Enzyme Activities of Two Recombinant Heme-Containing Peroxidases, TvDyP1 and TvVP2, Identified from the Secretome of Trametes versicolor

The secretome of T. versicolor BRFM 1218 grown on malt agar wood was analyzed by using proteomics (Table 1). Among the 200 proteins identified in the secretomes, 21 peroxidases were identified in the secretome and are listed in the order of relative abundance in Table 1. TvDyP1 and TvVP2 were selected to study the oxidative system of T. versicolor because (i) they represent new peroxidase models, as LiPs and MnPs from T. versicolor were extensively studied in previous works; (ii) they are cosecreted with other class II peroxidases, which suggests a physiological function important to the fungus; and (iii) of the DyP and VP representatives identified in the secretome, they were by far the most abundant.

For heterologous expression of TvDyP1, a 5-liter culture of Escherichia coli transformants was prepared, resulting in production of TvDyP1 as an active protein. After a lysis step, the enzyme was purified in two different chromatographic steps. As shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) monitoring of the purification process (Fig. 1A), the soluble fraction (lane 1) from the E. coli cultures was considerably enriched in the TvDyP1 (52 kDa) band after the first Mono Q chromatography step. However, several other proteins were still present.

These contaminating proteins were removed by the second Q-Sepharose chromatography step. Homogeneous TvDyP1 was obtained (lane 3) with a purification yield of 5 mg of protein/5-liter E. coli culture and a Reinheitzahl (Rz) A₄₀₅/A₂₈₀ ratio of ∼2.

For heterologous expression of TvVP2, a 5-liter culture of E. coli transformants was grown and after lysis, a large quantity of protein (about 120 mg · liter⁻¹) was found accumulated in insoluble inclusion bodies (Fig. 1B, lane 1). In vitro activation of E. coli-expressed TvVP2 was carried out under conditions previously optimized for other peroxidases (33), i.e., folding in 50 mM Tris-HCl (pH 9.5) containing 20 μM hemin, 0.16 M urea, 5 mM Ca²⁺, 0.5 mM oxidized glutathione, 0.1 mM dithiothreitol (DTT), and 0.1 mg · ml⁻¹ protein for ∼20 h at room temperature. The mixture was then concentrated, dialyzed, and centrifuged, allowing the elimination of folding additives and unfolded protein. TvVP2 was purified by a single anion-exchange chromatographic step with a Mono Q column (see Fig. S1B in the supplemental material). SDS-PAGE analysis of purified TvVP2 shows a single band of protein (lane 2) with a molecular mass of ∼37 kDa. A purification yield of 30 mg of purified and active protein/5-liter E. coli culture with an Rz A₄₀₅/A₂₈₀ ratio of ∼3 was obtained.

The N-terminal amino acid sequences were analyzed by sequential Edman degradation and confirmed the first five residues predicted from the protein sequence (Table 2), except for the first amino acid (methionine) of TvDyP1, which was missing. Mass spectrometry (MS) analysis (Table 2) showed two major peaks of TvDyP1 at 52.036 and 52.714 kDa, in agreement with the molecular mass calculated from the amino acid sequence (52,171 Da). A minor peak (∼10%) was recorded at 49,118 Da, which might correspond to the partial degradation of TvDyP1 during the purification procedure, although a protease inhibitor was added to the lysis buffer. Mass spectrometric analysis of TvVP2 (Table 2) showed a major peak corresponding a molecular mass of 36,375 Da, which is slightly lower than that of wild-type TvVP2 purified from fungal cultures (44.6 kDa) (18), probably because of the lack of glycosylation machinery in E. coli, but in agreement with that calculated from the amino acid sequence (36.25 kDa).

