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Laccase-mediator systems and their applications: A review

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DOI: 10.1134/S0003683807050055

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ISSN 0003-6838, Applied Biochemistry and Microbiology, 2007, Vol. 43, No. 5, pp. 523–535. © Pleiades Publishing, Inc., 2007.
Original Russian Text © O.V. Morozova, G.P. Shumakovich, S.V. Shleev, Ya.I. Yaropolov, 2007, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2007, Vol. 43, No. 5,
pp. 583–597.

Laccase–Mediator Systems and Their Applications: A Review

O. V. Morozova, G. P. Shumakovich, S. V. Shleev, and Ya. I. Yaropolov
Bach Institute of Biochemistry, Russian Academy of Sciences, Leninskii pr. 33, Moscow, 119071 Russia
e-mail: yaropolov@inbi.ras.ru
Received January 15, 2007

Abstract—The mechanism of operation of laccase–mediator systems (LMSs) in xenobiotic degradation medi-

ated by “true” redox mediators and laccase enhancing agents is considered. Structural formulae of most com-
mon laccase mediators and compounds that can be used as agents enhancing the enzyme operation are pre-
sented. Examples of LMS application in biotechnology are described.
DOI: 10.1134/S0003683807050055

Biocatalytic technologies find expanding applica-
tions in industry, probably owing to the environmental
safety of this approach [1–4]. Various enzymes are used
in industrial processes: proteases, lipases, xylanases,
hydrolases, isomerases, oxidoreductases, etc. The last
group includes laccase. This enzyme has a broad range
of substrates; therefore, it can be used in various bio-
technologies [1]. Moreover, the range of substrates of
the enzyme can be widened by its combination with
laccase mediators, which are simultaneously substrates
of the enzyme [2, 5, 6].
LACCASE PROPERTIES
Laccase (p-diphenol: oxygen oxidoreductase,
EC 1.10.3.2) belongs to copper-containing oxidases,
which catalyze reduction of molecular oxygen to water,
bypassing hydrogen peroxide formation [1, 7] accord-
ing to the equation
AH 2 + 1/2O 2
Laccase
A + H 2 O.
Laccase is among the few enzymes studied since the
late 19th century. It was first discovered in the Japanese
lacquer tree Rhus vernicifera. Later, other plant lacca-
ses were found and partly described: Rhus succedanea,
Acer pseudoplatanus, Pinus taeda, Populus eurameri-
cana, Liriodendron tulipifera, and Nicotiana tabacum
[8–13]. All plant laccases are monomeric proteins with
molecular weights within 90–130 kDa and high carbo-
hydrate contents (22–45%). Little is known about
higher plant laccases, probably because of their loca-
tion in cell walls.
Laccases are common in fungi [14]. Actually, they
have been found in most higher fungi. They occur in
fungal causative agents of soft rots, in most bracket
fungi causing white rot, in soil saprotrophs and plant
pathogens, and in many agarics, including cultivated
edible fungi: champignon, pleurotus, and medicinal
shiitake Lentinula edodes [15]. Other laccase producers
are wood-degrading fungi: Trametes (Coriolus) versi-
color, Trametes hirsuta, Trametes ochracea, Trametes
villosa, Trametes gallica, Cerrena maxima, Coriolop-
sis polyzona, Lentinus tigrinus, Plreurotus eryngii, etc.
Laccases occur in saprophytic ascomycetes inhabiting
composts (Myceliophthora thermophila and Chaeto-
mium thermophile) and in the soil hyphomycete Myce-
lia sterlia INBI 2-26. They appear to be involved in
humification [16–18].
Recent studies demonstrated laccase activity in bac-
teria [19–25].
Not all laccase functions are known yet [4, 8, 26]. It
is believed that plant laccases are involved in lignin pro-
duction, catalyzing polymerization of monomeric units
(p-coumaryl, coniferyl, and synapyl alcohols), follow-
ing the free radical mechanism. The main physiological
roles of fungal laccases are lignin degradation, fungus
development and morphogenesis, pathogenesis, and
detoxication [26–28].
Biochemical properties. By now, more than 100
laccases have been isolated and characterized to vari-
ous extents. Laccases are glycoproteins with molecular
weights 50–130 kDa. The carbohydrate portions of
plant laccases constitute up to 45% of the molecule
weight, whereas fungal laccases contain less carbohy-
drates (10–30%) [14]. It is believed that the carbohy-
drate portion of the molecule ensures the conforma-
tional stability of the globule and protects it from pro-
teolysis and inactivation by radicals [15, 29, 30].
Most fungi produce several laccase isoforms and
isoenzymes. The multiplicity of laccases is related to
the diversity of their roles: lignin synthesis/degrada-
tion, formation of fruit bodies, humification of organic
remains, pigment production, xenobiotic detoxication,
etc. [15].
The active center of laccases contains four copper
ions, classified into three types according to their spec-
troscopic and ESR parameters [31, 32]. The T1 center
shows a distinct absorption band at 600 nm (ε ~ 4500–

