Purification and Characterization of Highly Thermostable α-amylase from Thermophilic Alicyclobacillus acidocaldarius

In this study, the production of extracellular thermostable alpha-amylase by newly isolated thermophilic Alicyclobacillus acidocaldarius was detected on LB agar plates containing 1.0% soluble potato starch and incubated at 60 degrees C. This extracellular alpha-amylase was purified to homogeneity by ammonium sulphate precipitation followed by Sephadex and ion-exchange chromatography. The alpha-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 alpha-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 degrees C and 6.0 respectively; however, the enzyme activity was stable over a wide range of pH and temperatures. The alpha-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.