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研究生:楊銘鎌
研究生(外文):Ming-lian Yang
論文名稱:固定脂解酵素於幾丁聚醣之研究
論文名稱(外文):Studies of the immobilized lipase on chitosan Beads
指導教授:劉富雄劉富雄引用關係
指導教授(外文):Fu-Shung Liu
學位類別:碩士
校院名稱:國立雲林科技大學
系所名稱:工業化學與災害防治研究所
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:2003
畢業學年度:91
語文別:中文
論文頁數:66
中文關鍵詞:脂解酵素幾丁聚醣固定化酵素兩相水解反應
外文關鍵詞:lipasechitosanimmobilized enzymetwo-phase reaction
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脂質代謝是生物化學上佔有相當重要的環節,生物體以三酸甘油酯(triglyce- ride)的形式在體內儲存能量,為了代謝、轉換這些酯類,便產生酯質水解酵素(esterase)與脂肪分解酵素(lipase)。兩種酵素均能夠分解酯質,但是僅脂解酵素能夠催化非水溶性酯質的分解。由於脂解酵素具有酯類水解、酯類合成和轉酯化反應多項反應的能力,加上可與多種基質作用,在各項工業佔有極為重要的地位。

本實驗主要是研究固定Lipase珠狀去乙醯度80%的幾丁聚醣上時所需最適化條件和探討擔體對酵素影響。第一部分擔體最適化以3﹪幾丁聚醣和PEG2000 2﹪的條件為最佳。第二部分固定酵素環境最適化,當戊二醛濃度為0.1%、活化時間240min、固定時酵素溶液含量0.096%、且環境酸鹼值在pH6可得到每克幾丁聚醣酵素固定量達2.25mg、活性為99.42 U。第三部分為游離與固定化酵素水解橄欖油反應環境最適化部份,游離酵素在pH值7.0~8.0及溫度為30~40℃為最佳,固定化酵素最適化pH值跟溫度位移到8.0~9.0及50~60℃,固定化酵素在重複使用10次後,酵素的活性可保有原來的68﹪。最後部分探討動力學探討,實驗求得游離酵素Km及Vm的值分別為0.58 M和833.37 μmol/hr,脂解酵素固定在幾丁聚醣珠粒上的Km及Vm值分別為3.74 M和462.38 μmol/hr,在熱穩定性上固定酵素後比游離酵素的耐熱性佳,可能是因為固定化酵素的熱阻力比游離的大,失活的速率比游離酵素慢,而固定化酵素的失活常數kd大於游離酵素4倍以上,固定酵素較能在高溫下生產。
Lipid metabolism is an important part in biochemistry. Energy is stored as triglyceride in living cells that is to metabolize and to change ester. When needed, organisms produce esterase and lipase enzymes to hydrolyze these esters. It is interesting to know that only lipase is capable of decomposing water-insoluble ester. Lipase can also perform many other functions such as esterification and transterification. Since it can catalyze much different type of substrates, it plays an important role in many industries.

This research deals with immobilization of lipase with chitosan having 80﹪ deacetylation, and to understand optimal immobilization condition and effect of immobilization on enzyme activity. Firstly, the optimal support composition is: 3% chitosan and 2% of PEG 2000. Secondly, the optimal immobilization condition is: glutaraldehyde concentration 0.1%; the enzyme coupling time: 240 min; 0.096% enzyme (weight percentage). The enzyme activity of 99.42U was obtained at pH 6. Thirdly, hydrolysis of oil was studied with the free enzyme and the immobilized enzyme reactions using olive oil as substrate. The free enzyme has an optimal pH range of 7.0 ~ 8.0 and an optimal temperature range of 30 ~ 40℃. It shifted to an optimal pH rage of 8.0 ~ 9.0 and an optimal temperature range of 50 ~ 60℃ when the immobilized enzyme was used. With 10 repeated experiments, the enzyme activity still shows 68% of activity. The last, free enzyme and immobilization enzyme kinetics was discassed. Km and Vmax were calculated and founded to be 0.58M and 833.37 μmol/hr for free enzyme , and 3.74M and 462.38μmol/hr for immobilized enzyme, respectively. The thermal stability was increased after immobilization, because immobilized enzyme has higher thermal resistance. The immobilized enzyme has 4 times higher decay kinetics constant kd than free enzyme.
目 錄

