Enzyme and Microbial Technology 33 (2003) 854–862 Purification, characterization and sequence analysis of a laccase from the ascomycete Mauginiella sp. Hetti Palonen, Markku Saloheimo, Liisa Viikari, Kristiina Kruus∗ VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT, Finland Received 8 January 2003; accepted 9 July 2003 Abstract A laccase from the ascomycete Mauginiella sp. was purified to electrophoretic homogeneity and biochemically characterized. The molecular mass of the laccase was 63 kDa as determined by mass spectrometry and it existed as six isoforms with isoelectric points in the range of 4.8–6.4. The laccase showed activity towards the typical substrates: 2,2′ -azinobis-(3-ethylbenzothiazoline)-6-sulphonate (ABTS), guaiacol, dimethoxyphenol (2,6-DMP), and syringaldazine. The pH optima on guaiacol, 2,6-DMP and ABTS were 4, 3.5 and 2.4, respectively. The enzyme was strongly, 98%, inhibited by 1 mM NaN3 and 88% by 1 mM KCN. The laccase was stable at neutral pH, retaining 80% activity after 24 h at pH 6–8. The enzyme was sensitive to high temperatures: the half-life at 70 ◦ C was only 3 min. A fragment of the laccase gene was isolated and its nucleotide sequence was determined. The laccase gene showed high identity to the laccase genes lcc1 and lcc2 of the basidiomycetes Trametes versicolor and Trametes villosa, respectively. © 2003 Elsevier Inc. All rights reserved. Keywords: Laccase; Mauginiella sp.; Purification; Characterization; Amino acid sequence; Gene sequence 1. Introduction Laccases (p-diphenol: dioxygen oxidoreductase, EC 1.10.3.2) are multi-copper enzymes, which catalyze the oxidation of a variety of organic and inorganic substrates coupled to the reduction of molecular oxygen to water with the one-electron oxidation mechanism. Laccases catalyze the oxidation of a broad range of substrates e.g. polyphenols, substituted phenols, diamines, but also some inorganic compounds [1]. These enzymes are widely distributed enzymes in nature. Laccase or laccase-like activity has been demonstrated in higher plants, in some insects and in a few bacteria [2]. However, the best-known laccases are of fungal origin, especially those belonging to the class of white-rot fungi. Laccases of plant origin are reported to play an important, role in wound response and lignin synthesis [3], whereas in fungi they are involved in lignin degradation [4,5], as well as in several other functions, including pigmentation, fruiting body formation, sporulation, and pathogenesis [1]. Due to their interesting catalytic properties laccases have gained considerable interest in various industrial areas. The most intensively studied applications have included pulp delignification, textile dye bleaching, effluent ∗ Corresponding author. Tel.: +358-9-456-5143; fax: +358-9-456-2103. E-mail address: Kristiina.Kruus@vtt.fi (K. Kruus). 0141-0229/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0141-0229(03)00247-3 detoxification, detergent components, as well as biopolymer modification [2]. In order to further develop enzymatic oxidation technologies for industrial purposes, we screened for novel fungal laccases in the VTT culture collection. The strain VTT D-84228, deposited apparently incorrectly as Geotrichum candidum, L-3 [6], was found to be an efficient laccase producer. The strain was originally isolated from a North-American soil sample and was reported to produce an aniline oxidase, a peroxidase [7] and a laccase [8]. The culture filtrate of this microbe has been used for the detoxification of contaminated soil [8]. In addition, the strain has shown an ability to decolorize melanin produced by blue-stain fungi [9]. During the course of this work the fungal strain was re-identified as belonging to the genus Mauginiella, whose species has not yet been re-identified. Mauginiella, not a well-known fungus, has cell wall and hyphal septa characteristic of anamorphic ascomycete and may be related to Monilia [10]. Cavara [11] first described the fungus from Libya. The species was rediscovered in Morocco and renamed as Geotrichum scattae by Maire and Werner [12]. The Mauginiella strain used in this work was originally attributed apparently incorrectly as Geotrichum candidum, L-3 (ATCC 26195). The taxonomy of Geotrichum is complicated, since several species demonstrating