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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 梁博煌 | |
| dc.contributor.author | Barbara Yang | en |
| dc.contributor.author | 楊紫君 | zh_TW |
| dc.date.accessioned | 2021-06-16T13:25:15Z | - |
| dc.date.available | 2018-07-30 | |
| dc.date.copyright | 2013-07-30 | |
| dc.date.issued | 2013 | |
| dc.date.submitted | 2013-07-24 | |
| dc.identifier.citation | 1. Demain, A.L., Newcomb M., and Wu, J.H. (2005) Cellulase, Clostridia, and Ethanol. Microbiol Mol Biol Rev, 69(1), 124–154.
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(1996) Crystal structure of thermostable family 5 endocellulase E1 from Acidothermus cellulolyticus in complex with cellotetraose. Biochemistry, 35, 10648–10660. 63. Schagerlf, U., Schagerlf, H., Momcilovic, D., Brinkmalm, G., and Tjerneld, F. (2007) Endoglucanase sensitivity for substituents in methyl cellulose hydrolysis studied using MALDI-TOFMS for oligosaccharide analysis and structural analysis of enzyme active sites. Biomacromolecules, 8, 2358–2365. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/62053 | - |
| dc.description.abstract | 組成植物細胞壁的木質纖維素為生質能源的重要原料；纖維素和半纖維素中的木聚醣(xylan)是木質纖維素最主要的組成物。纖維素可藉由纖維素酶將之分解成葡萄糖單體；而木聚醣酶則可將木聚醣分解成木糖單體，進而發酵成酒精。為了有效分解組成複雜的木質纖維素，一般需要製備多種醣苷水解酶以共同協同作用。如能將所需的各種酵素活性合併於一多功能融合酵素，此方式將能簡化酵素製備程序，降低生產成本。在本篇論文中，透過基因融合方式將熱纖梭菌之纖維素酶/木聚醣酶雙功能酵素(CtCel5E)與來自細菌或真菌之葡萄糖苷酶(CcBglA或TrBgl2)接合成為具三種活性之融合酵素。藉由酵素活性試驗與薄膜層析法實驗顯示此二融合酵素確實保留了三種活性；並且，與雙功能酵素及葡萄糖苷酶(β-glucosidase)之酵素混和物相較，三功能融合酵素所展現的協同作用勝於酵素混和物，能更有效地分解人工纖維素成葡萄糖單體，而不會殘留雙醣。此外，CtCel5E-TrBgl2 P172L融合酵素能抑制原本TrBgl2之醣基轉移酶活性，避免已分解的葡萄糖合成回為寡醣。接著，進一步利用三功能融合酵素分解鹼處理之天然稻稈，結果顯示整個反應過程中，纖維二醣能有效地被水解為葡萄糖，酵素催化後之產物為葡萄糖與小分子木聚醣(雙醣、三醣為主)。若於三功能融合酵素催化之反應中外加木糖苷酶(β-xylosidase)，最終水解產物即為可發酵的葡萄糖與木糖。總結來說，多功能融合酵素所提供的水解效率以及製備的方便性，將有助於降低生質能源成本。另外，為了瞭解此天然雙功能酵素(CtCel5E)如何能於同一活性位置辨認及催化纖維素與木聚醣，多種小分子受質利用電腦模擬出其與酵素之結合情形。根據電腦模擬與單定點突變實驗，顯示CtCel5E第202號組胺酸(His202)能與位於-1 subsite之葡萄糖分子形成氫鍵，但是與木糖則無法；所以，突變202號位置對於纖維素酶活性的影響甚於對木聚醣酶活性的影響。假使能進一步得到該酵素與不同受質共結晶之結構，相信能提供更完整關於受質選擇性的資訊，找到哪些關鍵胺基酸後，將有助於酵素的改造工程。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2021-06-16T13:25:15Z (GMT). No. of bitstreams: 1 ntu-102-R00b46008-1.pdf: 3140630 bytes, checksum: 6d3ba5118442ce372ade385a1fa81f74 (MD5) Previous issue date: 2013 | en |
| dc.description.tableofcontents | Contents
中文摘要 vii Abstract viii Abbreviations x (1) Introduction 1 1.1 The demand for alternative energy feedstock 1 1.2 Structure of lignocellulosic biomass 2 1.3 Glycoside hydrolases 4 1.4 Overview of the conversion of biomass to biofuel 5 1.5 Specific aim of this study 7 (2) Materials and methods 9 2.1 Reagents 9 2.2 DNA source and bacterial strains 9 2.3 Construction of recombinant fusion proteins 10 2.3.1 Fusion genes between CtCel5E and CcBglA 10 2.3.2 Fusion genes between CtCel5E and TrBgl2 P172L or L167W 11 2.4 Site-directed mutagenesis 13 2.5 Expression and purification of recombinant proteins 13 2.5.1 The pHTPP13-constructs 14 2.5.2 The pHTPP15-Sso3032 15 2.6 Determination of enzyme activity 16 2.6.1 Substrates used for each enzyme activity 16 2.6.2 DNS assay 16 2.6.3 Optimal pH and temperature for enzyme activity 17 2.6.4 Determination of specific activity 17 2.6.5 Determination of enzyme activity for Sso3032 18 2.7 Determination of enzyme kinetics 18 2.8 Determination of enzyme thermal stability 19 2.9 Analysis of hydrolytic end produces 19 2.10 Pretreatment of rice straw 21 2.11 Docking of different substrates to CtCel5E 21 (3) Results 23 3.1 Design, construction and purification of fusion proteins 23 3.2 Characterization of the individual and fusion enzymes 25 3.3 Substrate specific activities of individual and fusion enzymes 26 3.4 Positive effects on the fused β-glucosidases 27 3.4.1 Kinetics parameters determination 27 3.4.2 TLC analysis of cellobiose degradation 28 3.4.3 Thermostability 28 3.5 Hydrolytic products from CMC, PASC and Beechwood xylan substrates 29 3.6 Hydrolytic products from degradation of biomass 32 3.6.1 Degradation of pretreated rice straw by tri-functional fusion enzymes 32 3.6.2 Characterization of β-xylosidase (Sso3032) 33 3.6.3 Complete degradation of pretreated rice straw by tri-functional fusion enzymes in the presence of β-xylosidase 34 3.7 Investigation of the substrate binding modes of CtCel5E 34 3.8 H202 may play a critical role for differential substrate recognition 36 (4) Discussion 39 4.1 Fusion enzymes represent practical strategy to improve hydrolysis efficiency and reduce the enzyme cost 39 4.2 Complete hydrolysis of both cellulose and hemicellulose increases the overall efficiency of biomass conversion to biofuels 41 4.3 Investigating the determinants of substrate specificity in CtCel5E 44 Tables 46 Figures 51 Reference 73 | |
| dc.language.iso | en | |
| dc.title | 設計建構多功能融合酵素分解纖維素/半纖維素以利於生產生質能源 | zh_TW |
| dc.title | Design and Construction of Multi-functional Fusion Enzymes to Degrade Cellulose/hemicellulose for Biofuel Production | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 吳世雄,趙裕展 | |
| dc.subject.keyword | 纖維素酶,木聚醣酶,葡萄糖苷,酶,融合酵素, | zh_TW |
| dc.subject.keyword | endoglucanase,xylanase,beta-glucosidase,fusion enzyme, | en |
| dc.relation.page | 80 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2013-07-24 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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