![: Laccase genes that have been shown to encode a biochemically characterized laccase protein [252].](https://www.researchgate.net/profile/Janardhan-Avilala/publication/262843177/figure/tbl2/AS:669199470981124@1536560995666/Laccase-genes-that-have-been-shown-to-encode-a-biochemically-characterized-laccase_Q320.jpg)
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Review Article
Fungal Laccases and Their Applications in Bioremediation
Buddolla Viswanath, Bandi Rajesh, Avilala Janardhan,
Arthala Praveen Kumar, and Golla Narasimha
Applied Microbiolog y Laboratory, Department of Virology, Sri Venkateswara University, Tirupati 517 502, India
Correspondence should be addressed to Buddolla Viswanath; buddolla@gmail.com
and Golla Narasimha; gnsimha@redimail.com
Received November ; Accepted April ; Published May
Academic Editor: David Ballou
Copyright © Buddolla Viswanath et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Laccases are blue multicopper oxidases, which catalyze the monoelectronic oxidation of a broad spectrum of substrates, for
example, ortho- and para-diphenols, polyphenols, aminophenols, and aromatic or aliphatic amines, coupled with a full, four-
electron reduction of O2to H2O. Hence, they are capable of degrading lignin and are present abundantly in many white-rot
fungi. Laccases decolorize and detoxify the industrial euents and help in wastewater treatment. ey act on both phenolic and
nonphenolic lignin-related compounds as well as highly recalcitrant environmental pollutants, and they can be eectively used in
paper and pulp industries, textile industries, xenobiotic degradation, and bioremediation and act as biosensors. Recently, laccase
has been applied to nanobiotechnology, which is an increasing research eld, and catalyzes electron transfer reactions without
additional cofactors. Several techniques have been developed for the immobilization of biomolecule such as micropatterning, self-
assembled monolayer, and layer-by-layer techniques, which immobilize laccase and preserve their enzymatic activity. In this review,
we describe the fungal source of laccases and their application in environment protection.
1. Introduction
Fungi can exploit marginal living conditions in large part
because they produce unusual enzymes capable of perform-
ingchemicallydicultreactions[,]. Interest in laccases
has increased recently because of their potential use in the
detoxication of pollutants and in bioremediation of phe-
nolic compounds [–]. ese fungal enzymes can convert
wood, plastic, paint, and jet fuel among other materials
into nutrients. Some of these enzymes have already been
harnessedinpulpandpaperprocessingandinthesynthesis
of ne chemicals []. Recent studies have suggested that
lignin-degrading or white-rot fungi (decay caused by these
species that gives wood a bleached appearance) such as
Phanerochaete chrysosporium and Trametes versicolor could
replace some of the chemical steps used in paper making
[,].
euseofenzymesforthetreatmentortheremovalof
environmental and industrial pollutants has attracted incre-
asing attention because of their high eciency, high selectiv-
ity, and environmentally benign reactions. Of these enzymes
studied for such purposes extracellular fungal peroxidases,
such as lignin peroxidase, manganese peroxidase, and fungal
laccases are the two major classes of enzymes that have been
evaluated for the removal of toxic phenolic compounds from
industrial wastewater and the degradation of recalcitrant
xenobiotics. Numerous reports have been published recently
on the improvements of the production of these enzymes,
such as discovery of new fungal strains, modication of
growth conditions, use of inducers, and use of cheaper growth
substrates such as agricultural and food wastes. e review
oftheliteraturegivenbelowisthereforeanaccountrelating
to laccases and their production, purication, biochemical
characterization, and their applications.
Laccase (EC ..., p-diphenol: dioxygen oxidoreduc-
tase) is one of a few enzymes that have been studied since
the nineteenth century. Yoshida rst described laccase in
when he extracted it from the exudates of the Japanese lacquer
Hindawi Publishing Corporation
Enzyme Research
Volume 2014, Article ID 163242, 21 pages
http://dx.doi.org/10.1155/2014/163242
Enzyme Research
tree, Rhus vernicifera []. In laccase was demonstrated
to be a fungal enzyme for the rst time by both Bertrand and
Laborde []. Laccases of fungi attract considerable attention
due to their possible involvement in the transformation of a
wide variety of phenolic compounds including the polymeric
lignin and humic substances []. Most lignolytic fungal
species produce constitutively at least one laccase isoenzyme
and laccases are also dominant among lignolytic enzymes in
the soil environment. In addition, laccase-mediated deligni-
cation allows increasing the nutritional value of agroindus-
trial byproducts for animal feed or soil fertilizer [].
e fact that they only require molecular oxygen for
catalysis makes them suitable for biotechnological applica-
tions for the transformation or immobilization of xenobiotic
compounds [].emajorroleoflaccasesinligninand
phenolic compound degradation has been evaluated in a
large number of biotechnological applications such as dye
degradation, bioremediation of some toxic chemical wastes
(e.g., chlorinated aromatic compounds, polycyclic aromatic
hydrocarbons, nitroaromatics, and pesticides) and biosensor
developments [–]. Commercially, laccases have been used
to delignify wood tissues, produce ethanol, and distinguish
between morphine and codeine. Research in recent years
has been intense, much of it elicited by the wide variety of
laccases, their utility, and their very interesting properties.
e current status of knowledge with regard to fungal laccase
and their applications to protect environment is reviewed.
2. Distribution and Physiological
Functions of Laccases
Laccases are common enzymes in nature and are found
widely in plants and fungi as well as in some bacteria and
insects []. e physiological functions of these biocatalysts,
which can be secreted or intracellular, are dierent in the
various organisms but they all catalyse polymerization or
depolymerization processes []. As mentioned earlier, the
rst laccase was reported in from Rhus vernicifera,the
Japanese lacquer tree from which the designation laccase
wasderived,andtheenzymewascharacterizedasametal
containing oxidase []. is makes it one of the earliest
enzymes ever described. Laccases have subsequently been
discovered from other numerous plants [] but the detection
and purication of plant laccases are oen dicult because
crude plant extracts contain a large number of oxidative
enzymes with broad substrate specicities [], which is
probablythereasonwhydetailedinformationaboutthe
biochemical properties of plant laccase is limited. However,
Rhus vernicifera laccase is an exception and has been exten-
sively studied, especially with regard to its spectroscopic
properties []. R. vernicifera laccase has also widely been
used in investigations of the general reaction mechanism of
laccases [,]. Plant laccases are found in the xylem, where
they presumably oxidize monolignols in the early stages of
lignication [], and also participate in the radical-based
mechanisms of lignin polymer formation []. In addition,
laccases have been shown to be involved in the rst steps of
healing in wounded leaves []. However, the occurrence of
laccases in higher plants appears to be far more limited than
in fungi [,].
Only a few bacterial laccases have been described hith-
erto. e rst bacterial laccase was detected in the plant root-
associated bacterium “Azospirillum lipoferum”[], where
it was shown to be involved in melanin formation [].
An atypical laccase containing six putative copper-binding
sites was discovered from Marinomonas mediterranea, but
no functional role has been assigned to this enzyme [].
Bacillus subtilis produces a thermostable CotA laccase which
participates in pigment production in the endospore coat
[]. Laccases have also been found from Streptomyces cya-
neus []andStreptomyces lavendulae []. Although there
arealsosomeotherreportsaboutlaccaseactivityinbacteria,
it does not seem probable that laccases are common enzymes
from certain prokaryotic groups []. Bacterial laccase-like
proteins are intracellular or periplasmic protein []. Laccases
producing bacteria from dierent environmental sources
with their possible physiological functions of laccase are
given below. B. licheniformis is a novel melanogenic soil
bacterium isolated from soil, which protects strain from UV
light and the oxidants []. It is involved in dimerization of
phenolic acids []. Bacillus endospores producing laccase
were isolated from soil and the enzyme involved in phenol
degradation [,].