considerably variation have been described in this genus [13]. Many Mauginiella sp. have been incorrectly H. Palonen et al. / Enzyme and Microbial Technology 33 (2003) 854–862 described as Geotrichum sp. For example, in Microfungus Collection & Herbarium, University of Alberta (UAMH), a number of strains, originally filed as Geotrichum sp. were later assigned to Mauginiella [13]. We report here the purification, characterization and sequence analysis of the major laccase isoform from Mauginiella sp. 2. Materials and methods 2.1. Fungal strain Mauginiella sp. (VTT D-84228), originally isolated from sand loam (USA), was maintained on potato dextrose agar plates at 4 ◦ C. The strain was originally identified as Galactomyces geotrichum (ATCC 26195; anamorph Geotrichum candidum). However during this study it was re-identified as Mauginiella sp. (Dr. D.W. Minter, CABI Bioscience Identification Service, UK). Members of this genus produce conidia in branched chains by fragmentation of pre-existing hyphae. 2.2. Media and culture conditions The fungus was cultivated in liquid media (YM Broth, Difco Laboratories, USA) containing (per litre) 3.0 g yeast extract, 3.0 g malt extract, 5.0 g peptone, 10.0 g dextrose and 0.04 l mineral stock solution containing (per litre) 1.0 g CaCl2 ·2H2 O (Merck, Germany), 1.0 g FeSO4 ·7H2 O (Merck, Germany), 0.1 g ZnSO4 ·7H2 O (Merck, Germany), 0.16 g CuSO4 ·5H2 O (Merck, Germany), 1.0 g Na2 EDTA (Merck, Germany). First, 100 ml of medium was inoculated with agar pieces, cut from well-grown mycelium on potato dextrose agar. After two days cultivation at 30 ◦ C under agitation (120 rpm), 50 ml of the culture was used to inoculate 500-ml culture medium and the fungus was further grown at 30 ◦ C under agitation (120 rpm). After 8 days the cells were removed by filtration trough filter paper (Whatman no. 1). The clear supernatant was frozen and used for purification. 2.3. Protein and enzyme activity determinations Protein concentration was determined using the Bio-Rad DC protein assay kit (Bio-Rad, USA) with bovine serum albumin as a standard. Laccase activity was determined according to Niku-Paavola et al. [14] using 2 mM ABTS, [2, 2′ -azino-bis-(3-ethylbenzothiazoline)-6-sulphonate], (Boehringer Mannheim, Germany) as a substrate. The oxidation of dimethoxyphenol (2,6-DMP) was performed by the method reported by Heinzkill et al. [15], oxidation of guaiacol was measured according to Paszczynski et al. [16], and oxidation of syringaldazine according to Leonowicz and Grywnowicz [17]. The activity towards tyrosine was measured according to Lerch and Ettlinger [18]. All the activity measurements were performed in 25 mM succinate buffer 855 (pH 4.5) at 25 ◦ C using a two-beam spectrophotometer (Lambda 20, Perkin-Elmer, Germany). The activities were expressed as katals (mol/s). 2.4. Purification of the laccase The proteins were precipitated from the cell-free fermentation broth with ammonium sulphate (80% saturation). The precipitate was dissolved in 20 mM Tris-buffer (pH 7.7) and the extra salt was removed by gel filtration (65 ml; Bio-Gel P-6DG Desalting Gel; Bio-Rad, USA). The sample in 20 mM Tris-buffer pH 7.7 was applied to a Q-Sepharose anion exchange column (106 ml; Pharmacia, Sweden), that was equilibrated with 20 mM Tris-buffer, pH 7.7. Proteins were eluted with a linear gradient (400 ml) of Na2 SO4 (0–300 mM) in Tris-buffer. The eluate was monitored for absorbance at 280 nm, conductivity and laccase activity. Laccase-positive fractions of the major peak were pooled and Na2 SO4 was added to the final concentration of 500 mM. The sample was applied to Phenyl Sepharose Fast Flow hydrophobic interaction chromatography column (33 ml, Pharmacia) equilibrated with 500 mM Na2 SO4 in 20 mM Tris-buffer (pH 7.7). Proteins were eluted with a decreasing (500–0 mM) Na2 SO4 gradient (120 ml) in Tris-buffer pH 7.7. Laccase-positive fractions were pooled and excessive salt was removed by ultrafiltration (Amicon 8010 ultrafiltration unit; PM10 membrane, Millipore, USA). Finally the enzyme was concentrated with ultrafiltration, as described above. The purified laccase preparation was further analyzed by anion exchange chromatography in a Mono-Q column (1 ml; Pharmacia, Sweden) using ÄKTA explorer (Pharmacia Biotech, Sweden) in order to separate the isoforms. The purified laccase sample was applied to the column, which was equilibrated with 10 mM Tris-buffer, pH 7.7. Proteins were eluted with a shallow, linear gradient of Na2 SO4 (0–135 mM; 20 ml). Purification was followed by SDS–PAGE and isoelectric focusing (IEF) analysis. All purification steps were performed at room temperature. 