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研究生:蔡明儒
研究生(外文):Ming-Ju Tsai
論文名稱:利用脂肪酵素進行甘油三丁酸酯之水解反應
論文名稱(外文):Hydrolysis of Tributyrin using Lipase
指導教授:吳和生吳和生引用關係
指導教授(外文):Ho-Shing Wu
學位類別:碩士
校院名稱:元智大學
系所名稱:化學工程學系
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:2003
畢業學年度:91
語文別:中文
論文頁數:76
中文關鍵詞:脂肪酵素水解反應固定化
外文關鍵詞:LipaseHydrolysisImmobilization
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脂肪酶(Lipase,EC 3.1.1.3)主要是水解三甘油脂成甘油及脂肪酸。廣泛地應用於脂肪、油品、食品及清潔劑工業。大多數的脂肪酶為適中溫性的酵素,無法水解在常溫下為固體的受質及對化學變性劑較無抗性,因此在工業應用上有所限制。但因其催化作用通常伴隨著高特定選擇和高鏡像選擇性,所以在生物技術上反而擁有巨大發展潛力。因此本實驗利用磁石攪拌器乳化油-水反應液以研究從 Pseudomonas fluorescenes CCRC-17015所的抽取的粗製脂肪酶,在批式反應器內催化甘油三丁酸酯的水解反應速率,並探討基質濃度、溫度、pH值、轉速等因素對水解速率之影響。而為了增加酵素的實用性,本實驗以多孔隙性的甲基丙烯醯胺共聚物為載體,此載體經由乙二胺改質、戊二醛活化後,做為酵素固定化的材質,並利用共價結合的方法,將鍵酵素結以形成固定化酵素。對此,固定化酵素以溫度、pH、轉速等不同的操作條件加以探討,並且計算出Km、Vmax。實驗結果顯示,脂肪酶固定化後擁有良好的熱穩定性與耐鹼性。
The primary purposes of lipase is use to hydrolyze triacylglyceol becomes the glycerol and the fatty acid. This enzyme has been widely used in fat, Food ingredients and Detergents industry. Most of lipases are mesophilic enzyme, which can’t hydrolyze substrate that exists in solid form at room temperature and are unstable when chemical solvents are present. This has severely restricted the application of lipase in industries. But in this catalysis reaction, it came with substrate specificity and enantioselectivity. Therefore, the the lipase is one of the most potential in biotechnological. The purpose of this study is research the hydrolysis rate of tributyrin in batch reactor use crude lipase from Pseudomonas fluorescenes CCRC-17015 and using magnetic stir reactor to emulsify oil-water phase. In order to increase the usability of enzyme, the study use lipase immobilization onto porous polymethyacrylamide by covalent fixation. The carrier modified with ethylenediamine and activated by glutaraldehyde. The primary purposes of this thesis are to study the effect of hydrolysis rate at different substrate concentrations, temperature, pH and stirring speed. Final, to determine the operating conditions of free enzyme and immobilized enzyme then we can get kinetic parameters Km and Vmax values. The experimental results showed lipase after immobilization own good thermal stability and greater stability at higher pH value.