523
524 MOROZOVA et al.
5000 å–1 cm–1), making enzyme solutions light blue,
and weak parallel hyperfine splitting in ESR spectra
(g|| = 2.30, Ä|| = (40–95) × 10–4 cm–1) [32, 33]. The
ligands coordinated by these ions (two histidine imida-
zoles and a cysteine thiol group) form a trigonal struc-
ture. The type I copper can be replaced by mercury or
cobalt [34–36]. The T3 center of laccases is a binuclear
copper center. Its two copper ions are antiferromagnet-
ically coupled via a bridge hydroxy group. For this rea-
son, the center is diamagnetic, and it cannot be detected
by ESR. It can be identified from a shoulder in the UV
spectrum at 330 nm [32]. The T2 center of laccases is a
mononuclear copper center, not detectable by absorp-
tion spectrometry but showing hyperfine ESR splitting
typical of copper ions in tetragonal complexes: (g|| =
2.24, Ä|| = (140–200) × 10–4 cm–1) [32, 33]. Selective
removal of the T2 center causes a substantial loss of the
enzyme activity [37]. Spectrometric studies of native
laccase and its T1Hg derivative indicate that the T2 and
T3 centers form a trinuclear copper cluster [38, 39]. It
was first proven by study of the crystal structure of
ascorbate oxidase [40] and, then, of other laccases [41–
44]. The T2 and T3 centers of the enzyme are located
close to each other (4 Å), and the T1 copper ion is 12 Å
apart from them [43, 44]. Eight histidine imidazoles are
ligands of the T2–T3 cluster [32, 45].
Laccases are nonspecific with regard to reducing
substrates. They catalyze oxidation of various organic
substances, including o- and p-diphenols, aminophe-
nols, polyphenols, polyamines, methoxy phenols,
lignins, aryl diamines, and some inorganic ions with
simultaneous direct reduction of dioxygen to water
without intermediate production of hydrogen peroxide
[1, 32, 46–48].
The organic substrates of laccases can be tentatively
divided into three groups: ortho-, meta-, or para-substi-
tuted compounds with a lone electron pair. Ortho-sub-
stituted compounds (e.g., guaiacol, o-phenylenedi-
amine, pyrocatechol, dihydroxyphenylalanine, pyro-
gallol, caffeic acid, gallic acid, and protocatechuic acid)
are the best substrates for most laccases, unlike para-
(p-phenylenediamine, p-cresol, and hydroquinone) or
meta-substituted compounds (m-phenylenediamine,
orcinol, resorcinol, and phloroglucin) [14, 49].
Apart from organic compounds, laccases catalyze
4–
oxidation of some inorganic ions: Mo(CN ) 8 ,
4– 4– 4–
Fe(CN ) 6 , Os(CN ) 6 , and W(CN ) 8 . They oxidize
Mn2+ with the presence of chelating agents [50–52].
Key indices of laccases include standard redox
potentials of the three copper centers of the enzyme:
T1, T2, and T3. The potential of T1 has been deter-
mined for many laccases. It varies within 430–780 mV
[32, 53]. Potentials of T2 are known only for the low-
potential plant laccase from R. vernisifera (390 mV) and
high-potential fungal laccase from T. hirsuta (400 mV).
The potentials of T3 of the laccases from R. ver-
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
nisifera and T. versicolor are 460 and 785 mV, respec-
tively [54–56].
On the grounds of the potential of T1, all copper-
containing oxidases are divided into three groups: high-,
medium-, and low-potential enzymes [57, 58]. Low-
potential laccases include enzymes with T1 potentials
below 430 mV; medium potential, 470–710 mV; and
high potential, above 710 mV. Laccases directly oxi-
dize only compounds whose ionization potentials do
not exceed (or insignificantly exceed) the redox poten-
tial of the T1 copper ion [59].
As noted above, laccases are not specific with regard
to the donor substrate, being able to oxidize a wide
range of inorganic and organic compounds. As the main
role of laccases in nature is lignin polymerization and
depolymerization, laccase substrates include aromatic
compounds. Many of these compounds are phenolic
fragments of lignin. Thus, they can be lignin model
compounds. Formerly, it was believed that the main
function of laccases in the course of lignin degradation
was the oxidation of hydroxy groups linked to the ben-
zene ring to form phenoxy radicals, involved in conden-
sation reactions thereafter. It has been shown that fur-
ther rearrangement of phenoxy radicals can occur with
synchronous cleavage of alkyl–aryl bonds, oxidation of
benzyl hydroxyls, cleavage of ëα–ëβ-bonds, or ring
opening in nonphenolic lignin model compounds [60].
It is believed that the main feature determining the abil-
ity of the enzyme to cleave nonphenolic lignin struc-
tures is the redox potential of the T1 center. The ioniza-
tion potentials of nonphenolic lignin structures, such as
veratryl alcohol, 1,2-dimethoxybenzene, and others,
are high (>1.4 V). It was thought for a while that they
were substrates of only high-potential enzymes (lignin
peroxidases) but not laccases, whose redox potential
did not exceed 800 mV [32, 61].
LACCASE REDOX MEDIATORS (ENHANCERS)
Since 1990, when the diammonium salt of 2,2'-
azine-bis(3-ethylbenzothiazoline-6-sulfonic acid
(ABTS) was found to serve as a laccase substrate medi-
ating or enhancing the enzyme action [62], the range of
compounds that can be converted by laccases has
increased dramatically. The notion of laccase function
in lignin production/degradation changed, and the
potential of laccase application to various biotechnolo-
gies became much wider. The same study demonstrated
that T. hirsuta laccase with the presence of ABTS and
Remazol blue as mediators could oxidize nonphenolic
lignin components with high redox potentials: veratryl
alcohol and 1-(3,4-dimethoxyphenyl)-2-(2-methox-
yphenoxy)propane-1,3-diol. Moreover, laccase with
ABTS can degrade a model lignin dimer, 1-(3,4-
dimethoxyphenyl)-2-phenoxyethane-1,2-diol, to yield
veratraldehyde and benzaldehyde.
An ideal redox mediator must be a good laccase
substrate; its oxidized and reduced forms must be stable
Vol. 43 No. 5 2007
 LACCASE–MEDIATOR SYSTEMS AND THEIR APPLICATIONS (REVIEW) 525

