在本研究中，使用電紡織法製備聚丙烯腈奈米纖維膜，
在最佳電紡操作參數下，成功地製備出直徑約在150到
300 nm的均勻奈米纖維。所製備出的奈米纖維膜經過
amidination reaction活化聚丙烯腈上的CN官能基，
與脂肪分解酶上的NH2官能基形成共價鍵結，而將酵素
固定於奈米纖維膜上。從SEM圖中可發現，所使用的活
化步驟與固定化程序對於纖維的構形並沒有造成太大
的改變。在酵素的固定化方面，固定化之後比活性約在
29.9（U/mg-protein）左右，而其蛋白質固定量為21.2
（mg/g-material），與其它材料相比之下，更突顯了
奈米載體高比表面積之特異性。在固定化酵素儲存20天
後，其水解活性並沒有任何損失，且經過10次之批次反
應後其活性仍維持在70%左右，其儲存安定性與操作安
定性有不錯的表現。在動力學參數部份，本研究之Km值
為0.55 mM，自由酵素為0.46 mM，經固定化後Km並沒有
太大的下降，證明酵素的構形經固定化後沒有太大的改
變。在三酸甘油酯之催化水解方面，本研究所製備而成
之固定化酵素水解速率相當快速，10分鐘便達到72%之轉
化率，與其它研究相比，由於水解速率較快，充份展現
奈米載體之優異性，更能降低工業上之成本。以大豆油
催化水解批次反應20次後，其轉化率仍能維持在第一次
反應的65 %，操作安定性較高。利用酵素製程生產脂肪
酸之高成本問題，也因為其水解速率較快、高儲存安定
性以及高操作安定性等特性而獲得改善。

Electrospinning, a novel process to make porous nanofiber,
was investigated to manufacture nanofibrous membrane as
a carrier for Candida rugosa lipase immobilization to
hydrolyze soybean oil. In this study, polyacrylonitrile
nanofiber with the mean diameter of 200 nm was used to
immobilize Candida rugosa lipase by covalent binding.
The immobilization efficiency using nanofiber was good.
The protein loading of immobilized enzyme on nanofiber
was about 60%, and the specific activity of immobilized
lipase was 81.3% of the free lipase. In comparison of
the kinetic parameter Km of the immobilized lipase with
that of the free lipase, the values of Km are similar.
It implies that the diffusion resistance of immobilized
enzyme is almost the same as that of free enzyme.
To compare with other types of immobilized lipase on
nanofibrous membrane, the Vmax index of our system was
higher than that of others. The effects of temperature,
pH and oil to water ratio on the conversion of oil hydrolysis
were also illustrated and the optimal operation condition
was determined. Under the optimal condition, temperature
at 30℃, pH at 7,and oil to water ratio of 2:1 for soybean
oil hydrolysis, a conversion of 72% was obtained in 10 minutes.

Bradford, M. M. A. “A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding,” Analytical Biochemistry, 72, 248-54 (1976).
Brockman, H. L.; Momsen, W. E.; Tsujita, T. “Lipid-lipid complexes: Properties and effects on lipase binding to surfaces,” Journal of American Oil Chemists’ Society, 65, 891-896 (1988).
Brzozowski, A. M.; Derewenda, U.; Dererwenda, Z. S.; Dodson, G. G.; Lawson, D. M.; Turkenburg, J. P.; Blorkling, F.; Huge-Jensen, B.; Patkar, S. A., Thim, L. “A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex,” Nature, 351, 491-495 (1991).
Buchko, C. J.; Chen, L. C.; Shen, Y.; Martin, D. C. “Processing and Microstructural Characterization of Porous Biocompatible Protein Polymer Thin Films,” Polymer, 40, 7397-7407 (1999).
Chiou, S. H.; Wu, W. T. “Immobilization of Candida rugosa lipase on chitosan with activation of the hydroxyl groups,” Biomaterials, 25, 197–204 (2004).
Colombie, S.; Tweddell, R. J.; Condoret, J. S.; Marty, A. “Water Activity Control: A Way to Improve the Efficiency of Continuous Lipase Esterification,” Biotechnology and Bioengineering, 60, 362-368 (1998).
Deitzel, J. M.; Kleinmeyer, J. D.; Harris, D.; Tan, N. C. B. “The Effect of Processing Variables on The Morphology of Electrospun Nanofibers and Textiles,” Polymer, 42, 261-272 (2001).
Demir, M. M.; Yilgor, I.; Yilgor, E.; Erman, B. “Electrospinning of Polyurethane Fibers,” Polymer, 43, 3303-3309 (2002).
Dube, M. A.; Tremblay, A. Y.; Liu, J. “Biodiesel production using a membrane reactor,” Bioresource Technology, 98, 639-647 (2007).
Fong, H.; Chun, I.; Reneker, D. H. “Beaded Nanofibers Formed During Electrospinning,” Polymer, 40, 4585-4592 (1999).
