|摘要: ||本研究乃探討石蓴脂氧合酶經固定化後之特性。石蓴粗抽出液經40-55%硫銨分劃、MacroPrep-Q離子交換及Sephacryl S-300膠過濾後,脂氧合酶活性(LOX)純化了103倍。八種不同抑制劑對脂氧合酶之影響以NDGA,BHA, esculetin,SnCl/sub 2/之抑制效果最高,脂氧合酶之催化機制似乎與其構造或自由基之產生有相當大之關係。於幾種固定化方法中以Chitosan-carbodiimide-glutaraldehyde之固定化方法效果較好。最適之Glutaraldehyde或Carbodiimide之用量分別為0.03及0.04%。經固定化後其最適溫度提高了約7.degree.C左右,最適pH不變;對熱、pH之安定性皆提高。經固定化後之LOX對溫度之耐性較未經固定化者高出甚多。固定化之海藻LOX於高溫下之操作穩定性較差,而於室溫或4.degree.C下之操作穩定性較高。|
A calcium stimulated-lipoxygenase (LOX) was isolated and purified 103-fold from sea algae (Ulva lactuca) using 40-55% saturation of ammonium sulfate fractionation, MacroPrep-Q ion exchange, and gel filtration on Sephacryl S-300. Eight synthetic LOX inhibitors including BHA, BHT, PG, esculetin, esculin, NDGA, SnCl/sub 2/, and HgCl/sub 2/ were used to study the activation mechanism of this partially purified LOX. Among them, NDGA showed the highest inhibition, followed by BHA, esculetin, and SnCl/sub 2/. It seems that the conformation change and the formation of free radical were involved in the activation process of algal LOX. The partially purified algal LOX was then immobilized with Chitosan-carbodiimide-glutaraldehyde (CN-EDC-GA) system, and the optimal condition for the immobilization was listed in the following: glutaraldehyde, 0.03%, carbodiimide, 0.04%. The optimal temperature of the immobilized algal LOX was 7.degree.C higher than that of soluble form, while the optimal pH was the same with soluble LOX. Moreover, the immobilized algal LOX exhibited higher pH stability and thermostability than those of soluble LOX. The immobilized algal LOX displayed the best operation stability with a denaturation half life of 360 h at 4.degree.C; 195 h at 26.degree.C. In conclusion, the immobilization of algal LOX on CN-EDC-GA system greatly enhanced the stability of the enzyme against thermal denaturation.