Cloning And Enzymatic Characterization Of A Novel Pyrethroid-Degrading Gene

Pyrethroids pesticides are widely used throughout the world. With the extensive use of this kind ofpesticides, their residuals have caused some negative impact on ecological environment and humanhealth. Great concerns have been raised about the persistence and degradation of pyrethroids pesticidesresiduals in environment. Therefore, it is urgent to develop an efficient strategy to eliminate pyrethroidsresiduals. In biodegradation of pesticides, microbial degradation is economical, efficient and with fewbyproducts and has become the research focus in degradation of pesticides residuals.Strain YZ-1capable of degrading pyrethroids was isolated by enrichment procedure from activatedsludge, which was collected from wastewater treatment system of a pyrethroids manufactory. On thebasis of morphological characteristics, Biolog test and16S rDNA gene sequence analysis,strain YZ-1was identified as Ochrobactrum anthropi.In liquid medium, the optimal degrading condition was of30℃, pH7.0and2.0%inoculum size.84%of lambda-cyhalothrin was degraded after6days of incubation under this optimized condition.Strain YZ-1could endure high concentration of lambda-cyhalothrin and grow well in MSM containing400mg/L of lambda-cyhalothrin. The degrading ability of strain YZ-1was enhanced obviously withadditional carbon or nitrogen source. In the presence of glucose, sucrose and yeast extract, degradationof lambda-cyhalothrin were increased to97%,97%和89%, respectively. YZ-1was a broad-spectrumdegrading isolate and was able to degrade beta-cypermethrin, beta-cyfluthrin, permethrin anddaltamethrin except lambda-cyhalothrin.3-phenoxybenzoic acid, a major metabolite of pyrethroids, wasalso degraded by strain YZ-1. Thin layer chromatography analysis of metabolite indicated the produceof3-phenoxybenzoic acid during the degradation of lambda-cyhalothrin.In order to clone the pyrethroid-degrading gene, genomic library of strain YZ-1was constructed.Approximate12,000clones were generated in constructed library. The library was screened with athree-step strategy for the target gene. As a result of final screening, one clone which showedpyrethroids degrading ability was obtained and designed as ZY-I. ORF analysis revealed that there weretwo ORFs in recombinant plasmid of ZY-I. These two ORFs were subcloned for functional verification,respectively. Only ORF1was capable of degrading pyrethroids and was named pytY.Blast search indicated that pytY shared the highest similarity of85%with a putative esterase fromOchrobactrum anthropi ATCC49188, but30-46%similarity to some other esterases. pytY belonged toesterase family and possessed esterase conserved domain. Multiple sequence alignment with relatedesterase sequences revealed that PytY had typical pentapeptide structure of "Gly-X-Ser-X-Gly". pytYshowed no sequence similarity with reported pyrethroids degrading gene. It was proved a newpyrethroid-degrading gene with the accession number JQ025998in GenBank.The recombinant protein PytY was expressed at a high level after induction with1mmol/L IPTG at30℃. Purified PytY showed a single band on SDS-PAGE with an approximate molecular mass of42kDa. PytY was a protein of specific expression and was confirmed by Western Blot. The optimaltemperature was35℃and more than80%relative activity was remained after keeping2h at each temperature in the range of15-35℃. The optimal pH was7.5and degradation could be performedefficiently in pH range of6.0-8.0. Na+and Fe2+had no effect on enzyme activity. Mg2+promoteddegradation a little. K+and Zn2+could inhibit enzyme activity slightly, but Ag+and Hg2+had a stronginhibitory effect. To some extent, Tween-20and Tween-80increased enzyme activity. However, SDS,PMSF and DEPC showed intense inhibition. Chelating agent EDTA and1,10-phenanthroline had noobvious effect on enzyme activity. Km and Vmax values of purified PytY were2.34mmol/L and56.32nmol/min respectively when lambda-cyhalothrin was used as substrate.