【作者】 王保战；

【导师】 李顺鹏；

【作者基本信息】 南京农业大学 ， 微生物学， 2010， 博士

【摘要】 拟除虫菊酯(以下简称菊酯)是人工合成的一类高效、广谱、神经致毒性杀虫剂,其结构与除虫菊(菊科小黄菊属中的一类植物)中的天然化学成分——“除虫菊素”类似。菊酯类杀虫剂被广泛应有于农业病虫害的防治和家庭卫生害虫的杀灭。目前菊酯类杀虫剂全球年销售额超过15亿美元,居世界杀虫剂市场的第2位；占我国农业杀虫剂使用面积的三分之一以上。随着对有机磷、有机氯等高毒农药的限制使用,菊酯类杀虫剂的使用量将进一步增加。相对传统的有机磷和有机氯农药,菊酯类杀虫剂对哺乳动物低毒,但最近的研究表明它对哺乳动物的神经、生殖、免疫、内分泌和心血管系统等仍有明显的毒副作用；此外,菊酯类杀虫剂对蜜蜂、鱼类和水生无脊椎动物等非靶标性生物高毒,比如对鱼的急性毒性浓度一般在μg/L级,安全系数为0.063左右。菊酯类杀虫剂在环境中的残留和降解越来越成为关注的焦点。研究表明,微生物的降解作用是环境中菊酯残留降解的主要途径。近些年报道了大量菊酯降解菌株,但是对菊酯在微生物体内降解途径、微生物降解菊酯的关键酶及其编码基因的研究还不够深入。本研究以实验室保存一株高效、广谱的菊酯降解菌株Sphingobium sp. JZ-1为研究材料。首先通过16S rDNA序列比对,发现它和Sphingobium faniae JZ-2T (98.6%)、Sphingobium cloacae JCM 10874T (98.5%)、Sphingobium vermicomposti VC-230T (97.4%)以及Sphingobium ummariense RL-3T (96.9%)同源性最高。DNA-DNA杂交结果显示,菌株JZ-1的DNA与以上四个菌株DNA同源性分别为Sphingobium faniae JZ-2T (30.2%)、Sphingobium cloacae JCM 10874T (23.3%)、Sphingobium vermicompostiVC-230T(10.9%)和Sphingobium ummariense RL-3T (7.9%)。菌株JZ-1主要的非极性脂肪酸是18：1ω7c,主要的2-羟基脂肪酸是14：0 2-OH；主要的极脂为乙醇酸磷酯、磷脂酰甘油、双磷脂酰甘油和神经鞘糖脂类；主要呼吸醌是Q-10；主要的多胺是亚精胺；G+C mol%含量为64.2%。综合以上对菌株生理生化、多胺、极性酯、呼吸醌和基因型(16S rDNA同源性分析、G+C mol%含量和DNA-DNA杂交)等特征的分析,将菌株JZ-1鉴定为Sphingobium中的新种,命名为文新鞘酯菌Sphingobium wenxiniae sp. nov. JZ-1T(=CGMCC 1.7748T=DSM 21828T)。通过液相色谱-质谱联机(HPLC-MS/MS)检测菌株JZ-1对氯氰菊酯的降解产物发现,氯氰菊酯首先通过断裂其结构核心——酯键,生成二氯菊酸3-苯氧基苯甲醛,3-苯氧基苯甲醛在醛脱氢酶的作用下转化成3-苯氧基苯甲酸。菊酯降解菌株Sphingobium sp. JZ-1及其细胞破碎后的粗酶液能在含150 mg/L氯氰菊酯农药平板上产生明显的水解圈。诱导实验证明菊酯水解酶为组成型酶,不需要菊酯底物诱导。菌株JZ-1能以氯氰菊酯为唯一碳源生长,能完全降解2～30 mg/L浓度的氯氰菊酯。通过构建基因组DNA文库,成功地从菊酯降解菌株Sphingobium wenxiniae JZ-1中克隆到一个新的菊酯水解酶基因,命名为pytH。该基因含840个碱基,编码280个氨基酸,G+C mol%含量为66.55%,没有信号肽序列。通过和PDB (Protein Data Bank)数据库中的氨基酸序列比对结果发现,PytH和α/β-水解酶折叠蛋白家族中的一些成员相似性最高：和来源于巴西橡胶树中的羟基乙腈裂解酶(PDB:2G4LA)有24%同源性；和来源于大肠杆菌羧酸酯酶BioH (PDB:1M33A)有23%同源性；和来源于金杆菌属的Y-内酰胺酶(1HKHA)有23%同源性；和来源于詹森菌属的咔唑水解酶(1J1IA)有20%的同源性,并具有α/β-水解酶家族蛋白的典型三联体催化活性位点Ser-His-Asp/Glu (Ser78、Asp202和His230)以及该家族蛋白的特征序列标签GXSXG (GHSLG从76位到80位)。通过PCR定点突变的方法对PytH可能的三联体催化位点和特征序列进行突变,突变体(Ser78Thr、Asp202Asn、His230Ile. Gly76Ser和Gly80Cys)酶活性完全丧失。因此推测PytH为α/β-水解酶折叠蛋白家族中的一个新成员。以pET29a为载体,构建表达质粒pET29a-pytH,实现了PytH蛋白在E. coli Rosseta(DE3)和E. coli BL21(DE3)中的融合表达,并用镍离子亲和层析柱对融合蛋白PytH实现了纯化。PytH是分子量为31 kD,等电点(pI)为4.85的单体酶。0.5 mmol/L的Ag+、Ni2+Cu2+、Hg2+和Zn2+；10 mmol/L的表面活性剂SDS和Tween80; 0.5 mmol/L的丝氨酸蛋白酶抑制剂PMSF,组氨酸变性剂DEPC以及巯基试剂pCMB和碘乙酰胺都能强烈的抑制PytH的活力。10 mmol/L的Triton X-100能抑制大约20～30%的酶活。10 mmol/L的金属离子螯合剂EDTA和1,10-二氮菲对酶抑制作用小于10%。菊酯的分子结构显著影响菊酯水解酶PytH对菊酯的降解效率,菊酯分子结构中酯键附近空间位阻越大,降解速率越慢。PytH对测定的菊酯降解速率顺序为：氯菊酯>甲氰菊酯>氯氰菊酯>功夫菊酯>氰戊菊酯>溴氰菊酯>联苯菊酯。PytH能水解短链的对硝基苯酚羧酸酯,其中对硝基苯酚乙酸酯的水解速率最高。随着羧酸链的增长,酶对底物催化效率不断下降。对超过6个碳原子的羧酸与对硝基苯酚形成的酯,PytH对其完全失去催化活力。由于脂肪酶更倾向于水解长链脂肪酸酯,而酯酶更倾向于水解短链羧酸酯,所以PytH为酯酶,而不是脂肪酶。PytH对氯氰菊酯、氯菊酯的降解没有顺反异构选择性,对甲氰菊酯有两种立体异构体(αR-,αS-)氰戊菊酯有四个立体异构体(aS,2R-,aR,2R-,aS,2S-,aR,2S-)也没有手性选择性,这不同于以往报道的菊酯水解酶。更多还原

