Thelephora terrestris, an ectomycorrhizal hymenomycete, produces extracellular laccase when grown in minimal liquid medium. The enzyme was characterized as a protein of 66 kDa. The optimal pH varied depending on the substrate utilized, being 5.0 for syringaldazine, 4.8 for guaiacol and 3.4 for ABTS. The Km was 2.52±0.4 μM for syringaldazine, 16±1.9 μM for ABTS and 120.6±4.9 μM for guaiacol. The secretion of laccase was biomass-dependent and independent of the glucose concentration in the medium. The addition of inducers did not increase laccase production which was dependent upon the ammonium phosphate concentration.
The interactions between symbionts during the establishment of symbiosis in mycorrhizas are poorly understood . Ectomycorrhizal fungi secrete phenol oxidases but their contribution to this interaction is controversial . Hutchison  suggested that laccase, a copper enzyme (benzenediol:oxygen oxidoreductase, EC 22.214.171.124), may be associated with the decomposition of phenolic compounds produced by the plant as a defence mechanism. He also pointed out that the enzyme may be involved in mycelium pigmentation. Laccases secreted by fungi have been intensively studied , particularly the role of laccase in lignin biodegradation [5–9]. Hutchison  grouped ectomycorrhizal fungi according to the presence of laccase in the medium. Telephora terrestris, a hymenomycete symbiont in the ectomycorrhizal association of conifers, produces laccase when cultivated in different agar media. The present work examines the physiological conditions for laccase production by T. terrestris.
2Materials and methods
2.1Organism and growth conditions
T. terrestris TT 223 (kindly supplied by Daison Silva from Universidade Federal de Viçosa, Brazil) was maintained on Melin Norkrans (MN) solid medium  supplemented with 2% (w/v) malt extract. The strain was characterized by the production of laccase as described by Hutchison . Cultures were grown in MN basal liquid medium supplemented with 2 mg l−1 of copper sulfate and variable concentrations of carbon or nitrogen sources and the pH was adjusted to 5.6 using 2 N HCl. In the routine assay, medium containing 27.7 mM glucose and 3.4 mM ammonium phosphate was used. The sterile medium was inoculated with a suspension of an exponential phase culture that was homogenized in a sterile Waring blender prior to inoculation. Cultures were grown at 28°C in a rotary shaker at 120 strokes min−1. Mycelial dry weights were estimated after oven drying at 60°C for 5 days. The cultures were grown for 96 h and the mycelium was removed by filtration through a muslin sheet with the filtrate used as the enzyme source.
The enzyme activity was measured as described by Petroski et al.  using 60 nmol of syringaldazine (N,N′-bis-(3,5-dimethoxy-4-hydroxybenzylidene hydrazine) (Sigma) as a substrate, 200 μmol of acetate buffer pH 5.0 in a final volume of 3 ml at 30°C in a Beckman DU 7400 spectrophotometer using a molar extinction coefficient of 65 000 M−1 cm−1 at 526 nm . For each assay a control with boiled enzyme was carried out with the data representing the median of results obtained from a kinetic assay performed at different protein concentrations. One unit of enzyme activity was defined as the amount of enzyme oxidizing 1 nmol of substrate min−1. Guaiacol (2-methoxyphenol) and ABTS (2,2′-azinobis-(3-ethyl-benzothiazoline-6-sulfonate) (Sigma) were also used as substrates. Oxidation of ABTS was monitored by determining the increase at OD420 (ε= 36 000) and the oxidation of guaiacol at OD470 (ε= 26 600). The kinetic constants were determined using the program ENZ-FIT.
2.3Analysis of chemical changes in the culture medium
The concentration of glucose in the culture medium was determined by the glucose oxidase assay , ammonium according to Chaney and Marbach  using ammonium sulfate as standard. Protein was determined by the Lowry method using bovine serum albumin as standard .
2.4Enrichment of laccase from T. terrestris culture filtrates
The extracellular fluid (350 ml) was concentrated 10 times using dry sucrose at 4°C without loss of enzyme activity. The supernatant was dialyzed overnight against water at the same temperature and concentrated to 1.3 ml by ultrafiltration using an Amincon membrane PM-30 with a cut-off at 30 000 Da. The concentrate was applied to a Superose 12 HR 10/30 column equilibrated with 50 mM phosphate buffer, pH 7.2, in a fast protein liquid chromatography (FPLC) system. The protein was eluted at room temperature with 15 mM NaCl at a flow rate of 0.4 ml min−1. Active laccase fractions were pooled and dialyzed before the electrophoretic analysis. SDS-polyacrylamide gel electrophoresis was performed using a gradient of 8–25% and the conditions of the Pharmacia Phast System. The gel was stained for laccase activity as described by Rehman and Thurston  when non-denaturing conditions were used.
3Results and discussion
3.1Physiology of laccase production
In the presence of 55.5 mM glucose as carbon source, T. terrestris grew with a doubling time of 11 h during exponential growth. Growth retardation in the stationary phase was due to ammonium deficiency which was exhausted within 48 h of growth. Culture filtrates of T. terrestris contained laccase activity. The time of appearance of exolaccase during growth is shown in Fig. 1. It was observed that the enzyme levels attained a peak at the end of the exponential phase, remaining relatively constant up to 48 h. The total protein secretion exhibited the same behavior whereas the glucose level was depleted only after 144 h of growth. Enzyme activity per mg mycelial dry weight did not vary with glucose concentration (20–55.5 mM). The secretion of laccase by the ectomycorrhizal fungus T. terrestris was biomass-dependent as observed by Freitag and Morrell  in lignocellulolytic fungi. Residual glucose levels were demonstrated to be proportional to carbon source concentration, suggesting that secretion was not regulated by catabolic repression as previously suggested for Phanerochaete chrysosporium.