The thermostability of TvDyP1 and TvVP2 was examined by measuring the oxidation of 2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS) after heat treatment of the enzymes at different temperatures. Both were found to be sensitive to high temperatures (60 to 70°C), with >80% of their activity lost after 30 min of incubation at 60°C, while no activity was measured for the same incubation time at 70°C (Fig. 2A and B). Similar patterns were observed with both enzymes at temperatures ranging from 30 to 50°C after 30 min of incubation, with 100 and 80% residual activity of TvDyP1 (Fig. 2A) and TvVP2 (Fig. 2B) measured, respectively. Beyond 30 min of incubation, activity decreased drastically, with the greater effect on TvVP2 with only 10 to 30% residual activity measured after 180 min of incubation at temperatures ranging from 30 to 50°C (Fig. 2B). Under the same conditions, TvDyP1 retained 20 to 80% of its initial activity (Fig. 2A). The thermal inactivation profiles of both enzymes (data not shown) showed that they were rather stable, with half-inactivation temperatures (60 min) of 51.7 and 52.2°C estimated for TvDyP1 and TvVP2, respectively.

The effect of pH on TvDyP1 and TvVP2 activities was initially examined with different substrates at pHs ranging from 2.0 to 7.5. As shown in Fig. 2C and D, both peroxidases were active at acidic pHs ranging from 2.0 to 5.0, although there were some exceptions; i.e., no TvDyP1 activity was recorded on VA, Reactive Black 5 (RB5), or Mn²⁺ at any pH tested (Fig. 2C), while maximum TvVP2 activity on Mn²⁺ was obtained at pH 7.0 (Fig. 2D). On the other hand, the specific activities and optimum pHs of both enzymes differed depending on the substrates tested. TvVP2 presents a broader substrate specificity than TvDyP1, and both enzymes were more active on ABTS than on any other substrate (9,892 and 2,074 nkat · mg⁻¹ for TvDyP1 and TvVP2, respectively). Oxidation of this substrate occurred at similar pHs with both enzymes (3.5 and 3.0 for TvDyP1 and TvVP2, respectively), and activities decreased drastically at pHs beyond 4.5.

Similar pH profiles of TvVP2 oxidation of VA and 2,6-dimethoxyphenol (DMP) were obtained, with an optimal pH of 2 to 2.5 and a 40% reduction of activity at pH 3.0. The optimum pH for RB5 oxidation was also pH 2.5, but in this case, the enzyme lost ∼75% of its activity at pH 3.0. DMP oxidation by TvDyP1 was, however, obtained at an optimal pH of 3.0. Reactive Blue 19 (RB19) decolorization by TvVP2 was optimal at pH 3.5. In contrast, the highest optimal pH of 7.0 was observed for Mn²⁺ oxidation.

The pH stability of TvDyP1 was studied by incubating the enzyme at pHs 2 to 6 at 4°C. As the protein was very stable in relation to pH, only results for 24 and 48 h of incubation are shown (Fig. 2E). The enzyme was found to be very stable at all of the pHs studied, since it retained ∼90% of its initial activity even after 48 h of incubation. For TvVP2 stability, shorter incubation times (30, 60, 120, and 180 min) were tested since the enzyme was more sensitive to pH (Fig. 2F). At acidic pHs (2 to 2.5), the enzyme was totally inactivated after the first 30 min of incubation at pH 2.0 and after 2 h of incubation at pH 2.5. At pHs ranging from 3.0 to 4.5, TvVP2 retained 50 to 70% of its initial activity after 3 h of incubation at 4°C. A more stable profile pattern of TvVP2 was observed at pHs 5.0 to 6.0, with >80% residual activity remaining after 3 h of incubation.

