Heterologous expression of laccase cDNA from Ceriporiopsis subvermispora yields copper-activated apoprotein and complex isoform patterns

Luis F. Larrondo1, Marcela Avila1, Loreto Salas1, Dan Cullen2 and Rafael Vicuña1

1 Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile and Instituto Milenio de Biología Fundamental y Aplicada, Santiago, Chile
2 USDA Forest Products Laboratory, Madison, WI 53705, USA

Correspondence
Rafael Vicuña
rvicuna{at}genes.bio.puc.cl


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Analysis of genomic clones encoding a putative laccase in homokaryon strains of Ceriporiopsis subvermispora led to the identification of an allelic variant of the previously described lcs-1 gene. A cDNA clone corresponding to this gene was expressed in Aspergillus nidulans and in Aspergillus niger. Enzyme assays and Western blots showed that both hosts secreted active laccase. Relative to the isozymic forms of the native C. subvermispora enzyme, the A. niger-produced laccase had a higher molecular mass and gave a single band on IEF gels. In contrast, A. nidulans transformants secreted several isoforms remarkably similar to those of the native system. Considered together with previously reported Southern blots and protein sequencing, expression in A. nidulans supports the view that C. subvermispora has a single laccase gene and that multiple isoforms result from post-translational processes. In addition, several lines of evidence strongly suggest that under copper limitation, A. nidulans secretes apoprotein which can be reconstituted by a short incubation with Cu(I) and to a lesser extent with Cu(II).


Abbreviations: ABTS, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate); MnP, manganese peroxidase

The GenBank accession numbers for the sequences reported in this paper are AY219235 and AY219236.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Ceriporiopsis subvermispora is one of the most widely used filamentous fungi in laboratory studies of lignin biodegradation. It secretes manganese peroxidase (MnP), a haem protein that catalyses the H2O2-dependent oxidation of Mn(II) to Mn(III) (Glenn & Gold, 1985; Paszczynski et al., 1985), and laccase (EC 1.10.3.2), a member of the multicopper oxidase family of proteins, which includes ascorbate oxidase and ceruloplasmin (Mayer, 1987; Reinhammer, 1984). Laccases catalyse one-electron oxidation of a variety of phenolic compounds, with the concomitant four-electron reduction of O2 to H2O. They are involved in lignin biogenesis in higher plants, and lignin depolymerization by fungi (Eggert et al., 1997; Kirk & Farrell, 1987). Laccases also participate in a broad range of cellular processes such as sporulation (Leatham & Stahman, 1981), fruit body formation and plant pathogenesis (Choi et al., 1992; Geiger et al., 1986; Marbach et al., 1985).

The active site of laccases possesses four copper ions, which can be classified according to their spectrophotometric properties. Type I copper (blue copper) exhibits an intense absorption at about 600 nm, owing to the charge transfer between Cu(II) and a cysteine residue. Type II copper shows a very weak absorption and functions as a one-electron acceptor. Type III copper contains two copper centres absorbing at 330 nm and functions as a two-electron acceptor (Jonsson et al., 1995; Reinhammer, 1984).

Several laccase isoforms can be identified in C. subvermispora cultures depending on the composition of the medium (Salas et al., 1995). Low-stringency Southern blots (Karahanian et al., 1998) and N-terminal sequences of laccases isoenzymes (Salas et al., 1995) suggest a single laccase gene, and its expression is strongly regulated by copper at the transcriptional level (Karahanian et al., 1998). The phenomenon of isoenzyme multiplicity is commonly observed among ligninolytic fungi, although its physiological significance is not known.

To further biochemical analysis of the C. subvermispora laccase we describe here the cloning and expression of Lcs-1 cDNA in Aspergillus nidulans and Aspergillus niger. The A. nidulans system produced multiple isoforms in a pattern that was similar to the native C. subvermispora enzyme, and copper was found to play a post-transcriptional role in laccase expression.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains.
Ceriporiopsis subvermispora strain FP-105752 was obtained from the Center for Mycology Research, Forest Products Laboratory, Madison, WI, USA. Aspergillus nidulans A722 (pyrG89, pabaA1; fwA1; uaY9) and Aspergillus niger A969 (cspA1; fwnA1; pyrG5; metB10) were obtained from the Fungal Genetic Stock Center (FGSC, Kansas City, KS, USA).

