In vitro synthesis and activity identification of Chromate reductase T

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.