2.5. Determination of molecular mass by mass spectrometry The molecular mass was determined by MALDI-TOF mass spectrometry on a BiflexTM time-of-flight instrument (Bruker–Daltonics, Germany) equipped with a nitrogen laser operating at 337 nm. The protein sample was desalted using Millipore C4 ZipTipTM (USA) and eluted with 0.1% trifluoroacetic acid in 60% acetonitrile. The eluate was analyzed in the linear positive ion mode employed with delayed extraction using saturated sinapic acid in a mixture of 0.1% trifluoroacetic acid and 50% acetonitrile (1:2) as a matrix. Samples were prepared by mixing 1 ␮l of protein with 1 ␮l of sinapic acid matrix on the target plate and dried under a gentle stream of warm air. The mass spectra 856 H. Palonen et al. / Enzyme and Microbial Technology 33 (2003) 854–862 were calibrated externally with bovine serum albumin as a standard. 2.10. Deglycosylation The optical absorption spectrum of the purified Mauginiella laccase was measured at 250–750 nm with a Varian Cary100 spectrophotometer (Varian Inc., Australia). The effect of deglycosylation on the isoelectric point and molecular mass of the laccase was analyzed. Deglycosylation was carried out using endo-H endoglycosidase (Glyko, USA) according to the manufacturer’s instructions. Native isoelectric focusing and SDS–PAGE analysis were performed from the deglycosylated samples. 2.7. Inhibition of laccase activity 2.11. Sequence analysis The effect of potential inhibitors (EDTA, H2 O2 , KCN, l-cysteine, NaN3 ) on laccase activity was tested by oxygen consumption method using oxygen electrode (Orion Research 081010) at room temperature under atmospheric air saturation in sealed and fully filled 30-ml Erlenmeyer flasks. Oxygen consumption rates were measured from solutions containing 2 mM ABTS, 20 ␮g laccase and 1 mM of various inhibitors in 50 mM citrate buffer (pH 5). The N-terminal amino acid sequence and two internal sequences of the tryptic digests of purified laccase were carried out by the Edman degradation method using a PE Biosystems Procise Sequencer (PE Biosystems, USA). The primer sequences for the PCR approach were based on the N-terminus and an internal amino acid sequence. Genomic DNA was isolated from 5-day-old mycelium. The mycelium were washed and lyophilized before DNA isolation by Easy DNA kit (Invitrogen, USA) following the manufacturers instructions. A fragment of the laccase gene was amplified by PCR. The PCR-primers were constructed using the published DNA sequence of T. villosa LCC2 [20]. The forward primer was 5′ -GCC ATC GGG CCG GTC GCC AGC CTC GT-3′ and the reverse primer 5′ -CCC GGC GAA CCC AAC CGT TCC GAA-3′ . PCR program had an initial denaturation at 94 ◦ C for 3 min followed by 25 cycles of 94 ◦ C for 0.75 min, 55 ◦ C for 0.5 min and 72 ◦ C for 1.5 min. A 1200 bp fragment was obtained from the PCR. It was purified from agarose gel (QIAquick PCR purification kit; Qiagen, USA) and cloned in to TOPO TA-cloning vector (Invitrogen, USA) following the manufacturer’s instructions. The plasmid was isolated using QIAprep Spin Miniprep-kit (Qiagen, USA). DNA sequencing of the plasmid was carried out in both directions with appropriate primers. The sequencing reactions were performed using the ABI PRISM dRhodamin Terminator Cycle Sequencing Kit (Perkin Elmer, Germany). 2.6. Spectrum 2.8. Thermal stability and pH dependency The pH optima of laccase on ABTS, 2,6-dimetoxyphenol (2,6-DMP) and guaiacol as substrates were determined within a pH range of 2.3–7.0 using McIlvaine universal buffer, diluted 1:4 with water. The stability of the enzyme at different pH values was determined by incubating a laccase solution (50 nkat/ml) in McIlvaine buffer at a pH range from 2.3 to 8.0, and in Tris-buffer at a pH range from 8.0 to 9.8. The residual activity was measured at pH 4.5 using ABTS as substrate. The temperature stability of the laccase was determined at 50, 60, and 70 ◦ C for various time periods. The enzyme solution (50 nkat/ml) was incubated in 60 mM citrate buffer (pH 6) and the residual enzyme activity was measured at pH 4.5 and temperature of 25 ◦ C using ABTS as substrate. 