International Journal of Biological Macromolecules 77 (2015) 344–349
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
A novel laccase from basidiomycete Cerrena sp.: Cloning, heterologous expression, and characterization Jie Yang a,b , Tzi Bun Ng c , Juan Lin a,b , Xiuyun Ye a,b,∗ a b c
College of Biological Sciences and Technology, Fuzhou University, Fuzhou, Fujian 350116, China Fujian Key Laboratory of Marine Enzyme Engineering, Fuzhou, Fujian 350116, China School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
a r t i c l e
i n f o
Article history: Received 23 January 2015 Received in revised form 19 March 2015 Accepted 20 March 2015 Available online 28 March 2015 Keywords: Cerrena sp. Laccase P. pastoris Decolorization
a b s t r a c t A novel laccase gene Lac1 and its cDNA were cloned from a white-rot fungus Cerrena sp. and characterized. The 1554-bp cDNA of Lac1 encoded a mature protein with 497 amino acids, preceded by a signal peptide of 20 amino acids. An unconventional intron splice site and incomplete splicing variants of Lac1 were observed. Lac1 was heterologously expressed in the yeast host Pichia pastoris, and a maximal laccase activity of 6.3 U mL−1 in the fermentation broth was achieved after fermentation for 9 days. The recombinant protein rLac1 was purified, and its enzymatic properties and functional characteristics were investigated. When ABTS was used as the substrate, the enzyme was most active at pH 3.5 and 55 ◦ C, and stable at pH 4–10 and 20–60 ◦ C. The Km and kcat values of rLac1 toward ABTS were 28.9 M and 332.4 s−1 , respectively. Furthermore, rLac1 was tolerant to common metal ions up to 100 mM concentration and capable of decolorizing structurally different dyes in the absence of a redox mediator. Hence, Lac1 may be useful for industrial applications, such as dye decolorization and bioremediation. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Laccases (EC 1.10.3.2) compose a family of copper-containing oxidases widely present in plants, fungi, insects, and bacteria. These enzymes can catalyze oxidation of a wide variety of phenolic and nonphenolic lignin-related compounds, accompanied by the reduction of oxygen to water. Being energy-saving and environmentally friendly, laccases have application values in various industrial sectors, such as wine and juice stabilization, biosensor development, dye decolorization, and bioremediation. Moreover, laccases hold tremendous potential in biofuel production to delignify biomass and remove phenolic inhibitors for subsequent hydrolysis of polysaccharides [1,2]. However, due to the high redox potential of nonphenolic units and the size of the lignin polymer, efficient lignin breakdown necessitates the concerted action of laccase and low-molecular-weight redox mediators [2]. Commonly used laccase mediators include 2,2 -azino-bis (3-ethylbenzothiazoline6-sulfonate) (ABTS), and 1-hydroxybenzotriazole (HBT).
∗ Corresponding author at: Fuzhou University, College of Biological Sciences and Technology, Fuzhou, Fujian 350116, China. Tel.: +86 591 22866376; fax: +86 591 22866376. E-mail address: [email protected] (X. Ye). http://dx.doi.org/10.1016/j.ijbiomac.2015.03.028 0141-8130/© 2015 Elsevier B.V. All rights reserved.
White-rot fungi have been extensively studied as they are the most efficient laccase producers [1]. Laccase genes have been cloned from Fome lignosus [3], Pleurotus ostreatus [4], Pycnoporus cinnabarinus [5], and various Trametes strains [6–8]. Although normally exhibiting low-to-moderate sequence identities, fungal laccases typically contain four copper-binding regions and highly conserved copper ligands [9]. Laccases often exist in gene families, and laccase isoenzymes in the same organism display diverse expression patterns and physicochemical characteristics [10]. In order to facilitate industrial laccase applications, it is necessary to discover and evaluate a large number of laccases. However, much published work has been focused on only one or a few predominant laccases secreted under laboratory culture conditions. Elucidation of the properties and function of individual laccase isozymes is often hindered by their low expression levels or mixed secretion [11,12]. Heterologous expression of individual laccase genes, on the other hand, has become a valuable approach to unravel the biochemical nature and structure-function relationships of these industrially important enzymes. Heterologously expressed laccases can also be engineered for enhancement of performance (e.g., improved activity and stability, and altered catalytic properties) with directed evolution or site-directed mutagenesis [13]. Yeasts including Pichia pastoris [3,7,11,14–16], and Saccharomyces cerivisiae [4,17], filamentous fungi such as Trichoderma reesei [18], and Aspergillus niger
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[19], as well as other hosts have been used to heterologously express laccase genes. In the present work, we report genetic and enzymatic characterization of a novel laccase, designated as Lac1, from a Cerrena sp. Several laccases have been purified and characterized from Cerrena [20–27], but molecular cloning and heterologous expression of Cerrena laccase genes have seldom been reported. This report would further our understanding of Cerrena laccases and facilitate their applications in biotechnology.
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2. Material and methods
Expression Kit User Manual. Putative transformants were screened on MM plates containing 0.2 mM ABTS, and positive transformants (X33/pPICZ␣-Lac1) were inoculated in YPD medium at 30 ◦ C with shaking at 200 rpm. After 24 h, the cells were harvested by centrifugation and resuspended to an OD600 of 2.0 with YP medium (pH 6.0) supplemented with 1 mM Cu2+ ions. Methanol at 1% (v/v) was added every 24 h, and secreted laccase activity was monitored in the supernatant after 1 mL culture had been centrifuged at 3000 g for 5 min. The strain transformed with the original pPICZ␣ vector (X33/pPICZ␣) was prepared as the negative control and analyzed in parallel.
2.1. Organisms and media
2.5. Purification of rLac1
The white-rot fungus Cerrena sp. HYB07 [28] was preserved in the culture collection of the College of Biological Sciences and Technology at Fuzhou University, Fuzhou, China and maintained on potato dextrose agar (PDA) medium. Fermentation of Cerrena sp. was carried out in 50 mL medium in 250 mL Erlenmeyer flasks at 30 ◦ C with shaking at 200 rpm. The fermentation medium contained (per liter): 60 g dextrin, 10 g peptone, 1.6 g ammonium tartrate, 6 g KH2 PO4 , 4.14 g MgSO4 ·7H2 O, 0.3 g CaCl2 , 0.18 g NaCl, 0.0625 g CuSO4 ·5H2 O, 0.018 g ZnSO4 ·7H2 O, and 0.015 g vitamin B1. P. pastoris host strain X33 was purchased from Invitrogen (USA). YP, YPD and MM media were prepared according to the instructions in the Pichia Expression Kit User Manual. E. coli DH5␣ was used for cloning.
rLac1 was precipitated by adding 70% NH4 SO4 to the fermentation broth and then resuspended in 50 mM Tris-HCl buffer (pH 8.5). After desalting by using a HiPrep Desalting column (GE Healthcare), the sample was applied to a 5 mL HiTrap DEAE FF column (GE Healthcare) pre-equilibrated with 50 mM Tris-HCl buffer (pH 8.5). The column was eluted with a linear gradient of 0–1 M NaCl in 50 mM Tris-HCl buffer (pH 8.5). Elution was monitored at 280 nm for detection of protein. The active fractions were pooled and stored at 4 ◦ C. The purity of the enzyme was checked by SDS-PAGE. Protein concentration was determined by using the Bradford assay with bovine serum albumin as the standard.
