INTRODUCTION

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Lignin is a three-dimensional biopolymer composed of oxygenated phenylpropanoid units. The most common linkage between the units is an arylglycerol--aryl ether or -O-4 bond (1). Degradation of recalcitrant lignin is an oxidative and nonspecific process carried out by white rot basidiomycetes, including the best-known ligninolytic organism, Phanerochaete chrysosporium (24). Some white rot fungi, such as Pleurotus eryngii and related species, have the capacity to remove lignin preferentially (i.e., with limited attack on cellulose) (30). This fact is relevant for environmentally friendly biotechnological delignification in paper pulp manufacture (7) and feed production (19).

The most prominent enzymes associated with lignin degradation are lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP), as well as laccases and H₂O₂-producing oxidases (24). LiP appears to be the key enzyme in the oxidation of nonphenolic phenylpropanoid units which leads to polymer fragmentation. This enzyme oxidizes aromatic nuclei to aryl cation radicals which undergo several nonenzymatic reactions, including cleavage of C-C and C-O linkages (14, 24). MnP oxidizes Mn²⁺ to Mn³⁺, which in turn may attack phenolic structures in lignin (10) as long as it is stabilized by suitable metal chelators secreted by fungi (25, 27); and also may attack nonphenolic structures via lipid peroxidation (6).

Laccase and aryl-alcohol oxidase (AAO) have been purified from cultures of P. eryngii in glucose-ammonium medium and have been characterized (13, 34), and it has been suggested that these enzymes are involved in lignin degradation via oxygen activation and redox mediators (12, 31, 34). However, significant levels of LiP and MnP have not been detected in cultures of P. eryngii in glucose-ammonium medium. Recently, a high level of Mn²⁺-oxidizing peroxidase activity was observed in this fungus when peptone was used as an N source (32), and the possibility that LiP was present was suggested by the results of a Western blot hybridization analysis performed with antibodies against P. chrysosporium LiP (37). Nevertheless, LiP activity has not been detected in P. eryngii, and although a LiP type of peroxidase has been found in Pleurotus ostreatus (3), a Southern blot hybridization analysis performed with a DNA probe from the P. chrysosporium gene lpo demonstrated that P. eryngii does not have a similar gene (18).

Veratryl alcohol oxidation, the most common test for LiP detection (42), can be inhibited by peptone constituents (40) or by aromatic fungal metabolites or lignin-derived products (43). Therefore, it would be useful to evaluate the production of ligninolytic enzymes in P. eryngii by using a different detection method. An assay which has been used to assess the potential for oxidative attack of the lignin macromolecule is the assay which measures ethylene release from -keto--thiomethylbutyric acid (KTBA) (21, 28). This assay is used to detect one-electron strong oxidants, such as LiP and the hydroxyl free radical (OH·) (Fig. 1A). A correlation between KTBA oxidation and ligninolytic activity has been observed in P. chrysosporium cultures (21). On the other hand, lignin model dimers with the most frequently found lignin intermonomer linkages (Fig. 1B and C) may be used to identify the specific degradation reactions of phenolic and nonphenolic phenylpropanoid units (the latter account for 80 to 90% of the total polymer) (9) produced by either ligninolytic organisms or enzymes (24).

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Oxidation of KTBA to ethylene (4) (A) and transformation of nonphenolic (B) and phenolic (C) -O-4 dimers were used to investigate the ligninolytic enzymes produced by P. eryngii. R· represents an aromatic radical from LiP or a hydroxyl radical from Fenton type reactions; generation of both of these chemical species requires H2O2, and generation of the hydroxyl radical also requires an Fe3+-reducing system. Me, methyl.

Both KTBA and lignin model dimers were used to study the mechanism of lignin degradation by P. eryngii. This study led to purification of KTBA-oxidizing enzymes from supernatants of liquid cultures and from wheat straw treated with the fungus under solid-state fermentation (SSF) conditions similar to the conditions found in the natural habitats of ligninolytic basidiomycetes. The ligninolytic potentials of these enzymes were evaluated by using different substrates, including phenolic and nonphenolic -O-4 dimers.

