Processing of protease under sub- and supercritical conditions for activity and stability enhancement

• This study is related to altering the activity and stability of Bacillus sp. protease under SC-CO2 conditions.
• The effects of various process parameters on the activity were evaluated.
• Best conditions were identified as 300 bar, 54 °C, 6 g/min CO2 flow and 120 min.
• An increase of 54.4% was achieved in comparison to the untreated enzyme.
• NMR, SEM, FTIR, SDS-PAGE and XPS analyses were conducted.

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 °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 °C, 6 g/min CO2 flow and 120 min of process time efficient 54.4% (417.50 μmol/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, SDS-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.