Site-Directed Mutagenesis And Kinetics Analysis Of Glyphosate N-acetyltransferase

Glyphosate (N-phosphonomethyl-glycine) is a kind of broad-spectrum and systemicconductibility herbicide which is low toxic to mammalian and low environmental impact.It has very high commercial value and abroad using all over the world. GAT is an enzymewhich can metabolize glyphosate to low toxion substance by a reaction of transferringacetyl. A new effect mechanism which is different from EPSPS approach is provided forglyphosate tolerant crop with glyphosate acetylated.The glyphosate N-acetyltransferase (GAT) gene was cloned from Bacillus.licheniformis (Weigmann) Chester AB 94036 to pGEX 6p-1 vector and transformed intoE.coli DH5αin this study. Glutamic acid in 67 site and Isoleucine in 132 site were bothmutated to Arginine by site-directed mutagenesis method for increasing GAT enzymeactivity. IPTG was used to induce the GST-GAT proteins (43 kD) of recombinant stains.Through the affinity chromatogram, about 17 kD purified GAT proteins were gained.Enzyme kinetic was menstruated by Thermo MULTISKAN SPECTRUM withcontinuous spectrophotometric assay and the Lineweaver-Burk method. Comparing withnative GAT (GAT_n), the result showed: GAT_n＜GAT_{Ile 132 Arg}＜GAT_{Glu 67 Arg} (the value ofk_cat (catalytic number)), and GAT_{Glu 67 Arg}＜GAT_n＜GAT_{Ile 132 Arg} (the value of K_M(Michaelis constant)), and we also found that the k_cat/K_M (catalytic efficiency) ofGAT_Glu67Arg and GAT_{Ile 132 Arg} rated 4 and 3-fold higher than that of GAT_n, respectively.It could be concluded that 67 and 132 sites of GAT had contribution to its catalyticefficiency of transferring acetyl, and the enzyme activity were increased. This resultconsequently established the foundation of further research of GAT enzyme directedevolution in vitro.