Eur J Biochem 271, 318–328 (2004) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03930.x Biochemical and molecular characterization of a laccase from the edible straw mushroom, Volvariella volvacea 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 We have isolated a laccase (lac1) from culture fluid of Volvariella volvacea, grown in a defined medium containing 150 lM CuSO4, by ion-exchange and gel filtration chromatography Lac1 has a molecular mass of 58 kDa as determined 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- 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 developmental cycle defined in this study, although the levels of transcription varied considerably depending upon the developmental phase Transcription of lac1 increased sharply during the latter phase of substrate colonization and reached maximum levels during the very early stages (primordium formation, pinhead stage) of fruit body morphogenesis Gene expression then declined to  20–30% of peak levels throughout the subsequent stages of sporophore development Volvariella volvacea, the edible straw mushroom, produces multiple forms of laccase (benzenediol:oxygen oxidoreductase, 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systems representative of the conditions used for industrial cultivation ([1]; S Chen, W Ge and J A Buswell, unpublished results) Whereas, in many other basidiomycetes enzyme biosynthesis is normally associated with primary growth [2–5], laccase production in V volvacea has the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] Furthermore, when the fungus is grown on cotton waste ÔcompostsÕ [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 antimicrobial 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 importance 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 compounds 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 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: lac1, laccase; 2,6-DMP, 2,6-dimethoxyphenol; HBT, 1-hydroxybenzotriazole; XYL, 2,5-xylidine; FA, ferulic acid Enzyme: benzenediol:oxygen oxidoreductase (EC 1.10.3.2) (Received 26 August 2003, revised November 2003, accepted 18 November 2003) Keywords: Volvariella volvacea; laccase; edible mushroom; gene expression Ó FEBS 2003 Laccase gene from Volvariella volvacea (Eur J Biochem 271) 319 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 fungus was cultivated at 32 °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 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  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 day-old cultures with mM (final concentration) of the test compound For purification of laccase (lac1), the fungus was grown in L flasks containing 600 mL basal medium with 150 lM CuSO4 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 °C for 30 After cooling, the compost was inoculated with fungal mycelium (from week-old compost cultures) and incubated at 32 °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, 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 °C prior to analysis Enzyme assay Laccase activity was determined using 2,2¢-azinobis-(3ethylbenzo-6-thiazolinesulfonic acid) (ABTS) as described previously [1,20] 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 The following procedures were all performed at °C Culture fluid obtained after filtration of 7-day cultures of V volvacea was centrifuged (10 000 g, 30 min) and concentrated  40-fold with the Pellicon ultrafiltration system (Millipore) using a 10-kDa molecular mass cut-off membrane Solid ammonium sulfate was added and the fraction precipitating at 80% saturation was collected by centrifugation, 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 mLỈmin)1 The active fractions ( 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 mLỈmin)1 and the combined active fractions ( 50 mL) concentrated to 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 mLỈmin)1 Pooled active fractions ( 30 mL) were concentrated with a Centriprep YM-10 centrifugal filter (Millipore) and stored at )20 °C Enzyme characterization 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 PhastGel 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 °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 specificity of purified lac1 was determined spectrophotometrically 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 °C for Ó FEBS 2003 320 S Chen et al (Eur J Biochem 271) N-terminal sequencing of laccase The N-terminal amino acid sequence of purified laccase was determined by electroblotting the enzyme on to an Immobilon poly(vinylidene difluoride) (PVDF) Millipore membrane (LKB Multiblot apparatus, Bio-Rad) followed by Edman degradation performed with a Hewlett-Packard G1005A Protein Sequencer coupled to a HPLC (HewlettPackard, Model 1090) for analysis of the phenylthiodantoin amino acids RNA manipulations, cDNA synthesis and cloning 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 °C for h in a 10-lL reaction volume containing: lL diethylpyrocarbonate-treated H2O, lL · First Strand Buffer (Gibco Invitrogen), 0.01 M dithiothreitol, 0.5 mM dNTPs, 0.5 lg oligo(dT), lg total RNA and 100 U SuperScript II (Gibco Invitrogen) The cDNA from the reaction was kept at )70 °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 (upper primer): 5¢-(CT)T(AGCT)AC(AGCT)AA(CT)GG(AGCT)TT(CT) GC-3¢ (encoding LTNGFA); primer (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 PCR amplification of the cDNA fragment encoding a portion of lac1 was carried out using a PTC-100 (MJ Research, Watertown, MA, USA) in 50 lL reaction volumes containing 1.25 U Taq DNA polymerase, lL 10· Mg-free reaction buffer, 200 lM dNTP, 2.5 mM MgCl2, lM primer or primer and 0.5 lL template Amplification conditions were: cycle of 94 °C for min, 50 °C for 30 s and 72 °C for min; 30 cycles of 94 °C for 30 s, 54 °C for 30 s, and 72 °C for min; then a final extension at 72 °C for 10 before storage at °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 °C overnight with U T4 DNA ligase (Promega), lL 10· buffer with 10 mM ATP (Promega) and 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 RACE was performed with the SMART RACE cDNA Amplification Kit (Clontech) to obtain full-length cDNA clones Using the 309-bp fragment sequence of lac1 obtained above, the gene-specific primer (5¢-GGCACT 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 fulllength cDNA of lac1 was then generated by 3¢-RACE using the primer 5¢-TCTCAACCGTCGACAGCAG 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 RT-PCR for semiquantification of the lac1 expression 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 °C for h in a total volume of 10 lL reaction solution containing lg total RNA or 30 ng mRNA, · First Strand Buffer, 10 mM dithiothreitol, 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 As a control for RNA loading, a 330 bp fragment of the V volvacea, V14 glyceraldehyde-3-phosphate dehydrogenase (gpd) gene was amplified with primers PGPDF (5¢-TAATGACGGCAAACTCGTGATC-3¢) and PGPDR (5¢-TGTATGACTTTGGCCAGAGGTG-3¢) (accession number AY280633) The PCR cycle programme was: 94 °C for min; 23 cycles of 94 °C for 20 s, 52 °C for 20 s and 70 °C for min; then a final extension at 72 °C for 10 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 °C for min; 28 cycles of 94 °C for 20 s, 52 °C for 20 s and 70 °C for min; then a final extension at 72 °C for 10 To validate the semiquantitative 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 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 Ó FEBS 2003 Laccase gene from Volvariella volvacea (Eur J Biochem 271) 321 Table Summary of the purification procedure for V volvacea lac1 Volume (ml) Crude enzyme Ultra filtration (NH4)2SO4 DEAE CL-6B Sephacryl S-300 Sephacryl S-100 Total activity (IU) Protein (mg) Specific activity (Img)1) Yield (%) Purification fold 2000 50 20 60 50 30 32.6 30.6 25 11.9 8.6 25 20 0.7 0.5 1.3 1.4 3.6 11.9 13 17.2 100 94 77 37 30 22 1.1 2.8 9.2 10 14 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 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 ( 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-3,4dimethoxyphenol 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 mg protein)1 Corresponding pH optima and specific activities for syringaldazine and 2,6-DMP were pH 5.6 and 3.4 mg protein)1 and pH 4.6 and 2.8 mg protein)1, respectively In standard assay mixtures, the velocity of ABTS oxidation was maximal at 45 °C The dependence of the rate of ABTS oxidation by lac1 on substrate concentration at pH 3.0 and 45 °C followed Michaelis–Menten kinetics A reciprocal plot revealed an apparent Km value of 0.03 mM and a Vmax of 16.4ỈU mg protein)1 Corresponding values for syringaldazine and 2,6-DMP under optimal conditions were 0.01 mM and 4.9 mg protein)1, and 0.57 mM and 5.6 mg 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 mM EDTA (20%) The N-terminal sequence of native protein was N-ALSSHTLTLTNGFASPD Cloning of full-length cDNA of lac1 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 showed highest overall identity with laccase (BAB84354) from Lentinula edodes (58%), laccase (AF170093) from Pycnoporus cinnabarinus (57%), laccase LCC3-1 (AF176230) from Polyporus ciliatus (56%) and laccase (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) demonstrating the workability of the RT-PCR assay for quantitating lac1 mRNA levels Regulation of lac1 expression by copper The effect of copper concentration on lac1 expression in cultures of V volvacea is shown in Fig Lac1 transcription increased with increasing concentrations of CuSO4 in the culture medium up to 200 lM Higher copper concentrations 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 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 Laccase induction in V volvacea by various aromatic compounds occurred at the level of gene transcription Figure shows the effect of different aromatic compounds on lac1 expression in V volvacea grown in nitrogen- 322 S Chen et al (Eur J Biochem 271) Ó FEBS 2003 Fig 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 Ó FEBS 2003 Laccase gene from Volvariella volvacea (Eur J Biochem 271) 323 Fig 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 sufficient medium without copper Highest levels of gene transcription were seen with FA, veratric acid, 4-hydroxybenzoic acid and 2,5-xylidine (XYL) Amounts of lac1 mRNA in the cultures increased with increasing concentrations of FA (1–10 mM) although transcript levels in mycelium grown with 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 lac1 during growth and fruiting of V volvacea on straw The duration of the developmental cycle of V volvacea when the mushroom was grown