Cloning and sequencing.
RT-PCR was used to isolate a full-length cDNA as described by Karahanian et al. (1998). Design of primers was based on the genomic sequence of lcs-1 (GenBank accession number AF053472). The upstream primer was located at position -30 from the ATG, whereas the downstream primer was at position +7 with respect to the stop codon. The PCR product, designated Lcs-1, was directly cloned in pBluescript KSII+ as described by Lobos et al. (1998). To obtain genomic clones, lcs-1 was amplified from C. subvermispora homokaryons 3 and 8 (Tello et al., 2001). Nucleotide sequence was determined with the ABI-Prism Dye terminator Cycle Sequencing kit (Perkin-Elmer Applied Biosystems) with an ABI373 DNA sequencer. Sequence editing and analysis were done with DNAstar software.

Aspergillus expression.
The expression vector pexLcs-1 was constructed by placing the Lcs-1 cDNA coding region, including the sequence for its signal sequence, under the control of the TAKA amylase promoter (Andersen et al., 1992) and the Aspergillus awamori glucoamylase terminator (Kersten et al., 1995) by the PCR overlap extension technique (Horton et al., 1989) using proofreading polymerase Pfu (Stratagene). The coding region and junctions of the expression cassette were sequenced. The selection marker, ppyrG, was obtained from the Fungal Genetic Stock Center.

A. nidulans A722 and A. niger A969 cotransformations were as described by Larrondo et al. (2001), except that A. niger protoplasts were prepared from germinated conidia after an overnight incubation at 300 r.p.m. at 30 °C. Protoplasts were cotransformed with 5 µg ppyrG and 5 µg pexLcs-1. Selection was based on complementation of uridine auxotrophy by the selectable marker pyrG. Transformants carrying the genetic construction pexLcs-1 were confirmed by PCR.

One hundred millilitres of Aspergillus minimal medium (AMM) (Cullen et al., 1987) (5 % maltose) or YEM (0·5 % yeast extract, 5 % maltose) was inoculated with 1x107 spores and incubated for 3 days at 30 °C in an orbital shaker (250 r.p.m.). In the case of A. niger A969, the medium was also supplemented with 1 mM methionine. Mycelium was harvested by filtration through Miracloth, snap frozen in liquid nitrogen and stored at -70 °C. For routine cultivations, the medium was supplemented with CuSO4 up to a final concentration of 100 µM. In copper-deficient cultures, only traces of copper were present (1–3 µM).

We screened rLcs-1 production by plate assay. Isolated transformants were inoculated and selected in agar-minimal medium plates containing maltose (5 %) and 1 mM 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS; Sigma). Characteristic ABTS oxidation could be observed after 2–3 days. Transformants were also evaluated for rLcs-1 production in liquid media by spectrophotometric measurements of ABTS oxidation

Enzyme purification and characterization.
One litre of extracellular fluid from A. nidulans Lcs-1 transformant (AnidL-6) culture was directly fractionated by chromatography on Q-Sepharose. One litre of supernatant from A. niger Lcs-1 transformant (AnigL-2) was concentrated 10-fold by filtration in a 185 ml Amicon cell possessing a 10 kDa cut-off membrane, and then dialysed twice against 500 ml 25 mM sodium acetate pH 4·5 and fractionated by chromatography on Q-Sepharose (Lobos et al., 1998). Enzyme activity was measured at 30 °C in a Shimadzu 160 UV-visible recording spectrophotometer. Reaction mixtures (1 ml) contained 50 mM glycine, pH 3·0, and 4·33 mM ABTS as substrate. Km values for ABTS were determined with 0·01 U laccase, using Eadie–Hofstee plots. For endoglycosidase treatment, 5 µg enzyme was treated as described by Larrondo et al. (2001).

