Modification of enzyme surface negative charges via covalent immobilization for tailoring the activity and enantioselectivity

With the hydrolytic resolution of (R,S)-mandelates in biphasic media via an esterase (SNSM-87) from Klebsiella oxytoca as the model system, increasing of the ionization constants of catalytic imidazolium moiety (i.e. pK_2R from 5.96 to 4.11 and pK_2S from 5.16 to 3.66) was confirmed if the enzyme was immobilized on a hexamethylenamino-activated support (Sepabeads^@ EC-HA) for decreasing the negative surface charges via multipoint attachments of the carboxylic acid groups to the support. A detailed kinetic analysis of varying the leaving alcohol moiety, substrate concentration and aqueous pH resulted in structure-reactivity correlations, concentration-activity profiles and pH-reactivity profiles that could be employed for the rationale of maximum enantioselectivity of V_S/V_R = 41.2 for (R,S)-methyl mandelate. Comparisons of the structure-reactivity correlations in terms of log(k_2R/K_mR)_int, log(k_2S/K_mS)_int and log(E_int) varied with the inductive parameter F for all enzyme preparations were made, showing the cooperative interaction among pH, F and ionization constants of catalytic imidazolium moiety on effectively manipulating the enzyme activity and enantioselectivity.