Characterization of the major laccase isoenzyme from Trametes pubescens and regulation of its synthesis by metal ionsa

Christiane Galhaup1, Sabine Goller2, Clemens K. Peterbauer3, Josef Strauss2 and Dietmar Haltrich1

Division of Biochemical Engineering, Institute of Food Technology1 and Centre of Applied Genetics2, University of Agricultural Sciences Vienna (Universität für Bodenkultur Wien, BOKU), Muthgasse 18, A-1190 Wien, Austria
Institute of Biochemical Technology and Microbiology, University of Technology (TU Wien), Getreidemarkt 9, A-1060 Wien, Austria3

Author for correspondence: Dietmar Haltrich. Tel: +43 1 36006 6275. Fax: +43 1 36006 6251. e-mail: haltrich{at}edv2.boku.ac.at


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The major laccase isoenzyme LAP2 secreted by the white-rot basidiomycete Trametes pubescens in response to high copper concentrations was purified to apparent electrophoretic homogeneity using anion-exchange chromatography and gel filtration. The monomeric protein has a molecular mass of 65 kDa, of which 18% is glycosylation, and a pI value of 2·6. The pH optima of the laccase depend on the substrates oxidized and show bell-shaped pH activity profiles with an optimum of 3–4·5 for phenolic substrates such as 2,6-dimethoxyphenol or syringaldazine, while the non-phenolic substrates ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)] and ferrocyanide show a monotonic pH profile with a rate increasing with decreasing pH. The catalytic efficiencies kcat/Km determined for some of its substrates were 48x106, 47x106, 20x106 and 7x106 M-1 s-1 for ABTS, syringaldazine, ferrocyanide and oxygen, respectively. Furthermore, the gene lap2 encoding the purified laccase was cloned and its nucleotide sequence determined. The gene consists of 1997 bp, with the coding sequence interrupted by eight introns and flanked by an upstream region in which putative CAAT, TATA, MRE and CreA consensus sequences were identified. Based on Northern analysis containing total RNA from both induced and uninduced cultures, expression of lap2 is highly induced by copper, which is also corroborated by an increase in laccase activity in response to copper. A stimulating effect of various other heavy metal ions on laccase synthesis was also observed. In addition to induction, a second regulatory mechanism seems to be repression of lap2 transcription by glucose.

Keywords: white-rot fungus, ligninolytic enzymes, polyphenol oxidase, induction, copper

Abbreviations: ABTS, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate); 2,6-DMP, 2,6-dimethoxyphenol; HSE, heat-shock element; MRE, metal-responsive element; STRE, stress-responsive element

a The GenBank accession numbers for the lap2 and lap1a genes reported in this paper are AF414807 and AF414808, respectively.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Laccases (benzenediol:oxygen oxidoreductases; EC 1.10.3.2), multicopper enzymes belonging to the blue oxidases, catalyse the one-electron abstraction from a wide variety of organic and inorganic substrates, including mono-, di- and polyphenols, aminophenols, methoxyphenols, metal complexes such as ferrocene, ferrocyanide or iodide, with the concomitant four-electron reduction of oxygen to water (Eggert et al., 1996 ; Reinhammar, 1984 ; Solomon et al., 1996 ; Thurston, 1994 ). Laccases are found in plants, insects and bacteria, but the most important sources of this enzyme are fungi. In these latter organisms, laccases are proposed to play a role in lignin degradation and/or the removal of potentially toxic phenols arising during this degradation, morphogenesis, sporulation, or phytopathogenesis and fungal virulence (Gianfreda et al., 1999 ). Typically, fungal laccases are extracellular, glycosylated proteins of 60–85 kDa of which 15–20% is carbohydrate (Thurston, 1994 ; Xu, 1999 ). In white-rot fungi, laccases are typically produced as multiple isoenzymes (Bollag & Leonowicz, 1984 ; Eggert et al., 1996 ) encoded by gene families (Kojima et al., 1990 ; Yaver & Golightly, 1996 ) and it has been suggested that genes encoding various isoenzymes are differentially regulated (Mansur et al., 1998 ), with some being constitutively expressed and others being inducible (Bollag & Leonowicz, 1984 ; Soden & Dobson, 2001 ).

Laccases are attractive, industrially relevant enzymes that can be used for a number of diverse applications, e.g. for biocatalytic purposes such as delignification of lignocellulosics and cross-linking of polysaccharides, for bioremediation such as waste detoxification and textile dye transformation (Gianfreda et al., 1999 ), for use in food technological, for personal and medical care applications (Xu, 1999 ) and for biosensor and analytical purposes (Yaropolov et al., 1994 ). Production of fungal laccase for these applications can be stimulated by the presence of various inducing substances, mainly aromatic or phenolic compounds related to lignin or lignin derivatives (Gianfreda et al., 1999 ). In addition, laccase production can be influenced by the nitrogen concentration in the culture medium (Gianfreda et al., 1999 ) and by the carbon source employed (Galhaup et al., 2002 ). Recently, an important effect of copper on laccase synthesis in Trametes versicolor, Pleurotus ostreatus and Ceriporiopsis subvermispora has been described (Collins & Dobson, 1997 ; Karahanian et al., 1998 ; Palmieri et al., 2000 ). In these studies it was shown that the addition of copper to the growth medium significantly increases laccase formation and that the regulation of the synthesis of several different laccase isoforms by copper occurs at the level of gene transcription.