Zymograms were as previously reported (Larrondo et al., 2001). Briefly, samples were applied with nonreducing denaturing loading buffer without boiling and subjected to SDS-PAGE at 4 °C. Gels were then fixed in a solution containing 10 % acetic acid and 40 % methanol for 10 min followed by incubation in 50 mM glycine buffer, pH 3·0, and 5 mM ABTS for 10 min, or until activity could be detected.

Reconstitution assays.
Q-Sepharose-purified laccase (0·5 µg) from low-copper-containing media was incubated at 25 °C with various concentrations of copper as indicated in the corresponding figure legend. Thereafter, samples were withdrawn at the indicated times to measure laccase activity.

Other methods.
Analytical IEF and protein concentration measurements were as described by Larrondo et al. (2001); RNA extraction and Northern blot hybridization were as described by Karahanian et al. (1998). The DNA probe corresponding to the entire coding region of Lcs-1 was labelled with [{alpha}-32P]dCTP by nick translation (Gibco-BRL).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Characterization of a new lcs-1 allele
In further attempts to isolate and characterize laccase genes from C. subvermispora, we have cloned laccase-like amplicons from homokaryotic strains. Each homokaryon harboured a highly conserved, but distinct sequence. Presumed to be allelic variants, the sequences are >99 % identical with all but one mismatch occurring in non-coding regions. The single coding region mismatch occurred in the third position of an alanine codon, (GCT to GCC), located in exon X, and did not affect translation. Designated lcs-1A and lcs-1B (accession number AY219235), the former corresponds to the previously deposited lcs-1 sequence AF053472 (Karahanian et al., 1998). (Minor sequencing errors in lcs-1A have been recently corrected.)

Cloning of lcs-1 cDNA
A unique and abundant product was obtained when RT-PCR was conducted with RNA extracted from dikaryotic strain FP105752 of C. subvermispora grown in liquid culture containing 100 µM CuSO4. The cDNA was cloned and sequenced, and based on the nucleotide difference mentioned above, it corresponded to lcs-1B (accession number AY219236).

Production of rLcs-1 in Aspergillus spp.
Transformants confirmed by PCR were screened for extracellular laccase activity when grown in minimal medium. Similar levels of enzyme activity were obtained for both Aspergillus hosts. No major variations in enzyme activity were detected when AMM or YEM media (both supplemented to 100 µM CuSO4 final) were used. Laccase activity appeared on day 2, reaching a peak of 0·23 U ml-1 on day 4 and then detected without major variation up to day 5 or 6. For purification purposes, cultures were routinely collected on day 3. The levels of rLcs-1 produced were about 1·5 mg per litre of culture.

Enzyme purification.
Q-Sepharose purification of laccase from A. nidulans-Lcs-1 cultures yielded one major protein (Anid-rLcs-1) with the same molecular mass and immunoreactivity as the native C. subvermispora enzyme (Fig. 1A). Zymograms showed the presence of only one major band, with a higher electrophoretic mobility than the enzyme under reducing conditions (Fig. 1B). The enzyme purified from A. niger-Lcs-1 cultures (Anig-rLcs-1) also reacted with antibodies against C. subvermispora laccase although its molecular mass was higher than expected. It was also detected as a single band in zymograms, with a lower electrophoretic mobility than Anid-rLcs-1 (Figs 1A and 1B, respectively).



View larger version (42K):
[in this window]
[in a new window]
 
Fig. 1. PAGE of native and recombinant laccases. (A) SDS-PAGE and Western blot of Anig-rLcs-1 (lane 1), Anid-rLcs-1 (lane 2) and native C. subvermispora laccase pool (lane 3). (B) Zymogram of 0·02 U Anig-rLcs-1 (lane 1), Anid-rLcs-1 (lane 2) and native C. subvermispora laccase pool (lane 3). Activity was developed with ABTS.

 
Isoelectric focusing.
IEF of Anid-rLcs-1 gave a pattern resembling the native Ceriporiopsis laccases. In contrast, Anig-rLcs-1 laccase yielded a single IEF band, which was similar in pI to the most basic isoform observed for C. subvermipora laccases (Fig. 2).



