臺灣博碩士論文加值系統

本文利用界面現象及界面活性劑去增進脂解酵素在有機溶劑的酯化活性，使其在有機溶劑的環境下進行酯化反應。實驗將採取三種方式進行研究：1.探討短鏈醇類對酵素活性的影響，活化結果以使用濃度在30%處理過後為最好，其進行丁酸乙酯酯化反應時，總活性高出原酵素3.4倍。2.利用界面活化法以兩性界面活性劑去開啟酵素活化部位，探討界面活性劑對酵素活化部位的影響，此法雖會達到活化的目的，但在實驗過程中會造成酵素失活使最終活性無法高於原值。3.以非離子型界面活性劑來進行包覆酵素實驗，並利用三種方式製備Surfactant-Coated-Lipase (SCL)：(1)自然沉降法(2)Water in Oil (W/O)型乳化法(3)Oil in Water (O/W)型乳化法 。觀察其活性及反應情形，發現界面活性劑的親疏水性，對於以何種方式製備SCL有重大的影響，且由實驗數據看來，以脂解酵素(100 mg)和 Span 60 (50 mg)在pH 6的MES緩衝液中，以均質速度7200 rpm製造出的SCL比活性高於原酵素12.1倍，且SCL長時間在有機溶劑中依然保有其活性，同時也有不錯的熱穩定性‧

In the study, we developed a method by utilizing surfactant to improve lipase esterification activity in organic solvent. The experiment was mainly divided into three parts. First, we discussed the effect of short-chain alcohols pretreatment on lipase activity.The best solvent is treatment with 30% 1-proanol and the total activity is 3.4 times higher than that of crude enzyme’s. Second, we also utilize surfactant to open up lipase’s lid which conceals the active site. This method is effective for enhancing lipase activity but cause enzyme inactive during the treatment process. Three methods were used to manufacture surfactant coated lipase (SCL): (1) gravity sedimentation (2) water in oil method (3) oil in water method.
Judging from esterification reaction, the result shows that the HLB of surfactant and the preparation method influence the activity of SCL. Among all SCL preparation methods, lipase(500 mg) and Span60(50 mg) in MES buffer of pH 6.0 gave the highest activity. The activity of SCL is 12.1 times higher than that of crude enzyme’s and exhibits high heat stability and high activity after long-time reaction in organic solvents.

第一章 緒論............................................1
1.1研究動機..........................................1
1.2脂肪分解酵素......................................3
1.3酵素使用在有機溶劑上的類型........................3
1.3.1固定化方法........................................4
1.3.2非固定化方法......................................6
1.4將界面活性劑使用在酵素之方法......................8
1.4.1 IAMI.............................................8
1.4.2自然沉降法........................................10
1.4.3W/O型乳化法製備SCL............................11
1.4.4 O/W型乳化法製備SCL...............................11
1.5界面活性劑種類....................................13
1.6 H.L.B值..........................................16
1.7微胞..............................................18
1.4.5以Span 60(sorbitan monostearate)製造界面活性劑包覆酵素.....................................................12
第二章 研究方法與步驟..................................19
2.1研究方法..........................................19
2.1.1實驗藥品......................................19
2.1.2實驗儀器......................................21
2.2實驗步驟..........................................23
2.2.1短鏈醇類活化步驟..............................23
2.2.2 IAMI步驟.....................................23
2.2.3製備SCL步驟...................................23
2.2.3.1自然沉降..................................23
2.2.3.2 W/O型包覆酵素法..........................26
2.2.3.3 O/W 型包覆酵素法...............................26
2.2.3.4以Span 60(sorbitan monostearate)製造界面活性劑包覆酵素..............................................27
2.3脂解酵素活性測定..................................32
2.3.1生成丁酸乙酯................................32
2.3.2醋酸香葉酯之酯化反應........................32
2.3.3不同醇類對油酸之酯化反應..........................32
2.4SCL之熱穩定性測定...................................33
2.5分析方法..........................................33
2.5.1 HPLC分析方法...............................33
2.5.2 GC分析方法.................................34
2.5.3滴定分析....................................39
2.5.4蛋白質分析..................................41
第三章 結果與討論......................................43
3.1利用短鏈醇類活化酵素..............................43
3.1.1酯化活性探討................................43
3.1.2酵素的蛋白質含量..................................45
3.1.3醇類添加量對酵素活化的影響........................50
3.1.4透析時間對短鏈醇類活化的影響......................50
3.1.5以短鏈醇類活化酵素合成醋酸香葉酯..................51
3.2以IAMI法活化脂解酵素..............................55
3.3界面活性劑包覆酵素（SCL）之結果...................59
3.3.1不同界面活性劑的影響..............................59
3.3.2緩衝液pH值對製備SCL之影響.........................63
3.3.3製備SCL時均質速度之影響...........................63
3.3.4不同製備方式的界面活性劑包覆酵素比較........67
3.3.5不同醇類進行酯化反應..............................69
3.3.6無溶劑反應系統 (Solvent-free system)反應..........69
3.3.7 SEM圖形比較......................................70
3.4以Span 60製造界面活性劑...........................74
3.4.1緩衝液pH對SCL之影響...............................74
3.4.2均質速度對SCL之影響...............................74
3.4.3界面活性劑添加量對SCL之影響.......................75
3.4.4以不同界面活性劑溶劑進行製備SCL...................81
3.4.5 SCL之熱穩定性....................................81
3.4.6改變製程濃度對活性的影響..........................84
3.4.7製備過程中界面活性劑對酵素的影響..................84
第四章 結論............................................87
參考文獻...............................................89
附錄 第六屆生化研討會論文..............................92

