DEACTIVATION AND CONFORMATIONAL-CHANGES OF CUTINASE IN REVERSE MICELLES

Deactivation data and fluorescence intensity changes were used to prob e functional and structural stability of cutinase in reverse micelles. A fast deactivation of cutinase in anionic (AOT) reverse micelles occ urs due to a reversible denaturation process. The deactivation and den aturation of cutinase is slower in small cationic (CTAB/1-hexanol) rev erse micelles and does not occur when the size of the cationic reverse micellar water-pool is larger than cutinase. In both systems, activit y loss and denaturation are coupled processes showing the same trend w ith time. Denaturation is probably caused by the interaction between t he enzyme and the surfactant interface of the reversed micelle. When t he size of the empty reversed micelle water-pool is smaller than cutin ase (at W-o 5, with W-o 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 inc rease in activity and shows only minor conformational changes relative to the native state. At W-o 20, the size of the empty water-pool was larger than cutinase and the data was well described by a two-state mo del for both anionic and cationic reverse micelles. For AOT reverse mi celles at W-o 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 W-o 20, the native cutinase was in equili brium 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. (C) 1998 John Wiley & Sons, Inc.