2.9. Electrophoresis and isoelectric focusing SDS–PAGE was performed using homogenous (12.5%) polyacrylamide slab gels on PhastSystem (Pharmacia, Sweden) and following the manufacture’s instruction. Alternatively, homogenous (12.0%) Tris–HCl Ready Gels (Bio-Rad, USA) were used according to Laemmli [19]. The low molecular weight calibration mixture (Pharmacia, Sweden) was used as a standard. Protein bands were visualized by staining with Silver stain (Bio-Rad, USA). The isoelectric point of the laccase was determined by IEF within a pH range of 3.5–9.5 (Ampholine PAGplates; Pharmacia, Sweden) on a LKB 2117 Multiphor II Electrophoresis System (LKB Pharmacia, Sweden) according to the manufacturer’s instructions. The pH gradient was measured with a surface electrode from the gel. Laccase activity was visualized by staining with 0.1% ABTS in 25 mM succinate buffer (pH 4.5). 3. Results 3.1. Enzyme production and purification The fungus Mauginiella produced substantially high amount of laccase in liquid culture. The organism was cultivated on a rich medium without any known inducers for laccase. The fungus grew well and the production of laccase started after four to five day’s cultivation. Within 8 days, a laccase activity of 27 nkat/ml was reached. A three-step purification procedure was developed for this enzyme (Table 1). Ammonium sulfate precipitation followed by anion exchange and hydrophobic interaction chromatography enriched the laccase by 82-fold. In anion exchange chromatography (Q-Sepharose), laccase activity was eluted at 30–150 mM Na2 SO4 concentration as two peaks. The H. Palonen et al. / Enzyme and Microbial Technology 33 (2003) 854–862 857 Table 1 Purification of laccase from Mauginiella sp. Purification step Volume (ml) Activity (nkat/ml) Protein (mg/ml) Specific activity (nkat/mg) Activity yield (%) Purification factor Culture filtrate (NH4 )2 SO4 precipitation Anion exchange HIC Concentrate 1300 65 57 42 9 26 419 286 310 1507 1.8 2.4 0.8 0.3 1.0 14 177 381 1192 1449 100 81 48 39 40 1 12 26 82 100 The specific activities were determined at pH 4.5. major laccase-containing fraction, eluted at 30–50 mM salt concentration, was further purified by hydrophobic interaction chromatography. The overall laccase activity yield was 40% (Table 1). The purified laccase showed intense blue color, typical for laccases. The other laccase-containing fraction from the anion exchange column eluted at 80–150 mM salt concentration, comprising about 5% of the total laccase activity, was not purified and characterized in this work. 3.2. Characterization of the purified laccase Purification of the laccase from Mauginiella resulted in a single protein band on SDS–PAGE with a molecular mass of 67 kDa as visualized by silver staining (shown in Fig. 1A). According to the mass spectrometry analysis the molecular mass of the laccase was 63.3 kDa. The crude enzyme showed at least eight active isoforms in the IEF (Fig. 2). The minor laccase-active fraction in the anion exchange chromatography contained the isoforms with pI at 2.9–3.5. The purified preparation showed six laccase-active bands in IEF with estimated pIs of 4.8, 5.0, 5.2, 5.6, 6.0 and 6.4 (Fig. 2). The laccase active band at pI 6.4 had the faintest color intensity, and the most intense bands were detected at a pI range 5.0–5.6. Silver staining showed also six bands (result not shown). Deglycosylation of laccase by Endo H resulted in about 5–7% decrease of molecular mass as determined on SDS–PAGE (Fig. 1A). Deglycosylation modified only slightly the isoelectric pattern. The number of the protein bands remained the same (Fig. 1B). Thus, the modifications of the N-glycan structures were obviously not the reason for the multiple isoforms shown on IEF. In order to further separate the isoforms, the purified laccase was loaded on Mono-Q column and the proteins were eluted with a very shallow salt gradient. The laccase active fractions eluted at 10–50 mM sodium sulfate concentration as six partially overlapping peaks. The isoelectric focusing revealed that the different isoforms were not completely separated (data not shown). 