(1).Klibanov, A. M. (1988) Chemtech. ,June,345-359
(2).歐靜枝編譯.1997.”乳化溶化技術實務”.復漢。
(3).Goto, M. , Kamiya, N. ,Miyata, M. and Nakashio, F.(1994) Biotechnol. Prog. 10,263-268.
(4).Tian, D. , Blacher, S. and Jerome. R. (1999) Polymer. 40,951-957.
(5).Okahata, Y. and Ijiro, K. (1988) J. Chem. Soc. Chem. Commun,
1392-1395
(6).CarmenBano, M. ,Braco, L. and Abad, C. (1991)Biochemistry,30,886-
894.
(7).Kamiya, N. ,Goto, M. and Nakashio, F. (1995) Biotechnol. Prog. 11,270-275.
(8).Kamiya, N. , Kasagi, H. ,Inoue, M. , Kusunoki, k. and Goto, M. (1999) Biotechnology and Bioengineering.65,227-231.
(9).Okazaki, S. , Kamiya, N. and Goto, M. (1997)Biotechnol. Prog. 13,551-556.
(10).Goto, M. , Chang, H. L. and Huang, S. Y. (1998) Enzyme and Microbial Technology. 22,552-557.
(11).Goto, M. , Kamiya, N. ,Miyata, M. and Nakashio,F. (1994)
Biotechnol. Prog. 10,263-268.
(12).González-Navarro, H. and Braco, L. (1997) J. of Molecular Catalysis B.3,111-119.
(13).Goto, M. , Kameyama, H. ,Goto, M. ,Miyata, M. and Nakashio, F. (1993) Journal of Chemical Engineering of Japan.26,109-111.
(14).Chou, S. T. and Chiang, B. H., 1998. J. Food Sci. 63(3): 399.
(15).蘇大榮.1999.”不同組成之熱敏感乳液粒子於酵素固定上之研究”。碩士論文，長庚大學。
(16).Okazaki, S. ,Kamiya, N. , Abe,K. and Goto, M. (1997)Biotechnology
and Bioengineering.55,454-460.
(17).張曉莉.1996.”酵素在有機溶劑中受界面活性劑保護下之逆微胞萃取操作策略及催化反應的增進”。博士論文，台灣大學。
(18).Tian, D. , Blacher, S. and Jerome. R. (1999)Polymer.40,951-957.
(19).Klibanov, A. M. (1988)chemtech. June. 345-359
(20).Luisi, P. L. (1985)Angew. Chem.Int. Ed. Engl. ,24,439-443
(21).Fukunaga, K. , Maruoka, N. , Sugimura, Y. , Nakao,K. and Shimizu,T. (1998)Biotechnology Letters. 20,1161-1165.
(22).Chou, S. T. and Chiang, B. H., 1998. J. Food Sci. 63(3): 399.
(23).Chiang, B. H. , Su, C. K. , Tsai, G. J. and Tsao.,G.T.,1993.J.Food Sci58(2):303.
(24).Mingarro, I. , Abad ,C. and Braco, L. (1994) Biochemistry, 33,4625-4660.
(25).Karra, M. ,Pulvin, S. ,Touraud, D. and Thomas, D. (1996) Biotechnology letters. 9,1083-1088.
(26).Mingarro, I. , Abad, C. and Braco, L. (1995) Proc. Natl .Acad. Sci. USA92,3308-3312.
(27).Mingarro, I. , González, N. H. and Braco, L. (1996)Biochemistry.35
9935-9944.
(28).王鳳英編譯.1993.”界面活性劑的原理與應用”高立。
(29).吳碩成.1995.”混合界面活性劑微胞之形成及其在苯乙烯乳化聚合反應中之角色”。碩士論文，台灣大學。