Baldrian 2006

Fungal laccases ^ occurrence and properties
Petr Baldrian
Laboratory of Biochemistry of Wood-Rotting Fungi, Institute of Microbiology ASCR, Prague, Czech Republic
Correspondence: Petr Baldrian, Laboratory Abstract

of Biochemistry of Wood-Rotting Fungi,
Institute of Microbiology ASCR, Vdenska
Laccases of fungi attract considerable attention due to their possible involvement
1083, 14220 Prague 4, Czech Republic. in the transformation of a wide variety of phenolic compounds including the
Tel.: 1420 2410 62315; fax: 1420 2410 polymeric lignin and humic substances. So far, more than a 100 enzymes have been
62384; e-mail: baldrian@biomed.cas.cz purified from fungal cultures and characterized in terms of their biochemical and
catalytic properties. Most ligninolytic fungal species produce constitutively at least
Received 27 May 2005; revised 1 September one laccase isoenzyme and laccases are also dominant among ligninolytic enzymes
2005; accepted 1 September 2005. in the soil environment. The fact that they only require molecular oxygen for
First published online 9 November 2005.
catalysis makes them suitable for biotechnological applications for the transforma-
tion or immobilization of xenobiotic compounds.
doi:10.1111/j.1574-4976.2005.00010.x
Editor: Jiri Damborsky
Keywords
biotechnology; ecology; humic substances;
laccase; lignin; soil; wood-rotting fungi.
nol mono-oxygenase tyrosinase (EC 1.14.18.1). Although

laccase was also called diphenol oxidase, monophenols
Introduction like 2,6-dimethoxyphenol or guaiacol are often better sub-
strates than diphenols, e.g. catechol or hydroquinone.
Laccase is one of the very few enzymes that have been Syringaldazine [N,N 0 -bis(3,5-dimethoxy-4-hydroxybenzyli-
studied since the end of 19th century. It was first demon- dene hydrazine)] is often considered to be a unique laccase
strated in the exudates of Rhus vernicifera, the Japanese substrate (Harkin et al., 1974) as long as hydrogen peroxide
lacquer tree (Yoshida, 1883). A few years later it was also is avoided in the reaction, as this compound is also oxidized
demonstrated in fungi (Bertrand, 1896). Although known by peroxidases. Laccase is thus an oxidase that oxidizes
for a long time, laccases attracted considerable attention polyphenols, methoxy-substituted phenols, aromatic dia-
only after the beginning of studies of enzymatic degradation mines and a range of other compounds but does not oxidize
of wood by white-rot wood-rotting fungi. tyrosine as tyrosinases do.
Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) Laccases are typically found in plants and fungi. Plant
belongs to a group of polyphenol oxidases containing copper laccases participate in the radical-based mechanisms of
atoms in the catalytic centre and usually called multicopper lignin polymer formation (Sterjiades et al., 1992; Liu et al.,
oxidases. Other members of this group are the mammalian 1994; Boudet, 2000; Ranocha et al., 2002; Hoopes & Dean,
plasma protein ceruloplasmin and ascorbate oxidases of 2004), whereas in fungi laccases probably have more roles
plants. Laccases typically contain three types of copper, one including morphogenesis, fungal plant-pathogen/host inter-
of which gives it its characteristic blue colour. Similar action, stress defence and lignin degradation (Thurston,
enzymes lacking the Cu atom responsible for the blue colour 1994). Although there are also some reports about laccase
are called yellow or white laccases, but several authors do activity in bacteria (Alexandre & Zhulin, 2000; Martins
not regard them as true laccases. Laccases catalyze the et al., 2002; Claus, 2003; Givaudan et al., 2004), it does not
reduction of oxygen to water accompanied by the oxidation seem probable that laccases are common enzymes from
of a substrate, typically a p-dihydroxy phenol or another certain prokaryotic groups. Bacterial laccase-like proteins
phenolic compound. It is difficult to define laccase by its are intracellular or periplasmic proteins (Claus, 2003).
reducing substrate due to its very broad substrate range, Probably the best characterized bacterial laccase is that
which varies from one laccase to another and overlaps isolated from Sinorhizobium meliloti, which has been de-
with the substrate range of another enzymethe monophe- scribed as a 45-kDa periplasmic protein with isoelectric
FEMS Microbiol Rev 30 (2006) 215242

c 2005 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
216 P. Baldrian
point at pH 6.2 and the ability to oxidize syringaldazine cetes such as Gaeumannomyces graminis (Edens et al., 1999),
(Rosconi et al., 2005). Magnaporthe grisea (Iyer & Chattoo, 2003) and Ophiostoma
The chemistry, function and biotechnological use of novo-ulmi (Binz & Canevascini, 1997), as well as from
laccases have recently been reviewed. The basic aspects of Mauginella (Palonen et al., 2003), Melanocarpus albomyces
laccase structure and function were reviewed by (Thurston, (Kiiskinen et al., 2002), Monocillium indicum (Thakker
1994), (Leonowicz et al., 2001) focused on the functional et al., 1992), Neurospora crassa (Froehner & Eriksson, 1974)
properties of fungal laccases and their involvement in lignin and Podospora anserina (Molitoris & Esser, 1970).
transformation and (Mayer & Staples, 2002) dealt with the It is difficult to say how many ascomycete species produce
latest results about the roles of laccases in vivo and its laccases as no systematic search has been undertaken. In
biotechnological applications. The physico-chemical prop- addition to plant pathogenic species, laccase production was
erties of multicopper oxidases have been comprehensively also reported for some soil ascomycete species from the
reviewed by (Solomon et al., 1996, 2001). An overview of genera Aspergillus, Curvularia and Penicillium (Banerjee &
technological applications of oxidases including laccase was Vohra, 1991; Rodriguez et al., 1996; Scherer & Fischer,
published by (Duran & Esposito, 2000) and (Duran et al., 1998), as well as some freshwater ascomycetes (Abdel-
2002) reviewed the literature concerning the use of immo- Raheem & Shearer, 2002; Junghanns et al., 2005). However,
bilized laccases and tyrosinases. the enzyme from Aspergillus nidulans was unable to oxidize
The main aim of this work is to summarize the rich syringaldazine (Scherer & Fischer, 1998) and the enzymes
literature data that has accumulated in the last years from from Penicillium spp. were not tested with this substrate,
the studies of authors purifying the enzyme from different leaving it unclear if they are true laccases.
fungal sources. In addition to a generally low substrate Wood-degrading ascomycetes like the soft-rotter Tricho-
specificity, laccase has other properties that make this derma and the ligninolytic Bothryosphaeria are ecologically
enzyme potentially useful for biotechnological application. closely related to the wood-rotting basidiomycetes produ-
These include the fact that laccase, unlike peroxidases, does cing laccase. Laccase activity has been described in both
not need the addition or synthesis of a low molecular weight genera, but whereas Bothryosphaeria produces constitutively
cofactor like hydrogen peroxide, as its cosubstrate oxygen a dimethoxyphenol-oxidizing enzyme that is probably a true
is usually present in its environment. Most laccases are laccase (Vasconcelos et al., 2000), only some strains of
extracellular enzymes, making the purification procedures Trichoderma exhibit a low level of production of a syringal-
very easy and laccases generally exhibit a considerable level dazine-oxidizing enzyme (Assavanig et al., 1992), mainly
of stability in the extracellular environment. The inducible associated with spores, which may act in the morphogenesis
expression of the enzyme in most fungal species also of this fungus (Assavanig et al., 1992; Holker et al., 2002).
contributes to the easy applicability in biotechnological Although no enzyme purification has been reported so far,
processes. This review should help to define the common laccases are probably also produced by wood-rotting xylar-
general characteristics of fungal laccases as well as the iaceous ascomycetes. Among the 20 strains tested, genes
unique properties of individual enzymes with a potential with sequences similar to basidiomycete laccases were de-
biotechnological use and contribute to the discussion on the tected in three strains, all of them Xylaria sp. Two strains of
occurrence and significance of laccase in the natural envir- Xylaria sp. and one of Xylaria hypoxylon exhibited syringal-
onment. dazine oxidation (Pointing S et al., 2005). In complex liquid
media, the fungi X. hypoxylon and Xylaria polymorpha
produced appreciable titres of an ABTS oxidizing enzyme,
Occurrence in fungi also present in the extracts of colonized beech wood chips
Laccase activity has been demonstrated in many fungal (Liers et al., 2005). Furthermore, ascomycete species closely
species and the enzyme has already been purified from tens related to wood-degrading fungi which participate in the
of species. This might lead to the conclusion that laccases are decay of dead plant biomass in salt marshes have been
extracellular enzymes generally present in most fungal shown to contain laccase genes and to oxidize syringaldazine
species. However, this conclusion is misleading as there are (Lyons et al., 2003).
many taxonomic or physiological groups of fungi that Yeasts are a physiologically specific group of both asco-
typically do not produce significant amounts of laccase or mycetes and basidiomycetes. Until now, laccase was only
where laccase is only produced by a few species. Laccase purified from the human pathogen Cryptococcus (Filobasi-
production has never been demonstrated in lower fungi, i.e. diella) neoformans. This basidiomycete yeast produces a
Zygomycetes and Chytridiomycetes; however, this aspect of true laccase capable of oxidation of phenols and amino-
these groups has not as yet been studied in detail. phenols and unable to oxidize tyrosine (Williamson, 1994).
There are many records of laccase production by ascomy- The enzyme is tightly bound to the cell wall and contri-
cetes. Laccase was purified from phytopathogenic ascomy- butes to the resistance to fungicides (Zhu et al., 2001;

