Mycol. Res. 107 (6): 727–735 (June 2003). f The British Mycological Society 727
DOI: 10.1017/S0953756203007937 Printed in the United Kingdom.
Identiﬁcation of a new laccase gene and conﬁrmation of
genomic predictions by cDNA sequences of Trametes sp. I-62
´ ´ ¨ ´
Tania GONZALEZ#, Marı´ a del Carmen TERRON, Ernesto ZAPICO$, Susana YAGUE, Alejandro TELLEZ·,
Howard JUNCA## and Aldo GONZALEZ*
Centro de Investigaciones Biolo´gicas, Vela´zquez 144, E-28006 Madrid, Spain.
E-mail : firstname.lastname@example.org
Received 21 October 2002; accepted 17 April 2003.
The strain Trametes sp. I-62 (CECT 20197) is a white-rot fungus with great potential for biotechnological applications in
the ﬁelds of industrial waste water decolorization and clean up. Three laccase genes: lcc1, lcc2 and lcc3 have been cloned
and sequenced from this basidiomycete. In this work, the coding regions of the corresponding cDNAs have been
synthesized, cloned, and sequenced. They are 1563, 1563 and 1575 bp in length, respectively. Former putative intron/
exon structures from genomic DNA are fully conﬁrmed by match analysis with our cDNA sequences. Using Polymerase
Chain Reaction – Restriction Fragment Length Polymorphism (PCR–RFLP) analysis, an additional laccase cDNA was
also identiﬁed, corresponding to a new gene, lcc1A, which displayed 99.6 % identity with lcc1 at protein level. Such high
similarity between lcc1 and lcc1A sequences, and the comparison with reports from other basidiomycete laccases, suggest
that in this strain these two genes are allelic variants.
INTRODUCTION & Eriksson 1976, Hatakka 1994, Bourbonnais et al.
1995, Call & Mucke 1997). Although the physiological
Laccases (benzenediol : oxygen oxidoreductases,
role of this enzyme has not as yet been deﬁnitively
EC126.96.36.199) are a heterogeneous group of multi-copper
clariﬁed, it is known that laccases catalyse the oxi-
oxidative enzymes widespread in plants and fungi
dation of diﬀerent aromatic compounds (mono-, di-,
(Thurston 1994). Although in bacteria laccase activity
and polyphenols, aminophenols, and diamines) by re-
has been rarely described, it has been detected in
ducing molecular oxygen to water (Reinhammar 1984).
Azospirillum lipoferum (Givaudan et al. 1993) and more
Laccases have great potential as industrial enzymes
recently in two marine bacteria and in Bacillus subtilis
due to their capacity to degrade a broad diversity of
endospore coat (Solano & Sanchez-Amat 1999, Mar-
natural and synthetic materials such as lignin (Kirk &
tins et al. 2002). Recently, a revision on the presence of
Farrell 1987, Hatakka, Mohammandi & Lundell 1989),
laccase in bacteria has been published (Alexandre &
chlorophenols (Roy-Arcand & Archibald 1991) poly-
Zhulin 2000). A variety of diﬀerent, and sometimes
cyclic aromatic hydrocarbons (Field et al. 1993), tan-
contradictory, physiological functions has been pro-
nins (Yague et al. 2000), melanoidins (Gonzalez et al.
posed for this type of oxidative enzyme. In fungi, lac-
2000) and azo dyes (reviewed by Husain & Jan 2000).
cases have been involved in pigmentation (Leatham
Diﬀerent applications for these enzymes would include
1985, Coll et al. 1993a, b), fruiting-body formation
the upgrading of animal feed (Hatakka et al. 1989,
(Wood 1980), pathogenicity (Larson, Choi & Nuss
Akin et al. 1993), pulp and paper production, textile
1992, Williamson 1997) and lignin degradation (Ander
dye bleaching, bioremediation and eﬄuent detoxiﬁca-
tion, washing powders components, removal of phenols
* Corresponding author. from wines (Servili et al. 2000), and transformation
Present addresses: # Instituto Cubano de Derivados de la Cana de
˜ of antibiotics and steroids (Breen & Singleton 1999).
Azucar, Havana, Cuba; $ Biotechnology Department, University
of Hamburg, D-21073, Hamburg, Germany; · Departmento de Bio- Several reviews on laccase function and applications
tecnologı´ a, Universidad Autonoma Metropolitana Iztapalapa, has been recently published (Leonowicz et al. 2001,
