【作者】 李晓慧；

【导师】 李顺鹏；

【作者基本信息】 南京农业大学 ， 微生物学， 2008， 博士

【摘要】 毒死蜱是一类中等毒性的有机磷杀虫剂,现已被许多国家广泛使用,由此引发的环境污染问题日益突出。环境污染的微生物修复具有其它修复方法无可比拟的优势,因此利用获得的能够高效降解毒死蜱的微生物资源,充分发挥其降解能力,进行毒死蜱残留污染环境的修复,具有非常重要的理论研究意义和实际应用价值。本研究的目标是分离、筛选能够以毒死蜱作为唯一碳源生长的降解菌株,并对该菌株在各种环境条件下降解毒死蜱的特性进行研究,以期为毒死蜱环境污染的修复工作提供科学依据；同时对其降解酶基因进行克隆和定向进化,为进一步研究其基因功能、构建具有更强降解能力的基因工程茵奠定基础。采用以毒死蜱为唯一碳源的选择性培养基,从毒死蜱污染的环境样品中分离到七株毒死蜱降解菌,分别命名为Dsp-1-Dsp-7.经生理生化鉴定和系统发育分析,七株茵属于5个不同的属。菌株Dsp-2鉴定为Sphingomonas sp.,菌株Dsp-4为Stenotrophomonas sp.,菌株Dsp-7为Brevundimonas sp.,菌株Dsp-6为Bacillus sp,其他菌株为Pseudomonas sp.。其中从Sphingomonas sp.和Brevundimonas sp.分离到毒死蜱农药降解茵尚属首次报道。对分离的毒死蜱降解菌进行基于16S rDNA序列的系统发育分析,对染色体ERIC-PCR旨纹图谱扩增及对降解特性进行比较研究,结果表明七株降解菌在遗传、生理和降解能力等方面表现出丰富的多样性。不同菌株对毒死蜱的降解能力有很大差异,其降解谱也表现出一定的差异。降解菌株Dsp-1-Dsp-4对多种有机磷类农药具有降解能力,与其他菌相比,菌株Dsp-2在液体培养基中拥有最快的降解速率,12h几乎完全降解100mg.L-1的毒死蜱,并且能降解有机磷农药丙溴磷。菌株Dsp-1在自然环境中对毒死蜱的降解效果最好。而菌株Dsp-5和Dsp-7只能降解毒死蜱农药,而不能降解其他有机磷农药。七株菌降解毒死蜱的中间产物均为3,5,6-三氯-2-吡啶酚(TCP),且积累TCP。根据已报道的有机磷水解酶基因序列设计引物,通过PCR的方法从降解茵Dsp-1-Dsp-4中克隆到了有机磷水解酶mpd基因,mpd结构基因共有996个碱基,编码331个氨基酸序列。通过序列比较发现,在基因水平上,四株降解菌的有机磷水解酶基因可以分为3种亚型,相互之间的同源性为99%；在氨基酸水平上,四株降解茵的有机磷水解酶可以分为3种亚型,相互之间的同源性为92%。将四株降解茵的mpd结构基因分别连接到表达质粒载体pET29a上,在E. coli BL21实现了基因的高效表达。通过致突变PCR对野生型的mpd基因进行定向进化,获得了5个毒死蜱降解能力增强的突变体。其中突变子Mn7,突变了5个氨基酸位点(N271T,I275S,G277S, A280V,P304S),水解毒死蜱的动力学参数Kcat和Kcat/Km比原始MPH分别提高10和118倍,水解3种供试的有机磷底物的相对活力都有明显的增强。通过定点突变的方法,对这些突变位点的作用进行进一步的研究。酶动力学参数的测定结果表明,单位点突变体N271I的Kcat/Km比原始MPH提高了15倍,A280V的Kcat/Km比原始MPH提高了32倍,是导致毒死蜱催化能力增强的重要位点。而F196I则使Kcat/Km下降了1倍,这可能与该位点是MPH酶作用的关键位点有关。对突变体的Mn7三维结构进行模拟分析表明,这些位点的突变并未对酶的三维结构产生明显的影响,但其二级结构分析表明a-螺旋和B-折叠的含量有所增加,这可能使得蛋白的构象更加稳定,从而提高了酶的水解活力。研究了菌株Dsp-1的最适生长条件,菌株Dsp-1的最适温度为30℃；最适生长初始pH在7左右；装液量小于150mL/250mL时,生长旺盛;Dsp-1生长不需要NaCl,无特殊生长因子要求。在供试的几种碳源中,Dsp-1对葡萄糖利用最好；在以葡萄糖为碳源时,以有机氮为氮源生长较好,在供试的几种无机氮源中,Dsp-1对NH4NO3利用最好；该菌株对多种常用抗生素敏感。在发酵培养基中,经过16h达到稳定期,菌体生长良好(>1011CFU·mL-1),能较好的满足后续试验的要求。降解菌剂的应用试验表明,在消除土壤和蔬菜毒死蜱残留的试验中,菌株Dsp-1对毒死蜱表现了较强的降解能力。人工接种毒死蜱降解菌Dsp-1到毒死蜱污染的三种不同的土壤(红粘土、灰潮土、高沙土),Dsp-1在3种土壤中都能有效降解毒死蜱,Dsp-1在pH中性的灰潮土中降解毒死蜱的速率最快,酸性的红粘土中降解效果较差,而碱性的高沙土中毒死蜱的自然降解作用也较明显；说明土壤pH值对降解效率的影响十分显著。相对较高的有机氮的含量也有利于毒死蜱的降解。在1-100mg-kg-1的浓度范围内Dsp-1都能有效降解土壤中的毒死蜱,对低浓度的毒死蜱降解率更高,达到90%以上。该结果显示了菌株Dsp-1具有对毒死蜱污染的土壤进行实地修复的潜力。应用Dsp-1分别对冬季和春季栽培的温室小青菜的毒死蜱残留进行降解试验,在环境条件适宜,使用推荐农药用量时,施用含1010CFU-mL-1活菌的降解菌剂可以发挥良好的作用,7天的降解效率达到80%以上,加快了毒死蜱的降解进程,从而保证在安全间隔期内达到国家的残留标准(0.1mg-kg-1).更多还原

【Abstract】 Chlorpyrifos [O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl) phosphorothioate] is a mid-toxic organophosphorus insecticide which has been widely used by many countries. Its vast use ultimately caused more serious environment pollution. Microbial remediation has been deemed to be much more advantageous method than the others. So it has been an important research item for us to exploit and utilize chlorpyrifos-degrading microbial resource to remove the environment pollution. This research aimed at isolating the bacteria that can use chlorpyrifos as sole carbon source, study their degrading characterizes of chlorpyrifos in different environments, and provided the theory base for the bioremediation of environment pollution with chlorpyrifos. At the