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Research in Microbiology ()
www.elsevier.com/locate/resmic

Purification of a new manganese peroxidase of the white-rot fungus

Irpex lacteus, and degradation of polycyclic aromatic hydrocarbons
by the enzyme
Petra Baborov a , Monika Mder b , Petr Baldrian a, , Kamila Cajthamlov a , Tom Cajthaml a
a Institute of Microbiology AS CR, Vdensk 1083, 14220 Prague 4, Czech Republic
b Department of Analytical Chemistry, UFZCentre for Environmental Research Leipzig-Halle, Permoserstrasse 15, 04318 Leipzig, Germany

Received 21 June 2005; accepted 1 September 2005

Abstract
The white-rot fungus Irpex lacteus has been reported to be an efficient degrader of polycyclic aromatic hydrocarbons, polychlorinated biphenyls
and pentachlorophenol. The fungus produces ligninolytic enzymes laccase, lignin peroxidase and manganese peroxidase (MnP), the latter being
the major one produced. MnP was purified using anion exchange and size exclusion chromatography. SDSPAGE showed the purified MnP to be
a monomeric protein of 37 kDa (37.5 kDa using MALDI-TOF) with an isoelectric point at 3.55. The pH optimum was relatively broad, from 4.0
to 7.0 with a peak at pH 5.5. Kinetic constants Km were 8 M for H2 O2 and 12 or 31 M for Mn2+ depending on the substrate. The enzyme did
not perform oxidation in the absence of H2 O2 or Mn2+ . MnP was active at 570 C with an optimum between 5060 C. At temperatures above
65 C the enzyme rapidly lost activity. Degradation of four representatives of PAHs (phenanthrene, anthracene, fluoranthene, and pyrene) was
tested and the enzyme showed the ability to degrade them in vitro. Major degradation products of anthracene were identified. The results confirm
the role of MnP in PAH degradation by I. lacteus, including cleavage of the aromatic ring.
2005 Elsevier SAS. All rights reserved.
Keywords: Anthracene; Biodegradation; Irpex lacteus; Manganese peroxidase; Polycyclic aromatic hydrocarbons

1. Introduction
Fungal oxidases and peroxidases have been suggested to
play a key role in lignin degradation [12,36] and to enable
their producersthe wood- and litter-decomposing fungito
detoxify xenobiotic compounds by partial degradation or complete mineralization [26]. Due to their broad substrate specificity, ligninolytic enzymes are capable of transforming many
structurally different pollutants including synthetic dyes, polychlorinated biphenyls, nitrated explosives, pesticides, polymers
and polycyclic aromatic hydrocarbons (PAHs) [25,26].
Among the pollutants degraded by white-rot fungi, PAHs
have attracted particular attention in the last decades, since carcinogenic substances may be formed during biotransformation
of PAHs in humans and microorganisms [8]. PAHs are formed
* Corresponding author.

E-mail address: baldrian@biomed.cas.cz (P. Baldrian).

0923-2508/$ see front matter 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.resmic.2005.09.001

by incomplete burning of fossil fuels and can enter the soil

via atmospheric deposition. Local contamination with PAHs
is particularly due to industrial activities such as old gasification plants and wood-preserving plants where creosote and
anthracene oil, partial distillates of oil with high concentrations
of PAHs, are used [31,39].
Ligninolytic enzymes perform non-specific one-electron
radical oxidation, producing cation radicals from PAHs followed by the appearance of quinones [3,37]. Using 14 C-labeled
compounds it was proven that ligninolytic fungi and isolated
ligninolytic enzymes are further able to mineralize PAHs to carbon dioxide [1,39]; however, only limited information is available about intermediates of fungal degradation, i.e., the ringcleavage products [2,5,11,17] or oxidized forms (quinones)
[20,32], and although the degradation by some purified enzymes has already been reported [4,10,28], the exact role
of individual ligninolytic enzymes in degradation is still not
clear [30]. One possible way of elucidating participation in

