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Purification and characterization of an extracellular laccase from the anthracene-degrading fungus Fusarium solani MAS2
Bioresource Technology 101 (2010) 9772–9777
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Purification and characterization of an extracellular laccase from the anthracene-degrading fungus Fusarium solani MAS2 Yi-Rui Wu a,*, Zhu-Hua Luo a,b, R. Kwok-Kei Chow a, L.L.P. Vrijmoed a,** a b
Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR, PR China Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, 178 Daxue Road, Xiamen 361005, PR China
a r t i c l e
i n f o
Article history: Received 21 April 2010 Received in revised form 14 July 2010 Accepted 21 July 2010 Available online 27 July 2010 Keywords: Polycyclic aromatic hydrocarbons (PAHs) ABTS Heavy metal tolerance Mangrove sediment
a b s t r a c t An extracellular laccase was purified from the culture medium of the non-white rot, anthracene-degrading fungal strain Fusarium solani MAS2. Both native PAGE and SDS–PAGE revealed one single band corresponding to a molecular weight of about 72 kDa. Treatment with endoglycosidase H reduced the molecular weight by 12%. The purified laccase maintained stable at pH 3–11 and up to 50 °C. The highest activity was detected at pH 3.0 and at 70 °C. The enzyme retained 46.2–97.2% of it activity in the presence of 20 mM Pb2+, Ni2+, Cr3+, and its activity was enhanced in the presence of 20 mM Hg2+. The laccase retained more than 50% of its activity in the presence of 5% acetone, acetonitrile, dimethyl sulphoxide (DMSO), ethanol and methanol. The kinetic constants (Km and kcat) showed that 2,6-dimethoxyphenol (DMOP) and 2,20 -azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt (ABTS) were the more effective substrates rather than catechol and guaiacol. The novel properties of this laccase suggest its potential for biotechnological and environmental applications. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Laccase (benzenediol: oxygen oxidoreductase; EC 1.10.3.2) is a polyphenol oxidase with the potential for bioremediation of related compounds such as kraft lignin bleaching, detoxification of environmental pollutants and wastewater treatment (Mayer and Staples, 2002; Xiao et al., 2003). Laccases can be divided into three different groups. Blue laccases belong to the multicopper-oxidase enzymes family with the maximum absorption spectrum at 600 nm (Messerschmidt and Huber, 1990), whereas the yellow and white laccases were lack of that absorption spectrum, and have a different oxidation status in their respective catalytic centers (Leontievsky et al., 1997; Palmieri et al., 1997; Pozdnyakova et al., 2006). Laccases are a kind of versatile carbohydrate-containing molecules with different structures from different species (Thurston, 1994). For example, laccase purified from Cantharellus cibarius were dimers with identical subunits (46 kDa) (Ng and Wang, 2004), but laccase from Gaeumannomyces graminis var. tritici had an apparent molecular mass of 190 kDa with three subunits (Edens et al., 1999). Some organisms, such as Podospora ansernia and Trametes sp., have two or more different isozymes (Durrens, 1981; Xiao et al., 2003). Both intracellular and extracellular laccase are known (Mayer and Staples, 2002; Nagai et al., 2003). * Corresponding author. Tel.: +852 97165286; fax: +852 27844093. ** Corresponding author. Tel.: +852 34429966; fax: +852 27844093. E-mail addresses: [email protected] (Y.R. Wu), [email protected] (L.L.P. Vrijmoed). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.07.091
Laccase synthesis can be constitutive or inducible, and the inducing substrates include a wide range of compounds, such as aromatic compounds, anilines, lignin and even various artificial dyes (Leonowicz et al., 2001; Xiao et al., 2003). The diversity of laccases reflects their various functions, including lignin degradation, pigment production, and even plant pathogenesis (Koschorreck et al., 2008). In the current study, a laccase was purified from the non-white rot fungal strain, Fusarium solani strain MAS2, and characterize with respect to its molecular weight, optimal temperature, pH and stability, tolerance to heavy metal ions, organic solvents as well as reaction with different aromatic compounds. The fungus produced this enzyme in the mineral salt medium with anthracene as the only carbon source substrate without producing detectable lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP). The enzyme differs from previously reported laccases (Xiao et al., 2003; Martin et al., 2007) with respect to its high tolerance against most of heavy metal ions, wide range of pH stability, especially the activity enhancement by mercuric ions. 2. Methods 2.1. Microorganism and cultivation Strain MAS2 was isolated from polycyclic aromatic hydrocarbons (PAHs)-contaminated mangrove sediments with the degradative ability of a 3-ring PAH-anthracene, and identified as