S. Rafael Atlixco nx 186, Col. Vicentina, C.O. 09340 Mexico D.F., Mayer & Staples 2002).
Mexico; ## Department of Environmental Microbiology, GBF-
National Research Centre for Biotechnology, D-38124 Braunsch- The biotechnological importance of laccases has
weig, Germany. encouraged the search for genes coding for them in
New fungal lcc gene, conﬁrmation of genomic DNAs 728
diﬀerent organisms. A laccase gene sequence was ﬁrst Pinar del Rı´ o, Cuba (Mansur et al. 1997). The voucher
described in the ascomycetous fungus Neurospora specimen from which this isolate was made is preserved
crassa (Germann & Lerch 1986). Two other gene in the National Research Institute on Tropical Agri-
sequences were reported, one of them in the hypho- culture (INIFAT), Habana ; it was determined as
mycete: Aspergillus nidulans (Aramayo & Timberlake Trametes sp. by Joost A. Stalpers (CBS) but could
1990), and the other, in the basidiomycete Coriolus not be named to species rank. The fungal culture was
hirsutus (Kojima et al. 1990). Perry et al. (1993) de- grown on agar plates with a modiﬁed Czapeck medium
scribed the presence of two laccase genes in the same ´
(Guillen, Martı´ nez & Martı´ nez 1990) for 7 d at 28 xC.
chromosome of the basidiomycete Agaricus bisporus, Submerged cultures of the fungus were prepared by
thus reporting the ﬁrst laccase gene family in fungi. the inoculation of eight plugs (1 cm2) from these plates,
Subsequently, four gene families have been described under sterile conditions, into 500 ml culture ﬂasks con-
in Rhizoctonia solani (Wahleithner et al. 1996) and taining 300 ml of the same growth medium and four
Pleurotus sajor-caju (Soden & Dobson 2001) ; three glass beads (1.5 cm diam). After incubation for 24 h at
in Pleurotus ostreatus (Giardina et al. 1995, 1996, 1999), 28 x in an orbital shaker (100 rpm), a 7.5 ml inoculum
a family of ﬁve genes in diﬀerent chromosomes of was transferred into 250 ml Erlenmeyer ﬂasks contain-
Trametes villosa (Yaver et al. 1996, Yaver & Golightly ing 75 ml (total volume) of Kirk medium (Kirk et al.
1996) and two genes in Trametes versicolor (Jonsson ¨ 1986). The culture was incubated for 7 d under the
et al. 1995, Mikuni & Morohoshi 1997, Ong, Pollock same conditions of temperature and agitation.
& Smith 1997).
At ﬁrst, the diversity of fungal laccase isozymes was
thought to be the result of post-translational modiﬁ-
cations of the same gene product. The characterization Laccase activity was determined in the extracellular
of the several laccase gene families mentioned above ﬂuid of fungal cultures (Wolfenden & Willson 1982),
suggested that at least in part, this biochemical diver- using ABTS (2,2k-azinobis-3-ethylbenzthiazoline-
sity would be the result of the multiplicity of laccase 6-sulphonate) as the substrate. One unit of laccase
genes in fungal genomes. However, in most fungi, lac- activity was deﬁned as that catalyzing formation of
case activity is produced at levels which are too low 1 mmol of oxidized ABTS minx1.
for industrial purposes, and it is clear that any realistic
application of these enzymes requires their production
from an inexpensive source. For this reason concerted
eﬀorts are being made dedicated to express cloned lac- Fresh mycelium samples (approx. 1 g) were harvested
case genes in heterologous hosts (Kojima et al. 1990, daily and those corresponding to the maximal laccase
Saloheimo & Niku-Paavola 1991, Gouka et al. 2001). activity were used for total RNA extraction, that was
A key step in the production of recombinant enzymes performed by using the Fast RNA kit-Red, following
from eukaryotes is the isolation, characterization and the indications of the manufacturer (BIO 101, Mon-
molecular cloning of their cDNAs. treal). Total RNA concentration was determined
The white-rot fungus Trametes sp. I-62 (CECT spectrophotometrically.
20197) is a strain with a wide biotechnological potential
(Pointing 2001). The high detoxiﬁcation capacity of dis-
tillery eﬄuents shown by this fungus, and the possible
role of laccases in this process have been studied in our First-strand cDNA synthesis was carried out from 2 mg
laboratory (Gonzalez et al. 2000). Moreover, Mansur of total RNA by using a cDNA synthesis kit (Roche)
and coworkers had already described the biochemical according to the manufacturer’s instructions.
diversity of laccases in this strain, and they cloned three For PCR ampliﬁcation, a 5 ml volume from each
laccase genes (lcc1, lcc2 and lcc3) of a probably larger RT reaction mixture was mixed with primers, Taq
family (Mansur et al. 1997). polymerase (PerkinElmer) and the rest of components
In the present work, cDNA sequences of the lcc1, used in standard PCR reactions (Sambrook, Fritsch
lcc2 and lcc3 laccase genes from Trametes sp. I-62 were & Maniatis 1989). cDNA ampliﬁcation was performed
cloned, sequenced and characterized. A new laccase in a Rapidcycler (Idaho Technology, Idaho Falls)
cDNA sequence was also identiﬁed, and evidence is thermocycler with a PCR temperature program of 95 x
shown that it corresponds to another laccase gene from for 5 min, followed by 35 cycles of 95 x for 45 s, 61 x
this strain. for 30 s, 72 x for 2 min, and a ﬁnal extension of
72 x for 7 min. MgCl2 ﬁnal concentration in all PCR
reactions was 2.5 mM. Speciﬁc primers to amplify the
MATERIALS AND METHODS complete codifying regions of cDNAs, and used in the
sequencing reactions were designed from the Trametes
Organism and culture conditions
sp. I-62 lcc1, lcc2 and lcc3 laccase gDNA sequences
The basidiomycete used in this study, Trametes sp. I-62 ´
previously reported (Mansur, Suarez & Gonzalez ´
(CECT 20197), was isolated from decayed wood of 1998). Fig. 1 shows primers sequences and annealing
T. Gonzalez and others 729
Fig. 1. Binding sites and sequences of primers used in the PCR reactions to amplify the coding sequence of lcc1, lcc2 and lcc3
laccase genes from Trametes sp. I-62. Primer annealing sites are indicated by arrows. Coding and intravening sequences are
represented by clear and dark regions respectively. ‘cd ’ primers were used in the ampliﬁcation of lcc gene cDNAs and
‘ icd ’ primers were used in the sequencing reactions.
