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 ...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 Ni^2＋-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 ℃, 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℃ are respectively 84, 32, 14, and 3 h, and it is quite stable in a pH range of 5.0-10.0. The Km and Vmax values of the recombinant β-glucosidase for the hydrolysis of pNPGlu at 80 ℃ 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.展开更多
基金Supported by the National High Technology Research and Development Program of China（No.2013AA102104） and the National Natural Science Foundation of China（Nos.20772046, 21072075）.
文摘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 Ni^2＋-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 ℃, 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℃ are respectively 84, 32, 14, and 3 h, and it is quite stable in a pH range of 5.0-10.0. The Km and Vmax values of the recombinant β-glucosidase for the hydrolysis of pNPGlu at 80 ℃ 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.
