Journal of Molecular Catalysis B: Enzymatic 13 (2001) 49–55 The dependence of a halophilic malate dehydrogenase on ω o and surfactant concentration in reverse micelles Sonsoles Piera-Velázquez ∗ , Frutos Marhuenda-Egea, Eduardo Cadenas División de Bioqu´ ımica, Facultad de Ciencias, Universidad de Alicante, Apartado 99, 03080 Alicante, Spain Received 5 February 1999; received in revised form 16 July 1999; accepted 31 August 2000 Abstract The halophilic malate dehydrogenase (hMDH) activity of Halobacterium salinarum was studied as a function of micelle size (ω o ), in cethyltrimethylammonium bromide (CTAB)/cyclohexane reverse micelles, with 1-butanol as cosurfactant. The velocity dependence of the ω o profile depends on the buffer used, the surfactant concentration, and the salt concentration. In phosphate buffer, the activity increases with increasing water content, while in Tris/HCl buffer a bell-shaped profile is generally observed. Despite a slight change in the ω o -activity profile, the enzymatic activity was higher at low salt concentration even when we employed a different buffer. The ω o value for the maximal activity (optimum ω o ) varies directly with the enzyme concentration. The hMDH activity in reverse micelles depends on the surfactant concentration and the dependence of the activity of this enzyme on the surfactant concentration, at constant ω o , is different for each ω o value. © 2001 Elsevier Science B.V. All rights reserved. Keywords: HMDH; Halophilic enzyme; Reverse micelles; CTAB; ω o 1. Introduction One of the most striking features in reverse mi- cellar enzymology is the fact that the enzymatic activities vary depending on the system’s water content, which is usually expressed in terms of ω o ((H 2 O)/(surfactant)). This factor controls the diame- ter of the water pool surrounding the enzymes. The numerous enzymes studied previously appear to dis- play a very similar dependence. This dependence can present in three different forms: (a) the satura- tion curve in which the ω o value does not have an effect on enzyme activity; (b) the bell shaped curve ∗ Corresponding author. Present address: Department of Medi- cine, Division of Rheumatology, Thomas Jefferson University, Philadelphia, PA 19107, USA. Tel.: +1-215-503-5565. E-mail address: sonsoles.piera@mail.tju.edu (S. Piera-Vel´ azquez). and (c) a curve in which enzyme activity decreases continuously as ω o increases, due to decreases in the conformational mobility of the enzyme at low ω o . Almost all enzymes have been found to exhibit a bell-shaped dependence of the activity on ω o . In gen- eral, the maximum activity is found to occur around that ω o value at which the size of the reverse micelle is somewhat larger than that of the entrapped enzyme. This characteristic is commonly attributed to confor- mational changes of the solubilized enzymes [1], the state of the solubilized water, and the ionic nature of the surfactant head groups [2]. The rates of reactions catalyzed by enzymes sol- ubilized in reverse micelles depend upon both the water and surfactant concentrations and not upon the degree of hydration or micellar diameter. However, it is possible that the reaction rate in a specific reverse micelle may be dependent on its diameter [3]. Buffer 1381-1177/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII:S1381-1177(00)00228-9