熒光標(biāo)記脂肪酶的固定化及其穩(wěn)定性

徐佳音，張馳，宋錫瑾，王杰

1 浙江大學(xué)化學(xué)工程與生物工程學(xué)系，杭州 310027 2 浙江大學(xué)化學(xué)系，杭州 310027

熒光標(biāo)記脂肪酶的固定化及其穩(wěn)定性

徐佳音1，張馳1，宋錫瑾1，王杰2

1 浙江大學(xué)化學(xué)工程與生物工程學(xué)系，杭州 310027 2 浙江大學(xué)化學(xué)系，杭州 310027

將標(biāo)記有熒光探針FITC(異硫氰基熒光素)的脂肪酶固定化，通過(guò)測(cè)定活性和熒光光譜，探究各種因素對(duì)固定化后熒光標(biāo)記脂肪酶性質(zhì)的影響，并分析活性、構(gòu)象和熒光光譜三者之間的聯(lián)系。研究結(jié)果表明：在固定化脂肪酶過(guò)程中，聚乙二醇 400二丙烯酸酯能形成合理的網(wǎng)格結(jié)構(gòu)，使酶活較高；配體誘導(dǎo)酶的催化構(gòu)象，使酶活性提高到未誘導(dǎo)酶的2倍以上；配體抽提能使脂肪酶活性中心得到釋放從而提高催化活力。固定化脂肪酶的穩(wěn)定性大大提高，在90℃、強(qiáng)酸強(qiáng)堿下固定化酶仍保有原酶70%、60%以上的活性；用鹽酸胍、脲等溶解變性劑浸泡15 d后，酶活性仍然可以保持初始活性的70%以上。熒光光譜能較好地反映脂肪酶的活性和構(gòu)象變化，最適pH和溫度下脂肪酶的熒光強(qiáng)度最低，在溶解變性劑中，熒光強(qiáng)度隨時(shí)間延長(zhǎng)而逐漸降低，這表明不同條件下脂肪酶構(gòu)象經(jīng)歷的去折疊過(guò)程不同。

脂肪酶，固定化，穩(wěn)定性，熒光光譜，構(gòu)象

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.

Keywords:lipase, immobilization, stability, fluorescence spectrum, conformation

脂肪酶(Lipase E.C.3.1.1.3)是一類(lèi)特殊的酯鍵水解酶，它能在油水界面上催化酯水解、酯合成、酯交換及立體異構(gòu)體拆分等有機(jī)合成反應(yīng)，因此工業(yè)用途廣泛，是目前被重點(diǎn)研究的生物催化劑。為了改善生物催化劑使用穩(wěn)定性差、易失活、適應(yīng)反應(yīng)介質(zhì)能力弱等缺點(diǎn)，常將脂肪酶固定化后使用。……