Production, purification and partial enzymatic and molecular characterization of a laccase from the wood-rotting ascomycete Xylaria polymorpha

Enzyme and Microbial Technology 41 (2007) 785–793

Production, purification and partial enzymatic and molecular characterization of a laccase from the wood-rotting ascomycete Xylaria polymorpha Christiane Liers a,∗ , Ren´e Ullrich a , Marek Pecyna b , Dietmar Schlosser b , Martin Hofrichter a a

International Graduate School Zittau, Environmental Biotechnology, Markt 23, D-02763 Zittau, Germany b Helmholtz Centre for Environmental Research—UFZ, Department of Environmental Microbiology, D-04318 Leipzig, Germany Received 29 March 2007; received in revised form 3 July 2007; accepted 3 July 2007

Abstract The hard wood-colonizing ascomycete Xylaria polymorpha, that is seemingly lacking peroxidases, produces laccase as sole ligninolytic oxidoreductase. The fungus secreted the enzyme preferably during the growth in complex media based on tomato juice. Addition of 2,5-xylidine considerably stimulated laccase production (up to 14,000 U l−1 ). The enzyme was purified to homogeneity by anion exchange and size exclusion chromatography and characterized by biochemical and molecular methods. Xylaria laccase has a molecular mass of 67 kDa, a pI of 3.1 and an absorption maximum at 605 nm that is characteristic for blue copper proteins. It oxidized all typical laccase substrates including ABTS, 2,6-dimethoxyphenol, guaiacol as well as syringaldazine (catalytic efficiencies 3 × 103 to 7 × 104 M−1 s−1 ). The deduced amino acid sequence of one amplified laccase gene sequence between the copper binding regions 1 and 3 showed a high level of identity to some other laccases from ascomycetes. Furthermore, the sequence of an internal peptide fragment of the purified laccase was identical with an amino acid sequence deduced from the nucleotide sequence of the laccase gene. Xylaria laccase was found to oxidize a non-phenolic ␤-O-4 lignin model compound in presence of 1-hydroxybenzotriazole into the corresponding keto-form. The results of this study show that – in addition to ligninolytic basidiomycetes – also wood-dwelling ascomycetes can produce high titers of laccase that may be involved in the oxidation of lignin. © 2007 Elsevier Inc. All rights reserved. Keywords: Xylariaceae; Soft-rot; Phenol oxidase; Lignin model compound; Redox mediator; Copper binding region

1. Introduction Laccase (benzenediol:oxygen oxidoreductases, EC 1.10.3.2) belongs to a family of copper-containing polyphenol oxidases that are widespread in fungi, plants, animals and bacteria [1–3]. In fungal physiology, laccases are involved in plant pathogenesis, pigmentation, detoxification and lignin degradation. All of these functions are related to the oxidation of various organic compounds by means of dioxygen (O2 ), including monophenols, polyphenols, aromatic amines and their derivatives, to free radicals, which in turn can undergo both spontaneous chemical and enzymatic reactions. The type I blue copper ion in the

∗

Corresponding author. Tel.: +49 3583 612754; fax: +49 3583 612734. E-mail address: [email protected] (C. Liers).

0141-0229/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2007.07.002

active site accepts electrons from the substrate and transfers them to a trinuclear copper center associated with the reduction of O2 to H2 O [1,4]. The unstable substrate radicals (e.g. phenoxyl radicals resulting from phenol oxidation) may further react with laccase or undergo non-enzymatic reactions including hydration, disproportionation and polymerization. So it is generally accepted that laccases are involved both in polymerization and depolymerization processes of lignin, melanin and humic substances [1,5]. Although laccases have comparatively low redox potentials (0.5–0.8 eV), they have been shown to oxidize in vitro non-phenolic lignin model compounds (LMC) and polymeric lignin in the presence of synthetic redox mediators [e.g. 1-hydroxybenzotriazole (HBT), 2,2 -azino-bis(3ethylbenzothiazoline-6-sulfonic acid) (ABTS)] [3,6,7]. Later it turned out that also natural lignin-derived compounds (e.g.

