A number of years ago we reported a two-step inactivation mechanism for -amylase (enzyme) on the basis of theoretical and experimental studies in aqueous solutions. In the first step the metal (Ca2+) ion dissociates reversibly from the enzyme followed by an irreversible thermal inactivation of the apoenzyme. In this study we report inactivation of the enzyme in the presence of ethanol-water solutions. We noticed that as the concentration of ethanol in the aqueous solution is increased, the thermal inactivation of the enzyme is suppressed with almost no inactivation (in 1 h, 30 degrees C) when 50% alcohol is present in the solution. These results are explained by the two-step inactivation model. (c) 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1271-1275, 2016