Biotechnol Lett DOI 10.1007/s10529-015-1865-6
ORIGINAL RESEARCH PAPER
Expression and characterization of LacMP, a novel fungal laccase of Moniliophthora perniciosa FA553 Huiping Liu . Chaofan Tong . Bing Du . Shuli Liang . Ying Lin
Received: 9 March 2015 / Accepted: 17 May 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Objective To characterize a putative laccase gene, LacMP, of Moniliophthora perniciosa FA553 that had been screened using a genome mining approach, then cloned and expressed in Pichia pastoris. Results The purified recombinant LacMP was *57 kDa with a maximum laccase activity of 232 U/ l. The optimal pH for oxidation reactions with 2,20 azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), syringaldazine, guaiacol and 2,6-dimethoxyphenol, were 6, 7.5, 6.5, and 6.5, respectively. The laccase activity
Electronic supplementary material The online version of this article (doi:10.1007/s10529-015-1865-6) contains supplementary material, which is available to authorized users. H. Liu C. Tong S. Liang Y. Lin (&) School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, Guangdong, China e-mail: [email protected] H. Liu e-mail: [email protected] C. Tong e-mail: [email protected] S. Liang e-mail: [email protected] B. Du College of Food Science and Engineering, South China Agricultural University, Guangzhou 510642, Guangdong, China e-mail: [email protected]
was optimal at 60, 45, 45, and 55 °C for the four respective substrates. LacMP was stable at pH 6–8 and \30 °C. Li?, K?, Co2?, Ni2?, Mn2?, and Mg2? at 1 mM had no obvious effect on its activity. Conclusion Given that LacMP has optimal activity under neutral and alkaline oxidation reaction conditions, it could be a potential candidate for industrial biocatalytic applications. Keywords Genome mining Laccase Moniliophthora perniciosa Pichia pastoris
Introduction Laccases (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) belong to the family of multi-copper oxidases (MCOs) and are widely distributed in fungi, bacteria, plants and insects (Hoegger et al. 2006). The fourelectron reduction of O2 to water is coupled with the oxidation of a wide variety of organic and inorganic compounds, including diphenols, polyphenols, diamines, and aromatic amines, via these enzymes (Kiiskinen et al. 2002). Laccases utilize a broad substrate range with water the sole by-product. Therefore there is considerable interest in the use of laccases for ‘‘green’’ biotechnological applications, such as dye degradation, synthesis of aromatic compounds, paper pulp biobleaching and pitch control (Dwivedi et al. 2011; Gutie´rrez et al. 2009; Hsu et al. 2012).
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The physiological roles of laccases are diverse and depend on their origin (Giardina et al. 2010). In fungi, laccases are involved in morphogenesis, fungal plant– pathogen host interactions, lignin mineralization, and pigment production (Baldrian 2006). Among fungal laccases, those produced by phytopathogenic fungi have not been widely investigated; therefore, there is a high likelihood that fungal laccases with novel functions remain to be identified (Kittl et al. 2012). In general, the activities of fungal laccases are diminished under alkaline conditions, limiting their use in biotechnological applications (Sharma et al. 2007). A desired catalytic property for laccases is that they remain active under neutral or alkaline conditions. The laccase from Coprinus comatus was previously reported to operate at a neutral pH, and could potentially degrade some recalcitrant synthetic dyes (Bao et al. 2013). In the current study, we identified LacMP, a novel laccase of Moniliophthora perniciosa FA553, a basidiomycete fungus that causes witches’ broom disease (WBD) in cocoa (Mondego et al. 2008). The amino acid sequence of this LacMP differed by over 30 % compared with the corresponding amino acid sequence of the C. comatus laccase. We cloned the LacMP gene of M. perniciosa FA553 and expressed the protein in Pichia pastoris for subsequent biochemical characterization. We found that neutral or alkaline conditions were optimal for LacMP oxidation reactions, leading us to believe that this laccase could be used in a wide range of biotechnological applications.
Materials and methods Strains, media and chemicals We transformed the pPICZaA cloning and expression vector (Invitrogen) into Escherichia coli Top10 (Invitrogen), while P. pastoris X33 (Invitrogen) was used for the heterologous expression of the recombinant laccase. All media and protocols used were the same as those outlined in the Invitrogen P. pastoris manual. 2,20 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), guaiacol, syringaldazine (SGZ), and 2,6dimethoxyphenol (2,6-DMP) were from SigmaAldrich. All other chemicals used were analytical grade.
