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

作　　者：胡开蕾[1] 韩剑[2] 刘伟丰[2] 王艳萍[1] 陶勇[2] 

机构地区：[1]天津科技大学食品工程与生物技术学院,天津300457 [2]中国科学院微生物研究所,北京100101

出　　处：《微生物学通报》2012年第7期891-900,共10页Microbiology China

基　　金：中国科学院微生物研究所微生物资源前期开发国家重点实验室开放课题(No.SKLMR-20100602)

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。[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 D-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 D-glucosidase are 65 ℃ and 7.0 respectively, the enzyme is stable for 4 h under the conditions of pH 5-10, 60 ℃, and it maintains its high enzymatic activity at the high salt concentration （up to 880 mmol/L K＋）. The recombinant [3-glucosidase is strongly activated by A13＋, while slightly inhibited by Co2＋. Under the optimal reaction con- dition, the enzyme specific activity of recombinant 13-glucosidase is 0.043 IU/mg. The [3-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 13-glucosidase from Geobacillus thermodenitrificans, and paved a way for further study of its catalytic mechanism and improvement its thermal stability of beta-glucosidase.

关 键 词：Β-葡萄糖苷酶 嗜热脱氮土壤芽孢杆菌 重组表达 耐热 耐盐 

分 类 号：Q93[生物学—微生物学]