臺灣博碩士論文加值系統

本實驗將Candida rugosa脂解酵素，以共價鍵結合法固定於PAN中空纖維表面。探討固定化脂解酵素對三酸甘油脂的催化反應。
首先，將PAN中空纖維以NaOH、己二胺溶液予以水解，再由戊二醛水溶液條件反應得PAN中空纖維的延伸臂，始可與酵素共價結合，形成固定化酵素。對此，固定化酵素施以溫度、pH、再用性、儲存穩定性加以探討。
實驗結果顯示，將NaOH、己二胺水溶液控制在50℃、1N下及戊二醛溶液為6 %形成的固定化酵素中空纖維模組其最適化溫度、pH值分別為40℃、6.5，較自由酵素30℃、7.0有較佳的適用性。本實驗所得之脂解酵素固定化PAN中空纖維擁有良好的熱穩定性(50℃下反應3hr活性尚有40%)、重複使用性(10次重複使用活性只下降32%)並可儲存7天(4℃)及比未經改質中空纖維有1.6倍的廓清率(pH=7, 37℃,4hr體外模擬操作)， 可用以提供進一步血液淨化之研究。

Surface of polyacrylonitrile (PAN) hollow fibers were hydrolyzed with NaOH(aq) and grafted with hexane diamine. Lipase of Candida rugosa origin was then covalently immobilized with glutaraldehyde onto the resulting hollow fibers. The catalytic activity to triglyceride (TG) of the lipase-immobilized hollow fiber dialyzer was evaluated under various pH, temperature. The reusability and shelf life were also measured. The results show that the optimal condition for the catalytic hydrolysis of TG for the immobilized lipase was at pH6.5 and 40°C, while that for free lipase was at pH7 and 30°C. Furthermore, immobilized lipase can retain 40% of activity at 50°C after 3 hr. After 10 times of reuse, the activity dropped about 32%. The shelf life was about 7 days. In a 4-hr in vitro simulation, the clearance of TG of lipase-immobilized dialyzer was about 1.6 times of that of ordinary dialyzer.

目錄
摘要I
AbstractII
誌謝III
目錄IV
圖表索引VII
第1.章緒論1
1.1.酵素1
1.2.研究背景及目的2
1.2.1.研究背景2
1.2.2.目的3
1.3.內容簡介3
第2.章文獻回顧5
2.1.酵素固定化5
2.1.1.酵素固定化之定義6
2.1.2.固定化方法6
2.2.固定化酵素的特性10
2.3.載體12
2.3.1.載體的種類12
2.3.2.載體的選擇13
2.4.中空纖維狀薄膜13
2.4.1.中空纖維狀薄膜表面修飾14
2.4.2.PAN的改質15
2.5.脂解酵素18
2.6.固定化酵素催化油脂應用19
2.7.血液透析20
2.7.1.透析原理20
2.7.2.透析膜 (40-41)23
2.7.3.透析器(42)26
第3.章實驗方法28
3.1.實驗材料28
3.2.實驗器材30
3.3.實驗步驟32
3.3.1.脂解酵素濃度測定32
3.3.2.三酸甘油脂濃度分析33
3.3.3.脂解酵素活性分析34
3.3.4.PAN中空纖維水解反應35
3.3.5.脂解酵素固定化方法37
3.3.6.最佳酵素固定化條件的探討38
3.3.7.固定化酵素基本性質的探討39
3.3.8.脂解酵素固定化中空纖維反應器的催化反應40
3.3.9.體外模擬實驗41
第4.章結果與討論42
4.1.PAN的水解反應探討42
4.2.水解條件對中空纖維膜的影響42
4.2.1.純水透過率之影響42
4.3.固定化條件之探討44
4.3.1.戊二醛濃度44
4.3.2.酵素濃度的探討46
4.4.固定化酵素活性之探討47
4.4.1.最適酸鹼值的探討47
4.4.2.最適溫度的探討48
4.4.3.固定化酵素熱穩定性的探討49
4.4.4.再用性的探討51
4.4.5.儲存活性52
4.5.固定化之脂解酵素中空纖維反應器對三酸甘油脂的催化反應52
4.5.1.不同溫度下三酸甘油脂的催化反應52
4.5.2.不同pH值下三酸甘油脂的催化反應54
4.6.體外模擬評估56
第5.章結論57
第6.章參考文獻58
圖表索引
Figure Caption
Fig 1-1中空纖維薄膜表面對於酵素之結合吸附 3
Fig 2-1酵素固定化之方法 9
Fig 2-2 PAN薄膜的酵素固定化流程 15
Fig 2-3 PAN水解反應機構 17
Fig 3-1實驗流程圖 31
Fig 3-2中空纖維純水滲透裝置圖 36
Fig 3-3脂解酵素中空纖維反應器之催化反應 40
Fig 3-4體外模擬裝置 41
Fig 4-1不同戊二醛濃度與酵素活性之關係 45
Fig 4-2不同戊二醛濃度與酵素固定率之關係 45
Fig 4-3不同脂解酵素濃度與活性的關係 46
Fig 4-4固定化酵素之最適酸鹼值 48
Fig 4-5固定化酵素的最適溫度 49
Fig 4-6固定化酵素在30℃、40℃、50℃的熱失活 50
Fig 4-7 固定化酵素在50℃時，不同戊二醛濃度的熱失活 50
Fig 4-8固定化酵素在不同戊二醛濃度的再使用性51
Fig 4-9中空纖維固定化酵素之儲存天數活性圖 52
Fig 4-10 戊二醛濃度= 0 % 時，不同溫度下三酸甘油脂的催化反應
53
Fig 4-11 戊二醛濃度=6% 時，不同溫度下三酸甘油脂的催化反應
53
Fig 4-12 戊二醛濃度 = 0 % 時，不同pH值下三酸甘油脂的催化率
55
Fig 4-13 戊二醛濃度 = 6 % 時，不同pH值下三酸甘油脂的催化率
55
Fig 4-14固定化脂解酵素對三酸甘油脂濃度與時間的作用關係 56
Fig 4-15固定化脂解酵素與未改質PAN對三酸甘油脂之廓清率圖 56
Table Caption
Table4-1純水流通量 43

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