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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 謝學真(Hsyue-Jen Hsieh) | |
dc.contributor.author | Jiun-Liang Lin | en |
dc.contributor.author | 林俊良 | zh_TW |
dc.date.accessioned | 2021-06-13T01:08:13Z | - |
dc.date.available | 2009-07-24 | |
dc.date.copyright | 2007-07-24 | |
dc.date.issued | 2007 | |
dc.date.submitted | 2007-07-23 | |
dc.identifier.citation | Adachi Y., Chen W. and Shang W.H., Development of a direct and sensitive detection method for DNA-binding proteins based on electrophoretic mobility shift assay and iodoacetamide derivative labeling , Analytical Biochemistry, 2005, 342, 348-351.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/29481 | - |
dc.description.abstract | 眾多因素會影響雞蛋白溶菌酶還原變性的程度,如: 蛋白質濃度、還原劑濃度與變性時間等等。 另外也有必要確認完全還原變性的溶菌酶具有哪些特徵。 在此篇研究中,利用自身螢光、水力半徑 (分子大小排斥層析)、螢光染劑IAF標記與活性回收率等方式來觀察溶菌酶變性的過程並且探討完全還原變性所需條件。 結果發現濃度 5 mg/mL 的溶菌酶必須溶在含有0.2 M二硫代蘇糖醇的變性溶液中經過30小時才能夠完全還原變性瓦解其立體結構。 而在還原變性過程中,觀察到了變性中間體的存在並且確認其結構屬於”殘存一個雙硫鍵的溶菌酶”。 經由這樣的發現,我們提出了”三階段還原變性”的動力學模式以下列步驟: 正確構型之溶菌酶至第一變性中間體,第一變性中間體至第二變性中間體,第二變性中間體至完全還原變性之溶菌酶此三步驟之反應速率常數各為: 0.61 mM-0.467h-1, 0.20 h-1以及0.0095 mM-0.333h-1。 而最後一步: 第二變性中間體至完全還原變性之溶菌酶則為反應速率決定步驟,另外經由螢光染劑的標記實驗結果得知: 第二變性中間體仍為殘存一個雙硫鍵的溶菌酶結構。 接下來我們以稀釋法進行復性發現當添加1.5 M的尿素至復性溶液中時,可以讓完全還原變性的溶菌酶逐漸復性達到80 % 的活性回收比且可以降低氧化態穀胱甘肽的使用量。 另外針對添加不同還原劑濃度或是不同變性時間所造成的不同還原變性程度之溶菌酶進行稀釋法復性時,若復性溶液中也添加了1.5 M 尿素,則可以觀察到不同變性程度的溶菌酶會有不同的復性速率與不同的活性回收比,因此復性效果優劣與變性的程度的高低密切相關。 一般而言,變性程度越高則復性速率越慢且活性回收比越低。 最後我們製備了巰基丙酸-戊二醛-幾丁聚醣微粒並用來促進溶菌酶的復性,配合透析復性法可以使溶菌酶的比活性增加到90 % 。 | zh_TW |
dc.description.abstract | Many factors affect the extent of reductive denaturation of hen egg white lysozyme, e.g., the concentration of reducing agent such as dithiothreitol (DTT), the concentration of lysozyme and the time length of denaturation. It is thus crucial to identify the fully reductively denatured lysozyme. In this study, the reductive denaturation process of lysozyme was monitored with intrinsic fluorescence、hydrodynamic radius (size exclusion chromatography)、5-iodoacetamido fluroescein (IAF) labeling and activity recovery to characterize the fully reductively denatured lysozyme. It was found that lysozyme (5 mg/mL) was fully reductively denatured after being incubated in denaturing buffer which contained 0.2 M DTT for 30 hrs. Further, a reductively denatured intermediate of lysozyme was also identified as one-disulfide bond-left lysozyme. According to these findings, we proposed a “three-step reductive denaturation” kinetic model: N (native lysozyme) to I1 (intermediate 1) to I2 (intermediate 2) to U (fully unfolded lysozyme) to describe the existence of intermediates during reductive denaturation of lysozymes. The kinetic constants were estimated by fitting the shift in the wavelength of maximum emission intensity during denaturation with the three-step kinetic model. The rate constants for each steps were found to be 0.61 mM-0.467h-1, 0.20 h-1, and 0.0095 mM-0.333h-1, respectively, suggesting that I2 to U is the rate-limiting step. Additionally, the reductively denatured lysozyme intermediate I2 still has one uncleaved disulfide bond as revealed by IAF labeling. After that, by the method of dilution refolding, addition of 1.5 M urea in refolding buffer highly enhanced the refolding yield to 80 % and minimized the cost of GSSG. The