Developing active immobilized enzymes and characterization of their use conditions is critically important prior to initiating studies of enzyme selectivity and substrate specificity in organic media. To this end, physical immobilization by hydrophobic interactions was performed with three well-characterized cutinases (Aspergillus oryzae Cutinase (AoC), Humicola insolens Cutinase (HiC), and Thielavia terrestris Cutinase (TtC)) using Lewatit VP OC 1600 as the macroporous support. We found that immobilization yields > 98% were achieved for all three cutinases under the following immobilization conditions: 100 mg/g loading ratio, immobilization buffers of 100 mM phosphate pH 8 (AoC and HiC) and 100 mM acetate pH 5 (TtC), mixing at 150 rpm and 30 degrees C for 24 h. Among the three cutinases, HiC has the highest tolerance towards solvents of increased polarity while TtC has the highest thermal stability (up to 80 degrees C) in a bulk reaction system that consists of the reactants butanol and lauric acid. In nonane, these cutinases retain > 64% of their activity at 90 degrees C. Furthermore, kinetic stability (residual activity as a function of time) analysis reveals that the cutinases retain > 75% residual activity at 70 degrees C in 3 h. Moreover, at 80 degrees C, the kinetic stability of TtC is higher than that of HiC and AoC. Collectively, the results herein set the stage for the in-depth evaluation of these catalysts for selective transformations in organic media.