L-岩藻糖异构酶和D-阿拉伯糖异构酶的性质鉴定及在稀少糖生产中的应用
发布时间：2022-09-27 17:03
L-岩藻酮糖和D-核酮糖都是稀少糖,在食品、农业和医药工业具有广泛的潜在应用价值。它们属于戊糖,戊糖包括醛戊糖和酮戊糖两大类。总共有八种醛戊糖和四种酮戊糖,除少数几种是天然存在的糖,其他大多数都是稀少糖,在自然界存在极少。稀少糖拥有很大的商业应用价值,尤其是在医药领域。由于在自然界中含量极少,且化学合成法难度较大,稀少糖的价格较高,且无法满足工业化生产的需求。通过生物酶法,将L-岩藻糖和D-阿拉伯糖分别转化为L-岩藻酮糖和D-核酮糖,是一种有效的生产方法,具有广阔的工业应用前景。L-岩藻糖异构酶（L-FI;E.C.5.3.1.25）和D-阿拉伯糖异构酶（D-AI;EC 5.3.1.3）属于同一家族蛋白酶,催化功能相似,且底物谱较广。两种酶具有相似的异构化机理,均可催化L-岩藻糖和D-阿拉伯糖。目前,由于缺乏合适的生物催化剂,通过生物法催化L-岩藻糖和D-阿拉伯糖生产L-岩藻酮糖和D-核酮糖还没有实现工业化。这也导致了这两种糖的产量低且生产成本高。戊糖“Izumoring”策略提供了一个完整的戊糖转化方法,通过使用不同种类的酶将各种戊糖联系起来。总体来说,利用L-FI和D-AI酶法生产L...
【文章页数】：140 页
【学位级别】：博士
【文章目录】：
Acknowledgements
Dedication
List of Abbreviations
Abstract
摘要
CHAPTER ONE:General Introduction and Literature Review
1.1. General introduction
1.2. Literature Review
1.2.1.H istory of L-FI & D-AI
1.2.2. Brief introduction of L-fucose and L-fuculose
1.2.2.1. Chemical structure of L-fuculose
1.2.2.2. Existing sources of L-fuculose
1.2.2.3. Extraction of L-fuculose
1.2.3. Enzymatic properties of L-FI
1.2.3.1. Identification and characterization of L-FI
1.2.3.2. pH
1.2.3.3. Temperature/thermostability/T_m
1.2.3.4. Metal ions
1.2.3.5. Molecular weight (M_W),Kinetic parameters,and Substrate specificities
1.2.3.6. Bioconversion ratio
1.2.3.7. Molecular structure
1.2.4. Application of L-FI in L-fuculose production
1.2.4.1. Isomerizing mechanism of L-FI from L-fucose to L-fuculose
1.2.5. Applications of L-fuculose
1.2.5.1. Agriculture and Food
1.2.5.2. Pharmaceuticals/medicine
1.2.5.2.1. Brain protein metabolism
1.2.5.2.2. Anti-inflammatory
1.2.5.2.3. Skin aging
1.2.5.3. Synthetic chemistry
1.2.6. Brief introduction of D-arabinose and D-ribulose
1.2.6.1. Chemical structure of D-ribulose
1.2.6.2. Existing sources of D-ribulose
1.2.6.3. Extraction of D-ribulose
1.2.7. Enzymatic properties of D-AI
1.2.7.1. Identification and characterization of D-AI
1.2.7.2. pH
1.2.7.3. Temperature and thermostability of D-AI
1.2.7.4. Metal ions
1.2.7.5. Kinetic parameters,substrate specificity,and M_W
1.2.7.6. Conversion ratio
1.2.7.7. Molecular structure
1.2.8. Application of D-AI in D-ribulose production
1.2.8.1. Isomerizing mechanism of D-AI from D-arabinose to D-ribulose
1.2.9. Applications of D-ribulose
1.2.9.1. Agriculture and Food
1.2.9.2. Pharmaceuticals/medicine
1.2.9.3. Synthetic chemistry
1.3. Objectives
1.4. References
CHAPTER TWO:Biochemical characterization of recombinant L-fucose isomerase fromCaldanaerobius polysaccharolyticus for L-fuculose production
2.1. Introduction
2.2. Materials and methods
2.2.1. Materials and chemicals
2.2.2. Microorganisms,culture conditions,and media
2.2.3. Amino acid sequence analysis in a database search
2.2.4. Gene cloning and expression of recombinant Caldanaerobius polysaccharolyticus(Capo)
2.2.5. Purification of Capo-Lflase
2.2.6. Molecular mass determination of Capo-Lflase
2.2.7. Determination of protein and enzyme assay
2.2.8. pH and temperature effects on Capo-Lflase activity
2.2.9. Metal ion effects on Capo-LfIase activity
2.2.10. Homology and molecular modeling of Capo-LfIase
2.2.11. Specific activities and substrate specificity of Capo-LfIase
2.2.12. Kinetic studies of Capo-LfIase
2.2.13. Amino acid sequence comparison of Capo-LfIase
2.2.14. Enzymatic production of L-fuculose
2.3. Results and discussions
2.3.1. Gene cloning,expression and amino acid sequence comparison of Capo-LfIase
