This work is aimed at assessing the influence of pressurized fluids treatment on the enzymatic activity of inulinases from Kluyveromyces marxianus NRRL Y-7571 immobilized in natural montmorillonite. The effects of system pressure, exposure time and depressurization rate, using propane and liquefied petroleum gas (LPG) on the enzymatic activity were evaluated through central composite designs (CCD) 2(3). In general, results showed that the enzyme activity changes significantly depending on the experimental conditions investigated, allowing the selection of proper operating conditions for advantageous application of this biocatalyst in hydrolysis reactions. Further, the stability of the enzyme after high-pressure pre-treatment was also experimentally monitored, and results demonstrated that the activity of the biocatalyst was always higher than the non-treated one. Results obtained here might be useful as a basis for the selection of appropriate process conditions, so that the catalyst can be applied in biotransformation reactions. (C) 2011 Elsevier Ltd. All rights reserved.