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F. solani according to the 18S rRNA gene sequencing as well as the microscopic morphology based on the method of Wu et al. (2009). The fungus was monthly transferred to fresh mineral salt medium (MSM) agar (1%) slants containing anthracene (50 mg l1) as the sole carbon source and stored at 25 °C. The MSM was modified from Hartmans et al. (1989), which contained (g l1): NaCl, 12.3; (NH4)2SO4, 2.0; K2HPO4, 1.55; NaH2PO42H2O, 0.85; MgCl26H2O, 2.53; CaCl22H2O, 0.73; KCl, 0.33; MgSO47H2O, 3.15; and (mg l1) NaHCO3, 90.0; ZnSO47H2O, 2.0; FeSO47H2O, 5.0; Na2MoO42H2O, 0.2; CuSO45H2O, 0.2; CoCl26H2O, 0.4; MnCl22H2O, 1.0; H3BO3, 1.0; KI, 0.5; KAl(SO4)212H2O, 0.5; NiCl26H2O, 0.5; pH 5.5 ± 0.1. For purification of the laccase, strain MAS2 was inoculated into the liquid MSM by incubating at 25 °C on a rotary shaker at 150 rpm for 25 days. 2.2. Enzyme purification The laccase was purified from a cell-free culture medium (250 ml) after filtration through qualitative filter paper (Grade 1, Whatman, UK) and centrifugation (10,000g, 30 min). The supernatant was mixed with three volumes of pre-cooled ethanol for 1 h at 20 °C. The precipitate was collected by centrifugation (5000g, 20 min), resuspended and dialyzed with 20 mM Tris/HCl buffer (pH 7.0) overnight. The suspension was applied to a 5-ml HiTrap DEAE FF column (GE Healthcare, USA) pre-equilibrated with the same buffer. Proteins were eluted with a linear gradient of sodium chloride (0–1.0 M) in Tris/HCl buffer (20 mM, pH 7.0) at a flow rate of 4.5 ml min1. Fractions of 1 ml were collected, and those with laccase activity were pooled and concentrated using an Amicon Ultra-4 tube (Millpore Corp., USA). The concentrate was then applied to a Hi-Prep Sephacryl S-200 column (30 cm length, 2 cm internal diameter) pre-equilibrated with Tris/HCl buffer (20 mM, pH 7.0) containing 150 mM sodium chloride and eluted with the same buffer at a flow rate of 0.5 ml min1. Fractions with laccase activity were also pooled, concentrated by Amicon Ultra-4 tube and the purified enzyme was store at 80 °C. The purification was carried out in the room temperature. The protein concentration was determined based on the Lowry procedure using bovine serum albumin (BSA) as the standard and the enzyme purity was assessed by SDS– PAGE. 2.3. Enzyme assay
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band by incubating the gel in 100 mM sodium tartrate buffer containing 5 mM ABTS (pH 4.5) at 25 °C. The determination of the carbohydrate content of the laccase was carried out by treating the denatured enzyme (10 lg) with 1 U of endoglycosidase H (NEB, USA) under 37 °C for 1 h. The optimal temperature and the thermal stability were investigated using 100 mM sodium tartrate buffer (pH 4.5) with ABTS at 0.1 mM as substrate. The optimal temperature was determined from 10 to 90 °C. The thermal stability was measured after incubating the proteins under different temperatures for 1 h before adding 0.1 mM ABTS reaction buffer. The optimal pH value and the pH stability was determined in 100 mM sodium tartrate buffer (pH 2.0–6.0), 100 mM sodium phosphate buffer (6.0–8.0), and 100 mM glycine–NaOH buffer (pH 9.0–12.0). The stability at pH 2.0–13.0 was tested after incubating the enzyme for 1 h at 30 °C. Metal ions (CdCl2, CoCl2, CrCl3, CuCl2, FeCl2, FeCl3, MnCl2, NiCl2, HgCl2, ZnCl2, AgNO3 and Pb(NO3)2, 20 mM), EDTA (2, 5 and 20 mM), SDS (2 and 5 mM), dithiothreitol (DTT, 2 mM) sodium azide (2 mM) as well as different organic solvents (5%, 20%, 50%) were mixed with the 0.1 mM ABTS reaction buffer to obtain the respective final concentrations, and the effect of these additives on the laccase activity was also determined by incubating at 30 °C for 1 h.
3. Results and discussions 3.1. Production and purification of extracellular laccase F. solani MAS2 is a non-white rot fungus, which can use anthracene as the sole carbon source for growth and simultaneously produce laccase extracellularly. During the 40 days of incubation, the amount of laccase production increased rapidly after 15 days and the maximum activity was recorded on day 35 (575 mU mg1 protein ) (Fig. 1). The purification was achieved a 69-fold increase in activity with a yield of 9.0% (Table 1). The purity was determined by SDS– PAGE (Fig. 2a). Both native and denatured PAGE indicated a molecular weight of 72 kDa, and this observation suggests that this enzyme is a monomeric protein (Fig. 2b). Deglycosylation by endoglycosidase H have shown that the deglycosylated laccase has a molecular mass of 64 kDa (Fig. 2c), suggesting that the enzyme contains about 12% carbohydrate. The purified laccase is thus different from those from Fusarium profileratum (54 kDa, before glycosylation (BD)), Myrioconium sp. (80 kDa, BD), Marasmius
The activity of laccase was spectrophotometrically determined by applying 50 ll of each test sample into 950 ll of 0.1 mM 2,20 azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt (ABTS) at 30 °C in 100 mM sodium tartrate buffer (pH 4.5) for 1 h based on the method from Novotny et al. (1999). Activity against catechol, guaiacol, 2,6-dimethoxyphenol (DMOP) and 4-hydroxy-3,5-dimethoxybenzaldehyde azine (syringaldazine) were also determined under the same reaction condition with different initial concentrations. The absorbance coefficients were: e420nm = 36,000 M1 cm1 for ABTS, e410nm = 2211 M1 cm1 for catechol, e468nm = 14,800 M1 cm1 for DMOP, e525nm = 65,000 M1 cm1 for syringaldazine (Pozdnyakova et al., 2006), and e470nm = 6740 M1 cm1 for guaiacol (Sengupta and Mukherjee, 1997). One unit (U) of activity was defined as the production of 1 lmol product per minute under the condition of 30 °C and pH 4.5. 2.4. Enzyme characterization SDS–PAGE (12% w/v polyacrylamide) was performed as described by Laemmli (1970). Protein bands were visualized by blue-silver staining (Candiano et al., 2004). The zymogram process was carried out by using native PAGE, and visualization of enzyme
Fig. 1. Production of extracellular laccase by F. solani MAS2 growing in MSM with 50 mg l1ANT as the sole carbon source. The activity of laccase was determined by 0.1 mM ABTS in 100 mM sodium tartrate (pH 4.5) at 30 °C for 1 h. Results represents means of three experiments, and error bars indicates ± SD.
