A strain of Punctularia atropurpurascens (H2126), grown and maintained on malt extract agar (Oxoid Ltd., Basingstoke, UK), from the collection of the Cátedra de Microbiología General Collection (CCMG) was used.

The presence of lignin-degrading enzymes in the Punctularia atropurpurascens strain was determined on solid media, as previously described [14] [15] . The Malt Extract Agar (MEA) medium was supplemented (separately) with 1) 0.02% Remazol Brilliant Blue (RBB, Sigma Chemical Co., St. Louis, MO), 2) 1% tannic acid (Sigma Chemical Co., St. Louis, MO), and 3) 1% gallic acid (Sigma Chemical Co., St. Louis, MO). The diameters of mycelia growth and decolorization or oxidation zones in the plates, were measured along the 10 days of the incubation at 28˚C.

The mycelia of Punctularia atropurpurascens were grown on MEA. To prepare the extracellular and the mycelial extracts, two 7-day-old culture fungal plugs (diameter, 9 mm ) were transferred to 500-ml Erlenmeyer flasks with 200 ml sterile malt extract broth (1.25%, w/v) and incubated at 28˚C at 100 rpm for 12 days. After the fermentation, the broth was centrifuged (15,000 g, 30 min, 4˚C) and the resulting clear supernatant (identified as extracellular extract, EE12) was used for the enzymatic assays. The mycelia were then ground in a mortar with liquid nitrogen and the frozen mycelial powder was suspended in phosphate-buffered saline pH 7.4 (PBS buffer). The resulting suspension was filtered through cheesecloth and the filtrate was centrifuged (15,000 g, 30 min, 4˚C). This mycelial extract was used for isolation of the lectin [16] .

Laccase, manganese peroxidase (MnP) and lignin peroxidase (LiP) activities were determined in the EE12 extract by monitoring the oxidation of syringaldazine [17] , MBTH/DMAB [18] and veratryl alcohol [19] as substrates, respectively. The enzyme unit (EU) was defined in each case, as the amount of enzyme that catalyses the formation of 1 µmol of the corresponding product per min.

Native PAGE was carried out with the Phast System equipment (Pharmacia, Uppsala, Sweden) using 8-25 gradient Phast gels. Proteins were stained using the silver protocol according to the manufacturer’s instructions and the laccase activity was detected on the gel by staining with 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). Isoelectric focusing (IEF) was done using the PhastGel IEF 3- 9. A broad pI calibration kit (3-9) was used as standard. Proteins were silver stained according to the manufacturer’s instructions (PhastSystem, Pharmacia).

The optimum temperature was determined on EE12 by performing enzyme activity assays at different temperatures between 30˚C and 60˚C. The optimum pH was determined by performing enzyme activity assays in buffers varying from pH 4.0 - 7.0, in the following pH ranges: 100 mM sodium acetate buffer (pH 4.0 - 6.0) and 100 mM phosphate buffer (pH 6.0 - 7.0). Each assay was run in duplicate.

The ability of the EE12 extract to enzymatically degrade Remazole Brilliant Blue R (RBBR, Sigma Chemical Co., St. Louis, MO) and Acid Blue 25 (Sigma Chemical Co., St. Louis, MO) dyes was evaluated. Tubes of 3 ml volume containing 0.25 mM dye and 300 µl of EE12 extract in 100 mM acetate buffer pH 4.5, were incubated on a rotation shaker at 37˚C for 48 h. In some cases the reaction was run in the presence of 20 mM MnSO₄ and 10 mM H₂O₂. Decolorization was followed spectrophotometrically at 590 nm and the decolorization efficiency (DE) was expressed as percentage of color removal calculated as C_i − C_r/C_i × 100, where C_i and C_r were initial and residual dye concentrations, respectively. Tubes containing dye without enzymes or with heat-inactivated enzymes (at 100˚C for 30 min) were used as controls.

