Purification and Characterization of α-Amylase from Bacillus simplex BCHCNZ282B

Microbial α-amylases are widely used in industrial biotechnology due to their catalytic efficiency, stability, and cost-effective production. Identifying sources of newly isolated bacterial strains with desirable biochemical properties remains important for improving industrial enzyme applications. In this study, a newly isolated strain, Bacillus simplex BCHCNZ282B, isolated from soil samples collected in Ergani (Diyarbakır, Turkey), was investigated for its extracellular α-amylase production. The enzyme production conditions were optimized, followed by purification involving ammonium sulfate precipitation, dialysis, and final purification by starch-affinity chromatography. The purified enzyme was biochemically characterized in terms of optimal activity conditions; stability; kinetic parameters; and the effects of metal ions, inhibitors, and detergents. Optimal α-amylase production was obtained at 35 °C and pH 7.0 after 36 h of incubation. The optimum activity of the purified enzyme was attained at 50 °C and pH 7.0. Sequential purification resulted in a six-fold increase in specific activity and a final recovery yield of 19%. The enzyme's molecular mass was 70 kDa. Increased enzyme activity was obtained in the presence of Mg²⁺ and Mn²⁺, while EDTA was inhibitory to enzyme activity. The purified enzyme maintained high relative activity from pHs 7.0 to 9.0 and was active at all temperatures studied. In addition, enzyme activity was observed even in the presence of some of the tested detergents. The α-amylase produced by B. simplex BCHCNZ282B possessed many biochemical properties, such as stability in a wide range of pH values, high-temperature activity, and resistance to several detergents, which indicate many potential biotechnological applications of the enzyme. Nevertheless, further research on genomic characterization and structural analysis should be conducted to establish the industrial application of the enzyme.