	MATERIALS AND METHODS

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Organisms and cultures. The fungal strains used were P. eryngii ATCC 90787 (= IJFM A169 = CBS 613.91) and P. chrysosporium ATCC 24725 (= VKM F-1767 = IJFM A547). Shaken cultures of P. eryngii in glucose-peptone medium were inoculated and grown as described elsewhere (32), and 5-ml samples were removed periodically for analysis. As indicated below, 0.1 mM MnSO₄ was sometimes added to the medium. P. chrysosporium shallow cultures were grown as described by Tien and Kirk (42) by using Falcon 25-cm² tissue culture flasks that contained 5 ml of medium inoculated with a spore suspension and were flushed with O₂ through rubber caps.

Enzyme assays. LiP activity was measured by monitoring the oxidation of 2 mM veratryl alcohol to veratraldehyde (₃₁₀, 9.3 mM¹ cm¹) in 0.1 M sodium tartrate (pH 3) supplemented with 0.4 mM H₂O₂. Controls without H₂O₂ were included. AAO activity was measured in reaction mixtures containing 5 mM veratryl alcohol and 0.1 M phosphate buffer (pH 6). MnP activity was measured by monitoring the formation of the Mn³⁺-tartrate complex (₂₃₈, 6.5 mM¹ cm¹) from 0.1 mM MnSO₄ in 0.1 M sodium tartrate (pH 5) supplemented with 0.1 mM H₂O₂. Mn-mediated oxidation of 0.1 mM 2,6-dimethoxyphenol (DMP) to the dimeric product coerulignone (₄₆₉, 27.5 mM¹ cm¹, referred to as the DMP concentration) (32) was estimated under the same conditions. Mn-independent peroxidase and laccase activities were measured by using 0.1 mM DMP in 0.1 M sodium tartrate (pH 5). In the case of Mn-independent peroxidase 0.1 mM H₂O₂ was added to the reaction mixture. One activity unit was defined as the amount of enzyme that oxidized 1 µmol of substrate per min. When purified enzymes were compared, the activities mentioned above (as well as the KTBA-oxidizing activity described below) were expressed as specific activities in units per milligram of protein, as estimated with the Bradford reagent obtained from Bio-Rad.

In vivo and in vitro ethylene release from KTBA. Assays to determine oxidation of KTBA (final concentration, 3.3 mM) (Fig. 1A) were performed in triplicate by using flasks closed with rubber caps, and the reactions were stopped by adding 0.5 to 1 ml of 3 M H₂SO₄. In vivo assays were performed with 5-ml samples from P. eryngii liquid cultures or with 1-g straw samples from SSF (in 5 ml of reaction mixture) that were placed in sterile 25-cm² tissue culture flasks and incubated for 5 to 8 h. In the case of P. chrysosporium, whole 5-ml cultures were used. As indicated below, 0.1 mM MnSO₄ or 0.5 mM H₂O₂ was sometimes added. The in vitro assays to determine KTBA oxidation were performed in 12-ml flasks containing 1-ml reaction mixtures at room temperature for 30 min. For enzyme purification (see below), the KTBA assay was performed with 0.1-ml aliquots obtained from chromatographic fractions. The reaction mixtures contained 1 mM veratryl alcohol, 0.4 mM H₂O₂, and 0.1 M sodium tartrate (pH 3). The assay to determine KTBA oxidation by peroxidases purified from P. eryngii cultures was also performed with 0.1 M sodium tartrate (pH 5) supplemented with 0.1 mM MnSO₄. The effects of H₂O₂ concentrations and pH were investigated. The possibility that KTBA oxidation could be increased by keeping the H₂O₂ concentration low was tested (the total H₂O₂ was added in six aliquots which were injected every 5 min). Oxidation of KTBA by LiP and MnP from P. chrysosporium was also analyzed. LiP was produced and purified as described previously (42), and MnP was a gift from B. Kurek. The LiP and MnP reactions were performed as described above for P. eryngii peroxidases in the presence of veratryl alcohol and Mn²⁺, respectively. KTBA oxidation by P. eryngii laccase (34) was examined by using up to 0.5 U of laccase per ml in 0.1 M sodium tartrate (pH 5) and adding 0.1 mM DMP or 10 mM 2,2-azinobis(3-ethylbenzothiazolin-6-sulfonate).

