【作者】 汤鸣强；

【导师】 尤民生；

【作者基本信息】 福建农林大学 ， 农药学， 2012， 博士

【摘要】 三唑磷是广泛使用的有机磷农药之一，作为甲胺磷等高毒有机磷农药的替代品，大量用于农业生产来防治病虫害。然而它毒性较高、半衰期长，大量频繁的使用使环境与农产品受到污染。如何降低和消除三唑磷及其它有机磷农药的副作用，成为人们亟待解决的问题。微生物降解具有高效、无毒、且操作简便、无二次污染、经济实用和应用范围广等特点，目前己成为去除农药残留污染的一种重要方法。基于上述认识，本课题拟以有机磷农药生产企业的污水处理池活性污泥及其周边土壤为样品，通过不断增加三唑磷选择压力的办法，驯化富集并最终分离三唑磷降解菌，并对分离菌株做进一步的研究，以期为有机磷农药污染的生物治理提供基础。主要内容如下：1．从福建建瓯福农生化有限公司污水处理池活性污泥及其出水口污泥中，通过富集培养基和基础培养基驯化培养分离到5株对三唑磷农药有较高降解率的细菌菌株INI-1、TAP-1、SS-1、Soil-1和Ter-2。在32°C、180r/min条件下培养5天，5株细菌对三唑磷（浓度为100mg/L）的降解率分别为53.8±1.42%、95.8±1.06%、66.7±2.21%、52.8±1.84%和46.8±1.15%。本论文对降解率最高的菌株TAP-1进行了进一步试验。2．形态与生理生化试验表明，TAP-1为革兰氏阳性杆菌，大小为(0.4~0.6)μm×(1.5~2.2)μm。菌株TAP-1能水解淀粉、葡萄糖、果糖、蔗糖、麦芽糖和乳糖。甲基红试验、接触酶试验和脲酶试验阳性。但不能液化明胶，V-P试验、H2S试验、苯丙氨酸裂解酶试验和柠檬酸盐试验阴性。TAP-1在牛肉膏蛋白胨培养基上，TAP-1菌落圆形，边缘整齐，中凹状，乳白色，不透明，较易从培养基上挑取。TAP-1生长的温度范围为10~37°C，生长的pH范围5.2~8.0，能耐受的最高NaCl浓度为6.5%。TAP-1对左氧氟沙星、克林霉素、罗红霉素和庆大霉素等抗生素敏感，而对头孢唑啉、头孢氨苄和头孢曲松等抗生素有抗性。16S rDNA同源性分析表明，TAP-1与Bacillus sp.cp-h58最为接近，同源性为100%，与其他14个芽孢杆菌菌株的同源性均为99%。系统进化分析表明，菌株TAP-1可能是芽孢杆菌属的一个新种。综合形态生理生化以及16S rDNA序列分析，菌株TAP-1鉴定为芽孢杆菌属细菌。其在Genbank上的登录号为Bacillus sp. TAP-1(HQ156466）。3．TAP-1对辛硫磷、毒死蜱、乐果和敌敌畏等有机磷农药都有不同程度的降解。研究了营养因子与环境因子对TAP-1生长与三唑磷降解的影响。结果表明，TAP-1在三唑磷为唯一碳、氮和磷源时，生长与降解能力较弱。葡萄糖浓度小于2%时，外加葡萄糖促进菌体生长与对三唑磷的降解。TAP-1以共代谢方式降解三唑磷农药。TAP-1降解三唑磷的适合温度与pH范围是28~33°C和pH6.5~8.0。4．在生物学研究的基础上，对TAP-1在茶园土壤中三唑磷的降解作用进行了研究。结果表明，在15d的试验周期里，灭菌土、灭菌土加菌、新鲜土、新鲜土加菌4个处理对三唑磷的降解率分别为39.9%、54.8%、73.3%和92.2%。新鲜土壤中三唑磷的降解速率明显快于灭菌土壤，而添加TAP-1则对茶园土壤中三唑磷的降解有显著的促进作用。5．降解酶定域试验表明，TAP-1三唑磷降解酶为组成型表达的胞内蛋白组分。研究了菌株TAP-1（Bacillus sp.）三唑磷降解酶的提取条件。采用超声波法破碎TAP-1细胞以获得三唑磷降解酶，通过单因素试验考察破碎功率、超声时间和菌体浓度对破碎效果的影响。进一步采用响应面分析法优化破碎条件。结果表明，当破碎功率为380W，超声时间12min，菌体浓度为200mg/mL时，酶活力达最高值1890U/mL。6．研究了菌株TAP-1（Bacillus sp. TAP-1）三唑磷降解酶的酶学特性。该酶降解三唑磷的最适pH6.5~7.0，在pH5.0~7.5之间，酶活力均能保持在最高酶活力的68%以上。该酶降解三唑磷的最适温度为35℃，在25~40℃的温度范围内能保75%以上的降解活性。以三唑磷为底物，该酶的米氏常数Km和最大反应速度Vmax分别为23.02μmol/L和0.738μmol/mg·min。7．研究了降解酶的固定化方法。选取包埋剂海藻酸钠、CaCl2及包埋比（酶液:海藻酸钠溶液）三个因素，设计正交实验。将TAP-1三唑磷降解酶进行固定化，测定组合条件制备的固定化酶对三唑磷的降解特性。研究表明，固定化酶的最佳固定化条件为海藻酸钠浓度为3.5%，酶液与海藻酸钠的包埋比为5%，氯化钙浓度为5.0%，固定化时间为2~4h。研究了固定化酶对三唑磷的降解特性。固定化酶对三唑磷的最适降解温度为35℃，在温度范围25~40℃内该固定化酶均能保持最高活力的83%以上。固定化酶的最适作用pH为7.0，但在pH6.0~7.5之间，酶活性都保持83%以上。稳定性试验表明，经过固定化，三唑磷降解酶的pH稳定范围往碱性方向偏移。以三唑磷为底物，粗酶液与固定化酶的Km分别为23.02μmol/L和84.95μmol/L，Vmax分别为0.738μmol/mg·min和2.076μmol/mg·min。与粗酶相比，固定化酶Km和Vmax均大于粗酶液，表明经过固定化，酶与底物的亲和力下降。8．菌株TAP-1三唑磷降解酶的分离纯化。通过硫酸铵沉淀、DEAE-SepharoseF.F.阴离子交换和Sephacryl S-200分子筛层析三个步骤，分离纯化到一个电泳纯的三唑磷降解酶，分子量约为83.3ku。酶的回收率为13.8%，纯化倍数为22.46。更多还原

【Abstract】 Triazophos is an organophosphate pesticide widely used for insect control inmany countries. However, as a substitute for other highly toxic pesticides includingmethamidophos, triazophos has caused serious problems to the environment andagricultural products because of its own toxicity and long half-life, thus making it apublic concern. The degradation of pesticides by microorganisms is one of the mostimportant methods for the detoxification of insecticide. This thesis includes eightchapters as follows:1. Five bacterial strains (INI-1, TAP-1, SS-1, Soil-1, and Ter-2) capable ofdegrading triazophos were isolated using enrichment culture