Characterization of polymeric buffers for operating membrane-trapped enzyme reactors in an electric field

A novel class of amphoteric, polymeric buffers, is described, consisting of grafting onto growing polyacrylamide chains weakly acidic and basic acryla midomonomers (called Immobilines; protolytic groups as N-substituents on th e nitrogen of the amido bond), for operating a membrane-immobilized enzyme reactor (MIER) in an electric field. With these soluble, polymeric buffers, it is possible to operate the membrane reactor at any optimum of pH activi ty, for any given enzyme, in the pH 3-10 scale. Such buffers, being amphote ric, are confined in the enzyme reaction chamber by the same isoelectric tr apping mechanism. The best buffers were found to be those polymerized in pr esence of 9% neutral monomer (acrylamide) and containing 20 mM Immobiline a s buffering ion. To decrease their viscosity in solution, the polymeric buf fers are synthesized at high temperatures (70 degrees C) and in presence of a chain-transfer agent. The weight average molecular size in these conditi ons has been found to be ca. 200,000 Da. These buffers exhibited excellent performance in a variety of enzyme reactions in the MIER, such as in the ca se of penicillin G acylase and histidine decarboxylase and were found to gr eatly stabilize enzyme activity, permitting operation of the MIER over exte nded periods of time. As an example, in a penicillin G acylase reactor, >75 % enzyme activity was maintained over a 10-d cycle of operation, while with conventional buffers more than 90% inactivation was experienced over the s ame period of time. This novel class of macromolecular, amphoteric buffers could also be exploited in other types of conventional bioreactors not base d on an isoelectric trapping mechanism. (C) 2000 John Wiley & Sons, Inc.