Producing natural flavours from isoamyl alcohol and fusel oil by using immobilised Rhizopus oryzae lipase

Enzymatic synthesis of short-chain esters (flavours) might enable their labelling as natural, increasing their value. Covalently immobilised Rhizopus oryzae lipase (EO-proROL) was used to synthesise isoamyl butyrate and acetate. In cyclohexane, the best performer reaction solvent, 1.8 times higher yield of isoamyl butyrate (ca. 100%) than isoamyl acetate (ca. 55%) was obtained. Optimum initial acid concentration (410 mM) and acid:alcohol mole ratio (0.5) were established by a central composite rotatable design to maximise isoamyl butyrate single-batch and cumulative production with reused enzyme. These conditions were used to scale up the esterification (150 mL) and to assess yield, initial esterification rate, productivity and enzyme operational stability. Commercial isoamyl alcohol and fusel oil results were found to be similar as regards yield (91% vs. 84%), initial reaction rate (5.4 µM min−1 with both substrates), operational stability (40% activity loss after five runs with both) and productivity (31.09 vs. 28.7 mM h−1). EO-proROL specificity for the structural isomers of isoamyl alcohol was also evaluated. Thus, a successful biocatalyst and product conditions ready to be used for isoamyl ester industrial production are here proposed.