Vol. 59, No. 12APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1993, p. 4017-40230099-2240/93/124017-07$02.00/0Copyright ©) 1993, American Society for Microbiology

Ubiquity of Lignin-Degrading Peroxidases among VariousWood-Degrading Fungi

ANN B. ORTH,lt DANIEL J. ROYSE,2 AND MING TIEN'*Departments ofMolecular and Cell Biology' and Plant Pathology,2 The Pennsylvania State University,

University Park, Pennsylvania 16802

Received 9 August 1993/Accepted 23 September 1993

Phanerochaete chrysosporium is rapidly becoming a model system for the study of lignin biodegradation.Numerous studies on the physiology, biochemistry, chemistry, and genetics of this system have beenperformed. However, P. chrysosporium is not the only fungus to have a lignin-degrading enzyme system. Manyother ligninolytic species of fungi, as well as other distantly related organisms which are known to producelignin peroxidases, are described in this paper. In this study, we demonstrated the presence of the peroxidativeenzymes in nine species not previously investigated. The fungi studied produced significant manganeseperoxidase activity when they were grown on an oak sawdust substrate supplemented with wheat bran, millet,and sucrose. Many of the fungi also exhibited laccase and/or glyoxal oxidase activity. Inhibitors present in themedium prevented measurement of lignin peroxidase activity. However, Western blots (immunoblots) revealedthat several of the fungi produced lignin peroxidase proteins. We concluded from this work that lignin-degrading peroxidases are present in nearly all ligninolytic fungi, but may be expressed differentially indifferent species. Substantial variability exists in the levels and types of ligninolytic enzymes produced bydifferent white rot fungi.

Lignin is a random phenylpropanoid polymer which isdifficult to degrade because of its complex, heterogeneousstructure. Up to 30% of plant material is composed of lignin,which gives plants their structural integrity and providesprotection from pests and pathogens. Two peroxidases dis-covered in the extracellular fluid of Phanerochaete chryso-sporium, lignin peroxidase (LP) (20, 49) and manganeseperoxidase (MnP) (19), are known to play important roles inthe initial degradation of lignin. Laccase also has beenimplicated in lignin degradation in some organisms. Theseenzymes and the organisms that produce them are widelyconsidered to have potential for industrial applications, suchas biodegradation of environmental pollutants (6), biocon-version of lignin (7), biobleaching and biopulping of woodchips (13), desulfurization of petroleum and coal (47), anddelignification of agricultural plant residues.Phanerochaete chrysosporium has been used as a model

system for the study of lignin biodegradation, and there havebeen numerous articles on the physiology, biochemistry,chemistry, and genetics of this system. These efforts havemade Phanerochaete chrysosporium by far the best charac-terized of all of the wood-rotting fungi (for a review, seereference 46). However, Phanerochaete chrysosporium isby no means the only fungus to have lignin-degradingcapacity and, hence, a lignin-degrading enzyme system.Many other ligninolytic species of fungi, as well as otherunrelated organisms, have been shown to produce LPs(Table 1). Many white rot fungi produce LPs, and recentreports have shown that LPs are present in brown rot fungias well (12). Peroxidase activity and genes that control thisactivity have even been found in actinomycete bacteria (56).Recent reports have raised the issue of the involvement of

LPs in the degradation of lignin (45). These reports have

* Corresponding author.t Present address: DowElanco, Discovery Research, Indianapo-

lis, IN 46268-1053.

cited evidence that the enzymes were not detectable incertain fungi that have appreciable ability to convert wheatstraw lignins to CO2. In this study, we found that the fungimentioned above do indeed produce lignin-degrading perox-idases in the presence of a wood substrate; in this paper wealso discuss many studies whose results support the conclu-sion that these peroxidases are ubiquitous in lignin-degrad-ing organisms. Below we summarize the current literatureregarding the presence of these peroxidases in wood-rottingorganisms. In this study we also confirmed the presence ofthe enzymes in nine species not previously investigated.

