Purification and Characterization of a Novel Laccase from Cerrena sp. HYB07 with Dye Decolorizing Ability

Laccases (EC 1.10.3.2) are a class of multi-copper oxidases with important industrial values. A basidiomycete strain Cerrena sp. HYB07 with high laccase yield was identified. After cultivation in the shaking flask for 4 days, a maximal activity of 210.8 U mL−1 was attained. A 58.6-kDa laccase (LacA) with 7.2% carbohydrate and a specific activity of 1952.4 U mg−1 was purified. 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) was the optimal substrate, with K m and k cat being 93.4 µM and 2468.0 s−1, respectively. LacA was stable at 60°C, pH 5.0 and above, and in organic solvents. Metal ions Na+, K+, Ca2+, Mg2+, Mn2+, Zn2+ enhanced LacA activity, while Fe2+ and Li+ inhibited LacA activity. LacA decolorized structurally different dyes and a real textile effluent. Its gene and cDNA sequences were obtained. Putative cis-acting transcriptional response elements were identified in the promoter region. The high production yield and activity, robustness and dye decolorizing capacity make LacA and Cerrena sp. HYB07 potentially useful for industrial and environmental applications such as textile finishing and wastewater treatment.


Introduction
Dyestuffs are lost in industrial effluents, posing health and environmental threats [1,2]. Traditional processes cannot remove all dyes or are costly [3]. Microbial treatments suggested for degradation of azo and triphenylmethane dyes often result in mutagenic or carcinogenic products under anaerobic conditions [1,4]. In contrast, laccases may provide a green and efficient alternative for decolorizing dyes before discharge into sewage treatment systems or the environment. By oxidation, laccases catalyze dye transformation, leading to dye decolorization and often detoxification as well [3][4][5][6][7].
White-rot fungi are the most efficient laccase producers and most intensively studied. Proposed roles of fungal laccases include morphogenesis, plant pathogenesis, pigment production and lignin degradation [8].
Although laccases have been studied for decades, their applications are hampered by low production yields and reduced performance under industrial conditions. Work is under way to explore sources of laccases with easy availability, high catalytic efficiency, broad substrate specificity, tolerance to alkaline conditions, high temperatures and organic solvents, etc. [5,[9][10][11] Pleurotus ostreatus and Trametes versicolor are two model organisms in laccase research [12,13]. Other fungi, such as those from the genera Cerrena and Ganoderma, may still provide laccases with remarkable qualities [5,9].
To date, publications on laccase-manufacturing Cerrena species mainly dealt with their isolation from the environment, fermentation medium and condition optimization, purification and biochemical characterization of laccases and potential applications in bioremediation and biodegradation [9,[14][15][16][17][18][19][20]. In the present work, a novel Cerrena sp. strain HYB07 with strong laccase activity was identified. A laccase, designated as LacA, was purified from the fermentation broth of HYB07. The biochemical characteristics, kinetic properties and dye/effluent decolorizing potentials of LacA were investigated, and the gene and cDNA sequences of LacA were cloned. The research presented herein provides a novel laccase with high yield and specific activity, thermo-and pH-stability, wide substrate range and strong dye decolorizing ability, which are important for industrial processes such as biodegradation and bioremediation.

Organisms and media
The strain Cerrena sp. HYB07 [21] was kept in the culture collection of College of Biological Sciences and Technology, Fuzhou University and maintained through periodic transfers on potato dextrose agar (PDA) (Difco, Franklin Lakes, New Jersey, USA) at 4uC. For laccase fermentation, five mycelial plugs (1 cm diameter) were removed from the peripheral region of 3-d-old PDA plates and inoculated in potato dextrose broth (PDB) (Difco, Franklin Lakes, New Jersey, USA). After growing for 3 d at 28uC and 150 rpm, an aliquot was taken to inoculate PDB medium supplemented with 0.5% yeast extract (PDY) and 0.4 mM CuSO 4 .

Phylogenetic analysis
Phylogeny of the strain was identified by 18S rDNA sequencing. Genomic DNA was extracted with a DNA Quick Plant System (TIANGEN, Beijing, China), and universal primers NS1 and NS8 (Table 1) were used for amplification of 18S rDNA [22]. The PCR product was ligated with the pMD18-T vector (Takara, Dalian, China), and the ligation products were transformed into E. coli TOP10 competent cells (Life Technologies, Grand Island, New York, USA). Four individual clones were sequenced. The 18S rDNA sequence has been submitted to GenBank with the accession number KM233493. The phylogenetic analysis (with 1,000 bootstraps) was performed with MEGA version 5.0 (http:// www.megasoftware.net/) by the neighbor-joining method. Other fungal 18S rDNA sequences used in this study were from GenBank.

