A 70-kDa extracellular laccase was purified from the rice blast fungus Magnaporthe grisea using gel filtration and ion exchange chromatography The procedure provided 282-fold purification with a specific enzyme activity of 225.91 U mg−1 and a yield of 11.92%. The enzyme oxidized a wide range of substrates. The highest level of oxidation was detected with syringaldazine as the substrate. Using syringaldazine as the substrate, the enzyme exhibited a pH optimum of 6 and temperature optimum of 30°C, and its Km was 0.118 mM. The enzyme was strongly inhibited by Cu-chelating agents.
Magnaporthe grisea, the rice blast fungus, is a major pathogen for rice and also infects other economically important crops such as barley, wheat, and millet. Rice blast is one of the most severe fungal diseases of rice throughout the world . Laccase (benzenediol:oxygen oxidoreductase, EC 184.108.40.206) is a copper-containing enzyme that catalyzes the oxidation of a phenolic substrate by coupling it to the reduction of oxygen to water. Fungal laccases display a wide substrate range, and are known to catalyze the polymerization, depolymerization, and methylation and/or demethylation of phenolic compounds . Laccases are involved in several physiological functions, such as degradation of lignin . The prerequisites are pH, temperature and availability of intermediate products for successful lignin breakdown . Additional roles of laccase in fungal development have been proposed. These include pigmentation in Aspergillus nidulans[5,6] and Aspergillus fumigatus, and morphogenesis of the edible mushroom Lentinula edodes. Laccases have been shown to be an important virulence factor in many diseases caused by fungi. For example, Dutch elm disease has been associated with laccase secretion by Ophiostoma ulmi and Ophiostoma novo-ulmi; strains of Cryphonectria parasitica with elevated levels of laccase caused severe chestnut blight disease  The function of laccase is still undetermined in the rice blast fungus. We report for the first time the purification, characterization, substrate and inhibitor studies of an extracellular laccase from the plant pathogen, M. grisea.
2Materials and methods
2.1Organism and culture conditions
M. grisea conidia were maintained on YEG agar plates (0.2% yeast extract, 1% dextrose, and 1.75% agar). 1.0×106 conidia ml−1 were inoculated in liquid complete medium containing 1% glucose, 0.2% peptone, 0.1% yeast extract, 0.1% trace element solution, 0.1% vitamin supplement, 0.6% NaNO3, 0.05% KCl, 0.05% MgSO4, and 0.15% KH2PO4, pH 6.5 , and incubated at 26°C on a rotary shaker (200 rpm). For copper induction experiments, the fungus was grown in Vogel's medium with and without CuSO4 to a final concentration of 400 μM. Fungal biomass was determined at specific time intervals by vacuum filtering mycelia through tarred 0.22-μm cellulose nitrate filter (Sartorius AG, Germany), washed with distilled water and dried to constant weight at 80°C. Laccase activity was monitored by removing 5 ml of culture filtrate at 24-h intervals for 8 days and centrifuging at 5000×g for 30 min at 4°C. 50 μl of the clear filtrate was assayed for laccase activity.
Laccase activity was determined at 30°C using 0.5 mM syringaldazine (?m=6.4×104 M−1 cm−1; Sigma, USA) in reaction mixture (1 ml) containing 0.05 M MES-NaOH (pH 6), and 10 μl of the culture supernatant for 5 min.
One unit of enzyme activity is defined as the amount of enzyme required to oxidize 1 μmol of syringaldazine  under standard assay conditions. Protein concentration was determined by the method of Bradford  using bovine serum albumin as standard.
2.3Enzyme purification and high performance liquid chromatography (HPLC) analysis
The culture supernatant was concentrated to a volume of 385 ml from 5 l with a tangential flow membrane filter using a 30-kDa filter cassette (Millipore, USA). The concentrated supernatant was dialyzed overnight against 10 mM MES-NaOH buffer (pH 6) with several changes and was loaded onto a Waters QMA-Accell anion exchange column (Millipore) previously equilibrated with 50 mM MES-NaOH buffer (pH 6). Proteins were eluted with a linear gradient of 0–0.4 M NaCl in MES-NaOH buffer pH 6. Laccase-positive fractions were pooled, concentrated with an Amicon ultrafiltration cell containing a 3-kDa molecular mass cutoff membrane (MWCO), and applied to a Sephadex G-75 (0.8×28 cm, Pharmacia) column previously equilibrated with 50 mM MES-NaOH buffer, pH 6. Fractions containing laccase activity were pooled, concentrated with an Amicon ultrafiltration cell containing 10-kDa MWCO and stored at −20°C. 1.5-ml fractions were collected, active fractions were pooled, and aliquots of 200 μl were loaded onto a Bio-sil TSK-250 (Bio-Rad) HPLC gel filtration column (300×7.5 mm), equilibrated in 20 mM Tris-MES buffer (pH 6.5), containing 100 mM NaCl and 0.1 mM dithiothreitol, at 1.0 ml min−1 flow rate. Active fractions were collected and used for the biochemical characterization of laccase activity. Protein standards (wheat germ phosphatase, 100 000 Da; bovine serum albumin, 66 000 Da; aldolase, 44 000 Da; and cytochrome c, 14 000 Da) were used to estimated the size of laccase.
