Cloning and functional analysis of a new laccase gene from Trametes sp. 48424 which had the high yield of laccase and strong ability for decolorizing different dyes

Bioresource Technology 102 (2011) 3126–3137 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech Cloning and functional analysis of a new laccase gene from Trametes sp. 48424 which had the high yield of laccase and strong ability for decolorizing different dyes Fangfang Fan a,b, Rui Zhuo a,b, Su Sun a, Xia Wan b, Mulan Jiang b, Xiaoyu Zhang a,⇑, Yang Yang a,b,⇑ a b College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China Key Laboratory of Oil Crops Biology of Ministry of Agriculture in China, Oil Crops Research Institute of Chinese Academy of Agricultural Sciences, Wuhan 430064, China a r t i c l e i n f o Article history: Received 13 June 2010 Received in revised form 15 October 2010 Accepted 18 October 2010 Available online 23 October 2010 Keywords: White-rot fungi Laccase gene Heterologous expression cis-Acting elements Decolorization of dyes a b s t r a c t The laccase gene lac48424-1 and its corresponding full-length cDNA were cloned and characterized from a novel white-rot fungi Trametes sp. 48424 which had the high yield of laccase and strong ability for decolorizing different dyes. The 1563 bp full-length cDNA of lac48424-1 encoded a mature laccase protein containing 499 amino acids preceded by a signal peptide of 21 amino acids. The deduced protein sequence of LAC48424-1 showed high similarity with other known fungal laccases and contained four copper-binding conserved domains of typical laccase protein. The functionality of lac48424-1 gene encoding active laccase was verified by expressing the gene in the yeast Pichia pastoris successfully. It was found that the recombinant laccase produced by the yeast transformant could decolorize different dyes. The 50 -flanking sequence upstream of start codon was obtained by Self-Formed Adaptor PCR. Many putative cis-acting responsive elements involved in the transcriptional regulation were identified in the promoter region of lac48424-1. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) is a group of copper-containing polyphenol oxidases which can catalyze the four-electron reduction of O2–H2O with the concomitant oxidation of phenolic compounds. Laccase is one of the important ligninolytic enzymes responsible for the strong ability of lignin degradation of white-rot fungi (Thurston, 1994; Baldrian, 2006). The research about the biological function of fungal laccases have suggested that this enzyme may be involved in lignin degradation, fungal morphogenesis, fungal virulence and pigmentation (Thurston, 1994; Baldrian, 2006). Laccase is also an very important and valuable enzyme for various biotechnological and industrial applications, such as biodegradation of lignin without polluting the environment, thorough degradation of different recalcitrant compounds, environmental protection and bioremediation, biological bleaching in paper industry, textile dye decolorization (Mayer and Staples, 2002). In an attempt to obtain lots of enzyme for biotechnological application and use laccase more efficiently in biotechnology, several laccase genes have been cloned from different fungal ⇑ Corresponding authors. Address: College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China. Tel.: +86 27 87792108. E-mail addresses: yangyang@mail.hust.edu.cn (Y. Yang), zhangxiaoyu@mail. hust.edu.cn (X. Zhang). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.10.079 sources (Thurston, 1994; Galhaup et al., 2002; Xiao et al., 2006) and heterologously expressed in Pichia pastoris (Soden et al., 2002), Saccharomyces cerivisiae (Bulter et al., 2003), Kluyveromyces lactis (Faraco et al., 2008), Aspergillus nidulans (Larrondo et al., 2003), and so on. Research about the regulation of laccase gene expression may be very useful for increasing the productivity of native laccase in fungi. It reveals that the transcription of laccase gene can be regulated by different factors such as metal ions (Collins and Dobson, 1997; Galhaup et al., 2002), various aromatic compounds related to lignin or lignin derivatives (Terrón et al., 2004), nutrient nitrogen (Collins and Dobson, 1997) and carbon (Soden and Dobson, 2001). Promising and valuable applications of laccase in biotechnology and industry have resulted in an increased interest and need for isolating new laccase genes from different sources. In addition, isolation of new laccase genes from novel white-rot fungi will greatly promote the precise elucidation of the biological function of laccase in fungi. In this study, the laccase gene lac48424-1 and its corresponding full-length cDNA were cloned and characterized from a novel white-rot fungi Trametes sp. 48424 isolated from China, which had the high yield of laccase and strong ability for decolorizing different dyes. The lac48424-1 gene was successfully expressed in the yeast Pichia pastoris, which confirmed the correct function of lac48424-1 gene encoding laccase in the view of gene expression. The recombinant laccase produced by the yeast transformant was found to possess the ability to decolorize different dyes. In addition, many putative cis-acting responsive elements 3127 F. Fan et al. / Bioresource Technology 102 (2011) 3126–3137 that may play a role in transcriptional regulation of laccase gene were found in the promoter region of lac48424-1. The category and number of cis-acting regulatory elements in the promoter region of lac48424-1 gene from Trametes sp. 48424 were quite different from that of other known laccase genes. Trametes sp. 48424 is a white-rot fungi strain with great potential for biotechnological applications. This research will lay good foundation for the further application of Trametes sp. 48424 in decolorization of different dyes, degradation of recalcitrant xenobiotic compounds, as well as enzymatic modification by directed evolution. 2. Methods 2.1. Strains and media Trametes sp. 48424 was a gift from Prof. Ping Xu in Shandong University and preserved in Institute of Environment & Resource Microbiology, Huazhong University of Science and Technology, Wuhan, China. Trametes sp. 48424 was maintained on potatodextrose agar (PDA) medium. Pichia pastoris GS115 and plasmid pPIC3.5K were purchased from Invitrogen. MD, BMGY, BMMY and BMM media were prepared according to the instruction of the Pichia Expression Kit manual (Invitrogen). Escherichia coli DH5a was used in all of the cloning procedures. 2.2. Isolation of genomic DNA and total RNA from fungi Trametes sp. 48424 was grown in the GYP medium: 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(Galhaup et al., 2002). The pH was adjusted to 5.0 with H3PO4 prior to sterilization. The mycelium was harvested from liquid culture at the peak of laccase activity. Genomic DNA was extracted using the E.Z.N.A. Fungal DNA kit (Omega, USA). Total RNA was extracted using the TRIZOL Reagent (Invitrogen) according to the instructions, followed by RNase-Free DNase (Promega) digestion. 