2.2. Nucleic acid manipulation Mycelia were harvested by pressure filtration. Genomic DNA was isolated from mycelia with the DNAquick Plant System (TIANGEN, China) according to the manufacturer’s instructions. Total RNA was extracted with Trizol (Life Technologies, USA). Contaminating genomic DNA was removed by using RNase-free DNase (Promega, USA), and the RevertAid First Strand cDNA Synthesis Kit (Fermentas, Canada) was used to synthesize first strands of cDNA. 2.3. Cloning of the Lac1 gene and cDNA The degenerate primers L-1F and L-4R (Table S1) were designed according to the conserved amino acid sequences of the first and fourth copper-binding motifs in fungal laccases, respectively [29]. A 1.8 kb PCR product was obtained from degenerate PCR with the genomic DNA of HYB07 as the template and inserted into pMD18-T vector (TAKARA, Japan). The fragment was sequenced and confirmed to be part of a novel laccase gene, which was named Lac1. Inverse PCR (iPCR) was used to amplify the 5 and 3 -flanking sequences of the 1.8 kb Lac1 fragment [14] with primers Lac11 and Lac1-2 (Table S1). The resulting product was purified and sequenced directly. The Lac1 cDNA was amplified with pfu polymerase (Fermentas, Canada) and the primer pair Lac1-RT-1 and Lac1-RT-2 (Table S1). The Lac1 gene has been deposited in GenBank with the accession number KC540913.
2.6. Enzyme assay Laccase activity was assayed with ABTS as the substrate by following changes in the absorbance at 420 nm for 5 min. One unit of enzyme activity (U) was defined as the amount of laccase required to oxidize 1 mol ABTS in 1 min. All assays were carried out in triplicate. 2.7. Effects of pH and temperature on rLac1 activity and stability To determine the optimum pH, laccase activity was measured in citric acid buffers between pH 3.0 and 6.0. The relative enzyme activity was calculated with the highest activity as 100%. pH stability was studied by incubating the purified enzyme at different pH values in 0.1 M citric acid–Na2 HPO4 buffers (pH 3.0–8.0), or 0.1 M Na2 HPO4 –NaOH buffers (pH 9.0–10.0) at room temperature for 24 h, and the residual laccase activity was quantified at the optimum pH and temperature. The activity of the untreated enzyme was taken as 100%. For the optimum temperature, laccase activity was measured at the optimum pH and various temperatures ranging from 20 to 65 ◦ C. Thermostability was analyzed by incubating the enzyme at various temperatures (20–70 ◦ C) for 2 h, and the residual enzyme activity was assayed at the optimum pH and temperature. All experiments were repeated three times with three replicates each time. 2.8. Effects of metal ions, inhibitors and surfactants on rLac1 activity
2.4. Heterologous expression of the Lac1 gene in P. pastoris Lac1 coding sequence, excluding the predicted signal sequence, was amplified with primers Lac1-Ex-F and Lac1-Ex-R (Table S1). The fragment was digested with XhoI and XbaI, and inserted into the yeast expression vector pPICZ␣ C to construct pPICZ␣-Lac1, which was sequenced to verify the correctness of the open reading frame. The recombinant pPICZ␣-Lac1 plasmid was linearized with BstxI and then electroporated into competent P. pastoris X33 cells with a Gene Pulser II apparatus (BioRad, USA) as described in the Pichia
Metal ions, including Na+ (Na2 SO4 ), K+ (K2 SO4 ), Ca2+ (CaSO4 ), Cu2+ (CuSO4 ), Mg2+ (MgSO4 ), Mn2+ (MnSO4 ), and Zn2+ (ZnSO4 ) ions, were used at 10 mM and 100 mM , respectively. Effects of inhibitors (L -cysteine, DTT, EDTA, NaN3 and kojic acid), and surfactants (SDS and Triton X-100) on rLac1 activity were also tested. Individual effector was incorporated in the standard enzyme activity assay, and the activity was determined at the optimal temperature and pH. The enzyme activity in the absence of an effector was set to 100%.
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2.9. Kinetic studies Substrate specificity of rLac1 for ABTS was determined at the optimum pH and temperature. The substrate concentration range used was 1–1500 M for ABTS. The kinetics parameters were estimated based on nonlinear regression of the Michaelis–Menten equation using GraphPad Prism version 5.0 (GraphPad Software, Inc., USA). 2.10. Dye decolorization by rLac1 Dye decolorization by rLac1 was carried out in the dark at 28 ◦ C for 24 h. The reaction mixture (2 mL) contained citric acid–Na2 HPO4 buffer (pH 6.0), rLac1 (0.4 U mL−1 ) and dye. When needed, kojic acid was included in the reaction mixture at the final concentration of 12.5 mM. For malachite green decolorization, metal ions were individually supplemented in the reaction at the final concentration of 10 mM. All reactions were carried out in triplicate. 3. Results 3.1. Structure of the Lac1 gene The Lac1-coding region was interrupted by 11 introns ranging from 59 to 120 bp in size. Although most splicing junctions in Lac1 adhered to the GT-AG rule, there was one exception (GT-TG) at the last intron. Furthermore, incomplete processing of introns was observed, resulting in transcripts of different lengths. For example, one splice variant had intron 1 remaining, and another one contained intron 6. In both cases, failure to remove all introns led to frame shifts that gave rise to premature stop codons. The cDNA of Lac1 was 1554 bp in length, encoding a polypeptide of 517 aa, with the first 20 aa predicted to be the signal peptide. The sequence has been submitted to GenBank with the accession number KC540913. The molecular weight of the mature Lac1 protein was predicted to be 54.14 kD. The deduced Lac1 protein was closest in amino acid sequence to laccase 1 from Spongipellis sp. FERM P-18171 (BAE79811) and LacA from Cerrena sp. HYB07 (AID59415) with 76% identity, followed by Cerrena sp. WR1 laccase (ACZ58369), and Panus rudis laccase A (AAW28932) with 74%, and 70% identities, respectively. 3.2. Heterologous expression of the Lac1 gene in P. pastoris The Lac1 gene was successfully expressed in P. pastoris and secreted into the culture medium. Laccase activity was only observed with X33/pPICZ␣-Lac1 transformants, but not X33/pPICZ␣ transformants (Fig. 1A). A maximum yield of 6.3 U mL−1 laccase activity was obtained after fermenting in the shake flask at 30 ◦ C for 9 days in YP medium supplemented with 1 mM CuSO4 and 1% methanol (Fig. 1B). The purified recombinant protein (rLac1) was obtained with a specific activity of 325.8 U mg−1 (Fig. 1C).
