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

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

摘要:

【目的】克隆嗜热脱氮土壤芽孢杆菌中的β-葡萄糖苷酶基因bglB, 在E. coli中异源表达, 纯化并研究其酶学性质。【方法】利用PCR技术从嗜热脱氮土壤芽孢杆菌的基因组DNA中克隆得到bglB基因, 将该基因克隆到表达载体pGEX-2TL上并在大肠杆菌BL21(DE3)中表达, 对纯化后的β-葡萄糖苷酶的酶学性质及寡聚状态进行分析。【结果】重组表达的β-葡萄糖苷酶最适温度为65 °C, 最适pH为7.0, 能在pH 5?10、60 °C下稳定存在4 h, 并能在较高的离子强度(880 mmol/L K+)下发挥其功能。Al3+离子对其有强烈的激活作用, Co2+有一定的抑制作用。最适反应条件下该酶比活力为0.043 IU/mg。该酶具有多种寡聚体形式, 这些寡聚体均有β-葡萄糖苷酶活性。【结论】获得一个耐热耐盐的中性β-葡萄糖苷酶, 为进一步研究β-葡萄糖苷酶的催化作用机理, 提高其热稳定性提供一定的帮助。

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.