Enzyme activity assay
Laccase activity was assayed according to a previously published method [23] with slight modifications. The reaction contained 25 mM citrate-phosphate buffer (pH 3.0) and 0.5 mM ABTS (Sigma-Aldrich, St. Louis, Missouri, USA), and increase in OD at 420 nm (e = 36,000 M 21 cm 21 ) was monitored for 5 min. One unit of enzyme activity (U) was defined as the amount of laccase required to oxidize 1 mmol ABTS per min. All assays were carried out in triplicate.

Protein purification
After cultivation in PDY medium for 4 d, the fermentation broth was harvested by paper filtration and then centrifuged (10,000 g, 20 min). The precipitate formed using 40 to 60% (NH 4 ) 2 SO 4 was collected by centrifugation (20,000 g, 20 min), resuspended in buffer A (25 mM Tris-HCl buffer, pH 7.5), and centrifuged (12,000 g, 5 min). The supernatant was desalted with a HiPrep 26/10 desalting column (GE Healthcare, Buckinghamshire, UK) and applied at 5 mL min 21 to a HiTrap DEAE column

SDS-PAGE analysis
SDS-PAGE [25] was conducted for molecular weight determination. For zymography analysis, nonreducing loading buffer (containing SDS but not DTT or b-mercaptoethanol) was employed, and the sample was not boiled before loading. After electrophoresis the gel was incubated in 10 mL sodium phosphate buffer (pH 6.0) containing 1% (v/v) Triton X-100 for 15-30 min for SDS removal [26] and then immersed in 10 mL fresh sodium phosphate buffer supplemented with 1 mM guaiacol for direct visualization of laccase activity on the gel.

Ultraviolet-visible (UV-Vis) absorption spectrum
The UV-Vis absorption spectrum of the purified laccase was recorded between 280 and 800 nm in buffer A.

2.7
Effects of pH and temperature on the activity and stability of purified LacA The effect of pH was determined between pH 2.0 to 7.0 at 25uC. pH stability was studied by incubating the enzyme at pH 2.0-10.0 at 25uC for 105 h. Residual laccase activity was quantified with ABTS as substrate. 50 mM glycine-HCl buffer (pH 2.0), citrate-phosphate buffer (pH 2.5-6.5), sodium phosphate buffer (pH 7.0-8.0) and glycine-NaOH buffer (pH 9.0-10.0) were used.
For ascertaining optimum temperature, laccase activity was measured at optimum pH and temperatures from 25 to 80uC. Thermostability was analyzed by incubating the enzyme at different temperatures over a duration of 150 min, and residual activity was assayed with substrate ABTS at optimum pH and temperature. All experiments were performed in triplicate.

Effects of inhibitors and metal ions on the activity of purified LacA
Inhibitors including L -cysteine, DTT, EDTA, NaN 3 , SDS and kojic acid, and metal ions including Na + , K + , Li + , Cu 2+ , Ca 2+ , Mg 2+ , Mn 2+ , Zn 2+ , Fe 2+ in form of sulfate, Cr 2+ and Co 2+ in form of nitrate, and Pb 2+ in form of subacetate were examined. Individual inhibitor or metal ion was incorporated in the enzyme assay, and activity was determined at optimal temperature and pH. Enzyme activity in absence of inhibitors or metal ions was regarded as 100%.
2.9 Effects of various organic solvents on the activity and stability of LacA 5%, 10% or 25% of the individual solvents, namely methanol, ethanol, acetonitrile, dimethyl sulfoxide (DMSO) and N,Ndimethylformamide (DMF) was added to standard enzyme activity assay. For enzyme stability in presence of organic solvents, LacA was pre-incubated with 5%, 10% or 25% of each solvent at 25uC for 4 h, and residual enzyme activity was determined by using enzyme assay with ABTS as substrate.

Kinetic studies
Substrate specificity of LacA was determined in triplicate by using 1-1500 mM ABTS, and 25-2500 mM guaiacol, 2,6dimethoxyphenol (2,6-DMP) and catechol at the respective optimum temperature and pH. Oxidation of the four substrates were measured for 5 min [27,28]. The kinetic parameters were estimated based on nonlinear regression of the Michaelis-Menten equation using GraphPad Prism version 5.0 (GraphPad Software, San Diego, California, USA).

