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KMID : 1094720100150030435
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Biotechnology and Bioprocess Engineering
2010 Volume.15 No. 3 p.435 ~ p.440
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Purification and Characterization of Highly Thermostable α-amylase from Thermophilic Alicyclobacillus acidocaldarius
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Kumar G. Satheesh
Chandra M. Subhosh
Mallaiah K. V.
Sreenivasulu P.
Choi Yong-Lark
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Abstract
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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 60oC. 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 60oC 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 Km and Vmax values were found to be 2.9 mg/mL and 7936 U/mL respectively.
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KEYWORD
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thermophilic Alicyclobacillus acidocaldarius, α-amylase, denaturing SDS-PAGE, non denaturing PAGE, characterization, stability
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