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (10% w/v gel) was performed according to Laemmli . Protein was silver stained as described  and compared with molecular mass markers (Gibco BRL). Native PAGE (8% w/v gel) was performed on aliquots of culture fluids at different time intervals in the presence of ABTS (0.1% in 50 mM phosphate-citrate buffer, pH 5.0). Protein bands exhibiting laccase activity stained green with ABTS (0.1% w/v) in 50 mM phosphate-citrate buffer, pH 5.0.
The pH optimum of the enzyme was determined within a pH range of 3–9 using syringaldazine as substrate in buffers: 50 mM citrate buffer for pH 3–6; 50 mM phosphate for pH 6–8; and 50 mM Tris–HCl for pH 8–9. Stability of the enzyme was determined by measuring the residual activity of the reaction mixtures by varying the pH using syringaldazine for 1 h at 30°C. The effect of temperature on enzymatic activity was determined at pH 6 in the range of 20–80°C. The temperature stability of the enzyme was determined at different temperatures for 1 h with syringaldazine. Oxidation rates of several substrates were monitored at their respective wavelengths after addition of 1.5 μg of laccase. The reaction mixtures contained 0.1 mM substrate in 100 mM MES-NaOH buffer (pH 6.0). The effect of various inhibitors on laccase activity was assayed at the optimal pH with syringaldazine with 50 mM MES-NaOH (pH 6). The effects of sodium azide, salicylhydroxyamic acid , kojic acid  and N-hydroxyglycine , on enzyme activity were determined after 15 min of pre-incubation at 30°C with the various inhibitors. Kinetic measurements were made at 30°C, with initial velocity measurements performed in 1-ml glass cuvettes. Reactions were initiated by the addition of laccase and initial rates were obtained from the linear portion of the progress curve. Kinetic data (Km and Vmax) for syringaldazine were fitted to the appropriate equation with the programs of Athel Cornish-Bowden  to obtain the desired kinetic parameters.
3Results and discussion
Laccases are ubiquitous enzymes in higher fungi and higher plants and have been shown to play important roles in the developmental cycle of various fungi. However, no comparable studies are known for M. grisea laccase. In this study, we focused on the purification and characterization of laccase from culture supernatant of M. grisea. Laccase activity was detected in liquid cultures as early as 24 h and was active at up to 96 h. Maximum activity of 21.0 U was detected at 24 h at which time the biomass level was 0.57 g l−1. Laccase activity was not detected as biomass increased to 5.26 g l−1 at 120 h (Fig. 1). Increase in biomass did not result in higher activity which indicated that laccase activity is independent of biomass production. Induction experiments indicate that laccase activity increased eight-fold after CuSO4 was added to a final concentration of 400 μM on the third day of incubation in Vogel's medium. Non-denaturing PAGE analysis of culture supernatant with ABTS as substrate detected more than one form of the native enzyme. The intensity of the laccase band at 24 h suggested there may be more than one isoform of laccase. ABTS-stained laccase bands with different electrophoretic mobilities compared to lane 1 (24 h) suggested the presence of different or modified forms of laccase (Fig. 2A). However, at later time points, we do not rule out the possibility of proteolytic cleavage of laccase resulting in differentially stained bands by ABTS. Periodic acid Schiff staining of the native gel indicated that the enzyme was glycosylated (data not shown). Additional forms have been reported to be present in C. parasitica. This heterogeneity could be due to differences in the extent of glycosylation . Many laccases have been reported to be produced as multiple isoforms, e.g. Trametes villosa produces at least three isoforms  and Pleurotus ostreatus four isoforms . Laccase activity was not detected from intracellular protein samples (data not shown).
