L. Castelletti et al., Characterization of polymeric buffers for operating membrane-trapped enzyme reactors in an electric field, BIOTECH BIO, 69(1), 2000, pp. 39-46
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.