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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 羅翊禎徐駿森 | zh_TW |
| dc.contributor.author | 郭宜蓓 | zh_TW |
| dc.contributor.author | Yi-Pei Kuo | en |
| dc.date.accessioned | 2021-07-11T15:37:38Z | - |
| dc.date.available | 2024-09-03 | - |
| dc.date.copyright | 2018-08-18 | - |
| dc.date.issued | 2018 | - |
| dc.date.submitted | 2002-01-01 | - |
| dc.identifier.citation | 王如邦. (2016). 探討Saccharomyces cerevisiae kre6Δ突變株及Dekkera bruxellensis 對羅漢果皂苷轉化之機制.國立台灣大學生物資源暨農學院食品科技研究所博士論文, 臺北, 臺灣.
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Stubbs, H. J., Brasch, D. J., Emerson, G. W., & Sullivan, P. A. (1999). Hydrolase and transferase activities of the beta-1,3-exoglucanase of Candida albicans. Eur J Biochem, 263(3), 889-895. Sunna, A., Moracci, M., Rossi, M., & Antranikian, G. (1997). Glycosyl hydrolases from hyperthermophiles. Extremophiles, 1(1), 2-13. Taylor, S. C., Ferguson, A. D., Bergeron, J. J. M., & Thomas, D. Y. (2004). The ER protein folding sensor UDP-glucose glycoprotein–glucosyltransferase modifies substrates distant to local changes in glycoprotein conformation. Nature Structural &Amp; Molecular Biology, 11, 128. Terpe, K. (2003). Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Applied Microbiology and Biotechnology, 60(5), 523-533. Varghese, J. N., Hrmova, M., & Fincher, G. B. (1999). Three-dimensional structure of a barley beta-D-glucan exohydrolase, a family 3 glycosyl hydrolase. Structure, 7(2), 179-190. Villaverde, A., & Mar Carrió, M. (2003). Protein aggregation in recombinant bacteria: biological role of inclusion bodies. Biotechnology Letters, 25(17), 138 Zhu, S., Gong, C., Ren, L., Li, X., Song, D., & Zheng, G. (2013). A simple and effective strategy for solving the problem of inclusion bodies in recombinant protein technology: His-tag deletions enhance soluble expression. Applied Microbiology and Biotechnology, 97(2), 837-845 | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79025 | - |
| dc.description.abstract | 外切-β-1,3-葡聚醣酶 (exo-β-1,3-glucanase, EC 3.2.1.58) 參與酵母菌細胞壁之代謝，主要功能為水解 β-1,3-glucan 與 β-1,6-glucan ，可將多醣從非還原端分解為葡萄糖。先前研究發現酒香酵母 (Dekkera bruxellensis) 對於羅漢果皂苷之轉化與其他種類酵母菌相比有其特殊性，並且確認 D. bruxellensis EXG1基因所產生之 exo-β-1,3-glucanase (DbExg1) 能夠專一性的將羅漢果五醣皂苷 (mogroside V，MG V) 轉化為四醣皂苷 (siamensoide I，SI) 。為了對 DbExg1 有更深的瞭解，因此本研究以 DbExg1 為研究對象，對其進行生化特性分析與蛋白結構模擬。實驗使用 E. coli BL21 (DE3) 為宿主表現重組蛋白並經過純化來獲得 DbExg1 。在酵素特性的部分， DbExg1 在酸性環境下有較好的酵素活性，最適反應 pH 值為 6.0 。在溫度方面則是 30-50℃ 之間可維持 50% 以上的酵素活性，最適反應溫度為40℃ 。而熱穩定性試驗指出 DbExg1在 pH 5-6 有較高之 Tm 值 (melting temperature) ，說明 DbExg1 在酸性溶液中蛋白結構較穩定。此外，酵素動力學的結果顯示 DbExg1 對於 laminarin (β-1,3-glucan) 之催化效率遠高於 pustulan (β-1,6-glucan) ，符合本身酵素特性。對於 MG V 之催化效率則介於兩者之間。在結構分析的部分，為採用同源模擬法 (homology modelling) 建立 DbExg1 之三級結構，模擬結果顯示 DbExg1 為 (β/α)8-TIM-barrel 之構形，並且針對可能影響 MG V 轉化差異之胺基酸進行定點突變，雖然並未找到關鍵胺基酸，實驗卻意外發現突變位置 G325S 造成 DbExg1 酵素活性之下降。綜合研究結果，我們對於 DbExg1 之基本酵素特性有更多的了解，並透過模擬建立 DbExg1 立體結構，而突變蛋白 DbExg1 G325S 酵素活性之降低，則推論可能與結構空間障礙相關。 | zh_TW |
