Annals of Botany 98: 1035–1042, 2006
doi:10.1093/aob/mcl184, available online at www.aob.oxfordjournals.org
Co-occurrence of the Multicopper Oxidases Tyrosinase and Laccase in
Lichens in Sub-order Peltigerineae
Z S A N E T T L A U F E R 1, R I C H A R D P . B E C K E T T 1,* and F A R I D A V . M I N I B A Y E V A 2
1
School of Biological and Conservation Sciences, University of KwaZulu-Natal, Private Bag X01,
Pietermaritzburg, Scottsville 3209, Republic of South Africa and 2Institute of Biochemistry
and Biophysics, Russian Academy of Science, PO Box 30, Kazan 420111, Russia
Received: 24 May 2006 Returned for revision: 19 June 2006 Accepted: 7 July 2006 Published electronically: 1 September 2006
Key words: Lichens, tyrosinase, laccases, peroxidases, Peltigerineae, Pseudocyphellaria, Peltigera.
INTROD UCTION
Tyrosinases are a family of multicopper oxidase proteins
found in some higher plants, fungi and prokaryotes (Seo
et al., 2003; Claus and Decker, 2006). Copper-containing
enzymes play important roles in the biological activation
of oxygen, necessary for the oxidation of a great variety of
different substrates. Multicopper oxidases include mononuclear copper enzymes (e.g. amine oxidase), coupled
binuclear copper enzymes (e.g. tyrosinase) and enzymes
containing trinuclear copper clusters (e.g. laccase and
ascorbate oxidase) (Solomon et al., 2001; Nakamura,
2005). These enzymes have overlapping substrate
specificities and can co-occur in the same samples,
making their identification difficult (Ratcliffe et al., 1994;
Baldrian, 2006). However, the particularity of tyrosinases
is their ability to catalyse the o-hydroxylation of
monophenols (cresolase activity or ‘monophenolase’)
and the subsequent oxidation of the resulting o-diphenols
into reactive o-quinones (catecholase activity or ‘diphenolase’), both reactions using molecular oxygen (Halaouli
et al., 2006; Marusek et al., 2006). Subsequently, the
o-quinones undergo non-enzymatic reactions with various
nucleophiles, producing intermediates which associate
spontaneously in dark brown pigments (Soler-Rivas et al.,
* For correspondence. E-mail rpbeckett@gmail.com
1997). In the fungi, tyrosinases are mainly associated with
browning as a result of melanin biosynthesis (Halaouli
et al., 2006). Melanins in the cell walls of Ascomycotina
are derived from 1,8-dihydroxynaphthalene (DHN) (Bell
and Wheeler, 1986). Melanin formation constitutes a
mechanism of defence and resistance to stress such as UV
radiation, free radicals, g-rays, dehydration and extreme
temperatures, and contributes to the fungal cell wall
resistance against hydrolytic enzymes in avoiding cellular
lysis (Bell and Wheeler, 1986). These pigments are also
involved in the formation and stability of spores (Mayer
and Harel, 1979), and in the defence and virulence
mechanisms (Soler-Rivas et al., 1997; Jacobson, 2000).
Perhaps surprisingly, no reports exist for tyrosinases in
an important group of fungi, the lichenized ascomycetes.
However, in lichens, strong evidence exists that melanins
act as screens for UV light (Gauslaa and Solhaug, 2001;
Stepanenko et al., 2002; Solhaug et al., 2003). Recently,
Laufer et al. (2006) demonstrated the widespread presence
of extracellular laccases in members of the lichenized
ascomycete sub-order Peltigerinae. To study the role of
cell wall redox enzymes in lichens further, testing was
started for the presence of other enzymes. A. Zavarzina
and A. Zavarzin (pers. comm.) recently carried out
some experiments showing that in the field lichens can
metabolize tyrosinase substrates, and this was confirmed
Ó The Author 2006. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
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Background and Aims Following previous findings of high extracellular redox activity in lichens and the presence
of laccases in lichen cell walls, the work presented here additionally demonstrates the presence of tyrosinases. Tests
were made for the presence of tyrosinases in 40 species of lichens, and from selected species their cellular location
and molecular weights were determined. The effects of stress and inhibitors on enzyme activity were also studied.
Methods Tyrosinase and laccase activities were assayed spectrophotometrically using a variety of substrates.
The molecular mass of the enzymes was estimated using polyacrylamide gel electrophoresis.
