The studies of ?-amylase from thermophilic Anoxybacillus sp. AH1

The purpose of this study was some properties of biotechnologically important ?-amylase enzyme produced by thermophilic Anoxybacillus sp. AH1 isolated from Dargeçit (Mardin) hot springs examined.Anoxybacillus sp. AH1 was grown on NB medium and ?-amylase activity was measured at differant incubation time. Maximum enzyme production was determinated 12-24 hours.The effect of pH and temperature on the ?-amylase activity was tested pH 4.0-11.0 and 30-90 oC both crude enzyme and partially purified enzyme respectively. The optimum pH and temprature for enzyme were found as 7.0 and 60 oC respectively.The effect of different medium, different nitrogen sources in 1% ratios and different carbon sources and starches in 0.5 % and 1% ratios on the production of ?-amylase was investigated. Maximum enzyme production observed on NB1 medium. The best nitrogen source was determinated as pepton and beef extract, While The best carbon source was determinated as maltose, glucose and lactose in % 0.5 ratios. Enzyme production was also enhanced in the presence of potato starch in % 0.5 and % 1 ratios and soluble starch in % 1 ratios.The effect of various concentration of CaCl2 on the enzyme production was examined. The enzyme production was increased in the presence of CaCl2. Maximum enzyme production was achieved at the presence of 20 mM CaCl2.?-Amylase in Anoxybacillus sp. AH1 was partially purified and the effect of some chemicals, metals and metal chelators and detergents on the enzyme activity was studied.MgCl2 (% 41 at 8 mM) and CaCl2 (% 70 at 8 mM) were found to increase ?-amylase activity at certain rates, while ZnCl2 (% 85 at 0.5 mM and % 93 at 1 mM), CuCl2 (% 76 at 0.5 mM and % 100 at 1 mM) and the metal chelators EDTA (% 63 at 10 mM) and 1,10-phenanthroline (% 22 at 10 mM) inhibited the enzymatic activity at high rates. ß-Mercaptoethanol (% 64 at 10 mM) and DTT (more than % 100 at 10 mM) were found to enhance ?-amylase activity great extent, while PCMB (% 52 at 4 mM) and PMSF (% 60 at 4 mM) inhibited the enzymatic activity at certain rates. Iodoacetamide and N-ethylmaleimide were found to have a little effect on the enzyme activity. The enzyme activity was found to increase in the presence of various detergants, while significantly inhibited by the concentrations of urea.The kinetic parameters such as Km and Vmax were determinated by the incubating the ?-amylase with various concentrations of soluble starch ranging from % 0.5 to % 3 (w/v) in buffer. Km and Vmax were calculated from Lineweaver-Bulk plot. as 0.102 mM and 0.929 µmol/min. respectively.The enzyme was found to be senstive to above 45 oC and the thermalstability was enhanced by glycerol and sorbitol. It was determinated enzyme retained % 99 and % 85 orijinal activity at 55 oC and 60 oC respectively after 120 minutes In the presence of % 30 glycerol.The enzyme electrophoretic analysis was performed by Nondenaturing polyacrylamide gel electrophoresis.Key Words: Anoxybacillus sp. AH1, Biotechnology, ?-amylase, enzyme production, enzyme characterization