中文摘要 ------------------------------------------------------- i
英文摘要 ------------------------------------------------------ ii
誌謝 ----------------------------------------------------- iii
目錄 ------------------------------------------------------ iv
圖表目錄 ------------------------------------------------------- v
第一章 緒 論 ------------------------------------------------------- 1
1. 1 幾丁質和幾丁聚醣-------------------------------------- 1
1. 1. 1 幾丁質之製備----------------------------------------- 1
1. 1. 2 幾丁聚醣之製備--------------------------------------- 2
1. 2 酵素-------------------------------------------------- 4
1. 2. 1 酵素的分類------------------------------------------- 4
1. 2. 2 酵素的簡介------------------------------------------- 5
1. 2. 3 酵素的應用------------------------------------------- 6
1. 2. 4 兩相催化之反應型態----------------------------------- 9
1. 3 脂解酵素---------------------------------------------- 10
1. 3. 1 脂解酵素的催化水解之反應機構-------------------------- 11
1. 3. 2 脂解酵素的特異性-------------------------------------- 14
1. 3. 3 脂解酵素之反應形式------------------------------------ 14
1. 4 酵素固定化-------------------------------------------- 15
1. 4. 1 固定化技術------------------------------------------- 17
1. 4. 2 固定化酵素的優缺點----------------------------------- 19
1. 5 橄攬油之水解------------------------------------------ 19
1. 6 文獻回顧---------------------------------------------- 20
1. 6. 1 脂解酵素--------------------------------------------- 20
1. 6. 2 操作脂肪酵素的穩定性和特異性------------------------- 21
第二章 實驗材料、設備與方法----------------------------------- 24
2. 1 藥品與材料-------------------------------------------- 24
2. 1. 1 藥品------------------------------------------------- 24
2. 1. 2 器材及設備------------------------------------------- 24
2. 2 實驗步驟簡圖------------------------------------------ 26
2. 3 連續式反應器裝置圖------------------------------------ 27
2. 4 幾丁聚醣珠粒製作-------------------------------------- 28
2. 5 擔體活化---------------------------------------------- 28
2. 6 酵素固定化-------------------------------------------- 28
2. 7 蛋白質檢測方法---------------------------------------- 28
2. 7. 1 藥品準備--------------------------------------------- 28
2. 7. 2 檢測方法--------------------------------------------- 29
2. 7. 3 檢量線製作------------------------------------------- 29
2. 8 酵速素活性分析---------------------------------------- 30
2. 9 實驗步驟---------------------------------------------- 30
2. 9. 1 擔體部分--------------------------------------------- 30
2. 9. 2 固定酵素部分----------------------------------------- 30
2. 9. 3 游離與固定化酵素部分--------------------------------- 31
2. 9. 3 酵素動力學部分--------------------------------------- 32
第三章 實驗結果及討論----------------------------------------- 33
3. 1 固定化擔體之製備-------------------------------------- 33
3. 2 擔體最適化-------------------------------------------- 39
3. 2. 1 以不同幾丁聚醣濃度製作擔體珠粒對酵素的影響----------- 39
3. 2. 2 以不同PEG的聚合度製作擔體珠粒對酵素的影響------------ 39
3. 3 固定酵素最適化---------------------------------------- 44
3. 3. 1 以不同戊二醛濃度活化幾丁聚醣珠粒對酵素的影響--------- 44
3. 3. 2 以不同活化時間對酵素的影響---------------------------- 44 3. 3. 3 在固定時以不同酵素濃度對酵素的影響------------------- 44
3. 3. 4 在固定時以不同反應環境pH值對酵素的影響--------------- 45
3. 4 游離與固定酵素的反應環靜最適化------------------------ 53
3. 4. 1 最適反應pH值----------------------------------------- 53
3. 4. 2 最適反應溫度----------------------------------------- 53
3. 4. 3 酵素反應時間----------------------------------------- 53
3. 4. 4 批式反應酵素的重覆使用性----------------------------- 53
3. 5 酵素動力學-------------------------------------------- 59
3. 5. 1 Michaelis-Menten constants的測定--------------------- 59
3. 5. 2 熱穩定性--------------------------------------------- 59
第四章 結論--------------------------------------------------- 63
第五章 參考文獻----------------------------------------------- 64