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Table 1 Steps in purifying to homogeneity the laccase activity detected in the supernatant of F. solani cultures containing 50 mg ml1 ANT.
Crude culture Precipitation DEAE FF Sephacryl S-200
Volume (ml)
Activity (mU ml1)
Total activity (mU)
Protein (mg ml1)
Specific activity (mU mg1)
Yield (%)
Fold purification
297 60 52 6.1
576.1 1536.7 1241.4 2511.1
171098.7 92202.6 64554.5 15317.6
0.111 0.100 0.012 0.007
5190.0 15367.1 103452.7 358726.9
100.0 53.9 37.7 9.0
1.0 3.0 19.9 69.1
Fig. 2. SDS–PAGE (a), native PAGE (b) and deglycosylation of purified laccase (c) from F. solani MAS2. (a) Lane 1: denatured protein marker, Lane 2: crude extracellular proteins, Lane 3: purified laccase; (b) Lane 1: purified laccase with zymogram staining, Lane 2: purified laccase with blue-silver staining, Lane 3: non-denatured protein marker; (c) Lane 1: deglycosylated laccase with endoglycosidase H treatment (arrow), Lane 2: non-deglycosylated laccase. Stained standard molecular weight markers are also indicated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
quercophilus (60 kDa, BD) and Xylaria polymorpha (67 kDa, BD; 62 kDa, after glycosylation (AD)) (Fernaud et al., 2006; Liers et al., 2007; Martin et al., 2007; Farnet et al., 2008), showing the occurrance of the extra amino acids in this enzyme.
3.2. Effects of pH value and temperature The laccase showed its highest activity at pH 3.0 and at 70 °C (Fig. 3). The optimum temperature was similar to that of the
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laccases from F. profileratum and Trametes sp. (60–75 °C) (Xiao et al., 2003; Fernaud et al., 2006), but much higher than that from the endophytic fungus Monotospora sp. (30 °C) (Wang and Hsu, 2006). The enzyme retained activity after incubation at 50 °C for 1 h, but the enzyme the activity sharply decreased when the temperature was increased to 60 °C and almost no activity was detected at 70 °C. The laccase can still oxidize ABTS when incubated at 70 °C for 5 min, but when the enzyme was incubated under the same condition without substrate, it lost almost all its activity. The results revealed that the presence of substrate protects the enzyme from being inactivated, which can explain that why the optimal temperature is not the same as the temperature of maximum enzymatic stability. Similar observations have been made with cellulase and xylanase (Wang and Hsu, 2006; Bendl et al., 2008). The enzyme remained active at pH values from 3.0 to 12.0, and its activity at 2.0 and 12.0 were 56.6% and 85.5% of that at 7.0, respectively. The range of pH stability is much wider than that of the laccases from Myrioconium sp. (Martin et al., 2007) and X. polymorpha (Liers et al., 2007).
Fig. 4. Effect of different organic solvents on the activity of purified laccase. All the experiments are performed with the same purified laccase, and activity without the addition of organic solvents was considered as 100%. Error bars shown are standard deviations of triplicate samples.
3.3. Effects of different organic co-solvents
activity was almost completely inhibited in all the tested solvents at concentrations of 50% (Fig. 4).
