The aim of this work was the preparation and application of highly hydrophobic epoxy-chitosan/alginate as a support to immobilize microbial lipases from Thermomyces lanuginosus commercially available as Lipolase (R) (TLL1) and Lipex (R) 100L (TLL2) and Pseudomonas fluorescens (PFL). The catalytic properties of the biocatalysts were assayed in olive oil hydrolysis and butyl butyrate synthesis. The results indicated that 12 h was enough for TLL1 to be immobilized on the support. Covalent attachment of TLL1 turned biocatalysts highly active and around 6-fold more stable than free lipase. Based on the results, a time of incubation of 24 h was selected for further studies about the maximum immobilized protein amount and butyl butyrate synthesis. Maximum protein loading immobilized was found to be 25.4 mg g(-1) support for TLL1, followed by TLL2 (20.5 mg g(-1)) and PFL (15.5 mg g(-1)) offering 80 mg protein g(-1) support. The immobilization of TLL1 and TLL2 resulted in highly active biocatalysts (around 1300 IU g(-1) gel), almost fivefold higher than PFL (272.4 IU g(-1) gel). In butyl butyrate synthesis, PFL showed similar activity to TLL1 and TLL2 derivatives, up to 60 mmol L-1. The biocatalysts displayed high activity after five successive cycles, retaining around 95% of the initial activity. (c) 2012 Elsevier Ltd. All rights reserved.