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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李昆達 | |
| dc.contributor.author | Zhi-Yu Wei | en |
| dc.contributor.author | 魏至宇 | zh_TW |
| dc.date.accessioned | 2021-06-15T07:05:47Z | - |
| dc.date.available | 2012-12-10 | |
| dc.date.copyright | 2010-12-10 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-12-03 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48635 | - |
| dc.description.abstract | 木酚素 (lignans)，為苯丙醇 (phenylpropanoid) 之二聚體衍生物，在生理學上具有植物雌激素功能 (phytoestrogen) 以及抗癌活性，存在於全麥食品、芝麻、亞麻籽。其中，裂環落葉松脂醇 (secoisolariciresinol) 及羅漢松樹脂醇 (matairesinol) 在哺乳類結腸中，可被轉換為屬於哺乳類木酚素 (mammalian lignans) 之腸內脂 (enterolactone) 及腸內二脂 (enterodiol)，而血清及尿液中高濃度腸內脂對於治療乳癌、骨質疏鬆症、及大腸癌都有正面效果。為建立木酚素高效率轉化系統，利用選殖自台灣八角蓮木酚素生合成酵素基因，松脂醇-落葉松脂醇還原酶 (pinoresinol-lariciresinol reductase, PLR) 以及裂環落葉松脂醇去氫酶(secoisolariciresinol dehydrogenase, SDH)，以非對稱重疊擴增聚合酶鏈鎖反應 (asymmetric overlap extension by polymerase chain reaction, AOE-PCR) 將上述酵素之基因藉由蛋白質連接序列 (linker) 連接形成雙功能性蛋白質融合基因，並於大腸桿菌 (Escherichia coli) 中表現。經聚丙烯醯胺膠體電泳 (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) 及西方點墨法 (Western blot) 蛋白質分析，選定經 0.01 mM IPTG 誘導12 小時，重組融合蛋白質可以於大腸桿菌中表現。於 pH 8.0、22OC 進行胞外生物轉化反應1小時，發現融合蛋白質 PLR-SDH，不僅同時具有兩種酵素活性，且反應效率高於重組 PLR 混和重組 SDH 之組別。PLR-SDH 之 matairesinol/pinoresinol總轉換率為 49.82 %，單位蛋白質轉換率為 3.41 % / | zh_TW |
| dc.description.abstract | Lignans, a class of dimeric phenylpropanoid derivatives, existing in whole-grain, sesame and flax seeds show anticancer activity and can react as phytoestrogen. Secoisolariciresinol and matairesinol, members of lignans, can be converted to enterolactone and enterodiol in mammalian proximal colon. Containing high concentration of enterolactone in serum and urine has positive effect for the treatment of breast cancer, osteoporosis and colon cancer. To establish a continuous bioconversion system for matairesinol formation from pinoresinol, pinoresinol reductase (plr) and secoisolariciresinol dehydrogenase (sdh) which were cloned from Podophyllum peltatum Hance were linked by asymmetric overlap extension by polymerase chain reaction (AOE-PCR), to form a bifunctional fusion gene (plr-sdh) and was expressed in Escherichia coli. The optimized conditions for recombinant fusion protein induction in E.coli were 0.01 mM IPTG, 12 h, 25oC determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. At pH 8.0, 22OC, 1 h reaction in vitro the results showed that the bioconversion efficiency of fusion protein PLR-SDH was higher than the mixture of individual rPLR and rSDH. The bioconversion rate of PLR-SDH was 49.82%, and 3.41% / | en |
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| dc.description.tableofcontents | 論文口試書 i
致謝 ii 中文摘要 iii Abstract iv Abbreviation vi 中英文專有名詞對照表 vii 目 錄 viii 圖表目錄 xi 第一章 前言 1 1.1 木酚素在臨床藥物上及自然界中所扮演的角色 2 1.2 鬼臼素及其衍生物 3 1.3 松脂醇、裂環落葉松脂醇及羅漢松脂醇 4 1.4 松脂醇-落葉松脂醇還原酶及裂環落葉松脂醇去氫酶 5 1.5 Escherichia coli M15 (pREP4) 表現系統 6 1.6 Fusion protein approach 融合蛋白質技術 7 1.7 研究目的 9 1.8 研究架構 9 第二章 材料與方法 11 2.1 載體及表現宿主 12 2.2 質體構築 12 2.2.1 plr-sdh及 sdh-plr融合蛋白質基因片段之構築 12 2.2.2 E.coli JM109質體保存株之熱休克轉形 17 2.2.3 重組菌株 JM109 之菌落聚合酶鏈鎖反應 17 2.2.4 pQE30Xa-plr-sdh 與 pQE30Xa -sdh-plr 表現載體之建立 18 2.2.5 表現株 M15 (pREP4) 之熱休克轉形 19 2.2.6 重組菌株 M15 之菌落聚合酶鏈鎖反應 20 2.3 融合蛋白質 rPLR-SDH 及 rSDH-PLR 於M15的表現 20 2.4 蛋白質純化 21 2.5 蛋白質分析 21 2.5.1 聚丙烯醯胺膠體電泳 21 2.5.2 His-tag 西方墨點轉印法 22 2.6 木酚素標準曲線 22 2.7 酵素活性分析 23 2.7.1 rPLR 酵素活性分析 23 2.7.2 rSDH 酵素活性分析 23 2.7.3 rPLR 及 rSDH 之共同轉化測試 24 2.7.4 融合蛋白質 rPLR-SDH 及 rSDH-PLR 活性測試 24 2.7.5 M15-pQE-PLR-SDH 之in vivo bioconversion 24 2.8 高效能液相層析 25 第三章 結果 26 3.1 rPLR 混和 rSDH 共同轉化測試 27 3.2 plr-sdh 及sdh-plr 融合蛋白質基因之構築 28 3.3 轉形株之建立 28 3.4 重組蛋白質之表現 29 3.5 重組蛋白質之純化 30 3.6 木酚素之標準曲線 30 3.7 融合蛋白質 in vitro bioconversion 31 3.8 M15-pQE-PLR-SDH 之 in vivo bioconversion 32 第四章 討論與展望 33 圖表 38 參考文獻 71 附 錄 79 | |
| dc.language.iso | zh-TW | |
| dc.subject | 融合蛋白質 | zh_TW |
| dc.subject | 松脂醇-落葉松脂醇還原酶 | zh_TW |
| dc.subject | 裂環落葉松脂醇去氫酶 | zh_TW |
| dc.subject | PLR | en |
| dc.subject | fusion protein | en |
| dc.subject | SDH | en |
| dc.title | 融合松脂醇-落葉松脂醇還原酶及裂環落葉去氫酶將松脂醇連續轉化為羅漢松脂醇 | zh_TW |
| dc.title | Fusion of Pinoresinol-lariciresinol reductase with Secoisolariciresinol dehydrogenase for converting Pinoresinol into Matairesinol | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蘇遠志,黃鵬林,楊啟伸,楊健志 | |
| dc.subject.keyword | 松脂醇-落葉松脂醇還原酶,裂環落葉松脂醇去氫酶,融合蛋白質, | zh_TW |
| dc.subject.keyword | PLR,SDH,fusion protein, | en |
| dc.relation.page | 81 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-12-03 | |
| 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 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-99-1.pdf 未授權公開取用 | 3.73 MB | Adobe PDF |
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