Research paper Improved activity of enzymes in mixed cationic reverse micelles with imidazolium-based surfactants Dibyendu Das, Debapratim Das, Prasanta Kumar Das * Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700 032, India Received 18 September 2007; accepted 9 November 2007 Available online 17 November 2007 Abstract This work reports the signiﬁcant enhancement in performance of interfacially active enzymes, Chromobacterium viscosum (CV) lipase and horseradish peroxidase (HRP) in mixed reverse micelles of cetyltrimethylammonium bromide (CTAB) and imidazolium-based amphiphiles having varying tail lengths. Lipase activity in these mixed systems was always higher than that in the individual cationic reverse micelles of CTAB or any imidazolium surfactant, highest being observed in the mixed system of CTAB (50 mM) and 6 (1-tetradecyl-3-methyl imidazolium bromide, 40 mM)/water/isooctane/n-hexanol (0.24 M), second-order rate constant, k 2 ¼ 1301  5 cm 3 g 1 s 1 , w200% higher compared to that in CTAB and w65% more than the most popular AOT-microemulsion. Activity increased with concentration of imidazolium surfactant and also with its alkyl tail length. To have a more profound view on the structureeactivity relationship, CTAB was replaced by cetyltriethylammonium bromide (CTEAB) and cetyltripropylammonium bromide (CTPAB) with subsequent increase in the headgroup size. The generalized inﬂuence of these mixed cationic systems on surface-active enzyme was also veriﬁed using HRP, where the activity improved w100%. This enhancement in enzyme activity is presumably due to the activating effect of the imidazolium cation in the enzymatic reactions by improving the nucleophilicity of interfacial water in vicinity of enzyme through hydrogen bonding. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Amphiphiles; Enzymes; Hydrophobic tail; Imidazole; Reverse micelles 1. Introduction Enzymology in self-organized aggregates [1e4] has been an area of wide interest over the past few decades. Activity of enzymes in reverse micelles has gained momentum primar- ily due to their potential technological and biotechnological applications [5e9]. Earlier works have shown enzymes to be highly active in anionic reverse micelle of Aerosol OT (AOT), conventionally regarded as superior host for surface- active enzyme [10e14]. In contrast, cationic systems due to their positive charge are known to inhibit [12,15e16] the sur- face-active enzymes by interacting at the active site. However, in our previous studies we have been progressively successful in utilizing cationic water-in-oil (w/o) microemulsions as proﬁcient hosts for hydrophilic (trypsin) and surface-active enzymes (lipase, HRP) [17e24] by changing the structure of surfactant. These modiﬁcations resulted marked improvement in activity, which is sometimes even signiﬁcantly better than that observed in AOT-reverse micelles. While, mixed reverse micelles [25e26] having cationic/anionic, and/or cationic/ nonionic components were also used to improve the enzyme’s efﬁciency [27e31] as it confronts reduced cationic charge [27,32]. However, binary systems of two different cationic am- phiphiles have not yet been explored in micellar enzymology since they are not expected to reduce the inhibiting cationic charge [12,15,16]. At this point, the role of exogenous imidaz- ole and its cation is recognized to enhance the activity of lipase and HRP by helping in the respective catalytic process [33e37]. For instance, Lin and coworkers showed notable improvement in lipase-catalyzed transesteriﬁcation in organic solvents by doping with both N-methylimidazole and its * Corresponding author. Tel.: þ91 33 24734971; fax: þ91 33 24732805. E-mail address: bcpkd@iacs.res.in (P.K. Das). 0300-9084/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2007.11.005 Available online at www.sciencedirect.com Biochimie 90 (2008) 820e829 www.elsevier.com/locate/biochi