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博碩士論文 etd-0205110-152503 詳細資訊
Title page for etd-0205110-152503
論文名稱
Title
利用嗜甲醇酵母菌表現阿拉伯芥內切型纖維素分解酶
Cloning and Expression of Arabidopsis endo-1,4-β-glucanase in Pichia pastoris
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
69
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2010-01-13
繳交日期
Date of Submission
2010-02-05
關鍵字
Keywords
纖維素、酵母菌
Pichia pastoris, endo-1, 4-β-glucanase
統計
Statistics
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中文摘要
人類進入工業化社會後,因過度使用石化燃料造成多種嚴重的環境污染,加上部分石化燃料在地球的存量即將用盡,促使替代能源的研究在近年發展,木質纖維素(包含木質素、纖維素、半纖維素)是自然界中最豐富的再生資源,廣泛應用在食品、紙及能源等方面,具有極高的經濟價值。近年來,將纖維素降解成最小單位-葡萄糖,再進而醱酵成乙醇成為生質能源的熱門話題。 本研究目的為選殖阿拉伯芥 (Arabidopsis) 的 endo-1,4- β-glucanase 基因,並轉殖到Escherichia coli(DH5α) 勝任細胞, 挑選出載體 (pPICZαA) 有成功轉入 DNA 片段的白色菌株,其後再將初步篩選菌株送交定序,並藉由細胞內表現載體pPICZαA 於酵母菌 Pichia. pastoris 中進行表現。以 AOX1 啟動子系統於重組酵母菌 Pichia pastoris 大量生產阿拉伯芥之endo-1,4-β-glucanase 基因,以甲醇誘導方式進行胞外表現,可得重組酵素蛋白質,而Western blotting的分析顯示具有此酵素活性的蛋白質At4g11050 pellet 分子量約為 89 KDa。再經由剛果紅和Dye- CMC assay 檢測酵素蛋白質之活性,結果發現At4g11050 pellet細胞內活性與 negative control (vector only) 相比有較明顯之活性。
Abstract
In recent years. as industrialized society developed. people have made a lot of environment pollution problems owing to overusing fossil fuel, moreover. fossil fuel is going to deplete. As the result. the study of the substitute energy is promoted.
Ligonocellulose (lignin, cellulose and hemicelluloses are included) are the most plentiful renewable resources in the nature, and it have high economic values which extensively use on food, paper and energy. Recent years, it is a hot issue that decomposing cellulose into the minimum unit called glucose, and further, fermenting into alcohol to generate biomass energy.
This research goal is cloning Arabidopsis endo-1,4-β-glucanase and transfer to Escherichia coli(DH5α). Selection pPICZαA the success transferr DNA Fragment. Then to sequence and because of cell in pPICZαA have expression in Pichia. pastoris. AOX1 promoter have Mass productions Arabidopsis endo-1,4-β-glucanase gene in Pichia pastoris. Carries on the extracellular expression by the methyl alcohol induction way. Can obtain recombinant DNA protein. However the Western blotting analysis demonstration has this enzyme active protein At4g11050 pellet Molecular weight about 89 KDa. Again by way of congo red and Dye-CMC assay activeness of the examination enzyme protein. The result discovers At4g11050 in the pellet cell activity compares with negative control has the obvious activity.
目次 Table of Contents
中文摘要………………………………………………………………..1
英文摘要………………………………………………………………..2
誌謝……………………………………………………………………..3
目錄……………………………………………………………………..5
圖索引…………………………………………………………………..6
前言……………………………………………………………………..7
材料與實驗方法………………………………………………………..20
結果……………………………………………………………………..36
討論……………………………………………………………………..40
圖表……………………………………………………………………..45
參考文獻………………………………………………………………..59
附錄……………………………………………………………………..66
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