Purification and characterization of manganese peroxidases from thelitter-decomposing basidiomycetes Agrocybe praecox

and Stropharia coronilla

Kari Timo Steffen*, Martin Hofrichter, Annele Hatakka

Department of Applied Chemistry and Microbiology, P.O. Box 56, Viikki Biocenter, 00014 University of Helsinki, Finland

Abstract

Extracellular manganese peroxidase (MnP) was purified from liquid cultures of the litter-decomposing basidiomycetes Agrocybe praecoxand Stropharia coronilla. Both fungi produced MnP increasingly in response to Mn2� in the medium. A. praecox secreted two MnP isoformswith similar isoelectric points (pI) of 6.3–7.0 and a molecular weight (MW) of 42 kDa. MnP activity was not observed in Mn2�-free culturesof A. praecox. In Mn2�-supplemented cultures, S. coronilla produced at least two MnPs, of which the main isoform MnP1 has a pI of6.3–7.1 and a MW of 41 kDa. In addition, S. coronilla possesses a partly constitutive MnP (MnP2) which was also detectable in Mn2�-freecultures, although its amount was considerably lower. MnP2 showed two distinct bands with acidic pIs of 3.5 and 3.7 in the IEF gel andhas a MW of 41 kDa. There are indications for the existence of a third, likewise Mn2�-inducible enzyme (MnP3), that could not beseparated from MnP2 but formed an additional band in eletrophoretic analyses (pI 5.1, MW 43 kDa). © 2002 Elsevier Science Inc.All rights reserved.

Keywords: Litter-decomposing fungi; Manganese peroxidase; Agrocybe praecox; Stropharia coronilla

1. Introduction

Litter-decomposing fungi are an ecologically importantgroup of microorganisms involved in the recycling of car-bon in soils [1]. Their habitat is the uppermost part of soil(litter layer) in forests and grasslands, which is rich inorganic matter and comprises cellulose, hemicelluloses andlignin from dead plant materials (leaves, needles, twigs,etc.). Litter-decomposing fungi, most prominently agaricbasidiomycetes, are capable of attacking all components ofthe lignocellulosic complex, including lignin. It has beenknown for decades that lignin and derived humic materialare efficiently degraded in soil litter by saprophytic basid-iomycetes, which results in the formation of white-rot hu-mus [1,2]. However, less information is available about theligninolytic system of these fungi. Tanesaka et al. [3] re-ported moderate lignin losses in beech wood caused byAgrocybe spp., Collybia spp. and Mycena spp., and later,nonspecific phenol-oxidizing activities were found in cul-tures of some of these fungi [4].

Manganese peroxidase (MnP, EC 1.11.1.13) is one of themain oxidoreductases involved in lignin degradation [5,6].It is an extracellular glycosylated heme protein produced byalmost all wood-colonizing white-rot fungi [7], e.g. byPhanerochaete chrysosporium [8,9], Phlebia radiata [10,11] and Nematoloma frowardii [12]. Recently, we havedemonstrated high activities of MnP in liquid cultures oftypical litter-decomposing fungi belonging to the generaAgrocybe and Stropharia [13]. MnP catalyzes the H2O2-dependent oxidation of Mn2� to Mn3�, which in turn acts asa nonspecific oxidant that attacks phenolic lignin structuresby one-electron abstraction. MnP has been shown todepolymerize and even mineralize lignins in vitro [14,15,16].

So far, only one MnP from a litter-decomposing fun-gus—that of the commercial white button mushroom (Agar-icus bisporus) has been investigated thoroughly [17,18]. Inthis study, we describe the partial purification and charac-terization of MnP from two litter-decomposing basidiomy-cetes, Agrocybe praecox and Stropharia coronilla, whichhave been shown in a recent publication [13] to mineralizea recalcitrant synthetic lignin (14C-ring-labeled DHP) to amoderate extent.

* Corresponding author. Tel.: �358-9-19159321; fax: �385-9-19159322.

E-mail address: kari.steffen@helsinki.fi (K.T. Steffen).

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2. Materials and methods

2.1. Organisms and culture conditions

Agrocybe praecox TM 70.84 and Stropharia coronillaTM 47–1 were obtained from the culture collection of theInstitute of Microbiology (University of Jena, Germany)[19] and maintained on 2% malt extract agar.

