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Studies on Enzymatic Synthesis of Functional Sugars

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Studies on Enzymatic Synthesis of Functional Sugars Studies on enzymatic synthesis of functional sugars A Dissertation Submitted to the Graduate School of Bioagricultural Sciences, Nagoya University in Partial Fulfillment of the Requirements for the Degree of Doctor of Agricultural Sciences March, 2013 Teruyo Ojima Laboratory of Molecular Biotechnology, Division of Biotechnology, Department of Bioengineering Sciences, Graduate School of Bioagricultural Sciences, Nagoya University Table of contents (page) Abbreviations ................................................................................................... 1 Chapter 1. General introduction ...................................................................... 3 1.1. Enzymatic synthesis of sugars ................................................................. 3 1.2. Value added sugars ................................................................................. 3 1.3. α-Glucosidase and glucoside ................................................................... 3 1.3.1 α-Glucosidase .................................................................................... 3 1.3.2. Industrial application of α-glucosidase .............................................. 5 1.3.3. Enzymatic synthesis of glucosides .................................................... 6 1.3.4. Problems in the synthesis of glucoside ............................................. 6 1.4. Cellobiose 2-epimerase and epilactose ................................................... 8 1.4.1. Research history of cellobiose 2-epimerase ...................................... 8 1.4.2. Enzymatic synthesis of epilactose ................................................... 10 1.5. The scope of this study .......................................................................... 10 Figures .......................................................................................................... 13 Chapter 2. Screening of α-glucosidase ........................................................ 16 2.1. Introduction ............................................................................................ 16 2.2. Materials and methods ........................................................................... 16 2.2.1. Isolation of bacterial strain. .............................................................. 16 2.2.2. Screening of α-glucosidase ............................................................. 17 2.2.3. Identification of bacterial strain. ....................................................... 18 2.3. Results ................................................................................................... 19 2.3.1. Selection of bacterial strain ............................................................. 19 2.3.2. Identification of bacterial strain ........................................................ 19 2.4. Discussion ............................................................................................. 20 Figures and Tables........................................................................................ 22 Chapter 3. Isolation and characterization of α-glucosidase from Halomonas sp. H11 ........................................................................................ 28 3.1. Introduction ............................................................................................ 28 3.2. Materials and methods ........................................................................... 29 3.2.1. Purification of α-glucosidase from Halomonas sp. H11 strain ......... 29 3.2.2. Enzyme activity assay ..................................................................... 30 3.2.3. Effects of pH and temperature on activity and stability .................... 31 3.2.4. Substrate specificity ........................................................................ 31 3.2.5. Effects of various salts on enzyme activity and stability .................. 32 3.2.6. Kinetic parameters for maltose and pNPG in the presence of monovalent cations ................................................................................... 32 3.2.7. Transglucosylation activity toward various alcohols ........................ 34 3.2.8. Enzymatic synthesis of α-glucosylglycerol ...................................... 34 3.3. Results ................................................................................................... 35 3.3.1. Purification of α-glucosidase from Halomonas sp. H11 ................... 35 3.3.2. Effects of pH and temperature......................................................... 36 3.3.3. Substrate specificity ........................................................................ 36 3.3.4. Effects of various salts on enzyme activity ...................................... 36 3.3.5. Kinetic analysis for activation of the enzyme by monovalent cations .................................................................................................................. 37 3.3.6. Effects of monovalent cations on pH–activity and temperature–activity .................................................................................................................. 38 3.3.7. Transglucosylation toward various alcohols .................................... 39 3.3.8. Enzymatic synthesis of α-D-glucosylglycerol ................................... 39 3.4. Discussion ............................................................................................. 40 Figures and Tables........................................................................................ 44 Chapter 4. Cloning and sequence analysis of α-glucosidase from Halomonas sp. H11 ........................................................................................ 58 4.1. Introduction ............................................................................................ 58 4.2. Materials and methods ........................................................................... 58 4.2.1. Analysis of N-terminal amino acid sequence ................................... 58 4.2.2. Analysis of internal amino acid sequence ........................................ 58 4.2.3. Cloning of the gene encoding α-glucosidase from Halomonas sp. H11 .................................................................................................................. 59 4.2.4. Recombinant expression in E. coli .................................................. 61 4.2.5. General properties of recombinant enzyme .................................... 62 4.3. Results ................................................................................................... 62 4.3.1. Amino acid sequence analysis ........................................................ 62 4.3.2. DNA sequence analysis .................................................................. 63 4.3.3. Expression, purification and characterization of recombinant enzyme .................................................................................................................. 64 4.4. Discussion ............................................................................................. 64 Figures and Tables........................................................................................ 67 Chapter 5. Enzymatic synthesis of α-glucosylated 6-gingerol ................... 73 5.1. Introduction ............................................................................................ 73 5.2. Materials and methods ........................................................................... 74 5.2.1. Enzymes ......................................................................................... 74 5.2.2. Enzyme assays ............................................................................... 75 5.2.3. Screening of enzymes for glucosylation of 6-gingerol ..................... 75 5.2.4. Purification of 5-α-Glc-gingerol and 5-α-Mal-gingerol ...................... 76 5.2.5. Structural analysis of glucosylated 6-gingerol ................................. 76 5.2.6. Time-course of production of 5-α-Glc-gingerol by α-Glucosidase from Halomonas sp. H11 ................................................................................... 77 5.2.7. Determination of physical properties of 5-α-Glc-gingerol ................ 78 5.3. Results ................................................................................................... 79 5.3.1. Glucosylation of 6-gingerol with various α-glucosidases and CGTases .................................................................................................................. 79 5.3.2. Chemoenzymatic synthesis of glucosylated 6-gingerol by α-Glucosidase from Halomonas sp. H11 ................................................... 80 5.3.3. Properties of 5-α-Glc-gingerol ......................................................... 82 5.4. Discussion ............................................................................................. 83 Figures and Tables........................................................................................ 87 Chapter 6. Cloning and characterization of cellobiose 2-epimerases from aerobic bacteria ............................................................................................ 101 6.1. Introduction .......................................................................................... 101 6.2. Materials and methods ......................................................................... 102 6.2.1. Bacterial strains ............................................................................. 102 6.2.2. Construction of the expression
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