Chemo-enzymatic methods constitute a promising approach to obtain various biologically active compounds, including enantiomerically pure substances. Entrapment in gels is one of the most convenient methods to stabilize enzymes for their application in water/organic media. Proteases and upases are widely used for enantioselective transformations of various organic compounds in water-poor media. In this study, chymotrypsin was entrapped into a composite poly(N-vinyl caprolactam)-calcium alginate (PVCL-CaAlg) and covalently attached to poly(vinyl alcohol) (PVA) cryogel beads. Lipase was immobilized by covalently attaching to aldehyde-bearing PVA cryogel beads. The activities of the entrapped biocatalysts were studied. Both entrapped α-chymotrypsin and lipase retained high activity in acetonitrile/water medium (water content 0.5-20%) and displayed high storage stability for several months. The high operational stability of immobilized α-chymotrypsin and lipase in a cyclic process (up to 912 h in total) was also demonstrated. Gel-immobilized enzymes were successfully used to obtain optically pure L-phenylalanine (ee 98.6 and 83% in the case of α-chymotrypsin and lipase, respectively) by enantioselective hydrolysis of Schiff's base of amino acid ethyl ester in an acetonitrile/water system. © 2005 IUPAC.