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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 劉?睿 | |
dc.contributor.author | Chih-Yuan Chu | en |
dc.contributor.author | 朱致遠 | zh_TW |
dc.date.accessioned | 2021-06-13T01:10:52Z | - |
dc.date.available | 2016-08-08 | |
dc.date.copyright | 2011-08-08 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-08-03 | |
dc.identifier.citation | 參考文獻
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/29574 | - |
dc.description.abstract | 中文摘要
纖維素為地球上最豐富的多醣且具有高度潛力成為再生能源,然而在自然界中的纖維素大多為結晶型。因此,具有對結晶型纖維素較高水解能力的酵素成為許多研究者追尋的目標。 由台灣水牛瘤胃真菌Piromyces rhizinflatus篩選出的β-聚葡萄糖酶基因cbhYW23-2,其序列長度為1557個含氮鹼基,表現出的酵素由519個胺基酸組成,分子量約為57 kDa。其胺基酸序列的C端具有屬於醣基水解酶第6族的酵素區,N端為屬於碳水化合物結合單元第1族的結合區,兩區由富含天門冬醯胺酸的胜肽連接。為測定酵素活性,將帶有His6的CbhYW23-2重組蛋白由Escherichia coli表現並以親和性管柱純化,再以反應曲面法結合中央合成設計及迴歸分析酵素之最適溫度及pH值,結果分別為46.4°C及pH 6.0,於此反應條件下,酵素比活性為2411.1±197.9 U/mg。CbhYW23-2也展現了對於Avicel、羧甲基纖維素 (carboxylmethyl cellulose, CMC)、lichenan、以及pachyman的活性。故證明CbhYW23-2對於結晶型纖維素具有極高的活性,因此具有應用於工業上的潛力。接著將cbhYW23-2基因構築於Saccharomyces cerevisiae表現質體上,並使CbhYW23-2接合於a凝集素,使CbhYW23-2能夠表現於S. cerevisiae的細胞壁上。經酵素活性測定、酵素擴散法、西方轉漬及間接免疫螢光染色分析,證實CbhYW23-2可成功表現並錨定在S. cerevisiae細胞表面。 綜上所述,本研究成功利用反應曲面法得到此β-聚葡萄糖酶之最適反應條件,並利用S. cerevisiae細胞表面蛋白a凝集素,將CbhYW23-2固定於S. cerevisiae表面並仍具有對β-聚葡萄糖之活性。 | zh_TW |
dc.description.abstract | Abstract
Cellulose is the most abundant renewable polysaccharide with a high potential for degradation to useful end products. In nature, most cellulose is produced as crystalline cellulose. Therefore, cellulases with high hydrolytic activity against crystalline cellulose are of great interest. In this study, a crystalline cellulose degradation enzyme was investigated. The cDNA encoding a β-glucanase, CbhYW23-2, was cloned from the ruminal fungus Piromyces rhizinflatus. The cDNA sequence of cbhYW23-2 contained an open reading frame of 1,557 bp encoding a protein of 519 amino acids with a predicted molecular weight of 57 kDa. A putative conserved domain of glycosyl hydrolase (GH) family 6 was observed at the C-terminus and a putative conserved domain of cellulose binding domain (CBD) family 1 was observed at the N-terminus of CbhYW23-2. These two domains were separated by an Asn-rich linker. To examine the enzyme activities, CbhYW23-2 was expressed in E. coli as recombinant fusion protein and purified by immobilized metal ion-affinity chromatography. Response surface modeling (RSM) combined with central composite design (CCD) and regression analysis were then employed for the planned statistical optimization of the β-glucanase activities of CbhYW23-2. The optimal conditions for the highest β-glucanase activity of CbhYW23-2 were observed at 46.4°C and pH 6.0. At this condition, the specific activity of CbhYW23-2 was 2411.1±197.9 U/mg. CbhYW23-2 also showed hydrolytic activities toward Avicel, carboxymethyl cellulose (CMC), lichenan, and pachyman. The results also proved that the highly activity of CbhYW23-2 