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acetosyringone) could act as mediating substances [8]. The laccase-mediator concept says that small molecules (e.g. phenolics, N–OH–type compounds) are oxidized by laccase to semi-stable radicals. They act as diffusible oxidants which can attack recalcitrant molecules being not susceptible to the direct oxidation by laccase (e.g. non-phenolic lignin moieties [9]). While laccases of wood-colonizing basidiomycetes (whiterot fungi) have been thoroughly studied (not least also with respect to laccase-mediator interaction), and many of them purified and characterized on the protein and gene level, less is known on the enzymatic and molecular properties of laccases from wood-dwelling ascomycetes. There are several reports on the extensive degradation of hard wood by ascomycetes of the family Xylariaceae [10–14]. These fungi cause a special type of wood destruction, nowadays referred to as soft-rot type II (earlier regarded as a kind of white-rot; [15,16]), which is accompanied by strong cellulose and hemicellulose degradation and a moderate loss in lignin [17]. In a previous study, we showed the partial mineralization and solubilization of 14 C-labelled lignin by Xylaria hypoxylon and Xylaria polymorpha as well as the secretion of laccase as sole ligninolytic oxidoreductase [18]. Here we report on the production of X. polymorpha laccase at larger scale in a bioreactor, its purification and characterization including the oxidation of a non-phenolic lignin model dimer in the presence of HBT. 2. Materials and methods 2.1. Fungal strain and culture conditions X. polymorpha strain (RJe-004) was isolated from a beech trunk in the Hainich National Park (Germany) and is deposited in the fungal culture collection of the International Graduate School of Zittau [18]. Stock cultures were maintained on 2% malt extract agar (MEA) at 4 ◦ C in the dark, and inoculation material for liquid cultures was pregrown on MEA plates at 25 ◦ C for 10–14 days. The content of an agar plate was homogenized in 80 ml of a sterile NaCl solution (0.9%), and the mycelial suspension used to inoculate liquid cultures (5%, v/v). The complex liquid medium (TJM) consisted of eco-tomato juice (Albi & Co., Germany) and distilled water (50:50, v/v). Fungal cultures were agitated in 500-ml flasks containing 200 ml of TJM on a rotary shaker at 100 rpm and 24 ◦ C for 20 days. To stimulate laccase production, 2,5-xylidine dissolved in 70% ethanol was added to certain cultures (final concentration 5 mM) after 8 days of cultivation. Samples (1 ml of the culture liquid) were taken every 1–2 days, and the activity of laccase and pH was measured. Whole cultures were harvested just after the activity had reached its maximum level, filtrated and used for subsequent purification studies. Larger amounts of X. polymorpha laccase were produced in a 10-l stirredtank bioreactor (Biostat B; Braun Biotech International GmbH, Melsungen, Germany) containing 6 l of TJM. The bioreactor was inoculated with 1 l of a homogenized fungal suspension pre-cultured as described above in nonsupplemented TJM. After 3 days of cultivation, 2,5-xylidine (5 mM) was added to the medium. Fermentation was carried out under following conditions: 200 rpm stirrer speed, 2 l min−1 aeration rate and 24 ◦ C; the pH was not regulated. Laccase activity and pH were determined every 1–2 days during a total fermentation period of 16 days.

2.2. Enzyme assays Laccase activity was determined in the culture liquid (after removing the fungal mycelium by centrifugation) by following the oxidation of 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) at 420 nm [2]. Activity of purified laccase was additionally measured with syringaldazine,

2,6-dimethoxyphenol (DMP) or guaiacol [19,20]. Enzyme assays were carried out in 100 mM citrate/phosphate buffer (pH 4.5), and activities are given in international units (1 unit = 1 ␮mol min−1 ). All chemicals used were obtained from Sigma–Aldrich (Weinheim, Germany) and Merck (Darmstadt, Germany).

2.3. Enzyme purification Culture flasks (200 ml culture liquid) or the bioreactor (10 l) were harvested on days 14 and 16, respectively, when maximum laccase titers were present (6 or 11 days after addition of 2,5-xylidine). Fungal mycelium was removed by filtration (filter GF6; Schleicher & Schuell, Dassel, Germany) and the culture liquid concentrated 10-fold and dialyzed at 4 ◦ C in a Pall-Filtron system (Dreieich, Germany) using a 10-kDa cut-off filter cassette. Crude laccase was further purified by two steps of fast protein liquid chromatography (FPLC) using anion exchange and size exclusion separation media. In the first step, the concentrated crude enzyme was applied to a DEAE-sepharose FF column (weak anion exchanger, 16 mm × 100 mm, Amersham Biosciences, Freiburg, Germany) and eluted with a linear gradient of 0–0.6 M NaCl in 10 mM sodium acetate buffer pH 5.0 at a flow rate of 5 ml min−1 . Laccase-containing fractions were pooled, concentrated, dialyzed against 10 mM sodium acetate (10 kDa cut-off, Filtron Microsep; Filtron Technology Corp.) and loaded onto a HiLoad Superdex 200 prep grade column (16 mm × 600 mm, Amersham Biosciences) equilibrated with 50 mM sodium acetate buffer containing 0.1 M NaCl. Enzyme was eluted with the same buffer at a flow rate of 1 ml min−1 . Fractions containing laccase activity were pooled, concentrated, dialyzed against 10 mM sodium acetate buffer and stored at −20 ◦ C.