| dc.description.abstract | Exo-β-1,3-glucanase (EC 3.2.1.58) is involved in the metabolism of yeast cell wall. It can hydrolyze β-1,3-glucan and β-1,6-glucan by breaking down polysaccharides from non-reducing ends. Compared to other yeast species, previous studies have shown the unique property of Dekkera bruxellensis on mogroside bioconversion and confirmed that exo-β-1,3-glucanase produced by the EXG1 gene from D. bruxellensis (abbreviated as DbExg1) can specifically transform mogroside V (MG V) into siamensoide I (SI). To get a better understanding of DbExg1, analysis on biochemical characterization and protein structure modelling was therefore performed. E. coli BL21 (DE3) was used to express recombinant proteins. Regards to the enzyme characteristics, DbExg1 shows better enzyme activity in acidic environments. Its optimal pH is 6.0 and optimal temperature is 40°C. More than 50% enzyme activity of DbExg1 remains under 30-50°C treatment. In thermal shift assay, DbExg1 has a higher Tm value at pH 5-6, indicating that DbExg1 is more stable in acidic solution. In addition, the results of enzyme kinetics showes that the catalytic efficiency of DbExg1 for laminarin is much higher than for pustulan.The results agrees with the natural properties of the protein. Interestingly, the efficiency of DbExg1 for MG V is between those of laminarin and pustulan. In the structural analysis, the tertiary structure of DbExg1 built by homology modeling showed (β/α)8-TIM-barrel folding. Moreover, the residues which may make a difference in MG V conversion was mutated. We surprisingly observed the decrease in enzyme activity of DbExg1 G325S mutant. Taken together, the basic enzyme properties and modeling structure of DbExg1 were studied. DbExg1 G325S mutant has low enzyme activity which may be related to form a spatial barrier in structure and lead to the reduced biotransformation. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T15:37:38Z (GMT). No. of bitstreams: 1 ntu-107-R05641002-1.pdf: 3635098 bytes, checksum: f67b6ade1f2095f77953d7a43df7ac7b (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 摘要 ii
Abstract iii 總目錄 iv 圖目錄 viii 表目錄 ix 壹、 前言 1 貳、 文獻回顧 2 第一節、 醣苷水解酶 2 1. 醣苷水解酶 (glycoside hydrolases ,GHs) 的定義與分類 2 2. Glycoside hydrolase family 5 (GH5) 之介紹 2 2.1 GH5 酵素之蛋白結構 4 2.2 GH5 酵素之催化機制 5 3. 外切-β-1,3-葡聚醣酶 (exo-β-1,3-glucanase) 之特性與來源介紹 6 3.1 酵母菌外切-β-1,3-葡聚醣酶之功能 8 3.2 酵母菌外切-β-1,3-葡聚醣酶之結構分析 8 第二節、 酒香酵母之特性 10 第三節、 羅漢果皂苷 11 1. 羅漢果皂苷 (mogroside) 之介紹 11 2. 羅漢果皂苷之生物轉化 13 參、 材料與方法 15 第一節、 實驗材料 15 1. 質體來源 15 2. 菌株 15 3. 載體 15 4. 抗生素 15 5. 標準品 15 6. 酵素 15 7. 