Since PAHs are hydrophobic, it is desirable to add organic solvents to improve their solubility in aqueous reaction system (Klibanov, 2001), however, these solvents can affect some enzymes (Redakiewicz-Nowak et al., 2000; Farnet et al., 2008). The F. solani MAS2 laccase retained approximately 75–80% of its initial activity in the presence of 5% acetonitrile and methanol (Fig. 4), suggesting that the enzyme could be suitable for use in reactions requiring such a concentration of these solvents. However, the inhibitory effect increased with increasing concentration of solvents, and the
3.4. Effects of heavy metal ions and enzyme inhibitors Heavy metal ions are common environmental pollutants and can affect the production and stability of the extracellular enzymes (Farnet et al., 2008). Table 3 shows the purified laccase was partially inhibited by 20 mM Cd2+, Co2+, Cr3+, Cu2+, Mn2+, Ni2+, Zn2+, and Fe2+, while Fe3+ and Ag+ completely inhibited the enzyme. Many studies have shown that laccase is a copper-containing oxidase (Thurston, 1994; Mayer and Staples, 2002), and that a Table 2 Kinetic constants of laccase from F. solani strain MAS2 reacting with different phenolic compounds. Substrate
Km (lM)
Vmax (lM min1 mg1)
kcat (s1)
kcat/Km (s1 M1)
ABTS Catechol DMOP Guaiacol
79.4 3433.6 8.6 2657.1
1483.1 620.6 1675.0 392.8
107.1 44.9 121.0 28.4
1.3 106 1.3 104 1.4 107 1.1 104
Table 3 Effects of metal ions, EDTA, sodium azide, DTT and SDS on laccase activity.
Fig. 3. Optima and stability of (a) temperature and (b) pH value for purified laccase reacting with ABTS. Optima curve (h); stability curve (j). The highest value of activity for each analytical curve was considered as 100%, and error bars shown are standard deviations of triplicate samples.
Compounds (mM)
Residual activity (%)
Control CdCl2 (20) CoCl2 (20) CrCl3 (20) CuCl2 (20) FeCl2 (20) FeCl3 (20) MnCl2 (20) NiCl2 (20) HgCl2 (20) ZnCl2 (20) Pb(NO3)2 (20) AgNO3 (20) SDS (20) SDS (2) EDTA (20) EDTA (5) EDTA (2) NaN3 (2) Dithiothreitol (2)
100 71.1 ± 0.7 61.9 ± 0.1 46.2 ± 0.4 68.9 ± 0.2 4.0 ± 0.2 0 49.1 ± 0.2 62.0 ± 0.1 130.7 ± 0.3 65.5 ± 0.1 97.2 ± 1.5 0 93.8 ± 1.0 131.1 ± 0.6 20.8 ± 1.5 33.9 ± 0.9 54.3 ± 1.2 0 0
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4. Conclusions The laccase produced from F. solani MAS2 is a monomeric glycoprotein with high pH stability. Both the stimulatory function of Hg2+ and the tolerance of Pb2+ have also indicated the novel characterizations of this enzyme. Further studies should be focused on the analysis on the laccase-encoded gene, and also investigation on whether the purified laccase could be involved in the direct oxidization of different PAH compounds in vitro, which could provide the potential on PAHs degradation and other biotechnological applications. Acknowledgements
Fig. 5. Effect of different concentrations of Hg2+ on the activity of purified laccase. Activity without addition of Hg2+ was considered as 100%. Error bars shown are standard deviations of triplicate samples.
low level of Cu2+ concentration (0.5–3.5 mM) enhanced the enzyme activity to some extent (Baldrian and Gabriel, 2002; Hatvani and Mecs, 2003; Rancano et al., 2003).The same was not the case for the MAS2 laccase as it was inhibited by Cu2+ ions even at 0.5 mM (3.9% of inhibition), whereas 20 mM Hg2+ increased the MAS2 laccase activity and toxic Pb2+ exerted almost no effect. Moreover, the stimulation of Hg2+ on laccase was increased by improving the Hg2+ concentration into 40 mM (Fig. 5), which suggests that the mercuric ions could be an activator for this enzyme, and could perhaps function like copper ions in other laccases (Rancano et al., 2003; Farnet et al., 2008). To our knowledge, the laccase activation by Hg2+ has not been reported, which also indicated the novelty of this laccase. The enzyme was totally inhibited by sodium azide (an inhibitor of oxidase), which indicates its function as an oxidase. DTT, a strong reducing agent on the disulphide bonds, was another strong inhibitor of the enzyme, suggesting the existence of the disulphide structure in its active domain. In addition, the laccase can be partially inhibited by EDTA (Table 3), which indicates that this laccase could contain a metal-binding domain. The addition of sodium dodecyl sulphate (SDS) had almost no effect on the enzyme activity, and it could even enhance the reaction rate at a lower concentration (Table 3). Similar results have been investigated by Jiang et al. (2003) on a purified polyphenol oxidase from tobacco leaf, which indicates that SDS could change the structure of the enzyme, and make access to the catalytically active center.
3.5. Oxidation of phenolic compounds by laccase Table 2 shows the Michaelis–Menten constants of laccase reacting with different substrates. The enzyme exhibited the highest activity with ABTS and DMOP, with respective apparent Km value of 8.6 and 79.4 lM determined from the Lineweaver–Burk plot. The apparent Km value determined for catechol and guaiacol were 3443.6 and 2657.1 lM, respectively. The kcat value for DMOP and ABTS was 107.1 and 121.0 s1, respectively, much higher than that from catechol and guaiacol. The laccase under study could also oxidize syringaldazine, but the reaction rate was very low under the condition provided. The differences of kcat value have also indicated that DMOP, ABTS are more effective substrates from F. solani MAS2 than catechol and guaiacol for the purified laccase. By comparisons on the chemical structure of these substrates, the differences is correlated to the number of reactive groups (–OH, –OCH3 or –SO3H), and substrate with more reactive groups could be oxidized by laccase in a lower concentration, which lead to a lower Km and kcat values (D’Annibale et al., 1996).
