纤维素外切酶CbhA在大肠杆菌中的重组表达、纯化及活性研究  

Recombinant Expression,Purification,and Activity of Cellulase CbhA in Escherichia coli

作　　者：乔想金 白丽娟 李文新 胡巍 韩丽 QIAO Xiang-jin;BAI Li-juan;LI Wen-xin;HU Wei;HAN Li(Engineering Technical Center of Biomedical Manufacturing,Lanzhou LS Energy Equipment Engineering Institute Co.,Ltd.,Lanzhou 730300,China)

机构地区：[1]兰州兰石能源装备工程研究院有限公司生物制造工程技术中心,甘肃兰州730300

出　　处：《化学与生物工程》2018年第4期42-47,共6页Chemistry & Bioengineering

基　　金：兰州兰石能源装备工程研究院有限公司科技创新项目(G17-22)

摘　　要：根据GenBank上纤维素外切酶CbhA的基因序列,设计并克隆目的基因;将目的基因克隆至表达载体pET32a,构建重组质粒pET32a-CbhA;再将重组质粒转化至大肠杆菌BL21(DE3)感受态细胞,成功构建了重组工程菌BL21(DE3)-pET32a-CbhA,探讨了诱导温度、IPTG浓度和诱导时间对重组工程菌表达量的影响。结果表明,所构建的重组工程菌在诱导温度为33℃、IPTG浓度为0.7 mmol·L^(-1)、诱导时间为8h的条件下,表达量可达到46.37 mg·L^(-1),具有较高的纤维素降解活性。该研究为纤维素酶在大肠杆菌中后续共表达和酶的放大发酵技术的建立奠定了重要基础。According to the original gene sequence of cellulase CbhA in GenBank,we designed and cloned the target gene CbhA.Then,we cloned gene CbhA into expression vector pET32a,and constructed the recombinant plasmid pET32a-CbhA.Finally,we transformed pET32a-CbhA into Escherichia coli BL21(DE3)competent cell,and constructed the recombinant strain BL21(DE3)-pET32a-CbhA.Meanwhile,we investigated the effects of induction temperature,IPTG concentration,and induction time on the expression of recombinant strain.The results indicate that the recombinant strain BL21(DE3)-pET32a-CbhA has stronger degradation activity to cellulose with expression of 46.37 mg·L-1 under the conditions as follows:the induction temperature of 33℃,IPTG concentration of 0.7 mmol·L-1,and the induction time of 8 h.This study lays an important foundation for the further co-expression of cellulase in Escherichia coli and the amplification fermentation of the enzyme.

关 键 词：纤维素酶 重组表达 发酵参数 降解活性 

分 类 号：Q753[生物学—分子生物学] Q78