【Abstract】 Synthetical pyrethroids (SPs), which are synthetic version of an extract from the chrysanthemum, are now the major class of insecticides used for insect control in agriculture and households as a replacement for more toxic and environmentally persistent organochlorine and organophosphorus pesticides. Currently, SPs annual sales are estimated to reach above 1.5 billion dollars and rank the second in global insecticide sales. In China, SPs account for 1/3 of total pesticides application area.Although pyrethroid pesticides generally have lower acute oral mammalian toxicity than organophosphate and organochlorine insecticides, exposure to pyrethroid pesticides might cause damage to reproductive, immune, endocrine and cardiovascular systems. Especially, most pyrethroid pesticides possess acute toxicity to some nontarget organisms, such as bees, fish, and aquatic invertebrates, with LC50 values less than 0.5μg/L and factor of safety lower than 0.063 to fish.Great concerns have been raised about the persistence and degradation of pyrethroid pesticides in the environment. Compared with other physicochemical methods, bioremediation is a cost-effective, convenient and security solution to eliminate the pyrethroid residues in soil, sediment, and agricultural products. Although many pyrethroid-degrading microorganisms have been isolated from the environment, there were only about five pyrethrod-hydrolyzing enzymes purified and three genes of pyrethrod-hydrolyzing enzymes were cloned.A SPs-degrading bacterium, designated JZ-1, isolated from activated sludge treating SPs-manufacturing wastewater, was determined using a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain JZ-1T belongs to the genus Sphingobium, as it showed highest sequence similarities to Sphingobium faniae JZ-2T (98.6%), Sphingobium cloacae JCM 10874T (98.5%), Sphingobium vermicomposti VC-230T (97.4%) and Sphingobium ummariense RL-3T (96.9%). Chemotaxonomic data also supported the affiliation of the strain to the genus Sphingobium:C18:1ω7c was the predominant non-hydroxylated fatty acid and C14:0 2-OH was the major hydroxylated fatty acid; Ubiquinone Q-10 was the main respiratory quinine; diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingoglycolipids (SGL) and two unidentified phospholipids was the major polar lipids; and spermidine was the major polyamine component. Strain JZ-1 showed low DNA-DNA relatedness values with Sphingobium faniae JZ-2T (30.2%), Sphingobium cloacae JCM 10874T (23.3%), Sphingobium vermicomposti VC-230T (10.9%) and Sphingobium ummariense RL-3T(7.9%), respectively. Based on the results of phenotypic, genotypic and phylogenetic properties, strain JZ-1T represents a novel species of the genus Sphingobium, for which the name Sphingobium wenxiniae sp. nov. is proposed. The type strain is JZ-1T (=CGMCC 1.7748T=DSM 21828T).The strain JZ-1 catalyzed the hydrolysis of cypermethrin to 2,2-dimethyl-3-(2,2-dichlorovinyl)-cyclopropanecarboxylic acid and 3-phenoxybenzaldehyde, which was converted to 3-phenoxybenzoic acid. The strain JZ-1 and cell-free extract produced a visible transparent halo of pyrethroid degradation on agar plates supplemented with 150 mg/L cypermethrin. Pyrethroid hydrolase activity was detected in both cell-free extracts prepared from cells grown