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Crit Rev Biotechnol, Early Online: 1–17

! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/07388551.2014.949617

REVIEW ARTICLE

Structure, functionality and tuning up of laccases for lignocellulose

and other industrial applications
Anna K. Sitarz, Jørn D. Mikkelsen, and Anne S. Meyer

Department of Chemical and Biochemical Engineering, Center for BioProcess Engineering, Technical University of Denmark, Lyngby, Denmark
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Abstract Keywords
Laccases (EC 1.10.3.2) are copper-containing oxidoreductases that have a relatively high redox Industrial applications, laccase, oxidation
potential which enables them to catalyze oxidation of phenolic compounds, including mechanism, phenolic oxidation, redox
lignin-derived phenolics. The laccase-catalyzed oxidation of phenolics is accompanied by potential
concomitant reduction of dioxygen to water via copper catalysis and involves a series of
electron transfer reactions balanced by a stepwise re-oxidation of copper ions in the active site History
of the enzyme. The reaction details of the catalytic four-copper mechanism of laccase-mediated
catalysis are carefully re-examined and clarified. The substrate range for laccase catalysis can be Received 20 December 2013
expanded by means of supplementary mediators that essentially function as vehicles for Revised 21 April 2014
electron transfer. Comparisons of amino acid sequences and structural traits of selected Accepted 30 June 2014
laccases reveal conservation of the active site trinuclear center geometry but differences in loop Published online 28 August 2014
conformations. We also evaluate the features and regions of laccases in relation to modification
and evolution of laccases for various industrial applications including lignocellulosic biomass
For personal use only.

processing.

