草甘膦N-乙酰转移酶反应动力学研究

Enzyme Kinetics Analysis of Glyphosate N-Acetyltransferase

草甘膦N-乙酰转移酶(glyphosate N-acetytransferase,GAT)是一种能够通过转乙酰基的反应将草甘膦代谢为低毒物质乙酰草甘膦的酶,在抗除草剂转基因作物中具有潜在的应用价值。通过将地衣芽孢杆菌Ba-cillus licheniformis(Weigmann)Chester AB 94036中的草甘膦N-乙酰转移酶(glyphosate N-acetyltransferase,GAT)基因克隆到大肠杆菌中,获得含有重组质粒pGEX 6p-1-gat的重组菌株。对重组菌株进行诱导表达,经过GST亲和层析纯化,获得约17 ku的目的蛋白。通过酶标仪和连续分光光度分析法,对GAT蛋白进行了酶反应动力学测定。研究表明GAT的催化常数kcat为6.420 min-1,米氏常数KM为1.162 mmol/L,由此得出其催化效率kcat/KM为5.52 L/(mmol.min)。

Glyphosate N-acetyltransferase （GAT） is an enzyme which can metabolize glyphosate to low toxion substance by the reaction of transfer acetyl, and has potential application value for glyphosate tolerance. The gat gene was cloned from Bacillus licheniformis （Weigmann） Chester AB 94036 to pGEX 6p-1 vector and transformed into E. coli DH5α. IPTG was used to induce the GST-GAT proteins（about 43 ku）of recombinant stains. Through the GST affinity chromatogram, about 17 ku purified GAT proteins were gained. Enzyme kinetic was measured by thermo multiskan spectrum,with continuous spectro- photometric assay and the line weaver-burk method. The result showed that the value of kcat was 6. 420 min^-1,the value of KM was 1. 162 mmol/L, and kcat/KM （catalytic efficiency） of GAT was 5.52 L/mmol min. It can be concluded that this study laid the foundation for further research of GAT evolution in vitro.