簡易檢索 / 詳目顯示

研究生: 張衾華
Chin-Hua Chang
論文名稱: 以枯草芽孢桿菌建構可感測並水解多醣生物質之智慧型菌株
Construction of smart Bacillus subtilis for sensing and hydrolyzing polysaccharide biomass
指導教授: 蔡伸隆
Shen-Long Tsai
口試委員: 李振綱
Cheng-Kang Lee
侯劭毅
SHAO-YI HOU
蔡伸隆
Shen-Long Tsai
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: 2020
畢業學年度: 108
語文別: 中文
論文頁數: 126
中文關鍵詞: SigI轉錄因子RsgI蛋白嗜熱梭狀芽胞桿菌枯草芽胞桿菌組成型啟動子誘導型啟動子綠螢光蛋白紅螢光蛋白碳水化合物結合位β-木聚糖酶β-木糖苷酶
外文關鍵詞: σI transcription factor, Anti-σI factor, Clostridium thermocellum, Bacillus subtilis, Constitutive promoters, Inducible promoters, Green fluorescent protein, Red fluorescent protein, Carbohydrate-binding module, β-1,4-xylanases, β-xylosidases
相關次數: 點閱:259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 iii Abstract iv 致謝 v 總目錄 vi 圖目錄 ix 表目錄 xii 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 3 第二章 文獻回顧 6 2.1 木質纖維素 6 2.2 纖維素水解酶 7 2.3 半纖維素水解酶 8 2.4 枯草芽孢桿菌(Bacillus subtilis) 9 2.5 組成型及誘導型啟動子 11 2.5.1 組成型啟動子(Constitutive promoters) 11 2.5.2 誘導型啟動子(Inducible promoters) 12 2.5.2.1 木糖誘導型啟動子 12 2.5.2.2 纖維二糖誘導型啟動子 13 2.6 轉錄調控基因與多醣生物質感測 13 2.7 綠螢光蛋白(GFP) 16 2.8 紅螢光蛋白(RFP) 16 2.9 藍色蛋白(BCP) 17 2.10 質體相容性 17 第三章 實驗材料與方法 19 3.1 菌種與質體 19 3.2 實驗藥品 21 3.3 實驗器材 25 3.4 實驗步驟 26 3.4.1 基因轉殖技術 26 3.4.1.1 質體純化法 26 3.4.1.2 聚合酶鏈反應 28 3.4.1.3 DNA瓊脂凝膠電泳 34 3.4.1.4 DNA瓊脂凝膠回收 36 3.4.1.5 限制酶酶切作用(Digestion) 37 3.4.1.6 核酸接合作用(DNA Ligation) 38 3.4.1.7 大腸桿菌勝任細胞(Competent cell)之製備 39 3.4.1.8 大腸桿菌轉型作用(Transformation) 40 3.4.1.9 電穿孔勝任細胞製備及電穿孔轉殖作用(枯草芽孢桿菌) 41 3.4.2 纖維水解酵素之蛋白表達 43 3.4.2.1 纖維素水解酶之蛋白表達 43 3.4.2.2 半纖維素水解酶之蛋白表達 44 3.4.3 螢光蛋白活性測試 46 3.4.3.1 紅螢光蛋白活性測定 46 3.4.3.2 綠螢光蛋白活性測定 47 3.4.4 纖維素水解酶蛋白活性測試 47 3.4.4.1 纖維素內切酶(CelA)活性測定 48 3.4.4.2 纖維素外切酶(CelS)活性測定 48 3.4.5 半纖維素水解酶蛋白活性測定 49 3.4.5.1 β-木聚糖酶(XynA)活性測定 49 3.4.5.2 細胞膜通透處理(Permeabilization) 50 3.4.5.3 β-木糖苷酶(XynB)活性測定 50 3.4.6 分析方法 51 3.4.6.1 DNS還原糖測定法 51 3.4.6.2 p-NPX呈色測定法 52 3.4.7 SigI6轉錄因子活性測試 53 3.4.8 SigI2轉錄因子活性測試 54 第四章 結果與討論 55 4.1質體建構Plasmid construction 55 4.1.1 雙質體系統建構所需考量之因素 55 4.1.1.1 質體相容性 55 4.1.1.2 抗生素選用 56 4.1.1.1.1 四環黴素(Tetracycline) 56 4.1.1.1.2 氯霉素(Chloramphenicol) 57 4.1.2 纖維素水解酶之質體建構 58 4.1.2.1 pChCACSm質體建構 58 4.1.2.2 pChCACSLR質體建構 62 4.1.3 半纖維素水解酶之質體建構 65 4.1.4 SigI6轉錄因子之質體建構 68 4.1.4.1 pMKGS6質體建構 68 4.1.5 SigI2轉錄因子之質體建構 71 4.1.5.1 pTU-PsigI2-RFP-PsigA-sigI2op質體建構 71 4.1.5.2 pMKRS2m質體建構 73 4.1.5.3 pMKRS2m(dR2)質體建構 76 4.1.5.4 pMKRS2P436H質體建構 79 4.1.5.5 pMKRS2P43(dR2)質體建構 81 4.1.5.6 pChRS2P436H質體建構 83 4.1.5.7 pChRS2P43(dR2)質體建構 85 4.1.5.8 pRB-BCP質體建構 87 4.1.5.9 