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degradation is by performing degradation with isolated enzymes together with identification of degradation products.
The white-rot basidiomycete Irpex lacteus has been reported
to be very efficient in binding pentachlorophenol to humic substances during soil bioremediation, as well as in the degradation
of polychlorinated biphenyls and polycyclic aromatic hydrocarbons [15,23]. Laccase, lignin peroxidase and Mn peroxidase
activities were found under different culture conditions [34,35]
and lignin peroxidase of the fungus has been purified and characterized [27]. The aim of this work was to isolate and characterize Mn peroxidase, produced by I. lacteus as the major
ligninolytic enzyme under most culture conditions, to test its
ability to degrade PAHs and to identify the degradation products.
2. Materials and methods
2.1. Chemicals
Anthracene, fluoranthene, phenanthrene, pyrene, 2,2 -azinobis-3-ethylbenzothiazoline-6-sulfonic acid, diammonium salt
(ABTS), ethylenediaminetetraacetate (EDTA), 3-methyl2-benzothiazolinone hydrazone (MBTH) and 3,3-dimethylaminobenzoic acid (DMAB) were purchased from Sigma
(USA). The purities of PAH compounds were 97% or higher.
Acetonitrile and acetone were of gradient grade (Merck, Germany). All other chemicals were of the highest grade available.
2.2. Organism and culture conditions
The wood-rotting basidiomycete I. lacteus CCBAS238 was
obtained from the Culture Collection of Basidiomycetes (Institute of Microbiology AS CR, Prague). The fungus was maintained on MEG agar slants (per liter: 5 g malt extract, 10 g
glucose, 15 g agar, pH 4.5). Static cultivations in the liquid
medium were performed in 250 ml Erlenmeyer flasks containing 20 ml of liquid MEG medium. Flasks were inoculated with
two 7-mm agar plugs with mycelium and the cultivation proceeded at 28 C in the dark without aeration. Each sampling
day 200 l samples of culture liquid were removed from the
cultures under sterile conditions, filtered and used for enzyme
assays. Measurements were performed in triplicate. For the purification of MnP, cultivation proceeded for 30 days under the
same conditions.
2.3. Enzyme assays
Activity of manganese peroxidase (EC 1.11.1.13, MnP) was
assayed in succinatelactate buffer (SLB, 100 mM, pH 4.5).
MBTH and DMAB were oxidatively coupled by the action of
the enzyme and formation of a purple indamine dye product
was followed spectrophotometrically at 590 nm [21]. The results were corrected by activities in test samples without manganese where manganese sulfate was substituted by EDTA to
chelate Mn present in the extract. Laccase (EC 1.10.3.2) activity was measured by monitoring the oxidation of ABTS in
100 mM citrate200 mM phosphate buffer, pH 5.0 [22]. The

formation of green dye was followed spectrophotometrically at

420 nm. Lignin peroxidase was assayed with veratryl alcohol as
a substrate [6]. One unit of enzyme activity was defined as the
amount catalyzing formation of 1 mol of the reaction product
per minute under assay conditions.
2.4. Purification of manganese peroxidase
Culture liquid from 30-day static culture was collected, filtered through Whatman paper, concentrated by ultrafiltration
and desalted in a stirred cell with a 10 kD membrane (Amicon, Millipore, US). The concentrate was loaded onto a DEAESepharose column (Pharmacia, HR 10/10) equilibrated with
20 mM phosphate buffer, pH 6.0. Proteins were eluted with
a gradient from 0 to 0.5 M NaCl in 20 min at a flow rate of
0.5 ml min1 . The MnP fractions were pooled, desalted and applied to a MonoQ anion-exchange column (Pharmacia, HR 5/5)
equilibrated with the same buffer and eluted with a gradient
from 0 to 0.5 M NaCl in 30 min at a flow rate of 0.5 ml min1 .
The pooled and desalted MnP fractions were applied again to
a MonoQ column equilibrated with 20 mM phosphate buffer
(pH 7.5) and chromatographed using the same procedure. The
MnP fractions were pooled, concentrated and loaded onto a
Superdex 75 column (Pharmacia, HR 10/30). Elution was performed with 50 mM sodium acetate buffer (pH 4.5) containing
0.15 M NaCl at a flow rate of 0.5 ml min1 . Finally, desalted
MnP fractions were applied to a MonoQ column equilibrated
with 50 mM sodium acetate buffer (pH 4.5) and eluted with
a gradient from 0 to 0.5 M NaCl in 30 min at a flow rate of
0.5 ml min1 . Purity of the enzyme was checked using SDS
PAGE. The concentrated MnP was desalted and stored frozen
(18 C).
2.5. Electrophoresis, protein assay
and molecular mass detection
SDSPAGE was performed using a 10% polyacrylamide gel.
Analytical isoelectric focusing (IEF) was carried out with a
Multiphor II electrophoresis system (Pharmacia, Sweden). The
IEF gel (7.5%) was prepared using ampholines of pI 2.55.0
and 3.510.0 (Pharmacia, Sweden). A low pI protein calibration
kit, pI 2.56.5 (Pharmacia, Sweden) was used for the estimation of isoelectric point. Gels were stained using silver staining
kit (BioRad, USA) and activity-stained with DMAB + MBTH.
Protein concentrations were measured with a BioRad protein
assay kit (BioRad, USA) using bovine serum albumin as a standard.
The molecular mass of the enzyme was estimated by two
methods: (1) SDSPAGE with protein ladder molecular weight
markers (Fermentas, The Netherlands); and (2) MALDI-TOF
mass spectrometry using a Voyager PRO DE (Applied Biosystems, Germany), calibrated with BSA in the mass range between 2 and 80 kDa at an accelerating voltage of 25 kV. The
used matrix was sinapic acid (3,5-dimethoxy-4-hydroxycinnamic acid) in the following preparation: 10 mg ml1 in 30%
acetonitrile with 0.1% trifluoroacetic acid.