Formhals, A. “Process and Apparatus for Preparing Artificial Threads,” US Patent, 1,975,504 (1934).
Formhals, A. “Method and Apparatus for Spinning,” US Patent, 2,160,962 (1939).
Formhals, A. “Artificial Thread and Method of Producing Same,” US Patent, 2,187,306 (1940).
Goldstein, L. “A new polymier carrier for immobilization of proteins of water insoluble derivaties of pepsin and trypsin,” Biochimica Et Biophysica Acta. Enzymology, 327,132-137 (1973).
Handa, T.; Hirose, A.; Yoshida, S.; Tsuchiya, H. “The Effect of Methylacrylate on the Activity of Glucoamylase Immobilized on Granular Polyacrylonitrile,” Biotechnology and Bioengineering, 24, 1639-1652 (1982).
Handa, T.; Hirose, A.; Akino, T.; Watanabe, K; Tsuchiya, H. “Preparation of Immobilized -Amylase Covalently Attached to Granular Polyacrylonitrile,” Biotechnology and Bioengineering, 25, 2957-2967 (1983).
Hermansyah, H.; Kubo, M.; Naomi, S. K.; Yonemoto, T. “Mathematical Model for Stepwise Hydrolysis of Triolein Using Candida rugosa Lipase in Biphasic Oil-Water System,” Biochemical Engineering Journal, 31, 125-132 (2006).
Holcapek, M.; Jandera, P.; Fisher, J.; Prokes, B. “Analytical monitoring of the production of biodiesel by high-performance liquid chromatography with various detection methods,” Journal of Chromatography A, 858, 13-31 (1999).
Huang, Z. M.; Zhang, Y. Z.; Kotaki, M.; Ramakrishna, S.; “A review on polymer nanofibers by electrospinning and their applications in nanocomposites,” Composites Science and Technology, 63, 2223–2253 (2003).
Huang, X. J.; Xu, Z. K.; Wan, L. S.; Innocent, C.; Seta, P. “Electrospun Nanofibers Modified with Phospholipid Moieties for Enzyme Immobilization,” Macromolecular Rapid Communications, 27, 1341-1345 (2006).
Jaeger, K. E.; Ransac, S.; Dijkstra, B. W.; Colson, C.; Heuvel, M. V.; Misset, O. “Bacterial lipase,” FEMS Microbiology Review, 15, 29-63 (1994).
Jaeger, K. E.; Reetz, M. T. "Microbial lipases form versatile tools for biotechnology," Trends in Biotechnology, 16, 396-403 (1998).
Jarusuwannapoom, T.; Hongrojjanawiwat, W.; Jitjaicham, S.; Wannatong, L.; Nithitanakul, M.; Pattamaprom, C.; Koombhongse, P.; Rangkupan, R.; Supaphol, P. “Effect of Solvents on Electro-spinnability of Polystyrene Solutions and Morphological Appearance of Resulting Electrospun Polystyrene Fibers,” European Polymer Journal, 41, 409-421 (2005).
Kazlauskas, R. J. “Elucidation Structure-mechanism Relationships in Lipases: Porspects for Prediction and Engineering Catalytic Properties,” Trends in Biotechnology, 12, 464-472 (1994).
Kumar, N. S. K.; Bhowmick, D. N. “Separation of Fatty Acids/Triacylglycerol by Membranes,” Journal of American Oil Chemists’ Society, 73, 399-401 (1996).
Lee, J. S.; Choi, K. H.; Ghim, H. D.; Kim, S. S.; Chun, D. H.; Kim, H. Y.; Lyoo, W. S. “Role of Molecular Weight of Atactic Poly(vinyl alcohol) (PVA) in The Structure and Properties of PVA Nanofabric Prepared by Electrospinning,” Journal of Applied Polymer Science, 93, 1638-1646 (2004).
Lotrakul, P.; Dharmsthiti, S. “Lipase production by Aeromonas sobria LP004 in a medium containing whey and soybean meal,” World Journal of Microbiology & Biotechnology, 13, 163-166 (1997).
Martinek, K.; Klibanov, A. M.; Goldmacher, V. S.; Berezin, I. V. “The principles of enzyme stabilization 1. Increase in thermostability of enzymes covalently bound to a complementary surface of a polymer support in a multipoint fashion,” Biochimica Et Biophysica Acta Enzymology, 485, 1-12 (1977).
Megelski, S.; Stephens, J. S.; Chase, D. B.; Rabolt, J. F. “Micro- and Nanostructured Surface Morphology on Electrospun Polymer Fibers,” Macromolecules, 35, 8456-8466, (2002)
Mit-uppatham, C.; Nithitanakul, M.; Supaphol, P. “Ultrafine Electrospun Polyamide-6 Fibers: Effect of Solution Conditions on Morphology and Average Fiber Diameter,” Macromolecular Chemistry and Physics, 205, 2327-2338 (2004).
