• ascomycetes;
  • aquatic hyphomycetes;
  • endocrine-disrupting chemicals;
  • technical nonylphenol;
  • laccase genes;
  • semi-quantitative PCR


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

We investigated the influence of potential laccase inducers with environmental relevance on extracellular laccase activity and removal of the xenoestrogen technical nonylphenol (tNP) by the aquatic hyphomycete Clavariopsis aquatica. Concomitantly, we identified two putative laccase gene fragments (lcc1 and lcc2) and have followed their expression during removal of tNP under different conditions. Our results indicate a significant effect of copper on extracellular laccase activity in supernatants of fungal cultures. Laccase activity was highest in the presence of copper when added together with vanillic acid, followed by copper when used alone. Only slight laccase activities were recorded in the presence of only vanillic acid, whereas in the absence of either compound laccase activities were negligible. Laccase activity was well correlated with the removal efficiency of tNP, indicating the involvement of laccase in tNP bioconversion. Overall, lcc2 was less expressed than lcc1. The expression of lcc1 and lcc2 correlated only partially with the measured laccase activity, suggesting the existence of cell-associated laccase fractions not detectable in fungal culture supernatants and/or the existence of additional laccase genes.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Technical nonylphenol (tNP) is used for the production of nonylphenol ethoxylate surfactants (NPEOs) and represents an isomeric mixture of mainly p-substituted phenols with various branched nonyl chains (Corvini et al., 2004, 2006). It is formed from NPEOs due to their incomplete biodegradation in wastewater treatment plants (WWTPs), and is discharged into aquatic ecosystems via WWTP effluents (Ying et al., 2002). Increasing concerns with respect to the largely uncertain environmental fate of tNP and its potentially adverse environmental and human health effects result from the reported ability of the compound to disrupt normal endocrine functions in vertebrates (Ying et al., 2002; Kim et al., 2004) and its resistance to biodegradation (Corvini et al., 2006). Consequently, microorganisms capable of eliminating tNP have increasingly gained attention (Corvini et al., 2006).

Aquatic hyphomycetes (AQH) represent a group of exclusively aquatic mitosporic fungi with diverse phylogenetic affiliations (Belliveau & Bärlocher, 2005). They have been demonstrated to metabolize various aquatic environmental pollutants including tNP (Junghanns et al., 2005, 2008; Martin et al., 2007a), and hence are relevant when microbial attack on organic pollutants in aquatic environments is considered.

Laccases are extracellular multicopper oxidases found frequently among fungi, which are able to oxidize certain lignin constituents and also many xenobiotic compounds quite unspecifically (Baldrian, 2006). Laccases from aquatic and terrestrial fungi have been shown to act on nonylphenols (Corvini et al., 2006). Laccase production by AQH has been shown (Abdel-Raheem & Ali, 2004; Junghanns et al., 2005). Clavariopsis aquatica, an AQH with a known ascomycete affiliation (Kirk et al., 2001), was shown to catalyze the biotransformation of tNP under conditions where extracellular laccase activity was absent (Junghanns et al., 2005). In addition, a laccase preparation isolated from this organism under laccase-producing conditions was demonstrated to oxidize tNP in cell-free systems (Junghanns et al., 2005). Hence, besides cell-bound reactions, extracellular laccase may contribute to tNP biotransformation by the entire organism when produced.

Despite numerous reports describing laccase gene structures in basidio- and ascomycetes including yeast (Hoegger et al., 2006; Tetsch et al., 2006; Martin et al., 2007b), no data on laccase genes in AQH and the regulation of their expression have been published so far.

The aim of the present study was to identify potential laccase gene sequences in C. aquatica, and to investigate the effect of potentially laccase-inducing compounds with environmental relevance on transcript levels of putative laccase genes during fungal removal of tNP. The influence of such compounds on extracellular laccase activity, fungal biomass, and nonylphenol removal was assessed comparatively.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Organism and cultures conditions

The origin, identification, and maintenance of the AQH C. aquatica De Wild. strain WD(A)-00-1 have been described previously (Junghanns et al., 2005).

