Expression, characterization, immobilisation by clea method of anoxybacillus flavithermus dsm 2641T α-amylase and α-glucosidase (oli̇go (1-6), (1-4) glucosidase

α-Amylase and α-Glucosidase are representatives of glyoxyl hydrolysis. In this study, cloning, expression, immobilization and characterization of α-glucosidase and α-amylase of Anoxybacillus flavithermus DSM 2641T were performed. α-amylase and α-glucosidase genes were produced recombinantly and purrified. Zymogram and Native PAGE analyses of α-amylase and α-glucosidase were performed, optimum pH, optimum temperature and kinetic parameters were determined. The effects of alkaline earth metals, alkali metals, potential inhibitors, ionic and non-ionic detergents, organic solvents on α-amylase and α-glucosidase activity were investigated. The optimum pH of α-amylase was between 6-8 in the presence of potato starch as a substrate. The optimum temperature of the enzyme was determined as 70 °C. For potato starch, the Km and Vmax values of α-amylase were 1.14 mg/ml and 31 U/mg by means of Lineweaver-Burk curve, respectively. The optimum pH for α-glucosidase in the presence of pNPG as substrate was found to be in the range of 6-8. The optimum temperature of the enzyme was 60 °C. The enzymes were immobilized separately and together by using the cross-linked enzyme aggregate (CLEA) method. The optimum pH for immobilized amylase was 8-9, the optimum temperature was 70 °C and the kinetic parameters were Km: 0.94 mg/ml and Vmax: 35 U/ml The optimum temperature for immobilized glucosidase was determined as 55 °C, the optimum pH as 6-8 and the kinetic parameters as Km: 0.66 mM and Vmax: 811 U/mg. The optimum pH for the co-immobilized enzymes was 55 °C and the optimum pH was 7.4.