but must not inhibit the enzymatic reaction. In addition, H3C

its redox conversion must be cyclic [63]. At first, medi- CH2 _
ators were considered to mean low-molecular-weight N S SO3
laccase substrates whose enzymatic oxidation gave rise N N
to stable high-potential intermediates. The latter took _
O3S S N
part in chemical (nonenzymatic) reactions with other H2C
compounds, not oxidizable by laccases alone, follow- ABTS CH3
ing the diffusion-controlled kinetics. The oxidized –e +e
mediator was reduced to the initial form by the com-
pound to be oxidized [64, 65], thereby closing the H3C
. CH2 _
cycle: + SO3
N S
O2 laccase mediatorox substrate N N
_
O3S S N
+ . H2C
H2O laccaseox mediator substrateox. ABTS CH3
–e +e
Ideally, without side reactions, a redox mediator can
perform many cycles without degradation. The oxi- H3C
dized mediator form produced in the course of the CH2 _
+ SO3
enzymatic reaction can nonenzymatically oxidize com- N S
pounds (including nonphenolic lignin structures) with N N
ionization potentials exceeding the potentials of lacca- _
O3S S N+
ses. However, the mechanism of these processes is H2C
insufficiently understood in many cases. The difference ABTS2+ CH3
between the redox potentials of the substrate to be oxi-
dized and T1 copper ions is the driving force of the Fig. 1. Oxidation of ABTS with the presence of laccase
reaction. However, in addition to thermodynamics, the [65].
kinetic factor should be taken into account, in cases
when compounds with potentials close to laccase redox
potentials are not laccase substrates. nism. It was shown that laccase-mediated ABTS oxida-
True redox mediators include a limited number of tion occurred in two stages [64, 65]. The fast first stage
+•
compounds, which, first of all, meet the requirement of was the formation of the ABTS cation radical, fol-
multiple reaction cycles in the redox process. These are lowed by slow oxidation of the cation radical to the
various complexes of transition elements (potassium ABTS2+ dication (Fig. 1).
octocyanomolybdate and octocyanotungstate) FeII
complexes with o-phenanthroline and 4,4'-dimethylbi- It is known that electrochemical oxidation of ABTS
+•
pyridine, and some organic laccase substrates, e.g., sequentially produces the ABTS cation radical and
ABTS and 2,2,6,6-tetramethyl-1-piperidinyloxyl the ABTS2+ dication. Cyclic voltammetry studies have
(TMPO). These compounds have sufficiently high shown that the redox states of ABTS are stable and
redox potentials and can perform multiple catalytic reversible, having formal redox potentials 0.472 V for
events without chemical degradation [65, 66]. The +•
mentioned transition metal complexes have high formal the ABTS/ABTS couple and 0.885 V for
+•
redox potentials and can directly oxidize nonphenolic ABT S /ABTS2+ against the Ag/AgCl reference elec-
lignin structures at relatively low concentrations in the trode [64]. With the presence of veratryl alcohol, the
reaction mixture. However, these compounds have ABTS2+ dication exhibits a catalytic action on its elec-
some drawbacks, which limit their industrial use. The trooxidation. However, no electrooxidation of this lig-
most significant of them are high cost in comparison nin model occurs in the potential range corresponding
with best organic mediators and insufficient environ- to the formation of the ABTS cation radical. The elec-
mental safety. trooxidation of veratryl alcohol with the presence of
The organic compound best fitting the term “redox ABTS2+ occurs at a slight overvoltage in comparison
mediator” is ABTS. Its use for oxidation of nonphe- with the potential of the start of veratryl alcohol elec-
nolic lignin structures gave impetus to search for new trooxidation on a glass–carbon electrode, which occurs
laccase mediators. at potentials above 1.25 V (Ag/AgCl). Homogeneous
Formerly, it was thought that just the ABTS cation oxidation of veratryl alcohol by the ABTS 2+ dication
radical, formed by enzymatic oxidation could oxidize displays second-order kinetics with the rate constant of
nonphenolic lignin structures. However, later studies, 170 M–1s–1. This relatively low constant rate is a conse-
mainly electrochemical and spectroelectrochemical, quence of the slow veratryl alcohol oxidation by the
showed that the process could involve another mecha- dication, which, in turn, is determined by the dramatic

