海栖热袍菌极耐热纤维二糖磷酸化酶在大肠杆菌中的克隆和表达

Cloning and Expression of a Thermostable Cellobiose Phosphorylase from Thermotoga Maritima

将来源于极端嗜热菌属海栖热袍菌(Thermotoga maritima)的编码纤维二糖磷酸化酶的结构基因cepA与新型热激质粒pHsh连接,得到重组质粒pHsh-cepA。将重组质粒pHsh-cepA转入大肠杆菌JM109进行表达。SDS-PAGE结果显示,该重组酶的分子量为90kD,与理论值相符。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 cepA from Thermotoga maritima MSB8 encoding cellobiose phosphorylase was amplified and ligated into pHsh,resulting in pHsh-cepA.Cellobiose phosphorylase was obtained after expressing pHsh-cepA in Escherichia coli JM109.SDS-PAGE analysis showed that the molecular mass of the expressed recombinant CepA was about 90 kD,which was exactly the size predicted.The expression vector system of the heat shock plasmid pHsh owned such advantages as high expression level and cheap induction.Moreover,the superior stability of the recombinant enzyme laid the base for the application of large-scale fermentation.