Recently, we found that the white-rot basidiomycete Trametes pubescens is an outstanding producer of this biotechnologically important enzyme (Galhaup & Haltrich, 2001 ; Galhaup et al., 2002 ). Extracellular laccase formation by this fungus can be greatly stimulated by the addition of copper in the millimolar range which has to be added to a simple, glucose-based medium during the growth phase of the organism to exert its maximal effect (Galhaup & Haltrich, 2001 ). In contrast, typically used aromatic inducers are hardly effective in increasing laccase synthesis by T. pubescens. Laccase production could be further increased when the substrate glucose was continuously fed to a cultivation of T. pubescens after almost complete consumption of the initially used substrate so that its concentration in the medium was always negligible. Under these conditions, T. pubescens routinely produced 750 U laccase ml-1, corresponding to approximately 700 mg laccase protein l-1 (Galhaup et al., 2002 ), which appears to be among the highest values reported for laccase production to date and even compares favourably with the heterologous expression of recombinant laccases (Gianfreda et al., 1999 ; Yaver et al., 1999 ). In addition, a purified laccase isoenzyme from T. pubescens was superior for biocatalytic applications investigated in our laboratory (Baminger et al., 2001 ). Because of potential uses of T. pubescens laccase in biocatalysis and biotechnology, we purified and characterized the major laccase isoenzyme secreted by this fungus under high copper growth conditions and studied its regulation in more detail.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Chemicals.
Chemicals were obtained from Sigma unless otherwise indicated and were of the highest purity available. Peptone from casein (pancreatic digest), 2,6-dimethoxyphenol (2,6-DMP) and MgSO4.7H2O were from Fluka, while yeast extract was from Merck. Enzymes for manipulating DNA were obtained from Promega. The cloning vector Bluescript SK+ was from Stratagene. Oligonucleotide primers were obtained from MWG Biotech and sequencing primers were synthesized at the Vienna Biocenter Genomics Instrumentation Facility of VBC Genomics.

Organisms and culture conditions.
T. pubescens MB 89 (=CBS 696.94; Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) was isolated from an ash tree (Fraxinus excelsior) in Gimbachtal, Upper Austria, by H. Prillinger (Institute of Applied Microbiology, University of Agricultural Science, Vienna) and was maintained through periodic transfer at 25 °C on potato dextrose agar plates. Shaken flask cultures of T. pubescens were grown at 25 °C with continuous agitation (110 r.p.m.) in baffled 1000 ml Erlenmeyer flasks containing 200 ml medium. The basal GYP medium used for cultures unless otherwise stated contained 20 g glucose l-1, 5 g yeast extract l-1, 5 g peptone from casein l-1 and 1 g MgSO4.7H2O l-1. The pH was adjusted to 5·0 with H3PO4 prior to sterilization. Several agar plugs cut from the actively growing, outer circumference of a fungal colony growing on potato dextrose plates were used as inocula. For stimulating laccase synthesis, CuSO4 . 5H2O was added after 64–96 h cultivation so that its final concentration in the medium was 2·0 mM (Galhaup & Haltrich, 2001 ). Inoculated flasks were cultivated for 8 days unless otherwise stated. Escherichia coli JM109 competent cells were obtained from Promega.

Purification of laccase.
For purification of extracellular laccase, T. pubescens was cultivated in a 15 l fermentation on GYP medium using 2 mM copper to stimulate laccase formation as described elsewhere (Galhaup & Haltrich, 2001 ). Mycelia were separated by centrifugation (20 min; 10000 g) after 200 h cultivation when laccase activity reached its maximum, and the culture supernatant was frozen, thawed and filtered to remove precipitated polysaccharides. The enzyme solution was then concentrated using a 30 kDa ultrafiltration membrane (Daicen). Any precipitate was removed by centrifugation (20 min; 10000 g). The clear supernatant was repeatedly dialysed against water and was applied to a Q-Sepharose Fast Flow column (50x250 mm; Amersham-Pharmacia), pre-equilibrated with 20 mM sodium acetate buffer, pH 5·0. The column was washed at a flow rate of 5 ml min-1 with 2000 ml buffer to remove unbound laccase isoforms and protein. Bound laccase was subsequently eluted from the column with a linear salt gradient (0–0·25 M NaCl in the same buffer) with a flow rate of 5 ml min-1. Elution was simultaneously monitored at 280 and 610 nm for protein and type-1 copper, respectively. Fractions containing laccase activity were pooled, concentrated as above, applied to a Superdex 75 prep grade column (800x16 mm; Amersham-Pharmacia) pre-equilibrated with 20 mM sodium acetate buffer, pH 5·0, containing 200 mM NaCl, and eluted at a flow rate of 0·5 ml min-1. Active fractions were pooled, desalted, filter-sterilized, and stored at 4 °C. For molecular mass determination of the native protein, a Superdex 75 HR 10/30 column (Amersham-Pharmacia), equilibrated with 50 mM phosphate buffer, pH 7·0, containing 100 mM KCl, was used. The column was calibrated with the standard proteins ribonuclease A (Mr 13700), carbonic anhydrase (Mr 29000), ovalbumin from chicken egg (Mr 45000), bovine serum albumin (Mr 66000) and transferrin (Mr 81000), each at a concentration of 2 mg ml-1. The flow rate for elution was 0·5 ml min-1.

