Abstract
The first step of the inactivation of the enzyme D-amino acid oxidase (DAAO) from porcine kidney at pH 5 and 7 is the enzyme subunit dissociation, while FAD dissociation has not a relevant role. At pH 9, both dissociation phenomena affect the enzyme stability. A strong effect of the buffer nature and concentration on enzyme stability was found, mainly at pH 7 and 9 (it was possible at the same temperature to have the enzyme fully inactivated in 5 mM of Hepes while maintaining 100% in 5 mM of glycine). The effect of the concentration of buffer on enzyme stability depended on the buffer: at pH 5, the acetate buffer had no clear effect, while Tris, Hepes and glycine (at pH 7) and carbonate (at pH 9) decreased enzyme stability when increasing their concentrations; phosphate concentration had the opposite effect. The presence of 250 mM of NaCl usually increased enzyme stability, but this did not occur in all cases. The effects were usually more significant when using low concentrations of DAAO and were not reverted upon adding exogenous FAD. However, when using an immobilized DAAO biocatalyst which presented enzyme subunits attached to the support, where dissociation was not possible, this effect of the buffer nature on enzyme stability almost disappeared. This suggested that the buffers were somehow altering the association/dissociation equilibrium of the enzyme.