Laccase activity has been demonstrated in many fun-
gal species belonging to ascomycetes and basidiomycetes,
and the enzyme has already been puried from many
species. ere are many records of laccase production by
ascomycetes. Phytopathogenic ascomycetes like Melanocar-
pus albomyces [], Cerrena unicolor [], Magnaporthe grisea
[], Trametes versicolor [], Trichoderma reesei [], and
Xylaria polymorpha []are examples for laccase production
and the enzyme was puried. Besides, in plant pathogenic
species, laccase production was also reported for some soil
ascomycete species from the genera Aspergillus, Curvularia,
and Penicillium []aswellassomefreshwaterascomycetes
[]. Yeasts are a physiologically specic group of both
ascomycetes and basidiomycetes. Until now, laccase was
onlypuriedfromthehumanyeastpathogenCryptococcus
(Filobasidiella)neoformans.isyeastproducestruelaccase
capable of oxidation of phenols and aminophenols and
unable to oxidize tyrosine []. e enzyme is tightly bound
to the cell wall and contributes to the resistance to fungicides
[]. Among physiological groups of fungi, laccases are typ-
ical of the wood-rotting basidiomycetes, which cause white
rot, and a related group of litter-decomposing saprotrophic
fungi, that is, the species causing lignin degradation. Almost
all species of white-rot fungi were reported to produce laccase
in varying degrees and the enzyme has been puried from
many species [].
e majority of laccases characterized so far have been
derived from white-rot fungi which are ecient lignin deg-
raders []. Many fungi contain several laccase-encoding
genes, but their biological roles are mostly not well under-
stood []. Agaricus bisporus [], Botrytis cinerea [],
Coprinus cinereus [], Phlebia radiata [], Pleurotus ostrea-
tus [], and Trametes versicolor [] were some examples
of basidiomycetes that produce laccases. In addition to
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plants, bacteria, and fungi, laccases or laccase-like activities
have been found in some insects, where they have been
suggestedtobeactiveincuticlesclerotization[]. Recently,
laccases have been represented as candidates for lignin
modication enzymes (“lignases”) in termites. Predominant
laccase activities against DMP and ABTS were detected in
the fungus combs and fungi isolated from the nests of three
genera of fungus-growing termites, that is, Macrotermes,
Odontotermes,andMicrotermes [–].
3. Screening of Fungal Species
Screening of laccase producing fungal species and their
variants is important for selecting suitable laccase producing
organisms. For this reason one usually relies on the use of
inexpensive, rapid, and sensitive testing methods. e screen-
ing strategy must aim to identify fungal strains and enzymes
that will work under industrial conditions []. Discovery
of novel laccases with dierent substrate specicities and
improved stabilities is important for industrial applications.
Fungi that produce laccase have been screened for either
on solid media containing coloured indicator compounds
that facilitate the visual detection of laccase production
[] or with liquid cultivations monitored with enzyme
activity measurements []. e use of coloured indicators
is generally simpler as no sample handling and measurement
are required. As laccases oxidize various types of substrates,
several dierent compounds have been used as indicators for
laccase production.
e traditional screening reagents tannic acid and gallic
acid [] have nowadays mostly been replaced with synthe-
tic phenolic reagents, such as guaiacol and syringaldazine
[,], or with the polymeric dyes Remazol Brilliant Blue
R (RBRR) and Poly R- []. RBRR and Poly R-
are decolourized by lignin degrading fungi []andthe
production of laccase is observed as a colourless halo around
microbial growth. With guaiacol a positive reaction is indi-
cated by the formation of a reddish brown halo [], while
with the tannic acid and gallic acid the positive reaction is a
dark-brown coloured zone []. Kiiskinen et al. []screened
novel laccase producing fungi by a plate method based on
polymeric dye compounds, guaiacol and tannic acid.
4. Cultural and Nutritional
Conditions for Laccase Production
Culture conditions and medium composition play a major
role in enzyme expression. Laccase production by fungi is
strongly aected by many fermentation parameters such as
time of cultivation, stationary or submerged cultures, organic
or inorganic compound concentrations, inducer concentra-
tion [], aeration [], and degradation or activation by
protease []. Laccases are generally produced during the
secondary metabolism of white-rot fungi growing on natural
substrate or in submerged culture []. Physiological demands
vary among white-rot fungi and considerable research has
been done on the inuence of agitation, pH, temperature,
carbon, nitrogen sources, and microelements and their levels.
Dong et al. [] compared laccase production by Trametes
gallica on twelve media under static or shaking conditions.
ey concluded that twelve culture media, in addition to
static and shaking conditions, show great inuence on
amount and pattern of laccase isoenzymes from Tra metes
gallica. Gayazov and Rodakiewicz-Nowak []reported
faster laccase production under semicontinuous cultivation
with high aeration and culture mixing compared to static
conditions. When using conical asks for cultivation it should
bebaedtoensureahighoxygentransfer[]. Simi-
larly, Piscitelli et al. [] described the inuence of various
physiological factors on laccase formation in a number of
white-rot fungi. Rened media are necessary for obtaining
largeamountoflaccasesthatmaybeusedinbiochemical
analysis and industrial application. Extracellular laccases of
white-rot fungi exist in isozymes that may be inducible or
constitutive. Ganoderma lucidum produced more than three
laccase isozymes in liquid culture []. Pleurotus pulmonarius
produced three laccase isozymes among which the lccand
lcc isoforms were produced in noninduced cultures, while
lcc was found only in induced-culture ltrates []. At least
seven laccase isozymes were found in the basidiomycete
CECT []. ree constitutive and four induced laccase
isozymes were found in Marasmius querocophilus strain
[]. At least nine constitutive laccase isozymes were
described for Pleurotus sp.[]. e pattern of isozymes has
been successfully applied in the identication of a number
of dierent microorganisms, particularly fungi such as ecto-
mycorrhiza [], deuteromycetes [], and basidiomycetes
[,].
A common technique for comparison of isozyme patterns
from dierent sources is the use of zymograms []. Praveen
et al. [] found that the production of high titres of the
laccase enzyme was not dependent on high biomass yields.
But laccase production was found to be highly related to the
conditions of cultivation of the fungus [,] and media
supporting high biomass did not necessarily support high
laccase yields []. e synthesis and activity of the laccase
were controlled during growth and can play an important
role in pigment and fruiting body formation [,]. Buswell
et al. [] reported that the production of laccase was
strongly aected by the nature and amounts of nutrients,
especially nitrogen and trace elements in the growth medium.
Laccases were generally produced in low concentrations by
laccase producing fungi [],buthigherconcentrationswere
obtainable with the addition of various supplements to media
[]. Laccase production by Phanerochaete chrysosporium
was not detected in low or high nitrogen medium with
glucose as the carbon source but was produced when the
organism was grown on low or high nitrogen medium with
cellulose as the carbon source [], whereas, in the white-
rot fungus Ganoderma lucidum, higher levels of laccases
were produced in high nitrogen medium with glucose as
thecarbonsource[]. Ligninolytic systems of white-rot
fungi were mainly activated during the secondary metabolic
phase and were oen triggered by nitrogen concentration
[]orwhencarbonorsulfurbecamelimited[]. e
addition of xenobiotic compounds such as xylidine, lignin,
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and veratryl alcohol was known to increase and induce lac-
case activity []. Towards maximization of laccase secretion
with culture additives, Sinegani et al. []studiedlaccase
production by Aspergillus terreus, Armillaria sp., Polyporus
sp., and Phanerochaete chrysosporium in liquid culture media
treated with N-ethyl aniline, N,N-dimethyl aniline and para-
bromoaniline as a laccase inducer.
Optimization of the production medium plays a major
role in higher laccase production. e Taguchi approach
of OA DOE constitutes a simple methodology that selects
the best conditions producing consistent performance. is
approachledtoanincreaseinlaccaseyieldtoU/L
from U/L. e increased production of laccase was
also conrmed by the dye decolorization experiment, which
showed an increased decolorization of reactive blue from
%to.%inthesameunitvolume[].
