【作者】 李康；

【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】 筛选分离毒死蜱（chlorpyrifos，CP）降解微生物解决毒死蜱农药残留给水体和土壤环境带来的污染问题是一个可行的生物修复技术，对于研究农药残留的生物降解具有重要意义。副球菌（Paracoccus sp.）TRP是一株对毒死蜱具有很强降解能力的菌株。本论文以其为研究对象，研究了菌株的基本特性，经多种基因克隆方法相结合获得一个新的毒死蜱降解酶基因，并研究了毒死蜱降解酶结构与功能的关系，研究内容如下：对本实验室筛选的TRP菌株进行复壮，菌株在第2天到第4天对CP的降解最快，到第7天对50mg/L CP的降解率可达83.3%。菌株对5～100μg/mL Amp具有抗性；对5～100μg/mL Tc及Gm不具有抗性。菌株不能够产生糖脂类阴离子生物表面活性剂。菌株组成型表达毒死蜱降解酶。采用Solexa测序技术测定了副球菌TRP的基因组序列（GenBank登录号AEPN00000000）。基因组大小约为3.9Mb，GC含量62.89%。预测所得CDS数为3961个。共得到155个代谢图，其中25个与异源物质代谢有关，包括除草剂阿特拉津代谢图和脱氮代谢图。在基因组序列上找到96个串联重复区域，200个转座子，147个IS，45个tRNA和5套rRNA。副球菌TRP与Paracoccusdenitrificans Pd1222在进化上关系较近，大部分基因的同源性较高。在副球菌TRP菌株中鉴定了与P. denitrificans Pd1222中类似的脱氮作用所需的所有四个氧化还原酶类基因。TRP菌株基因组的分析有助于鉴定多种不同的降解基因，进一步阐明微生物降解毒死蜱代谢途径。这是迄今为止关于毒死蜱降解副球菌菌株的基因组测序的首次报道。从副球菌TRP中克隆得到了毒死蜱降解基因cpd，基因全长927bp，GC含量60.73%。cpd基因与目前已发表的有机磷降解酶基因的同源性较低，是一个新的有机磷降解酶基因。CPD蛋白由308个氨基酸残基构成，分子量为33.8kDa。带电量为-7.0。等电点为4.8305。CPD蛋白是酯酶_脂酶超家族的成员之一，不仅具有Ser155-Asp251-His281催化三联体和模序GDSAG，还具有酯酶保守的底物结合口袋ILYIHGGGWSFCSA。在E.coli BL21（DE3）中表达的CPD融合蛋白大小约为53.9KDa，具有酯酶活性。CPD蛋白在行使酯酶功能时，不需要金属离子；1mmol/L PMSF、1mmol/L DEPC和0.025%SDS显著抑制其酯酶活力，0.025%Tween-20显著增强其酯酶活力；CPD蛋白行使酯酶功能时需要Ser和His。在15min内CPD酶液对50mg/L CP的降解率为63.5%±2.14。酶活力为59.62U/mg。将四环素Tc盒插入TRP菌株cpd基因中成功构建cpd基因敲除的TRP△cpd菌株。5天内，菌株TRP△cpd和TRP对50mg/L CP的降解率分别为27.3%和78.5%。TRP△cpd菌株对CP的降解率显著降低，说明cpd基因在菌株TRP对毒死蜱的降解中起到主要作用。CPD蛋白的三级结构是由α螺旋、β折叠及无规卷曲高度折叠形成的紧密的球状结构。活性位点Ser155，Asp251和His281均位于β折叠链和α螺旋之间柔韧性较强的loop区域。CPD与CP分子对接表明: Ser155的侧链羟基O原子与CP结构中P原子的空间距离较近，易于发生亲核攻击，将CP降解为TCP和DETP。通过PCR技术将Ser155突变为Ala（S155A）。15min内，CPD-S155A酶液对50mg/L CP的降解率为5.15%±1.03，酶活力为7.85U/mg。Ser155突变为Ala后，CPD蛋白对CP的降解率下降10余倍，说明Ser155在CPD对CP的降解中起到关键作用。更多还原

【Abstract】 A feasible way to remediate chlorpyrifos (CP) contaminated water and soil environments is toscreen chlorpyrifos degrading microorganisms because of its important significance in thebiodegradation of pesticide residues. Paracoccus sp. TRP is a strain capable of efficiently degradingchlorpyrifos. Paracoccus sp. TRP was selected as the research object in this study. The basiccharacteristics of the strain, the cloning of a new chlorpyrifos degrading gene, and the relationshipsbetween the structure and function of the chlorpyrifos degrading enzyme were investigated in this study.The contents are as follows:Strain TRP isolated in our lab was renovated.The strain can rapidly degrade chlorpyrifos from the2ndday to the4thday, and the degradation rate of50mg/L CP reached to83.3%at the7thday. The strainis resistant to5~100μg/mL Amp and not resistant to5~100μg/mL Tc and Gm. The strain can’t producesugar lipid biosurfactants.The chlorpyrifos degrading enzyme of the strain TRP was constitutive.Solexa sequencing technology was used to determine the genome sequence of Paracoccus sp. TRP(GenBank accession number AEPN00000000). Genome size is about3.9Mb with GC content of62.89%. The number of predicted CDS is3961.155metabolic maps were produced, of which25xenobiotic related metabolic maps were found, including metabolic maps for herbicide