TvDyP1 and TvVP2 were tested on six representative substrates, and the kinetic constants obtained are shown in Table 3. Significant differences between the two peroxidases were observed. Of the six substrates tested, TvDyP1 exhibited an affinity for only three, ABTS, DMP, and RB19 (K_m, 37.8 to 292.6 μM), while no activity was detected on RB5, VA, or Mn²⁺. As observed for other fungal DyPs, the highest turnover value of TvDyP1 from T. versicolor was found for ABTS (k_cat, 581.1 s⁻¹), followed by DMP (k_cat, 87.4 s⁻¹) and the anthraquinone dye RB19 (k_cat, 23.8 s⁻¹) (28). TvVP2 was found to possess an affinity for the six substrates, with the highest for RB5 and RB19. The oxidation of the low-redox-potential dye ABTS by TvVP2 was found to be lower (k_cat, 79.2 s⁻¹) than that by TvDyP1 (Table 3). Moreover, TvDyP1 presented a slightly higher apparent affinity for ABTS than TvVP2 (K_m, 292.2 and 415 μM, respectively), with an even better k_cat (582 and 79.2 s⁻¹, respectively); consequently, TvDyP1 was ∼10-fold more efficient than TvVP2 on this substrate (k_cat/K_m, 1,989.4 and 190.8 s⁻¹ mM⁻¹, respectively).

Peroxidases tend to lose activity in the presence of H₂O₂ through a mechanism-based process known as suicide inactivation (34). The optimum H₂O₂ concentration for TvDyP1 and TvVP2 depended on the substrate used (Fig. 3). Optimal H₂O₂ concentrations of 0.25 and 0.5 mM were obtained with ABTS as a substrate for TvVP2 and TvDyP1, respectively. Then the activity decreased gradually to <30% residual activity fof both enzymes at 5.0 mM H₂O₂. Interestingly, TvVP2 displayed significant activity in the absence of H₂O₂ at 7.5% of its maximal activity.

The capacity of both peroxidases to oxidize catechin (CAT) and quercetin (QUE), which are extractable from oak wood (35), was investigated. TvVP2, in the presence of hydrogen peroxide (0.1 mM), altered the 250- to 600-nm absorbance spectra of CAT recorded every 60 s over a 4-min period, while the modification of absorbance by TvDyP1 was weaker in the 400-nm region of the spectrum (Fig. 4A and B; Fig. S1 and S2A and B). A major increase in optical density at around 400 nm was observed, suggesting the formation of quinones (36). The same approach was used with QUE (Fig. S3). In this case, incubation of the flavonoid with both peroxidases in the presence of hydrogen peroxide led to strong modification of the observed spectra, in particular, a decrease of the 370 nm peak (Fig. 4C and D; Fig. S4A and B). As observed for CAT (Fig. S2C and D), the observed modification induced by TvVP2 seemed to be greater than that induced by TvDyP1 (Fig. S4C and D). Interestingly, the UV spectrum of QUE oxidation by TvVP2 showed a small increase in absorption at around 290 nm, which was not detected after oxidation by TvDyP1. This absorption band could match the production of 3,4-dihydroxybenzoic acid (37). Using reverse chromatography to analyze the reactional mixtures, we noticed that the CAT signal was dramatically decreased or lost after oxidation by TvDyP1 and TvVP2, respectively (data not shown). Nevertheless, no additional peaks were observed in chromatograms, suggesting that the quinones polymerized, and their detection was not possible by this method. The production of insoluble brown compounds was indeed observed, preventing any further characterization by chromatographic steps.

To study the abilities of TvDyP1 and TvVP2 to oxidize complex substrates, five industrial dyes were selected from different usages and chemical structures (acidic, basic, reactive, vat, and disperse dyes). As shown in Table 4, both peroxidases were active on Acid Black 172 (AB) dye, with a slightly higher decolorization percentage for TvVP2 than for TvDyP1. Regarding the other dyes, TvVP2 presented a wider range of action since it was also active on Basic Blue 41 (BB) and Reactive Black (RB).