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2.6. Effect of pH and temperature on MnP activity,

estimation of Michaelis constants
The effect of pH on MnP activity was examined in the pH
range 2.57.5 in 0.1 M citrate0.2 M phosphate buffer with
DMAB and MBTH as substrates. The effect of temperature on
enzyme activity and stability was determined in SLB. Michaelis
constants (Km ) for hydrogen peroxide and Mn2+ were calculated from LineweaverBurk plots using MBTH/DMAB and
ABTS as substrates (both in SLB). All measurements were performed in triplicates.
2.7. Degradation of polycyclic aromatic
hydrocarbons by manganese peroxidase
The degradation of PAHs (phenanthrene (PHE), anthracene
(ANT), fluoranthene (FLT), and pyrene (PYR)) was performed in 20% acetone in 50 mM malonate buffer (pH 4.5)
containing 15 g ml1 of each compound, 1 mM MnSO4 ,
30 mM glucose, 0.5 mM glutathione, 120 mU ml1 glucose
oxidase and 850 mU ml1 of the enzyme. The reaction proceeded at 25 C in the dark using agitation (90 rpm); five
replicate samples were used for each compound. Degradation was measured using a HewlettPackard 1040 HPLC
(HewlettPackard, The Netherlands) equipped with a diode
array detector. An isocratic program was applied with 85%
acetonitrile and 15% water, and PAHs were determined at
254 nm. PAHs were separated on a LichroCart PAH column
filled with LichroSphere (250 mm 5 mm, particle diameter 5 m) provided by Merck (Germany); 20 l of reaction
mixture was injected directly into HPLC. The data were expressed as recoveries compared to abiotic controls without
MnP.
For metabolite analysis, the reaction mixture was acidified
with HCl to pH 3 and the samples were extracted with
three portions of ethylacetate. The extracts were concentrated
using a rotary evaporator and injected into a gas chromatograph (GC). The intermediates were identified using gas chromatography coupled with mass spectrometry (GC-MS) with
an ion trap detector (GCQ, Finnigan, USA). The GC instrument was equipped with split/splitless injector and an HP-5
column was used for separation (30 m, 0.25 mm inner diameter, 0.25 m film thickness). The temperature program started
at 60 C and was held for 1 min in splitless mode. Then the
splitter was opened and the oven was heated to 150 C at a rate
of 25 C min1 . The second temperature ramp was up to 260 C
at a rate of 10 C min1 , with this temperature maintained for
20 min. The solvent delay time was set to 5 min. The transfer
line temperature was set to 280 C. Mass spectra were recorded
at 1 scan s1 under electron impact at 70 eV, mass range 50
350 amu. The excitation potential for the MS/MS product ion
mode applied was 0.5 V, and 0.9 V in the case of more stable
ions. Methane was used as a medium for chemical ionization
(CI).