Enzyme activity assay.
Laccase activity was determined with ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate)] as the substrate (Bourbonnais & Paice, 1990 ). The assay mixture contained 1 mM ABTS, 20 mM sodium acetate buffer (pH 3·5) and 10 µl aliquots of appropriately diluted enzyme sample. Oxidation of ABTS was monitored by following the increase in A436 ({epsilon} 29·3 mM-1 cm-1). One unit of laccase activity was defined as the amount of enzyme required to oxidize 1 µmol ABTS min-1 at 25 °C. To determine pH optima for various substrates, air-saturated sodium citrate buffer (20 mM, pH 2·5–6·5) was used. The optimum temperature was determined between 15 and 70 °C under standard assay condition using ABTS as the substrate.

Other analyses.
Protein concentrations were measured using the Bradford dye-binding assay (Coomassie blue, Bio-Rad) and bovine serum albumin (fraction V) as the standard. Glucose and fructose concentrations were determined using commercially available test kits (Boehringer Mannheim). The carbohydrate content of the purified laccase was estimated by the phenol sulfuric acid method using mannose as the standard (Dubois et al., 1956 ).

Electrophoretic analyses.
Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on the Amersham-Pharmacia Phast System using precast PhastGel 8–25% gels according to the manufacturer’s instructions. Samples of laccase LAP2 were denatured by dilution with an equal volume of Sample Buffer, Laemmli 2xconcentrate (Sigma) and incubation for 5 min at 95 °C. The molecular mass was estimated by comparison with marker proteins (Molecular Standard Mixture Recombinant, 15–150 kDa; Sigma) after proteins were visualized by silver staining. IEF was performed on the Multiphor II system (Amersham-Pharmacia) using precast dry gels (CleanGel IEF) rehydrated with carrier ampholytes (one part Pharmalyte pH 2·5–5 and one part Ampholine pH 4–6·5; Amersham-Pharmacia) as described by the manufacturer. The low-pI marker protein kit (pH 2·8–6·5; Amersham-Pharmacia) was used to determine pI values. Proteins were either stained with Coomassie blue or the laccase bands were visualized by activity staining. To this end the electrophoresis gel was incubated at room temperature with 20 mM sodium acetate buffer, pH 4·5, containing 10 mM ABTS, and gentle shaking until green bands were clearly visible.

Steady-state kinetic measurements.
All measurements were done at 25 °C at the pH optimum of the respective substrate using oxygen (air saturated solutions) as the electron acceptor. The extinction coefficients used are given in Table 2. Reactions were initiated by addition of laccase and initial rates were obtained from the linear portion of the progress curve. All kinetic constants were calculated by non-linear least-squares regression, fitting the observed data to the Henri–Michaelis–Menten equation. For the determination of the kinetic constants for oxygen, the rate of oxygen consumption was measured with an oxygen electrode placed in a thermostatically controlled vessel (Rank Brothers) at 25 °C using ABTS (1 mM in 20 mM sodium acetate buffer, pH 3·5) as the substrate.


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Table 2. Apparent kinetic constants of laccase LAP2 from T. pubescens for some of its substrates

 
N-terminal protein sequencing.
Purified laccase was electroblotted to PVDF membranes and sequenced by automated Edman degradation on a Protein Sequencer LF3600D (Beckman) at the Department of Biochemistry, Charles University, Prague, Czech Republic.

Molecular biological methods.
All manipulations of DNA and RNA were performed by established molecular biological methods (Sambrook et al., 1989 ).

Genomic DNA isolation.
Mycelia from 6-day-old cultures of T. pubescens grown in 200 ml GYP under continuous agitation were harvested by filtration, washed with sterile tap water and frozen in liquid nitrogen. The frozen mycelia were ground to a powder in a mortar containing liquid nitrogen. High-molecular-weight genomic DNA was isolated by organic extraction using phenol/chloroform/isoamyl alcohol 25:24:1 (by vol.) (Ausubel et al., 1990 ).

Oligonucleotide probes for T. pubescens laccase.
A specific degenerate forward primer 5'-GGB ATC GGB CCB GTB GCI GA-3' was designed according to the determined N-terminal protein sequence of purified laccase LAP2 (GIGPVADLTI-). A reverse degenerate primer 5'-TCV GTY TCG ATG ATV GTC-3' was designed corresponding to the amino acid sequence TIIET by homology comparison with the Trametes villosa lcc1 gene (Yaver et al., 1996 ) in a central position of the protein sequence. T. pubescens genomic DNA was used as the template for PCR. For PCR amplification, 200 ng template DNA was mixed with 5 µl 10x Taq buffer (Promega), 0·4 mM each dNTP, 20 pmol each primer, 2 U Taq polymerase, and adjusted to 50 µl with sterilized ultrapure water. The DNA amplification was performed in a DNA thermal cycler (PCR Sprint, Hybaid) with an initial cycle of denaturation (5 min at 95 °C) followed by 30 cycles of denaturation (1·5 min at 95 °C), annealing (2 min at 63 °C), extension (1·5 min at 72 °C), and then by a final incubation (10 min at 72 °C).