2.3. Cloning of the complete structural gene of lac48424-1 The degenerate primers CuI and CuIV (sequence shown in Table 1) were designed according to the conserved amino acids sequences of the copper-binding region I (HWHGFFQ) and IV (HCHIDFH) in fungal laccases, respectively. Degenerate PCR was performed using the genomic DNA of Trametes sp. 48424 as the template. A 1510 bp PCR fragment was obtained and cloned to pMD18-T vector (TAKARA) for sequencing. It was confirmed that this 1510 bp PCR fragment contained specific sequence of laccase gene by DNA sequencing. In order to obtain the complete structural gene encoding laccase, TAIL-PCR (thermal asymmetric interlaced PCR) (Liu et al., 1995) was performed to amplify the 50 and 30 -flanking sequence of the known 1510 bp laccase gene partial sequence using the genomic DNA of Trametes sp. 48424 as the template. The specific nested primers for amplify the 50 -flanking sequence of the known 1510 bp laccase gene were 48424-5-1, 48424-5-2 and 48424-5-3 (primer sequence shown in Table 1). The specific nested primers for amplify the 30 -flanking sequence of the known 1510 bp laccase gene were 48424-3-1, 48424-3-2, and 48424-3-3 (primer sequence shown in Table 1). The arbitrary degenerate primers (AD primers) used in TAIL-PCR were designed according to reference (Liu et al., 1995) and shown in Table 1. TAIL-PCR was performed according to the method described in Ref. Liu et al. (1995). The TAIL-PCR product was cloned to pMD18-T vector (TAKARA) for DNA sequencing. After obtaining the 50 and 30 -flanking sequence of the known 1510 bp laccase gene, high fidelity PCR was performed to amplify the complete structural Table 1 Oligonuleotide primers used in this study. D = A/G/T, N = A/G/C/T, R = A/G, Y = C/T. Primer Nucleotide sequence CuI CuIV CAYTGGCAYGGNTTYTTYCA TGRAARTCDATRTGRCARTG 48424-5-1 48424-5-2 48424-5-3 ccaatccacaagggtaatgacagtgtcgtc ccggatcattcgggtcgtaaacaacgaac cgagtgaccagatgagatcgggcactg 48424-3-1 48424-3-2 48424-3-3 ccatcaacatggcgttcaacttcaacgg cgactgtgcctgtcctgctccag caactacgacaaccccatcttccgcg 48424-f1 48424-r1 48424-f1-EcoRI 48424-r1-NotI lac-48424-SP1 lac-48424-SP2 lac-48424-SP3 lac-48424-SP4 AD1 AD2 AD3 AD4 AD5 AD6 atgtcgaggtttcactctcttctcgctttc ttactggtcgctcgggtcgag aaagaattcaccatgtcgaggtttcactctcttctcgc tttgcggccgcttactggtcgctcgggtcgag cgctagtgcgagccgagacgaaccatgttacccg ggtcggcgatgggaccgataccagcgtgg cttgaaggtagtgagtggNNNNNNNNNNatctcg cgcgcaacatcctttatacctcggcgaagccgg TG(A/T)GNAG(A/T)ANCA(G/C)AGA AG(A/T)GNAG(A/T)ANCA(A/T)AGG (G/C)TTGNTA(G/C)TNCTNTGC NTCGA(G/C)T(A/T)T(G/C)G(A/T)GTT NGTCGA(G/C)(A/T)GANA(A/T)GAA (A/T)GTGNAG(A/T)ANCANAGA gene using the genomic DNA of Trametes sp. 48424 as the template and 48424-f1, 48424-r1 as the specific primers (sequence shown in Table 1). The PCR product-1996 bp complete structural gene encoding laccase was cloned to pMD18-T vector (TAKARA) for DNA sequencing and designated as lac48424-1. 2.4. Cloning of the full-length cDNA of lac48424-1 According to the known 50 and 30 -end sequences of the laccase structural gene, primer 48424-f1 was designed to match the start codon ATG region and primer 48424-r1 was designed to match the sequence immediately downstream of the stop codon TAA (sequences of primer 48424-f1 and 48424-r1 were shown in Table 1). Using 48424-f1 and 48424-r1 as the specific primers, RT-PCR was then performed to amplify the full-length cDNA of lac484241 with PrimeSTAR™ HS DNA Polymerase (TAKARA). The 1563 bp full-length cDNA was cloned to pMD18-T vector (TAKARA) for DNA sequencing, resulting in the recombinant plasmid pMD18T-lac48424-1. 2.5. Cloning of the 50 -flanking sequence upstream of the start codon of lac48424-1 In order to obtain the promoter region of laccase gene, SelfFormed Adaptor PCR (SEFA PCR) (Wang et al., 2007) was performed to amplify the 50 -flanking sequence upstream of start codon of lac48424-1. The nested primers used for Self-Formed Adaptor PCR (lac-48424-SP1, lac-48424-SP2, lac-48424-SP3 and lac-48424SP4, the sequences of these primers were shown in Table 1) were designed based on the known structural gene sequence. lac48424-SP1, lac-48424-SP2, and lac-48424-SP4 were gene-specific primers and had high annealing temperatures (about 70 °C). lac-48424-SP3 was a partially degenerate primer. Self-Formed Adaptor PCR (SEFA PCR) was performed according to the method described in Ref. Wang et al. (2007). 2.6. Cloning of the ITS sequence of rDNA from Trametes sp. 48424 High fidelity PCR was performed to amplify the internal transcribed spacer (ITS) sequence of ribosome DNA using the genomic DNA of Trametes sp. 48424 as the template and ITS1, ITS4 as the specific primers (ITS1: tccgtaggtgaacctgcgg, ITS4: tcctccgcttattga- 3128 F. Fan et al. / Bioresource Technology 102 (2011) 3126–3137 tatgc). The PCR product was cloned to pMD18-T vector (TAKARA) for DNA sequencing. 2.7. Heterologous expression of lac48424-1 gene in Pichia pastoris Firstly the vectors for expression of lac48424-1 gene in Pichia pastoris were constructed. High fidelity PCR was performed to obtain the full-length lac48424-1 cDNA using the pMD18T-lac48424-1 as template and 48424-f1-EcoRI, 48424-r1-NotI as primers (sequences shown in Table 1, restriction sites for EcoRI and NotI were incorporated into upstream and downstream primers, respectively). Then the PCR product was purified and digested with EcoRI and NotI. This digested fragment was inserted into the same sites of pPIC3.5K vector (Invitrogen), resulting in the recombinant plasmid pPIC3.5K-lac48424-1 (containing the laccase native signal peptide). The pPIC3.5K-lac48424-1 and the control vector pPIC3.5K were linearized by SacI digestion and transformed into P. pastoris GS115 (Invitrogen), respectively by the electroporation method described in the instruction of Multi-Copy Pichia Expression Kit (Invitrogen). The electroporated cells were plated onto MD agar plates for selecting the His+ transformants. Some His+ transformants were selected randomly and grown on the BMGY agar plates at 28 °C for 2 days and then inoculated onto the BMM agar plates containing CuSO4 (0.1 mmol/l) and ABTS(0.2 mmol/l). Under the induction of methanol, secretion of active laccase was identified by the presence of a dark green zone around transformant colonies. After selection of positive transformants which could produce active laccase on ABTS plates, the positive transformants were then fermented with BMM liquid medium at 20 °C. The yeast transformants were inoculated into 20 ml BMG media in 250-ml Erlenmeyer flasks and incubated at 30 °C to OD600 of 10 with shaking at 200 rpm. Then the cultures were centrifuged at 3000g for 5 min and the cell pellets were suspended to OD600 of 2.0 with 30 ml BMM media (pH 6.0) containing 0.3 mM CuSO4 and 0.8% alanine. The cultures were grown at 20 °C with shaking at 200 rpm, with 0.5% (v/v) methanol being added daily. Secreted laccase activities in cultures were measured daily. Laccase activity was measured by means of ABTS method as described by reference(Galhaup et al., 2002). Native-PAGE was also performed according to the method described in reference(Xiao et al., 2006). All experiments were performed in triplicate. 