在本研究中，將脂肪分解酵素固定於聚丙烯腈披覆之氧化鐵（Fe3O4）磁性奈米粒子上，並應用於大豆油脂水解生成脂肪酸。首先利用化學共沉澱法製備Fe3O4磁性奈米粒子，然後將聚丙烯腈披覆於磁性奈米粒子上，透過amidination reaction活化方式，活化聚丙烯腈上之CN官能基，與脂肪分解酵素上之NH2官能基形成共價鍵結，完成固定化程序。由X-ray繞射光譜、穿透式電子顯微鏡與超導量子干涉儀分析結果得知，本研究所製備之Fe3O4磁性奈米粒子與固定化前後之聚丙烯腈磁性奈米粒子晶體結構相同、皆為奈米尺寸，且具有超順磁性。由傅立葉轉換紅外線光譜儀之分析，證明脂肪分解酵素以共價鍵結之方式固定於聚丙烯腈磁性奈米粒子之上。在聚丙烯腈高分子溶液濃度為1 wt%、活化時間為4分鐘、披覆聚丙烯腈之磁性奈米粒子添加量為70 mg/ml、固定化時間為20分鐘、固定化溫度為20℃與固定化pH值為5.0的酵素固定化條件下，固定化酵素具有較高之蛋白質固定量與最佳活性表現。應用於大豆油水解生產脂肪酸方面，本研究所製備之固定化酵素，在水解反應時間為8小時可達到75%之水解轉化率；而在最終水解時間30小時可達到97%之水解轉化率。在重複使用性部分，本研究所製備之固定化酵素，在重複使用5次後，其轉化率為起始轉化率之35%。

In this study, Fe3O4 magnetic nanoparticles coated with polyacrylonitrile (PAN) was activated by amidination reaction for immobilized Candida rugosa lipase with covalent binding. The immobilized lipase was employed to produce the fatty acids by the hydrolysis of soybean oil.
Firstly, the Fe3O4 magnetic nanoparticles were prepared by co-precipitating Fe3+ and Fe2+ ions. From the measurements of X-ray diffraction (XRD), transmission electron microscopy (TEM) and magnetic measurement (SQUID), the results showed that the structure and size of magnetic nanoparticles had no significantly change and maintained superparamagnetic characteristic after PAN coating and enzyme binding. The analysis of Fourier transform infrared (FTIR) spectroscopy confirmed that lipase was immobilized onto PAN-coated magnetic nanoparticles by covalent binding. The operation conditions of lipase immobilization were optimized to obtain the higher protein loading and the highest specific activity. The effects of oil-to-water ratio, temperature, and pH on hydrolysis of soybean oil were investigated to determine the optimal oil hydrolysis conditions. Under the optimal conditions of hydrolysis, the hydrolysis conversion of soybean oil was 75% after 8 hours and 97% after 30 hours. In reusability examination, it still keeps 35% of its initial conversion after being reused 5 times.

Bradford, M. M. (1976). "A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding." Analytical Biochemistry 72(1-2): 248-254.
Brzozowski, A. M., Derewenda, U., Derewenda, Z. S., Dodson, G. G., Lawson, D. M., Turkenburg, J. P., Bjorkling, F., Huge-Jensen, B., Patkar, S. A. and Thim, L. (1991). "A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex." Nature 351(6326):491-494.
Burns, P. C., Hawthorne, F. C., Libowitzky, E., Bordes, N. and Ewing, R. C.(1997). "Donathite discredited: A mixture of two spinels." Neues Jahrbuch Fur Mineralogie-Monatshefte(4): 163-174.
Chiou, S. H. and Wu, W. T. (2004). "Immobilization of Candida rugosa lipase on chitosan with activation of the hydroxyl groups." Biomaterials 25(2): 197-204.
Colombie, S., Tweddell, R. J., Condoret, J. S. and Marty, A. (1998). "Water activity control: A way to improve the efficiency of continuous lipase esterification." Biotechnology and Bioengineering 60(3): 362-368.
Gaur, R. and Khare, S. K. (2011). "Statistical optimization of palm oil hydrolysis by Pseudomonas aeruginosa PseA lipase." Asia-Pacific Journal of Chemical Engineering 6(1): 147-153.
Guerreiro, L., Castanheiro, J. E., Fonseca, I. M., Martin-Aranda, R. M., Ramos, A. M. and Vital, J. (2006). "Transesterification of soybean oil over sulfonic acid functionalised polymeric membranes." Catalysis Today 118(1-2): 166-171.
Handa, T., Hirose, A., Yoshida, S. and Tsuchiya, H. (1982). "The effect of methylacrylate on the activity of glucomylase immobilized on granular polyacrylonitrile." Biotechnology and Bioengineering 24(7): 1639-1652.
Handa, T., Hirose, A., Akino, T., Watanabe, K. and Tsuchiya, H. (1983)." Preparation of immobilized -Amylase covalently attached to granular polyacrylonitrile." Biotechnology and Bioengineering 25(12): 2957-2967.
Holcapek, M., Jandera, P., Fischer, J. and Prokes, B. (1999). "Analytical monitoring of the production of biodiesel by high-performance liquid chromatography with various detection methods." Journal of chromatography A 858(1): 13-31.
Kardash, E. and Tur'yan, Y. I. (2005). "Acid value determination in vegetable oils by indirect titration in aqueous-alcohol media." Croatica Chemica acta 78(1): 99-103.
Kazlauskas, R. J. (1994). "Elucidating structure-mechanism relationships in lipases: Prospects for predicting and engineering catalytic properties." Trends in Biotechnology 12(11): 464-472.
Kim, J., Grate, J. W. and Wang, P.(2008). "Nanobiocatalysis and its potential applications." Trends in Biotechnology 26(11): 639-646.
