Abstract
Amylase from Bacillus cereus MTCC 10205 was purified 20.41 with 11.82% recovery by ammonium sulfate precipitation, gel filtration chromatography through Sephadex G-100 and ion-exchange chromatography on diethylaminoethyl (DEAE)-cellulose. The final enzyme preparation was pure to near homogeneity as judged by native-polyacrylamide gel electrophoresis (PAGE). The enzyme had a molecular weight of 55 kDa as determined by gel filtration and a single band of 55 kDa as determined by sodium dodecyl sulfate-polycrylamide gel electrophoresis showing it to be a monomer. The purified enzyme had temperature optima of 55°C and pH optima of 5.5. The enzyme retained 72% of its original activity after 90 min of incubation and exhibited gradual loss in activity when incubated at higher temperature. At 60°C after 90 min of incubation, the enzyme was completely inactive. The enzyme appeared to be quite stable at 4°C as it could be stored upto five days with 10% loss in activity, whereas at 35°C, the enzyme lost 28% of its activity just after three days of storage. Inhibition studies revealed SH groups to be involved at the active site of the enzyme.
Highlights
Amylases are of ubiquitous occurrence and are holding maximum market share of enzyme sales (Sivaramakrishanan et al, 2006)
The final enzyme preparation was pure to near homogeneity as judged by native-polyacrylamide gel electrophoresis (PAGE)
Amylases that are active at acidic pH are generally used in the glucose syrup industry, whereas those active at basic pH are explored in detergent industries (Tonokova, 2006)
Summary
Amylases are of ubiquitous occurrence and are holding maximum market share of enzyme sales (Sivaramakrishanan et al, 2006). These hydrolyze starch molecules to give diverse products including dextrins and progressively smaller polymers composed of glucose units (Windish and Mhatre, 1965) and are used in a wide range of starch industries that is brewing, baking, starch liquefaction and distillery (Souza and Magalhães, 2010). The α-amylases (endo-1,4α-D glucan glucohydrolase, EC.3.2.1.3) are extra cellular enzymes that randomly cleave 1,4-α-D-glucosidic linkages between adjacent glucose units in linear amylose chain. We report here purification and characterization of acidic amylase from a new strain of bacteria having potential of being industrially used
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