Characterization Of Three Organophosphate Hydrolases And Directed Evolution Of Oph

Parathion hydrolase (OPH) is the first organophosphorus hydrolase which was studied profoundly. The methyl parathion hydrolase gene mpd cloned in our lab was a completely novel gene compared with ethyl parathion hydrolase gene opd. Their identity on amino acids level was less than20%. To characterize the differences between methyl parathion hydrolase (MPH) and parathion hydrolase (OPH), the parathion hydrolase gene (opd) from Flavobacterium sp. ATCC27551, and the methyl parathion hydrolase gene (mpd) from Plesiomonas sp. M6, and Pseudomonas putida DLL-E4were selected. Expression vector pET29a-mpd-dsp-his (M6), pET29a-mpd-dsp-his (DLL-E4) and pET29a-opd-dsp-his (Flavobacterium sp.) were constructed. Enzymes were expressed in Escherichia coli BL21(DE3), and purified with His-Tag affinity chromatography. Methods to terminate the enzymatic reaction were set up:using concentrated HCl to terminate reaction, then developing color in Glycine-NaOH buffer (pH10) to reduce the error when measuring the enzyme activity. Results showed that the optimum temperature of MPH (M6), MPH (DLL-E4), and OPH was30℃,35℃and40℃respectively; and the three enzymes all had highest stability at4℃. The optimum pH of MPH (M6), MPH (DLL-E4), and OPH was pH8.5, pH9.0, pH8.5respectively; and the three enzymes’ stability were relatively high under pH7.5-pH8.5(the remaining activity of three enzymes can achieve95%when they were stored at pH7.5, pH8.0, pH8.5buffer solution for3hours). Cu2+and Fe3+could promote the activity of MPH (M6), Ca2+and Zn+could promote the activity of MPH (DLL-E4), Co2+, Ni2+, and Zn2+could promote the activity of OPH, and high concentration of EDTA·2Na+(10mM) could inhibition three enzymes greatly, so we concluded that these three enzymes were metalloenzymes. Co2+, Cd2+and Li+could inhibit the activity of MPH (M6), Co2+and Cd2+could also inhibit the activity of MPH (DLL-E4), Cu2+could inhibit the activity of OPH. In addition, the higher concentration of imidazole(100mM) and NaCl(0.5M) also could inhibit the enzyme activity, so imidazole and NaCl should be removed after the Ni-NTA affinity chromatography. We identified a long-term preservation process of MPH:enzyme (8mg/mL) and "84" was mixed with equal volume, then the mixture was dissolved in15%NaCl and10%ethanol (the enzyme activity has no loss after three months), this proposal has a practical significance on developing detergent with pesticides-degrading enzymes as additives. Additionally, we measured the degradation spectrum of MPH and OPH, we found that these three enzymes all had broad degradation spectrum, and the degradation spectrum of OPH was broader than MPH; but the three enzymes could not degrade fenthion, and the structure of fenthion and methyl parathion were very similar. Therefore, we adopted Error-Prone PCR method to direct evolve OPH. We screened a mutant named M3-Q6which could degrade fenthion stably, and five mutants which could not degrade ethyl parathion. About50%of fenthion was degraded when the strain was cultured in a mineral salt medium containing50mg·L-1fenthion under the condition of30℃,150r·min-1,72h. With the analysis of the mutation, we found that the mutant sites which were on the surface of the OPH structure and far away from the reaction center could change the characterization of the OPH largely.