3. Results
The white-rot fungus I. lacteus produced the ligninolytic
enzymes laccase, lignin peroxidase and MnP during the stationary cultivation in MEG media. All three enzymes reached their
maxima after about 20 days of incubation. The fungus showed
a very low activity of laccase (12 U l1 ) and lignin peroxidase
(6 U l1 ). MnP was the dominant ligninolytic enzyme with an
average activity of 270 U l1 (results not shown).
The course of purification is summarized in Table 1. After all
purification steps MnP activity appeared as a single peak. The
purified enzyme appeared as a single band on SDSPAGE and
IEF. The molecular mass was 37 kDa by SDSPAGE, MALDITOF yielded a single peak with a molecular mass 37.5 kDa.
Isoelectric focusing showed one band around pH 3.55 (Fig. 1)
and activity staining confirmed that it was MnP.
MnP showed its highest activity at pH 5.56.5 while relatively strong activity (more than 60% of the maximum) was
present at a broad pH range 4.07.0. (Fig. 2). Temperature optimum was observed at 5060 C and the enzyme was not active
above 70 C (Fig. 3). A rapid loss of activity occurred at temperatures higher than 65 C.
The enzyme exhibited the following kinetic characteristics:
Km for H2 O2 was 8 M with both DMAB/MBTH and ABTS as
substrates, Km for Mn2+ was 12 M with DMAB/MBTH and
31 M with ABTS. The Km for ABTS was 1560 M and the
enzyme exhibited neither DMAB/MBTH nor ABTS oxidation
in the absence of hydrogen peroxide or Mn2+ .
To study degradation of selected PAHs we tested their solubility in a mixture of acetonewater with various ratios and we
Table 1
Purification of manganese peroxidase from the straw extract of I. lacteus
Purification step

Total
protein
(g)

Total
activity
(mU)

Specific
activity
(mU g1 )

Yield
(%)

Culture filtrate
DEAE Sepharose, pH 6.0
Mono Q HR 5/5, pH 6.0
Mono Q HR 5/5, pH 7.5
Superdex 75 HR 10/30, pH 4.5
Mono Q HR 5/5, pH 4.5

3829
1895
756
388
203
120

5550
4100
3200
1990
1100
960

1.4
2.2
4.2
5.1
5.3
8.0

100.0
73.8
57.6
35.8
19.4
17.3

Fig. 1. Isoelectric focusing of purified manganese peroxidase. Purified MnP

stained by activity staining with DMAB/MBTH (lane A) and with silver staining for protein detection (lane B). The numbers indicate pI values of marker
proteins used for calibration.

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Fig. 2. Effect of pH on the activity of MnP from I. lacteus. The activity was
examined in the pH range 2.57.5 in 0.1 M citrate0.2 M phosphate buffer with
DMAB and MBTH as substrates at 25 C. Relative activity is given (maximum
activity = 100%).

also measured MnP activity with respect to inhibition by acetone. Following these results (data not shown) we selected for
further experiments a mixture with 20% acetone that was sufficient to dissolve 15 g ml1 of each PAH compound and caused
less than 20% enzyme inhibition. The activity of the enzyme
during experiments in the watersolvent mixture was stable.
The degradation results of individual compounds are shown in
Table 2. Among all PAHs tested only ANT exhibited a concentration decrease after 168 h to 10.1 g ml1 in the control
treatment without the enzyme. The enzyme was able to decompose all the studied PAHs at different rates. While ANT and
PYR were degraded extensively to 0 and 8.3% of their original
concentration, recovery of PHE and FLT after 168 h of degradation was 56 and 58.1%, respectively.
We also tried to identify the major degradation products of the studied PAHs, but we were able to detect major distinct peaks only for ANT. Their mass spectral characteristics are given in Table 3 and they were identified as
anthrone, 9,10-anthracenedione, and 2-(2 -hydroxybenzoyl)benzoic acid, respectively. The first two degradation products
were also identified using a relevant chemical standard and
2-(2 -hydroxybenzoyl)-benzoic acid was characterized using
the MS/MS mode as shown in our previous work [5]. No formation of phthalic acid was detected. The sequence of degradation,
proposed on the basis of subsequent oxidation of identified
metabolites and the results of ANT degradation by whole cultures [5], is shown in Fig. 4. Degradation of the other PAHs did
not yield major peaks and it was thus impossible to identify the
products. Even pyrenediones or similar compounds that could
be anticipated were not detected.