2.4. Enzyme characterization Molecular mass of laccase was determined by SDS-PAGE (Novex Xcell SureLock mini cell; Invitrogen, Karlsruhe, Germany) as described by Laemmli [21]. After electrophoretical separation, the gel was stained and protein bands were visualized with Colloidal Blue Staining Kit (Invitrogen). A low-molecular mass protein calibration kit was used as the standard (MBI Fermentas, St. Leon Roth, Germany). The isoelectric point (pI) of the enzyme was detected using a polyacrylamide gel (5%) with a pH range from 2 to 6 (Fluka Chemie GmbH, Buchs, Switzerland) and a gel electrophoresis unit (Hoefer SE 600 Ruby, Amersham Biosciences). Activity staining was carried out by incubating the gel in a DMP solution (100 mM) at room temperature in citrate/phosphate buffer (pH 4.5). Protein concentration was determined by the method of Bradford using the Roti® -Nanoquant Protein Assay Kit (Roth, Karlsruhe, Germany) with serum albumin as the standard. UV–vis absorption spectra of purified laccase were recorded in 10 mM sodium acetate buffer in the range 250–800 nm using a Carry 50 spectrophotometer (Varian, Darmstadt, Germany).

2.5. Kinetic parameters Michaelis–Menten (Km ) and catalytic constants (kcat ) of purified laccase were determined for ABTS, DMP, syringaldazine and guaiacol in citrate/phosphate buffer (pH 4.5). Lineweaver–Burk plots were made from the initial rates obtained at varying substrate concentrations.

2.6. Effect of pH and temperature To estimate the pH optimum of X. polymorpha laccase, the above-mentioned assays with ABTS, DMP and syringaldazine as substrates were applied at a temperature of 25 ◦ C, and the pH of the citrate/phosphate buffer was varied between 2 and 8. Stability concerning pH was tested after storing the purified enzyme at pH 3, 7 or 10 in 100 mM citrate/phosphate buffer for 4 h. The influence of temperature on laccase activity was studied at pH 4.5 by varying the cuvette temperature in the spectrophotometer between 5 and 75 ◦ C using an integrated Peltier element (Varian). Temperature stability was determined after different pre-incubation times (30–240 min, at 20–60 ◦ C) as the residual activity detectable with ABTS in citrate/phosphate buffer (100 mM, pH 4.5). The influence of chloride was tested under the same conditions but varying the concentration of NaCl between 10 and 1500 mM.

C. Liers et al. / Enzyme and Microbial Technology 41 (2007) 785–793

2.7. Deglycosylation Carbohydrate content of the purified laccase was estimated after enzymatic deglycosylation using a kit from Sigma–Aldrich according to the instructions of the manufacturer. Laccase (50 ␮g protein) was boiled with denaturation solution for 5 min and afterwards incubated with the deglycosylation kit at 37 ◦ C for 3 h. The change in molecular mass of the deglycosylated enzyme was re-estimated after SDS-PAGE as described above.

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USA). The amino acid sequence was predicted using BioEdit 7.4 (Ibis Therapeutics, Carlsbad, USA). Sequence similarities were calculated from alignments with laccase protein sequences from NCBI Genbank database using ClustalW program and substitution matrix Blosum62 [24]. Xylaria laccase sequence was submitted to GenBank (accession number bankit922971 EF694057).

2.11. Oxidation of a non-phenolic lignin model compound (LMC)

Mycelium of X. polymorpha grown in TJM in the presence of 2,5-xylidine was harvested when laccase activity started to increase in the culture. The culture liquid was separated by filtration and the fungal biomass lyophilized in a rotatory evaporator (Christ Alpha 1-4, Osterode, Germany). Total mRNA was isolated from a small portion of lyophilized biomass of induced cells using the Trizol reagent (Invitrogen). cDNA was synthesized from 3 ␮g of total DNA-free RNA using “RevertAidTM H Minus M-MuLV” reverse transcriptase (Fermentas) according to the manufacture protocols. cDNA obtained was used as target for laccase-specific, degenerated primers.