Fig. 1. Heterologous expression of rLac1 in Pichia pastoris. (A) Positive Pichia transformants were screened on MM agar plates supplemented with ABTS. Colonies of Pichia transformants expressing rLac1 turned green while negative controls (transformed with pPICZ␣) did not. (B) Production of rLac1 by a laccase-positive Pichia transformant. The values represent means ± SD (n = 3).(C) SDS-PAGE analysis of the purified rLac1 protein.
At the optimal pH and temperature, the substrate binding constant (Km ) value of rLac1 for ABTS was 28.9 M, and the turnover rate (kcat ) and catalytic efficiency (kcat /Km ) values were 332.4 s−1 and 11.5 M−1 s−1 , respectively.
3.3. Effects of pH and temperature on rLac1 activity and stability
3.4. Effects of metal ions, inhibitors and surfactants on rLac1 activity
The activity and stability of rLac1 were studied with ABTS as the substrate. The optimum reaction pH of rLac1 was 3.5, and the enzyme was active between 3 and 4.5. Over 75% of the original enzyme activity was retained after incubating rLac1 at pH 4–10 for 24 h at 25 ◦ C (Fig. 2A). rLac1 was active between 30 and 60 ◦ C, and the maximal enzyme activity was observed at 55 ◦ C. Little rLac1 activity was lost during the 2 h incubation at 50 ◦ C. Approximately 50% of the original enzyme activity was retained after 2 h at 60 ◦ C, and the enzyme was inactivated at 70 ◦ C (Fig. 2B).
rLac1 activity was either not affected, or even promoted by most tested metal ions at both 10 and 100 mM concentrations (Table 1). One exception was Mn2+ ions, which at the high concentration of 100 mM decreased rLac1 activity by approximately 25%. The reducing reagent DTT nearly completely abolished laccase activity at 1 mM, and so did sodium azide. Also a reducing reagent, L -cysteine inhibited rLac1 activity by approximately 50% at 1 mM concentration. The laccase-specific inhibitor kojic acid [30] also negatively affected rLac1 activity, and the inhibition increased with concentration. On the contrary, only slight loss of activity was
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Table 1 Effects of cations, inhibitors and surfactants on rLac1 activity. The values represent means ± SD (n = 3). Metal ion
Concentration (mM)
Relative activity (%)
Na+
10 100 10 100 10 100 10 100 10 100 10 100 10 100
110.9 103.7 100.7 95.3 109.5 113.6 95.8 96.8 115.0 104.7 96.9 73.7 114.4 108.9
± ± ± ± ± ± ± ± ± ± ± ± ± ±
8
9
K+ Ca2+ Cu2+ Mg
2+
Mn2+ Zn2+
6.6 3.1 0.8 3.5 7.7 4.4 7.3 7.7 2.9 7.5 1.0 4.2 1.5 1.9
Inhibitor
Concentration (mM)
Relative activity (%)
L -Cysteine
0.1 1 0.1 1 1 10 0.1 1 10 100 0.05 0.25 0.1 1
107.0 44.0 92.2 6.1 97.5 92.8 69.9 12.9 66.5 15.3 92.5 47.4 119.0 133.6
DTT EDTA NaN3 Kojic acid SDS Triton X-100
± ± ± ± ± ± ± ± ± ± ± ± ± ±
8.4 1.0 7.0 0.3 1.0 2.2 3.6 0.1 1.2 0.2 1.6 2.7 2.2 1.3
A 120 Relative activity (%)
100 80 60 40 20 0
2
3
4
5
6
7
10
pH
B 120 Fig. 3. MG decolorization by rLac1 in the presence of various metal ions (10 mM). The values represent means ± SD (n = 3).
Relative activity(%)
100 80
Effects of metal ions on rLac1-mediated decolorization of malachite green were also evaluated. Most metal ions did not significantly affect the decolorization efficiency of rLac1 on MG with the exception of Mn2+ ions, which lowered the decolorization efficiency by approximately 40% (Fig. 3).
60 40 20 0 10
4. Discussion 20
30
40
50
60
70
Temperature (ºC) Fig. 2. Effects of pH and temperature on reactivity and stability of rLac1. (A) Effect of pH on the activity (triangle) and stability (circle) of rLac1. (B) Effect of temperature on the activity (triangle) and stability (circle) of rLac1. The values represent means ± standard errors (n = 3).
observed with the metal chelator EDTA at 10 mM concentration (Table 1). Surfactants SDS and Triton X-100 demonstrated opposite effects on rLac1 activity. Similar to the previous findings [11,31], the ionic surfactant SDS impaired rLac1 activity, whereas the nonionic surfactant Triton X-100 stimulated rLac1 activity. 3.5. Decolorization of dyes by rLac1 rLac1 decolorized seven structurally different dyes to different degrees (Table 2). Over 80% decolorization was achieved with aniline blue, brilliant green, Evans blue, indigo carmine, and malachite green. In contrast, incomplete RBBR decolorization was observed. Kojic acid substantially lowered decolorization efficiencies (Table 2), confirming that rLac1 was responsible for dye decolorization.
It is well known that fungal laccases often exist in families and that the family members can differ from each other in terms of physiological roles, enzymatic properties and application potentials. Therefore, investigating laccase isozymes not only is important for fundamental research, but also serves application purposes by providing a suitable laccase for each purpose [10,11]. We have recently reported the purification and characterization of a major laccase, LacA, from Cerrena sp. HYB07, a fungal strain with industrial value [24]. Here, by continuing our study on Cerrena laccases, we cloned and characterized another novel laccase gene, namely Lac1, with special gene features. The Lac1 gene contained one exceptional splice junction (GT-TG) at the last intron, different from LacA as well as other reported laccase genes containing only canonical intron splice sites [3,5,6,8,14]. TG dinucleotides have been discovered as an alternative 3 splice site in eukaryotic, including human genes [32]. Splicing alterations of Lac1 resulted in premature stop codons; this phenomenon of “altered splicing” might constitute an imperfect version of differential splicing seen in higher eukaryotes [33]. “Altered splicing” was reported for nonlaccase multicopper oxidase genes in Phanerochaete chrysosporium [33], and apparently was not correlated with the unconventional GT-TG splice site.
