The thermal stability of the Aes acetyl esterase from Escherichia coli has been investigated by means of differential scanning calorimetry and circular dichroism measurements. The calorimetric curves show a denaturation temperature of 68 degrees C for Aes and 61 degrees C for the single point mutant V20D-Aes. The same values are obtained from CD denaturation curves of the two proteins recorded in both the far-UV and near-UV regions. Even if the denaturation process is irreversible and characterized by a single calorimetric peak and a single inflection point in both far- and near-UV CD curves, the overall data indicate that the process is more complex than a two-state transition. This is in line with the presence of two structural domains in the 3D model of Aes, according to homology modelling. A comparison of the thermal stability of Aes with those of the homologous thermophilic EST2 and hyperthermophilic AFEST suggests that the optimization of charge-charge interactions should not be so effective in the case of the mesophilic enzyme. (C) 2005 Elsevier B.V. All rights reserved.