Research Of Methyl Parathion Hydrolase

A methyl parathion-degrading strain was isolated by our group in 2001 from Sanonda pesticides manufacture in Hubei province, China. The strain was identified as Pseudomonas sp. and named WBC-3. It can not only use the methyl-parathion as the sole C/N source to grow but also can degrade p-nitrophenol, the product of methyl parathion, completely. In the following research, the gene degrading methyl parathion was identified in a big plasmid and named mph. It is different from the familiar opd from Pseudomonas diminuta and Flavobacterium sp. described previously at both gene and protein level. Besides methyl parathion, MPH can also degrade fenitrothion, parathion, chlorpyrifos in degressive hydrolytic efficiency in turn. We found that purified MPH have a 20-fold higher hydrolytic efficiency to methyl parathion than to parathion. As the new charater is holded, MPH will occupy the distinctive place in the family of organophosphate hydrolase.In the present work, we try to express MPH in a heterogenous host, E.coil system AD494(DE3)/ pET32a(+). The resulted AD494(DE3)/ pET32a(+)-mph not only can express THMPA solubly and highly, but also changed character of the wild MPH: 1. under the definite protein concentration, the recombinant enzyme was more stable than wild enzyme at room temperature or 4 ℃; 2. the recombinant enzyme have 2-fold higher specific activity to parathion than wild enzyme. We have done a series of tests to prove it. 1. The protein MPH in the fusion form was more stable than MPH like other fusion proteins. 2. The fusion part thioredoxin might play a role of molecular chaperon to influence the process of folding and mature of recombinant MPH and make it form new structure fitted for parathion hydrolysis.With the new character of recombinant MPH , we set up a system to detect trace methyl parathion and parathion by enzyme absorbance assay. In a 96 cell microplatedetection system, special absorbance signals are linearly proportional to the concentration of the hydrolyzed methyl parathion and parathion substrates up to 100 μM, showing detection limits of 0.05 μM .The directed evolution of THMPA was done by error prone PCR using the recombinant THMPA. We hope get mutant with higher hydrolysis activity to one of the three subtrates. 30000 mutants in the libraries were screened. However, there wasn't any mutant with distinctive high hydrolysis activity. It implied that the recombinant THMPA had the most fitted structure with its fusion part, thus it is difficult to evolute it.