The EE12 extract was applied to a column (diameter 5 mm , height 30 mm ) packed with the affinity adsorbent Concanavalin A-Sepharose (2.4 ml of sedimented gel), previously equilibrated with 0.1 M acetate buffer pH 6.0 (adsorption buffer). The column was washed with the adsorption buffer and the bound enzymatic activities (laccase or MnP) were evaluated. A solution of the corresponding substrate, syringildazine (for the case of laccase activity) or MBTH/DMAB (for the case of MnP activity) was loaded onto the column and the bound activity was visualized by the change in the color of the halo produced by the oxidized products (pink or blue, respectively). The column was washed again with the adsorption buffer and elution of the adsorbed material was performed with a 0.45 M methylmannoside solution. Fractions of 1-ml were collected and gel filtered on PD-10 (Sephadex G-25, GE Healthcare) to remove the monosaccharide. The enzymatic activities (laccase or MnP) remaining were determined in these gel filtered eluted fractions, as described above.

In a parallel experiment the EE12 extract was adsorbed on the Con A-Sepharose column as before, and the degradation of a solution of RBBR dye was evaluated. A volume of 800 ml of dye was loaded onto the column and after 20 min, the bound enzymatic activity was visually registered, by the decolorization produced by the oxidized product. The column was washed again with adsorption buffer, elution of the adsorbed material was performed with a 0.45 M methylmannoside solution and the same procedure described above was followed, to determine the enzymatic activity remaining.

The lectin was purified by affinity chromatography on chitosan-Sepharose as described previously [16] . Briefly, the mycelium extract was loaded on the affinity column and the elution was performed with 100 mM N-acetyl- glucosamine in PBS buffer. Purified PAL was coupled to NHS-activated Sepharose via the amino groups, according to the procedure recommended by the manufacturer. An amount of 1.2 g of freeze dried powder was suspended in 1 mM HCl and washed on a sintered glass filter. The lectin solution (0.6 mg/ml) was adjusted to 0.2 M NaHCO₃ pH 8.3 buffer, supplemented with 0.5 M NaCl (coupling buffer) and mixed with the gel in an empty PD-10 column. The suspension was rotated end over end for 1 h at room temperature. The excess ligand was washed away on a glass filter with the coupling buffer. The derivative was washed with three cycles of alternating pH consisting of a wash with 0.5 M ethanolamine in coupling buffer, followed by a wash with 0.1 M acetate buffer, pH 4.0 supplemented with 0.5 M NaCl.

The lectin content of the gel was determined by using the bicinchoninic acid (BCA) technique as described by Giacomini et al. [20] . Immobilization yield was calculated as the ratio between the amount of immobilized lectin and the amount of the soluble lectin initially applied.

A glass mini-column (d = 0.35 cm) was packed with 0.5 ml of PAL-Sepharose and equilibrated with PBS buffer. In individual experiments, volumes of 200 ml of each of the following glycoproteins were applied to the column: bovine lactoferrin (LF), pregnant mare serum gonadotropin (PMSG) and fetuin from fetal calf serum (FT). In each experiment, the column was washed with PBS buffer and a competitive elution was performed with 100 mM N-acetyl-glucosamine in PBS. Fractions of 0.4 ml were collected on microtiter plates (flow rate: 0.5 ml/min) and analyzed for their protein content by recording the A 280 nm.

Test organisms used for bioautography assays [21] were Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 11105), Candida albicans (ATCC 10231) and Aspergillus niger (ATCC 16404). The purified lectin (4 mg/ml, 50 µl) was applied as small spots on silica plates. Gentamicin (20 µg/ml) and Nystatin (50 IU/ml) were used as positive controls for growth inhibition of bacteria and fungi, respectively. PBS (50 µl) was used as a blank control. The minimum inhibitory concentration (MIC) was defined as the minimum lectin concentration that inhibited the visible microorganism growth. The MIC was determined by the microdilution technique using 100 μl of Mueller-Hinton broth (Difco), 100 μl of two fold dilutions of the extracts (10 mg/ml) and 10 μl of a suspension of the microorganisms (10⁸ microorganisms/ml). The trays were incubated (24 h, 37˚) and developed with 0.1% p-iodonitrotetrazolium violet (Sigma Chemical Co., St. Louis, MO) solution. Nystatin and gentamicin were used as controls.

The ligninolytic activity of Punctularia atropurpurascens was determined using three different media. Decolorization haloes around the growth areas were observed when the medium was supplemented with RBB (Figure 1(a)). The halo diameters increased over the 10 days evaluated and correlated with the increase in the diameter of the mycelia growth during the same period. The oxidation zones, visualized as brown areas in the plates supplemented with tannic (Figure 1(b)) and gallic (Figure 1(c)) acids, also correlated with the mycelia growth.