c 2005 Federation of European Microbiological Societies FEMS Microbiol Rev 30 (2006) 215242
Fungal laccases occurrence and properties 217
Ikeda et al., 2003). A homologous gene has also been zine has never been detected (Gunther et al., 1998; Timonen
demonstrated in Cryptococcus podzolicus but not in other & Sen, 1998). It seems that tyrosinase is the major pheno-
heterobasidiomycetous yeasts tested (Petter et al., 2001) and loxidase of ECM, whereas syringaldazine oxidation has
there are some records of low laccase-like activity in some scarcely been reported (Burke & Cairney, 2002) and the
yeast species isolated from decayed wood (Jimenez et al., literature data reporting laccase activity in ECM fungi are
1991). The production of laccase was not demonstrated in usually based on the use of nonspecific substrates like ABTS
ascomycetous yeasts, but the plasma membrane-bound or naphtol (Gramss et al., 1998, 1999). The gene sequences
multicopper oxidase Fet3p from Saccharomyces cerevisiae are not found very frequently either (Chen et al., 2003).
shows both sequence and structural homology with fungal Laccases have been purified from a few fungi-forming
laccase. Although more closely related to ceruloplasmin, ectomycorrhiza: Cantharellus cibarius (Ng & Wang, 2004),
Fet3p has spectroscopic properties nearly identical to fungal Lactarius piperatus (Iwasaki et al., 1967), Russula delica
laccase, the configuration of their type-1 Cu sites is very (Matsubara & Iwasaki, 1972) and Thelephora terestris (Ka-
similar and both enzymes are able to oxidize Cu1 (Machon- nunfre & Zancan, 1998) or orchideoid mycorrhiza: Armil-
kin et al., 2001; Stoj & Kosman, 2003). laria mellea (Rehman & Thurston, 1992; Billal & Thurston,
Among physiological groups of fungi, laccases are typical 1996; Curir et al., 1997), as well as from the species of genera
for the wood-rotting basidiomycetes causing white-rot and a that contain both saprotrophic and mycorrhizal fungi
related group of litter-decomposing saprotrophic fungi, i.e. Agaricus, Marasmius, Tricholoma and Volvariella (Table 1).
the species causing lignin degradation. Almost all species of The activity of another ligninolytic enzyme, Mn-peroxidase,
white-rot fungi were reported to produce laccase to varying has thus far been confirmed only in Tylospora fibrillosa, a
degrees (Hatakka, 2001), and the enzyme has been purified species containing also a putative sequence of laccase
from many species (Table 1). In the case of Pycnoporus (Chambers et al., 1999; Chen et al., 2003) and possibly also
cinnabarinus, laccase was described as the only ligninolytic lignin peroxidase (Chen et al., 2001).
enzyme produced by this species that was capable of lignin
degradation (Eggert et al., 1996). Although the group of
brown-rot fungi is typical for its inability to decompose
Cellular localization
lignin, there have been several attempts to detect laccases in Due to the properties of their substrate, the enzymes
the members of this physiological group. A DNA sequence participating in the breakdown of lignin should be exclu-
with a relatively high similarity to that of laccases of white- sively extracellular. While this is without exception true for
rot fungi was detected in Gloeophyllum trabeum. Oxidation the lignin peroxidases and manganese peroxidases of white-
of ABTS (2,2 0 -azinobis(3-ethylbenzathiazoline-6-sulfonic rot fungi, the situation is not the same with laccases.
acid)) as an indirect indication of oxidative activity was also Although most laccases purified so far are extracellular
found in this fungus as well as in a few other brown-rot enzymes, the laccases of wood-rotting fungi are usually also
species (DSouza et al., 1996). Although no laccase protein found intracellularly. Most white-rot fungal species tested by
has been purified from any brown-rot species, the oxidation Blaich & Esser (1975) produced both extracellular and
of syringaldazine a reliable indication of laccase presence intracellular laccases with isoenzymes showing similar pat-
has recently been detected in the brown-rot fungus Con- terns of activity staining after isoelectric focusing. When
iophora puteana (Lee et al., 2004) and oxidation of ABTS was Trametes versicolor was grown on glucose, wheat straw and
reported in Laetiporus sulphureus (Schlosser & Hofer, 2002). beech leaves, it produced laccases both in extracellular and
The occurrence and role of laccases in brown-rot decay of intracellular fractions (Schlosser et al., 1997). The majority
wood is still unclear but it seems to be rare. of enzyme activity was produced extracellularly (98% and
Several attempts have been undertaken to detect lignino- 95% on wheat straw and beech wood, respectively). Traces of
lytic enzymes, including laccases in ectomycorrhizal (ECM) intracellular laccase activity were found in Agaricus bisporus,
fungi (Cairney & Burke, 1998; Burke & Cairney, 2002). Gene but more than 88% of the total activity was in the culture
fragments with a high similarity to laccase from wood- supernatant (Wood, 1980). The intra- and extracellular
rotting fungi have been found in several isolates of ECM presence of laccase activity was also detected in Phanerochaete
species including Amanita, Cortinarius, Hebeloma, Lactar- chrysosporium (Dittmer et al., 1997) and Suillus granulatus
ius, Paxillus, Piloderma, Russula, Tylospora and Xerocomus (Gunther et al., 1998). A fraction of laccase activity in N.
(Luis et al., 2004; Chen et al., 2003). In the case of Piloderma crassa, Rigidoporus lignosus and one of the laccase isoenzymes
byssinum, transcription of putative laccase sequence was of Pleurotus ostreatus is also probably localized intracellularly
confirmed by RT-PCR (Chen et al., 2003). However, a gene or on the cell wall (Froehner & Eriksson, 1974; Nicole et al.,
sequence does not necessarily correspond with the produc- 1992, 1993; Palmieri et al., 2000).
tion of an enzyme. In Paxillus involutus, a species containing The extracellular laccase activity of Lentinula edodes was
another putative laccase sequence, oxidation of syringalda- associated with a multicomponent protein complex of

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218
Table 1. Characteristics of laccases purified from fungi

pH optimum Km (mM)
Temperature
Species MW (kDa) pI ABTS DMP GUA SYR ABTS DMP GUA SYR optimum (1C) Reference
Agaricus bisporus 96 5.6 Wood (1980)
Agaricus bisporus 65 Perry et al. (1993)
Agaricus blazei 66 4.0 2.0 5.5 6.0 63 1026 4307 4 Ullrich et al. (2005)
Agrocybe praecox 66 4.0 Steffen et al. (2002)
Albatrella dispansus 62 4.0 70 Wang & Ng (2004b)
Armillaria mellea Lac I 59 4.1 3.5 178 Rehman & Thurston (1992)
Armillaria mellea Lac II Billal & Thurston (1996)
Armillaria mellea 80 3.1 Curir et al. (1997)
Aspergillus nidulans II 80 6.5 55 Scherer & Fischer (1998)

Botrytis cinerea 74 4.0 3.5 100 57 Slomczynski et al. (1995)
2005 Federation of European Microbiological Societies

Cantharellus cibarius 92 4.0 50 Ng & Wang (2004)
Ceriporiopsis subvermispora L1 71 3.4 3.0 4.0 3.0 30 2900 1600 Fukushima & Kirk (1995); Wang & Ng (2004b)
Ceriporiopsis subvermispora L2 68 4.8 3.0 4.0 5.0 20 7700 440 Fukushima & Kirk, (1995); Wang & Ng (2004b)
Cerrena maxima 5767 3.5 160300 50 Koroleva et al. (2001); Shleev et al. (2004)
Cerrena unicolor 66 4.0 Bekker et al. (1990)
Cerrena unicolor 58 Kim et al. (2002)
Chaetomium termophilum 77 5.1 190 96 400 34 60 Chefetz et al. (1998)
Chalara paradoxa 67 4.5 4.5 6.5 6.5 770 14 720 10 230 3400 Robles et al. (2002)
Colletotrichum graminicola 85 6.0 214 Anderson & Nicholson (1996)
Coniothyrium minitans 74 4.0 3.5 100 60 Dahiya et al. (1998)
Coprinus cinereus 58 4.0 4.0 6.5 26 6070 Schneider et al. (1999)
Coprinus friesii 60 3.5 5.0 8.0 41 Heinzkill et al. (1998)
Coriolopsis fulvocinnerea 5465 3.5 7090 Shleev et al. (2004); Smirnov et al. (2001)
Coriolopsis gallica 84 4.24.3 3.0 70 Calvo et al. (1998)
Coriolopsis rigida I 66 3.9 2.5 3.0 12 328 Saparrat et al. (2002)
Coriolopsis rigida II 66 3.9 2.5 3.0 11 348 Saparrat et al. (2002)
Coriolus hirsutus 55 4.0 Koroljova-Skorobogatko et al. (1998)
Coriolus hirsutus 78 4.2 8 45 Lee & Shin (1999)
Coriolus maxima 57 Smirnov et al. (2001)
Coriolus zonatus 60 4.6 55 Koroljova et al. (1999)
Cryptococcus neoformans 77 Williamson (1994)
Cyathus stercoreus 70 3.5 4.8 Sethuraman et al. (1999)
Daedalea quercina 69 3.0 2.0 4.0 4.5 7.0 38 48 93 131 70, 55 Baldrian (2004)
Dichomitus squalens c1 66 3.5 3.0 Perie et al. (1998)
Dichomitus squalens c2 66 3.6 3.0 Perie et al. (1998)
Fomes fomentarius 52 Rogalski et al. (1991)
Ganoderma lucidum 67 4 25 Lalitha Kumari & Sirsi (1972); Ko et al. (2001)
Ganoderma tsugae Eller et al. (1998)
Gaeumannomyces graminis 190 5.6 4.5 26 510 Edens et al. (1999)
Hericium echinaceum 63 5.0 50 Wang & Ng (2004c)
P. Baldrian

Junghuhnia separabilima 5862 3.43.6 Vares et al. (1992)
Lactarius piperatus 67 Iwasaki et al. (1967)
Lentinula edodes Lcc1 72 3.0 4.0 4.0 4.0 108 557 917 40 Nagai et al. (2002)
Lentinus edodes 65 3.0 Kofujita et al. (1991)
Magnaporthe grisea 70 6.0 118 30 Iyer & Chattoo (2003)
Marasmius quercophilus 60 4.04.4 5.0 80 Farnet et al. (2000)
Marasmius quercophilusw 60 4.85.1 5.0 80 Farnet et al. (2000)
Marasmius quercophilus 65 3.6 4.5 7 75 Dedeyan et al. (2000)
Marasmius quercophilusz 65 2.6 6.2 8 50 80 Farnet et al., (2002, 2004)

Marasmius quercophilus 60 4.0 4.5 113 4.2 80 Farnet et al. (2004)
Mauginiella sp. 63 4.86.4 2.4 3.5 4.0 Palonen et al. (2003)
Melanocarpus albomyces 80 4.0 3.5 5.07.5 6.07.0 65 Kiiskinen et al. (2002)
Fungal laccases occurrence and properties
Monocillium indicum 100 Thakker et al. (1992)

Myrothecium verrucaria 62 Sulistyaningdyah et al. (2004)
Neurospora crassa 64 Froehner & Eriksson (1974)
Ophiostoma novo-ulmi 79 5.1 2.8 6.0 6.0 Binz & Canevascini (1997)
Panaeolus papilionaceus 60 3.0 8.0 51 Heinzkill et al. (1998)
Panaeolus sphinctrinus 60 3.0 7.0 32 Heinzkill et al. (1998)
Panus tigrinus 64 2.93.0 Maltseva et al. (1991)
Panus tigrinus 63 Leontievsky et al. (1997)
Phanerochaete flavido-alba 94 3.0 30 Perez et al. (1996)
Phanerochaete chrysosporium 47 Srinivasan et al. (1995)
Phellinus noxius 70 Geiger et al. (1986)
Phellinus ribis 152 5.0 4.06.0 6.0 207 38 11 Min et al. (2001)
Phlebia radiata 64 3.5 Vares et al. (1995)
Phlebia tremellosa 64 Vares et al. (1994)
Pholiota mutabilis Leonowicz & Malinowska (1982)
Physisporinus rivulosus Lacc 1 66 3.3 2.5 3.0 3.5 3.5 Hakala et al. (2005)
c