Decolorization
Dye decolorizing ability was evaluated using 25 dyes. The reaction mixture in 20 mL was composed of (in final concentration): 0.1 M citrate-phosphate buffer (pH 6.0), 0.2 or 2 U mL 21 laccase, 0.1 mM ACE or 12.5 mM kojic acid (if needed) and dyes. Decolorization was carried out in the dark at 28uC and 150 rpm for 24 h. The negative control had no enzyme. Decolorization was followed at the wavelength for each dye, determined from its absorbance spectrum in 0.1 M citrate-phosphate buffer between    [29] were used to amplify laccase gene fragments from cDNA with high-fidelity DNA polymerase (Thermo Scientific, Waltham, Massachusetts, USA). PCR products were purified and inserted into pMD18-T vector. Recombinant vectors were transformed into E. coli TOP10 competent cells. Twenty clones were randomly selected and submitted to sequencing analysis (Life Technologies, Grand Island, New York, USA). All 20 clones corresponded to the same laccase gene, confirmed to be the gene encoding LacA by MALDI-TOF MS/ MS analysis. The 59-and 39-flanking genetic sequences of the amplified laccase fragment were obtained by thermal asymmetric interlaced PCR (TAIL-PCR) [30] with HYB07 genomic DNA as the template, LacA SP1-6 as gene-specific primers and AD1-7 as arbitrary degenerate primers (Table 1) [30]. The full-length cDNA sequence was amplified with primers LacA-RT-1 and LacA-RT-2 ( Table 1). The sequence has been submitted to GenBank with the accession number KF317949. SiteFinding PCR [31] was performed to further extend the promoter region of LacA, in which the gene-specific primers were LacA GSP1 and GSP2 (Table 1).

Species identification
A fragment of the 18S rDNA gene of the target fungal strain HYB07 was amplified by PCR and sequenced, and HYB07 was classified based on its 18S rDNA sequence. A phylogeny tree ( Fig. 1) was constructed, indicating HYB07 was closest to Cerrena unicolor (GenBank accession No. AY850007), sharing 99% sequence identity. The strain was named Cerrena sp. HYB07.

Production and purification of LacA
Laccase production by Cerrena sp. HYB07 in PDY liquid medium was monitored for 6 d in the shaking flask. With Cu 2+ as inducer, maximum laccase activity (210.8 U mL 21 ) was reached on day 4. Specific activity (806.3 U mg 21 ) peaked on day 3 ( Fig. 2A).
After (NH 4 ) 2 SO 4 precipitation, anion exchange and hydrophobic interaction chromatography, a monomeric laccase, designated as LacA, was purified 3.1-fold with 39.8% yield from the fermentation broth. The specific activity was 1952.4 U mg 21 ( Table 2). Deglycosylation with peptide N-glycosidase F indicated N-glycosylation. The molecular weight before and after deglycosylation was 58.6 and 54.5 kDa, respectively, thus its glycosylation level was 7.2%. The molecular weight of ,70 kDa on zymograph exceeded 58.6 kDa in SDS-PAGE (Fig. 2B).
The laccase solution was blue. UV-Vis absorption spectrum (Fig. 2C) showed a peak at 610 nm and a shoulder at 330 nm. The A280:A610 ratio was 20.6.

Effects of temperature and pH
The pH optimum was 3.0 for ABTS and 2,6-DMP, 4.0 for guaiacol and 4.5 for catechol (Fig. 3A). After storage at pH 4.0 for 105 h, 56% residual enzyme activity was retained. At pH 5.0 or higher, .80% activity remained. In contrast, LacA activity decreased to only approximately 1% and 20% at the end of 105-h incubation at pH 2.0 and 3.0, with t 1/2 being 18 h and 43 h, respectively (Fig. 3B).
Optimal temperature was 45uC with ABTS, 50uC with 2,6-DMP and 45-50uC with guaiacol and catechol. The enzyme displayed .50% of maximal activity between 25 and 65uC against the four substrates except that at 65uC for ABTS, the relative activity of LacA was only 45% (Fig. 3C). After incubation for 150 min at 50 and 60uC, approximately 85% and 56% of the original enzyme activity was retained, respectively. LacA was inactivated after 20 min at 70uC and 2 min at 80uC (Fig. 3D).