Fungi were routinely precultured on 2% malt extract agarfor 14 days. A basal liquid medium with or without supple-mented MnCl2 (�200 �M Mn2�) as described by Steffen etal. [13] was used for enzyme production. Cultivation wasperformed in 1-liter tissue culture flasks containing 200 mlof the liquid medium, which was inoculated with ten agarplugs (10 mm in diameter) of pregrown mycelium. Cultureswere incubated at 25°C in the dark for 10 weeks andsamples (300 �l) for enzymatic measurement were takenonce a week. Enzyme production for further purificationstudies was carried out under the same conditions, butcultures were harvested after 4 weeks. In all cases, threereplicate bottles were used.

2.2. Enzyme assays

MnP activity was measured at 270 nm by following theformation of Mn3�-malonate-complexes [20]. Laccase ac-tivity was determined by following the oxidation of ABTS(2,2�-azinobis(3-ethylbenzthiazoline-6-sulphonate)) at 420nm [21]. Lignin peroxidase (LiP) activity was measured at310 nm using the veratryl alcohol method [22]. All activitieswere expressed in units (U) defined 1 �mol of substrateoxidized per minute at 25°C.

2.3. Enzyme purification

Culture fluid (7.4 liter) was harvested after 4 weeks ofincubation, separated from the mycelium and concentratedas described by Vares et al. [23]. Extracellular proteins wereseparated by anion exchange chromatography on a Mono Qcolumn (Amersham Pharmacia Biotech, Uppsala, Sweden)with a 0.02 M–1 M NaCl gradient in 0.02 M sodium acetatebuffer (pH 5.5) for S. coronilla crude extracts and with a0.02 M–1 M sodium acetate (pH 5.5) gradient for those ofA. praecox. The elution of absorbing material from thecolumn was simultaneously monitored at 280 and 405 nm todetect total protein and heme, respectively. Fractions of 0.5ml were collected at a flow rate of 0.5 ml min�1. Fractionscontaining MnP were pooled, concentrated, and diafiltrated(10 kDa cut-off, Filtron Microsep) with the double amountof distilled water.

Protein concentrations were determined by the method ofBradford [24] using a Bio-Rad Protein Assay kit (Bio-RadLaboratories, Richmond, CA) with bovine serum albuminas a standard.

2.4. Enzyme characterization

Molecular weights (MW) of proteins were determined bysodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) as described in Laemmli [25]. After electro-phoresis, gels were stained and protein bands were visual-ized with Coomassie Brilliant Blue R-250. For the determi-nation of MW a low-molecular weight protein calibrationkit was used (Pharmacia).

Analytical isoelectric focussing (IEF) was carried outwith a Multiphor II electrophoresis system (Pharmacia).The IEF gel (7.5%) was prepared using ampholines of pI2.5–5.0 and pI 3.5–10.0 (Pharmacia). The pH gradient of thegel was measured with a surface electrode (Sentek; pHmeter from Orion, USA) and additionally a pI protein stan-dard kit (Pharmacia) was used. Gels were stained withphenol red according to Pease et al. [26].

The Km values of A. praecox MnP1 and S. coronillaMnP1, were determined for H2O2 and Mn2�. Lineweaver-Burke plots were made from the initial rates obtained atvarying substrate conditions while the concentration of thesecond substrate held constant was 0.1 mM. In addition, theKm-value of A. praecox MnP1 was determined for ABTS inreactions that lacked Mn2�.

3. Results

3.1. Production of MnP and other ligninolytic enzymes

MnP was the main oxidoreductase produced in liquidcultures of A. praecox and S. coronilla that were supple-mented with Mn2�. Additionally, both fungi producedlower levels of laccase, however, no LiP activity was ob-served. MnP activity in A. praecox cultures reached itsmaximum of approx. 400 U l�1 after 3 weeks, but thenactivity decreased rapidly from week 4 onwards so that after6 weeks, MnP activity was no longer detectable (Fig 1). NoMnP activity was found in A. praecox cultures that lackedsupplemented Mn2�. In addition, the fungus secreted lowamounts of laccase after one week in culture, but most of the

Fig. 1. MnP activity (squares) and laccase activity (circles) in liquidcultures of A. praecox and S. coronilla during a 10-week cultivation.Medium containing 200 �M Mn2� (closed symbols), Mn2�-free medium(open symbols). Arrows indicate the time of harvest of the cultures forenzyme purification.

551K.T. Steffen et al. / Enzyme and Microbial Technology 30 (2002) 550–555

activity in both Mn2�- and Mn2�-free cultures appearedrelatively late, when MnP activity had already started todecline. This result may indicate that laccase activity iscorrelated with cell lysis.