2.3.2. Purification of Capo-LfIase
2.3.3. Effects of pH, temperature, thermostability and melting temperature
2.3.4. The effect of metal ions on the activity of Capo-LfIase
2.3.5. Substrate specificity and kinetic study of Capo-LfIase
2.3.6. Homology and molecular modeling to identify active site residues
2.3.7. Bioconversion yield of L-fuculose from L-fucose
2.4. Conclusions
2.5. References
CHAPTER THREE:Characterization of a novel D-arabinose isomerase fromThermahaeromohas toyohehsis and its application for the production of D-ribulose and L-fuculose
3.1. Introduction
3.2. Materials and methods
3.2.1. Materials and chemicals
3.2.2. Gene cloning and expression
3.2.3. Protein purification and molecular mass determination of recombinant Thto-DaIase
3.2.4. Determination of protein concentration and enzyme assay
3.2.5. Effects of pH and temperature on the activity of recombinant Thto-DaIase
3.2.6. Effects of metal ions on recombinant Thto-Dalase activity
3.2.7. Homology modeling and amino acid sequence comparisons of recombinant Thto-DaIase
3.2.8. Specific activities,substrate specificities and kinetic parameters of recombinantThto-DaIase
3.2.9. Enzymatic production of D-ribulose and L-fuculose
3.3. Results and discussion
3.3.1. Homology and amino acid sequence comparison of putative Thto-DaIase amongother D-AIases
3.3.2. Overexpression and purification of the recombinant Thto-DaIase
3.3.3. Influences of pH, temperature, and thermostability on the activity and stability ofrecombinant Thto-DaIase
3.3.4. Effects of metal ions on recombinant Thto-DaIase activity
3.3.5. Substrate specificity of recombinant Thto-DaIase and enzyme kinetics
3.3.6. Bioproduction of D-ribulose and L-fuculose by recombinant Thto-DaIase
3.3.7. Docking and molecular modeling for active site residues of recombinant Thto-DaIase
3.4. Conclusions
3.5. References
CHAPTER FOUR: Characterization of L-fucose isomerase from Paenibacillusrhizosphaerae to produce L-fuculose from hydrolyzed fucoidan and commercial fucose
4.1. Introduction
4.2. Materials and methods
4.2.1. Chemicals,bacterial strains and plasmid
4.2.2. Extraction of fucoidan
4.2.2.1. Sampling and processing
4.2.2.2. Hydrolysis of fucoidan
4.2.2.3. HPAEC,GPC,and FTIR analysis of fucoidan
4.2.3. Characterization of recombinant Pa-LFI
4.2.3.1. Gene cloning,expression and purification
4.2.3.2. Determination of protein concentration and enzyme assay
4.2.3.3. Effects of pH, temperature and metal ions on recombinant Pa-LFI activity
4.2.3.4. Substrate specificity and kinetic parameters of recombinant Pa-LFI
4.2.3.5. Enzymatic production of L-fuculose by recombinant Pa-LFI
4.2.3.6. Molecular modeling and docking of recombinant Pa-LFI
4.3. Results and discussion
4.3.1. Extraction and M_W determination of fucoidan
4.3.2. Compositional and structural analysis of fucoidan
4.3.3. Amino acid sequence analysis of recombinant Pa-LFI
4.3.4. Expression and purification of the recombinant Pa-LFI
4.3.5. Effect of pH, temperature, and metal ions on the activity of recombinant Pa-LFI
4.3.6. Thermostability and melting temperature(T_m) of recombinant Pa-LFI
4.3.7. Specificity of substrates and kinetic parameters of recombinant Pa-LFI
4.3.8. Production of L-fuculose commercial fucose and fucoidan by recombinant Pa-LFI
