Báo cáo khóa học: Biochemical and molecular characterization of a laccase from the edible straw mushroom, Volvariella volvacea

doi:10.1046/j.1432-1033.2003.03930.x

Eur. J. Biochem. 271, 318–328 (2004) (cid:1) FEBS 2003

Biochemical and molecular characterization of a laccase from the edible straw mushroom, Volvariellavolvacea

Shicheng Chen, Wei Ge and John A. Buswell

Department of Biology and the Centre for International Services to Mushroom Biotechnology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China

tion in fungal mycelia grown on rice-straw revealed that, apart from during the early stages of substrate colonization, lac1 was expressed at every stage of the mushroom devel- opmental cycle defined in this study, although the levels of transcription varied considerably depending upon the developmental phase. Transcription of lac1 increased shar- ply during the latter phase of substrate colonization and reached maximum levels during the very early stages (primordium formation, pinhead stage) of fruit body mor- phogenesis. Gene expression then declined to (cid:1) 20–30% of peak levels throughout the subsequent stages of sporophore development.

Keywords: Volvariella volvacea; laccase; edible mushroom; gene expression.

We have isolated a laccase (lac1) from culture fluid of Vol- variella volvacea, grown in a defined medium containing 150 lM CuSO4, by ion-exchange and gel filtration chroma- tography. Lac1 has a molecular mass of 58 kDa as deter- mined by SDS/PAGE and an isoelectric point of 3.7. Degenerate primers based on the N-terminal sequence of purified lac1 and a conserved copper-binding domain were used to generate cDNA fragments encoding a portion of the lac1 protein and RACE was used to obtain full-length cDNA clones. The cDNA of lac1 contained an ORF of 1557 bp encoding 519 amino acids. The amino acid sequence from Ala25 to Asp41 corresponded to the N-terminal sequence of the purified protein. The first 24 amino acids are presumed to be a signal peptide. The expression of lac1 is regulated at the transcription level by copper and various aromatic compounds. RT-PCR analysis of gene transcrip-

Volvariella volvacea, the edible straw mushroom, produces multiple forms of laccase (benzenediol:oxygen oxidoreduc- tase, EC 1.10.3.2) when grown in submerged culture on defined media containing copper or various aromatic compounds, or in solid-state systems representative of the conditions used for industrial cultivation ([1]; S. Chen, W. Ge and J. A. Buswell, unpublished results). Whereas, in many other basidiomycetes enzyme biosynthesis is normally associated with primary growth [2–5], laccase production in V. volvacea has the relatively novel feature of occurring only in the later stages of primary growth i.e., when fungal biomass production has reached a maximum [1]. Further- more, when the fungus is grown on cotton waste (cid:1)composts(cid:2) [6], the very low levels of laccase observed throughout the substrate colonization phase increase sharply at the onset of fruit body initiation. This increase in laccase activity was observed only in those composts that produced fully developed sporophores [1].

Laccases have been assigned several different biological roles. In higher plants, laccases are involved in lignification of xylem tissues [7]. The enzyme has also been linked to pigment biosynthesis during conidial development and maturation in Aspergillus nidulans [8], the pathogenicity of the chestnut blight fungus Cryphonectria parasitica [9] and in the biosynthesis of cinnabarinic acid, a fungal metabolite produced by Pycnoporus cinnabarinus that exhibits anti- microbial activity against various bacterial species [10]. Of particular importance to our own research are the assigned roles of laccases in lignin degradation [11–13], in rendering phenolic compounds less toxic via oxidative coupling and polymerization [14] and in sporophore formation [15,16]. As all these latter three functions are of fundamental import- ance for the colonization of the various lignocellulosic substrates used in mushroom cultivation systems and for mushroom fruiting body development, we sought to learn more about the laccase component(s) of V. volvacea. This commercially important edible mushroom is grown and consumed in many parts of Asia, and currently ranks fifth among the major cultivated species in terms of annual production worldwide [17].

Our earlier studies established that two laccase isoforms were induced in submerged cultures of V. volvacea in response to addition of copper or various aromatic com- pounds to the culture medium [1]. In this study, we have purified and characterized one of the laccase isoforms, cloned and sequenced the cDNA encoding the enzyme protein, and examined the effect of copper and various

lac1, laccase; 2,6-DMP, 2,6-dimethoxyphenol; HBT,

Correspondence to J.A. Buswell, Edible Fungi Institute, 35 Nanhua Road, Shanghai 201106, China. Fax: + 86 21 62207544, Tel.: + 86 21 62208660, E-mail: jbuswell@saas.sh.cn Abbreviations: 1-hydroxybenzotriazole; XYL, 2,5-xylidine; FA, ferulic acid. Enzyme: benzenediol:oxygen oxidoreductase (EC 1.10.3.2). (Received 26 August 2003, revised 5 November 2003, accepted 18 November 2003)

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Protein determination

Protein in culture supernatants was determined by the method of Bradford [21] with bovine serum albumin as standard, and in column effluents by measuring A280.