Erlenmeyer flasks (250 mL) containing 75 mL of a 1% (w/v) liquid malt extract medium (pH 5.6–5.8) were inoculated with 1 mL of a mycelial suspension of C. aquatica prepared as described previously (Junghanns et al., 2005) and incubated at 120 r.p.m. and 14 °C in the dark. At culture day 4, tNP (purity 84%; Fluka, Neu-Ulm, Germany) was added aseptically from a methanolic stock solution to a final concentration of 25 μM (corresponding to 5.5 mg L−1), slightly above its reported water solubility of 4.9 mg L−1 (Brix et al., 2001). The final methanol concentration in fungal cultures was 1% (v/v). To improve the solubility of tNP, 0.1% (w/v) Tween 80 was also included. In order to stimulate laccase production differently and to investigate the resulting effects on tNP removal and the expression of putative laccase genes, fungal cultures were also additionally supplemented with the following compounds at culture day 4 (further on referred to as induction treatments): 50 μM CuSO4 (treatment Cu–tNP), 1 mM vanillic acid (treatment V–tNP), and 50 μM CuSO4+1 mM vanillic acid (treatment Cu–V–tNP). Cultures containing only tNP (treatment tNP) were used for comparison. Each treatment was carried out in triplicate. For each treatment, triplicate cultures inactivated with 0.5 g L−1 sodium azide at culture day 4 served as controls.

Determination of tNP concentrations in fungal cultures

Concentrations of tNP were determined upon harvesting fungal cultures at culture days 4, 5, 7, 13, 22, and 29 (treatment tNP) and at culture days 4, 5, 7, 10, 13, 15, 17, 22, and 29 (all other treatments), and applying samples from culture supernatants to HPLC. The sample preparation and a Merck-Hitachi HPLC system (Merck-Hitachi, Düsseldorf, Germany) equipped with a 125/4 LiChrospher 100 RP 18-5 column (Merck-Hitachi) used for analysis has been described previously (Junghanns et al., 2005). Gradient elution at a flow rate of 0.5 mL min−1 started with 40% (v/v) acetonitrile in distilled water, which was kept constant for 2 min, followed by a linear increase to 90% acetonitrile within 8 min. The 90% acetonitrile concentration was kept constant for 2 min, and then decreased back linearly to 40% acetonitrile within 8 min, and kept constant for another 5 min. The detection wavelength was 277 nm. Calibration was carried out with external standards.

Laccase activity determinations

Extracellular laccase activities in liquid culture supernatants were determined upon 2, 29-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) oxidation (Junghanns et al., 2005). Fungal cultures were harvested at culture days 0, 1, 4, 5, 7, 13, 22, and 29 (treatment tNP) and at culture days 0, 1, 4, 5, 7, 10, 13, 15, 17, 22, and 29 (all other treatments), and sampled for laccase activity determination. Enzyme activities are expressed as units (1 U corresponds to 1 μmol of product formed per minute).

Determination of fungal dry masses

For all treatments, additional triplicate fungal cultures were harvested at the time points used for determination of laccase activities.

Mycelia were removed from fungal cultures by filtration through filter papers (Whatman no. 6, Maidstone, England), washed with 50 mL distilled water, dried at 80 °C for 24 h, and weighted.

Determination of copper concentrations in malt extract media

Basal copper concentrations in malt extract media were determined upon inductively coupled plasma MS (ICP-MS), using an ELAN DRC-e instrument (PerkinElmer Sciex, Rodgau, Germany).

Statistical analyses

anova was conducted for the variables biomass, enzyme activity, and tNP concentration using the General Linear Model's module of the program sas (SAS Institute Inc., 1989). We analyzed our data using a two-way anova, where the two factors investigated were time and induction treatment. As we were interested in testing for significant differences between induction treatments over time, we tested for the interaction between the two parameters (time × induction treatment).