Pleurotus eryngii I 65 4.1 4.5 1400 7600 55 Munoz et al. (1997)
Pleurotus eryngii II 61 4.2 4.5 400 8000 55 Munoz et al. (1997)
Pleurotus florida 77 4.1 30 000 Das et al. (2000)
Pleurotus ostreatus 67 3.6 5.8 50 Hublik & Schinner (2000)
Pleurotus ostreatus POXA1b 62 6.9 3.0 4.5 6.0 370 260 220 Giardina et al. (1999)
Pleurotus ostreatus POXA1w 61 6.7 3.0 3.05.0 NA 6.0 90 2100 NA 130 4565 Palmieri et al. (1997)
Pleurotus ostreatus POXA2 67 4.0 3.0 6.5 6.0 6.0 120 740 3100 140 2535 Palmieri et al. (1997)
Pleurotus ostreatus POXA3a 8385 4.1 3.6 5.5 6.2 70 14 000 36 35 Palmieri et al. (2003)
Pleurotus ostreatus POXA3b 8385 4.3 3.6 5.5 6.2 74 8800 79 35 Palmieri et al. (2003)
Pleurotus ostreatus POXC 59 2.9 3.0 3.05.0 6.0 6.0 280 230 1200 20 5060 Palmieri et al. (1993, 1997); Sannia et al. (1986)
Pleurotus pulmonarius Lcc2 46 4.05.5 6.08.0 6.26.5 210 550 12 50 De Souza & Peralta (2003)
Pleurotus sajor-caju IV 55 3.6 2.1 92 Lo et al. (2001)
Podospora anserine 383 Molitoris & Esser (1970); Durrens (1981)
219

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Table 1. Continued.
220
pH optimum Km (mM)
Temperature
Species MW (kDa) pI ABTS DMP GUA SYR ABTS DMP GUA SYR optimum (1C) Reference
Polyporus anceps 5.05.5 Petroski et al. (1980)
Polyporus anisoporus 58 3.4 Vaitkyavichyus et al. (1984)
Polyporus pinsitus 66 3.0 5.0 22 Heinzkill et al. (1998)
Pycnoporus cinnabarinus 63 3.0 4.04.5 4.45.0 330 30 Schliephake et al. (2000)
Pycnoporus cinnabarinus 81 3.7 4.0 Eggert et al. (1996)
Pycnoporus coccineus 70 Oda et al. (1991)
Rhizoctonia solani 4 170 Iwasaki et al. (1967)
Rigidoporus lignosus B 55 3.7 3.0 6.2 80 480 Bonomo et al. (1998)
Rigidoporus lignosus S 60 3.1 3.0 6.2 49 108 Bonomo et al. (1998)
Russula delica 63 Matsubara & Iwasaki (1972)

Schizophyllum commune 6264 De Vries et al. (1986)
Sclerotium rolfsii SRL1 55 5.2 2.4 62 Ryan et al. (2003)
Sclerotium rolfsii SRL2 86 Ryan et al. (2003)
Stropharia coronilla 67 4.4 Steffen et al. (2002)
Stropharia rugosoannulata 66 2.5 3.5 Schlosser & Hofer (2002)
Thelephora terrestris 66 3.4 4.8 5.0 16 121 3 45 Kanunfre & Zancan (1998)
Trametes gallica Lac I 60 3.1 2.2 3.0 4.0 12 420 405 70 Dong & Zhang (2004)
Trametes gallica Lac II 60 3.0 2.2 3.0 4.0 9 410 400 70 Dong & Zhang (2004)
Trametes hirsute 6468 3.74.0 63 Shleev et al. (2004); Vares & Hatakka (1997)
Trametes multicolor II 63 3.0 Leitner et al. (2002)
Trametes ochracea 64 4.7 90 (Shleev et al. (2004)
Trametes pubescens LAP 2 65 2.6 14 72 360 6 Galhaup et al. (2002)
Trametes sanguinea 62 3.5 Nishizawa et al. (1995)
Trametes trogii 70 3.3; 3.6 30 410 Garzillo et al. (1998)
Trametes versicolor 68 2.5 3.5 4.0 37 15 55 Rogalski et al. (1990); Hofer & Schlosser (1999)
Trametes villosa 1 63 3.5 2.7 5.05.5 Yaver et al. (1996)
Trametes villosa 3 63 6.06.5 2.7 5.05.5 Yaver et al. (1996)
Trametes sp. AH28-2 A 62 4.2 4.5 25 25 420 50 Xiao et al. (2003)
Trichoderma sp. 71 Assavanig et al. (1992)
Tricholoma giganteum 43 4.0 70 Wang & Ng (2004a)
Volvariella volvacea 58 3.7 3.0 4.6 5.6 30 570 10 45 Chen et al. (2004)
ABTS, 2,2 0 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid); DMP, 2,6-dimethoxyphenol; GUA, 2-methoxyphenol (guaiacol); SYR. 4-hydroxy-3,5-dimethoxybenzaldehyde [(4-hydroxy-3,5-dimethoxyphe-
nyl)methylene]hydrazone (syringaldazine).
The species are listed under the names used in the original references.
NA, not active.
Strain 17, constitutive form.
w
Strain 17, induced with p-hydroxybenzoic acid.
z
Strain C7, constitutive form.

Strain 19, induced with ferulic acid.
P. Baldrian

660 kDa, which also exhibited peroxidase and b-glucosidase Typical fungal laccase is a protein of approximately
activities (Makkar et al., 2001). Although laccase activity was 6070 kDa with acidic isoelectric point around pH 4.0
not found in the cell wall fractions of the basidiomycete A. (Table 2). It seems that there is considerable heterogeneity
bisporus (Sassoon & Mooibroek, 2001), a substantial part of in the properties of laccases isolated from ascomycetes,
T. versicolor and P. ostreatus laccase is associated with the cell especially with respect to molecular weight.
wall (Valaskova & Baldrian, 2005). Laccase activity is almost Several laccase isoenzymes have been detected in many
exclusively associated with cell walls in the white-rot basi- fungal species. More than one isoenzyme is produced in
diomycete Irpex lacteus (Svobodova, 2005), the yeast most white-rot fungi. (Blaich & Esser, 1975) performed a
C. neoformans (Zhu et al., 2001) and in the spores of screening of laccase activity among wood-rotting fungi
Trichoderma spp. (Holker et al., 2002). The localization of using staining with p-phenylenediamine after isoelectric
laccase is probably connected with its physiological function focusing. All tested species, namely Coprinus plicatilis, Fomes
and determines the range of substrates available to the fomentarius, Heterobasidion annosum, Hypholoma fascicu-
enzyme. It is possible that the intracellular laccases of fungi lare, Kuehneromyces mutabilis, Leptoporus litschaueri, Panus
as well as periplasmic bacterial laccases could participate in stipticus, Phellinus igniarius, Pleurotus corticatus, P. ostreatus,
the transformation of low molecular weight phenolic com- Polyporus brumalis, Stereum hirsutum, Trametes gibbosa,
pounds in the cell. The cell wall and spores-associated T. hirsuta and T. versicolor, exhibited the production of
laccases were linked to the possible formation of melanin more than one isoenzyme, typically with pI in the range of
and other protective cell wall compounds (Eggert et al., pH 35.
1995; Galhaup & Haltrich, 2001). Several species produce a wide variety of isoenzymes. The
white-rot fungus P. ostreatus produces at least eight different
laccase isoenzymes, six of which have been isolated and
Structural properties characterized (Sannia et al., 1986; Palmieri et al., 1993, 1997,
Current knowledge about the structure and physico-chemi- 2003; Giardina et al., 1999). The main protein present in the
cal properties of fungal laccase proteins is based on the study cultures is the 59-kDa POXC with pI 2.7. The POXA2,
of purified proteins. Up to now, more than 100 laccases have POXB1 and POXB2 isoenzymes exhibit a similar molecular
been purified from fungi and been more or less character- weight around 67 kDa, while POXA1b and POXA1w are
ized (Table 1). Based on the published data we can draw smaller (61 kDa). The enzymes POXA3a and POXA3b are
some general conclusions about laccases, taking into ac- heterodimers consisting of large (61-kDa) and small (16- or
count that most enzymes were purified from wood-rotting 18-kDa) subunits. Although the POXC protein is the most
white-rot basidiomycetes; other groups of fungi-producing abundant in cultures both extra- and intracellularly, the
laccases (other groups of basidiomycetes, ascomycetes and highest mRNA production was detected in POXA1b, which
imperfect fungi) have been studied to a much lesser extent. is probably mainly intracellular or cell wall-associated as it is
Table 2. Properties of fungal laccases (data derived from Table 1)

Property n Median Q25 Q75 Min Max
Molecular weight (Da) 103 66 000 61 000 71 000 43 000 383 000
pI 67 3.9 3.5 4.2 2.6 6.9
Temperature optimum ( 1C) 39 55 50 70 25 80
pH optimum 49 3.0 2.5 4.0 2.0 5.0
ABTS
2,6-Dimethoxyphenol 36 4.0 3.0 5.5 3.0 8.0
Guaiacol 24 4.5 4.0 6.0 3.0 7.0
Syringaldazine 31 6.0 4.7 6.0 3.5 7.0
KM (mM)
ABTS 36 39 18 100 4 770
2,6-Dimethoxyphenol 30 405 100 880 26 14 720
Guaiacol 23 420 121 1600 4 30 000
Syringaldazine 21 36 11 131 3 4307
kcat (s 1)
ABTS 12 24 050 5220 41 460 198 350 000
2,6-Dimethoxyphenol 12 3680 815 6000 100 360 000
Guaiacol 10 295 115 3960 90 10 800
Syringaldazine 4 21 500 18 400 25 500 16 800 28 000
n, number of observations; Q25, lower quartile; Q75, upper quartile.

