國立中興大學 103 學年度第二學期 食品暨應用生物科技學系
碩士班一年級專題討論
Title：A thermostable α-amylase from a moderately thermophilic Bacillus subtilis
strain for starch processing.
Author：M. Asgher, M. Javaid Asad, S.U. Rahman, R.L. Legge
Impact Factor：2.576
5-Year Impact Factor：2.984
Speaker：陳柏亞
Moderator：王裕翔
Advisor：陳錦樹教授
Date： 2015/5/8（第七位）
Introduction
Amylases are enzymes which hydrolyse starch molecules to give diverse products including
dextrins and progressively smaller polymers composed of glucose units. Amylases having
approximately 25% of the enzyme market have almost completely replaced chemical hydrolysis of
starch in starch processing industry. Thermostability is a desired characteristic of most of the
industrial enzymes. Thermostable α-amylases are available from the mesophile Bacillus
licheniformis, Bacillus sp. ANT-6 and Bacillus sp. ASMIA-2. Each application of α-amylase
requires unique properties with respect to specificity, stability, temperature and pH dependence.
Material and Methods
 Selection and isolation of bacterial strain
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B. subtilis JS-2004 was a moderate thermophilic bacterium. The bacterial colonies appearing on
plate I were transferred to medium II. Amylase producing colonies were selected by flooding
the media II plates with iodine solution.
Enzyme production medium
The enzyme production was carried out in the basal medium of the following composition (%):
0.1 KH2PO4, 0.25 Na2HPO4, 0.1 NaCl, 0.2 (NH4)2SO4, 0.005 MgSO4 ∙ 7H2O, 0.005 CaCl2, 0.2
tryptone, and one waste potato starch powder.
Optimization of medium and culture conditions
Initially, the organism was grown in the liquid medium for 24–96 h at pH 7.0 and 40˚C and
then, supplemented with calcium (10 mM), yeast extract (0.5%), and glucose (0.5%) to study
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the effect of these nutrients on growth and enzyme production by B. subtilis JS-2004.
α-Amylase assay
One unit (U) is defined as the amount of enzyme which releases 1 μmol of reducing end groups
per minute in 0.1 M sodium phosphate buffer (pH 7.0) with 0.5% (w/v) soluble starch as
substrate at 37˚C.
Effect of pH on enzyme activity and stability
To determine the stability of α-amylase, the enzyme was pre-incubated in different buffers (pH
5–10) for 60 min.
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Effect of temperature on enzyme activity and stability
The effect of temperature on amylase stability was determined by measuring the residual
activity after 1 and 24 h of pre-incubation in 0.1 M sodium phosphate buffer (pH 7.0), at
temperatures ranging from 40 to 100˚C.
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Effect of metal ions on enzyme activity
Enzyme assay was performed after pre-incubation, at 60˚C (optimum) for 60 min, of the
enzyme with various metal ions each at a concentration of 2 mM.
Results
 Characterization of bacterial strain
The B. subtilis JS-2004 strain was gram positive. The pH for growth was 7.0 and optimum
temperature for growth was 50˚C. The strain B. subtilis JS-2004 possessed the ability to produce
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α-amylase and hydrolyze starch.
Amylase production
It was observed that maximum α-amylase production by B. subtilis JS-2004 occurred when cell
population reached the peak. Enhanced bacterial growth and enzyme activity may be the result
of increased availability of calcium ions. The result suggests that growth and synthesis of
α-amylase by B. subtilis JS-2004 is favored by yeast extract. A decrease in cell growth and
enzyme production was observed when glucose was added to the fermentation medium. There
was a stimulation of enzyme synthesis with an increase in pH from 5 to 7. Enzyme synthesis
occurred at temperatures between 30 and 50˚C.
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Characterization of crude α-amylase
The α-amylase of B. subtilis JS-2004 strain was found to be active in very broad pH range. The
optimum pH was found to be 8.0. Enzyme activity increased with temperature within the range
of 40–70˚C. Optimum temperature of B. subtilis JS-2004 α-amylase was 70˚C. Results suggest
that α-amylase did not require any ions for catalytic activity except Ca2+ and was activated
(relative activity 117%) by calcium.
Conclusions
The B. subtilis JS-2004 strain produced high levels of thermostable α-amylase with
characteristics suitable for application in starch processing and other food industries.
References
M. Asgher, M. Javaid Asad, S.U. Rahman, R.L. Legge. (2007). A thermostable α-amylase from a
moderately thermophilic Bacillus subtilis strain for starch processing. Journal of Food
Engineering, 79, 950–955.
Paula Monteiro de Souza, Pérola de Oliveira e Magalhães. (2010). Application of microbial
α-amylase in industry. Brazilian Journal of Microbiology, 41: 850-861.