Identification of laccase and β-actin gene fragments

Clavariopsis aquatica was grown in 1% malt broth for 15 days. Genomic DNA was isolated according to Nikolcheva & Bärlocher (2002) and fragments of laccase-like genes were amplified using the degenerate primer pair Cu1AF and Cu2R (Kellner et al., 2007 and Luis et al., 2004, respectively) (Invitrogen, Karlsruhe, Germany). These primers target a fragment of the laccase gene situated between the copper-binding regions I and II. The PCR reaction was performed on a Mastercycler Gradient Thermo Cycler (Eppendorf, Hamburg, Germany) in a total volume of 50 μL, containing 75 ng of DNA, and 60 μM of each primer. The PCR conditions were 3 min at 94 °C, followed by 35 cycles (30 s at 94 °C, 30 s at 48 °C, and 120 s at 72 °C) and a final elongation of 10 min at 72 °C. A fragment of c. 150 bp was extracted from agarose gel via the QIAEX II Gel Extraction Kit (Qiagen, Hilden, Germany) and cloned into the pCR4-Topo Vector (TOPO TA cloning kit, Invitrogen) following the manufacturer's protocol and transformed into TOP10 chemically competent Escherichia coli. Then, plasmids from 10 clones were extracted from E. coli (Perfectprep Plasmid Mini Kit, Eppendorf) and sequenced on an ABI PRISM 3100 Genetic Analyser (Applied Biosystems, Darmstadt, Germany) using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer's protocol. Two putative laccase gene fragments were identified and further on referred to as lcc1 and lcc2.

A fragment of 350 bp of the β-actin gene was amplified by means of the degenerate primers Act1_for (5′-CTGGGAYGAYATGGAKAAGAT-3′) and Act2_rev (5′-GYTCRGCSAGRATCTTCAT-3′) (MWG-BIOTECH, Ebersberg, Germany), which were derived from Bleve et al. (2003) and modified according to the DNA sequence of the phylogenetically close ascomycete Neurospora crassa (GenBank accession no. U78026). The fragment was then sequenced using the GATC Biotech AG (Konstanz, Germany). All sequences were submitted to GenBank and are available under the GenBank accession nos. EU747645, EU747646, and EU797523 for the lcc1, lcc2, and actin gene fragments, respectively.

We used the program blast of the NCBI to search for DNA and protein identities.

Analysis of laccase gene expression

Triplicate cultures of each induction treatment were filtered at the time points indicated in the text and freeze-dried overnight. Then, mycelia from identical treatment were ground in a mortar and 1 mg was used for total RNA isolation using the TRIzol® reagent (Invitrogen). The remaining traces of DNA were removed using DNAse I (DNAse I, RNAse-free, Fermentas). The quality of RNA was checked on agarose gel and the RNA concentration was estimated using a Nanodrop® ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE). Reverse transcription of 2 μg of DNA-free RNA was then performed using the Revert Aid H minus First Strand cDNA Synthesis Kit from Fermentas according to the manufacturer's protocol.

Semi-quantitative PCR analyses were performed with the primers designed for a specific amplification of lcc1 and lcc2. A fragment of the β-actin gene was concomitantly amplified as a control of RNA loading. The primer sequences were as follows: lcc1_for (5′-GGTCCACTGGCACGGTCTTC-3′), lcc1_rev (5′-CAAGTTCAAAGCCACACAATATGG-3′), lcc2_for (5′-GTCGGTGTGACCCAGTGCC-3′), lcc2_rev (5′-ACCAGAACGTTCCTGATTGTCC-3′), Actin_for (5′-CCAAAGTCCAACCGTGAAAAGAT-3′), and Actin_rev (5′-TGTCACTCACGTTGTCCCCATCTA-3′) (MWG-BIOTECH). PCR amplifications of the three fragments (ranging from 70 to 170 bp) were performed in independent tubes in a total volume of 20 μL, containing cDNA generated from 125 ng of RNA, 2.5 mM of MgCl2, and 10 pmol of each primer. Amplifications were run simultaneously on a Master cycler gradient system (Eppendorf) under the following conditions: 94 °C for 2 min, 35 cycles (94 °C for 30 s, 57 °C for 30 s, and 72 °C for 1 min), and 72 °C for 10 min. Thereafter, 6 μL of each PCR product was loaded onto a 2% (w/v) agarose gel. Electrophoresis was performed in Tris–acetate–EDTA buffer (0.5 M, pH 8.0) at 70 V cm−1 for 45 min. The gels were stained for 15 min with ethidium bromide (0.5 mg L−1) and DNA fragments were visualized and photographed under UV light.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Effects of potential laccase inducers on nonylphenol removal, laccase activity and fungal biomass

