Cloning And Prokaryotic Expression Of A Methyl Parathion Hydrolase Gene From Pseudomonas Sp. 1-7

Methyl parathion was widely used as a organophosphate insecticide throughout the world and had made a great contribution to agriculture and plant protection. It was used to control destructive insects for crop such as rice,cotton,corn,but it caused enormous damage to non-target organisms and have resulted in global environmental contamination especially in developping country.People found the bacterium in natural environment with the capability of degrading organophosphate pesticides, which provided us a good way to eliminate the pollution of organophosphate pesticides. At present,many strains with the capability of degrading methly parathion were found, including Pseudomonas sp.,Bacillus sp.,Flavobacterium sp.,Ochrobactrum sp.,Arthrobacter sp.,Bacillus cereus sp.,Serratia sp.and so on.Investigation showed that degrading microorganism could produce a enzyme which could degrade organophosphate insecticides by hydrolyze phosphate ester-bond .As so far ,Methyl parathion hydrolase was known most clearly ,which optimal substrate was methly parathion. Many methyl parathion hydrolase genes had been cloned and made prokaryotic expression. Besides methyl parathion hydrolase have been purified and characterized.In the paper, a bacterium with the capability of degrading methyl parathion, identified as Pseudomonas sp. by 16S rDNA.,was isolated from active sludge in organophosphate-treated aeration basin and designated Pseudomonas sp.1-7. The bacterium could excrete methyl parathion hydrolase constitutivly.Further more,PNP could also be degraded,which was primary metabolism production from methyl prarthion.A genome library was constructed using Pseudomonas sp.1-7 genome by shut gun method. The nucleotide sequence of methyl parathion hydrolase gene ophc3 was obstained from the library. The ophc3 gene was 975 bp long with G+C content of 63%, comprising one open reading frame encoding a polypeptide of 324 amino acids which included a signal peptide of 24 aa. Analysis of protein sequence of ophc3 showed that high similarity of 98% with organphosphorus hydrolase gene ophc2,but the ophc3 gene exhibited the low similarity below 50% with mpd gene . The ophc2 gene had been assigned and the GenBank accession number was FJ821775.The ophc3 with signal peptide was inserted into the vector pET-30a ,so prokaryotic expression recombinant vector pET-ophc3 was constructed and transfered into E.coli BL21.The expression products bored normal bioactivity.Eepressing recombinant protein was purified by Ni-NTA column and SDS-PAGE showed that purified protein was a single band. Characterization of methyl parathion hydrolase indicated that OPHC3 with a molecular weight of 36 kD had optimum activity for the reaction at 45℃and pH 8.0,Km was 78.9μmol/L,Vmax was 3.0μmol/L·min.Most metal ions and chemicals had no effect on the activity of OPHC3, but SDS inactivated this enzyme.