Pieterson, W. A.; Vidal, J. C.; Volwerk, J. J.; de Haas, G. H. “Zymogen-Catalyzed Hydrolysis of Monomeric Substrates and the Presence of a Recognition Site for Lipid-Water Interfaces in Phospholipase A” Biochemistry, 13, 1455-1460 (1974).
Pencreac’h, G.; Baratti, J. C. “Comparison of Hydrolytic Activity in Water and Heptane for Thirty-two Commercial Lipase Preparations,” Enzyme and Microbial Technol., 28, 473-479 (2001).
Ramakrishna, S.; Fujihara, K.; Teo, W. E.; Lim, T. C.; Ma, Z. “ An Introduction to Electrospinning and Nanofibers,” in Introduction & Electrospinning Process, 1st Edition, World Scientific, Singapore, 2005, Chapter 1&3.
Reneker, D. H.; Chun, I. “Nanometre Diameter Fibres of Polymer, Produced by Electrospinning,” Nanotechnology, 7, 216-223 (1996).
Reneker, D. H.; Yarin, A. L.; Fong, H.; Koombhongse, S. “Bending Instability of Electrically Charged Liquid Jets of Polymer Solutions in Electrospinning,” Journal of Applied Physics, 87, 4531-4547 (2000).
Reneker, D. H.; Fong, H. “Polymeric Nanofibers,” in Applications of Electrospun Nanofibers in Current and Future Material, 1st Edition, American Chemical Society, Washington, 2006, Chapter 9.
Rutledge, G. C.; Li, Y.; Fridrikh, S.; Warner, S. B.; Kalayci, V. E.; Patra, P. “Electrostatic Spinning and Properties of Ultrafine Fibers” National Textile Center Annual Report, Project M98-D01, 1-10 (2000).
Sharma, R.; Chisti, Y.; Banerjee, U.C. “Production, purification, characterization, and applications of lipases,” Biotechnology Advances, 19, 627-662 (2001).
Shenoy, S. L.; Bates, W. D.; Frisch, H. L.; Wnek, G. E. “Role of Chain Entanglements on Fiber Formation During Electrospinning of Polymer Solutions: Good Solvent, Non-specific Polymer-polymer Interaction Limit,” Polymer, 46, 3372-3384 (2005).
Tan, S. H.; Inai, R.; Kotaki, M.; Ramakrishna, S. “Systematic parameter study for ultra-fine fiber fabrication via electrospinning process,” Polymer, 46, 6128–6134 (2005).
Tan, T; Chen, B. Q.; Ye, H. "Enzymatic synthesis of 2-ethylhexyl palmitate by lipase immobilized on fabric membranes in the batch reactor," Biochemical Engineering Journal, 29, 41–45 (2006).
Ting, W. J.; Tung, K. Y.; Giridhar, R.; Wu, W. T. “Application of Binary Immobilized Candida rugosa Lipase for Hydrolysis of Soybean Oil,” Journal of Molecular Catalysis B: Enzymatic, 42, 32-38 (2006).
Valivety, R. H.; Halling, P. J.; Peilow, A. D.; Macrae, A. R. “Relationship Between Water Activity and Catalytic Activity of Lipases in Organic Media,” European Journal of Biochemistry, 222, 461-466 (1994).
Verger, R.; Mieras, M. C. E.; de Hass, G. H. “Action of Phospholipase A at Interfaces,” The Journal of Biological Chemistry, 248, 4023-4034 (1973).
Wang, Z. G.; Wang, J. Q.; Xu, Z. K. “Immobilization of Lipase from Candida rugosa on Electrospun Polysulfone Nanofibrous Membranes by Adsorption,” Journal of Molecular Catalysis B: Enzymatic, 42, 45-51 (2006).
Ye, P.; Xu, Z. K.; Wu, J.; Innocent, C.; Seta, P. “Nanofibrous Poly(acrylonitrile-co-maleic acid) Membranes Functionalized with Gelatin and Chitosan for Lipase Immobilization,” Biomaterials, 27, 4169-4176 (2006).
Zhao, S. L.; Wu, X. H.; Wang, L. G.; Huang, Y. “Electrospinning of Ethyl-cyanoethyl Cellulose/Tetrahydrofuran Solutions,” Journal of Applied Polymer Science, 91, 242-246 (2004).
Zhong, X. H.; Kim, K. S.; Fang, D. F.; Ran, S. F.; Hsiao, B. S.; Chu, B. “Structure and Process Relationship of Electrospun Bioabsorbable Nanofiber Membranes,” Polymer, 43, 4403-4412 (2002).
呂鋒洲、林仁混，「基礎酵素學」，聯經出版事業公司，臺灣，1991，第十八章。
邱少華，「幾丁聚醣在固定化技術上之應用」，博士論文，清華大學，臺灣新竹 (2003)。
洪佃玠，「利用幾丁聚醣固定脂肪分解酵素之研究」，碩士論文，清華大學，臺灣新竹 (2002)。