222 P. Baldrian
cleaved by an extracellular protease (Palmieri et al., 1997; cleaved. SDSPAGE and MALDI-MS analyses of purified
Giardina et al., 1999). The production of laccase isoenzymes POXA3a and POXA3b laccases from P. ostreatus reveal the
in P. ostreatus is regulated by the presence of copper and the presence of three different polypeptides of 67, 18 and
two dimeric isoenzymes have only been detected in the 16 kDa, whereas the native proteins behave homogeneously,
presence of copper (Palmieri et al., 2000, 2003). Isoenzymes as demonstrated by the presence of a single peak or band in
of laccase with different molecular weight and pI were also gel filtration chromatography, isoelectric focusing and na-
detected in the litter-decomposing fungus Marasmius quer- tive-PAGE analysis. All the other laccase isoenzymes isolated
cophilus (Farnet et al., 2000, 2002, 2004; Dedeyan et al., from P. ostreatus were characterized as monomeric proteins
2000). A study with 17 different isolates of this fungus (Palmieri et al., 2003).
showed that the isoenzyme pattern was consistent within Like most fungal extracellular enzymes, laccases are
different isolates. Moreover, all isolates showed the same glycoproteins. The extent of glycosylation usually ranges
isoenzyme pattern (one to three laccase bands on SDS- between 10% and 25%, but laccases with a saccharide
PAGE) after the induction of laccase with different aromatic content higher than 30% were found: e.g. Coriolopsis
compounds (Farnet et al., 1999). fulvocinnerea 32% (Shleev et al., 2004) and P. pulmonarius
Some fungal species, e.g. Coriolopsis rigida, Dichomitus 44% (De Souza & Peralta, 2003). Even higher saccharide
squalens, Physisporinus rivulosus and Trametes gallica, pro- contents were found in Botrytis cinnerea, the monomeric
duce isoenzymes that are closely related both structurally enzyme of the strain 6134 containing 49% sugars (Slomc-
and in their catalytic properties (Table 1). Different proper- zynski et al., 1995). Other preparations from the same
ties of laccases purified from the same species and reported species exhibited as much as 6580% of saccharides includ-
by different authors can be explained as a result of both the ing arabinose, xylose, mannose, galactose and glucose (Gigi
production of different isoenzymes and different laccase et al., 1981; Marbach et al., 1984; Zouari et al., 1987). On the
properties in different strains of the same fungus (Table 1). other hand, very low extent of glycosylation was detected in
In P. chrysosporium, production of different laccase isoen- Pleurotus eryngii, where laccase I contained 7% and laccase
zymes was detected in cell extract and in the culture medium II only 1% of bound sugars (Munoz et al., 1997). The
(Dittmer et al., 1997); however, since laccase gene was not glycans are N-linked to the polypeptide chain (Ko et al.,
found in the complete genome sequence of this fungus 2001; Brown et al., 2002; Saparrat et al., 2002). The most
(Martinez et al., 2004), these are probably multicopper detailed structure of laccase glycan is available for R. lignosus
oxidases rather than true laccases (Larrondo et al., 2003). laccase, which is also glycosylated with N-bound mannose
The molecular basis for the production of different iso- (Garavaglia et al., 2004). The glycosylation of fungal laccases
enzymes is the presence of multiple laccase genes in fungi is one of the biggest problems for the heterologous produc-
(see e.g. Chen et al., 2003). tion of the enzyme, which is extremely difficult to overcome.
Most fungal laccases are monomeric proteins. Several It was proposed that in addition to the structural role,
laccases, however, exhibit a homodimeric structure, the glycosylation can also participate in the protection of laccase
enzyme being composed of two identical subunits with a from proteolytic degradation (Yoshitake et al., 1993).
molecular weight typical for monomeric laccases. This is the Laccases belong to the group of blue multicopper oxi-
case of the wood-rotting species Phellinus ribis (Min et al., dases (BMCO) that catalyze a one-electron oxidation con-
2001), Pleurotus pulmonarius (De Souza & Peralta, 2003) comitantly with the four-electron reduction of molecular
and Trametes villosa (Yaver et al., 1996), the mycorrhizal oxygen to water (Solomon et al., 1996, 2001; Messerschmidt,
fungus C. cibarius (Ng & Wang, 2004) and the ascomycete 1997). The catalysis carried out by all members of this family
Rhizoctonia solani (Wahleithner et al., 1996). The ascomy- is guaranteed by the presence of different copper centres in
cetes G. graminis, M. indicum and P. anserina also produce the enzyme molecule. In particular, all BMCO are character-
oligomeric laccases. In M. indicum a single band of 100 kDa ized by the presence of at least one type-1 (T1) copper,
after gel filtration resolved into three proteins (24, 56 and together with at least three additional copper ions: one type-
72 kDa) on SDS-PAGE (Thakker et al., 1992): G. graminis 2 (T2) and two type-3 (T3) copper ions, arranged in a
produces a trimer of three 60-kDa subunits (Edens et al., trinuclear cluster. The different copper centres can be
1999); P. anserina laccase is a heterooligomer (Molitoris & identified on the basis of their spectroscopic properties.
Esser, 1970); and one of the laccases purified from A. mellea The T1 copper is characterized by a strong absorption
has a heterodimeric structure (Curir et al., 1997). According around 600 nm, whereas the T2 copper exhibits only weak
to Wood (Wood, 1980), A. bisporus laccase consists of absorption in the visible region. The T2 site is electron
several polypeptides of 2356 kDa. (Perry et al., 1993), on paramagnetic resonance (EPR)-active, whereas the two
the basis of Western blot analyses, suggested that the native copper ions of the T3 site are EPR-silent due to an
Lac2 of the same species is produced as a dimer of identical antiferromagnetic coupling mediated by a bridging ligand.
polypeptides, one of which is then partially proteolytically The substrates are oxidized by the T1 copper and the

belong to the blue copper proteins because it lacks Cu1 and

contains one Mn atom per molecule. The structural differ-
ences are probably also responsible for the relatively high pH
optimum for ABTS oxidation (Min et al., 2001). The white
laccase POXA1 from P. ostreatus contains only one copper
atom, together with two zinc and one iron atoms per
molecule (Palmieri et al., 1997). Future structural studies
will probably show that laccases are a more structurally
heterogeneous group of proteins than expected.
Catalytic properties
Laccase catalyses the reduction of O2 to H2O using a range
of phenolic compounds (though not tyrosine) as hydrogen
donors (Thurston, 1994; Solomon et al., 1996). Unfortu-
nately, laccase shares a number of hydrogen donors with
tyrosinase, making it difficult to assign unique descriptions
to either enzyme. A further complication is the overlap
Fig. 1. Catalytic cycle of laccase. in activity between monophenol monooxygenase and cate-
extracted electrons are transferred, probably through a chol oxidase (1,2-benzenediol: oxygen oxidoreductase, EC
strongly conserved His-Cys-His tripeptide motif, to the T2/ 1.10.3.1). The broad range of substrates accepted by laccase
T3 site, where molecular oxygen is reduced to water as hydrogen donors notwithstanding, oxidation of syringal-
(Messerschmidt, 1997) (Fig. 1). Some enzymes lack the T1 dazine in combination with the inability to oxidize tyrosine,
copper and some authors hesitate to call them true laccases. has been taken to be an indicator of laccase activity (Harkin
Others use the term yellow laccases because these enzymes et al., 1974; Thurston, 1994). Unambiguous determination
lack the characteristic absorption band around 600 nm of laccase activity is best achieved by purification of the
(Leontievsky et al., 1997, 1997). protein to electrophoretic homogeneity followed by deter-
Until recently, the three-dimensional structure of five mination of KM or kcat with multiple substrates. Ideally,
fungal laccases has been reported: Coprinus cinereus (in a these should include substrates such as syringaldazine, ABTS
copper type-2-depleted form) (Ducros et al., 1998), T. or catechol, for which laccase has a high affinity, and some
versicolor (Bertrand et al., 2002; Piontek et al., 2002), P. (e.g. tyrosine) for which laccase has little or no affinity
cinnabarinus (Antorini et al., 2002), M. albomyces (Hakuli- (Edens et al., 1999; Shin & Lee, 2000). In common with
nen et al., 2002) and R. lignosus (Garavaglia et al., 2004), the catechol oxidase and tyrosinase, laccase catalyzes the four-
latter four enzymes with a full complement of copper ions. electron reduction of O2 to H2O. In the case of laccase, at
Moreover, the three-dimensional structure of the CoA lac- least, this is coupled to the single-electron oxidation of the
case from Bacillus subtilis endospore has also recently been hydrogen-donating substrate (Reinhammar & Malmstrom,
published (Enguita et al., 2003, 2004). Despite the amount 1981). Since four single-electron substrate oxidation steps
of information on laccases as well as other BMCO, neither are required for the four-electron reduction of water, the
the precise electron transfer pathway nor the details of analogy of a four-electron biofuel cell has been proposed to
dioxygen reduction in BMCO are fully understood (Gar- explain this complex mechanism (Thurston, 1994; Call &
avaglia et al., 2004). A detailed structural comparison Mucke, 1997; Barriere et al., 2004). Laccases are known to be
between a low redox potential (E0) C. cinereus laccase and a highly oxidizing. E0 ranges from 450480 mV in Myce-
high E0 T. versicolor laccase showed that structural differ- liophthora thermophila to 760790 mV in Polyporus pinsitus
ences of the Cu1 coordination possibly account for the (Solomon et al., 1996; Xu, 1996; Xu et al., 2000) and the
different E0 values (Piontek et al., 2002). This was later presence of four cupric ions, each co-ordinated to a single
confirmed by the study of R. lignosus laccase with a high polypeptide chain, is an absolute requirement for optimal
redox potential (Garavaglia et al., 2004). However, more activity (Ducros et al., 1998). There have been few measure-
effort will be needed to elucidate the relation between the ments of the redox potentials of tyrosinase or catechol
structure of the catalytic site and the substrate preference of oxidase; however, (Ghosh & Mukherjee, 1998) estimated
different laccase enzymes. the E0 of a tyrosinase model system to be 260 mV, consider-
Unlike the laccases described above, the enzyme from P. ably lower than that reported for laccase, suggesting that this
ribis with catalytic features typical for laccases does not class of enzyme is much less oxidizing than laccase.

c2005 Federation of European Microbiological Societies
224 P. Baldrian
Due to the difficulties with distinguishing laccases from Esser, 1975). Similar results were also obtained with
other oxidases, the data in this review are based exclusively T. versicolor and the ascomycetes P. anserina and Pyricularia
on the reports concerning purified enzymes. However, the oryzae, whereas laccase from Ganoderma lucidum catalyzed
direct comparison of biochemical data reported for different the oxidation of only ortho and para dihydroxyphenyl
fungal laccases that would be extremely important for the compounds, p-phenylenediamine and polyphenols, not the
biotechnological applicability is difficult, as different test meta hydroxymethyl compounds or ascorbic acid (Fahraeus,
conditions have been used in different reports. There are only 1961; Fahraeus & Ljunggren, 1961; Schanel & Esser, 1971;
a few works comparing laccase properties of enzymes from Lalitha Kumari & Sirsi, 1972). More than 70% oxidation of
different sources, e.g. the work of (Shleev et al., 2004) focusing o-substituted compounds was obtained with laccase from
on physico-chemical and spectral characteristics of four M. indicum, whereas p-compounds and the m-phenol
different laccases. However, this comparison is rather limited. phloroglucinol were oxidized at a relatively low rate (Thak-
A very wide range of substrates has been shown to be ker et al., 1992). The relative oxidation rates for different
oxidized by fungal laccases (Table 3) but the catalytic substrates in relation to the oxidation of 2,6-dimethoxyphe-
constants have been reported mostly for a small group of nol are summarized in Table 4. The data demonstrate the
substrates e.g. the non-natural test substrate ABTS and the high activity with ABTS (with the exception of Myrothecium
phenolic compounds 2,6-dimethoxyphenol (DMP), guaia- verrucaria) and a generally high variation with other sub-
col and syringaldazine. KM ranges from 10 s of mM for strates.
syringaldazine and ABTS to 100 s of mM for DMP and In addition to the oxidation of phenols, laccases have also
guaiacol. The catalytic performance expressed as kcat spans been recently demonstrated to catalyze the oxidation of
several orders of magnitude for different substrates and is Mn21 in the presence of chelators. Laccase from the white-
usually characteristic for a specific protein (Table 3). Lac- rot fungus T. versicolor oxidized Mn21 to Mn31 in the
cases in general combine high affinity for ABTS and presence of pyrophosphate (Hofer & Schlosser, 1999). The
syringaldazine with high catalytic constant, whereas the same was also later demonstrated for the enzyme of
oxidation of guaiacol and DMP is considerably slower and the litter-decomposer Stropharia rugosoannulata with oxalic
the respective KM constants higher. Low KM values are typical and malonic acids as chelators (Schlosser & Hofer, 2002).
for sinapic acid, hydroquinone and syringic acid, whereas The chelators probably decrease the high redox potential of
relatively high values were found for para-substituted phe- the Mn21/Mn31 couple. Mn21 oxidation involved conco-
nols, vanillic acid or its aldehyde. For the species capable of mitant reduction of laccase type-1 copper, thus providing
oxidizing polycyclic aromatic hydrocarbons or pentachlor- evidence that it occurs via one-electron transfer to type-1
ophenol, only very low catalytic constants were detected for copper as usual for substrate oxidation by blue laccases
these xenobiotic compounds; the KM value is also high for (Schlosser & Hofer, 2002). A P. ribis laccase devoid of type-1
pentachlorophenol with T. versicolor laccase (Table 3). copper was unable to catalyze the same reaction (Min et al.,
Some fungi produce isoenzymes with similar KM and kcat 2001). It was proposed that laccase and Mn-peroxidase can
values. In wood-rotting basidiomycetes that are usually co-operate. In the presence of Mn21 and oxalate, laccase
dikaryotic this fact probably indicates that allelic variability produces Mn31-oxalate. The latter initializes a set of follow-
is responsible for the production of isoenzymes rather than up reactions leading to H2O2 formation, which may initiate
the evolution of enzymes adapted to the special needs of the or support peroxidase reactions (Schlosser & Hofer, 2002).
fungus. In the case of P. ostreatus, however, the isoenzymes The production of H2O2 and Mn31 was also described in
show the KM and kcat values for 2,6-dimethoxyphenol or P. eryngii for the oxidation of hydroquinone (Munoz et al.,
guaiacol differing by several orders of magnitude and the 1997).
POXA1 isoenzyme is not active with guaiacol at all (Table 3). Fungal laccases typically exhibit pH optima in the acidic
Even very early reports showed that different laccase pH range. While the pH optima for the oxidation of ABTS
enzymes differ considerably in their catalytic preferences. are generally lower than 4.0, phenolic compounds like DMP,
Laccases can be grouped according to their preference for guaiacol and syringaldazine exhibit higher values of between
ortho-, meta- or para- substituted phenols. Ortho-substi- 4.0 and 7.0 (Table 2). pH optima of different fungal enzymes
tuted compounds (guaiacol, o-phenylenediamine, caffeic for hydroquinone and catechol are 3.64.0 and 3.56.2,
acid, catechol, dihydroxyphenylalanine, protocatechuic respectively (Lalitha Kumari & Sirsi, 1972; Shleev et al.,
acid, gallic acid and pyrogallol) were better substrates 2004). It was proposed that the bell-shaped pH profile of
than para-substituted compounds (p-phenylenediamine, phenolic compounds is formed by two opposing effects. The
p-cresol, hydroquinone) and the lowest rates were obtained oxidation of phenols depends on the redox potential differ-
with meta-substituted compounds (m-phenylenediamine, ence between the phenolic compound and the T1 copper
orcinol, resorcinol and phloroglucinol) with crude laccase (Xu, 1996). The E0 of a phenol decreases when pH increases
preparations from L. litschaueri and P. brumalis (Blaich & due to the oxidative proton release. At a rate of DE/

