博碩士論文 943204010 詳細資訊




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姓名 鍾武傑(Wu-Jie Jhong)  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 利用雙功能吸附基材進行蛋白復性-蛋白吸附狀態對復性的影響
(Protein refolding by Dual-Functional adsorbents:The effect of adsorbed-proteins state)
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摘要(中) 由雙功能吸附基材在幫助澱粉分解酶-Bacillus amyloliquefaciens α-amylase復性的過程中,我們發現到在高鹽濃度下,蛋白彼此之間會因鹽離子所造成的靜電遮蔽效應較強,使蛋白在尚未吸附在基材之前而形成部分聚集,再以此種較少疏水區域暴露在外的構形吸附在基材表面,且因為部分聚集的團聚物可和基材配體作用的疏水區域不多或吸附力不強,所以只需很低的靜電排斥力便可將其從基材上脫附下來,。
 在蛋白在較低鹽濃度進行吸附時,我們發現需要較高的靜電排斥力才能將吸附在基材上的蛋白脫附下來,可見蛋白與基材之間吸附頗強,蛋白的吸附型態是以單一伸展型的蛋白不同數目的疏水基進行吸附,才使得蛋白與基材之間的吸附力強到需要較高的靜電排斥力與高濃度的變性劑才能使蛋白脫附下來。我們發現蛋白與單一個疏水基的吸附型態,其蛋白的再摺疊過程中也較不會收到干擾,比較容易形成正確的構形,我們在兩種低鹽濃度下([(NH4)2SO4]=0.05 M 、0.1 M)其以此種吸附型態吸附的脫附蛋白比活性測試分別得為1.5與4.86Unit/mg;螢光測試中,兩者脫附蛋白的三級結構再摺疊也顯示出蛋白確實有摺疊回部份正確的結構。
而我們也發現從脫附蛋白的相對比活性中,經由層析復性的蛋白比活性仍太低,可見蛋白和基材之間的疏水性吸附過強,使蛋白在基材表面進行再摺疊時遭遇到疏水性區域無法再摺疊回正確的構形內,才會造成蛋白復性效果不如預期。
摘要(英) The main purpose of this research is to study the relation between the absorbed state of Bacillus amyloliquefaciens α-amylase adsorbed on a dual-functional absorbent and different conditions of refolding. Further, we also study the effect of different absorbed states on the activity recovery of eluted protein.
From our results, we find that denatured proteins will partially aggregate before proteins absorbed on the adsorbent under high salt concentration. During refolding process and the hydrophobic area of the partial-aggregate structure isn’t sufficient to maintain the interaction, the proteins are easily eluted by increasing pH value. Because the structure of proteins are mis-folded during absorbed on the adsorbent, the eluted proteins without native-tertiary structure measured by fluorescence spectrum.
We also find proteins be eluted under higher pH value at the refolding condition of low salt concentration. It is obvious to realize that the hydrophobic interaction is quite strong. So the absorbed states of proteins could be single denatured protein adsorbed on a few ligands or more protein’s hydrophobic area adsorbed on plenty of ligands. And the absorbed proteins aren’t interrupted during refolding step and form a correct structure compared with proteins adsorbed on adsorbents under high salt concentration . From the amylase activity test and fluorescence spectrum of proteins of tertiary structure,it showed that relative activity of eluted proteins are 1.5 and 4.86 Unit/mg under salt concentration of 0.1 M and 0.05 M; each refolding ratio of tertiary structure is about 70%. It points out indeed that eluted proteins have been refolded to partial native structure. According to relative activity of eluted proteins, we find that the activity of proteins refolded with hydrophobic interaction chromatography is too low.
It demonstrates that the hydrophobic interaction between proteins and substrate is still too high to make hydrophobic area refold to native structure. This affect refolded activities of proteins. If we lower the hydrophobicity of substrate by decreasing the number of ligand or ligand density, it may could improve this problem.
關鍵字(中) ★ 蛋白復性
★ 吸附型態
★ 疏水性層析
關鍵字(英) ★ refolding
★ hydrophobic interaction chromatography
論文目次 總目錄
中文摘要.........................I
英文摘要........................III
總目錄..........................V
圖目錄.........................VIII
表目錄..........................X
第一章 緒論........................1
1.1. 研究動機..................... 1
1.2. 研究目的..................... 3
第二章 文獻回顧......................4
2.1. 蛋白質簡介.................... 4
2.1.1. 蛋白質結構介紹................ 5
2.1.2. 不具生物活性的蛋白質--內聚體的形成......12
2.1.3. 蛋白質變性................. 14
2.1.4. 蛋白質復性................. 15
2.2. 蛋白質層析復性之介紹.............. 17
2.2.1. 分子大小排斥層析.............. 19
2.2.2. 離子交換層析................ 24
2.2.3. 親和性層析................. 29
2.2.4. 疏水性層析................. 33
2.3. 雙功能吸附基材介紹............... 35
2.3.1. 雙功能吸附基材其吸脫附原理......... 35
2.3.2. 雙功能吸附基材的合成............ 36
2.4. 澱粉分解酶的介紹................ 38
第三章 實驗設備、藥品、方法............... 40
3.1. 實驗藥品............... ..... 40
3.1.1. 蛋白質............... .... 40
3.1.2. 變性劑............... .... 40
3.1.3. 緩衝溶液............... ... 40
3.1.4. 活性分析試劑................ 41
3.1.5. 其他藥品與實驗用品............. 41
3.2. 實驗設備............... ..... 42
3.3. 實驗方法............... ..... 44
3.3.1. 蛋白質濃度測定................44
3.3.2. 蛋白質活性測定............. .. 44
3.3.3. 澱粉分解酶的變性.............. 46
3.3.4. 利用批次吸附方式以雙功能吸附基材探Bacillus
amyloliquefaciens α-amylase吸附情況... 47
3.3.5. 利用層析方式以雙功能吸附基材復性Bacillus
amyloliquefaciens α-amylase......... 49
第四章 結果與討論............... .....51
4.1. 利用批次吸附方式以雙功能吸附基材探討Bacillus
amyloliquefaciens α-amylase吸附情況..... 51
4.1.1. 蛋白吸附情況之探討............. 51
4.2 利用層析方式以雙功能吸附基材復性Bacillus
amyloliquefaciens α-amylase.......... 58
4.2.1. 在不同硫酸銨濃度下探討吸附狀態與Bacillus
amyloliquefaciens α-amylase脫附時的波峰位置、強
度關係.............. ..... 62
4.2.2. 在不同硫酸銨濃度下吸附對脫附蛋白構型的影響. 67
第五章 結論與建議............... .....71
參考文獻.................... .. ..73
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指導教授 阮若屈(Ruoh-Chyu Ruaan) 審核日期 2007-7-23
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