食碱戈登氏菌YC-RL2降解邻苯二甲酸酯的分子机制
发布时间：2022-04-23 14:38
邻苯二甲酸酯（phthalic acid esters,PAEs）的毒性和致癌性受到越来越多的关注。PAEs在不同环境介质中普遍检出,甚至极地偏远地区如北极圈内也有检出。因其半挥发性和疏水性,PAEs能够长距离运输并在环境中存留。PAEs可以在厌氧和营养不良的环境中存留多年。Gordonia属（放线菌门）在PAEs降解中尤其重要。虽然微生物降解PAEs的途径已被阐明,但菌株和酶的基因信息有限且能够水解高分子量PAEs的酶很少被报道。利用Biosciences RS II平台和单分子实时（SMRT）技术对分离的食碱戈登氏菌YC-RL2基因组进行测序。利用NCBI原核生物基因组注释程序进行注释。生成的基因组序列为4979656 bp,平均G+C含量为67.45%。平均核苷酸一致性证实菌株YC-RL2确实是G.alkanivorans。在COG数据库中进行检索;3132个CDSs聚类到COG家族,预测有1808个CDSs参与了111个通路。95个KEGG注释基因被预测参与了外源物质降解。未发现PAEs降解操纵子,表明菌株YC-RL2具有一种新PAEs降解途径。203个CDSs和22个CDSs...
【文章页数】：137 页
【学位级别】：博士
【文章目录】：
摘要
abstract
List of Abbreviations
Chapter1:General Introduction
1.1 Background
1.2 Problem statement
1.3 Justification of the study
1.4 General objective
1.5 Specific objectives
Chapter2:Literature review
2.1 Occurrence of PAEs
2.1.1 Surface waters and sediments
2.1.2 Occurrence in Air
2.1.3 Agricultural soils
2.1.4 Wastewater treatment plants and sludge
2.1.5 Occurrence in landfills
2.1.6 Food,beverages and personal care products
2.2 Eco-toxicological effects of PAEs
2.2.1 Effect of PAEs on soil properties and crops
2.2.2 Effect on aquatic organisms
2.2.3 Effects on terrestrial organisms
2.2.4 Effect on humans
2.3 Microbial removal of PAEs
2.3.1 PAEs biodegradation pathways in bacteria
2.3.2 Role of genus Gordonia in degradation of PAEs
Chapter3:Genome sequencing,annotation and comparative analysis of strain YC-RL
3.1 Introduction
3.2 Materials and methods
3.2.1 Chemicals and media
3.2.2 Substrate utilization tests and intermediate analysis
3.2.3 DNA extraction,genome library preparation and sequencing
3.2.4 Phylogenetic and comparative genomic analysis
3.2.5 Prediction of secondary metabolite potential
3.2.6 Heavy metal tolerance tests
3.2.7 Accession numbers
3.3 Results
3.3.1 Genome sequence and annotation results
3.3.2 Classification
3.3.3 COG analysis and KEGG analysis
3.3.4 Analysis of phthalate catabolic genes
3.3.5 Secondary metabolite biosynthesis potential
3.3.6 Heavy metal resistance
3.3.7 Genetic basis for colony morphology changes
3.4 Discussion
3.5 Conclusion
Chapter4:Cloning and expression of a novel carboxylesterase Car259 capable of hydrolyzing Di-2-ethylhexyl phthalate
4.1 Introduction
4.2 Materials and methods
4.2.1 Chemicals and reagents
4.2.2 Cloning,expression and purification of Car
4.2.3 Enzyme assay
4.2.4 Substrate specificity
4.2.5 Detection of degradation intermediates
4.2.6 Homology modeling and model verification
4.2.7 Protein-ligand docking and visualization
4.2.8 Construction of mutants
4.3 Results
4.3.1 Expression and purification of Car
4.3.2 Multiple sequence alignment and phylogenetic analysis
4.3.3 Effect of pH,temperature,ions,solvents and detergents on Car259 activity
4.3.4 Substrate specificity
4.3.5 Enzyme Kinetic parameters
4.3.6 Degradation intermediates
4.3.7 Homology modeling and docking results
4.4 Discussion
4.5 Conclusion
Chapter5:Biochemical and structural characterization of a monoethylhexyl phthalate hydrolase(MehpH)
5.1 Introduction
5.2 Materials and methods
5.2.1 Chemicals,solutions and media
5.2.2 Protein purification
5.2.3 Enzyme activity assays
5.2.4 Enzyme structure modeling
5.3 Results
5.3.1 Protein purification
5.3.2 Enzyme activity assays
5.3.3 Enzyme structure modeling
5.4 Discussion
5.5 Conclusion
Chapter6:General conclusions and recommendations
6.1 Conclusions
6.2 Recommendations
References
Acknowledgements
Author’s Resume
【参考文献】：
