Purification and characterization of highly thermostable α-amylase from thermophilic <Emphasis Type="Italic">Alicyclobacillus acidocaldarius</Emphasis> |
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Authors: | G Satheesh kumar M Subhosh Chandra K V Mallaiah P Sreenivasulu Yong-Lark Choi |
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Institution: | (1) CAS in Marine Biology, Annamalai University, Parangipettai, 608502, India;(2) Department of Chemical and Biological Sciences, Polytechnic University, Six Metrotech Center, Brooklyn, NY 11201, USA |
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Abstract: | In this study, the production of extracellular thermostable α-amylase by newly isolated thermophilic Alicyclobacillus acidocaldarius was detected on LB agar plates containing 1.0% soluble potato starch and incubated at 60°C. This extracellular α-amylase
was purified to homogeneity by ammonium sulphate precipitation followed by Sephadex and ion-exchange chromatography. The α-amylase
was purified to 8.138 fold homogeneity with a final recovery of 58% and a specific activity of 3,239 U/mg proteins. The purified
α-amylase appeared as a single protein band on SDS-PAGE with a molecular mass of 94.5 kDa. Non-denaturing PAGE analysis showed
one major band associated with enzyme activity, indicating the absence of isoenzymes. A TLC analysis showed maltose as major
end product of the enzyme. The optimum assay temperature and pH for enzyme activity were 60°C and 6.0 respectively; however,
the enzyme activity was stable over a wide range of pH and temperatures. The α-amylase retained its activity in the presence
of the denaturing agents — SDS, Triton X-100, Tween-20, Tween-80, and was significantly inhibited by EDTA and urea. Calcium
ions increased the enzyme activity, while Hg2+, Zn2+, and Co2+ had inhibitory effects. The K
m and V
max values were found to be 2.9 mg/mL and 7936 U/mL respectively. |
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Keywords: | |
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