The effects of either CuSO4, vanillic acid, or both compounds on tNP concentrations and laccase activities in fungal cultures are depicted in Fig. 1. Most efficient tNP removal was obtained with the treatment Cu–V–tNP, where a remaining tNP concentration of about 4 μM was observed. A less effective tNP removal (final tNP concentration about 11 μM) was recorded for the treatment Cu–tNP. In the Cu–tNP and V–tNP treatments, declines in tNP concentrations proceeded with similar rates until culture day 13. Later on, the tNP removal became stagnant in the V–tNP treatment (final tNP concentration c. 20 μM). The lowest initial tNP removal rate was observed in the presence of only tNP, where the decline in the tNP concentration started clearly later than in the other treatments (final tNP concentration c. 13 μM). No significant tNP removal was observed in NaN3-inactivated fungal controls (Fig. 1). On average, tNP concentrations differed significantly over time between all induction treatments (Tables 1 and 2). With respect to the averaged efficiency of tNP removal, the treatments followed the rank order Cu–V–tNP>Cu–tNP>V–tNP>tNP. The fungal dry mass as averaged over 29 days of cultivation also differed significantly between induction treatments and was highest in the treatment Cu–tNP, followed by the Cu–V–tNP, V–tNP, and tNP treatments (Tables 1 and 2). Therefore, corresponding extracellular laccase activities were dry mass based in Fig. 1 and for further statistical analyses (Tables 1 and 2).


Figure 1.  Time-courses of tNP concentrations (solid curves) and laccase activities (bars) in supernatants of cultures of Clavariopsis aquatica under different induction treatments. (a) tNP; (b) Cu–tNP; (c) V–tNP; and (d) Cu–V–tNP. The laccase activity is based on fungal dry mass. Dashed curves correspond to the time-courses of tNP concentrations in C. aquatica cultures inactivated by addition of 0.5 g L−1 sodium azide (controls). All values represent means from triplicate cultures and SEs.

Download figure to PowerPoint

Table 1.   Descriptive statistics of variables biomass, enzyme activity, and nonylphenol concentration measured in Clavariopsis aquatica cultures under different induction treatments
 Induction treatment
  1. Average values of the variables obtained throughout 29 days of cultivation are shown. The biomass is expressed in mg fungal dry mass per mL medium, the laccase activity is expressed in units per g fungal dry mass, and the nonylphenol concentration is expressed in μM. (N) represents the sample size, and (SEM) represents the SE of the mean.

Laccase activity0.46240.2710.33332.172.35331.2932.07334.59
tNP concentration21.55181.0219.40271.4021.37271.137.85270.88
Table 2.   Effect of the different induction treatments (tNP, Cu–tNP, V–tNP, Cu–V–tNP) and time on the variables biomass, biomass-weighted laccase activity, and tNP concentration in liquid cultures of Clavariopsis aquatica
 dfMean squaresFP
  1. The table summarizes the results of a two-way anova showing the degrees of freedom (df), the mean squares, the F-statistic values (F) and the level of significance (***P<0.001). The interaction ‘inductor × time’ tests for significant differences between treatments over time for a measured variable.

 Inductor × time270.1519.62***
Laccase activity
 Inductor × time27534.4420.27***
tNP concentration
 Inductor × time2121.744.88***

Laccase activities differing significantly between treatments followed the rank order Cu–V–tNP>Cu–tNP>V–tNP>tNP and were well correlated with the averaged efficiency of tNP removal (Fig. 1; Tables 1 and 2), which implies an extracellular laccase contribution to tNP removal. Evidence for the involvement of laccase in nonylphenol bioconversion has also been accumulated for white-rot fungi such as Trametes versicolor (Soares et al., 2005, 2006).

