Greed Phagocytosis Variovorax Boronicumulans J1 Degradation Of Neonicotinoid Insecticide Thiamethoxam Lin Mechanism Research

This research mainly investigated the molecular mechanism of degradation of neonicotinoid insecticide thiacloprid (THI) by bacterium Variovorax boronicumulans Jl, Then the nitrile hydratase gene (NHase) and the corresponding activator gene (J) of V.boronicumulans J1were cloned and overexpressed in Escherichia coli BL21(DE3). The resting cells of E. coli BL21(DE3) overexpressed NHase were tested its biotransformation ability.In our previous study, a neonicotinoid insecticide thiacloprid-degrading bacterium J1was isolated from soil and identified as Variovorax boronicumulans by16S rRNA gene sequence analysis in previous study. Liquid chromatography-masss pectrometry and nuclear magnetic resonance analysis indicated the major pathway of thiacloprid (THI) metabolism by V.boronicumulans J1involved hydrolysis of the N-cyanoimino group to form an N-carbamoylinino group containing metabolite, THI amide, which may be mediated by nitrile hydratase. So the nitrile hydratase of V.boronicumulans J1was cloned and expressed in E. coli BL21. A2.6kb gene cluster from V.boronicumulans J1that includes the full length of the nitrile hydratase gene and the full length of the corresponding activator gene (J) was cloned and investigated by degenerate primer polymerase chain reaction (PCR) and inverse PCR. The nitrile hydratase gene has a length of1304bp and codes a cobalt-type nitrile hydratase with an a-subunit of213amino acids and a β-subunit of221amino acids, the corresponding activator gene (J) has a length of378bp and codes128amino acids. Bioinformatic analysis of the nitrile hydratase gene sequence show that the nitrile hydratase is a cobalt-type nitrile hydratase.The nitrile hydratase gene and the corresponding activator gene (J) were recombined into plasmid pET28a and over expressed in Escherichiacoli BL21(DE3). The successful expression of the target protein was detected by SDS-PAGE and Western Blot. The enzyme activity was detected by HPLC. The resting cells of recombinant E.coli BL21(DE3)-pET28a-NHase and E.coli BL21(DE3)-pET28a-NHJ with expression of nitrile hydratase transformed thiacloprid to its amide metabolite, whereas resting cells of the control E.coli BL21(DE3)-pET28a did not. Therefore, the major hydration pathway of thiacloprid is mediated by nitrile hydratase. The corresponding activator protein could improve NHase activity. Biotransformation of THI by V.boronicumulans J1and the study on nitrile hydratase of V.boronicumulans J1have not been reported. This research investigated the nitrile hydratase of V.boronicumulans J1for the first time, which is very valuable for the study of metabolism of neonicotinoid insecticide thiacloprid and other nitrile compounds by microorganism.