簡易檢索 / 詳目顯示

研究生: 張家瑜
Chia-Yu Chang
論文名稱: 纖維素水解酵素協同作用之研究
The study of the synergistic effect between cellulose hydrolases
指導教授: 蔡伸隆
Shen-Long Tsai
口試委員: 李振綱
Cheng-Kang Lee
王勝仕
Sheng-Shih Wang
學位類別: 碩士
Master
系所名稱: 工程學院 - 化學工程系
Department of Chemical Engineering
論文出版年: 2021
畢業學年度: 109
語文別: 中文
論文頁數: 97
中文關鍵詞: 纖維素水解纖維水解酵素酵素協同作用熱休克啟動子解纖維梭菌
外文關鍵詞: Cellulose hydrolysis, Enzyme synergism, Heat shock promoter, Clostridium cellulolyticum, Cel9Ec, Cel9G, BglA
相關次數: 點閱:215下載:0
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報


摘要 I ABSTRACT II 致謝 III 總目錄 IV 圖目錄 VII 表目錄 IX 第一章. 緒論 1 1.1. 研究背景 1 1.2. 研究動機與目的 1 1.3. 研究內容 2 第二章. 文獻回顧 4 2.1. 纖維素 4 2.2. 纖維素水解酵素 5 2.2.1. 內切型纖維素水解酵素Cel9Gf 6 2.2.2. 外切型纖維素水解酵素Cel9Ec 6 2.2.3. β-葡萄糖苷酵素BglA 9 2.3. 熱激啟動子表達載體pHsh 10 第三章. 實驗材料與方法 11 3.1. 材料與儀器 11 3.1.1. 實驗藥品 11 3.1.2. 生物製劑及套組 13 3.1.3. 儀器設備 14 3.1.4. 菌種與質體 15 3.2. 實驗方法 16 3.2.1. 基因轉殖技術 16 3.2.1.1. 質體DNA純化法Mini-prep(鹼性溶裂法) 16 3.2.1.2. 質體DNA純化法Mini-prep(TOOLS Plasmid Miniprep) 18 3.2.1.3. 質體DNA純化法Mini-prep(ZR Plasmid Miniprep) 20 3.2.1.4. 聚合酶鏈反應(Polymerase Chain Reaction, PCR) 22 3.2.1.5. DNA瓊脂凝膠電泳(DNA Agarose Gel Electrophoresis) 23 3.2.1.6. DNA瓊脂凝膠回收(DNA Recovery) 25 3.2.1.7. 酶切(Digestion) 26 3.2.1.8. 核酸接合作用(DNA Ligation) 27 3.2.1.9. 大腸桿菌勝任細胞(Competent cell)之製備 28 3.2.1.10. 大腸桿菌轉殖作用(Transformation) 30 3.2.2. 纖維素水解酵素表達與純化 31 3.2.2.1. 纖維素水解酵素之蛋白表達 31 3.2.2.2. 熱激啟動子(pHsh)表達β-葡萄糖苷酵素(BglA) 33 3.2.2.3. 金屬親和層析法(Immobilized metal affinity chromatography, IMAC) 35 3.2.3. 分析方法 38 3.2.3.1. 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(SDS-PAGE) 38 3.2.3.2. ImageJ圖像分析法 40 3.2.3.3. Bradford蛋白質定量法 42 3.2.3.4. DNS還原糖測定法 44 3.2.3.5. β-葡萄糖苷酵素(BglA)活性測試 45 3.2.3.6. 苯酚-硫酸法 46 3.2.4. 纖維素水解酵素協同水解作用 48 第四章. 結果與討論 50 4.1. 纖維素水解酵素表達與純化 50 4.1.1. Cel9Gf與Cel9Ec酵素蛋白表達 50 4.1.2. Cel9Gf與Cel9Ec酵素蛋白純化條件測試 51 4.1.2.1. Cel9Ec酵素蛋白純化條件測試 51 4.1.2.2. Cel9Gf酵素蛋白純化條件測試 53 4.1.3. Cel9Gf與Cel9Ec酵素蛋白純化 55 4.2. 纖維素水解酵素協同水解反應 56 4.2.1. 纖維素水解能力 56 4.2.1.1. Cel9Gf及Cel9Ec對不同纖維素底物水解能力測試 56 4.2.1.2. Cel9Gf及Cel9Ec協同水解能力測試 59 4.2.2. 酵素間協同作用 65 4.3. β-葡萄糖苷酵素 71 4.3.1. 質體建立 72 4.3.2. 熱激啟動子表達質體(pHsh) 72 4.3.3. β-葡萄糖苷酵素(pHsh-BglA) 75 4.3.4. BglA酵素蛋白表達與可溶性測試 78 4.3.5. BglA酵素活性測試 80 第五章. 結論 81 文獻回顧 82 附錄 84 附錄 1. BglA酵素以pET系統表達之情形 84 附錄 2. 底物抑制動力學模型 85