Purification of lac1

aromatic compounds on gene expression. We have also determined the transcription pattern for the laccase gene in V. volvacea grown on paddy-straw throughout various stages of the mushroom developmental cycle and detected large increases in lac1 gene transcription late in the substrate colonization phase and during the early stages of fruit body morphogenesis. A good correlation existed between total laccase activity and lac1 expression under these growth conditions. A better understanding of the role(s) played by individual laccase isoforms in sporophore development should aid the development of strategies for improving mushroom growth yields.

Experimental procedures

Organism and growth conditions

V. volvacea V14 was obtained from the culture collection of the Centre for International Services to Mushroom Biotechnology (accession no. CMB 002) [1].

The following procedures were all performed at 4 (cid:2)C. Culture fluid obtained after filtration of 7-day cultures of V. volvacea was centrifuged (10 000 g, 30 min) and con- centrated (cid:1) 40-fold with the Pellicon ultrafiltration system (Millipore) using a 10-kDa molecular mass cut-off mem- brane. Solid ammonium sulfate was added and the fraction precipitating at 80% saturation was collected by centrifu- gation, redissolved in 20 mL 10 mM phosphate buffer, pH 5.8, and dialysed overnight against two changes of fresh buffer. Precipitated material was removed by centrifugation (10 000 g, 30 min) and the supernatant was applied to a column (2.5 · 20 cm) of DEAE/Sepharose pre-equilibrated with the same buffer. After washing with 350 mL of 10 mM phosphate buffer, the enzyme was eluted with a linear gradient of 0–1.0 M NaCl in 500 mL of this buffer at a flow rate of 0.5 mLÆmin)1. The active fractions ((cid:1) 60 mL) were pooled, concentrated to 10 mL by ultrafiltration using a Centriprep YM-10 centrifugal filter (Millipore) and applied to a Sephacryl-S300 column (1.5 · 90 cm) pre-equilibrated with 10 mM potassium phosphate buffer, pH 6.5. The enzyme was eluted with the same buffer at a flow rate of 0.5 mLÆmin)1 and the combined active fractions ((cid:1) 50 mL) concentrated to 5 mL by ultrafiltration. This pooled material was applied to a Sephacryl-S100 column (1.5 · 90 cm) pre-equilibrated with 10 mM potassium phosphate buffer, pH 6.5, and the enzyme was eluted with the same buffer at a flow rate of 0.5 mLÆmin)1. Pooled active fractions ((cid:1) 30 mL) were concentrated with a Centriprep YM-10 centrifugal filter (Millipore) and stored at )20 (cid:2)C.

The fungus was cultivated at 32 (cid:2)C in stationary 250 mL Erlenmeyer flasks containing 50 mL basal medium [18]. Nitrogen was added as NH4NO3 and L-asparagine at concentrations equivalent to 26 mM-N [19]. The effects of ferulic acid (FA; Sigma, St Louis, MO, USA) on lac1 transcription were studied by growing the fungus on basal medium without FA for 6 days before supplementing cultures with different concentrations of FA as indicated. Total RNA was extracted from mycelia after 24 h further incubation. A basal medium prepared with a modified trace element solution [18] lacking the Cu component (equivalent to (cid:1) 1.5 lM Cu) was used to examine the effect of Cu on laccase gene expression. The effect of different aromatic compounds on laccase production was determined after 36 h following supplementation of 6 day-old cultures with 2 mM (final concentration) of the test compound. For purification of laccase 1 (lac1), the fungus was grown in 2 L flasks containing 600 mL basal medium with 150 lM CuSO4.

Enzyme characterization

Gene expression during the mushroom developmental cycle was determined in rice-straw compost cultures prepared as follows: 150 g rice-straw was soaked overnight in 450 mL of distilled water. After draining off any remaining free water, the straw was mixed with lime (15 g) and wheat bran (15 g) and the material distributed into cellophane bags and autoclaved at 121 (cid:2)C for 30 min. After cooling, the compost was inoculated with fungal mycelium (from 2 week-old compost cultures) and incuba- ted at 32 (cid:2)C and 90–95% relative humidity. The bags were removed after 14 days to promote fruiting. Samples were taken from duplicate cultures at different stages of the mushroom developmental cycle: early, middle and late substrate colonization stages (4, 8 and 12 days); pinhead stage (day 14), button stage (day 18), egg stage (day 21), elongation stage (day 22) and mature stage (day 23). After collection, compost material was stored immediately at )70 (cid:2)C prior to analysis.