4.3.9. Molecular modeling and docking to identify the active site residues
4.4. Conclusions
4.5. References
CHAPTER FIVE:General Conclusion and Recommendation
5.1. Conclusion
5.2. Key innovation of thesis
5.3. Recommendations
List of Publications
本文编号：3681260
【文章页数】：140 页
【学位级别】：博士
【文章目录】：
Acknowledgements
Dedication
List of Abbreviations
Abstract
摘要
CHAPTER ONE:General Introduction and Literature Review
1.1. General introduction
1.2. Literature Review
1.2.1.H istory of L-FI & D-AI
1.2.2. Brief introduction of L-fucose and L-fuculose
1.2.2.1. Chemical structure of L-fuculose
1.2.2.2. Existing sources of L-fuculose
1.2.2.3. Extraction of L-fuculose
1.2.3. Enzymatic properties of L-FI
1.2.3.1. Identification and characterization of L-FI
1.2.3.2. pH
1.2.3.3. Temperature/thermostability/T_m
1.2.3.4. Metal ions
1.2.3.5. Molecular weight (M_W),Kinetic parameters,and Substrate specificities
1.2.3.6. Bioconversion ratio
1.2.3.7. Molecular structure
1.2.4. Application of L-FI in L-fuculose production
1.2.4.1. Isomerizing mechanism of L-FI from L-fucose to L-fuculose
1.2.5. Applications of L-fuculose
1.2.5.1. Agriculture and Food
1.2.5.2. Pharmaceuticals/medicine
1.2.5.2.1. Brain protein metabolism
1.2.5.2.2. Anti-inflammatory
1.2.5.2.3. Skin aging
1.2.5.3. Synthetic chemistry
1.2.6. Brief introduction of D-arabinose and D-ribulose
1.2.6.1. Chemical structure of D-ribulose
1.2.6.2. Existing sources of D-ribulose
1.2.6.3. Extraction of D-ribulose
1.2.7. Enzymatic properties of D-AI
1.2.7.1. Identification and characterization of D-AI
1.2.7.2. pH
1.2.7.3. Temperature and thermostability of D-AI
1.2.7.4. Metal ions
1.2.7.5. Kinetic parameters,substrate specificity,and M_W
1.2.7.6. Conversion ratio
1.2.7.7. Molecular structure
1.2.8. Application of D-AI in D-ribulose production
1.2.8.1. Isomerizing mechanism of D-AI from D-arabinose to D-ribulose
1.2.9. Applications of D-ribulose
1.2.9.1. Agriculture and Food
1.2.9.2. Pharmaceuticals/medicine
1.2.9.3. Synthetic chemistry
1.3. Objectives
1.4. References
CHAPTER TWO:Biochemical characterization of recombinant L-fucose isomerase fromCaldanaerobius polysaccharolyticus for L-fuculose production
2.1. Introduction
2.2. Materials and methods
2.2.1. Materials and chemicals
2.2.2. Microorganisms,culture conditions,and media
2.2.3. Amino acid sequence analysis in a database search
2.2.4. Gene cloning and expression of recombinant Caldanaerobius polysaccharolyticus(Capo)
2.2.5. Purification of Capo-Lflase
2.2.6. Molecular mass determination of Capo-Lflase
2.2.7. Determination of protein and enzyme assay
2.2.8. pH and temperature effects on Capo-Lflase activity
2.2.9. Metal ion effects on Capo-LfIase activity
2.2.10. Homology and molecular modeling of Capo-LfIase
2.2.11. Specific activities and substrate specificity of Capo-LfIase
2.2.12. Kinetic studies of Capo-LfIase
2.2.13. Amino acid sequence comparison of Capo-LfIase
2.2.14. Enzymatic production of L-fuculose
2.3. Results and discussions
2.3.1. Gene cloning,expression and amino acid sequence comparison of Capo-LfIase
2.3.2. Purification of Capo-LfIase
2.3.3. Effects of pH, temperature, thermostability and melting temperature
2.3.4. The effect of metal ions on the activity of Capo-LfIase
2.3.5. Substrate specificity and kinetic study of Capo-LfIase
2.3.6. Homology and molecular modeling to identify active site residues