Purification of KTBA-oxidizing enzymes from P. eryngii. The supernatants from liquid cultures were concentrated 15-fold and dialyzed (5-kDa cutoff membrane) against 10 mM sodium tartrate (pH 4.5). The contents of the SSF bottles were extracted with 3 liters of water by shaking the bottles at 200 rpm for 1 h, and the extracts were filtered, concentrated 20-fold, and dialyzed. The concentrates were loaded onto a Bio-Rad Q-cartridge (1 ml/min), and the retained fractions were eluted with 1 M NaCl. The fractions capable of oxidizing KTBA in the presence of veratryl alcohol or Mn²⁺ were applied to a Sephacryl S-200 HR (Pharmacia K16/100) column. The KTBA-oxidizing fractions were chromatographed with a Mono-Q column (Pharmacia HR 5/5) by using 10 mM sodium tartrate (pH 5) and a 0 to 0.25 M NaCl gradient (0.8 ml/min, 30 min).

Transformation of lignin model dimers by the enzymes. The two dimers used, veratrylglycerol--guaiacyl ether (nonphenolic -O-4 dimer) and guaiacylglycerol--guaiacyl ether (phenolic -O-4 dimer) (Fig. 1B and C) were synthesized by the methods described by Adler et al. (2) and Nakatsubo (35). The reaction mixtures used for degradation of the nonphenolic dimer contained peroxidase (0.5 U/ml, measured on the basis of the veratraldehyde formed), the dimer at a concentration of 0.25 mM, 0.1 M sodium tartrate (pH 3.5), and 0.1 mM H₂O₂ (added every 5 min). The effect of adding 0.25 mM veratryl alcohol or 0.1 mM MnSO₄ was also determined. Phenolic dimer degradation was assayed like nonphenolic dimer degradation except that the pH was 4.5 (with or without 0.1 mM Mn²⁺) and the peroxidase concentration was 0.25 to 1 U/ml (measured on the basis of the Mn³⁺-tartrate formation). The reactions were monitored by high-performance liquid chromatography by using standard calibration curves for both the substrates (the phenolic and nonphenolic dimers) and the reaction product (veratraldehyde). Samples (20 µl) were injected into a Pharmacia system equipped with a Spherisorb S50DS2 column. The analyses were carried out at 30°C with a flow rate of 1 ml/min by using methanol-water as the eluent (starting with 20% methanol and ending with 40% methanol after 30 min). The UV detector was operated at 210 and 250 nm or at 280 and 310 nm. Fourier transform infrared spectra were obtained with a model IFS 28 Equinox spectrometer (Brucker) by using a DTGS-type detector and 100 scan accumulation.

	RESULTS

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Action of fungal cultures on KTBA. The ability to oxidize KTBA was investigated during growth of P. eryngii under both liquid and SSF culture conditions. The profiles obtained with liquid cultures were compared with the profiles obtained with P. chrysosporium (Fig. 2). Similar amounts of ethylene were released from KTBA by the two fungi despite the differences in the enzymatic activities produced. LiP was detected only in P. chrysosporium, and laccase and AAO were found only in P. eryngii. In both fungi the highest levels of activity were the levels of peroxidase that oxidize Mn²⁺ to Mn³⁺. The highest level of LiP activity appeared after the highest levels of MnP activity were observed, and the peak matched the KTBA oxidation activity in the P. chrysosporium culture (day 5). In P. eryngii, the broad peroxidase activity peak, the laccase activity peak, and the onset of AAO activity coincided roughly with the time when the maximum amount of ethylene was released from KTBA (day 9). The same enzymes were detected in SSF cultures of P. eryngii, and it was not possible to correlate ethylene release with an individual enzymatic activity.

View larger version (20K): [in this window] [in a new window]   FIG. 2.   Patterns of enzyme production and KTBA oxidation by P. eryngii (A) and P. chrysosporium (B) cultures. Symbols: , ethylene released from KTBA; , LiP activity; , MnP activity; , laccase activity; , AAO activity.