View larger version (60K):
[in this window]
[in a new window]
 
Fig. 2. IEF of recombinant and native laccase: 0·04 U of Anig-Lcs-1 (lane 1), Anid-Lcs-1 (lane 2) and C. subvermispora laccase (lane 3) was loaded and after running the gel, activity was developed with 4-chloronaphthol.

 
Glycosylation.
Treatment of Anid-rLcs-1 with endoglycosidase H revealed N-glycosylation of about 25 % of the original molecular mass, a composition similar to the native C. subvermispora laccase. A comparable degree of glycosylation was observed with Anig-rLcs-1, but its molecular mass after the treatment remained higher than that of native C. subvermispora laccase (Fig. 3) (Salas et al., 1995).



View larger version (42K):
[in this window]
[in a new window]
 
Fig. 3. Endoglycosidase H treatment of laccases. Western blot of endoglycosidase H treated (End+) and untreated (End-) laccases. Anig-rLcs-1 End+ (lane 1) and End- (lane 2); Anid-rLcs-1 End+ (lane 3) and End- (lane 4); C. subvermispora Lcs1 End+ (lane 5) and End- (lane 6).

 
Km for ABTS.
The Km values determined for the oxidation of ABTS were 0·042 mM for Anid-rLcs-1 and 0·038 mM for Anig-rLcs-1, which is in accordance with the value of 0·03 mM reported for this enzyme (Fukushima & Kirk, 1995).

Effect of copper on rLcs-1 production
Copper dramatically affected laccase titres in YEM cultures of A. nidulans Lcs-1. YEM cultures supplemented with 100 µM CuSO4 yielded laccase levels up to 200 U l-1, as compared to less than 20 U l-1 obtained in unsupplemented growth medium. When normalized by biomass, this represents a sixfold stimulation by copper (Fig. 4A). A similar result was observed when A. niger transformants were analysed (data not shown). This effect was not exerted at the transcriptional or the translational levels, as determined by Northern (Fig. 4C) and Western (Fig. 4B) blots, respectively. This suggests that apoprotein is efficiently produced and secreted at low copper levels. The effect of copper was also assessed in AMM, with and without added copper. In the absence of externally added copper (copper levels of 1–3 µM), three and five times less activity was observed in A. nidulans and A. niger transformants, respectively.



View larger version (44K):
[in this window]
[in a new window]
 
Fig. 4. Effect of copper supplementation (100 µM) on rLcs-1 expression in A. nidulans. (A) Specific laccase activity from 3-day-old YEM cultures. (B) Western blot analysis of samples from each culture, containing 2·5 µg protein. (C) Northern blot analysis of lcs-1. (D) Loading control: ethidium-bromide-stained formaldehyde gel shows approximately equal amount of rRNA.

 
Partial reconstitution of laccase activity
Copper could be incorporated into the apoprotein, giving rise to active enzyme. When supernatant or Q-Sepharose supernatant pools of Anid-Lcs1 cultures grown at low copper levels were incubated with different copper concentrations, laccase activity could be recovered in a concentration- and time-dependent manner. Concentrations up to 5 mM CuSO4 were tested. At this copper level, a fourfold increase in activity was achieved (Fig. 5). A larger effect was observed when copper was present as Cu(I) due to incubation of Cu(II) with ascorbate. Maximal reconstitution levels were achieved after 20 min incubation of apoprotein in a solution containing 5 mM CuSO4 and 1 mM ascorbate. Under these conditions, a ninefold increase in activity was reached, which constitutes about 75 % of the maximal activity present in the Q-Sepharose pool obtained from A. nidulans-Lcs-1 grown in YEM medium in the presence of 100 µM CuSO4 (data not shown). Incubation of the latter enzyme preparation with copper and ascorbate resulted in only a slight increase of activity (15 %), which is consistent with the loss of copper during the purification procedure.