MATERIALS AND METHODS

Strains and culture conditions. The fungal strains used inthis study and their sources are shown in Table 2.Spawn (inoculum) of each fungus was prepared as de-

scribed by Jodon and Royse (27). Rye grain (Secale cerealeL.) and hardwood sawdust were weighed (80 and 13 g,respectively) and were placed into 500-ml Erlenmeyer flasks.Then 1 g of CaSO4 was added to each flask along with 120 mlof warm (37°C) tap water. The flasks and contents wereautoclaved for 45 min at 121°C. After autoclaving, the flaskswere allowed to cool and were inoculated with strains of thefungi maintained on potato dextrose-yeast extract mediumas described by Jodon and Royse (27). The spawn wasinoculated at room temperature (23 + 2°C) and was shakentwice each week for 2 weeks to prevent the formation ofclumps.The finished spawn was used to inoculate a moistened

(60%) substrate (2,270 g [wet weight]; 908 g [dry weight])contained in autoclavable polypropylene bags, each ofwhichhad a microporous breather strip. The substrate containedthe following ingredients (per bag): 742 g (dry weight) of oaksawdust, 80 g (dry weight) of wheat bran, 80 g (dry weight)of white millet, and 6 g (dry weight) of sucrose.

Extraction of enzymes. After incubation for 30 days, thecultures were harvested by pressing the substrate with a

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TABLE 1. Organisms capable of producing LP and/or MnP

Organism Enzyme(s) produced Method(s) of detection Reference(s)

Bjerkandera adusta (Polyporous adustus) LP Enzyme activity, hybridization, gene cloned 26, 32, 53Ceniporiopsis subvermispora MnP Enzyme activity, hybridization 43Chrysonilia sitophila LP Enzyme activity 15Chrysosponium pruinosum LP Enzyme activity 53Conolopsis occidentalis LP Enzyme activity 36Coriolopsis polyzona MnP Enzyme activity 37Conolus consors LP Hybridization 32Coriolus hirsutus LP Enzyme activity 37, 48Daedaleopsis confragosa MnP Enzyme activity 10Dichomitus squalens MnP Enzyme activity 37, 41Fomes lignosus (Rigidoporous LP Enzyme activity 26, 53

microsporous)Ganoderma valesiacum MnP Enzyme activity 37Lentinula edodes MnP Enzyme activity, antibodies 8, 17Panus tigrinus MnP Enzyme activity 34Phanerochaete chrysosporium LP, MnP Enzyme activity, genes cloned 19, 20, 49Phellinus pini LP Antibody 4Phlebia brevispora LP, MnP Enzyme activity, hybridization 43Phlebia radiata LP, MnP Enzyme activity, hybridization, LP cloned 25, 30, 38, 44Pleurotus ostreatus LP Enzyme activity 53Polyporus ostreiformis LP Enzyme activity 12Pycnoporus cinnabaninus MnP Enzyme activity 37Rigidoporus lignosus MnP Enzyme activity 18Stereum hirsutum MnP Enzyme activity 37Streptomyces vinidosporus LP Enzyme activity, gene cloned 42, 54Trametes gibbosa LP, MnP Enzyme activity 37Trametes versicolor (Coriolus versicolor) LP, MnP Enzyme activity, antibody, hybridization, 1, 3, 4, 26, 28, 29, 53

gene clonedTrametes villosa MnP Enzyme activity 10

hydraulic press that generated a pressure of 660 lb/in2, whichextruded most of the fluid from the solid substrate. No bufferwas added to the cultures before the fluid was pressed out.The exudate (total volume, approximately 600 ml for eachculture) was filtered through several layers of cheesecloth toremove particulate matter and was centrifuged at 12,000 x gfor 30 min at 4°C. The clarified extract was concentrated at4°C approximately 10-fold by ultrafiltration through a typePM10 Dialflo ultrafilter having a 10,000-dalton cutoff (Ami-con). Samples were dialyzed against 10 mM sodium acetatebuffer (pH 5.5). The resulting preparation was frozen in

