Cloning, Expression And Characterization Of β-Glucosidase From The Deep-Sea Exiguobacterium Oxidotolerans A011

β-glucosidase is widely distributed in nature, existing in almost all organisms. Their different sources make them reveal different nature. They involved in the metabolism of organisms, playing an important role in the maintenance of normal physiological function of organisms. Study found that, (3-glucosidase involved in EMP glycolytic pathway, one enzyme of which involved in glucose metabolism in Bifidobacterium. Humans and mammals lactase/phlorizin (LPH) hydrolase also contains an aryl-β-glucosidase, as LPH relate to adult-type lactase deficiency-a common human genetic disorders which received extensive attention. In addition,β-glucosidase is able to hydrolyze flavor precursor in fruits, vegetables, and tea to rich natural flavor aroma, in the saccharification of cellulose,β-glucosidase is to hydrolyze cellulose disaccharide and oligosaccharide into glucose. Therefore, the research on P-glucosidase has important theoretical and practical value.We isolated a deep-sea bacterium Exiguobacterium oxidotolerans A011 producedβ-glucosidase from the marine bacteria supplied by Third institute of Oceanography State Oceanic Administration. And constructed the genomic DNA library of Exiguobacterium oxidotolerans A011 by shotgun library, for the first time we completed itsβ-glucosidase cloning and prokaryotic expression, its enzymatic properties were studied.Experiment with pUC18 as the carrier, the P-glucosidase gene(bgl1c) contains 1347bp, encoding a 488-residue polypeptid with predicted protein molecular weight of 51.6 kDa. Bgl1C appears the highest amino acid sequence homology with other known P-glucosidases is 68%, belonging to glycoside hydrolase family 1. bgl1c was cloned into the pGEX-6p-1, expression is fine after the induction of IPTG for 8 hours. The value of optimum pH and the pH stability, optimum temperature and thermal stability, impact of gold ions and chemical reagents with pNPGlu as a substrate. Optimal conditions for activity were pH 7 and a temperature of 35℃and Bgl1C was stable in buffers ranging from pH 6.6-9. The specific activity, Km, and Vmax for the substrate p-nitrophenyl-β-D-glucopyranoside were 41 U mg-1,1.72 mg ml-1 and 0.45μg ml-1 s-1, respectively. Na+, Ca2+, EDTA andβ-mercaptoethanol had no effect on the activity, while Hg2+, Cu2+,Co2+ strongly inhibited it. It is noteworthy that Bgl1C is a cold active enzyme that retains about 61% of its maximum activity at 10℃. Bgl1C can partly hydrolyze cellobiose, cellotriose and cellotetraose, and also possessing highβ-galactosidase activity, is a bifunctional enzyme. It is worth mentioning that Bgl1C remain high activity at low temperature, low temperature enzyme activity and the nature of its dual functions in industrial applications have potential application.