Influence of Water on Enzymatic Esterification of Racemic Ketoprofen with Ethanol in a Solvent-Free System

The effect of water added in the kinetic resolution of rac-ketoprofen with ethanol catalyzed with Candida antarctica lipase B was investigated under reaction conditions and at a molecular level. The reaction was performed at 45 °C for 24 h with the commercial biocatalyst known as Novozym® 435. The addition of water from 0.5 to 10% v/v to the reaction medium enhances the specific activity (from 2 to 4 μmol mg−1 h−1), enantiomeric ratio E (1 towards 5), enantiomeric factor (EF 0.1 towards 0.5) and the enantiomeric excess towards dexketoprofen (above 50%). At a molecular level, the rate of H/D isotopic exchange of the amide hydrogen of the lipase B of C. antarctica demonstrated that the enzyme exposed to dehydrated ethanol possesses highly flexible sites (45% of the sites are exchanged with a rate of 30 min−1) and the addition of water diminishes the rate of H/D exchange altering the flexibility (the exchange rate falls down to 2.7–0.06 min−1). Additional studies on the secondary structure suggest that the β-sheet structure lead to slowest exchange H-amide sites.