臺灣博碩士論文加值系統

由於脂肪酶是一種在油水界面進行催化的酵素，微環境的疏水性可能影響其催化能力。為了了解這個影響性，本研究利用一次合成法製備出一系列的巨孔有機二氧化矽，包括帶有乙基、辛基、十二烷基、苯基及甲基丙烯醯基等官能基，探討其性質，並用於吸附脂肪酶。由水的接觸角分析顯示，這些有機二氧化矽的疏水程度依序為十二烷基衍生二氧化矽＞辛基衍生二氧化矽＞苯基衍生二氧化矽＞乙基衍生二氧化矽＞甲基丙烯醯氧丙基衍生二氧化矽。由Pseudomonas cepacia脂肪酶在這些巨孔有機二氧化矽擔體上的固定量顯示酵素以在乙基衍生二氧化矽上的吸附量最多，在辛基衍生二氧化矽的吸附量最少。固定化脂肪酶的催化能力評估則是藉由橄欖油的水解反應以及(R,S)-1-苯乙醇與醋酸乙烯酯的鏡像選擇性轉酯化反應進行。酵素催化橄欖油水解反應與轉酯化反應的比活性以固定在十二烷基衍生二氧化矽的脂肪酶最高，但固定在純二氧化矽擔體的脂肪酶在轉酯化反應中表現出最佳的鏡像選擇性，顯示Pseudomonas cepacia脂肪酶在高疏水性環境中的反應活性較高，但在低疏水性環境中的鏡像選擇性較佳。固定在苯基衍生二氧化矽的脂肪酶有較佳的操作穩定性，在連續6次，每次反應3小時後，還保有96%的初始反應活性。

Because lipase catalyses reaction at water-oil interface, the hydrophobicity of the microenvironment surrounding enzyme may affect the catalytic capability of lipase. To investigate the influence of microenvironment, a series of macroporous organosilicas containing various organic groups, including ethyl, octyl, dodecyl, phenyl, and methacryloyloxy groups, were prepared, characterized, and applied to the immobilization of Pseudomonas cepacia lipase. Based on the contact angle data of water on these materials, the hydrophobicity followed the order of dodecyl-silica > octyl-silica > phenyl-silica > ethyl-silica > methacryloyloxy-silica. The loading of Pseudomonas cepacia lipase on these materials indicated that ethyl-silica had the highest capacity and octyl-silica had the lowest capacity. The catalytic ability of immobilized lipase was evaluated by the hydrolysis of olive oil and transesterification between (R,S)-1-phenylethanol and vinyl acetate. Lipase immobilized on dodecyl-silica showed highest activity in hydrolysis and transesterification. However, the lipase immobilized on pure silica showed a better enantioselectivity in transesterification. This result revealed that Pseudomonas cepacia lipase had a higher reaction activity in a more hydrophobic environment while it had a better enantioselectivity in a less hydrophobic environment. The lipase immobilized on phenyl-silica exhibited a better stability, as it retained 96% of its initial activity after 6 repeats of a three-hour reaction.

中文摘要 I
英文摘要 II
誌謝 III
目錄 IV
表目錄 VIII
圖目錄 IX
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 酵素 3
2.1.1 脂肪酶 5
2.1.1.1 Pseudomonas sp.脂肪酶 9
2.1.1.1.1 Pesudomonas cepacia脂肪酶 9
2.1.2 酵素於有機溶劑中之反應 10
2.2 固定化酵素技術 13
2.2.1 固定化方法 14
2.2.2 影響固定化酵素性能的因素 20
2.3 多孔性固定化擔體 22
2.3.1 巨孔材料 24
2.3.2 三維整齊排列巨孔材料合成 25
2.3.2.1 膠體晶體模板法 26
2.3.2.1.1 巨孔無機氧化物的製備 27
2.3.2.2 生物模板法 32
2.3.2.3 其他模板法 33
2.4 有機-無機巨孔材料 33
2.4.1 表面接枝改質法 33
2.4.2 一次合成法 34
2.5 聚苯乙烯模板合成技術 36
2.5.1 乳化聚合反與無乳化劑乳化聚合反應 38
2.5.2 無乳化劑乳化聚合反應機構 39
2.5.3 乳化聚合法與無乳化劑乳化聚合法之比較 41
2.6 鏡像異構物 42
2.6.1 鏡像異構物的光學分割 44
2.6.2 固定化酵素催化1-苯乙醇的轉酯化反應 46
第三章 實驗方法 50
3.1 實驗藥品 50
3.2 實驗設備 52
3.3 實驗步驟 54
3.3.1 固定化擔體的製備 54
3.3.1.1 無機二氧化矽擔體的製備 54
3.3.1.2 有機二氧化矽擔體的製備 58
3.3.1.3 無機與有機二氧化矽薄膜製備 59
3.3.2 固定化脂肪酶的製備 59
3.3.2.1 以吸附法固定脂肪酶 59
3.3.3 利用固定化脂肪酶催化醋酸苯乙酯的合成 60
3.3.3.1 單一光學活性醋酸苯乙酯的製備 60
3.4 分析方法 61
3.4.1 聚苯乙烯分析 61
3.4.1.1 聚苯乙烯粒徑分析 61
3.4.1.2 聚苯乙烯熱重分析 61
3.4.2 巨孔有機二氧化矽擔體表面分析 62
3.4.2.1 氮氣吸/脫附法 62
3.4.2.2 場發射掃瞄式電子顯微鏡觀察 62
3.4.2.3 傅利葉紅外線光譜分析 63
3.4.2.4 有機二氧化矽與水的接觸角分析 63
3.4.3 蛋白質定量分析 65
3.4.4 固定化酵素水解活性檢測 65
3.4.5 1-苯乙醇與醋酸-1-苯乙酯鏡像異構物的分析 66
第四章 結果與討論 70
4.1 固定化擔體的合成 70
4.1.1 聚苯乙烯模板合成 70
4.1.2 二氧化矽擔體合成 76
4.1.3 有機二氧化矽擔體之疏水特性分析 91
4.2 固定化脂肪酶 93
4.2.1 脂肪酶在多孔二氧化矽及有機二氧化矽的吸附 93
4.2.2 不同擔體對固定化脂肪酶水解活性之影響 95
4.3 單一光學活性合成 96
4.3.1 1-苯乙醇與乙酸乙烯酯的不對稱轉酯化反應 96
4.3.2 有機溶劑對單一光學活性合成探討 99
4.3.3 固定化酵素穩定性探討 104
4.3.4 固定化酵素熱穩定性探討 106
第五章 結論 108
第六章 未來展望 109
參考文獻 110
附錄 118
A. 動態光散射粒徑分析 118
B. 氣體等溫吸脫附曲線 120
作者簡介 126

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