Isolation, purification and characterization of extracellular ?-amylase from Geobacillus stearothermophilus

In this study, production, purification and characterization of ?-amylase from the strain Geobacillus stearothermophilus obtained from DSMZ were examined.Optimal conditions of bacterial growth were determined at 16th hour, 55°C and pH 7.0, respectively. Maximum enzyme production time was determined as 24th hour and bacteria production was conducted in these conditions. ?-Amylase enzyme activity was tested in the supernatant from produced bacteria and optimum temperature of enzyme activity was determined as 70°C, and pH was 7.0. The effect of carbon and nitrogen sources on the production of enzyme was investigated. It was found that carbon sources, sucrose and lactose inhibited the enzyme production. It was observed that nitrogen sources did not influence the enzyme production. The effect of various concentration of CaCl2 on the enzyme production were examined. The enzyme production was increased in the presence of CaCl2. Maximum enzyme production was achieved at the presence of 10 mM CaCl2. ?-Amylase was purified by gel filtration and ion exchange chromatography. Purification coefficient was determined to be 65, and yield as 46%. Molecular weight of the purified enzyme was found to be 63 kDa by SDS-PAGE.The effect of some inhibitors such as EDTA, PMSF, DTT, ß-mercaptoethanol, Etanol and on purified enzyme was studied. At the end of this study, the highest inhibitory effect 1 mM EDTA (65%) and 10 mM EDTA (89%) was obtained. It was determined that the inhibitory effect on enzyme activity increased in parallel with the increase in the concentration. To determine the effect of some metals on pure enzyme activity of pure enzyme, 1,5 mM CaCl2, CuCl2, MgCl2 ZnCl2, HgCl2, MnCl2, CoCl2, and FeCl2 were tested. Enzyme activity was increased in presence of CaCl2 (123%) and MnCl2 (122%), while activity of the enzyme was inhibited in presence of CuCl2 (81%) and HgCl2 (83%). It was determined that the purified enzyme was stable at 50oC and 60oC, and pH 7.0. Km and Vmax were calculated as 0,051 mM and 1,424 ?mol/min respectively according to the Lineweaver-Burk plot.