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Table 2 Decolorization of structurally different dyes by rLac1. The values represent means ± SD (n = 3). Dye
Class
Concentration (mg L−1 )
max (nm)
Decolorization (%) 0.4 U mL−1 6h
Acid violet 7 Aniline blue Brilliant green Evans blue Indigo carmine Malachite green RBBR
Azo Triphenylmethane Triphenylmethane Azo Indigo Triphenylmethane Anthraquinone
25 100 25 25 50 25 100
522 585 622 606 610 620 594
The deduced Lac1 protein showed 76% sequence identity to LacA from the same fungal strain. Similar to LacA, Lac1might also have a high redox potential (E0 ), suggested by the presence of Phe461 in the polypeptide sequence [5,34], as well as Glu458 and Ser113, responsible for a long Cu N bond [35]. In Cerrena sp. HYB07, Lac1 was expressed at a much lower level compared to LacA. Lac1 was expressed in P. pastoris to facilitate characterization of the enzyme. The maximum yield of rLac1 (6.3 U mL−1 ) was approximately 20-fold of that of rLacA (Yang et al., unpublished data). Heterologous expression of both Cerrena enzymes did not require modification of the N-terminal sequence, unlike the case of Coprinus comatus laccases [11]. With ABTS as the substrate, higher optimal pH and temperature of rLac1 (pH 3.5 and 55 ◦ C, respectively) than those of LacA (pH 3.0 and 45 ◦ C, respectively), were observed. This was different from the situation in Cerrena unicolor C-139, in which the four laccase isoforms isolated demonstrated uniform optimal pH (5.5) and temperature (50 ◦ C) [23]. The optimal pH of rLac1 was higher than those of the two laccases purified from C. unicolor VKMF-3196 [22] and C. unicolor strain 137 [25]. Furthermore, rLac1 was most reactive toward dyes such as indigo carmine and MG at pH values 5–6, consistent with previous observations that laccases often display different pH optima against different substrates [36]. Although rLac1 had an optimal temperature which is 10 ◦ C above that of LacA, it exhibited a wide reacting temperature range, which helps to reduce its application costs at ambient temperatures. For instance, the relative activity of rLac1 at 30 ◦ C (approximately 82%) was higher than that of laccases from C. unicolor strain 137 [25], and C-139 [23]. rLac1 had a higher affinity for ABTS (28.9 M) compared to LacA (93.4 M ) from Cerrena sp. HYB07, but within the range (3.27–57.1 M) reported for other Cerrena laccases [22,25–27]. Despite the different pH and temperature optima, rLac1 and LacA displayed similar, beneficial pH- and thermo-stability. rLac1 was stable between pH 4–10; it was more stable than Lac IId from C. unicolor (at pH values below 8.0) [27], as well as laccase from C. unicolor C-139 (at pH values below 6.0) [37]. In addition, rLac1 manifested augmented thermostability compared with Lacc I from C. unicolor strain 137 [25], LacC2 from C. unicolor VKMF-3196 [22], and Lcc3 from Cerrena sp. WR1 [26]. It was also more stable at 60 ◦ C relative to a laccase from Fomitopsis pinicola [38], along with an elevated specific activity. rLac1 was tolerant to common environmental metal ions, which would be advantageous for its practical applications since many laccases are inactivated by metal ions present in industrial processes, such as treatment of effluents [12,36]. Metal tolerance of rLac1 was comparable to or even more pronounced (in the case of Cu2+ ) than a recently reported Lcc9 from Coprinopsis cinerea [12]. rLac1 was more recalcitrant to inhibitors (e.g., L -cysteine, DTT, NaN3 and SDS) than its isozyme, LacA. Compared with Lac IId from C. unicolor [27], rLac1 had increased resistance to NaN3 , Kojic acid, and SDS.
0.4 U mL−1 + Kojic acid 12 h
47.2 61.7 39.9 82.2 60.6 62.1 21.8
± ± ± ± ± ± ±
3.4 1.6 4.3 1.8 0.6 1.3 1.5
71.1 85.8 58.9 87.9 91.2 83.6 26.9
24 h ± ± ± ± ± ± ±
2.5 0.9 2.4 2.3 2.2 1.1 2.3
73.4 86.6 83.2 94.3 97.5 87.0 37.2
6h ± ± ± ± ± ± ±
1.5 0.4 0.5 4.5 2.1 1.1 1.3
0.5 3.7 2.8 4.7 10.8 0.5 0.5
± ± ± ± ± ± ±
0.2 0.1 0.8 1.0 0.7 0.2 0.4
12 h
24 h
1.4 ± 0.2 13.9 ± 0.3 10.2 ± 0.6 11.9 ± 1.0 16.6 ± 3.1 6.1 ± 0.7 1.4 ± 0.2
4.4 ± 0.8 19.4 ± 3.2 21.1 ± 1.4 24.6 ± 1.2 28.7 ± 6.5 18.3 ± 5.4 5.6 ± 1.2
rLac1 could efficiently decolorize various dyes, including textile and food dyes indigo carmine and malachite green, without the presence of a laccase mediator. This characteristic resembles LacA from the same fungal strain, reinforcing the potent dye decolorizing ability of Cerrena sp. HYB07. In contrast, many laccases require a redox mediator for efficient dye decolorization. For example, ABTS or HBT was necessary during indigo carmine decolorization with a Trametes versicolor laccase [39], two recombinant laccases expressed in P. pastoris, derived from Trametes sp. 420 [14], and F. lignosus [40], respectively, and a recently reported Lcc9 from C. cinerea [12]. Although laccase-mediator systems are regarded to be promising in the decolorization of recalcitrant dyes, mediators are expensive, can be toxic, and may lead to loss of enzyme activity [12,40]. For this reason, rLac1 is a more attractive candidate in industrial applications than laccases requiring a mediator for efficient dye decolorization. However, rLac1 probably had a different substrate range from LacAfrom Cerrena sp. HYB07 [24] or Lcc3 from Cerrena sp. WR1 [26], since RBBR was not a preferred substrate for rLac1, unlike these two other laccases. More work is needed to elucidate the substrate ranges of the laccase isozymes and the molecular structure underlying such difference. 5. Conclusions A novel laccase gene, namely Lac1, was cloned and characterized in this study. rLac1 was active over a wide range of temperatures (30–60 ◦ C) and stable at pH 4–10 and 20–60 ◦ C. rLac1 was tolerant to metal ions and decolorized various dyes in the absence of a redox mediator. The stability, metal-tolerance, and dye decolorization ability of rLac1, make it a promising candidate for industrial applications, such as wastewater treatment. Work should be carried out to reveal the physiological roles and biochemical properties of laccases in Cerrena sp. HYB07. Understanding the structural determinants of enzyme properties would be valuable for protein engineering to design improved laccases tailor-made for special application purposes. Acknowledgements The work was funded by grants from Natural Science Foundation of China (41306120) and Oceanic Public Welfare Industry Special Research Project of China (201305015). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijbiomac. 2015.03.028. References [1] D.S. Arora, R.K. Sharma, Appl. Biochem. Biotechnol. 160 (2010) 1760–1788.
