Gene cloning and molecular characterization of a β-glucosidase from Thermotoga naphthophila RUK-10: an effective tool for synthesis of galacto-oligosaccharide and alkyl galactopyranosides

Abstract

A novel thermostable β-glucosidase(Tnap0602) with β-galactosidase activity was cloned from Thermotoga naphthophila RUK-10 and overexpressed in Escherichia coli BL21(DE3) with the aid of pET28b(+) vector. The recombinant β-glucosidase was purified to homogeneity by heat precipitation and Ni²⁺-affinity chromatography. The molecular weight of the recombinant enzyme was estimated to be 51 kDa by SDS-PAGE analysis. The optimum temperature for the hydrolyses of p-nitrophenyl-β-D-glucopyranoside and o-nitrophenyl-β-D-galactopyranoside by the recombinant β-glucosidase were both above 95 °C, and the corresponding optimum pH value was found to be the same as 7.0. Thermostability studies show that the half-lives of the recombinant enzyme at 75, 80, 85 and 90 °C are respectively 84, 32, 14, and 3 h, and it is quite stable in a pH range of 5.0–10.0. The K _m and V _max values of the recombinant β-glucosidase for the hydrolysis of pNPGlu at 80 °C are 0.127 mmol/L and 18389.1 μmol·min⁻¹·mg⁻¹, the corresponding values are 0.625 mmol/L and 6250 μmol·min⁻¹·mg⁻¹ for the hydrolysis of oNPGal, respectively. The enzyme also display the hydrolysis activity for lactose and cellobiose. Galacto-oligosaccharide and alkyl galactopyranosides could be synthesized from Tnap0602 when lactose was used as the transglycosylation substrate, indicating that the thermostable β-glucosidase could be a candidate for industrial application.