Copper is a well-known laccase inducer acting on the gene transcription level as has been demonstrated for asco- (Litvintseva & Henson, 2002) and basidiomycetes (Palmieri et al., 2000; Soden & Dobson, 2001). Reported copper concentrations in the range of 11–430 mg kg−1 (corresponding to about 173–6763 μmol kg−1) (Sridhar et al., 2008) in the upper layers of bottom sediments of brooks and rivers, which represent preferred habitats of aquatic fungi, are well above the CuSO4 concentration of 50 μM applied within the present study. Vanillic acid is a plant-related organic acid (Huang & Sheu, 2006) considered as a phenolic lignin model compound (Bollag et al., 1982), which is relevant for AQH because these organisms grow on plant-derived detritus in their natural freshwater environments and, hence, would be expected to come in contact with plant-related organic laccase inducers. Vanillic acid was shown to have species-specific effects on extracellular laccase titers, examples for this being the stimulation of laccase production in the soil deuteromycete Paecilomyces inflatus (Kluczek-Turpeinen et al., 2003), or no effect on laccase production in Volvariella volvacea (Chen et al., 2003). Only little extracellular laccase activity was detected when vanillic acid was added to C. aquatica cultures (treatment V–tNP, Fig. 1c). The highest laccase activities were observed upon application of a mixture of CuSO4 and vanillic acid (treatment Cu–V–tNP, Fig. 1d), thereby suggesting a synergistic rather than a simple additive effect of CuSO4 and vanillic acid on laccase activity when comparing the laccase titers in the Cu–tNP (Fig. 1b), V–tNP (Fig. 1c), and Cu–V–tNP treatments (Fig. 1d). In T. versicolor, either copper, 2,5-xylidine, or both compounds caused increased laccase gene transcript levels within a short time (Collins & Dobson, 1997). However, corresponding increases in extracellular laccase activity were only observed if copper was present, suggesting that the synthesized enzyme remained inactive in the absence of copper. Summarizing, AQH such as C. aquatica could also be expected to produce laccase under natural conditions. Although the natural functions of these enzymes in AQH have not been clarified finally, they could be involved in degradation of lignocellulose-containing plant litter or in fungal defense towards toxic plant compounds. Lignin solubilization and laccase production has been reported for several tropical freshwater fungi (Bucher et al., 2004). Owing to their unspecificity, laccases may concomitantly contribute to the bioconversion of environmental pollutants if such compounds are present.

Identification of laccase and β-actin gene fragments

Clavariopsis aquatica strain WD(A)-00-1 originated from a conidiospore (Junghanns et al., 2005) and therefore represents the haploid stage of the organism. Accordingly, different PCR products derived from the primer pair Cu1AF/Cu2R indicate different nonallelic laccase genes.

The deduced amino acid sequences of lcc1 and lcc2 present the typical laccase signatures L1 (H-W-H-G-X9-D-G-X5-QCPI) and L2 (G-T-X-W-Y-H-S) identified by Kumar et al. (2003) (Fig. 2). The deduced amino acid sequences of lcc1 and lcc2 matched 61% and 70% in identity to laccases of the ascomycetes Fusarium oxysporum (GenBank accession EF990895) and Gyromitra esculenta (GenBank accession AJ715432), respectively. To our knowledge, lcc1 and lcc2 are the first laccase gene fragments amplified in aquatic hyphomycetes to date.


Figure 2.  Alignment of the amino acid sequences of PCR products lcc1 and lcc2 amplified with degenerated laccase primers Cu1AF and Cu2R. The fragments lcc1 and lcc2 contained the complete region of the copper-binding site I (framed amino acids) and seven residues of the N-terminus of the copper-binding site II region (gray boxes). Within each copper-binding site, amino acids homologous to amino acids of the L1 and L2 regions defined by Kumar et al. (2003) are underlined (H-W-H-G-X9-D-G-X5-QCPI and G-T-X-W-Y-H-S, respectively).

Download figure to PowerPoint

Additionally, a β-actin gene fragment was identified in C. aquatica. On the amino acid level, this fragment presents 97% identity with a β-actin gene of the ascomycete Aspergillus fumigatus (GenBank accession XM_742511.1).

Semi-quantitative reverse transcriptase (RT)-PCR analysis of laccase mRNA transcripts and enzyme activity

The β-actin gene was constitutively expressed in all treatments, which was not observed for lcc1 and lcc2 (Fig. 3). Additionally, lcc1 and lcc2 displayed different expression patterns over time, with lcc2 being overall less expressed than lcc1. This suggests a differential regulation of these genes as often reported for different laccases of one organism (Mansur et al., 1998; Smith et al., 1998).