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 43 No. 5 2007

526 MOROZOVA et al.
difference of their redox potentials and poor solubility
of veratryl alcohol.
A similar catalytic action was observed in the course
of electrooxidation of softwood kraft lignin by the elec-
trochemically generated ABTS 2+ dication on a glass
carbon electrode. It was shown that the laccase activity
with respect to oxidation of lignin phenol groups
increased with the presence of ABTS, probably owing
to diffusion of the resulting intermediates inside the
wood [67]. These compounds cannot be oxidized by
+•
laccase without ABTS. The ABTS cation radical
intensely interacts with vanillyl alcohol, but ABTS is
not completely recovered on the electrode during the
catalytic event. It appears that part of the produced
+•
ABT S cation radicals interacted with vanillyl alcohol
+•
radicals [64]. Thus, ABTS can interact only with lig-
nin phenolic groups, and the presence of ABTS2+ is
required for degradation of nonphenolic lignin struc-
tures.
The first study dedicated to mediators showed that
+•
the S cation radical alone could not oxidize veratryl
alcohol or some dimeric nonphenolic lignin model
compounds [62]. This process occurs only with the
presence of both the mediator and laccase. It is sug-
gested that, during oxidation of nonphenolic lignin
structures by the laccase–ABTS system, the enzyme
catalyzes oxidation of the mediator to the dication. The
laccase from the basidiomycete T. versicolor, used in
[64], belongs to high-potential laccases. The potential
of its T1 copper center is 785 mV against the standard
hydrogen electrode [55], which corresponds to 585 mV
against the Ag/AgCl electrode. This potential is about
300 mV lower that the redox potential of the
+•
ABTS /ABTS2+ couple. However, the authors of the
study applied the Nernst equation
Ö = Ö0 + 2.3RT/nF log ( c ox /c red ) ,
which links the redox potential of the system, the stan-
dard redox potential of the couple, and the ratio
between the oxidized and reduced forms of the redox
compound, and showed that laccase could slowly oxi-
dize ABTS to ABTS2+, thereby allowing slow oxidation
of nonphenolic lignin structures. It is assumed that, at
the first oxidation stage of high-potential substrates,
including nonphenolic lignin structures (e.g., veratryl
alcohol), removal of a hydrogen atom produces an
active radical, subsequently oxidized to aldehyde.
Recent studies of enzymatic ABTS oxidation cast
doubt on this mechanism of the reaction. A combina-
tion of spectrometrical, electrochemical, and enzymatic
+•
methods showed that the ABTS cation radical had
five absorption maximums in electron spectra at 214,
394, 414, 646, and 728 nm [68]. At 728 nm, the spec-
trum of the cation radical does not overlap the spectra
of the initial ABTS or the dication. Complete oxidation
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
of ABTS to the ABTS2+ dication decolorizes the solu-
tion. The fungal laccase from M. thermophila catalyzes
ABTS oxidation to the mixture of the initial substrate
+•
and the stable ABTS cation radical, accompanied by
formation of the ABTS2+ dication. However, the redox
potential of the T1 copper center of this laccase is low,
0.3–0.4 V against the Ag/AgCl electrode [69]. Thus, the
question of formation of the high-potential
+•
ABT S /ABTS2+ couple during enzymatic ABTS oxi-
dation remains open. It is conceivable that use of high-
potential laccases results in the formation of the dica-
tion, which then interacts with nonphenolic lignin mod-
els. The authors of [68] also showed that the resulting
+•
ABT S cation radical could mediate degradation of
many organic dyes, including indigo. In this process, it
is reduced to the initial species.
Although many studies considered degradation of
nonphenolic lignin structures by the laccase/ABTS sys-
tem, the mechanism of the process is not fully under-
stood. In particular, it is suggested that ABTS2+, as well
+•
as ABTS , cannot directly participate in the oxidation
of benzyl alcohols, and the degradation of nonphenolic
lignin structures is performed by intermediates formed
by enzymatic ABTS oxidation [70].
Also, redox properties of another laccase mediator,
hydroxybenzotriazole (HBT), have been studied [64].
The cyclic voltammetric curves of HBT at low potential
scanning speeds show only one oxidation peak at 878 mV
and a poorly pronounced cathode maximum at 463 mV.
The great difference between the peaks of HBT oxida-
tion and reduction of the products and the difference
between the values of the cathode and anode currents
indicate that the radical formed by HBT electrooxida-
tion is unstable: it decomposes into electrochemically
inactive compounds. Addition of veratryl alcohol into
the cell with HBT gives rise to the catalytic current of
veratryl alcohol oxidation within the potential range of
the HBT radical formation. It is suggested that electro-
chemical oxidation of HBT generates the benzotriaz-
ole-1-oxyl radical. The second-order rate constant of
the reaction between HB í • and veratryl alcohol was
calculated to be 2.5 Må–1 s–1. Thus, the paper discussed
is the first to demonstrate the possibility of the cyclic
voltammetric test of compounds as mediators by the
examples of ABTS and HBT.
Attempts to use polyoxometals, stable mainly in
strongly acidic solutions, as laccases are described in
[71–73]. The polyoxometallate [SiW11V1O40]5– is stable
at neutral and weakly acidic pH values, but it is rapidly
inactivated by oxygen during reoxidation. Neverthe-
less, [SiW11V1O40]5– is reactivated by laccases. It can
serve as a laccase mediator in lignin degradation [72].