4.1. Inuence of pH on Laccase Production. e pH of the cul-
ture medium is critical and plays a signicant role in the
growth and laccase production of the organism. ere is
not much information available on the inuence of pH on
laccase production, but when fungi are grown in a medium
with pH . laccase will be produced in excess []. Most
reports indicated initial pH levels set between pH and
prior to inoculation, but the levels were not controlled
during most cultivations [,]. e optimum pH of laccase
production, as reported in many fungi, falls between .
and . [,,]. Maximum titres of laccase and biomass
were observed in the medium adjusted to pH . by Fomes
sclerodermeus, white-rot basidiomycetes. e optimal range
for the laccase isoforms secreted by Tram e tes pubes ce n s
fungal strain has been reported between pH . and .,
potentially indicating that laccase may be produced and
function optimally under conditions that are not favourable
to growth []. Laccases from fungi have been found in wide
applications ranging from the pharmaceutical sector to the
pulp and paper industry, but eukaryotic laccases generally
prefer low pH for better functioning. In contrast, bacterial
laccases can act and are more stable at wider pH range
[,]. With the advantage of immense environmental
adaptability and biochemical versatility, prokaryotes deserve
tobestudiedfortheirpossibleapplicationsinindustryaswell
as in medical science [].
4.2. Inuence of Temperature on Laccase Production. Temp e r -
ature, like any other physical parameters, plays a vital role
in growth and laccase production of the organism. It has
been found that the optimal temperature for fruiting body
formation and laccase production is ∘C in the presence of
light but ∘Cforlaccaseproductionwhentheculturesare
incubated in the dark []. In general the fungi were cultivated
at temperatures between ∘Cand
∘Cforoptimallaccase
production []. When cultivated at temperatures higher than
∘C, the activity of laccase was reduced []. e wood-
decaying basidiomycete Steccherinum ochraceum isolate
was reported to produce three highly thermostable laccase
isoforms with maximum activities in the region –∘C
[,]. erefore, it is proved that optimum production of
laccase can dier greatly from one strain to another.
4.3. Inuence of Carbon on Laccase Production. Carbon is a
part of all living organisms. Breakdown of carbon sources
liberates energy, which is utilized by the organism for growth
anddevelopment.emostreadilyusablecarbonsourceby
white-rot fungi is glucose []. Collins and Dobson []
reported that glucose at g/L enhanced the growth and lac-
case production by Coriolus versicolor.InTrametes versicolor,
glucose at higher concentration ( g/L) favoured laccase
production []. In Ganoderma lucidum, glucose at g/L
increased the mycelial growth but at g/L favoured expres-
sion of enzyme []. e maximum titres of extracellular
laccase in cultures of Lentinula edodes and Grifola frondosa
were grown in liquid medium with g/L glucose [,].
Glucoseatg/Lintheliquidmediumsupportedlaccase
production by Tram e tes gallic a []. Maximum laccase pro-
duction was obtained using response surface methodology
with glucose (. g/L) as the carbon source for Pleurotus
orida NCIM [].
Among several carbon sources tested, malt extract turned
out to be the best carbon source in the medium for pro-
nounced laccase production by Phlebia oridensis,P. b r e v i s -
pora,P.radiata,and P. fascicularia []. D’Souza-Ticlo et al.
[] screened dierent carbon sources for maximum laccase
production by Botryosphaeria sp. ey have screened glucose,
fructose, galactose, galacturonic acid, xylose, lactose, sucrose,
mannitol, pectin, and inulin and found increased laccase
production with most carbon sources studied except inulin
and galacturonic acid. Revankar and Lele []obtained
highest laccase activities with Trametes versicolor MTCC
using dierent carbon sources, namely, glucose, fructose,
sucrose, lactose, starch, and glycerol. ey observed a -
fold increase of laccase production when glucose was used
instead of fructose, and starch further improved laccase
production by %. e carbon source mannitol increased
laccase enzymatic activity to .% in Ganoderma lucidum
strain - in Pichia pastoris at a concentration of mM but
had no eect at . mM [].
4.4. Inuence of Nitrogen Sources on Laccase Production.
Laccases of white-rot fungi are mainly activated during the
secondary metabolic phase of the fungus and are oen
triggered by nitrogen depletion [],butitwasalsofound
that in some strains nitrogen concentrations had no eect on
laccase activity []. ese contradictory observations were
ascribed to dierences between the strains of Phanerochaete
chrysosporium and Lentinus edodes []. Buswell et al. []
foundthatlaccaseswereproducedathighnitrogenconcen-
trations although it is generally accepted that a high carbon
to nitrogen ratio is required for laccase production. Laccase
was also produced earlier when the fungus was cultivated
in a substrate with a high nitrogen concentration and these
changes did not reect dierences in biomass. Heinzkill et al.
[] also reported a higher yield of laccase using nitrogen
rich media rather than the nitrogen limited media usually
employed for induction of oxidoreductase. In another study
by [],ariseinnitrogenconcentration(from.to.g/L)
enhanced laccase synthesis yield. Higher nitrogen levels are
oen required in order to enhance laccase production []
but with certain fungi nitrogen-limited culture conditions
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simulate the formation of laccase enzyme [,]. ey
stated that the culture parameter with the most deleterious
eect on extracellular enzyme activity was of high level
nitrogen concentration.
e optimum nitrogen concentration for obtaining
the highest laccase activity from Pycnoporus sanguineus
( mU mL−1) is provided by a sucrose-asparagine medium
containing times as much asparagine as Kirk’s medium;
in fact, such a medium provided a . times higher laccase
activity than reference medium. ese conditions yielded
maximum laccase activity []. Laccase was best produced
at the concentration of mg NH4Cl and mg malt extract
with gross yield of . U/mL. []. e nitrogen source that
improved laccase synthesis to the greatest extent was peptone
(.-fold increase) []. Revankar and Lele []obtained
highest laccase activities by Trametes versicolor MTCC
using a complex nitrogen source (yeast extract). Leatham and
Kent Kirk [] screened dierent nitrogen sources, namely,
KNO3, glutamic acid, glycine, beef extract, and corn steep
liquor,andfoundthatglutamicacidwithlowconcentration
yielded higher amounts of laccase.
4.5. Inuence of Aromatic Compounds on Laccase Production.
Low molecular weight aromatic compounds have shown
signicant inuence on the growth and activity of lignocel-
lulolytic microorganisms []. Several compounds with a
methylated p-phenolic group are the products of ferulic and
syringic acid metabolism in Phanerochaete chrysosporium
[]. Aromatic compounds which are structurally related
to lignin, such as xylidine, ferulic acid, and veratric acid,
are routinely added to fungal cultures to increase laccase
production [,].Xylidineisknowntoincreaselaccase
transcription in Tramete s v i l l o s a [], Trametes versicolor
[], and P. s a j o r - c a j u []. It has been reported that one of
the possible functions for fungal laccase is the polymerization
of toxic aromatic compounds formed during the degradation
of lignin []. A dark precipitate was observed in xylidine-
induced cultures of T. versicolor and has been suggested that
it may represent a laccase polymerized form of aromatic
compounds []. Earlier studies on white-rot fungi have
shown that methylation of lignin-related aromatics inhibits
fungal growth only at higher concentrations ( and mM)
with stimulation occurring at mM concentration [].
Veratryl (, -dimethoxybenzyl) alcohol is an aromatic com-
pound known to play an important role in the synthesis and
degradation of lignin.
e addition of veratryl alcohol to cultivation media
ofmanywhite-rotfungihasresultedinanincreasein
laccase production []. Some of these compounds aect the
metabolism or growth rate [] while others, such as ethanol,
indirectly trigger laccase production []. Many of these
compounds resemble lignin molecules or other phenolic
chemicals []. ere are many reports describing the dier-
enteectsofaromaticcompoundsonlaccaseactivity.Highest
laccase activity was observed in Botryosphaeria rhodina when
veratryl alcohol was added to the nutrient medium at the
beginning of fermentation [].R.lignosusshows maxi-
mum laccase activity with compound with phenylhydrazine
[]. In a study by Elisashvili and Kachlishvili, , , -tri-
nitrotoluene (TNT) supplemented medium at appropriate
concentration signicantly accelerated Cerrena unicolor lac-
case production and -fold increased laccase specic activity
[]. Xylidine is known to increase laccase transcription in
Trametes villosa []andinTrametes versicolor [,].
e addition of veratryl alcohol to cultivation media of many
white-rot fungi has resulted in an increase in laccase pro-
duction [,]. e aromatic compound hydroquinone
increased -fold T. versicolor laccase activity while decreas-
ing-and-foldyieldsofMnPandendoglucanase[].