pRB-mCherry質體建構 89 4.2 報告基因活性測試 91 4.2.1 綠螢光蛋白活性測試 91 4.2.2 紅螢光蛋白活性測試 94 4.3 半纖維素水解酶蛋白活性測試 95 4.3.1 β-木聚糖酶 (XynA)活性測定 95 4.3.2 β-木糖苷酶 (XynB)活性測定 96 4.4 半纖維水解酵素之表達系統 96 4.5 纖維素水解酶蛋白活性測試 101 4.6 SigI6木聚醣感測活性測試 102 4.7 SigI2纖維素感測活性測試 103 第五章 結論 105 5.1以單雙糖誘導型啟動子調控基因表現(木糖/纖維二糖) 105 5.1.1木糖啟動子調控基因表現 105 5.1.2 纖維二糖啟動子調控基因表現 106 5.2以SigI轉錄因子調控基因表現(SigI2/SigI6) 107 5.2.1 SigI2轉錄因子調控基因表現 107 5.2.2 SigI6轉錄因子調控基因表現 108 參考文獻 109

1. Banerjee, S., G. Mishra, and A. Roy, Metabolic Engineering of Bacteria for Renewable Bioethanol Production from Cellulosic Biomass. Biotechnology and Bioprocess Engineering, 2019: p. 1-21.
2. Melro, E., et al., A brief overview on lignin dissolution. Journal of Molecular Liquids, 2018. 265: p. 578-584.
3. Moraïs, S., et al., Assembly of xylanases into designer cellulosomes promotes efficient hydrolysis of the xylan component of a natural recalcitrant cellulosic substrate. MBio, 2011. 2(6): p. e00233-11.
4. Yaniv, O., et al., Fine-structural variance of family 3 carbohydrate-binding modules as extracellular biomass-sensing components of Clostridium thermocellum anti-sigma(I) factors. Acta Crystallographica Section D-Structural Biology, 2014. 70: p. 522-534.
5. Yokoyama, S. and Y. Matsumura, The Asian biomass handbook: a guide for biomass production and utilization. Thermochemical conversion of biomass, 1st (Ed) part, 2008. 4: p. 71.
6. Sun, Y. and J. Cheng, Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresource technology, 2002. 83(1): p. 1-11.
7. Gaudin, C., et al., CelE, a Multidomain Cellulase fromClostridium cellulolyticum: a Key Enzyme in the Cellulosome? Journal of bacteriology, 2000. 182(7): p. 1910-1915.
8. Sajjad, M., et al., Influence of positioning of carbohydrate binding module on the activity of endoglucanase CelA of Clostridium thermocellum. Journal of biotechnology, 2012. 161(3): p. 206-212.
9. Teeri, T.T., Crystalline cellulose degradation: new insight into the function of cellobiohydrolases. Trends in biotechnology, 1997. 15(5): p. 160-167.
10. Fujita, Y., et al., Synergistic saccharification, and direct fermentation to ethanol, of amorphous cellulose by use of an engineered yeast strain codisplaying three types of cellulolytic enzyme. Appl. Environ. Microbiol., 2004. 70(2): p. 1207-1212.
11. Fujita, Y., et al., Direct and efficient production of ethanol from cellulosic material with a yeast strain displaying cellulolytic enzymes. Appl. Environ. Microbiol., 2002. 68(10): p. 5136-5141.
