Isolation And Characteristics Of Methomyl-degrading Strains,Cloning Of The Gene Responsible For Methomyl Hydrolysis

Methomyl is a highly toxic oxime carbamate insecticides.It is widely used in agriculture as common and compound agents,which can control pests by inhibiting acetylcholinesterase(AChE).In 1997,methomyl was listed as suspicious endocrine disruptor by the World Wildlife Fund,and it could damage antioxidant system of the body.In addition,it may cause genetic toxicity.In recent years,many physical and chemical methods have been used to treat methomyl.A few biodegradation ways have been reported.Biodegradation is the main method to eliminate pesticide in the environment,which has the advantages of high efficiency,easy operation and green economy.Therefore,isolating of the methomyl-degrading strains and cloning of the genes responsible for its degradtion have an important role in the remediation of methomyl.The main results of this study are as follows:1.The bacterial strains named MDW-2 and MDW-3 were isolated by using the sludge from the wastewater-treating system of a pesticide manufacturer.They were preliminarily identified as Aminobacter sp.and Afipia sp.,respectively.They could degrade 50 mg/L methomyl completely within 3 d by the cooperative metabolism.Methomyl was first converted to an intermediate metabolite by strain MDW-2,but the metabolite could not be sequentially degraded by strain MDW-2.However,it could be degraded by strain MDW-3.2.Strain MDW-2 degraded methomyl through the hydrolysis of its carbamate bond,which was the detoxification and key step during the degradation of methomyl.So the growth and degradation characteristics of strain MDW-2 were primarily studied.The optimum growth temperature and pH of strain MDW-2 were 30? and 7.0,respectively.The strain MDW-2 could degrade 200 mg/L methomyl in 18 h with the inoculation amount of 1%,and the optimum degradation temperature range and pH were 30?35? and 7.0,respectively.3.On the basis of HPLC-MS and the co-existence of strains MDW-2 and MDW-3 during the degradation process,the combined degradation pathway of methomyl was proposed.Methomyl was hydrolyzed to methomyl oxime and methylcarbamic acid by strain MDW-2,and methylcarbamic acid was utilized as the carbon source for the growth of strain MDW-2.Then strain MDW-3 could degrade methomyl oxime and use it as the carbon source.4.By ammonium sulfate precipitation,DEAE-Sepharose fast flow anion exchange chromatography,Q-Sepharose fast flow aion exchange chromatography and Sephadex-200 gel chromatography,the hydrolase responsible for the degradtion of methomyl was purified from crude enzyme of strain MDW-2.And it was further analyzed by MALDI-TOF-MS spectrometry.An orf06623 was obtained by analyzing the amino acid sequences of MALDI-TOF-MS spectrometry and genome.The cloned gene was named ameH which was 951 base pairs in length and could encode a protein composed of 316-amino acid.By analyzing the amino acid sequence,it was found that AmeH showed the highest identity with the putative formamidase C869.04 from Schizosaccharomyces pombe ATCC 24843(27%),acetamidase from Mycobacterium smegmatis NCTC 8159(28%)and formamidase from Methylophilus methylotrophus(26%).It indicated that ameH may was be a novel gene.5.The ameH was expressed in Escherichia coli BL21(DE3)and the recombinant protein was purified by Ni2+-NTA affinity chromatography.The single strip was about 33 kDa.And the activity of pure enzyme was detected by HPLC-MS,which could transform methomyl to methomyl oxime.It indicated that ameH gene could encode the hydrolase responsible for the degradtion of methomyl.