on rice-straw ÔcompostsÕ 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 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 developmental 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 transcripts 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 324 S Chen et al (Eur J Biochem 271) Ó FEBS 2003 Fig Validation of semiquantitative RTPCR 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 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 concentrations 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 understand these effects at the molecular level, we have now Fig RT-PCR analysis of total RNA isolated from V volvacea, V14, grown in defined medium supplemented with mM aromatic compounds Fungal mycelia were cultured for days prior to addition of aromatic compound and harvested after a further 36 h incubation 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) 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 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 Ó FEBS 2003 Laccase gene from Volvariella volvacea (Eur J Biochem 271) 325 Fig 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 extracted from rice-straw composts by suspending the substrate in 50 mM KHPO4 buffer (pH 6.5) and shaking (150 r.p.m.) for h at room temperature represents the first report on the cloning of a laccase gene and the factors affecting its transcription in this economically important mushroom 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 enzyme In both cases, lac1 resembles the ÔwhiteÕ 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 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 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 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 according to the categorization proposed by Eggert et al [34] Other class 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] 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 the different samples and Southern blot analysis using the probe prepared with the lac1 primers also revealed a single band Furthermore, all of these generated PCR products were found to comprise of sequences identical to those present in the lac1 cDNA 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]., Podospora 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] 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 copperdeficient cultures Stimulation of laccase production by 326 S Chen et al (Eur J Biochem 271) Ó FEBS 2003 Fig Comparison of N-terminal sequences of various fungal laccases 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 originating 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  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-hydroxybenzotriazole (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] Transcription 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 extracellular 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, 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 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 In straw cultures of Pleurotus cornucopiae var citrinopileatus, 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 ÔpinningÕ 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 days between the appearance of the first fruit body initials and fruit body maturation [50] Moreover, laccase gene expression 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 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 bioconversion 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 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 ÔcompostsÕ 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] Ó FEBS 2003 Laccase gene from Volvariella volvacea (Eur J Biochem 271) 327 Acknowledgements 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 20 References Chen, S.C., Ma, D., Ge, W & Buswell, J.A (2003) Induction of laccase activity in the edible straw mushroom, Volvariella volvacea FEMS Microbiol Lett 218, 143–148 Collins, P.J & Dobson, A.D.W (1997) Regulation of laccase gene transcription in 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edodes on supplemented sawdust FEMS Microbiol Lett 210, 111–115 55 Ohga, S., Cho, N.S., Thurston, C.F & Wood, D.A (2000) Transcriptional regulation of laccase and cellulase genes in Lentinula edodes on a sawdust-based substrate Mycoscience 41, 149–153 56 Cai, Y.J., Buswell, J.A & Chang, S.T (1994) Production of cellulases and hemicellulases by the straw mushroom, Volvariella volvacea Mycol Res 98, 1019–1024 57 Bu’lock, J.D (1967) Fungal metabolites with structural function In Essays in Biosynthesis and Microbial Development: E.R Squibb Lectures on Chemistry of Microbial Products, pp 1–18 John Wiley, New York 58 Leatham, G.F & Stahmann, M.A (1981) Studies on the laccase of Lentinus edodes: specificity, localization and association with the development of fruiting bodies J Gen Microbiol 125, 147–157  ... 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. .. primers The putative N-glycosylation site is boxed; *; stop codon The putative polyadenylation signals (TATAAA and CATAAA) are in white on a black background Ó FEBS 2003 Laccase gene from Volvariella. .. min; then a final extension at 72 °C for 10 The primers for lac1 PLAC1F (5¢-AGCTTT CATTCCCAGTGATTG-3¢) and PLAC1R (5¢-AACGAG CTCAAGTACAAATGACT-3¢) were designed according to our cloned cDNA (GenBank