FEMS Microbiology Letters 206 (2002) 69^74

www.fems-microbiology.org

Copper and cadmium increase laccase activity in Pleurotus ostreatus

Petr Baldrian *, Jir­¨| Gabriel
Laboratory of Biochemistry of the Wood-rotting Fungi, Institute of Microbiology, ASCR, V|¨den­skä 1083, 14220 Prague 4, Czech Republic

Received 14 August 2001 ; received in revised form 26 October 2001; accepted 31 October 2001

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First published online 28 November 2001

Abstract

Addition of copper (0.5^5 mM) or cadmium (1^5 mM) to the white rot fungus Pleurotus ostreatus cultivated in liquid nitrogen-limited
medium for 12 days increased the activity of laccase. The addition of 2 mM Cd led to an 18.5-fold increase of activity, 1 mM Cu increased
the activity eight-fold. When added earlier than 12 days, the activation of laccase was delayed (Cu) or decreased (Cd). Ag, Hg, Pb, Zn, and
H2 O2 decreased laccase activity. To study the effect on native enzymes, purified laccase was incubated with Cd, Cu, and Hg. The addition of
Hg decreased the activity of laccase immediately and reduced the temporal stability of the enzyme, while the addition of Cu (0.05^50 mM)
increased both enzyme activity and stability. Laccase extracted at different stages of straw colonisation differed in its response to heavy
metals. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords : Biotechnology ; Heavy metal; Laccase; Manganese peroxidase; Pleurotus ostreatus

1. Introduction ported [5^7]. There is a range of compounds that regulate

Laccase (benzenediol:oxygen oxidoreductase, EC
1.10.3.2) is one of the blue copper oxidases which catalyse
the four-electron reduction of oxygen to water. Laccase
has a broad substrate speci¢city towards aromatic com-
pounds containing hydroxyl and amine groups. As a
component of the lignin-degrading complex of the white
rot fungi, laccase participates in the degradation of
wood components. In the presence of redox mediators,
laccase can also oxidise non-phenolic substrates [1] and
thus it is proposed for applications in degradation of
xenobiotics.
Pleurotus ostreatus is a white rot basidiomycete whose
ligninolytic system consists of laccase and manganese per-
oxidase. Its ability to grow in soil and to compete with
indigenous micro£ora, as well as its excellent performance
in biodegradation tests, made this fungus a model for the
degradation of xenobiotics. P. ostreatus produces several
laccase isoenzymes, which have been puri¢ed [2^4].
The regulation of laccase gene expression and enzyme
production in response to culture conditions has been re-
* Corresponding author. Tel. : +420 (2) 475 2315;
Fax: +420 (2) 475 2396.
E-mail address : baldrian@biomed.cas.cz (P. Baldrian).
0378-1097 / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 5 1 9 - 5
laccase activity including aromatic substrates of the en-
zyme. Heavy metals are also an important group of en-
zyme activity modulators, which are present in the envi-
ronment either naturally (Cu) or as a result of human
activities (Cd, Hg, Pb). Heavy metals may pose a serious
problem for in situ biodegradation of polycyclic aromatic
hydrocarbons (PAHs) or polychlorinated biphenyls, since
the sites often contain high concentrations of heavy metals
[8]. Currently, positive regulation of laccase by copper
has been reported [9,10]. In our previous studies we re-
ported the negative e¡ect of heavy metals on the growth
of several wood-rotting fungi including P. ostreatus
[11]. The presence of Cd inhibited the decolorisation of
the dye Poly R-478 and decreased the activity of Mn-per-
oxidase and laccase in Stereum hirsutum and Phanero-
chaete chrysosporium [12]. However, the biodegradation
of PAHs by P. ostreatus in non-sterile soil was not signi¢-
cantly a¡ected by Cd, and elevated concentrations of
laccase were found in the presence of low concentrations
of Cd [13].
The aim of this work was to study the e¡ect of heavy
metals on the production of laccase by P. ostreatus
cultures and on the activity of the enzyme. The work fo-
cused on cadmium as an important industrial pollutant
and copper, the biogenic heavy metal that can play a
role in the modulation of laccase activity under natural
conditions.