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
A novel laccase from basidiomycete Cerrena sp.: Cloning, heterologous expression, and characterization Jie Yang a,b , Tzi Bun Ng c , Juan Lin a,b , Xiuyun Ye a,b,∗ a b c
College of Biological Sciences and Technology, Fuzhou University, Fuzhou, Fujian 350116, China Fujian Key Laboratory of Marine Enzyme Engineering, Fuzhou, Fujian 350116, China School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
a r t i c l e
i n f o
Article history: Received 23 January 2015 Received in revised form 19 March 2015 Accepted 20 March 2015 Available online 28 March 2015 Keywords: Cerrena sp. Laccase P. pastoris Decolorization
a b s t r a c t A novel laccase gene Lac1 and its cDNA were cloned from a white-rot fungus Cerrena sp. and characterized. The 1554-bp cDNA of Lac1 encoded a mature protein with 497 amino acids, preceded by a signal peptide of 20 amino acids. An unconventional intron splice site and incomplete splicing variants of Lac1 were observed. Lac1 was heterologously expressed in the yeast host Pichia pastoris, and a maximal laccase activity of 6.3 U mL−1 in the fermentation broth was achieved after fermentation for 9 days. The recombinant protein rLac1 was purified, and its enzymatic properties and functional characteristics were investigated. When ABTS was used as the substrate, the enzyme was most active at pH 3.5 and 55 ◦ C, and stable at pH 4–10 and 20–60 ◦ C. The Km and kcat values of rLac1 toward ABTS were 28.9 M and 332.4 s−1 , respectively. Furthermore, rLac1 was tolerant to common metal ions up to 100 mM concentration and capable of decolorizing structurally different dyes in the absence of a redox mediator. Hence, Lac1 may be useful for industrial applications, such as dye decolorization and bioremediation. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Laccases (EC 1.10.3.2) compose a family of copper-containing oxidases widely present in plants, fungi, insects, and bacteria. These enzymes can catalyze oxidation of a wide variety of phenolic and nonphenolic lignin-related compounds, accompanied by the reduction of oxygen to water. Being energy-saving and environmentally friendly, laccases have application values in various industrial sectors, such as wine and juice stabilization, biosensor development, dye decolorization, and bioremediation. Moreover, laccases hold tremendous potential in biofuel production to delignify biomass and remove phenolic inhibitors for subsequent hydrolysis of polysaccharides [1,2]. However, due to the high redox potential of nonphenolic units and the size of the lignin polymer, efficient lignin breakdown necessitates the concerted action of laccase and low-molecular-weight redox mediators [2]. Commonly used laccase mediators include 2,2 -azino-bis (3-ethylbenzothiazoline6-sulfonate) (ABTS), and 1-hydroxybenzotriazole (HBT).
∗ Corresponding author at: Fuzhou University, College of Biological Sciences and Technology, Fuzhou, Fujian 350116, China. Tel.: +86 591 22866376; fax: +86 591 22866376. E-mail address: [email protected] (X. Ye). http://dx.doi.org/10.1016/j.ijbiomac.2015.03.028 0141-8130/© 2015 Elsevier B.V. All rights reserved.
White-rot fungi have been extensively studied as they are the most efficient laccase producers [1]. Laccase genes have been cloned from Fome lignosus [3], Pleurotus ostreatus [4], Pycnoporus cinnabarinus [5], and various Trametes strains [6–8]. Although normally exhibiting low-to-moderate sequence identities, fungal laccases typically contain four copper-binding regions and highly conserved copper ligands [9]. Laccases often exist in gene families, and laccase isoenzymes in the same organism display diverse expression patterns and physicochemical characteristics [10]. In order to facilitate industrial laccase applications, it is necessary to discover and evaluate a large number of laccases. However, much published work has been focused on only one or a few predominant laccases secreted under laboratory culture conditions. Elucidation of the properties and function of individual laccase isozymes is often hindered by their low expression levels or mixed secretion [11,12]. Heterologous expression of individual laccase genes, on the other hand, has become a valuable approach to unravel the biochemical nature and structure-function relationships of these industrially important enzymes. Heterologously expressed laccases can also be engineered for enhancement of performance (e.g., improved activity and stability, and altered catalytic properties) with directed evolution or site-directed mutagenesis [13]. Yeasts including Pichia pastoris [3,7,11,14–16], and Saccharomyces cerivisiae [4,17], filamentous fungi such as Trichoderma reesei [18], and Aspergillus niger
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[19], as well as other hosts have been used to heterologously express laccase genes. In the present work, we report genetic and enzymatic characterization of a novel laccase, designated as Lac1, from a Cerrena sp. Several laccases have been purified and characterized from Cerrena [20–27], but molecular cloning and heterologous expression of Cerrena laccase genes have seldom been reported. This report would further our understanding of Cerrena laccases and facilitate their applications in biotechnology.
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2. Material and methods
Expression Kit User Manual. Putative transformants were screened on MM plates containing 0.2 mM ABTS, and positive transformants (X33/pPICZ␣-Lac1) were inoculated in YPD medium at 30 ◦ C with shaking at 200 rpm. After 24 h, the cells were harvested by centrifugation and resuspended to an OD600 of 2.0 with YP medium (pH 6.0) supplemented with 1 mM Cu2+ ions. Methanol at 1% (v/v) was added every 24 h, and secreted laccase activity was monitored in the supernatant after 1 mL culture had been centrifuged at 3000 g for 5 min. The strain transformed with the original pPICZ␣ vector (X33/pPICZ␣) was prepared as the negative control and analyzed in parallel.
2.1. Organisms and media
2.5. Purification of rLac1
The white-rot fungus Cerrena sp. HYB07 [28] was preserved in the culture collection of the College of Biological Sciences and Technology at Fuzhou University, Fuzhou, China and maintained on potato dextrose agar (PDA) medium. Fermentation of Cerrena sp. was carried out in 50 mL medium in 250 mL Erlenmeyer flasks at 30 ◦ C with shaking at 200 rpm. The fermentation medium contained (per liter): 60 g dextrin, 10 g peptone, 1.6 g ammonium tartrate, 6 g KH2 PO4 , 4.14 g MgSO4 ·7H2 O, 0.3 g CaCl2 , 0.18 g NaCl, 0.0625 g CuSO4 ·5H2 O, 0.018 g ZnSO4 ·7H2 O, and 0.015 g vitamin B1. P. pastoris host strain X33 was purchased from Invitrogen (USA). YP, YPD and MM media were prepared according to the instructions in the Pichia Expression Kit User Manual. E. coli DH5␣ was used for cloning.
rLac1 was precipitated by adding 70% NH4 SO4 to the fermentation broth and then resuspended in 50 mM Tris-HCl buffer (pH 8.5). After desalting by using a HiPrep Desalting column (GE Healthcare), the sample was applied to a 5 mL HiTrap DEAE FF column (GE Healthcare) pre-equilibrated with 50 mM Tris-HCl buffer (pH 8.5). The column was eluted with a linear gradient of 0–1 M NaCl in 50 mM Tris-HCl buffer (pH 8.5). Elution was monitored at 280 nm for detection of protein. The active fractions were pooled and stored at 4 ◦ C. The purity of the enzyme was checked by SDS-PAGE. Protein concentration was determined by using the Bradford assay with bovine serum albumin as the standard.
