Screening Of Bacteria With The Capability Of Degrading Methyl Parathion And Cloning Of A Methyl Parathion Hydrolase Gene

Organphosphorus (OP) compounds which were invented in 1940s are widely used as highly efficient and broad-spectrum insecticides. OPs have made great contribution to the development of human society during the half of a century. 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. Organphosphorus hydrolase can destroy O-P bond of OPs, and consequently the toxicity of OPs is eliminated. The products of highly efficient and cheap organphosphrous hydrolase enzyme by the method of biotechnology are the best potential choice to solve those problems.A bacterium named M231 with the capability of degrading methyl parathion, identified as Ochrobactrum by 16SrDNA, is isolated from OP-polluted soil. The bacterium can excrete methyl parathion hydrolase constitutivly.The full-length methyl parathion hydrolase mphxw is cloned by PCR. The mphxw gene is 993 bp long with G+C content of 67.27%, comprising one open reading frame encoding a polypeptide of 330 amino acids with a molecular weight of 35 kD which includes a signal peptide of 35 aa. Although the protein sequence of mphxw shows high similarity of 98% with those methyl parathion hydrolases from Ochrobactrum, mphxw lacks one amino acid. The mphxw gene exhibits the low similarity of 43.7% with ophc2 which was repoted previously by our lab. The mphxw gene has been assigned and the GenBank accession number is EU596456.The prokaryotic expression recombinant vector pET-mphxw and the eukaryotic expressin recombinant pPIC9γ-mphxw are constructed respectively. The two recombinant vectors are transfered into E.coli and Pichia pastoris respectively and both expression products bore normal bioactivity.The proteins from Ochrobactrum sp. M231 and the prokaryotic recombinant protein have been purified, and the aa sequence of purified protein from Ochrobactrum sp. M231 accords with recombinant protein by the analysis of mass spectrum. The optional pH and temperature of the two proteins are nearly the same, with the optional pH 9-10 and the optional temperature 20℃. But the prokaryotic recombinant protein shows better stability in pH and temperature than the protein from Ochrobactrum sp. M231 . However, the non-recombinant protein shows high specific enzyme activity 148U/mg which is about 30 folds higher than the recombinant protein.We hopefully could explain the mechanism of thermostability and catalysis of methyl parathion hydrolase through the study of the molecular structure of MPHXW and OPHC2 which are very different in sequences and enzymatic activity.