Enzymatic reactions were carried out in 10-ml test vials covered with parafilm and incubated on a magnet stirrer at 24 ◦ C. The reaction mixture (1 ml) contained 50 mM sodium acetate buffer (pH 5.0), 0.5 mM 1-hydroxybenzotriazole and 0.1 mM of a non-phenolic ␤-O-4 LMC [1-(3,4dimethoxyphenyl)-1-oxo-2-(2-methoxyphenoxy)-1,3-dihydroxypropane] that was synthesized as described by Hatakka [25]. The reaction was initiated by adding 1 U of purified X. polymorpha laccase, and additional enzyme (1 U) was added at 8-h intervals over a total reaction period of 24 h. Controls did not contain laccase. For comparison, a high-redox potential laccase from the whiterot basidiomycete Pycnoporus cinnabarinus (JenaBios GmbH, Jena, Germany) was used under identical conditions. The reaction was stopped by addition of 1 ml acetonitrile, and then the samples were centrifuged and analyzed by HPLC using a reversed phase C18 -column (Phenomenex® Synergi, 4.6 mm × 125 mm, Phenomenex Inc., Torrance, CA) and a HP 1100 liquid chromatograph (HewlettPackard, Waldbronn, Germany). Reaction products were eluted under isocratic conditions with 30% acetonitrile in 15 mM phosphoric acid at a flow rate of 1 ml min−1 and their identity was proven by comparing their retention times and UV spectra with authentic reference compounds (veratric acid, veratraldehyde, guaiacol and the keto-form of LMC; [26]). LC-MS studies were carried out using an Agilent LC-MSD system (Agilent Technologies, USA), and mass detection occurred in the positive API-ES (atmospheric pressure electro spray ionization) mode in the mass range of 100–1000.

2.10. PCR conditions and sequencing

3. Results

2.8. Protein sequencing The deglycosylated enzyme was loaded onto a SDS-PAGE gel. After electrophoresis, the protein was transferred to a polyvinylidene fluoride membrane (Amersham Biosciences) by electro-blotting and stained. N-terminal amino acid sequencing by Edman degradation and de-novo peptide sequencing using MALDI-MS-MS after partial digestion with trypsin were performed by Proteome Factory AG (Berlin, Germany).

2.9. mRNA extraction and cDNA synthesis

The degenerated primer pair Cu1AF (5 -ACM WCB GTY CAY TGG CAY GG-3 ) and Cu3R (5 -TG ICC RTG IAR RTG IAN IGG RTG-3 , B = CGT, I = inosine, M = CG, N = ACGT, R = AG, W = AT, Y = CT) was used for amplification of laccase gene fragments between the copper binding region cbr1 and cbr3. Primers were designed by Kellner et al. [22] and purchased from MWG Biotech (Ebersberg, Germany). For each amplification, 1 ␮l of cDNA (0.25 ␮g) was added to a 25 ␮l reaction mixture containing 12.5 ␮l PCR Master Mix (2×, Promega, Madison, USA) and 0.25 ␮l for each primer (1 ␮M). Gradient PCR was performed in a Tetrad® 2 Gradient Cycler (Bio-Rad, Hercules, USA) with initial denaturation (3 min at 95 ◦ C), 40 cycles (45 s at 95 ◦ C; 45 s Gradient 46–56 ◦ C; 3 min at 72 ◦ C) and a final elongation (10 min at 72 ◦ C). Amplified products were visualized onto 0.8% agarose gels and stained with ethidium bromide. After purification using the Wizard SV kit (Promega), the PCR products were cloned using pCR2.1 TA cloning kit (Invitrogen) according to manufacture’s instructions. Plasmids derived from cloning were verified by colony PCR [23] and sequenced afterwards. Sequencing was performed on ABI 3100 equipment using BigDye Sequencing Chemistry (both Applied Biosystems, Foster City,

3.1. Laccase production X. polymorpha produced moderate amounts of laccase in a complex medium based on tomato juice (TJM). After 1 week of growth in agitated culture, the fungus formed characteristic pellets and the medium changed its color from deep red to pale reddish. Secretion of laccase started on day 11 and its appearance was accompanied by the black coloring of TJM as well as by a drastic increase in pH from 4.5 to 7.8 (Fig. 1A). The maximum level of laccase activity (1100 U l−1 ) was reached on day 14. Supplementation of the medium with 2,5-xylidine on day 8 considerably stimulated laccase production and resulted in a maximum level of 10,500 U l−1 . Also in this case, laccase production was accompanied by a drastic change in color (from

Fig. 1. Time-course of laccase production by X. polymorpha in TJM; (A) agitated liquid cultures in 500-ml flasks containing 200 ml medium. (B) 10-liter bioreactor containing 6 l TJM. Black diamonds: activities without induction, black dashed line: pH; black circles: activities after addition of 2,5-xylidine, black solid line: pH; Data points represent means of three replicates with standard deviation (<5%). Arrows indicate the time of 2,5-xylidine addition.

