Isolation And Identification Of Organophosphorus Degrading Strain And Characterization Of The Relevant Methyl Parathion Hydrolase

Methyl parathion(MP),a common organophosphorus pesticide,is applied extensively for crop protection especially in developing countries.However,it has toxic effects on the environment including water,air,and soil.Therefore,the isolation of high and MP-degrading strains,cloning of methyl parathion degradation gene(mpd)and expression of methyl parathion hydrolase(MPH)play important roles in solving the problem on the residue of MP.In this study,a MP-degrading bacterial strain,designated as MP-1,was isolated from a former pesticide-manufacturing waste land.Polyphasic taxonomic studies showed that MP-1 is a Gram-negative,non-spore-forming,rod-shaped,and motile bacterium.Phylogenetic analysis based on 16S rRNA gene sequences demonstrated that MP-1 belongs to the genus Burkholderia,showing the highest sequence similarity to Burkholderia grimmiae R27T(98.45%).In addition,the gyrB and recA gene segments of strain MP-1 exhibited less than 89.03%and 95.05%similarities with the above most-related type strains,respectively.The G+C content of strain MP-1 was 62.6 mol%.The major isoprenoid quinone was ubiquinone Q-8.The predominant polar lipid was composed of phosphatidyl ethanolamine,phosphatidyl glycerol,aminolipid,and phospholipid.The principal fatty acid in strain MP-1 was C18:1?7c/C18:1?6c(23.33%).The DNA-DNA relatedness values between strain MP-1 and three type strains were ranging from 24.60%to 37.40%.In accordance with phenotypic and genotypic characteristics,strain MP-1 represents a novel species of the genus Burkholderia,for which the name Burkholderia jiangsuensis sp.nov.MP-1T was proposed.Strain MP-1 was able to metabolize MP and employed MP as the sole carbon source for growth.Strain MP-1 could efficiently degrade MP,ethyl-paraoxon,malathion,fenitrothion,dichlorvos,chlorpyrifos,methamidophos,phoxim and diazinon.The degradation rate of MP was up to 173 ± 7 mg l-1 h-1.The gene Bjmpd was cloned from MP-1.The BLAST sequence search results indicated that the amino acid sequence of enzyme BjMPH(encoded by gene Bjmpd)has only three animo acid residues difference with MPH(from Pseudomonas sp.strain WBC-3),including Ser166,Val281,Ala301.The enzyme BjMPH has a broad substrate spectrum,its hydrolytic efficiency for MP was significantly greater than MPH.Among these,Ala301 is located in activity pocket which has the distance of 4.7 A from the catalytic center?-metal ion.The kcat/Kmof mutant T98N was improved 4 times comparing to the BjMPH towards MP.