TABLE 2. Fungal strains used in this study and their sources

Organism Sourcea Strain

Cyathus stercoreus ATCC ATCC 36910Dichomitus squalens FPL FPL 6691Ganoderma lucidum PSUMCC PSUMCC 510Gnfola frondosa PSUMCC PSUMCC 483Lentinula edodes PSUMCC PSUMCC R26Perenniporia medulla-panis FPL FPL 11024Phanerochaete ATCC ATCC 24725 (=

chrysosporium BKM-F-1767)Pleurotus sapidus PSUMCC PSUMCC 528Pleurotus eryngii PSUMCC PSUMCC 515Pleurotus pulmonarius PSUMCC PSUMCC R29Polyporus versicolor Merrill PSU 48Trametes cingulata ATCC ATCC 26747

a ATCC, American Type Culture Collection, Rockville, Md.; FPL, ForestProducts Laboratory, U.S. Department of Agriculture, Madison, Wis.;PSUMCC, Pennsylvania State University Mushroom Culture Collection,Pennsylvania State University, University Park; Merrill, Williams Merrill,Pennsylvania State University, University Park.

small aliquots for use in all subsequent enzyme assays andgels.Enzyme assays. Glyoxal oxidase activity was determined

by measuring the amount of H202 produced with a peroxi-dase-coupled assay, as modified by Kersten and Kirk (31).Each reaction mixture contained 50 mM sodium 2,2-dimeth-ylsuccinate (pH 6.0), 10 mM methylglyoxal as the substrate,0.01% phenol red, 10 ,ug of horseradish peroxidase (type II;Sigma Chemical Co., St. Louis, Mo.) per ml, and up to 300,u of extracellular fluid per ml of reaction mixture. A 5-mlmixture was prepared, and the reaction was initiated byadding methylglyoxal. The H202-dependent oxidation ofphenol red (£ = 22,000. M`- cm-1) by horseradish perox-idase was determined by monitoring the change in A610. Thereaction was carried out at room temperature. The reactionmixture was sampled every 30 s for 4 min by removing 1 mlof the mixture and adding it to 50 RI of 2 N sodiumhydroxide.MnP activity was determined by monitoring the oxidation

of phenol red spectrophotometrically at 610 nm (19). Eachreaction mixture contained 50 mM sodium succinate (pH4.5), 50 mM sodium lactate (pH 4.5), 0.1 mM MnSO4, 3 mgof gelatin per ml, 50 ,uM H202, and 0.1 mM phenol red. Thereaction was initiated by adding H202, and the reactionmixture was incubated at 30°C. The reaction mixture wassampled by removing 1 ml of the 5-ml mixture and adding the1-ml sample to 40 ,u1 of 5 N sodium hydroxide every minutefor 4 min.Laccase activity was determined by the method of Bollag

and Leonowicz (5) by monitorin~ the oxidation of syring-aldazine (£ = 65,000. M-1 cm- ) at 525 nm and 24°C.LP activity was measured by monitoring the oxidation of

veratryl alcohol to veratraldehyde (£ = 9,300 M-1. cm-')

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TABLE 3. MnP activities found in supplemented oak sawdustculture extracts of various fungi incubated for 30 days at 23°C