2.8. Purification of recombinant laccase produced by Pichia pastoris transformants The Pichia Pastoris transformant in which lac48424-1 gene was successfully expressed was cultivated in BMG medium at 28 °C, 250 rpm for two days (OD600: 20). The cell were centrifugated at the speed of 1500 g for 5 min and then resuspended in BMM medium(containing 0.3 mM Cu2+ and 0.8% alanine). The cultures were grown at 20 °C with shaking at 200 rpm, with 0.5% (v/v) methanol being added daily for ten days. 200 ml of culture was centrifugated at 10,000g for 20 min. The supernatant was concentrated into 10 ml using PEG20000 and then dialysed against PBS buffer (pH6.8, 0.05 mol/l) for 24 h by ice bath. The precipitate was abandoned by centrifugation (12,000g/min). The supernatant was repeatedly dialysed against phosphate buffer (pH6.8, 0.05 mol/l) and then applied to a DEAE-cellulose column (DEAE52,100  200 mm), which was pre-equilibrated with 0.05 mol/l PBS, pH6.8. The column was washed at a flow rate of 3 ml min 1 with 2L PBS buffer to remove unbound protein. Bound laccase was eluted from the column with salt gradient (0.05, 0.15, 0.25, 0.35, 0.45 M NaCl) in the same buffer with a flow rate of 3 ml min 1. Elution was simultaneously monitored at 280 nm for protein detection. Active fractions were pooled, desalted, filter- sterilized, and stored at 4 °C. Protein concentrations was measured using the Bradford dye-binding assay (Coomassie brilliant blue) and bovine serum albumin as the standard. The homogeneity of the purified recombinant laccase was detected by SDS–PAGE. 2.9. Decolorization of dyes by Trametes sp. 48424 The culture supernatants prepared from Trametes sp. 48424 were used to decolorize four dyes: methyl orange, malachite green, bromophenol blue, and crystal violet. The assays were carried out at 30 °C. The reaction mixture in a total volume of 1 ml contained (final concentration): acetate buffer (25 mM, pH 4.5), dyes (methyl orange, malachite green, bromophenol blue: 50 mg/l; crystal violet: 20 mg/l), and 100 ll culture supernatant (containing 0.1 U laccase). The decolorization of dye, expressed as dye decolorization (%), was calculated by means of the formula: decolorization (%) = [(Ci Ct)/Ci]  100, where, Ci: initial concentration of the dye, Ct: dye concentration along the time (Lorenzo et al., 2006). Kojic acid was added into the culture supernatants of Trametes sp. 48424 at a final concentration of 20 mM. Then the decolorization of dyes was performed again as described above. 2.10. Decolorization of dyes by the purified recombinant laccase produced by Pichia pastoris transformants The decolorization of different dyes by purified recombinant laccase was determined over a 12 h period. The reaction mixture respectively contained 50 mg/l methyl orange, malachite green, bromophenol blue or 20 mg/l crystal violet, acetate buffer (25 mM, pH 4.5) and purified enzyme (0.02 U). During incubation at 30 °C, the time course of decolorization was detected every 3 h by measuring the absorbance at 618 nm for malachite green, 462 nm for methyl orange, 592 nm for bromophenol blue, 584 nm for crystal violet. The decolorization of dye, expressed as dye decolorization (%), was calculated by means of the formula: decolorization (%) = [(Ci Ct)/Ci]  100, where, Ci: initial concentration of the dye, Ct: dye concentration along the time (Lorenzo et al., 2006). The decolorization efficiency of each dye was shown as dye decolorization (%). Kojic acid was added into the reaction mixture at a final concentration of 20 mM. Then the decolorization of dyes was performed again as described above. 2.11. Bioinformatic analysis of gene sequence Analysis of the homology between the protein encoded by lac48424-1 and other known laccase proteins was performed using BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The molecular weight and isoelectric point of protein were predicted with Compute pI/Mw tool (http://www.expasy.org/tools/pi_tool.html). N-glycosylation sites (Asn-X-Ser/Thr) were identified with ScanProsite program (http://www.expasy.ch/tools/scanprosite/). Signal peptides were predicted with SignalP 3.0 (http://www.cbs.dtu.dk/ services/SignalP/). The Conserved Domains of protein were predicted and analyzed with Conserved Domain Database (CDD) (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The secondary structure of protein was predicted by ANTHEPROT software. Alignments of multiple DNA and amino acid sequences were generated with ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/ index.html). The putative cis-acting elements in the promoter region of laccase gene were predicted and identified with SoftBerry-NSITE/ Recognition of Regulatory motifs (http://www.softberry.ru/berry. phtml?topic=nsite&group=programs&subgroup=promoter). The putative cis-acting elements in the promoter region of laccase gene were also examined using the MatInspector software (http:// www.genomatix.de/products/MatInspector/). 3129 F. Fan et al. / Bioresource Technology 102 (2011) 3126–3137 3.2. Cloning of full-length cDNA of the laccase gene from Trametes sp. 48424 and bioinformatic analysis The 1563 bp full-length cDNA of the laccase gene containing intact ORF was cloned from Trametes sp. 48424. This laccase gene Dye decolorization (%) A 48424/malachite green 48424/kojic acid/malachite green Time (hour) Dye decolorization (%) B 48424/methyl orange 48424/kojic acid/methyl orange Time (hour) C Dye decolorization (%) The laccase production of Trametes sp. 48424 was detected continuously. It was found that the secreted laccase activity of Trametes sp. 48424 could be stimulated significantly by various concentrations of Cu2+. The highest laccase activity of Trametes sp. 48424 was enhanced to be 15273.4 U L 1 by 1 mM Cu2+, while the laccase activity of Trametes sp. 48424 growing in the same medium without Cu2+ was only 16.68 U L 1. It suggested that Cu2+ could greatly stimulate the production of laccase by Trametes sp. 48424. Molecular taxonomy based on internal transcribed spacer (ITS) sequence of ribosome DNA was further used to identify this fungal strain. The ITS sequence of 48424 strain was cloned and sequenced. Sequence analysis revealed that the 623 bp ITS sequence of 48424 strain was most similar to the ITS sequence of Trametes versicolor strain CTB 863 (GenBank accession no: EF524042) with 99% identity. Although Trametes sp. 48424 was not a novel species, the laccase production of Trametes sp. 48424 was very high compared with other Trametes strains already reported. Collins and Dobson have studied the regulation of laccase gene transcription in Trametes versicolor 290. The highest laccase activity measured in cultures grown in the presence of 400 lM Cu2+ was about 2500 U L 1 (Collins and Dobson, 1997). Galhaup and Haltrich have studied the stimulatory effects of copper on the laccase production by different Trametes strains. The highest laccase activity of Trametes versicolor MB 52, Trametes versicolor MB 54, Trametes suaveolens MB 51 induced by 1 mM Cu2+ were about 9000, 10000, 7000 U L 1, respectively (Galhaup and Haltrich, 2001). Using glucose as the sole carbon source, higher concentrations of Cu2+ (1–2 mM) significantly stimulated laccase synthesis in Trametes sp. AH28-2. The highest laccase activity induced by 1 mM Cu2+ was observed to reach a value of 175 U L 1 (Xiao et al., 2006). Lorenzo et al. studied the effect of copper on the laccase production from Trametes versicolor (CBS100.29). The highest values were obtained by the cultures supplemented with 2 mM Cu2+, showing maximum values around 6000 U L 1(Lorenzo et al., 2006). In our research, the highest laccase