【作者】 康占海；

【导师】 张金林；

【作者基本信息】 河北农业大学 ， 农药学， 2012， 博士

【摘要】 利用生物修复法来降低环境中的农药是行之有效的措施之一,在诸多的农药生物修复方法中以微生物修复的研究和应用最为广泛,尤其微生物修复环境中长残效农药的研究较多。甲嘧磺隆是一种超高效、广谱、持效期长的磺酰脲类除草剂,在世界范围内广泛应用于林业及非耕地杂草的防除。甲嘧磺隆性质稳定、不易降解、在环境中持效期长,因此对敏感作物很容易造成药害。甲嘧磺隆的水溶性较高,在土壤中的移动性较强,易造成对地下水的污染,因此,对甲嘧磺隆微生物降解作用的研究具有一定的实践意义。而关于甲嘧磺隆的微生物降解作用研究鲜见报道,本研究通过富集培养的方法从土壤和水中分离筛选出19株可降解甲嘧磺隆的微生物,对其中降解能力较强的JH2和JH3菌株进行了降解特性及降解机理研究,主要研究结果如下:1.降解甲嘧磺隆微生物的筛选。利用富集培养技术从生产甲嘧磺隆农药厂的废水中分离得到了19株能够降解甲嘧磺隆的微生物,通过测定不同菌株对甲嘧磺隆的降解率发现:供试菌株对低浓度甲嘧磺隆的降解率要显著高于高浓度的降解率,在培养基中甲嘧磺隆的含量为5 mg·L^-1时,JH3菌株的降解率最高,为98.9%,其次是JH2菌株,降解率为95.3%。对其中降解作用较强的7个菌株进行鉴定,其中JH3菌株为睾丸酮丛毛单胞菌（Comamonas testosteroni）;JH2菌株为食酸丛毛单胞菌（Delftia subsp）;JH10菌株为枯草芽孢杆菌（Bacillus subtilis）;JH6菌株为解淀粉芽孢杆菌（Bacillus amyloliquefaciens）;SH3菌株为枯草芽孢杆菌（Bacillus subtilis）;SH5菌株为枯草芽孢杆菌（Bacillus subtilis）;SH4菌株为沙门氏菌（salmonella sp.）。2. JH2、JH3菌株的降解特性及降解机理研究。结果表明,JH2菌株的最适降解培养基为基础培养基Ⅲ,以甲嘧磺隆浓度为5 mg·L^-1时JH2、JH3菌株降解率最高;JH2菌株对甲嘧磺隆降解的适宜温度为30℃,JH2菌株接种量为1×108 CFU·mL^-1,转速为150 r·min^-1,培养120 h对甲嘧磺隆的降解率最高;JH3菌株对甲嘧磺隆降解的适宜温度为35℃,接种量为1×107 CFU·mL^-1,转速为150 r·min^-1,培养72 h时对甲嘧磺隆的降解率最高;通过对JH2、JH3菌株进行作用机理研究,两菌株降解产物的特征峰基本相同,降解产物的主要碎片离子为m/z198、分子式为C8H7O4S的化合物,其紫外检测的保留时间为3.0 min,推断甲嘧磺隆结构式中的另一半结构可能降解为链状烷烃,没有紫外吸收,也不能离子化,因此没有特征峰。以此判断JH2、JH3对甲嘧磺隆降解的作用位点在S-N键。3. JH2、JH3菌株降解酶活性研究。通过JH2、JH3菌株对甲嘧磺隆降解酶定域表达试验表明,两菌株的胞内酶和胞外酶均有活性,降解甲嘧磺隆的关键酶都主要是胞外酶且降解酶诱导表达。在LB液体培养基中,当甲嘧磺隆浓度为5 mg·L^-1、接种量为2.3×108 CFU·mL^-1、初始pH值为8.0、温度30℃、转速为150 r·min^-1、培养时间120 h时JH2菌株胞外粗酶的酶比活最高达到85.8 U·mg^-1;其酶促反应体系的甲嘧磺隆最适浓度为5 mg·L^-1、温度30℃、pH值为8.0时胞外酶对甲嘧磺隆的降解率最大为86.9%。在LB液体培养基中,当甲嘧磺隆浓度为5 mg·L^-1、接种量为2.3×107 CFU·mL^-1、初始pH值为9.0、温度35℃、转速为150 r·min^-1、培养时间72 h时JH3菌株胞外粗酶的酶比活最高达到88.4 U·mg^-1;酶促反应体系的甲嘧磺隆最适浓度为5mg·L^-1、温度35℃、pH值为9.0时胞外酶对甲嘧磺隆的降解率最大为91.3%。两菌株甲嘧磺隆降解酶在25℃ 45℃范围内热稳定性较好,pH值为7 9碱性条件下能够保持较高降解活性。4. JH2、JH3菌株降解甲嘧磺隆活性酶的纯化。胞外粗蛋白经过DEAE SepHarose Fast Flow阴离子交换柱分离,在280 nm下JH2菌株得到5个蛋白吸收峰,降解圈法检测发现3个洗脱峰P1^-1、P1-3、P1-5均有降解活性;JH3菌株得到2个蛋白吸收峰,降解圈法检测发现仅P2-2洗脱峰具有降解活性;SDS-PAGE电泳检测P1^-1、P1-3、P1-5和P2-2的分子量分别是43 kDa、32 kDa、15 kDa和46 kDa。将甲嘧磺隆降解酶P1^-1、P1-3、P1-5和P2-2利用生物质谱技术测定了氨基酸序列,得到4种降解酶分别为:信号转导组氨酸蛋白激酶、预苯酸脱氢酶、未知蛋白和糖基转移酶。根据得到的编码氨基酸的基因序列设计合成了简并引物,通过PCR分别从JH2、JH3菌株基因组中克隆出甲嘧磺隆降解酶基因G1^-1、G1-3、G1-5和G2-2,编码基因全长分别是1191 bp、886 bp、415 bp、1274 bp;G1^-1、G1-3、G1-5和G2-2分别与载体PMD19-Vector连接转入大肠杆菌,筛选得到阳性克隆并测序,通过Blast软件分析,所得结果与已测序得到的编码氨基酸的基因序列基本吻合。5.降解甲嘧磺隆微生物菌剂加工及应用的研究。本论文初步研制出JH2、JH3菌株的水剂和颗粒剂配方,筛选出了对甲嘧磺隆敏感的植物是小麦,生物活性测定甲嘧磺隆对小麦的IC50值为0.014 mg·kg^-1。在分别使用JH2和JH3菌株水剂20 d和15 d后能将土壤中甲嘧磺隆浓度为5 mg·kg^-1和1 mg·kg^-1的残留降解为对小麦无明显药害水平,通过HPLC检测处理后土壤中的甲嘧磺隆发现,土壤中甲嘧磺隆残留量小于0.0077 mg·kg^-1,降解率达到了99%以上。更多还原

