敏捷食酸菌腈水解酶基因在大肠杆菌中的表达  

Expression of Nitrilase Gene from Acidovorax facilis in Escherichia coli

作　　者：管启旺 马江锋[1] 贺爱永[1] 姜岷[1] 宫长斌[1] 

机构地区：[1]南京工业大学生物与制药工程学院材料与化学工程国家重点实验室,南京211816

出　　处：《生物技术通报》2015年第1期181-185,共5页Biotechnology Bulletin

基　　金：国家自然科学基金项目(21076105);国家重点基础研究发展计划("973"计划)(2009CB724701);江苏高校优势学科建设工程项目

摘　　要：以含有敏捷食酸菌的腈水解酶基因的克隆质粒为模板,通过PCR扩增获得长度为1 080 bp的腈水解酶(Nitrilase)基因片段。使用表达质粒p ET-28-a构建表达载体,获得重组质粒p ET-Nit。对所表达的基因进行测序对比发现与Gen Bank所公布的基因序列比较,相似性99%,读码框出现2 bp的突变,但并未引起相应氨基酸突变,故不影响酶的表达与特性。将重组质粒转化到表达宿主Escherichia coli Rosetta(DE3)感受态中,使用诱导剂IPTG对菌株进行诱导表达,获取菌液进行SDS-PAGE分析,得知目的蛋白分子量约为41.28 k D,与预期所想的一致。酶活力分析表明,上清的比活力为3 U/mg。进一步对诱导条件进行优化,包括诱导温度,IPTG浓度,诱导时间等一系列条件。在最优条件下,扩大培养体积,比活力可达15 U/mg,活力提高了5倍左右。Plasmid containingAcidovorax facilis gene, was used as a template, and length of 1 080 bp nitrilase（Nitrilase）gene fragment was obtained by PCR. Expressed genes were sequenced and compared to gene sequences found in GenBank. There are 2 bp mutation was found, which can not affect the expression and characterization of the enzyme. The recombinant plasmid was transformed into theEscherichia coliRosetta（DE3）competent cell, which was induced IPTG for strain- expression. SDS-PAGE analysis was used and showed that the protein molecular weight was about 41.28 kD. Enzyme activity analysis showed that the specific activity of the supernatant was 3 U/mg. Induction conditions were optimized, including induction temperature, IPTG concentration, induction time and a series of conditions. Under optimal conditions and with the expansion of the culture volume, the specific activity can up to 15 U/mg, and activity increased by about five times.

关 键 词：腈水解酶 敏捷食酸菌 大肠杆菌 克隆 

分 类 号：Q78[生物学—分子生物学]