View larger version (34K):
[in this window]
[in a new window]
 
Fig. 5. Reconstitution of laccase activity with copper. Q-Sepharose-purified laccase (0·5 µg) from low-copper medium (LC) and copper-supplemented medium (C) was incubated with various amounts of copper, with or without ascorbate (Asc), and samples were taken at the indicated times to measure laccase activity.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Laccase isoenzyme multiplicity is a phenomenon observed in several fungi, and in the case of Pleurotus sajor-caju (Soden & Dobson, 2001), the basidiomycete CECT 20197 (Mansur et al., 1997), Pleurotus ostreatus (Palmieri et al., 2000), Trametes villosa (Yaver & Golightly, 1996), Agaricus bisporus (Perry et al., 1993) and Rhizoctonia solani (Wahleithmer et al., 1995), families of structurally related genes have been described. In the case of C. subvermispora, over four isoforms are observed, with different substrate specificity, but only one gene, lcs-1, has been identified (Karahanian et al., 1998; Salas et al., 1995). The search for a new laccase gene in C. subvermispora led to the identification of the allelic variant lcs-1B. This allele cannot contribute to the isoform multiplicity observed in this fungus, because the two alleles are predicted to encode identical proteins. Taken together with previously reported low-stringency Southern blots and N-terminal protein sequencing, the results support the existence of a single laccase gene in C. subvermispora.

We were able to successfully express Lcs-1 cDNA in A. nidulans and A. niger. To our knowledge, this is the first report of expression of a white-rot fungus laccase in A. nidulans and the second instance of using A. niger (Record et al., 2002) as a host. Enzyme titres were substantially lower than those reported for other laccases in Aspergillus oryzae systems (Wahleithmer et al., 1995; Yaver et al., 1996, 1999; Record et al., 2002; Berka et al., 1997).

The molecular mass obtained for Anid-rLcs-1 was similar to that of the native enzyme, whereas Anig-rLcs-1 had a higher molecular mass. After treatment with endoglycosidase H, the molecular mass of Anig-rLcs-1 remained higher than that of the native enzyme, which suggests that modifications other than N-glycosylation may alter molecular mass. This was also associated with an altered pI, although the Km for ABTS remained unchanged.

The IEF pattern of Anid-rLcs-1 was strikingly similar to that obtained with laccase activity from C. subvermispora. The phenomenon of isoform multiplicity was previously observed with recombinant Cs-MnP1, as well as recombinant Phanerochaete chrysosporium MnP1, both expressed in A. nidulans (Larrondo et al., 2001). We have also observed multiple isoforms for P. chrysosporium multicopper oxidase when expressed in Aspergillus (L. F. Larrondo and others, unpublished). Thus, our experimental data clearly show that multiple isoforms can arise from a single cDNA, which is consistent with the presence of a single laccase gene in C. subvermispora. The precise nature of isoform modifications in A. nidulans and C. subvermispora remains unknown. Possibilities of post-translational modification include glycosylation and phosphorylation. Notably, IEF of A. niger-produced laccase revealed a single isoform, indicating that post-translational modifications differ among species of Aspergillus. Further work is required to understand the biochemical basis of this divergence. Prior to this work, there had been a single report of laccase expression in A. niger. In that study, the recombinant protein had the same characteristics as the native enzyme (Record et al., 2002).

It has been reported that the expression of the Trichoderma reesei {beta}-mannanase gene in yeast gives rise to two proteins with different pIs, which match the pIs described for the same activity in T. reesei (Stalbrand et al., 1995). A detailed study of T. reesei cellobiohydrolase I (CBHI) secretion revealed several pI forms during processing in the secretion pathway (Pakula et al., 2000). Both N- and O-glycans might be responsible for the final IEF pattern, but at least in the case of CBHI, it seems plausible that isoform multiplicity is due to phosphorylation of O-glycans. In addition, sulfatation of the glycopeptide region, as described in CBHI produced by the T. reesei strain ALKO2788 (Harrison et al., 1998), can not be excluded.

