耐热纤维素酶在大肠杆菌中的高效表达及酶学特性分析

High expression of a thermostable cellulase gene from Clostridium thermocellum in Escherichia coli and characterization of its recombinant enzyme

将来源于热纤梭菌（Clostridium thermocellum ATCC 27405）的编码内切β-1,4-葡聚糖酶的结构基因（celD）分别插入到pHsh和pET两个表达系统,分别得到质粒pHsh-celD和pET-20b-celD。将重组质粒转化入大肠杆菌Escherichia coli BL21-CodonPlus（DE3）-RIL中表达,在pET系统表达时形成包涵体,采用热激表达系统pHsh在大肠杆菌表达时实现了纤维素酶的可溶性表达。SDS-PAGE结果显示,该重组酶的分子质量为66kD,与理论值相符。纯化的纤维素酶的最适反应pH为5.4,在特性不同的pH条件下60℃保温30 min,重组纤维素酶在5.4-7.8的pH范围内比较稳定,在75℃下酶的半衰期约为1 h,Ca^2＋可以增强酶活,而EDTA则会抑制酶的活性。基于热激载体pHsh的重组表达系统具有诱导表达简便、诱导方式廉价的优点,且重组酶热稳定性非常好,这对该酶的大规模发酵应用具有重要意义。

The structure gene from Clostridium thermocellum ATCC 27405 encoding endoglucanase was cloned into expression vectors pET-20b（ ＋ ） and pHsh to construct pHsh-celD and pET-2Ob-celD. In comparison, celD was over-produced in E. coli BL21-CodonPlus（ DE3）-RIL by using plasmid pHsh in soluble formation, while it formed inclusion-body by using plasmid pET-20b （ ＋ ）. The results from SDS - PAGE showed that the molecular mass of the expressed recombinant celD was about 66 kD, which was exactly the predicted size. celD was purified by heat treat- ment and Ni-NTA affinity chromatography. The purified celD exhibited the highest activity at pH 5.4 and 60℃, and retained more than 50% activity after incubated at 75℃ for 1 h. The cellulase activity of celD was significantly enhanced by Ca^2＋ and inhibited by EDTA. The expression vector system of the heat shock plasmid pHsh owned such advantages as high expression level and low cost for induction. Moreover the superior stability of the recombinant enzyme laid the base for the application of large scale fermentation.