Introduction related compounds as electron donors, with oxygen as the

Laccases (benzenediol: dioxygen oxidoreductases, EC
1.10.3.2) belong to the family of blue multi-copper oxidases.
This type of enzymes are characterized by the presence of at
least four copper ions, referred to as T1, T2, T3a and T3b, in
the active site, and these copper ions are located in different
cupredoxin domains in the enzyme protein structure
(Hakulinen et al., 2002).
In nature, laccases play diverse roles by catalyzing
reactions involved in lignification, delignification, oxidative
in planta stress management, fungal morphogenesis and
virulence. Laccases are produced by bacteria (Beloqui et al.,
2006), insects (Dittmer et al., 2004), plants (O’Malley et al.,
1993) and fungi (Basidomycota and Ascomycota). Laccases
produced by white-rot fungi (Basidiomycota) currently
receive particular attention due to their apparent ability to
catalyze the modification and depolymerization of lignin and
in this way assist in the decomposition of plant material.
Laccases exhibit broad substrate specificity with respect to
the electron donor, and principally act on diphenol hydroxyls,
but apparently also on hydroxyl groups of monophenols and
Address for correspondence: Anne S. Meyer, Department of Chemical
and Biochemical Engineering, Center for BioProcess Engineering,
Technical University of Denmark, Building 229, DK-2800 Lyngby,
Denmark. E-mail: am@kt.dtu.dk
electron acceptor according to the overall reaction:
4 Benzenediol þ O2 ! 4 Benzosemiquinone þ 2 H2 O
(or as discussed later: 4 monophenols + O2 ! 4 quinones + 2
H2O).
This catalytic reaction thus consists of two half-reactions:
(1) the oxidation of phenolic substrates and (2) the reduction
of dioxygen to water. Due to the broad substrate specificity
with respect to the electron donor, laccases can catalyze
oxidation of an array of different substrates (phenols,
polyphenols, benzenothiols, polyamines, hydroxyindols and
aryldiamines), and their substrate range can be further
expanded by the use of redox mediators, i.e. diffusible
electron carriers from natural or synthetic sources (Cañas &
Camarero, 2010; Maté et al., 2010). The specificity for
dioxygen as the electron acceptor, in contrast to requiring
hydrogen peroxide as acceptor, as is the case for peroxidases,
is an advantage with respect to various applications.
Laccases can be classified as low redox potential (LRP) or
high redox potential (HRP) laccases depending on their redox
potential measured at the Type 1 copper site (T1Cu; to be
discussed later). The redox potentials for the laccase-mediated
catalysis range from +0.43 V versus normal hydrogen elec-
trode (NHE) in bacterial and plant laccases to +0.79 V versus
NHE in certain fungal laccases (Alcalde, 2007; Rodgers et al.,
2009). The higher the redox potential of a laccase is the
broader is the (donor) substrate versatility without the use of
mediators.
 2 A. K. Sitarz et al.
Laccases have been explored to substitute chemical
catalysis in various processes, particularly in pulp and paper
processing. Currently, laccases are intensely investigated for
pre-delignification of biomass to develop more ‘‘green’’
processing regimes, e.g. in bioethanol production from
lignocellulosic biomass (Lu et al., 2010; Qiu & Chen, 2012;
Ryu et al., 2013; Sitarz et al., 2013).
Recent developments in the structural elucidation of novel
laccases allow comparisons of structure–function relation-
ships and investigation of the significance of specific
structural traits of laccases for industrial functionality. The
objective of this review is to: (1) re-examine the detailed
mechanism of the multicopper oxidase biocatalysis reaction,
notably with respect to the reduction of dioxygen to water,
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and via that provide an improved base for identifying the
specific structural traits of significance for employing
laccases to catalyze lignocellulosic and other bioprocessing
reactions; (2) emphasize the recent developments concerning
protein-structural features of importance for the catalytic
reaction and enzyme robustness for industrial use; and (3)
explore how laccases may be optimized for improved
functional properties for industrial applications.
Oxidative lignin modification by multicopper
oxidases
Oxidation of phenolic substrates
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Lignin is a heterogeneous, water insoluble arrangement of
phenyl-propanoid units present in secondary plant cell walls.
Lignin is principally made up of three monolignols:
p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol
(Figure 1). The oxidation of the phenolic substrate is the first
step in laccase catalyzed lignin modification. As discussed in
Figure 1. Schematic representation of three main monolignols forming a
three-dimensional network of lignin.
Figure 2. Formation of resonance-stabilized phenoxy radicals of one of the building blocks of lignin – p-coumaryl alcohol.
Crit Rev Biotechnol, Early Online: 1–17
more detail later, this first catalytic step takes place outside
the core active site pocket of the enzyme, i.e. proximal to the
T1Cu site, where the substrate interacts with amino acids of
the binding loops of the laccase. Laccase-catalyzed oxidation
of phenolic hydroxyl groups of lignin is based on a one-
electron reaction which generates resonance-stabilized phe-
noxy radicals (Figure 2; Kawai et al., 1988). These phenoxy
radicals may undergo a second enzyme-catalyzed oxidation,
which may also be catalyzed by fungal laccases, or react
further in non-enzymatic reactions (hydration, disproportion-
ation or polymerization). The factors that determine the
preference for either one of these mechanisms are not known.
The laccase-catalyzed cleavage of bonds in lignin model
compounds, i.e. the catalytic reaction(s) which may take place
after the initial oxidation of the phenol-hydroxyl groups,
can follow one of the three primary mechanisms: (a) Ca–Cb
cleavage, (b) Ca–oxidation or (c) alkyl–aryl cleavage
(Figure 3).
The laccase-catalyzed oxidation of phenols, anilines and
benzenethiols correlates with the redox potential difference
between the T1Cu site in the active site of the laccase and the
substrate. The phenoxy group is less prone to oxidation when
bulky o- and p-substituents are present. This is due to the
ability of this type of substituents to reduce electron density at
the phenoxy group making the phenol less reactive in
surrendering the electron to the T1Cu of laccase. Moreover,
bulky substituents may cause steric hindrance and thus hinder
proper docking of the substrate resulting in decreased enzyme
catalysis (Xu, 1996).
The structure of fungal laccases
All fungal laccases consist of a polypeptide chain of
approximately 520–550 amino acids, where 20–22 residues
make up the N-terminal signal peptide (Smith et al., 1997),
which is not a part of the mature protein. In rare cases, a
propeptide (Berka et al., 1997) and a C-terminal signal
peptide exist (Hakulinen et al., 2006). The fungal laccases
are extracellular, monomeric proteins of approximately
60–70 kDa, typically having an isoelectric point (pI) around
4.0 (Giardina et al., 2010). Glycosylation can increase the
molar weight significantly as shown for the Rhus vernicifera
laccase (Reinhammar, 1997).
Fungal laccases differ from other members of the multi-
copper oxidase family by the presence of a set of four
ungapped sequence regions, R1–R4, containing conserved
 DOI: 10.3109/07388551.2014.949617
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For personal use only.
Figure 3. Possible degradation mechanisms of lignin units, presented on a phenolic b-1 lignin model compound. The suspected primary degradation
reactions include: (a) Ca–Cb cleavage, (b) Ca oxidation and (c) aryl–alkyl cleavage. Model compound was adapted from (Kawai et al., 1988).
residues of 1 cysteine and 10 histidines that are involved in
binding of the four copper atoms (Table 1 and Supplemental
Figure S1; Thurston, 1994). Alignment of sequences was
made using ClustalW 2.0 software (Goujon et al., 2010;
Larkin et al., 2007) and ESPript for final output (Gouet et al.,
1999). The signal peptides were cleaved off prior to alignment
using SignalP software (Emanuelsson et al., 2007). Eight out
of the 10 histidine residues in these sequence regions appear
in a highly conserved pattern of four HXH motifs in the
enzyme (Table 1; an X in this motif represents an undefined
residue). The HXH motifs are separated by segments of
between 25 and 175 residues (Kumar et al., 2003) but are
likely brought into close proximity in the composite catalytic
enzyme by protein folding. In addition to the R1–R4
sequence segments, laccases have four loop regions, which
have been identified on the basis of 3D structures (Larrondo
Structure, functionality and tuning up of laccases 3
et al., 2003) – these loop segments are designated I–IV, and
are involved in the substrate binding (discussed further
below).
The molecular architecture of laccases revolve around
three tightly associated cupredoxin-like domains, each of
which has a Greek key b-barrel topology (Murphy et al.,
1997) and the 3D crystallographic structure of the Trametes
versicolor laccase, one of the most widely studied fungal
laccases (PDB ID: 1KYA chain C; Figure 4b), has revealed
that the four catalytically important copper ions are situated in
domains 1 and 3. The T1 copper site is thus located in domain
3 with a copper atom (T1Cu) situated in a shallow cleft on the
surface of the enzyme, whereas the T2 and the T3 copper
sites, which have one (T2Cu) and two (T3aCu and T3bCu)
copper atoms, respectively, that form a trinuclear copper
cluster (T2/T3), are positioned at the interface between
 4 A. K. Sitarz et al. Crit Rev Biotechnol, Early Online: 1–17

Table 1. Copper signature sequences in fungal laccases.