Hadibarata et al. [] have proved the importance of extra-
cellular laccase of Armillaria sp. F in the transformation
of anthracene into anthraquinone, benzoic acid, and other
products such as -hydroxy--naphthoic acid and coumarin.
4.6. Inuence of Amino Acids on Laccase Production. Lac-
case production by white-rot fungi is strongly aected by
the presence of amino acids in the media []. Various
amino acids and their analogues have shown stimulatory as
well as inhibitory eects on laccase production by Cyathus
bulleri []. According to this study, DL-methionine, DL-
tryptophan, glycine, and DL-valine stimulated laccase pro-
duction, while L-cysteine monohydrochloride completely
inhibited the enzyme production. Dong et al. []used
amino acid mixture that contained the following amino acids:
L-arginine, L-histidine, L-valine, L-threonine, L-isoleucine,
L-tyrosine, L-methionine, L-serine, L-asparagine, L-lysine,
L-aspartic acid, L-tryptophan, and L-cysteine, at % (w/v)
concentration. is amino acid mixture favoured the laccase
production by Tram e tes galli c a . It is possible that amino acids
act as inducer for laccase production by many white-rot fungi
[,]. Sun et al. []reportedthatallthesixamino
acids (alanine, histidine, glycine, arginine, aspartate, and
phenylalanine) at mM concentration increase the catalytic
abilityofthelaccaseenzymefromGanoderma lucidum
strain - when expressed in Pichia pastoris.Aminoacid
tryptophan also induces laccase production in Crinipellis sp.
RCK- [].
4.7. Inuence of Copper on Laccase Production. Copper is an
indispensable micronutrient for most living organisms and
copper requirements by microorganisms are usually satised
by low concentrations of the metal []. In its free form
cupric ion at higher concentration is extremely toxic to
microbialcells.ebindinganduptakeofcopperinfungi
usually comprise two phases: metabolism-independent sur-
face binding followed by an energy-dependent metal inux
[]. e stimulatory eect of copper on laccase synthesis
was also eective for several other basidiomycetes and hence
couldbeusedasasimplemethodtoimprovetheproduction
of this enzyme []. Using northern blot analysis it was
determined that increased production of laccase activity
wouldbeobtainedinthecoppersupplementedculturesof
Pleurotus ostreatus [].
Copper has been reported to be a strong laccase inducer
in several species, for example, Neurospora crassa [],
Trametes versicolor [], Phanerochaete chrysosporium [],
Panus osteratus [], Pleurotus sajor-caju [], Trametes
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trogii [], Volv a r ie l la volva c e a [], Lentinula edodes [],
and Grifola frondosa []. Huber and Lerch []reported
that Trame t e s pubescens grown at . mM CuSO4exhibited
high laccase activity ( U/mL) and using western blot
analysis they further demonstrated that the synthesis of the
laccase protein was linked to the presence of copper ions
in the culture medium. In addition, both the time and the
concentration of copper supplementation were important for
obtaining high levels of laccase. According to a recent study
on the white-rot fungus Tramet e s t rogii [], the addition
of copper strongly stimulated lignolytic enzyme production,
and higher decolorization of polymeric dyes-poly R- was
observed as well. However, higher copper concentrations
( mM) inhibited the growth and notably decreased man-
ganese peroxidase production although they did not aect
secretion of laccase []. e addition of low concentrations
of copper to the cultivation media of laccase producing fungi
stimulated laccase production []. Palmieri et al. []found
that the addition of 𝜇M copper sulphate to the cultivation
media can result in a yfold increase in laccase activity
compared to a basal medium. Copper has been reported to
be a strong laccase inducer in several species, for example,
Neurospora crassa [], Paecilomyces sp. WSH-L [],
Shiraia bambusicola strain GZK [], Tr ametes t rogii TEM
H[], Pleurotus orida NCIM [], Peniophora sp.
[], and so forth.
5. Laccases Inducers
e promoter regions of the genes encoding for laccase con-
tain various recognition sites that are specic for xenobiotics
and heavy metals []. ese xenobiotics and heavy metals
can bind to the recognition sites of the gene when present in
the medium and induce laccase production. White-rot fungi
were very diverse in their responses to tested inducers for
laccase. e addition of certain inducers can increase the
concentration of a specic laccase or induce the production
of new isoforms of the enzyme []. Some inducers interact
variably withdierent fungal strains. Lu et al. []foundthat
the addition of xylidine as inducer had the most pronounced
eect on laccase production. e addition of 𝜇M xylidine
aer h of cultivation gave the highest induction of laccase
activity and increased laccase activity by ninefold. At higher
concentrations the xylidine had a reduced eect probably due
to toxicity. Laccase oers protection for the fungus against
toxic phenolic monomers of polyphenols []. Dhawan and
Kuhad [] investigated the inducing eect of alcohols on
the laccase production by Trametes versicolor. e enhanced
laccase activity was comparable to those obtained using ,
-xylidine and veratryl alcohol []. It was hypothesized
that the addition of ethanol to the cultivation medium
caused a reduction in melanin formation. e monomers,
when not polymerised to melanin, then acted as inducers
for laccase production []. e addition of ethanol as an
indirect inducer of laccase activity oers a very economical
way to enhance laccase production. Garzillo et al. []
found that there was a strong correlation between hyphal
branching and the expression and secretion of laccase. e
addition of cellobiose can induce profuse branching in certain
Pycnoporus species and consequently increase laccase activity
[]. e addition of cellobiose and lignin can increase the
activity of extracellular laccases without an increase in total
protein concentration [].
An important distinction may be drawn between laccases;
they may either be inducibleor constitutively expressed [].
e constitutive, or noninducible, group does not react read-
ily to dissolved compounds that exhibit properties similar
to their substrates, and no inducer producing signicant
improvements in their yield has as yet been isolated. Single
inducers may not elicit the desired response in laccase pro-
duction, and a complex mixture of inducers may be required
[].
Several compounds may elicit a positive response on
laccase production; these compounds known as inducers
which include the metal ions, copper or cadmium [],
cycloheximide [], and low molecular weight aromatic or
organic acids, such as veratric acid [] and ferulic acid
[]aswellasotherphenolicoraromaticcompoundssuch
as , -xylidine []andveratrylalcohol[]. Soden and
Dobson []provedthatCu
2+ ions have the ability to induce
laccase production by forming an integral prosthetic group.
In the same way, natural substrates such as aromatic/phenolic
compounds and lignin derivatives such as veratryl alcohol
and , -xylidine induced laccase production []. ere
is evidence in Trametes versicolor that these compounds
causeanincreaseinmRNAlevels,butonlycopperwas
involved in increasing laccase mRNA translation []. e
exact action of inducers is, however, unknown. It has been
demonstrated that fungi may possess several isozymes of
laccase encoded by several laccase genes, and these may be
dierentially regulated []. It has further been suggested
that the action of certain inducers may be a direct result
of their toxicity to the fungus and the capability of laccase
to polymerize and detoxify them []. e use of inducers
does, however, suer from several disadvantages including
their toxicity and the extra expense associated with the
addition of an inducer. e white-rot fungus Tramet e s sp.
AH- can synthesize extracellular laccase by induction in
cellobiose-based liquid culture medium []. Both yields and
composition of laccase isoenzymes, produced by Trame t e s sp.
AH-, would be quite dierent with induction by dierent
small-molecule aromatic compounds, o-toluidine, guaiacol,
and , -dihydroxytoluene, which aected microbial growth
and the synthesis of laccase isoenzymes dierentially [].
