中间苍白杆菌尼古丁降解相关基因ocnH的克隆及功能分析  

Cloning and functional analysis of ocnH gene involved in nicotine degradation in Ochrobactrum intermedium strain

作　　者：王萌蕾 许倍滔 夏珍珍 王力 张建国 李晓华[1] WANG Menglei;XU Beitao;XIA Zhenzhen;WANG Li;ZHANG Jianguo;LI Xiaohua(Hubei Provincial Engineering and Technology Research Center for Resources and Utilization of Microbiology&College of Life Sciences,South-Central Minzu University,Wuhan 430074,China;Jiangsu Shenli Ecological Agricultural Science and Technology Co.Ltd.,Yixing 214221,China)

机构地区：[1]中南民族大学生命科学学院&微生物资源与利用湖北省工程技术研究中心,武汉430074 [2]江苏神力生态农业科技有限公司,宜兴214221

出　　处：《中南民族大学学报（自然科学版）》2023年第4期457-461,共5页Journal of South-Central University for Nationalities：Natural Science Edition

基　　金：国家自然科学资金资助项目(31070087);中央高校基本科研业务费专项资金资助项目(CZY19018);企业委托资助项目(HZY20042,HZY21015)。

摘　　要：通过PCR扩增获得中间苍白杆菌SCUEC4菌株中与尼古丁降解相关的ocnH基因,构建重组质粒pET28a(+)-ocnH,转化大肠杆菌BL21(DE3)菌株进行异源表达,并对表达条件进行优化,用高效液相色谱法测定重组表达菌株的马来酸异构酶活性.结果表明:ocnH基因大小为585 bp,编码蛋白分子量约为20.6 kDa.重组表达菌株在IPTG浓度为0.6 mmol·L^(-1)、诱导温度为25℃、诱导表达时间为20 h时,OcnH蛋白的表达量较高.转入pET28a(+)-ocnH的大肠杆菌BL21(DE3)菌株具有将马来酸转化为富马酸的功能.The ocnH gene involved in nicotine degradation in Ochrobactrum intermedium SCUEC4 was amplified with polymerase chain reaction,and the recombinant plasmid pET28a(+)-ocnH was constructed and transformed into Escherichia coli BL21(DE3)for heterologous expression.Meanwhile,the effects of isopropylβ-D-thiogalactoside concentration,induction temperature and time on the expression of the OcnH protein were studied,while the potential maleate isomerase enzymatic activities of the heterologous expression strain was analyzed by high performance liquid chromatography.The results indicated that the size of ocnH gene was 585 bp,the molecular weight of the encoded protein was about 20.6 kDa.Under the optimized expression conditions of IPTG concentration of 0.6 mmol·L^(-1),induction temperature at 25℃and induction expression time of 20 h,the expression of the OcnH protein was relatively high.The strain of E.coli BL21(DE3)carrying the pET28a(+)-ocnH recombinant plasmid have the function of transforming maleic acid into fumaric acid.

关 键 词：中间苍白杆菌SCUEC4菌株 尼古丁降解 ocnH基因 克隆与表达 功能分析 

分 类 号：Q93[生物学—微生物学]