【Abstract】 Sulfometuron-methyl is a member of the sulfonylurea herbicides, and has been applied in the woodlands. Sulfometuron-methyl has been widely used in the world because of its low-dosage and good effect. Sulfometuron-methyl has a long period of validity, was sensitive to crops, has a strong transferability, and can contaminate groundwater. Especially, high concentration of Sulfometuron-methyl exists in industry wastewater from the manufactory of producing this kind of herbicide. Therefore, studying on the degradation of Sulfometuron-methyl is very significant. In order to screen and obtain the microorganisms degrading Sulfometuron-methyl, nineteen strains of microorganisms degrading Sulfometuron-methyl were obtained and two strains of them were found to have the highest degradation rate and of which were studied the degrading characteristics and pathways. The main results were summarized as follows:1. The microorganisms degrading Sulfometuron-methyl were screened. The nineteen strains of microorganisms degrading Sulfometuron-methyl were obtained. All the microorganisms were cultured in medium that containing different concentrations of Sulfometuron-methyl, then put in swing bed at 30℃and shook at 150 r·min^-1 for 5 days, detected the concentration of Sulfometuron-methyl by using HPLC and calculated the degradation efficiency. It was shown that the degradation rates of the seven strains of microorganisms were higher in low concentration than in high concentration for Sulfometuron-methyl, with JH3 having the highest degradation rate of 98.9%, secondly, JH2 having the degradation rate of 95.3%. It was also identified that JH3 was Comamonas testosteroni, JH2 was Delftia subsp, JH10 was Bacillus subtilis, JH6 was Bacillus amyloliquefaciens, SH3 was Bacillus subtilis, SH5 was Bacillus subtilis and SH4 was salmonella sp.2. The study on the degrading characteristics and pathways of Sulfometuron-methyl degrading JH2 and JH3 strain. The results showed that （1） the optimal culture medium was the basic mediumⅢ, （2） among the Sulfometuron-methyl concentrations tested, the highest degradation rates of JH2 and JH3 strain were found in treatments with 5 mg·L^-1, （3） the appropriate temperatures for Sulfometuron-methyl degradation by JH2 was 30 oC, the appropriate inoculating quantity was 1×108 CFU·mL^-1, the appropriate rotation rates was 150 r·min^-1 and the appropriate cultured time was 120 hours, （4） the appropriate temperatures for Sulfometuron-methyl degradation by JH3 was 35oC, the appropriate inoculating quantity was 1×107 CFU·mL^-1, the appropriate rotation rates was 150 r·min^-1 and the appropriate cultured time was 72 hours, （4） with mass Sulfometuron-methyl spectrometric analysis on the degradation products of JH2 and JH3 strain, it was proved that the degradation products of JH2 and JH3 strain was same. Only one degradation product spectrometric peak was obtained. The main ion was m/z198 and retention time of the spectrometric peak was 3.0 min.3. Study on extracellular enzyme activity of JH2 and JH3 strain. The key enzyme（s） involved in the initial biodegradation of Sulfometuron-methyl was localized to extracellular proteins and shown to be induced expressed. JH2’enzyme specific activity was up to 86.9 U·mg^-1 at pH 8.0 and 30℃, incubation at 150 r·min^-1 