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研究生： 江亭瑩
Ting-Ying Jiang
論文名稱： 澱粉吸附區與配體交互作用之功能性分析(Functional Characterization of Interaction between Starch Binding Domain and Ligands)
Functional Characterization of Interaction between Starch Binding Domain and Ligands
指導教授： 張大慈
Margaret Dah-Tsyr Chang
口試委員:
學位類別： 碩士
Master
系所名稱： 生命科學暨醫學院 - 分子與細胞生物研究所
Institute of Molecular and Cellular Biology
論文出版年： 2008
畢業學年度： 96
語文別： 英文
論文頁數： 63
中文關鍵詞： 葡糖澱粉酶 、澱粉吸附區 、醣類吸附模組 、原子力顯微鏡
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    • 米根黴菌 (Rhizopus oryzae) 的葡糖澱粉酶 (glucoamylase、GA) 屬於一種醣類水解酶，其整體結構包含胺基端的澱粉吸附區 (starch-binding domain、RoSBD) 與羧基端的催化區。RoSBD隸屬於醣類吸附模組 (carbohydrate-binding module, CBM) 家族二十一，能與顆粒狀生澱粉結合。SBD的主要功能為促進配體與GA結合、增加催化區活化位置的配體濃度以提升酵素水解效率。近年來許多研究指出SBD能利用非水解的方式讓生澱粉配體的結構鬆弛，使之更容易讓催化區接近而提高酵素的催化活性。SBD與配體的結合主要靠芳香環胺基酸與醣類的六角環形成交互作用，以及極性胺基酸與多醣的羧基形成氫鍵結合。根據核磁共振與結晶繞射解析出的米根黴菌葡糖澱粉酶之澱粉結合區 (RoSBD) 三級結構，推測天門冬胺酸29、酪胺酸32、離胺酸34、色胺酸47、天門冬胺酸50、苯丙胺酸58、酪胺酸67、穀胺酸68、酪胺酸83、酪胺酸93、酪胺酸94、天門冬胺酸96和天門冬胺酸101可能為參與配體結合的重要胺基酸。本實驗室過去的研究中曾以單點突變、旋光儀分析蛋白質二級結構，並以定量配體結合試驗結檢視此B型CBM和多醣之間的疏水性作用力與氫鍵結合力。結果顯示芳香環與極性胺基酸與配體作用時扮演不同的角色，前者主要負責RoSBD與配體結合，後者則於澱粉結合後，促使澱粉支鏈鬆弛而暴露出更多的表面積，使得更多RoSBD得以與配體結合。為了證實RoSBD與澱粉結合後將讓配體產生結構的改變，本研究以原子力顯微鏡觀察RoSBD與澱粉醣 (amylose) 之間的作用。初步研究結果發現RoSBD與澱粉醣結合後會改變澱粉醣的形態，而單點突變重要胺基酸的RoSBD其改變澱粉醣型態的能力會明顯地減弱。由不同的單點突變RoSBD的觀察結果本論文首先提出RoSBD改變澱粉醣形態時，其可能經歷不同階段的反應模式。

    中文摘要 I Abstract II Acknowledgement IV Table of Contents V List of Figures VII List of Tables IX Abbreviations X Chapter 1 Introduction 1 1. 1 Starch and Glucoamylase 1 1. 2 Atomic force microscopy 2 1. 3 Research motivation 4 Chapter 2 Materials and Methods 10 2. 1 Enzymes, reagents and chemicals 10 2. 2 Microbial strains, plasmids and culture conditions 11 2. 3 Construction of plasmids 12 2. 4 Competent cell preparation and transformation of E. coli 14 2. 5 In situ PCR 15 2. 6 Mini-preparation of plasmid and DNA sequencing 15 2. 7 Expression of recombinant SBDs by E. coli 17 2. 8 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting 17 2. 9 Purification of recombinant SBDs and determination of protein concentration 18 2. 10 Samples preparation for atomic force microscopy and imaging procedures 19 Chapter 3 Results 21 3. 1 Expression and purification of recombinant RoSBD and AnSBD 21 3. 2 Visualisation of the complexes between amylose and RoSBD 23 3. 3 Visualizations of the complexes between amylose typeIII and RoSBD derivatives 24 3. 3. 1 Wild-type RoSBD and wild-type RoSBD with fusion tag 24 3. 3. 2 RoSBD-6xHis mutant with point mutation of aromatic residue 25 3. 3. 3 RoSBD-6xHis mutant with point mutation of polar residue 25 Chapter 4 Discussion 46 4. 1 Preparation of AFM samples 46 4. 2 Different contribution of aromatic and polar residues in amylose disruption 47 4. 3 Different conformational change of amylose in the presence of RoSBD 47 4. 4 Conservation of ligand binding residues in CBM21 members 48 4. 5 Visualization of the complexes of amylose/AnSBD and amylose/RoSBD 49 References 51 Appendix 56

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