Degradation Characteristics Of Pseudomonas Stutzeri YC-YH1 For The Organophosphorus Pesticide

Extensive use of organophosphorus pesticides(OPs)hasbrought about general organophosphorus pesticide residues and environmental pollution though OPs could effectively protect the crops and vegetables.Air, soil, water and other environments have been polluted in defferent degrees, seriously harming human safety. Finding fast and effective way to remove pesticide residues in the environment has become a hot research. Using microbial enzyme to degrade organophosphorus pesticides is non-toxic, no residue, no secondary pollution,etc. It is a safe, effective and inexpensive method of degradating organophosphorus pesticides. The enzymatic reaction is a quick process, and most of the organic phosphorus degrading enzymesare intracellular enzymes, which would make organic phosphorus into the cellular processes become the rate-limiting step, severely limitting the rate of degradation.In this study, two organophosphorus hydrolase genes mpd and ophc2 were cloned from an organophosphorus pesticide-degrading bacteria Pseudomonas stutzeri YC-YH1.The recombinant plasmidpET-mpdand pET-ophc2 were acquired by inserting the two genes into the expression vector pET-32 a and then they were transformed into E. coliBL21(DE3) respectively. We used Ni-affinity column to purify enzymes and then study the nature of the two enzymes. It was suggested that the optimal temperature of MPH is 40℃, and the MPH displays high activity in the pH range of 8 to 12. The optimal temperature of OPHC2 is 30℃. In the pH ranging of 8 to12, OPHC2 has higher activity. The mixture of MPH and OPHC2 in the proportion of 1: 1 shows higher activity in the range of 30℃ to 40℃, the enzyme shows more than 95% activity in the range of pH8~12. A variety of metalions affect the activity of the enzymes and the enzymes are sensitive to SDS and EDTA, but the compound could reduce the sensitivity to the metalions.In addition,the twin-arginine signal peptide of trimethylamine N-oxide reductase(TorA) from E. coliand mpd from Pseudomonas stutzeri YC-YH1 were cloned. Vector pHSG299(sTorA-mpd)was constructed anda method of electroporation transformation of the strain YC-YH1 was established. MPH periplasmic secretion was measured. Compared with wild-type strain YC-YH1, extracellular enzyme activity increased 36.4% biodegradation.The degredation capacity increased 8.7% within 48 h and supernatant degradation capacity increased 44.4% within 12 h. Methyl parathion hydrolase was transported to the periplasm throughTat Pathway, which could overcome the limitations of substrate absorption.MPH periplasmic secretion improved degradation. It is important for bioremediation of the polluted environment and industrial organophosphorus degradation.