Isolation And Characterization Of Serratia Marcescens And Cloning Of The Methyl Parathion Hydrolase Gene

Organophosphorus pesticides?OPs?,the most widely used class of pesticides,can effectively reduce field pests and improve crop yields but also cause potential hazards to the environment and human health.Commonly used treatments of pesticide-contaminated wastewater include flocculation and sedimentation,chemical hydrolysis,oxidation,photocatalysis and biodegradation.Among these,biological processing through microbial strains has the advantages of being non-toxic and not forming residues or secondary pollution,making it an attractive alternative option for eliminating and degrading pesticides.The isolation of functional strains and microbial enzymes that could degrade a broad-spectrum of OPs efficiently is the basis of bioremediation.Nine kinds of organophosphorus pesticide degrading bacteria were isolated,MEW96 was chosen to further study due to its high degradation capacity of MP and less researches about pesticides degradation.MEW96 cells are gram-negative,short rods,0.9-1.2?m×1.5-2.0?m.Possesses motile,peri-flagellum,no-flagellum,no spores,and no capsule.Colonies on LB plates are red,circular and smooth and moistened with surface,slightly serrated on the edge.Combined with morphological observation,physiological and biochemical identification and 16S rDNA sequencing analysis,MEW96 was identified as S.marcescens subsp.marcescens.MEW96 could grow well in LB supplement with 50 mg/L pesticeds,and can eliminate methyl parathion within 5 days.The degradation of paraoxon was lower than that of MP,MEW96 degraded paraoxon?50 mg/L?by 90%after 5 d,and PNP gradually accumulates which was consistent with the reduction of paraoxon in the process of paraoxon degradation;MEW96 is also able to degrade dimethoate at a slightly lower rate,87.2%of dimethoate was degraded within 7 d.These results revealed its efficiency in pesticides degradation.MEW96 has many functions,such as chitin and lecithin hydrolysis,fungistasis toward fusarium oxysporum,botryosphaeria dothidea,thanatephorus cucumeris and botrytis cinerea etc.and inorganic phosphorus enrichment;Therefore,MEW96 can be applied for biocontrol,but also has good potential in bioremediation.An analysis of the‘MEW96'genome revealed a possible organophosphorous hydrolase gene,designated mpd,and the gene's expression was up-regulated with MP inoculations,as verified by real-time RT-PCR.Furthermore,mpd gene with size 945bp,encoding an methyl parathion hydrolase?MPH?,was cloned through the construction of recombinant MBW300.A clear IPTG inducible band corresponding to about 35 kDa was observed in SDS–PAGE.Furthermore,we realized the meaningful production of MPH,more than 2 g/L of soluble recombinant MPH was obtained through Ni-chelating affinity chromatography within 1.2 L shaking ferment liquid under the best induced condition?37 ^oC,0.3 mM IPTG,8 h?.With MP as a substrate,the optimum temperature and pH for MPH activity were³5°C and 11.0,respectively.Additionally,the activity was enhanced 23.5%by Co²⁺,60.5%by Fe²⁺and 18.4%by Cd²⁺,suggesting that it is a metallase.Cu²⁺,Zn²⁺,Mg²⁺,Mn²⁺and Ca²⁺ions had venial effects on the enzyme activity.The specific MPH activity was 3.66 U/mg under the conditions of 35°C and pH 9.0,with MP as the substrate.Accordingly,the catalytic constants Km and kcat were 430.5±49.5?M and1,467.2±6.1 min^–1.Thus,the kcat/Km value was 3.41?M^-1min^-1.An HPLC analysis revealed that strain MEW06's MPH converted MP to p-nitrophenol by hydrolyzing the P-O bond.When the enzyme concentration was 300?g/mL,methyl parathion was completely transformed to PNP after 24 h.MBW300 can eliminate methyl parathion within 60h,that reverals its ability toward methyl parathion degradation is higher than MEW96.