Enzyme assay

Laccase activity was determined using 2,2¢-azinobis-(3- ethylbenzo-6-thiazolinesulfonic acid) (ABTS) as described previously [1,20].

The molecular mass of purified laccase was determined by SDS/PAGE (15% w/v acrylamide gels) using low molecular mass standards (Bio-Rad). The isoelectric point of the enzyme was determined with the Phastsystem using Phast- Gel IEF 3–9 operated for 410 Vh and standard pI markers (Pharmacia). The standard assay conducted in 0.1 M NaAc buffer (pH 5.0) over the range 30–65 (cid:2)C was used to determine the optimal temperature, and the optimal pH was established using 0.1 M Na2HPO4-citrate (pH 2.2–7.0) and 0.1 M acetate (pH 4.0–7.0) buffer systems. Substrate speci- ficity of purified lac1 was determined spectrophotometri- cally in sodium citrate buffer (0.1 M, pH 5.0) using the specific wavelength of each substrate. Michaelis–Menten constants for ABTS, 2,6-dimethoxyphenol (2,6-DMP) and syringaldazine were determined from Lineweaver–Burk plots of data obtained by measuring the reaction rate under optimal conditions using substrate concentration ranges of 0.005–1 M, 0.01–1 M and 0.0025–0.025 M, respectively. The effect of putative laccase inhibitors was determined in standard assay reaction mixtures following incubation of lac1 with individual inhibitors (0.1 mM or 1.0 mM in sodium citrate buffer, pH 5) at 32 (cid:2)C for 5 min.

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N-terminal sequencing of laccase

the primer

GAGTGACGAAGGCAGGACCATC-3¢), was designed for the 5¢-RACE reaction to generate the 5¢-cDNA end fragment of lac1. The 5¢-cDNA end fragment was cloned into pGEM T-vector and sequenced as above. The full- length cDNA of lac1 was then generated by 3¢-RACE 5¢-TCTCAACCGTCGACAGCAG using TGTTCGTG-3¢ designed from the sequence of the extreme 5¢ end of lac1. The full-length cDNA of lac1 was cloned and sequenced as above.

The N-terminal amino acid sequence of purified laccase was determined by electroblotting the enzyme on to an Immo- bilon poly(vinylidene difluoride) (PVDF) Millipore mem- brane (LKB Multiblot apparatus, Bio-Rad) followed by Edman degradation performed with a Hewlett-Packard G1005A Protein Sequencer coupled to a HPLC (Hewlett- Packard, Model 1090) for analysis of the phenylthiodantoin amino acids.

RNA manipulations, cDNA synthesis and cloning

RT-PCR for semiquantification of the lac1expression levels

Total RNA extracted from liquid cultures and mRNA extracted from compost cultures using the polyA TRACT mRNA isolation system II kit (Promega), was reverse transcribed into cDNA at 42 (cid:2)C for 1 h in a total volume of 10 lL reaction solution containing 1 lg total RNA or 30 ng mRNA, 1 · First Strand Buffer, 10 mM dithiothrei- tol, 0.5 mM each dNTP, 0.5 lg oligo-dT and 100 U SuperScript II (Gibco Invitrogen). PCR was performed in a volume of 25 lL consisting of PCR buffer, 0.2 mM each dNTP, 2.5 mM MgCl2, 0.2 lM each primer, and 0.5 U of Taq polymerase. One microlitre of RT reaction was used in each PCR reaction.

Mycelium from V. volvacea cultures grown for 12 days in defined medium with 200 lM copper was harvested, frozen with liquid nitrogen and ground to a fine powder with a mortar and pestle. Total RNA was isolated from this material using the Tri-Reagent (Molecular Research Center, Inc. Cincinnati, OH, USA) and used to synthesize cDNA. Reverse transcription was carried out at 42 (cid:2)C for 2 h in a 10-lL reaction volume containing: 2 lL diethylpyrocarbo- nate-treated H2O, 2 lL 5 · First Strand Buffer (Gibco Invitrogen), 0.01 M dithiothreitol, 0.5 mM dNTPs, 0.5 lg oligo(dT), 2 lg total RNA and 100 U SuperScript II (Gibco Invitrogen). The cDNA from the reaction was kept at )70 (cid:2)C and used for PCR amplification using degenerate primers designed on part of the N-terminal amino acid sequence and a conserved copper-binding region. The sequences and primers were: primer 1 (upper primer): 5¢-(CT)T(AGCT)AC(AGCT)AA(CT)GG(AGCT)TT(CT) GC-3¢ (encoding LTNGFA); primer 2 (lower antisense primer): 5¢-(AG)(AG)TG(AGCT)(GC)(AT)(AG)TG(AG) TACCA(AG)AA-3¢ (encoding FWYHSHL). Different primers were designed with any one of the bases shown in parentheses.

