利用pHsh载体克隆与表达多功能半纤维素酶  

Cloning and expression of multi-functional hemicellulase using pHsh as vector in Escherichia coli

作　　者：彭静静[1] 

机构地区：[1]泰山学院生物与酿酒工程学院,山东泰安271021

出　　处：《江苏农业学报》2014年第4期875-879,共5页Jiangsu Journal of Agricultural Sciences

基　　金：泰安市科技发展计划(20132094);泰山学院博士科研启动基金(Y-01-2013001)

摘　　要：将来源于嗜热厌氧乙醇菌(Thermoanaerobacter ethanolicus JW200)的双活性阿拉伯/木糖苷酶(XarB)基因构建到新型热激质粒pHsh上,得到质粒pHsh-xarB。将来源于疏棉状嗜热丝孢菌(Thermomyces lanuginosus DSM5826)的木聚糖酶A(XynA)基因构建到pHsh-xarB上,得到表达质粒pHsh-xarB-xynA。将重组质粒pHsh-xarB-xynA转入大肠杆菌Escherichia coli JM109进行表达。SDS-PAGE结果显示,该重组酶的分子量为108 000,与理论值相符。Xyn-SDS-PAGE显示该融合酶具备木聚糖酶的活性。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,且重组酶热稳定性好,这对该酶的大规模发酵应用具有重要意义。The structure gene from Thermoanaerobacter ethanolicus JW200 encoding XarB was amplified and ligated into pHsh, resulting in pHsh-xarB. The structure gene from Thermomyces lanuginosus encoding xylanase （ XynA） was am-plified and ligated into pHsh-xarB, resulting in pHsh-xarB-xynA. The multi-functional enzyme （ XarB-XynA） was achieved after the expression of pHsh-xarB-xynA in Escherichia coli JM109. SDS-PAGE analysis showed that the molecular mass of the expressed recombinant XarB-XynA was about 108 000, which was exactly the size predicted. The recombinant XarB-XynA exhibited xylanase activity. The expression vector system based on the heat shock vector pHsh owned such advantages as high expression level, cheap induction, and thermo-stability of the recombinant enzyme, which were crucial for the application of large-scale fermentation.

关 键 词：阿拉伯 木糖苷酶 木聚糖酶 pHsh 多功能半纤维素酶 克隆 表达 

分 类 号：Q557.1[生物学—生物化学]