Cloning and expression of Afbgl gene from, Anoxybacillus flavithermus and biochemical characterization of thermophilic β-glucosidase

β-glucosidases (3.2.1.21) hydrolyse celluse which is the most abundant and renewable source of energy on Earth. Also this enzyme completes the hydrolysis by converting cellobiose and cello-oligosaccaharides into glucose monomers. In this study, we aimed to clone, expression, and biochemical characterization of thermophilic β- glucosidase gene from thermophilic bacterium, Anoxybacillus flavithermus. The gene was cloned, sequenced, and expressed in E.coli Afbgl was cloned and overexpressed in E.coli BL21 Star (DE3) host cells with Pet100 Directional TOPO Expession Kit. Then, we purified to homogenity by heat precipitation and Ni+2 affinity chromatography. The molecular mass of the recombinant enzyme was 53 kDa. The optimum temperature and ph of the purified enzyme were 65 ºC and 6,8, respectively. The specifity of beta-glucosidase enzyme was determined used pNPG, pNPC, pNPX and oNPG sustrates. At these points, the enzyme had 5,13 x 105 µmol/dak/mg protein Vmax and 422,3 µM of Km towards p-nitrophenyl β-D-glucoside. The effects of K+, Fe+2, Mn+2,Mg+2, Zn+2, Cu+2 and Al+3, and which are commonly seen heavy metals in the nature on purified beta-glucosidase enzyme was investigated in vitro. It was determined that from this heavy metals Mg+2 has affected as activation on ezyme activity. However, it was determined that the other heavy metals K+, Cu+2, Zn+2, Mn+2, Fe+2 and Al+3have inhbition effects on enzyme activity. Keyword: Anoxybacillus flavithermus, β-glucosidase, thermophilic enzyme, pNPG, E.Coli.