市售套組 16 8. 化學藥品 16 9. 儀器設備與用具 17 第二節、 藥品配製 20 1. LB 液態培養基 20 2. LB-kanamycin 固態培養基 20 3. 1 % 瓊脂膠體 20 4. 蛋白質電泳膠體 20 5. 5X protein loading dye 21 6. SDS-PAGE staining buffer 21 7. SDS-PAGE destaining buffer 21 8. Transfer buffer (pH 8.3 ) 21 第三節、 實驗方法 22 1. DbExg1之基因構築 22 1.1 引子設計 22 1.2 放大目標基因 22 1.3 限制酵素酶切 23 1.4 ligation 23 1.5 熱休克轉形至DH5α 24 1.6 colony PCR 24 1.7 質體抽取與定序 25 2. DbExg1之蛋白表達與純化 25 2.1 熱休克轉形至BL21(DE3) 25 2.2 小量表現 25 2.3 蛋白質電泳分析 (SDS-PAGE) 26 2.4 大量表現 26 2.5 Ni-column純化 26 2.6 透析與濃縮 27 2.7 蛋白質濃度測定 27 2.8 膠體過濾層析法 (Gel filtration chromatography) 28 2.9 離子交換層析法 (Ion exchange chromatography) 28 2.10 西方墨點法 (western blotting) 確認蛋白大小 28 2.11 動態光散射儀 (Dynamic Light Scattering, DLS) 評估分子粒徑 29 3. DbExg1之生化特性分析 29 3.1 轉化羅漢果五醣皂苷 29 3.2 最適反應pH值 31 3.3 最適反應溫度與溫度耐受性 31 3.4 圓二色光譜 (Circular Dichroism, CD ) 實驗 31 3.5 差示掃描螢光法 (differential scanning fluorimetry) 評估蛋白穩定性 32 3.6 酵素活性試驗 32 3.7 酵素動力學 33 4. DbExg1之結構分析 35 4.1 晶體培養 35 4.2 同源模擬 36 4.3 胺基酸序列比對 36 4.4 定點突變（Site-directed mutagenesis） 36 肆、 結果與討論 38 第一節、 DbExg1之建構與表現 38 1. 基因構築 38 2. 評估最適表現溫度 39 3. 蛋白大量表現與純化 39 4. DbExg1 monomer 與 oligomer 之獲得 40 5. DbExg1 monomer 與 oligomer 之二級結構比較 41 6. DbExg1 monomer 與 oligomer之活性分析 42 第二節、 DbExg1之生化特性 42 1. MG V轉換確認 42 2. pH值對 DbExg1 之影響 42 3. 溫度對 DbExg1 之影響 43 4. DbExg1 酵素動力學測定 44 第三節、 DbExg1 之結構模擬與分析 45 1. DbExg1 晶體培養 45 1.1 以 thrombin 去除 DbExg1 N 端 His-tag 46 1.2 參考同源蛋白養晶條件 47 1.3 陰離子交換純化 DbExg1 47 1.4 膠體過濾層析法純化 DbExg1 47 1.5 改變緩衝系統 47 2. 以同源模擬法 (Homoligy modelling) 建立 DbExg1 立體結構 48 3. DbExg1 突變位點之選擇 49 4. 點突變蛋白轉化 MG V 49 5. 點突變蛋白活性分析 50 伍、 結論 51 陸、 圖表 52 柒、 參考文獻 80 捌、 附錄 85 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 酵素學 | zh_TW |
| dc.subject | 外切-β-1 | zh_TW |
| dc.subject | 3-葡聚醣? | zh_TW |
| dc.subject | 酒香酵母 | zh_TW |
| dc.subject | 結構模擬 | zh_TW |
| dc.subject | 3-glucanase | en |
| dc.subject | Exo-β-1 | en |
| dc.subject | Dekkera bruxellensis | en |
| dc.subject | enzymology | en |
| dc.subject | homology modelling | en |
| dc.title | 酒香酵母外切-β-1,3-葡聚醣酶之生化特性與結構分析 | zh_TW |
| dc.title | Biochemical properties and structural analysis of an exo-β-1,3-glucanase from Dekkera bruxellensis | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 106-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 方翠筠;陳勁初;黃學聰;呂廷璋 | zh_TW |
| dc.contributor.oralexamcommittee | ;;; | en |
| dc.subject.keyword | 外切-β-1,3-葡聚醣?,酒香酵母,酵素學,結構模擬, | zh_TW |
| dc.subject.keyword | Exo-β-1,3-glucanase,Dekkera bruxellensis,enzymology,homology modelling, | en |
| dc.relation.page | 114 | - |
| dc.identifier.doi | 10.6342/NTU201802391 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2018-08-14 | - |
| dc.contributor.author-college | 生物資源暨農學院 | - |
| dc.contributor.author-dept | 食品科技研究所 | - |
| 顯示於系所單位： | 食品科技研究所 | |
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