云芝NADPH-细胞色素P450还原酶在大肠杆菌中表达及酶学特性分析  

Expression and characterization of NADPH-cytochrome P450 reductase from Trametes versicolor in Escherichia coli

作　　者：孙雪娃 何超 方泽民[1,2,3] 肖亚中 Xuewa Sun;Chao He;Zeming Fang;and Yazhong Xiao(School of Life Sciences, Anhui University, Hefei 230601, Anhui, China;Anhui Key Laboratory of Modern Biomanufacturing, Hefei 230601, Anhui, China;Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, Hefei 230601, A nhui, China)

机构地区：[1]安徽大学生命科学学院,安徽合肥230601 [2]现代生物制造安徽省重点实验室,安徽合肥230601 [3]安徽省微生物与生物催化工程技术研究中心,安徽合肥230601

出　　处：《生物工程学报》2018年第7期1156-1168,共13页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(Nos.31370114;31170056)资助~~

摘　　要：云芝Trametes versicolor具有很强的环境有机污染物降解能力,其烟酰胺腺嘌呤二核苷酸-细胞色素P450还原酶(NADPH-cytochrome P450 reductase,CPR)为细胞色素P450酶(Cytochrome P450s,CYPs)提供电子,参与有机污染物的降解过程。序列分析显示,云芝基因组拥有1个潜在CPR序列和多个潜在CYP序列。为深入研究云芝CPR参与细胞降解有机污染物的分子机制,实验进行了云芝CPR在大肠杆菌中异源表达和酶学特性分析。结果表明,经IPTG诱导后,去除预测的N端膜锚定区域(氨基酸残基1–24)的CPR蛋白(CPRΔ24)可在重组菌中实现可溶性表达,且表达蛋白的分子量与理论值(78 kDa)一致。镍离子亲和层析和分子筛层析纯化后测得其比活性为5.82 U/mg。酶学性质分析显示,重组CPRΔ24的最适温度和pH分别为35℃和8.0,并对一些金属离子及有机溶剂具有不同程度的耐受性。酶在35℃、pH 8.0反应条件下对NADPH的动力学参数K_m和k_(cat)分别为19.7μmol/L、3.31/s;对底物细胞色素c的动力学参数K_m和k_(cat)分别为25.9μmol/L、10.2/s。以上研究为探究云芝CPR在环境有机污染物降解途径中的功能机制奠定基础。Trametes versicolor has strong ability to degrade environmental organic pollutants. NADPH-cytochrome P450 reductase（CPR） of T. versicolor transfers electron to cytochrome P450s（CYPs） and participates in the degradation process of organic pollutants. Sequence analysis showed that the genome of T. versicolor contains 1 potential CPR and multiple potential CYP sequences. To further study the molecular mechanism for the involvement of T. versicolor CPR in the cellular degradation of organic pollutants, a CPR gene from T. versicolor was cloned and heterologously expressed in Escherichia coli. Subsequently, the main properties of the recombinant enzyme were investigated. A truncated CPR protein lacking the predicted membrane anchor region（residues 1-24）, named CPRΔ24, was overexpressed as a soluble form in E. coli. The recombinant CPRΔ24 protein showed a molecular weight consistent with the theoretical value of 78 kDa. Recombinant CPRΔ24 was purified using a Ni^（2＋）-chelating column followed by size exclusion chromatography. The specific activity of the purified CPRΔ24 was 5.82 U/mg. The CPRΔ24 enzyme displayed the maximum activity at 35 and pH 8.0. It has different ℃degrees of tolerance against several types of metal ions and organic solvents. The apparent Km and kcat values of recombinant CPRΔ24 for NADPH were 19.7 μmol/L and 3.31/s, respectively, and those for the substrate cytochrome c were 25.9 μmol/L and 10.2/s, respectively, under conditions of 35 ℃and p H 8.0. The above research provides the basis for exploring the functional mechanism of T. versicolor CPR in the degradation pathway of environmental organic pollutants.

关 键 词：云芝 NADPH-细胞色素P450还原酶 大肠杆菌 异源表达 酶学特性 

分 类 号：Q55[生物学—生物化学]