Key Results Extracellular tyrosinase and laccase activity was measured in 40 species of lichens from different
taxonomic groupings and contrasting habitats. Out of 20 species tested from the sub-order Peltigerineae, all
displayed significant tyrosinase and laccase activity, while activity was low or absent in other species tested.
Representatives from both groups of lichens displayed low peroxidase activities. Identification of the enzymes
as tyrosinases was confirmed by the ability of lichen thalli or leachates derived by shaking lichens in distilled water
to metabolize substrates such as L-dihydroxyphenylalanine (DOPA), tyrosine and epinephrine readily in the absence
of hydrogen peroxide, the sensitivity of the enzymes to the inhibitors cyanide, azide and hexylresorcinol, activation
by SDS and having typical tyrosinase molecular masses of approx. 60 kDa. Comparing different species within the
Peltigerineae showed that the activities of tyrosinases and laccase were correlated to each other. Desiccation and
wounding stimulated laccase activity, while only wounding stimulated tyrosinase activity.
Conclusions Cell walls of lichens in sub-order Peltigerineae have much higher activities and a greater diversity of
cell wall redox enzymes compared with other lichens. Possible roles of tyrosinases include melanization, removal of
toxic phenols or quinones, and production of herbivore deterrents.
1036
Laufer et al. — Polyphenol Oxidases in Lichenized Ascomycetes
T A B L E 1. Laccase activity, measured using the substrates ABTS and syringaldazine, and tyrosinase activity, detected as oxidation
of epinephrine to adrenochrome in a range of lichens
Species
Laccase activity
syringaldazine (mmol g
dry mass h 1)
1
Tyrosinase
activity (mmol g
dry mass h 1)
Drakensberg, RSA
Drakensberg, RSA
Sortavala, Russia
Drakensberg, RSA
White Sea, Russia
Lahemma, Estonia
Sortavala, Russia
Sortavala, Russia
Sortavala, Russia
Sortavala, Russia
Drakensberg, RSA
Sortavala, Russia
Sortavala, Russia
Sortavala, Russia
Sortavala, Russia
Sortavala, Russia
28
1
16
68
37
25
83
130
162
9
156
25
62
113
76
120
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
9
1
2
7
14
3
22
10
11
2
22
13
13
33
16
12
1.3
0.4
1.1
3.0
0.1
0.7
3.0
2.3
2.2
1.0
6.8
1.6
1.0
2.4
1.2
2.4
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
0.3
0.2
0.4
1.9
0.0
0.4
0.3
0.2
0.3
0.4
1.9
0.3
0.4
0.5
0.4
0.7
56
24
22
25
131
62
109
145
224
55
234
134
85
175
151
138
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
13
7
3
2
14
9
11
11
11
17
28
28
15
10
16
18
Sortavala, Russia
Nottingham Road, RSA
Nottingham Road, RSA
Pietermaritzburg, RSA
121
19
6
54
6
6
6
6
19
4
1
17
2.6
1.7
3.8
2.5
6
6
6
6
0.5
0.6
0.6
0.1
227
60
78
94
6
6
6
6
31
13
7
13
Sararemma, Estonia
Sortavala, Russia
362
060
0.0 6 0.0
0.3 6 0.0
Sortavala, Russia
Sortavala, Russia
Sortavala, Russia
Sortavala, Russia
Sortavala, Russia
Drakesnberg, RSA
Sortavala, Russia
Umgeni Valley, RSA
Sortavala, Russia
1
3
1
6
2
2
5
1
5
0.1
0.1
0.0
0.0
0.0
0.2
0.1
0.5
0.0
Sortavala, Russia
Pietermaritzburg, RSA
Sortavala, Russia
Sortavala, Russia
Umgeni Valley, RSA
Sortavala, Russia
Sortavala, Russia
Sortavala, Russia
Pietermaritzburg, RSA
Mean for sub-order Peltigerineae (n = 20)
Mean for non-sub-order
Peltigerineae (n = 20)
6
6
6
6
6
6
6
6
6
0
0
0
2
1
1
2
0
1
261
13 6 3
1
1
0
0
0
0
2
6
6
6
6
6
6
6
0
0
0
0
0
0
2
66 6 12
261
6
6
6
6
6
6
6
6
6
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.2
0.0
0.1 6 0.0
1.5 6 0.4
0.0
0.1
0.0
0.1
0.1
0.2
0.1
6
6
6
6
6
6
6
0.0
0.0
0.0
0.1
0.0
0.0
0.1
2.1 6 0.5
0.2 6 0.0
1
11 6 4
460
1
6
3
5
1
4
4
6
5
6
6
6
6
6
6
6
6
6
0
1
1
1
0
2
1
1
2
461
13 6 4
4
3
4
4
3
7
3
6
6
6
6
6
6
6
1
1
2
1
1
1
1
111 6 14
561
Figures are given 6s.d., n = 5.
by our own preliminary results. The aims of the work
presented here were to verify the presence of tyrosinases
in lichens, distinguish them from laccases on the bases
of cellular location, substrate specificity and molecular
weight, and determine the relationship between laccase
activity and tyrosinases activity in a range of species.