2.3.7. Bioconversion yield of L-fuculose from L-fucose
2.4. Conclusions
2.5. References
CHAPTER THREE:Characterization of a novel D-arabinose isomerase fromThermahaeromohas toyohehsis and its application for the production of D-ribulose and L-fuculose
3.1. Introduction
3.2. Materials and methods
3.2.1. Materials and chemicals
3.2.2. Gene cloning and expression
3.2.3. Protein purification and molecular mass determination of recombinant Thto-DaIase
3.2.4. Determination of protein concentration and enzyme assay
3.2.5. Effects of pH and temperature on the activity of recombinant Thto-DaIase
3.2.6. Effects of metal ions on recombinant Thto-Dalase activity
3.2.7. Homology modeling and amino acid sequence comparisons of recombinant Thto-DaIase
3.2.8. Specific activities,substrate specificities and kinetic parameters of recombinantThto-DaIase
3.2.9. Enzymatic production of D-ribulose and L-fuculose
3.3. Results and discussion
3.3.1. Homology and amino acid sequence comparison of putative Thto-DaIase amongother D-AIases
3.3.2. Overexpression and purification of the recombinant Thto-DaIase
3.3.3. Influences of pH, temperature, and thermostability on the activity and stability ofrecombinant Thto-DaIase
3.3.4. Effects of metal ions on recombinant Thto-DaIase activity
3.3.5. Substrate specificity of recombinant Thto-DaIase and enzyme kinetics
3.3.6. Bioproduction of D-ribulose and L-fuculose by recombinant Thto-DaIase
3.3.7. Docking and molecular modeling for active site residues of recombinant Thto-DaIase
3.4. Conclusions
3.5. References
CHAPTER FOUR: Characterization of L-fucose isomerase from Paenibacillusrhizosphaerae to produce L-fuculose from hydrolyzed fucoidan and commercial fucose
4.1. Introduction
4.2. Materials and methods
4.2.1. Chemicals,bacterial strains and plasmid
4.2.2. Extraction of fucoidan
4.2.2.1. Sampling and processing
4.2.2.2. Hydrolysis of fucoidan
4.2.2.3. HPAEC,GPC,and FTIR analysis of fucoidan
4.2.3. Characterization of recombinant Pa-LFI
4.2.3.1. Gene cloning,expression and purification
4.2.3.2. Determination of protein concentration and enzyme assay
4.2.3.3. Effects of pH, temperature and metal ions on recombinant Pa-LFI activity
4.2.3.4. Substrate specificity and kinetic parameters of recombinant Pa-LFI
4.2.3.5. Enzymatic production of L-fuculose by recombinant Pa-LFI
4.2.3.6. Molecular modeling and docking of recombinant Pa-LFI
4.3. Results and discussion
4.3.1. Extraction and M_W determination of fucoidan
4.3.2. Compositional and structural analysis of fucoidan
4.3.3. Amino acid sequence analysis of recombinant Pa-LFI
4.3.4. Expression and purification of the recombinant Pa-LFI
4.3.5. Effect of pH, temperature, and metal ions on the activity of recombinant Pa-LFI
4.3.6. Thermostability and melting temperature(T_m) of recombinant Pa-LFI
4.3.7. Specificity of substrates and kinetic parameters of recombinant Pa-LFI
4.3.8. Production of L-fuculose commercial fucose and fucoidan by recombinant Pa-LFI
4.3.9. Molecular modeling and docking to identify the active site residues
4.4. Conclusions
4.5. References
CHAPTER FIVE:General Conclusion and Recommendation
5.1. Conclusion
5.2. Key innovation of thesis
5.3. Recommendations
List of Publications
本文编号：3681260
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