期刊论文
[1]MehpH的生化与结构特性（英文）[J]. Ruth Nahurira,贾阳,任磊,王俊欢,乔铖,樊双虎,王海胜,闫艳春. 微生物学报. 2018(02)
[2]邻苯二甲酸酯在不同类型土壤-植物系统中的累积特征研究[J]. 宋广宇,代静玉,胡锋. 农业环境科学学报. 2010(08)
[3]城市污泥和化肥对水稻土种植的通菜中邻苯二甲酸酯（PAEs）的影响[J]. 蔡全英,莫测辉,朱夕珍,吴启堂,王伯光,蒋成爱,李海芹. 应用生态学报. 2003(11)
本文编号：3647382
【文章页数】：137 页
【学位级别】：博士
【文章目录】：
摘要
abstract
List of Abbreviations
Chapter1:General Introduction
1.1 Background
1.2 Problem statement
1.3 Justification of the study
1.4 General objective
1.5 Specific objectives
Chapter2:Literature review
2.1 Occurrence of PAEs
2.1.1 Surface waters and sediments
2.1.2 Occurrence in Air
2.1.3 Agricultural soils
2.1.4 Wastewater treatment plants and sludge
2.1.5 Occurrence in landfills
2.1.6 Food,beverages and personal care products
2.2 Eco-toxicological effects of PAEs
2.2.1 Effect of PAEs on soil properties and crops
2.2.2 Effect on aquatic organisms
2.2.3 Effects on terrestrial organisms
2.2.4 Effect on humans
2.3 Microbial removal of PAEs
2.3.1 PAEs biodegradation pathways in bacteria
2.3.2 Role of genus Gordonia in degradation of PAEs
Chapter3:Genome sequencing,annotation and comparative analysis of strain YC-RL
3.1 Introduction
3.2 Materials and methods
3.2.1 Chemicals and media
3.2.2 Substrate utilization tests and intermediate analysis
3.2.3 DNA extraction,genome library preparation and sequencing
3.2.4 Phylogenetic and comparative genomic analysis
3.2.5 Prediction of secondary metabolite potential
3.2.6 Heavy metal tolerance tests
3.2.7 Accession numbers
3.3 Results
3.3.1 Genome sequence and annotation results
3.3.2 Classification
3.3.3 COG analysis and KEGG analysis
3.3.4 Analysis of phthalate catabolic genes
3.3.5 Secondary metabolite biosynthesis potential
3.3.6 Heavy metal resistance
3.3.7 Genetic basis for colony morphology changes
3.4 Discussion
3.5 Conclusion
Chapter4:Cloning and expression of a novel carboxylesterase Car259 capable of hydrolyzing Di-2-ethylhexyl phthalate
4.1 Introduction
4.2 Materials and methods
4.2.1 Chemicals and reagents
4.2.2 Cloning,expression and purification of Car
4.2.3 Enzyme assay
4.2.4 Substrate specificity
4.2.5 Detection of degradation intermediates
4.2.6 Homology modeling and model verification
4.2.7 Protein-ligand docking and visualization
4.2.8 Construction of mutants
4.3 Results
4.3.1 Expression and purification of Car
4.3.2 Multiple sequence alignment and phylogenetic analysis
4.3.3 Effect of pH,temperature,ions,solvents and detergents on Car259 activity
4.3.4 Substrate specificity
4.3.5 Enzyme Kinetic parameters
4.3.6 Degradation intermediates
4.3.7 Homology modeling and docking results
4.4 Discussion
4.5 Conclusion
Chapter5:Biochemical and structural characterization of a monoethylhexyl phthalate hydrolase(MehpH)
5.1 Introduction
5.2 Materials and methods
5.2.1 Chemicals,solutions and media
5.2.2 Protein purification
5.2.3 Enzyme activity assays
5.2.4 Enzyme structure modeling
5.3 Results
5.3.1 Protein purification
5.3.2 Enzyme activity assays
5.3.3 Enzyme structure modeling
5.4 Discussion
5.5 Conclusion
Chapter6:General conclusions and recommendations
6.1 Conclusions
6.2 Recommendations
References
Acknowledgements
Author’s Resume
【参考文献】：
期刊论文
[1]MehpH的生化与结构特性（英文）[J]. Ruth Nahurira,贾阳,任磊,王俊欢,乔铖,樊双虎,王海胜,闫艳春. 微生物学报. 2018(02)
[2]邻苯二甲酸酯在不同类型土壤-植物系统中的累积特征研究[J]. 宋广宇,代静玉,胡锋. 农业环境科学学报. 2010(08)
[3]城市污泥和化肥对水稻土种植的通菜中邻苯二甲酸酯（PAEs）的影响[J]. 蔡全英,莫测辉,朱夕珍,吴启堂,王伯光,蒋成爱,李海芹. 应用生态学报. 2003(11)
本文编号：3647382
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