Determination of the taxonomic distribution of tyrosinase
activity and characterization of the enzymes will be
essential for elucidating the biological roles of these redox
enzymes in lichen physiology.
MATERIA LS A ND METHODS
Lichen material
Table 1 lists the species of lichens examined in this study
and their collection localities. Specimens collected from
the field in a fully hydrated state were equilibrated in a
controlled environment chamber for 48 h at 15 C and
a photosynthetic photon fluence rate (PPFR) of 75 mmol
photons m 2 s 1 of continuous fluorescent light. All light
intensities were measured across photosynthetically
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Lichens from sub-order Peltigerineae
Collema flaccidum (Ach.) Ach.
Leptogium saturninum (Dicks.) Nyl.
Lobaria pulmonaria (L.) Hoffm.
Lobaria scrobiculata (Scop.) DC.
Nephroma arcticum (L.) Torss.
Nephroma parile (Ach.) Ach.
Peltigera apthosa (L.) Willd.
Peltigera canina (L.) Willd.
Peltigera didactyla (With.) J R Laundon
Peltigera horizontalis (Huds.) Baumg.
Peltigera hymenina (Ach.) Delise
Peltigera leucophlebia (Nyl.) Gyelnik
Peltigera malacea (Ach.) Funck
Peltigera neopolydactyla (Gyelnik) Gyelnik
Peltigera polydactylon (Necker) Hoffm.
Peltigera praetextata (Flörke ex
Sommerf) Zopf.
Peltigera scabrosa Th. Fr.
Pseudocyphellaria aurata (Ach.) Vaino
Sticta fuliginosa (Dicks.) Ach.
Sticta limbata (Sm.) Ach.
Lichens from non-sub-order Peltigerineae
Anaptychia ciliaris (L.) Körb.
Bryoria simplicior (Vanio) Brodo and
D. Hawksw.
Cetraria islandica (L.) Ach.
Cladonia cariosa (Ach.) Spreng.
Cladonia stellaris (Opiz) Pouzar and Vezda
Evernia prunastri (L.) Ach.
Flavocetraria nivalis (L.) Karnefelt and Thell
Heterodermia speciosa (Wulf.) Trevis.
Hypogymnia physodes (L.) Nyl.
Parmelia cetrarioides (Duby) Nyl.
Platismatia glauca (L.) W. L. Culb. and
C. F. Culb.
Pseudevernia furfuracea (L.) Zopf
Ramalina celastri (Sprengel) Krog
and Swinscow
Ramalina farinacea (L.) Ach.
Ramalina pollinaria (Westr.) Ach.
Roccella montagnei Bél.
Stereocaulon tomentosum Fr.
Umbilicaria deusta (L.) Baumg.
Umbilicaria pustulata (L.) Hoffm.
Usnea undulata Stirton
Collection locality
Laccase activity
ABTS (mmol g 1
dry mass h 1)
Laufer et al. — Polyphenol Oxidases in Lichenized Ascomycetes
Colorimetric laccase, peroxidase and tyrosinase assays
Enzyme activities were measured using thallus disks,
leachates derived from shaking lichens in water, and from
cellular fractionation experiments (described below).
Assays were always carried out under optimal conditions
as determined in preliminary experiments for Peltigera and
Pseudocyphellaria.
