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 acrylamido-monomers (called Immobilines; protolytic groups as N-substituents on the 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 activity, for any given enzyme, in the pH 3-10 scale. Such buffers, being amphoteric, are confined in the enzyme reaction chamber by the same isoelectric trapping mechanism. The best buffers were found to be those polymerized in presence of 9% neutral monomer (acrylamide) and containing 20 mM Immobiline as buffering ion. To decrease their viscosity in solution, the polymeric buffers are synthesized at high temperatures (70 degrees C) and in presence of a chain-transfer agent. The weight average molecular size in these conditions 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 case of penicillin G acylase and histidine decarboxylase and were found to greatly stabilize enzyme activity, permitting operation of the MIER over extended 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 same period of time. This novel class of macromolecular, amphoteric buffers could also be exploited in other types of conventional bioreactors not based on an isoelectric trapping mechanism.
Castelletti
L
L
University of Verona, Department of Industrial and Agricultural Biotechnologies, Strada Le Grazie, Ca' Vignal, 37134 Verona, Italy.
Bossi
A
A
Righetti
P G
PG
D019149:Bioreactors; D002021:Buffers; D004560:Electricity; D019075:Electrophoresis, Capillary; D006640:Histidine Decarboxylase / Q000378:metabolism*; D010399:Penicillin Amidase / Q000378:metabolism*; D011108:Polymers
D002021:Buffers; D011108:Polymers; D010399:Penicillin Amidase; D006640:Histidine Decarboxylase