Thermostable alpha-amylase enzyme production from Bacillus laterosporus: Statistical optimization, purification and characterization

Production and characterization of a thermostable alpha-amylase enzyme from Bacillus laterosporus isolate was investigated in this study. Box-Behnken design (BBD) of response surface methodology (RSM) was applied to evaluate the optimal level of four medium components (yeast extract, starch, peptone and NaCl). R2 value of 0.867 showed that model was well fitted with the experimental data and linear effect of starch, yeast extract and peptone were found significant on alpha-amylase production. Optimum values of yeast extract, starch, peptone and NaCl were predicted as 0.58%, 2.44%, 2.34% and 0.11%, respectively, with maximum enzyme activity of 4.838 U/ml. Enzyme purification was carried out by ammonium sulphate precipitation and size exclusion chromatography (SEC). Maximum purification was obtained by SEC with 4.71 fold. Effect of temperature and pH were studied on purified enzyme and the maximum activity was observed at optimal conditions of temperature (60 °C) and pH 7. Presence of Ca2+ ions and EDTA does not affect the enzyme activity, however reduced activity of the enzyme was observed in presence of Mg2+ ions, SDS and β-mercaptoethanol.