Isolation And Characterization Of Organophosphate Pesticides Degrading Strains, Cloning And Expression Of Ophc2 Gene

Phoxim-degrdading strain XSP-1 and methyl parathion-degrading strain SMSP-1 were isolated from organophosphate pesticides-contaminated soil. These bacterium were preliminarily identified as Delftia sp. and Stenotrophomonas sp. based on the physiological and biochemical analysis and 16S rRNA gene series source analysis. They were designated as XSP-1 (GenBank Accession No. EF061135) and SMSP-1 (GenBank Accession No. EU312979) respectively.Biological properties were analyzed respectively. The optimal pH value for the growth of strain XSP-1 was 5.0-9.0, the optimal temperature was 30℃. The optimal osmotic pressure for strain XSP-1 was 5-35g NaCl/L. The optimal medium for the growth of strain XSP-1 was fructose as carbon source and organic nitrogen as nitrogen source. Strain XSP-1 could completely degrade phoxim within 7h, the optimal phoxim-degrading condition was pH7.0, 30℃. Strain XSP-1 could also degrade fenitrothion, methyl parathion and fenthion rapidly, but had little degrading ability towards chlorpyrifos.Strain SMSP-1 could grow well through pH5.0-10.0, the optimal temperature were 30℃and 35℃, the optimal osmotic pressure was 5-45g NaCl/L. The optimal medium for growth was glucose as carbon source and organic nitrogen as nitrogen source. SMSP-1 could completely hydrolyze methyl parathion to p-nitrophenol (PNP) within 48h, but it had no further degrading ability towards PNP. The optimal MP-degrading condition was pH7.0, 30℃. Strain SMSP-1 could also degrade ethyl parathion, fenthion, fenitrothion and phoxim, but could not hydrolyse chlorpyrifos. Through the construction of genomic DNA library of strain SMSP-1, we obtained ophc2 gene encoding the organic phosphorus hydrolyzing enzyme. According to the analysis of online-software and GenBank database, this sequence had 98% similarity to ophc2 gene (GenBank Accession No. AJ605330) and contained a 24-aa signal peptide, and it was the second report of cloning the ophc2 gene and the first report of this gene from the genus of Stenotrophomonas up to now. The ophc2 gene with and without signal peptide were ligated into expression vector pET29a, then transformed into E.coli BL21 (DE3) and overexpessed. The map of SDS-PAGE showed that there might exist a protease-cleavage site other than the signal peptide site, which could be recognized by some kind of proteases in BL21 (DE3) and cleaved, and this cleavage site was located at the C-terminal of OPHC2.