Agrobacterium Tumefaciens Lba4404 Glucose Tolerance In Beta-grape Glycosidases Cloning, Expression And Recombinant Enzyme In Nature

β-glucosidases catalyze the transfer of glycosyl group between oxygen nucleophiles and can be applied in the energy, food and medicine area. Mostβ-glucosidases are highly inhibited by the glucose. To date, the glucose-tolerantβ-glucosidase is not found in prokaryocyte, and the glucose-tolerantβ-glucosidases which have been found are not highly expressed. So it is very important to find the highly expressed glucose-tolerantβ-glucosidases for the industry requirement.Aβ-glucosidase gene bgl from the Agrobacterium tumefaciens str. LBA4404 was cloned into the expression vector pET-28b(+) and transformed into Escherichia coli RP (DE3). Bacteria containing positive clone were routinely grown and IPTG was added to induce the expression of recombinant protein. Theβ-glucosidase activity of crude extracts was found up to 36.7μmol min-1 mg of protein-1. Enzymatic study was performed on the recombinantβ-glucosidase purified with Ni column and found that this Bgl bore high substrate affinity and low substrate specificity, which belonged to the carbohydrate hydrolase superfamily 1. Thisβ-glucosidase displayed quite high activity at pH 5-8 and 40℃, and was able to be stored quite stablely at pH 5-10 and under 40℃. Using pNP-β-Glc as the substrate, the optimum pH and temperature of the hydrolysis reaction were revealed to be 6.4 and 60℃, and the Km of Bgl was 0.09 mmol/L at pH 6.4 and 37℃. It was inhibited by the competitive inhibitor glucono-δ-lactone (Ki 0.03 mmol/L) and resistant to inhibition by glucose(Ki 75 mmol/L). We also found that Ag+ and Zn2+ strongly inhibited the activity of Bgl. Its Km for pNP-β-Gal and pNP-a-Glc were respectively 3.61 mmol/L and 14.31 mmol/L.It is very significant that the highly expressed glucose-tolerantβ-glucosidase is found in prokaryocyte.