on LB with and without cypemethrin, indicating that pyrethroid hydrolase was expressed constitutively in strain JZ-1. The strain was capable of utilizing cypermethrin as carbon source and degrading 2-30 mg/L cypermethrin completely.A novel esterase gene, pytH, encoding a pyrethroid-hydrolyzing carboxylesterase was cloned from Sphingobium sp. JZ-1. The gene contained an open reading frame of 840 bp encoding for a protein of 280 amino acids. The G+C mol%content was 66.55%. No potential signal sequence was found. The deduced protein was compared with the known enzymes available from the Protein Data Bank (NCBI database). PytH showed the highest homology with someα/β-hydrolase fold proteins, e.g., a hydroxynitrile lyase from Hevea brasiliensis (24% identity), a carboxylesterase BioH from E. coli (23% identity), an (-) y-lactamase from Aureobacterium sp. (23% identity), and a hydrolase from Janthinobacterium sp. (20% identity). PytH contains the typical catalytic centers Ser-His-Asp/Glu and highly conserved pentapeptide GXSXG ofα/β-hydrolase fold proteins. The mutant enzymes Ser78Thr, Asp202Asn and His230Ile (catalytic centers Ser-His-Asp mutations), and Gly76Ser and Gly80Cys (highly conserved pentapeptide GXSXG mutations) did not show any specific activity against pyrethroids at all. Thus, the enzyme PytH was proposed to be a novel member ofα/β-hydrolase fold proteins.PytH was expressed in Escherichia coli BL21 (DE3) and Escherichia coli Rosseta (DE3), and was purified using Ni-nitrilotriacetic acid affinity chromatography.PytH was a monomeric structure with a molecular mass of approximately 31 kDa and a pI of 4.85. No cofactors were required for enzyme activity. The enzyme was strongly inhibited by many metal ions (Ag+, Ni2+, Cu2+, Hg2+and Zn2+; 0.5 mM), the surfactants SDS and Tween 80 (10 mM), the Ser protease inhibitor PMSF, His modifier DEPC (0.5 mM), and thiol reagent pCMB and iodoacetamide (0.5 mM); while Triton X-100 (10mM) showed only slight inhibition (20-30% inhibition), and chelating agents EDTA and 1, 10-phenanthroline (10 mM) had little effect on the enzyme activity (less than 10% inhibition).PytH was capable of hydrolyzing all of the pyrethroids tested, and hydrolysis efficiencies were dependent on the pyrethroid molecular structure, with the hydrolysis rates descending as follows:permethrin> fenpropathrin> cypermethrin> cyhalothrin> fenvalerate> deltamethrin> bifenthrin. PytH showed the highest activity with p-nitrophenyl acetate, and the activities decreased with the increase of the aliphatic chain length. No lipolytic activity was observed with esters containing an aliphatic chain length longer than six carbon atoms, indicating that PytH was an esterase and not a lipase. The specific activities for trans- and cis-permethrin (or cypermethrin) were found to be relatively similar, and stereoisomers of fenpropathrin or fenvalerate were hydrolyzed at an approximately equal rate, indicating that PytH lacked isomer selectivity for pyrethroid pesticides.更多还原