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研究生： 林勇義
Giyanto - Wijaya Salim
論文名稱： Facile Hydrophobic Modification of Alginate by using Paper Sizing Agent Alkyl Ketene Dimer (AKD) for Enzyme Immobilization and Refolding
Facile Hydrophobic Modification of Alginate by using Paper Sizing Agent Alkyl Ketene Dimer (AKD) for Enzyme Immobilization and Refolding
指導教授： 李振綱
Cheng-kang Lee
口試委員: 劉懷勝
Hwai-shen Liu
王孟菊
Meng-jiy Wang
學位類別： 碩士
Master
系所名稱： 工程學院 - 化學工程系
Department of Chemical Engineering
論文出版年： 2010
畢業學年度： 98
語文別： 英文
論文頁數： 92
中文關鍵詞： alginate 、alkyl ketene dimer 、D-hydantoinase 、protein refolding 、hydrophobic modification
外文關鍵詞： alginate, alkyl ketene dimer, D-hydantoinase, protein refolding, hydrophobic modification
相關次數： 點閱：149  下載：0 
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    • As the sustainable developments were demanded, the research in bio-based materials needs to be conducted. Alginate, as one of the biopolymers, offers not only the benefit of other bio-based material, but also the ability to be employed in the protein immobilization. In order to increase the stability of the alginate as the protein immobilization support, a paper sizing agent, alkyl ketene dimer (AKD), had been grafted to the alginate. The hydrophobic modification process involved an alginate-AKD emulsification, solvent exchange, and extraction of the unreacted AKD. The analyses on the swelling kinetics, protein release, FTIR, and water contact angle showed the increased hydrophobicity of the AKD-modified alginate. To obtain the high hydrophobically modified alginate, 25% AKD concentration at the reaction temperature of 50°C had been employed. At this condition, the water contact angle rose to 93.8°, as well as the swelling ratio decreased to 1.13. The hydrophobic properties of the alginate-AKD enabled the application of the alginate on the protein refolding. The protein refolding was performed by complex precipitation and bead-dialysis methods using D-hydantoinase as the model protein. The enzymatic activity was regained after the refolding by using both of the two mentioned methods.

    As the sustainable developments were demanded, the research in bio-based materials needs to be conducted. Alginate, as one of the biopolymers, offers not only the benefit of other bio-based material, but also the ability to be employed in the protein immobilization. In order to increase the stability of the alginate as the protein immobilization support, a paper sizing agent, alkyl ketene dimer (AKD), had been grafted to the alginate. The hydrophobic modification process involved an alginate-AKD emulsification, solvent exchange, and extraction of the unreacted AKD. The analyses on the swelling kinetics, protein release, FTIR, and water contact angle showed the increased hydrophobicity of the AKD-modified alginate. To obtain the high hydrophobically modified alginate, 25% AKD concentration at the reaction temperature of 50°C had been employed. At this condition, the water contact angle rose to 93.8°, as well as the swelling ratio decreased to 1.13. The hydrophobic properties of the alginate-AKD enabled the application of the alginate on the protein refolding. The protein refolding was performed by complex precipitation and bead-dialysis methods using D-hydantoinase as the model protein. The enzymatic activity was regained after the refolding by using both of the two mentioned methods.

    ABSTRACT ii 碩士學位論文指導教授推薦書 iii 碩士學位考試委員審定書 iv 誌謝 v TABLE OF CONTENTS vi LIST OF FIGURES viii LIST OF TABLES x CHAPTER I INTRODUCTION 1 I.1. Background 1 I.2. Objectives 3 I.3. Thesis Organization 3 CHAPTER II LITERATURE REVIEW 5 II.1. Hydrophobically Modified Alginate (HMA) 5 II.2. Alkyl Ketene Dimers (AKD) 7 II.3. D-hydantoinase 10 II.4. Immobilization of Enzyme in the Hydrophobically Modified Alginate 17 II.5. Protein Refolding 21 II.5.1. Refolding by Dilution 22 II.5.2. On-column Refolding 22 II.5.3. Alginates as Protein Refolding Agent 23 II.5.4. Analytical Methods for Refolding 25 CHAPTER III METHODOLOGY 26 III.1. Materials 26 III.2. Equipments 28 III.3. Experimental Methods 29 III.3.1. Media and Stock Solution Preparations 31 III.3.2. Preparation of AKD-Modified Alginate 34 III.3.3. Growing the Culture of Escherichia coli Expressing EGFP 35 III.3.4. Growing The Culture of Escherichia coli pET-Ch-HDT Culture 36 III.3.5. Isolation and Solubilization of Inclusion Bodies 36 III.3.6. Immobilized Metal-ions Affinity Chromatography (IMAC) 37 III.3.7. Protein Refolding 38 III.3.8. Analyses 41 CHAPTER IV RESULTS AND DISCUSSION 47 IV.1. Fourier Transform Infra Red (FTIR) Spectra of Sodium Alginate 47 IV.2. Effect of Temperature on the Preparation of Alginate-AKD 54 IV.3. Effect of AKD Concentration on the Preparation of Alginate-AKD 56 IV.4. Optimum Condition for Alginate Modification 59 IV.5. Protein Refolding 66 IV.5.1. On-column Refolding 67 IV.5.2. Complex Precipitation Refolding 69 IV.6. Bead-dialysis Refolding 73 CHAPTER V CONCLUSION & RECOMMENDATION 76 V.1. Conclusion 76 V.2. Recommendation 77 REFERENCES 78 國立臺灣科技大學博碩士論文授權書 82

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