As a control for RNA loading, a 330 bp fragment of the V. volvacea, V14 glyceraldehyde-3-phosphate dehydro- genase (gpd) gene was amplified with primers PGPDF (5¢-TAATGACGGCAAACTCGTGATC-3¢) and PGPDR (5¢-TGTATGACTTTGGCCAGAGGTG-3¢) (accession number AY280633). The PCR cycle programme was: 94 (cid:2)C for 2 min; 23 cycles of 94 (cid:2)C for 20 s, 52 (cid:2)C for 20 s and 70 (cid:2)C for 2 min; then a final extension at 72 (cid:2)C for 10 min. The primers for lac1 PLAC1F (5¢-AGCTTT CATTCCCAGTGATTG-3¢) and PLAC1R (5¢-AACGAG CTCAAGTACAAATGACT-3¢) were designed according to our cloned cDNA (GenBank Accession No. AY249052). The PCR cycle programme was: 94 (cid:2)C for 2 min; 28 cycles of 94 (cid:2)C for 20 s, 52 (cid:2)C for 20 s and 70 (cid:2)C for 2 min; then a final extension at 72 (cid:2)C for 10 min. To validate the semiquanti- tative RT-PCR reactions, a series of RT-PCR reactions were sampled at different cycles and analysed by electrophoresis to ensure that product abundance was evaluated in the exponential phase of the reaction (half of the maximum product). To further validate the assays, the optimized cycle number (23 and 28 for gpd and lac1, respectively) was used to amplify serially diluted gpd and lac1 DNA templates. After electrophoresis on 2% agarose gel, the PCR products were stained with ethidium bromide and visualized in a UV-transilluminator. The signal intensity was quantified with the Gel-DOC 100 system using the MOLECULAR ANALYST SOFTWARE (Bio-Rad). The specificity of PCR and RT-PCR amplification was confirmed by cloning the products into pGEM T-vector (Promega) followed by sequencing.

Results

PCR amplification of the cDNA fragment encoding a portion of lac1 was carried out using a PTC-100 (MJ in 50 lL reaction Research, Watertown, MA, USA) volumes containing 1.25 U Taq DNA polymerase, 5 lL 10· Mg-free reaction buffer, 200 lM dNTP, 2.5 mM MgCl2, 1 lM primer 1 or primer 2 and 0.5 lL template. Amplifi- cation conditions were: 1 cycle of 94 (cid:2)C for 3 min, 50 (cid:2)C for 30 s and 72 (cid:2)C for 1 min; 30 cycles of 94 (cid:2)C for 30 s, 54 (cid:2)C for 30 s, and 72 (cid:2)C for 1 min; then a final extension at 72 (cid:2)C for 10 min before storage at 4 (cid:2)C. Amplification products were fractionated by electrophoresis in 2.0% agarose/Tris borate/EDTA gels and appropriate bands excised and purified from the gel using the NucleoTrap Gel Extraction Kit (Clontech). The purified DNA was precipitated with ethanol and resuspended in 10 lL H2O. An aliquot (4 lL) was incubated at 4 (cid:2)C overnight with 3 U T4 DNA ligase (Promega), 1 lL 10· buffer with 10 mM ATP (Promega) and 1 lL pGEM T-vector in a total volume of 10 lL and transformed into E.coli DH5a. Plasmids encoding the lac1 fragment were isolated using the Wizard Miniprep Kit (Promega) and sequenced by the dideoxy chain-termination method using an automated ABI310 sequencer (Perkin Elmer) according to the manufacturer’s instructions.

Purification of lac1

Lac1 was separated as a single peak by a final gel filtration step using Sephacryl S-100 and shown to be homogeneous

RACE was performed with the SMART RACE cDNA Amplification Kit (Clontech) to obtain full-length cDNA lac1 clones. Using the 309-bp fragment sequence of obtained above, the gene-specific primer (5¢-GGCACT

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Table 1. Summary of the purification procedure for V. volvacea lac1.

Purification fold Yield (%) Volume (ml) Total activity (IU) Protein (mg) Specific activity (IUÆmg)1)

1 1.1 2.8 9.2

cinnabarinus

(57%),

laccase

by SDS/PAGE and by isoelectric focusing combined with silver staining (data not shown). After a five-step purification protocol, the specific activity of lac1 was increased 14-fold with a 22% recovery yield (Table 1).

