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研究生:曾韋寧
研究生(外文):Wei-Ning Tseng
論文名稱:以天絲纖維不織布固定化金屬親和吸附基材進行純化及固定化過程
論文名稱(外文):Tencel nonwoven fabric-based immobilized metal affinity chromatography adsorbents for enzyme purification and immobilization
指導教授:林松池
指導教授(外文):Sung-Chyr Lin
口試委員:楊芳鏘邱信程
口試委員(外文):Fan-Chiang YangHsin-Cheng Chiu
口試日期:2016-07-27
學位類別:碩士
校院名稱:國立中興大學
系所名稱:化學工程學系所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:72
中文關鍵詞:天絲纖維不織布海藻糖合成酶蛋白質純化固定化金屬親和層析法酵素固定化
外文關鍵詞:Tencelnonwoven fabrictrehalose synthaseprotein purificationimmobilized metal affinity chromatographyenzyme immobilization
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本研究以天絲纖維不織布做為固定化金屬親和吸附材之固定載體,接著以化學合成法依序接上環氧氯丙烷及螯合劑亞胺二乙酸(IDA),最後再接上過渡金屬離子,即完成固定化金屬吸附基材。
此金屬螯合親和吸附材之過渡金屬離子可以與含有poly His-tag 基因重組海藻糖合成酶的poly His-tag形成配位共價鍵,以達到純化及固定化海藻糖合成酶的目的。研究結果指出最適化的基材活化條件是在 6.2N 環氧氯丙烷與 3N 的氫氧化鈉於35°C持續反應六小時,得到的銅離子鍵結量為6771 μmol Cu(II)/ g-support;比較由六種不同金屬離子固定化後的海藻糖合成酶,以Zn (II)-loaded adsorbent呈現出最高的蛋白質吸附量為23.9 mg protein/g adsorbent與比活性為 15.9 U/ mg protein;粗酵素液含有300 mM NaCl具有最佳的選擇性,並且發現在吸附時的粗酵素液含有50 mM imidazole的純化效果比未添加時的純化效果更佳:以四種不同比例的Tencel/PET吸附海藻糖合成酶,發現添加PET會減少蛋白質吸附量但是可能會增加與蛋白質之間的吸附力。固定化海藻糖合成酶經過重複批次的使用,會因為酵素脫落問題導致活性下降。


In this study, Tencel nonwoven fabric-based immobilized metal affinity chromatography (IMAC) adsorbents were prepared upon activation with epichlorohydrin and conjugation with iminodiacetic acid. The results showed that the optimum surface activation was achieved with 6.2 N epichlorohydrin, 3 N sodium hydroxide at 35 ° C for six hours, giving a metal chealting capacity of 6771 μmol Cu(II)/ g-support. Among the six metal ions tested, the Zn (II) –loaded IMAC adsorbent exhibited the highest absorptive capacity and superior selectivity for the model protein, the poly(His)-tagged trehalose synthase, 23.9 mg protein/g adsorbent with a specific activity of 15.9 U/ mg protein. The Optimal selectivity was observed with 300 mM NaCl in the adsorption buffer. Furthermore, it was found that the inclusion of 50 mM imidazole in the adsorption buffer or wash buffer could significantly increase the purity of trehalose synthase. It was found that PET can reduced the adsorption capacity of the nonwoven fabric-based adsorbents but might enhance adsorption. Based on the above results, we can used for purification and immobilization of poly(His)-tagged proteins. A residual activity of approximately 71% was maintained after 20 cycles in a repeated-batch operation. The decline in residual activity was primarily due to the desorption of the trehalose synthase.

