嗜热真菌纤维素酶的CBD与海栖热袍菌的纤维素酶融合表达

Expression and Characterization of the Fusion Protein CBD-CelB

摘要:

将嗜热真菌毛壳菌纤维素酶Cel7A的纤维素结合结构域编码区与极端嗜热厌氧菌海栖热袍菌的纤维素酶CelB基因进行融合, 构建重组质粒pHsh-CBD-CelB, 并在大肠杆菌中表达。对融合蛋白进行纯化, 通过热处理和离子交换层析, 纯化到的融合蛋白SDS-PAGE 电泳图谱显示为单一条带。对融合蛋白的特性研究, 结果表明融合蛋白降解CMC的最适反应温度为90°C, 结晶纤维素吸附实验表明该融合蛋白具有结合结晶纤维素的能力, 并且融合蛋白降解CMC与结晶纤维素的能力得到提高。

关键词:海栖热袍菌, 嗜热毛壳菌, 纤维素结合结构域 (CBD), 融合蛋白

Abstract:

The CBD-encoding region of family 7 cellobiohydrolases gene from the thermophilic fungus Chaetomium thermophilum was inserted into the upstream of the CelB gene from the hyperthermophilic bacterium Thermotoga maritime. The recombinant plasmid pHsh-CBD-CelB was expressed in Escherichia coli JM109. CBD-CelB fusion protein was purified by a simple heat treatment followed by DEAE-Sepharose FF column. The optimal temperature of CBD-CelB fusion protein for CMC activity was 90°C, and the fusion protein had the ability of binding crystalline cellulose. Enzyme activity of CMC and Avicel was increased.

Key words:Thermotoga maritime, Chaetomium thermophilum, Cellulose binding domain (CBD), Fusion protein