2.2. Nucleic acid manipulation Mycelia were harvested by pressure filtration. Genomic DNA was isolated from mycelia with the DNAquick Plant System (TIANGEN, China) according to the manufacturer’s instructions. Total RNA was extracted with Trizol (Life Technologies, USA). Contaminating genomic DNA was removed by using RNase-free DNase (Promega, USA), and the RevertAid First Strand cDNA Synthesis Kit (Fermentas, Canada) was used to synthesize first strands of cDNA. 2.3. Cloning of the Lac1 gene and cDNA The degenerate primers L-1F and L-4R (Table S1) were designed according to the conserved amino acid sequences of the first and fourth copper-binding motifs in fungal laccases, respectively [29]. A 1.8 kb PCR product was obtained from degenerate PCR with the genomic DNA of HYB07 as the template and inserted into pMD18-T vector (TAKARA, Japan). The fragment was sequenced and confirmed to be part of a novel laccase gene, which was named Lac1. Inverse PCR (iPCR) was used to amplify the 5 and 3 -flanking sequences of the 1.8 kb Lac1 fragment [14] with primers Lac11 and Lac1-2 (Table S1). The resulting product was purified and sequenced directly. The Lac1 cDNA was amplified with pfu polymerase (Fermentas, Canada) and the primer pair Lac1-RT-1 and Lac1-RT-2 (Table S1). The Lac1 gene has been deposited in GenBank with the accession number KC540913.
2.6. Enzyme assay Laccase activity was assayed with ABTS as the substrate by following changes in the absorbance at 420 nm for 5 min. One unit of enzyme activity (U) was defined as the amount of laccase required to oxidize 1 mol ABTS in 1 min. All assays were carried out in triplicate. 2.7. Effects of pH and temperature on rLac1 activity and stability To determine the optimum pH, laccase activity was measured in citric acid buffers between pH 3.0 and 6.0. The relative enzyme activity was calculated with the highest activity as 100%. pH stability was studied by incubating the purified enzyme at different pH values in 0.1 M citric acid–Na2 HPO4 buffers (pH 3.0–8.0), or 0.1 M Na2 HPO4 –NaOH buffers (pH 9.0–10.0) at room temperature for 24 h, and the residual laccase activity was quantified at the optimum pH and temperature. The activity of the untreated enzyme was taken as 100%. For the optimum temperature, laccase activity was measured at the optimum pH and various temperatures ranging from 20 to 65 ◦ C. Thermostability was analyzed by incubating the enzyme at various temperatures (20–70 ◦ C) for 2 h, and the residual enzyme activity was assayed at the optimum pH and temperature. All experiments were repeated three times with three replicates each time. 2.8. Effects of metal ions, inhibitors and surfactants on rLac1 activity
2.4. Heterologous expression of the Lac1 gene in P. pastoris Lac1 coding sequence, excluding the predicted signal sequence, was amplified with primers Lac1-Ex-F and Lac1-Ex-R (Table S1). The fragment was digested with XhoI and XbaI, and inserted into the yeast expression vector pPICZ␣ C to construct pPICZ␣-Lac1, which was sequenced to verify the correctness of the open reading frame. The recombinant pPICZ␣-Lac1 plasmid was linearized with BstxI and then electroporated into competent P. pastoris X33 cells with a Gene Pulser II apparatus (BioRad, USA) as described in the Pichia
Metal ions, including Na+ (Na2 SO4 ), K+ (K2 SO4 ), Ca2+ (CaSO4 ), Cu2+ (CuSO4 ), Mg2+ (MgSO4 ), Mn2+ (MnSO4 ), and Zn2+ (ZnSO4 ) ions, were used at 10 mM and 100 mM , respectively. Effects of inhibitors (L -cysteine, DTT, EDTA, NaN3 and kojic acid), and surfactants (SDS and Triton X-100) on rLac1 activity were also tested. Individual effector was incorporated in the standard enzyme activity assay, and the activity was determined at the optimal temperature and pH. The enzyme activity in the absence of an effector was set to 100%.
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2.9. Kinetic studies Substrate specificity of rLac1 for ABTS was determined at the optimum pH and temperature. The substrate concentration range used was 1–1500 M for ABTS. The kinetics parameters were estimated based on nonlinear regression of the Michaelis–Menten equation using GraphPad Prism version 5.0 (GraphPad Software, Inc., USA). 2.10. Dye decolorization by rLac1 Dye decolorization by rLac1 was carried out in the dark at 28 ◦ C for 24 h. The reaction mixture (2 mL) contained citric acid–Na2 HPO4 buffer (pH 6.0), rLac1 (0.4 U mL−1 ) and dye. When needed, kojic acid was included in the reaction mixture at the final concentration of 12.5 mM. For malachite green decolorization, metal ions were individually supplemented in the reaction at the final concentration of 10 mM. All reactions were carried out in triplicate. 3. Results 3.1. Structure of the Lac1 gene The Lac1-coding region was interrupted by 11 introns ranging from 59 to 120 bp in size. Although most splicing junctions in Lac1 adhered to the GT-AG rule, there was one exception (GT-TG) at the last intron. Furthermore, incomplete processing of introns was observed, resulting in transcripts of different lengths. For example, one splice variant had intron 1 remaining, and another one contained intron 6. In both cases, failure to remove all introns led to frame shifts that gave rise to premature stop codons. The cDNA of Lac1 was 1554 bp in length, encoding a polypeptide of 517 aa, with the first 20 aa predicted to be the signal peptide. The sequence has been submitted to GenBank with the accession number KC540913. The molecular weight of the mature Lac1 protein was predicted to be 54.14 kD. The deduced Lac1 protein was closest in amino acid sequence to laccase 1 from Spongipellis sp. FERM P-18171 (BAE79811) and LacA from Cerrena sp. HYB07 (AID59415) with 76% identity, followed by Cerrena sp. WR1 laccase (ACZ58369), and Panus rudis laccase A (AAW28932) with 74%, and 70% identities, respectively. 3.2. Heterologous expression of the Lac1 gene in P. pastoris The Lac1 gene was successfully expressed in P. pastoris and secreted into the culture medium. Laccase activity was only observed with X33/pPICZ␣-Lac1 transformants, but not X33/pPICZ␣ transformants (Fig. 1A). A maximum yield of 6.3 U mL−1 laccase activity was obtained after fermenting in the shake flask at 30 ◦ C for 9 days in YP medium supplemented with 1 mM CuSO4 and 1% methanol (Fig. 1B). The purified recombinant protein (rLac1) was obtained with a specific activity of 325.8 U mg−1 (Fig. 1C).