Table 3. Substrates and inhibitors of fungal laccases. The numbers in brackets indicate Michaelis constant (KM, mM) or rate constant (kcat, s 1), multiple
values for the same species refer to different isoenzymes. Only compounds that undergo transformation without the presence of redox mediators are
listed as substrates
Species
Substrate
(3,4-Dimethoxyphenyl)methanol (veratryl alcohol) Ts, Tv
(4-Hydroxy-3-methoxyphenyl)acetic acid Pe
1,2,4,5-Tetramethoxybenzene Cs (KM: 6900; kcat: 1680), Cs (KM: 900; kcat: 3360)
1,2,4-Benzenetriol Bc
1,2-Benzenediol (catechol) Ab, Am, Bc, Cf (KM: 85; kcat: 90), Ch, Cm (KM: 120; kcat: 320), Cn, Cr, Ds, Gg (KM: 250), Gl (KM:
55), Le (KM: 220), Le (KM: 22 400), Lp, Mi, Mq, Pc, Pe (KM: 2200), Pe (KM: 4100), Pr, Rl, Sr, Th
(KM: 142; kcat: 390), To (KM: 110; kcat: 80), Tp (KM: 470; kcat: 27 600), Ts, Tt
1,3-Dihydroxybenzene (resorcinol) Cn, Ts
1,4-Benzohydroquinone Am, Bc, Cf (KM: 68; kcat: 110), Ch, Cn, Cm (KM: 100; kcat: 290), Cr, Ct (KM: 36), Ds, Gl (KM: 29),
Lp, Le (KM: 110), Pc, Pe (KM: 2500), Pe (KM: 4600), Pi, Pn, Rl, Th (KM: 61; kcat: 450), To (KM: 74;
kcat: 110), Tp (KM: 390; kcat: 19 200), Ts, Tt
1-Naphthol Ab, Bc, Gl
2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-4H- Am
chromen-4-one
2-Chlorophenol Tv
2,2 0 -Azinobis(3-ethylbenzothiazoline-6-sulfonic Al (kcat 21), Cr (kcat: 4680), Cr (kcat: 4620), Cs (kcat: 5760), Cs (kcat: 6060), Po (kcat: 16 000),
acid) Po (kcat: 350 000), Po (kcat: 90 000), Rl (kcat: 34 700), Rl (kcat: 32 100), Tp (kcat: 41 400),
Tr (kcat: 41 520), Tt (kcat: 198)
2,3-Dichlorophenol Tv
2,3-Dimethoxyphenol Ds
2,3,6-Trichlorophenol Tv
2,4,6-Trichlorophenol Tv
2,4,6-Trimethylphenol Pe
2,4-Dichlorophenol Tt, Tv
2,5-Dihydroxybenzoic acid Pi
2,6-Dichlorophenol Tt, Tv
2,6-Dimethoxy-1,4-benzohydroquinone Cr (KM: 107; kcat: 8580), Cr (KM: 89; kcat: 11 220)
2,6-Dimethoxyphenol Al (kcat: 15), Cr (kcat: 6360), Cr (kcat: 5640), Cs (kcat: 1380), Cs (kcat: 4560), Po (kcat: 100),
Po (kcat: 250), Po (kcat: 360 000), Rl (kcat: 2800), Rl (kcat: 2000), Tp (kcat: 24 000), Tr (kcat: 4860),
Tt (kcat: 109)
2,7-Diaminofluorene Rl
2-Amino-3-(3,4-dihydroxyphenyl)propanoic acid Am, Cn, Ct (KM: 100), Le (KM: 650), Lp, Ma, Pa (KM: 3300), Ts, Tv (KM: 15 600)
2-Amino-3-hydroxybenzoic acid Pc
2-Amino-4-methylphenol Tt
2-Amino-4-nitrophenol Tt
2-Aminophenol Tt
2-Aminophenylamine Cn
2-Chlorobenzene-1,4-diol Tt
2-Chlorophenol Le (KM: 1350), Mq, Tt
2-Methoxy-1,4-benzohydroquinone Cr (KM: 216; kcat: 7620), Cr (KM: 229; kcat: 6300), Pe
2-Methoxy-4-[prop-1-enyl]phenol Ts
2-Methoxy-4-methylphenol Tt
2-Methoxyaniline Tt
2-Methoxyphenol (guaiacol) Al (kcat: 159), Cf (kcat: 95), Cm (kcat: 160), Cs (kcat: 3120), Cs (kcat: 3960), Po (kcat: 150),
Th (kcat: 430), To (kcat: 90), Tp (kcat: 10 800), Tr (kcat: 4140), Tt (kcat: 115)
2-Methoxy-1,4-benzohydroquinone Pe
2-Methyl-1,4-benzohydroquinone Pe (KM: 1600), Pe (KM: 2100)
2-Methylanthracene Cg (kcat: 0.082)
2-Methylphenol Bc, Tt
2-Naphthol Bc, Gl
2,4-Dichlorophenol Mq
2,4,6-Trichlorophenol
3-(3,4-Dihydroxyphenyl)acrylic acid (caffeic acid) Am, Bc, Cs, Le (KM: 40), Mi, Mq, Ts, Tt

226 P. Baldrian
Table 3. Continued.
Species
3-(4-Hydroxy-3,5-dimethoxyphenyl)acrylic acid Cf (KM: 21, kcat: 140), Cm (KM: 24; kcat: 330), Cs, Ff, Le (KM: 110), Pn, Pr, Rl, Th
(KM: 24; kcat: 580), To (KM: 11; kcat: 170), Tv
3-(4-Hydroxy-3-methoxyphenyl)acrylic acid (ferulic Am, Cf (KM: 20), Ch, Cs, Ct (KM: 270), Ff, Le (KM: 240), Le (KM: 2860), Mi, Mq, Pc, Pn, Pr, Rl, Sr,
acid) Tt (KM: 40; kcat: 145), Tv
3-(4-Hydroxyphenyl)acrylic acid Le (KM: 240), Pn, Rl, Tt
3,3 0 -Dimethoxy-1,1 0 -biphenyl-4,4 0 -diamine Mi (KM: 25), Pr, Tc
3,4,5-Trihydroxybenzoic acid (gallic acid) Ab, Am, Bc, Ct (KM: 130), Le (KM: 130), Mq, Nc, On
3,4-Dihydroxybenzoic acid Cr, Mi, Mq, Pe, Tt
3,5-Cyclohexadiene-1,2-diol Pe
3,5-Dimethoxy-hydroxy-benzaldazine Bc
3-{[3-(3,4-Dihydroxyphenyl)prop-2-enoyl]oxy}- Am, Ct
1,4,5-trihydroxycyclohexanecarboxylic acid
3-Amino-4-hydroxybenzenesulphonic acid Tt
3-Methoxyphenol Pr
4-(Hydroxymethyl)-2-methoxyphenol Cs (KM: 1600; kcat: 2820), Cs (KM: 610; kcat: 2220), Pe
4-[3-Hydroxyprop-1-enyl]-2,6-dimethoxyphenol Mq
4-[3-Hydroxyprop-1-enyl]-2-methoxyphenol Pe, Rl
(coniferyl alcohol)
4-[3-Hydroxyprop-1-enyl]-phenol Mq
4-Amino-2,6-dichlorophenol Bc, Tt
4-Aminophenol Pe (KM: 1000), Pe (KM: 800), Tt
4-Aminophenylamine Am (KM: 1690), Bc, Cn, Gl, Lp, Le (KM: 256), Pe, Tc, Ts
4-Hydroxy-3,5-dimethoxybenzaldehyde Cr
4-Hydroxy-3,5-dimethoxybenzaldehyde [(4-hydroxy-
3,5-dimethoxyphenyl)methylene]
hydrazone (syringaldazine) Al (kcat: 5), Po (kcat: 23 000), Po (kcat: 28 000), Po (kcat: 20 000), Tp (kcat: 16 800)
4-Hydroxy-3,5-dimethoxybenzoic acid (syringic acid) Cr, Cs (KM: 100; kcat: 4680), Cs (KM: 130; kcat: 1860), Ds, Ff (KM: 30), Mi, Pe, Pr, Tv (KM: 60)
4-Hydroxy-3-methoxybenzaldehyde Cr, Cs (KM: 6300; kcat: 1560), Cs (KM: 9000; kcat: 600), Pe
4-Hydroxy-3-methoxybenzoic acid (vanillic acid) Cf (KM: 160), Cs (KM: 1000; kcat: 3960), Cs (KM: 1100; kcat: 2220), Ct (KM: 150), Ff (KM: 970),
Mi, Pe, Pr, Tt, Tv (KM: 130)
4-Hydroxybenzoic acid Mi
4-Hydroxyindole Ch, Pc
4-Chlorophenol Le (KM: 1740), Tt
4-Methoxyaniline Cr, Mi, Pe (KM: 3100), Pe (KM: 3300), Tp (KM: 1600; kcat: 7800), Tt
4-Methoxyphenol Cr, Le (KM: 330), Pe (KM: 800), Pe (KM: 900), Pr, Tt
4-Methylbenzene-1,2-diol Am, Bc, Ch, Cy, Le (KM: 170), Mq, Pc, Rl
4-Methylphenol Bc, Le (KM: 2200), Tt
4-Nitrobenzene-1,2-diol Tt
5-(1,2-Dihydroxyethyl)-3,4-dihydroxyfuran-2-one Ab, Am, Bc, Lp, Mi, Nc, Pa (KM: 190)
(ascorbic acid)
9-Methylanthracene Cg (kcat: 4)
Acenaphthene Cg (kcat: 0.167)
Anthracene Cg (kcat: 0.087), Po, Tv
Benzcatechin Pa (KM: 2270)
Benzene-1,2,3-triol (pyrogallol) Ab, Bc, Ch, Cy, Gg (KM: 310), Gl, Le (KM: 30), Le (KM: 417), Lp, Nc, Pc, Rl, Sr, Ts
Benzene-1,3,5-triol (phloroglucinol) Mi
Benzo[a]pyrene Cg (kcat: 1.38)
Biphenylene Cg (kcat: 0.063)
Fluoranthene Po
K4[Fe(CN)6] Am (KM: 1720), Cf (KM: 170; kcat: 130), Cm (KM: 115; kcat: 450), Lp, Pa (KM: 1030), Pi,
Th (KM: 180; kcat: 400), To (KM: 96; kcat: 150), Tp (KM: 43; kcat: 51 000)
Mn21 St, Tv (KM: 186)
N,N 0 -Dimethylbenzene-1,4-diamine Ab, Am, Bc, Cf, Pc
o-Tolidine Gl (KM: 402)
o-Vanillin Cf (KM: 3900)
Pentachlorophenol Tv (KM: 3000; kcat: 0.023)
Phenanthrene Cg (kcat: 0.013)
Phenylhydrazine Rl

