Deactivation and conformational changes of cutinase in reverse micelles

✍ Scribed by E. P. Melo; C. M. L. Carvalho; M. R. Aires-Barros; S. M. B. Costa; J. M. S. Cabral

• Publisher: John Wiley and Sons
• Year: 1998
• Tongue: English
• Weight: 75 KB
• Volume: 58
• Category: Article
• ISSN: 0006-3592
• DOI: 10.1002/(sici)1097-0290(19980520)58:4<380::aid-bit5>3.0.co;2-f

✦ Synopsis

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 waterpool 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 W 0 5, with W 0 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 W 0 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 W 0 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/1hexanol reverse micelles at W 0 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/1hexanol reverse micelles.