Purification of KTBA-oxidizing enzymes. The enzymes responsible for KTBA oxidation were purified by using 8-day-old liquid cultures and 21-day-old SSF cultures of P. eryngii; these cultures exhibited maximal activity with KTBA. The ethylene release tests during protein purification were performed in the presence of H₂O₂ and veratryl alcohol (as described above for P. chrysosporium LiP detection), as well as in the absence of these compounds. Supernatants from liquid cultures and extracts from straw treated with the fungus were concentrated and dialyzed, and the colored compounds, which were especially abundant in the straw extracts, were removed by using a Q-cartridge. After molecular size exclusion gel permeation of samples from liquid cultures, a protein peak with strong KTBA-oxidizing peroxidase activity, as well as a high level of absorbance at 410 nm, was obtained (Fig. 3A). Mono-Q chromatography of this peak revealed two proteins (peroxidases PL1 and PL2) that were capable of oxidizing KTBA in the presence of H₂O₂ and veratryl alcohol (Fig. 3B). Despite the fact that all of the fractions obtained from Sephacryl chromatography of straw extracts had high levels of absorbance at 280 nm, a wide KTBA oxidation peak was identified, which corresponded to the peak in the 410-nm profile (Fig. 3C). Two 410-nm peaks were obtained by Mono-Q chromatography; however, only one of these peaks (peroxidase PS1) was able to oxidize KTBA, while the second peak (designated peroxidase PS3 because the peroxidase PS1 tail was designated peroxidase PS2) was not capable of oxidizing KTBA under the conditions described above (Fig. 3D). However, when KTBA oxidation was assayed in the presence of Mn²⁺ and H₂O₂, some ethylene was released with the four peroxidases obtained. These peroxidases, which were purified from liquid and SSF cultures, were different, as shown by their N-terminal sequences (Table 1) and the catalytic properties discussed below. Their molecular masses (42 to 45 kDa as determined by SDS-PAGE and 36 to 39 kDa as determined by MALDI-TOF) and isoelectric points (pI 3.65 to 3.80) differed only slightly.

View larger version (30K): [in this window] [in a new window]   FIG. 3.   Purification of KTBA-oxidizing peroxidases from liquid (A and B) and SSF (C and D) cultures of P. eryngii: isolation of peroxidases PL1, PL2, PS1, and PS3 by Mono-Q chromatography (B and D) of the KTBA-oxidizing fractions (elution volumes, 50 to 55 ml in panel A and 50 to 60 ml in panel B) from Sephacryl S-200 gel permeation (A and C) of concentrated extracellular proteins. , 280-nm profile; ..., 410-nm profile; , amount of ethylene released from KTBA in the presence of 1 mM veratryl alcohol; ----, NaCl gradient.

KTBA oxidation by P. eryngii enzymes. Information on the mechanism of oxidation of KTBA by P. eryngii peroxidases was obtained by monitoring ethylene release by gas chromatography and veratryl alcohol oxidation by UV spectrophotometry (data not shown). In the absence of KTBA, veratryl alcohol oxidation to veratraldehyde by peroxidases PL1 and PL2 (and to some extent by peroxidase PS1) was revealed by increased absorbance at 310 nm. However, when KTBA was added to the reaction mixture, ethylene was obtained, but there was no increase in absorbance at 310 nm. This observation was attributed to the presence of an indirect oxidation mechanism. The mediator formed by the action of the enzyme, probably the veratryl alcohol cation radical, oxidized KTBA, and there was concomitant production of ethylene. In the absence of enzyme, mediators (veratryl alcohol or Mn²⁺), or cofactors (hydroperoxides), no KTBA oxidation was observed. Laccase from P. eryngii was not capable of releasing ethylene from KTBA, even in the presence of 2,2-azinobis(3-ethylbenzothiazolin-6-sulfonate) or DMP as a potential mediator.

View this table: [in this window] [in a new window]   TABLE 2.   Comparison of the four KTBA-oxidizing peroxidases isolated from liquid cultures (peroxidases PL1 and PL2) and SSF cultures (peroxidases PS1 and PS3) of P. eryngii: enzymatic activities, KTBA oxidation, and degradation of -O-4 dimers (effects of Mn2+ and veratryl alcohol)

View larger version (13K): [in this window] [in a new window]   FIG. 4.   Inhibition of P. eryngii peroxidase PL2 activity () by different amounts of H2O2, compared with P. chrysosporium MnP activity (----).

Transformation of lignin model dimers by the P. eryngii peroxidases. We investigated degradation of both phenolic and nonphenolic -O-4 dimers by peroxidases from liquid and SSF cultures of P. eryngii. The phenolic dimer was degraded by peroxidases PL1 and PL2 in the absence of Mn²⁺ (Table 2). However, the reaction was enhanced by Mn²⁺ (Fig. 5A), and the highest degradation rate was observed at pH values around 4.5. On the other hand, peroxidases PS1 and PS3 from SSF cultures required Mn²⁺ in order to attack the phenolic dimer (Fig. 5B). Despite the fact that both of these enzymes exhibited Mn-independent activity with DMP, they differed from peroxidases obtained from liquid cultures by exhibiting lower levels of activity with guaiacol (the specific activities with 5 mM guaiacol were only 10 to 20% of the specific activities of the liquid culture peroxidases). During the peroxidase reaction with phenolic dimers, a pale brown precipitate was formed, which was characterized by Fourier transform infrared signals at 1,727, 1,628, 1,502, 1,464, 1,259, 1,215, and 1,125 cm¹ and probably resulted from condensation of the aromatic radicals formed.

