中间苍白杆菌SCUEC4菌株中尼古丁降解相关ocnH基因的克隆表达及其功能分析
Cloning, expression and functional analysis of ocnH gene involved in nicotine degradation in Ochrobactrum intermedium SCUEC4
投稿时间:2022-03-15  修订日期:2022-03-15
DOI:
中文关键词: 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
英文关键词: Ochrobactrum intermedium SCUEC4  nicotine degradation  ocnH gene  cloning and expression  functional analysis
基金项目:国家自然科学基金资助项目(31070087);中央高校基本科研业务费专项基金资助项目(CZY19018);企业委托资助项目(HZY20042,HZY21015)
作者单位邮编
王萌蕾 中南民族大学 生命科学学院 
许倍滔 中南民族大学 生命科学学院 
夏珍珍 中南民族大学 生命科学学院 
王力 江苏神力生态农业科技有限公司 
张建国 江苏神力生态农业科技有限公司 
李晓华 中南民族大学 生命科学学院 430074
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中文摘要:
      本文对中间苍白杆菌SCUEC4菌株中与尼古丁降解相关的ocnH基因进行克隆、表达及其功能分析,并对OcnH蛋白的表达条件进行优化. 通过PCR扩增获得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.
英文摘要:
      The study aimed to clone, express and functional identification of the ocnH gene involved in nicotine degradation in Ochrobactrum intermedium SCUEC4 and optimize the conditions of the expression of the OcnH protein. The ocnH gene 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 isopropyl β-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.
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