and the streak platemethod from the sewage sludge of a wastewater treatment system fororganophosphate pesticides produced by the Funong Group Co. in Jianou, Fujian,southeastern China. The biodegradation rates of triazophos (100mg/L) at32°C withshaking (180r/min) for INI-1, TAP-1, SS-1, Soil-1, and Ter-2were53.8±1.42%,95.8±1.06%,66.7±2.21%,52.8±1.84%and46.8±1.15%, respectively. Further studieswere performed on the TAP-1strain, which had the highest biodegradation rate.2. Morphological and physiological tests showed that TAP-1is a Gram-positive rodapproximately1.5~2.2μm in length and0.4~0.6μm in width during late exponentialphase. When grown on TBG medium, TAP-1generally produced round, beige,mucoid, smooth-edged colonies with entire margins and opaque surfaces; underprolonged incubation times, the edges of the colonies became rough. Colonies testedpositive for catalase, starch hydrolysis and acid production from carbohydrates, butnegative for the Voges-Proskauer (V.P.) test, gelatin liquefaction, H2S production,indole and the phenylalanine ammonia-lyase test. Growth was observed attemperature range of10~37°C and a pH range of5.2~8.0. The highest concentrationof NaCl tolerated by TAP-1was6.5%. TAP-1showed sensitivity to antibiotics such aslevofloxacin, clindamycin, roxithromycin and gentamycin, but was resistant tocefazolin, cephalexin and ceftriaxone. The analysis of16S rDNA sequences revealed100%similarity between TAP-1and Bacillus sp. cp-h58and99%similarity betweenTAP-1and the other14Bacillus strains analyzed. The phylogenetic tree constructed using the16S rDNA sequence data was closest to that of Bacillus. Thus, TAP-1wasproposed to be a new species of Bacillus based on phenotypic, physiological andbiochemical characteristics, as well as the phylogenetic analysis of16S rDNAsequences. The GenBank number for TAP-1is Bacillus sp. TAP-1(HQ156466).3. TAP-1was also found to degrade other organophosphate pesticides such asphoxim, chlorpyrifos, dimethoate, and dichlorvos. The effects of nutritional andenvironmental factors on the growth of TAP-1and its biodegradation of triazophoswere investigated under laboratory conditions. The results showed that the growth ofthe strain as well as its degradation rate were poor if triazophos was used as the solesource of carbon, nitrogen and phosphorous. Increasing the glucose concentration ofthe medium enhanced both the growth and degradation rate of TAP-1so long as theconcentration of glucose remained below2%. Thus, TAP-1could degrade triazophosthrough co-metabolism. The results showed that TAP-1degrades triazophos veryefficiently between28°C~33°C and at a pH range of6.5~8.0.4. We also studied the degradation of triazophos by TAP-1in soil from a tea garden.The results of15-day tests showed that the degradation rates for sterilized soil,sterilized soil with TAP-1, native soil, and native soil with TAP-1, were39.9%,54.8%,73.3%, and92.2%, respectively. Compared with sterilized soil, greater degradationrates were observed in native soil. Thus, TAP-1could greatly enhance the degradationof