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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 劉懷勝(Hwai-Shen Liu) | |
| dc.contributor.author | Yu-An Sheim | en |
| dc.contributor.author | 冼祐安 | zh_TW |
| dc.date.accessioned | 2021-06-17T00:21:02Z | - |
| dc.date.available | 2017-06-29 | |
| dc.date.copyright | 2012-06-29 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-06-21 | |
| dc.identifier.citation | Artymiuk P., Blake C., Grace D., Oatley S., Phillips D., Sternberg M. (1979) Crystallographic studies of the dynamic properties of lysozyme.
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Physical properties and kinetics of aggregation. Biochemistry 18:5567-5571. 林煒泰. 2010 溶菌酶復性程序中氧化還原對之研究, 台灣大學碩士論文 林右晨. 2009 殘留之二硫代蘇糖醇與尿素復性之影響, 台灣大學碩士論文 周若莟. 2008 變性劑各成分在透析操作中移除方式的設計對溶菌酶復性效果之 影響, 台灣大學碩士論文 張棨翔. 2007二硫代蘇糖醇對溶菌酶變性及復性程序之影響, 台灣大學碩士論文 林昱祺. 2006 復性緩衝液中氧化還原對之動力學分析, 台灣大學碩士論文 張哲魁. 2005 以大小排組層析法進行蛋白質復性之研究, 台灣大學博士論文 張學凱. 2004 復性緩衝液中的成分對蛋白質復性之研究, 台灣大學碩士論文 廖彗媚. 2003 復性緩衝液中氧化還原反應對溶菌酶復性之研究, 台灣大學碩士論文 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66076 | - |
| dc.description.abstract | 變性蛋白質的復性通常是利用不同方法移除變性成分進而使其恢復活性。傳統的直接稀釋法是利用大量的復性劑，稀釋變性成分對蛋白質的作用。本實驗則藉由簡單的氧化還原反應，即利用GSSG以四倍稀釋與變性劑中所殘餘的DTTRed反應降低DTTRed對蛋白質的破壞。由實驗結果顯示採用低倍率直接稀釋法亦可得由如同高倍率稀釋之效果，因而進一步探討因變性殘於的DTTRed與GSSG於復性環境中對復性之影響。
低倍率直接稀釋溶菌酶突破傳統高倍率直接稀釋的方法，在實驗中除了展示四倍稀釋對復性之高效率，更確立DTTRed、GSSG共同存在可提供良好的復性環境。由實驗結果檢視活性回復率受復性系統中的變因包括DTTRed與GSSG及氧化還原試劑對活性之影響。例如因DTTRed過量而無法呈現活性之原因歸為還原態DTT控制所致；並將所有操作溶菌酶濃度依其受復性劑成分影響所呈現的活性回復結果，區分為氧化還原控制區、氧化還原至聚集體過度區、聚集體控制區。於尿素2M可將氧化還原區之溶菌酶，添以適當的GSSG濃度復性可得80%之復性回收率。例如經四倍稀釋後於2M尿素中，最終溶菌酶濃度小於1.5g/L之活性回復率皆可達到80%以上。 聚集體控制區之溶菌酶因濃度過高，故於復性環境中加入尿素可有效地抑制聚集體產生而提升此區溶菌酶的復性回復率，例如3g/L的溶菌酶於2M尿素之環境活性回復率僅有60%，但於3M尿素中則可達70%以上。 | zh_TW |
| dc.description.abstract | Protein refolding is mainly carried out by eliminating denaturant through various methods. Direct dilution method is a traditional one by diluting the denaturant with a large amount of refolding buffer. However, this research added GSSG to react with carry-over DTTRed which was a crucial hinderence to successful protein refolding. The experimental results indicated that low dilution refolding method was as good as, if not better than, that of high dilution refolding method with this strategy. Therefore, this research further discussed the impacts of refolding environmental factors including GSSG and carry-over DTTRed.
