Polymeric biocatalytic membranes with immobilized thermostable phosphotriesterase G. Vitola a , R. Mazzei a,n , E. Fontananova a,n , E. Porzio b , G. Manco b , S.N. Gaeta c , L. Giorno a a Institute on Membrane Technology, National Research Council, ITM-CNR, via P. Bucci,17/C at University of Calabria Campus, 87036 Rende (Cosenza), Italy b Institute of Protein Biochemistry, National Research Council, via P. Castellino 111, 80131 Naples, Italy c GVS S.P.A., Via Roma 50, 40069 Zola Predosa (Bologna), Italy article info Article history: Received 6 April 2016 Received in revised form 9 June 2016 Accepted 10 June 2016 Available online 11 June 2016 Keywords: Biocatalytic membrane Enzyme covalent immobilization Organophosphorus compounds decontami- nation Polymeric membranes Membrane functionalization abstract In this work the catalytic performance of a mutant (named SsoPox W263F) of the thermophilic Phos- photriesterase-like lactonase (PLL) isolated from Sulfolobus solfataricus (SsoPox) was reported both on free and immobilized membrane systems using as substrate the pesticide paraoxon. The single mutant SsoPox W263F was immobilized on/into hydrophilic (Non standard grade Polyethersulphone, NGS-PES) and hydrophobic (Polyvinylidenefluoride, PVDF) membranes. Biocatalytic membrane systems able to work in liquid and vapour phase were developed aimed at decontamination of water and air, respec- tively. The work aimed at membranes characterization in terms of vapour and water permeability, free and immobilized enzyme catalytic and specific activity. Enzyme stability was also monitored for more than 5 months in order to evaluate the stability of developed systems. Results demonstrated that although free SsoPox W263F showed a higher performance up to 30 days it completely loses its activity after two months. On the contrary biocatalytic membranes showed an initial loss of activity in the first days (residual specific activity of about 54%when immobilized on/into NSG-PES and 5% when immobilized on/into PVDF) but any change in stability during more than 5 months. In the case of PVDF, the residual specific activity can be enhanced tuning the amount of immobilized SsoPox W263F, in fact it was demonstrated that there is a trade-off between amount of immobilized enzyme and catalytic performance. & 2016 Elsevier B.V. All rights reserved. 1. Introduction Organophosphates triesters (OPs) are a class of toxic com- pounds that have been employed in the agricultural industry as pesticides and insecticides; the warfare nerve agents (sarin, soman and VX) are also OPs [1]. The mechanism of action of these com- pounds is realized by the irreversible inhibition of the enzyme acetylcholinesterase, a key enzyme of the nervous system, re- sulting in accumulation of acetylcholine which interferes with muscular responses leading to death. OPs poisoning may occur through: (1) consumption of liquids or foods contaminated with nerve agents, (2) contact with con- taminated surfaces (3) terrorist acts or industrial accidents. Therefore, there is a strong necessity of different strategies-sys- tems for OPs treatment and a growing public concern regarding the protection from these substances. To date, several strategies for organophosphate decontamination have been reported in literature [2]. Current method for OPs decontamination can be divided into physical, chemical or enzymatic treatments. Physical methods aims just to remove the OPs contaminant from any given site, without operate theirs destruction. Chemical methods permit to operate the OPs decontamination in particular for materials surfaces, whereas they are not fully safe for victims. These methods consist of chemical reactions such as: hydrolysis [3], oxidation [4] or reduction [5]. Enzymatic methods are able to operate the OPs decontamination in less harsh conditions compared to previously reported methods. Owing to, the high costs or environmental concerns of the physical and chemical methods used for destruction of OPs, the enzymatic detoxification has become of great interest [6] also for theirs in situ monitoring. Bacterial phosphotriesterase are appealing due to their broad substrate specificity and high catalytic rate [7], but their stability in solution is low [8]. It is very well known that immobilization of enzyme gives the possibility to improve the catalytic stability of the biocatalyst. Membranes are the optimal support for the phosphotriesterase immobilization because they can mimic native microenvironment [9,10]. Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/memsci Journal of Membrane Science http://dx.doi.org/10.1016/j.memsci.2016.06.020 0376-7388/& 2016 Elsevier B.V. All rights reserved. n Corresponding authors. E-mail addresses: r.mazzei@itm.cnr.it (R. Mazzei), e.fontananova@itm.cnr.it (E. Fontananova). Journal of Membrane Science 516 (2016) 144–151