MnP Protein Sp actOrganism activity concn (/go(U/mI of (mg/ml of protein)

extract) extract) poen

Cyathus stercoreus 0.04 0.40 0.10Dichomitus squalens 16.20 1.91 8.49Ganoderma lucidum 5.7 2.30 2.48Gnfola frondosa 0.05 1.11 0.04Lentinula edodes 12.4 1.05 11.8Perenniporia medulla-panis 16.4 2.06 7.98Phanerochaete chrysosporium 3.4 0.65 5.2Pleurotus sapidus 14.7 1.07 13.7Pleurotus eryngii 4.1 0.92 4.47Pleurotus pulmonarius 22.3 1.17 19.0Polyporus versicolor 4.2 1.00 4.25Trametes cingulata 0.33 1.68 0.20

as indicated by an increase in A310 (50). Each assay mixturecontained 25 mM sodium tartrate (pH 2.5), 2 mM veratrylalcohol, 0.4 mM H202, and 50 to 275 ,ul of extracellular fluidin a total volume of 0.5 ml. The reaction was initiated byadding H202.One unit of enzyme activity was defined as 1 ,umol of

product formed per min.Protein contents were determined by a protein assay

(Bio-Rad, Richmond, Calif.), using bovine serum albumin asthe standard.HPLC analysis. Oxidation of veratryl alcohol to veratral-

dehyde was analyzed by high-performance liquid chroma-tography (HPLC) as well as spectrophotometrically. Assayswere performed as described above for veratryl alcoholoxidation. The LP isozyme H2 used in the assay was purifiedas previously described (51). The assay mixture was injectedonto a reverse-phase C18 column (Vydac) in an SSI model210 Guardian HPLC system. The reaction mixture waseluted from the column by using water and methanol gradi-ents as follows: 0 to 70% methanol for 48 min and then 70 to100% methanol for 12 min at a flow rate of 1 ml/min. Theelution profile was monitored at A280 and A310 with a Gilsonmodel 1l1B UV detector. The products were quantified byusing veratryl alcohol as the standard.SDS-PAGE and Western blotting. A prepared extract (50

,ul) from each culture was subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE)(10% acrylamide) under denaturing conditions as describedby Laemmli (33). Western blots (immunoblots) were visual-ized by using rabbit polyclonal antibody to isozyme H2 andgoat anti-rabbit antibody conjugated to alkaline phos-phatase. Antisera were obtained from New Zealand Whiterabbits immunized with LP isozyme H2 as previously de-scribed (52).

RESULTS

A total of 12 strains were analyzed for their extracellularenzymes after growth on an oak sawdust substrate. Themost abundant activity found in these 12 strains was MnPactivity (Table 3). All 12 strains exhibited MnP activity, andsome strains clearly exhibited higher levels of activity thanothers. The highest level of activity was found in Pleurotuspulmonarius (19 U/mg of protein), followed by Pleurotussapidus (13.7 U/mg), Lentinula edodes (11.8 U/mg), andDichomitus squalens (8.49 U/mg). All of these fungi exhib-

0.08

0.06

¢)0.04

0.02 -

0 1 2 3 4 5Time, min

FIG. 1. Extracellular MnP activity found in a supplemented oaksawdust culture extract of Cyathus stercoreus. Symbols: 0, H202not present; 0, H202 present. MnP activity was measured bydetermining the oxidation of phenol red as described in Materialsand Methods.

ited higher levels of activity than Phanerochaete chryso-sporium (5.2 U/mg) grown under similar conditions. Al-though two of the strains (Cyathus stercoreus and Grifolafrondosa) exhibited much lower levels of activity than theother strains, they still exhibited appreciable levels of activ-ity, as shown in Fig. 1.The fungi used in this study exhibited much less laccase

activity than MnP activity. Laccase is another enzymeimplicated in the biodegradation of lignin (Table 4). Manyorganisms exhibited no laccase activity at all, as is the casewith Phanerochaete chrysosporium. Cyathus stercoreus(0.68 nmol/min/mg of protein) and L. edodes (0.43 nmol/min/mg) exhibited the highest levels of laccase activity.