Enzyme characterization

showed highest overall identity with laccase 1 (BAB84354) from Lentinula edodes (58%), laccase (AF170093) from Pycnoporus LCC3-1 (AF176230) from Polyporus ciliatus (56%) and laccase 1 (AAC498287) from Trametes versicolor (56%) (Fig. 2). The lac1 protein contains only one potential N-glycosylation site (Asn-X-Thr/Ser in which X is not proline). The calculated isoelectric point for the cloned cDNA product is 4.5. All the amino acid residues that serve as Cu2+ ligands (10 His residues and one Cys residue) are present in the lac1 coding sequence (Fig. 2).

Validation of semiquantitative RT-PCR assay for lac1 and gpd

A series of RT-PCR reactions for lac1 (Fig. 3: left panel) and gpd were performed at different cycles and analysed by electrophoresis. Using the cycle number that generated the half-maximal PCR amplification, PCR reactions were performed on serially diluted lac1 and gpd template DNA. A clear linear relationship between the amount of template inputs and PCR amplification was obtained for both lac1 (Fig. 3: right panel) and gpd (data not shown) demonstra- ting the workability of the RT-PCR assay for quantitating lac1 mRNA levels.

Regulation of lac1 expression by copper

The molecular mass of purified lac1 was estimated by SDS/ PAGE to be 58 kDa and the isoelectric point of the enzyme was 3.7. In addition to ABTS, syringaldazine and 2,6-DMP were also oxidized by lac1 ((cid:1) 17% of the activity observed with ABTS). Guaiacol, catechol and 2,6-DMP were poor substrates for the enzyme (< 5% compared with ABTS) and no activity was detected with tyrosine, FA, L-3,4- dimethoxyphenol and dihydroxyphenylalanine (L-DOPA). With ABTS as the substrate, lac1 displayed a pH optimum of 3.0 corresponding to a specific activity of 13.5 UÆmg protein)1. Corresponding pH optima and specific activities for syringaldazine and 2,6-DMP were pH 5.6 and 3.4 UÆmg protein)1 and pH 4.6 and 2.8 UÆmg protein)1, respectively. In standard assay mixtures, the velocity of ABTS oxidation was maximal at 45 (cid:2)C. The dependence of the rate of ABTS oxidation by lac1 on substrate concentration at pH 3.0 and 45 (cid:2)C followed Michaelis–Menten kinetics. A reciprocal plot revealed an apparent Km value of 0.03 mM and a Vmax of 16.4ÆU mg protein)1. Corresponding values for syring- aldazine and 2,6-DMP under optimal conditions were 0.01 mM and 4.9 UÆmg protein)1, and 0.57 mM and 5.6 UÆmg protein)1, respectively. Lac1 is inhibited (100%) by thioglycollic acid (1 mM), dithiothreitol (0.1 mM), azide (0.1 mM) and cysteine (0.1 mM) but less so by 1 mM EDTA (20%). The N-terminal sequence of native protein was N-ALSSHTLTLTNGFASPD.

Cloning of full-length cDNA of lac1

The effect of copper concentration on lac1 expression in cultures of V. volvacea is shown in Fig. 4. Lac1 transcrip- tion increased with increasing concentrations of CuSO4 in the culture medium up to 200 lM. Higher copper concen- trations were inhibitory and approximately 70% fewer transcripts were observed in hyphae grown in the presence of 300 lM copper sulfate. No lac1 transcripts were detected in fungal cells grown in the absence of copper. A constant level of control gpd fragment amplification was observed in each reaction thereby confirming the uniform efficiency of PCR amplification of RT reaction products. In time-course experiments, transcripts of lac1 were detected in fungal hyphae after 6 days growth in cultures supplemented with 200 lM copper sulfate and reached peak levels after 14 days (data not shown).

Regulation of lac1 expression by aromatic compounds

The cDNA contained a predicted ORF of 1557 bp encoding 519 amino acids (Fig. 1). The amino acid sequence from Ala25 to Asp41 corresponded to the N-terminal sequence of the purified protein, and the putative presequence of 24amino acids is a hydrophobic signal peptide as predicted by the computer program PLOT.AHYD using the method described by Kyte and Doolittle [22]. The remaining 495 amino acids are considered to constitute the lac1 mature protein giving a calculated molecular mass of 53 212 Da. Two putative polyadenylation signals (TATAAA and CATAAA) were identified near the 3¢-end, suggesting possible differential splicing over the 3¢-untranslated region after transcription.

Alignment of the deduced amino acid sequence of lac1 with deduced amino acid sequences of other fungal laccases

Laccase induction in V. volvacea by various aromatic compounds occurred at the level of gene transcription. Figure 5 shows the effect of different aromatic compounds on lac1 expression in V. volvacea grown in nitrogen-

Crude enzyme Ultra filtration (NH4)2SO4 DEAE CL-6B Sephacryl S-300 Sephacryl S-100 2000 50 20 60 50 30 32.6 30.6 25 11.9 9 8.6 25 20 7 1 0.7 0.5 1.3 1.4 3.6 11.9 13 17.2 100 94 77 37 30 22 10 14

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Fig. 1. Nucleotide and deduced amino acid sequences of V. volvacea lac1. Dashed underline, signal peptide; solid line, N-terminal sequence; double solid line, amino acid sequences used to design degenerate primers. The putative N-glycosylation site is boxed; *; stop codon. The putative polyadenylation signals (TATAAA and CATAAA) are in white on a black background.

