Fungal laccase – a versatile enzyme for biotechnological applications

1. Introduction
Laccase (benzenediol:oxygen oxidoreductase, EC belongs to the small group of enzymes called
the blue copper proteins or the blue copper oxidases along with the plant ascorbate oxidase and the
mammalian plasma protein ceruloplasmin [1,2] among others. These proteins are characterized by
containing 4 catalytic copper atoms. One copper is placed at the T1 site, where reducing substrate binds,
and it is responsible in the characteristic blue-greenish colour in the oxidizing resting state Cu2+ [1,3].
The other three coppers are clustered in the called T2/T3 site in which molecular oxygen binds.
Laccase is widely distributed in higher plants and fungi [4] and has been found also in insects and
bacteria. Recently a novel polyphenol oxidase with laccase like activity was mined from a metagenome
expression library from bovine rumen microflora [5].
Yoshida first described laccase in 1883 when he extracted it from the exudates of the Japanese lacquer
tree, Rhus vernicifera [1,6]. In 1896 laccase was demonstrated to be present in fungi for the first time by
both Bertrand and Laborde [1,6]. Since then, laccases have been found in Ascomycetes, Deuteromycetes
and Basidiomycetes; being particularly abundant in many white-rot fungi that are involved in lignin
metabolism [7,8]. Fungal laccases have higher redox potential than bacterial or plant laccases (up to
+800 mV), and their action seems to be relevant in nature finding also important applications in
biotechonology. Thus, fungal laccases are involved in the degradation of lignin or in the removal of
potentially toxic phenols arising during lignin degradation [1]. In addition, fungal laccases are
hypothesized to take part in the synthesis of dihydroxynaphthalene melanins, darkly pigmented polymers
that organisms produce against environmental stress [9] or in fungal morphogenesis by catalysing the
formation of extracellular pigments [10].
Concerning their use in the biotechnology area, fungal laccases have widespread applications, ranging
from effluent decolouration and detoxification to pulp bleaching, removal of phenolics from wines,
organic synthesis, biosensors, synthesis of complex medical compounds and dye transfer blocking
functions in detergents and washing powders, many of which have been patented [11]. The
biotechnological use of laccase has been expanded by the introduction of laccase-mediator systems, which are able to oxidise non-phenolic compounds that are otherwise hardly or not oxidised by the
enzyme alone.
2. Occurrence and location of laccases
Laccase is the most widely distributed of all the large blue copper-containing proteins, as it is found in a
wide range of higher plants and fungi [8,12] and recently some bacterial laccases have also been
characterized from Azospirillum lipoferum [13], Bacillus subtilis [14], Streptomyces lavendulae [15], S.
cyaneus [16] and Marinomonas mediterranea [17]. The occurrence of laccases in higher plants appears
to be far more limited than in fungi. Laccases in plants have been identified in trees, cabbages, turnips,
beets, apples, asparagus, potatoes, pears, and various other vegetables [6]. The classical demonstration of
laccase in R. vernicifera is well documented [18]. In addition, the lacquer tree is a member of the
Anacardiaceae family, appear to contain laccase in the resin ducts and in the secreted resin [18]. Cell
cultures of Acer pseudoplatanus have been shown to contain eight laccases, all expressed predominantly
in xylem tissue [19]. Other reports are those of Wosilait et al. [20] on the presence of a laccase in leaves
of Aesculus parviflora and in green shoots of tea [21]. Other higher plant species also appear to contain
laccases, although their characterization is less convincing [22]. Laccases have been isolated from
Ascomyceteous, Deuteromyceteous and Basidiomyceteous fungi [23]. In the fungi, Ascomycetes and
Deuteromycetes have not been a clear focus for lignin degradation studies as much as the white-rot
Basidiomycetes. Laccase from Monocillium indicum was the first laccase to be characterised from an
Ascomycete showing peroxidative activity [24]. This chapter will focus on laccases isolated from the
white-rot fungi.
The white-rot basidiomycetes are the most efficient degraders of lignin and also the most widely
studied. The enzymes implicated in lignin degradation are: (1) lignin peroxidase, which catalyses the
oxidation of both phenolic and non-phenolic units, (2) manganese-dependent peroxidase, (3) laccase,
which oxidises phenolic compounds to give phenoxy radicals and quinones; (4) glucose oxidase and
glyoxal oxidase for H2O2 production, and (5) cellobiose-quinone oxidoreductase for quinone reduction
[24]. The veratryl alcohol oxidase and some esterases may also play roles in the complex process of
natural wood decay. The different degrees of lignin degradation with respect to other wood components
depend on the environmental conditions and the fungal species involved. It has been demonstrated that
there is no unique mechanism to achieve the process of lignin degradation and that the enzymatic
machinery of the various microorganisms differ. Pleurotus ostreatus, for instance, belongs to a subclass
of lignin–degrading microorganisms that produce laccase, manganese peroxidase and veratryl alcohol
oxidase but no lignin peroxidase [25]. Pycnoporus cinnabarinus has been shown to produce laccase as
the only ligninolytic enzyme [26] and Pycnoporus sanguineus produces laccase as the sole phenol
oxidase [27]. In plants, laccase plays a role in lignification, whereas in fungi laccases have been
implicated in many cellular processes, including delignification, sporulation, pigment production,
fruiting body formation and plant pathogenesis [1,11]. Only a few of these functions have been
experimentally demonstrated [28].
Ligninolytic enzymes have mostly been reported to be extracellular but there is evidence in literature
of the occurrence of intracellular laccases in white–rot fungi [29]. Intracellular as well as extracellular
laccases were identified for Neurospora crassa by Froehner and Eriksson [30], who suggested that the
intracellular laccase functioned as a precursor for extracellular laccase as there were no differences
between the two laccases other than their occurrence.

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