Glyoxal oxidase is one enzyme that is responsible for theproduction of the H202 needed for the ligninolytic peroxi-dases to function (31). Although not detected in all extracts(Table 5), glyoxal oxidase activity was observed at appre-ciable levels in D. squalens (0.04 U/mg), Ganoderna luci-dum (0.14 U/mg), Perenniporia medulla-panis (0.27 U/mg),Polyporus versicolor (0.05 U/mg), and Trametes cingulata(0.22 U/mg).The veratryl alcohol oxidation assay is the standard assay

used to detect LP activity. However, we did not detect LP

TABLE 4. Laccase activities found in supplemented oak sawdustculture extracts of various fungi incubated for 30 days at 23°C

Laccase activity Sp actOrganism (nmol/min/ml of (nmol/min/mg

extract) of protein)

Cyathus stercoreus 0.27 0.68Dichomitus squalens 0.43 0.23Ganoderma lucidum 0.06 0.03Gnfola frondosa 0.2 0.18Lentinula edodes 0.45 0.43Perenniporia medulla-panis 0.00 0.00Pleurotus sapidus 0.21 0.20Pleurotus eryngii 0.00 0.00Pleurotus pulmonarius 0.00 0.00Polyporus versicolor 0.00 0.00Trametes cingulata 0.00 0.00

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TABLE 5. Glyoxal oxidase activities found in supplemented oaksawdust culture extracts of various fungi incubated for 30 days

at 23°C

Glyoxal Activity in

Organism oxidase methylglyoxal Spmactactivity (U/ml control (U/ml protein)of extract) of extract) poen

Cyathus stercoreus NDa NDDichomitus squalens 0.08 0.0 0.04Ganoderma lucidum 0.33 0.01 0.14Gnfola frondosa ND NDLentinula edodes ND NDPerenniponia medulla-panis 0.56 0.05 0.27Pleurotus sapidus ND NDPleurotus eryngii ND NDPleurotus pulmonarius ND NDPolyporus versicolor 0.05 0.01 0.05Trametes cingulata 0.37 0.00 0.22

a ND, not detected.

activity in any of the fungal extracts collected. To confirmthis result, we assayed for the appearance of veratraldehydeby monitoring veratraldehyde elution from an RP-C18 HPLCcolumn. We detected no veratraldehyde production whenthe extracts were used individually as enzyme sources (datanot shown). To determine whether there was an enzyme-inhibiting substance in the sawdust extracts, we performedthe assay with purified isozyme H2 from Phanerochaetechrysosporium. As shown in Table 6, addition of this extractto the reaction mixture inhibited the oxidation of veratrylalcohol to veratraldehyde. After a 90-min reaction, theconcentration of veratraldehyde in the solution was 1.04 mMwhen isozyme H2 was used. However, when extracts ofPhanerochaete chrysosporium grown on sawdust mediumwere used, the veratraldehyde concentration was the sameas the concentration in the control lacking H202.A Western blot analysis of extracts to determine LP

activity was performed by using the antibody for H2 (Fig. 2).This analysis revealed that at least four of the fungal strains(Pleurotus sapidus, Pleurotus eryngii, Pleurotus pulmonar-ius, Polyporus versicolor) could produce LP under theseconditions. In each case, the mobility of at least one proteinon the SDS-PAGE gel was identical to the mobility of thePhanerochaete chrysosporium enzyme used as a standard.A second LP appeared to be present in Polyporus versicolor,as shown by its strong reactivity with the H2 antibody.

TABLE 6. Inhibition of veratryl alcohol oxidation bysupplemented oak sawdust culture extracts

of Phanerochaete chrysosporium

Reaction conditionsa Veratryl alcohol Veratraldehyde

Extract H202 concn (mM) concn (mM)b

- - 0.71 0.10- + 0.63 1.04+ - 0.77 0.11+ + 0.69 0.07

a Isozyme H2 of LP was present in each reaction mixture. The extract wasan extract from a culture fluid of Phanerochaete chrysosporium that waspressed and processed as described in Materials and Methods.

b Concentration of veratraldehyde produced by purified isozyme H2 of LPin each reaction mixture after a 90-min reaction as described in Materials andMethods.

