The Stability Of Methyl Parathion Hydrolase And Secretion

Methyl parathion hydrolase can biodegrade organophosphorus pesticides. Due to its special application value, more and more researchers pay attention to it. In this study, we found that wild-type MPH-Och from Ochrobactrum sp. can not be secreted in Pichia pastoris. In this paper, in order to increase the secretion level, we improved the thermal stability and acid stability of the MPH.The MPH-S274 Q and MPH-S274 Q mutant, which thermal stability was increased, was expressed in Pichia pastoris. After recombinant strain was induced by the shaking culture for 120 hours, the enzyme activity of culture supernatant reached0.7 U/mL. It was 14 times higher than the wild type MPH-Och. The analysis of SDS-PAGE electrophoresis found that the culture supernatant of mutant MPH-S274 Q had a significant protein band. By RT-PCR analysis showed that the mph transcript level of mutant MPH-S274 Q was 2.1 times than the wide-type MPH. The hac1 and kar2 level were respecively higher 2.4 and 0.4 folds than MPH. This was partly revealed that the MPH-S274 Q secreted more easily, because S274 Q mutation sythesized more protein folding factors in the endoplasmic reticulum.Since the induction medium of Pichia pastoris was inconsistented with the optimal pH of MPH, so we changed the polarity MPH surface amino acids. Our strategy to build the acid-stable mutant MPH-K208 E, MPH-K277 D and double-point mutant MPH-S274QK277 D in order to iproving the acid stability of MPH. The results showed that the protein secretion of mutant MPH-K277 D was 14.8 folds than MPH.By SDS-PAGE analysis, protein secretion was significantly improved. The RT-PCR results of MPH-K277 D was similar to MPH-S274 Q.This study shows that improving the thermal and acid stability of the protein can be used as an effective way to increase the secretion of exogenous protein in Pichia pastoris.The target genes expressed successfully with eGFP, it provided an experimental foundation for further exploration of the mutant Pichia pastoris secretion mechanismof subcellular localization.