Characterization of an extracellular laccase, PbLac1, purified from Polyporus brumalis Keiko NAKADE 1 , Yuko NAKAGAWA, Akira YANO, Toshitsugu SATO, Yuichi SAKAMOTO* Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024-0003, Japan article info Article history: Received 23 January 2009 Received in revised form 11 May 2010 Accepted 11 May 2010 Available online 24 May 2010 Corresponding Editor: Marc Stadler Keywords: Biodegradation Dye decolorization Laccase Polyporus brumalis Redox potential abstract Polyporus brumalis (strain ibrc05015) secreted high amounts of laccases (Lacs) in liquid medium. With 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) as a substrate, Lac activity was 7.72 U ml 1 and this strain secreted a maximum 0.23 mg ml 1 of total protein. The enzyme, PbLac1 was purified to homogeneity using hydrophobic and anion-exchange chromatography. The purified PbLac1 had a molecular mass of 63.4 kDa as determined by polyacrylamide-gel electrophoresis. PbLac1 oxidized a wide range of substrates such as 3,4-dihydroxy L-phenylalanine (L-DOPA) and catechol, but not tysorine. The activity of PbLac1 was increased by addition of 10.0 mM Cu 2þ . PbLac1 could decolorize several industrial dyes, such as Remazol Brilliant Blue R known as model dyes of environmental xenobiotics. In addition, PbLac1 decolorized a wide range of sub- strates, such as the carcinogen, Poly R-478, in the presence of violuric acid as mediator. The E  value of PbLac1 was 0.80 V  0.01 versus normal hydrogen electrode, which is a very high redox potential compared to those of other basidiomycetous Lacs. These results suggest the potential utility of PbLac1 for industrial applications. ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Introduction Laccase (Lac; p-benzenediol:dioxygen oxidoreductase, EC 1.10.3.2) belongs to a group of polyphenol oxidases that con- tain copper atoms in their catalytic centers. Lac catalyzes the oxidation of single-electron from phenolic substrates or aromatic amines. Many organisms possess Lac-encoding genes with a wide range of biological roles. Plant Lacs are in- volved in the formation of the lignin polymers by radical- based mechanisms (Sterjiades et al. 1992; Liu et al. 1994; Ranocha et al. 2002; Hoopes & Dean 2004). Fungal Lacs are in- volved in various biological processes, such as lignin degradation (Leonowicz et al. 2001), fruiting-body formation (De Vries et al. 1986), and human and plant pathogenesis (Burke & Cairney 2002; Nosanchuk & Casadevall 2003; Langfelder et al. 2003). In addition, Lacs have potential indus- trial applications for processes such as pulp bleaching (Bourbonnais et al. 1995; Call & Mu ¨ cke 1997), dye decoloriza- tion (Chivukula & Renganathan 1995), polymer synthesis, and biosensing (Gomes et al. 2004). Lacs also have the ability to degrade environmentally persistent xenobiotics such as chlorophenols (Ullah et al. 2000; Ahn et al. 2002), pesticides (Amitai et al. 1998; Maruyama et al. 2006), and endocrine-dis- rupting chemicals (Fukuda et al. 2001; Tsutsumi et al. 2001; * Corresponding author. Tel.: þ81 197 68 2911; fax: þ81 197 68 3881. E-mail addresses: knakade@ibrc.or.jp (K. Nakade), sakamoto@ibrc.or.jp (Y. Sakamoto) Abbreviations: ABTS, 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt; BPB, Bromophenol Blue; L-DOPA, 3,4-dihydroxy L-phenylalanine; EDTA, ethylenediaminetetraacetic acid; ITS, internal transcribed spacer; Lac, Laccase; NBB, Naphthol Blue Black; NHE, normal hydrogen electrode; RBBR, Remazol Brilliant Blue R; 2,6-DMP, 2,6-dimethoxyphenol. 1 Tel.: þ81 197 68 2911; fax: þ81 197 68 3881. journal homepage: www.elsevier.com/locate/funbio fungal biology 114 (2010) 609 e618 1878-6146/$ e see front matter ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2010.05.002