中文摘要 I
ABSTRACT II
誌謝 III
目錄 IV
Captions of Figures VIII
Captions of Tables XI
符號說明 XII
第一章 緒論 1
1-1前言 1
1-2酵素的應用與發展 2
1-2-1生物觸媒─酵素(酶) 2
1-2-2影響酵素催化反應速率的因素 3
1-2-2.1 溫度之影響 3
1-2-2.2 pH值之影響 3
1-2-3 酵素的應用發展 3
1-3 脂肪酶的發展和應用 4
1-3-1 脂肪酶的簡介 4
1-3-2 脂肪酶在食品工業上的應用 7
1-3-3 脂肪酶在化學合成工業上的應用 7
1-3-4 脂肪酶在清潔劑工業上的應用 9
1-3-5 脂肪酶在油脂工業上的應用 9
1-3-6 脂肪酶在廢水處理上的應用 9
1-4 脂肪酶的結構特性與活化現象 10
1-5 固定化酵素 12
1-5-1 固定化的發展與應用 12
1-5-2 固定化酵素的特性 13
1-5-3 固定化的方法 14
1-5-4 載體的分類與材質的選擇 16
1-5-5 高分子載體的製備 17
1-6 文獻回顧 18
1-7 脂肪酶的模式與理論 20
1-8 研究動機與目的 22
第二章 實驗部份 23
2-1實驗儀器 23
2-2操作設備 23
2-3實驗藥品與材料 24
2-4實驗步驟 25
2-4-1 菌體的培養與酵素的取得 25
2-4-1.1 菌種來源 25
2-4-1.2 培養基組成 25
2-4-1.3 菌種培養與保存方法 26
2-4-1.4粗抽酵素液的取得 26
2-4-2 定量方法 26
2-4-2.1 菌體生長曲線 26
2-4-2.2 菌液中細胞乾重測定 26
2-4-2.3 酵素含量測定 27
2-4-2.4 反應物之分析 27
2-4-3 酵素水解實驗 27
2-4-3.1 反應條件之最適化 28
2-4-3.1(a) 不同pH值 28
2-4-3.1(b) 不同溫度 28
2-4-3.1(c) 不同轉速 28
2-4-3.2 不同基質濃度對酵素活性之影響 28
2-4-4 低基質濃度下酵素活性的最適化 29
2-4-4.1 不同溫度下酵素活性之關係 29
2-4-4.2 不同pH值下酵素活性之關係 29
2-4-4.3 Lipase經不同pH值處理後酵素活性之比較 29
2-4-5 界面活性劑對酵素活性之影響 29
2-4-6 懸浮細胞之水解實驗 30
2-4-6.1 不同溫度下水解實驗 30
2-4-6.2 不同pH值水解實驗 30
2-5 固定化酵素的製備 30
2-5-1 固定化載體(PMAA)的製備 30
2-5-2 載體之改質 31
2-5-3 載體之活化 31
2-5-4 酵素固定化 33
2-5-5 計算固定化效率 34
2-6 固定化酵素基本性質探討 34
2-6-1 溫度對固定化酵素之影響 34
2-6-2 轉速對固定化酵素之影響 34
2-6-3 pH值對固定化酵素之影響 35
2-6-4 動力學常數之測定 35
2-7 本研究之反應器系統介紹 35
第三章 目前的結果與討論 36
3-1 Pseudomonas fluorescenes的生長曲線 36
3-2 測試酵素活性之反應條件實驗 38
3-2.1 改變反應條件之pH值 38
3-2.2 改變反應條件之溫度 39
3-2.3 改變反應條件之轉速 41
3-3 溫度對酵素活性之影響 43
3-4 不同基質濃度下對反應產率之影響 44
3-5 低基質濃度下酵素活性的最適化實驗 48
3-5.1 反應溫度的影響 48
3-5.2 pH值的影響 50
3-5.3 酵素的保存 52
3-6 界面活性劑對酵素活性之影響 54
3-7 懸浮細胞之脂解實驗 55
3-7-1 溫度對懸浮細胞之影響 55
3-7-2 pH值對懸浮細胞之影響 56
3-8 自製載體之酵素固定量測試 58
3-9 固定化酵素基本性質探討 59
3-9-1 溫度對固定化酵素之影響 59
3-9-2 轉速對固定化酵素之影響 60
3-9-3 pH對固定化酵素之影響 62
3-9-4 自由酵素和固定化酵素之動力學常數測定 63
第四章 結論與未來展望 67
4-1 結論 67
4-1 未來展望 68
第五章 參考文獻 69
附錄A、酵素活性定義 74
附錄B、磷酸鹽緩衝液的配製 75
附錄C 76
Captions of Figures
Figure 1.1、The catalytic action of lipases 5
Figure 1.2、The structure of the lipase from Pseudomonas aeruginosa in a model built using
the X-ray 11
Figure 1.3、Interfacial activation of lipase 10
Figure 1.4、Model for description of interfacial kinetics with a water-soluble lipase enzyme
acting on insoluble substrate…………………………………………………..19
Figure 1.5、Two phase system model of interfacial kinetics for lipase. 21
Figure 2.1、Synthesis reaction of support. 32
Figure 2.2、Diagram showing modification, activation and immobilization of support 33
Figure 2.3、Schematic diagram of batch reactor 35
Figure 3.1、The growth curves of Pseudomonas fluorescenes 37
Figure 3.2(a)、Conversion of tributyrin vs. time curves at different pH for lipase. 38
Figure 3.2(b)、Specific activity of lipase at different pH. 38
Figure 3.3(a)、Conversion of tributyrin vs. time curves at different temperature for lipase. 40
Figure 3.3(b)、Specific activity of lipase at different reaction temperature 40
Figure 3.4(a)、Conversion of tributyrin vs. time curves at different stirring speed for lipase. 41
Figure 3.4(b)、Specific activity of lipase at different stirring speed. 42
Figure 3.5(a)、Activation energy(Ea) of lipase. (20~30℃) 44