Genomic Southern blot analysis and cloning of laccase genes.
Genomic DNA was digested with several restriction enzymes, separated by agarose electrophoresis and blotted onto a Hybond-N+ membrane (Amersham-Pharmacia). The DNA fragments used as probes were labelled with digoxigenin-dUTP by using the DIG High Prime DNA labelling and detection kit (Roche Diagnostics) and were used following the manufacturer’s instructions. Based on the results obtained from Southern analysis, gene restriction maps of the lap2 genomic locus were established. A BamHI–HindIII fragment of approximately 5 kb apparently containing the complete coding region as well as the 5' regulatory and the 3' terminatory region was selected for cloning. Total genomic DNA was digested with BamHI/HindIII, separated by agarose electrophoresis and a fragment of the corresponding size was cut out of the gel, eluted by using the Qiagen gel extraction kit and ligated into vector Bluescript SK+. Resulting clones were screened by PCR using the same primers as above. Positive clones were subjected to restriction mapping and sequencing.

DNA sequencing and sequence analysis.
DNA sequencing was performed by VBC Genomics using a LI-COR 4200 L-1 sequencer. Sequence similarities were calculated by alignment of laccase gene sequences from EMBL/GenBank databases using the CLUSTAL V algorithm (GENEDOC).

RNA isolation.
T. pubescens cultures were grown on the basal GYP medium, in which glucose was replaced by fructose (FYP) at 25 °C. CuSO4 was added after 8 days so that its final concentration was 2 mM. Fungal mycelia were collected by filtration before and 10, 24 and 48 h after the addition of CuSO4. Control cultures without additional CuSO4 were treated accordingly. Mycelia were washed twice in sterile tap water and frozen in liquid nitrogen. RNA was isolated after grinding mycelia under liquid nitrogen with the RNeasy Plant Mini kit (Qiagen).

Northern blot analysis.
For Northern analysis, total RNA was separated on a 1·2% (w/v) agarose gel, transferred to Hybond-N membranes (Amersham-Pharmacia) and hybridized with radiolabelled probes under high stringency conditions (hybridization at 37 °C overnight; two washes each for 10 min at 42 °C using 2x SSC with 0·1% SDS, 1xSSC with 0·1% SDS and 0·5x SSC with 0·1% SDS). Radiolabelling of probes was performed by using Ready-To-Go DNA labelling beads (-dCTP) (Amersham-Pharmacia). RNA load and intensity of hybridization signals were monitored using a STORM 860 light scanning densitometer (Amersham-Pharmacia) supported by Image QuanNT software.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Purification of laccase II (LAP2)
Isolation of the main laccase isoenzyme (LAP2) was performed from the culture supernatant of a T. pubescens laboratory batch fermentation with glucose as the main substrate and stimulating laccase formation by the addition of copper to the actively growing culture (Galhaup & Haltrich, 2001 ). Under these conditions approximately 62000 U laccase activity and 350 mg extracellular protein l-1, of which a large fraction is laccase, are routinely formed by the fungus. As is evident from IEF of culture supernatant and activity staining with ABTS, T. pubescens secreted at least eight multiple isomeric forms of laccase under the growth conditions investigated which could be distinguished because of differences in their pI values, ranging from 2·6 to 6·0 (Fig. 1). Interestingly, the pattern of laccase isoenzymes and relative intensities of laccase bands were identical during the entire laccase production phase. The laccase isoform, which is predominantly formed by T. pubescens under the growth conditions chosen in this study and accounts for roughly 40% of total laccase activity, has a pI of 2·6 and was selected for purification and cloning.



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Fig. 1. IEF of culture supernatants obtained at different times of a cultivation of T. pubescens grown on a glucose-based medium; laccase bands were visualized by activity staining using ABTS as the substrate. Lanes: A, sample obtained 3 h after the addition of CuSO4; B, 10 h; C, 24 h; D, 48 h after the addition of CuSO4.

 
The purification protocol established for the selected laccase isoenzyme termed laccase II (LAP2) is based on anion-exchange chromatography and gel filtration. Anion-exchange chromatography of the concentrated culture filtrate gave two peaks of laccase activity. The first peak (LAP1) contained several unbound laccase isoenzymes, which were recovered with the equilibrating buffer. These isoenzymes had pI values above 3 as was proven by IEF followed by activity staining with ABTS (data not shown). The second peak, which eluted at a NaCl concentration of approximately 65 mM, contained only one laccase isoform with a pI of 2·6 (LAP2). Table 1 presents a summary of a typical purification of LAP2. This two-step procedure yielded a bright blue protein that was apparently homogeneous as judged by IEF and SDS-PAGE (data not shown). The purified LAP2 had a specific activity of 1100 U mg-1 under standard assay conditions using ABTS and oxygen (air) as substrates. The molecular mass of LAP2 was 65 kDa as determined by SDS-PAGE and 57 kDa by gel filtration, indicating a monomeric structure of this enzyme. The carbohydrate content of LAP2 was estimated to be 18% using the phenol/sulfuric acid method with mannose as the standard.


