Cutinase stability in AOT reversed micelles: system optimization using thefactorial design methodology

Fusarium solani pisi cutinase microencapsulated in reversed micelles of AOT (bis(2-ethylhexyl) sodium sulfosuccinate) was used to catalyse the alcohol ysis 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 st art 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 desi gn expanded further to a central composite design (CCD), the effects of eac h factor mentioned above (with exception of temperature and acetate concent ration) on the stability were calculated and the interactions were determin ed. The surface methodology applied to the results showed that cutinase, in cubated at 40 degrees C retained a high activity level (90%) after 3 days w hen 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 s tability. A hexanol concentration of 500-600 mM was essential to prevent de activation 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 a ctivity and stability. Cutinase in AOT reversed micelles has been usually r eported to be very sensitive to temperature, however, in this work, at an i ncubation temperature of 40 degrees C, cutinase displayed half-lives signif icantly greater than previously stated. The main reasons for this are the p resence of hexanol and the reduced wafer content that promote a stabilisati on of cutinase structure inside the micelles. The thermostability experimen ts performed with the selected conditions led to a half-life of 53 days, co nfirming the model predictions. (C) 1999 Elsevier Science me. All rights re served.