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sufficient medium without copper. Highest levels of gene transcription were seen with FA, veratric acid, 4-hydroxy- benzoic acid and 2,5-xylidine (XYL). Amounts of lac1 mRNA in the cultures increased with increasing concentra- tions of FA (1–10 mM) although transcript levels in mycelium grown with 5 mM and 10 mM of the aromatic compound were not significantly different (data not shown). No transcription was detectable in controls without added FA.

Transcriptional regulation of lac1during growth and fruiting of V.volvaceaon straw

primordia (day 12), appearance of pinheads (day 14), button stage (day 18), egg stage (day 21), elongation stage (day 22), and mature fruiting body (day 23). The results of RT-PCR analysis of gene transcription in fungal mycelia grown on rice-straw and analysed at various stages of the developmen- tal cycle are shown in Fig. 6A. No transcripts were detected in the early stages of substrate colonization and lac1 was first expressed only when substrate colonization was virtually complete. A large increase in the number of transcripts was seen at the stage of primordia formation and transcription levels were still high when pinheads appeared. Transcription levels declined at the button stage and then remained relatively stable throughout the remaining stages of fruiting body development. Approximately 70–80% fewer tran- scripts were detected throughout these stages compared with peak levels observed in the initial stages of sporophore formation. Extracellular laccase activity in the same compost samples are shown in Fig. 6B.

The duration of the developmental cycle of V. volvacea when the mushroom was grown on rice-straw (cid:1)composts(cid:2) was approximately 23 days. For the purpose of this study, eight separate stages were identified as follows: half-colonization of substrate (day 4), full-colonization (day 8), formation of

Fig. 2. Alignment of deduced amino acid sequences of lac1 and other fungal laccases. *, Amino acid conserved among all of the sequences. Ten His residues and one Cys residue represent the amino acids that serve as Cu2+ ligands and are shown in white on a black background.

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Fig. 3. Validation of semiquantitative RT- PCR assays for lac1. Left panel: Kinetics of PCR amplification with the electrophoretic image shown at the top. The cycle number (28·) that generates half maximal reaction was used to analyse the expression of the gene. Right panel: PCR amplification of the cloned lac1 cDNA using the cycle number obtained from the left panel. Each value represents the mean ± SD of three PCR reactions.

Fig. 4. RT-PCR analysis of transcription patterns of lac1 induced by different concentrations of copper. Total RNA was isolated from V. volvacea, V14, grown in defined medium with various concentra- tions of copper sulfate. PCR products were electrophoresed in a 2% agarose gel and stained with ethidium bromide. The expression levels were normalized by using the relative mRNA ratio (lac1/gpd).

Discussion

Basidiomycetes typically produce multiple laccase isoforms [5,23–29] and V. volvacea produces at least two protein bands with laccase activity when grown in submerged culture under different conditions [1]. Previous physiological studies have shown that, as in other basidiomycetes, laccase production by V. volvacea is induced by copper and by various aromatic compounds. However, unlike most other basidiomycetes, enzyme activity can be detected in the extracellular culture fluids of V. volvacea only during the latter stages of primary growth. In order to better under- stand these effects at the molecular level, we have now

purified and characterized one of these laccases, lac1, and cloned and sequenced the cDNA encoding the protein. RT-PCR was used to study the regulation of lac1 gene expression in V. volvacea when the fungus was grown in submerged culture in the presence of known laccase inducers, and in solid-state systems representing conditions used for industrial cultivation. To our knowledge, this

Fig. 5. RT-PCR analysis of total RNA isolated from V. volvacea, V14, grown in defined medium supplemented with 2 mM aromatic compounds. Fungal mycelia were cultured for 6 days prior to addition of aromatic compound and harvested after a further 36 h incubation. PCR pro- ducts were electrophoresed in a 2% agarose gel and stained with ethidium bromide. The expression levels were normalized by using the relative mRNA ratio (lac1/gpd). A, Vanillic acid; B, syringic acid; C, 4-hydroxybenzoic acid; D, veratric acid; E, 4-hydroxybenzaldehyde; F, ferulic acid; G, 3,4,5-trimethoxybenzoic acid; H, 2,5-xylidine; I, vanillin; J, 3,4-dimethoxybenzaldehyde; K, 3,4-dimethoxybenzyl alcohol; L, p-coumaric acid.

