Enzyme and Microbial Technology 38 (2006) 914–920 Analysis of solvent-free ethyl oleate enzymatic synthesis at equilibrium conditions V. Bucal´ a a , M.L. Foresti a , G. Trubiano a,∗ , M.L. Ferreira a , M. Briozzo b , S. Bottini a a PLAPIQUI-CONICET, Camino La Carrindanga Km 7 CC 717, 8000 Bah´ ıa Blanca, Argentina b Department of Chemical Engineering, Universidad Nacional del Sur, 8000 Bah´ ıa Blanca, Argentina Received 2 May 2005; received in revised form 6 August 2005; accepted 10 August 2005 Abstract This work reports experimental equilibrium data for the esterification of pure oleic acid and a fatty acid mixture with ethanol, using an immobilized Candida antarctica B lipase as catalyst. Reactions are performed in a solvent-free system, containing a mixture of substrates and different amounts of distilled water. According to the initial amount of water and the extent of the reaction, one or two liquid phases are present. Therefore, when the equilibrium is achieved, the liquid–liquid and chemical reaction equilibria have to be simultaneously satisfied. Several reports dealing with enzymatic reactions performed in two-phase systems have found that the value of the reaction equilibrium constant calculated from overall experimental concentrations varies not only with temperature but also with substrate ratio and water content. Although this approach is a valuable way to explore equilibrium shifts in biphasic systems, it is limited to ideal systems with constant partition coefficients. The aim of this work is to consider the biphasic nature of the reactive mixture through a computational procedure that simultaneously takes into account liquid–liquid and reaction equilibria. This approach enables the determination of a classical temperature-dependent thermodynamic equilibrium constant, which accurately fits experimental equilibrium conversions over a wide range of operating conditions. © 2005 Elsevier Inc. All rights reserved. Keywords: Enzymatic esterification; Liquid–liquid equilibrium; Chemical reaction equilibrium; Biphasic constant 1. Introduction In the last years, many enzymes were found to work properly in organic media, as well as in liquid–liquid two-phase systems [1–6]. This work is specially focused on the equilibrium condi- tions that govern enzymatic reactions performed in liquid–liquid biphasic systems. Several reports dealing with enzymatic reactions performed in two-phase systems have found that the value of the equilib- rium constant calculated from overall experimental concentra- tions varies not only with temperature but also with substrate ratio and water content [7,8]. The variations of the experimental equilibrium constant have been generally handled by represent- ing chemical equilibrium in terms of the so-called apparent biphasic equilibrium constant. The apparent biphasic constant is defined in terms of the overall concentrations of the two-phase system, which makes this equilibrium constant to depend not ∗ Corresponding author. Tel.: +54 291 4861700/1600; fax: +54 291 4861700/1600. E-mail address: gtrubiano@plapiqui.edu.ar (G. Trubiano). only on temperature, as expected, but also on the initial amount of reactants and products, and on the relative amount of the two liquid phases. The concept of the biphasic constant was first introduced by Martinek et al. [9] and later used by Antczak et al. [10]. Following a more rigorous approach, Eggers et al. [11] also applied the concept of an apparent biphasic constant to enzymatic reactions performed in dilute aqueous solutions in equilibrium with a hydrophobic organic phase. Working with aqueous/organic two-phase systems, Martinek et al. [9] proved that, depending on the values of the phase volume ratios and the partition coefficients of reactants and products, the apparent biphasic constant can be higher than the equilibrium constant of a one-phase system, highlighting the benefits of using biphasic reaction media. Antczak et al. [10] correlated the equilibrium constant of the organic phase with the phase volume ratio of the biphasic system. Even though this correlation allowed the authors to fit the experimental data accurately, the approach is theoretically incorrect, since the one-phase chemical equilib- rium constant should only vary with temperature. On the other hand, the models of Martinek et al. [9] and Eggers et al. [11] are only suitable for dilute two-phase systems, with constant partition coefficients. 0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.08.017