臺灣博碩士論文加值系統

Glucoamylase hydrolyzes starch and polysaccharides to β-D-glucose. Rhizopus oryzae glucoamylase (RoGA) consists of two functional domains, an N-terminal starch binding domain (SBD) and a C-terminal catalytic domain and these two domains are connected by an O-glycosylated linker. RoSBD belongs to carbohydrate-binding module 21 (CBM21). In previous study, two crystal structures of RoSBD complexes with linear maltoheptaose (RoSBD-G7) and β-cyclodextrin (RoSBD-βCD) with α-(1,4) glycosidic linkage have been solved in our lab. The overall structures belong to an immunoglobulin-like folding and two carbohydrate-binding sites were observed. Site I is created by several conserved aromatic residues, Trp47, Tyr83, and Tyr94, forming a broad, flat, and firm hydrophobic binding surface. Site II is built up by Tyr32 and Phe58, producing a protruded and narrow binding environment.
In this study, two crystal structures of the RoSBD complexes with α-(1,6) gluco-oligosaccharide, isomaltotriose (RoSBD-isoG3) and isomaltotetraose (RoSBD-isoG4), were determined at 1.2 and 1.3A respectively. Two unique polyN loops play an important role in distinguish difference between α-(1,4) and α-(1,6) gluco-oligosaccharide upon protein-carbohydrate interaction. Although there are two carbohydrate-binding sites available and exist in RoSBD, only one binding site, site II, was observed in the RoSBD-isoG3 complex. Site II (Tyr32) might act as the initial and recognition binding site and bind to carbohydrate first. Site I (Trp47) could not bind to carbohydrate independently; it participated and supported the binding after that Site II (Tyr32) has been bound with carbohydrate. When the carbohydrate length is longer than three glucosyl units the second binding site, site I would be activated and promoted the carbohydrate binding. Two binding sites would assist each other to reinforce the binding ability as well as stabilize the binding environment when bind to the long gluco-oligosaccharide as well as starch.

Chapter 1 Introduction 1
1.1. Starch 1
1.2. Glucoamylase 1
1.3. Carbohydrate-binding modules (CBMs) 2
1.4. Starch-binding domain (SBD) 3
1.5. Isomaltotriose (isoG3) and isomaltotetraose(isoG4) 4

Chapter 2 Materials and methods 6
2.1. Protein expression and purification 6
2.2. Analytical gel filtration chromatography 7
2.3. Crystallization 7
2.4. X-ray data collection 8
2.5. Matthew coefficient 9
2.6. Phase determination 10
2.7. Structure refinement 10

Chapter 3 Results 12
3.1. Structure-based multiple sequence alignment 12
3.2. Structures of RoSBD-isoG3 and RoSBD-isoG4 complexes 12
3.2.1. Overall structure of RoSBD-isoG3 complex 12
3.2.2. Overall structure of RoSBD-isoG4 complex 14
3.3. Comparison of structures among unliganded and liganded RoSBDs 14
3.4. Ligand binding residues in RoSBD-isoG3 complex 15
3.5. Ligand binding residues in RoSBD-isoG4 complex 18
3.6. Comparison of ligand binding residues among unliganded and
Liganded RoSBD complexes 20
3.7. Crystal packing of RoSBD complexes 21
3.8. Structure comparison of RoSBD-G7 and TvSBD-G6 complexes 22
3.9. Structural correlation between RoSBD and human antibody 2G12 23
3.10. Structural modeling of Man4 binding sites on RoSBD 24
Chapter 4 Discussion 26
Figures 29
Tables 52
Reference 60

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