Table 3. Continued
Species
Inhibitor
Ca21 Le
Cd21 Dq, Le
Co21 Dq
Fe21 Ct, Po, Lp, Tc
Hg21 Ct, Dq, Le, Pu
Mn21 Dq, Pu
Rb1 Le
Sn21 Le
Zn21 Le, Po
1-Phenyl-2-thiourea Ct
2-Mercaptobenzothiazole Ct
2-Mercaptoethanol Ct, Pu, Te
3-(4-Hydroxyphenyl)acrylic acid Le
4-Nitrophenol Pz
8-Hydroxyquinoline Lp, Te
Ascorbic acid Ct, Tc
Cetylpyridinium bromide Ab
Cetyltriammonium bromide Ab, Tc
CN- Ab, Bc, Ct, Gl, Lp, Ma, Me, Mi, Pn, Po, Pz, Rl, Tg, Tr, Ts
Cysteine Ct, Ch, Dq, Gl, Le, Mq, Pc, Py, Sr, Te, Vv
Diethyldithiocarbamic acid Bc, Ch, Gl, Lp, Mi, Pp, Pc, Ps, Sr
Dithiothreitol Ch, Dq, Le, Pc, Py, Vv
EDTA Ct, Ma, Mq, Vv
Glutathione Dq, Gl
Humic acid Pt
Hydroxylamine Po
KCl Le
Kojic acid Dq, Le, Po
NaCl Sr
NaF Ds, Me, Sr, Tt
NaN3 Ab, Bc, Ch, Ct, Dq, Ds, Gl, Le, Ma, Me, Mi, Pa, Pc, Po, Pp, Pr, Ps, Pu, Pz, Sr, Tc, Te, Tg, Tr, Ts, Vv
Salicylaldoxime Gl
SDS Ds, Mq, Pu, Tr
Thiamine Sr
Thiogylcolic acid Ct, Mi, Po, Pr, Sr, Vv
Thiourea Ct, Dq
Trifluoroacetic acid Tr
Tropolone Ch, Le, Pc
Ab, Agaricus bisporus (Wood, 1980); Al, Agaricus blazei (Ullrich et al., 2005); Am, Armillaria mellea (Rehman & Thurston, 1992; Curir et al., 1997); Bc,
Botrytis cinerea (Zouari et al., 2002); Cf, Coriolopsis fulvocinnerea (Smirnov et al., 2001; Shleev et al., 2004); Cg, Coriolopsis gallica (Pickard et al., 1999); Ch,
Coriolus hirsutus (Eggert et al., 1996); Cm, Cerrena maxima (Shleev et al., 2004); Cn, Cryptococcus neoformans (Williamson, 1994); Cr, Coriolopsis rigida
(Saparrat et al., 2002); Cs, Ceriporiopsis subvermispora (Fukushima & Kirk, 1995; Salas et al., 1995); Ct, Chaetomium termophilum (Chefetz et al., 1998;
Ishigami et al., 1998); Cy, Cyathus stercoreus (Sethuraman et al., 1999); Dq, Daedalea quercina (Baldrian, 2004); Ds, Dichomitus squalens (Perie et al., 1998);
Ff, Fomes fomentarius (Rogalski et al., 1991); Gg, Gaeumannomyces graminis (Edens et al., 1999); Gl, Ganoderma lucidum (Lalitha Kumari & Sirsi, 1972; Ko
et al., 2001); Le, Lentinula edodes (DAnnibale et al., 1996; Nagai et al., 2002); Lp, Lactarius piperatus (Iwasaki et al., 1967); Ma, Mauginiella sp. (Palonen
et al., 2003); Me, Melanocarpus albomyces (Kiiskinen et al., 2002); Mi, Monocillium indicum (Thakker et al., 1992); Mq, Marasmius quercophilus (Dedeyan
et al., 2000; Farnet et al., 2004); Nc, Neurospora crassa (Froehner & Eriksson, 1974); On, Ophiostoma novo-ulmi (Binz & Canevascini, 1997); Pa, Podospora
anserina (Molitoris & Esser, 1970); Pc, Pycnoporus cinnabarinus (Eggert et al., 1996, 1995); Pe, Pleurotus eryngii (Munoz et al., 1997, 1997); Pi, Polyporus
anisoporus (Vaitkyavichyus et al., 1984); Pn, Phellinus noxius (Geiger et al., 1986); Po, Pleurotus ostreatus (Palmieri et al., 1997; Giardina et al., 1999;
Pozdnyakova et al., 2004; Das et al., 2000); Pp, Panaeolus papilionaceus (Heinzkill et al., 1998); Pr, Phellinus ribis (Min et al., 2001); Ps, Panaeolus sphinctricus
(Heinzkill et al., 1998); Pt, Panus tigrinus (Zavarzina et al., 2004); Pu, Pleurotus pulmonarius (De Souza & Peralta, 2003); Py, Pycnoporus coccineus (Oda et al.,
1991); Pz, Pyricularia oryzae (Neufeld et al., 1958); Rl, Rigidoporus lignosus (Geiger et al., 1986; Bonomo et al., 1998; Cambria et al., 2000); Sr, Sclerotium
rolfsii (Ryan et al., 2003); St, Stropharia rugosoannulata (Schlosser & Hofer, 2002); Tc, Trichoderma sp. (Assavanig et al., 1992); Te, Thelephora terrestris
(Kanunfre & Zancan, 1998); Tg, Trametes gallica (Dong & Zhang, 2004); Th, Trametes hirsuta (Shleev et al., 2004); To, Trametes ochracea (Shleev et al., 2004);
Tp, Trametes pubescens (Galhaup et al., 2002); Ts, Trametes sanguinea (Nishizawa et al., 1995); Tr, Trametes sp. AH28-2 (Xiao et al., 2003); Tt, Trametes trogii
(Garzillo et al., 1998); Tv, Trametes versicolor (Bourbonnais & Paice, 1990; Rogalski et al., 1991; Salas et al., 1995; Johannes et al., 1996; Collins et al., 1996;
Dawel et al., 1997; Hofer & Schlosser, 1999; Itoh et al., 2000; Ullah et al., 2000; Leontievsky et al., 2001); Vv, Volvariella volvacea (Chen et al., 2004).

228 P. Baldrian
Table 4. Reactivity of fungal laccases with different substrates. The numbers indicate the rate of substrate oxidation (%) compared to the oxidation of
2,6-dimethoxyphenol
Species
Substrate Am Ct Ch Cy Ds Ma Me Mv Pr Pe Pc Sr Ts Tt Tv
2,6-Dimethoxyphenol 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0
2-Amino-3-(3,4-dihydroxy- 55.9 46.0
phenyl)propanoic acid
4-Aminophenol 109.8 26.7
4-Aminophenylamine 62.9 14.7 194.8 56.0
4-Hydroxyindole 87.6 107.6
4-Methoxyphenol 7.3 9.8 19.8
4-Methylcatechol 69.3 31.4 103.5
ABTS 76.5 271.7 114.4 800.0 288.3 1.4 97.4 284.1 452.0 136.7 27.7
Caffeic acid 18.6 95.0 80.2 24.5
Catechol 74.2 44.9 59.2 34.9 33.1 21.6 74.3 18.3 76.0 27.9 110.7
Ferulic acid 111.8 48.4 8.5 76.0 116.3
Guaiacol 59.7 73.5 107.8 37.9 92.0 122.4 31.0 39.1 9.7 140.9 35.0 64.0 55.8 38.4
Hydroquinone 50.0 105.3 80.8 12.9 25.5 62.0 69.0 30.2 56.0
N,N-dimethyl-1,4-phenylenediamine 74.8 1.8 23.4 19.9 237.1
o-Anisidine 45.5 9.3
p-Anisidine 19.3 3.5
Pyrogallol 24.0 11.8 12.3 34.5 76.0 6.3
Syringaldazine 51.6 120.6 79.2 131.7 126.5
Syringic acid 115.2 46.9 14.1
Vanillic acid 61.8 7.3 8.1
Am, Armillaria mellea (Rehman & Thurston, 1992); Ct, Chaetomium thermophilum (Chefetz et al., 1998); Cy, Cyathus stercoreus (Sethuraman et al.,
1999); Ds, Dichomitus squalens c1 (Perie et al., 1998); Ma, Mauginiella sp. (Palonen et al., 2003); Me, Melanocarpus albomyces (Kiiskinen et al., 2002);
Mv, Myrothecium verrucaria (Sulistyaningdyah et al., 2004); Pr, Phellinus ribis (Min et al., 2001); Pe, Pleurotus eryngii (Munoz et al., 1997); Pc,
Pycnoporus cinnabarinus (Eggert et al., 1996); Sr, Sclerotium rolfsii SRL1 (Ryan et al., 2003); Ts, Trametes sanguinea (Nishizawa et al., 1995); Tt,
Trametes trogii (Garzillo et al., 1998); Tv, Trametes versicolor (Sulistyaningdyah et al., 2004).
DpH = 59 mV at 25 1C, a pH change from 2.7 to 11 would optimum of the fungi. The temperature stability varies
result in a E0 decrease of 490 mV for the phenol. However, considerably. The half life at 50 1C ranges from minutes in
over the same pH range, the E0 changes for the fungal B. cinnerea (Slomczynski et al., 1995), to over 23 h in
laccases studied (T. villosa, R. solani and M. thermophila) L. edodes and A. bisporus (Wood, 1980; DAnnibale et al.,
were much smaller ( 100 mV) (Xu, 1997). The enzyme 1996), to up to 5070 h in Trametes sp. (Smirnov et al.,
activity at higher pH is decreased by the binding of a 2001). While the enzyme from G. lucidum was immediately
hydroxide anion to the T2/T3 coppers of laccase that inactivated at 60 1C, the thermostable laccase from M.
interrupts the internal electron transfer from T1 to T2/T3 albomyces still exhibited a half life of over 5 h and thus a
centres (Munoz et al., 1997). Not only the rate of oxidation very high potential for selected biotechnological applica-
but also the reaction products can differ according to pH as tions (Lalitha Kumari & Sirsi, 1972; Kiiskinen et al., 2002).
pH may affect abiotic follow-up reactions of primary A very wide range of compounds has been described to
radicals formed by laccase. Laccases from Rhizoctonia prati- inhibit laccase (Table 3). In addition to the general inhibi-
cola and T. versicolor formed different products from syr- tors of metal-containing oxidases like cyanide, sodium azide
ingic and vanillic acids at different pH values, but both or fluoride, there are some selective inhibitors for individual
enzymes generated the same products at a particular pH oxidases. Carbon monoxide, 4-hexylresorcinol or salicylhy-
(Xu, 1997). The stability of fungal laccases is generally droxamic acid are examples of specific inhibitors of tyrosi-
higher at acidic pH (Leonowicz et al., 1984), although nases but not laccases (Petroski et al., 1980; Allen & Walker,
exceptions exist (Mayer, 1987; Baldrian, 2004). 1988; Dawley & Flurkey, 1993) that may facilitate estimation
Temperature profiles of laccase activity usually do not of laccase activity when protein purification is not success-
differ from other extracellular ligninolytic enzymes with ful. Inhibition by diethyl dithiocarbamate and thioglycolic
optima between 501 and 70 1C (Table 2). Few enzymes with acid could be supposed to be due to the presence of copper
optima below 35 1C have been described, e.g. the laccase in the catalytic centre of the enzyme, and several sulfhydryl
from G. lucidum with the highest activity at 25 1C (Ko et al., organic compounds have been described as laccase inhibi-
2001). This has, however, no connection with the growth tors: e.g. dithiothreitol, thioglycolic acid, cysteine and

