Deactivation and conformational changes of cutinase in reverse micelles

Abstract

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. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:380–386, 1998.

Stability of cutinase, wild type and mutants, in AOT reversed micellar system—effect of mixture components of alkyl esters production

“…1(a), even for incubation times up to around 1 h. The deactivation of the cutinase in the micellar system was more obvious when a pre‐incubation of 3 h was performed; in this case not only did the initial activity decreased by around 45%, but also the reaction stopped after 15 min and a methyl esters conversion of only 14% was achieved. The loss of the enzyme activity is explained by the denaturing effect that AOT has towards cutinase, which has already been observed in previous work 11, 18, 19. Also, Ternstrom et al explained that when cutinase interacts with AOT its active site region suffers a conformational change, as does the region around tryptophan residue 20…”

Entrapment of Glucose Oxidase in Reverse Micelle Microemulsion Systems for Glucose Detection in Lipid Based Food Products

“…However, the recovery yield and the separation factor of an ionic reversed micellar extraction are usually low because of the intensive electrostatic interactions and the low selectivity of electrostatic interactions [27]. Furthermore, the intensive electrostatic interactions often lead to enzyme inactivation or protein denaturation resulting from protein unfolding [28][29][30]. Therefore, for enzyme catalysis applications we chose foodgrade non-ionic surfactant (SPAN 85) for the entrapment of GO x with HLB value less than 7 which promotes W/O emulsions [31].…”