FEMSLE 10263 3-1-02

70 P. Baldrian, J. Gabriel / FEMS Microbiology Letters 206 (2002) 69^74
2. Materials and methods
2.1. Organism and culture conditions
The wood-rotting basidiomycete P. ostreatus CCBAS-
477 was from the Culture Collection of Basidiomycetes
(CCBAS, Institute of Microbiology, Academy of Sciences
of the Czech Republic, Prague, Czech Republic). The fun-
gus was maintained on GC agar slants (per litre : 20 g
glucose, 7 g corn steep, 25 g agar pH 6.0). Static cultiva-
tions in the liquid mineral medium were performed in 250-
ml conical £asks containing 40 ml of nitrogen-limited me-
dium [14]. Flasks were inoculated with two 7-mm agar
plugs from the edge of an actively growing colony, pre-
cultivated on the same medium (25 g l31 agar) for 8 days
at 28³C. The liquid stationary cultivation proceeded at
28³C in the dark. At de¢ned times, the £asks were sup-
plemented with Cd(NO3 )2 , CuSO4 , AgNO3 , HgCl2 ,
Pb(NO3 )2 , ZnSO4 , or H2 O2 . Five replicates of each treat-
ment were used. The cultivation of P. ostreatus on natural
substrate proceeded in 100-ml conical £asks containing 5 g
air-dried milled wheat straw (particle size 2^8 mm). The
straw was moistened with 15 ml distilled water (¢nal water
content 75%), stopped with cotton plugs, autoclaved
(2U121³C for 30 min) and inoculated with two agar plugs
with mycelium. The cultures were incubated at 28³C in the
dark.
2.2. Sampling procedure and treatment of extracts
Each sampling day, 500-Wl samples were removed from
liquid cultures, ¢ltered and used for enzyme assays. At the
end of cultivation, cultures were ¢ltered and dry mass of
mycelia estimated after drying at 105³C until constant
weight. The straw cultures of P. ostreatus were incubated
for 8, 22, and 28 days and after incubation, £asks were
supplemented with 40 ml of acetate bu¡er (80 mM, pH
5.0). The straw was mixed with the bu¡er for 3 h at 4³C
with mixing. Extracts from ¢ve £asks were pooled and
¢ltered through Whatman paper ¢lter. The ¢ltrate was
supplemented with an equal amount of Cd(NO3 )2 , CuSO4 ,
or HgCl2 solutions in the bu¡er to give a ¢nal concentra-
tion range of 0.01^50 mM, incubated for 18 h at 28³C in
the dark and then used for enzyme assays. Two replicates
were used for each combination.
2.3. Enzyme puri¢cation
The culture liquid from a 10-day culture of P. ostreatus
grown on nitrogen-limited medium was ¢ltered and con-
centrated by ultra¢ltration. The concentrate was loaded
onto a DEAE-Sepharose column (Pharmacia LKB, HR
10/10) equilibrated with 20 mM phosphate bu¡er, pH
7.0. Proteins were eluted with a gradient from 0 to 1 M
NaCl in 20 min at a £ow rate of 1 ml min31 . The laccase
fractions were pooled, desalted on Sephadex G-25 and
FEMSLE 10263 3-1-02
applied to a MonoQ anion exchange column (Pharmacia
LKB, HR 5/5) equilibrated with 20 mM phosphate bu¡er,
pH 7.0. Proteins were eluted with a gradient from 0 to 0.5
M NaCl in 20 min at a £ow rate of 1 ml min31 . The
laccase-containing fractions were pooled, concentrated us-
ing ultra¢ltration and applied to a Superdex 75 column
(Pharmacia LKB, HR 10/30). Elution was performed with
20 mM phosphate bu¡er, pH 7.0 containing 0.15 M NaCl
at a £ow rate of 0.5 ml min31 . The laccase fractions were
