博碩士論文 89321004 詳細資訊




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姓名 周茂村(Mao-Tsun Chou)  查詢紙本館藏   畢業系所 化學工程與材料工程學系
論文名稱 不同化學變性劑、溫度和還原劑對溶菌
(The studies of the lysozyme structure change with different chemical denaturants, temperatures and reduced reagent)
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摘要(中) 重組蛋白質在回收過程中,常會因為胜
摘要(英) In the process of recombinant protein production, we often get inactive and insoluble inclusion bodies instead of target proteins due to high peptide concentration. In industrial application, denaturants such as urea or guanidine hydrochloride are used to solve these inclusion bodies to renature and purify/separate proteins and then denaturants are removed to get the protein we want.
Factors to stabilize protein structures include hydrophobic interactions, hydrogen bonding, electrostatic interaction and covalent disulfide bonding. Urea is mainly used to break hydrophobic interactions and guanidine hydrochloride is to destroy hydrophobic and electrostatic interactions. Sometimes, salts such as sodium chloride/lithium chloride are added in urea as electrostatic interaction destroyer to enhance the denaturant power. Another commonly used reduced reagent (dithiothreitol, DTT) is to reduce disulfide bonding within protein. All these different mechanisms bring various influences to protein structures.
In our study, we examine secondary and tertiary structure changes of lysozyme in various conditions (different concentrations of urea / guanidine hydrochloride / salts (sodium chloride), experimental temperature, with/without reduced reagent) with the aid of circular dichroism and fluorescence spectrophotometer to investigate the relationship between above factors and spectrum structure.
When reduced reagent exists, guanidine hydrochloride causes greater changes in protein structure than urea and sodium chloride. Without reduced reagent, only high concentration of guanidine chloride causes significant protein structure changes. The results of fluorescence experiments indicates that tryptophan inside protein didn’’t expose easily when disulfide bond remained unbroken.
Besides, from temperature manipulation experiments, we see the denature reaction with denaturants and reducing agents added is an exothermic reaction. At the mean time, the entropy of system decreases and melting temperature (Tm) is lower than that in buffer solution.
From the above experiments, we know that if the disulfide bond is not reduced, we have to use denaturant with the capacity to break hydrophobic and electrostatic interactions in high concentration to induce significant changes in lysozyme structure. When disulfide bond is reduced, denaturants causes changes of lysozyme structure easier that indicate lysozyme is a hard protein itself.
It’s possible to investigate the relationship between activity and structure of enzymes with the aid of ultraviolet spectrometer. In further studies, comparing reaction enthalpy in various conditions measured by isothermal titration calorimeter with thermodynamic data from spectrometer, we can learn the pathway of lysozyme denature process to see it’s two state or multi-state phase transformation reaction.
關鍵字(中) ★ 溶菌
★ 螢光光譜儀
★ 圓二色光譜儀
★ 二硫代蘇糖醇
★ 胍鹽酸
★ 尿素
★ 還原劑
★ 結構
★ 變性劑
★ 溫度
關鍵字(英) ★ temperature
★ denaturant
★ structure
★ lysozyme
★ fluorescence spectrophotometer
★ circular dichroism
★ reduced reagent
★ urea
★ guanidine hydrochloride
★ dithiothreitol
論文目次 目 錄
中文摘要......................................................................Ⅰ
Abstract......................................................................Ⅲ
目錄..........................................................................Ⅴ
圖目錄........................................................................Ⅷ
表目錄......................................................................ⅩⅠ
第一章 緒論..................................................................1
第二章 實驗原理與文獻回顧....................................................3
2.1 蛋白質介紹............................................................3
2.1.1 蛋白質的組成、功能及穩定結構之作用力..............................3
2.1.2 蛋白質結構........................................................9
2.1.3 穩定態的構形與特性...............................................14
2.1.4 蛋白質摺疊模型...................................................15
2.2 蛋白質之變性.........................................................17
2.2.1 破壞蛋白質穩定力量的方式.........................................17
2.2.2 變性劑與還原劑對蛋白質分子之作用.................................18
2.3 常見的分析方法.......................................................20
2.4 圓二色光譜儀.........................................................21
2.4.1 圓二色光譜儀發展歷史.............................................21
2.4.2 光的吸收與光學活性的關係.........................................24
2.4.3 圓二色光譜儀測量原理.............................................28
2.4.4 蛋白質或多胜
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指導教授 陳文逸(Wen-yih Chen) 審核日期 2002-7-4
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