Laccase was purified to near homogeneity by ultrafiltration, ion exchange and gel filtration chromatography as shown in Table 1. An overall 282-fold purification and activity recovery of 11.92% with specific activity of 225.91 U mg−1 of protein was achieved. The molecular mass of the laccase as determined by SDS–PAGE was 70 kDa (Fig. 2B). The molecular mass of the purified laccase is consistent with the molecular masses of most other fungal laccases, which have been reported to be between 60 000 and 80 000 Da . The relationship between laccase activity and substrate concentration produced a typical Michaelis–Menten curve. Syringaldazine, which is considered a specific substrate for laccase , exhibited a Km of 0.118 mM with the purified enzyme.
Table 1. Purification steps of laccase produced by M. grisea
Specific activity (U mg−1)
Total activity (U)
Total protein (mg)
Ultrafiltration (30-kDa MWCO)
The optimal pH of enzyme oxidation of syringaldazine as the substrate was determined at pH 6 (Fig. 3A). The enzyme exhibited broad pH optima between 6 and 8. A slight change in the oxidation rate of syringaldazine by the enzyme was observed between pH 7 and 8. The enzyme showed 67% of its maximum activity at pH 8 (Fig. 3B), suggesting that the enzyme was active at alkaline pH. The activity of the enzyme was determined at various temperatures (20–70°C) using syringaldazine as substrate. The enzyme showed a temperature optimum of 30°C (Fig. 4A). The enzyme showed approximately 50% reduction in specific activity at 40°C (Fig. 4A) and the activity of the enzyme dropped sharply at elevated temperatures of 60–80°C, relative to enzyme activity at 30°C (100%). Thermal stability of the assay reaction mixture was measured after 1 h of pre-incubation (Fig. 4B). The enzyme retained approximately 45–55% of relative activity at temperatures of 50–60°C (Fig. 4B). The enzyme was totally inactivated beyond 60°C, after 1 h of pre-incubation.
3.1Substrate specificity and inhibitor studies
Table 2 presents the relative rate of oxidation of purified M. grisea laccase with various substrates. The enzyme exhibited oxidative activity towards a wide range of substrates (o- and p-phenols, ferulic acid, and 3,4-dihydroxyphenylalanine), α-naphthol and syringaldazine. The affinity for various substrates was of the following order: syringaldazine>l-3,4-dihydroxyphenylalanine (DOPA)>ferulic acid>α-naphthol>hydroquinone>guaiacol>p-cresol>catechol>4-methylcatechol. Among the ascomycetes group, M. grisea laccase enzyme was able to serve as electron donor with guaiacol, in contrast to laccase II of A. nidulans which was unable to oxidize syringaldazine, guaiacol and ABTS . Although a direct comparison to other lignin-degrading fungi cannot be made due to differences in the reaction conditions, the enzyme in this study was able to oxidize phenolic substrates suggesting a possible role in lignin degradation. Based on the substrate specificity, laccase from M. grisea oxidized a wide range of substrates like other fungal laccases . To characterize the enzyme further, several potential inhibitors for laccase were tested using syringaldazine as the substrate (Table 3). Tropolone, a compound which is known to inhibit the copper-containing tyrosinases , did not affect laccase activity at up to 8.0 mM (unpublished data). However, the enzyme was inhibited at 1 mM sodium azide (99%) suggesting the presence of metal ion in the catalytic center. In keeping with the general properties of fungal laccases , the enzyme was inhibited by 1 mM salicylhydroxyamic acid (97%), 5 mM kojic acid (74%) and 0.1 mM N-hydroxyglycine (87%).
Table 2. Substrate specificity of M. grisea laccase
aRelative activities were obtained by comparing change in optical density to syringaldazine (100%). All values are the means of duplicate experiments, and the coefficients of variation were less than 5%.
Relative activity (%)a
Table 3. Effect of various inhibitors on oxidation of syringaldazine by M. grisea laccase
In conclusion, a 70-kDa laccase was purified from M. grisea which was able to oxidize several lignin substrates. Further, it was also inhibited by metal-chelating inhibitors. Hence, purification of a laccase is an important step to raise specific antibodies to study the expression of laccase in gene disruption and biochemical experiments.
Financial support of the Rockefeller Foundation is gratefully acknowledged. We thank Dr. André Fleissner and Dr. Rajeev Gupta for critical reading and valuable discussions. G.I. was supported by a fellowship from the University Grants Commission.