Region of Consensus motifsa in signature sequences of

consensus basidiomyceteous laccases Residue (coordinating copper)
64 87 64
R1 HWHG-F/L-FQ-X-GTNWADG-X2-F/G-X-NQCPI H(T2Cu), 66H(T3bCu)
104
R2 GTFWYHSH-L/F-S/G-TQYCDGLRGP-F/MV125 109
H(T3bCu), 111H(T1Cu)
395
R3 HPFHLHGH402 395
H(T1Cu), 398H(T2Cu), 400H(T3aCu)
447
R4 GPWF-L/F-HCHI-D/E-FHL-E/M-X-G-F/L-A-V/I-V-X-A468 452
H(T3aCu), 454H(T3bCu), 458H(T1Cu)
b
Loop sequence motif

Loop I Loop II Loop III Loop IV

159
GPAFPLGAD166 261
ANPNFGNVGFTGGIN275 332
FNGTNFF338 386
ATAAAPGAP393
a
The start position of the signature sequence is shown with respect to T. versicolor (PDB ID:1KYA c) laccase (Bertrand et al., 2002).
Bold H indicates the 10 conserved histidines involved in copper binding, including the four HXH motifs (see text).
b
The range of amino acids forming a loop in G. lucidum’s laccase was predicted based on T. versicolor (PDB ID: 1KYA c) laccase (Larrondo, 2003).
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Figure 4. Graphical representation of two superimposed laccase structures from T. versicolor (PDB ID: 1KYA chain C) and G. lucidum CBS229.93
illustrated using the PyMol Molecular Graphic System Version 1.5 Schrödinger, LLC. (a) The red colored 3D structure of G. lucidum laccase was
predicted by submitting its full amino acid sequence to the 3D comparative protein modeler – 3D-JIGSAW (http://bmm.cancerresearchuk.org/
3djigsaw/) (Bates et al., 2001) where the crystallographic structure of T. versicolor was used as a template. The green sticks represent cysteine
residues responsible for formation of disulphide bridges. (b) The 3D structure of T. versicolor representing a base for prediction of 3D structure for
G. lucidum laccase. The four copper ions (brown spheres) are located in domain 3 (T1Cu) and between domain 3 and 1 (T2Cu, T3aCu and T3bCu).
The orange sticks represent cysteine residues that form disulphide bridges. Domain 1 (residues 1–131 and 476–499) is black, domain 2 (residues 132–
300) is blue and domain 3 (residues 301–475) is green. The range of amino acid residues being part of each domain was predicted according to
(Aleksandrov & Shinddyalov, 2003) and found at www.pdb.org under T. versicolor’s sequence. The ‘‘floating’’ yellow and blue spheres illustrate the
oxygen and water channels, respectively (calculated using CAVER a plug-in for PyMol; Medek et al., 2007). (c) Two superimposed laccases from
G. lucidum (a) and T. versicolor (b) with their potential position of disulphide bridges.

