Cloning Of The Glyphosate N-acetyltransferase Gene,Characterization Of Enzyme Activity And Analysis Of The Saturate Mutagenesis At AlbD Enzyme S40 Site

Glyphosate N-acetyltransferase (GAT) is capable of detoxifying glyphosate by N-acetylation, which could potentially be used in herbicide resistence transgenic crops. The glyphosate N-acetyltransferase gene was amplified from soil total DNA by PCR. Recombinant plasmid pGEX-gat was constructed by inserting objective fragment into the expression vector pGEX-6p-1. The recombinant plasmid was transformed into Escherichia.coli DH5a. and the engineered strain capable of expressing GST-GAT fusion protein was obstained. The sequencing results showed that the homology were 99.3%, 93% and 95.2% comparing with published DNA sequence of AX543338, AX543339 and AX543340 which came from Bacillus licheniformis. The GAT enzyme was purified by affinity chromatography. After SDS-PAGE, gat gene expression protein about 17kDa can be observed, which identical to the deduced molecular weight. The GAT enzyme was characterized and the results showed that the optimal temperature of this enzyme was 30℃, the optimal pH of the reaction was 7. Effects of 6 metal ions on the activity of GAT enzyme were investigated. The effect of univalent ions (K⁺ and Na⁺) on GAT enzyme activity was stronger than that of divalent ions (Mg²⁺ and Mn²⁺), among which Na⁺ was the strongest, it increased the enzyme activity 48.3%. But Ca²⁺ was obvious inhibitor for activity of this enzyme, it decreased about 63.7%.Leaf scald is one of the major diseases in sugarcane, which is caused by phytotoxin albicidin. Our lab isolated the AlbD enzyme from Erwinia herbicola, which can digest albicidin. Based on sequence alignment, AlbD enzyme and esterase had the same motifLXXXGXXG--GXSXG. Moreover, AlbD enzyme was found to possesses themulti-substrate esterase activity for p-nitrophenyl esters with C₂-C₈ length of fatty acid chain. Based on previous research, the S40 site might be one part of active site, enzyme activity might be different when S40 site changed. In order to find out the effect of S40 site on the enzyme activity, 19 mutants was obtained throngh saturate mutagenesis of S40 site by using of mutagenesis PCR. The detoxification and esterase activity of different mutants was characterized. The results showed that S40A and S40R mutants increased the AlbD enzyme detoxification 1.1-fold and 1.2-fold respectively. S40Q enhanced the esterase activity 1.2-fold when p-nitrophenyl butyrate was used as the substrate; S40G increased the esterase activity 1.2-fold when p-nitrophenyl valerate was used. S40N,S40L and S40W decreased both detoxification and esterase activity obviously. The research results lay a foundation to the action mechanism and further study.