铬还原酶T体外合成及活性鉴定

In vitro synthesis and activity identification of Chromate reductase T

摘要:

目的：克隆沙雷氏菌CQMUS2中铬还原酶T（Chromate reductase T，ChrT）的全基因，构建其原核表达载体并转化至大肠杆菌中表达蛋白，分析表达产物的活性。方法：应用PCR技术，以耐铬沙雷氏菌CQMUS2基因组DNA为模板扩增ChrT全基因，将该基因连接至表达载体pET-28a（+）并转化进大肠杆菌感受态细胞E.coli BL21（DE3）表达，用酶促反应鉴定重组ChrT酶的活性和Cr（Ⅵ）还原能力，探索其最适pH和温度。结果：克隆出的ChrT全基因长为567 bp，编码188个氨基酸，分子量约20 kD；IPTG诱导10 h后，在大肠杆菌中成功表达了目的蛋白，其碱基序列和分子量与ChrT酶一致；纯化后，ChrT酶能还原酶促反应体系中的Cr（Ⅵ），使实验组Cr（Ⅵ）含量明显低于对照组（P＝0.000），其酶活可达997 U/L；ChrT酶的最适pH为6.5~7.5，最适温度为37 ℃。结论：ChrT酶具有Cr（Ⅵ） 还原活性，为进一步研究高效除铬基因工程菌奠定了基础。

Abstract:

Objective：To clone the full length gene of Chromate reductase T（ChrT） from serratia sp. CQMUS2，to construct the prokary－otic expression vector and to transform it into E.coli to express protein，and to analyze the activity of expression products. Methods：The ChrT gene was cloned from DNA template of serratia sp. CQMUS2，a chromium-resistant bacteria by PCR and was connected with prokaryotic vector pET-28a（+）. Then the recombinant vector was transformed into the E.coli BL21（DE3） to express protein. The activity and the reduction ability of hexava－lent chromium of recombinant ChrT enzyme were determined by the enzymatic reaction. The optimal pH and temperature of recombi－nant ChrT enzyme were also studied. Results：The ChrT gene was an ORF of 567 bp that encoded a 188-aa protein with a relative molecular mass of 20 kD At. 10 h after the induction with IPTG，the target protein was successfully expressed in E.coli and the base sequences and relative molecular mass were consistent with ChrT enzyme. After the purification，ChrT enzyme reduced Cr（Ⅵ） in the enzymatic system and the Cr（Ⅵ） concentration was significantly lower in experiment group than in control group（P=0.000）. The ac－tivity of ChrT enzyme was up to 997 U/L，the optimum pH of ChrT enzyme was 6.5-7.5 and the optimum temperature was 37 ℃. Conclusion：ChrT enzyme can reduct Cr（Ⅵ），which provide solid foundation for further study on the high-level expression of Cr（Ⅵ）-removal engineering bacteria.