DTT, lysozyme, glutathione, hemin, DMP, VA, RB19, CAT hydrate (98% pure; molecular weight [MW], 290.27), QUE (95% pure; MW, 302.24), hydrogen peroxide solution (50% [wt/wt] in water, stabilized), reduced l-glutathione (98% pure; MW, 307.32), and dibasic sodium phosphate (99% pure; BioReagents) were purchased from Sigma-Aldrich (Steinheim, Germany); isopropyl-β-d-thiogalactopyranoside (IPTG) was from Calbiochem (Meudon, France); ABTS was from Boehringer Mannheim (Saint-Quentin Fallavier, France); and sodium phosphate monobasic dehydrate was obtained from Euromedex (Souffelweyersheim, France). The industrial dyes AB, RB5, Disperse Blue 79 (DB), BB, and Vat Green 1 (VG) were supplied by the SETAS Company (Tekirdağ, Turkey).

The dikaryon T. versicolor was deposited in the CIRM-CF collection under no. BRFM 1218. T. versicolor BRFM 1218 was collected on oak (Quercus pubescens) in Goult (Vaucluse, France). To confirm the morphological identification, molecular identification by internal transcribed spacer (ITS; ITS1, ITS2, and 5.8S rRNA) sequencing was performed. The sequence was compared by similarity analysis (BLASTn) to those in GenBank and FunGene-DB (49), and it corresponded unequivocally to the species T. versicolor. The fungus was grown on 4% malt extract agar medium, and three oak sapwood wood blocks (25 by 15 by 5 mm) were then placed on the grown mycelium after 2 weeks of incubation. The blocks were dried for 48 h at 103°C before use. Incubation was carried out at 25°C in the dark. Oak blocks colonized for 1 month were removed carefully from the agar plates, and the mycelium surrounding the blocks was excised. The secretome was extracted by soaking the wood chips in potassium acetate buffer (100 mM, pH 4.5) for 120 min at room temperature. The secretome obtained was stored at −20°C before further analysis. The total amount of proteins was assessed with the Bradford assay (Protein Assay Dye Reagent Concentrate; Bio-Rad, Ivry, France) with bovine serum albumin standard concentrations that ranged from 0.2 to 1 mg · ml⁻¹.

Short SDS-PAGE runs (precast 4 to 12% Bis-Tris minigels; Invitrogen, France) were performed, allowing proteins diafiltered from the secretome (15 μg) to migrate on a 0.5-cm length, and gels were stained with Coomassie blue (Bio-Rad, Marnes-la-Coquette, France). Each gel electrophoresis lane was cut into two slices (2 mm wide), and protein identification was performed by the PAPPSO (Plate-forme d'Analyze Protéomique de Paris Sud-Ouest) platform facilities. In-gel digestion was carried out in accordance with a standard trypsinolysis protocol. Gel pieces were washed twice with 50% (vol/vol) acetonitrile (ACN)–25 mM NH₄CO₃ and incubated in the presence of 10 mM DTT for 1 h at 56°C. After cooling, the supernatant was removed and the samples were incubated with 55 mM iodoacetamide at room temperature in the dark. Gel plugs were washed with ACN and then dried in a vacuum concentrator (Thermo Fisher Scientific, Villebon sur Yvette, France). Digestion was performed for 8 h at 37°C with 200 ng of modified trypsin (Promega, Charbonnières-les-Bains, France) dissolved in 25 mM NH₄CO₃. Tryptic peptides were extracted first with 50% (vol/vol) ACN–0.5% (vol/vol) trifluoroacetic acid (TFA) and then with pure ACN. Peptide extracts were dried in a vacuum speed concentrator (Thermo Fisher Scientific, Villebon sur Yvette, France) and suspended in 25 μl of 2% (vol/vol) ACN–0.05% (vol/vol) TFA–0.08% (vol/vol) formic acid. HPLC was performed on a NanoLC-Ultra system (Eksigent, Les Ulis, France). Tryptic digestion products were first concentrated and desalted on a precolumn cartridge (PepMap 100 C₁₈, 0.3 by 5 mm, Dionex; Thermo Fisher Scientific) with 0.1% formic acid at 7.5 μl min⁻¹ for 3 min. The precolumn cartridge was connected to the separating column (C₁₈, 0.075 by 15 cm; Biosphere Nanoseparations, Nieuwkoop, The Netherlands), and the peptides were eluted with a linear gradient of 5 to 35% ACN in 0.1% formic acid for 40 min at 300 nl · min⁻¹. On-line analysis of peptides was performed with a Q-exactive mass spectrometer (Thermo Fisher Scientific, United States) with a nanoelectrospray ion source. Ionization (1.8-kV ionization potential) was performed with a stainless steel emitter (30-μm inner diameter; Thermo Electron, Villebon sur Yvette, France). Peptide ions were analyzed with Xcalibur 2.1 software (Thermo Scientific, Villebon sur Yvette, France) with the following data-dependent acquisition steps: step 1, full MS scan (mass-to-charge ratio [m/z] 400 to 1,400; resolution, 70,000); step 2, tandem MS (MS/MS; normalized collision energy, 30%; resolution, 17,500). Step 2 was repeated for the eight major ions detected in step 1. Dynamic exclusion was set to 40 s. The raw mass data were first converted to mzXML format with the ReAdW software (SPC Proteomics Tools, Seattle, WA). Protein identification was performed by querying the MS/MS data against databases, together with an in-house contaminant database, with the X!Tandem software (X!Tandem Cyclone, Jouy en Josas, France) and the following parameters: one trypsin missed, cleavage allowed, alkylation of cysteine, conditional oxidation of methionine, and precursor and fragment ion set at 2 ppm and 0.005 Da, respectively. A refined search with similar parameters was added, except that semitryptic peptides, possible N-term acetylation, and histidine mono- and dimethylations were also searched. All peptides matched with an E value of <0.05 were parsed with X!Tandem pipeline software. Proteins identified with at least two unique peptides and a log E value of <−2.6 were validated.