Previous investigations have stressed the importance of copper in the regulation of laccase at the transcriptional level (Karahanian et al., 1998; Soden & Dobson, 2001; Palmieri et al., 2000; Galhaup et al., 2002). We previously demonstrated that under low-copper conditions, lcs-1 is not efficiently transcribed in C. subvermispora (Karahanian et al., 1998). Here, we assessed the influence of copper on the laccase titres in a foreign host. Copper limitation decreased production of fully active laccase, but transcript levels remained constant. At low concentrations of this metal, laccase activity was barely detectable. Interestingly, when AMM medium was used, the effect of copper limitation was not as striking as in YEM medium. Biomass and the total extracellular protein produced in YEM cultures were not drastically affected by supplementation with 100 µM CuSO4. We corroborated this observation by partially recovering activity from supernatants by incubation with copper concentrations in the millimolar range. This implies that the apoprotein lacks some or all of the structural copper atoms necessary for its activity.

There has been some interest in the study of laccase copper centres, by depletion and reconstitution after addition of this metal (Hanna et al., 1988). Usually, this has been achieved by extensive dialysis against cyanide ion. In one case, apoprotein was produced under low copper levels (Bligny et al., 1986), but the activity could not be reconstituted by incubation with copper. On the other hand, reconstitution of laccase from the Chinese lacquer tree requires the addition of Cu(I) and not Cu(II) (Hauenstein & McMillin, 1978). Omura (1961) also reported that Cu(I), but not Cu(II), could be used to reconstitute the apoprotein, results that were confirmed by Ando (1970). In the case of C. subvermispora laccase produced by A. nidulans, spectroscopic studies may help to clarify whether all three types of copper or only a single one are affected in cultures with limiting concentrations of this metal.

The expression systems reported here should be useful for an array of biochemical investigations, including studies of post-transcriptional modification. The results also highlight the importance of adequate copper levels in the production of fully active laccase.


   ACKNOWLEDGEMENTS
 
This work was financed by grants 8990004 and 20 00076 from FONDECYT-Chile, DIPUC to L. F. L, and by the US Dept of Energy grant DE-FG02-87ER13712. L. F. L is a Predoctoral Fellow supported by Fundacion Andes.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Andersen, H. D., Jensen, E. B. & Welinder, K. G. (1992). A process for producing heme proteins. European Patent Application 0505311A2.

Ando, K. (1970). Preparations and properties of apo- and reconstructed Rhus-laccases. J Biochem 68, 501–508.[Medline]

Berka, R. M., Schneider, P., Golightly, E. J., Brown, S. H., Madden, M., Brown, K. M., Halkier, T., Mondorf, K. & Xu, F. (1997). Characterization of the gene encoding an extracellular laccase of Myceliophthora thermophila and analysis of the recombinant enzyme expressed in Aspergillus oryzae. Appl Environ Microbiol 63, 3151–3157.[Abstract]

Bligny, R., Gaillard, J. & Douce, R. (1986). Excretion of laccase by sycamore (Acer pseudoplatanus L.) cells. Effects of a copper deficiency. Biochem J 237, 583–588.[Medline]

Choi, G. H., Larson, T. G. & Nuss, D. L. (1992). Molecular analysis of the laccase gene from the chestnut blight fungus and selective suppression of its expression in an isogenic hypovirulent strain. Mol Plant-Microbe Interact 5, 119–128.[Medline]

Cullen, D., Gray, G., Hayenga, K., Lamsa, M., Norton, S., Rey, M., Wilson, L. & Berka, R. (1987). Controlled expression and secretion of bovine chymosin by Aspergillus nidulans. Bio/Technology 5, 369–376.