sites for each gene. DNA samples were electrophoresed RESULTS
on 1% agarose gels, and visualized under UV light after
Synthesis, cloning and sequencing of lcc gene cDNAs
staining with ethidium bromide.
In order to isolate RNA for cDNA synthesis, Trametes
sp. I-62 was grown in conditions that result in high
cDNA cloning and restriction analysis
levels of laccase, with the highest enzymatic activity
PCR products were puriﬁed from agarose gels using the being reached at the ﬁfth day of culture. The mycelium
‘Ultra Clean DNA puriﬁcation Kit ’ from MOBIO, and harvested at this day was used to purify total RNA.
cloned in the pGEM-T vector (‘pGEM-T Vector Sys- This was reverse transcribed into single-stranded
tem ’, Promega). Prior to sequencing, the clones corre- cDNA, that was ﬁnally ampliﬁed by PCR using speciﬁc
sponding to lcc1, lcc2 and lcc3 cDNAs, were checked primers, as described above.
by digestion with endonuclease enzymes that were As a result of the PCR reactions, a single product
expected to produce characteristic restriction patterns was obtained for each of the three laccase genes of
according to the restriction maps of corresponding Trametes sp. I-62. The size of the products was ap-
gDNA sequences. proximately 1600 bp, as expected from the putative
cDNA deduced from the genomic DNA sequences re-
ported by Mansur et al. (1997). The ampliﬁed cDNAs
DNA sequencing and analysis
were cloned and the resulting constructions were
Nucleotide sequences of inserts from selected clones subjected to restriction analysis as a ﬁrst step in the
were determined in the Sequencing Facility at Centro sequence analysis of these inserts. All cDNA clones
de Investigaciones Biologicas (Madrid) by using an corresponding to lcc2 and lcc3 genes showed the ex-
automated 3700 DNA sequencer (PerkinElmer, Ap- pected restriction pattern. However, lcc1 cDNA clones
plied Biosystems, Hitachi) and the sequencing kit Big were segregated into two diﬀerent groups (both having
Dye based on ﬂuorescence labelling reaction from the same proportion). One of them produced identical
the same manufacturer. All DNAs were sequenced on restriction patterns as expected from the gDNA se-
both strands. 5k and 3k ends of the cDNA cloned in the quence, but the other group showed unexpected pat-
pGEM-T vector were sequenced using the T7 and M13 terns upon digestion with ApaI and BamHI enzymes.
reverse ‘universal ’ primers. Sequencing of internal re- Inserts from the last group of clones could not be
gions was done using speciﬁc primers for each cDNA digested with ApaI, as it should be if they had the
(Fig. 1). cDNA and putative protein sequences were expected sequence ; and the digestion with BamHI
analysed with ALIGN, BESTFIT and BLAST pro- produced only two restriction fragments instead of
grams integrated in the macroprogram ‘GCG Wis- the three that should be obtained. These results indicate
consin ’ developed by Devereux, Haeberli & Smithies the lost of both the single ApaI site, and of one of the
(1984). The sequences of Trametes sp. I-62 lcc1, lcc1A, BamHI recognition sites in the cDNAs from this group
lcc2 and lcc3 cDNAs, reported in this paper, have of clones.
been assigned the GenBank accession nos. AF548032, Two clones carrying cDNA of each lcc2 and lcc3
AF548033, AF548034, AF548035, respectively. genes, together with two further clones from both
New fungal lcc gene, conﬁrmation of genomic DNAs 730
Fig. 2. Alignment of a new laccase cDNA sequence (lcc1A) from Trametes sp. I-62, isolated by RT–PCR (on top), with the
cDNA sequence of the lcc1 gene from this fungus. Diﬀerences are indicated by *.