activity of Trametes sp. 48424 could be stimulated to be 15273.4 U L 1 by 1 mM Cu2+. It suggested that the laccase production of Trametes sp. 48424 induced by copper was relatively very high compared with other Trametes strains described above. Further research revealed that the culture supernatants prepared from Trametes sp. 48424 could decolorize different dyes efficiently. Decolorization of dyes by Trametes sp. 48424 was performed according to the method described in Section 2. But the ability to decolorize four dyes was inhibited by adding kojic acid (Fig. 1A–D). Previous research have proved that kojic acid was a specific inhibitor of fungal laccase (Murao et al., 1992; Kim et al., 2008). Our result also confirmed that the laccase activity of Trametes sp. 48424 could be significantly inhibited by kojic acid (Fig. 1E). Thus above results indicated that laccase played a very important role in the decolorization of different dyes by Trametes sp. 48424. In order to better utilize this novel laccase-high producing fungus for the decolorization of different dyes, the laccase gene of Trametes sp. 48424 and its full-length cDNA were cloned and characterized in the following work. 48424/bromophenol blue 48424/kojic acid/bromophenol blue Time (hour) D 48424/crystal violet Dye decolorization (%) 3.1. Laccase production of Trametes sp. 48424 and its ability for decolorizing different dyes was designated as lac48424-1 (GenBank accession no. HM483869). The coding region of lac48424-1 consisted of a 1560 bp ORF encoding 520 aa with a 21 aa as the signal peptide sequence. The product of lac48424-1 was predicted to be a mature protein of 499 aa 48424/kojic acid/crystal violet Time (hour) E Laccase activity(U/L) 3. Results and discussion Kojic acid(mM) Fig. 1. Decolorization of different dyes by Trametes sp. 48424. The culture supernatants prepared from Trametes sp. 48424 were used to decolorize four dyes: malachite green, methyl orange, bromophenol blue, and crystal violet. The ability for decolorizing four dyes by Trametes sp. 48424 could be inhibited by adding kojic acid (20 mM) which was a specific inhibitor of fungal laccase. In the figure, it was shown as 48424/kojic acid/different dyes (Fig. 1A–D). The laccase activity could be inhibited by adding 20 mM kojic acid (Fig. 1E). 3130 F. Fan et al. / Bioresource Technology 102 (2011) 3126–3137 Fig. 2. Alignments of multiple amino acid sequences of LAC48424-1 and other laccases protein (indicated as the GenBank accession number of each laccase protein). It showed that LAC48424-1 protein contained four copper-binding conserved domains of typical laccase: CuI (HWHGFFQ), CuII (HSHLSTQ), CuIII (HPFHLHG), and CuIV (HCHIDFHL), which were indicted with box in the figure. 3131 F. Fan et al. / Bioresource Technology 102 (2011) 3126–3137 Table 2 The levels of identify between LAC48424-1 and other laccase proteins from Trametes. The percent identify between LAC48424-1 and other laccases were calculated by using the ClustalW method with data for the deduced or determined proteins. The identity was indicated by percentage. 1. LAC48424-1. 2. AAB47734: Trametes villosa laccase. 3. AAC41687: Trametes villosa laccase. 4. AAC49828: Trametes versicolor laccase I. 5. AAC49829: Trametes versicolor laccase IV. 6. AAL00887: Trametes versicolor laccase 1. 7. AAM18408: Trametes pubescens laccase 1A. 8. AAM66349: Trametes sp. C30 laccase 2. 9. AAQ12267: Trametes sp. I-62 laccase. 10. AAW28934: Trametes sp. AH28-2 laccase C. 11. AAW28935: Trametes sp. AH28-2 laccase D. 12. AAW29420: Trametes versicolor laccase 1. 13. AAW31597: Trametes sp. AH28-2 laccase B. 14. ABB21020: Trametes sp. 420 laccase E. 15. ACK77785: Trametes versicolor laccase protein. 16. ACO53431: Trametes sp. C30 laccase 5. 17. ACO53432: Trametes sp. C30 LACCASE HYBRID. 18. BAA23284: Trametes versicolor laccase. 19. BAD98306: Trametes versicolor laccase2. 20. BAD98307: Trametes versicolor laccase3. 21. BAD98308: Trametes versicolor laccase4. 22. CAA59161: Trametes versicolor laccase. 23. CAM12361: Trametes versicolor MULTICOPPER OXIDASE. residues with a calculated molecular mass of 55.5 KDa and isoelectric point of 4.75. LAC48424-1 contained seven potential N-glycosylation sites (Asn-X-Ser/Thr). The deduced amino acid sequence of LAC48424-1 protein showed high homology with other known laccases of fungi. The conserved domains of LAC48424-1 protein were predicted and analyzed with Conserved Domain Database (CDD). It revealed that LAC48424-1 protein contained typical conserved domains of multicopper oxidases. Alignments of multiple amino acid sequences of LAC48424-1 and other laccase protein were generated with ClustalW2. It showed that LAC48424-1 protein contained four copper-binding conserved domains of typical laccase: CuI (HWHGFFQ), CuII (HSHLSTQ), CuIII (HPFHLHG), and CuIV (HCHIDFHL) (Fig. 2). Ten conserved histidine residues and one cysteine residue were located in the copper-binding centers of LAC48424-1 protein. The deduced amino acid sequence of LAC48424-1 protein was compared with the sequences of 22 other laccases available in the GenBank database (Table 2). LAC48424-1 protein was closest to Trametes sp. I-62 laccase (AAQ12267), which had 78.7% identity with the amino acid sequence of Trametes sp. I-62 laccase, followed by Trametes sp. AH28-2 laccase D (AAW28935) and Trametes versicolor laccase I (AAC49828) with 78.6% and 77.6% identity, respectively. The percent identity of LAC48424-1 compared with other laccases ranged from 78.7% to Table 3 The predicted secondary structure of LAC48424-1 was compared with that of other laccase from Trametes (indicated by GenBank accession no.). Laccase Helix (%) Sheet (%) Turn (%) Coil (%) LAC48424-1 AAB47734 AAC49828 AAL00887 AAM18408 AAM66349 AAQ12267 AAW28934 AAW29420 AAW31597 ABB21020 ACO53431 BAD98307 10 9 10 10 9 11 9 8 11 10 9 11 11 28 28 30 29 28 29 31 30 29 29 31 30 28 6 6 5 5 6 6 7 6 6 6 7 7 7 55 56 55 56 57 54 53 56 54 55 54 53 54 3132 F. Fan et al. / Bioresource Technology 102 (2011) 3126–3137 69.1%. The secondary structure of LAC48424-1 protein was also predicted. It revealed that this protein contained 10% helix, 28% sheet, 6% turn and 55% coil. We compared the predicted secondary structure of LAC48424-1 with that of other laccase (Table 3). There were little difference between the secondary structure of LAC48424-1 and that of other known laccase from Trametes. 