3.3. Spectrum The purified laccase had an intense blue color characteristic for blue-copper proteins. The ultraviolet-visible adsorption spectrum showed two peaks at 279 and 610 nm and a shoulder at 330 nm. The peak at 610 nm is typical for the type I copper and the shoulder at 330 nm indicates the presence of the type III binuclear copper pair. 3.4. Inhibition and substrate specificity pattern The Mauginiella laccase showed activity towards the typical substrates of laccases. Table 2 presents the specific Fig. 1. The effect of deglycosylation on the molecular mass of purified Mauginiella laccase as analyzed by SDS–PAGE and silver staining (A) and isoelectric points as analyzed by IEF and subsequent active staining (B). Lanes: 1. Molecular weight marker (6.5, 16.5, 25, 32.5, 47.5, 62, 83, 175 kDa); 2 and 4; purified laccase and 3 and 5; deglycosylated laccase. Fig. 2. Isoelectric focusing and subsequent active staining with ABTS. Lane 1: culture filtrate of Mauginiella sp. (0.38 nkat), Lane 2: laccase after anion exchange (0.29 nkat). H. Palonen et al. / Enzyme and Microbial Technology 33 (2003) 854–862 Table 2 Specific activities of Mauginiella laccase towards different substrates: ABTS, guaiacol and 2,6-DMP at the optimum pH, pH 2.4, 4.0 and 3.5, respectively and syringaldazine, DOPA and tyrosine at pH 4.5 Substrate Specific activity (nkat/mg) Relative specific activity (%) ABTS Guaiacol 2,6-DMP Syringaldazine DOPA Tyrosine 2870 440 360 283 2.5 0 100 15.3 12.5 9.9 0.1 0 100 Residual activity (%) 858 80 60 40 20 0 0 30 60 90 1 20 Time (min) Table 3 Inhibition of Mauginiella laccase by some selected inhibitors at 1 mM concentration Inhibitor Inhibition (%) EDTA H2 O2 KCN l-Cysteine NaN3 48 54 88 0 98 activities of the purified laccase on various substrates. The specific activity was highest with ABTS, 2870 nkat/mg. Also guaiacol, 2,6-DMP, syringaldazine and DOPA were oxidized, although the specific activity with DOPA was very low. The Mauginiella laccase did not oxidize tyrosine. The Mauginiella laccase was strongly inhibited (98–88%) by 1 mM sodium azide and KCN (Table 3). It was also sensitive to H2 O2 and EDTA. l-Cysteine was not an inhibitor, actually it increased the oxygen consumption rate in the reaction mixture with laccase and ABTS as substrate. 3.5. Thermal stability and pH dependency Relative activity (%) The pH optima for the purified laccase were determined with ABTS, guaiacol and 2,6-DMP as substrates (Fig. 3). The laccase had optimal activity at the lowest limit of the pH range, pH 2.4, when ABTS was used as a substrate. 100 80 60 40 20 0 2 3 4 5 6 7 pH Fig. 3. The pH optima of Mauginiella laccase with ABTS (䉱), 2,6-DMP (䊊) and guaiacol (䊏). The value is the average of three independent experiments. Fig. 4. Thermal stability of Mauginiella laccase at 40 ◦ C (䉫), 50 ◦ C (䊊), 60 ◦ C (䊐) and 70 ◦ C (䉱). The pH was 6. The value is the average of three independent experiments. Oxidation of 2,6-DMP showed pH optimum around pH 3.5. The pH optimum for guaiacol was also in the acidic pH range, at pH 4.0. The laccase showed practically no activity at pH values higher than six with any of the tested substrates. However, the purified laccase remained stable within the pH range of 4.0–8.0 after 24-h incubation. At lower pH values the enzyme retained lower activity, e.g. at pH 2.4 approximately 40% residual activity was observed after a 24-h incubation (data not shown). The laccase was not stable at higher temperatures during prolonged incubation. Although the enzyme retained almost full activity at 40 ◦ C after 2-h incubation, it showed half-lives of about 40 min and 3 min at 60 and 70 ◦ C, respectively (Fig. 4). 3.6. Sequence analysis of the laccase protein and gene The N-terminal as well as the two internal amino acid sequences of the purified Mauginiella laccase were determined, altogether 52 amino acid residues (Fig. 5). When compared to the amino acid sequences of known laccases, 100% identity was found with two Trametes laccase sequences, namely T. villosa lcc2 gene (GenBank accession number L49377) coding a laccase form 3 [20] and T. versicolor laccase I (lccI cDNA, GenBank accession number U44430) [21]. In order to compare longer sequences of these laccases, a gene fragment encoding the Mauginiella-laccase (1200 bases), corresponding to about 52% of the laccase gene of T. versicolor laccase