View larger version (13K): [in this window] [in a new window]   FIG. 5.   Differences between P. eryngii peroxidases PL2 (A) and PS3 (B), as shown by oxidation of a phenolic -O-4 dimer in the presence () and in the absence (...) of 0.1 mM Mn2+ (0.5 U of peroxidase per liter, estimated as Mn3+-tartrate, and 0.1 M tartrate [pH 4.5] were used).

	DISCUSSION

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Detection of the ligninolytic activity of P. chrysosporium peroxidases was related to the use of KTBA and lignin model dimers as substrates. When Kuwahara et al. (28) purified the two ligninolytic peroxidases secreted by this fungus, LiP was detected on the basis of its ability to release ethylene from KTBA in the presence of veratryl alcohol. Its ability to degrade a nonphenolic -O-4 dimer was considered a reflection of its ligninolytic capabilities (41). In the present study we found that P. eryngii, a fungus that was investigated because of its ability to degrade wheat lignin selectively (30), oxidizes KTBA to ethylene when it is grown in peptone-containing media, as well as during treatment of straw under the SSF conditions used for biomechanical and biosemichemical pulping (7, 11, 30). LiP activity was not detected in the cultures mentioned above, and the oxidation of veratryl alcohol observed was due to Pleurotus AAO. However, the KTBA oxidation observed was similar to that found in P. chrysosporium cultures grown under ligninolytic conditions. KTBA oxidation in the absence of a typical LiP has also been observed in some other fungi (22, 29).

The search for ligninolytic enzymes in P. eryngii resulted in identification of four proteins that have the ability to release ethylene from KTBA (Table 2). Three of these proteins (one protein from SSF cultures and two proteins from liquid cultures) were detected under the conditions used to identify LiP in P. chrysosporium (20) (i.e., they degraded KTBA in the presence of H₂O₂ and veratryl alcohol, which in the absence of KTBA was oxidized to veratraldehyde). These peroxidases also oxidized Mn²⁺, as well as different phenols in the absence of Mn²⁺. Phenolic compounds inactivate LiP (15) and can interfere with veratryl alcohol oxidation by P. eryngii peroxidases because of competitive inhibition. This explains why peroxidase activity with veratryl alcohol was not detected in peptone-containing medium or in SSF extracts, which contained different phenols (40, 43). The KTBA-oxidizing enzymes obtained from liquid cultures were the same peroxidases as those described previously by Martínez et al. (32) as two MnP isoenzymes that exhibit Mn-independent activity with some aromatic substrates. In the present study we observed some of the LiP characteristics of these enzymes, such as the ability to degrade nonphenolic -O-4 dimers (yielding veratraldehyde) and the ability to release ethylene from KTBA in the presence of veratryl alcohol. It is interesting that the P. eryngii peroxidases oxidized both dimers in the absence of mediators, Mn²⁺, or veratryl alcohol, whereas KTBA oxidation was dependent on the presence of these compounds. The third enzyme that could oxidize KTBA in the presence of veratryl alcohol and H₂O₂ (peroxidase PS1) was found in the SSF cultures. This peroxidase exhibited high levels of activity with Mn²⁺ and DMP, lower levels of activity with veratryl alcohol and guaiacol, and no activity with the dimers used. It differs from P. chrysosporium MnP mainly by its Mn-independent activity with DMP and veratryl alcohol. The lack of activity with the two model dimers revealed that this enzyme, isolated for the first time in this study, is different from the peroxidases obtained from liquid cultures (as confirmed by N-terminal sequencing). The three peroxidases discussed above were identified on the basis of their abilities to oxidize KTBA in the presence of veratryl alcohol. However, when assessing KTBA oxidation by P. eryngii cultures, we found that adding Mn²⁺ to the reaction mixtures enhanced ethylene release. A similar result suggested that MnP may be involved in KTBA oxidation by Trametes versicolor (4). This finding led to identification and purification of a fourth peroxidase in the SSF cultures of P. eryngii (peroxidase PS3), which oxidized KTBA in the presence of Mn²⁺. The catalytic properties of this enzyme, which was isolated for the first time in this study, are similar to those of previously described Mn-dependent peroxidases (it does not oxidize veratryl alcohol or nonphenolic dimers, and oxidation of the phenolic dimer is strictly dependent on Mn²⁺).