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Cloning of LacMP and vector construction A protein BLAST search (http://blast.ncbi.nlm.nih. gov/Blast.cgi) for uncharacterized laccases was conducted using a laccase (GenBank Accession Number AFD97050) from Coprinus comatus as the search term. Of the numerous homologs, a protein (XP_002388482) annotated as ‘‘hypothetical protein MPER_12490’’ from M. perniciosa FA553 was selected. The sequence and location of signal peptide cleavage sites in this protein were predicted using SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/). The molecular weight and isoelectric point (pI) of hypothetical protein MPER_12490 was predicted by the Expasy Proteomics server (www.expasy.org). The sequence of the selected gene was optimized for P. pastoris expression using OptimumGene technology and synthesized by GenScript Biotech Inc. (Nanjing, China). Amplification of the putative laccase gene was performed by PCR using primers PMPF/ PMPR (Supplementary Table 1), with the synthesized laccase gene used as the template. PCR products were purified using a gel extraction kit and digested with EcoRI and NotI. The purified and digested PCR products were then ligated into pPICZaA, which had also been digested with EcoRI and NotI to yield pPICZaA-LacMP. For expression of the laccase gene containing an additional N-terminal amino acid tag (ETEAEF) (Mate et al. 2013), the a-factor signal sequence fused with the extending peptide (ETEAEF) at the EcoRI site was amplified by PCR using primers a-factor-6AAF and a-factor-6AAR (Supplementary Table 1), and pPICZaA as the template. The purified amplicon and pPICZaA-LacMP were digested with BstBI and EcoRI prior to ligation and then transformed into E. coli Top 10. The resulting expression plasmid was designated pPICZaA-6AA-LacMP. Heterologous expression of LacMP in P. pastoris The expression plasmid pPICZaA-6AA-LacMP was linearized with PmeI and transformed into electrocompetent P. pastoris X33 cells. Transformants were grown on YPD plates (1 % yeast extract, 2 % peptone, 0.4 % glucose and 1.5 % agar) supplemented with 100 lg zeocin/l and screened on minimal methanol (MM) plates containing 0.2 mM ABTS and 0.1 mM CuSO4. Transformants that were dark green indicating that they were highly expressing were selected and
Biotechnol Lett
cultivated in 50 ml flasks containing 10 ml buffered glycerol complex (BMGY) medium [1 % yeast extract, 2 % peptone, 1.34 % yeast nitrogen base (YNB), 100 mM potassium phosphate (pH 6.0) and 1 % (v/v) glycerol] at 30 °C and 250 rpm. When the OD600 of cultures was *6, cells were harvested by centrifugation (60009g, 4 °C, 5 min) and then resuspended in 25 ml BMMY medium [1 % yeast extract, 2 % peptone, 1.34 % yeast nitrogen base (YNB), 100 mM potassium phosphate (pH 6.0) and 1 % (v/v) methanol] in 250 ml flasks containing CuSO4 at an initial OD600 of 1. For extracellular laccase production, these cultures were incubated at 25 °C and 250 rpm for 5 days with the daily addition of 1 % (v/ v) methanol. To investigate the effects of Cu2? on laccase yield, CuSO4 (0, 0.2, 0.4, 0.5, 0.6, and 0.8 mM) was added to the induction medium. Laccase activity assay Laccase activity was assayed according to the method of Kittl et al. (2012), with some minor modifications. LacMP activity was determined at 45 °C by monitoring the oxidation of 1 mM ABTS at 420 nm (e420 = 36,000 M-1 cm-1). The 1 ml reaction mixture contained an appropriate dilution of LacMP, 1 mM ABTS and 50 mM Na2HPO4/NaH2PO4 buffer (pH 6.0). Other substrates for the measurement of laccase activity were 2 mM guaiacol (e465 = 12,000 M-1 cm-1), 100 lM SGZ (e525 = 65,000 M-1 cm-1) and 2 mM 2,6-DMP (e468 = 49,600 M-1 cm-1), respectively. One unit of enzyme was defined as the amount of enzyme that oxidizes 1 lmol substrate per min. All assays were performed in triplicate. Purification and characterization of LacMP Supernatants of BMMY cultures were collected by centrifugation (60009g, 4 °C, 5 min). Recombinant LacMP was purified by immobilized metal affinity chromatography (IMAC), using a HisTrap FF column with an AKTA-FPLC system (GE Healthcare). Purified LacMP was stored at 4 °C until required. The purity of LacMP was assessed by 10 % SDS–PAGE. Following electrophoresis, gels were stained with Coomassie Brilliant Blue R-250. The concentration of purified LacMP was estimated using the Bradford assay with bovine serum albumin as the standard.
The optimum pH and temperature for LacMP were determined using ABTS, guaiacol, 2,6-DMP and SGZ as substrates, from pH 3.5 to 9, in a universal buffer (40 mM H3BO3, 40 mM H3PO4, 40 mM acetic acid ? NaOH to give the required pH) and from 30 to 70 °C. LacMP was incubated at 30 °C for 24 h in the same universal buffer over a pH range of 4–8; residual activities were determined with ABTS. The thermostability of LacMP was determined by measuring residual activities with substrate ABTS after pre-incubation of LacMP at various temperatures (30–50 °C) for 0–40 min. The kinetic parameters for LacMP were measured using various concentrations of the substrates ABTS (62.5–1000 lM), SGZ (3.125–200 lM), guaiacol (125–2000 lM), and 2,6-DMP (62.5–4000 lM) at their optimal temperature and pH. The experimental results were fitted to Lineweaver–Burk plots. The effects of various metal ions at 1 mM, and inhibitors (1 mM SDS or 1 mM EDTA) on enzyme activity were investigated by incubating LacMP with each effector at 4 °C for 15 min. The residual activity was determined using ABTS as a substrate under standard assay conditions (Gu et al. 2014). The effects of halides on LacMP activity were determined by adding NaCl (0–200 mM), NaBr (0–200 mM), and NaF (0–100 mM) to the enzyme assay mixture. Residual activity was assayed as described above with ABTS.