domains 1 and 3 that provide the ligand residues for the
coordination of the copper atoms (Figure 4b). This position of
the copper atoms fits well with the structural motifs available
for other fungal laccases as well – even those produced by
fungi which are phylogenetically distant to T. versicolor. For
example, the first X-ray structure of Coprinus cinerreus
laccase (Ducros et al., 1998) and the superimposed structure
of Ganoderma lucidum laccase (Figure 4a). We have recently
observed that the G. lucidum laccase enhances the glucose
release during cellulose-catalyzed degradation of lignocellu-
lose and have suggested that this effect could be due to the
G. lucidum laccase catalyzing modification of the lignin
 DOI: 10.3109/07388551.2014.949617
(Sitarz et al., 2013). A closer analysis shows that the 3D
structure from T. versicolor has more than 80% homology at
the conformational level with the laccase from G. lucidum.
The laccases from T. versicolor and G. lucidum immediately
appear to be very similar when superimposed: for instance,
both have a hypothetical water and oxygen channel, which are
located between domains 1 and 3, and 3 and 2, respectively
(Figure 4). However, when compared more closely, some key
differences in the topology of these two enzymes become
apparent and slight conformational differences can thus be
observed in loops II and III, where the T. versicolor laccase
contains a fragment of a b-strand (Supplemental Figure S2).
One of the intriguing aspects in the 3D structure of the
G. lucidum laccase, that might have implications during
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catalysis, is moreover the existence of an additional a-helix in
the vicinity of the trinuclear copper site. The presence of this
a-helix results in an additional residue (TRP107) in the
vicinity of the T2/T3 copper site (Figure 5).
The tertiary structure of basidiomycete laccases is
stabilized by two disulfide bridges between domain 1 and 2,
and between domain 1 and 3 (Figure 4a–c). The two
disulphide bridges are formed between CYS85 and CYS488,
and between CYS117 and CYS205, and are present in the
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Figure 5. Graphical representation of the three copper centers in
laccases for two superimposed laccases from T. versicolor (PDB ID:
1KYA) – green sticks, and G. lucidum CBS229.93 – red sticks,
respectively. Copper sites are shown as brown spheres. Two coordinating
water molecules are presented as red spheres. The TRP107 residue (red)
of the G. lucidum laccase is in the vicinity of 3.7 Å from the T3b copper
center. The T. versicolor laccase lacks this residue in that position. The
interatomic distances of residues belonging to the G. lucidum laccase, in
the vicinity of each copper site, are shown as black nicked lines and
values are given in Angstrom [Å]. The amino acid nomenclature and
their position, according to the G. lucidum laccase, are written in black.
Nd1, colored blue, represents a nitrogen atom from which a proton shifts
to the carbonyl of Cys453, when assisting in the electron transfer from
T1Cu(+) to T2Cu(++) while the dioxygen is bound to the T2/T3 copper
cluster. The T1–Cys453–His452–T3b pathway, which enables the
electron transfer from T1Cu(++) to T2/T3(++) copper cluster is
shown via signed single amino acids.
Structure, functionality and tuning up of laccases 5
exact same place for T. versicolor and G. lucidum laccases
(Figure 4c). The position and number of disulfide bridges is
not conserved throughout fungal multicopper oxidases,
however, but they can still be arranged into two groups;
basidiomycetes with two and ascomycetes with three
disulphide bridges (Hakulinen et al., 2002).
The active site of fungal laccases
The T1 copper center (blue copper center)
The T1 copper has a characteristic absorbance at 614 nm and
can be detected by electronic paramagnetic resonance (EPR;
so-called ‘‘paramagnetic blue copper’’). The charge transfer
transition SCys ! Cu(++) is responsible for the deep blue
color of the enzyme (Lee et al., 2002). The T1Cu ion has a
trigonal coordination (Figure 6) with two histidines and one
cysteine as conserved equatorial ligands (Claus, 2004). In
addition, two non- (or weakly) coordinating residues, one of
them a methionine residue, have also been observed in the
vicinity (4 Å) of the T1Cu(++) center (Rodgers et al., 2009).
The methionine residue has been suggested to be a fourth
ligand, but since the electron donating ability of the thioether
group is considerably lower than the sulphur in cysteine
and the imidazole nitrogen, the coordinating property may
be low (Gray et al., 2000). A non-coordinating phenylalanine
or a leucine may also be found in this position in several
fungal laccases (Figure 6). The lack of the fourth coordinating
axial ligand in fungal laccases may be an advantage and
considered an important factor determining the higher redox
potential values (E0), displayed by these laccases (Figure 7),
Figure 6. A graphical representation of the T1 copper site (brown
sphere) for two superimposed laccases from T. versicolor (PDB ID:
1KYA) – green sticks, and G. lucidum CBS229.93 – red sticks. The
interatomic distances [Å] are based on the location of residues belonging
to G. lucidum’s laccase. The amino acid nomenclature and their position
according to G. lucidum laccase are written in black. PHE463 and
ILE455 are non-coordinating amino acids and the dashed lines do not
represent the binding to the copper ion.
 6 A. K. Sitarz et al. Crit Rev Biotechnol, Early Online: 1–17
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Figure 7. Potentials of the T1 copper site of some oxidoreductases. The values in the boxes represent the redox potential and the given amino acid
residues represent residues in the axial position of the T1 copper. Zuccini (Caburbita pepo), Myrothecium verrucaria, Homo sapiens, and Trametes
villosa (Alcalde, 2007; Kumar et al., 2003), Rhus vernicifera (Alcalde, 2007; Yaropolov et al., 1994), Myceliophthora thermophila (Alcalde, 2007;
Alcalde et al., 2005; Kumar et al., 2003), Scytalidium thermophilum (Alcalde, 2007; Kumar et al., 2003; Xu et al., 1995), Coprinus cinereus (Alcalde,
2007; Kumar et al., 2003; Schneider et al., 1999), Pleurotus ostreatus and Trametes trogii (Garzillo et al., 2001; Morozova et al., 2007), Rhizoctania
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soalni (Alcalde, 2007; Kumar et al., 2003; Wahleithner et al., 1996), Neurospora crassa (Alcalde, 2007; Piontek et al., 2002), Cerrena maxima
(Koroleva et al., 2001; Morozova et al., 2007a), Trametes versicolor (Alcalde, 2007; Alcalde et al., 2005).