One of the most efficient laccase mediators of the poly-
oxometal group is [SiW11MnIII(H2O)O39]5–. Its use for
lignocellulose degradation includes two stages: chemi-
cal and enzymatic. Lignin oxidation by this mediator
Vol. 43 No. 5 2007
 LACCASE–MEDIATOR SYSTEMS AND THEIR APPLICATIONS (REVIEW)
occurs at 110°ë, and reoxidation by laccase, at 45°ë.
The degree of lignin degradation in the samples reaches
50% [72].
Catalytic oxidation of 2-methoxy-1,4-benzohydro-
quinone and 2,6-dimethoxy-1,4-benzohydroquinone,
modeling guaiacyl and syringyl lignin structures, by the
laccase from P. eryngii is intermediated by formation of
a semiquinone [74]. Autooxidation of semiquinones by
dioxygen generates the superoxide radical:
–• –•
Q + O2 Q + O2 .
The superoxide anion radical can also be an electron
donor or acceptor; in addition, it can dismutate to pro-
duce hydrogen peroxide:
–• +
2O 2 + 2H H2 O2 + O2 .
In the presence of iron ions, the Haber–Weiss reaction
–• • –
O2 + H2 O2 OH + OH + O 2 ,
generates the highly reactive OH radical, able to react
with nonphenolic lignin structures. With Mn2+, present
in wood in catalytic quantities, the superoxide anion
radical is reduced to hydrogen peroxide:
–• 2+ + 3+
O 2 + Mn + 2H H 2 O 2 + Mn .
In this process, chelated Mn3+ can take part in a succes-
sion of nonenzymatic events ultimately degrading non-
phenolic lignin structures. Laccase and manganese ions
have been shown to be much more important for oper-
ation of the enzymatic lignin-degrading complex of
wood-degrading fungi than it was thought before [50–
52]. Divalent manganese ions with dicarboxylic acids
(oxalic, malonic, or tartaric) as chelating agents are lac-
case substrates, oxidizable by dioxygen to Mn3+. The
trivalent manganese ions can then react with dicarbox-
ylic acids to yield hydrogen peroxide in the following
reaction series:
–•
Mn3+ + çééë–ëééç Mn2+ + ë O 2 + ëé2 + 2H+
–• –•
ë O 2 + é2 O 2 + ëé2
–•
O 2 + Mn2+ + 2H+ Mn3+ + ç2é2
–•
2 O 2 + 2H+ ç2é2 + é2.
The resulting hydrogen peroxide can generate the
hydroxyl radical, as mentioned above. In addition,
hydrogen peroxide is a substrate of other important
enzymes of the lignin-degrading complex of basidio-
mycetes: manganese peroxidase and lignin peroxidase.
Thus, laccase can initiate reactions catalyzed by per-
oxidases. The redox potential of the Mn2+/Mn3+ couple
against the standard hydrogen electrode is 1.51 V;
therefore, oxidation of Mn2+ by laccase (the redox
potential of its T1 copper center being ca. +0.8 V) is
thermodynamically impossible. However, the presence
of natural chelating agents, such as dicarboxylic
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
527
anions, abundant in natural samples, reduces the redox
potential of the complex of the dicarboxylic acids with
manganese ions by ca. 0.4 to 0.5 V, allowing enzymatic
oxidation of Mn2+ by dioxygen with laccase. The
resulting Mn3+ ions coordinated with dicarboxylic
acids have high potentials and can directly oxidize non-
phenolic lignin structures (e.g., veratryl alcohol to ver-
atryl aldehyde), as shown in experimental studies [52].
Hence, manganese ions work as laccase redox media-
tors.
Unfortunately, at present, no compounds are known
that would perfectly meet the requirements imposed on
laccase redox mediators. The compounds described as
redox mediators actually are not mediators at all or can
be assigned to such with great reserve. First of all, it is
related to low stability of the intermediates formed dur-
ing the enzymatic reaction and, as a result, the small
number of redox cycles during catalytic oxidation of
nonphenolics. For this reason, the term “enhancer” (of
the catalytic action of laccases) has been coined. Apart
from true redox mediators, preserved during multiple
cyclic events, whose enzymatic oxidation products
have high redox potentials, the term can denote com-
pounds whose oxidation gives rise to an active radical,
experiencing further transformations. Few compounds
able to perform considerable numbers of redox cycles,
although not being ideal (true) redox mediators, have
been described [62, 65, 66].
Many recent papers are dedicated to the search for
new laccase enhancers and the study of their operation
mechanisms. Such laccases should be nontoxic, inex-
pensive, and highly efficient. Most of them are not true
redox mediators, because they are eliminated from the
reaction by secondary chemical transformations after
one or several cycles. The most commonly used
enhancers are compounds containing the structural
groups >N–OH or >N–O. They include HBT, TMPO,
N-hydroxyphthalimide (HPI), violuric acid (VA), and
N-hydroxyacetanilide (HAA) (Fig. 2) [65, 75–81].
Experiments with four enhancers of the >N–OH
type (HBT, HPI, VA, and HAA) in the reaction of cleav-
age of the C–H bond followed by oxidation of alkylare-
nes by T. villosa laccase showed that HBT was the most
efficient laccase enhancer [77]. As with phenolic com-
pounds, oxidized by laccase to phenoxy radicals, the
enzymatic oxidation of >N–OH compounds is medi-
ated by formation of the highly active nitroxyl radical
>N– O • owing to the removal of an electron followed by
release of a proton. Like the ABTS cation radical, the
nitroxyl radical can remove a hydrogen atom from the
high-potential substrate to produce a benzyl radical
(Fig. 3). This stage limits the reaction rate. The benzyl
radical is then oxidized by dioxygen to produce a mix-
ture of oxidation products [77].
Three mediators of the >N–OH type were tested in
oxidation of lignin model compounds by seven fungal
laccases [82]. The mediators were the following: HBT,
VA, and HAA (Fig. 2), with redox potentials against the
Vol. 43 No. 5 2007
528 MOROZOVA et al.