diethyldithiocarbamic acid. However, experiments with T. and acetosyringone belong to the main products of both
versicolor laccase showed that the inhibitory effect found biological and enzymatic degradation of syringyl-rich lig-
with these compounds is probably due to the methodology nins (Kirk & Farrell, 1987).
using ABTS as the enzyme substrate (Johannes & Majcherc-
zyk, 2000) and that these compounds, contrary to sodium
azide, do not decrease the oxygen consumption by laccase
Laccases in the natural environment
during the catalysis. The considerable attention devoted to white-rot basidiomy-
Given the natural occurrence of laccases in soil, the cetes and their ligninolytic system in the past might lead to
inhibition by heavy metals and humic substances must be the conclusion that decaying wood is the most typical
taken into account (Zavarzina et al., 2004). While some environment for laccase production. The possible mechan-
laccases exhibit a sensitivity towards heavy metals (Table 3), isms involved in lignocellulose degradation by laccases have
others, e.g. the enzyme from G. lucidum, are completely been studied in detail and a comprehensive recent review is
insensitive (Lalitha Kumari & Sirsi, 1972). In the complex available on this topic (Leonowicz et al., 2001). Far less is
environment of soil or decaying lignocellulosic material, known about the occurrence, properties and roles of laccases
many different substrates of laccase are usually present that occurring in other types of natural lignocellulose-containing
can compete for the oxidation and thus competitively material like forest litter or soil. Compared to wood, soil or
inhibit the transformation of other compounds (Itoh et al., litter is a more complex and heterogeneous environment,
2000). Thus it is difficult to estimate the transformation which may hamper the detection and estimation of enzyme
rates of laccase substrates in soils based on laboratory results activities. Another problem is to link the enzyme activities in
and these rates can significantly differ in different soils. soil to a specific species producing it, if this is at all possible.
Some low molecular weight compounds that can be Several works [e.g. (Lang et al., 1997, 1998; Baldrian et al.,
oxidized by laccase to stable radicals can act as redox 2000)] followed the production of enzymes by fungi intro-
mediators, oxidizing other compounds that in principle are duced into soils and a number of protocols for laccase
not substrates of laccase due to its low redox potential. In extraction have been proposed to optimize the extraction
addition to enabling the oxidation of compounds that are yield. These include direct incubation with guaiacol as
not normally oxidized by laccases (e.g. the nonphenolic laccase substrate (Nannipieri et al., 1991), extraction with
lignin moiety), the mediators can diffuse far away from the surfactants or calcium chloride (Criquet et al., 1999) or the
mycelium to sites that are inaccessible to the enzyme itself. most widely used extraction with phosphate buffer (Lang
Several compounds involved in the natural degradation of et al., 1997), depending on the nature of the substrate (forest
lignin by white-rot fungi may be derived from oxidized litter, agricultural soil, compost).
lignin units or directly from fungal metabolism and act as Relatively high activities of laccase compared to agricul-
mediators (Camarero et al., 2005). (Eggert et al., 1996) tural or meadow soils can be detected in forest litter and
proposed that 3-hydroxyanthranilate can be a mediator of soils in both broadleaved (Rosenbrock et al., 1995; Criquet
lignin degradation by P. cinnabarinus, the fungus lacking et al., 2000; Carreiro et al., 2000) and coniferous forests
ligninolytic peroxidases. Other naturally occurring media- (Ghosh et al., 2003), where laccase is the dominant lignino-
tors include phenol, aniline, 4-hydroxybenzoic acid and lytic enzyme (Criquet et al., 2000; Ghosh et al., 2003). The
4-hydroxybenzyl alcohol (Johannes & Majcherczyk, 2000). laccase activity reflects the course of the degradation of
Recently, some phenols, including syringaldehyde and organic substances and thus it varies with time. Laccase
acetosyringone, have been described as laccase mediators activity was found to increase during leaf litter degradation
for indigo decolorization (Campos et al., 2001) as well as for in Mediterranean broadleaved litter (Fioretto et al., 2000) and
the transformation of the fungicide cyprodinil (Kang et al., the pattern of detected isoenzymes varied during the succes-
2002) and hydrocarbon degradation (Johannes & Majcherc- sion (Di Nardo et al., 2004). In evergreen oak litter, laccase
zyk, 2000). A comprehensive screening for natural media- activity was found to reflect the annual dynamics of fungal
tors was performed by (Camarero et al., 2005). Among 44 biomass that is probably driven by the seasonal drying
tested natural lignin-derived compounds they selected 10 (Criquet et al., 2000). The annual variation of laccase activity
phenolic compounds derived from syringyl, guaiacyl, and p- in temperate forests is also great and probably reflects the
hydroxyphenyl lignin units, characterized by the presence of seasonal input of fresh litter (P. Baldrian, unpublished data).
two, one or no methoxy substituents, respectively (in ortho The activity of laccase also reflects the presence of fungal
positions with respect to the phenolic hydroxyl). Syringal- mycelia. Significantly increased laccase activity was detected
dehyde, acetosyringone, vanillin, acetovanillone, methyl in the fairy rings of Marasmius oreades along with the
vanillate and p-coumaric acid have been found to be the production of organic acids and a high concentration of
most effective for mediated oxidation using laccases of P. available nitrogen and carbon due to the degradation of plant
cinnabarinus and T. villosa. Among them, syringaldehyde roots by the fungus (Gramss et al., 2005). Along with the

230 P. Baldrian
vertical gradient of fungal distribution in soil profiles, the Laccases are also actively produced during the compost-
laccase activity decreases with increasing depth. The decrease ing process. Of 34 isolates of fungi from woody compost, 11
of laccase activity is also reflected in the decrease of laccase were able to oxidize syringaldazine (Chamuris et al., 2000).
gene diversity with soil depth (Chen et al., 2003). Laccase was isolated both from compost-specific fungi and
Laccases as the most abundant ligninolytic enzymes in the compost itself (Chefetz et al., 1998, 1998) and it seems
soil also attracted the attention of ecologists studying its role that it participates in both degradation of lignin and humic
in the carbon cycle, especially with respect to the nitrogen acids and humic acid formation (Chefetz et al., 1998;
input. Several studies documented a significant decrease of Kluczek-Turpeinen et al., 2003, 2005). In water-saturated
laccases and peroxidases in forest soils subjected to elevated environments, laccase activity is driven by the concentration
nitrogen doses (Carreiro et al., 2000; Gallo et al., 2004), with of oxygen. Laccase activity in peatlands is thus low due to
the simultaneous increase in the litter layer (Saiya-Cork low oxygen availability (Pind et al., 1994; Williams et al.,
et al., 2002). This phenomenon was accompanied by the 2000) but increases dramatically when the oxygen concen-
decrease of fungal biomass and the fungal: bacterial biomass tration increases. The burst of laccase activity can lead to the
ratio in soil as well as by increased incorporation of vanillin depletion of phenolic compounds that inhibit organic
as a model lignin-derived substrate into fungal biomass; matter degradation by oxidative and hydrolytic enzymes
hence it seems that nitrate deposition directs the flow of (Freeman et al., 2004) and it can be assumed that the
carbon through the heterotrophic soil food web (DeForest oxygen-regulated laccase activity plays an important role in
et al., 2004, 2004). On the other hand, an increase of carbon cycling in this environment. In the water environ-
phenolic compounds in forest soil after burning increased ment, laccase was demonstrated to participate in the degra-
laccase activity (Boerner & Brinkman, 2003). dation of wood as well as humic substances (Claus & Filip,
Similar to the situation in other lignocellulose-containing 1998; Hendel & Marxsen, 2000). Its activity is dependent on
substrates, laccases probably also participate in the transfor- the succession step of substrate decay and it can exhibit a
mation of lignin contained in the forest litter. It is also seasonal pattern of activity dependent on the input of its
generally presumed that laccases are able to react with soil substrate (Artigas et al., 2004).
humic substances that can be directly formed from lignin Although the breakdown of lignin and the metabolism of
(Yavmetdinov et al., 2003). This is supported by the fact that humic acids may be the main ecological processes where
humic acids induce laccase activity and mRNA expression laccases are involved, there are probably more roles that
(Scheel et al., 2000). However, the interaction of laccases these enzymes can play. One of them is the interaction of
with humic substances probably leads both to depolymer- fungi with different microorganisms including soil fungi
ization of humic substances and their synthesis from mono- (e.g. Trichoderma sp.) and bacteria, a process usually accom-
meric precursors; the balance of these two processes can be panied by a strong induction of laccase (Freitag & Morrell,
influenced by the nature of the humic compounds (Zavarzi- 1992; Savoie et al., 1998; Savoie, 2001; Velazquez-Cedeno
na et al., 2004). (Fakoussa & Frost, 1999) observed the et al., 2004) that is probably general for laccase-producing
decolorization and decrease of molecular weight of humic basidiomycetes (Iakovlev & Stenlid, 2000; Baldrian, 2004)
acids, accompanied by the formation of fulvic acids during but was also demonstrated in R. solani challenged with
the growth of T. versicolor cultures producing mainly laccase, Pseudomonas strains producing antifungal compounds
and humic acid synthesis was also documented in vitro using (Crowe & Olsson, 2001). Since laccase and their products
the same enzyme (Katase & Bollag, 1991). Adsorption of do not have a direct effect on soil bacteria or fungi (Baldrian,
laccases to soil humic substances or inorganic soil constitu- 2004) it is probably involved in the passive defence by the
ents changes their temperature and activity profiles (Criquet formation of melanins or similar compounds (Eggert et al.,
et al., 2000) and inhibits its activity (Claus & Filip, 1990). 1995; Baldrian, 2003). Laccase can probably also contribute
(Zavarzina et al., 2004) estimated inhibition constants for to the degradation of phenolic antibiotics that inhibit fungal
humic acids towards Panus tigrinus laccase. The Ki ranged growth like 2,4-diacetylphloroglucinol. The role of laccases
from 0.003 mg mL 1 for humic acids from peat soils to 0.025 in the defence against heavy metals was also proposed in
mg mL 1 for humic acids from chernozems. Recently, spite of the fact that different heavy metals induce its activity
(Keum & Li, 2004) demonstrated that humic substances do and is connected with the production of melanins (Galhaup
not strongly bind laccase and the inactivation is thus not due & Haltrich, 2001; Baldrian, 2003).
to binding but to the dissociation of copper that is chelated
by humic substances. This introduces another difficulty for
the determination of laccase activities in soil or forest litter,
Laccases in environmental biotechnology
as different extraction methods extract different amounts of Laccases offer several advantages of great interest for bio-
humic acids together with soil proteins enzymes (Criquet technological applications. They exhibit broad substrate
et al., 1999). specificity and are thus able to oxidize a broad range of