denaturation of lysozyme in various extent could be generated by the addition of different concentrations of reducing agent (DTT) or different denaturing times under the influence of 1.5 M urea. The refolding of reductively denatured lysozyme was highly affected by the extent of denaturation. In general, a high DTT concentration and a long denaturing time resulted in slow refolding with low activity recovery. Finally, the beads of chitosan and its thiol derivative (MPA-GLA-chitosan) were prepared and used to facilitate the refolding of lysozyme. The use of MPA-GLA-chitosan beads as refolding additives increased the specific activity of lysozyme to 90 % after dialysis refolding. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T01:08:13Z (GMT). No. of bitstreams: 1 ntu-96-D88524009-1.pdf: 1524879 bytes, checksum: 8d8cc6007de44a34b52e21df68af3e60 (MD5) Previous issue date: 2007 | en |
dc.description.tableofcontents | 誌謝I
中文摘要III 英文摘要V 目錄VII 圖目錄XII 表目錄XVII 符號與簡寫說明XVIII 中英名詞對照XX 第一章 緒論1 第二章文獻回顧 2-1 蛋白質摺疊技術發展的起因6 2-2 溶解包涵體的目地及破壞蛋白質立體結構的方式9 2-3 目標蛋白:雞蛋白溶菌酶(hen egg white lysozyme)之簡介11 2-4 蛋白質變性過程的觀察14 2-4-1 以螢光觀測變性時蛋白質三級結構的變化17 2-4-2 以水力半徑判斷蛋白質變性程度19 2-4-3 還原變性過程中氫硫基的測量20 2-5 蛋白質的復性方式21 2-5-1 降低變性劑與蛋白質濃度22 2-5-2 將變性劑與蛋白質,蛋白質與蛋白質進行隔離23 2-5-2-1 層析法復性24 2-5-2-2 以其他吸附材或復性添加物隔離復性中之蛋白質25 2-5-3 以雙硫鍵為主的復性方式27 2-6 幾丁聚醣的簡介及其應用30 第三章 藥品、器材與實驗方法 3-1 藥品33 3-2 儀器34 3-3 實驗方法 3-3-1 雞蛋白溶菌酶還原變性過程測定 3-3-1-1 自身螢光法 (Intrinsic fluorescence)35 3-3-1-2大小排斥層析法 (SEC)36 3-3-1-3 螢光染劑 (IAF) 標記法36 3-3-1-4 不同變性程度下之lysozyme復性差異(不同還原劑濃度)37 3-3-1-5不同變性程度下之復性差異 (不同變性時間)38 3-3-2 完全還原變性之雞蛋白溶菌酶之稀釋復性條件最適化 3-3-2-1氧化態榖胱甘肽 (GSSG) 濃度對稀釋復性效果之響38 3-3-2-2 最終lysozyme濃度對稀釋復性效果之影響39 3-3-2-3 Urea濃度於復性溶液中之添加量對稀釋復性效果之影響39 3-3-2-4在1.5 M urea濃度下GSSG濃度對復性效果之影響.40 3-3-3 幾丁聚醣顆粒之製備40 3-3-4 幾丁聚醣顆粒之交聯與化學修飾 3-3-4-1幾丁聚醣顆粒 (chitosan bead) 之交聯40 3-3-4-2 經GLA交聯後的chitosan beads (GLA-Chi)之化學修飾41 3-3-5 以chitosan beads 幫助lysozyme復性 3-3-5-1以未經交聯之chitosan beads 幫助復性42 3-3-5-2以GLA-Chi 或MPA-GLA-Chi 幫助復性 3-3-5-2-1 透析法復性43 3-3-5-2-2 以層析法復性44 第四章 實驗結果與討論 4-1 溶菌酶變性過程的觀察 4-1-1 以螢光光譜觀測溶菌酶的變性過程與變性程度45 4-1-2 以分子大小排斥層析分析lysozyme的變性程度52 4-1-3 Lysozyme變性中間體的標記58 4-1-4 結論68 4-2 溶菌酶變性動力學模式之探討 4-2-1 還原變性過程之動力學模式建構70 4-2-2 還原變性動力學之迴歸77 4-2-3 結論82 4-3 完全變性lysozyme復件條件之最佳化 4-3-1 復性時各種復性添加物濃度對稀釋復性效果的影響84 4-3-2 還原變性程度對復性效果之影響93 4-3-3 結論99 4-4 以幾丁聚醣 (Chitosan) 作為復性添加物增進溶菌酶之復性 4-4-1 以幾丁聚醣作為復性添加物之構想100 4-4-2 幾丁聚醣顆粒之製備及性質測定101 4-4-3 幾丁聚醣顆粒之交聯與化學修飾106 4-4-4 以幾丁聚醣顆粒與其化學修飾之衍生物作為復性添加物109 4-4-5 結論113 第五章 總結 5-1 總結115 5-2未來研究方向118 參考文獻 119 附錄A137 附錄B164 | |
dc.language.iso | zh-TW | |
dc.title | 溶菌酶還原變性之觀察、變性動力學之分析以及復性策略之探討 | zh_TW |
dc.title | Observation and Kinetic Analysis on the Reductive Denaturation of Lysozyme and Investigation on the Strategy of Lysozyme Refolding | en |
dc.type | Thesis | |
dc.date.schoolyear | 95-2 | |
dc.description.degree | 博士 | |
dc.contributor.coadvisor | 阮若屈(Ruoh-Chyu Ruaan) | |
dc.contributor.oralexamcommittee | 黃世佑(Shih-Yow Huang),陳文逸(Wen-Yih Chen),陳秀美(Hsiu-Mei Chen),劉懷勝(Hwai-Shen Liu) | |
dc.subject.keyword | 溶菌酶,還原變性,動力學模式,復性,雙硫鍵, | zh_TW |
dc.subject.keyword | Lysozyme,Reductive Denaturation,Kinetic Model,Renatutration,Disulfide bond, | en |
dc.relation.page | 166 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2007-07-23 | |
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 |
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