Isolation,Identification Of Monocrotophos Degrading Strains & Degradation Mechanism

Monocrotophos is a highly toxic organophosphorus insecticide,which was prohibited in China in 2007,but food residue of monocrotophos was still detected in recent years.Great concerns have been raised about the environmental pollution and ecological destruction of monocrotophos.The degradation of monocrotophos has three forms in the environment,including photochemical degradation,chemical degradation and biodegradation.Bioremediation based on the degradation ability of microorganisms plays an important role in the degradation of monocrotophos.The aim of this study was to isolate monocrotophos-degrading strains,elucidate the degradation pathway,clone the degradation-related genes,which would be helpful to study the gene function and utilize microbial resource to remove environment pollution.1.Isolation,identification of monocrotophos-degrading bacteriaUsing monocrotophos as the target substrate,two enrichments designated as Hunan enrichment and Jiangdu enrichment were obtained by a successive enrichment method with the soil sample collected from the rice field in Hunan province and Jiangsu province.Two strains named YW1 and YW16 were isolated from Jiangdu enrichment.YW6 was isolated from Hunan enrichment.UV and HPLC assay showed that YW6 and YW16 could degrade monocrotophos,but YW1 could not degrade monocrotophos.YW1 and YW6 could use N-methylacetoacetamide as the sole carbon source for growth,but YW16 could not.We speculated that YW1 could use intermediate metabolite?N-methylacetoacetamide?produced by YW16 during the degradation of monocrotophos for gowth.Because HPLC and UV could not detecte the small molecular weight of N-methylacetoacetamide,the degradation of YW1 is not studied in this study.We will study the degrding mechanism of monocrotophos by YW6 and YW16.According to the morphological observation,physiological biochemical tests,16S rRNA gene phylogenetic analysis,strains YW1,YW6 and YW16 were identified as Comamonas sp.,Starkeya sp.,and Sphingobium sp.,respectively.YW1 was a potential new species of Comamonas from the results of 16S rRNA gene phylogenetic analysis.Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain YW1 had the highest similarities with Comamonas aquatica LMG 2370T?98.5%?,followed by Comamonas kerstersii LMG 3475T?97.7%?,and Comamonas terrigena LMG 1253T?97.7%?,and showed less than 97.0%sequence similarities with other strains from the family of Comamonadaceae.Strain YW1 contained C16:0?30.1%?,C14:0?5.8%?,C17:0 cyclo?7.4%?,summed feature 3?C16:1?6c and/or C16:1?7c;25.4%?and summed feature 8?C18:1?6c and/or C18:1?7c;15.3%?as the main cellular fatty acids.The predominant ubiquinone was Q-8.Diphosphatidylglycerol?DPG?;phosphatidylglycerol?PG?;phosphatidylethanolamine?PE?;unknown phospholipids?PL?;unknown lipids?L?were the predominant polar lipids.The DNA G+C content of the total DNA was 65.3 mol%.DNA-DNA hybridization results showed that strain YW1 had low genomic relatedness with C.aquatica LMG 2370T?31.5±3.9%?,C.kerstersii LMG 3475T?21.6±6.6%?,and C.terrigena LMG 1253T?17.2±4.4%?,respectively.On the basis of the above results,we propose that the strain YW1T represents a novel species in the genus Comamonas,and designated as Comamonas jiangduensis sp.nov..2.Degradation characteristics of monocrotophos by strain YW16 and cloning&expression of organophosphorus insecticide hydrolase gene opdAThe optimal temperature and pH for the degradation of monocrotophos by strain YW16 were 30?,7.0-7.5 respectively.It was found that the addition of other carbon sources,such as sodium citrate,maltose,and sucrose could promote the degradation of MCP.When the concentration of monocrotophos was more than 2mM,it had a strong inhibitory effect on the degradation.Strain YW16 could utilize dimethyl phosphate as the sole carbon source for growth,it was also able to hydrolyze other organophosphorus insecticides,including clorpyrifos,fenitrothion,phoxim,parathion,and triazophos etc.Strain YW16 could hydrolyze monocrotophos into dimethylphosphate and N-methylacetoacetamide,and utilize dimethylphosphate as the sole source of carbon for growth,but could not degrade N-methylacetoacetamide further.We speculated that the degradation of monocrotophos was not complete.The hydrolysis of clorpyrifos by strain YW16 could form a clear transparent halo around the colonies LB agar containing clorpyrifos.Using this approach,a 7107-bp DNA fragment,including an organophosphorus hydrolase encoding gene opdA,was cloned from strain YW16 using the shotgun technique combined with SEFA-PCR.Six ORFs were found in the DNA fragment.The first ORF encoding a putative transposase,shares highest similarity with Agrobacterium tumefaciens P230?100%?and Escherichia coli?99%?.The second ORF encoding a putative integrase,shares highest similarity with Agrobacterium tumefaciens P230?95%?and Sphingomonas wittichii RW1?88%?.The third ORF encoding a putative ATP-binding protein,shares highest similarity with Agrobacterium tumefaciens P230?99%?and Sphingomonas wittichii RW1?88%?.The fourth ORF encoding a putative organophosphorus hydrolase,shares highest similarity with Agrobacterium tumefaciens P230?99%?,named opdA.The fifth