荧光标记脂肪酶的固定化及其稳定性

Immobilization of lipase labeled with fluorescent probe and its stability

摘要:

将标记有荧光探针FITC (异硫氰基荧光素) 的脂肪酶固定化，通过测定活性和荧光光谱，探究各种因素对固定化后荧光标记脂肪酶性质的影响，并分析活性、构象和荧光光谱三者之间的联系。研究结果表明：在固定化脂肪酶过程中，聚乙二醇400二丙烯酸酯能形成合理的网格结构，使酶活较高；配体诱导酶的催化构象，使酶活性提高到未诱导酶的2倍以上；配体抽提能使脂肪酶活性中心得到释放从而提高催化活力。固定化脂肪酶的稳定性大大提高，在90℃、强酸强碱下固定化酶仍保有原酶70％、60％以上的活性；用盐酸胍、脲等溶解变性剂浸泡15 d后，酶活性仍然可以保持初始活性的70％以上。荧光光谱能较好地反映脂肪酶的活性和构象变化，最适pH和温度下脂肪酶的荧光强度最低，在溶解变性剂中，荧光强度随时间延长而逐渐降低，这表明不同条件下脂肪酶构象经历的去折叠过程不同。

Abstract:

The lipase labeled with the fluorescein isothiocyanat (FITC) was immobilized on the derivatives of the polyethylene glycol. The article discussed the effect of factors on the characters of lipase and analyzed the relationships among the activity of lipase, conformation, and fluorescence spectrum while the activity and the fluorescence spectrum of immobilized lipase were determined. The results demonstrated that polyethylene glycol 400-diacrylate could form appropriate network to improve the activity of enzyme. Adding ligand induced the lipase’s catalytic conformation to increase the activity twice more than before. The active centre of lipase could be released by the extraction of ligand thus increasing the activity. After immobilization, the stability of labeled lipase improved greatly: immobilized lipases retained more than 70% and 60% of initial activity under conditions of 90°C and strong acid or alkali, respectively. After immersing immobilized lipases into guanidine hydrochloride or urea for 15 days, the lipases retained upwards of 70% activity. The fluorescence spectrum could obviously reflect the changes of the activity and conformation of lipase. The fluorescence intensity was the minimum in the optimal pH and temperature. In the denaturing agent it declined as time passed. These results indicated that the unfolded processes of immobilized lipases are different under different conditions.