triazophos in the soil from tea gardens.5. Enzyme distribution experiments showed that the TAP-1enzyme responsible fortriazophos degradation appears to be a constitutive intracellular component. Theextraction conditions for the TAP-1triazophos-hydrolase were subsequentlydetermined. Sonication was used to lyse the TAP-1cells to obtain thetriazophos-hydrolase. Three factors were investigated, including disruption power,disruption time and cell concentration, for their effects on enzyme activity;concurrently, single-factor tests were combined with the Response Surface Analysis(RSA) method to optimize the treatment conditions. The results showed that optimumconditions were achieved using a disruption power of380W, a disruption time of12min and a cell concentration of200mg/mL. Under these conditions, crude enzymeactivity could reach1,890U/mL. 6. The characteristics of the crude triazophos-hydrolase were determined. Theenzyme demonstrated greatest enzymatic activity in the pH range of6.5~7.0with itshighest activity occurring in the pH range of5.5~7.5. Enzymatic activity occurred atan optimum temperature of30°C for the degradation of triazophos; activity remainedabove75%of the maximum over a temperature range of25°C~40°C. The Km valueand the maximal degradation rate of the triazophos-hydrolase for triazophos were23.02μmol/L and0.738μmol/mg·min, respectively.7. Immobilization methods for the free enzyme were also studied. The free enzymeextracted from TAP-1was immobilized using an L9(43) orthogonal array whose threefactors were sodium alginate, calcium chloride and a proportion of crude enzyme andsodium alginate. The ability of the immobilized enzyme to degrade triazophos wasthen determined. The results indicated that the free enzyme was immobilized for2to4h with3.5%sodium alginate content,5.0%calcium chloride content and a5%proportion of crude enzyme and sodium alginate. The characteristics of triazophosdegradation by the immobilized enzyme were determined. Enzymatic activity of theimmobilized enzyme occurred at an optimum temperature of35°C and remainedabove83%of the maximal activity across the temperature range of25~40°C. Theimmobilized enzyme also showed maximal degradative activity against triazophos atpH7.0but retained greater than83%of the maximal activity over a pH range of6.0~7.5. Additional experimental evidence suggested that the immobilized enzymewas more stable at alkaline pH than the free enzyme. The immobilized enzymeshowed Km and Vmax values for triazophos of84.95μmol/L and2.076μmol/mg·min,respectively, both of which were higher than for the free enzyme, indicating a declinein affinity between the enzyme and triazophos during immobilization.8. The triazophos-hydrolase from Bacillus sp. TAP-1was isolated and purified inthree steps: ammonium sulfate precipitation, DEAE Sepharose Fast Flow anionexchange chromatography and Sephacryl S-200filtration. The electrophoreticallyhomogeneous triazophos-hydrolase was obtained with an apparent molecular weightof approximately83.3ku, a yield of13.8%and a22.4-fold purification.更多还原