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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 梁博煌(Po-Huang Liang) | |
| dc.contributor.author | Chun-Hsu Chen | en |
| dc.contributor.author | 陳俊旭 | zh_TW |
| dc.date.accessioned | 2021-06-16T09:24:35Z | - |
| dc.date.available | 2027-06-04 | |
| dc.date.copyright | 2017-07-12 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-06-19 | |
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(2002) Regulation of endo-acting glycosyl hydrolases in the hyperthermophilic bacterium Thermotoga maritima grown on glucan- and mannan-based polysaccharides. Appl Environ Microbiol, 68(2), 545–554. 48. Vlasenko, E., Schülein, M., Cherry, J., and Xu, F. (2010) Substrate specificity of family 5, 6, 7, 9, 12, and 45 endoglucanases. Bioresour Technol, 101(7), 2405–2411. 49. Lawoko, M., Nutt, A., Henriksson, H., Gellerstedt, G., and Henriksson, G. (2000) Hemicellulase activity of aerobic fungal cellulases. Holzforschung, 54, 497–500. 50. Lo Leggio, L., and Larsen, S. (2002) The 1.62 Å structure of Thermoascus aurantiacus endoglucanase: completing the structural picture of subfamilies in glycoside hydrolase family 5. FEBS Lett, 523, 103–108. 51. Hilge, M., Gloor, SM., Rypniewski, W., Sauer, O., Heightman, T.D., Zimmermann, W., Winterhalter, K., and Piontek, K. 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Biochim Biophys Acta, 1814(12), 1832–1840. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59464 | - |
| dc.description.abstract | 木質纖維素可被酵素水解並轉換成生質燃料。在實驗室先前的研究中，有一個從熱纖梭菌純化出來的雙功能水解酶(CtCel5E)，其具有的活性可分解兩種最主要組成植物細胞壁的多醣，分別為纖維素(為葡萄糖的多聚醣)和聚木醣(為木糖聚合而成的半纖維素主要構造)，使之最終分解為纖維二醣與木二醣。在之前碩士論文中，上述的雙功能酵素將與來自細菌或真菌的葡萄醣苷酶(CcBglA or TrBgl2 P172L)接合形成具cellulase, xylanase, 及β-glucosidase三種活性之融合酵素。在另一篇碩士論文中，CtCel5E中的一個flexible loop被另一來自T. maritima可分解纖維素及甘露醣的雙功能酵素TmCel5A置換而稱為CtCel5E_Tmloop，因此增加了甘露聚糖酶 (mannanase)活性。而在本篇論文發現，CtCel5E-CcBglA此融合酵素還增加了熱穩定性。CtCel5E還與另一個具木糖苷酶與葡萄糖苷酶活性的雙功能酵素接合，比起兩個雙功能酵素的混合反應，此融合酵素一樣擁有更好的活性與熱穩定性。此論文證明了一種有效與便捷的方法來合併不同酵素活性以利於生質原料的處理。為了更進一步達成統合生物加工過程(consolidated bioprocessing)，融合酵素被篩選並轉殖於嗜甲醇酵母菌內。我們的最終目標是利用生物工程來創造一個能同時使用葡萄糖與木糖的釀酒酵母發酵系統，來生產生質酒精。另外，為了增進三功能酵素(CtCel5E_Tmloop)的甘露聚糖酶活性，我們利用電腦軟體疊合一些甘露聚糖酶與CtCel5E_Tmloop的結構，並藉此結構分析來預測酵素工程的突變位置，接著使用點突變與DNA片段插入刪除實驗來驗證。 | zh_TW |
| dc.description.abstract | Lignocellulosic biomass can be enzymatically degraded and converted into biofuel. We have studied a bifunctional cellulase/xylanase from thermophilic C. thermocellum (CtCel5E), which can digest two major plant cell wall polysaccharides, cellulose (a polymer of glucose) and xylan (a major hemicellulose composed of xylose), into disaccharides cellobiose and xylobiose. In the previous thesis, the bifunctional enzyme was fused with a bacterial β-glucosidase from C. cellulovorans (CcBglA) or a fungal β-glucosidase from Trichoderma reesei (TrBgl2 P172L) to form tri-functional fusion enzymes. In another thesis, a flexible loop of CtCel5E was replaced with the corresponding loop in a bifunctional cellulase/mannanase from T. maritima, called TmCel5A, to form a tri-functional enzyme with additional mannanase activity. In this thesis, we found CtCel5E-CcBglA showed enhanced thermostability. Furthermore, CtCel5E was fused with a bifunctional β-glucosidase/β-xylosidase (RuBG3A) to degrade cellulose/hemicellulose into glucose and xylose also with better efficiency than the mixture of two parental enzymes and enhanced thermostability. This study demonstrates an efficient and convenient way of consolidating different enzyme activities for synergistic biomass processing. A further step, to achieve consolidated bioprocessing, suitable fusion enzymes were selected to be incorporated into Pichia pastoris . Our final goal is to engineer a glucose/xylose-utilizing Saccharomyces cerevisiae fermentative system, to produce bioethanol. In addition, to improve the mannanase activity of the tri-functional CtCel5E_Tmloop, superimposing some structure of β-mannanase with CtCel5E_Tmloop by computer software was achieved for analyzing the strategy of enzyme engineering. Enzyme engineering was performed by site-directed mutagenesis and sequence deletion/insertion experiments. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T09:24:35Z (GMT). No. of bitstreams: 1 ntu-106-R04b46028-1.pdf: 2712407 bytes, checksum: 067552cd58a16ff94ca469d17abc1eb0 (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 中文摘要 (viii)
