Supercritical carbon dioxide (SC-CO2) has been successfully employed in a variety of applications due to its numerous advantages. Despite extensive investigations on the relationship between the activity of enzymes treated with supercritical fluids and supercritical operating conditions, there are no experimental studies that have addressed the effects of supercritical pretreatment on enzyme denaturation. in this study, we have explored the impact Of SC-CO2 pretreatment on the activity and stability of hen egg-white lysozyme during its course of denaturation. Our data indicated no noticeable enhancement in enzyme activity and stability in the presence of SC-CO2 pretreatment for lysozyme samples denatured in 8 M urea at 50 degrees C and pH 6.2. However, SC-CO2 pretreated lysozyme samples in 0.067 M phosphate buffer containing dithiothreitol (DTT) (0.1 M DTT, pH 6.2, 25 degrees C or 0.01 M DTT, pH 6.2, 50 degrees C) at 2500 psi and 50 degrees C had better residual activity relative to samples that were not pretreated. In addition, when denaturing at 65 degrees C and pH 9.0, the pretreatment in SC-CO2 at 2500 psi and 50 degrees C resulted in the best stability of lysozyme. The result of this study may provide supporting evidence that supercritical fluids serve as potential media for enhancing the activity of enzymes used in a variety of biochemical applications. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.