pooled, desalted on Sephadex G-25 and chromatographed
once more using a MonoQ column under conditions de-
scribed above. Resulting laccase-containing fractions were
pooled and checked for homogeneity using SDS^PAGE.
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The concentrated laccase fractions were transferred to cit-
rate-phosphate bu¡er (pH 5.0) and used for enzyme as-
says.
The puri¢ed laccase was incubated in Cd(NO3 )2 ,
CuSO4 , or HgCl2 solutions in citrate-phosphate bu¡er at
¢nal concentrations of 0.01^50 mM and 0 mM at 20³C.
Samples for enzyme assays were taken immediately after
mixing and then at di¡erent time intervals. Two replicates
were used for each combination.
2.4. Enzyme assays
Laccase activity was measured by monitoring the oxi-
dation of 2,2P-azinobis-3-ethylbenzothiazoline-6-sulfonic
acid in citrate-phosphate (100 mM citrate, 200 mM phos-
phate) bu¡er (pH 5.0) [15]. Activity of manganese peroxi-
dase (MnP) was assayed according to Ngo and Lenho¡
[16] in succinate-lactate bu¡er (100 mM, pH 4.5). All mea-
surements were done in 10-mm quartz cuvettes using a
UV-Vis spectrophotometer (Lambda 11, Perkin-Elmer)
and evaluated using UV-Winlab software from the same
manufacturer. One unit of enzyme activity was de¢ned as
the amount catalysing the production of one Wmol of col-
oured product per ml per min.
3. Results
3.1. Activity of laccase in the presence of cadmium
The cultures of P. ostreatus growing in nitrogen-limited
medium were supplemented with Cd(NO3 )2 to ¢nal con-
centrations of 0, 0.1, 0.2, 0.5, 1, 2, and 5 mM on day 12.
During the ¢rst week of cultivation, only negligible
amounts of laccase ( 6 3 U ml31 ) were produced by the
fungus in control £asks. The activity began to increase
rapidly after 10 days of cultivation. At Cd concentrations
up to 0.2 mM, there was no signi¢cant e¡ect of the metal
on laccase activity. At higher Cd concentrations laccase
activity increased compared to the control (Fig. 1). The
highest increase was apparent at 2 mM, where laccase
activity was 18.5-fold higher than in the control on day
14; the increase of enzyme activity at 5 mM Cd was slow-
 P. Baldrian, J. Gabriel / FEMS Microbiology Letters 206 (2002) 69^74
Fig. 1. Time course of laccase activity during the growth of P. ostreatus
in nitrogen-limited medium containing 0.0, 1.0, 2.0 and 5.0 mM cadmi-
um (¢nal concentration). Cd was added to growing cultures on day 12.
Averages of ¢ve replicates and S.E.M. are shown.
er. The di¡erences in laccase activities decreased with
time ; however, they were still signi¢cant 9 days after metal
addition. The addition of Cd decreased the growth rate of
the mycelium. At the end of the experiment, the dry mass
of mycelium in 1 mM Cd was only about 50% of the
control value.
The e¡ect of metal addition was di¡erent, when cadmi-
um was added to cultures at di¡erent stages of fungal
growth (¢nal concentration 2 mM). When added before
inoculation, and on day 4 or 7, laccase activity in Cd-
treated replicates was lower compared to the control,
although there was also a slow increase of enzyme activity
after day 10. It has to be noted that the growth of the
fungus ceased when cadmium was added during the ¢rst
days of growth: after 30 days of cultivation, the dry
