International Journal of Science and Research (IJSR), India Online ISSN: 2319-7064

Isolation, Optimization and Characterization of α-

Amylase from Bacillus alcalophilus
Roshan Kumar1 Archana Mehta2
1
Microbiology Lab, Department of Botany, School of Biological Sciences,
Dr. Hari Singh Gour Central University, Sagar - 470003, India

Abstract: Amylase is one of the most important enzymes widely used in paper, textile, pharmaceutical industries etc. In the present
investigation a thermostable, alkalophilic amylase was isolated from underlying soil samples of domestic wastes, and was screened on
starch agar medium. The amylase activity was measured using colorimetric method at 540nm. Among forty five isolates, thirteen
demonstrated clear zone of hydrolysis around the colonies when flooded with Gram’s iodine solution. Maximum zone of hydrolysis was
8 mm, and tentatively identified as Bacillus alcalophilus. Its maximum activity was 9.210 + 0.14 U/ml at pH 9 and 80°C temperature. It
retains 70% activity after 1 h at 80°C and 60% activity after 1 hour at 90°C. With these promising features B. alcalophilus may be
utilized in starch processing, detergent manufacturing brewing, and sugar production.

Keywords: Amylase, Bacillus alcalophilus, thermophilic, thermostable, domestic waste

1. Introduction Isolation of microorganisms was done by adding sample to

Starch broth medium (HIMEDIA) containing: Meat extract
Amylases are enzymes which breakdown starch molecule to 0.3%, Peptic digest of animal tissue 0.5% and Starch soluble
give diverse products such as dextrins and some small 0.2%. After 24 hours of growth medium was serial dilution
polymer composed of glucose [1]. They are produced by method and spread on Starch Agar plates. After 48 hours of
plants, animals and microorganisms but, those produced by incubation at 37°C, plates were flooded with Gram’s Iodine
microorganisms is still most preferred. Various fungal and solution. Organism showing zone of clearance around the
bacterial species have been explored and many are still in colony represents degradation of starch i.e. presence of
this process to be explored [2, 3]. Amylase isolated from the amylase activity. All isolates were preserved in 15% glycerol
bacteria has found more applications in food, textile, in at 4°C. Identification of isolates was done by performing
brewing, detergent and distilling industries [1]. Mainly two staining techniques, biochemical tests, advanced bacterial
types of amylases are produced by the bacteria: α-amylase identification software (ABIS) and Bergey’s manual.
and glucoamylase. α–amylase has found much commercial
importance than glucoamylases. It degrades α–1-4 glucosidic 2.3 Optimization of Culture Conditions
linkage of starch and related species [4]. Amylases (E.C.
3.2.1.1.) have approximately 25% of the enzyme market and Screened strains of bacteria were subjected to various culture
have completely replaced chemical hydrolysis of starch in conditions to derive optimum culture condition for amylase
starch processing industries. It offers various advantages to production. Growth and amylase production was estimated;
industries i.e. risk of contamination, cost of external cooling, incubation period (6, 12, 18, 24, 30, 36, 42 and 48 hours),
increase diffusion rate, resistant to denaturing agents and temperature (30, 40, 50, 60, 70, 80, 90, 100°C), pH (3, 4, 5,
proteolytic enzymes [4-9]. Thermostable alkaline amylases 6, 7, 8, 9, 10 and 11), Carbon source (Glucose, starch and
have extensive commercial applications with pH values sucrose) and Nitrogen source (peptone, yeast extract,
higher than 8.0 and in starch saccharification, industries, as tryptone). All experiments were carried out in 250ml
an ingredient in detergent for automatic dish washers and Erlenmeyer flask containing 100ml medium containing
laundries. α–amylase requires unique properties with respect peptone 0.5%, meat extract 1%, starch 1%, CaCl2 0.05% and
to specificity, stability, temperature and pH dependencies NaCl 0.1%. Sterile medium was inoculated with 1ml (105
[10-13]. CFU). Inoculated flasks were incubated at 37°C at 120 rpm
for 48 hours.
In this present investigation, thermostable and alkalophilic
amylase producing Bacillus alcalophilus was isolated and 2.4 Extraction and Purification
identified as potent organism for enzyme production.
After 48 hours of incubation culture broth was centrifuged at
2. Material and Methods 6000 rpm for 15 minutes. Supernatant was divided into two
parts. One part of supernatant was used as crude enzyme
2.1 Sample Collection whereas the other part was 60% saturated with ammonium
sulphate. The precipitate obtained was dialysed with 20mM
Various underlying soil samples were collected from potassium phosphate buffer and used as purified enzyme.
dumping sites/domestic wastes from local area of Sagar. Protein content of both crude and purified form of amylase
Samples were collected in pre-sterilized bottles and brought was estimated [14,15].
to the lab for investigation.
2.5 Enzyme Assay
2.2 Isolation and Screening

