Cloning, expression and characterization of a beta-glucosidase from Geobacillus thermodenitrificans

Abstract:

[Objective] Cloning of β-glucosidase gene bglB from Geobacillus thermodenitrificans, 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 expressed 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 condition, 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 filtration 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.