weight of mycelia from such treatments was less than 0.6
mg ml31 , whereas in control £asks the dry weight was
higher than 5.1 mg ml31 . Later addition of Cd (on days
11^25) led to an increase of the enzyme activity.
3.2. Activity of laccase in the presence of copper
The cultures of P. ostreatus growing on nitrogen-limited
medium were supplemented with CuSO4 to ¢nal concen-
trations of 0.2, 0.5, 1, and 5 mM on day 12 (Cu concen-
Table 1
Activity of P. ostreatus laccase in the presence of 1 mM AgNO3 , HgCl2 ,
Pb(NO3 )2 , ZnSO4 , or 0.5 mM H2 O2
Treatment Activity (U ml31 )
Control 8.37 þ 1.48
Ag 0.26 þ 0.24
Hg 0.05 þ 0.10
Pb 2.00 þ 0.45
Zn 4.96 þ 3.15
H2 O 2 5.31 þ 0.97
The metal salts and hydrogen peroxide were added to the growing fun-
gal cultures on day 12 and the enzyme activities were estimated on day
15. Averages of ¢ve replicates and S.E.M. are shown.
FEMSLE 10263 3-1-02
71
tration in the unsupplemented medium was 0.4 WM). The
addition of Cu led to an increase of laccase activity, how-
ever at Cu concentration 0.2 M, the increase was not sig-
ni¢cant. The maximum di¡erence between the control and
metal-supplemented £asks was detected later than with
Cd, on day 19 (Fig. 2). The highest increase was recorded
at 1 mM Cu, where the peak activity was eight times high-
er than in control £asks; at 0.5 and 5 mM, the increase
was 4.7- and 3.7-fold, respectively. The addition of copper
resulted in slower mycelial growth: at 9 days after metal
addition, the dry weight of control mycelia increased by
3.1 mg ml31 , whereas it was only 1.2^1.6 mg ml31 at 0.5^5
mM Cu.
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The addition of Cu led to an increase of laccase activity
independent of the time of addition. However, when Cu
was added during the ¢rst days of growth, the increase of
laccase activity was not immediate ^ after Cu addition on
day 3, the activity began to increase around day 9, when
the growth of the cultures began to decelerate. Unlike
cadmium, after Cu addition the elevated laccase activity
lasted for the duration of cultivation (30 days).
3.3. Activity of laccase in the presence of other heavy
metals and H2 O2
The increase of laccase activity after the addition of Cd
and Cu led us to question the e¡ect of other heavy metals
on the activity of this enzyme. Since the toxic e¡ect of
both Cd and Cu is connected with the induction of oxi-
dative stress, the e¡ect of hydrogen peroxide on laccase
production was followed as well. The fungal cultures were
supplemented with AgNO3 , HgCl2 , Pb(NO3 )2 and ZnSO4
on day 12 to a ¢nal concentration of 1 mM, H2 O2 was
added to a ¢nal concentration of 0.5 M. On the day of
metal addition, the activity of laccase was 8 U ml31 . In all
combinations, the addition of these metals and H2 O2 led
to the decrease of laccase activity within 3 days (Table 1),
and the inhibition lasted until the end of cultivation (day
Fig. 2. Time course of laccase activity during the growth of P. ostreatus
in nitrogen-limited medium containing 0.0, 0.5, 1.0 and 5.0 mM copper
(¢nal concentration). Cu was added to growing cultures on day 12.
Averages of ¢ve replicates and S.E.M. are shown.
72 P. Baldrian, J. Gabriel / FEMS Microbiology Letters 206 (2002) 69^74