compared to other multicopper oxidases (Giardina et al.,
2010; Gray et al., 2000). Phenylalanine may determine a
higher redox potential of the T1Cu center (Eggert et al.,
1998). Therefore, it is also expected that the redox potential of
G. lucidum’s laccase will be high, due to the presence of a
non-coordinating PHE463 residue in the vicinity of the T1Cu
center (Figure 6).
A non-coordinating ILE455 is also found in the vicinity of
the T1Cu site (Figure 6). Moreover, due to its high redox
potential (0.5–0.8 V; Xu et al., 1996; versus NHE), the T1Cu
is the primary electron acceptor site where substrate oxidation
takes place and HIS458, occurring in fungal laccases,
is believed to be the entrance door for the electrons
abstracted from a reducing substrate during their transfer to
T1Cu(++).
The T2 copper site (normal copper center)
T2 copper confers no color (so-called: paramagnetic
‘‘non-blue’’ copper), but can still be detected by EPR
(Claus, 2004). Moreover, it is strategically proximal to the
T3 coppers and coordinates with two histidine residues and
one oxygen atom as an OH ligand, forming a trigonal
coplanar configuration (Figure 5; Xu et al., 2000).
The T3 copper site (coupled binuclear copper center,
containing two Cu atoms)
T3 copper center is a pair of copper atoms, T3aCu and
T3bCu that gives a weak absorbance in the near UV
spectrum (330 nm) and it does not have any EPR signal
(Claus, 2004). Each of the T3 coppers coordinates with
three histidine residues and a bridging oxygen atom
having a distorted tetrahedral geometry (Figure 5; Xu et al.,
2000).
The T2/T3 copper site (trinuclear copper center)
The T2/T3 copper center is a complex domain comprising
both the T2 and T3 copper sites. It contains three copper
atoms, and is localized approximately 13 Å away from the
T1Cu site (Reinhammar & Malmstrom, 1981). The T2/T3
copper site carries out the reduction of molecular oxygen
using the electrons transferred from the T1Cu site. The T3
copper ions are symmetrically connected to six imidazole side
chain residues from histidines and T2 copper is connected to
two imidazoles (Figure 5). The geometry of this structure is
trigonal planar. The conserved residues of G. lucidum and
T. versicolor are well aligned to each other. Moreover, one
additional residue, TRP107, is present only in the
G. lucidum’s laccase and correlates to the observed a-helix
from Figure 4(c) that is positioned in the vicinity of the
catalytic coppers. However, further evidence will require an
elucidation of the crystallographic structure of G. lucidum’s
laccase.
The trinuclear center has access to the solvent through a
water channel (Figure 4a and b), leading to the T2 and T3
copper atoms. The water channel leading to the T3 coppers
contain residues like Asp and Glu, which are capable of
donating protons (Bento et al., 2006), to form a hydrogen
 DOI: 10.3109/07388551.2014.949617 Structure, functionality and tuning up of laccases 7
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f)
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Figure 8. A two 2-electron mechanism of O2 reduction to H2O by multicopper oxidases, adapted from (Augustine et al., 2010; Solomon et al., 2008).
Red arrows indicate steps taking place in the catalytic cycle of multicopper oxidases, while black arrows indicate steps that can be experimentally
observed but which are not a part of the catalytic cycle. (a) The fully oxidized state has four copper atoms in their (++) oxidation state and two OH
molecules (one is bridging between T3bCu(++) and T3aCu(++) and the second one bound to the T2Cu(++) center). The fully oxidized enzyme
receives, in total, 4 e from its T1Cu(++) center, which are transferred, one by one, over ca. 13 Å to the T2/T3 trinuclear cluster, forming a fully
reduced active site structure (b) that can accept an O2 molecule. O2 preferentially binds to the T3bCu(+) ion and is then reduced by 2e, one from
T3bCu(+) and one from T2Cu(+) forming a peroxide intermediate (PI; c) with its 2e reduced O2. The driving force for the 2e reduction is a
negatively charged D94 residue (D94 deprotonates by donating a H+ to the OH ligand bound to the T2 center – step a), which lowers the potential of the
T3b–T2 edge. After the first 2e reduction of O2, one electron is transferred intramolecularly from T1Cu(+) to T2Cu(++) via T1–Cys–His–T3b
pathway. This transfer is assisted by an H-bond shunt between the carbonyl oxygen and T1Cu–Cys ligand and Nd1 on the His483 ligand bounded to the
T3bCu, thus forming a PI + e intermediate (d). As a result, the 1e reduced T2Cu(++) loses its strong bond to peroxide and a H+ from E487 is
transferred to the peroxide. The conversion of H2O to OH, while protonating the D94 residue, lowers the redox potential of the T2Cu center and
increases the driving force for another 1e transfer to peroxide, thus assisting in O–O bond cleavage, producing a fully oxidized native intermediate
(NI) with two oxide bridges in the catalytic cavity (e). In excess of the reducing substrate (RH), NI can be reduced to a fully reduced form of the enzyme
by accepting 4 e and 4H+ (3H+ will protonate two oxide bridges and release 2 H2O molecules, 1H+ will protonate E487 residue and 1H+ will transfer
from D94 residue to the OH ligand bounded to the T2Cu center). In lack of RH, NI will accept only 2H+ which will protonate only one of the oxide
bridges, releasing only one H2O molecule. (f) A graphical representation of the electron transfer pathway (T1–Cys484–His483–T3b) and copper
centers in Fet3p protein (PDB ID: 1ZPU, chain D). The 3D structure of copper sites was modeled using the PyMol Molecular Graphic System Version
1.5 Schrödiner, LLC. According to the modeling pathway after knowing the crystal structure, electrons (that are used to reduce O2 to H2O) go through
T1Cu ! Cys-S ! Cys-C ¼ O ! H-bond (of the Nd1 of His483), N"2 of His483, then to the T3b (red arrows).
 8 A. K. Sitarz et al.
network with the water molecules in the channel and therefore
allows for fast access of dioxygen and also effective release of
water after performing a ‘‘ping-pong’’ reaction (Bukh et al.,
2006).
Reaction details of laccase catalysis
Reduction of dioxygen to water
The second step, or the parallel half-reaction, which occurs
simultaneously with the phenol oxidation, is the laccase-
catalyzed reduction of dioxygen to two molecules of water
(Dwivedi et al., 2011; Quintanar et al., 2005; Rulišek et al.,
2005; Solomon et al., 2008). This reaction takes place in
the catalytic pocket of the enzyme, at the so-called T2/T3
copper site (discussed in more detail below), and requires
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four reduced copper ions, i.e. that all four copper ions are
Cu(+). The oxidation of the copper ions occurs only
when four single electrons are abstracted from the hydroxyl
groups of the phenolic substrate (hence referred to as the
reducing substrate; Figure 8a and b). The overall stoichiom-
etry is:
aÞ 4e bÞ
4 CuðþþÞoxid: ! 4 CuðþÞred:
c, dÞ
þ O2 ! 2½CuðþþÞCuðþÞoxid:=red:
eÞ 4e 4e bÞ
! 4 ½CuðþþÞoxid: ! 4CuðþÞred: þ2H2 O
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Superscript letters a, b, c, d and e represent the individual
steps in the catalytic cycle (Figure 8), and stoichiometric
forms in square brackets are the intermediate forms generated
during the redox reaction.
The most convincing model for the catalytic mechanism of
the multicopper oxidases, including improvements of the
intermediate state structure, has been proposed by Solomon’s
group (Augustine et al., 2010; Solomon et al., 2008). This
model (Figure 8) is based on the catalytic mechanism of the
Fet3p protein [a multicopper oxidase from Saccharomyces
cerevisiae (PDB ID: 1ZPU)] functioning in iron transport
(Taylor et al., 2005). A comparison of the trinuclear cluster of
Fet3p and its environment with that of other known structures
of this enzyme family, e.g. the T. versicolor laccase, has thus
shown that the trinuclear cluster is structurally highly
conserved (Piontek et al., 2002); in the context, the
‘‘trinuclear cluster’’ refers to the organization of three of
the four copper ions in three reaction ‘‘nuclei’’ sites
composed of the T2 and T3 sites in which the T3
encompasses the pair of two copper ions referred to as T3a
and T3b, respectively (Augustine et al., 2010; Figure 8). The
tripeptide His–Cys–His, involved in the electron transfer
pathway between the T1 copper and the T2/T3 in the cluster is
also retained when comparing the Fet3p and the T. versicolor
laccase structures (Piontek et al., 2002).
Therefore, the mechanism of the copper oxidation and
dioxygen reduction is presumed to be common among all blue
multi-copper oxidases.
Details of the electron transfer steps
The mechanism of copper oxidation and dioxygen reduction
to water taking place in the active site of laccases can be
Crit Rev Biotechnol, Early Online: 1–17
divided into five steps (Figure 8): The first step is initiated by
abstraction of a single electron from a reducing substrate: in
total, four single electrons from four reducing substrate
moieties (i.e. phenol hydroxyls) are required in the overall
process (Figure 8a). The laccase stores the electrons as a
battery during the first step of the oxidation half-reaction so
that these electrons can be used during the dioxygen
reduction. The initial phase of electron abstraction takes
place at the T1Cu site of a fully oxidized (resting) form of the
enzyme (Figure 8a), i.e. where all copper atoms are at the
(++) oxidation state and where one OH group is bridging
between the T3aCu(++) and the T3bCu(++) copper ions and