Results and discussion Cloning and expression of LacMP A novel laccase of M. perniciosa FA553 was identified by protein Blast using a laccase sequence of C. comatus as the search term. Of numerous homologs, the ‘‘hypothetical protein MPER_12490’’ identified in the fully sequenced genome of M. perniciosa FA553 was selected for cloning and expression in P. pastoris. This uncharacterized putative laccase gene was designated LacMP. The ORF of LacMP was composed of 1548 nucleotides predicted to encode a protein of 516 amino acids, with a putative signal peptide of 22 amino acids. The theoretical molecular weight and pI of the predicted LacMP were 57 kDa and 4.9, respectively. The ligands which bind
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Recombinant LacMP was purified by IMAC, and SDS–PAGE analysis revealed that it was approx.
57 kDa, which was in agreement with its predicted theoretical molecular mass (Fig. 1c). The optimal pH for LacMP activity on ABTS, SGZ, guaiacol, and 2,6-DMP was 6, 7.5, 6.5, and 6.5, respectively (Fig. 2a). LacMP was active at a higher pH than the laccase from C. comatus (Bao et al. 2013). The optimum pH for most fungal laccases has been noted to fall between 3.5 and 5 (Santhanam et al. 2011). The activity of most fungal laccases is very low, or lost, above a pH of 7–8. However, LacMP activity was maintained under neutral and alkaline conditions, with 44, 50, 65, and 67 % of its maximum activity on ABTS, SGZ, guaiacol and 2,6-DMP, respectively at pH 8.0 (Fig. 2a). Furthermore, LacMP was found to be stable at pH 6–8, maintaining more than 70 % of its original activity following incubation at 30 °C for 24 h (Fig. 2b). In certain industrial processes, such as decolorization of textile wastewater or treatment of kraft pulps, alkaline conditions prevail, therefore a laccase that has activity under these conditions would be preferable (Gunne and Urlacher 2012). The optimum temperatures for LacMP activity on ABTS, SGZ, guaiacol, and 2,6-DMP were 60, 45, 45, and 55 °C, respectively (Fig. 3a). These results were similar to those for a laccase from C. comatus (Bao et al. 2013). LacMP was stable at temperatures \30 °C but at higher temperatures the loss of enzymatic activity was more pronounced with a half-life of 35 and 7.5 min at 40 and 50 °C, respectively (Figs. 3b, 4).
Fig. 1 Time course of laccase production (a), effect of addition of copper salt in induction medium (b) and SDS-PAGE of the purified LacMP from P. pastoris (c). a Laccase activity over time in P. pastoris cultured at 25 °C in the presence of 0.4 mM CuSO4. b Laccase activity of LacMP in P. pastoris exposed to
various concentrations of CuSO4 at the beginning of the induction phase. c The purified recombinant LacMP was analyzed by SDS–PAGE. Lane M protein marker; Lane 1 purified LacMP. Data points are the average of triplicate measurements. Error bars represent the standard deviation
T1 copper and trinuclear copper in laccases were all conserved in LacMP. To express the LacMP in P. pastoris, the expression plasmid pPICZaA-6AA-LacMP carrying the laccase gene with an additional N-terminal tag (ETEAEF) was generated. LacMP could not be expressed in P. pastoris that only contained in pPICZaA (data not shown). However, Mate et al. (2013) observed that an N-terminal ETEAEF tag improved laccase expression in P. pastoris. LacMP was successfully expressed in P. pastoris by fusing this additional ETEAEF tag at the N-terminal, demonstrating its essential role. Thus, the N-terminal ETEAEF tag, as consequence of an alternative processing at the Golgi compartment, is beneficial for secretion without jeopardizing the biochemical laccase properties (Mate et al. 2013). The P. pastoris clones showing a deeper color on the selection plates were selected to produce the recombinant laccase using liquid medium (Fig. 1). Maximum laccase activity of 232 U/l was observed 4 days after 0.4 mM CuSO4 was added (Fig. 1a, b). Laccase activity was not detected when CuSO4 was not added (data not shown), indicating the essential role of copper for LacMP expression in P. pastoris. These observations were similar to those seen for Cyathus bulleri laccase expression in P. pastoris (Garg et al. 2012). Characterization of recombinant LacMP
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Fig. 2 Effects of pH on the activity (a) and stability (b) of purified recombinant LacMP. a Relative activities of LacMP over a pH range on ABTS (pH 3.5–8), SGZ (pH 4.5–8.5), guaiacol (pH 3.5–8), and 2,6-DMP (pH 5–9). b Stability of
LacMP over a pH range with ABTS used as the substrate. Data points are the average of triplicate measurements. Error bars represent the standard deviation
Fig. 3 Effects of temperature on the activity (a) and stability (b) of purified recombinant LacMP. a Optimal temperatures were determined when ABTS (40–70 °C), SGZ (30–65 °C), guaiacol (30–65 °C) and 2,6-DMP (40–70 °C) were used as
substrates. b Residual laccase activity of LacMP was determined after incubation at 30, 40, and 50 °C for 40 min. Values presented are the average from triplicate measurements. Error bars represent the standard deviation
The kinetic characteristics of LacMP oxidation on ABTS, SGZ, guaiacol, and 2,6-DMP oxidation were determined at its optimal pH and temperature (Table 1). The highest catalytic efficiency of LacMP was observed for ABTS, with higher kcat and kcat/Km values than those for the other substrates investigated. Metal ions, SDS, EDTA and halides are inhibitors of laccases, with the levels of inhibition exerted by these substances varying widely. The inhibition of laccases by certain inhibitors, such as metal ions and chloride, hampers their use in dye decolorization in saltcontaining effluents and in biofuel cells (Kittl et al.