Table 2
Activity of puri¢ed P. ostreatus laccase incubated with Hg, Cd, and Cu
Treatment Activity (U ml31 ) Activity (%)
0 1h 1 day 7 days after 7 days
Control 18.4 þ 0.5 15.5 þ 1.2 8.9 þ 0.7 4.9 þ 1.0 27

Cd 0.05 mM 19.6 þ 0.7 16.3 þ 1.0 9.7 þ 0.0 5.6 þ 1.5 29

1.00 mM 20.3 þ 0.7 17.2 þ 0.7 9.7 þ 0.0 4.9 þ 1.5 24
50.00 mM 18.2 þ 1.7 16.0 þ 0.5 7.3 þ 1.5 4.3 þ 0.7 24

Cu 0.05 mM 20.5 þ 1.5 17.0 þ 0.5 10.6 þ 0.7 5.9 þ 2.0 29

1.00 mM 19.8 þ 1.5 16.7 þ 1.5 9.9 þ 0.3 6.3 þ 0.5 32
50.00 mM 21.7 þ 2.2 17.0 þ 1.5 12.5 þ 1.0 9.4 þ 0.0 43

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Hg 0.05 mM 15.5 þ 0.3 10.2 þ 0.3 3.0 þ 0.3 1.6 þ 0.3 10
1.00 mM 4.2 þ 0.0 3.1 þ 0.0 0.5 þ 0.3 0.0 þ 0.0 0
50.00 mM 0.0 þ 0.0 0.0 þ 0.0 0.0 þ 0.0 0.0 þ 0.0 0
Averages of two replicates and S.E.M. are shown

21). The addition of Ag and Hg almost led to the disap-
pearance of enzyme activity. These two metals also imme-
diately stopped the growth of the fungus. The addition of
Pb resulted in a substantial decrease of laccase activity,
whereas the growth of the fungus was una¡ected. The
addition of Zn and H2 O2 led to faster growth of the fun-
gus, but the activity of laccase was decreased.
3.4. E¡ect of Cd, Cu and Hg on produced extracellular
laccase
Since the experiments with liquid cultures showed tem-
poral di¡erences in the response to metals, extracts from
straw cultures at di¡erent growth stages were used: the
early stage of straw colonisation (day 8), the day of com-
plete substrate colonisation (day 22), and the stage of the
fastest straw degradation (day 28). Addition of mercury to
the extracts led to a decrease in laccase activity even at low
concentrations. At 0.05^0.1 mM Hg, the activity was re-
duced to 50% and at 0.1^0.5 mM Hg, the extract showed
only 10% laccase activity compared to the control. The
extract from 8-day-old culture was the most sensitive. In
the case of cadmium, metal addition did not a¡ect the
enzyme activity at concentrations up to 10 mM. After
incubation with copper the metal-supplemented extracts
exhibited in general higher laccase activity than the control
(Fig. 3). Signi¢cant di¡erences were found between the
extracts from cultures of di¡erent ages. In the extract
from 8-day-old culture, elevated laccase activity was found
only with 0.5^10 mM Cu. Maximum activity was found at
10 mM Cu where it was 40% higher than in the control. In
extracts from 22- and 28-day-old cultures, laccase activity
was higher than the control at all Cu concentrations.
Highest activities were recorded in extracts containing
1 mM Cu, where the activity after incubation was 3.2-
fold (22 days) or 6.9-fold (28 days) compared to respective
controls.
To assess the e¡ect of metals on laccase produced by P.
ostreatus, the puri¢ed enzyme was incubated with di¡erent
concentrations of Cd, Cu, and Hg. The metals in£uenced
the activity of laccase immediately (Table 2). The addition
of mercury at more than 0.01 mM strongly decreased the
activity of laccase as well as its temporal stability. At more
than 0.1 mM Hg, no laccase activity was detected after 7
days of incubation. Both Cd and Cu moderately increased
the enzyme activity. The stability of laccase was increased
in the presence of 0.5^50.0 mM Cu. During 7 days, the
activity of laccase decreased by 55^68% in the presence of
copper, while the control lost 73% of its activity.

