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  1. 1. 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 laccase family ´ ´ ¨ ´ 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 : aldo@cib.csic.es 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 EC1.10.3.2) 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
  2. 2. 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 Laccase activity 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 RNA preparation 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- RT–PCR 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
  3. 3. ´ 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
  4. 4. 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,
  5. 5. ´ T. Gonzalez and others 731 (A) (B) (C) 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
  6. 6. New fungal lcc gene, confirmation of genomic DNAs 732 of the laccase gene family in this biotechnologically important fungus. 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- DISCUSSION 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
  7. 7. ´ 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
  8. 8. 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: vestigation. 655–663. Givaudan, A., Effosse, A., Faure, D., Potier, P., Bouillant, M. L. & Bally, R. (1993) Polyphenol oxidase from Azospirillum lipoferum. ACKNOWLEDGEMENTS 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. 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