AbstractBACKGROUNDIt is vitally important to understand the effect of sub‐/super‐critical CO2 pretreatment on enzyme conformation and its activity, which should improve the understanding of enzymatic biotransformation applications in nonaqueous media. This study evaluated the effect of sub‐/super‐critical CO2 treatment, including pressure (6 and 10 MPa), exposure time (20, 30 and 150 min) and temperature (35 and 40 °C), on the conformation (e.g. 1D, 2D and 3D structures) and catalytic properties (e.g. residual activity, kinetics constants (Km and Vmax), activation energies (Eα), thermo‐stability and organic solvent tolerance) of two commercial enzymes, CALB and lipase PS in their solution forms.RESULTSCompared with the blank control, the 1D structure of both treated lipases was unchanged, whereas their 2D and 3D structures were altered to some extent. The highest relative activities were 105% and 116% for CALB and lipase PS, respectively. For CALB, Vmax/Km value significantly increased, while for lipase PS, Vmax/Km value was almost constant. Both treated enzymes showed high thermo‐stability with recovery of enzyme activity up to 76% and 80%, respectively. Also, both treated enzymes presented high organic solvent tolerance.CONCLUSIONSIt was speculated that 2D, 3D structure alterations were probably responsible for the satisfactory catalytic properties of enzymes treated with sub‐/super‐critical CO2. © 2013 Society of Chemical Industry