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Purification and characterization of an extracellular laccase from the anthracene-degrading fungus Fusarium solani MAS2 Yi-Rui Wu a,*, Zhu-Hua Luo a,b, R. Kwok-Kei Chow a, L.L.P. Vrijmoed a,** a b
Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR, PR China Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, 178 Daxue Road, Xiamen 361005, PR China
a r t i c l e
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Article history: Received 21 April 2010 Received in revised form 14 July 2010 Accepted 21 July 2010 Available online 27 July 2010 Keywords: Polycyclic aromatic hydrocarbons (PAHs) ABTS Heavy metal tolerance Mangrove sediment
a b s t r a c t An extracellular laccase was purified from the culture medium of the non-white rot, anthracene-degrading fungal strain Fusarium solani MAS2. Both native PAGE and SDS–PAGE revealed one single band corresponding to a molecular weight of about 72 kDa. Treatment with endoglycosidase H reduced the molecular weight by 12%. The purified laccase maintained stable at pH 3–11 and up to 50 °C. The highest activity was detected at pH 3.0 and at 70 °C. The enzyme retained 46.2–97.2% of it activity in the presence of 20 mM Pb2+, Ni2+, Cr3+, and its activity was enhanced in the presence of 20 mM Hg2+. The laccase retained more than 50% of its activity in the presence of 5% acetone, acetonitrile, dimethyl sulphoxide (DMSO), ethanol and methanol. The kinetic constants (Km and kcat) showed that 2,6-dimethoxyphenol (DMOP) and 2,20 -azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt (ABTS) were the more effective substrates rather than catechol and guaiacol. The novel properties of this laccase suggest its potential for biotechnological and environmental applications. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Laccase (benzenediol: oxygen oxidoreductase; EC 1.10.3.2) is a polyphenol oxidase with the potential for bioremediation of related compounds such as kraft lignin bleaching, detoxification of environmental pollutants and wastewater treatment (Mayer and Staples, 2002; Xiao et al., 2003). Laccases can be divided into three different groups. Blue laccases belong to the multicopper-oxidase enzymes family with the maximum absorption spectrum at 600 nm (Messerschmidt and Huber, 1990), whereas the yellow and white laccases were lack of that absorption spectrum, and have a different oxidation status in their respective catalytic centers (Leontievsky et al., 1997; Palmieri et al., 1997; Pozdnyakova et al., 2006). Laccases are a kind of versatile carbohydrate-containing molecules with different structures from different species (Thurston, 1994). For example, laccase purified from Cantharellus cibarius were dimers with identical subunits (46 kDa) (Ng and Wang, 2004), but laccase from Gaeumannomyces graminis var. tritici had an apparent molecular mass of 190 kDa with three subunits (Edens et al., 1999). Some organisms, such as Podospora ansernia and Trametes sp., have two or more different isozymes (Durrens, 1981; Xiao et al., 2003). Both intracellular and extracellular laccase are known (Mayer and Staples, 2002; Nagai et al., 2003). * Corresponding author. Tel.: +852 97165286; fax: +852 27844093. ** Corresponding author. Tel.: +852 34429966; fax: +852 27844093. E-mail addresses: [email protected] (Y.R. Wu), [email protected] (L.L.P. Vrijmoed). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.07.091
Laccase synthesis can be constitutive or inducible, and the inducing substrates include a wide range of compounds, such as aromatic compounds, anilines, lignin and even various artificial dyes (Leonowicz et al., 2001; Xiao et al., 2003). The diversity of laccases reflects their various functions, including lignin degradation, pigment production, and even plant pathogenesis (Koschorreck et al., 2008). In the current study, a laccase was purified from the non-white rot fungal strain, Fusarium solani strain MAS2, and characterize with respect to its molecular weight, optimal temperature, pH and stability, tolerance to heavy metal ions, organic solvents as well as reaction with different aromatic compounds. The fungus produced this enzyme in the mineral salt medium with anthracene as the only carbon source substrate without producing detectable lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP). The enzyme differs from previously reported laccases (Xiao et al., 2003; Martin et al., 2007) with respect to its high tolerance against most of heavy metal ions, wide range of pH stability, especially the activity enhancement by mercuric ions. 2. Methods 2.1. Microorganism and cultivation Strain MAS2 was isolated from polycyclic aromatic hydrocarbons (PAHs)-contaminated mangrove sediments with the degradative ability of a 3-ring PAH-anthracene, and identified as