groups of lcc1 cDNA clones were sequenced. Clones
Partial cloning of the new laccase gene by PCR–RFLP
of the two diﬀerent groups derived from lcc1 cDNA
restriction analysis have a very similar nucleotide se- Fig. 3 shows the strategy designed to isolate a fragment
quence (Fig. 2). They diﬀer in nine nucleotides, result- of gDNA corresponding to the possible new laccase
ing only in a two amino acid change between the gene. The basis of the analysis is the diﬀerence in the
putative proteins. These small diﬀerences prompted restriction patterns obtained from the digestion with
us to perform an additional analysis to verify that the ApaI and BamHI endonucleases. At ﬁrst, the 5k half of
new cDNA sequence corresponded to another laccase the lcc1 gene from genomic DNA was PCR-ampliﬁed,
gene, and that nucleotide changes were not due to using the speciﬁc primers shown in Fig. 3. This per-
mistakes introduced in the cDNA synthesis and/or mitted the cloning of a PCR product of approximately
ampliﬁcation. 1 kb (Fig. 4A) that was puriﬁed and digested with ApaI,
T. Gonzalez and others 731
Fig. 3. Strategy to conﬁrm the presence of the Trametes sp. I-62 lcc1A laccase gene. Coding and intravening sequences are
represented by clear and dark regions respectively. The coding region diﬀers from that of lcc1 in only nine base substitutions.
(A) These base changes cause the lost of an ApaI recognition site and of one of the two BamHI sites. (B) The 5k half of the gene
is ampliﬁed, and the PCR product is digested with ApaI, BamHI and KpnI. (C) Last enzyme must produce the same pattern for
both genes. Undigested DNA bands obtained upon treatment with ApaI and BamHI must correspond to the new gene (lcc1A).
In order to verify this assumption, this DNA was cloned and sequenced.
BamHI, and KpnI enzymes. As it was expected, part of
Characterization of the lcc cDNA sequences
the PCR product from the lcc1 gene ampliﬁcation was
digested with ApaI (Fig. 4B) and BamHI (Fig. 4C) and The DNA sequences of lcc1, lcc2 and lcc3 cDNAs
fragments of the expected size were obtained. However, obtained by RT–PCR consisted of 1563, 1563 and
approximately half of the total amount of this product 1575 bp, respectively. That is, lcc2 and lcc3 cDNA
remained undigested, as would occur if another laccase lengths agree with those predicted from the genomic
gene which diﬀers from lcc1 in these two restriction DNA sequences (Mansur et al. 1997). That from lcc1
sites actually exists. Digestion with KpnI endonuclease, diﬀers in the presence of three additional bases that
which has two recognition sites in this fragment, was do not cause any reading frame displacement. They
done in order to conﬁrm that the PCR product derived only introduce a new amino acid (valine) in the com-
exclusively from both lcc1 and the new laccase gene, position of the putative protein. Comparison between
and not from other diﬀerent sequences. In this case cDNA and genomic DNA sequences of the lcc1, lcc2
the PCR product was completely digested, and the and lcc3 genes conﬁrmed the structure proposed by
three expected restriction fragments were obtained these authors in relation to the number and position
(Fig. 4D). of intron/exons in each one of those genes (data not
Fragments of approximately 1 kb undigested with shown).
ApaI and BamHI, corresponding to the putative new The mature proteins encoded by lcc1, lcc2 and lcc3
laccase gene were subsequently puriﬁed and cloned cDNAs are predicted to be 499 amino acids in length.
in the pGEM-T vector. Clones containing both inserts The rest of the characteristics of these deduced protein
were sequenced. The sequences of the new lcc cDNA sequences, such as the signal peptide, the potential sites
isolated in this work, and that of the genomic DNA of N-glycosylation and the copper-binding motifs were
fragment entirely coincide in the exons regions. This as proposed by Mansur et al. (1997), and they are in-
conﬁrms that they correspond to the same gene: a dicated in the amino acid sequences shown in Fig. 5.
laccase gene diﬀerent from lcc1, which was assigned Furthermore, lcc1 and lcc3 products display 71.9 %
as lcc1A. identity at the amino acid level, whereas lcc2 product
New fungal lcc gene, conﬁrmation of genomic DNAs 732
of the laccase gene family in this biotechnologically
The comparison between the sequence of each cDNA
and the corresponding genomic sequence allowed us
to: (1) conﬁrm the predicted structures according to
the number and position of intron/exons; and (2)
deduce the sequences of mature proteins. Analysis of
the structural characteristic of lcc1, lcc2 and lcc3 genes
from Trametes sp. I-62, and their evolutive relation-
ship with laccase genes from other basidiomycetes were
broadly discussed by Mansur et al. (1997). Taking into
account the structural analysis of the three lcc genes,
and the diﬀerences in the nucleotide sequences and in
the number and position of introns, these authors
concluded that they were not alleles. Moreover, the
results obtained by Southern Blot analysis suggested
that the laccase family of Trametes sp. I-62 is formed
by up to ﬁve diﬀerent genes.