Figure 3.  Visualization of the semi-quantitative PCR of lcc1, lcc2, and β-actin used as a housekeeping gene as observed during different induction treatments. PCR products were separated using 2% agarose gel electrophoresis.

Download figure to PowerPoint

The transcript levels of lcc1 and lcc2 correlated only partially with laccase activities measured in the culture media. Laccase mRNA transcripts were detected until the end of cultivation in the induction treatments tNP and V–tNP (Fig. 3) but laccase activity was measured only at culture days 5 (tNP) and 5 and 7 (V–tNP) (Fig. 1a and c). In the white-rot basidiomycetes T. versicolor and Ceriporiopsis subvermispora, laccase gene expression was induced with 2,5-xylidine and Ag+, respectively, but no significant increase in laccase activity could be measured under copper limitation (Collins & Dobson, 1997; Manubens et al., 2007). A similar mechanism may apply to C. aquatica as supported by the expression pattern of especially lcc1 and concomitantly enhanced laccase activities observed for the Cu–tNP treatment (Figs 1b and 3). Alternatively, lcc1 and lcc2 may encode for laccase proteins localized elsewhere than in the culture supernatant, and basal levels of copper of 3 μg L−1 detected in the used malt extract medium without further amendments may be sufficient to ensure catalytic activity of such laccase proteins. Intracellular and also extracellular laccase activities remaining associated with cell surfaces have been detected in numerous fungi (Linden et al., 1991; Schlosser et al., 1997; Klonowska et al., 2001 and Tetsch et al., 2005, 2006; Valaskova & Baldrian, 2006). Extracellular laccase located within extracellular glucan sheaths has been detected in the basiomycete Rigidoporus lignosus (Nicole et al., 1992, 1993) and in the ascomycete Botrytis cinerea (Gil-ad et al., 2001). In aquatic fungi, cell-associated laccase activities have not been investigated up to now. However, compared with terrestrial fungi, aquatic fungi have to cope with the potential problem of losing extracellular enzymes through water flow and turbulences. Hence, the ability to keep laccases associated with the cell surface, for example embedded within a slime mucilage, might represent an adaptive strategy of these fungi to circumvent the problems associated with their natural environments.

A further type of discrepancy was represented by only low expression levels of lcc1 and lcc2 but the highest extracellular laccase activities observed in the treatment Cu–V–tNP (Figs 1d and 3). This result might suggest the existence and expression of other laccase genes in C. aquatica not yet identified. Indeed, laccases are produced as a number of isoenzymes that are encoded by gene families in numerous fungi (Hoegger et al., 2006). Work addressing the potential existence and expression of other laccase genes in C. aquatica upon amplification and isolation of laccase genes on the cDNA level under induced culture conditions is currently in progress in our laboratory.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

We wish to thank H.-J. Stärk and R. Wennrich (UFZ-Leipzig) for performing the ICP-MS analysis of malt extract media. H.K. and F.B. are grateful to the German Science Foundation (DFG) for financial support (Program SPP1090, Grant no. BU 941/2-3) and M.S. and D.S. are grateful to the DFG-Graduate College ‘Adaptive Physiological and biochemical reactions to ecological important substances’ at the Martin-Luther-University Halle-Wittenberg (Germany).


