch and corn dust. This saccharifying ability of the enzyme may
increase its applicability in industries related to sugar production.
The end product analysis indicated that the main end product of amylase action on tamarind
kernel starch and potato starch correspond to maltotriose rather to glucose or maltose, a result
similar to the observation of Kobayashi et al.,1992(8) and Ben Abdelmalek-Khedher et al, 2008
(6).
Immobilization of enzyme: The enzyme immobilized in 7% (w/v) gelatin powder showed the
best result as most of the enzyme was lost from immobilized matrix due to lack of dense packing
in 5% gelatin entrapped enzyme, whereas the substrate did not get access to the enzyme
immobilized with 10% gelatin powder.
CONCLUSION
The amylase secreted by Bacillus sp KR11 was inactivated in presence of phytic acid that
revealed its alpha amylolytic nature (20). On the other hand presence of maltotriose and absence
of glucose as end product of hydrolysis indicated that the enzyme in question was maltotriose
producing amylase. The thermostability and pH stability, saccharifying potential made it
applicable in industries.
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