2012). Further research into the effects of numerous laccase inhibitors on LacMP activity was conducted to determine its usefulness in various applications. The effect of different metal ions, SDS, and EDTA on LacMP activity was investigated with ABTS used as the substrate (Table 2). Some metal ions, such as Li?, K?, Co2?, Ni2?, Mn2? and Mg2? had little to no effect on LacMP activity, while Pb2? and Zn2? reduced LacMP activity by 24 and 34 %, respectively. In addition, LacMP was sensitive to SDS and EDTA, with laccase activity inhibited by around 87 and 68 % in the presence of SDS and EDTA, respectively (Table 2).
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Fig. 4 Inhibition of laccase activity by halides. LacMP was incubated in 50 mM Na2HPO4/NaH2PO4 (pH 6.0) using ABTS as the substrate. Data points are the average of triplicate measurements. Error bars represent the standard deviation
Table 1 Kinetic properties of purified recombinant LacMP Substrate
Km (lM)
Kcat (s-1)
ABTS
170 ± 0.5
21.2 ± 0.18
SGZ
Kcat/Km (s-1 lM-1) 0.12 ± 0.001
21 ± 0.8
0.45 ± 0.01
0.02 ± 0.0003
Guaiacol
180 ± 5.7
7.3 ± 0.07
0.04 ± 0.002
2,6-DMP
108 ± 3.6
0.3 ± 0.001
0.003 ± 0.0001
Table 2 Effects of metal ions and inhibitors on LacMP activity Metal ions
Relative activity (%)a
None
100
?
Li
100 ± 0.8
K?
101 ± 1.5
Co2?
101 ± 1.5
Pb2?
76 ± 1.2
Ni2?
97 ± 1.4
Mn2?
104 ± 1.0
Mg
2?
92 ± 1.8
Zn2?
66 ± 0.9
SDS
13 ± 0.43
EDTA
32 ± 0.44
a
Residual activity (%) was measured using ABTS as the substrate after adding each metal ion to the assay mixture to achieve the final concentration for each effector. The values are presented as the mean ± SD of triplicate tests
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Compared with other laccases, LacMP was resistant to most metal ions investigated; however its activity was inhibited by SDS and EDTA (Bao et al. 2013; Gu et al. 2014; Liu et al. 2015). The inhibition of enzymatic activity by F-, Cl- and Br- was measured at pH 6 using ABTS as substrate. LacMP activity was reduced by about 50 % with 100 mM NaCl or 20 mM NaF and exhibited about 65 % activity by 200 mM NaBr. These findings clearly indicate that lower molecular weight halides were more efficient inhibitors of LacMP activity (fluoride [ chloride [ bromide), which could be attributed to limited access of the laccase’s T2/T3 trinuclear copper cluster site (Xu 1996). The I50 value (the concentration of an inhibitor that results in a 50 % reduction in its activity) of Cl- ranges between 0.4 and 1400 mM for fungal laccases (Kittl et al. 2012). We found that LacMP was moderately resistant to Cl-, with an I50 of about 100 mM. In conclusion: We identified a novel fungal laccase of M. perniciosa FA553 and expressed a recombinant form of this laccase in P. pastoris. Characterization of LacMP revealed it to be tolerant to alkaline conditions, with its oxidative capacity maintained at an alkaline or neutral pH. These findings regarding LacMP suggest that it could be a potential candidate for use in industrial biocatalytic applications. Acknowledgments This work was supported by the Chinese High-Tech Development Program (National ‘‘863’’ Project No. 2011AA100905). Supporting information Supplementary Table 1 – Oligonucleotide primers used for plasmid generation.
References Baldrian P (2006) Fungal laccases–occurrence and properties. FEMS Microbiol Rev 30:215–242 Bao S, Teng Z, Ding S (2013) Heterologous expression and characterization of a novel laccase isoenzyme with dyes decolorization potential from Coprinus comatus. Mol Biol Rep 40:1927–1936 Dwivedi UN, Singh P, Pandey VP, Kumar A (2011) Structure– function relationship among bacterial, fungal and plant laccases. J Mol Catal B-Enzym 68:117–128 Garg N, Bieler N, Kenzom T, Chhabra M, Ansorge-Schumacher M, Mishra S (2012) Cloning, sequence analysis, expression of Cyathus bulleri laccase in Pichia pastoris and characterization of recombinant laccase. BMC Biotechnol 12:75
Biotechnol Lett Giardina P, Faraco V, Pezzella C, Piscitelli A, Vanhulle S, Sannia G (2010) Laccases: a never-ending story. Cell Mol Life Sci 67:369–385 Gu C, Zheng F, Long L, Wang J, Ding S (2014) Engineering the expression and characterization of two novel laccase isoenzymes from Coprinus comatus in Pichia pastoris by fusing an additional ten amino acids tag at N-terminus. PLoS One 9:e93912 Gunne M, Urlacher VB (2012) Characterization of the alkaline laccase Ssl1 from Streptomyces sviceus with unusual properties discovered by genome mining. PLoS One 7:e52360 Gutie´rrez A, Jose´ C, Martı´nez AT (2009) Microbial and enzymatic control of pitch in the pulp and paper industry. Appl Microbiol Biotechnol 82:1005–1018 Hoegger PJ, Kilaru S, James TY, Thacker JR, Ku¨es U (2006) Phylogenetic comparison and classification of laccase and related multicopper oxidase protein sequences. FEBS J 273:2308–2326 Hsu C-A, Wen T-N, Su Y-C, Jiang Z-B, Chen C-W, Shyur L-F (2012) Biological degradation of anthraquinone and azo dyes by a novel laccase from Lentinus sp. Environ Sci Technol 46:5109–5117 Kiiskinen L-L, Viikari L, Kruus K (2002) Purification and characterisation of a novel laccase from the ascomycete Melanocarpus albomyces. Appl Microbiol Biotechnol 59:198–204
