Optimisation of the enantioselective biocatalytic hydrolysis of naproxen ethyl ester using ChiroCLEC-CR

In a biocatalytic reaction the immobilized lipase ChiroCLEC-CR enantioselectively hydrolysed a naproxen ethyl ester racemate, yielding (S)-naproxen with an enantiomeric excess of more than 98%, an enantiomeric ratio (E) of more than 100, and substrate conversion in excess of 40%. Statistically designed experiments were performed to optimise temperature, enzyme to substrate ratio, substrate concentration, agitation, reaction time, pH, buffer concentration and co-solvent addition. Optimisation efforts resulted in more than 20-fold improvement of activity, while the excellent enantioselectivity of the enzymes was maintained. In particular, the addition of PEG 1000 as a co-solvent improved conversion rates 10-fold. The kinetic parameters Vmax and KM were determined to be 0.359 μmol/min/mg and 17.6 mM, respectively. The optimised reaction conditions were 10% (m/v) substrate, and enzyme to substrate ratio of 1:50, at 50 °C and pH 5 with addition of 41% PEG 1000. In spite of these kinetic improvements, the stability of the biocatalytic activity under these conditions was poor, limiting the number of possible recycles.