According to the experiments, there was a relationship between refolding environments and lysozyme activity recovery depending on its concentrations. Namely, it was classified into three regions, including redox control region, transition region, and aggregation control region. For example, in redox control region, the activity of lysozyme up to final concentration of 1.5g/L would regain 80% in 2M urea condition with appropriate GSSG refolding buffer. In addition, lysozyme of higher concentration in aggregation control region usually needed the help of urea to minimize aggregation, and thus obtain better activity recovery. For instance, the activity recovery of the 3g/L final lysozyme concentration by 4-times dilution in 2M urea condition was merely 60%; nevertheless, the activity recovery could be enhanced to 70% in 3M urea. | en |
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| dc.description.tableofcontents | 致謝 I
摘要 II Abstract III 目錄 V 圖目錄 VIII 表目錄 XIII 第一章 緒論 1 第二章 文獻回顧 2 2-1 蛋白質功能與組成 2 2-1-1 胺基酸 3 2-1-2蛋白質結構 5 2-2 蛋白質間之作用力 9 2-3 溶菌酶 12 2-3-1溶菌酶簡介 12 2-3-2 溶菌酶的結構 14 2-3-3 溶菌酶的活性與偵測 16 2-4 蛋白質之變性 18 2-4 蛋白質之復性 21 2-4-1蛋白質復性機制 24 2-4-2蛋白質聚集體產生 27 2-5 蛋白質復性方法 30 2-5-1直接稀釋法(direct dilution) 30 2-5-2透析法(dialysis) 36 2-5-3 大小排阻層析法(size exclusion chromatography, SEC) 38 2-5-4 逆微胞法(reversed micelles) 42 2-6 常見蛋白質復性劑 44 2-6-1二硫代蘇糖醇(dithiothreitol，DTT) 44 2-6-2穀胱甘肽（glutathione,GSH） 45 2-6-3氧化態穀胱甘肽(Oxidized Glutathione,GSSG) 46 第三章 實驗裝置、藥品、步驟 47 3-1實驗裝置 47 3-2實驗藥品 47 3-3實驗步驟 49 3-3-1【實驗試劑備製步驟】 49 3-3-2【溶菌酶之活性測定】 50 3-3-3【96孔盤活性測試】 51 3-3-4【溶菌酶之變性】 52 3-3-5【殘餘活性之測量】 52 3-3-6【直接稀釋法復性溶菌酶隨時間變化】 53 3-3-7【直接稀釋法】 53 3-3-8【氧化態DTT校正曲線】 55 3-3-9【還原態DTT殘留測試】 55 第四章 微量盤式分析儀活性測試 56 4-1 微量盤式分析儀與分光光譜儀之比較 57 4-2 微量盤式分析儀對溶菌酶活性測試 58 第五章 直接稀釋法 68 5-1 實驗動機 68 5-2 溶菌酶之變性與殘餘活性 68 5-2-1代表符號 68 5-2-2實驗方法 69 5-2-3實驗結果與討論 69 5-3 復性時間 72 5-3-1 實驗方法 72 5-3-2 實驗結果與討論 72 5-4 測量殘留DTTred 74 5-4-1 實驗方法 74 5-4-2 氧化態DTTOxi濃度校正曲線 75 5-4-3 溶菌酶經變性24小時後殘留DTTred 77 5-5 還原態DTT對活性之影響 80 5-5-1代表符號 80 5-5-2實驗方法 80 5-5-3實驗結果與討論 81 第六章 還原態DTT與GSSG對溶菌酶四倍稀釋之復性 83 6-1 實驗動機 83 6-2 實驗方法 83 6-3 實驗結果與討論 86 6-3-1 復性環境中(DTTred)Refolding、(GSSG)Refolding與活性回復率之關係 86 6-3-2 不同濃度之溶菌酶(DTTred)Refolding、(GSSG)Refolding與活性回復率關係 88 6-3-3 探討復性環境之反應 95 6-3-4 反應過後各成分對活性之影響 98 6-3-5 氧化還原控制區與聚集體控制區之比較 110 第七章 尿素對溶菌酶復性的影響 112 7-1 實驗動機 112 7-2 實驗方法 112 7-3 實驗結果與討論 113 7-3-1 尿素對溶菌酶之復性 113 7-3-2 探討反應後尿素對溶菌酶復性之影響 118 7-3-3蛋白質復性之應用 130 第八章 結論 131 第九章 參考文獻 134 | |
| dc.language.iso | zh-TW | |
| dc.subject | 二硫代蘇糖醇 | zh_TW |
| dc.subject | 溶菌酶 | zh_TW |
| dc.subject | 蛋白質復性 | zh_TW |
| dc.subject | 直接稀釋法 | zh_TW |
| dc.subject | direct dilution | en |
| dc.subject | DTT | en |
| dc.subject | lysozyme | en |
| dc.subject | protein refolding | en |
| dc.title | 低倍率之溶菌酶復性 | zh_TW |
| dc.title | Low Dilution Refolding for Lysozyme | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李振綱(Cheng-Kang Lee),王孟菊(Meng-Jiy Wang) | |
| dc.subject.keyword | 溶菌酶,蛋白質復性,直接稀釋法,二硫代蘇糖醇, | zh_TW |
| dc.subject.keyword | lysozyme,protein refolding,direct dilution,DTT, | en |
| dc.relation.page | 141 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-06-21 | |
| dc.contributor.author-college | 工學院 | zh_TW |
| dc.contributor.author-dept | 化學工程學研究所 | zh_TW |
| 顯示於系所單位： | 化學工程學系 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-101-1.pdf 未授權公開取用 | 12.78 MB | Adobe PDF |
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