Melo EP, Carvalho CM, Aires-Barros MR, Costa SM, Cabral JM
Centro de Engenharia Biologica e Quimica, Laboratorio Engenharia Bioquimica, Instituto Superior Tecnico, 1000 Lisbon, Portugal.
Biotechnol Bioeng. 1998 May 20;58(4):380-6. doi: 10.1002/(sici)1097-0290(19980520)58:4<380::aid-bit5>3.0.co;2-f.
Deactivation data and fluorescence intensity changes were used to probe functional and structural stability of cutinase in reverse micelles. A fast deactivation of cutinase in anionic (AOT) reverse micelles occurs due to a reversible denaturation process. The deactivation and denaturation of cutinase is slower in small cationic (CTAB/1-hexanol) reverse micelles and does not occur when the size of the cationic reverse micellar water-pool is larger than cutinase. In both systems, activity loss and denaturation are coupled processes showing the same trend with time. Denaturation is probably caused by the interaction between the enzyme and the surfactant interface of the reversed micelle. When the size of the empty reversed micelle water-pool is smaller than cutinase (at W0 5, with W0 being the water:surfactant concentration ratio) a three-state model describes denaturation and deactivation with an intermediate conformational state existing on the path from native to denaturated cutinase. This intermediate was clearly detected by an increase in activity and shows only minor conformational changes relative to the native state. At W0 20, the size of the empty water-pool was larger than cutinase and the data was well described by a two-state model for both anionic and cationic reverse micelles. For AOT reverse micelles at W0 20, the intermediate state became a transient state and the deactivation and denaturation were described by a two-state model in which only native and denaturated cutinase were present. For CTAB/1-hexanol reverse micelles at W0 20, the native cutinase was in equilibrium with an intermediate state, which did not suffer denaturation. 1-Hexanol showed a stabilizing effect on cutinase in reverse micelles, contributing to the higher stabilities observed in the cationic CTAB/1-hexanol reverse micelles. Copyright 1998 John Wiley & Sons, Inc.
失活数据和荧光强度变化被用于探究角质酶在反胶束中的功能和结构稳定性。由于可逆的变性过程，角质酶在阴离子（AOT）反胶束中快速失活。在小阳离子（CTAB/1-己醇）反胶束中，角质酶的失活和变性较慢，当阳离子反胶束水池的尺寸大于角质酶时则不会发生失活和变性。在这两种体系中，活性丧失和变性是耦合过程，随时间呈现相同趋势。变性可能是由酶与反胶束表面活性剂界面之间的相互作用引起的。当空反胶束水池的尺寸小于角质酶时（在W0 = 5时，W0为水与表面活性剂的浓度比），一个三态模型描述了变性和失活过程，在从天然角质酶到变性角质酶的路径上存在一个中间构象状态。通过活性增加清楚地检测到了这个中间体，并且相对于天然状态它仅显示出微小的构象变化。在W0 = 20时，空水池的尺寸大于角质酶，对于阴离子和阳离子反胶束，数据都可以用双态模型很好地描述。对于W0 = 20的AOT反胶束，中间状态变成了一个瞬态，失活和变性由一个双态模型描述，其中只存在天然和变性的角质酶。对于W0 = 20的CTAB/1-己醇反胶束，天然角质酶与一个中间状态处于平衡，该中间状态不会发生变性。1-己醇对角质酶在反胶束中表现出稳定作用，这导致在阳离子CTAB/1-己醇反胶束中观察到更高的稳定性。版权所有1998约翰威立父子公司。
