J. of Supercritical Fluids 40 (2007) 93–100 On the importance of the supporting material for activity of immobilized Candida antarctica lipase B in ionic liquid/hexane and ionic liquid/supercritical carbon dioxide biphasic media Pedro Lozano a , Teresa De Diego a , Tanja Sauer a , Michel Vaultier b , Said Gmouh b , Jos´ e L. Iborra a,∗ a Departamento de Bioqu´ ımica y Biolog´ ıa Molecular B e Inmunolog´ ıa, Facultad de Qu´ ımica, Universidad de Murcia, P.O. Box 4021, E-30100 Murcia, Spain b Universit´ e de Rennes-1, UMR-CNRS 6510, Campus de Beaulieu, Av. G´ en´ eral Leclerc, F-35042 Rennes, France Received 4 October 2005; received in revised form 23 March 2006; accepted 29 March 2006 Abstract A commercial solution of free Candida antarctica lipase B (Novozyme 525L) has been immobilized by adsorption onto 12 different silica supports modified with specific side chains (e.g. alkyl, amino, carboxylic, nitrile, etc.). The immobilized derivatives were assayed for the kinetic resolution of rac-1-phenylethanol in both ionic liquid/hexane and ionic liquid/supercritical carbon dioxide biphasic media. The best results were obtained for the supports modified with non-functionalized alkyl chains and when the in water activity increased from 0.33 to 0.90 (e.g. the CALB/butyl-silica activity was enhanced up to five times). Coating immobilized enzyme particles with ionic liquids (butyltrimethylammonium bistriflimide or trioctylmethylammoniun bistriflimide) resulted in a decrease in activity (10 times), although half-life times were enhanced (up to six times) in hexane media at 95 ◦ C. However, immobilized derivatives coated with ionic liquids clearly improved their synthetic activity in supercritical CO 2 by up to six times with respect to the hexane medium, which agrees with the “philicity” between alkyl chain lengths of both the silica support and the cation of ionic liquid. © 2006 Elsevier B.V. All rights reserved. Keywords: Lipase; Ionic liquids; Supercritical fluids; Enzyme immobilization; Biotransformation; Green chemistry 1. Introduction The use of supercritical fluids as green non-aqueous media for enzyme-catalyzed synthetic reactions has been widely stud- ied during the last decade, due to their excellent recognized properties [1–3]. The greenness of enzymatic biotransforma- tions in scCO 2 begins with the enzyme, because enzymes are catalytic proteins of living systems, and so, acts as environ- mentally benign catalysts, with having high chemo-, regio- and enantioselectivity [4]. However, enzymes have been designed to work in aqueous solutions within a narrow range of environ- mental conditions (pH, temperature, pressure, etc.), and their use in non-aqueous media is seriously limited by the denatura- tive action of these media on the proteins. In this way, scCO 2 ∗ Corresponding author. Tel.: +34 968367398; fax: +34 968364148. E-mail address: jliborra@um.es (J.L. Iborra). has been described as a solvent with a strong deactivating effect on enzymes [1,5,6], making it necessary to develop enzyme sta- bilization strategies, e.g. covalent immobilization on supports coated with hydrophilic polymers [7]. On the other hand, ionic liquids (ILs) have recently emerged as exceptionally interesting green non-aqueous reaction media for enzymatic transformations [8]. They are salts and therefore entirely composed of ions that are liquids below 100 ◦ C or close to room temperature. Their interest as green solvents resides in their high thermal stability and very low vapor pressure, which mitigates the problem of releasing volatile organic solvents into the atmosphere. Moreover, the physical properties of ILs (den- sity, viscosity, melting points, polarity, etc.) can be finely tuned by the appropriate selection of anions and/or cations. ILs can be designed to be miscible or immiscible with water or organic solvents (e.g. hexane, toluene, ether, 1-propanol, etc.), making it easy to recover products from the reaction mixture [9]. Water immiscible ILs have been shown to be excellent non-aqueous 0896-8446/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.supflu.2006.03.025