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Table 1. Purification of laccase LAP2 from T. pubescens

 
Kinetic properties of LAP2
In accordance with results on laccases from other fungal sources, T. pubescens LAP2 oxidizes a wide range of different substrates. Table 2 lists some of these substrates and their apparent kinetic constants determined spectrophotometrically when using oxygen (air-saturated solutions) as the electron acceptor. The highest catalytic efficiencies kcat/Km were found for the model substrates ABTS (48x106 M-1 s-1) and syringaldazine (47x106 M-1 s-1). For ABTS, which was investigated in more detail, the apparent Michaelis constant Km increased with increasing pH, hence a lower pH seems to favour binding of this substrate, while the apparent catalytic constant kcat decreased approximately twofold when the pH was increased from 3·0 to 4·5. Furthermore, LAP2 exhibited high activity with either hydroxy- or methoxy-substituted phenols. LAP2 also catalysed the oxidation of inorganic ions such as ferrocyanide very efficiently (kcat/Km 20x106 M-1 s-1). In addition, kinetic constants for oxygen are given (Table 2). A Km of 0·41 mM was determined for this laccase substrate. Considering the low solubility of oxygen in aqueous solutions under standard assay conditions, approximately 0·26 mM, laccase typically is employed with non-saturating concentrations of its electron acceptor oxygen.

The effects of several known laccase inhibitors on the activity of LAP2 were examined using ABTS as substrate at pH 3·5. The enzyme was totally inhibited by 0·02 mM azide and 1·0 mM fluoride, while 0·01 mM EDTA resulted in 13% inhibition. The inhibiting effects of halides, well-known inhibitors of laccases, were studied in more detail. The I50 values, the concentration of an inhibitor causing 50% activity reduction, for the sodium halides NaF, NaCl, and NaBr were found to be 0·031, 16 and 230 mM, respectively.

The activity of LAP2 as a function of the pH was studied for different substrates. The pH optimum varied between pH 2·5 and 5, depending on the substrate employed. While the phenolic substrates such as catechol or p-anisidine showed a typical bell-shaped pH activity profile with pH optima of 3·0–4·5, the two non-phenolic substrates ABTS and ferrocyanide showed monotonic pH profiles in which the rate decreased with increasing pH. The reaction velocity of the laccase-catalysed oxidation of ABTS increased with increasing temperature, showing a broad maximum at 50–60 °C (3 min, standard assay conditions at pH 3·5). Thermal stability of LAP2 was studied at pH 5·0 where it showed maximal stability. The half-life times of activity at 4, 30, 40, 50, 60 and 70 °C were 8·6 months, 14 days, 4 days, 2·9 h, 1·5 h and 3·7 min, respectively.

Cloning of the lap2 gene
To facilitate the cloning of the gene encoding LAP2, the amino acid sequence of the N terminus of the protein was determined and was GIGPVADLTI-. This N-terminal sequence was identical to that of T. villosa LCC1 (Yaver et al., 1996 ). Using T. pubescens total genomic DNA as a template with degenerate oligonucleotides based on the N-terminal sequence of LAP2 and on intergenic sequences homologous to T. villosa lcc1, a PCR product of 1·0 kb length was obtained and cloned. The deduced amino acid sequence of the product was determined from the DNA sequence and showed high homology to T. villosa LCC1 when aligned and analysed by pairwise comparison. This PCR product was further used as probe in Southern analysis under high stringency conditions. According to the results obtained from Southern analysis, gene mapping of the genomic locus was established and a BamHI–HindIII fragment of approximately 5 kb, which should contain the complete coding region as well as the 5'-regulatory and the 3'-terminatory region, was selected for cloning. PCR screening of obtained E. coli colonies allowed the identification of eight clones, all of them containing the same 5 kb fragment. Of these, one was selected for sequencing and contained the entire lap2 gene. A gene encoding another laccase isoform, LAP1A, was cloned by using a similar strategy (BankIt accession number AF414808). The predicted protein sequence of LAP1A structural gene showed 95·2% identity with T. villosa LCC5 (Yaver & Golightly, 1996 ), whilst the degree of identity between lap1a and lap2 is only 65%. LAP1A as a minor laccase isoenzyme was not purified from the T. pubescens culture supernatant.

Structure of the lap2 gene
In addition to the entire sequence of the LAP2 structural gene, the resulting clone contained 1418 bp of the 5' promoter region (Fig. 2) and 1500 bp of the 3' terminatory sequence (data not shown). The lap2 gene contains eight introns with splicing junctions and internal lariat formation sites adhering to the GT-AG rule (Padgett et al., 1984 ). The coding region of lap2 consists of a 1569 bp ORF encoding 523 aa with a 21 aa signal sequence. The product of lap2 is predicted to be a mature protein of 501 aa residues with a calculated molecular mass of 53·6 kDa. LAP2 contains seven potential N-glycosylation sites (Asn-X-Thr/Ser) and has 95·0% identity with the sequence of T. villosa LCC1 (Yaver & Golightly, 1996 ).



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Fig. 2. Nucleotide sequence of the T. pubescens lap2 promoter. Putative CAAT and TATA promoter elements are boxed. Several putative CreA-binding sites, one general stress responsive element (STRE) and two metal responsive elements (MRE) are indicated.

