International Journal of Biological Macromolecules 43 (2008) 339–345 Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac The influence of the support nature on the kinetics parameters, inhibition constants and reactivation of immobilized acetylcholinesterase Katya Gabrovska a , Ivaylo Marinov a , Tzonka Godjevargova a,∗ , Marianna Portaccio b,c , Maria Lepore c , Valentina Grano c , Nadia Diano c , Damiano Gustavo Mita b,c a University “Prof. Dr. A. Zlatarov”, Department of Biotechnology, Prof. Y. Yakimov Street 1, 8010 Bourgas, Bulgaria b Department of Experimental Medicine, Second University of Naples, Via Costantinopoli 16, 80138 Naples, Italy c Institute of Genetics and Biophysics of CNR, Via P. Castellino 111, 80129 Naples, Italy article info Article history: Received 13 May 2008 Received in revised form 2 July 2008 Accepted 3 July 2008 Available online 15 July 2008 Keywords: Acetylcholinesterase Inhibition Modification abstract Acetylcholinesterase (AChE) was immobilized on two different composite membranes constituted by a chemically modified poly-acrylonitrile (PAN) membrane plus a layer of tethered chitosan of different molecular weight, 10 kDa or 400 kDa. AChE was also directly immobilized on a chemically modified PAN membrane with NaOH and ethylenediamine (EDA) without chitosan. To know how the different supports affected the enzyme activity and the kinetic parameters, the AChE activity was studied in the soluble form and in the insoluble form with all the three types of modified PAN membranes. The best performance was obtained by the modified PAN membrane having the chitosan with the lower molecular weight. The results concerning the AChE inhibition by methyl-paraoxon and the subsequent reactivation by pyridine-2-aldoxime methochloride (2-PAM) are presented and discussed. The composite membrane having chitosan with the lower molecular weight appeared to be potentially useful for applications in the field of biosensors. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Enzymes, usually used as biocatalysts in biotechnological pro- cesses, are preferred to chemical catalysts because they are more selective and more efficient. Immobilization is considered an important technique since it enhances enzyme stability. Actually, the enzyme stability is greatly dependent on the immobilization strategy [1–3]. Several polymers have been selected for enzyme immobilization intended for a variety of applications. Among these, polyacryloni- trile (PAN) is very versatile and convenient due to its hydrophilic nature, good blood compatibility, high chemical and mechanical stability, and resistance toward microbial and enzymatic attacks [4–6]. One of the trends in the preparation of supports suitable for enzyme immobilization is to complement the mechanical and chemical stability of PAN in the presence of functional groups in biopolymers such as chitosan and gelatin. Application of chi- tosan in ultrafiltration membranes has been reported by several authors [7–9]. Musale et al. [10] prepared PAN/chitosan composite ∗ Corresponding author. Tel.: +359 56 858 353; fax: +359 56 820 249. E-mail address: godjevargova@yahoo.com (T. Godjevargova). ultrafiltration membranes by filtrating chitosan solution through PAN base membrane and subsequent curing and treatment with NaOH. A few publications deal with covalent immobilization of enzymes onto chemically modified membranes of AN copolymer [11]. Among immobilization techniques, covalent attachment has a higher commercial potential because it ensures more time stabil- ity with a longer retention of catalytic activity. The method also offers reusability of expensive supports after the inactivation of immobilized enzyme [12–14]. The kinetics of immobilized enzyme has been extensively stud- ied [15,16]. The charge of the carrier is of a great importance for enzyme kinetics [11]. The matrix to which enzyme is ultimately attached, indeed, has an important influence on the properties of the enzyme such as activity, reaction rate, substrate specificity, inhibitors action, kinetic constants, etc. Acetylcholinesterase (AChE, E.C. 3.1.1.7) plays in vivo a key role in cholinergic transmission by catalysing the rapid hydrolysis of the neurotransmitter acetylcholine (ACh) into acetate and choline [17]. The enzyme effectively terminates the chemical impulse at rates that are similar to a diffusion-controlled process allowing a rapid and repetitive response [18–20]. In the brain, acetylcholine is involved in learning and memory. In Alzheimer’s disease there is a shortage of acetylcholine [21]. Presently, in the frame of the 0141-8130/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2008.07.006