12. Roy, N. and M.R. Habib, Isolation and characterization of Xylanase producing strain of Bacillus cereus from soil. Iranian Journal of Microbiology, 2009: p. 49-53.
13. Lindner, C., J. Stülke, and M. Hecker, Regulation of xylanolytic enzymes in Bacillus subtilis. Microbiology, 1994. 140(4): p. 753-757.
14. Godoy, M.G., et al., Agricultural Residues as Animal Feed. 2018, Elsevier. p. 235-256.
15. Simonen, M. and I. Palva, Protein secretion in Bacillus species. Microbiology and Molecular Biology Reviews, 1993. 57(1): p. 109-137.
16. Zhang, X.-Z., et al., High-level expression and secretion of methyl parathion hydrolase in Bacillus subtilis WB800. Appl. Environ. Microbiol., 2005. 71(7): p. 4101-4103.
17. Zhang, X.Z. and Y.H.P. Zhang, One‐step production of biocommodities from lignocellulosic biomass by recombinant cellulolytic Bacillus subtilis: Opportunities and challenges. Engineering in Life Sciences, 2010. 10(5): p. 398-406.
18. Joliff, G., et al., Inducible secretion of a cellulase from Clostridium thermocellum in Bacillus subtilis. Appl. Environ. Microbiol., 1989. 55(11): p. 2739-2744.
19. Lina, C.-C., et al., Deciphering characteristics of the designer cellulosome from Bacillus subtilis WB800N via enzymatic analysis. 2017.
20. Anderson, T.D., et al., Assembly of minicellulosomes on the surface of Bacillus subtilis. Appl. Environ. Microbiol., 2011. 77(14): p. 4849-4858.
21. Helianti, I., et al., Production of Xylanase by Recombinant Bacillus subtilis DB104 Cultivated in Agroindustrial Waste Medium. HAYATI Journal of Biosciences, 2016. 23(3): p. 125-131.
22. Garvey, M., et al., Cellulases for biomass degradation: comparing recombinant cellulase expression platforms. Trends in Biotechnology, 2013. 31(10): p. 581-593.
23. Zuber, U., K. Drzewiecki, and M. Hecker, Putative sigma factor SigI (YkoZ) of Bacillus subtilis is induced by heat shock. Journal of bacteriology, 2001. 183(4): p. 1472-1475.
24. Anderson, T.D., et al., Recombinant Bacillus subtilis that grows on untreated plant biomass. Appl. Environ. Microbiol., 2013. 79(3): p. 867-876.
25. Morimoto, T., et al., Enhanced recombinant protein productivity by genome reduction in Bacillus subtilis. DNA research, 2008. 15(2): p. 73-81.
26. Liu, J.-M., et al., Cloning of thermostable cellulase genes of Clostridium thermocellum and their secretive expression in Bacillus subtilis. Applied biochemistry and biotechnology, 2012. 166(3): p. 652-662.
27. Bien, T.L.T., et al., Secretion of heterologous thermostable cellulases in Bacillus subtilis. The Journal of general and applied microbiology, 2014. 60(5): p. 175-182.
28. Phan, T.T.P., H.D. Nguyen, and W. Schumann, Development of a strong intracellular expression system for Bacillus subtilis by optimizing promoter elements. Journal of biotechnology, 2012. 157(1): p. 167-172.
29. Coelho, R.V., et al., Bacillus subtilis promoter sequences data set for promoter prediction in Gram-positive bacteria. Data in brief, 2018. 19: p. 264-270.
30. Zhou, C., et al., Promoter engineering enables overproduction of foreign proteins from a single copy expression cassette in Bacillus subtilis. Microbial cell factories, 2019. 18(1): p. 1-11.
31. Jordan, S., et al., Regulation of LiaRS-dependent gene expression in Bacillus subtilis: identification of inhibitor proteins, regulator binding sites, and target genes of a conserved cell envelope stress-sensing two-component system. Journal of bacteriology, 2006. 188(14): p. 5153-5166.
32. Yansura, D.G. and D.J. Henner, Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis. Proceedings of the National Academy of Sciences, 1984. 81(2): p. 439-443.
33. Kim, L., A. Mogk, and W. Schumann, A xylose-inducible Bacillus subtilis integration vector and its application. Gene, 1996. 181(1-2): p. 71-76.
34. Singh, K.D., et al., Carbon Catabolite Repression in <em>Bacillus subtilis</em>: Quantitative Analysis of Repression Exerted by Different Carbon Sources. Journal of Bacteriology, 2008. 190(21): p. 7275.