Kittl R, Mueangtoom K, Gonaus C, Khazaneh ST, Sygmund C, Haltrich D, Ludwig R (2012) A chloride tolerant laccase from the plant pathogen ascomycete Botrytis aclada expressed at high levels in Pichia pastoris. J Biotechnol 157:304–314 Liu H, Cheng Y, Du B, Tong C, Liang S, Han S, Zheng S, Lin Y (2015) Overexpression of a novel thermostable and chloride-tolerant laccase from Thermus thermophilus SG0. 5JP17-16 in Pichia pastoris and its application in synthetic dye decolorization. PLoS One 10:e0119833 Mate DM, Gonzalez-Perez D, Kittl R, Ludwig R, Alcalde M (2013) Functional expression of a blood tolerant laccase in Pichia pastoris. BMC Biotechnol 13:38 Mondego JM, Carazzolle MF, Costa GG, Formighieri EF, Parizzi LP, Rincones J, Cotomacci C, Carraro DM, Cunha AF, Carrer H (2008) A genome survey of Moniliophthora perniciosa gives new insights into witches’ broom disease of cacao. BMC Genom 9:548 Santhanam N, Vivanco JM, Decker SR, Reardon KF (2011) Expression of industrially relevant laccases: prokaryotic style. Trends Biotechnol 29:480–489 Sharma P, Goel R, Capalash N (2007) Bacterial laccases. World J Microbiol Biotechnol 23:823–832 Xu F (1996) Oxidation of phenols, anilines, and benzenethiols by fungal laccases: correlation between activity and redox potentials as well as halide inhibition. Biochemistry 35:7608–7614
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ORIGINAL RESEARCH PAPER
Expression and characterization of LacMP, a novel fungal laccase of Moniliophthora perniciosa FA553 Huiping Liu . Chaofan Tong . Bing Du . Shuli Liang . Ying Lin
Received: 9 March 2015 / Accepted: 17 May 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Objective To characterize a putative laccase gene, LacMP, of Moniliophthora perniciosa FA553 that had been screened using a genome mining approach, then cloned and expressed in Pichia pastoris. Results The purified recombinant LacMP was *57 kDa with a maximum laccase activity of 232 U/ l. The optimal pH for oxidation reactions with 2,20 azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), syringaldazine, guaiacol and 2,6-dimethoxyphenol, were 6, 7.5, 6.5, and 6.5, respectively. The laccase activity
Electronic supplementary material The online version of this article (doi:10.1007/s10529-015-1865-6) contains supplementary material, which is available to authorized users. H. Liu C. Tong S. Liang Y. Lin (&) School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, Guangdong, China e-mail: [email protected] H. Liu e-mail: [email protected] C. Tong e-mail: [email protected] S. Liang e-mail: [email protected] B. Du College of Food Science and Engineering, South China Agricultural University, Guangzhou 510642, Guangdong, China e-mail: [email protected]
was optimal at 60, 45, 45, and 55 °C for the four respective substrates. LacMP was stable at pH 6–8 and \30 °C. Li?, K?, Co2?, Ni2?, Mn2?, and Mg2? at 1 mM had no obvious effect on its activity. Conclusion Given that LacMP has optimal activity under neutral and alkaline oxidation reaction conditions, it could be a potential candidate for industrial biocatalytic applications. Keywords Genome mining Laccase Moniliophthora perniciosa Pichia pastoris
Introduction Laccases (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) belong to the family of multi-copper oxidases (MCOs) and are widely distributed in fungi, bacteria, plants and insects (Hoegger et al. 2006). The fourelectron reduction of O2 to water is coupled with the oxidation of a wide variety of organic and inorganic compounds, including diphenols, polyphenols, diamines, and aromatic amines, via these enzymes (Kiiskinen et al. 2002). Laccases utilize a broad substrate range with water the sole by-product. Therefore there is considerable interest in the use of laccases for ‘‘green’’ biotechnological applications, such as dye degradation, synthesis of aromatic compounds, paper pulp biobleaching and pitch control (Dwivedi et al. 2011; Gutie´rrez et al. 2009; Hsu et al. 2012).
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The physiological roles of laccases are diverse and depend on their origin (Giardina et al. 2010). In fungi, laccases are involved in morphogenesis, fungal plant– pathogen host interactions, lignin mineralization, and pigment production (Baldrian 2006). Among fungal laccases, those produced by phytopathogenic fungi have not been widely investigated; therefore, there is a high likelihood that fungal laccases with novel functions remain to be identified (Kittl et al. 2012). In general, the activities of fungal laccases are diminished under alkaline conditions, limiting their use in biotechnological applications (Sharma et al. 2007). A desired catalytic property for laccases is that they remain active under neutral or alkaline conditions. The laccase from Coprinus comatus was previously reported to operate at a neutral pH, and could potentially degrade some recalcitrant synthetic dyes (Bao et al. 2013). In the current study, we identified LacMP, a novel laccase of Moniliophthora perniciosa FA553, a basidiomycete fungus that causes witches’ broom disease (WBD) in cocoa (Mondego et al. 2008). The amino acid sequence of this LacMP differed by over 30 % compared with the corresponding amino acid sequence of the C. comatus laccase. We cloned the LacMP gene of M. perniciosa FA553 and expressed the protein in Pichia pastoris for subsequent biochemical characterization. We found that neutral or alkaline conditions were optimal for LacMP oxidation reactions, leading us to believe that this laccase could be used in a wide range of biotechnological applications.