S. coronilla produced up to 900 U l�1 MnP, and this highactivity was reached within 7 weeks of culturing (Fig. 1). Aswith A. praecox, MnP activity decreased afterwards, butsubstantial levels were detectable until the end of the ex-periment. Contrary to the results obtained with A. praecox,MnP was also produced in S. coronilla cultures, which werenot supplemented with Mn2�. However, the peak activitywas only about one quarter of that found in cultures thatwere supplemented with Mn2�. Laccase was produced in

similar amounts as by A. praecox, but the activity did notdecline with time. Production of laccase was very low in S.coronilla cultures that lacked supplemented Mn2�.

3.2. Purification of MnP

To separate MnP from the concentrated culture filtrate,anion exchange chromatography with an FPLC apparatuswas employed. Final specific activities of purified MnPfrom S. coronilla and A. praecox was 692 U mg�1 (yield53%) and 128 U mg�1 (yield 8%), respectively.

In Mn2�-containing cultures, A. praecox expressed atleast two forms of MnP with MnP1 eluting first and clearly

Fig. 2. FPLC protein profile of 4-week old, concentrated culture filtrate from A. praecox grown in the absence of Mn2� (upper two pictures) or in the presenceof Mn2� (lower two pictures). Sodium acetate (NaAc) gradient is marked as dotted line. Absorbance at 405 nm (bold lines) and 280 nm (thin lines) describesthe elution of heme-containing proteins and total protein, respectively, along with the activities of laccase (open circles) and MnP (closed circles). Inset:SDS-PAGE of pooled MnP peaks; the left lanes represent protein standards.

552 K.T. Steffen et al. / Enzyme and Microbial Technology 30 (2002) 550–555

being the major enzyme (Fig. 2). As activity measurementsalready suggested, no indication for the presence of MnPwas found in Mn2�-free cultures.

S. coronilla expressed in Mn2�-containing cultures atleast two, possibly three MnP forms (Fig. 3). As was thecase with A. praecox, the main isoform MnP1 was elutedfirst. Another MnP (MnP3) was probably hidden within theelution peak of MnP2. These forms could not be separatedunder the conditions applied, but electrophoresis and iso-electric focusing results supported this assumption (see be-low). In Mn2�-free cultures of S. coronilla, only one iso-form—MnP2—was observed (Fig. 3). Thus, it can be

concluded that S. coronilla MnP1 is an Mn2�-inducible enzyme,whereas MnP2 seems to be—at least in part—constitutive.

3.3. Characterization of MnP

The main MnPs of both fungi had nearly neutral pIvalues of 6.3–7.0 for A. praecox and 6.3–7.1 for S. coronilla(Fig. 4). Their MWs were also similar: about 42 kDa forMnP1 of A. praecox and 41 kDa for MnP1 of S. coronilla(Fig. 2 and 3). There were clear differences regarding MnP2of both fungi. Whereas A. praecox MnP2, consisting prob-ably of two isoforms with pIs of 6.3 and 6.7 (MW 42 kDa),

Fig. 3. FPLC protein profile of 4-week old, concentrated culture filtrate from S. coronilla grown in the absence of Mn2� (upper two pictures) or in the presenceof Mn2� (lower two pictures). NaCl gradient is marked as dotted line. Further explanations are given in Fig. 2.

553K.T. Steffen et al. / Enzyme and Microbial Technology 30 (2002) 550–555

showed properties similar to those of its MnP1, MnP2 fromS. coronilla consisted of two acidic forms that had distinctpIs of 3.5 and 3.7 and an identical MW of 41 kDa. Indica-tions of a third S. coronilla MnP (MnP3) with a pI of 5.1and a MW of 43 kDa (Fig. 3) were found in the SDS-PAGEand IEF analyses (Fig. 4, lane 3).

The laccases of both fungi had pIs of 4.0 and 4.4 andMWs of 66 and 67 kDa for A. praecox and S. coronilla,respectively (data not shown).

The Michaelis-Menten constants (Km) of both main MnPforms (S. coronilla MnP1 and A. praecox MnP1) for H2O2

and Mn2� were relatively low, which indicates high affinityof these enzymes for their substrates (Table 3). In additionto Mn2� oxidation, A. praecox MnP1 showed an Mn2�-independent activity that oxidized ABTS directly. The affinity ofthe enzyme to ABTS, however, was about 40 times lower thanthat to Mn2� according to the estimated Km-values (Table 1).