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enzyme. In both cases, lac1 resembles the (cid:1)white(cid:2) laccase (POXA1), isolated earlier from Pleurotus ostreatus [30] and the laccase produced by Phellinus ribis [31]. Furthermore, the N-terminal sequence of the lac1 protein exhibits very low homology with sequences of other basidiomycete laccases (Fig. 7).

Although the spectral characteristics of the lac1 protein suggest the absence of a type 1 copper site, this is not borne out by analysis of the deduced amino acid sequence of the enzyme. Thus, the 10 histidines and one cysteine residue required to coordinate the four copper atoms at the active site of the enzyme [32] were all conserved in the V. volvacea gene (Fig. 4). It is possible that depletion of type 1 copper may have occurred during purification. However, attempts to reconstitute the copper to the purified enzyme were unsuccessful. Laccases also have an additional residue involved in the coordination of type 1 copper atoms, located 10 residues downstream of the single cysteine. This residue, which appears to have a role in determining the redox potential of the enzyme [33], can be methionine, leucine or phenylalanine. Therefore, lac1 with a leucine residue at position 458 should be assigned to class 2 according to the categorization proposed by Eggert et al. [34]. Other class 2 enzymes include laccases from the basidiomycetes, Agaricus bisporus [27], Podospora anserina [35], Phlebia radiata [36], the ascomycetous fungi, G. graminis [23], Neurospora crassa [37], Cryphonectria parasitica [9] and the yeast, Cryptococcus neoformans [38]. Two laccase genes described in Lentinula edodes also have a leucine residue in the analogous downstream position. However, the cysteine residue believed to be critical for coordination of the copper atoms is apparently not conserved in these genes and is replaced by tryptophan [39].

the different

Although V. volvacea produces at least two laccase isoforms [1], the primers used in this study appear to be specific for lac1 as in every case, only one band was amplified from each of samples and Southern blot analysis using the probe prepared with the lac1 primers also revealed a single band. Further- more, all of these generated PCR products were found to comprise of sequences identical to those present in the lac1 cDNA.

represents the first report on the cloning of a laccase gene and the factors affecting its transcription in this economi- cally important mushroom.

Copper regulates lac1 transcription in V. volvacea and the correlation between copper concentration and lac1 transcription corresponds well with previously observed effects of copper on total extracellular laccase activity in copper-supplemented cultures of V. volvacea [1]. A similar regulatory role for copper has been proposed for some laccase genes in several other basidiomycete fungi including Pleurotus spp. [5,26,40,41]., Trametes spp. [2,42]., Podos- pora anserina [35] and Ceriporiopsis subvermispora [43]. In P. ostreatus, copper not only regulated laccase gene expression but also positively affected the activity and stability of the enzyme [40]. The effect of copper on enzyme stability may be related to the inhibitory effect of the metal on the activity of an extracellular protease produced by P. ostreatus (PoSl) that is reported to degrade laccase [44].

The purified lac1 protein is unusual in itself in that concentrated solutions lack the typical blue colour and the spectral maxima near 600 nm that characterize all the blue oxidases. Furthermore, guaiacol is a poor substrate for the

Copper does not appear to be essential for the activation of lac1 transcription in V. volvacea as several aromatic compounds also induce gene transcription in copper- deficient cultures. Stimulation of laccase production by

Fig. 6. Transcription analysis of lac1 by RT-PCR (A) and total laccase activity (B) during various stages of V. volvacea fruitbody development. mRNA was extracted from the mycelium harvested at the following stages: substrate colonization stages (4, 8, 12 days), pinhead (day 14), button stage (day 18), egg stage (day 21), elongation stage (day 22) and mature stage (day 23). The expression levels were normalized by using the relative mRNA ratio (lac1/gpd). Extracellular protein was extrac- ted from rice-straw composts by suspending the substrate in 50 mM KHPO4 buffer (pH 6.5) and shaking (150 r.p.m.) for 2 h at room temperature.

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In straw cultures of Pleurotus cornucopiae var. citrinopile- atus, laccase activity was maximal during the vegetative growth and declined rapidly at the onset of fruiting [48]. Laccase activity in cultures of A. bisporus grown under standard composting conditions increased in the compost until just after the (cid:1)pinning(cid:2) stage (height of sporophores, 1.0 cm) of development and was correlated strongly with the loss of lignin from the compost [49]. Enzyme activity then declined rapidly during the later stages of fruit body development [16,50,51], decreasing by 87% in the 7 days between the appearance of the first fruit body initials and fruit body maturation [50]. Moreover, laccase gene expres- sion measured as mRNA levels was maximal at the fully colonized stage prior to fruiting and then declined to very low levels during fruiting [52]. Similarly, the activity of laccase was regulated strongly during the development of L. edodes fruit bodies [53,54]. Laccase gene expression (measured as mRNA levels) during growth of the fungus on a sawdust-based substrate was maximal at the fully colonized stage prior to fruiting and declined to very low levels during fruiting [53,54].