Materials and methods Strains, media and chemicals We transformed the pPICZaA cloning and expression vector (Invitrogen) into Escherichia coli Top10 (Invitrogen), while P. pastoris X33 (Invitrogen) was used for the heterologous expression of the recombinant laccase. All media and protocols used were the same as those outlined in the Invitrogen P. pastoris manual. 2,20 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), guaiacol, syringaldazine (SGZ), and 2,6dimethoxyphenol (2,6-DMP) were from SigmaAldrich. All other chemicals used were analytical grade.
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Cloning of LacMP and vector construction A protein BLAST search (http://blast.ncbi.nlm.nih. gov/Blast.cgi) for uncharacterized laccases was conducted using a laccase (GenBank Accession Number AFD97050) from Coprinus comatus as the search term. Of the numerous homologs, a protein (XP_002388482) annotated as ‘‘hypothetical protein MPER_12490’’ from M. perniciosa FA553 was selected. The sequence and location of signal peptide cleavage sites in this protein were predicted using SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/). The molecular weight and isoelectric point (pI) of hypothetical protein MPER_12490 was predicted by the Expasy Proteomics server (www.expasy.org). The sequence of the selected gene was optimized for P. pastoris expression using OptimumGene technology and synthesized by GenScript Biotech Inc. (Nanjing, China). Amplification of the putative laccase gene was performed by PCR using primers PMPF/ PMPR (Supplementary Table 1), with the synthesized laccase gene used as the template. PCR products were purified using a gel extraction kit and digested with EcoRI and NotI. The purified and digested PCR products were then ligated into pPICZaA, which had also been digested with EcoRI and NotI to yield pPICZaA-LacMP. For expression of the laccase gene containing an additional N-terminal amino acid tag (ETEAEF) (Mate et al. 2013), the a-factor signal sequence fused with the extending peptide (ETEAEF) at the EcoRI site was amplified by PCR using primers a-factor-6AAF and a-factor-6AAR (Supplementary Table 1), and pPICZaA as the template. The purified amplicon and pPICZaA-LacMP were digested with BstBI and EcoRI prior to ligation and then transformed into E. coli Top 10. The resulting expression plasmid was designated pPICZaA-6AA-LacMP. Heterologous expression of LacMP in P. pastoris The expression plasmid pPICZaA-6AA-LacMP was linearized with PmeI and transformed into electrocompetent P. pastoris X33 cells. Transformants were grown on YPD plates (1 % yeast extract, 2 % peptone, 0.4 % glucose and 1.5 % agar) supplemented with 100 lg zeocin/l and screened on minimal methanol (MM) plates containing 0.2 mM ABTS and 0.1 mM CuSO4. Transformants that were dark green indicating that they were highly expressing were selected and
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cultivated in 50 ml flasks containing 10 ml buffered glycerol complex (BMGY) medium [1 % yeast extract, 2 % peptone, 1.34 % yeast nitrogen base (YNB), 100 mM potassium phosphate (pH 6.0) and 1 % (v/v) glycerol] at 30 °C and 250 rpm. When the OD600 of cultures was *6, cells were harvested by centrifugation (60009g, 4 °C, 5 min) and then resuspended in 25 ml BMMY medium [1 % yeast extract, 2 % peptone, 1.34 % yeast nitrogen base (YNB), 100 mM potassium phosphate (pH 6.0) and 1 % (v/v) methanol] in 250 ml flasks containing CuSO4 at an initial OD600 of 1. For extracellular laccase production, these cultures were incubated at 25 °C and 250 rpm for 5 days with the daily addition of 1 % (v/ v) methanol. To investigate the effects of Cu2? on laccase yield, CuSO4 (0, 0.2, 0.4, 0.5, 0.6, and 0.8 mM) was added to the induction medium. Laccase activity assay Laccase activity was assayed according to the method of Kittl et al. (2012), with some minor modifications. LacMP activity was determined at 45 °C by monitoring the oxidation of 1 mM ABTS at 420 nm (e420 = 36,000 M-1 cm-1). The 1 ml reaction mixture contained an appropriate dilution of LacMP, 1 mM ABTS and 50 mM Na2HPO4/NaH2PO4 buffer (pH 6.0). Other substrates for the measurement of laccase activity were 2 mM guaiacol (e465 = 12,000 M-1 cm-1), 100 lM SGZ (e525 = 65,000 M-1 cm-1) and 2 mM 2,6-DMP (e468 = 49,600 M-1 cm-1), respectively. One unit of enzyme was defined as the amount of enzyme that oxidizes 1 lmol substrate per min. All assays were performed in triplicate. Purification and characterization of LacMP Supernatants of BMMY cultures were collected by centrifugation (60009g, 4 °C, 5 min). Recombinant LacMP was purified by immobilized metal affinity chromatography (IMAC), using a HisTrap FF column with an AKTA-FPLC system (GE Healthcare). Purified LacMP was stored at 4 °C until required. The purity of LacMP was assessed by 10 % SDS–PAGE. Following electrophoresis, gels were stained with Coomassie Brilliant Blue R-250. The concentration of purified LacMP was estimated using the Bradford assay with bovine serum albumin as the standard.