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F. solani according to the 18S rRNA gene sequencing as well as the microscopic morphology based on the method of Wu et al. (2009). The fungus was monthly transferred to fresh mineral salt medium (MSM) agar (1%) slants containing anthracene (50 mg l1) as the sole carbon source and stored at 25 °C. The MSM was modified from Hartmans et al. (1989), which contained (g l1): NaCl, 12.3; (NH4)2SO4, 2.0; K2HPO4, 1.55; NaH2PO42H2O, 0.85; MgCl26H2O, 2.53; CaCl22H2O, 0.73; KCl, 0.33; MgSO47H2O, 3.15; and (mg l1) NaHCO3, 90.0; ZnSO47H2O, 2.0; FeSO47H2O, 5.0; Na2MoO42H2O, 0.2; CuSO45H2O, 0.2; CoCl26H2O, 0.4; MnCl22H2O, 1.0; H3BO3, 1.0; KI, 0.5; KAl(SO4)212H2O, 0.5; NiCl26H2O, 0.5; pH 5.5 ± 0.1. For purification of the laccase, strain MAS2 was inoculated into the liquid MSM by incubating at 25 °C on a rotary shaker at 150 rpm for 25 days. 2.2. Enzyme purification The laccase was purified from a cell-free culture medium (250 ml) after filtration through qualitative filter paper (Grade 1, Whatman, UK) and centrifugation (10,000g, 30 min). The supernatant was mixed with three volumes of pre-cooled ethanol for 1 h at 20 °C. The precipitate was collected by centrifugation (5000g, 20 min), resuspended and dialyzed with 20 mM Tris/HCl buffer (pH 7.0) overnight. The suspension was applied to a 5-ml HiTrap DEAE FF column (GE Healthcare, USA) pre-equilibrated with the same buffer. Proteins were eluted with a linear gradient of sodium chloride (0–1.0 M) in Tris/HCl buffer (20 mM, pH 7.0) at a flow rate of 4.5 ml min1. Fractions of 1 ml were collected, and those with laccase activity were pooled and concentrated using an Amicon Ultra-4 tube (Millpore Corp., USA). The concentrate was then applied to a Hi-Prep Sephacryl S-200 column (30 cm length, 2 cm internal diameter) pre-equilibrated with Tris/HCl buffer (20 mM, pH 7.0) containing 150 mM sodium chloride and eluted with the same buffer at a flow rate of 0.5 ml min1. Fractions with laccase activity were also pooled, concentrated by Amicon Ultra-4 tube and the purified enzyme was store at 80 °C. The purification was carried out in the room temperature. The protein concentration was determined based on the Lowry procedure using bovine serum albumin (BSA) as the standard and the enzyme purity was assessed by SDS– PAGE. 2.3. Enzyme assay
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band by incubating the gel in 100 mM sodium tartrate buffer containing 5 mM ABTS (pH 4.5) at 25 °C. The determination of the carbohydrate content of the laccase was carried out by treating the denatured enzyme (10 lg) with 1 U of endoglycosidase H (NEB, USA) under 37 °C for 1 h. The optimal temperature and the thermal stability were investigated using 100 mM sodium tartrate buffer (pH 4.5) with ABTS at 0.1 mM as substrate. The optimal temperature was determined from 10 to 90 °C. The thermal stability was measured after incubating the proteins under different temperatures for 1 h before adding 0.1 mM ABTS reaction buffer. The optimal pH value and the pH stability was determined in 100 mM sodium tartrate buffer (pH 2.0–6.0), 100 mM sodium phosphate buffer (6.0–8.0), and 100 mM glycine–NaOH buffer (pH 9.0–12.0). The stability at pH 2.0–13.0 was tested after incubating the enzyme for 1 h at 30 °C. Metal ions (CdCl2, CoCl2, CrCl3, CuCl2, FeCl2, FeCl3, MnCl2, NiCl2, HgCl2, ZnCl2, AgNO3 and Pb(NO3)2, 20 mM), EDTA (2, 5 and 20 mM), SDS (2 and 5 mM), dithiothreitol (DTT, 2 mM) sodium azide (2 mM) as well as different organic solvents (5%, 20%, 50%) were mixed with the 0.1 mM ABTS reaction buffer to obtain the respective final concentrations, and the effect of these additives on the laccase activity was also determined by incubating at 30 °C for 1 h.
3. Results and discussions 3.1. Production and purification of extracellular laccase F. solani MAS2 is a non-white rot fungus, which can use anthracene as the sole carbon source for growth and simultaneously produce laccase extracellularly. During the 40 days of incubation, the amount of laccase production increased rapidly after 15 days and the maximum activity was recorded on day 35 (575 mU mg1 protein ) (Fig. 1). The purification was achieved a 69-fold increase in activity with a yield of 9.0% (Table 1). The purity was determined by SDS– PAGE (Fig. 2a). Both native and denatured PAGE indicated a molecular weight of 72 kDa, and this observation suggests that this enzyme is a monomeric protein (Fig. 2b). Deglycosylation by endoglycosidase H have shown that the deglycosylated laccase has a molecular mass of 64 kDa (Fig. 2c), suggesting that the enzyme contains about 12% carbohydrate. The purified laccase is thus different from those from Fusarium profileratum (54 kDa, before glycosylation (BD)), Myrioconium sp. (80 kDa, BD), Marasmius
The activity of laccase was spectrophotometrically determined by applying 50 ll of each test sample into 950 ll of 0.1 mM 2,20 azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt (ABTS) at 30 °C in 100 mM sodium tartrate buffer (pH 4.5) for 1 h based on the method from Novotny et al. (1999). Activity against catechol, guaiacol, 2,6-dimethoxyphenol (DMOP) and 4-hydroxy-3,5-dimethoxybenzaldehyde azine (syringaldazine) were also determined under the same reaction condition with different initial concentrations. The absorbance coefficients were: e420nm = 36,000 M1 cm1 for ABTS, e410nm = 2211 M1 cm1 for catechol, e468nm = 14,800 M1 cm1 for DMOP, e525nm = 65,000 M1 cm1 for syringaldazine (Pozdnyakova et al., 2006), and e470nm = 6740 M1 cm1 for guaiacol (Sengupta and Mukherjee, 1997). One unit (U) of activity was defined as the production of 1 lmol product per minute under the condition of 30 °C and pH 4.5. 2.4. Enzyme characterization SDS–PAGE (12% w/v polyacrylamide) was performed as described by Laemmli (1970). Protein bands were visualized by blue-silver staining (Candiano et al., 2004). The zymogram process was carried out by using native PAGE, and visualization of enzyme
Fig. 1. Production of extracellular laccase by F. solani MAS2 growing in MSM with 50 mg l1ANT as the sole carbon source. The activity of laccase was determined by 0.1 mM ABTS in 100 mM sodium tartrate (pH 4.5) at 30 °C for 1 h. Results represents means of three experiments, and error bars indicates ± SD.