Laccase activity was investigated using the oxidation
of 2,2-azino-bis 3-ethybenz-thiazoline-6-sulfonic acid
(ABTS; Fluka, Buchs, Switzerland) to the stable cation
radical (ABTS+) (Min et al., 2001) and that of the natural
compound syringaldazine (Sigma) to its corresponding
quinone (Medeiros et al., 1999). For whole thallus assays
using ABTS, 30 mg of fresh mass of lichen tissue was
shaken at 60 rpm in 5 mL of 1 mM ABTS dissolved in
25 mM phosphate buffer, pH 5, for 15 min at 30 C. To
assay leachates or cellular fractions, 10–100 mL of extract,
100 mL of 10 mM ABTS (final concentration 1 mM) and
25 mM phosphate buffer pH 5 to give a final volume
of 1 mL were incubated for 15 min at 30 C. The
extinction coefficient of the product measured at A420 is
36 mM 1 cm 1. For syringaldazine, the same conditions
were used with 10 mM syringaldazine in 25 mM phosphate
buffer pH 65. The extinction coefficient of the product at
A525 is 65 mM 1 cm 1. Peroxidase activity was estimated
for cellular fractions only as the increase in the rate of
reaction when H2O2 was added to give a final concentration
of 10 mM in a 1 mM ABTS assay solution at pH 6.
Tyrosinase activity was estimated by the oxidation of
epinephrine (Sigma) to adrenochrome (Sugumaran et al.,
1997), the oxidation of L-dihydroxyphenylalanine (DOPA;
Sigma) to 2-carboxy-2,3-dihydroindole-5,6-quinone (dopachrome; Horowitz et al., 1970) and the oxidation of
L-tyrosine (Mueller et al., 1996). For whole thallus assays,
150 mg of fresh mass of lichen tissue was shaken at 60 rpm
in 35 mL of 1 mM epinephrine, pH 7 (pH adjusted with
HCl and NaOH), for 15 min in the dark at 25 C. The
extinction coefficient of adrenochrome measured at A490 is
447 mM 1 cm 1. To assay leachates or cellular fractions,
50–100 mL of extract, 200 mL of 10 mM DOPA (final
concentration 2 mM) and 50 mM phosphate buffer pH 6 to
give a final volume of 1 mL were incubated for 10–15 min
at 25 C. The extinction coefficient of the product measured
at A475 is 36 mM 1 cm 1. As an additional test for the
ability of lichens to metabolize tyrosinase substrates,
150 mg of fresh mass of lichen tissue was shaken at 60 rpm
in 35 mL of 2 mM L-tyrosine, pH 6, for 15 min at 25 C. For
leachates, 200 mL of extract, 200 mL of 10 mM L-tyrosine
(final concentration 2 mM) and 50 mM phosphate buffer
pH 6 to give a final volume of 1 mL were incubated for
30 min at 25 C. The extinction coefficient of the product
measured at A490 is 33 mM 1 cm 1.
Cellular location of enzymes in Pseudocyphellaria aurata and
Flavocetraria nivalis
The cellular location of redox enzymes was determined
using a modification of the method of Rast et al. (2003).
Fresh material (35 g) was freeze-dried, ground in 35 mL of
ice-cold 025 M Tris–HCl buffer pH 8, and then centrifuged
at 4 C for 15 min at 4000 g, the supernatant representing
the cytosolic fraction (‘C’). Initial experiments with
P. aurata showed that shaking lichens in distilled water
for 1 h only released between 04 and 08 % of total
enzyme activity in solution. Therefore, this fraction was
not analysed separately, but was included within the
C fraction. The pellet was suspended in 20 mL of 50 mM
phosphate buffer pH 7, and then centrifuged as above.
This was repeated three times, the sum of the supernatants representing enzymes loosely bound to the cell
wall, e.g. by hydrogen bonds (‘B1’). The pellet was resuspended in 15 mL of phosphate buffer and solid digitonin
(Sigma) added to give a final concentration of 03 %.
The solution was stirred for 3 h at 4 C, centrifuged as
above, and then the step was repeated. The combination of
the two supernatants represented enzymes bound by van
der Waals forces and hydrophobic interactions (‘B2’). The
pellet was re-suspended in 15 mL of phosphate buffer, and
NaCl added to give a final concentration of 2 M. The
solution was then stirred for 3 h at 4 C, centrifuged as
above, and then the step was repeated. The combination
of the two supernatants represents enzymes bound by
strong electrostatic forces (‘B3’). Finally, the remaining
pellet was re-suspended in 10 mL of phosphate buffer,
and represents enzymes bound by covalent linkages. This
last fraction was used directly in assays (‘B4’). The cell
wall fragments were pelleted after the assay to allow
measurement of absorption. All the above fractions
were assayed for laccase, tyrosinase and peroxidase
activity as described above. To test for the presence of
latent enzyme forms, assays for tyrosinases were carried
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active wavebands using the light meter in a Parkinson leaf
chamber designed for an Analytical Development Corporation Mark III portable infrared gas analyser (Hoddeston, UK). Lichens collected dry were gradually rehydrated
using air at a relative humidity of 100 % for 48 h (over
distilled water) at 15 C and a PPFR of 75 mmol m 2 s 1,
followed by contact with wet filter paper for a further 24 h.