Effects of inhibitors and metal ions
Relative laccase activity was dramatically reduced in the presence of L -cysteine (to 5.5%), DTT (to 5.9%), sodium azide (to 9.2%) and SDS (to 11.4%) at the concentration of 0.1 mM. The laccase-specific inhibitor kojic acid at 12.5 mM reduced LacA activity by 50%. No significant activity loss was observed with 10 mM EDTA (Table 3).

Effects of various organic solvents
When individual water-miscible organic solvent was added at 5 and 10% final concentration, 102.5-72.7% activity was retained. Activity was compromised in all solvents at 25%; .70% activity remained with DMF and ethanol, approximately 60% activity remained with other three organic solvents (Table 4).
LacA was generally stable in the presence of the organic solvents. Residual activity was determined after incubation with each solvent at 5, 10 or 25% for 4 h at 25uC. An exception was exposure to 25% acetonitrile which was detrimental to activity. LacA was more stable in methanol and DMSO than in aqueous solution.

Kinetic properties of purified LacA
Initial rate kinetic analyses were performed with ABTS, guaiacol, 2,6-DMP and catechol at the respective optimal temperature and pH values ( Table 5). The lowest K m value was found for ABTS (93.4 mM), so were the highest turnover rate (k cat ) and catalytic efficiency (k cat /K m ) values (2468.0 s 21 and 26.4 mM 21 s 21 , respectively). Catalytic efficiency on ABTS was 7.5 times of that on 2,6-DMP, 22 times of that on catechol and 65 times higher than that on guaiacol. Catechol had the highest Km value (528.8 mM), followed by guaiacol (299.8 mM) and 2,6-DMP (200.4 mM).

Dye and real textile effluent decolorization by LacA
Decolorizing capability of purified LacA on industrial and laboratory dyes was evaluated at a low enzyme activity of 0.2 U mL 21 (Table 6). Without the help of small redox mediators, LacA was able to effectively decolorize dyes of different classes, some of which were notoriously recalcitrant to biodegradation, e.g., the indigo dye carmine, triphenylmethane dye malachite green, anthraquinone dye Remazol Brilliant Blue R (RBBR), and azo dyes acid violet 7 and orange II. Among the 25 dyes studies, 13 could be oxidized by 0.2 U mL 21 LacA with efficiencies higher than 50%. It was worth pointing out that although 24 h was arbitrarily selected for decolorization measurements, decolorization of some dyes proceeded faster than others. For example, decolorization of RBBR, indigo carmine and Evans blue was completed within 30 min, but took approximately 2 hours for malachite green and brilliant green (data not shown). A higher enzyme activity of 2.0 U mL 21 helped improve decotablorization efficiencies to different extents. For instance, the decolorization rate increased from 28.8% to 92.4% for Coomassie Brilliant Blue R-250, whereas for Congo red, only a modest improvement (from 33.0% to 57.2%) was seen. On the contrary, for four dyes, namely basic fuchsin, crystal violet, methylene blue and Rhodamine B, there were no significant changes. Therefore, LacA alone at 2.0 U mL 21 could decolorize 19 dyes with efficiencies higher than 50%. For the nine dyes with decolorization efficiencies lower than 60%, we further evaluated their removal by LacA in the presence of 0.1 mM ACE, a natural laccase mediator. Seven of the nine dyes were efficiently decolorized by the LacA-ACE system except for Rhodamine B and methylene blue. For dyes such as methyl orange and neutral red which could already be partially decolorized by LacA, their decolorization rates were enhanced by ACE (from 53.2% and 28.0% to 92.3% and 80.1%, respectively). In the cases of crystal violet and basic fuchsin, however, ACE was required for their decolorization, since neither was decolorized by LacA alone at 2.0 U mL 21 . Even for dyes with over 80% decolorization efficiencies, inclusion of the laccasespecific inhibitor kojic acid in reactions decreased decolorization efficiencies to 4.2-28.4%, suggesting that LacA was responsible for dye decolorization.
Decolorization of the real textile effluent by LacA predominantly occurred within the first 3 d, accompanied by flattening of the absorption spectrum (Fig. 4A). A negative control was conducted in parallel, and there was little decrease in the absorbance in the absence of LacA (Fig. 4B).