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Fig. 2. Purification of X. polymorpha laccase by FPLC. Ion exchange chromatography on DEAE-sepharose (A) and size exclusion chromatography on a Superdex column (B), solid lines: absorbance at 280 nm, black circles: laccase activity assayed with ABTS, dashed lines: NaCl gradient.

red to purple), and the pH increased from 4.3 to 5.7 (Fig. 1A). X. polymorpha did not show any activity of peroxidase during the growth in TJM neither in the presence nor in the absence of xylidine. Laccase production at larger scale was performed in a 10l stirred-tank bioreactor and the highest activity was again observed when the inductor xylidine was added (on the 3rd day of fermentation). The scale up of the process, from 0.2 to 6 l, did not have a negative effect on the laccase level and on contrary, the latter even increased by 36%. Laccase production started in the bioreactor on day 13 (152 U l−1 ) and increased rapidly within the next 3 days to reach a final activity of 14,380 U l−1 on day 16 (Fig. 1B). Again, enzyme production was accompanied by an increase in pH (from 4.3 to 6.4) and the purple coloring of pellets. 3.2. Enzyme purification One liter fractions of the harvested culture liquid were concentrated, dialyzed and further purified by two chromatographic steps using an FPLC-system. In the first step, ruby colored

Fig. 3. SDS-PAGE (right) and native isoelectric focusing (left) of purified X. polymorpha laccase (arrows). Lane 1: IEF gel of purified laccase (pI 3.1), lane 2: purified laccase, lane 3: deglycosylated laccase, lane 4: protein standard.

pigments (probably tomato ingredients and/or xylidine polymerization products) were removed by a DEAE-sepharose passage (a weak anion exchanger where the pigments were tightly bound) (Fig. 2A). The second separation step was carried out using a Superdex 200 size exclusion column and resulted in a distinct protein peak in the elution profile and a greenish-blue fraction with high laccase activity (after concentration 823 U mg−1 protein) (Fig. 2B). The purification procedure led to an activity loss of approximate 70%, which may be explained by the binding of laccase to TJM components, which were removed during purification (Table 1). 3.3. Laccase characterization The electrophoretic analysis of purified laccase showed a single protein band with a molecular mass of 67 kDa and a distinct acidic pI of 3.1. After deglycosylation, the molecular mass decreased by 5 kDa indicating a carbohydrate content of 7.5% (Fig. 3). The UV–vis absorption spectrum of the enzyme showed two characteristic absorption maxima at 280 and 605 nm, which

Table 1 Purification of laccase from X. polymorpha Purification step

Protein (mg)

Culture liquid Ultrafiltration DEAE-sepharose FF Superdex 200

112.6 81.5 26.2 5.0

Total activity (U) 14,380 10,760 10,830 4,460

Specific activity (U mg−1 ) 127.7 132.0 412.4 823.2

Yield (%) 100 75 75 31

Purification (x-fold) 1.0 3.2 6.4

C. Liers et al. / Enzyme and Microbial Technology 41 (2007) 785–793

Fig. 4. UV–vis spectrum of purified X. polymorpha laccase (160 ␮M) in 10 mM sodium acetate buffer.

indicates the presences of type I copper (Cu2+ ) that is responsible for the blue color of laccases. A shoulder at 330 nm indicating the presence of a type III binuclear copper pair was not observed (Fig. 4). Purified Xylaria laccase was relatively stable at 20 ◦ C and lost about 5% of its activity within 4 h; higher temperatures caused a more rapid inactivation of the enzyme. So the loss of activity at 40 ◦ C was almost linear and amounted to 10% per hour, whereas at 60 ◦ C, the enzyme lost its activity almost completely within

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1 h (Fig. 5A). On the other hand, X. polymorpha laccase was stable at neutral and basic pH (pH 10) with activity losses of 3 and 14% within 4 h, respectively, whereas an acidic pH of 3 caused a rapid decrease in laccase activity (69% within the first 30 min, almost 100% within 4 h) (Fig 5B). There was no clear activity maximum concerning the oxidation of ABTS; highest activity was observed at pH 2.5, then it decreased towards higher pH values to become zero at pH 7.0. The curve for DMP looks similar though it shows a maximum at pH 3, and the optimum of syringaldazine oxidation is between pH 4 and 5 (Fig. 5E). The influence of temperature on the activity of purified laccase was studied in the range from 5 to 75 ◦ C using ABTS as the substrate (Fig. 5D). Highest activity was observed between 55 and 60 ◦ C and the enzyme reached still 20% of its maximum activity at 5 ◦ C. NaCl had an inhibitory effect on laccase; a concentration of 6 mM caused a decrease in enzyme activity of 10%. On the other hand, the enzyme remained partly stable over a wide range of NaCl concentrations and exhibited still 20% of its initial activity at a concentration as high as 1500 mM NaCl (Fig. 5C). 3.4. Amino acid sequence and molecular analyses Determination of the N-terminal sequence was unsuccessful probably due to the N-terminal blocking by pyroglutamic acid [27]. Comparison of an amino acid fragment of purified Xylaria laccase (I/LVNTAIDTMFK) obtained by de-novo pep-