atrazine anddenitrification.96tandem repeats,200transposons,147IS,45tRNA and5sets of rRNA were alsofound in the genome sequence. Paracoccus sp. TRP and Paracoccus denitrificans Pd1222are closelyrelated in evolutionary distance. Most of the genes are highly homologous. All the four genes of redoxenzymes for denitrification similar to those of P. denitrificans Pd1222were identified in Paracoccus sp.TRP. The analysis of the genome of strain TRP can help us identify more different degrading genes andfurther clarify microbial degradation pathways for chlorpyrifos. This is so far the first report about thegenome sequencing of Paracoccus sp. capable of biodegrading chlorpyrifos.Chlorpyrifos degrading gene cpd was cloned from Paracoccus sp.TRP. Gene size is927bp withGC content of60.73%. The homology between cpd gene and other organophosphorus degrading genesalready published is low, indicating that it is a new organophosphorus degrading gene. CPD proteinconsists of308amino acid residues. Molecular weight is33.8kDa. Charge is-7.0. Isoelectric point is4.8305. CPD protein, a member of the esterase_lipase superfamily, not only has Ser155-Asp251-His281catalytic triad and motif GDSAG but also has the conserved substrate pocket ILYIHGGGWSFCSA.Thesize of CPD fusion protein expressed in the E.coli BL21(DE3) is about53.9KDa.CPD protein hasesterase activity. CPD protein esterase activity needs no metal ions. CPD protein esterase activity wassignificantly inhibited by1mmol/L PMSF,1mmol/L DEPC and0.025%SDS. CPD protein esteraseactivity was significantly enhanced by0.025%Tween-20. CPD esterase activity needs His and Ser.Within15min, the degradation rate of50mg/L CP by CPD enzyme is63.5%±2.14. The enzymeactivity is59.62U/mg. Strain TRP△cpd, in which cpd gene was knocked out, was successfullyconstructed by inserting tetracycline box into the cpd gene of strain TRP. Within5days, the degradationrate of50mg/L CP by strain TRP△cpd and strain TRP were27.3%and78.5%, respectively.The degradation rate of CP by strain TRP△cpd significantly reduced, indicating that cpd gene plays animportant role in chlorpyrifos degradation of strain TRP.The3D structure of CPD protein is a tightly folded globular structure consisting of the alphahelixes, beta strands and coils. The active site Ser155, Asp251and His281in catalytic triad located atthe flexible loops between β strand and α helix. Molecular docking between CPD and CP showed thatspace distance between O atom from hydroxyl group of active site Ser155and P atom from CP is near,which facilitates to produce nucleophilic attack to degrade CP to form TCP and DETP. The mutation ofactive site Ser155to Ala was performed by PCR mediated site-directed mutagenesis technology, namelyS155A. Within15min, the degradation rate of50mg/L CP by CPD-S155A enzyme is5.15%±1.03,enzyme activity is7.85U/mg. Ser155mutation for Ala of the CPD protein reduced the degradation rateof CP by more than10times, indicating that Ser155plays a key role in the degradation of CP by CPDenzyme.更多还原