Fusarium solani pisi cutinase microencapsulated in reversed micelles of AOT (bis(2-ethylhexyl) sodium sulfosuccinate) was used to catalyse the alcoholysis of butyl acetate (acyl dorter) with hexanol. In the present study the stability of cutinase is evaluated comparatively with the initial activity in order to obtain a set of conditions to fully optimise the system. The start conditions were selected based on a synergy of the results assigned in previous work, where relevant parameters for the cutinase activity, such as temperature, buffer molarity, pH, amount of water, surfactant, hexanol and butyl acetate concentrations were studied. Using a 2((5-1)) factorial design expanded further to a central composite design (CCD), the effects of each factor mentioned above (with exception of temperature and acetate concentration) on the stability were calculated and the interactions were determined. The surface methodology applied to the results showed that cutinase, incubated at 40 degrees C retained a high activity level (90%) after 3 days when microencapsulated at W-0 (molar ratio of water to surfactant) 2.7. The W-0 values in the range 5-8 led to optimal-specific activities with lower stability. A hexanol concentration of 500-600 mM was essential to prevent deactivation and at the same time allowed relatively high initial rates. A pH value of 8.0-8.55 and a buffer molarity of 200 mM were suitable for both activity and stability. Cutinase in AOT reversed micelles has been usually reported to be very sensitive to temperature, however, in this work, at an incubation temperature of 40 degrees C, cutinase displayed half-lives significantly greater than previously stated. The main reasons for this are the presence of hexanol and the reduced wafer content that promote a stabilisation of cutinase structure inside the micelles. The thermostability experiments performed with the selected conditions led to a half-life of 53 days, confirming the model predictions. (C) 1999 Elsevier Science me. All rights reserved.