圖 表 目 錄

圖目錄

圖1 幾丁質、幾丁聚醣及纖維素之結構--------------------------------- 3
圖2 澱粉加工之酵素應用--------------------------------------------- 7
圖3 P. aeruglumae lipase的活性中心結構----------------------------- 12
圖4 脂解酵素水解油脂之反應機構------------------------------------- 13
圖5 吸附法固定化酵素----------------------------------------------- 16
圖6 酵素包埋法----------------------------------------------------- 16
圖7 酵素微包法----------------------------------------------------- 16
圖8 磷脂粒酵素固定法----------------------------------------------- 17
圖9 血液透析纖維包埋法固定化酵素----------------------------------- 17
圖10 中空纖維包埋法固定化酵素--------------------------------------- 18
圖11 實驗步驟簡圖--------------------------------------------------- 26
圖12 連續式反應器裝置圖--------------------------------------------- 27
圖13 蛋白質標準曲線------------------------------------------------- 29
圖14 幾丁聚醣珠粒內部放大1500倍的SEM圖------------------------------ 34
圖15 幾丁聚醣珠粒內部放大2500倍的SEM圖------------------------------ 34
圖16 以PEG400製成幾丁聚醣珠粒表面放大1500倍的SEM圖------------------ 35
圖17 以PEG400製成幾丁聚醣珠粒表面放大2500倍的SEM圖------------------ 35
圖18 以PEG4000製成幾丁聚醣珠粒表面放大1500倍的SEM圖----------------- 36
圖19 以PEG4000製成幾丁聚醣珠粒表面放大2500倍的SEM圖----------------- 36
圖20 以2.5%幾丁聚醣珠粒表面放大1500倍的SEM圖------------------------ 37
圖21 以2.5%幾丁聚醣珠粒表面放大2500倍的SEM圖------------------------ 37
圖22 以4%幾丁聚醣珠粒表面放大1500倍的SEM圖-------------------------- 38
圖23 以4%幾丁聚醣珠粒表面放大2500倍的SEM圖-------------------------- 38
圖24 不同幾丁聚醣含量的珠粒對酵素固定量之影響----------------------- 40
圖25 不同幾丁聚醣含量珠粒對負載活性之影響--------------------------- 41
圖26 以不同PEG聚合度製作幾丁聚醣珠粒對酵素固定量之影響-------------- 42
圖27 以不同PEG聚合度製作幾丁聚醣珠粒對負載活性之影響---------------- 43
圖28 以不同戊二醛濃度活化幾丁聚醣珠粒對酵素固定量的影響------------- 46
圖29 以不同戊二醛濃度活化幾丁聚醣珠粒對酵素活性的影響--------------- 47
圖30 不同活化時間的珠粒對酵素固定量之影響--------------------------- 48
圖31 不同活化時間的珠粒對酵素活性之影響----------------------------- 49
圖32 在固定化時不同酵素濃度對固定量之影響--------------------------- 50
圖33 在固定化時不同酵素濃度對酵素活性之影響------------------------- 51
圖34 在固定化時不同反應環境pH值對酵素固定量之影響------------------- 52
圖35 在固定化時不同反應環境pH值對酵素活性的影響--------------------- 53
圖36 pH值對游離酵素及固定酵素之影響--------------------------------- 55
圖37 反應溫度對游離酵素及固定酵素之影響----------------------------- 56
圖38 反應時間對游離酵素及固定酵素之影響----------------------------- 57
圖39 重複次數對固定化酵素之影響------------------------------------- 58
圖40 游離酵素的Lineweaver-Burk Plot -------------------------------- 60
圖41 固定化酵素的Lineweaver-Burk Plot ------------------------------ 61
圖42 游離酵素及固定化酵素在60℃時的熱穩定性------------------------- 62

表目錄

表1 皮革製造程序酵素之應用----------------------------------------- 8
表2 常見的脂解酵素------------------------------------------------- 10
表3 細菌中脂解酵素在生物技術上應用--------------------------------- 11
表4 游離與固定酵素M-M模式動力學參數-------------------------------- 59
表5 熱失活速率常數------------------------------------------------- 59
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