The Study On The Properties Of Methyl Parathion Hydrolase And Cloning Of The Mpd Gene From Strain G1

A bacteria strain Stenotrophomonas acidaminiphila G1 has been isolated in this lab before.The strain can degrade chlorpyrifos(CP),methyl parathion(MP),phoxim,diazine phosphorus,triazophos(TAP),profenofos.The mpd gene from strain G1 was cloned into E.coli in this paper,then the influences of temperature,pH,organic solvents of methanol to the activity of MPH were studied,including the degradation rate of crude enzyme from strain G1 and that from the engineering bacteria on methyl parathion,chlorpyrifos and triazophos.A length of 996 bp of mpd gene from strain G1 was cloned to construct a recombination plasmid,and to transfer the E.coli.The recombinant plasmid pET28 a was expressed in E.coli BL21(DE3),a clear IPTG inducible band corresponding to about 30 kDa was observed in SDS-PAGE,and its enzyme activity was tested.The result showed that the recombinant protein could degrade MP.The degradation rate of MP was arrived at the highest point at temperature 47 ? when the influence of temperature to the activity of the MPH was studied.It showed that the optimal reaction temperature of MPH was 47 ?.The degradation rate of MP in alkaline conditions was higher than in neutral condition,and this showed that the high activity of MPH was in alkaline conditions,and the optimal reaction pH of MPH was 9.1.The whole experiment system designed in the volume of 5 mL when the effect of organic solvents methanol on the activity of MPH was studied.The results showed that methano could improve the activity of MPH when adding volume of methanol in the system was between 3%~50%.The degradation rate of the substrate significantly higher than that without any methanol;but the activity of MPH was suppressed when adding volume of methanol was more than 50%,and the degradation of the substrate was very little,far less than the control group without methanol.The result of the experiment(the infiuence of PNP to the activity of MPH)indicated that the degradation product PNP of MP had no effect on the activity of MPH.In the degradation experiment of crude enzyme from strain G1 and the engineering bacteria on two kinds of pesticide,the crude from the engineering bacteria had a better hydrolytic efficiency than the crude from the strain G1 through the comparisons of the degradation rates of the two different crudes on CP and TAP.The crude from the strain G1 had a higher degradation rate on CP than on TAP when the same amount of protein was added.The degradation ability of the strain G1 declined compared the results of MP degradation by the strain G1 before and crude enzyme from the strain G1 in this paper.In the degradation experiment,the crude enzyme from the engineering bacteria had a higher activity when the same amount of total protein was added,and the degradation rate of TAP was higher than MP.The reason might be that the metabolites of MP,O,O-dimethyl sulfur phosphate,has inhibitory effect on MPH.