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研究生:賴雋仁
論文名稱:結合逆流層析與反微胞技術萃取及濃縮蛋白質分子
論文名稱(外文):Combining countercurrent chromatography and reversed-micelle technologies to extract and concentrate protein molecules
指導教授:余艇
學位類別:碩士
校院名稱:國立交通大學
系所名稱:應用化學系
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
中文關鍵詞:逆流層析逆流層析儀反微胞逆微胞萃取濃縮蛋白質蛋白質分子
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本實驗選擇含有陰離子型界面活性劑sodium bis(2-ethylhexyl) sulfosuccinate (AOT) 的正己烷溶液為反微胞有機相,以有機相為靜相,水溶液相為動相,並以高速逆流層析儀(high speed counter-current chromatograph)當作一高效率之萃取裝置。當含有生物分子的水溶液流經管柱中的靜相時,蛋白質分子會被萃取至反微胞相中而滯留在分離管柱中;待正向萃取完成後,注入高pH值的鹼性溶液,藉由動相的帶動改變反微胞相的環境,使蛋白質分子轉移至此一水溶液中,完成反向萃取,並在過程中達成濃縮蛋白質分子。在對單一成分蛋白質cytochrome c之回收效率探討中,我們使用50 mM AOT萃取100 mg/l cytochrome c可以達到95.4%之回收率;而我們也使用未事先經過平衡之動靜相溶劑系統針對20 mg/l cytochrome c進行萃取,結果發現:在同樣以90 mM AOT為靜相之條件下,此系統所得之回收率(78.7%)高於採用經過平衡之溶劑系統所得到之回收率(62%);最後我們嘗試針對酵素混合溶液,在逆流層析儀中進行選擇性的分離萃取。
總 目 錄
中文摘要………………………………………………………….. i
英文摘要………………………………………………………….. ii
謝誌…………………………………………………………... iii
總目錄…………………………………………………………….. iv
表目錄…………………………………………………………….. vii
圖目錄…………………………………………………………….. viii
第一章 緒論……………………………………………………… 1
第二章 研究背景與理論………………………………………… 3
2.1 逆流層析…………………………………………………….. 3
2.1-1 簡介……………………………………………………. 3
2.1-2 起源與發展……………………………………………. 3
2.1-3 高速逆流層析…………………………………………. 12
2.2 反微胞萃取法……………………………………………….. 15
2.2-1 界面活性劑……………………………………………. 15
2.2-2 反微胞…………………………………………………. 15
2.2-3 反微胞萃取……………………………………………. 17
2.2-4 常見的反微胞萃取法…………………………………. 20
2.2-5 反微胞萃取生物分子的主要驅動力………………….. 21
2.2-6 影響蛋白質在反微胞所處之有機相與水溶液間分佈
傳遞的因素…………………………………………….. 21
2.2-7 反微胞技術的其他應用………………………………. 26
2.3 反微胞技術所使用的儀器裝置…………………………….. 30
第三章 實驗…..…………………………………………………. 32
3.1 試藥………………………………………………………….. 27
3.1-1 藥品……………………………………………………... 32
3.1-2 溶劑……………………………………………………... 32
3.2 實驗儀器……………………………………………………. 32
3.2-1 儀器裝置圖…………………………………………….. 33
3.2-2 儀器設備說明…………………………………………... 33
3.3 溶劑系統的製備…………………………………………….. 35
3.3-1 靜相溶劑-反微胞有機相…….………………………... 35
3.3-2 動相溶劑-水溶液相…..……………………………….. 35
3.3-3 動靜相溶劑系統的平衡……………………………….. 36
3.3-4 樣品溶液-蛋白質及酵素水溶液………..…………….. 36
3.3-5 高pH鹼性溶液……………………………………….. 37
3.4 實驗操作步驟……………………………………………….. 37
3.4-1包含正向萃取與反向萃取之完整反微胞萃取步驟…… 37
3.4-2單純正向萃取之操作方法……………………………… 39
3.4-3對酵素混合樣品進行反微胞選擇性分離萃取………… 39
3.5 樣品收集方法……………………………………………... 43
3.6 定量方法…………………………………………………... 43
第四章 結果與討論..…………………………………………… 45
4.1採用平衡過的動/靜相溶劑系統進行反微胞萃取………….. 44
4.1-1蛋白質濃度效應………………………………………… 47
4.1-2界面活性劑濃度效應…………………………………… 48
4.2採用未經過平衡處理之動/靜相溶劑系統進行反微胞萃取.. 50
4.3緩衝溶液對回收率的影響…………………………………... 52
4.4高pH值鹼性溶液與蛋白質萃取物活性的探討…………… 54
4.5酵素混合溶液的選擇性分離萃取………………………….. 55
4.5-1批次反向萃取…………………………………………… 56
4.5-2連續式反向萃取………………………………………… 60
4.5-3再循環式萃取…………………………………………… 62
第五章 結論……………………………………………………… 67
參考文獻………………………………………………………….. 68
表目錄
表(一)分別以 50、70、90 mM AOT / n-hexane 萃取濃縮
20 mg/l cytochrome c……………………………………45
表(二)分別以 50、70、90 mM AOT / n-hexane 萃取濃縮
50 mg/l cytochrome c……………………………………45
表(三)分別以 50、70、90 mM AOT / n-hexane 萃取濃縮
100mg/l cytochrome c……………………………………46
表(四)分別以未經過分布平衡之 50、70、90 mM AOT / n-hexane
萃取濃縮 20 mg/l cytochrome c…………………………51
表(五)四種酵素之基本資料………………………………………56
圖目錄
圖(一)液滴式逆流層析(DCCC)示意圖………………….………….5
圖(二)螺管在不同轉速下兩不互溶溶劑的相分佈………………..8
圖(三)反扭轉機構的七個模式…………………………….……….11
圖(四)高速逆流層析之旋轉機構…………………………………..13
圖(五)IV型同步行星式逆流層析管柱內部混合液的分佈情形…..14
圖(六)界面活性劑、微胞與反微胞的示意圖………………………16
圖(七)反微胞萃取示意圖…………………………………………..17
圖(八)利用反微胞技術進行蛋白質再摺疊………………………..27
圖(九)利用GSH/GSSG 氧化還原系統完成蛋白質雙硫鍵合成…. 28
圖(十)Hentsch(1992)在探討界面活性劑濃度時所採用的裝置….31
圖(十一)孔洞旋轉盤接觸器………………………………………..31
圖(十二)儀器裝置圖…………………………………………………33
圖(十三)高速逆流層析儀……………………………………………34
圖(十四)結合pH gradient之儀器設備圖………………………….42
圖(十五)典型的萃取過程訊號圖……………………………………44
圖(十六)ribonuclease a、trypsin及lysozyme 混合溶液之
分離萃取圖 …………………………………………………58
圖(十七)針對ribonuclease a、trypsin及lysozyme混合溶液分離萃 取之HPLC 層析圖…………………………………………59
圖(十八)ribonuclease a及lysozyme混合溶液之分離萃取圖……61
圖(十九)連續式反向萃取實驗結果之MALDI-MS分析圖……………62
圖(二十)再循環式萃取實驗之第一階段反向萃取圖………………63
圖(二十一)再循環式萃取實驗之第一階段反向萃取收集
MALDI-MS分析圖…………………………………………64
圖(二十二)再循環式萃取實驗之第二階段反向萃取圖……………65
圖(二十三)再循環式萃取實驗之第二階段反向萃取收集
MALDI-MS分析圖…………………………………………65
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