In the present work, a cDNA sequence which only
Fig. 4. Conﬁrmation of lcc1A gene by PCR–RFLP. (A) diﬀers from that of lcc1 in nine base changes has been
Fragment of lcc1 gene ampliﬁed from Trametes sp. I-62 isolated. This represents a 99.6 % identity at the protein
genomic DNA. (B–C) Restriction analysis of the ampliﬁed level. A fragment of the genomic sequence of this gene,
fragment digested with ApaI and BamHI, respectively, which named lcc1A, was isolated by PCR–RFLP analysis.
cut lcc1 gene, but not lcc1A. Changes in the sequence of lcc1A The multiplicity of genes having only slight diﬀer-
cause lost of both restriction sites. (D) Complete digestion of ences in their nucleotide sequences is a phenomenon
the ampliﬁed fragment with KpnI conﬁrms that all the PCR frequently found in fungi. Nevertheless, the great simi-
product is derived from lcc1 and lcc1A genes. It has two rec-
larity between sequences can be the product of the
ognition sites for this enzyme that generate three character-
istic bands. M=molecular weight marker.
duplication of ancestral genes that constitute families,
or simply the result of allelic diﬀerences. As mentioned
in the introduction of the present work, laccase genes
shares 67.4 and 75.5 % identity with Lcc1 and Lcc3 have been described in diﬀerent species of basidio-
proteins, respectively. With regard to lcc1A, as men- mycete fungi. Although less frequent, there are some
tioned previously, its cDNA sequence diﬀers from that reports on the allelic sequences of these genes. The
of lcc1 only in nine nucleotide changes, located in the existence of a laccase family in a single organism could
5k region of the gene (Fig. 2). The two proteins deduced represent an evolutionary mechanism to expand its
from their cDNA sequences have identical amino acid catabolic capabilities associated with the adaptation to
number. Only two of the nucleotide changes resulted the environment. Similar enzymes can evolve by the
in amino acid changes : a phenylalanine instead of introduction of changes in their active or structural
leucine in the signal-peptide region from Lcc1A, and sites, and are selected according to their degradation
an isoleucine is replaced by valine in the corresponding potential against a wide range of toxic compounds to
mature protein. It should be noted that as these four the fungus. This variability can be also enhanced and/
amino acids belong to the group of neutral and hydro- or assessed in allelic copies. Coincidently, the ﬁrst de-
phobic amino acids, the inﬂuence of these changes in scription of laccase genes from a basidiomycete fungus
the ﬁnal protein characteristics should be less than if involved the isolation of cDNA and genomic DNA
they were from diﬀerent groups. The rest of nucleotide sequences coding two allelic forms of Coriolus hirsutus
changes correspond to silent mutations. As shown in laccases (Kojima et al. 1990). The coding regions of
Fig. 5, the most important characteristics of both pro- these alleles diﬀered in only 18 bases changes rep-
teins are almost identical. The two amino acid changes resenting a single amino acid diﬀerence in the corre-
represent only a 0.4 % divergence in the deduced amino sponding putative proteins. In the basidiomycete
acid sequence of these gene products. Rhizoctonia solanii, from which a family of four laccase
genes has been reported, two cDNA sequences that
diﬀer in a few nucleotides with respect to the corre-
sponding genomic DNAs have also been described.
The identiﬁcation and genomic cloning of three laccase This produces changes in only ﬁve and four amino
genes from Trametes sp. I-62 was reported by Mansur acids of the protein sequences deduced from the lcc3
et al. in 1997. Nevertheless, the cDNA sequences of and lcc4 genes, respectively. Therefore, the authors
these genes had not been yet isolated. Here, we report proposed that they represented allelic forms of both
on the cloning of the lcc1, lcc2 and lcc3 together with genes (Wahleithner et al. 1996). Finally, Zhao & Kwan
a novel lcc1A, cDNA permitting a fuller examination (1999) have described two genomic sequences that
T. Gonzalez and others 733
Fig. 5. Alignment of the putative Lcc1, Lcc1A, Lcc2 and Lcc3 amino acid sequences deduced from the corresponding cDNA
sequences of these Trametes sp. I-62 genes (they are shown from the upper side to the bottom, in this order). Areas of dark
background indicate common amino acids. Copper-binding domains are indicated in boxes. The predicted cleavage sites of the
signal peptides are indicated by arrows.
represent allelic variants of the lcc1 gene from Lentinula other copies of lcc2 and lcc3 must be also present in
edodes. These alleles diﬀer in 45 nucleotides producing Trametes sp. I-62. Nevertheless, looking at the high
seven amino acid changes between the deduced pro- similarity between lcc1 and lcc1A, it could be not sur-
teins. Mansur et al. (1997) found that all the three lac- prising that the allelic copies have not any detectable
case genes they sequenced had the highest similarities change in their coding sequences respect to those of
with laccases from other basidiomycetes, not between lcc2 and lcc3. The real importance of our hypothesis
them ; on the contrary, the sequence we are reporting about the existence of allelic copies in this fungus
here, lcc1A, has the highest identity with laccase lcc1 then, is that its laccase family would be less numerous
from the same organism, Trametes sp. I-62. If the dif- than expected from the ﬁrst studies by Mansur et al.