4. Discussion

Fig. 3. Activity of laccase in the extracts from P. ostreatus cultures

grown on wheat straw for 8, 22, and 28 days, after 18-h incubation in
the presence of 0.01^50 mM Cu. Laccase activity in controls : 158.44 U
ml31 (day 8), 9.32 U ml31 (day 22), and 5.36 U ml31 (day 28). Aver-
ages of two replicates are shown.
Di¡erent compounds in£uence the stability of lignino-
lytic enzymes in the extracellular environment. Inactiva-
tion of MnP was observed due to the presence of hydro-
gen peroxide in the culture liquid [17]. Also the metals

FEMSLE 10263 3-1-02

 P. Baldrian, J. Gabriel / FEMS Microbiology Letters 206 (2002) 69^74
present in the environment in£uence the stability of se-
creted enzymes ^ a protective role of Mn2‡ was proposed
[18], as well as the thermal stabilisation of MnP by Cd2‡
and Ca2‡ [19]. The induction of laccase by copper has
already been documented in P. ostreatus [20]. The authors
cultivated the fungus in nutrient-rich medium supple-
mented with 150 WM CuSO4 at the time of inoculation.
Here we report the induction of laccase by copper when
added to the fungus growing on nitrogen-limited medium,
which better re£ects the nutrient availability under natural
conditions. This is the ¢rst report of induction of laccase
by cadmium, a non-essential heavy metal. The induction
only occurs when cadmium is added during the later stages
of growth. The sensitivity of the fungus towards Cu and
Cd changes with time. The same has been shown with the
extracts from fungal cultures growing on wheat straw. It
might be due to production of di¡erent isoenzymes during
straw colonisation. Although all known isoenzymes are
positively regulated by copper, the extent of induction is
di¡erent [20]. It also has to be noted that the extract from
day 8 showed lower laccase activity than the extracts from
later days. The experiments with puri¢ed laccase showed
that Cu not only induces laccase by the expression of lac-
case genes, but it also positively a¡ects the activity and
stability of the enzyme.
Recently, a novel extracellular protease of P. ostreatus,
PoSl, has been puri¢ed and shown to be responsible for
the degradation of laccase [21]. The activity of PoSl is
decreased to 77% in the presence of 1 mM Cu. This might
explain the higher positive e¡ect of Cu on enzyme stability
in culture extracts than using puri¢ed enzyme. Since cop-
per is a biogenic metal present in the environment, its
concentration may have an important role in enzyme per-
formance and stability.
The presented results show that the increase of laccase
activity in P. ostreatus cultures after Cu addition is due to
both increased laccase production [20] and the stabilisa-
tion of the enzyme in the extracellular environment. In the
case of Cd, since there is no e¡ect of the metal on enzyme
stability, the increased levels of laccase after metal addi-
tion are due to increased enzyme production. The negative
e¡ect of mercury is mainly due to the inactivation of en-
zyme by the metal.
Heavy metals do not in£uence only the activity of lac-
case. The activity of MnP is very sensitive to the presence
of heavy metals [12] and the inhibition of its activity in
Cd-supplemented soil probably leads to limited PAH deg-
radation [13]. In the experiments described here, the activ-
ity of MnP was strongly decreased by Cd. Interestingly,
Cu and also Hg increased MnP activity slightly. When
MnP extracted from fungal cultures was incubated in the
presence of all three metals, the activity was decreased
even at low concentrations of Cd, Cu, and Hg.
The results show that the presence of heavy metals in
the environment plays an important role in the regulation
of extracellular enzyme activities. For practical bioremedi-
FEMSLE 10263 3-1-02
73
ation processes, also other e¡ects of heavy metals should
be taken into consideration, including the inhibition of
fungal growth [22] or substrate colonisation [13]. However,
the activation and stabilisation of produced laccase by
copper seems to be useful in biotechnological applications
using immobilised enzyme or other laccase-based prepara-
tions.
Acknowledgements
This study was supported by the Grant Agency of the
Czech Republic (204/99/1528) and by Institutional Re-
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search Concept AV0Z5020903 of the Institute of Micro-
biology, Academy of Sciences of the Czech Republic.
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