3.3. Heterologous expression of lac48424-1 gene in Pichia pastorisfunctional verification of lac48424-1 gene encoding laccase The plasmid pPIC3.5K-lac48424-1 in which the full-length cDNA of lac48424-1 was cloned into pPIC3.5K (Invitrogen) and the control vector pPIC3.5K were transformed into P. pastoris GS115, respectively. His+ transformants were screened and confirmed to be correct transformants by PCR (data not shown), which were named as GS115(pPIC3.5K-lac48424-1) and GS115(pPIC3.5K). The expression of lac48424-1 gene in transformants was then detected. The BMM agar plates containing CuSO4 and ABTS were used to screen the positive transformants which could produce active laccase. Under the induction of methanol, dark green zones were present around the colonies of GS115(pPIC3.5K-lac48424-1) after three days growth. On the contrary, no dark green zones appeared around the colonies of the negative controlGS115(pPIC3.5K) (shown in Fig. 3A). After selection of positive transformants which could produce active laccase on ABTS plates, the laccase-positive transformants as well as the negative controlGS115(pPIC3.5K) were then fermented with BMM liquid medium at 20 °C and induced by adding 0.5% (v/v) methanol daily. Laccase activities in cultures were measured daily (shown in Fig. 3B). The highest yield of laccase of GS115(pPIC3.5K-lac48424-1) was reached following a 7-days growth at 20 °C. The highest laccase activity was 104.45U L 1. However, no extracellular laccase activity was detected in culture supernatants of the negative control-GS115(pPIC3.5K). Native-PAGE was also performed using the culture supernatants of yeast transformants to test the recombinant laccase protein. Protein band with the expected laccase activity was significantly detected in the culture supernatant of GS115(pPIC3.5K-lac48424-1) by ABTS staining on the native PAGE gel, while no signal was detected in the culture supernatant of GS115(pPIC3.5K) (Fig. 3C). All of above results demonstrated that lac48424-1 gene could be successfully expressed in Pichia pastoris, the active laccase could be produced and secreted properly. The function of lac48424-1 gene encoding laccase was further verified by means of gene expression. A 3.4. Decolorization of dyes by the purified recombinant laccase produced by Pichia pastoris transformants in which lac48424-1 gene was successfully expressed B Laccase activity (U/L) GS115(pPIC3.5K) GS115(pPIC3.5Klac48424-1) The recombinant laccase, produced by Pichia pastoris transformants in which lac48424-1 gene was successfully expressed, was purified according to the method described in Section 2. The purified recombinant laccase which was designated as rLAC48424-1 appeared as a single protein band in SDS–PAGE (Fig. 4). The purified rLAC48424-1 had a specific laccase activity of 49.32 U mg 1. Then the purified rLAC48424-1 was analyzed for its ability to decolorize four dyes: methyl orange, malachite green, bromophenol blue and crystal violet. As shown in Fig. 5, all of the four dyes could be decolorized by purified rLAC48424-1. It suggested that the recombinant laccase produced by the transformant in which the la48424-1 gene was successfully expressed could confer the ability to decolorize different dyes. However, different efficiency of decolorization was observed among these dyes. The highest decolorization efficiency for 12 h of incubation was detected to Time (day) C Fig. 3. Detection of the expression of lac48424-1 gene in yeast transformants. A: The BMM agar plates containing CuSO4 and ABTS were used to screen the positive transformants which could produce active laccase. It showed that dark green zones were present around the colonies of GS115(pPIC3.5K-lac48424-1) after 3 days growth. B: The laccase-positive transformant GS115(pPIC3.5K-lac48424-1) as well as the negative control-GS115(pPIC3.5K) were fermented with BMM liquid medium at 20 °C, with 0.5% (v/v) methanol being added daily. Laccase activities in cultures were measured daily. C: Native-PAGE was also performed for detecting the laccase activity in the culture supernatants of yeast transformants. The amount of laccase applied onto lanes 2 and 4 was 0.01 U. Lane 1: protein marker, Lane 2: GS115(pPIC3.5K-lac48424-1), Lane 3: GS115(pPIC3.5K), Lane 4: GS115(pPIC3.5Klac48424-1). (For interpretation of the references in color in this figure legend, the reader is referred to the web version of this article.) Fig. 4. SDS–PAGE of purified recombinant laccase produced by Pichia pastoris transformants. Lane 1: protein marker, Lane 2: purified rLAC48424-1 stained with Coomassie Brilliant Blue. Laccase activity (U/l) F. Fan et al. / Bioresource Technology 102 (2011) 3126–3137 Dye decolorization (%) Kojic acid (mM) rLAC48424-1/malachite green rLAC48424-1/20 mM kojic acid/malachite green Time (hour) Dye decolorization (%) rLAC48424-1/methyl orange rLAC48424-1/20mM kojic acid /methyl orange Time (hour) Dye decolorization (%) rLAC48424-1/bromophenol blue rLAC48424-1/20 mM kojic acid/bromophenol blue Time (hour) Dye decolorization (%) rLAC48424-1/crystal violet rLAC48424-1/20 mM kojic acid/crystal violet Time (hour) Fig. 5. Decolorization of dyes by the purified recombinant laccase rLAC48424-1. The purified rLAC48424-1 was analyzed for its ability to decolorize four dyes: methyl orange, malachite green, bromophenol blue and crystal violet. The ability for decolorizing four dyes by rLAC48424-1 could be inhibited by adding kojic acid (20 mM) which decreased the laccase activity significantly. In the figure, the ability for decolorizing four dyes was shown as dye decolorization (%). rLAC48424-1/ 20 mM kojic acid/different dyes indicated that kojic acid was added at the final concentration of 20 mM. rLAC48424-1/different dyes indicated that no kojic acid was added. be 95.66% for malachite green, 78.34% for methyl orange, 90.28% for bromophenol blue and 68.36% for crystal violet (Fig. 5). Kojic acid was found to be a specific inhibitor of fungal laccase (Murao et al., 1992; Kim et al., 2008). Therefore, it was used to 3133 further detect whether the decolorization of dyes was dependent on laccase. Kojic acid was added into the reaction mixture at a final concentration of 20 mM, which inhibited the laccase activity significantly (Fig. 5). Then the decolorization of dyes was performed again. It was found that the ability for decolorizing four dyes by rLAC48424-1 was obviously inhibited by adding kojic acid. After addition of kojic acid (20 mM), the decolorization efficiency (12 h) of malachite green, methyl orange, crystal violet and bromophenol blue decreased to 44.5%, 55%, 41.8%, and 20.3% of the control without adding kojic acid, respectively (Fig. 5). This result further demonstrated that laccase was responsible for the decolorization of different dyes. We further compared the decolorization capability of the recombinant laccase rLAC48424-1 with that of other laccase reported previously. The purified laccase derived from Pleurotus ostreatus was used to decolorize malachite green (50 mg/l). The highest decolorization efficiency for 24 h of incubation was detected to be 70% for malachite green (Yan et al., 2009). It was found that purified laccase from the white-rot fungus Ganoderma lucidum was able to decolorize 40.7% malachite green (at 25 mg/l) after 24 h of incubation. The decolorization level after 24 h for the samples (containing 50 mg/l malachite green), which contained catechol, phenol, guiacol, and 2,4-dimethoxy phenol as the redox mediator was 89.7%, 84.7%, 79.0%, and 47.0%, respectively. If no mediator was added, very poor decolorization occurred up to 12 h, and further incubation up to 24 h yielded only 12% of malachite green decolorization upon treatment with purified laccase (Murugesan et al., 2009). In our research, we found that the recombinant laccase derived from Trametes sp. 48424 had the more powerful capability for decolorizing malachite green compared with some other known laccase (Yan et al., 2009; Murugesan et al., 2009). The highest decolorization efficiency for 12 h of incubation was detected to be 95.66% for malachite green (50 mg/l) even without adding any mediator. It was found that laccase from Trametes