 
Analysis of the promoter region
The lap2 promoter region extending about 1420 bp upstream of the start codon ATG is shown in Fig. 2. A TATA box, TATAAA, at nt position -100, as well as seven CAAT boxes located at nt positions -5, -346, -355, -633, -915, -1201 and -1277 upstream from the translation start site are found. A long pyrimidine-rich region, typical for strong fungal promoters (Ballance, 1991 ), is present between the TATA box and the translation start site. Two putative metal-responsive elements (MREs) adhering to the consensus sequence TGCRCNC (Thiele, 1992 ), all of them being present in direct orientation with respect to the direction of transcription, were detected and are centred at nt positions -798 and -1114. Four putative CreA-binding sites (GCGGGG) (Arst & MacDonald, 1975 ) are clustered between nt positions -927 and -1270 (-927, -947, -1207 and -1270). Furthermore, 27 potential heat-shock elements (HSEs) composed of the repeated 5 bp NGAAN element in either orientation (Mager & De Kruijff, 1995 ) are spread between nt positions -1180 and -10 relative to the start ATG. A ‘general’ stress-responsive promoter element (STRE) with the consensus core sequence CCCCT located at nt position -144 has been identified in the upstream region of lap2. The 3'-untranslated region of the lap2 gene contains a putative polyadenylation site AAACAA, which is a slight variation of the consensus polyadenylation signal sequence AATAAA (Proudfoot, 1991 ) 153 bp downstream of the termination codon.

Regulation of laccase expression
The formation of laccase activity in T. pubescens cultures growing on either GYP or FYP is presented in Fig. 3. The fungus was cultivated for 8 days before CuSO4 was added to a final concentration of 2 mM. At this point the fructose concentration in the medium was 8·3 g l-1, whereas glucose was almost depleted (1 g l-1). When using fructose as the main carbon source, a sudden onset of laccase production was observed following copper addition with 0·6 U ml-1 laccase activity detectable after 3 h which further increased to reach a maximum of 72 U ml-1 after 19 days total cultivation time. In contrast, laccase secretion was much slower on glucose medium, starting only 10 h after the addition of copper (0·14 U ml-1). A significant increase in laccase activity was only observed when glucose was completely depleted from the medium (1·1 U ml-1 after 24 h) to reach a final value of 65 U ml-1 after 19 days.



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Fig. 3. Time-course of cultivations of T. pubescens using glucose (GYP medium) or fructose (FYP medium) as the substrate. CuSO4 (2 mM final concentration) was added at the time indicated (dashed line). Mean values are shown for duplicate cultivations; the standard deviation did not exceed 5%. {circ}, Glucose concentration; {square}, fructose concentration; {bullet}, laccase activity on GYP; {blacksquare}, laccase activity on FYP.

 
The synthesis of laccase activity in T. versicolor is induced by copper (Collins & Dobson, 1997 ). To examine the inducing effect of copper on T. pubescens lap2 expression, Northern analysis and enzyme assays of induced and non-induced cultures were performed (Fig. 4). Cultures were grown on FYP to avoid possible repression by glucose and copper was added after 8 days, but not to control flasks. Fungal biomass was harvested and total RNA was isolated before the addition of CuSO4 as well as 10, 24 and 48 h after its addition. Aliquots of induced and non-induced culture supernatants were assayed for laccase activity (Fig. 3).



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Fig. 4. Northern blot analysis of total RNA isolated from T. pubescens grown on a fructose-based medium to which 2 mM CuSO4 was added after 8 days of cultivation (+Cu2+) and control flasks without copper (-Cu2+). a, hybridization with lap2-specific probe; b, hybridization with A. nidulans actin; c, total RNA. Samples were taken after the addition of copper at the times indicated.

 
For the detection of lap2-specific mRNA, the same fragments as for the Southern hybridizations were used as probes. As a loading control, hybridization with the Aspergillus nidulans actin gene was performed (Mansur et al., 1998 ). The values for the intensity of the lap2 transcript were normalized to the actin hybridization signals. The abundance of lap2 mRNA increased dramatically shortly after the addition of CuSO4 (Fig. 4). A single transcript was detected when copper was present (+Cu), but not when it was omitted from the medium (-Cu). Ten hours after copper addition, clearly visible levels of the lap transcript were detected in fungal cells, corresponding to a measurable laccase activity in the culture medium (Table 3). The lap2 transcript continued to accumulate markedly until 48 h after copper addition. Enzyme activities parallel the formation of specific mRNA. In control cultures without additional CuSO4, neither specific mRNA nor laccase activity was detected at any time tested.


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Table 3. Correlation between laccase activity in the culture supernatant of T. pubescens grown on FYP medium supplemented with 2 mM CuSO4 and relative intensities of hybridization signals obtained from Northern blot analysis using a lap2 specific probe

 
In addition to copper, several other heavy metal ions stimulated laccase formation when added to actively growing culture of T. pubescens (Fig. 5). In these experiments the basal GYP medium contained 5 µM Cu2+; metal ions (Ag+, Cd2+, Hg2+) were added on the fourth days of cultivation in various low amounts (5–125 µM). In particular, 5 µM Cd2+ and Hg2+ were very effective in stimulating laccase synthesis, increasing this enzyme activity approximately 2·7- and 1·9-fold, respectively, compared to the blank that contained only 5 µM Cu2+. These low concentrations of the heavy metal ions did not affect growth (data not shown). Higher concentrations of Cd2+ and Hg2+ (25 and 125 µM) did not stimulate or stimulated only to a lower extent laccase formation; the latter was found for 25 µM Cd2+, which increased laccase synthesis 1·9-fold relative to the blank. Apparently these higher concentrations of the heavy metals are toxic for the fungus as was evident from reduced growth. While the lower Ag+ concentrations tested (5 and 25 µM) did not affect laccase secretion by T. pubescens, 125 µM increased laccase yields approximately 1·7-fold (Fig. 5).



