Enzymatic Characterization of a Novel Glucose-tolerant β-glucosidase from Agrobacterium tumefaciens

Abstract:

To date, the glucose-tolerant β-glucosidase has not been found in prokaryocyte. In the present study, the β-glucosidase gene bg1 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. The β-glucosidase activity of crude extracts was found up to 36.7 μmol/(min·mg). Enzymatic study was performed on the recombinant β-glucosidase purified with Ni column and found that this Bg1 bore high substrate affinity and low substrate specificity, which belonged to the carbohydrate hydrolase superfamily 1. This β-glucosidase displayed quite high activity at pH 5-8 and 40°C, and was able to be stored quite stablely at pH 5-10 and under 40°C. Using pNP-β-Glc as the substrate, the optimum pH and temperature of the hydrolysis reaction were revealed to be 6.4 and 60°C, and the Km of Bg1 was 0.09 mmol/L at pH 6.4 and 37°C. It was inhibited by the competitive inhibitor glucono-δ-lactone (Ki 0.03 mmol/L) and resistant to the inhibition of glucose (Ki 75 mmol/L). We also found that Ag+ and Zn2+ strongly inhibited the activity of Bg1. Its Km for pNP-β-Gal and pNP-α-Glc were respectively 3.61 mmol/L and 14.31 mmol/L.

Key words:β-glucosidase, Agrobacterium tumefaciens str. LBA4404, Glucose tolerance