Colloids and Surfaces B: Biointerfaces 73 (2009) 132–139 Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb Interaction of cellulase with cationic surfactants: Using surfactant membrane selective electrodes and fluorescence spectroscopy Ali Asghar Rastegari a , Abdol-Khalegh Bordbar b,∗ , Asghar Taheri-Kafrani b a Department of Biological Science, Science & Research Branch, Islamic Azad University (IAU), Tehran, Iran b Laboratory of Biophysical Chemistry, Department of Chemistry, University of Isfahan, Hezar Jereeb Street, Isfahan 81746-73441, Iran article info Article history: Received 1 December 2008 Received in revised form 7 May 2009 Accepted 12 May 2009 Available online 19 May 2009 Keywords: Cellulase n-Alkyl trimethyl ammonium bromides Binding isotherm Surfactant selective electrode Binding capacity Fluorescence spectroscopy abstract The interaction of cationic surfactants, n-alkyl trimethyl ammonium bromides (C n TAB, n = 12 and 14), with cellulase from Aspergillus niger has been investigated at 25 ◦ C and various pH, using C n TAB-membrane selective electrodes as a simple, fast, cheap and accurate technique and fluorescence spectroscopy. The regions of C 1 (the surfactant concentration at which binding is initiated) and C 2 (enzyme saturated by surfactant) were determined using potentiometric measurements. The obtained binding isotherms have been analyzed using Scatchard plot and binding capacity concept. The results were interpreted on the basis of nature of forces which interfered in the interaction and represent two binding sets system for all of the studied conditions. Hill equation parameters have been estimated and used for calculation of intrinsic Gibbs free energy that decreases with extension of binding. The effect of C n TAB binding on cellulase intrinsic fluorescence spectra was also examined. A biphasic behavior was observed for quenching process of endoglucanase by C n TAB that confirms the results of binding studies correspond to the existence of two types of binding sites for C n TAB on cellulase. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Cellulases are the group of hydrolytic enzymes which are able to hydrolyze insoluble cellulose to soluble glucose. Cellulose, a polymer of -1,4-linked glucose units, is the major polysaccharide constituent part of plant cell walls and one of the most abun- dant organic compounds in the biosphere and bioenergy processes. This enzyme is produced by microorganisms, plants, and animals (in this case by symbiotic microorganisms). The cellulase sys- tem consists of several distinct enzymes. There are three types of enzymes that have been traditionally assigned to the cellulase system: endoglucanases (endo-1,4--glucanases or 1,4--d-glucan 4-glucanohydrolases, EC 3.2.1.4), cellobiohydrolases (exo-1,4-- glucanases or 1,4--d-glucan cellobiohydrolases, EC 3.2.1.9l), and cellobiases (-glucosidases or -d-glucoside glucohydrolases, EC 3.2.1.21) [1,2]. Enhancement of enzymatic cellulose hydrolysis by adding sur- factants has been reported by several authors [3–6]. It is believed that surfactants adsorb to the cellulose during hydrolysis. This is in agreement with Ooshima et al. [5], who claimed that the adsorption of the cationic surfactant Q-86W on cellulose obeys the ∗ Corresponding author. Tel.: +98 311 7932710/913 167 7331 (mobile); fax: +98 311 6689732. E-mail addresses: bordbar@chem.ui.ac.ir, khalegh bordbar@yahoo.com (A.-K. Bordbar). Langmuir isotherm. Kim et al. suggested that surfactants adsorb at the air–liquid interface and may thus prevent enzyme denatura- tion during agitation in the hydrolysis mixture [7]. Based on kinetic analysis, Kaar and Holtzapple have indicated that surfactants can promote the availability of reactant sites, which would increase the hydrolysis rate [8]. The surfactant can also increase the stability of the enzymes and thus, reduce enzyme denaturation during the hydrolysis [9]. The following mechanism is usually proposed for interaction of ionic surfactants with globular proteins; at first, the binding of charged head groups of the surfactants to sites with opposite charge at the surface of the protein occurred. This phenomenon is subse- quently followed by unfolding and exposure of the hydrophobic interior sites and numerous hydrophobic binding sites [10]. There is experimental evidence indicates that the resultant unfolded protein–surfactant complex consists of the protein chain has been wrapped around the micelle-like surfactant aggregates [11]. Several methods such as calorimetry, electromotive force mea- surement, nuclear magnetic resonance (NMR), and fluorescence spectra employing different probes to comprehend the aggregation behavior of biopolymer and ionic surfactants [12,13]. Fluorescence spectroscopy has been an important tool in the study of the protein dynamics conformation. Tryptophan fluorescence is sensitive to its environments and is thus a useful structural probe. For evaluation the mechanism of surfactant denaturation, a very accurate measurement of the binding data is essential [14]. Poten- tiometry is a valuable technique that has been frequently used to 0927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.05.010