The optimum pH and temperature for LacMP were determined using ABTS, guaiacol, 2,6-DMP and SGZ as substrates, from pH 3.5 to 9, in a universal buffer (40 mM H3BO3, 40 mM H3PO4, 40 mM acetic acid ? NaOH to give the required pH) and from 30 to 70 °C. LacMP was incubated at 30 °C for 24 h in the same universal buffer over a pH range of 4–8; residual activities were determined with ABTS. The thermostability of LacMP was determined by measuring residual activities with substrate ABTS after pre-incubation of LacMP at various temperatures (30–50 °C) for 0–40 min. The kinetic parameters for LacMP were measured using various concentrations of the substrates ABTS (62.5–1000 lM), SGZ (3.125–200 lM), guaiacol (125–2000 lM), and 2,6-DMP (62.5–4000 lM) at their optimal temperature and pH. The experimental results were fitted to Lineweaver–Burk plots. The effects of various metal ions at 1 mM, and inhibitors (1 mM SDS or 1 mM EDTA) on enzyme activity were investigated by incubating LacMP with each effector at 4 °C for 15 min. The residual activity was determined using ABTS as a substrate under standard assay conditions (Gu et al. 2014). The effects of halides on LacMP activity were determined by adding NaCl (0–200 mM), NaBr (0–200 mM), and NaF (0–100 mM) to the enzyme assay mixture. Residual activity was assayed as described above with ABTS.
Results and discussion Cloning and expression of LacMP A novel laccase of M. perniciosa FA553 was identified by protein Blast using a laccase sequence of C. comatus as the search term. Of numerous homologs, the ‘‘hypothetical protein MPER_12490’’ identified in the fully sequenced genome of M. perniciosa FA553 was selected for cloning and expression in P. pastoris. This uncharacterized putative laccase gene was designated LacMP. The ORF of LacMP was composed of 1548 nucleotides predicted to encode a protein of 516 amino acids, with a putative signal peptide of 22 amino acids. The theoretical molecular weight and pI of the predicted LacMP were 57 kDa and 4.9, respectively. The ligands which bind
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Recombinant LacMP was purified by IMAC, and SDS–PAGE analysis revealed that it was approx.
57 kDa, which was in agreement with its predicted theoretical molecular mass (Fig. 1c). The optimal pH for LacMP activity on ABTS, SGZ, guaiacol, and 2,6-DMP was 6, 7.5, 6.5, and 6.5, respectively (Fig. 2a). LacMP was active at a higher pH than the laccase from C. comatus (Bao et al. 2013). The optimum pH for most fungal laccases has been noted to fall between 3.5 and 5 (Santhanam et al. 2011). The activity of most fungal laccases is very low, or lost, above a pH of 7–8. However, LacMP activity was maintained under neutral and alkaline conditions, with 44, 50, 65, and 67 % of its maximum activity on ABTS, SGZ, guaiacol and 2,6-DMP, respectively at pH 8.0 (Fig. 2a). Furthermore, LacMP was found to be stable at pH 6–8, maintaining more than 70 % of its original activity following incubation at 30 °C for 24 h (Fig. 2b). In certain industrial processes, such as decolorization of textile wastewater or treatment of kraft pulps, alkaline conditions prevail, therefore a laccase that has activity under these conditions would be preferable (Gunne and Urlacher 2012). The optimum temperatures for LacMP activity on ABTS, SGZ, guaiacol, and 2,6-DMP were 60, 45, 45, and 55 °C, respectively (Fig. 3a). These results were similar to those for a laccase from C. comatus (Bao et al. 2013). LacMP was stable at temperatures \30 °C but at higher temperatures the loss of enzymatic activity was more pronounced with a half-life of 35 and 7.5 min at 40 and 50 °C, respectively (Figs. 3b, 4).