the second OH group is bound to the T2Cu(++) copper
center. In addition, the fully oxidized structure of a multi-
copper oxidase is stabilized by hydrogen bonding between a
deprotonated glutamic acid (E487), an oxide bridge of the
T3bCu(++) and the T3aCu(++), and a water molecule
between the two latter molecules (Augustine et al., 2010).
There are two highly conserved amino acid residues, namely,
aspartic acid (D94) and glutamic acid (E487), in the vicinity
of the catalytic pocket, which act together to create a flow of
protons from the T3Cu site (involving both the T3aCu and the
T3bCu) of the trinuclear cluster to the T2Cu site, a flow that
drives the cleavage of the O–O bond in the subsequent steps
of the catalytic reaction (Augustine et al., 2007). The E487
amino acid residue is deprotonated and is located near the T3
center and appears to be responsible for first accepting a
hydrogen during the initial reduction reactions of the four
copper ions (Figure 8a and b), and subsequently facilitating
the donation of a H+ during the reductive cleavage of O–O
bond in the peroxy intermediate stage of the enzyme catalysis
(Figure 8c and d).
The other highly conserved amino acid in the active site is
a protonated aspartic acid residue (D94), located near the
T2Cu, which plays a key role in the reaction of the reduced
trinuclear center with dioxygen. The D94 presumably assists
in driving the electron transfer from the T2Cu center to cleave
the O–O bond by deprotonating the T2Cu water ligand
[Cu(++)–H2O to Cu(++)–OH; Figure 8(d); Augustine et al.,
2007].
The four one-electron-reduction of the four copper ions is a
slow process mainly because only one electron is moved
around the catalytic site at a time and travels from the
T1Cu(++) to the T2/T3(++) trinuclear cluster over a distance
of 13 Å (Figure 5). After receiving one electron on each of
T3Cu’s, the bond between T3bCu(++), T3aCu(++) and the
hydroxide is weakened, and the hydroxide is being protonated
by H+ from the bulk (aqueous) environment (Figure 8a
and b). That the hydroxide becomes protonated by H+ from a
water molecule agrees with the T2/T3 trinuclear center being
positioned in a water channel (Figure 4a and b). As a result, a
protonated hydroxide dissociates as a water molecule through
a T2/T3 water channel leaving the catalytic cavity in a fully
reduced form (Figure 8b). However, before the catalytic
cavity is able to accept a dioxygen molecule, glutamic acid
(E487) needs to be protonated by H+ from the bulk solution
and aspartic acid (D94) needs to be deprotonated. D94 loses
its H+ to the OH ligand bound to the T2 copper center and
forms a H2O molecule (Figure 8b). Now, the fully reduced
enzyme is ready to accept a dioxygen molecule, which has a
 DOI: 10.3109/07388551.2014.949617
preference to bind to T3bCu(+), due to its geometry.
Computational studies have shown that the T3bCu(+)
copper ion has a trigonal pyramidal geometry, which is
more reactive to bind a fourth ligand than a more energet-
ically stable trigonal planar geometry of the T3aCu(+) copper
ion (Augustine et al., 2010).
Reaction details of the oxygen reduction in the active
site of laccases
When the dioxygen has docked into the catalytic cavity, the
redox potential of the T3bCu(++) and the T2Cu(++) is being
lowered by a negative charge from the deprotonated D94
residue. This provides the driving force necessary to reduce
the dioxygen by 2 electrons (the 2e are coming from the
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Tennessee on 09/11/14
T3bCu(+) and T2Cu(+) copper ions and not from a reducing
substrate), thereby forming a peroxy intermediate (PI;
Figure 8c). The 2e reduced dioxygen is now tightly bound
to the trinuclear cluster as coordinated side-on to the
T3bCu(++) and also bonds to both the T2Cu(++) and the
T3aCu(+) copper ions (Figure 8c). This coordinated bonding
helps to stabilize the peroxide and is facilitated by the
structural flexibility of the T3a–T2 edge which moves from a
distance 45 Å in the reduced state to 4 Å in the PI
(Augustine et al., 2010).
The next step in the dioxygen reduction to water, based on
Fet3p protein, is a rapid, single electron transfer from the
For personal use only.
T1Cu(+) to T2Cu(++) via the T1–Cys484–His483–T3b
pathway (Figure 8f; or T1–Cys453–His454–T3b pathway in
the laccase from Ganoderma lucidum CBS229.93, Figure 5).
The transfer of an electron is assisted by an H-bond shift
(shunt) between the carbonyl oxygen (C–C¼O) of T1Cu–
Cys484 ligand (Cys453 in the G. lucidum laccase) and Nd1 of
the His483ligand (His454 in the G. lucidum laccase Figure 5)
of the T3bCu, forming a PI + e intermediate (Figure 8d).
In contrast, the T1–Cys484–His485–T3a pathway (and pos-
sibly the T1–Cys453–His452–T3a pathway in the G. lucidum
laccase) does not have a comparable H-bond (Augustine et al.,
2008, 2010; Taylor et al., 2005). As a result of this
intramolecular transfer, the T2Cu(++) copper center is
being reduced and loosens its bond to the peroxide
(Solomon et al., 2008). Afterwards, two single electrons
from T3aCu(+) and T2Cu(+) are transferred onto the
antibonding orbitals of the peroxide causing an increase in
the energy and making the peroxide structure very unstable
(Figure 8d). Therefore, to lower the bonding order, the O–O
bond is cleaved and the proton from E487 is transferred to the
peroxide (Figure 8e). In addition, the pKa decreases for the
water molecule and a proton from the T2Cu(+)-bound water
is transferred to the D94. This conversion of H2O to OH
lowers the redox potential of the T2 copper center and
increases the driving force for an electron transfer to peroxide,
thus assisting in O–O bond cleavage (Augustine et al., 2007)
and decay of PI, producing a fully oxidized Native
Intermediate (NI; Figure 8e).
When the NI forms, i.e. when the fully oxidized form of
the multicopper oxidase with two bridging oxides is formed, it
can either (1) return to a fully reduced form of the enzyme by
accepting 4H+ (Figure 8e ! b) and releasing two water
molecules or (2) it can return to the fully oxidized state by
Structure, functionality and tuning up of laccases 9
releasing one water molecule (Figure 8e ! a). The latter
occurs when laccase does not have enough of the reducing
substrate to oxidize. On the other hand, to be able to release
two water molecules, a laccase needs 4H+. 3H+ of the four are
needed directly for the release of two H2O molecules since 1
H+ (not accounted for in the gross reaction) is transferred
from the protonated D94 residue to the OH ligand on the T2
copper center. The last fourth H+ protonates a deprotonated
E487 residue and 4e from four molecules of a reducing
substrate reduce the four coppers in the active site of laccase
simultaneously releasing two water molecules, or it can
release one water molecule and return to its fully oxidized
form (Figure 8a). Release of two water molecules is possible
due to the flexibility of the T3a–T3b edge which facilitates an
increase in the Cu–Cu distance from 53.5 Å in NI back to
45 Å in the reduced form. Opening of the trinuclear cluster
enables the elimination of two water molecules (Augustine
et al., 2010) formed probably by protons provided by the bulk
solvent in the T2/T3 channel. Therefore, the way the reaction
follows is determined by the availability of a reducing
substrate. In excess of it, laccase will return to its fully
reduced form and be ready to accept a dioxygen molecule
(Augustine et al., 2010). However, if the amount of substrate
is insufficient, the enzyme will return to its resting state and
remain in that form until it can be oxidized again.
Other models for the catalysis
Other catalytic models, in particular for the dioxygen
reduction steps, have been presented in the literature:
Andréasson et al. (1976) studied the kinetics of laccase by
mixing the fully reduced enzyme with dioxygen and
visualized the intermediates formed by EPR and UV–VIS
spectroscopy. On this basis, a catalytic model was proposed,
which we believe lacks a thorough evaluation, in which the
dioxygen is supposedly reduced to water directly by accepting
four electrons. Accordingly, the two first electrons were
suggested to be transferred from the T3 pair of copper ions
giving a peroxide end-on coordinated to the T3bCu(++). The
third electron was proposed to be donated from the T1Cu(+)
to reduce the peroxide end-on coordinate and in turn form an
oxygen radical and the last, fourth electron was believed to be
transferred from the T2Cu(++) to the previously formed
oxygen radical, which showed a slow decay in the EPR signal.
However, the reaction transfer from T2Cu(++) was too slow
to be catalytically significant, and therefore it was suggested
that during this turnover reaction, the last electron was
donated to the oxygen radical from a reducing substrate
molecule via the re-reduction of the T1Cu(+). Moreover, the
oxidized laccase form was proposed to have an oxide bridge
between T3aCu(++) and T3bCu(++) (Andréasson et al.,
1976; Bukh et al., 2006; Malmström et al., 1969). A refined
model was based on Magnetic Circular Dichroism studies of
the long-lived optical intermediate (Clark & Solomon, 1992).
According to this model, proposed by Shin et al. (1996) and
improved by Quintanar et al. (2005), dioxygen was also
reduced to water by accepting four electrons. The first two
electrons were proposed to be donated from the T3 and T2
copper sites. The peroxide was then presumed reduced to a
hydroxide ion via the simultaneous donation of two electrons
 10 A. K. Sitarz et al. Crit Rev Biotechnol, Early Online: 1–17