hirsuta could decolorize methyl orange. But compared with other dyes, methyl orange (65% of degradation in 24 h) was more resistant to degradation (Moldes et al., 2003). In our research, we found that the recombinant laccase derived from Trametes sp. 48424 could decolorize methyl orange more efficiently. When methyl orange containing solutions (50 mg/l) were treated with the purified laccase (0.02 U), enzymatic decolorization of methyl orange in 6 h was achieved to the level of 75.9% without any mediator. The highest decolorization efficiency for 12 h of incubation was detected to be 78.34% for methyl orange (Fig. 5). Tong et al. have studied the decolorization of bromophenol blue by the purified laccase LacE from Trametes sp. 420. The decolorization efficiency for bromophenol blue (50 mg/l) slowly increased to 45% within 8 h in the presence of a redox mediator, ABTS. If no mediator was added, the decolorization efficiency for bromophenol blue only reached 20% within 8 h (Tong et al., 2007). In our research, the recombinant laccase derived from Trametes sp. 48424 could decolorize bromophenol blue very rapidly. The decolorization efficiency for bromophenol blue (50 mg/l, without any mediator) reached 90% within 9 h. The highest decolorization efficiency for 12 h of incubation was detected to be 90.28% for bromophenol blue (Fig. 5). The previous research about the decolorization of crystal violet by the free laccase from Trametes versicolor have revealed that the degradation rate of crystal violet by free laccase was slow in the absence of the mediator. A high degradation efficiency of crystal violet by free laccase was observed in the presence of ABTS. The decolorization of crystal violet (at 10 mg/l) in 5 h was achieved to the level of 90% in the presence of ABTS (Dai et al., 2010). In our research, the purified recombinant laccase rLAC48424-1 was able to decolorize 68.36% crystal violet (at 20 mg/l) after 12 h of incubation in the absence of the mediator. If adding ABTS in the reaction mixture at the final concentration of 0.003 mM, the decolorization 3134 F. Fan et al. / Bioresource Technology 102 (2011) 3126–3137 Fig. 6. The 1718 bp 50 -flanking sequence upstream of the start codon ATG in la48424-1 gene. The putative cis-acting responsive elements in the promoter region were underlined and indicated by the following abbreviations. CreA: CreA-binding sites; MRE: metal-responsive elements; XRE: xenobiotic-responsive elements; ACE: ACE element; STRE: stress-responsive element; NIT2: consensus sequences for binding of NIT2 transcription factor; HSE: Heat shock element. CAAT box and TATA box were also underlined and bolded. The first nucleotide of the start codon ATG was designated as +1. efficiency for crystal violet after 12 h of incubation could increase to 82.45%. But the decolorization efficiency for crystal violet was still relatively lower compared with the decolorization efficiencies for the other three dyes: methyl orange, malachite green, and bromophenol blue. The diversity of the chemical structures of dyes might result in these differences in the decolorization efficiencies (Moldes et al., 2003). In summary, the decolorization capability of the recombinant laccase rLAC48424-1 derived from Trametes sp. 48424 were compared with that of some other laccase reported previously. The recombinant laccase from Trametes sp. 48424 possess stronger capacity for decolorizing different dyes such as malachite green, methyl orange, and bromophenol blue compared with some other known laccase (Yan et al., 2009; Murugesan et al., 2009; Moldes et al., 2003; Tong et al., 2007). Thus, rLAC48424-1 derived from Trametes sp. 48424 exhibits great potential and promising application in decolorizing and detoxifying industrial dyes. 3.5. Cloning and analysis of the 50 -flanking sequence upstream of the start codon ATG of lac48424-1 gene The 1996 bp complete structural gene of lac48424 was cloned and sequenced as described in Section 2. The lac48424-1 structural gene contained eight introns and nine exons. All of the splicing 3135 F. Fan et al. / Bioresource Technology 102 (2011) 3126–3137 2002; Colao et al., 2003; Xiao et al., 2006), some other cis-acting regulatory elements, that were not reported in the promoter of known laccase genes from Trametes, were present in the promoter region of lac48424-1 gene (Fig. 6). Firstly, two putative ironresponsive element (Kosman, 2003) located at position 81 and 220. Previous research have revealed that the laccase for melanin synthesis in the pathogen Cryptococcus neoformans could be regulated by iron (Jacobson and Compton, 1996). Some research have revealed that the expression of laccase gene from Trametes could be regulated by some metal ions, such as Cu2+, Ag+, Cd2+, Hg2+ (Galhaup et al., 2002). But there are few report that the expression of laccase gene from white-rot fungi such as Trametes could be regulated by iron. The presence of putative iron-responsive element in the promoter region implied that the expression of lac48424-1 gene in Trametes sp. 48424 may be regulated by iron. But it needs to be proved. Further work about the regulation of laccase gene expression by iron are ongoing. Secondly, three putative cAMPresponsive elements were detected at positions 382, 601, and 1429, respectively. Previous research have suggested that cAMP could induce the production of laccase from plant-pathogenic fungus Rhizoctonia solani (Crowe and Olsson, 2001). Boominathan and Reddy also have found that cAMP played a key role in the regulation of production of lignin peroxidase and manganese-dependent peroxidase in white-rot fungi Phanerochaete chrysosporium (Boominathan and Reddy, 1992). But as for Trametes, there are few report about the regulation of laccase gene expression by cAMP. The presence of putative cAMP-responsive elements in the promoter region suggested that the expression of lac48424-1 gene in Trametes sp. 48424 may be regulated by cAMP. Thirdly, three putative carbon source-responsive elements were found at positions 486, 735, and 1181, respectively. It suggested that the different carbon source may regulate the laccase gene expression in Trametes sp. 48424. Fourthly, two putative Skn7 response regulator element was detected at position 1646 and 369. The Skn7 response regulator could control the gene expression in the oxidative stress response of Saccharomyces cerevisiae (Morgan et al., 1997). The presence of putative Skn7 response regulator element in the promoter region implied that the expression of lac48424-1 gene in Trametes sp. 48424 could probably be regulated in response to oxidative stress through the transcription factor similar to Skn7. We further compared the category and number of cis-acting regulatory elements of lac48424-1 gene with that of other known laccase genes from Trametes: lap2 from Trametes pubescens (Galhaup et al., 2002), lacA, lacB, and lacC from Trametes sp. AH28-2 (Xiao et al., 2006), lcc1 from Trametes trogii (Colao et al., 2003) (Table 4). Although the cDNA sequence of lac48424-1 gene from Trametes sp. 48424 was similar to that of other laccase genes from Trametes (the percent identity of lac48424-1 cDNA with junctions and internal lariat formation sites of the introns adhered to the GT-AG rule of eukaryotic gene. The 