致謝 ii
中文摘要 iii
Abstract iv
目錄 v
圖目錄 x
表目錄 xiii
第一章 緒論 1
1.1 前言 1
1.2 研究動機 2
第二章 文獻回顧 3
2.1 蛋白質純化 3
2.1.1 蛋白質簡介 3
2.1.2 蛋白質純化技術 4
2.1.2.1膠體過濾法 (gel filtration) 5
2.1.2.2離子交換法 (ion exchange) 6
2.1.2.3親和層析法 (affinity chromatography) 6
2.1.2.4鹽溶鹽析 (salt in / salt out) 7
2.2 固定化金屬離子親和層析法 (Immobilized Metal Ion Affinity Chromatography) 9
2.2.1 固定化金屬離子親和層析介紹 9
2.2.1.1 吸附基材 (matrix) 10
2.2.1.2 延伸臂 (spacer arm) 11
2.2.1.3 螯合劑 (chelating agent) 11
2.2.1.4 過渡金屬離子 (transition metal ion) 13
2.2.2 固定化金屬親和層析 (IMAC) 與 polyhistidine tag 13
2.2.3 影響固定化金屬親和層析法的吸附探討 14
2.2.4 影響蛋白質脫附之探討 15
2.2.4.1 質子化 (protonation) 15
2.2.4.2 配位基交換法 (ligand exchange) 15
2.2.4.3 螯合消除法 (chelate annihilation) 15
2.3 酵素固定化與固定化方法 16
2.3.1 酵素固定化 16
2.3.2 酵素固定化方法 17
2.3.2.1 交聯法 (cross-linking) 17
2.3.2.2 包埋法 (entrapment) 17
2.3.2.3 鍵結法 (binding to a support or carrier) 18
2.3.3 酵素穩定性 19
2.4 研究系統簡介 20
2.4.1 天絲纖維不織布 (Tencel non-woven fabric) 20
2.4.2 環氧氯丙烷 (Epichlorohydrin) 21
2.4.3 海藻糖 (Trehalose) 21
2.4.4 Picrophilus torridus海藻糖合成酶 (Picrophilus torridus trehalose synthase, PTTS) 23
第三章 實驗藥品與儀器 24
3.1 實驗藥品 24
3.2 實驗儀器與設備 26
第四章 實驗方法 27
4.1 酵素製備 27
4.1.1 菌株介紹 27
4.1.2 LB固態洋菜培養基的製備 27
4.1.3 LB液態培養基的製備 27
4.1.4 菌株培養方法 28
4.1.5 菌體回收與粗酵素液製備 28
4.1.6 利用商業化膠體進行酵素純化 29
4.2 天絲纖維不織布親和吸附材之製備 30
4.2.1 基材表面的活化反應 30
4.2.2 基材表面的螯合反應 30
4.2.3 金屬離子的鍵結 31
4.3 最佳純化條件探討 32
4.3.1 海藻糖合成酶的純化 32
4.3.2 基材表面活化反應的最適化 32
4.3.2.1 活化反應溫度最適化 32
4.3.2.2 活化反應氫氧化鈉濃度最適化 33
4.3.2.3 活化反應時間最適化 34
4.3.3 基材表面活化條件對海藻糖合成酶吸附之影響 34
4.3.4 金屬離子對海藻糖合成酶吸附之影響 36
4.3.5 添加鹽類對海藻糖合成酶吸附之影響 37
4.3.6 添加咪唑(imidazole)對海藻糖合成酶吸附之影響 38
4.3.7 四種不同比例基材對海藻糖合成酶吸附之影響 39
4.4 重複批次操作對固定化酵素活性之探討 41
4.4.1 海藻糖合成酶固定化反應 41
4.4.2 固定化海藻糖合成酶重複使用穩定性之探討 41
4.5 分析方法 42
4.5.1 全反射傅立葉轉換紅外線光譜分析 (ATR-FTIR) 42
4.5.2 蛋白質濃度分析 42
4.5.2.1 蛋白質檢量線 42
4.5.2.2 待測樣品濃度分析 43
4.5.3 銅離子濃度分析 43
4.5.3.1 銅離子檢量線 43
4.5.3.2 待測樣品濃度分析 43
4.5.4 SDS-PAGE電泳分析 43
4.5.4.1 製備SDS-PAGE膠體 43
4.5.4.2 樣品製備 44
4.5.4.3 電泳分析 44
4.5.5 醣類分析 45
第五章 實驗結果與討論 47
5.1 全反射傅立葉轉換紅外線光譜 (ATR-FTIR) 47
5.2 表面活化反應的最適化 48
5.2.1 活化反應溫度最適化 48
5.2.2 活化反應氫氧化鈉濃度最適化 49
5.2.3 活化反應時間最適化 50
5.2.4 最適化表面活化反應條件 50
5.3 天絲纖維金屬螯合吸附基材之基本性質測試 51
5.3.1 基材表面活化條件基本性質測試 51
5.3.1.1 銅離子鍵結量的量測 51
5.3.1.2 金屬螯合劑密度的量測 52
5.3.2 不同組成比例基材基本性質測試 53
5.3.2.1 銅離子鍵結量的量測 53
5.3.2.2 金屬螯合劑密度的量測 54
5.4 最佳純化條件探討 55
5.4.1 基材表面活化條件對海藻糖合成酶吸附之影響 55
5.4.2 金屬離子對海藻糖合成酶吸附之影響 56
5.4.3 添加鹽類對海藻糖合成酶吸附之影響 58
5.4.4 添加咪唑(imidazole)對海藻糖合成酶吸附之影響 61
5.4.5 四種不同比例基材對海藻糖合成酶吸附之影響 63
5.5 固定化海藻糖合成酶操作穩定性之探討 66
第六章 結論 67
第七章 參考文獻 68

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