Eggert, C., Temp, U. & Eriksson, K. (1997). Laccase is essential for lignin degradation by the white-rot fungus Pycnoporus cinnabarinus. FEBS Lett 407, 89–92.[CrossRef][Medline]

Fukushima, Y. & Kirk, T. K. (1995). Laccase component of the Ceriporiopsis subvermispora lignin-degrading system. Appl Environ Microbiol 61, 872–876.[Abstract]

Galhaup, C., Goller, S., Peterbauer, C. K., Strauss, J. & Haltrich, D. (2002). Characterization of the major laccase isoenzyme from Trametes pubescens and regulation of its synthesis by metal ions. Microbiology 148, 2159–2169.[Abstract/Free Full Text]

Geiger, J. P., Nicole, M., Nandris, D. & Rio, B. (1986). Root diseases of Hevea brasiliensis. Physiological and biochemical aspects of root aggregation. Phytochemistry 24, 2559–2561.[CrossRef]

Glenn, J. K. & Gold, M. H. (1985). Purification and characterization of an extracellular Mn(II)-dependent peroxidase from the lignin-degrading basidiomycete Phanerochaete chrysosporium. Arch Biochem Biophys 242, 329–341.[Medline]

Hanna, P. M., McMillin, D. R., Pasenkiewicz-Gierula, M., Antholine, W. E. & Reinhammar, B. (1988). Type 2-depleted fungal laccase. Biochem J. 253, 561–568.

Harrison, M. J., Nouwens, A. S., Jardine, D. R., Zachara, N. E., Gooley, A. A., Nevalainen, H. & Packer, N. H. (1998). Modified glycosylation of cellobiohydrolase I from a high cellulase-producing mutant strain of Trichoderma reesei. Eur J Biochem 256, 119–127.[Abstract]

Hauenstein, B. L., Jr & McMillin, D. R. (1978). On the reconstitution of laccase from the Chinese lacquer tree. Biochem Biophys Res Commun 85, 505–510.[Medline]

Horton, R., Hunt, H., Ho, S., Pullen, J. & Pease, L. (1989). Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 61–68.[CrossRef][Medline]

Jonsson, L., Sjostrom, K., Haggstrom, I. & Nyman, P. O. (1995). Characterization of a laccase gene from the white-rot fungus Trametes versicolor and structural features of basidiomycete laccases. Biochim Biophys Acta 1251, 210–215.[Medline]

Karahanian, E., Corsini, G., Lobos, S. & Vicuña, R. (1998). Structure and expression of a laccase gene from the ligninolytic basidiomycete Ceriporiopsis subvermispora. Biochim Biophys Acta 1443, 65–74.[Medline]

Kersten, P. J., Witek, C., Vanden Wymelenberg, A. & Cullen, D. (1995). Phanerochaete chrysosporium glyoxal oxidase is encoded by two allelic variants: structure, genomic organization and heterologous expression of glx1 and glx2. J Bacteriol 177, 6106–6110.[Abstract]

Kirk, T. K. & Farrell, R. L. (1987). Enzymatic "combustion": the microbial degradation of lignin. Annu Rev Microbiol 41, 465–505.[CrossRef][Medline]

Larrondo, L. F., Lobos, S., Stewart, P., Cullen, D. & Vicuña, R. (2001). Isoenzyme multiplicity and characterization of recombinant manganese peroxidases from Ceriporiopsis subvermispora and Phanerochaete chrysosporium. Appl Environ Microbiol 67, 2070–2075.[Abstract/Free Full Text]

Leatham, G. & Stahman, M. A. (1981). Studies on the laccase of Lentinus edodes; specificity. localization and the development of fruiting bodies. J Gen Microbiol 125, 147–157.

Lobos, S., Larrondo, L., Salas, L., Karahanian, E. & Vicuña, R. (1998). Cloning and molecular analysis of a cDNA and the Cs-mnp1 gene encoding a manganese peroxidase isoenzyme from the lignin-degrading basidiomycete Ceriporiopsis subvermispora. Gene 206, 185–193.[CrossRef][Medline]

Mansur, M., Suarez, T., Fernandez-Larrea, J. B., Brizuela, M. A. & Gonzalez, A. E. (1997). Identification of a laccase gene family in the new lignin-degrading basidiomycete CECT 20197. Appl Environ Microbiol 63, 2637–2646.[Abstract]

Marbach, I., Harel, E. & Mayer, A. M. (1985). Pectin, a second inducer for laccase production by Botrytis cinerea. Phytochemistry 24, 2559–2561.[CrossRef]

Mayer, A. M. (1987). Polyphenol oxidases in plants – recent progress. Phytochemistry 26, 11–20.[CrossRef]