Dissolved oxygen has been shown to influence the levels of laccase in different ways depending on the fungus [19, 20]. The growth of T. terrestris in shaken and static cultures was compared using the same carbon and nitrogen concentrations. Under static conditions the exolaccase activity levels and protein secretion decreased but the level of enzyme per mg mycelial mass was 1.48 times higher (Table 1).
Table 1. Laccase production in shaken and static cultures
Initial condition: 100 ml of MN medium containing 27.7 mM glucose and 3.4 mM ammonium phosphate pH 5.6 was innoculated with exponential phase mycelium (14.6 mg dry weight) and cultivated at 28°C for 97 h. The values represent the means of triplicate assays.
Residual glucose (mM)
Residual ammonium (mM)
Total extracelullar protein (mg)
Mycelial dry weight (mg)
Total laccase activity (nmol min−1)
Laccase activity (mg−1 mycelial dry weight)
The level of enzyme activity was determined as a function of copper concentration in the medium: no significant variation was observed at concentrations up to 2 mg l−1 CuSO4. In T. terrestris the expression of exolaccase was not affected by the presence of inducers such as 1 mM veratryl alcohol, 2.8 μM cycloheximide and 1 mM 2,4-dihydroxybenzoic acid, as verified in other fungi [9, 21–23].
The time course of exolaccase production under different ammonium phosphate concentrations is shown in Fig. 2. Extracellular ammonium was completely depleted at 72 h of growth and mycelial dry weight became stationary at the same time. The level of exolaccase activity was proportional to the ammonium concentration in the medium as was observed with the production of laccase by ligninolytic fungi [6, 7, 24].
The results from changing the nutrient ratio indicate that nitrogen limitation affected laccase production (Table 2) and mycelial growth. The secretion of exolaccase was indirectly proportional to the C/N ratio when the enzyme activity was related to mycelial growth. The expression of laccase in T. terrestris was regulated by a low C/N ratio in spite of the growth rate. High C/N ratios increased the production of laccase in Pycnoporus cinnabarinus whereas in Lentinus edodes, Phanerochaete chrysoporium and Phanerochaete flavido-alba the opposite was observed.
Table 2. Effect of C/N ratio on the secretion of the exolaccase by T. terrestris
Residual glucose (mM)
Total extracelullar protein (mg)
Laccase activity (nmol min−1 mg−1)
Aliquots (50 ml) of MN medium containing different C/N ratios were inoculated with 7.8 mg dry weight mycelium and cultivated at 28°C, 120 rpm for 120 h. The parameters were determined according to Section 2.
Laccases are not substrate-specific . When the optimal exolaccase pH of T. terrestris was analyzed (Fig. 3), maximal activity was obtained at acid pH values, being pH 5.0 for syringaldazine, pH 4.8 for guaiacol and pH 3.4 for ABTS. Variation of optimal pH depending on the substrate was also observed with laccases isolated from other fungi [5, 8]. The effect of temperature on laccase activity was measured and maximal activity was obtained at 45°C. The kinetic parameters determined at the optimal pH for each substrate showed that the best substrate was syringaldazine (2.52±0.4 μM), followed by ABTS (16±1.9 μM) and guaiacol (120.6±4.9 μM). The oxidation of syringaldazine was independent of hydrogen peroxide since the addition of excess catalase (85–1360 units) to the reaction did not affect it, as observed in P. flavido-alba.
The effects of various compounds on enzyme activity were examined. The laccase activity was 50% inhibited by 1 mM hydroxyquinoline but not by 1 mM EDTA like the enzymes of Trametes sanguinea and Pycnoporus coccineus. The enzyme was inactivated by either 1 mM cysteine, 1 mM mercaptoethanol or 1 mM sodium azide as observed with other fungal enzymes [21, 22]. Copper atoms participate in the catalysis and dithiocarbamate, a specific copper chelator, inactivated the exolaccase activity of T. terrestris only at 7.5 mM, suggesting that the copper atom might be inaccessible to the chelator.
The exolaccase of T. terrestris was unstable after freezing and thawing. The enzyme lost 50% of its activity in 8 days when the culture filtrate was kept at −18°C. The activity was preserved for 30 days when the filtrate was kept at 4°C under an argon atmosphere. The enzyme was stable for 3 h at 60°C and was inactivated after a 40-min exposure to 70°C.
In order to determine the enzyme molecular mass, the extracellular fluid was collected from a static culture, concentrated and analyzed by chromatography. As can be seen in Fig. 4, the fungal secretion analyzed by gel permeation showed the presence of several proteins. The fraction containing the laccase activity was eluted as a protein of 61 kDa and was partially purified (12.5 times). The fraction subjected to SDS-PAGE showed one band, with a molecular mass of 66 kDa, which corresponded to enzyme activity when the gel was run in non-denaturing condition. This molecular mass is similar to that of several other laccase enzymes isolated from different fungi [4, 22, 23].
Considering that laccase is normally secreted by the ectomycorrhizal fungus T. terrestris under different nutritional conditions, it is reasonable to suggest that the enzyme may participate in the detoxification of phenolic compounds with concomitant pigmentation of the mycelium since such phenolic compounds polymerize . Further studies must be performed to elucidate the role of laccase in ectomycorrhizal formation.
This work was supported by Grant 60.92.012.100 from the Financiadora de Estudos e Projetos (FINPEP) and C.F.K. was a fellow of CNPq (Conselho Nacional de Desenvolvimento Cientıćfico e Tecnológico).