They were then used immediately. Exceptions to the above
were the lichens collected in Russia and Estonia. If
collected moist, they were allowed to dry slowly between
sheets of newspaper. All specimens were then stored
refrigerated for up to 6 weeks after collection, and then
rehydrated as above.
For experiments on whole lichens, to reduce variability,
in species with large lobes, 6 mm disks were cut from the
thalli. For species in which disks could not be cut, thalli
were cut into 1 cm strips. In all cases, large collections of
lichens were made, and the thalli used were randomly
sampled from those thalli that appeared most healthy.
Disks or thallus strips were stored as above for least 12 h
before an experiment to minimize any effect of mechanical
disruption on enzyme activity, and then used immediately.
For experiments that used lichen leachates, typically 1 g of
fresh mass of lichens was shaken for 1 h in 10 mL of
distilled water, with the pH adjusted to 70 with HCl and
NaOH. The leachates were filtered through Whatman no. 1
filter paper, and used immediately.
1037
out with and without 2 mM SDS as recommend by Moore
and Flurkey (1990).
Electrophoretic investigation of tyrosinases and laccases
Effect of wounding and desiccation on laccase and tyrosinase
activity in Pseudocyphellaria aurata
Pseudocyphellaria aurata was collected dry, stored
for 2 d at 15 C at 100 % relative humidity, then for 1 d on
wet paper. Disks (6 mm) were then cut and stored for a
further 12 h. Each treatment comprised five replicates,
and each replicate comprised five disks. Three treatments
were used. Treatment one comprised undesiccated and
unwounded material. Treatment two comprised material
in which enzyme activities (with ABTS and epinephrine as
substrates) were measured for 05 h, the disks cut into
quarters using a scalpel, and then the activity measured
over selected 15 min intervals for 25 h. Treatment three
comprised material in which enzyme activity was
measured for 05 h, the material allowed to dry over 25 h
to a relative water content of 005, rehydrated by the
addition of liquid water, then the enzyme activity measured
over selected 15 min intervals for 25 h. Leakage of
cytosolic enzymes was quantified by measuring the
proportion of the strictly cytosolic enzyme glucose-6phosphate dehydrogenase (G-6-PD) released into the
medium following stress. The assay mixture contained
160
A
120
80
40
0
8
B
6
4
2
0
0
150
200
100
Tyrosinase activity
(mmol product g−1 dry mass h−1)
50
250
F I G . 1. Rates of metabolism of the laccase substrates ABTS (A) and
syringaldazine (B) in a range of lichens are positively correlated to rates
of metabolism of the tyrosinase substrate epinephrine (P < 0001). Each
symbol denotes a single species; filled circles indicate lichens in sub-order
Peltigerineae, and open circles lichens from other suborders.
glucose-6-phosphate (1 mM), NADP (02 mM), MgCl2
(1 mM), Tris–HCl buffer pH 80 (01 mM) and sample, and
production of NADPH was measured at 340 nm.
Effects of enzyme inhibitors on tyrosinase and laccase activity in
Pseudocyphellaria aurata
Lichen leachates were incubated in 1 mM KCN,
2 mM sodium azide, 40 mM NaF or 02 mM hexylresorcinol
(Fluka) for 20 min, substrate was added (ABTS for
laccase activity and DOPA for tyrosinase activity), and
then the formation of product was measured over the next
20 min.
RESULTS
In the survey involving assays with whole thalli, laccase
and tyrosinase activities were detected in almost all lichens
in the sub-order Peltigerineae, but in other species the
enzymes had very low activities or were absent (Table 1).
Lichens and leachates derived from them also readily
metabolized L-tyrosine, although at only approx. 10 % of
the typical rates observed with epinephrine and DOPA. For
example, in Pseudocyphellaria, rates were 7 6 3 mmol g 1
dry mass h 1 for thallus disks, and 096 6 012 mmol g 1
dry mass h 1 for leachates derived by shaking lichens
in water (figures are given 6 s.d., n = 5). Within the
Peltigerineae, the activities of the enzymes were significantly correlated to each other (Fig. 1).