ferences between the pairs of alleles reported from the (1997). Southern Blot analysis performed by these
three basidiomycetous species mentioned above are authors using speciﬁc probes to detect lcc1, lcc2 and
compared with those existing between lcc1 and lcc1A lcc3 genes under restrictive hybridization conditions,
genes, it is possible to suggest that these two genes suggested the existence of other laccase genes which
are alleles. This would be entirely passing given that diﬀered from these 3 ones. One of the additional hy-
Trametes sp. I-62 is an heterokaryon strain. So that, bridization bands was detected in the genomic DNA
New fungal lcc gene, conﬁrmation of genomic DNAs 734
digested with BamHI endonuclease. The lost of one Devereux, J., Haeberli, P. & Smithies, O. (1984) A comprehensive
BamHI recognition site was one of the features found set of sequence analysis programs for the VAX. Nucleic Acids
Research 12: 387–395.
to distinguish lcc1A from lcc1. Then, the additional Field, J. A., de Jong, E., Feijoo-Costa, G. & de Bont, J. A. M. (1993)
band observed, having a higher size than that corre- Screening for ligninolytic fungi applicable to the biodegradation
sponding to lcc1 would be the result of the hybridiz- of xenobiotics. Trends in Biotechnology 11: 44 – 49.
ation of lcc1A. In order to conﬁrm this hypothesis, it Germann, U. A. & Lerch, K. (1986) Isolation and partial nucleotide
is necessary to obtain a monokaryotic mycelium from sequence of the laccase gene from Neurospora crassa: amino acid
sequence homology of the protein to human ceruloplasmin. Pro-
Trametes sp. I-62 and to study the segregation of genes ceedings of the National Academy Sciences, USA 83: 8854 –8858.
in monokaryons. Physical maps, sequencing of new Giardina, P., Cannio, R., Martirani, L., Marzullo, L., Palmieri, G. &
laccase genes, analysis of laccase activity using speciﬁc Sannia, G. (1995) Cloning and sequencing of a laccase gene from
substrates, and induction studies, among others, will the lignin-degrading basidiomycete Pleurotus ostreatus. Applied
support a theoretical frame to correlate laccase cod- and Environmental Microbiology 61: 2408–2413.
Giardina, P., Aurilia, V., Cannio, R., Marzullo, L., Amoresano, A.,
ing sequences with their possible role on biological Siciliano, R., Pucci, P. & Sannia, G. (1996) The gene, protein and
ﬁtness, biotechnological applications, and rational en- glycan structures of laccase from Pleurotus ostreatus. European
zyme design. This will contribute to a better under- Journal of Biochemistry 235: 508–515.
standing of a gene family that, due to its great potential Giardina, P., Palmieri, G., Scaloni, A., Fontanella, B., Faraco, V.,
in biotechnological applications, deserves future in- Cennamo, G. & Sannia, G. (1999) Protein and gene structure of
a blue laccase from Pleurotus ostreatus. Biochemistry Journal 341:
Givaudan, A., Eﬀosse, A., Faure, D., Potier, P., Bouillant, M. L. &
Bally, R. (1993) Polyphenol oxidase from Azospirillum lipoferum.
FEMS Microbiology Letters 108 : 205–210.
We are grateful to Gloria del Solar, Manuel Espinosa and Alan D. W. ´ ´
Gonzalez, T., Terron, M. C., Yague, S., Zapico, E., Galletti, G. C. &
Dobson for their critical reading of the manuscript. We wish also to ´
Gonzalez, A. E. (2000) Pyrolysis/gas chromatography/mass
acknowledge the valuable help of Juan Pascual and Lisandro Rodon spectrometry monitoring of fungal-biotreated distillery wastewater
in the design of some of the ﬁgures. Authors wish to thank the CI- using Trametes sp. I-62 (CECT 20197). Rapid Communications in
CYT (Madrid) BIO 95-2065-E and BIO 97-0655 for the ﬁnancial Mass Spectrometry 14: 1417–1424.
support. Tania Gonzalez acknowledges support from a Mutis Pro- Gouka, R. J., van der Heiden, M., Swarthoﬀ, T. & Verrips, C. T.
gramme doctoral grant from AECI (Spain), and Marı´ a C. Terron, a (2001) Cloning of a phenol oxidase gene from Acremonium
postdoctoral grant from Conserjerı´ a de Educacion y Cultura de la murorum and its expression in Aspergillus awamori. Applied and
Comunidad Autonoma de Madrid. Environmental Microbiology 67: 2610–2616.
Guillen, F., Martı´ nez, A. T. & Martı´ nez, M. J. (1990) Production of
hydrogen peroxide by aryl-alcohol oxidase from the ligninolytic
fungus Pleurotus eryngii. Applied Microbiology and Biotechnology
REFERENCES 32: 465– 469.