from the T1 and T2 copper ions. The optical intermediate was
suggested to arise from the hydroxide ion bridging T3 and T2.
The oxide/hydroxide ion bridging the T3 pair was then
proposed to derive from the solution, whereas the hydroxide
ion or water molecule coordinated to the T2 copper ion, in the
fully oxidized enzyme was proposed to originate from
reduced dioxygen (Bukh et al., 2006; Shin et al., 1996). The
optical intermediate was formed simultaneously with the
oxidation of the T1Cu site.
In summary, all the available models (Andréasson et al.,
1976; Augustine et al., 2010; Chalupský et al., 2006;
Messerschmidt et al., 1993; Quintanar et al., 2005; Rulišek
et al., 2005; Shin et al., 1996; Solomon et al., 2001, 2008)
each have their own interpretation of the intermediate
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Tennessee on 09/11/14
oxidized intermediates are electrochemically unstable
(Morozova et al. 2007b). In addition, a redox potential of a
mediator seems to play a negligible role in the catalytic
efficiency of lignin oxidation. Their effectiveness is likely to
depend on the chemical reactivity of the radical formed after
their initial oxidation step (González et al. 2009). In general,
mediators are low molecular weight compounds able to
generate stable radicals (in the oxidized form) while avoiding
laccase inactivation. For industrial use, the ideal mediator
should be recyclable without degeneration, be available at low
cost and environmentally safe (Cañas & Camarero, 2010).
Artificial mediators
The two most commonly used artificial mediators are

