【作者】 宋金龙；

【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， 2013， 博士

【摘要】 磺酰脲类除草剂的大量使用给生态环境和人类健康带来诸多危害，如何清除其在环境中的残留已成为众多科学家关注的热点问题。利用降解菌及其产生的降解酶降解农药具有高效、无二次污染等特点，是修复农药污染环境和去除食品中农药残留的重要手段之一。本文从烟嘧磺隆生产厂废水处理系统的活性污泥中分离到一株烟嘧磺隆高效降解菌LZM1，该菌株能够利用烟嘧磺隆为唯一氮源生长。通过形态学特征和ITS序列分子进化分析，将菌株LZM1鉴定为黄篮状菌（Talaromyces flavus）。利用单因素试验结合响应面法研究菌株LZM1的最适降解条件，结果表明：菌株LZM1在CSM培养基中的最适降解条件为29℃、pH6.1和接种量0.55g/L，该条件下培养5d，可将100mg/L烟嘧磺隆完全降解。菌株LZM1可耐受并降解高达700mg/L的烟嘧磺隆，同时对供试的六种磺酰脲类除草剂苄嘧磺隆、氯磺隆、砜嘧磺隆、胺苯磺隆、苯磺隆和醚磺隆均具有良好的降解效果。运用高效液相色谱质谱联用检测菌株LZM1降解烟嘧磺隆所产生的代谢产物，获得8种中间代谢产物，并推断了可能的代谢途径。这些降解特性表明菌株LZM1在磺酰脲类除草剂残留物的生物修复上具有很大的应用潜力。运用硫酸铵沉淀、离子交换层析、疏水层析和凝胶过滤层析技术，在菌株LZM1胞内粗酶液中纯化得到烟嘧磺隆降解酶FMO。研究降解酶FMO的酶活力、温度、pH、金属离子、化学试剂对降解酶酶活力的影响，结果表明降解酶FMO最适反应温度为40℃，在20-45℃的温度范围内可有效降解烟嘧磺隆，20-40℃时稳定性良好，保温1h后剩余相对酶活力均大于80%。最适反应pH为7.0且在pH6.5-8.0范围内均可有效降解烟嘧磺隆。Na⁺、K⁺、Fe²⁺对酶活性无明显作用，Mg²⁺对酶活性有一定促进作用，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对降解酶FMO具有少许促进作用。使用飞行时间质谱测定降解酶FMO的部分氨基酸序列，设计简并引物扩增到部分cDNA序列后，结合3’和5’RACE技术，获得了降解酶基因fmo的3’和5’末端序列，运用DNAMAN软件对三段序列进行了拼接，降解酶基因fmo全长cDNA为1716bp，开放阅读框1449bp（121-1569bp），5’非编码区为120bp，3’非编码区有144bp（不包含终止密码子TGA），编码一个483个氨基酸的蛋白质。经BLAST比对后发现，该蛋白编码基因与Aspergillus flavus NRRL3357产生的黄素单加氧酶编码基因相似性为92%。运用ExPASy网站提供的生物信息学软件结合NCBI数据库和SWISS-MODEL数据库，推测和分析了降解酶基因fmo编码氨基酸序列，以及降解酶蛋白的一级、二级、三级结构。将降解酶基因fmo插入到pET32a表达载体中，成功构建了重组表达载体pET32a-fmo，并转化至E.coli BL21（DE3）。经IPTG诱导，重组蛋白FMO实现了高水平表达，15min内对50mg/L烟嘧磺隆的降解率达71.7%，比活力42.3U/mg，说明降解酶FMO具有良好的工业化生产前景和实际应用价值。更多还原

【Abstract】 With the extensive use of sulfonylurea herbicides, their residuals have caused a lot of negativeimpact on ecological environment and human health. Great concerns have been raised about thepersistence and degradation of sulfonylurea herbicides residuals in environment. Pesticides degradationwhich is caused by degradative enzymes produced by degradative microbe has the characteristics ofefficiency and pollution-free, which is an important way for environmental remediation and to removethe pesticide residues in the food.Strain LZM1capable of degrading nicosulfuron was isolated by enrichment procedure fromactivated sludge, which was collected from wastewater treatment system of a nicosulfuron manufactory.On the basis of morphological characteristics, ITS sequence analysis，strain LZM1was identified asTalaromyces flavus.Single factor experiments and response surface methodology were used to optimize the parametersthat significantly affected nicosulfuron biodegradation by LZM1and their interactions. The results showthat the optimal degrading condition was of29℃, pH6.1and0.55g/L inoculum size, repectively. All ofnicosulfuron was degraded after5days of incubation under this optimized condition. Strain LZM1could endure high concentration of nicosulfuron and grow well in CSM containing700mg/L ofnicosulfuron. Strain LZM1was also found to be highly efficient in degrading tribenuron methyl,chlorsulfuron, bensulfuron methyl, ethametsulfuron methyl, cinosulfuron, and rimsulfuron. Metabolitesfrom nicosulfuron degradation were identified by liquid chromatography mass spectrometry, and apossible degradation pathway was deduced. The results indicate that strain LZM1can be used in thebioremediation of nicosulfuron-contaminated environments.Ammonium sulfate precipitation, desalting chromatography, ion-exchange chromatography,hydrophobic interaction chromatography and gel filtration were used to isolate and purfy degradativeenzyme. A degradative enzyme named FMO was obtained finally. Some parameters （temperature, pH,metallic ion, chemical reagent） which could affect degradative enzyme activity were carried out. Theresults suggest that the optimal temperature was40℃and more than80%relative activity was remainedafter keeping1h at each temperature in the range of20-45℃. The optimal pH was7.0and degradationcould be performed efficiently in pH range of6.5-8.0. Na⁺K⁺and Fe²⁺had no effect on enzyme activity.Mg²⁺was useful to promoted degradation rate and Ag⁺and Hg²⁺had a strong inhibitory effect. To someextent, Tween-20and Tween-80increased enzyme activity.The amino acid sequence of degradative enzyme FMO was determined by mass spectrum andoligonucleotide primers were designed. RT-PCR.combined with3’RACE and5’RACE technologieswere used to obtain the whole cDNA sequences of degradative enzyme FMO. The upstream,downstream and internal cDNA sequences were amplified and were combined by using DANMANsoftware. The whole cDNA sequence was1716bp,5’UTR was120bp,3’UTR was144bp, respectively.In this sequence, the ORF encodes a483amino acid protein. The protein alignment indicated thatdeduced amino acid of fmo shared homology of92%with a flavin-containing monooxygenase which was produced by Aspergillus flavus NRRL3357. Softwares on ExPASy website combined withSWISS-MODEL and NCBI data base were used to duce the frimary structure, secondary structure,tertiary structure of deduced amino acid of fmo.Encoding gene fmo was inserted to vector pET32a, and then tranfected into E.coli BL21（DE3）.The recombinant protein FMO was expressed at a high level after induction with IPTG and has highdegradative efficiency after puried. Within15min, the degradation rate of50mg/L nicosulfuron byFMO is71.7%, enzyme activity is42.3U/mg.The results provided theoretical basis and reference datafor recombinant protein FMO use in industrial production and application.更多还原