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 (R) 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. (C) 2008 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.