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  • Abdel-Raheem AM & Ali EH (2004) Lignocellulolytic enzyme production by aquatic hyphomycetes species isolated from the Nile's delta region. Mycopathologia 157: 277286.
  • Baldrian P (2006) Fungal laccases – occurrence and properties. FEMS Microbiol Rev 30: 215242.
  • Belliveau MJR & Bärlocher F (2005) Molecular evidence confirms multiple origins of aquatic hyphomycetes. Mycol Res 109: 14071417.
  • Bleve G, Rizzotti L, Dellaglio F & Torriani S (2003) Development of reverse transcription (RT)-PCR and real-time RT-PCR assays for rapid detection and quantification of viable yeasts and molds contaminating yogurts and pasteurized food products. Appl Environ Microbiol 69: 41164122.
  • Bollag J-M, Liu S-Y & Minard RD (1982) Enzymatic oligomerization of vanillic acid. Soil Biol Biochem 14: 157163.
  • Brix R, Hvidt S & Carlsen L (2001) Solubility of nonylphenol and nonylphenol ethoxylates. On the possible role of micelles. Chemosphere 44: 759763.
  • Bucher VVC, Pointing SB, Hyde KD & Reddy CA (2004) Production of wood decay enzymes, loss of mass, and lignin solubilization in wood by diverse tropical freshwater fungi. Microb Ecol 48: 331337.
  • Chen S, Ma D, Ge W & Buswell JA (2003) Induction of laccase activity in the edible straw mushroom, volvariella volvacea. FEMS Microbiol Lett 218: 143148.
  • Collins P & Dobson A (1997) Regulation of laccase gene transcription in Trametes versicolor. Appl Environ Microbiol 63: 34443450.
  • Corvini P, Schaeffer A & Schlosser D (2006) Microbial degradation of nonylphenol and other alkylphenols – our evolving view. Appl Microbiol Biol 72: 223243.
  • Corvini PFX, Vinken R, Hommes G, Schmidt B & Dohmann M (2004) Degradation of the radioactive and non-labelled branched 4(3′,5′-dimethyl 3′-heptyl)-phenol nonylphenol isomer by Sphingomonas ttnp3. Biodegradation 15: 918.
  • Gil-ad NL, Bar-Nun N & Mayer AM (2001) The possible function of the glucan sheath of Botrytis cinerea: effects on the distribution of enzyme activities. FEMS Microb Lett 199: 109113.
  • Hoegger PJ, Kilaru S, James TY, Thacker JR & Kuess U (2006) Phylogenetic comparison and classification of laccase and related multicopper oxidase protein sequences. FEBS J 273: 23082326.
  • Huang W-Y & Sheu S-J (2006) Separation and identification of the organic acids in Agelicae radix and Ligustici rhizoma by HPLC and CE. J Sep Sci 29: 26162624.
  • Junghanns C, Moeder M, Krauss G, Martin C & Schlosser D (2005) Degradation of the xenoestrogen nonylphenol by aquatic fungi and their laccases. Microbiology 151: 4557.
  • Junghanns C, Krauss G & Schlosser D (2008) Potential of aquatic fungi derived from diverse freshwater environments to decolourise synthetic azo and anthraquinone dyes. Bioresour Technol 99: 12251235.
  • Kellner H, Luis P & Buscot F (2007) Diversity of laccase-like multicopper oxidase genes in Morchellaceae: identification of genes potentially involved in extracellular activities related to plant litter decay. FEMS Microbiol Ecol 61: 153163.
  • Kim Y-S, Katase T, Sekine S, Inoue T, Makino M, Uchiyama T, Fujimoto Y & Yamashita N (2004) Variation in estrogenic activity among fractions of a commercial nonylphenol by high performance liquid chromatography. Chemosphere 54: 11271134.
  • Kirk PM, Cannon PF, David JC & Staplers JA (2001) Ainsworth and Bisby's Dictionary of the Fungi. CABI Publishing, Wallingford, UK.
  • Klonowska A, Le Petit J & Tron T (2001) Enhancement of minor laccases production in the basidiomycete Marasmius quercophilus C30. FEMS Microbiol Lett 200: 2530.
  • Kluczek-Turpeinen B, Tuomela M, Hatakka A & Hofrichter M (2003) Lignin degradation in a compost environment by the deuteromycete Paecilomyces inflatus. Appl Microbiol Biol 61: 374379.
  • Kumar SVS, Phale PS, Durani S & Wangikar PP (2003) Combined sequence and structure analysis of the fungal laccase family. Biotechnol Bioeng 83: 386394.
  • Linden RM, Schilling BC, Germann UA & Lerch K (1991) Regulation of laccase synthesis in induced Neurospora crassa cultures. Curr Genet 19: 375381.
