2.4. Enzymatic esterification reaction
Esterification reactions catalyzed by T. fusca cutinase were car-
ried out in a total volume of 10 ml inside 25 ml screw-top vials.
Unless otherwise stated in the text, ethyl caproate was used as
the model ester for optimizing the reaction conditions of ester syn-
thesis at an acid concentration of 0.1 M and ethanol/acid molar
ratio of 2.0. The substrate concentrations of 0.05–1 M were used
for investigating the effects of substrate molar ratio and concentra-
tion on cutinase-catalyzed ester synthesis. Acid, alcohol and water
were thoroughly mixed in the organic solvent isooctane, and then
lyophilized cutinase was added to initiate ester synthesis. The
reaction mixtures were incubated in a water bath/orbital shaker
at 200 rpm. All of the organic reagents were previously dried over
4 Å molecular sieves. The rate of the reaction was calculated by
determining the formation of the product formed per hour
(de Barros et al., 2011). The ester yield was calculated using the
molar ratio between the ester and its respective limiting substrate.
Each experiment was performed in triplicate, except the experiments (quintuplicate) for the effect of chain lengths on T. fusca
cutinase-catalyzed ester synthesis.