It has been suggested that the addition of veratryl alcohol
may not elicit an inductive eect; rather it may act as a pro-
tective agent against inactivation by hydrogen peroxide
produced endogenously by the fungus [], thereby indi-
rectly eliciting a higher enzyme production. e addition
of surfactants or detergents, for example, Tween or ,
has resulted in higher yields of ligninolytic enzymes in
certain fungi. ere is evidence that these detergents result
in higher permeability of oxygen and extracellular enzyme
transport through the cell membranes of fungi []. Eective
induction of laccase from Pleurotus orida with anionic and
cationic surfactants has been demonstrated [].
Enzyme Research
Inspiteofaninitialinhibitoryeectonmycelialgrowth,
ethanolwasshowntobeaverystronginducerforlaccase
expression by Pycnoporus cinnabarinus [].Shankar and
Shikha [] reported that veratryl alcohol induced maxi-
mum laccase production giving .U/mL laccase activity
by Peniophora sp., whereas . mM xylidine was used as
an inducer to optimized production of laccase by Coriolop-
sis caperata RCK under solid state fermentation [].
Xylidine is the most widely reported inducer of laccase
production and enhanced laccase specic production by -
fold in Coriolopsis polyzona [].
ere have been many studies regarding the eects of
inducers using a plethora of fungal genera, species, and
even strains. Dierences in laccase stimulation were already
observed in very early studies more than half a century ago.
Ethanol has improved laccase synthesis signicantly when
used as a carbon source [] for a monokaryotic strain
Pycnoporus cinnabarinus. Later work by this group indicated
that ethanol improved gene expression and inhibited protease
activity, thereby playing an important regulatory role in
laccase production by the fungus [].
6. Purification and Biochemical
Properties of Laccases
Production of extracellular laccase is a common feature of
many fungi, particularly those associated with wood decay
or the terminal stages of decomposition of leaf litter. Current
knowledge about the structure and biochemical properties
of fungal laccase proteins is based on the study of puried
proteins.Morethanlaccasesfromvariousfungihave
been puried and characterized by researchers for more
than years []. Most of the white-rot fungi produce
laccase in multiple isoforms []. Several purication steps
are required to obtain a preparation free of both pigment and
other contaminant proteins. Multiple steps like ultraltration,
precipitation using ammonium sulphate or organic solvents,
and ion exchange and size exclusion chromatography have
been used for the purication of laccases from the culture
ltrate. Typical fungal laccase is a protein of approximately
– kDa with acidic isoelectric point around . [].
Several laccase isoenzymes have been detected in many
fungal species. More than one isoenzyme is produced in
most white-rot fungi []. is has been demonstrated
by p-phenylenediamine staining the laccase activity in all
tested wood rot fungi aer isoelectric focusing. All tested
species, namely, Coprinus plicatilis, Fomes fomentarius, Het-
erobasidion annosum, Hypholoma fasciculare, Kuehneromyces
mutabilis, Leptoporus litschaueri, Panus stipticus, Phellinus
igniarius, Pleurotus corticatus, P. ostreatus, Polyporus bru-
malis, Stereum hirsutum, Trametes gibbosa, T. hirsuta, and
T. versicolor, exhibited the production of more than one
isoenzyme,typicallywithpIintherangeofpHto[]. e
white-rot fungus P. ostreatus produces at least eight dierent
laccase isoenzymes, six of which have been isolated and
characterized [,]. e production of laccase isoen-
zymes in P. ostreatus is regulated by the presence of copper,
and the two dimeric isoenzymes have only been detected
inthepresenceofcopper[]. Isoenzymes of laccase with
dierent molecular weight and pI were also detected in the
litter-decomposing fungus Marasmius quercophilus []. A
study with dierent isolates of this fungus showed that the
isoenzyme pattern was consistent within dierent isolates.
Moreover, all isolates showed the same isoenzyme pattern
(one of the three laccase bands on SDS PAGE) aer induction
of laccase with dierent aromatic compounds [].
ecatalyticactionofanenzymeisquantitatively
described by the Michaelis constant 𝐾𝑀and the catalytic
eciency constant 𝑘cat. ese constants have been measured
foralargenumberoflaccases,andrathergreatvariancecan
be observed among them (Tab l e ). e 𝐾𝑀values of laccases
are generally in the range of . 𝜇M depending on the enzyme
source and the reducing substrate (Table ). e lowest 𝐾𝑀
values have been measured with syringaldazine, which is a
dimer of two molecules of , -dimethoxyphenol linked by
an azide bridge. Either the azide bridge or the dimer form
is apparently benecial for the anity of syringaldazine to
laccases because the 𝐾𝑀values measured for monomeric ,
-dimethoxyphenol are generally higher than those obtained
with syringaldazine (Tab l e ). e comparison of 𝐾𝑀values
also shows that laccases from dierent source organisms
have dierent substrate preferences []. e specicity for
oxygen is less dependent on the enzyme, and 𝐾𝑀values
of – 𝜇MforO
2have been reported for several laccases
[,].
Very signicant variance has also been observed in the
catalytic eciencies (𝑘cat) of various laccases. Dierences as
high as -fold can be seen in the 𝑘cat values between
dierent laccases with the same substrates (Table ). On the
other hand, the 𝑘cat values for a single laccase do not generally
dier more than –-fold between dierent substrates,
which reects the fact that 𝑘cat describes the rate of the
electron-transfer reactions taking place inside the enzyme
aer substrate binding [].However,thevarianceinassay
conditions must always be taken into account when the
catalytic constants measured in dierent laboratories are
compared. e constants in Ta bl e have been measured
under varying pH, ionic strength, and temperature condi-
tions and by using dierent protein concentrations, all of
which have a great eect on the results. In addition, dierent
molar extinction coecients for oxidation products have
sometimes been used in spectrophotometric assays because
the nature of the actual oxidation products is oen complex
or poorly understood. is aects particularly the numerical
values of 𝑘cat.
In addition to the kinetic constants, the catalytic per-
formance of laccases by catalytic activity and stability in
dierent pH and temperature conditions has been described.
e pH activity proles of laccases are oen bell-shaped,
with optima around ., when measured with phenolic
substrates []. e decrease in laccase activity in neutral or
alkaline pH values is aected by increasing hydroxide anion
inhibition because, as a small anion, hydroxide ion is also a
laccase inhibitor. On the other hand, increasing pH decreases
the redox potential of the phenolic substrate, making the
substrate more susceptible to oxidation by laccase [].
Oxidation of nonphenolic substrates, such as ABTS, does not
Enzyme Research
T : Kinetic constants of laccases at specied pH.
Substrate 𝐾𝑀𝑘cat pH Laccase Reference
(M) (min−)
Guaiacol
Pleurotus ostreatus POXC []
n.r∗Pleurotus ostreatus POXA []
n.r Chaetomium thermophilum []
. Tr ame t es tro g i i POXL []
n.r . Gaeumannomyces graminis []
Tramet e s p ubes c e ns LAP []
. Pleurotus sajor-caju Lac []
ABTS
. Tram e tes vill o s a Lcc []
n.r . Rhizoctonia solani Lcc []
Pleurotus ostreatus POXA []
n.r Pleurotus ostreatus POXA []
Pleurotus ostreatus POXC []
n.r Chaetomium thermophilum []
. Tramet e s t rogi i POXL []
n.r Panaeolus sphinctrinus []
n.r Coprinus friesii []
. Coprinus cinereus Lcc []
Tramete s p ubes c e ns LAP []
. Trichophyton rubrum []
n.r Pycnoporus cinnabarinus []
. Pleurotus sajor-caju Lac []
Myceliophthora thermophila []
Syringalda-zine
. . Tram e tes vill o s a Lcc []
n.r . Rhizoctonia solani Lcc []
Pleurotus ostreatus POXC []
Pleurotus ostreatus POXA []
n.r Pleurotus ostreatus POXA []
n.r Chaetomium thermophilum []
. Coprinus cinereus Lcc []
. Tramet e s p ubes c e ns LAP []
. Pleurotus sajor-caju Lac []
. Myceliophthora thermophila []
,-DMP
n.r . Botrytis cinerea []
Pleurotus ostreatus POXC []
Pleurotus ostreatus POXA []
n.r . Pleurotus ostreatus POXA []
. Tramet e s trog i i POXL []
n.r Chaetomium thermophilum []
n.r . Gaeumannomyces graminis []
Tram etes pu b escen s LAP []
Pleurotus sajor-caju Lac [].
