L-巖藻酮糖和D-核酮糖都是稀少糖,在食品、農(nóng)業(yè)和醫(yī)藥工業(yè)具有廣泛的潛在應(yīng)用價值。它們屬于戊糖,戊糖包括醛戊糖和酮戊糖兩大類。總共有八種醛戊糖和四種酮戊糖,除少數(shù)幾種是天然存在的糖,其他大多數(shù)都是稀少糖,在自然界存在極少。稀少糖擁有很大的商業(yè)應(yīng)用價值,尤其是在醫(yī)藥領(lǐng)域。由于在自然界中含量極少,且化學(xué)合成法難度較大,稀少糖的價格較高,且無法滿足工業(yè)化生產(chǎn)的需求。通過生物酶法,將L-巖藻糖和D-阿拉伯糖分別轉(zhuǎn)化為L-巖藻酮糖和D-核酮糖,是一種有效的生產(chǎn)方法,具有廣闊的工業(yè)應(yīng)用前景。L-巖藻糖異構(gòu)酶（L-FI;E.C.5.3.1.25）和D-阿拉伯糖異構(gòu)酶（D-AI;EC 5.3.1.3）屬于同一家族蛋白酶,催化功能相似,且底物譜較廣。兩種酶具有相似的異構(gòu)化機(jī)理,均可催化L-巖藻糖和D-阿拉伯糖。目前,由于缺乏合適的生物催化劑,通過生物法催化L-巖藻糖和D-阿拉伯糖生產(chǎn)L-巖藻酮糖和D-核酮糖還沒有實現(xiàn)工業(yè)化。這也導(dǎo)致了這兩種糖的產(chǎn)量低且生產(chǎn)成本高。戊糖“Izumoring”策略提供了一個完整的戊糖轉(zhuǎn)化方法,通過使用不同種類的酶將各種戊糖聯(lián)系起來。總體來說,利用L-FI和D-AI酶法生產(chǎn)L... 

【文章頁數(shù)】：140 頁

【學(xué)位級別】：博士

【文章目錄】：
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



本文編號：3681260