Enzyme and Microbial Technology 46 (2010) 153–158 Contents lists available at ScienceDirect Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt Factors affecting the activation of pulps with laccase A. Suurnäkki a,∗ , T. Oksanen a , M. Orlandi b , L. Zoia b , C. Canevali c , L. Viikari d a VTT Technical Research Centre of Finland, P.O. Box 1000, Espoo FI-02044 VTT, Finland b Dipartimento di Scienze dell’ Ambiente e del Territorio, Università di Milano-Bicocca, Piazza della Scienza 1, 20126 Milano, Italy c Dipartimento di Scienza dei Materiali, Università di Milano-Bicocca, Via R. Cozzi, 53 Milano, Italy d Department of Applied Chemistry and Microbiology, P.O. Box 27, FI-00014 University of Helsinki, Finland article info Article history: Received 7 April 2009 Received in revised form 13 November 2009 Accepted 22 November 2009 Keywords: Activation of fibres Reactivity of pulps Laccase treatment Phenoxy radicals EPR abstract Activation of fibres by radical formation is the first step when aiming at oxidative coupling of new func- tional groups on the fibre bound lignin. In this work, factors affecting the amount of phenoxy radicals created to unbleached TMP, CTMP, softwood kraft and hardwood kraft pulp fibres in the laccase catalysed oxidation were determined by EPR. Laccase was able to catalyse the oxidation of all the pulps studied. The reactivity of the pulp was found to be affected by both the physical accessibility of lignin in the fibres and the chemistry of the surface lignin accessible to laccase. Laccase dosage, use of extra oxygen in the laccase catalysed radicalization reaction, treatment time and also the amount and type of low-molecular weight compounds (LMWC) present in the pulp were all found to contribute to the radical content of pulp fibres measured after the enzymatic reaction. It could not been excluded that two types of reac- tions take place during the radical formation in fibres. Within the fibre matrix there may be both fibre material bound and soluble lignin fragments differing with respect to accessibility, molecular weight or chemical structure which can be radicalized at various rates, and the formed radicals may also undergo cross-coupling reactions reducing the amount of the total radicals. © 2009 Elsevier Inc. All rights reserved. 1. Introduction Wood fibres contain unique properties currently exploited mainly in paper and paperboard manufacture. Fibre modification enhancing the existing fibre properties or even creating completely novel properties to fibres can be expected to broaden the applica- tion areas of wood fibres. In addition to traditional chemical and physical modification methods, enzymatic and chemo-enzymatic methods can be used for fibre modification. Recently, bond- ing of chemical components to lignin-rich wood fibres by using enzymatic functionalisation has been reported [1–4]. In the enzy- matic functionalisation oxidative enzymes are used both for the activation of fibre material and further bonding of additional func- tionalities to the fibres. Fibre activation is the first step in functionalisation of wood fibres [3,4]. Oxidative enzymes, such as laccases or peroxidases can be used to activate the surface lignin of lignin-rich pulps. Laccase catalyses an one-electron oxidation of phenolic hydroxyl groups to phenoxy radicals with concomitant reduction of oxygen to water [5,6]. Stoichiometrically, four moles of the phenolic substrate are oxidized in the one electron abstracting oxidative reaction lead- ing to the production of four phenoxy radicals and the reduction ∗ Corresponding author. E-mail address: anna.suurnakki@vtt.fi (A. Suurnäkki). of one mole of oxygen into water. Thus, the oxygen consumption in the reaction should be directly proportional with the production of phenoxy radicals. A linear correlation between the radical for- mation and oxygen consumption in beech wood fibres has indeed been reported [7]. In mechanical pulp, this correlation has not, however, always been confirmed [3]. It has been observed that these fibre-bound radicals are fairly stable [7,3]. The half-lives of radicals in larger lignin structures and in wood pulp can be from hours up to weeks because of the immobilization of the radicals in the lignin matrix [8,9]. In the oxidation of complex wood fibre materials laccases initiate the radical formation in lignin, lignans, and different types of lipophilic extractives that can further react non-enzymatically [3,10–13]. Due to the high reactivity of these radicals (either with each other or with a secondary substrate), further reactions leading to polymerisation, depolymerisation, co- polymerisation or grafting can be targeted. The radicals exploitable for further functionalisation of pulps are proposed to be the lignin- bound phenoxy radicals formed during laccase treatment rather than those delocalised to the lignin structure [4]. The enzymatic oxidation and activation of different fibrous substrates has previously been studied. Thermomechanical pulps, TMPs, [3,4], medium density fibreboard fibres, MDF [7,11], and kraft pulps with various kappa numbers [2,14] have been the target for oxidation. Various factors affect the radical formation in lignin rich pulp fibre materials by laccase treatment. In mechanical pulps, dissolved and colloidal substances (DCS) have been proposed to 0141-0229/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2009.11.009