1 2 3 4 5 6 7 8 9 10 1 112

FIG. 2. Western blot analysis of LPs present in wood chipextracts of various white rot fungi. The proteins in the extracts wereseparated by SDS-PAGE, transferred to nitrocellulose, and visual-ized by using polyclonal anti-H2. Lane 1, purified Phanerochaetechrysosporium H2; lane 2, Dichomitus squalens; lane 3, T. cingu-lata; lane 4, Polyporus versicolor; lane 5, Pleurotus eryngii; lane 6,Pleurotus sapidus; lane 7, L. edodes; lane 8, Grifola frondosa; lane9, Cyathus stercoreus; lane 10, Ganoderma lucidum; lane 11,Pleurotus pulmonanus; lane 12, Perenniporia medulla-panis.

Weaker reactivity with some of the other fungal extracts wasfound as well.

DISCUSSIONIn this study, we demonstrated the ubiquity of the lignin-

degrading peroxidases in fungi known to degrade lignin. Inall of the fungi tested, including nine species never investi-gated previously, MnP activity was found when the organ-isms were cultured on a supplemented sawdust substrate. Insome of these fungi, glyoxal oxidase activity was also found,and the LP H2 antibody exhibited a positive reaction withseveral fungi as well. A thorough search of the literatureconfirmed that these peroxidases are present in virtuallyevery lignin-degrading fungus that has been studied (at least25 different species) (Table 1). More detailed work withsome of these organisms has also revealed the genes whichcode for the proteins.The significance of extracellular peroxidases in lignin

biodegradation is well documented. Mutants that exhibitlower levels of extracellular peroxidase activity and alsolower levels of total ligninolytic activity have been generated(21). More significantly, these peroxidases have been shownto catalyze the depolymerization of synthetic lignins (23, 24,55) and methylated native lignins in vitro (49). Finally, theseperoxidases require H202 for activity, which is also secretedby the fungi (16), and addition of catalase to ligninolyticcultures inhibits lignin degradation (14).

Previous workers have speculated that the extracellularperoxidases play only a minor role in lignin degradation,citing as support for this argument the fact that some whiterot fungi, including T. cingulata and Fomes lignosus, do notproduce these peroxidases (45). These authors cited a studyperformed by Waldner et al. (53) as evidence for theirconclusion. However, Sarkanen and his coworkers (45)seem to have interpreted the data presented in this studydifferently than the original authors, who state that "thetested fungi have similar lignin degrading systems with minordifferences... Peroxidase production was common to allfungi." In addition, investigators who discount the impor-tance of LPs in the degradation of lignin have not provided

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positive evidence for the presence of another lignin-degrad-ing enzyme.

In our study, we showed both experimentally and throughan examination of previous work that the fungi examined doindeed produce peroxidases in significant quantities. In T.cingulata, we observed appreciable levels of MnP activity,as well as glyoxal oxidase activity, in fungal extracts of anatural substrate. The requirement for an extracellularH202-producing enzyme in this organism provides additionalevidence that white rot fungi use peroxidases as their pri-mary ligninolytic system. In F. lignosus, the assertion thatno peroxidases are produced (45) is difficult to understand inlight of research which predates this claim. In addition to thestudy in which it was shown that F. lignosus is capable ofproducing LP activity (53), a second investigation providedevidence for the presence of an LP gene through hybridiza-tion to a cloned Phanerochaete chrysosponum LP geneprobe (26).