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 International Journal of Science and Research (IJSR), India Online ISSN: 2319-7064

Amylase enzyme activity was assayed by using 3,5 –

dinitrosalicylic acid (DNS) method as described by of Miller
(1959) [16]. By reacting 0.5 ml of enzyme to 0.5 ml of
soluble starch and incubating the enzyme mixture at 37°C
for 15 minutes. The reaction was stopped by addition of 1ml
of DNS reagent and boiled for 15 minutes. The Absorbance
was read at 540 nm and results obtained were compared with
standard of maltose. One unit of enzyme was defined as one
micromoles of maltose released per ml of enzyme.

2.6 Effect of Temperature and Metals on Enzyme

Activity
Figure 2: Effect of pH on growth and enzyme activity
The effect of temperature on enzyme activity was measured
by incubating the enzyme at different time interval of 10, 20,
30, 40, 50 and 60 minutes in 50mM Tris buffer. Effect of
different metals such as FeCl2, CuCl2, NaCl, MgCl2, CaCl2,
EDTA and SDS at 1mM concentration was used to check its
effect on enzyme activity. The activity of enzyme without
adding any metals was taken as control.

2.7 Molecular weight determination by SDS-PAGE

Molecular weight of amylase was determined by performing

SDS-PAGE with 10% polyacrylamide gel following the
method described by Laemmli. The purified enzyme was
loaded on wells parallel with standard protein markers of
known molecular weights. Protein bands were stained by
using coomassie brilliant blue and was destained. Molecular Figure 3: Effect of temperature on growth and enzyme
weight was calculated using Total lab ver. 10. activity

3. Results Media supplemented with lactose, starch, maltose and

fructose as sole carbon source, increase the enzyme activity
Optimum growth of Bacillus alcalophilus and enzyme to 6.38 U/mL (4.42 %), 6.32 U/mL (3.44 %) 6.29 U/mL
production was at stationary phase (fig. 1). Maximum (2.95 %) and 6.25 U/mL (2.29 %) respectively. Glucose and
growth and enzyme production was also obtained at pH – 9 sucrose exhibited slight increase in the activity from (0.05 to
(fig. 2) which signifies towards alkaline nature. 0.12 %). In another case where the medium was
Thermotolerant nature was proved when cell growth and supplemented with peptone and tryptone as nitrogen source,
enzyme production were highest at 60-80°C (fig. 3). maximum enzymatic activity was 6.28 U/mL (2.78 %) and
6.24 U/mL (2.13 %) as compared from the control (Fig. 4).

Figure 4. Effect on Carbon/Nitrogen source on enzyme

Figure 1: Effect of incubation period on growth and
activity
enzyme activity
When MnCl2, CaCl2 and NaCl metal ions were used 3.60 %,
2.95 % and 2.29 % increase in the amylase activity was
reported whereas with SDS, EDTA and Urea, 1.31 %, 3.27
% and 2.29 % decrease in the enzymatic activity was
observed (Fig. 5). Subsequently decrease up to 90°C and
beyond 90°C no enzyme activity were found. With purified

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enzyme high specific activity of 24.95 U/mg was found. Acknowledgement

Molecular weight determination of the purified amylase on
SDS-PAGE showed a single band with molecular weight of This work was supported by a grant from the University
52 kDa indicating the purity of amylase. Grant Commission, New Delhi and Department of Botany,
Dr. H. S. Gour University, Sagar (M.P.).

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Author Profile
Roshan Kumar received the B.Sc. and M.Sc. degrees
in Biotechnology from R. T. M. University, Nagpur in
2005 and 2008, respectively. From 2008 till date, he is
pursuing Ph.D. in Department of Botany, Dr. H. S. Gour
University and also is a recipient of Junior research
fellow of UGC.

Archana Mehta received the B.Sc. and M.Sc. degree

in Botany from Dr. H. S. Gour University, sagar in
1975 and 1977 respectively. She has done Ph.D. from
the same department in 1983. She is recipient of JRF
(UGC), SRF (CSIR), Pool Scientist (CSIR), RA
(CSIR) while working in the same Department. She has also
received one UNESCO and three INSA fellowships to work with
the best scientist around the world. Currently, engaged in teaching
and research at Department of Botany, Dr. H. S. Gour Central
University, Sagar.

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