Journal of Molecular Catalysis B: Enzymatic 97 (2013) 203–208 Contents lists available at ScienceDirect Journal of Molecular Catalysis B: Enzymatic jo ur nal home p age: www.elsevier.com/locate/molcatb A spectroscopic characterization of a phenolic natural mediator in the laccase biocatalytic reaction Andrea Martorana a , Lorenzo Sorace b , Harry Boer c , Rafael Vazquez-Duhalt d , Riccardo Basosi a,∗ , Maria Camilla Baratto a,∗∗ a Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A. Moro 2, 53100 Siena, Italy b Department of Chemistry and INSTM RU, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy c VTT Technical Research Centre of Finland, P.O. BOX 1000, FI-02044 Espoo, Finland d Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico, Apartado Postal 150-3, Cuernavaca, Moorelos 62210, Mexico a r t i c l e i n f o Article history: Received 25 March 2013 Received in revised form 21 August 2013 Accepted 22 August 2013 Available online 4 September 2013 Keywords: Acetosyringone Radical intermediate Laccase Multifrequency ESR NALDI-TOF MS a b s t r a c t Multi-frequency ESR combined with NALDI-TOF MS has been used for the characterization of 3,5- dimethoxy-4-hydroxyacetophenone radical intermediate and by-products formed during the Coriolopsis gallica laccase catalytic reaction. A stable radical species is formed and an intense and well-structured ESR spectrum was detected and fully characterized at S-, X- and W-bands. The presence of by-products generated as the result of by-reactions has been investigated and analyzed through NALDI-TOF MS, per- forming the experiments versus time. The superior radical stability of such phenoxy radical, due to steric hindrance in ortho to the phenol group and the great delocalization of the unpaired electron on the acetyl substituent, makes acetosyringone particularly interesting for biotechnological applications. This represents a good example for the development of new stable laccase mediator molecules. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Several studies have been carried out using 3,5-dimethoxy- 4-hydroxyacetophenone, also called acetosyringone, as natural laccase mediator. Acetosyringone and some other natural lignin- derived phenols, such as syringaldehyde, are promising eco- friendly mediators for dye degradation in terms of both efficiency and velocity of oxidation [1,2]. Among the dimethoxy phenols, acetosyringone resulted the first compound showing a considerable radical stability and could represent a good example for the production of new mediator molecules. The large amount of free radical produced has allowed the identification and full characterization of the radical interme- diate generated during the catalytic reaction mediated by laccase. In the past, evidence of such a stable radical was observed at the X-band ESR (Electron Spin Resonance) [3,4], but it was produced by horseradish peroxidase and ligninase in the presence of H 2 O 2 . In this work, for the first time, the radical was generated by laccase ∗ Corresponding author. Tel.: +39 0577 234240; fax: +39 0577 234239. ∗∗ Corresponding author. Tel.: +39 0577 234247; fax: +39 0577 234239. E-mail addresses: riccardo.basosi@unisi.it (R. Basosi), mariacamilla.baratto@unisi.it (M.C. Baratto). and O 2 and an accurate determination of the magnetic parameters, through a multifrequency (S-, X-, W-band) ESR approach, has been achieved. Laccase (p-diphenol:oxygen oxidoreductase E.C.1.10.3.2) belongs to copper-containing oxidases that catalyze the oxi- dation of a wide range of substrates using molecular oxygen as co-substrate. During its catalytic process, laccase removes a single electron from the substrate generating free radicals, with the concomitant reduction of oxygen to water [5]. Due to steric hindrance factors and to its redox potential between 0.5 and 0.8 V, laccase is unable to oxidize non-phenolic lignin units which have a high-redox potential (>1.5 V) and it can only oxidize phenolic units at the substrate surface. Furthermore it is known that fatty acids present in lignocellulosic pulps are responsible for “pitch prob- lems” which can be overcome using laccase, modifying liphophilic extractives [6–9]. To overcome the accessibility problem and to extend the ability of non-phenolic lignin unit oxidation, laccase is often used in the presence of an oxidizing small molecule, called mediator, which acts as a sort of electron shuttle between the enzyme and the lignin [10–14] (see Scheme 1). Once oxidized, the mediator (Med ox ), represented by the phenolic compound, diffuses away from the enzymatic pocket and in turn oxidizes other molecules, extending the range of substrates susceptible to the enzymatic action. In this way, laccases are able to oxidize 1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molcatb.2013.08.013