Downloaded from http://aob.oxfordjournals.org/ by guest on February 9, 2016
Electrophoretic studies were carried using Peltigera
malacea, P. rufescens and Pseudocyphellaria aurata.
These species were chosen because they were locally
abundant species with good enzyme activity. Fractions
prepared as above were concentrated by dialysis against
solid sucrose. Typically, samples prepared by concentrating fraction B1 were relatively free of cytosolic
contaminants, and were used in subsequent electrophoresis.
To determine the molecular mass of the active form of the
tyrosinases, fractions were loaded into 10 % (w/v)
polyacrylamide gels (Laemmli, 1970). Running buffer
and gels contained the normal concentrations of SDS
(01 %), but samples were not heated, and mercaptoethanol
was omitted from the loading buffer. After electrophoresis,
bands were visualized by incubating gels in 10 mM DOPA
(Sigma) in 01 M phosphate buffer pH 6, and 10 mM
epinephrine in 01 M phosphate buffer pH 7. After approx.
20 min, black (dopachrome) and orange (adrenochrome)
bands appeared at the position of the tyrosinase. Gels were
also stained with the ‘Silver Plus’ stain (Bio-Rad, Hercules,
CA, USA) with and without heating with mercaptoethanol.
To determine the molecular mass of the active form of
the laccases, fractions were loaded onto 6 % (w/v)
polyacrylamide gels (Laemmli, 1970). After electrophoresis, laccase bands were visualized by incubating gels in
10 mM 2,6-dimethoxyphenol (DMP) (Sigma) in 02 M MES
buffer, pH 6. After approx. 30 min, a yellow-brown colour
appeared at the position of the laccase. Both gels were
run with molecular mass markers (‘Broad Range’,
Bio-Rad) which were stained using Coomassie brilliant
blue G250 (Sigma).
Laccase activity
(µmol product g−1 dry mass h−1)
Laufer et al. — Polyphenol Oxidases in Lichenized Ascomycetes
Laccase activity
(µmol product g−1 dry mass h−1)
1038
1039
Laufer et al. — Polyphenol Oxidases in Lichenized Ascomycetes
% activity in each fraction
100
A
75
Pseudocyphellaria
aurata
50
25
0
100
B
75
50
25
0
C
B1
B2
Fraction
B3
B4
F I G . 2. Cellular location in Flavocetraria nivalis (A) and Pseudocyphellaria aurata (B) of laccases (open bars), tyrosinases without SDS
(light grey), tyrosinases with 2 mM SDS (dark grey) and peroxidases
(solid bars). Values are expressed as percentages of the following fractions:
C, cytosolic fraction; B1, loosely bound to the cell wall, e.g. by hydrogen
bonds; B2, bound by van der Waals forces and hydrophobic interactions;
B3, bound by strong electrostatic forces; B4, bound by covalent linkages.
Table 2 gives the actual enzyme activities. Error bars indicate the standard
deviation, n = 5.
Low activities of laccase and peroxidase, but not
tyrosinase, were detected in Flavocetraria nivalis
(Fig. 2A, Table 2). Laccase activity was mostly intracellular, while peroxidase activity was equally distributed
between the intracellular and the hydrophobic cellular
locations. Pseudocyphellaria aurata displayed activities of
all three enzymes (Fig. 2B, Table 2). Laccase was located
mostly in the loosely and hydrophobically bound cell
wall fractions. Compared with the laccases, a greater
proportion of tyrosinases occurred intracellularly. Adding
SDS stimulated tyrosinase activity in all fractions except
those bound by electrostatic interactions (fraction B3,
Table 2). Separate experiments showed that SDS had no
effect on laccase activity measured with ABTS (data
not shown).
Following electrophoresis, tyrosinase visualization with
DOPA and epinephrine revealed one main band with
a molecular mass of approx. 60 kDa (Fig. 3). Peltigera
malacea also possessed a smaller amount of an enzyme
with a molecular mass of approx. 160 kDa. The molecular
masses of the isoforms were the same in the cytoplasmic
and cell wall fractions (data not shown). Silver staining
also revealed a protein in B1 with a molecular mass
identical to that of the activity stain. The position of this
band did not change if samples were heated in mercaptoethanol before loading, suggesting that estimates of
Laccase activity
C
B1
B2
B3
B4
Tyrosinase activity –SDS
C
B1
B2
B3
B4
Tyrosinase activity +SDS
C
B1
B2
B3
B4
Peroxidase activity
C
B1
B2
B3
B4
Flavocetraria
nivalis
0.60
0.01
0.02
0.10
0
9
36
23
3
5
6
6
6
6
6
1
2
1
0
0
46
51
48
18
28
6
6
6
6
6
1
16
2
1
4
0
0
0
0
0
370
79
141
9
79
6
6
6
6
6
6
10
7
3
5
0
0
0
0
0
4.3 6 0.3
0
0.5 6 0.0
0
0.5 6 0.0
6
6
6
6
0.01
0.01
0.01
0.01
0
0.02 6 0.01
0
0.02 6 0.01
0
Activities of tyrosinases are given with and without the addition of 2 mM
SDS to the assay medium. Figures are all mmol product g 1 dry mass h 1, and
are given 6s.d., n = 5.
