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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃慶璨(Ching-Tsan Huang) | |
| dc.contributor.author | Ying-Jie Wu | en |
| dc.contributor.author | 吳盈潔 | zh_TW |
| dc.date.accessioned | 2021-06-08T04:16:29Z | - |
| dc.date.copyright | 2011-08-22 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-08-17 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22376 | - |
| dc.description.abstract | 嗜甲醇酵母菌Hansenula polymorpha是常用的異源蛋白質表現系統，可藉由強力且可調控的甲醇氧化酶啟動子 (methanol oxidase promoter, MOXp) 來啟動異源蛋白質表現。MOXp除了能使用甲醇進行誘導外，還能藉由甘油、葡萄糖、木糖為單一碳源之碳源受限 (carbon source starvation) 方式，使MOXp發生去抑制 (derepression) 作用進而表現下游基因。目前工業上嗜甲醇酵母菌大規模的醱酵應用，大多使用甲醇做為誘導物，但使用甲醇的缺點包括甲醇具有毒性及易燃性，對於工業化大量生產會增加風險及管控成本、濃度過高的甲醇對菌體造成毒性或造成蛋白質產物降解、生產醫藥性蛋白質可能有甲醇殘留的疑慮。因此，發展一個非甲醇誘導之誘導策略實屬必要。本研究以不同的溶氧模式調控甘油添加，使MOXp發生去抑制作用表現木聚醣酶。研究發現，以固定溶氧值之調控模式，溶氧值設定越低可得到較高的酵素活性，將溶氧值固定於80%、60%及40%，所生產的木聚醣酶最高活性分別為4368.30±85.65、6903.73±152.21和7512.47±147.13 U/ml (n=3)。並且在不同誘導模式中發現甘油的添加量、鹼的添加量及木聚醣酶活性增加量都呈現正相關，因此在醱酵過程中可做為重組蛋白質產量的良好指標。 | zh_TW |
| dc.description.abstract | The methylotrophic yeast Hansenula polymorpha is one of the most commonly used heterologous protein expression systems. Its methanol oxidase promoter (MOXp) is a strong and tightly regulated promoter, and can be used in heterologous protein expression. Except methanol, the MOXp can also be derepressed by single carbon source starvation such as glycerol, glucose or xylose for expressing downstream genes. Methanol is a commonly used inducer at large-scale fermentation of methylotrophic yeasts in industrial application. The disadvantages of using methanol include that methanol is toxic and flammable, the large-scale production of menthanol would increase the risk, and the cost for managing is high. In addition, high methanol concentration may hurt cells or cause target protein to be degrade. Since therapeutic proteins may face the risk of methanol remanet, it is necessary to develop a methanol-free induction strategy. In this study, using MOXp derepression to express xylanase through different pO2 control modes for regulating glycerol starvation. We found that lower pO2 control can get higher enzyme activity at fixed pO2 control.When pO2 control at 80%, 60% and 40%, the highest xylanase activity are 4368.30±85.65, 6903.73±152.21 and 7512.47±147.13 U/ml (n=3). And the addition of glycerol, base and xylanase activity correlated well with different pO2 control modes, suggesting that the glycerol and base addition can be a good indicator of recombinant protein production in fermentation course. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T04:16:29Z (GMT). No. of bitstreams: 1 ntu-100-R98b47414-1.pdf: 2268495 bytes, checksum: cef0696a95f5d8c4f2a5d7ec22a3a7d4 (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 中文摘要 I
Abstract II 圖目錄 V 表目錄 VII 第一章 前言 1 1. 異源蛋白質表現系統 1 2. 嗜甲醇酵母菌表現系統 3 2-1. Pichia pastoris 4 2-2. Pichia methanolica 6 2-3. Hansenula polymorpha 7 3. 來自瘤胃真菌Neocallimastix frontalis的木聚醣酶 11 4. 研究動機與目的 13 第二章 材料與方法 16 1. 菌株與培養條件 16 1-1. H. polymorpha MOXZa-xyn11B’ 1B2-17 16 1-2. 培養條件 16 2. 醱酵槽大量生產目標蛋白質 17 2-1. 前置培養 17 2-2. 批式饋料培養 17 2-3. 甘油受限誘導培養 17 2-4. 甲醇誘導培養 17 3. 木聚醣酶活性測定 18 3-1. 木聚醣酶活性測定原理 18 3-2. 木聚醣酶活性測定方式 19 4. 生長曲線 20 4-1. 生菌數測定 20 4-2. A600吸光值測定 20 5. 蛋白質定量 20 第三章 結果 21 1. 細胞高密度培養 21 2. 甲醇誘導 21 3. 溶氧調控甘油添加之碳源受限誘導 22 第四章 討論 50 1. 細胞高密度培養 50 2. 甲醇誘導 50 3. 溶氧調控甘油添加之碳源受限誘導 51 4. 去抑制作用之基因調控 54 第五章 結論 55 第六章 未來展望 56 第七章 參考資料 57 附錄 64 | |
| dc.language.iso | zh-TW | |
| dc.title | 以非甲醇策略誘導漢遜氏酵母菌表現重組木聚醣酶 | zh_TW |
| dc.title | A non-methanol induction strategy for recombinant xylanase expression in Hansenula polymorpha | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 許瑞祥,楊啟伸,李昆達,陳浩仁 | |
| dc.subject.keyword | 漢遜氏酵母菌,異源蛋白質,甲醇氧化酶,啟動子,去抑制作用,木聚醣酶, | zh_TW |
| dc.subject.keyword | Hansenula polymorpha,heterologous proteins,MOX promoter,derepression,xylanase, | en |
| dc.relation.page | 70 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2011-08-18 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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| ntu-100-1.pdf 目前未授權公開取用 | 2.22 MB | Adobe PDF |
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