Fig. 1. Heterologous expression of rLac1 in Pichia pastoris. (A) Positive Pichia transformants were screened on MM agar plates supplemented with ABTS. Colonies of Pichia transformants expressing rLac1 turned green while negative controls (transformed with pPICZ␣) did not. (B) Production of rLac1 by a laccase-positive Pichia transformant. The values represent means ± SD (n = 3).(C) SDS-PAGE analysis of the purified rLac1 protein.
At the optimal pH and temperature, the substrate binding constant (Km ) value of rLac1 for ABTS was 28.9 M, and the turnover rate (kcat ) and catalytic efficiency (kcat /Km ) values were 332.4 s−1 and 11.5 M−1 s−1 , respectively.
3.3. Effects of pH and temperature on rLac1 activity and stability
3.4. Effects of metal ions, inhibitors and surfactants on rLac1 activity
The activity and stability of rLac1 were studied with ABTS as the substrate. The optimum reaction pH of rLac1 was 3.5, and the enzyme was active between 3 and 4.5. Over 75% of the original enzyme activity was retained after incubating rLac1 at pH 4–10 for 24 h at 25 ◦ C (Fig. 2A). rLac1 was active between 30 and 60 ◦ C, and the maximal enzyme activity was observed at 55 ◦ C. Little rLac1 activity was lost during the 2 h incubation at 50 ◦ C. Approximately 50% of the original enzyme activity was retained after 2 h at 60 ◦ C, and the enzyme was inactivated at 70 ◦ C (Fig. 2B).
rLac1 activity was either not affected, or even promoted by most tested metal ions at both 10 and 100 mM concentrations (Table 1). One exception was Mn2+ ions, which at the high concentration of 100 mM decreased rLac1 activity by approximately 25%. The reducing reagent DTT nearly completely abolished laccase activity at 1 mM, and so did sodium azide. Also a reducing reagent, L -cysteine inhibited rLac1 activity by approximately 50% at 1 mM concentration. The laccase-specific inhibitor kojic acid [30] also negatively affected rLac1 activity, and the inhibition increased with concentration. On the contrary, only slight loss of activity was
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Table 1 Effects of cations, inhibitors and surfactants on rLac1 activity. The values represent means ± SD (n = 3). Metal ion
Concentration (mM)
Relative activity (%)
Na+
10 100 10 100 10 100 10 100 10 100 10 100 10 100
110.9 103.7 100.7 95.3 109.5 113.6 95.8 96.8 115.0 104.7 96.9 73.7 114.4 108.9
± ± ± ± ± ± ± ± ± ± ± ± ± ±
8
9
K+ Ca2+ Cu2+ Mg
2+
Mn2+ Zn2+
6.6 3.1 0.8 3.5 7.7 4.4 7.3 7.7 2.9 7.5 1.0 4.2 1.5 1.9
Inhibitor
Concentration (mM)
Relative activity (%)
L -Cysteine
0.1 1 0.1 1 1 10 0.1 1 10 100 0.05 0.25 0.1 1
107.0 44.0 92.2 6.1 97.5 92.8 69.9 12.9 66.5 15.3 92.5 47.4 119.0 133.6
DTT EDTA NaN3 Kojic acid SDS Triton X-100
± ± ± ± ± ± ± ± ± ± ± ± ± ±
8.4 1.0 7.0 0.3 1.0 2.2 3.6 0.1 1.2 0.2 1.6 2.7 2.2 1.3
A 120 Relative activity (%)
100 80 60 40 20 0
2
3
4
5
6
7
10
pH
B 120 Fig. 3. MG decolorization by rLac1 in the presence of various metal ions (10 mM). The values represent means ± SD (n = 3).
Relative activity(%)
100 80
Effects of metal ions on rLac1-mediated decolorization of malachite green were also evaluated. Most metal ions did not significantly affect the decolorization efficiency of rLac1 on MG with the exception of Mn2+ ions, which lowered the decolorization efficiency by approximately 40% (Fig. 3).
60 40 20 0 10
4. Discussion 20
30
40
50
60
70
Temperature (ºC) Fig. 2. Effects of pH and temperature on reactivity and stability of rLac1. (A) Effect of pH on the activity (triangle) and stability (circle) of rLac1. (B) Effect of temperature on the activity (triangle) and stability (circle) of rLac1. The values represent means ± standard errors (n = 3).
observed with the metal chelator EDTA at 10 mM concentration (Table 1). Surfactants SDS and Triton X-100 demonstrated opposite effects on rLac1 activity. Similar to the previous findings [11,31], the ionic surfactant SDS impaired rLac1 activity, whereas the nonionic surfactant Triton X-100 stimulated rLac1 activity. 3.5. Decolorization of dyes by rLac1 rLac1 decolorized seven structurally different dyes to different degrees (Table 2). Over 80% decolorization was achieved with aniline blue, brilliant green, Evans blue, indigo carmine, and malachite green. In contrast, incomplete RBBR decolorization was observed. Kojic acid substantially lowered decolorization efficiencies (Table 2), confirming that rLac1 was responsible for dye decolorization.
It is well known that fungal laccases often exist in families and that the family members can differ from each other in terms of physiological roles, enzymatic properties and application potentials. Therefore, investigating laccase isozymes not only is important for fundamental research, but also serves application purposes by providing a suitable laccase for each purpose [10,11]. We have recently reported the purification and characterization of a major laccase, LacA, from Cerrena sp. HYB07, a fungal strain with industrial value [24]. Here, by continuing our study on Cerrena laccases, we cloned and characterized another novel laccase gene, namely Lac1, with special gene features. The Lac1 gene contained one exceptional splice junction (GT-TG) at the last intron, different from LacA as well as other reported laccase genes containing only canonical intron splice sites [3,5,6,8,14]. TG dinucleotides have been discovered as an alternative 3 splice site in eukaryotic, including human genes [32]. Splicing alterations of Lac1 resulted in premature stop codons; this phenomenon of “altered splicing” might constitute an imperfect version of differential splicing seen in higher eukaryotes [33]. “Altered splicing” was reported for nonlaccase multicopper oxidase genes in Phanerochaete chrysosporium [33], and apparently was not correlated with the unconventional GT-TG splice site.