4. Discussion

MnP is the main extracellular oxidoreductase producedby A. praecox and S. coronilla in liquid culture. Both fungi

expressed at least two different isoforms, S. coronilla prob-ably even more. Supplementation of the culture mediumwith Mn2� stimulated MnP production by both fungi con-siderably.

Purification of the MnPs from both fungi showed somedifferences between the individual enzymes. Thus, MnPfrom A. praecox was less stable than that of S. coronillawhich is evident from the purification yields. The purifica-tion of S. coronilla MnP was easier since the fungus pro-duced, under the same conditions, more MnP than A. prae-cox did. Because both fungi were found to mineralize 14C-labeled lignin nearly to the same extent [13], other factorsthan the amount of MnP alone apparently determine theextent of lignin degradation.

The pI values reported in this paper show that MnPisoforms produced by one fungus can differ noticeably. SopIs of MnP from S. coronilla varied between 3.5 and 7.1.The fungus produced the neutral isoform only in the pres-ence of supplemented Mn2�, whereas the acidic ones werealso observed in absence of Mn2�. Acidic pIs have beenalso reported for MnPs from the litter decomposing fungusA. bisporus ranging from 3.25–3.3 [17] to 3.5 [18]. Alsomost MnPs from wood-colonizing white-rot fungi are acidicproteins [6]. Thus, the agaric white-rot fungus N. frowardii,that belongs to the same family (Strophariaceae) as S.coronilla does, produces an MnP with pI 3.2 [12], and thepIs of P. radiata MnP range from 3.6 to 4.85 [11,27]. Aneutral pI, as reported in this work for MnP2 from S.coronilla (pI 7.1), has so far been described only for thecoprophilic fungus Panaeolus sphinctrinus (pI of 7.2),which belongs to the Strophariaceae as well [28].

MWs of MnPs from both litter-decomposing fungi stud-ied in this article ranged from 41–43 kDa, which is commonfor this enzyme [7]. The litter-decomposer A. bisporus wasshown to produce MnP with a MW of 40 kDa during growthon solid compost [17] and the manure-colonizers P. sphinc-trinus one with a MW of 42 kDa in liquid culture [28]. MWsof MnPs from white-rot fungi tend to be higher and canreach 50 kDa [7]. More common for white-rot fungi are,however, MnPs with MWs around 45 kDa as for P. chryso-sporium (46 kDa) [8] and Bjerkandera sp. BOS55 (44–45kDa) [29]. All in all, the MW range of MnPs from white-rotfungi seems to be broader than that of litter-decomposingfungi.

Fig. 4. IEF analysis of pooled MnP fractions after Mono-Q separation.MnP1 (lane 2) and MnP2 (lane 1) from A. praecox, MnP1 (lane 4), MnP2and MnP3 (lane 3) from S. coronilla.

Table 1Michaelis-Menten constants (Km) of MnP1, the main MnP isoform fromA. praecox and S. coronilla. Reactions were performed in 50 mM Namalonate at pH 4.5 and 25°C

Substrate A. praecox MnP1Km (�M)

S. coronilla MnP1Km (�M)

H2O2 (0.1 mM Mn2�) 2 2Mn2� (0.1 mM H2O2) 17 12ABTS (0.1 mM H2O2,

without Mn2�)667 n.d.

554 K.T. Steffen et al. / Enzyme and Microbial Technology 30 (2002) 550–555

Because litter-decomposing basidiomycetes—at leastthose mineralizing lignin to moderate extent [13]—produceMnP and laccase, but obviously no LiP, MnP might be thekey enzyme in lignin degradation by this eco-physiologicalgroup of fungi. Degradation of lignin in their natural habitat(e.g. in twigs or leaves) may help these fungi to get accessto carbohydrates that are usually not available for other soilmicroorganisms such as molds or saprophytic bacteria. Inthis context, litter-decomposers resemble white-rot fungiwhich follow a similar ecological strategy in wood. Takinginto account that a substantial part of lignin in the biosphereoriginates from small annual plants and grasses, as well asleaves and needles from trees, lignin degradation by litter-decomposing fungi is probably important for the recyclingof aromatic carbon in soil environments.

Future work will focus on lignin and humus degradation,and ligninolytic enzyme production by litter-decomposingfungi in their natural habitat, i.e. litter and soil.

Acknowledgements

We thank Ralf Bortfeldt for his help in the laboratory.The work was supported by a grant of “The Finnish Grad-uate School of Environmental Science and Technology” andby the Academy of Finland (projects no. 39906 and 52063).

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