Improving The Thermostability Of Methyl Parathion Hydrolase MPH By Site-directed Mutagenesis

Organphosphorus (OP) compounds have been widely used, but meantime it made a great pollution for environment and threatened the human health. In the early 1970s, the bacterium in natural environment can secrete organophosphorus hydrolase with the capability of degrading OPs, which can destroy the phosphide ester bond of OPs, and eliminated the toxicity of OPs. It provided us a good way to eliminate the pollution of OPs.This study aimed at the methyl parathion hydrolase (MPH) isolated from Ochrobactrum sp. M231. The enzyme could degrade the methyl parathion with high efficiency, but have poor thermostability. Meanwhile, another methyl parathion hydrolase, OPHC2 (CAE53631) from Pseudomonas pseudoalcaligenes C2-1 has a greater thermostability than MPH. In order to improve the thermostability of MPH, the sequences of MPH and OPHC2 were aligned. The sequence identity and similarity between OPHC2 and MPH were 48% and 66%, respectively. Based on the alignment restult, the residues (P41 and P43) on the structure surface in the unaligned region were selected to be mutated into the charged amino acids through the methods of site-directed mutagenesis. Different mutants were analyzed to test and verify the effects of mutations.Finally, four mutants have been constructed and the thermostability of the mutants was different to the wild type enzyme. The thermostability of the mutant MPHP41D_P43K was better than the wild type enzyme. The mutant MPHP41D_P43K remain more than 70% enzyme activity after incubate 10 minutes at 65℃, which the half-life of the mutant MPHP41D_P43K was 2-fold and 1.5-fold than the wild MPH at 65℃and 70℃, respectively. Meanwhile, the specific activity of the MPHP41D_P43K was about 1.8-fold higher than wild MPH. By replacing the most rigid residues of proline with two charged residues in the MPH, an ion pair has been generated in the protein surface and might be the reason for improving the thermostability of MPH.The thermostability of methyl parathion hydrolase (MPH) was improved by protein engineering methods in this study. We hopefully could provide the theoretical basis for the application in the large-scale.