LacA gene cloning and analysis
A fragment of LacA between its first and fourth copper-binding motifs was amplified from Cerrena sp. HYB07 cDNA. TAIL-PCR was adopted to clone the 59 and 39-flanking genetic sequences of the fragment, based on which the full-length gene sequence of LacA was obtained. The 3,685-bp DNA sequence consisted of an 892 bp 59-uncoding region, a 2168-bp gene sequence and a 625bp 39-uncoding region. The full-length cDNA sequence (1,551 bp) was subsequently obtained. The LacA cDNA corresponded to purified LacA, as confirmed by MALDI-TOF MS/MS analysis (Fig. S1). The coding region was interrupted by 11 introns between 50-65 bp, and all splicing junctions adhered to the GT-AG rule.
LacA consisted of 516 amino acids, with the first 21 residues being the signal peptide, and 3 putative N-glycosylation sites at positions 453, 489 and 495. The molecular weight of mature LacA was predicted to be 52.9 kDa, close to the observed 54.5 kDa. Calculated pI was 5.6.
SiteFinding PCR was adopted to further extend the 59-flanking region of LacA, rendering a region of 1,544 bp upstream of the start codon, referred to as the LacA promoter. Bioinformatics analysis revealed multiple putative cis-acting transcription regulation sites within the LacA promoter sequence in both orientations (Fig. 6). A TATA box was located 92 bp upstream from the start codon ATG, and three CCAAT boxes were found at positions 2324, 2405 and 21134. The LacA promoter contained two metal response elements (MREs) with the consensus sequence TGCRCNC at positions 21013 and 21227 and one xenobiotic response element (XRE) with the core sequence TNGCGTG [38] at 21223. Apart from MREs and XREs, LacA had many ACE1 copper-responsive transcription factor binding sites, consisting of the HWHNNGCTGD or NTNNHGCTGN core [39], at positions 211, 2456, 2774 and 21336, respectively. In addition, one antioxidant response element (ARE) adhering to the consensus sequence TGACNNNGC [40] was present at 21035 in the LacA promoter. Putative response elements (PREs) [38], often found in basidiomycete laccase promoter sequences, were also present within the 59-flanking sequence of LacA: a TGGGT was located at position 21360, an inverted one at 21377, and two ATATC at 2122 and 2795. Furthermore, two heat shock response elements (HSEs) composed of alternately oriented NGAAN repeats [41] were found at 2422 and 2857 in the LacA promoter. LacA also contained multiple transcription factor binding sites involved in nitrogen and carbon regulation. Two putative CreA-binding sites (SYGGRG) were found at 2648 and 2706, and five NIT2 binding sites adhering to sequence TATCDH [5] were scattered at 2120, 2525, 2793, 21139 and 21286. No stress response elements (STREs) with the consensus sequence of CCCCT or Sp-1 transcription factor recognition site (GGGCGG) [39] were identified in the LacA promoter sequence.