xenobiotic compounds including chlorinated phenolics an immediately following reaction or when its redox poten-
(Royarcand & Archibald, 1991; Roper et al., 1995; Ullah tial is lowered, for instance by chelation.
et al., 2000; Schultz et al., 2001; Bollag et al., 2003), synthetic Another possibility for the oxidation of compounds
dyes (Chivukula & Renganathan, 1995; Rodriguez et al., with high redox potentials is the use of redox mediators.
1999; Wong & Yu, 1999; Abadulla et al., 2000; Nagai et al., From the description of the first laccase mediators, ABTS
2002; Claus et al., 2002; Soares et al., 2002; Peralta-Zamora (Bourbonnais & Paice, 1990), to the more recent use of
et al., 2003; Wesenberg et al., 2003; Zille et al., 2003), the -NOH- type, synthetic mediators such as 1-hydroxyben-
pesticides (Nannipieri & Bollag, 1991; Jolivalt et al., 2000; zotriazole, violuric acid and N-hydroxyacetanilide or TEM-
Torres et al., 2003) and polycyclic aromatic hydrocarbons PO, a large number of studies have been performed on the
(Johannes et al., 1996; Collins et al., 1996; Majcherczyk mechanisms of oxidation of nonphenolic substrates (Bour-
et al., 1998; Majcherczyk & Johannes, 2000; Cho et al., bonnais et al., 1998; Xu et al., 2000; Fabbrini et al., 2002;
2002; Pozdnyakova et al., 2004). They can bleach Kraft pulp Baiocco et al., 2003), the search for new mediators (Bour-
(Reid & Paice, 1994; Paice et al., 1995; Bourbonnais & Paice, bonnais et al., 1997; Fabbrini et al., 2002), and their
1996; Call & Mucke, 1997; Monteiro & de Carvalho, 1998; de applications in the degradation of aromatic xenobiotics
Carvalho et al., 1999; Sealey et al., 1999; Balakshin et al., (Bourbonnais et al., 1997; Johannes et al., 1998; Kang et al.,
2001; Lund et al., 2003; Sigoillot et al., 2004) or detoxify 2002; Keum & Li, 2004). Nevertheless, the laccase-mediator
agricultural byproducts including olive mill wastes or system has yet to be applied on the process scale due to the
coffee pulp (DAnnibale et al., 2000; Tsioulpas et al., 2002; cost of mediators and the lack of studies that guarantee the
Velazquez-Cedeno et al., 2002) (for review see Duran & absence of toxic effects of these compounds or their deriva-
Esposito, 2000; Duran et al., 2002; Mayer & Staples, 2002). tives. The use of naturally occurring laccase mediators
Unlike ligninolytic peroxidases they use molecular oxygen, would present environmental and economic advantages.
which is usually available in the reaction system as the final Their capability to act as laccase mediators has recently been
electron acceptor, instead of hydrogen peroxide, which that demonstrated. The possibility of obtaining mediators from
must be produced by the fungus. Laccases are usually natural sources and the low mediator/substrate ratios of
constitutively produced in at least some stages of the growth 14 (Camarero et al., 2005) or 2040 (Eggert et al., 1996;
of a particular fungus. They can be extracted from lignocel- Campos et al., 2001) increase the feasibility of the laccase-
lulosic substrates colonized by fungi as well as from soil or mediator system for use in biotechnology.
forest litter, or used in the form of spent substrate from the In addition to substrate oxidation, laccase can also
cultivation of edible mushrooms (Eggen, 1999; Lau et al., immobilize soil pollutants by coupling to soil humic sub-
2003; Law et al., 2003). The possibility of increasing the stances a process analogous to humic acid synthesis in soils
production of laccase by the addition of inducers to fungal (Bollag, 1991; Bollag & Myers, 1992). The xenobiotics that
cultures and a relatively simple purification process are can be immobilized in this way include phenolic com-
other advantages. Last but not least, the considerable pounds and anilines such as 3,4-dichloroaniline, 2,4,6-
amount of data concerning the properties of fungal laccases trinitrotoluene or chlorinated phenols (Tatsumi et al., 1994;
accumulated in the past years allows us to select a protein Dawel et al., 1997; Dec & Bollag, 2000; Ahn et al., 2002). The
suitable for a specific application (e.g. temperature-resistant immobilization lowers the biological availability of the
or pH-stable). xenobiotics and thus their toxicity.
However, the low redox potential of laccases The current development in laccase catalysis research and
(450800 mV) compared to those of ligninolytic peroxidases the design of mediators along with the research on its
(4 1 V) only allows the direct degradation of low-redox- heterologous expression opens a wide spectrum of possible
potential compounds and not the oxidation of more recal- applications in the near future. Moreover, laccase can also
citrant aromatic compounds, including some synthetic dyes offer a simple and convenient alternative to supplying
or polycyclic aromatic hydrocarbons (PAH) (Xu et al., peroxidases with H2O2, because laccases are available on an
1996), although there is some evidence that PAH can be economically feasible scale.
oxidized by some laccases to a considerable degree; yellow
laccase from P. ostreatus (YLPO) degraded PAH anthracene
(95% within 2 days) and fluoranthene (14% within 2 days)
Conclusions
with an optimum pH of 6.0 without redox mediators This review summarizes the available data about the bio-
(Pozdnyakova et al., 2004), whereas blue laccases from chemical properties of fungal laccases, their occurrence
other fungi were not capable of efficient oxidation (Jo- under natural conditions and possible biotechnological use.
hannes et al., 1996; Majcherczyk et al., 1998; Kottermann However, it leaves many important questions open: Why do
et al., 1998). The compounds with higher redox potential fungi produce laccase? What are the respective roles of
can only be transformed if the reaction product is subject to different isoenzymes? Do their biochemical characteristics

232 P. Baldrian
differ with respect to their function? The understanding of laccase from Trichoderma. Appl Microbiol Biotechnol 38:
the physiological role of laccase has not increased signifi- 198202.
cantly since it was reviewed by (Thurston, 1994) and (Mayer Baiocco P, Barreca AM, Fabbrini M, Galli C & Gentili P (2003)
& Staples, 2002). The problem is its low substrate specificity Promoting laccase activity towards non-phenolic substrates: a
and a very wide range of reactions that it can potentially mechanistic investigation with some laccase-mediator systems.
catalyze. Despite many efforts to address the involvement of Org Biomol Chem 1: 191197.
laccase in the transformation of lignocellulose, it is still not Balakshin M, Capanema E, Chen CL, Gratzl J, Kirkman A &
completely clear how important a role laccase plays in lignin Gracz H (2001) Biobleaching of pulp with dioxygen in the
degradation and if it contributes more to the formation or laccase-mediator systemreaction mechanisms for
decomposition of humic substances in soils. In this sense it degradation of residual lignin. J Mol Catal B-Enzym 13: 116.
is even more difficult to estimate its involvement and role in Baldrian P (2003) Interactions of heavy metals with white-rot
carbon cycling or during interspecific interactions of soil fungi. Enzyme Microb Technol 32: 7891.
fungi. Hopefully, these questions will attract more attention Baldrian P (2004) Purification and characterization of laccase
of researchers in the future. from the white-rot fungus Daedalea quercina and
decolorization of synthetic dyes by the enzyme. Appl Microbiol
Biotechnol 63: 560563.
Acknowledgements Baldrian P (2004) Increase of laccase activity during interspecific
interactions of white-rot fungi. FEMS Microbiol Ecol 50:
This work was supported by the Grant Agency of the Czech
245253.
Academy of Sciences (B600200516), by the Grant Agency of
Baldrian P, in der Wiesche C, Gabriel J, Nerud F & Zadrazil F
the Czech Republic (526/05/0168) and by the Institutional
(2000) Influence of cadmium and mercury on activities of
Research Concept no. AV0Z50200510 of the Institute of
ligninolytic enzymes and degradation of polycyclic aromatic
Microbiology, ASCR.
hydrocarbons by Pleurotus ostreatus in soil. Appl Environ
Microbiol 66: 24712478.
Banerjee UC & Vohra RM (1991) Production of laccase by
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Fungal laccases ^ occurrence and properties

Correspondence: Petr Baldrian, Laboratory Abstract

Editor: Jiri Damborsky

nol mono-oxygenase tyrosinase (EC 1.14.18.1). Although

FEMS Microbiol Rev 30 (2006) 215242

FEMS Microbiol Rev 30 (2006) 215242

Table 1. Characteristics of laccases purified from fungi

Published by Blackwell Publishing Ltd. All rights reserved

2005 Federation of European Microbiological Societies

FEMS Microbiol Rev 30 (2006) 215242

FEMS Microbiol Rev 30 (2006) 215242

Monocillium indicum 100 Thakker et al. (1992)

2005 Federation of European Microbiological Societies

Published by Blackwell Publishing Ltd. All rights reserved

FEMS Microbiol Rev 30 (2006) 215242

Table 2. Properties of fungal laccases (data derived from Table 1)

n, number of observations; Q25, lower quartile; Q75, upper quartile.

FEMS Microbiol Rev 30 (2006) 215242

belong to the blue copper proteins because it lacks Cu1 and

FEMS Microbiol Rev 30 (2006) 215242

FEMS Microbiol Rev 30 (2006) 215242

FEMS Microbiol Rev 30 (2006) 215242

FEMS Microbiol Rev 30 (2006) 215242

FEMS Microbiol Rev 30 (2006) 215242

FEMS Microbiol Rev 30 (2006) 215242

FEMS Microbiol Rev 30 (2006) 215242

FEMS Microbiol Rev 30 (2006) 215242

FEMS Microbiol Rev 30 (2006) 215242

FEMS Microbiol Rev 30 (2006) 215242

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