In this study we found that P. eryngii produces three types of extracellular peroxidases. One of these peroxidases, which is found only in SSF cultures, is related to the MnP of P. chrysosporium. The second peroxidase is a polyvalent peroxidase that is isolated from liquid cultures and exhibits both LiP and MnP types of catalytic properties, including Mn²⁺ oxidation and direct oxidation of phenolic and nonphenolic dimers. It differs from P. chrysosporium MnP and LiP with respect to oxidation of phenols (P. chrysosporium MnP requires Mn²⁺ to oxidize phenols [10], and LiP requires veratryl alcohol [26]). The third type of peroxidase, which is isolated from SSF cultures, exhibits activity with Mn²⁺ and phenols, a low level of activity with veratryl alcohol, and no activity with dimers, suggesting that the redox potential is intermediate between the redox potentials of the other two P. eryngii peroxidases. Similar Mn-oxidizing peroxidases are produced by other Pleurotus species grown in liquid media or on lignocellulosic substrates under SSF conditions (Table 1). Based on their catalytic properties and N-terminal sequences (or whole protein sequences when available), the peroxidases could be classified into the following four groups: (i) group i, including P. eryngii peroxidase PS1 from wheat straw cultures, P. ostreatus peroxidase MnP2 from sawdust (38), and P. pulmonarius peroxidase from wheat straw (8) (N terminus, VTCATGQTT); (ii) group ii, including P. eryngii peroxidase from liquid cultures (32) (including two allelic variants), P. ostreatus peroxidases from sawdust (MnP1) (38) and liquid cultures (39), and P. pulmonarius peroxidase from liquid cultures (8) (N terminus, ATCDDGRTT); (iii) group iii, including P. eryngii peroxidase PS3 from wheat straw (N terminus, VTCADGNTV); and (iv) group iv, including the P. ostreatus peroxidase described by Asada et al. (5) from liquid cultures (N terminus, ATCAGGQVT). The peroxidases in groups iii and iv can be considered close relatives of P. chrysosporium MnP, whereas the peroxidases in groups i and ii correspond to two different versatile peroxidases which have been found in the three Pleurotus species. Similar peroxidases have been found recently in Bjerkandera adusta (16) and Bjerkandera sp. (33); the latter was described as a LiP-MnP "hybrid" peroxidase. These Pleurotus and Bjerkandera Mn²-oxidizing peroxidases can oxidize typical LiP substrates, such as veratryl alcohol and dimethoxybenzenes, and inhibition studies have shown that they have different binding sites for Mn²⁺ and veratryl alcohol or dyes (17, 33).

Molecular models for the new polyvalent peroxidases produced by P. eryngii (peroxidases PL1 and PS1) have been constructed recently (Brookhaven Protein Database entries 1A20 and 1BQW), and these models reveal greater structural (and sequence) homology with P. chrysosporium LiP than with MnP. This could explain the LiP type properties observed and the crossed-reactivity with the anti-LiP antibodies used by Orth et al. (37). Moreover, these P. eryngii peroxidases have a putative Mn interaction site (which includes inner heme propionate and peroxidase PL1 Glu-36, Glu-40, and Asp-175 residues or peroxidase PS1 Glu-36, Glu-40, and Asp-181 residues) that is involved in Mn²⁺ oxidation (17). It is necessary to mention that Mn²⁺ oxidation by both wild-type and recombinant LiP isoenzyme H2 has been reported (23, 36), but this isoenzyme does not have an Mn-binding site (Brookhaven Protein Database entry 1QPA) and the mechanism involved is not known. The versatile catalytic properties of some Pleurotus peroxidases can explain why these enzymes have been reported by different authors to be either LiP (3) or MnP (32, 39). Additional studies on the regulation and structure-function relationships of these enzymes are under way in order to better understand their role in lignin degradation by P. eryngii and other Pleurotus species which do not produce typical LiP.