tyrosinase molecular mass are reasonably accurate.
Laccase visualization with DMP revealed a single brown
band with a molecular mass of approx. 340 kDa in the two
Peltigera species, and 160 kDa in P. aurata (data not
shown). As for the tyrosinases, the molecular masses of the
laccase isoforms were the same in the cytoplasmic and cell
wall fractions (data not shown).
Wounding stress strongly stimulated both laccase and
tyrosinase activity, while desiccation stress increased
laccase but not tyrosinase activity (Fig. 4). The total
activity of G-6-PD was 150 6 9 U g 1 dry mass h 1, of
which 37 6 28 U were released by shaking disks in water
for 1 h. Shaking wounded disks in water for 1 h increased
the release to 64 6 30 U.
The inhibitors cyanide, azide and sodium fluoride all
strongly reduced both tyrosinase and laccase activity.
Tyrosinase activity was more resistant to cyanide and
azide, and while these inhibitors reduced tyrosinase activity
equally, laccase activity was more sensitive to azide than
cyanide. The tyrosinase-specific inhibitor hexylresorcinol
(02 mM) reduced tyrosinase activity by approx. 20 %.
DISCUSSION
The main findings of this study are that high extracellular
tyrosinase activity occurs in lichens in the Peltigerineae
(Table 1, Fig. 1), extending an earlier report of laccase
activity from this group (Laufer et al., 2006). Demonstration of tyrosinase activity was based on several
Downloaded from http://aob.oxfordjournals.org/ by guest on February 9, 2016
% activity in each fraction
T A B L E 2. Activities of the enzymes laccase, tyrosinase and
peroxidase in the lichens Pseudocyphellaria aurata and Flavocetraria nivalis in various cellular fractions (see text for details)
Laufer et al. — Polyphenol Oxidases in Lichenized Ascomycetes
1040
200−
200−
200−
160−
116−
97−
116−
97−
116−
97−
66−
66−
66−
58−
61−
61−
60−
45−
45−
45−
31−
31−
31−
DOPA
DOPA
Peltigera malacea
Peltigera rufescens
DOPA
Epinephrine
Pseudocyphellaria
aurata
Stress
40
30
20
10
0
150
B
Stress
Laccase activity
(µmol product g−1 dry mass h−1)
Tyrosinase activity
(µmol product g−1 dry mass h−1)
A
50
100
50
0
0
1
2
Time (h)
3
4
F I G . 4. The effect of wounding and desiccation on (A) laccase activity
(measured with ABTS as a substrate) and (B) tyrosinase activity (measured
with epinephrine as a substrate) in Pseudocyphellaria aurata. The arrow
indicates the time that material was stressed, either by cutting disks into
quarters (filled circles) or by desiccating it to a relative water content of 005
over 25 h then suddenly rehydrating it (open triangles). Open circles represent control (unstressed) material. Error bars indicate s.d., n = 5.
observations, all consistent with the characteristics of
tyrosinases from free-living fungi (Halaouli et al., 2006).
These include the ability of lichens and their leachates
readily to metabolize classic tyrosinase substrates such as
DOPA, epinephrine and L-tyrosine, the sensitivity of the
enzymes to cyanide, azide and hexylresorcinol, and typical
molecular masses (Fig. 3). As no reports of tyrosinases
exist in free-living cyanobacteria or algae, it seems most
likely that enzymes are produced exclusively by the fungal
symbionts of the lichens studied here.
