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Title: | 厭氣性真菌Neocallimastix patriciarum J11纖維素分解酶複合體之研究 Studies of anaerobic fungus Neocallimastix patriciarum J11 cellulosome system |
Authors: | Hui-Chang Wang 王惠昌 |
Advisor: | 許瑞祥(Ruey-Shyang Hseu) |
Keyword: | 厭氣性真菌,纖維素分解?,纖維素分解?複合體,錨固蛋白質,生質酒精, anaerobic fungi,cellulase,cellulosome,dockerin,bioethanol, |
Publication Year : | 2014 |
Degree: | 博士 |
Abstract: | 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/5065 |
Fulltext Rights: | 同意授權(全球公開) |
Appears in Collections: | 生化科技學系 |
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