Koneracká, M., Kopcanský, P., Antalík, M., Timko, M., Ramchand, C. N., Lobo, D., Mehta, R. V. and Upadhyay, R. V. (1999). "Immobilization of proteins and enzymes to fine magnetic particles." Journal of Magnetism and Magnetic Materials 201(1-3): 427-430.
Lee, D. G., Ponvel, K. M., Kim, M., Hwang, S., Ahn, I. S. and Lee, C. H. (2009). "Immobilization of lipase on hydrophobic nano-sized magnetite particles." Journal of Molecular Catalysis B-Enzymatic 57(1-4): 62-66.
Li, S. F., Fan, Y. H., Hu, R. F. and Wu, W. T. (2011). "Pseudomonas cepacia lipase immobilized onto the electrospun PAN nanofibrous membranes for biodiesel production from soybean oil." Journal of Molecular Catalysis B: Enzymatic 72(1-2): 40-45.
Lotrakul, P. and Dharmsthiti, S. (1997). "Lipase production by Aeromonas sobria LP004 in a medium containing whey and soybean meal." World Journal of Microbiology and Biotechnology 13(2): 163-166.
Mateo, C., Palomo, J. M., Fernandez-Lorente, G., Guisan, J. M. and Fernandez-Lafuente, R. (2007). "Improvement of enzyme activity, stability and selectivity via immobilization techniques." Enzyme and Microbial Technology 40(6): 1451-1463.
Mehta, R. V., Upadhyay, R. V., Charles, S. W. and Ramchand, C. N. (1997). "Direct binding of protein to magnetic particles." Biotechnology Techniques 11(7): 493-496.
Pencreac'h, G. and Baratti, J. C. (2001). "Comparison of hydrolytic activity in water and heptane for thirty-two commercial lipase preparations." Enzyme and Microbial Technology 28(4-5): 473-479.
Ramani, K., John Kennedy, L., Ramakrishnan, M. and Sekaran, G. (2010). "Purification, characterization and application of acidic lipase from Pseudomonas gessardii using beef tallow as a substrate for fats and oil hydrolysis." Process Biochemistry 45(10): 1683-1691.
Rodrigues, R. C., Bolivar, J. M., Palau-Ors, A., Volpato, G., Ayub, M. A. Z., Fernandez-Lafuente, R. and Guisan, J. M. (2009). "Positive effects of the multipoint covalent immobilization in the reactivation of partially inactivated derivatives of lipase from thermomyces lanuginosus." Enzyme and Microbial Technology 44(6-7): 386-393.
Schmid, R. D. and Verger, R. (1998). "Lipases: Interfacial Enzymes with Attractive Applications." Angewandte Chemie International Edition 37(12): 1608-1633.
Sharma, R., Chisti, Y. and Banerjee, U. C.(2001). "Production, purification, characterization, and applications of lipases." Biotechnology Advances 19(8): 627-662.
Shaw, S. Y., Chen, Y. J., Ou, J. J. and Ho, L. (2006). "Preparation and characterization of Pseudomonas putida esterase immobilized on magnetic nanoparticles." Enzyme and Microbial Technology 39(5): 1089-1095.
Valivety, R. H., Halling, P. J., Peilow, A. D. and Macrae, A. R. (1994). "Relationship between water activity and catalytic activity of lipases in organic media." European Journal of Biochemistry 222(2): 461-466.
Verger, R., Mieras, M. C. and de Haas, G. H. (1973). "Action of phospholipase A at interfaces." The Journal of biological chemistry 248(11): 4023-4034.
Yigitoglu, M. and Temoçin, Z. (2010). "Immobilization of Candida rugosa lipase on glutaraldehyde-activated polyester fiber and its application for hydrolysis of some vegetable oils." Journal of Molecular Catalysis B: Enzymatic 66(1-2): 130-135.
Yong, Y., Bai, Y., Li, Y., Lin, L., Cui, Y. and Xia, C. (2008). "Preparation and application of polymer-grafted magnetic nanoparticles for lipase immobilization." Journal of Magnetism and Magnetic Materials 320(19): 2350-2355.
Zhang, W. X., Wang, Y. Z. and Sun, C. F. (2007). "Characterization on oxidative stabilization of polyacrylonitrile nanofibers prepared by electrospinning." Journal of Polymer Research 14(6): 467-474.
呂鋒洲、林仁混，「基礎酵素學」，聯經出版事業公司，1991。
李昇峰，「聚丙烯腈奈米纖維薄膜於脂肪分解酵素固定化之應用」，博士
論文，國立成功大學化學工程研究所，2009。
陳國誠，「生物固定化技術與產業應用」，茂昌圖書有限公司，2000。
馬振基，「奈米材料科技原理與應用」，全華科技出版社，2003。
莊萬發，「超微粒子理論應用」，復漢出版社，1998。
陳育裕，「鐵氧超微磁粉之製備研究」，碩士論文，國立成功大學化學工
程研究所，1998。
黃世宏，「氧化鐵磁性奈米粒子在酵素固定化及分離上之應用研究」，碩
士論文，國立成功大學化學工程研究所，2003。
張松文，「以氧化鐵磁性奈米粒子分離伴刀豆球蛋白之研究」，碩士論文，
國立成功大學化學工程研究所，2007。
王思婷，「具核殼結構的磁性微脂粒之熱效應研究」，碩士論文，國立成
功大學化學工程研究所，2008。
洪佃玠，「利用幾丁聚醣固定脂肪分解酵素之研究」，碩士論文，國立
清華大學化學工程研究所，2002。
李智傑，「酸性觸媒在生質柴油製程之研究」，碩士論文，國立成功大
學化學工程研究所，2008。