Different lipases (lipase B from Candida Antarctica, CAL-B, lipase from Thermomyces lanuginose, TLL and lipase from Bacillus thermocatenulatus, BTL) and a phospholipase (Lecitase(R) Ultra) were immobilized by interfacial activation on four different hydrophobic supports (hexyl- and butyl-toyopearl and butyl- and octyl-agarose) and their properties were compared. The results suggested that selection of different supports yielded very different results in terms of recovered activity (ranging from a sevenfold hyperactivation to almost fully inactive biocatalysts), stability, specificity and adsorption strength. Even more interestingly, the enantioselectivity of the enzymes in the hydrolysis of (+/-)-2-O-butyryl-2-phenylacetic acid was strongly dependent on the support utilized. For example, BTL immobilized on octylagarose was fully enantiospecific towards the hydrolysis of (S)-2-O-butyryl-2-phenylacetic acid (E > 100), whereas when immobilized on hexyl-toyopearl, the enantiomeric value of the immobilized lipase was only E = 8. However, there is not an optimal support; it depends on the lipase and on the studied parameter. In the asymmetric hydrolysis of phenylglutaric acid diethyl diester, BTL immobilized on hexyl-toyopearl was the most enantioselective catalyst with ee > 99% (A factor > 100) in the production of S-monoester product, whereas the enzyme immobilized on butyl-toyopearl only exhibited an A factor of 3. Finally, butyl-agarose was chosen as the most specific support on the lipase adsorption - compared to other proteins - at low ionic strength yielding the best purification of BTL from crude preparations. (C) 2008 Elsevier Ltd. All rights reserved.