指導教授：許瑞祥臺灣大學：生化科技學系王惠昌Wang, Hui-ChangHui-ChangWang2014-11-262018-07-062014-11-262018-07-062014http://ntur.lib.ntu.edu.tw//handle/246246/261608本研究自以結晶性纖維Avicel為碳源的厭氣性真菌Neocallimastix patriciarum J11培養上清液中純化分子量大於669 kDa且具纖維素分解酶活性之大分子蛋白聚合物，經變性聚丙烯醯胺膠體電泳及酵素活性染色分析，結果呈現結果呈現12條蛋白質，其中8條具纖維素分解酶活性、4條具木聚醣分解酶活性。由此結果顯示，N. patriciarum J11當以結晶性纖維培養時可以誘導類似厭氣性細菌之纖維素分解酶複合體生成。西方雜合分析結果顯示，厭氣性真菌特有之錨固區蛋白質能結合至N. patriciarum J11纖維素分解酶複合體中分子量約79 kDa之蛋白質。因此，該複合體組成份中應包含類似厭氣性細菌纖維素分解酶複合體之支架蛋白質而將各分解酶組合成一複合體。此外，錨固區蛋白質與相對應結合之支架蛋白質的結合作用不需鈣離子參與。點突變錨固區蛋白質結合滴定實驗顯示，N. patriciarum J11之纖維素分解酶複合體系統中，保守性高的Trp-30應以疏水性結合力與支架蛋白質上相對應的結合區蛋白質結合。碳源誘導纖維素分解酶複合體生成的實驗結果顯示，不同碳源的誘導可影響N. patriciarum J11纖維素分解酶複合體生成量及其組成份比例。本研究藉由串連式質譜分析鑑定了N. patriciarum J11纖維素分解酶複合體的6個組成份，其包含了內切型(endo- type)、非還原端外切型(exo-type, non-reducing end)及還原端外切型(exo-type, reducing end)三種纖維素分解酶。此三類型分解酶的組合使纖維素分解酶複合體能以有效的協同作用方式分解結晶性纖維。基於上述結果，本研究首度發表了厭氣性真菌纖維素分解酶複合體的相關組成份。In this study, we purified the cellulosome of Neocallimastix patriciarum J11 from a broth through cellulose affinity purification. The cellulosome with molecular weight more than 669 kDa, and the cellulase and xylanase activities were detected by polyacryamide gel electrophoresis and zymogram. The cellulosome is composed of at least 12 comprised proteins, based on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Eight and four of these constituents have demonstrated cellulase and xylanase activites on zymogram analysis, respectively. Western hybridization analysis revealed that the fungal dockerin could bind to the protein with molecular weight of 79 kDa, and did not need calcium ion for mediation. This postulated fungal scaffoldin responded to the gathering of the cellulolytic components. Binding titration revealed that the high conserved Trp30 residue in fungal dockerin could participate in binding with scaffoldin by the hydrophobic interaction. The levels and subunit ratio of the cellulosome from N. patriciarum J11 might have been affected by their utilized carbon sources, whereas the components of the cellulosome were consistent. In this study, we identified six components of N. patriciarum J11cellulosome using liquid chromatography/mass spectrometry. The trypsin-digested peptides of six proteins were matched to the sequences of cellulases originating from rumen fungi, based on identification through LC/MS/MS, revealing that at least three types of cellulase, including one