35. Tobisch, S., et al., Identification and characterization of a new beta-glucoside utilization system in Bacillus subtilis. Journal of bacteriology, 1997. 179(2): p. 496-506.
36. Tobisch, S., J. Stülke, and M. Hecker, Regulation of the lic Operon ofBacillus subtilis and Characterization of Potential Phosphorylation Sites of the LicR Regulator Protein by Site-Directed Mutagenesis. Journal of bacteriology, 1999. 181(16): p. 4995-5003.
37. Sierro, N., et al., DBTBS: a database of transcriptional regulation in Bacillus subtilis containing upstream intergenic conservation information. Nucleic Acids Research, 2007. 36(suppl_1): p. D93-D96.
38. Kahel-Raifer, H., et al., The unique set of putative membrane-associated anti-σ factors in Clostridium thermocellum suggests a novel extracellular carbohydrate-sensing mechanism involved in gene regulation. FEMS microbiology letters, 2010. 308(1): p. 84-93.
39. Nataf, Y., et al., Clostridium thermocellum cellulosomal genes are regulated by extracytoplasmic polysaccharides via alternative sigma factors. Proceedings of the National Academy of Sciences, 2010. 107(43): p. 18646-18651.
40. Lamed, R., Carbohydrate-sensing system - suggested mode of operation. 2013.
41. Yagi, K., Applications of whole-cell bacterial sensors in biotechnology and environmental science. Applied Microbiology and Biotechnology, 2007. 73(6): p. 1251-1258.
42. Phillips, G.J., Green fluorescent protein–a bright idea for the study of bacterial protein localization. FEMS microbiology letters, 2001. 204(1): p. 9-18.
43. Liu, X., et al., Whole-cell fluorescent biosensors for bioavailability and biodegradation of polychlorinated biphenyls. Sensors, 2010. 10(2): p. 1377-1398.
44. Alieva, N.O., et al., Diversity and evolution of coral fluorescent proteins. PloS one, 2008. 3(7): p. e2680.
45. Shintani, M., Z.K. Sanchez, and K. Kimbara, Genomics of microbial plasmids: classification and identification based on replication and transfer systems and host taxonomy. Frontiers in Microbiology, 2015. 6(242).
46. Udo, E. and W. Grubb, A new incompatibility group plasmid in Staphylococcus aureus. FEMS microbiology letters, 1991. 78(1): p. 33-36.
47. Del Solar, G., et al., Replication and control of circular bacterial plasmids. Microbiology and molecular biology reviews, 1998. 62(2): p. 434-464.
48. Khan, S.A., Plasmid rolling-circle replication: highlights of two decades of research. Plasmid, 2005. 53(2): p. 126-136.
49. Guglielmetti, S., D. Mora, and C. Parini, Small rolling circle plasmids in Bacillus subtilis and related species: organization, distribution, and their possible role in host physiology. Plasmid, 2007. 57(3): p. 245-264.
50. Biedendieck, R., et al., A sucrose-inducible promoter system for the intra- and extracellular protein production in Bacillus megaterium. Journal of Biotechnology, 2007. 132(4): p. 426-430.
51. Malten, M., et al., A Bacillus megaterium plasmid system for the production, export, and one-step purification of affinity-tagged heterologous levansucrase from growth medium. Applied and environmental microbiology, 2006. 72(2): p. 1677-1679.
52. Bernhard, K., H. Schrempf, and W. Goebel, Bacteriocin and antibiotic resistance plasmids in Bacillus cereus and Bacillus subtilis. Journal of Bacteriology, 1978. 133(2): p. 897.
53. Vary, P., et al., Bacillus megaterium – from Simple Soil Bacterium to Industrial Protein Production Host. Applied microbiology and biotechnology, 2007. 76: p. 957-67.
54. Michod, R. and M. Wojciechowski, DNA repair and the evolution of transformation IV. DNA damage increases transformation. Journal of Evolutionary Biology, 1994. 7: p. 147-175.
55. Sullivan, M.A., R.E. Yasbin, and F.E. Young, New shuttle vectors for Bacillus subtilis and Escherichia coli which allow rapid detection of inserted fragments. Gene, 1984. 29(1): p. 21-26.

無法下載圖示 全文公開日期 2025/08/20 (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