Electrically immobilized enzyme reactors: Bioconversion of a charged substrate. Hydrolysis of penicillin G by penicillin G acylase

The possibility of using the multicompartment immobilized enzyme reactor (M IER) in presence of a charged substrate is here explored. Penicillin G acyl ase is used to convert penicillin G (a free acid, with a pK of 2.6) into tw o charged products: phenyl acetic acid (PAA, with a pK of 4.2) and 6-aminop enicillanic acid (6-APA, a zwitterion with a pi of 3.6). The enzyme is trap ped by an isoelectric mechanism in a chamber of the electrolyzer delimited by a pi 5.0 and a p/9.0 amphoteric, isoelectric membranes. Under normal ope rating conditions (continuous substrate feeding in the presence of an elect ric field), only a low substrate conversion can be achieved, due to rapid e lectrophoretic transport of unreacted penicillin G out of the reaction cham ber towards the anode. Excellent conversion rates (>96%) are obtained under a "doubly-discontinuous" operation mode: a time-lapse substrate feeding, a ccompanied by short times (4-8 min) of electric field interruption. The pro duct of interest (6-APA, a precursor of semisynthetic penicillins), by virt ue of its amphoteric nature, is trapped in a chamber delimited by a p/3.5 m embrane and a p/5.5 membrane, adjacent to the reaction chamber on its anodi c side. The other contaminant product (PAA) first accumulates in the same c hamber and then progressively vacates it to collect in the anodic reservoir , leaving behind a pure 6-APA solution. In this operation mode, vanishing a mounts of unreacted substrate (penicillin G) leave the reaction chamber to contaminate the adjacent, anodic chambers. A novel class of zwitterionic bu ffers is additionally reported, able to cover very thoroughly any pH value along the pH 3-10 interval: polymeric, zwitterionic buffers, synthesized wi th the principle of the Immobiline (acrylamido weak acids and bases) chemic als. Enhanced enzyme reactivity is found in this macromolecular buffers as compared to conventional ones. (C) 1999 John Wiley & Sons, Inc.