Discussion
Although laccases have industrial and environmental health implications, commercial applications are limited by low enzyme yields, high costs and tolerance to extreme conditions. Herein, a new Cerrena sp. strain HYB07 produced over 200 U mL 21 laccase activity after cultivation for only 3 d with Cu 2+ as the inducer. The high laccase yield and short production period of HYB07 would be advantageous for application and commercialization, since many fungal laccase producers require longer production periods [14]. For C. unicolor VKMF-3196, 15 U mL 21 was obtained on the 8 th day of cultivation [20]. Enhanced laccase production by Cerrena unicolor strain MTCC 5159 was observed with inducers, effluents and synthetic dyes, and a textile effluent was the most effective inducer, resulting in 85.8 U mL 21 after 12 d [18]. For Cerrena sp. WR1, maximal production was 202 U mL 21 after 13-d cultivation under Cu 2+ and 2,5-xylidine induction [9]. For Cerrena unicolor C-139 [15,16], a production of 250.0 U mL 21 was reached after submerged culture for 7 d, and activity culminated at 450 U mL 21 after 14 d. We aim at optimizing the fermentation medium and conditions of HYB07, and even higher laccase yields are anticipated. Apart from easy production, the novel laccase LacA purified from the fermentation broth of HYB07 showed low substrate specificity and strong decolorizing ability. LacA is a blue multicopper oxidase; the absorption peak at 610 nm and shoulder around 330 nm indicated the presence of type I (responsible for the blue color) and type III copper ions. The A280/A610 ratio of 20.6 is similar to other laccases [42].
Like other laccase promoters [5,39,41,46], putative regulatory elements found in the promoter sequence suggest regulation of LacA transcription by metal ions, aromatic compounds, phenolic antioxidants, etc. Nitrogen and carbon may play a role in LacA expression, as inferred from CreA and NIT2 binding sites in the promoter region. LacA expression may be repressed by glucose since CreA is a major regulator in carbon catabolite repression and NIT2 activates gene expression when nitrogen is limiting [39,41]. LacA promoter is reminiscent of reported laccases whose transcription is regulated by metal ions, aromatic compounds related to lignin or lignin derivatives and nutrient nitrogen and carbon [18,39,41] and forms the basis for elucidating the functional correlation between possible regulatory elements and LacA expression.
LacA had a broad substrate range, as manifested by the kinetic and decolorization studies. Among the four compounds used for kinetic experiments, ABTS was the preferred substrate for LacA, and LacA had the lowest affinity for catechol. The specific activity of LacA against ABTS was 1.9-13.9 times as high as the specific activity of laccases from Cerrena unicolor (CFC-120) [17], Cerrena unicolor strain 137 [14] and Cerrena sp. WR1 [9]. In addition, compared with an alkali-resistant and metal-tolerant laccase Tplac from Trametes pubescens [11], the specific activity of LacA was over 100-fold higher, along with a higher affinity and catalytic efficiency towards ABTS. Despite the common use of ABTS in laccase assays, LacA also reacted with guaiacol and 2,6-DMP, two lignin building blocks. Though the catalytic efficiency of LacA on ABTS was only 1/10 of Lcc3 from Cerrena sp. WR1 (which has a small K m value), its catalytic efficiencies on guaiacol, 2,6-DMP and catechol were approximately 2.4, 12.5 and 9.2-fold of the corresponding efficiencies of Lcc3 [9]. Furthermore, the catalytic efficiency of LacA on 2,6-DMP was also higher than those of both laccases from Cerrena unicolor strain 137 [14]. The speculated high redox potential and low substrate specificity of LacA can be beneficial for its practical application, which was corroborated by LacA-catalyzed breakdown of structurally different dyes. Since dyes in the same class may not respond equally to the same laccase [47], it may be necessary to test the decolorizing efficiencies of individual dyes of interest with specific laccases. This was also true for LacA. LacA alone decolorized malachite green with .90% efficiency, whereas LacA degraded crystal violet only in presence of a laccase mediator. Besides degrading a variety of dyestuffs, LacA also decolorized a real textile effluent. Hence, LacA holds great promise for applications in biodegradation and bioremediation, especially treatment of dye effluents.
The activity of LacA over 25-65uC helps reducing application costs. LacA displayed strong decolorizing ability at 28uC. LacA was more thermostable than alkali-resistant and metal-tolerant Trametes pubescens Tplac [11] whose unusual instability at 10/ 20uC causes handling and storage problems. LacA was also more robust at 60uC than Cerrena sp. WR1 Lcc3 [9], Shiraia sp. SUPER-H168 laccase [10] and Streptomyces sviceus Ssl1 [27], whose t 1/2 at 60uC are 40, 120 and 88 min, respectively. LacA was more stable at pH 4.0 than Cerrena sp. WR1 Lcc3 [9] and Trametes pubescens Tplac [11] and at various pH values than laccases from Cerrena unicolor C-139 [16] and MTCC5159 [19]. Furthermore, LacA was highly tolerant of organic solvents, a quality valuable for industrial processes such as organic synthesis. The high production yield, robustness, broad substrate specificity, and remarkable dye decolorizing capacity make LacA and fermentation broth of Cerrena sp. HYB07 an attractive and affordable candidate for applications in a diversity of industries including biodegradation and bioremediation, textile, paper and pulp.

Conclusion
A new Cerrena sp. strain HYB07 was reported, which produced over 200 U mL 21 laccase activity after 3-d cultivation in the shaking flask. The high laccase yield and short production period of HYB07 are advantageous for application and commercialization. A major laccase, namely LacA, was purified from the fermentation broth of HYB07 and showed sequence homology to other Cerrena laccases. The promoter sequence of LacA contained many putative regulatory elements. Biochemical characterization revealed that LacA had high specific activity, low substrate specificity, strong decolorizing ability, thermo-and pH-stability, and tolerance of organic solvents. Further work to explore LacA's application potentials in various industrial processes, such as biodegradation, dye effluent decolorization and detoxification, textile finishing and organic synthesis, is warranted.