I, was cloned and sequenced (Fig. 5). This fragment covered the gene from the N-terminus to the site encoding the more distal of the two peptide sequences obtained from internal peptides. The cloned fragment of the laccase gene of Mauginiella showed the highest identity, 97% at the nucleotide level, to T. versicolor lcc1 gene. In addition, the fragment displayed 94% identity with the T. villosa lcc2 gene at the nucleotide level. The predicted protein coded by Mauginiella laccase gene was different from the T. versicolor laccase I only in one amino acid: an asparagine (N) residue near the N-terminus H. Palonen et al. / Enzyme and Microbial Technology 33 (2003) 854–862 859 Fig. 5. Nucleotide sequence of Mauginiella laccase (M-lac) aligned with the sequence of laccase I from T. versicolor (T-lac; GenBank accession number U44430). Vertical lines show the conserved base pairs. The peptide sequences obtained from the isolated Mauginiella laccase are underlined. The difference to T-lac in the predicted amino acid sequence is marked with a rectangle. 860 H. Palonen et al. / Enzyme and Microbial Technology 33 (2003) 854–862 was replaced by an aspartic acid (D). The asparagine residue is a potential N-glycosylation site in the Trametes laccase sequence [21]. The laccase coded by the Mauginiella laccase gene differed from the laccase coded by T. villosa lcc2 gene (L49377) by three amino acid residues. The Mauginiella laccase gene appeared to contain seven introns in the sequenced region at positions conserved with the Trametes genes. 4. Discussion The results reported here show that Mauginiella produces multiple extracellular laccase isoforms that can be separated by anion exchange chromatography into two fractions. The main activity with pI values ranging from 4.8 to 6.4 was purified and characterized in this work. The purified Mauginiella laccase had characteristics typical of fungal laccases. It was able to oxidize typical substrates for laccases: various phenolic compounds and ABTS. The highest activity was observed on ABTS, as reported for many other laccases like those from Pycnoporus cinnabarinus and Coriolus hirsutus [22] and for Trichophyton rubrum laccase [23]. The second highest activity was observed with guaiacol. For example, Melanocarpus albomyces laccase showed highest activity towards ABTS, followed by syringaldazine, 2,6-dimethoxyphenol, and guaiacol [24]. Mauginiella laccase was inhibited by typical inhibitors of laccase, however, l-cysteine did not inhibit enzyme activity. Inhibition of laccase activity was tested by monitoring the oxygen consumption in the reaction because it has been shown [25] that the observed inhibition effect of many sulfhydryl-containing compounds (e.g. l-cysteine) were actually caused by the reduction of the oxidized substrate and not by true inhibition of the enzyme. The pH optima of Mauginiella laccase were similar to those of other fungal laccases, which typically preferred an acidic environment [26]. Besides Mauginiella laccase, also laccases from Polyporus pinsitus, Rhizonia solani and Myceliophthora thermophila showed highest activity on ABTS at the lower limit of the pH range tested (pH 2.7) [27]. The enzyme was not stable at temperatures higher than 50 ◦ C during prolonged incubation. As a comparison, the laccase from Melanocarpus albomyces was reported to have a half-life of 5 h at 60 ◦ C [24], whereas the half-life of the Mauginiella laccase was only 40 min. Ligninolytic fungi often express either constitutively or under inductive conditions multiple laccase isoforms [26]. Separate genes encode the true isoforms and the genes can be divided into different gene families [28]. For example, Coprinus cinereus laccase has two isoforms [29], Pleurotus ostreatus at least four isoforms [30] and Cerrena maxima have three isoforms [31]. Trametes versicolor secreted several isoforms of laccases and the genes and/or cDNAs encoding six isoforms have been isolated [32]. The purified laccase from Trametes trogii had two isoforms with a similar molecular mass and identical N-terminal amino acid sequence [33]. Two laccase genes were cloned from P. cinnabarinus and two isoforms with pIs of 5.4 and 3.7 could be detected in culture filtrates of this fungus grown on cellulose [34,35]. Also heterogeneity in glycosylation has been