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

Abstract

The possibility of using the multicompartment immobilized enzyme reactor (MIER) in presence of a charged substrate is here explored. Penicillin G acylase is used to convert penicillin G (a free acid, with a pK of 2.6) into two charged products: phenyl acetic acid (PAA, with a pK of 4.2) and 6‐aminopenicillanic acid (6‐APA, a zwitterion with a pI of 3.6). The enzyme is trapped by an isoelectric mechanism in a chamber of the electrolyzer delimited by a pI 5.0 and a pI 9.0 amphoteric, isoelectric membranes. Under normal operating conditions (continuous substrate feeding in the presence of an electric field), only a low substrate conversion can be achieved, due to rapid electrophoretic transport of unreacted penicillin G out of the reaction chamber towards the anode. Excellent conversion rates (>96%) are obtained under a “doubly‐discontinuous” operation mode: a time‐lapse substrate feeding, accompanied by short times (4–8 min) of electric field interruption. The product of interest (6‐APA, a precursor of semisynthetic penicillins), by virtue of its amphoteric nature, is trapped in a chamber delimited by a pI 3.5 membrane and a pI 5.5 membrane, adjacent to the reaction chamber on its anodic side. The other contaminant product (PAA) first accumulates in the same chamber and then progressively vacates it to collect in the anodic reservoir, leaving behind a pure 6‐APA solution. In this operation mode, vanishing amounts of unreacted substrate (penicillin G) leave the reaction chamber to contaminate the adjacent, anodic chambers. A novel class of zwitterionic buffers is additionally reported, able to cover very thoroughly any pH value along the pH 3–10 interval: polymeric, zwitterionic buffers, synthesized with the principle of the Immobiline (acrylamido weak acids and bases) chemicals. Enhanced enzyme reactivity is found in this macromolecular buffers as compared to conventional ones. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 383‐391, 1999.

Differential down‐regulation of CD95 or CD95L in chronically HIV‐infected cells of monocytic or lymphocytic origin: cellular studies and molecular analysis by quantitative competitive RT‐PCR

“…1) or polyclonal antibodies (not shown). Recently, it was shown that, before degranulation, CD95L can be stored in specialised secretory lysosomes in both CD4+ and CD8+ T lymphocytes and natural killer cells [37]. We thus analysed the amount of intracellular molecule by a £ow cytometric approach, and indeed could detect the presence of CD95L either in A301 or in its HIV+ clone, ACH-2.…”

Enzyme immobilization on/in polymeric membranes: status, challenges and perspectives in biocatalytic membrane reactors (BMRs)

“…These membranes have isoelectric point values far apart from each other and the isoelectric point of the enzyme lies in between those of the membranes. 32 All these types of retention have their own advantages and have been included in other reviews. 5,33,34 However, this category falls outside the scope of this review.…”

Use of Enzymes in the Production of Semi-Synthetic Penicillins and Cephalosporins: Drawbacks and Perspectives

“…Many studies [46][47][48][49][50][51][52][53][54] address the reaction components separation or an inhibitory component removal by an electrodialysis process using usually soluble enzymes. Electrodialysis can be defined as a type of technology that arranges ion-exchange membranes (cation-selective, anion-selective and/or bipolar membranes) alternately in a direct current field [55][56][57][58][59].…”

“…Suga et al [48] applied electrodialysis membranes to separate reaction components of penicillin G hydrolysis, reducing to 24.7% the time required to reach 98% conversion. Penicillin G hydrolysis with simultaneous products separation was studied also by Bossi et al [53] who used a multicompartment immobilized enzyme reactor where the enzyme was isoelectrically trapped in a reaction chamber. High substrate conversion values (>96%) were achieved under a discontinuous operation and a product of high purity was obtained.…”