Screening Of Methamidophos Degrading Bacteria And Molecular Cloning And Recombinant Expression Of Organophosphate Hydrolase Gene

Organophosphorus pesticides(OPs),invented in 1940s,are highly produced and widely used as highly efficient and broad-spectrum insecticides.OPs have greatly improved the crop yields and contributed greatly to the development of society.However,more and more problems about environment pollution,human health and continuable development begin to come out along with long-time and wide use of Ops and have widely led to public attention.How to solve the problem has become a public concern.Bioremediation has more merits than any other degrading ways.This research includes two parts:(1) Screening and characterization of methamidophos degrading bacterium.Twelve degrading methamidophos bacteria were isolated from activated sludge of a pesticide factory in Shandong province.These strains could use methamidophos as sole carbon and nitrogen source.The residual MAP was analyzed by gas chromatography-mass spectrometry,and the results showed that the MAPD-4 and MAPD-10 had better degrading activities and been chosen for further research.Blast analysis showed that the MAPD-4 and MAPD-10 were belonged to Staphylococcus and Alcaligens respectively.The optimal temperature of MAPD-4 and MAPE-10 was determined at 30℃and the optimal pH were 7.0 and 6.0,respectively.64%and 49%methamidophos could be degraded by the two strains within 15 days using methamidophos as the sole carbon source.(2) Molecular cloning and recombinant expression of organophosphate hydrolase gene. Total DNA was purified by Soil DNA Isolation Kit from activated sludge of a pesticide factory in Shandong province.A 1003 bp DNA fragment was amplified by nested PCR with the primers designed according to organophosphate hydrolase genes.Sequencing result indicated that the fragment contained a 993 bp full open reading frame.Blast analysis showed that the homology between the fragment and methyl parathion hydrolase gene was high to 99%.The open reading frame was subcloned into pET-30a plasmid and transferred into E.coli BL21(DE3).After induced by IPTG a 41.7 kD protein with molecular weight of 41.7 kD was largely expressed in the cells.The result of optimization of recombinant expressing conditions showed that the optimal concentration of IPTG was 0.1 mM and the optimal inducing period was 5 hours.