In earlier publications (Minibayeva and Beckett, 2001;
Beckett et al., 2003), it was shown that the oxidation of
epinephrine to adrenochrome can be used to indicate O–
2
production in lichens. This oxidation was sensitive to
superoxide dismutase (SOD), the main enzyme involved in
O–
2 detoxification. However, further work showed that in
most species the oxidation of epinephrine was SOD
insensitive or SOD had very little effect, suggesting that
epinephrine oxidation in lichens can happen via superoxide-dependent and -independent pathways. Purified fungal
tyrosinases can also directly oxidize epinephrine to
adrenochrome (Sugumaran et al., 1997). For these reasons,
the presence of tyrosinases complicates the original
epinephrine assays for O–
2 , and alternative methods are
therefore recommended. For example, the recent
approaches to quantifying O–
2 production with fluorescent
dyes are probably more specific (Weissman et al., 2005).
While tyrosinase activity was correlated with laccase
activity (Fig. 1, Table 1), these enzymes could be
distinguished by their molecular masses, substrate specificities, cellular location, sensitivity to inhibitors, and
activation by SDS and stress. Firstly, PAGE separation of
cytosolic and cell wall fractions from P. malacaea,
P. rufescens and P. aurata showed that a band with a
molecular mass of approx. 60 kDs appeared within minutes
of incubation in the tyrosinase substrates DOPA or
epinephrine (Fig. 3). This mass is similar to those reported
for other ascomycete tyrosinases (van Gelder et al., 1997;
Halaouli et al., 2006; Marusek et al., 2006). The molecular
mass of the laccases from the two Peltigera species were
much higher at well over 300 kDa, in agreement with the
earlier finding for P. malacea (Laufer et al., 2006), while
the laccase from P. aurata had a molecular mass of
160 kDa. Interestingly, after incubating gels for several
hours with DOPA, faint bands appeared at the same
location as those of the laccase bands visualized by DMP,
Downloaded from http://aob.oxfordjournals.org/ by guest on February 9, 2016
F I G . 3. Characterization of tyrosinases from Peltigera malacea, P. rufescens and Pseudocyphellaria aurata. A native 10 % polyacrylamide gel was used;
tyrosinase activity staining with DOPA and epinephrine. Molecular mass markers were stained using Coomassie brilliant blue G250. Weights of the
molecular markers and tyrosinase bands are indicated in kDa.
Laufer et al. — Polyphenol Oxidases in Lichenized Ascomycetes
Roles of tyrosinases in lichen biology
In free-living fungi, the roles of tyrosinases remain
unclear but, as discussed in the Introduction, they can
certainly catalyse melanization (van Gelder et al., 1997;
Seo et al., 2005; Halaouli et al., 2006). While melanins
are present in Peltigeralean lichens such as Lobaria
pulmonaria (Gauslaa and Solhaug, 2001) where they
appear to protect the photobiont from excessive radiation,
they are also present in non-Peltigeralean lichens with very
low laccase and tyrosinase activities such as Cetraria
islandica (Nybakken et al., 2004). It is assumed that, as
for fungi (Butler and Day, 1998), various mechanisms of
melanin synthesis exist in lichens, and presumably some
use other oxidases. A further role of these enzymes could
be to remove harmful quinone radicals and phenols in the
soil or bark on which lichens grow. These compounds
are produced as by-products of delignification (Rimmer,
2006). Interestingly, Krastanov (2000) recommended the
use of co-immobilized laccases and tyrosinases in polyclar
columns used to remove phenolic xenobiotics, because
their activities on different substrates complement each
other. Stimulation of the activity of these enzymes
following stress (Fig. 4) is consistent with a role in
removal of stress-induced toxic compounds. Future
progress in understanding the roles of these enzymes in
lichens will depend on the elucidation of these reactions.
Finally, Van Alstyne et al. (2006) recently showed that
DOPA in green algae can deter herbivores, and possibly
tyrosinase-catalysed DOPA formation may have the same
role in lichens. Certainly, members of the Peltigerineae do
not contain the normal secondary metabolites possessed by
lichens outside of this sub-order (Huneck and Yoshimura,
1996). However, the widespread occurrence of tyrosinases
in lichens in the Peltigerineae suggests that they play an
important role in the biology of these lichens.
ACKNOWLEDGEMENTS
The University of KwaZulu-Natal and the Russian
Foundation for Basic Research (06-04-48143) are thanked
for financial support. We particularly thank Anna Zavarzina and Alexei Zavarzin for extremely valuable discussions
and for making available some of their unpublished data.
The following kindly collected lichens for us: Yasmina
Minibayeva, Kazan region, Russia; and Richard D Beckett,
Estonia. Clive Dennison is also thanked for his help with
electrophoresis.
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Farida Minibayeva