Figure 3.5(b)、The death activation energy(Ed) of lipase. (30~40℃) 44
Figure 3.6、Conversion of tributyrin vs. time curves at different concentrations of substrate for lipase. 45
Figure 3.7、pH vs. time curves at 1.0 g/cm3(3.308 M) concentrations of substrate. 46
Figure 3.8、Specific activity of lipase at different concentrations of substrate. 48
Figure 3.9(a)、Conversion of tributyrin vs. time curves at different temperature for lipase. 49
Figure 3.9(b)、Specific activity of lipase at different reaction temperature. 49
Figure 3.10(a)、Conversion of tributyrin vs. time curves at different pH for lipase. 50
Figure 3.10(b)、Specific activity of lipase at different pH. 51
Figure 3.11、After still 24 hr later, Conversion of tributyrin vs. time curves at different pH for lipase keep in 4℃ 52
Figure 3.12、After still 48 hr later, Conversion of tributyrin vs. time curves at different pH for lipase keep in 4℃ 53
Figure 3.13、Specific activity of lipase at different pH and lipase keep in 4℃ 53
Figure 3.14、Conversion of tributyrin vs. time curves for lipase with added different surfactant 54
Figure 3.15(a)、Conversion of tributyrin vs. time curves at different temperature for suspension cells. 55
Figure 3.15(b)、Specific activity of suspension cells(Pseudomonas fluorescenes) at different temperature 56
Figure 3.16(a)、Conversion of tributyrin vs. time curves at different pH for suspension cells
57
Figure 3.16(b)、Specific activity of suspension cells(Pseudomonas fluorescenes) at different pH 57
Figure 3.17(a)、Conversion of tributyrin vs. time curves at different temperature for immobilized lipase 59
Figure 3.17(b)、Specific activity of immobilized lipase at different reaction temperature 60
Figure 3.18(a)、Conversion of tributyrin vs. time curves at different stirring speed for immobilized lipase 61
Figure 3.18(b)、Specific activity of immobilized lipase at different stirring speed 61
Figure 3.19(a)、Conversion of tributyrin vs. time curves at different pH for immobilized lipase 62
Figure 3.19(b)、Specific activity of immobilized lipase at different pH 63
Figure 3.20(a)、Conversion of tributyrin vs. time curves at different concentrations of substrate for immobilized lipase 64
Figure 3.20(b)、Specific activity of immobilized lipase at different concentrations of substrate 64
Figure 3.20(c)、Lineweaver-Burk plot for immobilized enzyme 65
Figure 3.20(d)、Specific activity of free lipase at different concentrations of substrate 65
Figure 3.20(e)、Lineweaver-Burk plot for free enzyme 66
FigureC.1、Optical density vs. dry cell weight Figure with Pseudomonas fluorescenes 76
FigureC.2、Standard Curve of Net Absorbance vs. protein sample concentration(using BSA as the standard protein) 76
Captions of Tables
Tables 1.1、Examples of commercially available microbial lipase ...……………………6
Tables 1.2、Specificity of lipase…………………………………… ………………8
Tables 1.3、Compare the advantage and fault with different immobilization 14
Tables 3.1、Specific activity of lipase at different concentrations of substrate 47
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