Improvement of enzyme stability for alkyl esters synthesis in miniemulsion systems by using media engineering

BACKGROUND: Oil-in-water miniemulsions have been suggested to display potential for application as a ‘green’ system for enzymatic alkyl ester synthesis. Still, a realistic assessment of the feasibility of this approach for practical applications requires further insight, namely the relation between enzyme stability and reaction operational conditions. The objective of this work is within such scope. Accordingly, it extends previous research on esterification catalyzed by Fusarium solani pisi cutinase, by addressing medium-chain length substrates, and aims to contribute to the optimization of reaction conditions and towards the design of an efficient set-up. RESULTS: The esterification yield was significantly enhanced (e.g. 50% increase for octyl octanoate system) through the correction of the initial pH of the reaction medium to pH 6. Under such environment, conformational changes of the enzyme were negligible after 24 h, resulting in improved enzyme stability for all systems under study. Maximum esterification rate (3.59 mmol min^-1) and product yield (91%) were achieved for octyl decanoate synthesis. Fed-batch mode of operation allowed for the production of 1 mol L^-1 hexyl octanoate, while retaining enzyme activity over 5 days. Consecutive batch runs allowed for the cumulative production of 1.1 mol L^-1 hexyl octanoate after four cycles. CONCLUSIONS: Medium engineering enabled the use of miniemulsion systems for the synthesis of medium-chain alkyl esters and increase in the final concentration of the intended product. Through a fed-batch mode the enzyme stability and reutilization were tuned allowing long-term utilization of the free enzyme which could not be achieved through a repeated batch system.