Additional stabilization of penicillin G acylase-agarose derivatives by controlled chemical modification with...

We have tested the effect of chemical modifications with formaldehyde on the activity/stability of immobilized derivatives of the enzyme penicillin G acylase (PGA). These derivatives were previously stabilized through enzyme-support multipoint covalent attachment. We carried out very different chemical treatments of our derivatives by testing the effect of different variables which control the intensity and the nature of these amine-formaldehyde reactions. The variables tested were: formaldehyde concentration, pH, time, and temperature. We also developed a colorimetric titration of the free amine groups on immobilized PGA in order to evaluate the extension of the reaction between formaldehyde and the amine groups of the enzyme. As a consequence of these studies, we have been able to get additional stabilizations of our previously stabilized-immobilized derivatives: e.g. a factor of 24-fold was achieved in terms of stabilization against irreversible thermal inactivation. The integrated effect of additional chemical modification plus previous multipoint covalent attachment has allowed us to prepare PGA derivatives which are 50,000 more thermostable than native PGA as well as most of the commercial PGA derivatives.