1718 bp 50 -flanking sequence upstream of the start codon ATG in lac48424-1 gene was obtained by Self-Formed Adaptor PCR (SEFA PCR) and then analyzed for the presence of putative cis-acting elements involved in transcriptional regulation. The putative promoter region of lac48424-1 extending 1718 bp upstream of the start codon was shown in Fig. 6. The TATA box was located at nt positions 102 bp upstream from the start codon ATG. Three CAAT box were located at 212, 357, and 907 upstream from the start codon ATG, respectively. Some putative response elements were also found in the promoter region (shown in Fig. 6). Eight putative CreA-binding sites (SYGGRG) were located at positions 60, 392, 858, 919, 945, 1095, 1205, and 1593. CreA was a DNA-binding protein belonging to Cys2-His2 zinc finger class. It was identified as a repressor involved in the glucose repression in Aspergillus nidulans (Strauss et al., 1999). The presence of eight putative CreA-binding sites in the promoter region of the lac48424-1 implied that the expression of lac48424-1 gene may be repressed by glucose. Six putative metal-responsive elements (MREs) adhering to the consensus sequence TGCRCNC which conferred the ability to respond to heavy metal (Thiele, 1992) were present at positions 401, 930, 959, 1112, 1136, and 1536, respectively. Two putative xenobiotic-responsive elements (XREs) matching the consensus sequence CACGCW (Rushmore et al., 1990) were also detected at positions 278, and 1667. XREs were important cis-acting elements which could mediate the transcriptional activation of eukaryotic genes by aromatic compounds (Rushmore et al., 1990). Two potential stress-responsive elements (STREs) with the consensus sequence of CCCCT (Treger et al., 1998) were also detected at positions 145 and 1688. Six ACE elements adhering to the consensus sequence HWHNNGCTGD or NTNNHGCTGN were present at positions 409, 644, 828, 1124, 1130, and 1238, respectively. ACE element was the target sequence of the ACE1 copper-responsive transcription factor originally found in Saccharomyces cerevisiae. Recent research have found that expression of genes encoding laccase and manganesedependent peroxidase in the fungus Ceriporiopsis subvermispora was mediated by an ACE1-like copper-fist transcription factor (José et al., 2009). Two putative consensus sequences (TATCDH) for the binding of NIT2 transcription factor, which was the major positive regulatory protein involved in the nitrogen regulation of gene expression in fungi (Fu and Marzluf, 1990), were also present at positions 767 and 1286, respectively. Two potential heatshock elements (HSEs) composed of the repeated 5 bp NGAAN element in either orientation were present at positions 560, 1441, respectively. Besides above regulatory elements which have also been found in the promoters of other known laccase genes (Galhaup et al., Table 4 The category and number of putative cis-acting elements of lac48424-1 gene were compared with that of other known laccase genes from Trametes: lap2 from Trametes pubescens (Galhaup et al., 2002); lacA, lacB and lacC from Trametes sp. AH28-2 (Xiao et al., 2006); lcc1 from Trametes trogii (Colao et al., 2003). Promoter Iac48424-1 (Trametes sp. 48424) Iap2 (Trametes pubescens) lacA (Trametes sp. AH28-2) lacB (Trametes sp. AH28-2) lacC (Trametes sp. AH28-2) lcc1 (Trametes trogii) cis-Acting element CreA-binding sites MRE XRE STRE ACE element NIT2 HSE IRE CRE CSRE SKRE Number 8 4 9 1 2 NR 6 2 5 2 4 1 2 NR 7 2 2 NR 2 1 2 2 2 NR 6 NR NR NR NR 1 2 NR NR NR NR NR 2 27 NR NR NR NR 2 NR NR NR NR NR 3 NR NR NR NR NR 3 NR NR NR NR NR 2 NR NR NR NR NR MRE: metal-responsive elements; XRE: xenobiotic-responsive elements; ACE: ACE element; STRE: stress-responsive element; NIT2: consensus sequences for binding of NIT2 transcription factor; HSE: heat shock element; IRE: iron-responsive element; CRE: cAMP-responsive element; CSRE: carbon source-responsive element; SKRE: Skn7 response regulator element. NR: not reported. 3136 F. Fan et al. / Bioresource Technology 102 (2011) 3126–3137 lap2 from Trametes pubescens (AF414807), lcc1 from Trametes trogii(AJ294820), lacA, lacB, and lacC from Trametes sp. AH282(AY839936, AY846842, AY839937) were 92.1%, 74.8%, 83.4%, 72.7%, and 75.3%, respectively), the promoter region of lac484241 gene was quite different from that of other known laccase genes. lac48424-1 gene differed from other known laccase genes of Trametes mainly on the category and number of cis-acting regulatory elements in the promoter. The comparison result was summarized in Table 4. For example, the number of MRE and ACE element in the promoter of lac48424-1 gene were the biggest among the six laccase genes. Several other special cis-acting regulatory elements, which were not reported in the promoter of known laccase genes from Trametes (Galhaup et al., 2002; Colao et al., 2003; Xiao et al., 2006), were found in the promoter region of lac48424-1 gene. These regulatory elements included iron-responsive element, cAMP-responsive element, carbon source-responsive element and Skn7 response regulator element. What is the significance of these differences? What is the relationship between the cis-acting regulatory elements in the promoter of lac48424-1 gene and the high yield of laccase by Trametes sp. 48424? Further work is underway for solving these interesting problems in our laboratory. The difference of number and locus of cis-acting elements in the promoter region may result in the difference of regulation of gene expression. The special category and number of regulatory elements in the promoter of lac48424-1 may be closely related to the high production of laccase by Trametes sp. 48424 and may result in the special regulatory mechanism different from other known laccase genes. In summary, the presence of many putative response cis-acting elements in the promoter region of lac48424-1 suggested that the expression of lac48424-1 gene may be regulated by metal ions (such as copper, iron), xenobiotic compounds, glucose, nitrogen, cAMP and oxidative stress at the level of transcription. These response elements may be involved in the regulation of lac48424-1 gene transcription. But the precise molecular mechanism of regulating laccase gene expression through these putative cis-acting elements is still unknown and needs to be fully elucidated in the future work. 4. Conclusions In this study, the laccase gene lac48424-1 and its corresponding full-length cDNA were cloned and characterized from a novel white-rot fungi Trametes sp. 48424 isolated from China, which had the high yield of laccase and strong ability for decolorizing different dyes. The correct function of lac48424-1 gene encoding active laccase was confirmed by the successful expression of lac48424-1 in Pichia pastoris. The recombinant laccase produced by the yeast transformant was found to possess the ability to decolorize different dyes. Many putative cis-acting responsive elements involved in the transcriptional regulation of laccase gene were found in the promoter region of lac48424-1. Acknowledgements This work was supported by the Open Fund of Key Laboratory of Oil Crops Biology of Ministry of Agriculture in China (2010–2011), National Natural Science Foundation of China (Nos. 30800007 and 31070069), Doctoral Fund of the New Teacher Program of Ministry of Education of China (No. 200804871024), Research Fund of Independent Innovation of Huazhong University of Science and Technology (No. M2009046), Natural Sciences Foundation of Hubei Province (No. 2009CDB009), Major S&T Projects on the Cultivation of New Varieties of Genetically Modified Organisms (Grant 2009ZX08009-120B). References Baldrian, P., 2006. Laccases-occurrence and properties. FEMS Microbiol. Rev. 30, 215–242. Boominathan, K., Reddy, C.A., 1992. CAMP-mediated differential regulation of lignin peroxidase and manganese-dependent peroxidase production in the white-rot basidiomycete Phanerochaete chrysosporium. Proc. Nati. Acad. Sci. USA 89, 5586–5590. Bulter, T., Alcalde, M., Sieber, V., Meinhold, P., Schlachtbauer, C., Arnold, F.H., 2003. Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution. Appl. Environ. Microbiol. 69, 987–995. Collins, P.J., Dobson, A.D.W., 1997. Regulation of laccase gene transcription in Trametes versicolor. Appl. Environ. Microbiol. 63, 3444–3450. Colao, M.C., Garzillo, A.M., Buonocore, V., Schiesser, A., Ruzzi, M., 2003. Primary structure and transcription analysis of a laccase-encoding gene from the basidiomycete Trametes trogii. Appl. Microbiol. Biotechnol. 63, 153–158. Crowe, J., Olsson, S., 2001. Induction of laccase activity in Rhizoctonia solani by antagonistic Pseudomonas fluorescens strains and a range of chemical treatments. Appl. Environ. Microbiol. 67, 2088–2094. Dai, Y.R., Niu, J.F., Liu, J., Yin, L.F., Xu, J.J., 2010. In situ encapsulation of laccase in microfibers by emulsion electrospinning: preparation, characterization, and application. Bioresour. Technol. 101, 8942–8947. Faraco, V., Ercole, C., Festa, G., Giardina, P., Piscitelli, A., Sannia, G., 2008. Heterologous expression of heterodimeric laccases from Pleurotus ostreatus in Kluyveromyces lactis. Appl. Microbiol. Biotechnol. 77, 1329–1335. Fu, Y.H., Marzluf, G.A., 1990. Nit-2, the major positive-acting nitrogen regulatory gene of Neurospora crassa, encodes a sequence-specific DNA-binding protein. Proc. Natl. Acad. Sci. USA 87, 5331–5335. Galhaup, C., Haltrich, D., 2001. Enhanced formation of laccase activity by the whiterot fungus Trametes pubescens in the presence of copper. Appl. Microbiol. Biotechnol. 56, 225–232. Galhaup, C., Goller, S., Peterbauer, C.K., Strauss, J., Haltrich, D., 2002. Characterization of the major laccase isoenzyme from Trametes pubescens and regulation of its synthesis by metal ions. Microbiology 148, 2159–2169. Jacobson, E.S., Compton, G.M., 1996. Discordant regulation of phenoloxidase and capsular polysaccharide in Cryptococcus neoformans. J. Med. Vet. Mycol. 34, 289–291. José, M.Á., Paulo, C., Rodrigo, A.M., Rubén, P., Paulina, A.S., Rafael, V., 2009. Expression of genes encoding laccase and manganese-dependent peroxidase in the fungus Ceriporiopsis subvermispora is mediated by an ACE1-like copper-fist transcription factor. Fungal Genet. Biol. 46, 104–111. Kim, Y., Yeo, S., Song, H.G., Choi, H.T., 2008. Enhanced expression of laccase during the degradation of endocrine disrupting chemicals in Trametes versicolor. J. Microbiol. 46, 402–407. Kosman, D.J., 2003. Molecular mechanisms of iron uptake in fungi. Mol. Microbiol. 47, 1185–1197. Larrondo, L.F., Avila, M., Salas, L., Cullen, D., Vicuna, R., 2003. Heterologous expression of laccase cDNA from Ceriporiopsis subvermispora yields copperactivated apoprotein and complex isoform patterns. Microbiology 149, 1177– 1182. Liu, Y.G., Mitsukawa, N., Oosumi, T., Whittier, R.F., 1995. Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J. 8, 457–463. Lorenzo, M., Diego Moldes, M., Sanroman, A., 2006. Effect of heavy metals on the production of several laccase isoenzymes by Trametes versicolor and on their ability to decolourise dyes. Chemosphere 63, 912–917. Mayer, A.M., Staples, R.C., 2002. Laccase, new functions for an old enzyme. Phytochemistry 60, 551–565. Moldes, D., Gallego, P.P., Couto, S.R., Sanroman, A., 2003. Grape seeds: the best lignocellulosic waste to produce laccase by solid state cultures of Trametes hirsute. Biotechnol. Lett. 25, 491–495. Morgan, B.A., Banks, G.R., Toone, W.M., Raitt, D., Kuge, S., Johnston, L.H., 1997. The Skn7 response regulator controls gene expression in the oxidative stress response of the budding yeast Saccharomyces cerevisiae. EMBO J. 16, 1035– 1044. Murugesan, K., Yang, I.H., Kim, Y.M., Jeon, J.R., Chang, Y.S., 2009. Enhanced transformation of malachite green by laccase of Ganoderma lucidum in the presence of natural phenolic compounds. Appl. Microbiol. Biotechnol. 82, 341– 350. Murao, S., Hinode, Y., Matsumura, E., Numata, A., Kawai, K., Ohishi, H., Jin, H., Oyama, H., Shin, T., 1992. A novel laccase inhibitor, N-hydroxyglycine, produced by Penicillium citrinum YH-31. Biosci. Biotech. Biochem. 56, 987–988. Rushmore, T.H., King, R.G., Paulson, K.E., Pickett, C.B., 1990. Regulation of glutathione S-transferase Ya subunit gene expression, identification of a unique xenobiotic-responsive element controlling inducible expression by planar aromatic compounds. Proc. Natl. Acad. Sci. USA 87, 3826–3830. Soden, D.M., Dobson, A.D.W., 2001. Differential regulation of laccase gene expression in Pleurotus sajor-caju. Microbiology 147, 1755–1763. Soden, D.M., O’Callaghan, J., Dobson, A.D.W., 2002. Molecular cloning of a laccase isozyme gene from Pleurotus sajor-caju and expression in the heterologous Pichia pastoris host. Microbiology 148, 4003–4014. Strauss, J., Horvath, H.K., Abdallah, B.M., Kindermann, J., Mach, R.L., Kubicek, C.P., 1999. The function of CreA, the carbon catabolite repressor of Aspergillus nidulans, is regulated at the transcriptional and post-transcriptional level. Mol. Microbiol. 32, 169–178. F. Fan et al. / Bioresource Technology 102 (2011) 3126–3137 Terrón, M.C., González, T., Carbajo, J.M., Yagüe, S., Arana-Cuenca, A., Téllez, A., Dobson, A.D., González, A.E., 2004. Structural close-related aromatic compounds have different effects on laccase activity and on lcc gene expression in the ligninolytic fungus Trametes sp. I-62. Fungal Genet. Biol. 41, 954–962. Thiele, D.J., 1992. Metal regulated transcription in eukaryotes. Nucl. Acid. Res. 20, 1183–1191. Thurston, C.F., 1994. The structure and function of fungal laccases. Microbiology 140, 19–26. Treger, J.M., Magee, T.R., McEntee, K., 1998. Functional analysis of the stress response element and its role in the multistress response of Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 243, 13–19. 3137 Tong, P.G., Hong, Y.Z., Xiao, Y.Z., Zhang, M., Tu, X.M., Cui, T.G., 2007. High production of laccase by a new basidiomycete, Trametes sp. Biotechnol. Lett. 29, 295–301. Wang, S.M., He, J., Cui, Z.L., Li, S.P., 2007. Self-formed adaptor PCR, a simple and efficient method for chromosome walking. Appl. Environ. Microbiol. 73, 5048– 5051. Xiao, Y.Z., Hong, Y.Z., Li, J.F., Hang, J., Tong, P.G., Fang, W., Zhou, C.Z., 2006. Cloning of novel laccase isozyme genes from Trametes sp. AH28-2 and analyses of their differential expression. Appl. Microbiol. Biotechnol. 71, 493–501. Yan, K.L., Wang, H.X., Zhang, X.Y., Yu, H.B., 2009. Bioprocess of triphenylmethane dyes decolorization by Pleurotus ostreatus BP under solid-state cultivation. J. Microbiol. Biotechnol. 19, 1421–1430.