O reaction. Laccases from T. villosa and Pycnoporus cin-

N nabarinus oxidized the dimer with the presence of VA
N faster than with HBT; however, laccase from Botrytis
N OH
N cinerea oxidized the dimer with VA much slower than
OH with HBT. The rate of dimer oxidation depended on
O
HBT HPI both the kcat of laccases and of their stability. During the
(1-hydroxybenzotriazole) (N-hydroxyphthaleimide) reaction, HBT and VA were inactivated, because part of
the radicals interacted with amino acids of the protein,
O H3C and the rate of this interaction was significantly higher
HN OH C O with VA. Presence of the dimer of Phenol Blue in the
O N N reaction mixture somewhat reduced enhancer inactiva-
HN OH tion in the course of the enzymatic reaction.
O The mechanism of oxidation of 4-methoxybenzyl
VA HAA alcohol, a lignin model compound, with the presence of
(violuric acid) (N-hydroxyacetanilide) laccase from T. villosa and various mediators was stud-
ied in [65]. It was shown that oxidation of this model by
various laccase mediators followed two mechanisms:
H3C CH3 radical (hydrogen atom transfer) and electron transport
N (Fig. 4).
H3C CH3
O. Compounds of the >N–OH type (HBT and HPI) fol-
TMPO low the former mechanism, to produce corresponding
(2,2',6,6'-tetramethylpiperidine-1-oxyl) ketones from nonphenolic structures. In the electron-
transport mechanism, oxidation of nonphenolics is
Fig. 2. Structural formulae of some organic laccase media- mediated by formation of their cation radicals followed
tors of the >N–OH type. by cleavage of the ëα–ëβ bond to yield an aldehyde as
a final product (Fig. 4). The mediators oxidizing non-
phenolic lignin structures by this mechanism include
standard hydrogen electrode at pH 4.0 equaling 1.1, TMPO, ABTS, and coordinated transition metals [6,
0.91, and 0.83 V, respectively. In the pH range from 4 65].
to 10, the redox potential of HBT was virtually con-
stant, and the potentials of VA and HAA decreased by As shown in [65], TMPO is more efficient than
100 and 200 mV, respectively. The efficiency of cata- ABTS, HBT, VA, HPI, or the natural laccase mediator
lytic oxidation of various >N–OH compounds 3-hydroxyanthranilic acid. The TMPO mediator is
expressed as log(kcat/Km) depends on the difference present in the solution in the form of a relatively stable
between the redox potential of the >N–OH compound N-oxyl radical, which can perform primary modifica-
and the potential of the T1 copper of the laccase. tion of some high-potential substrates even without lac-
case [65]. The action of TMPO in oxidation of nonphe-
Comparison of various fungal laccases with regard nolic structures as suggested in [6, 65] is schematically
to oxidation of nonphenolic lignin models with the presented in Fig. 5. Laccase oxidizes TMPO to produce
presence of HBT and VA was reported in [61]. The the oxo-ammonium ion, which reacts with the sub-
dimer 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphe- strate. Proton removal yields the oxidized product and
noxy)-propane-1,3-diol was used as a nonphenolic lig- the reduced (N–OH) form of TMPO. The reduced
nin model, and Phenol Blue, as a phenolic one. The TMPO is converted by laccase to the oxidized form and
rates of the dimer oxidation by laccases with both HBT then to the oxoammonium ion (Fig. 5).
and VA decreased in the following order: T. villosa > Other classes of organic compounds are also used as
Pycnoporus cinnabarinus > Botrytis cinerea > M. ther- laccase enhancers: nitroso compounds and phenothiaz-
mophila. Both enhancers were partially expended in the ine derivatives (for structural formulae see Fig. 6) [83,

H3C H3C • H3C OH H3C O

CH2 CH CH C
N O
O2
+
–
N OH
OMe OMe OMe OMe

Fig. 3. Mechanism of the interaction between the nitroxyl radical and nonphenolic lignin structures [77].

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 43 No. 5 2007

 LACCASE–MEDIATOR SYSTEMS AND THEIR APPLICATIONS (REVIEW) 529

O structures by P. cinnabarinus laccase [85]. The com-

MeO C H
2 (in particular, Dichlorophenol red) are mediators of
OH β MeO
α
MeO C CMe3
MeO H
1 O
MeO C CMe3
MeO
Fig. 4. Oxidation of nonphenolic lignin model compounds
by laccase mediators according to the (1) radical and (2)
electron-transport mechanisms [65].
84]. One of phenothiazine derivatives, phenothiazine-
10-propionic acid, is present as a laccase mediator in
the commercial formula DeniLite®, manufactured by
Novozymes (Denmark) for indigo decolorization.
Enhancers of the high-potential laccase from T. ver-
sicolor include phenols and aromatic amines [63]. Such
mediators as phenol, aniline, p-hydroxybenzoate, and
p-hydroxybenzyl alcohol cooperate with this enzyme
as efficiently as ABTS or HBT. Oxidation efficiency
linearly increases with the redox potential of a media-
tor. Also, methionine, cysteine, and reduced glutathione
are efficient enhancers of T. versicolor laccase.
Natural metabolites, e.g., benzoic and 3-hydroxyan-
thranilic acids can be mediators [85, 86]. 3-Hydroxyan-
thranilic acid, produced by white rot fungi, is an effi-
cient mediator for oxidation of nonphenolic lignin
monly known indicator Phenol red and its derivatives
P. pinsitus laccase. Their efficiency in oxidation of the
nonphenolic compound 4-methoxybenzyl alcohol is
tenfold higher than with the natural laccase mediator
3-hydroxyanthranilic acid [87].
Unidentified compounds were detected in laccases
grown on natural lignocellulose substrates [88, 89].
These laccases were named “yellow” because of the
yellow-brown color of their homogeneous concentrated
solutions. Such a laccase was first detected as multiple
forms in experiments on solid-phase growth of the fun-
gus Panus tigrinus on wheat straw. Submerged growth
of this fungus in liquid medium yields a common
“blue” laccase. Both enzymes have identical molecular
weights, close isoelectric point values, four copper ions
in active centers, and similar kinetic indices of the reac-
tions of phenolic substrate oxidation. However, solu-
tions of yellow laccases do not have the absorption
band at 600 nm, typical of blue laccases. These laccases
can oxidize nonphenolic model lignin structures (vera-
tryl alcohol or dimeric compounds) without any redox
enhancers. Solid-phase growth of Pleurotus ostreatus
yields a yellow laccase able to oxidize anthracene with-
out a mediator to produce anthraquinone [89]. It is sug-
gested that solid-phase growth involves enzymatic oxi-
dation of natural laccase mediators, giving rise to radi-
cals, which interact with amino acids of the protein
globule, thereby modifying the enzyme. However, the
authors do not present any data indicative of the struc-
ture of the low-molecular-weight modifier and its effi-
ciency in the oxidation of nonphenolic lignin struc-
tures.

Laccase
+
N N
O
. O

+
CH2OH N CHO
+ HO O
+ –BH+
+
N C H N
O R H .. Ç R OH

Laccase
Laccase

N
O
.

