Isolation And Study Of Bacterial Strains That Is Capable Of Degradation Of Organophosphate Pesticides

Parathion, p-nitrophenylthiophosphate and its derivatives, are a group of organophosphate pesticides. They are highly toxic and widely used for insects control in many countries. However, these pesticides also cause serious problems to the environment and food safety. How to solve the problem has become a public concern.It has been known that parathion can be mineralized by some microorganisms through co-metabolism, i.e., one strain hydrolyzed parathion to produce p-nitrophenol that is whereafter break down by other strains. We have isolated several pesticid.~sdegrading bacteria and found one is capable of complete degradation of parathion.The strain was isolated from the polluted soil around ShaShi Pesticides Factory, China. Through chemotaxonomic characterizations and phylogenetic inference based on 1 6s rDNA sequence analysis, the strain was identified as a member of the genus Pseudomonas. This strain grows well under pH7-8 and 23-30C. It can tolerate highconcentration methylparathion up to 800ppm in basic medium and up to 2,000ppm in 0.1% glucose medium. The strain can metabolize methylparathion as its sole carbon and nitrogen sources. It can break down 300ppm methylparathion thoroughly in 22 hours.The strain possesses two plasmids. The smaller one has a molecular weight of 20kb. After treatment of mitomycin C, the strain lost its plasmids and so did parathion degradative capability. The gene coding for organophosphate hydrolase was abtained from its 20kb plasmid by PCR. DNA sequening shows that similarity of opd gene between the new species and Flavobacterium species reached 99%. An expression vector pET28-opd was constructed and transformed in E. coil BL2 I (DE3). The target protein was successfully expressed, leading to a similiar enzyme activity compared with the native strain.The organophosphate hydrolase was purified from the periplasm of WBC-3 to-2-homogeneity, through a whole process consisting of ammonium sulfate precipitation, Sephadex G-75 gel filtration, Q Sepharose FF ion-exchange column chromatography. The purified enzyme showed a single band on SDS-PAGE gel with an approximatemolecular weight of 33 X iO~. The optimal temperature and pH for the enzyme are 40450(2 and pH8.5. The enzyme can be can be activated by Ca2~ and Cd2~, but inhibitedby sulfhydryl reagents such as DTT and 2-mercaptoethanol and some heavy metal salts such as Mn2~ ~. Zn2~ .~. Fe2~ . Hg2~, as well as EDTA. Treating with SDS resulted in a complete lose of enzyme activity.Compared with the Flavobacterium sp. ATCC 27551 or Pseudomonas diminuta MG, which have been known as high efficient organophosphate pesticides degradating bugs, WBC-3 is capable of complete degradation of organophosphate pesticides such as parathion and has a higher tolerance to high-concentration pesticides. Moreover, the organophosphate hydrolase locates in the periplasm, which enables more convenient extraction and purification. During the treatment to lake pollution caused by aromatic compounds, WBC-3 showed a stable, powerful degradative capability. So the strain was regarded as a high-efficiency, broadspectrum degradative bacterium. It抯 worthwhile for a further research and exploitation.