same time, the degrading gene of chlorpyrifos was cloned and evolved, which would be helpful to research the gene function and establish the foundation of construct the genetic engineered strain by enhanced degrading capacity.Seven chlorpyrifos-degrading bacteria, named Dsp-1to Dsp-7were isolated from chlorpyrifos-contaminated samples using selective culture medium with chlorpyrifos as sole carbon source. These isolated strains were identified based on morphological, physiological and biochemical tests with reference to Bergey’s Manual of Determinative Bacteriology combined with16S rDNA sequence analysis. Based on these analyses, strains Dsp-2, Dsp-4, Dsp-6and Dsp-7were identified as Sphingomonas sp., Stenotrophomonas sp., Bacillus sp. and Brevundimonas sp., respectively, while all other strains were members of Pseudomonas sp. This is the first report of isolating chlorpyrifos-degrading bacteria from Sphingomonas sp.and Brevundimonas sp. Comparative studies were performed to study their phylogenetic relationship based on their16S rDNA sequence. ERIC-PCR fingerprints and the degrading capability were also compared. These results revealed high biodiversity of chlorpyrifos-degrading bacteria in many aspects. Degrading characteristics of seven degrading bacteria were investigated. However, significant differences in the ability of degrading chlorpyrifos and other organophosphate pesticides were observed among these strains. Strain Dsp-l-Dsp-4can degrade several organophosphate pesticides. Contrast to orther six strains, strain Dsp-2can degrade profenofos and had the fastest degrading rate in liquid medium,100mg-L" chlorpyrifos was degraded to an undetectable level within12h. Strain Dsp-1instead of Dsp-2had the highest degrading rate in soil. Strain Dsp-5and Dsp-7can not degrade any supplied pesticides except chlorpyrifos. All these isolates could utilize chlorpyrifos as sole carbon source with3,5,6-trichloro-2-pyridinol (TCP) as intermediate.The primers were constructed based on the published sequences of the organophosphorus hydrolase gene opd and mpd. The methylparathion hydrolase gene mpd was cloned by PCR from Dsp-l-Dsp-4. The analysis and alignment of the four organophosphate pesticides hydrolase genes revealed that this gene has996bases, which coding for the organophosphate pesticides hydrolase contained331amino acids. The four genes were dividing into three subgroups at the level of gene base sequences, the similarity values between them were99%. At the level of amino acid sequences, the four organophosphate pesticides hydrolase also were dividing into three subgroups, the similarity values between them were92%.Each mpd gene from the four strains was linked to the expression plasmid vector pET29a, the expression strains of the four genes were gained by transfer of the expression plasmid into strain BL21(DE3). SDS-PAGE analysis of the total proteins of the four the expression strains identified the hydrolase.Error Prone PCR was used to generate MPH variants improvement in hydrolysis of chlorpyrifos and five mutations