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Table 1 Steps in purifying to homogeneity the laccase activity detected in the supernatant of F. solani cultures containing 50 mg ml1 ANT.
Crude culture Precipitation DEAE FF Sephacryl S-200
Volume (ml)
Activity (mU ml1)
Total activity (mU)
Protein (mg ml1)
Specific activity (mU mg1)
Yield (%)
Fold purification
297 60 52 6.1
576.1 1536.7 1241.4 2511.1
171098.7 92202.6 64554.5 15317.6
0.111 0.100 0.012 0.007
5190.0 15367.1 103452.7 358726.9
100.0 53.9 37.7 9.0
1.0 3.0 19.9 69.1
Fig. 2. SDS–PAGE (a), native PAGE (b) and deglycosylation of purified laccase (c) from F. solani MAS2. (a) Lane 1: denatured protein marker, Lane 2: crude extracellular proteins, Lane 3: purified laccase; (b) Lane 1: purified laccase with zymogram staining, Lane 2: purified laccase with blue-silver staining, Lane 3: non-denatured protein marker; (c) Lane 1: deglycosylated laccase with endoglycosidase H treatment (arrow), Lane 2: non-deglycosylated laccase. Stained standard molecular weight markers are also indicated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
quercophilus (60 kDa, BD) and Xylaria polymorpha (67 kDa, BD; 62 kDa, after glycosylation (AD)) (Fernaud et al., 2006; Liers et al., 2007; Martin et al., 2007; Farnet et al., 2008), showing the occurrance of the extra amino acids in this enzyme.
3.2. Effects of pH value and temperature The laccase showed its highest activity at pH 3.0 and at 70 °C (Fig. 3). The optimum temperature was similar to that of the
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laccases from F. profileratum and Trametes sp. (60–75 °C) (Xiao et al., 2003; Fernaud et al., 2006), but much higher than that from the endophytic fungus Monotospora sp. (30 °C) (Wang and Hsu, 2006). The enzyme retained activity after incubation at 50 °C for 1 h, but the enzyme the activity sharply decreased when the temperature was increased to 60 °C and almost no activity was detected at 70 °C. The laccase can still oxidize ABTS when incubated at 70 °C for 5 min, but when the enzyme was incubated under the same condition without substrate, it lost almost all its activity. The results revealed that the presence of substrate protects the enzyme from being inactivated, which can explain that why the optimal temperature is not the same as the temperature of maximum enzymatic stability. Similar observations have been made with cellulase and xylanase (Wang and Hsu, 2006; Bendl et al., 2008). The enzyme remained active at pH values from 3.0 to 12.0, and its activity at 2.0 and 12.0 were 56.6% and 85.5% of that at 7.0, respectively. The range of pH stability is much wider than that of the laccases from Myrioconium sp. (Martin et al., 2007) and X. polymorpha (Liers et al., 2007).
Fig. 4. Effect of different organic solvents on the activity of purified laccase. All the experiments are performed with the same purified laccase, and activity without the addition of organic solvents was considered as 100%. Error bars shown are standard deviations of triplicate samples.
3.3. Effects of different organic co-solvents
activity was almost completely inhibited in all the tested solvents at concentrations of 50% (Fig. 4).
Since PAHs are hydrophobic, it is desirable to add organic solvents to improve their solubility in aqueous reaction system (Klibanov, 2001), however, these solvents can affect some enzymes (Redakiewicz-Nowak et al., 2000; Farnet et al., 2008). The F. solani MAS2 laccase retained approximately 75–80% of its initial activity in the presence of 5% acetonitrile and methanol (Fig. 4), suggesting that the enzyme could be suitable for use in reactions requiring such a concentration of these solvents. However, the inhibitory effect increased with increasing concentration of solvents, and the
3.4. Effects of heavy metal ions and enzyme inhibitors Heavy metal ions are common environmental pollutants and can affect the production and stability of the extracellular enzymes (Farnet et al., 2008). Table 3 shows the purified laccase was partially inhibited by 20 mM Cd2+, Co2+, Cr3+, Cu2+, Mn2+, Ni2+, Zn2+, and Fe2+, while Fe3+ and Ag+ completely inhibited the enzyme. Many studies have shown that laccase is a copper-containing oxidase (Thurston, 1994; Mayer and Staples, 2002), and that a Table 2 Kinetic constants of laccase from F. solani strain MAS2 reacting with different phenolic compounds. Substrate
Km (lM)
Vmax (lM min1 mg1)
kcat (s1)
kcat/Km (s1 M1)
ABTS Catechol DMOP Guaiacol
79.4 3433.6 8.6 2657.1
1483.1 620.6 1675.0 392.8
107.1 44.9 121.0 28.4
1.3 106 1.3 104 1.4 107 1.1 104
Table 3 Effects of metal ions, EDTA, sodium azide, DTT and SDS on laccase activity.
