短小芽孢杆菌β-1,4-葡聚糖内切酶基因的克隆及在大肠杆菌中的分泌表达  

Cloning of β-1,4-Endoglucanase Gene from Bacillus Pumilus and Its Secretory Expression in Escherichia Coli

作　　者：郭成栓[1] 欧阳蒲月[1] 崔堂兵[2] 郭勇[2] 

机构地区：[1]广东食品药品职业学院中药和生物系,广东广州510520 [2]华南理工大学生物科学与工程学院,广东广州510640

出　　处：《化学与生物工程》2010年第12期53-55,68,共4页Chemistry & Bioengineering

基　　金：广东省工业科技攻关计划项目(2005B10401051);广东食品药品职业学院自然科学青年基金资助项目(2009YZ005)

摘　　要：从一株产碱性纤维素酶的短小芽孢杆菌H12中克隆了编码β-1,4-葡聚糖内切酶的基因,对其基因序列及其酶的结构域进行了分析预测,同时将该酶的基因构建于大肠杆菌表达载体pET20b中,获得重组表达载体pET20b-EglA,将其转化至大肠杆菌BL21(DE3)菌株中进行表达。结果表明,该基因大小为1980 bp,共编码659个氨基酸;对β-1,4-葡聚糖内切酶结构域分析表明,该酶由两个不连续的结构域组成,其一为N-端催化结构域,由糖基水解酶家族9组成,其二为C-端底物结合结构域,由碳水化合物绑定结构域家族3组成;平板实验结果表明,β-1,4-葡聚糖内切酶基因在重组大肠杆菌中得到了良好的分泌表达;SDS-PAGE电泳图谱表明该酶的分子大小约为73 kDa。The β-1,4-endoglucanase gene was cloned from Bacillus pumilus H12. The gene sequence and domain of β-1,4-endoglucanase were analyzed. The gene was cloned into Escherichia coli expression vector pET20b and the construct was transformed to a E. coli BL21（DE3） strain. The results showed that the gene had a sequence of 1980 bp and it coded 659 amino acids. The predicted protein,EglA,consisted of a N-terminal catalytic domain belonging to glycoside hydrolase family 9 and a C-terminal cellulose-binding domain belonging to carbohydrate 3. EglA was secretedly expressed in Escherichia coli and the molecular weight.approximately was 73 kDa.

关 键 词：β-1 4-葡聚糖内切酶 大肠杆菌 短小芽孢杆菌 克隆 表达 

分 类 号：Q78[生物学—分子生物学]