Enzymatic reactions with supercritical carbon dioxide (SC-CO2) have received increased attention during the last decade. The objective of this study was to alter the activity and stability of Bacillus sp. protease under SC-CO2 conditions. The activity and stability of protease were evaluated according to the effects of operational variables, temperature (28-80 degrees C), pressure (60-300 bar), CO2 flow (2-10 g/min) and process duration (60-180 min). Best conditions were identified as 300 bar, 54 degrees C, 6 g/min CO2 flow and 120 min of process time efficient 54.4% (417.50 mu mol/ml/min) as against to the untreated enzyme. Activity enhancement was observed with the raise of pressure while low flow rates decreased the activity by 5.1% with a flow rate of 2 g/min under supercritical conditions. Potential mechanisms for pressure stimulated activation and stabilization were investigated by NMR, SEM, FTIR, SOS-PAGE and XPS analyses. As a result, applications of SC-CO2 medium for enzymatic processes are expected to become sustainable and important with economical synthetic and environmentally friendly protocols. (C) 2014 Elsevier B.V. All rights reserved.