Fig. 3. Optima and stability of (a) temperature and (b) pH value for purified laccase reacting with ABTS. Optima curve (h); stability curve (j). The highest value of activity for each analytical curve was considered as 100%, and error bars shown are standard deviations of triplicate samples.
Compounds (mM)
Residual activity (%)
Control CdCl2 (20) CoCl2 (20) CrCl3 (20) CuCl2 (20) FeCl2 (20) FeCl3 (20) MnCl2 (20) NiCl2 (20) HgCl2 (20) ZnCl2 (20) Pb(NO3)2 (20) AgNO3 (20) SDS (20) SDS (2) EDTA (20) EDTA (5) EDTA (2) NaN3 (2) Dithiothreitol (2)
100 71.1 ± 0.7 61.9 ± 0.1 46.2 ± 0.4 68.9 ± 0.2 4.0 ± 0.2 0 49.1 ± 0.2 62.0 ± 0.1 130.7 ± 0.3 65.5 ± 0.1 97.2 ± 1.5 0 93.8 ± 1.0 131.1 ± 0.6 20.8 ± 1.5 33.9 ± 0.9 54.3 ± 1.2 0 0
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4. Conclusions The laccase produced from F. solani MAS2 is a monomeric glycoprotein with high pH stability. Both the stimulatory function of Hg2+ and the tolerance of Pb2+ have also indicated the novel characterizations of this enzyme. Further studies should be focused on the analysis on the laccase-encoded gene, and also investigation on whether the purified laccase could be involved in the direct oxidization of different PAH compounds in vitro, which could provide the potential on PAHs degradation and other biotechnological applications. Acknowledgements
Fig. 5. Effect of different concentrations of Hg2+ on the activity of purified laccase. Activity without addition of Hg2+ was considered as 100%. Error bars shown are standard deviations of triplicate samples.
low level of Cu2+ concentration (0.5–3.5 mM) enhanced the enzyme activity to some extent (Baldrian and Gabriel, 2002; Hatvani and Mecs, 2003; Rancano et al., 2003).The same was not the case for the MAS2 laccase as it was inhibited by Cu2+ ions even at 0.5 mM (3.9% of inhibition), whereas 20 mM Hg2+ increased the MAS2 laccase activity and toxic Pb2+ exerted almost no effect. Moreover, the stimulation of Hg2+ on laccase was increased by improving the Hg2+ concentration into 40 mM (Fig. 5), which suggests that the mercuric ions could be an activator for this enzyme, and could perhaps function like copper ions in other laccases (Rancano et al., 2003; Farnet et al., 2008). To our knowledge, the laccase activation by Hg2+ has not been reported, which also indicated the novelty of this laccase. The enzyme was totally inhibited by sodium azide (an inhibitor of oxidase), which indicates its function as an oxidase. DTT, a strong reducing agent on the disulphide bonds, was another strong inhibitor of the enzyme, suggesting the existence of the disulphide structure in its active domain. In addition, the laccase can be partially inhibited by EDTA (Table 3), which indicates that this laccase could contain a metal-binding domain. The addition of sodium dodecyl sulphate (SDS) had almost no effect on the enzyme activity, and it could even enhance the reaction rate at a lower concentration (Table 3). Similar results have been investigated by Jiang et al. (2003) on a purified polyphenol oxidase from tobacco leaf, which indicates that SDS could change the structure of the enzyme, and make access to the catalytically active center.
3.5. Oxidation of phenolic compounds by laccase Table 2 shows the Michaelis–Menten constants of laccase reacting with different substrates. The enzyme exhibited the highest activity with ABTS and DMOP, with respective apparent Km value of 8.6 and 79.4 lM determined from the Lineweaver–Burk plot. The apparent Km value determined for catechol and guaiacol were 3443.6 and 2657.1 lM, respectively. The kcat value for DMOP and ABTS was 107.1 and 121.0 s1, respectively, much higher than that from catechol and guaiacol. The laccase under study could also oxidize syringaldazine, but the reaction rate was very low under the condition provided. The differences of kcat value have also indicated that DMOP, ABTS are more effective substrates from F. solani MAS2 than catechol and guaiacol for the purified laccase. By comparisons on the chemical structure of these substrates, the differences is correlated to the number of reactive groups (–OH, –OCH3 or –SO3H), and substrate with more reactive groups could be oxidized by laccase in a lower concentration, which lead to a lower Km and kcat values (D’Annibale et al., 1996).
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