在本研究中，利用電紡織法的方式製備出聚丙烯腈奈米纖維薄膜，作為脂肪分解酵素之固定化載體。於最佳電紡織操作條件下，成功地製備出纖維直徑約在150~300 nm並且結構均勻之聚丙烯腈奈米纖維薄膜。完成薄膜製備後，透過amidination reaction活化聚丙烯腈上的硝基官能基，與脂肪分解酵素上的胺基官能基形成共價鍵結，完成酵素固定化程序。由FTIR的結果可直接證明脂肪分解酵素成功地共價鍵結於聚丙烯腈奈米纖維薄膜之上。在活化時間為5分鐘、固定化時間為60分鐘、固定化溫度為30℃與固定化pH值為7.0的條件之下，所製備之固定化脂肪分解酵素具有較高的蛋白質固定量與較佳的酵素活性表現。製備之固定化脂肪分解酵素，其動力學參數Km值與Vmax值分別為0.55 mM與31.2 U/mg，與自由酵素所測得Km值（0.46 mM）與Vmax值（39.7 U/mg）相近，證實在固定化程序之後，脂肪分解酵素仍能保持其原有構形；與其他文獻研究結果相比，證明本研究所製備之固定化脂肪分解酵素有較為優異的表現。在穩定性測試方面，本研究所製備之固定化脂肪分解酵素在儲存20天後，仍保有起始活性的95%，且經過10次之批次反應後，其活性仍維持起始活性的70%左右，由此可證明本研究所製備之固定化脂肪分解酵素，具有儲存安定性佳與可重複使用等優點。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脂肪分解酵素，在水解反應時間為10分鐘時可達到72%之水解轉化率；而水解反應時間為90分鐘時可達到85%之平衡轉化率。利用大豆油催化水解進行批次反應20次後，其轉化率仍能維持在起始轉化率的65%，證明本研究所製備之固定化脂肪分解酵素，其穩定性相當優異。以期藉由本研究所製備之固定化脂肪分解酵素，其水解速率快、儲存安定性佳以及重複使用性優異等優點，改善酵素製程應用於工業發展上成本過高的問題。

Polyacrylonitrile (PAN) nanofibrous membrane with fiber diameter in the range of 150–300 nm could be manufactured by electrospinning, providing huge specific surface area for enzyme immobilization. Lipase from Candida rugosa was covalently immobilized onto the PAN nanofibrous membrane by amidination reaction. Aggregates of enzyme molecules were found on the nanofiber surface from field emission scanning electron microscopy. In addition, covalent bond formation between enzyme molecule and the nanofiber was confirmed from FTIR measurements. After lipase was immobilized onto the PAN nanofibrous membrane, the mechanical strength of the nanofibrous membrane was improved in doubly the tensile stress at break and Young’s modulus. Besides, the activity retention of the immobilized lipase was 87.5% of the free enzyme under optimal immobilization conditions. The kinetic parameters, Km and Vmax, of the immobilized lipase were determined to be 0.55 mM and 31.2 U/mg, respectively. The immobilized lipase retained over 95% of its initial activity, when stored in the buffer at 30℃ for 20 days, and still retained 70% of its initial activity after being reused 10 times.
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 hydrolysis reaction conditions, the hydrolysis conversion of soybean oil was 72% after 10 minutes and 85% after 90 minutes. In reusability examination, it still retained 65% of its initial conversion after being reused 20 times. These results imply that the proposed scheme for immobilization of lipase has the potential in industrial applications for oil hydrolysis.

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