In addition to finding that each of the fungi which wetested exhibited MnP activity, we also demonstrated thepresence of LP in some, but not all, of these fungi byWestern blotting. Our findings are in agreement with theresults of a number of other studies which have revealedgreat variability in the ligninolytic enzymes produced byvarious wood-rotting fungi. A study in which workers inves-tigated enzyme activities in a variety of basidiomycete fungidemonstrated the presence of LP only in Phanerochaetechrysosporium, as was found in our study (10). However, inanother report, MnP was the dominant enzyme of Phanero-chaete chrysosporium extracts cultured on mechanical pulpof aspen wood, even though LP, MnP, and glyoxal oxidasewere all found by an immunoblot analysis under theseconditions (9). The authors concluded that the genes encod-ing lignin-degrading enzymes are expressed differently dur-ing solid-state fermentation than during liquid culture. An-other study whose results support the hypothesis thatdifferential expression of the enzymes occurs under differentconditions revealed that MnP, laccase, and glyoxal oxidaseactivities are present in Phlebia brevispora and Cenponiop-sis subvermispora, but that there was no LP activity inagitated liquid cultures (43). However, Ruttimann et al.demonstrated the presence of LP genes in both of these fungiby Southern hybridization by using a probe from Phanero-chaete chrysosporium. Thus, these fungi appear to be capa-ble of producing LP under the proper conditions. Indeed,when one fungal strain used in our study, the Polyporusversicolor strain, was cultured in the same artificial mediumused to induce LP production in Phanerochaete chrysos-ponum (51), respectable levels of LP activity (200 U/liter)were found; these levels were similar to those found inPhanerochaete chrysosponum. We speculate that in studiessuch as ours, in which extracts obtained from a naturallydecaying substrate are assayed, the extracellular enzymeprofile could vary depending on the state of substrate decay.It seems reasonable that differential expression of enzymeshaving different specificities may be the most efficient wayfor an organism to optimally degrade its substrate in order toobtain nutrients.The results of many other studies confirm the variability of

production of the ligninolytic enzymes. Perez and Jeffries(40) have demonstrated that there is a correlation betweensynthetic lignin mineralization and LP production, but notlaccase or MnP production, in Phanerochaete chrysospo-rium and Phlebia brevispora in synthetic medium. Blanch-ette et al. (4) used antisera to isozymes H8 and H2 of LPfollowed by immunogold labeling to detect LP outside and

inside fungal hyphae within delignified walls of birch woodafter 12 weeks of decay by Phanerochaete chrysosponum orPhellinuspini. These two fungi exhibited preferential degra-dation of lignin in this study rather than continuous degra-dation of all cell wall components. In the same work,although T. versicolor has been very well studied in thelaboratory and is known to produce LP (Table 1), very littleH8 and no H2 were found in the birch wood decayed by thisfungus. Similarly, although Phanerochaete chrysosporium isknown to produce LP, in this study we found only MnPactivity when the organism was grown on the sawdustsubstrate. This is consistent with the results of other inves-tigations (2, 39) performed with T. versicolor, in which it wasfound that laccase production and MnP production (but notLP production) are closely associated with pulp bleaching,demethylation, and delignification of kraft pulp. The resultsof these investigations confirm the variability of expressionof the enzymes in different organisms under different condi-tions.

Extracellular enzyme production in wood-rotting fungi hasproven to be quite diverse. An as-yet-undefined peroxidaseactivity has been found in Pycnoporus cinnabarinus (22); amanganese-inhibited peroxidase has been found in one Bjer-kandera sp. (11); only laccase was found in some Pleurotusspecies (35), although the authors did not look for MnP; andin Rigidoporus lignosus, an unusual requirement for syner-gistic action of MnP and laccase has been proposed (18). Weconclude that the potential to produce lignin-degrading per-oxidases is present in nearly all ligninolytic fungi. However,the nature of ligninolytic enzyme expression and the roles ofthe various isozymes in vivo have yet to be determined andwarrant further study.

ACKNOWLEDGMENTS

This work was supported in part by U.S. Department of Energygrant DE-FG02-87ER13690 and Public Health Service grant1-P42ES04922-01 from the National Institute of EnvironmentalHealth. Ann B. Orth is a postdoctoral fellow supported by NationalResearch Service Award 1-F32-ES05503 from the National Instituteof Environmental Health.

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