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Table 2 Decolorization of structurally different dyes by rLac1. The values represent means ± SD (n = 3). Dye
Class
Concentration (mg L−1 )
max (nm)
Decolorization (%) 0.4 U mL−1 6h
Acid violet 7 Aniline blue Brilliant green Evans blue Indigo carmine Malachite green RBBR
Azo Triphenylmethane Triphenylmethane Azo Indigo Triphenylmethane Anthraquinone
25 100 25 25 50 25 100
522 585 622 606 610 620 594
The deduced Lac1 protein showed 76% sequence identity to LacA from the same fungal strain. Similar to LacA, Lac1might also have a high redox potential (E0 ), suggested by the presence of Phe461 in the polypeptide sequence [5,34], as well as Glu458 and Ser113, responsible for a long Cu N bond [35]. In Cerrena sp. HYB07, Lac1 was expressed at a much lower level compared to LacA. Lac1 was expressed in P. pastoris to facilitate characterization of the enzyme. The maximum yield of rLac1 (6.3 U mL−1 ) was approximately 20-fold of that of rLacA (Yang et al., unpublished data). Heterologous expression of both Cerrena enzymes did not require modification of the N-terminal sequence, unlike the case of Coprinus comatus laccases [11]. With ABTS as the substrate, higher optimal pH and temperature of rLac1 (pH 3.5 and 55 ◦ C, respectively) than those of LacA (pH 3.0 and 45 ◦ C, respectively), were observed. This was different from the situation in Cerrena unicolor C-139, in which the four laccase isoforms isolated demonstrated uniform optimal pH (5.5) and temperature (50 ◦ C) [23]. The optimal pH of rLac1 was higher than those of the two laccases purified from C. unicolor VKMF-3196 [22] and C. unicolor strain 137 [25]. Furthermore, rLac1 was most reactive toward dyes such as indigo carmine and MG at pH values 5–6, consistent with previous observations that laccases often display different pH optima against different substrates [36]. Although rLac1 had an optimal temperature which is 10 ◦ C above that of LacA, it exhibited a wide reacting temperature range, which helps to reduce its application costs at ambient temperatures. For instance, the relative activity of rLac1 at 30 ◦ C (approximately 82%) was higher than that of laccases from C. unicolor strain 137 [25], and C-139 [23]. rLac1 had a higher affinity for ABTS (28.9 M) compared to LacA (93.4 M ) from Cerrena sp. HYB07, but within the range (3.27–57.1 M) reported for other Cerrena laccases [22,25–27]. Despite the different pH and temperature optima, rLac1 and LacA displayed similar, beneficial pH- and thermo-stability. rLac1 was stable between pH 4–10; it was more stable than Lac IId from C. unicolor (at pH values below 8.0) [27], as well as laccase from C. unicolor C-139 (at pH values below 6.0) [37]. In addition, rLac1 manifested augmented thermostability compared with Lacc I from C. unicolor strain 137 [25], LacC2 from C. unicolor VKMF-3196 [22], and Lcc3 from Cerrena sp. WR1 [26]. It was also more stable at 60 ◦ C relative to a laccase from Fomitopsis pinicola [38], along with an elevated specific activity. rLac1 was tolerant to common environmental metal ions, which would be advantageous for its practical applications since many laccases are inactivated by metal ions present in industrial processes, such as treatment of effluents [12,36]. Metal tolerance of rLac1 was comparable to or even more pronounced (in the case of Cu2+ ) than a recently reported Lcc9 from Coprinopsis cinerea [12]. rLac1 was more recalcitrant to inhibitors (e.g., L -cysteine, DTT, NaN3 and SDS) than its isozyme, LacA. Compared with Lac IId from C. unicolor [27], rLac1 had increased resistance to NaN3 , Kojic acid, and SDS.
0.4 U mL−1 + Kojic acid 12 h
47.2 61.7 39.9 82.2 60.6 62.1 21.8
± ± ± ± ± ± ±
3.4 1.6 4.3 1.8 0.6 1.3 1.5
71.1 85.8 58.9 87.9 91.2 83.6 26.9
24 h ± ± ± ± ± ± ±
2.5 0.9 2.4 2.3 2.2 1.1 2.3
73.4 86.6 83.2 94.3 97.5 87.0 37.2
6h ± ± ± ± ± ± ±
1.5 0.4 0.5 4.5 2.1 1.1 1.3
0.5 3.7 2.8 4.7 10.8 0.5 0.5
± ± ± ± ± ± ±
0.2 0.1 0.8 1.0 0.7 0.2 0.4
12 h
24 h
1.4 ± 0.2 13.9 ± 0.3 10.2 ± 0.6 11.9 ± 1.0 16.6 ± 3.1 6.1 ± 0.7 1.4 ± 0.2
4.4 ± 0.8 19.4 ± 3.2 21.1 ± 1.4 24.6 ± 1.2 28.7 ± 6.5 18.3 ± 5.4 5.6 ± 1.2
rLac1 could efficiently decolorize various dyes, including textile and food dyes indigo carmine and malachite green, without the presence of a laccase mediator. This characteristic resembles LacA from the same fungal strain, reinforcing the potent dye decolorizing ability of Cerrena sp. HYB07. In contrast, many laccases require a redox mediator for efficient dye decolorization. For example, ABTS or HBT was necessary during indigo carmine decolorization with a Trametes versicolor laccase [39], two recombinant laccases expressed in P. pastoris, derived from Trametes sp. 420 [14], and F. lignosus [40], respectively, and a recently reported Lcc9 from C. cinerea [12]. Although laccase-mediator systems are regarded to be promising in the decolorization of recalcitrant dyes, mediators are expensive, can be toxic, and may lead to loss of enzyme activity [12,40]. For this reason, rLac1 is a more attractive candidate in industrial applications than laccases requiring a mediator for efficient dye decolorization. However, rLac1 probably had a different substrate range from LacAfrom Cerrena sp. HYB07 [24] or Lcc3 from Cerrena sp. WR1 [26], since RBBR was not a preferred substrate for rLac1, unlike these two other laccases. More work is needed to elucidate the substrate ranges of the laccase isozymes and the molecular structure underlying such difference. 5. Conclusions A novel laccase gene, namely Lac1, was cloned and characterized in this study. rLac1 was active over a wide range of temperatures (30–60 ◦ C) and stable at pH 4–10 and 20–60 ◦ C. rLac1 was tolerant to metal ions and decolorized various dyes in the absence of a redox mediator. The stability, metal-tolerance, and dye decolorization ability of rLac1, make it a promising candidate for industrial applications, such as wastewater treatment. Work should be carried out to reveal the physiological roles and biochemical properties of laccases in Cerrena sp. HYB07. Understanding the structural determinants of enzyme properties would be valuable for protein engineering to design improved laccases tailor-made for special application purposes. Acknowledgements The work was funded by grants from Natural Science Foundation of China (41306120) and Oceanic Public Welfare Industry Special Research Project of China (201305015). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijbiomac. 2015.03.028. References [1] D.S. Arora, R.K. Sharma, Appl. Biochem. Biotechnol. 160 (2010) 1760–1788.
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