Cloning, expression and characterization of a beta-glucosidase from Geobacillus thermodenitrificans
嗜热脱氮土壤芽孢杆菌β-葡萄糖苷酶的克隆与重组表达及其酶学性质研究

Objective] Cloning of β-glucosidase gene bglB from Geobacillus thermodenitrifi-cans,heterologous expression in E.coli,purification and characterization of its enzymatic properties.Methods] Molecular cloning of the β-glucosidase encoding gene(bglB) from Geobacillus thermodenitrificans was performed by using a PCR technique.The gene was ex-pressed in BL21(DE3) of Escherichia coli.After purification,the enzymatic properties and the protein aggregation of β-glucosidase was investigated.Results] The optimum temperature and optimum pH of the recombinant β-glucosidase are 65 °C and 7.0 respectively,the enzyme is stable for 4 h under the conditions of pH 5?10,60 °C,and it maintains its high enzymatic activity at the high salt concentration(up to 880 mmol/L K+).The recombinant β-glucosidase is strongly activated by Al3+,while slightly inhibited by Co2+.Under the optimal reaction con-dition,the enzyme specific activity of recombinant β-glucosidase is 0.043 IU/mg.The β-glucosidase GST fusion protein exists in different oligomers by a Superdex G-200 gel filtra-tion analysis,and the different oligomers of enzymes all have hydrolase activity.Conclusion] We successfully obtained a heat-and salt-resistant neutral recombinant β-glucosidase from Geobacillus thermodenitrificans,and paved a way for further study of its catalytic mechanism and improvement its thermal stability of beta-glucosidase.