1.Gal, L., et al., CelG from Clostridium cellulolyticum: a multidomain endoglucanase acting efficiently on crystalline cellulose. 1997. 179(21): p. 6595-6601.
2.Gaudin, C., et al., CelE, a Multidomain Cellulase fromClostridium cellulolyticum: a Key Enzyme in the Cellulosome? 2000. 182(7): p. 1910-1915.
3.Ravachol, J., et al., Characterization of all family-9 glycoside hydrolases synthesized by the cellulosome-producing bacterium Clostridium cellulolyticum. The Journal of biological chemistry, 2014. 289(11): p. 7335-7348.
4.Tsai, S.L., et al., Functional Assembly of Minicellulosomes on the Saccharomyces cerevisiae Cell Surface for Cellulose Hydrolysis and Ethanol Production. Applied and Environmental Microbiology, 2009. 75(19): p. 6087-6093.
5.Fierobe, H.-P., et al., Action of designer cellulosomes on homogeneous versus complex substrates controlled incorporation of three distinct enzymes into a defined trifunctional scaffoldin. 2005. 280(16): p. 16325-16334.
6.Teeri, T.T., Crystalline cellulose degradation: new insight into the function of cellobiohydrolases. Trends in Biotechnology, 1997. 15(5): p. 160-167.
7.Shieh, J.-C. 3. 纖維乙醇之製程(Cellulosic ethanol process). 2007; Available from: http://www.taiwan921.lib.ntu.edu.tw/mypdf/be03-3.pdf.
8.陳文恆‧郭家倫‧黃文松‧王嘉寶. 纖維酒精技術之發展. [internet] 2007; Available from: http://www.biotaiwan.org.tw/mag/image_doc/9/09%E7%BA%96%E7%B6%AD%E9%85%92%E7%B2%BE%E6%8A%80%E8%A1%93%E4%B9%8B%E7%99%BC%E5%B1%95(1).pdf.
9.Sun, Y. and J. Cheng, Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresource technology, 2002. 83(1): p. 1-11.
10.Gaudin, C., et al., CelE, a multidomain cellulase from Clostridium cellulolyticum: a key enzyme in the cellulosome? Journal of bacteriology, 2000. 182(7): p. 1910-1915.
11.Gal, L., et al., Characterization of the cellulolytic complex (cellulosome) produced by Clostridium cellulolyticum. Applied and environmental microbiology, 1997. 63(3): p. 903-909.
12.Aït, N., N. Creuzet, and J.J.M. Cattaneo, Properties of β-glucosidase purified from Clostridium thermocellum. 1982. 128(3): p. 569-577.
13.Gräbnitz, F., et al., Structure of the beta-glucosidase gene bglA of Clostridium thermocellum. Sequence analysis reveals a superfamily of cellulases and beta-glycosidases including human lactase/phlorizin hydrolase. Eur J Biochem, 1991. 200(2): p. 301-9.
14.Bhatia, Y., S. Mishra, and V.S. Bisaria, Microbial beta-glucosidases: cloning, properties, and applications. Crit Rev Biotechnol, 2002. 22(4): p. 375-407.
15.contributors, C. Glycoside Hydrolase Family 1. 28 November 2012 01:10 UTC; Available from: http://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_1&oldid=7911.
16.Ait, N., et al., Characterization and purification of thermostable β-glucosidase from Clostridium thermocellum. 1979. 90(2): p. 537-546.
17.Schwarz, W., K. Bronnenmeier, and W.L.J.B.l. Staudenbauer, Molecular cloning ofClostridiumthermocellum genes involved in β-glucan degradation in bacteriophage lambda. 1985. 7(12): p. 859-864.
18.Gräbnitz, F. and W.L.J.B.l. Staudenbauer, Characterization of two β-glucosidase genes fromClostridiumthermocellum. 1988. 10(2): p. 73-78.
19.Wu, H., et al., pHsh vectors, a novel expression system of Escherichia coli for the large-scale production of recombinant enzymes. Biotechnology Letters, 2010. 32(6): p. 795-801.
20.蒋钰瑶, et al., 新型大肠杆菌高效表达载体 pHsh 的构建与应用. 2012. 39(3): p. 394-400.
21.Roncarati, D. and V. Scarlato, Regulation of heat-shock genes in bacteria: from signal sensing to gene expression output. FEMS Microbiology Reviews, 2017. 41(4): p. 549-574.
22.Peng, J., et al., Enhanced soluble expression of a thermostable cellulase from Clostridium thermocellum in Escherichia coli. 2011. 63(6): p. 523.
23.Le, Y., et al., An approach to the production of soluble protein from a fungal gene encoding an aggregation-prone xylanase in Escherichia coli. 2011. 6(4): p. e18489.
24.Berg, J., J. Tymoczko, and L. Stryer, Section 8.4-The Michaelis-Menten Model Accounts for the Kinetic Properties of Many Enzymes. Biochemistry, fifth ed. WH Freeman, New York. Available from: http://www. ncbi. nlm. nih. gov/books/NBK22430, 2002.
25.Ahmed, S.S., et al., Soluble Production, Characterization, and Structural Aesthetics of an Industrially Important Thermostable β-Glucosidase from Clostridium thermocellum in Escherichia coli. 2019. 2019.
26.Singh, S.M., A.K.J.J.o.b. Panda, and bioengineering, Solubilization and refolding of bacterial inclusion body proteins. 2005. 99(4): p. 303-310.
27.Singh, A., et al., Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. 2015. 14(1): p. 1-10.

無法下載圖示 全文公開日期 2026/01/28 (校內網路)
全文公開日期 本全文未授權公開 (校外網路)
全文公開日期 本全文未授權公開 (國家圖書館:臺灣博碩士論文系統)
QR CODE