Fig. 1 Time course of laccase production (a), effect of addition of copper salt in induction medium (b) and SDS-PAGE of the purified LacMP from P. pastoris (c). a Laccase activity over time in P. pastoris cultured at 25 °C in the presence of 0.4 mM CuSO4. b Laccase activity of LacMP in P. pastoris exposed to
various concentrations of CuSO4 at the beginning of the induction phase. c The purified recombinant LacMP was analyzed by SDS–PAGE. Lane M protein marker; Lane 1 purified LacMP. Data points are the average of triplicate measurements. Error bars represent the standard deviation
T1 copper and trinuclear copper in laccases were all conserved in LacMP. To express the LacMP in P. pastoris, the expression plasmid pPICZaA-6AA-LacMP carrying the laccase gene with an additional N-terminal tag (ETEAEF) was generated. LacMP could not be expressed in P. pastoris that only contained in pPICZaA (data not shown). However, Mate et al. (2013) observed that an N-terminal ETEAEF tag improved laccase expression in P. pastoris. LacMP was successfully expressed in P. pastoris by fusing this additional ETEAEF tag at the N-terminal, demonstrating its essential role. Thus, the N-terminal ETEAEF tag, as consequence of an alternative processing at the Golgi compartment, is beneficial for secretion without jeopardizing the biochemical laccase properties (Mate et al. 2013). The P. pastoris clones showing a deeper color on the selection plates were selected to produce the recombinant laccase using liquid medium (Fig. 1). Maximum laccase activity of 232 U/l was observed 4 days after 0.4 mM CuSO4 was added (Fig. 1a, b). Laccase activity was not detected when CuSO4 was not added (data not shown), indicating the essential role of copper for LacMP expression in P. pastoris. These observations were similar to those seen for Cyathus bulleri laccase expression in P. pastoris (Garg et al. 2012). Characterization of recombinant LacMP
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Fig. 2 Effects of pH on the activity (a) and stability (b) of purified recombinant LacMP. a Relative activities of LacMP over a pH range on ABTS (pH 3.5–8), SGZ (pH 4.5–8.5), guaiacol (pH 3.5–8), and 2,6-DMP (pH 5–9). b Stability of
LacMP over a pH range with ABTS used as the substrate. Data points are the average of triplicate measurements. Error bars represent the standard deviation
Fig. 3 Effects of temperature on the activity (a) and stability (b) of purified recombinant LacMP. a Optimal temperatures were determined when ABTS (40–70 °C), SGZ (30–65 °C), guaiacol (30–65 °C) and 2,6-DMP (40–70 °C) were used as
substrates. b Residual laccase activity of LacMP was determined after incubation at 30, 40, and 50 °C for 40 min. Values presented are the average from triplicate measurements. Error bars represent the standard deviation
The kinetic characteristics of LacMP oxidation on ABTS, SGZ, guaiacol, and 2,6-DMP oxidation were determined at its optimal pH and temperature (Table 1). The highest catalytic efficiency of LacMP was observed for ABTS, with higher kcat and kcat/Km values than those for the other substrates investigated. Metal ions, SDS, EDTA and halides are inhibitors of laccases, with the levels of inhibition exerted by these substances varying widely. The inhibition of laccases by certain inhibitors, such as metal ions and chloride, hampers their use in dye decolorization in saltcontaining effluents and in biofuel cells (Kittl et al.
2012). Further research into the effects of numerous laccase inhibitors on LacMP activity was conducted to determine its usefulness in various applications. The effect of different metal ions, SDS, and EDTA on LacMP activity was investigated with ABTS used as the substrate (Table 2). Some metal ions, such as Li?, K?, Co2?, Ni2?, Mn2? and Mg2? had little to no effect on LacMP activity, while Pb2? and Zn2? reduced LacMP activity by 24 and 34 %, respectively. In addition, LacMP was sensitive to SDS and EDTA, with laccase activity inhibited by around 87 and 68 % in the presence of SDS and EDTA, respectively (Table 2).
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Fig. 4 Inhibition of laccase activity by halides. LacMP was incubated in 50 mM Na2HPO4/NaH2PO4 (pH 6.0) using ABTS as the substrate. Data points are the average of triplicate measurements. Error bars represent the standard deviation
Table 1 Kinetic properties of purified recombinant LacMP Substrate
Km (lM)
Kcat (s-1)
ABTS
170 ± 0.5
21.2 ± 0.18
SGZ
Kcat/Km (s-1 lM-1) 0.12 ± 0.001
21 ± 0.8
0.45 ± 0.01
0.02 ± 0.0003
Guaiacol
180 ± 5.7
7.3 ± 0.07
0.04 ± 0.002
2,6-DMP
108 ± 3.6
0.3 ± 0.001
0.003 ± 0.0001
Table 2 Effects of metal ions and inhibitors on LacMP activity Metal ions
Relative activity (%)a
None
100
?
Li
100 ± 0.8
K?
101 ± 1.5
Co2?
101 ± 1.5
Pb2?
76 ± 1.2
Ni2?
97 ± 1.4
Mn2?
104 ± 1.0
Mg
2?
92 ± 1.8
Zn2?
66 ± 0.9
SDS
13 ± 0.43
EDTA
32 ± 0.44
a
Residual activity (%) was measured using ABTS as the substrate after adding each metal ion to the assay mixture to achieve the final concentration for each effector. The values are presented as the mean ± SD of triplicate tests
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Compared with other laccases, LacMP was resistant to most metal ions investigated; however its activity was inhibited by SDS and EDTA (Bao et al. 2013; Gu et al. 2014; Liu et al. 2015). The inhibition of enzymatic activity by F-, Cl- and Br- was measured at pH 6 using ABTS as substrate. LacMP activity was reduced by about 50 % with 100 mM NaCl or 20 mM NaF and exhibited about 65 % activity by 200 mM NaBr. These findings clearly indicate that lower molecular weight halides were more efficient inhibitors of LacMP activity (fluoride [ chloride [ bromide), which could be attributed to limited access of the laccase’s T2/T3 trinuclear copper cluster site (Xu 1996). The I50 value (the concentration of an inhibitor that results in a 50 % reduction in its activity) of Cl- ranges between 0.4 and 1400 mM for fungal laccases (Kittl et al. 2012). We found that LacMP was moderately resistant to Cl-, with an I50 of about 100 mM. In conclusion: We identified a novel fungal laccase of M. perniciosa FA553 and expressed a recombinant form of this laccase in P. pastoris. Characterization of LacMP revealed it to be tolerant to alkaline conditions, with its oxidative capacity maintained at an alkaline or neutral pH. These findings regarding LacMP suggest that it could be a potential candidate for use in industrial biocatalytic applications. Acknowledgments This work was supported by the Chinese High-Tech Development Program (National ‘‘863’’ Project No. 2011AA100905). Supporting information Supplementary Table 1 – Oligonucleotide primers used for plasmid generation.
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