Fig. 5. Putative mechanism of substrate oxidation with the presence of laccase and TMPO [65].

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 43 No. 5 2007

530 MOROZOVA et al.
H CH2CH2COOH
N N
S S
phenothiazine phenothiazine-10-propionic
acid
CH2CH2CH2N(CH3)2 CH2CH2CH2N(CH3)2
N N Cl
S S
promazine chloropromazine
SO3H
HO3S SO3H
OH
N systems (LMSs) with HPLC analysis of the products
N
O O OH
1-nitroso-2-naphthol- 2-nitroso-1-naphthol-
3,6-disulfonic acid 4-sulfonic acid
Fig. 6. Phenothiazine derivatives and nitroso compounds
used as laccase mediators.
The method of selection of candidate laccase medi-
ators and their experimental testing proposed in [90]
includes four stages: (1) selection of compounds by
consideration of their structural formulae (presence of
conjugated bonds, heterocyclic atoms, OH groups, and
NH2 groups), (2) analysis of the cyclic voltammetric
curves of selected compounds with and without lignin
model compounds, (3) determination of catalytic indi-
ces of the compounds in homogeneous reactions cata-
lyzed by laccase, and (4) direct experiment involving
the analysis of products of lignin model oxidation by
the mediator/laccase system. This method allowed dis-
covery of a new class of potential laccase mediators:
1-phenyl-3-methyl-pyrazolones-5. These compounds
are heterocycles containing two electron-donor substi-
tutes: methyl and phenyl groups. They occur in solu-
tions in two tautomeric forms. Two compounds of this
class were studied by spectral and electrochemical
methods: sodium 1-phenyl-3-methyl-4-methylamino-
pyrazolone-5- N(4)-methanesulfonate (NaPhP) and 1-(3'-
sulfophenyl)-3-methyl-pyrazolon-5 (mSPhP) [90, 91]. It
was shown that electrochemical and enzymatic oxida-
tion of these compounds yielded high-potential inter-
mediates able to oxidize the model lignin compound
veratryl alcohol.
About 20 compounds of different structures, includ-
ing heterocycles, >N–OH compounds, and benzoic
acid derivatives, were studied in [92]. Some representa-
tives of each class were characterized by electrochemi-
cal and enzymatic methods. Candidate laccase media-
tors were chosen by studies of homogeneous reactions
catalyzed by T. hirsuta laccase. The following com-
pounds proved to be laccase substrates: 1-phenyl-3-
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
methyl-pyrazolone-5 (PhP), its sulfo (mSPhP and
pSPhP) and amino (PhAP and NaPhP) derivatives,
HPI, and 3-amino-(6-hydroxy)-benzoic acid (AHBA).
Their structural formulae are shown in Fig. 7. The
kinetic indices of oxidation of the sulfo and amino
derivatives of 1-phenyl-3-methyl-pyrazolone by lac-
case were comparable with the indices of ABTS oxida-
tion, whereas the rate of enzymatic oxidation of HPI
was much lower. Electrochemical studies showed that
oxidation of 1-phenyl-3-methyl-pyrazolone derivatives
yielded high-potential intermediates, able to oxidize
veratryl alcohol. Compounds whose oxidation half-
wave potential in heterogeneous electrochemical oxi-
dation exceeded 500 mV against the Ag/AgCl electrode
proved to be most efficient in the reaction with veratryl
alcohol. Direct experiments on oxidation of veratryl
alcohol in homogeneous solutions by laccase–mediator
showed that the degree of veratryl alcohol oxidation by
the systems laccase–pSPhP or mSPhP and laccase–
HBT was high, reaching 30, 35, and 70% after 48 h of
the reaction.
Laccase–mediator systems in biotechnology.
Numerous articles and patents are dedicated to the use
of laccases and LMSs to biotechnology. The Danish
company Novozymes manufactures commercial for-
mulae containing a laccase and a mediator: DeniLite®
for fabric bleaching, Suberash® for cork modification,
and Novozymes® 51003 for paper pulp delignification
(http://www.novozymes.com).
Many articles and reviews concerning applications
of laccases and LMSs have been published recently
[3, 4, 5, 14, 93–96].
Lignin removal from wood is considered in many
papers. First of all, this fact is related to the importance
of the process for the wood-pulp and paper industry,
where enormous quantities of lignocellulose material
are processed annually. The technology of paper pro-
duction is diverse. It involves many enzymes, mainly
for partial replacement of chlorine bleaching of cellu-
lose pulp. In addition to glycosyl hydrolases, attempts
at using redox enzymes for lignocellulose delignifica-
tion are made [2, 97–101]. Delignification of paper
pulp with LMSs is now performed in pilot-scale plants.
Commercial use of this approach is expected in the
years immediately ahead. Such plants have been devel-
oped in Germany, the United States, Canada, and Fin-
land.
Extensive studies were carried out for years in order
to optimize the conditions of delignification and
bleaching of lignocellulose matter with LMSs [2]. The
laccase from the basidiomycete T. versicolor was used
as a biocatalyst, and compounds of the >N–OH group,
as mediators. The best results were obtained with HBT
as a mediator. The optimum technological parameters
of the process proved to be the following: temperature
40–65°ë, pH 4.5, air pressure 10–14 bar, exposure time
1–4 h, mediator concentration 0.1–2% of the dry
Vol. 43 No. 5 2007
 LACCASE–MEDIATOR SYSTEMS AND THEIR APPLICATIONS (REVIEW) 531