The mature protein-coding sequences of a DyP and a VP (GenBank accession numbers 19415892 and 19412643) encoding the proteins with Joint Genome Institute (JGI) protein ID no. 48870 and 26239 from T. versicolor (FP-101664 SS1, corresponding to TvDyP1 and TvVP2, respectively) were synthesized by GeneArt Gene Synthesis (Life Technologies, Germany) after codon optimization for E. coli expression.

The TvDyP1 coding sequence was cloned into the pET23b(+) vector (Novagen, Darmstadt, Germany), and the resulting plasmid (pET23b-TvDyP1) was used for expression in E. coli BL21(DE3) (Invitrogen, GeneArt, Regensburg, Germany). Cells were grown for 3 h at 37°C in terrific broth (TB) containing 100 μg · ml⁻¹ ampicillin, induced with 1 mM IPTG, and grown further for 48 h at 16°C in the presence of 20 μM hemin. Cells were harvested by centrifugation at 8,000 rpm for 10 min at 4°C. The bacterial pellet was resuspended in 100 ml of lysis buffer (20 mM Tris-HCl [pH 8.0] containing 1 mM EDTA and 5 mM DTT) supplemented with lysozyme (Sigma-Aldrich) at 2 mg · ml⁻¹, DNase I, and a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). After 1 h of incubation, cells were sonicated and then centrifuged at 12,000 rpm for 30 min to remove cell debris. The resulting supernatant was dialyzed first against 20 mM acetate, pH 4.3, and then further dialyzed against 20 mM Tris, pH 7.5. Insoluble material was removed after each dialysis step (12,000 rpm for 10 min). TvDyP1 was purified with an ÄKTA Express purification system (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) in two consecutive steps. First, the TvDyP1 solution was loaded onto a 1-ml Mono Q 5/50 GL column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) in 20 mM Tris (pH 7.5) buffer at a flow rate of 1 ml · min⁻¹. After column washing with the buffer described above, two successive linear NaCl concentration gradients (0 to 0.15 and 0.15 to 0.5 M NaCl) were applied for 20 and 10 min, respectively.