Extracellular extracts of Punctularia atropurpurascens (EE12) showed MnP and laccase activities starting at the fifth day of fermentation (Figure 1(d)); no LiP activity was detected. When the EE12 extract was reversibly immobilized on a ConA-Sepharose column, the MnP and laccase activities of the extract were visualized as colored bands on the columns, after the addition of substrates (Figure 2(a)). A broad pink zone was observed at approximately 0.5 cm from the top of the column (left) as a result of the laccase activity using syringaldazine as

substrate. In the case of the MnP activity, a tenuous and diffuse blue zone was observed at approximately 1 cm from the top of the column (right) produced by the oxidation of the MBTH/DMAB substrate. These results demonstrate that both enzymes present in the EE12 are active in the gel. As the interaction with the Con A lectin is mediated by alpha-D-mannopyranosil or alpha-D glucopyranosyl or sterically related residues in glycoconjugates, this also reveals that these enzymes are glycosylated. Moreover, the reversibility of the lectin-glycoen- zyme interaction was confirmed when the enzymes were eluted from the column with the methyl mannoside. After gel filtration on PD-10 columns to eliminate the competitive carbohydrate, the enzymes were still active (data not shown).

The PAGE profile of the extracellular extract EE12 showed the presence of a defined protein band coincident with the migration front and a retarded diffused and smeared zone (Figure 3(a), right). When the gel was stained with the enzymatic substrate ABTS (Figure 3(a), left) two colored bands are observed, the main active one is coincident with the silver stained band. The other green band (just below the main band), does not coincide with any protein band in the silver stained gel.

There was no change in the specific stain when the gel was submerged in a MnSO₄ and H₂O₂ solution (data not shown). These results may indicate the presence of at least two laccases in the extracellular extract. IEF analysis of the EE12 extract showed a clear band of pI lower than 3.0 and a smeared band centred at pI 5.2 (Figure 3(b), left). However, the stain with ABTS indicated that only this latter band was revealed by the specific detection (Figure 3(b), right).

Temperature profiles of MnP activity showed the optimum temperature at 50˚C, while two peaks at 35˚C and 50˚C of laccase activity were detected (Figure 4(a)). The pH profile (Figure 4(b)) showed maximum activity at 4.5 for MnP and 6.0 for laccase.

The ability of the enzymes of the extracellular extract EE12 to degrade Remazole Brilliant Blue R (RBBR) and Acid Blue 25 was evaluated. Both dye solutions were almost completely decolored (90%, calculated as indicated in Methods) after 48 h of incubation while no changes in the decolorization efficiency were observed after the addition of Mn and H₂O₂ suggesting that the laccase, but not the MnP, activity may be responsible for the decolorization effect. The decolorization of the RBBR dye was also evaluated using the enzymes immobilized on Con A-Sepharose. In this case, the enzymatic activities of the adsorbed EE12 extract was evaluated by visualizing the disappearance of the initial blue band present in the column (Figure 2(b)). The colored band disappeared after 20 minutes of incubation (data not shown) indicating that the enzyme retained its activity when adsorbed on the affinity gel.

The lectin was purified from the P. atropurpurascens mycelium extract (PAL) as previously reported [16] and then immobilized on NHS-activated Sepharose. The resulting PAL-Sepharose adsorbent contained 0.8 mg PAL/ ml sedimented gel, determined by the BCA optimized protocol (as described in Materials and methods) using a calibration curve (y = 0.024x + 0.0025, r² = 0.994). The chromatographic behaviors of three glycoproteins (LF, PMSG and FT) were evaluated on columns packed with PAL-Sepharose. The adsorbent displayed high selective binding of LF, as 60% of the applied material was eluted from the column, but there was no interaction with FT or PMSG (Figure 5).

The purified lectin (PAL) was evaluated against four microorganisms: S. aureus, E. coli, C. albicans and A. niger . Under the experimental conditions PAL was not effective at inhibiting the growth of E. coli, S. aureus or C. albicans. However, a clear inhibition halo was obtained against A. niger . The MIC against A. niger was determined as 0.08 mg of PAL/ml.