While the higher levels of

aromatics is well-documented and has led to the suggestion that one role of the enzyme is as a defence mechanism against oxidative stress caused by oxygen radicals origin- ating from aromatic compounds [14,45]. However, wide variations are seen with respect to the induction of laccase gene transcription by aromatic compounds with both inducible and noninducible forms described [46], suggesting that only certain isoenzymes serve in a protective capacity. Transcription of one laccase gene, lcc1, from Trametes villosa was induced (cid:1) 17-fold by the addition of XYL but a second gene (lcc2) was constituitive under the conditions tested [29]. Amounts of laccase mRNA and laccase activity in 10-day-old cultures of T. versicolor were a direct function of concentrations of the laccase inducers, 1-hydroxybenzo- triazole (HBT) and XYL but no induction was observed after the addition of FA and veratric acid (VA) [2]. Two laccase genes, lcc1 and lcc2 present in an unidentified basidiomycete I-62 were both inducible by veratryl alcohol but at different stages of growth, whereas transcriptional levels of a third gene, lcc3, were unaffected [24]. HBT, XYL, FA and VA all induced extracellular laccase production in P. sajor-caju and transcription levels of three laccase genes were increased by FA and XYL [5]. Higher levels of laccase mRNA were also reported in cultures of three white-rot fungi supplemented with aromatic compounds [47]. Tran- scription of only one of two laccase genes in T. versicolor was induced by 2,5-dimethylaniline [30]. Several aromatic compounds increase transcription of V. volvacea lac1 to varying degrees. FA was the most effective inducer of transcription and correspondingly high levels of extracellu- lar laccase activity were observed in cultures supplemented with this aromatic compound [1]. Ferulic acid is toxic to V. volvacea and radial mycelial growth on agar plates containing 1, 5 and 10 mM FA is inhibited by 37.6%, 71.8% and 100%, respectively (data not shown) suggesting that induction of this laccase isoform may in part, at least, be a detoxification response.

laccase gene expression observed during vegetative growth of A. bisporus and L. edodes, together with supporting biochemical data, indicate that laccases are involved directly in lignin biocon- version by these fungi [49,53,54], such a function in V. volvacea seems unlikely. Indeed, there is little evidence showing that V. volvacea is actually capable of degrading the lignin polymer and it has been suggested that an inability to do so accounts for the relatively poor mushroom yields achieved on lignified growth substrates [55]. Instead, the appearance of extracellular laccase activity in submerged cultures of V. volvacea at the onset of secondary metabolism [1], the temporal correlation observed between laccase production and sporophore formation when the mushroom was grown on cotton waste (cid:1)composts(cid:2) which were virtually devoid of any lignin component [1] and the pattern of lac1 transcription reported here, all provide strong evidence indicating that at least one enzyme isoform plays a key role in the morphogenesis of the V. volvacea fruit body. It has been proposed that phenoloxidases such as laccase could crosslink hyphal walls into coherent aggregates during primordium initiation [56] and may continue to act on the hyphal surfaces throughout fruit body development [57]. Further studies on a possible link between laccase and sporophore development in V. volvacea are underway as part of our overall aim to develop strategies for improved mushroom production through the control of fruit body growth, flush yield and flush timing [52].

Both laccase activity and lac1 gene transcription in compost cultures of V. volvacea were detected late in the substrate colonization phase when a sharp increase in both parameters was recorded (Fig. 6A,B). There was also good correlation between total laccase activity and lac1 expression although, as V. volvacea produces at least one other laccase isoform in addition to lac1 [1], this and possibly other laccase isoforms may be contributing to the total laccase activity detected at the various developmental stages. The pattern of laccase gene expression observed in compost grown cultures of V. volvacea contrasts sharply with activity profiles reported for other mushroom species.

Fig. 7. Comparison of N-terminal sequences of various fungal laccases.

Laccase gene from Volvariella volvacea (Eur. J. Biochem. 271) 327

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Acknowledgements

variella volvacea, the edible straw mushroom. Appl. Environ. Microbiol. 65, 553–559.

This work was supported by a grant from the Hong Kong Research Grants Council (grant CUHK 4163/01 M). We thank Dr Shao-jun Ding for providing the solid-state samples for transcriptional analysis. 19. Buswell, J.A., Mollet, B. & Odier, E. (1984) Ligninolytic enzyme production by Phanerochaete chrysosporium under conditions of nitrogen sufficiency. FEMS Microbiol. Lett. 25, 295–299.

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