Fig. 3. Effect of temperature on the activity of MnP from I. lacteus. The enzyme
reaction was performed in succinate-lactate buffer, pH 4.5 using DMAB and
MBTH as substrates. Relative activity is given (maximum activity = 100%).

4. Discussion

Table 2
Recovery of individual PAHs (in percent of the original concentration) after
3168 h incubation with MnP from I. lacteus
Time (h)
3
6
10
24
146
168

Recovery (%)
Phenanthrene

Anthracene

Fluoranthene

Pyrene

92.7 1.0
90.2 1.1
87.2 1.5
82.0 6.9
65.5 9.9
56.0 2.6

6.0 0.7
5.8 0.8
2.4 0.2
0.2 0.2
0.1 0.0
0.0 0.0

78.2 8.3
74.9 6.0
72.7 6.4
72.1 8.8
61.3 6.8
58.1 5.9

57.6 5.2
46.2 2.8
39.8 3.9
31.2 4.4
10.7 1.6
8.3 1.4

Initial concentration of PAHs was 15 g ml1 and 150 mU ml1 MnP was
used. The data represent means and standard errors of five replicate estimations.

Mn peroxidase from I. lacteus was the major ligninolytic enzyme produced in liquid culture. It ranged among the smallest
MnPs, with its molecular weight similar to Abortiporus biennis
and Trametes spp. [13], belonging to the group of MnPs with
acidic isoelectric points, e.g., Agaricus bisporus, Bjerkandera
adusta and Phanerochaete flavido-alba [7,16,24]. The pH optimum of the enzyme was broad and slightly higher than that
of other MnPs purified so far; also, the temperature optimum
was relatively high, although the stability at elevated temperatures was low. The Km for both H2 O2 and Mn2+ was similar to
that of Pleurotus ostreatus and Phanerochaete chrysosporium
enzymes [24,29], while that for ABTS was much higher than
the values reported for other fungi [33,38].

Table 3
Gas chromatographymass spectrometry identification of metabolites of anthracene degradation by MnP from I. lacteus: retention data and mass spectral characteristics of anthracene metabolites
No.

tR
(min)

MW according
to CI

m/z of fragment ions

(relative intensity)

Structural suggestion

1
2
3

13
13.51
14.61

194
208
242

194 (100), 165 (98.4), 139 (49.6), 81 (37.1)

208 (100), 180 (64.2),152 (58.8), 126 (4.4), 76 (5.9)
242 (14.3), 224 (100), 196 (41), 168 (32.8), 139 (14.1)

Anthrone
9,10-Anthracenedione
2-(2 -Hydroxybenzoyl)-benzoic acid

tR retention time, MWmolecular weight, CIchemical ionization.

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Acknowledgements
This work was supported by the grants KJB6020308 and
KJB600200516 from the Grant Agency of the Academy of Science of the Czech Republic and by the Institutional Research
Concept No. AV0Z50200510 of the Institute of Microbiology,
ASCR.
References

Fig. 4. The sequence of anthracene degradation by MnP from I. lacteus.

The degradation of PAH by MnP was first described in

Phanerochaete chrysosporium as a lipid peroxidation-dependent process [18]. It was later demonstrated that PAH degradation by some MnPs also occurs directly: MnP from Nematoloma frowardii degrades anthracene, phenanthrene, pyrene,
fluoranthene and benzo[a]pyrene, leading to partial mineralization [14,29]. Benzo[a]pyrene and anthracene are also mineralized by MnP from the litter-decomposing fungus Stropharia
coronilla. The oxidation of benzo[a]pyrene leads to benzo[a]
pyrene-1,6-quinone as a temporal intermediate [32]. PAH
degradation experiments showed that MnP isolated from I. lacteus was able to efficiently degrade three and four ring PAHs
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The MnP from I. lacteus had a tolerance to acetone comparable to that of MnP from Bjerkandera adusta and Phanerochaete
chrysosporium [9,40].
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2-(2 -hydroxybenzoyl)-benzoic acid indicates that MnP is able
to cleave the aromatic ring of a PAH molecule even without lipid peroxidation [18]. Thus MnP probably plays a more
important role than simply that of the initial oxidation and production of quinones.

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