endoglucanase and two exogluanases, could be found in the N. patriciarum J11cellulosome. The cellulolytic subunits could hydrolyze synergistically on both the internal bonds and the reducing and nonreducing ends of cellulose. Based on our research, our findings are the first to depict the composition of the cellulosome produced by N. patriciarum J11, and this complex is composed of scaffoldin and three types of cellulase.摘要 I ABSTRACT II 目錄 III 圖目錄 VII 表目錄 VIII 第一章 前言 1 1.1 替代性能源與生質酒精現況 1 1.2纖維素與半纖維素 3 1.3 纖維素分解酶與木聚醣分解酶 5 1.4 纖維素分解酶與木聚醣分解酶的應用 8 1.5 纖維素分解酶與木聚醣分解酶來源 11 1.6 纖維素分解酶複合體-Cellulosome 13 (1) Cellulosome 13 (2) 支架蛋白質-Scaffoldin 15 (3) 結合區-Cohesin 17 (4) 錨固區-Dockerin 19 (5) 結合區與錨固區結合作用-Cohesin-dockerin interaction 20 (6) 纖維素分解酶複合體基因群-cellulosome gene clusters 22 (7) 重組纖維素分解酶複合體-Cellulosome chimeras 23 1.7 厭氣性真菌 25 1.8 厭氣性真菌纖維素分解酶系統研究 28 (1) 纖維素分解酶 28 (2) 纖維素分解酶複合體 29 1.9 研究動機 30 1.10 實驗架構及流程 31 第二章 材料與方法 32 2.1 藥品、試劑與儀器 32 (1) 藥品與試劑 32 (2) 儀器 34 2.2 厭氣性真菌培養 35 (1) 菌種 35 (2) 厭氣基礎培養基 35 (3) 各類操作培養基 35 (4) 培養方法與生長測試 36 2.3 Cellulosme純化 38 2.4 膠體過濾法 39 2.5 纖維素分解酶活性分析 39 2.6 變性及原態聚丙烯醯胺膠體電泳 40 (1) 膠體電泳製備 40 (2) 變性聚丙烯醯胺膠體電泳條件 40 (3) 原態聚丙烯醯胺膠體電泳條件 40 (4) 聚丙烯醯胺膠片染色 40 (5) 酵素活性染色 (zymogram) 41 2.7 錨固區蛋白質表達與純化 42 (1) 表達質體建構 42 (2) 重組蛋白質表達 45 (3) 重組蛋白質純化 45 2.8 西方雜合分析 46 2.9 支架蛋白質基因釣取 47 (1) cDNA基因庫免疫篩選法 47 (2) 基因組篩選法 48 2.10 單一錨固區定點突變 49 (1) 引子設計點突變方法 49 (2) Kunkel氏定位突變法 50 2.11 蛋白質結合滴定法 53 (1) Cellulosome製備液 53 (2) 實驗方法 53 2.12 基因定序與保存 53 2.13 蛋白質定量分析 54 2.14 蛋白質鑑定 54 2.14 蛋白質胺基端定序 54 第三章 結果 55 3.1 N. patriciarum J11之纖維素分解酶複合體生成 55 (1) 菌體生長與酵素誘導生成 55 (2) N. patriciarum J11 cellulosome生成 56 (3) N. patricirum J11 scaffoldin偵測 60 3.2 N. patriciarum J11 支架蛋白質基因釣取 97 (1) cDNA基因庫免疫篩選法 97 (2) 基因組篩選 97 3.3 N. patriciarum J11 纖維素分解酶複合體誘導生成 101 (1) 纖維素分解酶複合體誘導生產與純化 101 (2) 纖維素分解酶複合體組成分圖譜 107 3.4 N. patriciarum J11 纖維素分解酶複合體組成蛋白質鑑定 109 (1) LC/MS/MS蛋白質鑑定 109 (2) 蛋白質胺基端定序 81 (3) N. patriciarum J11 cellulosome組成份基因選殖 115 第四章 討論 122 4.1 N. patriciarum J11之纖維素分解酶複合體生成 122 (1) 菌體生長與酵素誘導生成 122 (2) N. patricirum J11 cellulosome生成 125 (3) N. patricirum J11 scaffoldin偵測 126 4.2 N. patriciarum J11 支架蛋白質基因釣取 95 4.3 N. patriciarum J11 纖維素分解酶複合體誘導生成 96 4.4 N. patriciarum J11 纖維素分解酶複合體組成蛋白質鑑定 97 第五章 總結 98 第六章 未來展望 100 參考文獻 1012821329 bytesapplication/pdf論文公開時間：2019/08/25論文使用權限：同意無償授權厭氣性真菌纖維素分解酶纖維素分解酶複合體錨固蛋白質生質酒精thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/261608/1/ntu-103-D94b47403-1.pdf