The protective effect of organic media on protein conformation of covalent immobilization enzyme
首发时间:2013-04-12
Abstract:In order to know the feasibility of protecting active conformation of enzyme during the covalent-immobilization, three lyophilized enzymes catalase (CAT), trypsin (TRY) and laccase (LAC) are immobilized under organic media and aqueous media, and kinetic constants are investigated. Specific activity of CAT immobilized in chloroform is 1.67 times larger with Kcat 2.43 times higher and Kmapp is 0.74 times smaller than that of CAT immobilized in aqueous phase. Specific activity of TRY immobilized in ethyl acetate is 5.33 times larger with Kcat 55.58 times higher and Kmapp is 0.40 times smaller than that of TRY immobilized in aqueous phase. Specific activity of LAC immobilized in ethyl acetate is 0.24 times larger with Kcat 0.29 times higher and Kmapp is 0.27 times smaller than that of LAC immobilized in aqueous phase.Results show that organic media has more advantages over aqueous media to remarkably improve activities and kinetic constant values. Organic media makes the enzymes under〝rigid〞state, and this "rigid" state helps enzymes maintain their original active conformation and prevent enzymes from conformation disorder during the modification reaction occurring.
keywords: Enzyme Covalent immobilization Conformation Organic media
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有机溶剂对共价固定化酶构象的保护
摘要:蛋白质共价固定化时,容易发生构象的不可逆变化而造成活性的丧失。为了使蛋白质在共价固定化过程中保持活性构象,本研究将冻干后的过氧化氢酶、胰蛋白酶、漆酶作为研究对象,将其在不同的有机溶剂中进行共价固定化,通过酶活力和动力学常数来探讨有机溶剂对蛋白质构象的保护效应。结果显示:与传统的水相固定化酶相比较,氯仿固定化过氧化氢酶的比活提高了1.67倍,Kcat提高了2.43倍,Km降低了0.74倍;乙酸乙酯固定化胰蛋白酶的比活提高了5.33倍,Kcat提高了55.58倍,Km降低了0.4倍;乙酸乙酯固定化漆酶的比活提高了0.24倍,Kcat提高了0.29倍,Km降低了0.27倍.可见与水相固定化相比较,有机溶剂有利于改善固定化酶的比活与动力学常数。由此表明有机溶剂有助于蛋白质在共价固定化过程中维持其活性构象,阻止其构象发生紊乱。
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