Reaction kinetics of immobilized α chymotrypsin in organic media 2. Effects of substrate partition

The reaction kinetics of αchymotrypsin (EC 3.4.21.1.) catalyzed esterification of N-protected phenylalanine with ethanol were studied. The enzyme was deposited on Chromosorb and reactions were performed mainly in water-saturated mixtures of ethyl acetate and heptane, but also other media were used, such as mixtures of ethyl acetate and acetonitrile. The hydrophobicity of the substrate was varied by using different N-protecting groups (acetyl, Cbz and Fmoc). The apparent K_m obtained for the three substrates were 1.1, 7.8 and 17 mM, respectively. The apparent V_max decreased as the hydrophobicity of the substrates increased. The reaction medium greatly affected the apparent kinetic parameters, K_m and V_max. Nonpolar media (increasing proportion of heptane in mixtures of ethyl acetate and heptane) increased the apparent K_max and decreased the apparent K_m. The effects on the apparent K_m values could be correlated with the partitioning of the substrates between the reaction medium and an aqueous phase. In mixtures of acetonitrile and ethyl acetate both the apparent K_m and the apparent V_max decreased as the proportion of acetonitrile increased. The apparent K_m and V_max were also dependent on the water content in the reaction media as well as the buffer concentration and buffer pH.