∗n.r: not reported.
ABTS: ,-azinobis-(-ethylbenzthiazoline--sulphonate).
,-DMP: ,-dimethoxyphenol.
involveprotonexchange,andthereforenearlymonotonicpH
activity proles with highest activities at pH values of . are
obtained []. In contrast to their activity, the stability of
laccases is generally the highest at pH values around - [,
]. Temperature stabilities of laccases vary considerably,
depending on the source organism. In general, laccases are
stable at –∘C and rapidly lose activity at temperatures
above ∘C[,].
Enzyme Research
T : Laccase genes that have been shown to encode a biochemically characterized laccase protein [].
Organism
Gene Protein encoded by the gene
Name EMBL Length MW
+
pI
Acc. Number (aa)∗∗ (kDa)
Ceriporiopsis subvermispora lcs-1 AY .
Coprinus cinereus lcc1 AF .–.
Cryptococcus neoformans CNLAC1 L n.d.∗
Gaeumannomyces graminis LAC2 AJ .
Marasmius quercophilus lac1 AF .
Myceliophthora thermophila lcc1 AR .
Neurospora crassa alleles M- .
Phlebia radiata X lac1 —.
Pleurotus ostreatus poxa1b AJ .
Pleurotus ostreatus poxc Z .
Basidiomycete PM(CECT) lac1 Z .
Podospora anserina lac2 Y .
Populus euramericana lac90 Y .
Rhizoctonia solani lcc4 Z .
Streptomyces lavendulae — AB n.d.∗
Tramet e s pube s c ens la p 2 AF .
Tramet e s trog i i lc c 1 Y .–.
Trametes versicolor lccI L n.d.∗
Trametes versicolor lcc2 U .–.
Tramet e s v illos a lc c 1 L .
Tramet e s v illos a lcc 2 AY .–.
∗n.d.: not determined.
+Molecular weights determined by SDS-PAGE.
∗∗Amino acids.
7. Molecular Biology of Laccases
e rst laccase genes were isolated and sequenced about
years ago from the fungi Neurospora crassa [], Aspergillus
nidulans [,], and Phlebia radiata []. Since then,
sequencing of laccase genes has increased considerably. How-
ever, the number of laccase genes of which the corresponding
protein products have been experimentally characterized is
signicantly lower. To date, there are about such enzymes,
most of which are fungal laccases (Ta b l e ). A typical laccase
gene codes for a protein of – amino acids and the
molecular weights of laccases are usually in the range of to
kDa when determined by SDS-PAGE (Table ). Dierence
between the molecular weight predicted from the peptide
sequence and the experimentally obtained molecular weight
is caused by glycosylation, which typically accounts for about
–% of the total MW []. e isoelectric points of
microbial laccases are generally around . (Ta b l e ). Several
fungal genomes contain more than one laccase gene [].
e expression levels of dierent laccase genes typically
depend on cultivation conditions []. For example, high
nitrogen content of the medium has been shown to induce
transcription of laccase genes in the Basidiomycete I-
(CECT ) and in Pleurotus sajor-caju [].
Copper is also oen a strong inducer of laccase gene
transcription, and this has been suggested to be related to
a defense mechanism against oxidative stress caused by free
copper ions []. In addition to copper, other metal ions
such as Mg2+,Cd
2+,orHg
2+ can also stimulate laccase exp-
ression []. Certain aromatic compounds that are struc-
turally related to lignin precursors, such as , -xylidine or
ferulic acid, have also been shown to increase laccase gene
transcription in Tramete s v i l l o s a ,Trametes versicolor, and
Pleurotus sajor-caju []. On the other hand, Tramete s v i l l o s a
and Pleurotus sajor-caju have also been shown to contain
constitutively expressed laccase genes, and this may be related
to dierent physiological roles of the various laccases in the
fungi []. A clear understanding of expression laccase gene
mayleadtooverproductionoflaccaseenzyme.
7.1. Heterologous Production of Laccases. e natural hosts
produce very low yields of laccases for commercial pur-
poses. erefore, to improve the production, the cloning
of laccase gens and heterologous expression are employed.
Recent advances in the eld of genetic engineering have
allowed the development of ecient expression vectors for
the production of functional laccase. Laccase gene was cloned
in the most commonly used organisms, Pichia pastoris [],
Aspergillus oryzae [], A. niger [,], A. nidulans [],
Trichoderma reesei [], and Yarrowia lipolitica []. Lac-
case production levels have oen been improved signicantly
Enzyme Research
T : Laccase production in heterologous hosts [].
Laccase gene Production host Laccase production (mgL−1)∗
Ceriporiopsis subvermispora lcs-1 Aspergillus nidulans .
Aspergillus niger .
Coprinus cinereus lcc1 Aspergillus oryzae
Myceliophthora thermophila lcc1 Aspergillus oryzae
Saccharomyces cerevisiae
Phlebia radiata lac1 Trichoderma reesei
Pleurotus sajor-caju lac4 Pichia pastoris .
Pycnoporus cinnabarinus lac1
Pichia pastoris
Aspergillus niger
Aspergillus oryzae
∗e reported production levels have been obtained in shake ask cultivations, except in the case of Phlebia radiata laccase which was produced in a laboratory
fermentor.
by expression in heterologous hosts, but the reported levels
have still been rather low for industrial applications (Tabl e ).
e common problems associated with heterologous expres-
sion of fungal enzymes are incorrect folding and inecient
codon usage of expression organisms, resulting in nonfunc-
tional or low yields of enzyme. e incorrect substitution of
carbohydrate residues during glycosylation of proteins, which
is due to preferential utilization of specic carbohydrates by
the expression organism, may pose an additional problem to
heterologous expression. ese problems are being overcome
by using more advanced organisms as expression vectors
whosecodonusageandmolecularfoldingapparatusare
suitable for correct expression of these proteins.
Production of heterologous laccase has oen been impro-
ved by varying the cultivation conditions. For example, better
production of heterologous laccase has been achieved in yeast
systems by controlling the pH of the culture medium and
by lowering cultivation temperatures [,]. Buering of
the culture medium to maintain the pH above has been
proposed to be important in stability of secreted laccases
and inactivation of acidic proteases [], whereas lowered
cultivation temperatures may result in better production due
to improved folding of heterologous proteins []. In addi-
tion, over expression of Ssop, a membrane protein involved
in the protein secretion machinery []. Larsson et al. has
been shown to improve heterologous laccase production in
S. cerevisiae [,].
e addition of copper to the culture medium has also
proved to be important for heterologous laccase production
in Pichia pastoris and Aspergillus sp. [,]. In contrast to
homologous laccase production, in which copper addition
oen aects laccase gene expression, the increased laccase
production by copper addition is probably related to improv-
ing folding of the active laccase in heterologous production
[]. e importance of adequate copper concentration for
proper laccase folding was further corroborated by studies
in which two genes related to copper-tracking in Trame t es
versicolor were overexpressed in S. cerevisiae expressing T.
versicolor lacIII gene; the heterologous laccase production by
S. cerevisiae was improved up to -fold []. e eect was
suggestedtoresultfrommoreecienttransportofcopperto
the Golgi compartment [].
Directed evolution has also been used for improving
heterologous laccase production. Mutations in the Myce-
liophthora thermophila laccase gene resulted in the high-
est reported laccase production level in S. cerevisiae [].
e most important obstacles to commercial application of
laccases are the lack of sucient enzyme stocks and the
costofredoxmediators.us,eortshavetobemadein
order to achieve cheap overproduction of these biocatalysts
in heterologous hosts and also their modication by chemical
means of protein engineering to obtain more robust and
active enzymes.
