Various physical, chemical and genetic approaches have been applied in order to enhance enzyme stability and activity. In this study, the aim was to investigate the capability of sub- and supercritical carbon dioxide to alter the stability and activity of a-amylase as an alternative technique. The effects of operational parameters such as pressure (50-300 bar), temperature (28-80 degrees C), CO(2) flow (2-10 gmin(-1)) and time (60-180 min) were evaluated in regard to the activity and stability of fungal based alpha-amylase from Aspergillus oryzea. The activity of untreated enzyme was determined as 17,726 mu mol/ml/min. While both sub- and supercritical conditions enhanced the activity, the increase in flow rate had an adverse effect and the activity was decreased by 28.9% at a flow rate of 10 gmin(-1) under supercritical conditions. Nuclear magnetic resonance (NMR) spectra of untreated enzyme and treated samples exhibiting the lowest and the highest activities were almost identical except for the chemical shifts observed at the lowest activity sample from 4.0 to 4.4 ppm which were assigned to protons of hydrogen-bonded groups. Optimum conditions were determined as 240 bar, 41 degrees C, 4 gmin(-1) CO(2) flow and 150 min of process duration yielding 67.7% (29,728 mu mol/ml/min) higher activity than the untreated enzyme providing fundamental basis for enzymatic applications. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.