Hatakka, A. I., Mohammandi, O. K. & Lundell, T. K. (1989) The
Akin, D. E., Sethuraman, A., Morrison III, W. H., Martin, S. A. & potential of white-rot fungi and their enzymes in the treatment
Eriksson, K. E. (1993) Microbial deligniﬁcation with white rot of lignocellulosic feed. Food Biotechnology 3: 45– 48.
fungi improves forage digestibility. Applied and Environmental Hatakka, A. (1994) Lignin-modifying enzymes from selected white-
Microbiology 59 : 4272– 4282. rot fungi: production and role in lignin degradation. FEMS
Alexandre, G. & Zhulin, I. B. (2000) Laccase are widespread in Microbiology Review 13: 125–135.
bacteria. TIBTECH 18: 41– 42. Husain, Q. & Jan, U. (2000) Detoxiﬁcation of phenols and aromatic
Ander, P. & Eriksson, K. E. (1976) The importance of phenol oxidase amines from polluted wastewater by using phenol oxidases. Journal
activity in lignin degradation by the white rot fungus Sporotrichum of Sciences of Indian Research 59 : 286–293.
pulverulentum. Archives of Microbiology 109 : 1–8. Jonsson, L., Sjostrom, K., Haggstrom, I. & Nyman, P. O. (1995)
¨ ¨ ¨ ¨
Aramayo, R. & Timberlake, W. E. (1990) Sequence and molecular Characterization of a laccase gene from the white-rot fungus
structure of the Aspergillus nidulans yA (laccase I) gene. Nucleic Trametes versicolor and structural features of basidiomycete
Acids Reserach 18 : 3415. laccases. Biochimica and Biophysica Acta 1251: 210–215.
Bourbonnais, R., Paice, M. G., Reid, I. D., Lanthier, P. & Yaguchi, Kirk, T. K., Croan, S., Tien, M., Murtagh, K. E. & Farrell, R. L.
M. (1995) Lignin oxidation by laccase isozymes from Trametes (1986) Production of multiple ligninases by Phanerochaete chryso-
versicolor and role of the mediator 2,2k-azinobis (3-ethylbenzthia- sporium: eﬀect of selected growth conditions and use of a mutant
zoline-6-sulfonate) in kraft lignin depolymerization. Applied and strain. Enzyme Microbiology and Technology 8: 27–32.
Environmental Microbiology 61: 1876–1880. Kirk, T. K. & Farrell, R. (1987) Enzymatic ‘combustion’: the
Breen, A. & Singleton, F. L. (1999) Fungi in lignocellulose break- microbial degradation of lignin. Annual Review of Microbiology
down and biopulping. Current Opinion Biotechnology 10: 252–258. 41: 465–505.
Call, H. P. & Mucke, I. (1997) History, overview and applications of
¨ Kojima, Y., Tsukuda, Y., Kawai, Y., Tsukamoto, A., Sugiura, J.,
mediated ligninolytic systems, especially laccase-mediator-systems Sakaino, M. & Kita, Y. (1990) Cloning, sequence analysis, and
(Lignozym1 -process). Journal of Biotechnology 53 : 163–202. expression of ligninolytic phenoloxidase genes of the white-rot
Coll, P. M., Fernandez-Abalos, J. M., Villanueva, J. R., Santamarı´ a, basidiomycete Coriolus hirsutus. Journal of Biological Chemistry
R. & Perez, P. (1993a) Puriﬁcation and characterization of a 265: 15224 –15230.
phenoloxidase (laccase) from the lignin-degrading basidiomycete Larson, T. G., Choi, G. H. & Nuss, D. L. (1992) Regulatory
PM1 (CECT 2971). Applied and Environmental Microbiology 59: pathways governing modulation of fungal gene expression by a
2607–2613. virulence-attenuating mycovirus. EMBO Journal 11: 4539– 4548.
Coll, P. M., Tabernero, C., Santamarı´ a, R. & Perez, P. (1993b) Leatham, G. F. (1985) Extracellular enzymes produced by the
Characterization and structural analysis of the laccase I gene from cultivated mushroom Lentinus edodes during degradation of a
the newly isolated ligninolytic basidiomycete PM1 (CECT 2971). lignocellulosic medium. Applied and Environmental Microbiology
Applied and Environmental Microbiology 59 : 4129–4135. 50: 859–867.
T. Gonzalez and others 735
Leonowicz, A., Cho, N. S., Luterek, J., Wilkolazka, A., Wojtas- Soden, D. M. & Dobson, A. D. W. (2001) Diﬀerential regulation of
Wasilewska, M., Matuszewska, A., Hofrichter, M., Wesenberg, D. laccase gene expression in Pleurotus sajor-caju. Microbiology 147:
& Rogalski, J. (2001) Fungal laccase: properties and activity on 1755–1763.
lignin. Journal of Basic Microbiology 41: 185–227. Solano, F. & Sanchez-Amat, A. (1999) Studies on the phylogenetic
Mansur, M., Suarez, T., Fernandez-Larrea, J. B., Brizuela, M. A. & relationships of melanogenic marine bacteria. Proposal of Mar-
Gonzalez, A. E. (1997) Identiﬁcation of a laccase gene family in inomonas mediterranea sp. nov. International Journal of Systematic
the new lignin-degrading basidiomycete CECT 20197. Applied and Bacteriology 49: 1241–1246.
