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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 劉?睿 | |
dc.contributor.author | Ya-Shan Cheng | en |
dc.contributor.author | 鄭雅珊 | zh_TW |
dc.date.accessioned | 2021-05-19T18:03:56Z | - |
dc.date.available | 2022-12-24 | |
dc.date.available | 2021-05-19T18:03:56Z | - |
dc.date.copyright | 2013-02-01 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-12-25 | |
dc.identifier.citation | Adams PD, Mustyakimov M, Afonine PV, Langan P (2009) Generalized X-ray and neutron crystallographic analysis: more accurate and complete structures for biological macromolecules. Acta Crystallogr D Biol Crystallogr 65: 567-573
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/8039 | - |
dc.description.abstract | Cellulose is the major component of plant cell wall and the most abundant renewable biomass on earth. Cellulases which can hydrolyze cellulose into simple sugar have been used in many industrial applications such as biofuel production and food industry for a long time. Despite cellulases have been widely utilized, most of current industrial cellulases are from mesophile and do not have good thermostability. Cellulase 12A, which was isolated from an anaerobic hyperthermophilic bacterium Thermotoga maritima (TmCel12A), belongs to glycoside hydrolase family 12. It is a hyperthermostable β-1,4-endoglucanase which can randomly degrade cellulose molecules into smaller fragments, facilitating further utilization of carbohydrate. In this study, the crystal structures of TmCel12A and its complex form with oligosaccharides were both determined by X-ray crystallography. The TmCel12A structure shows a typical β-jelly roll protein folding of the GH12 enzymes, with two antiparallel β-sheets and a central active-site cleft for substrate binding. A unique surface loop A3-B3 that contains Arg60 and Tyr61 interacting with the substrate by hydrogen bonds and aromatic residue stacking is observed in the complex structures of TmCel12A with cellobiose and cellotetraose. The role of Arg60 and Tyr61 in the substrate binding and catalytic reaction of TmCel12A was further investigated by site-directed mutagenesis. The results showed that the Y61G mutant had highest enzymatic activity when compared with the wild-type enzyme and other mutants. It also exhibited wider range of working temperature than the wild type, while its hyperthermostability resembled to the wild type. Besides, the kcat and Km values of Y61G were both higher than those of the wild type. In combination with the crystal structure of Y61G-substrate complex, the kinetic data suggested that the higher endoglucanase activity of Y61G is probably due to easier dissociation of cleaved sugar moiety at the reducing end. Further structural analyses of Y61GG and Y61del mutants indicated that the insertion and deletion mutations of the Tyr61 site did not affect the overall protein structure. However, their local perturbations might reduce the substrate binding affinity. It seems that the catalytic reaction of TmCel12A is a subtle modulation of substrate binding and product release. To summarize, the enhancement of enzymatic activity by single mutation of Y61G is a good example of enzyme engineering for industrial application. It also provides information regarding developing a better industrial enzyme by subtle changes of protein structure. | en |
dc.description.provenance | Made available in DSpace on 2021-05-19T18:03:56Z (GMT). No. of bitstreams: 1 ntu-101-D98642017-1.pdf: 6896015 bytes, checksum: cfbd147ec11310988dc4f513c364bb23 (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | 中文摘要 II
ABSTRACT IV TABLE OF FIGURES VIII TABLE OF TABLES VIII CHAPTER I 12 BACKGROUND 12 1.1 PLANT CELL WALL AND ITS PRIMARY COMPONENTS 12 1.1.1 Cellulose 12 1.1.2 Other components of plant cell wall 13 1.2 CELLULOLYTIC MICROORGANISMS 13 1.2.1 Thermotoga maritima 14 1.3 ENZYMATIC BREAKDOWN OF CELLULOSE BIOMASS 15 1.4 CLASSIFICATION OF CELLULOLYTIC ENZYMES 16 1.5 PROTEIN STRUCTURE OF CELLULASES 16 1.6 CATALYTIC MECHANISM OF GLYCOSIDE HYDROLASE 17 1.7 APPLICATION OF CELLULASES IN VARIOUS INDUSTRIES 18 1.8 BENEFIT OF THERMOSTABLE CELLULASES 19 1.9 PROTEIN ENGINEERING OF CELLULASES 20 1.9.1 Rational design 21 1.9.2 Directed evolution 22 CHAPTER II 27 CRYSTAL STRUCTURE AND SUBSTRATE-BINDING COMPLEX OF CELLULASE 12A FROM THERMOTOGA MARITIMA 27 2.1 INTRODUCTION 27 2.2 MATERIALS AND METHODS 29 2.2.1 Gene cloning, protein expression and purification 29 2.2.2 Crystallization and data collection 30 2.2.3 Structural determination and refinement 31 2.3 RESULTS AND DISCUSSIONS 33 2.3.1 Structural feature of the TmCel12A protein 33 2.3.2 Comparison with other GH 12 enzymes 34 2.3.3 Sugar-bound TmCel12A complex 35 CHAPTER III 44 ENHANCED ACTIVITY OF CEL12A FROM THERMOTOGA MARITIMA BY MUTATING A UNIQUE SURFACE LOOP 44 3.1 INTRODUCTION 44 3.2 MATERIALS AND METHODS 46 3.2.1 Gene cloning, protein expression and purification 46 3.2.2 Cellulase activity and kinetics analysis 47 3.2.3 Optimum temperature and thermostability analysis 47 3.2.4 Crystallization and data collection 48 3.2.5 Structural determination and refinement 48 3.3 RESULTS AND DISCUSSIONS 50 3.3.1 Enzyme activities of wild-type TmCel12A and its mutants 50 3.3.2 Kinetic analysis of wild-type TmCel12A and the Y61G mutant 51 3.3.3 Optimal reaction temperature and thermostability of wild-type TmCel12A and the Y61G mutant 52 3.3.4 Structural analysis of the Y61G, Y61GG and Y61del crystals 53 3.3.5 Comparison with other studies of GH11 and GH12 enzymes 55 CONCLUSION 65 REFERENCES 67 APPENDIX 75 I. Crystal structure and substrate-binding mode of cellulase 12A from Thermotoga maritima 76 II. Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop 88 | |
dc.language.iso | en | |
dc.title | 海棲熱袍菌纖維素酶 Cel12A 之結晶結構研究及其基因改造 | zh_TW |
dc.title | Crystal structural studies and genetic engineering of Thermotoga maritima cellulase Cel12A | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-1 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 郭瑞庭,陳純琪,劉?德,楊?伸 | |
dc.subject.keyword | 耐熱纖維素酶,蛋白結構,基因改造,定點突變, | zh_TW |
dc.subject.keyword | thermostable cellulase,endoglucanase,protein structure,X-ray crystallography,protein engineering,enzymatic activity,site-directed mutagenesis, | en |
dc.relation.page | 96 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2012-12-26 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 生物科技研究所 | zh_TW |
顯示於系所單位: | 生物科技研究所 |
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