Purification and characterization of α-amylase from Bacillus simplex BCHCNZ282B

In this study, soil samples were taken from Makam Mountain in Ergani district of Diyarbakır. Bacteria were isolated from samples taken from soil. Biochemical tests and 16S rRNA analyzes were performed to identify the isolate obtained from soil samples. As a result of the analysis, the isolate was identified as Bacillus simplex BCHCNZ282B. The effect of different parameters was investigated to determine the optimum conditions for the production of extracellularly secreted α-amylase by Bacillus simplex BCHCNZ282B. The optimum conditions for bacterial growth were determined to be 12 hours, 37°C and pH 7,0. Bacteria production was carried out by determining the time for maximum enzyme production as the 36th hour. By determining α-amylase activity in the supernatant taken from the bacterial culture grown for 36 hours, the optimum temperature and pH of the enzyme were determined to be 50°C and pH 7,0, respectively. The effects of 1% different carbon, nitrogen sources and media on enzyme production were examined. It was determined that sucrose, a carbon source, inhibited enzyme production, while nitrogen sources did not affect enzyme production. It was determined that the medium in which maximum enzyme production occurred was NB (Nutrient Broth) medium. α-amylase produced by Bacillus simplex BCHCNZ282B was purified 6,13-fold in 19,50% yield by 80% ammonium sulfate [(NH4)2SO4] precipitation, dialysis, and starch affinity chromatography. The molecular weight of the purified α-amylase was determined as ~70 kDa by SDS-PAGE. The effect of some inhibitors such as EDTA, PMSF, 2-Mercaptoethanol, Ethanol and Methanol on purified α-amylase activity was investigated. It was determined that 5 mM, 10 mM EDTA (79%, 84%, respectively) and 10 mM PMSF (61%) caused a strong inhibition of enzyme activity. The effect of some metals such as ZnCl2, HgCl2, CuCl2, MgCl2, CaCl2 and MnCl2 at 1,5 mM concentration on pure α-amylase activity was investigated. It was determined that MgCl2 (115%) and MnCl2 (112) increased the enzyme activity, while HgCl2 (85) inhibited the enzyme activity slightly compared to the control. The effect of some detergents such as 0,5% Tween-40, Tween-60, Tween-80, TritonX-100, SDS, Brij and commercially available Omo matik, which does not contain enzymes, on pure α-amylase activity was investigated. It was determined that SDS (20%) caused a strong inhibition of enzyme activity. . It was determined that pure α-amylase showed stability at pH 8,0 and 40°C-50°C for 60 minutes. According to the Lineweaver–Burk plot, it was calculated as Km and Vmax values of pure α-amylase are 0,002 mM and 2,125 μmol/min respectively. Keywords: Bacterial isolation, Bacillus simplex, α-Amylase, Purification, Starch affinity chromatography