for 120 h,inoculum 2.3×108 CFU·mL^-1 in Luria-Bertani liquid medium with Sulfometuron-methyl of 5 mg·L^-1. The maximum degradation rate of extracellular crude enzymes on Sulfometuron-methyl was 86.9% at pH 8.0, 30℃in the enzymatic reaction system with Sulfometuron-methyl of 5 mg·L^-1. JH3’enzyme specific activity was up to 88.4 U·mg^-1 at pH 9.0 and 35℃, incubation at150 r·min^-1 for 72 h,inoculum 2.3×107 CFU·mL^-1 in Luria-Bertani liquid medium with Sulfometuron-methyl of 5 mg·L^-1. The maximum degradation rate of extracellular crude enzymes on Sulfometuron-methyl was 91.3% at pH 9.0, 35℃in the enzymatic reaction system with Sulfometuron-methyl of 5 mg·L^-1. The degrading enzyme（s） was not sensitive to high temperature and kept high activity under alkaline conditions.4. The activity enzyme of JH2 and JH3 strain degrading Sulfometuron-methyl were purified. In this study,the extracellular crude proteins of JH2 were eluted by DEAE SepHarose Fast Flow anion exchange column and five peaks were found. The proteins were detected by by a clear zone, three peaks had activity. The extracellular crude proteins of JH3 were eluted by DEAE SepHarose Fast Flow anion exchange column and two peaks were found. The proteins were detected by a clear zone, only one peak had activity. Four components (P1^-1, P1-3, P1-5, P2-2) had obviously activity. The molecular weight of four components were 43 kDa, 32 kDa, 15 kDa and 46 kDa through SDS-PAGE respectively. The amino acid sequences of Sulfometuron-methyl degrading enzymes (P1^-1, P1-3, P1-5, P2-2) derived from JH2 and JH3 strain were determined by MALDI-TOF-MS and four degrading enzyme were signal transduction histidine kinase, prephenate dehydrogenase, unnamed protein, and glycosyltransferase, respectively. According to the gene sequence encoding amino acids (G1^-1, G1-3, G1-5, G2-2) the degenerate primers were designed. The Sulfometuron-methyl degrading enzyme genes from the genome of JH2 and JH3 were amplified by PCR and full-length gene were 1191 bp, 886 bp, 415 bp and 1274 bp, respectively. G1^-1, G1-3, G1-5 and G2-2 were connected with PMD19-Vector and transfered to E. coli DH5α. Positive clones were screened out by ampicillin and determined the sequences. The obtained results were compared with gene sequence reported in the Genbank and kept accordance with gene sequence encoding amino acids (G1^-1, G1-3, G1-5, G2-2) by Blast analysis.5. Preliminarily studied on bacterial agent of JH2 and JH3. The formulation of AS and GR of JH2, JH3 were obtained. The plants sensitive with Sulfometuron-methyl were screened by activity determination. The result showed that wheat is sensitive with Sulfometuron-methyl and IC50 was 0.014 mg·kg^-1. The residual amount of the soil contained Sulfometuron-methyl of 5 mg·kg^-1 and 1 mg·kg^-1 that degraded respectively by JH2 and JH3 AS for 15 days and 20 days were lower than 0.0077 mg·kg^-1.更多还原