H3C
H3C H3C
N
N OH N
N N
N OH OH

SO3H SO3H
PhP pSPhP oSPhP
(1-phenyl- (1-(4'-sulfophenyl)- (1-(3'-sulfophenyl)-
3-methylpyrazolone-5) 3-methylpyrazolone-5) 3-methylpyrazolone-5)

CH3
H3C NH2 H3C N
CH2SO3Na
N N
H3C N O N OH

PhAP NaPhP
(1-phenyl-2,3-dimethyl- (1-phenyl-3-methyl-4-methylamino-
aminopyrazolone-5) pyrazolone-5-N(4)-methane sulfonate)

O NH2
C
N OH HO
C
C
O HO O
HPI AHBA
(N-hydroxyphthaleimide) (3-amino-(6-hydroxy)-benzoic acid)

Fig. 7. Structural formulae of organic substances regarded as potential laccase mediators.

weight of the pulp, and the enzyme amount 20 U/g
pulp.
Codexis Inc., United States, has patented a binary
mediator system, which, in combination with laccase,
efficiently bleaches cellulose [102]. It includes prooxi-
dants and prodegraders, as termed by the applicant, and
a fungal laccase of the genus Aspergillus. Urea, thio-
urea, sulfaminic acid, sulfamide, guanidine, methylsul-
fonic acid, and their mixtures are used as prodegraders.
These substances are not laccase enhancers by them-
selves. Ascorbic, salicylic, and nicotinic acids; their
salts; lignin sulfonates; and mixtures of these sub-
stances are patented as prooxidants. They are efficient
laccase enhancers by themselves, in both oxidation of
some dyes (e.g., Chicago blue) and delignification of
paper pulp. However, simultaneous use of prooxidants
and prodegraders allows more efficient paper pulp
delignification than with prooxidants not supplemented
with prodegraders.
Small quantities of coordination compounds of tran-
sition metals are also used as redox mediators in lac-
case–mediator complexes for pulp delignification [66].
Lignin oxidation with these mediators follows the elec-
tron mechanism, as with >N–OH compounds. These
mediators are laccase substrates. They are recoverable
in the redox cycle; therefore, delignification does not
demand their high concentrations. The best results in
softwood pulp bleaching were obtained with potassium
octocyanomolybdate. After treatment with the laccase–
mediator complex, whiteness reached 36.9% in com-
parison with 19.2% in control samples. This index was
10% lower with the next efficient mediator, FeII(4,4'-
dipyridyl)3. An important advantage of these mediators
over organic enhancers is that they can be repeatedly
used in lignin oxidation cycles. For example, use of one
mediator in two successive redox cycles allowed the
reduction of the content of residual lignin (kappa num-
ber) in both cycles from 15.6 to 7.4 at the mediator
amount 0.1% of the pulp weight.
Another application of laccases and LMSs is the
modification of lignin, lignocellulose, or lignocellulose
materials to endow them with new properties. The
selective oxidation of 6-hydroxymethylene groups of
cellulose to carbonyl and carboxy groups with laccase

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 43 No. 5 2007

532 MOROZOVA et al.
and nitroxyl compounds (e.g., TMPO) is described in
[103]. The resulting material, bearing active groups,
can be further modified by compounds bearing amino
or other groups.
The surface charges of lignocellulose materials can
be increased and the surface of paper or cellulose fiber
can be made hydrophobic by using laccases or LMSs
[104–108].
The environmentally safe process with the use of
LMSs can partially replace the chlorine bleaching of
pulp. In spite of the great promise of commercial use of
LMSs for pulp delignification and advances in this
field, the process is hampered by the high cost of medi-
ators and enzymes.
The first laccase preparation commercially used in
the fabric industry is DeniLite® (Novozyme, Denmark).
It is applied to indigo degradation in the course of jeans
fabric processing. The active principle of this prepara-
tion is the laccase obtained by submerged growth of a
genetically modified species of the genus Aspergillus.
In addition to the enzyme, the preparation contains a
redox enhancer, phenothiazine-propionate (Fig. 6), and
nonionic surfactants.
Use of laccase from various sources and other
enzyme mediators for degrading indigo and other tex-
tile dyes is also described in [109–112].
Crude laccase preparations supplemented with vari-
ous mediators are very promising for linen bleaching,
especially in Russia. Methods of bleaching jute and
linen fiber were developed on the base of a crude prep-
aration of the laccase from P. tigrinus and purified lac-
case with a mediator [113]. The degree of fiber deligni-
fication reached 70–75%.
A method of biodegradation of dioxins in ash and
soil with laccase and mediators (ABTS, HBT, 1-naph-
thoso-2-naphthol-3,6-disulfonic acid) was proposed in
[114].
Phosphororganic compounds, to which many insec-
ticides and nerve poisons belong, are very toxic. Their
nonenzymatic hydrolysis is slow. The purified laccase
from P. ostreatus with the presence of ABTS performs
rapid and complete oxidative degradation of nerve
gases VX and Russian VX and the insecticide diisopro-
pylamiton, the specific activities being 2200, 667, and
1833 nmol/(min mg), respectively [115]. Methods of
the degradation of polyurethane and polyolefine with
laccase–mediator systems have been patented [116,
117].
Other applications of laccases and LMSs include
dyeing of keratin fibers, bleaching of tooth enamel, use
as a binder for production of biodegradable polymers,
enzymatic synthesis, and modification of synthetic and
polymeric materials [118–121].
In conclusion, it should be mentioned that new com-
pounds that could be used as redox mediators or laccase
enhancers and that meet the requirements imposed on
ideal mediators have been sought after since the discov-
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
ery of laccase mediators in 1990. The number of com-
pounds patented as laccase redox mediators is con-
stantly increasing [84, 99, 122–125]. However, few of
them are really commercially valuable.
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