on crystalline cellulose makes it a promising candidate enzyme for biotechnological and industrial applications. Then cbhYW23-2 was constructed into the expression vector of S. cerevisiae. CbhYW23-2 was displayed on the cell surface of S. cerevisiae via a-agglutinin. The localization of a-agglutinin-CbhYW23-2 fusion protein on the cell surface was confirmed by analysis of β-glucanase activity assay, enzyme diffusion method, western blot, and immunofluorescence microscopy. In summary, the results suggested that RSM combined with CCD and regression analysis were effective in determining optimized temperature and pH conditions for the enzyme activity of CbhYW23-2. The a-agglutinin-CbhYW23-2 fusion protein was expressed on the cell surface of S. cerevisiae successfully and still remained the β-glucanase activity. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T01:10:52Z (GMT). No. of bitstreams: 1 ntu-100-R98626022-1.pdf: 4358144 bytes, checksum: 8845b2855080c7ab517a35e8c57bf162 (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | 目錄
中文摘要 1 英文摘要 2 第一章、序言 4 第二章、文獻探討 5 一、台灣水牛簡介 5 二、瘤胃真菌簡介 5 (一) 絕對厭氧真菌之發現 5 (二) 絕對厭氧真菌之分離源 6 (三) 瘤胃真菌生活史 6 (四) 瘤胃真菌之分類 7 (五) 絕對厭氧真菌在瘤胃中扮演的角色 9 三、木質纖維素的組成及來源 9 (一) 木質纖維素的組成 9 (二) 木質纖維素的來源 10 四、纖維素酶之種類及應用 10 (一) 纖維水解酵素之種類 10 (二) 纖維素酶的應用 12 五、Saccharomyces cerevisiae細胞表面展現系統 14 六、以S. cerevisiae生產生質酒精 16 (一) 原料之前處理方式 17 (二) 纖維素糖化 18 七、生質酒精之生產方式 19 (一) 同步糖化發酵法 19 (二) 聯合生物加工法 20 第三章、材料與方法 28 一、CbhYW23-2之基因來源及選殖 28 (一) 基因來源 28 (二) cbhYW23-2之選殖 28 二、CbhYW23-2之胺基酸序列與結構區域分析 29 三、以E. coli表現CbhYW23-2 29 (一) 纖維水解酶表現質體構築 30 (二) 組胺酸標幟融合蛋白純化 31 (三) CbhYW23-2蛋白質濃度測定 33 (四) 聚丙烯醯胺膠體電泳分析 34 (五) 酵素活性測定 37 (六) CbhYW23-2之最適作用pH值與溫度 38 (七) CbhYW23-2之熱穩定性 40 (八) CbhYW23-2之受質專一性及酵素動力學測定 40 四、以S. cerevisiae表現CbhYW23-2 41 (一) E. coli-S. cerevisiae穿梭載體建構與選殖 41 (二) 穿梭載體於S. cerevisiae Invsc1之轉形及選殖 43 (三) CbhYW23-2於S. cerevisiae細胞表現 44 (四) 酵素擴散法 45 (五) 西方轉漬分析 46 (六) 間接免疫螢光染色 48 第四章、結果與討論 58 一、CbhYW23-2之胺基酸序列與結構區域分析 58 二、CbhYW23-2於E. coli之表現及純化 60 三、CbhYW23-2酵素之最適作用pH值與溫度 61 (一) pH值及溫度對酵素活性之影響 61 (二) 反應曲面法模式分析 61 四、CbhYW23-2 β-聚葡萄糖酶之受質專一性與酵素動力學 62 五、CbhYW23-2之熱穩定性 63 六、以S. cerevisiae表現CbhYW23-2 64 七、S. cerevisiae細胞表面展現CbhYW23-2之β-聚葡萄糖酶活性分析 64 八、S. cerevisiae表現CbhYW23-2之蛋白質表現分析 65 九、S. cerevisiae及其轉型株之間接免疫螢光染色分析 66 第五章、結論 87 參考文獻 88 | |
dc.language.iso | zh-TW | |
dc.title | 瘤胃真菌Piromyces rhizinflatus之外切聚葡萄糖酶基因選殖及其外源性表現 | zh_TW |
dc.title | Molecular cloning of a cellobiohydrolase from Piromyces rhizinflatus and heterologous expression | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳明汝,劉啟德,陳小玲,謝建元 | |
dc.subject.keyword | Piromyces rhizinflatus,β-聚葡萄糖酶,Saccharomyces cerevisiae, | zh_TW |
dc.subject.keyword | Piromyces rhizinflatus,β-glucanase,Saccharomyces cerevisiae, | en |
dc.relation.page | 99 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-08-03 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 動物科學技術學研究所 | zh_TW |
顯示於系所單位: | 動物科學技術學系 |
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