Omura, T. (1961). Studies on lacases of lacquer trees. Reconstitution of laccase from its protein and copper. J Biochem 50, 389–393.[Medline]

Pakula, T. M., Uusitalo, J., Saloheimo, M., Salonen, K., Aarts, R. J. & Penttila, M. (2000). Monitoring the kinetics of glycoprotein synthesis and secretion in the filamentous fungus Trichoderma reesei: cellobiohydrolase I (CBHI) as a model protein. Microbiology 146, 223–232.[Abstract/Free Full Text]

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, 920–924.[Abstract/Free Full Text]

Paszczynski, A., Huynh, V.-B. & Crawford, R. L. (1985). Enzymatic activities of an extracellular, manganese-dependent peroxidase from Phanerochaete chrysosporium. FEMS Microbiol Lett 29, 37–41.[CrossRef]

Perry, C., Smith, M., Britnell, C., Wood, D. & Thurston, C. (1993). Identification of two laccase genes in the cultivated mushroom Agaricus bisporus. J Gen Microbiol 139, 1209–1218.[Medline]

Record, E., Punt, P. J., Chamkha, M., Labat, M., van Den Hondel, C. A. & Asther, M. (2002). Expression of the Pycnoporus cinnabarinus laccase gene in Aspergillus niger and characterization of the recombinant enzyme. Eur J Biochem 269, 602–609.[Abstract/Free Full Text]

Reinhammer, B. (1984). In Copper Proteins and Copper Enzymes, pp. 1–35. Edited by R. Lontie. Boca Raton, FL: CRC Press.

Salas, C., Lobos, S., Larrain, J., Salas, L., Cullen, D. & Vicuña, R. (1995). Properties of laccase isoenzymes produced by the basidiomycete Ceriporiopsis subvermispora. Biotechnol Appl Biochem 21, 323–333.[Medline]

Soden, D. M. & Dobson, A. D. (2001). Differential regulation of laccase gene expression in Pleurotus sajor-caju. Microbiology 147, 1755–1763.[Abstract/Free Full Text]

Stalbrand, H., Saloheimo, A., Vehmaanpera, J., Henrissat, B. & Penttila, M. (1995). Cloning and expression in Saccharomyces cerevisiae of a Trichoderma reesei beta-mannanase gene containing a cellulose binding domain. Appl Environ Microbiol 61, 1090–1097.[Abstract]

Tello, M., Seelenfreund, D., Lobos, S., Gaskell, J., Cullen, D. & Vicun~a, R. (2001). Isolation and characterization of homokaryotic strains from the ligninolytic basidiomycete Ceriporiopsis subvermispora. FEMS Microbiol Lett 199, 91–96.[CrossRef][Medline]

Wahleithmer, J. A., Xu, F., Brown, K., Brown, S., Golightly, E., Halkier, T., Kauppinen, S., Pederson, A. & Schneider, P. (1995). The identification and characterization of four laccase genes from the plant pathogenic fungus Rhizoctonia solani. Curr Genet 29, 395–403.[CrossRef]

Yaver, D. & Golightly, E. (1996). Cloning and characterization of three laccase genes from the white-rot basidiomycete Trametes villosa: genomic organization of the laccase gene family. Gene 181, 95–102.[CrossRef][Medline]

Yaver, D., Xu, F., Golightly, E. & 7 other authors (1996). The purification, characterization, molecular cloning and expression of two laccase genes from the white-rot basidiomycete Trametes villosa. Appl Environ Microbiol 62, 834–841.[Abstract]

Yaver, D. S., Overjero, M. D., Xu, F., Nelson, B. A., Brown, K. M., Halkier, T., Bernauer, S., Brown, S. H. & Kauppinen, S. (1999). Molecular characterization of laccase genes from the basidiomycete Coprinus cinereus and heterologous expression of the laccase lcc1. Appl Environ Microbiol 65, 4943–4948.[Abstract/Free Full Text]

Received 26 November 2002; revised 13 February 2003; accepted 14 February 2003.