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Fig. 5. Formation of extracellular laccase activity by T. pubescens in response to different metal ions. These were added to the basal GYP medium containing 5 µM CuSO4 so that their final concentration was as indicated. Data shown are the mean values from two independent replicates±standard deviations. {bullet}, Blank; {square}, 5 µM Ag+; {blacksquare}, 125 µM Ag+; {triangleup}, 5 µM Cd2+; {circ}, 5 µM Hg2+. The dashed line marks the addition of the various metal ions.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Laccases are typically produced as multiple isoenzymes in white-rot fungi (Bollag & Leonowicz, 1984 ; Eggert et al., 1996 ), which was also found to be true for T. pubescens. By IEF and activity staining with ABTS, at least eight bands with laccase activity could be detected in crude culture supernatants of high copper cultivations. Whether all these bands represent true laccase isoforms that are transcribed from separate genes, or are the result of post-translational modifications of the extracellular polypeptide by proteolysis (Palmieri et al., 2001 ) or differences in glycosylation (Yaver et al., 1996 ), remains unclear at present. Judged from cloning of two isoforms as well as purification and determination of the N-terminal amino acid sequence of a third, neutral isoform with a pI of 6·0 (data not shown), we conclude that T. pubescens forms at least three true laccase isoenzymes.

Based on anion-exchange chromatography, laccases from T. pubescens can be separated into two fractions, LAP1, containing several isoforms, and LAP2, representing a single, acidic isoenzyme with a molecular mass of 65 kDa and a pI of 2·6. LAP2 oxidized typical laccase substrates such as ABTS, ferrocyanide and hydroxy- or methoxy-substituted phenols very efficiently as is evident from the high values determined for the catalytic constant kcat and the catalytic efficiencies kcat/Km for some selected substrates. Interestingly, the introduction of a second methoxyl group as found in 2,6-DMP resulted in an almost 10-fold increase in kcat/Km as compared to the monomethoxylated substrate guaiacol. This can be explained by the strong electron-donating effect of the two methoxyl substituents and the favourable redox potential of the substrate 2,6-DMP (Jovanovic et al., 1991 ; Xu, 1996 ). LAP2 shows a pH dependence similar to that described for other fungal laccases, i.e. bell-shaped pH activity profiles for the phenolic substrates and monotonic curves for the non-phenolic substrates. It has been suggested that both OH inhibition at the type-2/type-3 trinuclear copper cluster of laccase and the redox potential difference between a reducing substrate and the type-1 copper cause these different, substrate-dependent pH activity profiles (Xu, 1997 ). In addition, this distinct pH dependence of fungal laccases has been explained with structural features of the enzyme (Garzillo et al., 2001 ).

With regard to the amino acid sequences, highly conserved regions among all laccases exist which are involved in the co-ordination of the copper ions to form a redox centre (Ducros et al., 2001 ; Soden & Dobson, 2001 ). Therefore, PCR primers based on these conserved sequences around the two pairs of histidines in the copper-binding regions can easily be designed. By combining a specific N-terminal forward primer and an intergenic reverse primer selected according to the conserved regions mentioned above, a rather specific fragment can be amplified by PCR. The T. pubescens lap2 gene was cloned following a novel cloning strategy without preparing a cDNA or genomic library. This cloning strategy is based on mapping of the genomic locus of the gene of interest according to restriction analysis obtained from Southern blots hybridized with gene-specific fragments (the specific PCR products) under high stringency conditions.

The deduced protein sequence of the lap2 gene has a high degree of identity with sequences of other basidiomycete laccases; the highest identities of 95·0% and 90·0% were found for T. villosa lcc1 and Coriolus hirsutus laccase, respectively. The percentage identities to other laccases from the closely related fungi T. villosa and T. versicolor were in the range 63–77%, for the white-rot fungi Phlebia radiata and Pleurotus ostreatus in the range 53–60%, whilst they were fairly low (27%) compared with laccase from the ascomycete Botrytis cinerea.

It has been predicted that the type-1 copper axial ligand, the amino acid residue 10 aa downstream of the conserved cysteine, has an important effect on the redox potential of the type-1 copper in the active site (Canters & Gilardi, 1993 ), thus providing the basis for assigning laccases to class 1 (Met), 2 (Leu) or 3 (Phe) (Eggert et al., 1998 ). Laccases harbouring a Phe residue at this position have a type-1 copper centre with a high redox potential of 0·71–0·8 V (Palmer et al., 1999 ; Xu et al., 1996 ). LAP2 bearing such a Phe residue at this position can thus be categorized as a ‘high E0 laccase.

The lap2 gene contains eight introns with splicing junctions and internal lariat formation sites adhering to the GT-AG rule, i.e. the 5'-consensus sequence GTRNGT and the 3'-consensus splice sites (c/t AG), respectively (Padgett et al., 1984 ). The position of the introns was inferred from comparison with other genes and the consensus sequences but were not verified by the cDNA sequence. Only intron one (T at position 3) exhibits a slight variation. The size of the introns ranging from 50 to 64 is typical for most fungal introns (Gurr et al., 1987 ).

