【作者】 翟逸；

【导师】 闫艳春；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2012， 博士

【摘要】 拟除虫菊酯杀虫剂的大量使用给生态环境和人类健康带来很大的危害，已日益引起世界各国科学家的关注。如何清除环境中拟除虫菊酯杀虫剂残留已成为一个迫切需要解决的问题。农药残留的微生物降解因成本低廉、有效、二次污染小等特点，已成为农药残留生物修复的有效措施。本文从一拟除虫菊酯生产厂废水处理系统的活性污泥中分离到1株拟除虫菊酯高效降解菌YZ-1。通过形态学特征、生理生化实验和16S rDNA分子进化分析，将菌株YZ-1鉴定为人苍白杆菌（Ochrobactrum anthropi）。菌株YZ-1在液体培养基中的最适降解条件为30℃、pH7.0和2.0%的接种量。在此条件下6天内可将50mg/L的高效氯氟氰菊酯降解84%。菌株YZ-1可耐受并降解400mg/L的高效氯氟氰菊酯。添加葡萄糖、蔗糖和酵母提取物等外加碳氮源后，菌株YZ-1对高效氯氟氰菊酯的降解率分别提高到97%，97%和89%；除高效氯氟氰菊酯外，YZ-1还可有效降解多种菊酯类杀虫剂及其中间代谢产物3-苯氧基苯甲酸。代谢产物的薄层层析分析表明，在高效氯氟氰菊酯的降解过程中有3-苯氧基苯甲酸产生。为克隆拟除虫菊酯水解基因，构建了菌株YZ-1的基因组文库。在所构建的基因组文库中大约产生了12,000个转化子。通过三步筛选策略获得一个具有菊酯降解活性的阳性转化子，将其命名为ZY-I。测序结果的ORF分析表明，重组质粒中插入片段包含两个ORFs。亚克隆后的功能分析显示仅ORF1具有拟除虫菊酯水解功能，将其命名为pytY。pytY基因与Ochrobactrum anthropi ATCC49188中一个假定的酯酶有85%的相似性，而与其它酯酶序列相似性约为30-46%。蛋白家族和保守结构域分析表明，PytY属于酯酶家族，具有典型的“Gly-X-Ser-X-Gly”保守五肽结构。pytY基因与已报道菊酯降解基因在序列上相似性极低，是一个新的菊酯水解酯酶基因，在GenBank中的登录号为JQ025998。重组PytY蛋白在30℃时经1mmol/L IPTG诱导实现了高水平表达。SDS-PAGE分析显示在42kDa处出现了特异表达的蛋白条带。表达蛋白经Western Blot验证为诱导表达的特异蛋白。纯化的PytY重组蛋白最适反应温度为35℃，在20-45℃的温度范围内可有效降解高效氯氟氰菊酯，在15-35℃范围较稳定，保温2h后其剩余的相对酶活力均大于80%。PytY在pH6.0-8.0内均可有效降解拟除虫菊酯，最适反应pH为7.5。Na+和Fe2+对酶活性无明显的作用，Mg2+对酶活性有一定的促进作用。K+和Zn2+对酶活性表现出轻微抑制，而Ag+和Hg2+对酶活力产生了强烈的抑制作用。表面活性剂Tween-20和Tween-80对PytY的活性有一定的促进作用。SDS，丝氨酸蛋白酶抑制剂PMSF和组氨酸修饰物DEPC对降解酶的活性有强烈的抑制作用。螯合剂EDTA和1,10-phenanthroline对酶活性的影响不明显。当以高效氯氟氰菊酯为底物时，纯化PytY的Km=2.34mmol/L，最大反应速率Vmax=56.32nmol/min。更多还原

【Abstract】 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.更多还原