Peroxidase activity was monitored by measuring ABTS oxidation in the presence of H₂O₂ as described below. The active fractions were pooled, dialyzed against 20 mM Tris (pH 7.5), and loaded into a 20-ml Q-Sepharose column (GE Healthcare Bio-Sciences AB) in the same buffer at a flow rate of 1 ml · min⁻¹. Proteins were then eluted with a 0 to 0.5 M NaCl gradient for 20 min. TvDyP1 purification was checked by SDS-PAGE in a 12% gel stained with Coomassie brilliant blue R-250 (Sigma). The electronic absorption spectrum of the purified enzyme was recorded with an Agilent 8453 diode array UV-visible spectrophotometer (Agilent Technologies, Santa Clara, CA).

The TvVP2 coding sequence was cloned into the pET23b(+) vector (Novagen), and the resulting plasmid (pET23b-TvVP2) was used for expression in E. coli BL21(DE3) (Invitrogen). Cells were grown for 3 h at 37°C in TB containing 100 μg · ml⁻¹ ampicillin, induced with 1 mM IPTG, and grown for 4 h. TvVP2 accumulated in inclusion bodies, as observed by SDS-PAGE, and was solubilized with 8 M urea. In vitro refolding was performed in 0.16 M urea–5 mM Ca²⁺–20 μM hemin–0.5 mM oxidized glutathione–0.1 mM dithiothreitol–0.1 mg · ml⁻¹ protein at pH 9.5 as previously described (28).

TvVP2 was purified by a one-step chromatographic procedure with an ÄKTA Express purification system (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and a Mono Q 5/50 GL column (GE Healthcare Bio-Sciences AB) with a 0 to 0.5 M NaCl gradient (1 ml · min⁻¹, 40 min) in 10 mM tartrate (pH 5.5) containing 1 mM CaCl₂.

To determine the N-terminal sequence of the purified peroxidases and those of the fragments generated by partial proteolysis, the protein material was subjected to protein sequence analysis on an Applied Biosystems 476A sequencer by the proteomic platform of the Institut de Microbiologie de la Méditerrranée, CNRS, Aix-Marseille Université. Matrix-assisted laser desorption ionization–time of flight MS of samples was carried out on a Microflex II time of flight mass spectrometer (Bruker Daltonik, Germany).

The optimal pH for substrate oxidation by each of the two T. versicolor peroxidases was determined by measuring the enzymatic activity with saturating concentrations of RB5 (30 μM), RB19 (200 μM), ABTS (5 mM), DMP (20 and 60 mM for TvVP2 and TvDyP1, respectively), VA (20 mM), and Mn²⁺ (10 mM) in 0.1 M tartrate buffer as described below.

The kinetic constants of T. versicolor TvDyP1 and TvVP2 were estimated from the absorbance changes observed during substrate oxidation at the optimal pH at 30°C in a Uvikon XS spectrophotometer (BioTek Instruments, Colmar, France) (29). Depending on the substrate, different concentrations of H₂O₂ were added to initiate the reaction. Oxidation of ABTS was determined by measuring the generation of its cation radical (ε₄₃₆ = 29,300 M⁻¹ cm⁻¹). RB5 and RB19 oxidation was monitored for colorant disappearance (ε₅₉₈ = 30 mM⁻¹ cm⁻¹ and ε₅₉₅ = 10 mM⁻¹ cm⁻¹, respectively). Oxidation of Mn²⁺ was determined by monitoring Mn³⁺-tartrate complex (ε₂₃₈ = 6.5 mM⁻¹ cm⁻¹) formation. VA oxidation was determined for veratraldehyde (ε₃₁₀ = 9.3 mM⁻¹ cm⁻¹) formation. DMP oxidation was monitored for dimeric coerulignone (ε₄₆₉ = 55 mM⁻¹ cm⁻¹). All enzymatic activities were measured as initial velocities taking linear increments (decreases for RB5 and RB19). Mean apparent affinity constant (Michaelis constant, K_m) and enzyme turnover (catalytic constant, k_cat) values and standard errors were obtained by nonlinear least-squares fitting to the Michaelis-Menten model. Fitting of these constants to the normalized Michaelis-Menten equation υ = (k_cat/K_m)[S]/(1+[S]/K_m) yielded enzyme efficiency values (k_cat/K_m) with their standard errors.