Within the lap2 5'-regulatory region extending about 1400 bp upstream of the ATG, the usual promoter elements, i.e. a TATA box TATAAA and seven CAAT motifs, are present. CAAT motifs play a pivotal role in determining the efficiency of the promoter. A long pyrimidine-rich region, typical for strong fungal promoters (Ballance, 1991 ), is found between the TATA box and the translation start site. Two putative MREs (Thiele, 1992 ) were detected in the promoter region. These cis-acting MREs have been discovered as multiple copies in the Saccharomyces cerevisiae metallothionein promoter, there being essential for efficient metal-inducible transcription (Thiele, 1992 ). Metallothioneins have been proposed to be involved in a number of cellular processes including metal storage and detoxification (Karin, 1985 ). A range of heavy metals induces the expression of the metallothionein genes, with regulation operating via a metal-regulatory protein which functions both as a metal receptor and a transcription factor. The presence of one MRE confers the ability to respond to heavy metal; a greater level of induction may be achieved by the inclusion of multiple elements (Lewin, 1997 ). The increased synthesis of laccase in T. pubescens in response to several heavy metal ions suggests that these MREs in the lap2 promoter have physiological significance. Furthermore, 27 potential HSEs (Mager & De Kruijff, 1995 ) and a ‘general’ STRE have been identified in the upstream region of lap2. In yeast, HSEs were also proven to transcriptionally activate metallothionein genes in response to heat shock (Tamai et al., 1994 ). HSE and STRE might be involved in stress-regulated lap2 gene expression caused by, for example, high concentrations of Cu2+. Interestingly, neither xenobiotic response elements (XREs) nor antioxidant response elements (ARE) could be detected in the 5'-untranscribed region of lap2. These elements are known to mediate transcriptional activation by aromatic substances in eukaryotic genes (Li & Jaiswal, 1992 ; Rushmore et al., 1991 ); amongst others, they have been suggested to be involved in the induction of fungal laccases by aromatic compounds (Soden & Dobson, 2001 ). This lack of XRE elements in the promoter region of T. pubescens lap2 is in agreement with data obtained from T. pubescens shaken flask cultures to which various aromatic substances, typically used laccase inducers such as 2,5-xylidine or catechol, were added. In these experiments none of the aromatic compounds tested stimulated laccase formation (Galhaup & Haltrich, 2001 ; Galhaup et al., 2002 ). Apparently, this stimulating effect of copper and other metal ions is not simply based on growth inhibition as has been suggested for other fungi (Gianfreda et al., 1999 ). One possible explanation could be a protective mechanism based on the stimulation of melanin synthesis, which we previously observed for T. pubescens in the presence of increased concentrations of copper (Galhaup & Haltrich, 2001 ).

Four putative CreA-binding sites were found in the 5'-non-coding region of the lap2 gene. CreA has been identified in A. nidulans as a GC-box binding repressor involved in glucose repression (Arst & MacDonald, 1975 ; Dowzer & Kelly, 1991 ; Strauss et al., 1999 ). This glucose effect – repression of genes that are used in the metabolism of alternative carbon sources – is widely known in fungi and yeasts (Ronne, 1995 ). Laccase production in T. pubescens is induced by copper. When using glucose as the carbon source, laccase formation only starts when this substrate is almost completely depleted from the medium, no matter when copper is added (Galhaup & Haltrich, 2001 ; Galhaup et al., 2002 ). Apparently, glucose, when present in the culture medium above a certain concentration, represses laccase synthesis in the fungus. This is further corroborated since lap2 mRNA was not detectable on Northern blots of RNA isolated from copper-induced cultures still containing glucose (data not shown). Since the depletion of the carbon source results in a drastic increase of the culture pH and apparently also autolysis of the organism (Galhaup & Haltrich, 2001 ), we were not successful in isolating RNA from these mycelia and were thus unable to analyse them for lap transcripts at later stages of growth. However, when T. pubescens was grown on the typically non-repressing substrate fructose as the carbon source, laccase activity was detectable shortly after the addition of CuSO4 even though fructose was still present in higher concentrations (Fig. 3). Northern blot analyses of RNA isolated from these samples clearly revealed that the addition of 2 mM copper has a marked effect on induction of T. pubescens lap2 gene transcription. Northern blot analyses revealed the sudden onset of laccase-specific mRNA only a few hours after the addition of copper and an increase for the next 2 days. Again, analysis of the laccase transcripts could not be continued as fructose was completely consumed from the medium at this time (48 h after addition of copper), after which RNA could not be adequately prepared from the mycelia.

In conclusion, the results presented here indicate that the expression of at least the major T. pubescens laccase isoenzyme gene is regulated by several factors at the level of gene transcription. Because of the high levels of laccase production by the wild-type strain of T. pubescens on very simple media and the ease of its induction, these laccase preparations should find wide use in various biotechnological applications.


   ACKNOWLEDGEMENTS
 
We thank Hansjörg Prillinger (University of Agricultural Sciences Vienna) for the fungal strain. This work was supported by a grant from the Austrian Science Foundation (FWF project P14537).


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Received 31 December 2001; revised 15 March 2002; accepted 25 March 2002.