ORF encoding a putative aromatic hydrolase,shares highest similarity with Sphingobium fuliginis ATCC 27551?91%?and Sphingomonas wittichii RW1?88%?.The sixth ORF encoding a conjugal transfer protein,shares highest similarity with Sphingobium sp.SYK-6?69%?.The combination of a putative transposase and Tral suggests that opdA may transfer from strain YW16 to other bacteria through conjugation.OpdA was expressed in Escherichia coli BL21 and purified using Ni-nitrilotriacetic acid affinity chromatography.OpdA was able to hydrolyse a wide range of organophosphate pesticides.The optimum initial temperature of the enzyme was 40?and the initial pH was 9.0.1 mM Cu²⁺ could significantly inhibit the activity of OpdA,Zn²⁺ and Co²⁺ could promote the activity of enzymes,OpdA could catalyze the hydrolysis of many kinds of organic phosphorus pesticides.It was found that the degradation rate of different pesticides was different,and we could guess that this might be related to the conformation of the pesticide,and it should be further studied.3.Degradation characteristics of monocrotophosby strain YW6 and its metabolic pathwayStrain YW6 could completely degrade monocrotophos.The maximum removal of monocrotophos was observed at 30?.The optimal pH was 7.0-7.5.Addition of maltose and glucose resulted in slowing down of the initial rate of degradation of MCP.The concentration of monocrotophos affected the degradation of YW6.The concentration higher than 0.6 mM inhibited the degradation strongly.In addition to the degradation of MCP,strain YW6 was also able to degrade a wide range of OPs containing P-O-C bond.A MCP degradation pathway was proposed on the basis of metabolite production patterns and identification of the metabolites.?E?-Dimethyl 1-methyl-3-?methylamino?-3-oxo-1-propenyl phosphate?compound??was transformed to?Z?-Dimethyl 1-methyl-3-?methylamino?-3-oxo-1-propenyl phosphate?compound??,?Z?-Dimethyl 1-methyl-3-?methylamino?-3-oxo-1-propenyl phosphate was hydrolyzed to N-methyl acetoacetamide?compound??by phosphotriesterase.N-methyl acetoacetamide?compound??was transformed to N-methyl-4-oxo-pentanamide?compound??by insertion of an carbon atom between the carbon atoms of the carbonyl group in N-methyl acetoacetamide.N-methyl-4-oxo-pentanamide?compound??was transformed to 5-?methylamino?-5-oxo-pentanoic acid?compound??.5-?methylamino?-5-oxo-pentanoic acid was cleaved by amidase to glutaric acid?compound??and methylamine.4.Cloning and expression of monocrotophos-degrading related mmH of strain YW6As strain YW6 could use the intermediate N-methylacetoacetamide as the sole carbon and nitrogen source for growth.Also,it could hydrolyze the amide bond of p-acetaminophenol?a colorless compound?and produce a purple-red color product in LB plate supplemented with p-acetaminophenol.Both N-methylacetoacetamide and p-acetaminophenol have amide bond.So,p-acetaminophenol was used as the substrate to construct the genomic library,and a positive clone presenting amidase activity from the genomic library and giving a color change around the colony,was obtained.A novel amidase gene,mmH,which was responsible for the catalyzing the amide bonds cleavage in the amide pesticides,was cloned from the strain YW6,mmH contains a 1476 bp open reading frame that encodes a 492-amino acid protein.The deduced molecular weight of MmH was 52,467 Da.The sequence of mmH was compared with other known enzymes available in the NCBI database.MmH shows the highest identity?50%identity?with a well-characterized amidase from strain Pseudomonas sp.CSBL00001.The highly conserved catalytic triad of amidase signature?AS?enzyme family,Ser-Ser-Lys and the basic element G-?GAV?-S-?GS?2-GX-?GSAE?-?GSAVYCT?-X-?LIVMT?-?GSA?-X6-?GSAT?-X-?GA?-X-?DE?-X-?GA?-X-S-?LIVM?-R-X-P-?GSACTL?were found in the amino acid sequence of MmH.Thus,we concluded that MmH from strain YW6 was a new member of the amidase signature family.To confirm the correlation of mmH to the degradation of monocrotophos in strain YW6,mmH was disrupted through a single-crossover,and the resultant strain YW-DM could degrade monocrotophos,but lost the ability to grow with monocrotophos as the nitrogen source,indicating that MmH catalyze the transformation of the downstream intermediate of monocrotophos.MmH was expressed in Escherichia coli BL21 and was homogenously purified using Ni-nitrilotriacetic acid affinity chromatography.The enzyme yielded a single band with a molecular weight of 53 kDa on SDS-PAGE,which is in good agreement with the molecular mass deduced from amino acid sequence?52,467 Da?.MmH displays maximum enzymatic activity at 40? and pH 8.0.1 mM Hg²⁺ was able to severely inhibit its activity,and the addition of Mg²⁺,Mn²⁺ increased the enzymatic activity by 10%.Co²⁺ and Ca²⁺ had slight inhibitory effect on the amidase activity of MmH.Cu²⁺,Ni²⁺ and Zn²⁺ showed 30-40%inhibition of MmH hydrolytic activity.In addition,the inhibition of metal ion chelating agent EDTA was not obvious.SDS,polyethylene glycol octyl phenyl ether,Tween80,mercaptoethanol and iodine acetamide showed inhibition effect on the enzyme activity.MmH could also efficiently hydrolyze a variety of amine compounds such as 5-?methylamino?-5-oxo-pentanoic acid,propanil,4-acetaminophenol,and chlorpropham.