To study the effect of pH on T. versicolor peroxidase stability, the enzymes were incubated in 0.1 M tartrate buffer at pH 2 to 6 and kept at room temperature for different time periods. Residual activities were measured after 1 min of incubation (to evaluate the initial survival of the enzyme at each pH) and 30, 60, 120, and 180 min of incubation for TvVP2 and 24 and 48 h for TvDyP1. The activity obtained with the sample incubated for 1 min at pH 5 was taken as a reference (maximum activity). Activity was determined by oxidation of a saturating concentration of ABTS in 0.1 M tartrate at the optimal pH of each enzyme (3.0 for TvVP2 and 3.5 for TvDyP1) under the conditions described above.

To study the thermal stability of T. versicolor peroxidases, the enzymes were incubated in 0.1 M tartrate buffer (pH 5.0) over a range of 25 to 70°C. Residual activity was determined after 1, 30, 60, 120, and 180 min after a cooling step on ice. The activity of the enzyme incubated at 4°C was taken as a reference (maximum activity) to calculate the percentage of residual activity at any time.

The effects of different H₂O₂ concentrations on peroxidase activity were determined under standard assay conditions at the optimal pH for each enzyme (3.0 for TvVP2 and 3.5 for TvDyP1).

Oxidation of CAT hydrate and QUE was determined at room temperature by analyzing the spectra of both flavonoids between 250 and 600 nm. Additional kinetic experiments were conducted by determining the absorbance at 400 and 370 nm, corresponding to the characteristic peaks of CAT hydrate and QUE, respectively. These experiments were carried out with a Cary 50 spectrophotometer (Agilent Technologies). CAT and QUE were used at 20 and 10 μM, respectively. For both compounds, reactions were carried out in 100 mM phosphate buffer (pH 5.8). For some conditions, CAT and QUE were incubated in the presence of 1 mM H₂O₂. For these assays, the concentration of the peroxidases tested was 0.2 μM.

To test the abilities of peroxidases to decolorize industrial dyes, five dyes, AB, RB5, DB, BB, and VG, were selected. Dyes were kindly provided by the SETAS Company (Çerkezköy, Turkey). They were prepared from either powder dyes, i.e., AB (0.005% [vol/vol]), RB (0.005% [vol/vol]), and DB (0.01% [vol/vol]), prepared in 0.1 M sodium tartrate buffer (pHs 2.5, 3.0, and 3.5), or liquid dyes, i.e., BB (0.002% [vol/vol]) and VG (0.01% [vol/vol]), in each buffer (pHs 2.5, 3.0, and 3.5). Enzymatic dye decolorization capacity was assayed in 96-well microplates by adding 2 μl of H₂O₂ and 0.4 μg of enzyme to 200 μl of dye solution in sodium tartrate buffer at pHs 2.5, 3, and 3.5. Two controls were performed under the same conditions without H₂O₂ or without enzyme. The decolorization of AB, RB5, DB, BB, and VG was measured at 37°C at wavelengths of 560, 610, 530, 610, and 640 nm, respectively. Percent decolorization was calculated with the following formula: % decolorization = [(Abs t₀ − Abs t)/Abs t₀] × 100, where Abs t₀ is the optical density at the beginning of the experiment (t₀) and Abs t is the optical density after 1 h of incubation, as indicated in the legend of Table 4.

The ITS sequence obtained in this study was deposited in GenBank under accession number MG554226.