8. Mode of Action
Laccases are mostly extracellular glycoproteins []andare
multinuclear enzymes [] with molecular weights between
and kDa []. Most monomeric laccase molecules
containfourcopperatomsintheirstructurethatcanbe
classied in three groups using UV/visible and electron
paramagnetic resonance (EPR) spectroscopy []. e type
Icopper(T)isresponsiblefortheintensebluecolourof
the enzymes at nm and is EPR-detectable, the type II
copper (T) is colourless but is EPR-detectable, and the type
III copper (T) consists of a pair of copper atoms that give a
weak absorbance near the UV spectrum but no EPR signal
[]. e T and T copper sites are close together and form
atrinuclearcentre[] that are involved in the catalytic
mechanismoftheenzyme[].
Laccase only attacks the phenolic subunits of lignin, lead-
ing to C𝛼oxidation, C𝛼-C𝛽cleavage, and aryl-alkyl cleavage.
Laccases are able to reduce one molecule of dioxygen to two
molecules of water while performing one-electron oxidation
of a wide range of aromatic compounds [], which includes
polyphenols [], methoxy-substituted monophenols, and
aromatic amines [].isoxidationresultsinanoxygen-
centred free radical, which can then be converted into a
second enzyme-catalysed reaction to quinone. e quinone
and the free radicals can then undergo polymerization [].
Enzyme Research
Laccases are similar to other phenol-oxidising enzymes,
which preferably polymerise lignin by coupling of the phe-
noxy radicals produced from oxidation of lignin phenolic
groups []. Due to this specicity for phenolic subunits
in lignin and their restricted access to lignin in the bre
wall, laccase has a limited eect on pulp bleaching []. e
substrate range of laccase can be extended to nonphenolic
subunits of lignin by the inclusion of a mediator such as ,
-azinobis-(-ethylbenzthiazoline--sulfonate) (ABTS).
9. Biotechnological Applications of Laccase
Laccases of fungi are of particular interest with regard to
potential industrial applications because of their capability
to oxidize a wide range of industrially relevant substrates.
Oxidation reactions are comprehensively used in industrial
processes, for instance, in the textile, food, wood process-
ing and pharmaceutical and chemical industries. Enzymatic
oxidation is a potential substitute to chemical methods since
enzymes are very specic and ecient catalysts and are
ecologically sustainable. Laccases are currently studied inten-
sively for many applications and they are already used in large
scale in the textile industry. Together with low molecular
weight redox-mediator compounds, laccases can generate a
desired worn appearance on denim by bleaching indigo dye
[]. e potential use of laccase for bleaching has been
investigated and this has even led to the esoteric suggestion
of using laccase in the presence of hydroxyl stilbenes as
hair dyes []. Another potential environmental application
for laccases is the bioremediation of contaminated soils as
laccases are able to oxidize toxic organic pollutants, such as
polycyclic aromatic hydrocarbons []andchlorophenols
[]. e most useful method for this application would
probably be inoculating the soil with fungi that are ecient
laccaseproducers because the use of isolated enzymes is not
economically feasible for soil remediation in large scale. e
current practical applications of the use of laccase have led
to a search for source of the enzyme from white-rot fungi
andtheuseofmediators,whichpromoteorfacilitateenzyme
action.
10. Laccases Role in Bioremediation
Oneofthemajorenvironmentalproblems,facedbytheworld
today, is the contamination of soil, water, and air by toxic
chemicals. With industrialization and the extensive use of
pesticides in agriculture, the pollution of the environment
with mandate organic compounds has become a serious
problem. Eighty billion pounds of hazardous organopo-
llutants are produced annually in the United States and only
% of these are disposed of safely []. Certain hazardous
compounds, such as polycyclic aromatic hydrocarbons
(PAH), pentachlorophenols (PCP), polychlorinated biphe-
nyls (PCB), ,,-trichloro-,-bis(-chlorophenyl) ethane
(DDT), benzene, toluene, ethylbenzene, and xylene (BTEX)
as well as trinitrotoluene (TNT), are persistent in the env-
ironment and are known to have carcinogenic and/or
mutanogenic eects. e ability of fungi to transform
a wide variety of hazardous chemicals has aroused interest in
using them in bioremediation []. Enzymatic treatment is
currently considered an alternative method for the removal
of toxic xenobiotics from the environment [].
10.1. Degradation of Xenobiotics. Laccases exhibit broad sub-
strate specicity and is thus able to oxidize a broad range
of xenobiotic compounds including chlorinated phenolics
[], pesticides [], and polycyclic aromatic hydrocarbons
[]. Moreover, polycyclic aromatic hydrocarbons, which
arise from natural oil deposits and utilization of fossil
fuels, were also found to be degraded by laccases [].
Laccase puried from a strain of Coriolopsis gallica oxidized
carbozole, N-ethylcarbozole, uorine, and dibenzothiophene
in presence of -hydroxybenzotriazole and .-azinobis (-
ethylbenzthiazoline)--sulfonic acid as free radical mediators
[]. Laboratory experiments have demonstrated that phe-
nols and aromatic amines may be removed from water by
the application of laccase []. e underlying mechanism
of the removal involves enzymatic oxidation of the pollutants
to free radicals or quinones that undergo polymerization and
partial precipitation []. Laccase from white-rot fungus,
Trametes hi r suta, has been used to oxidize alkenes []. e
oxidation is the eect of a two-step process in which the
enzyme rst catalysed the oxidation of primary substrate,
a mediator added to the reaction, and then the oxidized
mediator oxidizes the secondary substrate, the alkene, to the
corresponding ketone or aldehyde. In addition to substrate
oxidation, laccase can also immobilize soil pollutants by
coupling to soil humic substances—a process analogous to
humic acid synthesis in soils []. e xenobiotics that can
be immobilized in this way include phenolic compounds
including chlorinated phenols and anilines such as , -
dichloroaniline, , , -trinitrotoluene, or chlorinated phe-
nols []. e immobilization lowers the biological avail-
ability of the xenobiotics and thus their toxicity. A laccase
produced in the yeast, Pichia pastoris, was engineered by site-
directed mutagenesis to improve the rate of electron transfer
betweenthecopper-containingactivesiteoflaccaseandan
electrode []. us laccase may be usefully engineered
to improve the eciency of particular bioremediation pro-
cesses.
10.2. Decolourisation of Dyes. e textile industry accounts
for two-thirds of the total dyestu market and consumes
large volumes of water and chemicals for wet processing of
textiles []. e chemical reagents used are very diverse in
chemical composition, ranging from inorganic compounds
to polymers and organic products []. ere are about
,, commercially available dyes with over ×5tonnes
of dyestu produced annually []. Due to their chemical
structure dyes are resistant to fading on exposure to light,
water, and dierent chemicals and most of them are dicult
to decolourise due to their synthetic origin.
Government legislation is becoming more and more
stringent, especially in the more developed countries, regard-
ing the removal of dyes from industrial euents []. Con-
cern arises as several dyes are made from known carcinogens
Enzyme Research
such as benzidine and other aromatic compounds [].
Most currently existing processes to treat dye wastewater are
ineective and not economical. erefore, the development
of processes based on laccases seems an attractive solution
due to their potential in degrading dyes of diverse chemical
structure [] including synthetic dyes currently employed
in the industry [].euseoflaccaseinthetextile
industry is growing very fast since, besides decolorizing
textile euents as commented above, laccase is used to bleach
textiles and even to synthesize dyes []. Flavodon avus
decolourized several synthetic dyes such as Azure B and
Brilliant Blue R in low nitrogen medium []. Alternatively,
laccase, along with stabilizers, may be suitable for treatment
of wastewater [,]. Partial decolorization of two azo
dyes and complete decolorization of two triphenylmethane
dyes (bromophenol blue and malachite green) was achieved
by cultures of Pycnoporus sanguineus producing laccase as the
sole phenoloxidase [,]. Saratale et al. [] demon-
strated employing HPLC analysis, the degradation of the dye
“NavyblueHER”byfungusTrichosporon beigelii NCIM-
aer one day under static conditions for the identication
of metabolic products from dye degradation. Enayatizamir
et al. [] observed degradation of % in the Azo Black
Reactive dye by P. c h r y s o s p o r i u m aer days of treatment.
