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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 梁博煌(Po-Huang Liang) | |
| dc.contributor.author | Wen-Jou Jung | en |
| dc.contributor.author | 鍾雯州 | zh_TW |
| dc.date.accessioned | 2021-06-15T01:14:32Z | - |
| dc.date.available | 2012-08-03 | |
| dc.date.copyright | 2009-08-03 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-07-29 | |
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H., Synergistic Effects on Crystalline Cellulose Degradation between Cellulosomal Cellulases from Clostridium cellulovorans. J. Bacteriol., 2002. 184(18): p. 5088-5095. 28.Murashima, K., Chen, C. L., Kosugi, A., Tamaru, Y., Doi, R. H., Wong, S. L., Heterologous Production of Clostridium cellulovorans engB, Using Protease-Deficient Bacillus subtilis, and Preparation of Active Recombinant Cellulosomes. J. Bacteriol., 2002. 184(1): p. 76-81. 29.Bolam, D. N., Ciruela, A., McQueen-Mason, S., Simpson, P., Williamson, M. P., Rixon, J. E., Boraston, A., Hazlewood, G. P., Gilbert, H. J., Pseudomonas cellulose- binding domains mediate their effects by increasing enzyme substrate proximity. Biochem. J., 1998. 331(3): p. 775-781. 30.Hall, J., Black, G. W., Ferreira, L. M., Millward- Sadler, S. J., Ali, B. R., Hazlewood, G. P., Gilbert, H. J., The non-catalytic cellulose-binding domain of a novel cellulase from Pseudomonas fluorescens subsp. cellulosa is important for the efficient hydrolysis of Avicel. Biochem. J., 1995. 309(3): p. 749-756. 31.Irwin, D., Shin, D. H., Zhang, S., Barr, B. K., Sakon, J., Karplus, P. A., Wilson, D. B., Roles of the Catalytic Domain and Two Cellulose Binding Domains of Thermomonospora fusca E4 in Cellulose Hydrolysis. J. Bacteriol., 1998. 180(7): p. 1709-1714. 32.Sakon, J., Irwin, D., Wilson, D. B., Karplus, P. A., Structure and mechanism of endo/exocellulase E4 from Thermomonospora fusca. Nat. Struct. Biol., 1997. 4(10): p. 810-818. 33.Meinke, A., Gilkes, N. R., Kilburn, D. G., Miller, R. C. Jr., Warren, R. A., Cellulose-binding polypeptides from Cellulomonas fimi: endoglucanase D (CenD), a family A beta-1,4-glucanase. J. Bacteriol., 1993. 175(7): p. 1910- 1918. 34.Fukumura, M. B., A. Kruus, K. Wu, J. H. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42480 | - |
| dc.description.abstract | Clostridium thermocellum endoglucanase CelT hydrolyzes internal β-1,4-glucosidic linkages along the cellulose chains to release shorter fragments. The CelT consists of a family-9 catalytic domain of the glycoside hydrolases, and a dockerin domain responsible for cellulosome assembly, but lacks a cellulose-binding domain, which is often found with family 9 catalytic domains.
This study is focused on improving cellulolytic efficiency of CelT. The family-3 cellulose binding domain (CBD) from C. thermocellum cel9I was fused to the catalytic domain of CelT to generate CelT-CBD fusion protein. CelT-CBD is more active toward insoluble celluloses than the CelT (devoid of the region encoding the dockerin domain). The result indicates that the construction of a fusion protein using CBD from another thermophilic endoglucanase represents a possible strategy for obtaining higher activity toward insoluble cellulose substrates. In the case of the CelT-CBD catalyzed hydrolysis of phosphoric acid-swollen cellulose (PASC), the soluble sugars were released at a 4-fold higher rate to compared with wild type CelT. Due to the heterogeneity in the composition and the structure of the plant cell wall, a wide range of enzymes is required for the biodegradation of these polysaccharides. Previous studies also showed that cellulosomal enzymes work synergistically for efficient lignocelluloses degradation. To improve efficiently of plant cell wall degradation, the synergistic interactions of endoglucanse (CelT△doc) and β-mannanase (ManCthe0032△doc) were determined on the degradation of rice straw biomass. The results showed an increased rate of reducing sugar production by the addition of CelT△doc and ManCthe0032△doc when comparing to the sum of the individual activities of the corresponding enzymes. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T01:14:32Z (GMT). No. of bitstreams: 1 ntu-98-R96b46031-1.pdf: 17553680 bytes, checksum: faf8cba3874404931deb9fb1b577cd18 (MD5) Previous issue date: 2009 | en |
| dc.description.tableofcontents | 中文摘要 ………………………………………………………………1
ABSTRACT……………………………………………………………… 3 ABBREVIATION………………………………………………………… 5 (1) INTRODUCTION 1-1 Need for Alternative Energy Source……………………6 1-2 Cell Wall Polysaccharides ………………………………7 1-3 Clostridium thermocellum…………………………………8 1-4 Glycoside hydrolases………………………………………10 1-5 Cellulose Binding Domains(CBD) ………………………10 1-6 Specific Aims of This Study ……………………………12 (2) MATERIALS AND METHODS 2-1 Chemicals ……………………………………………………14 2-2 Strains and vectors ………………………………………14 2-3 Cloning of recombinant proteins ………………………15 2-4 Recombinant fusion gene construction…………………17 2-5 Protein determination ……………………………………19 2-6 Western blotting …………………………………………19 2-7 Determination of enzyme activity………………………20 2-8 Expression of recombinant proteins……………………22 2-9 Purification of recombinant proteins…………………23 2-10 Temperature and pH optimum………………………………24 2-11 End-product determinationv………………………………25 2-12 Effect of metal ions and organic reagents …………26 (3) RESULTS 3-1 Construction of the studied gene………………………27 3-2 Expression and purification of three truncated forms of CelT, ManCthe0032, ManCthe2811 and chimeric protein CelT-CBD……………………………………………28 3-3 Characterization of recombinant CelT and fusion protein CelT-CBD …………………………………………29 3-4 Characterization of recombinant mannanase ManCthe0032 and ManCthe2811 ……………………………31 3-5 Synergism between endoglucanase and mannanase in degrading biomass …………………………………………33 (4) DISCUSSION 4-1 Cellulose-binding domain enhance activity against insoluble cellulose ………………………………………35 4-2 Synergism between endoglucanase and mannanase ……37 (5) REFERENCE ……………………………………………………40~45 (6) FIGURE…………………………………………………………46~68 (7) TABLE …………………………………………………………69~73 | |
| dc.language.iso | en | |
| dc.subject | 纖維素水解酶 | zh_TW |
| dc.subject | 酵素 | zh_TW |
| dc.subject | 生質能 | zh_TW |
| dc.subject | enzyme | en |
| dc.subject | bioethanol | en |
| dc.subject | cellulase | en |
| dc.title | 改造嗜熱菌株之纖維素水解酶與甘露糖水解酶並研究其酵素特性以及分解纖維素產生生質能之應用 | zh_TW |
| dc.title | Expression, characterization, and engineering of endoglucanase and mannanase from Clostridium thermocellum for degrading cellulose and biofuel production | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蔡蔭和(Inn-Ho Tsai),林俊宏(Chun-Hung Lin) | |
| dc.subject.keyword | 酵素,生質能,纖維素水解酶, | zh_TW |
| dc.subject.keyword | cellulase,bioethanol,enzyme, | en |
| dc.relation.page | 73 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2009-07-29 | |
| 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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