suggested to produce multiple isoforms of laccases. For example, two minor active isoforms (pIs 2.85 and 2.65) were detected by isoelectric focusing of the homogenous, purified Pycnoporus cinnabarinus laccase and may have represented glycosylated isoforms of the enzyme. The predominant isoform had a pI of 3.0 [36]. SDS-electrophoresis of the Mauginiella laccase showed a single protein of 67 kDa. The purified laccase showed repeatedly six bands in the isoelectric focusing between pIs 4.8 and 6.4, indicating the presence of multiple isoforms. The most intense bands appeared at pI range from 5 to 5.6. Sequencing of the protein, however, resulted in one N-terminal sequence and homogeneous inner sequences thus implicating the existence of a single homogeneous polypeptide in the sample. The different isoforms of Mauginiella laccase were partially separated by anion exchange chromatography on Q-Sepharose and Mono-Q columns. Modification, or different amounts of N-glycans were obviously not the reason for several isoforms detected in the IEF, since deglycosylation of the laccase did not change the pI pattern. In conclusion, it was likely that the different pI forms of Mauginiella laccase were caused by post-translational modifications of a single polypeptide chain, and not by the heterogeneity of the N-glycans. The N-terminal and the internal amino acid sequences from Mauginiella laccase, altogether 52 amino acid residues, displayed 100% identity to the peptides corresponding to Trametes versicolor lcc1 and Trametes villosa lcc2 genes. Also the genomic laccase gene fragment, representing about 52% from the total Mauginiella laccase gene, was nearly identical to the corresponding regions of the Trametes genes (Fig. 5). This was unexpected, since the laccase sequences of Ascomycetes usually have identities of 20–30% compared to basidiomycete laccases. One possible explanation for our findings could be that Mauginiella could have obtained the laccase gene from a basidiomycete strain by horizontal gene transfer at a much later time than that of the separation between Ascomycetes and Basidiomycetes. Horizontal gene transfer is widely accepted with prokaryotes [37]. It has been recently shown to play a role in the evolution of fungi as well [38,39]. The Mauginiella laccase showed the highest level of identity to T. versicolor laccase I (U44430) [21]. T. versicolor laccases can be divided into two different groups (laccases A and B) based upon their pI values, and have been extensively characterized in terms of their spectroscopic properties: Laccase A contains isoelectric forms with low pI (3.07–3.27; two forms) and laccase B with high pI (4.64–6.76; 10 forms) [40,41]. The laccase I (U44430) is obviously a laccase B isoform [42]. Bourbonnais et al. [43] have purified and partially characterized two laccase H. Palonen et al. / Enzyme and Microbial Technology 33 (2003) 854–862 isoforms, namely laccase 1 and laccase 2, from 2.5-xylidine induced T. versicolor culture supernatant. Mauginiella laccase resembled the laccase 1 in terms of molecular weight and specific activity determined on ABTS. The Mauginiella laccase gene also showed high similarity to the lcc2 gene encoding for the laccase form 3 of T. villosa. T. villosa secretes at least three laccase forms and five laccase genes have been cloned [20,28]. The laccase form 3 coded by lcc2 has been reported to have pI values of 6.2, 6.5, and 6.8 and a pH optimum at pH 6 on ABTS [20], which differed considerably from the pH optimum of Mauginiella laccase (at pH 2.4). The T. villosa laccase form 3 has not been properly characterized, thus we are not able to conclude whether the Mauginiella laccase is similar in terms of other biochemical properties. Bordeleau and Bartha [6,7] have reported that Geotrichum candidum (ATCC 26195) produced heat sensitive aniline oxidase and peroxidase in liquid culture. Although aniline oxidase activity is not mentioned in the Enzyme Nomenclature, some laccases have been reported to oxidize substituted anilines [44]. Aniline oxidase activity might thus refer to laccase activity. Indeed, the same strain was later reported to produce an extracellular laccase [8], however, purification or characterization of this laccase has not been reported. 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