P. c h r y s o s p o r i u m URM and Curvularia lunata URM
strains decolourize euent containing textile indigo dye by
approximately % for days of treatment []. Laccase
puried from the fungus Trame t es hirsut a was able to degrade
triarylmethane, indigoid, azo, and athraquinonic dyes used in
dyeing textiles []aswellasindustrialdyes[].
10.3. Euent Treatment. Laccases from fungi oer several
advantages of great interest to biotechnological applications
of industrial euent treatment. As they exhibit broad sub-
strate specicity, they can bleach Kra pulp or detoxify
agricultural byproducts including olive mill wastes or coee
pulp [].Laccaseofanisolateofthefungus,Flavodon
avus, was shown to decolourize the euent from a Kra
paper mill bleach plant []. Laccase puried from white-
rot basidiomycete, Trametes villosa, degrades bisphenol A,
an endocrine-disrupting chemical []. Nonylphenols have
increasingly gained attention because of their potential to
mimic the action of natural hormones in vertebrates [].
ey result from incomplete biodegradation of nonylphenol
polyethoxylates (NPEOs), which have been widely used as
nonionic surfactants in industrial processes. Both nonylphe-
nols and NPEOs are discharged into the environment, mainly
due to incomplete removal of wastewater treatment facilities
[]. Nonylphenols are more resistant to biodegradation
than their parent compound and hence are found worldwide
in wastewater treatment plant euents and rivers []. Due
to their hydrophobicity, they tend to be absorbed onto surface
waterparticlesandsedimentsandaccumulateinaquatic
organisms. Consequently, nonylphenols represent a serious
environmental and human health risk. Laccases from aquatic
hyphomycete, Clavariopsis aquatica, have proved to degrade
xenoestrogen nonylphenol []. In addition to the potential
role of such degradation processes for natural attenuation
processes in freshwater environments, this enzyme laccase
also oers new perspectives for biotechnological applications
such as wastewater treatment.
10.4. Laccases: Pulp and Paper. Pulp bleaching is currently
achieved by treating pulps with chlorine-based chemicals.
is results in the formation of chlorinated aliphatic and aro-
matic compounds that could be acutely toxic, mutagenic, and
carcinogenic []. In recent years there have been intensive
studies performed to develop enzymatic, environmentally
benign, bleaching technologies []. e use of laccase-
mediated systems has shown potential for the biobleaching
of pulp, but the feasibility of its use is hindered by the lack
of an inexpensive mediator []. e bioremediatory role
of laccases in the pulp and paper industry is hindered by the
alkalinity of the euent. us several researchers have spent
considerable eort in identifying laccases that could be suit-
able for this type of remediation. e laccase from Coriolopsis
gallica has been implicated in the decolourisation of alkaline
euents such as the euent from the pulp and paper industry
[].Laccaseshavealsobeenshowntobeapplicabletothe
bioremediation of pulp and paper industry waste by eecting
direct dechlorination [] and the removal of chorophenols
and chlorolignins from bleach euents []. Other uses of
laccases for the pulp and paper industry include reduction
of the kappa number of pulp [] and an improvement in
the paper making properties of pulp []. Fungal laccases
can be used for the treatment of euents from pulp mills or
from other industries containing chlorolignins or phenolic
compounds []. Laccases render phenolic compounds less
toxic via degradation or polymerization reactions and/or
cross-coupling of pollutant phenol with naturally occurring
phenols [].
10.5. Laccases: Biosensors and Biofuel Cells. e use of laccase
in biosensor technology is mainly attributed to its broad
substrate range allowing for the detection of a broad range
of phenolics; this does however disallow the detection of
specic constituents [,]. Biosensors that utilize laccase
include an electrode that may be used for the detection of
phenols, such as catechols in tea [], phenolic compounds
in wine, and lignins and phenols in wastewaters [].
Fogel and Limson []developedarapid,simplemethod
of electrochemically predicting a given phenolic substrate’s
ability by amperometric laccase biosensors. Novel biosensors
have been developed using benecial properties of laccase,
such as the potentiometric immunosensor for the detection
of antigens []. Laccase has displayed a signicant potential
foritsuseinbiofuelcells[]. e major reason for this
interest is the use of oxygen as a substrate, which is converted
into water. e obvious advantage of this is the potential use
in nanotechnology for medical applications in living animals
since oxygen may be scavenged from the bloodstream, while
the byproduct (water) is benign. Slomczynski et al. []
developed a zinc-laccase biofuel cell adapting the zinc-air
cell design conguration. Unlike most biofuel cells, this zinc-
laccase cell operated under open ambient conditions. In this
single chamber, membraneless cell design was utilized and
laccase biocatalyst was le to be freely suspended (i.e., not
Enzyme Research
immobilized) in quasineutral potassium dihydrogen phos-
phate buer (pH .) electrolyte. Despite its simple design
features and not operating under controlled conditions, the
zinc-laccase system studied demonstrated power output of
comparable performance to biofuel cell system utilizing a
much more complex design immobilized enzyme and elec-
tron transfer mediator, controlled temperature and humidity,
oxygenated electrolyte, and so forth []. e drawback
of using laccase in this technology is its inability to reduce
oxygen at the physiological pH of blood, a technical hurdle
that must be overcome [].
10.6. Food and Beverage Industry. e beverage industry is
also set to be a benefactor of laccase. Laccase may prevent
undesirable changes such as discoloration, clouding, haze,
or avour changes in beer, fruit juices, and wine, improving
their shelf life by removing phenols such as coumaric acids,
avans, and anthocyanins [,]. e practical applica-
tions of laccases have led to a search for sources of the
enzyme from white-rot fungi and the use of mediators, which
promote or facilitate enzyme action. is review summarizes
the available data about the biological properties of fungal
laccases, their occurrence, and biotechnological applications.
Itisclearfromtheforgonesurveyofliteraturethatfocus
has been paid to a few laccase producing fungi, particularly
Phanerochaete chrysosporium, Trametes versicolor, Pleurotus
ostreatus,andsoforth,buttherearestilllargenumbers
of fungal organisms not covered for laccase production.
Search and screening of uncovered fungal organisms are
needed along with the known and reference culture to select
the potential culture with production of laccase in larger
amounts. Further understanding of kinetic parameters of
laccases will be useful to application of laccases for practical
purposes.
11. Conclusion
Because of their specic nature, laccases are receiving much
attention from researchers around the globe. e interest
in utilizing laccases for biotechnological applications has
increased rapidly since the discovery of these enzymes in
white-rot fungi. Emerging technologies include selective
delignication for production of cellulosics in pulp bleaching,
conversion of lignocellulosics into feed and biofuel, and
treatment of environmental pollutants and toxicants gen-
erated in various industrial processes. erefore, laccases
have been widely studied for various applications, including
the functionalization of lignocellulosic materials, wood ber
modication, and the remediation of soil and contaminated
euentsaswellastheiruseinbiosensors.isreviewshows
that laccase has a great potential application in environment
protection.However,muchmoreresearchisrequiredtomake
use of laccases to protect environment and other industrial
applications.
Conflict of Interests
e authors declare that there is no conict of interests
regarding the publication of their paper.
Acknowledgments
e corresponding author, Buddolla Viswanath, is thank-
ful to Science and Engineering Research Board (SERB),
Department of Science and Technology (DST), Government
of India, for awarding Young Scientist Scheme (SR/FT/LS-
/) to the project entitled “Environmental geochem-
istry,humaninvitrobioaccessibilityandecosystemhealth
eects of scarce, technologically important metals (STIM).”
e authors are all thankful to Professor Bontha Rajasekhar
Reddy, S.K. University, Anantapur, India, and other anony-
mous reviewers for their valuable suggestions to improve this
review.
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