Abstract (ix) Abbreviations (xi) (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 (6) 1.5 Specific aim of this study (7) (2) Materials and methods (10) 2.1 Reagents (10) 2.2 DNA source (10) 2.3 Bacterial and yeast strains (11) 2.4 Cloning of RuBG3A (11) 2.5 Construction of fusion genes (11) 2.6 Expression and purification of recombinant proteins from E.coli (12) 2.7 Expression of recombinant proteins from P.Pastoris (14) 2.7.1 Purification of recombinant proteins with methanol-inducible expression vector pPICZα (14) 2.7.2 Purification of recombinant proteins with non-inducible expression vector pGAPZα (15) 2.8 Site-directed mutagenesis and sequence deletion/insertion (16) 2.9 Determination of enzyme activity (17) 2.9.1 Substrates used for each enzyme activity (17) 2.9.2 DNS assay (18) 2.9.3 Glucose assay (18) 2.9.4 Determination of enzyme activity for RuBG3A and Sso3032 (19) 2.9.5 Optimal pH and temperature for enzyme activity (20) 2.9.6 Determination of specific activity (20) 2.9.7 Determination of enzyme kinetics (21) 2.10 Determination of enzyme thermal stability (21) 2.11 Measurement of melting temperatures (21) 2.12 Pretreatment of rice straw (22) 2.13 Analysis of hydrolytic end produces (23) 2.14 Growth curve of recombinant P.pastoris strains in medium with different carbon sources (24) 2.15 Product inhibition assay of CtCel5E (24) 2.16 Investigation of the substrate binding modes of CtCel5E and CtCel5E_Tmloop for mannanase activity by structure superimposition (25) (3) Results (26) 3.1 Design, construction and purification of fusion proteins (26) 3.2 Characterization of the individual and fusion enzymes (27) 3.3 Kinetics of the CtCel5E and β-glucosidase fusion enzymes (28) 3.4 Kinetics of the CtCel5E and β-glucosidase/β-xylosidase fusion enzymes (30) 3.5 Elevation of melting temperature by the fusion enzymes (31) 3.6 Increased thermostability by the fusion enzymes (32) 3.7 Favorable hydrolytic behavior of the fusion enzyme compare to the enzyme mixture as revealed by TLC analysis of products (33) 3.8 Degradation of alkali-treated rice straw by tetra-functional fusion enzyme (34) 3.9 Incorporation of fusion enzymes into P. pastoris (35) 3.10 Investigation of the substrate binding modes of CtCel5E and CtCel5E_Tmloop for improving mannanase activity (36) (4) Discussion (40) 4.1 Fusion enzymes represent practical strategy to improve hydrolysis efficiency (40) 4.2 The multi-functional fusion enzymes can be potentially applied to consolidated bioprocessing (41) 4.3 Improving the mannanase activity of CtCel5E and CtCel5E_Tmloop requires further studies (43) Tables (45) Figures (51) Reference (71) | |
| dc.language.iso | en | |
| dc.subject | 半纖維素? | zh_TW |
| dc.subject | 統合生物加工過程 | zh_TW |
| dc.subject | 融合酵素 | zh_TW |
| dc.subject | 纖維素? | zh_TW |
| dc.subject | 生質能源 | zh_TW |
| dc.subject | biofuel | en |
| dc.subject | consolidated bioprocessing | en |
| dc.subject | fusion enzymes | en |
| dc.subject | cellulase | en |
| dc.subject | xylanase | en |
| dc.title | 建構與特性分析具有提高功效與熱穩定性之多功能纖維素/半纖維素分解酶 | zh_TW |
| dc.title | Construction and characterization of multi-functional cellulose/hemicellulose-degrading enzymes with improved efficacy and thermostability | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃慶璨,何孟樵 | |
| dc.subject.keyword | 生質能源,纖維素?,半纖維素?,融合酵素,統合生物加工過程, | zh_TW |
| dc.subject.keyword | biofuel,cellulase,xylanase,fusion enzymes,consolidated bioprocessing, | en |
| dc.relation.page | 77 | |
| dc.identifier.doi | 10.6342/NTU201700838 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-06-19 | |
| 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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