  • Litvintseva AP & Henson JM (2002) Cloning, characterization, and transcription of three laccase genes from Gaeumannomyces graminis var. Tritici, the take-all fungus. Appl Environ Microbiol 68: 13051311.
  • Luis P, Walther G, Kellner H, Martin F & Buscot F (2004) Diversity of laccase genes from basidiomycetes in a forest soil. Soil Biol Biochem 36: 10251036.
  • Mansur M, Suarez T & Gonzalez AE (1998) Differential gene expression in the laccase gene family from basidiomycete I-62 (CECT 20197). Appl Environ Microbiol 64: 771774.
  • Manubens A, Canessa P, Folch C, Avila M, Salas L & Vicuna R (2007) Manganese affects the production of laccase in the basidiomycete Ceriporiopsis subvermispora. FEMS Microbiol Lett 275: 139145.
  • Martin C, Moeder M, Daniel X, Krauss G & Schlosser D (2007a) Biotransformation of the polycyclic musks hhcb and ahtn and metabolite formation by fungi occurring in freshwater environments. Environ Sci Technol 41: 53955402.
  • Martin C, Pecyna M, Kellner H, Jehmlich N, Junghanns C, Benndorf D, Von Bergen M & Schlosser D (2007b) Purification and biochemical characterization of a laccase from the aquatic fungus Myrioconium sp. Uhh 1-13-18-4 and molecular analysis of the laccase-encoding gene. Appl Microbiol Biol 77: 613624.
  • Nicole M, Chamberland H, Geiger JP, Lecours N, Valero J, Rio B & Ouellette GB (1992) Immunocytochemical localization of laccase L1 in wood decayed by Rigidoporus lignosus. Appl Environ Microbiol 58: 17271739.
  • Nicole M, Chamberland H, Rioux D, Lecours N, Rio B, Geiger JP & Ouellette GB (1993) A cytochemical study of extracellular sheaths associated with rigidoporus-lignosus during wood decay. Appl Environ Microbiol 59: 25782588.
  • Nikolcheva LG & Bärlocher F (2002) Phylogeny of Tetracladium based on 18S rDNA. Czech Mycol 53: 285295.
  • Palmieri G, Giardina P, Bianco C, Fontanella B & Sannia G (2000) Copper induction of laccase isoenzymes in the ligninolytic fungus Pleurotus ostreatus. Appl Environ Microbiol 66: 920924.
  • Schlosser D, Grey R & Fritsche W (1997) Patterns of ligninolytic enzymes in Trametes versicolor. Distribution of extra- and intracellular enzyme activities during cultivation on glucose, wheat straw and beech wood. Appl Environ Biol 47: 412418.
  • Smith M, Shnyreva A, Wood DA & Thurston CF (1998) Tandem organization and highly disparate expression of the two laccase genes lcc1 and lcc2 in the cultivated mushroom Agaricus bisporus. Microbiology 144: 10631069.
  • Soares A, Jonasson K, Terrazas E, Guieysse B & Mattiasson B (2005) The ability of white-rot fungi to degrade the endocrine-disrupting compound nonylphenol. Appl Microbiol Biol 66: 719725.
  • Soares A, Guieysse B & Mattiasson B (2006) Influence of agitation on the removal of nonylphenol by the white-rot fungi Trametes versicolor and Bjerkandera sp. bol 13. Biotechnol Lett 28: 139143.
  • Soden DM & Dobson ADW (2001) Differential regulation of laccase gene expression in Pleurotus sajor-caju. Microbiology 147: 17551763.
  • Sridhar KR, Bärlocher F, Wennrich R, Krauss G-J & Krauss G (2008) Fungal biomass and diversity in sediments and on leaf litter in heavy metal contaminated waters of Central Germany. Fund and Appl Limnol/Archiv für Hydrobiol 171: 6374.
  • Tetsch L, Bend J, Janßen M & Hölker U (2005) Evidence for functional laccases in the acidophilic ascomycete Hortaea acidophila and isolation of laccase-specific gene fragments. FEMS Microbiol Lett 245: 161168.
  • Tetsch L, Bend J & Hölker U (2006) Molecular and enzymatic characterisation of extra- and intracellular laccases from the acidophilic ascomycete Hortaea acidophila. Anton Leeuw Int J G 90: 183194.
  • Valaskova V & Baldrian P (2006) Estimation of bound and free fractions of lignocellulose-degrading enzymes of wood-rotting fungi Pleurotus ostreatus, Trametes versicolor and Piptoporus betulinus. Res Microbiol 157: 119124.
  • Ying G-G, Williams B & Kookana R (2002) Environmental fate of alkylphenols and alkylphenol ethoxylates – a review. Environ Int 28: 215226.