Fig. 5. Stability of purified X. polymorpha laccase at different temperatures (A), pH values (B) and NaCl concentrations (C); (A) 20 ◦ C (diamonds), 40 ◦ C (circles), 60 ◦ C (squares) in 50 mM citrate/phosphate buffer (pH 4.5). (B) pH 7 (circles), pH 10 (squares), pH 3 (diamonds) in 50 mM citrate/phosphate buffer at 25 ◦ C. (C) 0–1500 mM NaCl in 50 mM citrate/phosphate buffer (pH 4.5). Effect of temperature (D) and pH (E) on the activity of purified X. polymorpha laccase; (D) temperature of 5–75 ◦ C in 50 mM citrate/phosphate buffer (pH 4.5). (E) ABTS (diamonds), DMP (circles), syringaldazine (squares) in 50 mM citrate/phosphate buffer (pH 4.5). Data points represent means of three replicates with standard deviations <5%.

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Fig. 6. Deduced protein sequence between copper binding region cbr1 and cbr3 encoded by a laccase gene fragment of X. polymorpha. Copper binding regions are marked in dark-grey with white letters. An internal peptide fragment identical with a de-novo peptide sequence of purified Xylaria laccase is written in bold italics.

tide sequencing (MALDI-MS-MS) with the enzyme database BRENDA showed 91% sequence identity with laccase fragments encoded by the ascomycetes Gaeumannomyces graminis var. graminis (IVNTAIDTHFK) and G. graminis var. tritici (LVNTAIDTHFK), respectively. Only one transcribed laccase gene was found in the cDNA which encoded a polypeptide of 334 amino acids. The deduced amino acid sequence between cbr1 and cbr3 (Fig. 6) showed the highest level of identity and similarity with laccases of other ascomycetous fungi (53/69.5% G. graminis var. tritici, 52/70% G. graminis var. graminis [28]; 51/68.2% Cryphonectria parasitica [29]; 51/65.6% Glomerella cingulata [30]; 38/56% Melanocarpus albomyces [31]). Furthermore, we could clearly identify the de-novo peptide fragment (I/LVNTAIDTMFK) obtained from the purified Xylaria laccase within an internal sequence (LVNTAIDTMFK) of the deduced polypeptide from the induced and transcribed laccase gene (Fig. 6). Only one amino acid position was uncertain due to the impossibility to distinguish between leucine and isoleucine by mass spectrometry. 3.5. Kinetic constants X. polymorpha laccase oxidized a number of substrates including monoaromatic (DMP, guaiacol) and complex (syringaldazine) phenols as well as the non-phenolic heterocyclic compound ABTS (Table 2). The Michaelis–Menten constant (Km ) for ABTS oxidation was calculated to be 20 ␮M, which is relatively high compared to Km -values of other fungal laccases. The enzyme showed the highest affinity (Km = 3.5 ␮M) to and catalytic efficiency (kcat /Km = 56,809 s−1 mM−1 ) for syringaldazine. In contrast, the Km -values for DMP (97.5 ␮M) and guaiacol (2625 ␮M) were noticeably higher indicating a lower affinity of the enzyme to smaller and less substituted phenols. The catalytic constants (kcat ) ranged between 200 s−1 for syringaldazine and 7400 s−1 for guaiacol. 3.6. Oxidation of a non-phenolic LMC by the couple laccase/HBT HPLC analyses demonstrated the ability of X. polymorpha laccase to oxidize a non-phenolic ␤-O-4 LMC in presence of Table 2 Kinetic constants of purified laccase from X. polymorpha Substrate

Km (␮M)

kcat (s−1 )

kcat /Km (s−1 mM−1 )

