米根黴菌 (Rhizopus oryzae) 的葡糖澱粉酵素 (glucoamylase, GA) 為含579個胺基酸之醣類水解酶，其結構包括澱粉吸附區 (starch-binding domain, RoSBD) 、酵素催化區、及一段高度醣基化的連接片段。其中催化區之功能及結構的研究成果眾多，本研究旨在探討連接片段及澱粉吸附區的功能，以瞭解此酵素之基本結構、生物功能及應用的潛力。
許多葡糖澱粉酵素皆有連接片段聯結不同的功能區域，但對於連接區域的分子功能則較少著墨。本研究以不同長度、不同醣基化修飾之連接片段為材料，觀察其功能及穩定性的變化，以釐清該片段對於不同功能區域的影響。實驗結果顯示連接片段的確會影響該酵素的表現及功能，並與蛋白質結構的穩定性關係密切。
醣類吸附模組 (carbohydrate-binding module, CBM) 對於不溶性多醣的水解具重要的調節功能。本研究以隸屬醣類吸附模組家族二十一的RoSBD為材料，進行生物化學與生物物理特性分析、及功能性應用之研究，並剖析RoSBD與其他類似功能CBM家族間的關聯性。位於胺基端之RoSBD為含106個胺基酸的獨立結構，能與顆粒狀生澱粉進行親合性的結合。SBD的主要功能為促使配體與GA結合、增加催化區活化位置的糖基質濃度、及提高酵素水解效率。研究蛋白質-醣類交互作用，不僅可由分子層面揭示蛋白質的功能，深入了解醣類基質的結合機制，更能進一步運用於重組蛋白質工程，使基礎研發成果與產業需求結合，具體貢獻於生技領域的發展。

Fungal glucoamylase (GA; 1,4-□-D-glucan glucohydrolase, EC. 3.2.1.3) from Rhizopus oryzae consists of 579 amino acids with starch binding domain (SBD) and a catalytic domain connected by a highly glycosylated linker region. The function and structure of the catalytic domain have been thoroughly investigated; here this study focuses on the characterization and application of linker and ligand binding domain.
Many GAs have linker regions separating the SBD from the catalytic domain. The extent of interaction between the catalytic modules in GAs with carbohydrate binding module family 21 (CBM21) remains an open question. Our data provide direct evidence that although the length, composition, and glycosylation of the interdomain linker vary in structural organization and in flexibility of the functional domains in GAs, presence of the linker indeed promotes the hydrolytic action towards the natural substrates.
Insoluble polysaccharides can be degraded by hydrolytic enzymes appended to one or more non-catalytic CBMs. The CBM21 of RoGA (RoSBD) containing 106 amino acid residues is situated N-terminally and retained functionality in its isolated form. The properties and applications of recombinant RoSBD are described here and compared with other SBD-containing CBM families, including 20, 25, 26, 34, 41, 45, 48 and 53. Understanding of the CBMs properties and mechanisms in ligand binding may provide the basis for new strategies in functional predictions and industry applications.

論文封面………………1
中文摘要………………2
Abstract………………3
Acknowledgements………………4
Table of Contents………………5
List of Tables………………8
List of Figures………………9
List of Appendix………………11
Abbreviations………………12

Chapter 1
Glucoamylase – overview and the linker domain…13
The glycans in the linker domain play an essential role in protein function
Background and Introduction
Results and Discussion
The linker region of RoGA is required for protein expression in Saccharomyces cerevisiae
The linker region of RoGA is modified by both N- and O-linked glycosylation
The N-linked carbohydrates in the linker play important roles in RoGA function
The linker region of RoGA is response of the structure stability
The linker region of RoGA is related to starch-degrading activity
Conclusion

Chapter 2
Glucoamylase – the starch binding domain…33
Diversity, structure, and function of the starch binding domain
Background and Introduction
Results and Discussion
Structural properties of RoSBD
Characteristic properties of RoSBD
Determination of residues important in ligand binding
SBD and starch recognition
Adsorption and hydrolysis of various starches
Conclusion

Chapter 3
Application and innovation of the starch binding domain…56
Background and Introduction
Results and Discussion
Purification of RoSBD by amylose affinity chromatography
RoSBD as a purification handle in E. coli expression system
RoSBD as a purification tag in P. pastoris expression system
The role of the glycosylated linker in SBD engineering
Structural correlation between SBD and 2G12
Specificity between RoSBD and HIV-1 glycoprotein
Specific mannan binding activity of RoSBD
Mapping Man4 binding sites on RoSBD
Conclusion

Chapter 4
Protein-carbohydrate interactions…81
Background and Introduction
Results and Discussion
Feature-incorporated alignment (FIA) illustration
Prediction of ligand-binding residues in CBM45 and CBM53Carbohydrate labeling with europium
Lanthanide-based time-resolved fluorescence
Synthesis of fluorescence-labeled carbohydrates
Fluorescence spectrometry in studies of protein-carbohydrate interactions
Isothermal titration calorimetry (ITC) in studies of protein-carbohydrate interactions
Conclusion

Chapter 5
Materials and Methods…119
Strains, media, and plasmids
Construction of the recombinant plasmids
Construction of deletion mutants of RoGA
Site-directed mutagenesis of amino acid residues in the linker
Halo assay for GA activity
Expression of recombinant protein in E. coli
Expression of recombinant protein in S. cerevisiae
Expression of recombinant protein in P. pastoris
Purification of recombinant RoGA
Purification of SBD and SBD-fusion proteins
Electrophoresis and Western blot analysis
RoGA activity toward soluble starch
Deglycosylation assays
Circular dichroism (CD) spectroscopy and thermal stability
Association rate assay of SBD
Adsorption assay
Saturation binding assay
Effects of pH on SBD binding ability
Effects of starch on SBD adsorption
Sandwich ELISA
Glycan array screening of RoSBD
Magnetic reduction binding assay
Molecular modeling of the putative Man4 binding site
Phenol-sulfuric acid method
Carbohydrate determination by High pH Anion Exchange Chromatography-Pulsed Amperometric Detection (HPAE-PAD)
Labeling of carbohydrates with europium
Determination of DTPA derivatives
Determination of DAP-DTPA derivatives
Determination of europium content
Preparation of fluorescent carbohydrate derivatives
Fluorescence measurements
Fluorescence Polarization (FP) measurement
Isothermal titration calorimetry (ITC)

Publication list………………140
References………………143

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