were obtained. One variant, Mn7which had five site mutations (N271T, I275S, G277S, A280V, P304S), displayed a10-fold increase in Arcat and118-fold increase in kcat/Km values. It also exhibited increase special activity for methylparathion chlorpyrifos and triazophos compared to wild type-MPH. We obtained8site-mutation variants by site-directed mutagenesis. Study of their kinetic parameters for hydrolysis of chlorpyrifos was performed. Results showed that N271I exhibited a15-fold increase in kcat/Km values and A280V exhibited a15-fold increase in kcat/Km values. These results showed that these two sites are the key sites for improved hydrolysis of chlorpyrifos. F196I which showed one fold reduction in kcat/Km values may because that Phe196is the key acid amino that form an aromatic cluster at the entrance of the catalytic center. No noticeable difference between MPH from Mn7and that from M6was observed when analysis of enzymatic3D-structure. But secondary structure analysis showed that the content of a-helix and β-pleated sheet was increased, suggesting that is likely to contribute to stabilized the conformation, so that improved the efficiency of catalysis.The optimal growth conditions of strain Dsp-1were studied. The optimal growth temperature and initial pH of strain Dsp-1are30℃and7.0, respectively. The oxygen had little effect on the growth of strain Dsp-1. Strain Dsp-1could growth without NaCl and special growth factor. Glucose was the best carbon source and organic nitrogen was better nitrogen source. The optimal inorganic nitrogen was NH4NO3. Strain Dsp-1was sensitive to many antibodies. In the ferment medium, after16hours, Dsp-1cell could reach to1011mL-1. Addition of strain Dsp-1to three test soils (Red soil. Brown soiland Sandy soil) treated with chlorpyrifos resulted in a high degradation rate of chlorpyrifos. The highest degradation rate was showed in brown soil, which was netrual pH. The degradation effect was not satisfactory in red soil which was acidic pH and the physical degradation of chlorpyrifos was obvisely in sandy soil because of its alkaline pH. However, the soil pH had notability effect on degradation. The moderate pH, moisture and nutrients could promote the degradation. Strain Dsp-1worked well when the concentration of chlorpyrifos in soil ranged in1-100mg kg-1. The degradation rate reached to90%at low concentration. The fine characteristic of Dsp-1will help it to play well in different chlorpyrifos-contamination soil. The research showed that strain Dsp-1had the potential to cleanup chlorpyrifos contaminated environment. The degradation patterns of chlorpyrifos on pakchoi in the greenhouse were performed in spring and winter separately. When using1010CFU-mL-1zymolytic strain Dsp-1, the degradation rates of chlorpyrifos on pakchoi reached to80%after7days. The results suggested that the strain Dsp-1could efficiently remove or detoxify chlorpyrifos residues on pakchoi in the greenhouse, making sure that the residual values of chlorpyrifos on pakchoi were lower than the China MRL (0.1mg-kg-1).更多还原