Environmental Microbiology 63: 2637–2646. Thurston, C. F. (1994) The structure and function of fungal laccases.
Mansur, M., Suarez, T. & Gonzalez, A. E. (1998) Diﬀerential Microbiology 140 : 19–26.
gene expression in the laccase gene family from basidiomycete Wahleithner, J. A., Xu, F., Brown, K. M., Brown, S. H., Golightly,
I-62 (CECT 20197). Applied and Environmental Microbiology 64: E. J., Halkier, T., Kauppinen, S., Pederson, A. & Schneider, P.
771–774. (1996) The identiﬁcation and characterization of four laccases from
Martins, L. O., Soares, C. M., Pereira, M. M., Teixeira, M., the plant pathogenic fungus Rhizoctonia solani. Current Genetics
Costa, T., Jones, G. H. & Henriques, A. O. (2002) Molecular 29: 395–403.
and biochemical characterization of a highly stable bacterial Williamson, P. R. (1997) Laccase and melanin in the pathogenesis
laccase that occurs as a structural component of the Bacillus of Cryptococcus neoformans. Frontiers in Bioscience 2: 99–107.
subtilis endospore coat. Journal of Biological Chemistry 277: Wolfenden, B. S. & Willson, R. L. (1982) Radical-cations as reference
18849–18859. chromogens in kinetic studies of one-electron transfer reactions:
Mayer, A. M. & Staples, R. C. (2002) Laccase: new functions for an pulse radiolysis studies of 2,2k-azinobis-(3-ethylbenzthiazoline-6-
old enzyme. Phytochemistry 60 : 551–565. sulphonate). Journal of the Chemical Society. Perkin Transactions.
Mikuni, J. & Morohoshi, N. (1997) Cloning and sequencing of a II: 805–812.
second laccase gene from the white-rot fungus Coriolus versicolor. Wood, D. A. (1980) Inactivation of extracellular laccase during
FEMS Microbiology Letters 155 : 79–84. fruiting of Agaricus bisporus. Journal of General Microbiology 117:
Ong, E., Pollock, W. B. R. & Smith, M. (1997) Cloning and sequence 339–345.
analysis of two laccase complementary DNAs from the ligninolytic ´ ´
Yague, S., Terron, M. C., Gonzalez, T., Zapico, E., Bocchini, P.,
basidiomycete Trametes versicolor. Gene 196: 113–119. ´
Galletti, G. C. & Gonzalez, A. E. (2000) Biotreatment of a tannin-
Perry, C. R., Smith, M., Britnell, C. H., Wood, D. & Thurston, C. F. rich beer-factory wastewater with the white-rot basidiomycete
(1993) Identiﬁcation of two laccase gene in the cultivated mush- Coriolopsis gallica monitored by pyrolysis/gas chromatography/
room Agaricus bisporus. Journal of Genetic Microbiology 139: mass spectrometry. Rapid Communications in Mass Spectrometry
1209–1218. 14: 905–910.
Pointing, S. B. (2001) Feasibility of bioremediation using white rot Yaver, D. S. & Golightly, E. J. (1996) Cloning and characterization
fungi. Applied Microbiology and Biotechnology 57 : 20–33. of three laccase genes from the white-rot basidiomycete Trametes
Reinhammar, B. (1984) Laccase. In Copper Proteins and Copper villosa: genomic organization of the laccase gene family. Gene 181:
Enzymes. (R. Lontie, ed.): 1–35. CRC Press, Boca Raton. 95–102.
Roy-Arcand, L. & Archibald, F. S. (1991) Direct dechlorination of Yaver, D. S., Xu, F., Golightly, E. J., Brown, K. M., Brown, S. H.,
chlorophenolic compounds by laccases from Trametes (Coriolus) Rey, M. W., Schneider, P., Halkier, T., Mondorf, K. & Dalbøge,
versicolor. Enzyme Microbiology and Technology 13 : 194–203. H. (1996) Puriﬁcation, characterization, molecular cloning, and
Saloheimo, M. & Niku-Paavola, M.-L. (1991) Heterologous expression of two laccase genes from the white rot basidiomycete
production of a ligninolytic enzyme: expression of the Phlebia Trametes villosa. Applied and Environmental Microbiology 62:
radiata laccase gene in Trichoderma reesei. Biotechnology 9: 834–841.
987–990. Zhao, J. & Kwan, H. S. (1999) Characterization, molecular cloning
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular cloning: and diﬀerential expression analysis of laccase genes from the edible
a laboratory manual. Cold Spring Harbor Laboratory Press, Cold mushroom Lentinula edodes. Applied and Environmental Micro-
Spring Harbor, NY. biology 65 : 4908–4913.
Servili, M., DeStefano, G., Piacquadio, P. & Sciancalepore, V. (2000)
A novel method for removing phenols from grape must. American
Journal of Enology and Viticulture 51: 357–361. Corresponding Editor: S. B. Pointing