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摘要：3,5-二溴-4-羟基苯甲酸（3,5-dibromo-4-hydroxybenzoate, DBHB）是一种溴代芳烃，易存在于化工、食品、医药工业的废水中，并且达到可观的浓度，目前是主要的环境污染物，但对于DBHB的降解研究很少。本实验室前期从长期受卤代芳烃污染的土壤中分离筛选到一株高效降解DBHB的菌株Pigmentiphaga sp.H8，经过比较转录组学和比较蛋白组学的研究，推断菌株Pigmentiphaga sp.H8基因组中orf420-426参与DBHB的降解。本文通过基因敲除和回补以及异源表达实验证明orf420（odcA）是降解DBHB的关键基因，获得了纯的OdcA蛋白。通过体外酶学转化实验和LC-MS分析，鉴定OdcA降解DBHB的产物是2,6-二溴对苯二酚。通过酶促反应研究了OdcA的酶学特性。35711
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关键词：3,5-二溴-4-羟基苯甲酸；Pigmentiphaga sp.H8；odcA；菌株Pigmentiphaga sp. H8中3,5-二溴-4-羟基苯甲酸关键降解酶基因odcA的功能验证
Functional verification of the key gene odcA for 3,5-dibrimo-4-hydroxybenzoate catabolism in strain Pigmentiphaga sp. H8
Abstract：3,5-dibromo-4-hydroxybenzoic acid is a mental type of environmental pollutants belonging to brominated aromatic compound, which can be easily found in medical treatment, chemical industry and food industry. However, few studies on the microbial degradation of DBHB have been done. Pigmentiphaga sp. H8 is a strain isolated from long-term contaminated soil with halogenated aromatic compounds. It can mineralize DBHB quickly and effectively. We can find genes involved in the degradation of DBHB by comparing the differences in mRNA transcription and proteomic expression between DBHB-induced and non-induced strains. The results show that orf420-426 of the strain H8 genome may involved in degradation DBHB. This study uses the methods of gene knockout , complementary experiments and heterologous expression to prove odcA is the key gene of metabolic pathway of DBHB in strain H8. We get the purified OdcA. By in vitro enzymatic transformation experiments and LC-MS analysis, we identified the product of OdcA degradation of DBHB was 2,6-dibromohydroquinone. Studies about the best situation of OdcA’ s enzymatic reactions are also been done.
Key words: 3,5-dibromo-4-hydroxybenzoate；Pigmentiphaga sp.H8；odcA；
目 录
摘要.1
关键词.1
Abstract.1
Key words1
引言.2
1材料与方法3
1.1材料3
1.1.1菌株、质粒、因物.3
1.1.2培养基和试剂4
1.2方法.4
1.2.1大肠杆菌一步法感受态的制备与转化4
1.2.2质粒的提取5
1.2.3细菌总DNA的提取.5
1.2.4静息细胞法5
1.2.5odcA基因的敲除5
1.2.6odcA基因的回补.5
1.2.7odcA基因的异源表达.6
1.2.8OdcA蛋白的纯化.6
1.2.9DBHB的检测.6
1.2.10 SDS聚丙烯酰胺凝胶电泳（SDS-PAGE）.6
1.2.11 OdcA的酶学特性6
2 结果与分析.7
2.1基因odcA缺失菌株H8ΔodcA和回补菌株H8ΔodcA(pMCS-odcA)降解DBHB结果.7
2.2 OdcA异源表达和纯化7
2.3 OdcA催化DBHB降解产物的测定8
2.4 OdcA的酶学特性研究结果.8
2.4.1辅因子对OdcA的酶活影响8
2.4.2温度对OdcA的酶活影响.9
2.4.3pH对OdcA的酶活影响.9
2.4.4金属离子对OdcA的酶活影响.9
2.4.5OdcA酶促反应动力学常数的测定.10
3讨论和总结.10
3.1本实验研究前提.10
3.2本研究总结.10
3.3本研究创新点与不足.10
致谢11
参考文献11
引言
卤代芳烃类化合物具有稳定性好、阻燃、耐疲劳、耐热等优点，广泛运用于化工中间体、阻燃剂、农药、医药[1,2]等生产。由于卤代芳烃具有难降解、高毒性等特点，很多被列为“持久性有机污染物”（ Persistent Organic Pollutants, POPs）和“优先控制污染物”（Priority pollutants）[3,4]。卤素原子的强电子吸附效应使得卤代芳烃易与生命细胞内的酶系统结合进而对细胞产生毒性。因此，卤代芳烃造成的环境污染对生态环境和人类健康造成严重的威胁[5]，卤代芳烃的微生物降解与污染修复已引起学术界的广泛关注。研究卤代芳烃的微生物降解与污染修复具有重要的意义。