ABTS 2,6-DMP Syringaldazine Guaiacol

20 97.5 3.5 2625

550 247 199 7384

27,504 2,535 56,809 2,813

the redox mediator HBT (Fig. 7A, LMC: peak 1). The laccase/HBT couple oxidized about 18% of the LMC within 24 h; laccase alone was not able to convert the model compound (data not shown). P. cinnabarinus laccase tested for comparison was more efficient and oxidized 68% under identical conditions. The keto-form of LMC [1-(3,4-dimethoxyphenyl)-1-oxo-2-(2methoxyphenoxy)-3-hydroxypropane] was identified in both cases as the major oxidation product by comparing its retention time and UV spectrum with the authentic reference substance (Fig. 7A, peak 2). Moreover, the molecular mass of the predicted keto-form of LMC was confirmed by LC-MS analysis (Fig. 7B). The mass spectrum of the oxidation product shows a molecule mass signal of m/z 333.2 that fits well to the protonated LMC-ketone (Mw 333.2 Da). Other potential metabolites such as 3,4-dimethoxybenzaldehyde, 3,4-dimethoxybenzoic acid or guaiacol could not be observed but an unidentified hydrophilic product probably originating from HBT was found in the HPLC elution profile (Fig. 7A, peak 3). LMC was not converted when laccase or HBT was omitted from the reaction mixture and also other potential mediators, such as unsaturated fatty acids (linoleic acid or its derivative Tween 80) did not enhance LMC oxidation by the Xylaria laccase/HBT couple (data not shown). 4. Discussion The hardwood-dwelling ascomycete X. polymorpha, that was earlier found to convert a 14 C-labelled lignin into 14 CO2 and water-soluble 14 C-fragments [18], secretes laccase as the only potential ligninolytic enzyme. High amounts of this laccase were produced in diluted tomato juice in stirred-tank bioreactors. Its purification required anion exchange and size exclusion chromatographic steps and gave an enzyme preparation with typical laccase characteristics and the ability to oxidize a non-phenolic lignin model compound in a mediator-coupled reaction. To our best knowledge, laccases from other xylariaceous fungi have not been purified and characterized so far. Thus, the findings reported here may help to better understand the degradative system of these most efficient wood-rotters among the ascomycetes. Stimulation of fungal growth and enzyme production in plantbased complex media (e.g. tomato juice) is a phenomenon that has been reported for other fungi as well. Such media contain hundreds of different ingredients (e.g. tannins, flavonoids, aromatic glycosides), many of which may somehow promote fungal activities and hence rather reflect natural conditions [1,32–34]. Noticeable stimulation of Xylaria laccase production was achieved by the addition of 2,5-xylidine to TJM at a relatively high concentration (5 mM). The necessity to convert this toxic aniline may stimulate laccase production on the molecular

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Fig. 7. HPLC elution profile (A) and LC-MS mass spectrum (B) of oxidation products obtained after treatment of a non-phenolic LMC with X. polymorpha laccase and in the presence of HBT. Non-phenolic LMC (1) and its major oxidation product (2) [1-(3,4-dimethoxyphenyl)-1-oxo-2-(2-methoxyphenoxy)-3-hydroxypropane] after 24 h of incubation, as well as an unidentified reaction product (3).

level and finally lead to the oxidative coupling and detoxification of the compound [2,36,37]. Although laccases are mostly secreted in multiple forms [32,33], SDS-PAGE and isoelectric focusing indicated the presence of only one acidic isoform of Xylaria laccase, whose molecular mass (67 kDa), isoelectric point (pI 3.1), carbohydrate content (7.5%) and spectral properties are in the typical range of fungal laccases [1,33,34]. Thus, the Xylaria laccase has similar activity optima as well as stability criteria towards pH and temperature as laccases from other ascomycetes and basidiomycetes. Laccases from the thermophilic ascomycete M. albomyces or the wood-rotting fungi Pleurotus ostreatus and Cerrena unicolor also tolerate alkaline conditions (pH 8–9) and show moderate activities towards phenolic compounds [35–37]. Since laccase secretion by X. polymorpha was accompanied with a drastic increase in pH from 4.5 to 7.8 (Fig. 1A), it can be assumed that neutral and slightly alkaline conditions are suitable for the enzyme’s stability and catalytic activity. The activity loss of Xylaria laccase with respect to the NaCl concentration was relatively low (still 20% of the maximum activity was observed at 1.5 M NaCl) whereas other laccases were reported to loose their activities almost completely at NaCl concentrations above 0.2 M [38–40]. This remarkable property in combination with the stability under alkaline conditions may be of interest from the biotechnological point of view because some potential applications of laccase refer to extreme environments (e.g. wastewater, pulps) [41]. The kinetic parameters of Xylaria laccase for ABTS (20 ␮M, 550 s−1 ) indicate that the enzyme has a higher affinity to and efficiency for this substrate than the laccases from the ascomycetes M. albomyces (280 ␮M, 75 s−1 ; [42]) and Chaetomium thermophilum (190 ␮M, <10 s−1 ; [43]) as well as the “average laccase” (39 ␮M, 400 s−1 ) described by Baldrian [33]. The Km -

