Purification, Characteristics's Study Of OPD Enzyme And Preliminary Research On Its Gene Clone

The purification, characteristics of degrading enzyme from strain JS018 and its gene clone were studied in this paper, which is a kind of bacterium with highly degrading organophosphate pesticides and isolated by our lab:1 , Purification and homology of the organophosphorous pesticides degrading enzyme(OPD) produced by JS018The parathion-methyl degrading enzyme produced from the strain JS018 was purified by ammonium sulfate precipitation and Sephadex G-100 gel filtration. The purified enzyme appeared on PAGE with four bands. It was, then, further purified to homogeneity by chromatography on DEAE -52. The molecular weight of the enzyme was estimated to be 37K Da by means of SDS-PAGE.The N-terminal amino acid sequence was determined as follow : GAPFQNDVPPACYRFR. The closest relatives (29% relatedness) to the OPD of JS018 was those of AAK44461 and NP-334647. It suggests that the OPD might be a new organophosphorous pesticide degrading enzyme.2, Properties and kinetics of OPDTests showed that the optimal temperature and pH for OPD was 35℃ and pH 8.5, respectively. The OPD activity was stable at 15³5℃ and pH 5.5⁹.0. The OPD activity could be increased by the addition of 1% C₂H₅OH.EDTA at lmmol.L^-1 had a negligible effect, but inhibited at higher concentrations, on the enzyme's activity. The higher the EDTA concentration, the stronger the enzymatic inhibition was. When the EDTA concentration reached beyond 25 mmol.L^-1, the enzymatic activity was completely stopped. Tween-20 and Tween-80 (1mmol.L^-1) reduced the enzyme's activity by 21.6% and 16.1%. One mmol.L^-1 Fe²⁺ and Ca²⁺increased the activity by 28% and 14.1%, respectively. One mmol.L^-1 K⁺, Co²⁺, Li⁺, Na⁺ and Mg²⁺ had no effect on the enzyme. One mmol.L^-1 Ba²⁺,Fe³⁺, Ag⁺, Al³⁺,Cu²⁺,SDS, Hg²⁺ or Cu²⁺ strongly inhibited the enzyme's activity. Metal ions showed varying effects on OPD activity that suggested that the OPD was possibly a metallic enzyme. With methyl parathion as a substrate, the K_m was 63.559μmol.L^-1...