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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/24270完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃慶璨 | |
| dc.contributor.author | Chia-En Wu | en |
| dc.contributor.author | 吳佳恩 | zh_TW |
| dc.date.accessioned | 2021-06-08T05:20:18Z | - |
| dc.date.copyright | 2005-08-01 | |
| dc.date.issued | 2005 | |
| dc.date.submitted | 2005-07-27 | |
| dc.identifier.citation | 王正仁, 陳孟伶, 林畢修平. 1999. 水解酵素在工業上的利用. 生物產業 10:1-11.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/24270 | - |
| dc.description.abstract | 植物細胞壁之主要成分纖維素，為自然界蘊藏最豐的碳源與多醣類，亦為最充裕的再生資源。由於纖維素結構緊密，其內含的醣類難以利用，須以化學或生化反應的方式將其水解，以提高纖維素於生物能源再利用率，而纖維分解酵素扮演了極重要的角色。
纖維分解酵素為一群水解酵素的總稱，可經協同(synergic)作用將纖維素分解，目前已知具有纖維素分解能力的生物包括細菌、真菌、原生動物等。其用途相當廣泛，已應用於眾多產業上，主要為食品與釀酒、動物飼料、紡織與衣物清洗、造紙工業及農業，其他用途包括：廢棄物處理、製藥工業、基因工程及污染防治等。目前已有多種同源(homologous)或異源(heterologous)表達系統，如細菌、酵母菌及絲狀真菌等，可生產出具功能性之纖維分解酵素。 本研究以巨型桿菌(Bacillus megaterium)表達Piromyces rhizinflatus之內切型纖維分解酵素(endoglucanase, EglA)。以T7啟動子替代商品化選殖載體pWH1520之木糖啟動子，並導入巨型桿菌β-澱粉水解酵素之訊息胜肽，以期得到強力啟動子且具外泌能力之載體，更進一步比較不同表達質體表現纖維分解酵素EglA之效果。 目前已完成外泌性載體pWH1520-S、T7 RNA聚合殖載體pWH1520-T7R之建構，表現質體pWH1520-C，亦完成T7啟動子之釣取，待接入目標基因eglA後，即可進行纖維分解酵素之生產及比較不同表達系統之功效。可成功地於巨型桿菌B. megaterium WH1520-C中穩定表達纖維分解酵素EglA，其酵素活性達5 U/mL；亦可於B. megaterium WH1520-T7R中表達T7 RNA聚合酶。 | zh_TW |
| dc.description.abstract | Cellulose, the major component of plant cell walls, is the most abundant renewable biomass in nature and a sustainable clean resource for energy. Cellulase is responsible for the first step of degradation of cellulose. The application potential of cellulase includes food, brewery and wine, animal feed, textile and laundry, pulp and paper, as well as agricultures. Several homologous or heterologous expression systems, such as bacteria, yeasts or filamentous fungi, have been developed for production of cellulase.
Bacillus megaterium has several advantages over E. coli for a broad industrial use in the production of recombinant proteins. First, ease of culture, non-pathogenic and no inclusion body. Second, there is no alkaline proteases. Last, the recombinant plasmids in B. megaterium is stable structurally and segregationally. In this study, we described the establishment of a B. megaterium based recombinant protein production and export system for the cellulase EglA from Piromyces rhizinflatus. We modified the B. megaterium by adapting E. coli T7 system for over-production of proteins and inserting a signal peptide from homologous β-amylase gene for protein secretion. The goal of this study is to develop an alternative expression system which could secret the desired protein into the culture supernatant. We have constructed secretory cloning vector pWH1520-S, xylose-inducible T7 RNA polymerase vector pWH1520-T7R, expression plasmid pWH1520-C. We also cloned T7 promoter gene and inserted eglA gene downstream. We have successfully express cellulase EglA in B. megateirum WH1520-C, and 5 Units/mL of crude extraction were measured. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T05:20:18Z (GMT). No. of bitstreams: 1 ntu-94-R92b47411-1.pdf: 4226902 bytes, checksum: b1a0bf6b48177943d38481494662fde1 (MD5) Previous issue date: 2005 | en |
| dc.description.tableofcontents | 表目錄 IV
圖目錄 V 摘 要 VI Abstract VII 第一章、緖言 1 一、纖維素與纖維分解酵素 1 二、表達系統 13 2.1 枯草桿菌屬 14 2.2 枯草桿菌屬異源表達系統 14 2.3 枯草桿菌表達系統 17 2.3.1 選殖載體(cloning vector)的來源 17 2.3.2 構築表達質體 18 2.3.3 啟動子 18 2.3.4 訊息胜肽 22 2.3.5 枯草桿菌表達系統的缺點 23 2.4 巨型桿菌之表達系統 23 2.4.1 簡介 23 2.4.2 異源表達系統 26 2.4.3 選殖載體 27 2.4.4 商品化之選殖載體及表達系統 29 三、研究動機與目的 32 第二章 材料與方法 35 一、實驗材料 35 菌株、質體及培養基 35 二、實驗方法 38 1. 質體建構 38 1.1 pWH1520-S 38 1.2 pWH1520-C及pWH1520-SC 39 1.3 pWH1520-T7R 39 1.4 pYE-T7P、pYE-T7PC及pYE-T7PSC 39 1.5 pWH1520-T7RC及pWH1520-T7RSC 40 2. 質體轉形 46 2.1 質體DNA製備 46 2.2 巨型桿菌原生質體製備及DNA轉形 46 3. 巨型桿菌轉形株檢定 47 3.1 纖維分解酵素表現之偵測 47 3.2 轉形株質體DNA萃取 47 3.3 聚合酶連鎖反應分析 48 3.4 限制酶截切分析 48 4. 纖維分解酵素之表達 48 4.1三角瓶培養及誘導表達 48 4.2 菌體生長量之測定 49 5. 纖維分解酵素活性分析 49 5.1 粗酵素液製備 49 5.2 十二烷基磺酸鈉-聚丙烯胺膠體電泳 50 5.3 膠體酵素活性染色 50 5.4 T7 RNA聚合酶西方雜合分析 50 5.5 蛋白質定量分析 51 5.6 纖維分解酵素活性分析 51 三、實驗使用之套組(kit) 51 第三章、實驗結果 53 1. 表現質體之建構 53 1.1 質體pWH1520-S 53 1.2 質體pWH150-C及pWH1520-SC 55 1.3 質體pWH1520-T7R 57 1.4 質體pYE-T7P、pYE-T7PC及pYE-T7PSC 59 2. 巨型桿菌之轉形 62 2.1 原生質體製備 62 2.2 聚乙二醇轉形法 63 3. 巨型桿菌轉形株檢定 64 3.1 纖維分解酵素表現之偵測 64 3.2 轉形株質體DNA萃取及分析 65 3.4 西方雜合偵測T7 RNA聚合酶於巨型桿菌之表現 66 4. 纖維分解素之表達 68 4.1 B. megaterium WH1520-C 68 4.1.1菌體生長量之測定 68 4.1.2纖維分解酵素活性分析 70 第四章、討論 74 1. T7 RNA聚合酶異源表達系統 74 2. 外泌性異源表達系統 75 第五章、結論 77 第六章、未來工作 78 參考文獻 79 | |
| dc.language.iso | zh-TW | |
| dc.subject | 纖維分解酵素 | zh_TW |
| dc.subject | 巨型桿菌 | zh_TW |
| dc.subject | T7 RNA聚合酶 | zh_TW |
| dc.subject | 蛋白質外泌 | zh_TW |
| dc.subject | 異源表達 | zh_TW |
| dc.subject | Bacillus megaterium | en |
| dc.subject | cellulase | en |
| dc.subject | heterologous expression | en |
| dc.subject | secretion | en |
| dc.subject | T7 RNA polymerase | en |
| dc.title | 利用基因重組巨型桿菌生產纖維分解酵素EglA之研究 | zh_TW |
| dc.title | Production of cellulase EglA by recombinant Bacillus megaterium | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 93-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 許瑞祥,常玉強 | |
| dc.subject.keyword | 巨型桿菌,T7 RNA聚合酶,蛋白質外泌,異源表達,纖維分解酵素, | zh_TW |
| dc.subject.keyword | Bacillus megaterium,T7 RNA polymerase,secretion,heterologous expression,cellulase, | en |
| dc.relation.page | 86 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2005-07-28 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-94-1.pdf 未授權公開取用 | 4.13 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。