value of Xylaria laccase for syringaldazine (3.5 ␮M) is very low and comparable to that of M. albomyces laccase (1.3 ␮M; [42]), indicating a preference for highly substituted phenols (in comparison, the Km -values for the phenolics DMP and guaiacol were found to be 97.5 and 2625 ␮M, respectively). Different physiological studies clearly indicate that laccase is the only ligninolytic oxidoreductase secreted by X. polymorpha and related fungi [1,18,33,36,44–46]; and ligninolytic peroxidases, which are thought to be the key enzymes of lignin biodegradation by basidiomycetes [5,47,48], are seemingly lacking in ascomycetes. Thus, if laccase is the only biocatalyst, that can oxidatively attack lignin, the enzyme will have to “manage” somehow the problem of recalcitrant non-phenolic lignin structures (not least against the background of moderate lignin mineralization observed for Xylaria spp.; [18]). Though laccases are not able to oxidize non-phenolic lignin structures directly, they can do it in the presence of suitable redox mediators [49,50]. Xylaria laccase oxidized a non-phenolic LMC when HBT was present, even if it was not as efficient as the high-redox potential laccase of P. cinnabarinus (−0.78 V [3]) that was tested for comparison. Maybe there are other factors (e.g. kinetic parameters and enzyme stability) which influence the efficiency of laccase-mediator couples and enable Xylaria laccase to oxidize the non-phenolic LMC [6,51,52]. Further investigations are warranted to ascertain the existence of natural mediators. The deduced amino acids of Xylaria laccase show high levels of homology to laccases of phytopathogenic ascomycetes of the related order Diaporthales (X. polymorpha belongs to the order of Sphaeriales, [53]). So the highest identity level for both the identified sequence between cbr1 and cbr3 and the internal peptid fragment was found for the protein sequence of a laccase from G. graminis var. tritici, a phytopathogen that causes the

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chestnut blight and forms heavily melanized hyphae during the infection process [28,44]. In conclusion, our study indicates a certain potential of X. polymorpha laccase to modify non-phenolic lignin structures in the presence of suitable mediators. Furthermore, in addition to partial ligninolysis, this enzyme may be involved in the melanization process (formation of melanin lamellae in wood) that is characteristic for xylariaceous ascomycetes [15,18,54]. Future studies will focus on the search for natural mediators (e.g. in wood extracts) as well as on the role of Xylaria laccase in melanization. Acknowledgements Financial support by the Federal State of Saxony (HWP program), the integrated EU project BIORENEW, the DBU project “Fungal secretoms”, and the administration of the International Graduate School Zittau (R. Konschak) is gratefully acknowledged. We thank Martin Kluge (Inge) and Matthias Kinne (Konrad) for useful comments and Ulrike Schneider and Monika Brandt for technical assistance. Furthermore, we thank Dr. Magali Sol´e, Dr. B¨arbel Kiesel and Dr. Sabine Kleinsteuber from the Helmholtz Centre for Environmental Research for providing access to the laboratory facilities and technical equipments and Harald Kellner for appropriation of degenerated primers. References [1] Thurston CF. The structure and function of fungal laccases. Microbiology 1994;140:19–26. [2] Eggert C, Temp U, Dean JF, Eriksson KE. A fungal metabolite mediates degradation of non-phenolic lignin structures and synthetic lignin by laccase. FEBS Lett 1996;391:144–8. [3] Li K, Xu F, Eriksson KE. Comparison of fungal laccases and redox mediators in oxidation of a nonphenolic lignin model compound. Appl Environ Microbiol 1999;65:2654–60. [4] Riva S. Laccases: blue enzymes for green chemistry. Trends Biotechnol 2006;24:219–26. [5] Hatakka A. Biodegradation of Lignin. Biopolymers: lignin, humic substances and coal, vol. 1. Weinheim: Wiley-VCH; 2001. pp. 129– 180. [6] Li K, Helm RF, Eriksson KEL. Mechanistic studies of the oxidation of a non-phenolic lignin model compound by the laccase/1hydroxybenzotriazole redox system. Biotechnol Appl Biochem 1998;27:239–43. [7] Bourbonnais R, Paice MG, Freiermuth B, Bodie E, Borneman S. Reactivities of various mediators and laccases with kraft pulp and lignin model compounds. Appl Environ Microbiol 1997;63:4627–32. [8] Camarero S, Ibarra D, Martinez MJ, Martinez AT. Lignin-derived compounds as efficient laccase mediators for decolorization of different types of recalcitrant dyes. Appl Environ Microbiol 2005;71:1775–84. [9] Baiocco P, Barreca AM, Fabbrini M, Galli C, Gentili P. Promoting laccase activity towards non-phenolic substrates: a mechanistic investigation with some laccase-mediator systems. Org Biomol Chem 2003;1:191–7. [10] Merrill W, French DW, Wood FA. Decay of wood by species of the Xylariaceae. Phytopathology 1964;54:56–8. [11] Sutherland JB, Crawford DL. Lignin and glucan degradation by species of the Xylariaceae. Trans Br Mycol Soc 1981;76:335–7. [12] Nilson T, Daniel G, Kirk TK, Obst JR. Chemistry and microscopy of wood decay by some higher ascomycetes. Holzforschung 1989;43: 11–8. [13] Blanchette RA. Delignification by wood-decay fungi. Annu Rev Phytopathol 1991;29:381–98.

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