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 mu M 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.

Automated summary

Enzymatic synthesis of short-chain esters using covalently immobilised Rhizopus oryzae lipase was investigated. The best yield of isoamyl butyrate (ca. 100%) was obtained in cyclohexane as the reaction solvent. Optimum initial acid concentration and acid:alcohol mole ratio were determined to maximize the production of isoamyl butyrate with reused enzyme. The biocatalyst and reaction conditions proposed in this study could be used for industrial production of isoamyl esters.