本研究乃探討香蕉葉脂氧合酶之固定化方法及其特性。香蕉葉粗抽出液經25-50％硫銨分劃、hydroxyapatite管柱層析及Superdex pg 200膠過濾分離後，脂氧合活性純化了327倍，回收率為 9.9％。香蕉葉脂氧合於不同之固定化方法中，以海藻酸鹽及hydroxyapatite 之固定化效果較佳。若以多點式結合之chitosan+ glutadialdehyde方法來處理，其固定化之效果並不理想。香蕉葉脂氧合經海藻酸鹽固定化後，以凍結乾燥之活性保留最多。所得之固定化酵素，其最適溫度及pH分別為45℃及pH 6.8。將該固定化酵素於4℃放置68天後，仍保持80％以上活性。連續與基質作用6次後仍保持50％酵素活性。除此之外，本研究中首度採用hydroxyapatite之固定方法，於磷酸鹽10 mM濃度下其固定化效果約為海藻酸鹽者之兩倍，後續之研究目前仍進行中。 The lipoxygenase (LOX, linoleate:oxygen oxidoreductase, E.C. 1.13.11.12) from banana leaf (Giant Cavendishii, AAA) were isolated and purified 327-fold using 25-50% saturation of ammonium sulfate fractionation, hydroxyapatite column separation, and gel filtration on Superdex 200. MW of the purified LOX was 85 kD The banana leaf LOX was further immobilized with sodium alginate, chitosan, talc, DEAE Sepharose, marco-prep 50Q, and hydroxylapatite. Among them, sodium alginate and hydroxylapatite showed higher LOX activity during immobilization. The multi-point-attachment immobilization with chitosan and glutadialdehyde seemed to be not suitable to the LOX from banana leaf. LOX activity from different drying methods of immobilized lipoxygenase with sodium alginate method was compared. LOX from freeze drying showed the highest enzyme activity followed by those from suction drying and air drying.. The optimal temperature of the immobilized LOX was 45 ℃ and optimal pH was pH 6.8. The immobilized LOX retained 80 % enzyme activity for 68 days at 4℃, and more than 50 % LOX activity for 6 reused cycles at 28℃. Moreover, LOX activity immobilized with hydroxylapatite was 2-fold higher that with sodium alginate method. Further studies on the immobilization with hydroxylapatite are proceediing.