structures formed during the oxygen reduction to water. We

2,20 -azinobis(3-ethylbenzthiazoline-6-sulphonate) (ABTS;
believe that the model described here (Figure 8), which is
Bourbonnais & Paice, 1990) and 1-hydroxybenzotriazole
based on an H-bond shunt, gives the most reasonable
(HBT; Call & Mücke, 1997; Hirai et al., 2006; Kawai et al.,
explanation to what really happens in the catalytic pocket
2002). They both oxidize non-phenolic substrates of lignin
during the redox reaction. However, the three major steps that
(representing 80–90% of lignin; Kawai et al., 2004) but follow
are independent of the intermediate structures seem to be
different mechanisms. Laccase/mediator catalyzed oxidation
common for all models. Those are (Witayakran & Ragauskas,
of non-phenolic substrates (etc. 4-methoxybenzalcohol) can
2009):
proceed via two different mechanisms depending on the
(1) The T1 copper ion, in its resting state, is reduced by
chemical structure of the mediator rather than the difference in
accepting electrons from the reducing substrate.
redox potential between the target substrate and the mediator
(2) Electrons are transferred internally ca. 13 Å from the
(Galli & Gentilli, 2004). The mediators containing a N–OH
T1 copper to the T2/T3 trinuclear cluster.
structural feature, such as HBT, violuric acid, 3-hydroxyantha-
(3) Molecular oxygen is activated and reduced to water at the
nilic acid and N-hydroxy-phthalimide, favor a radical
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T2/T3 trinuclear center.

Hydrogen Atom Transfer (HAT) pathway (Figure 9a). In this
type of mechanism, the conversion of non-phenolic lignin
Oxidation of non-phenolic substrates – laccase/ substrates into a product is initiated by an enzymatic,
mediator-catalyzed oxidation monoelectron oxidation of a mediator which yields in a
formation of the radical cation, which in turn deprotonates
Laccases have a lower redox potential (0.5–0.8 V versus
NHE) than ligninolytic peroxidases (41 V versus NHE) and it
was initially thought that laccases would only be able to
oxidize phenolic substrates which represent less than 20% of
lignin (Galli & Gentilli, 2004; Kersten et al., 1990). Phenols
are typical laccase substrates because their redox potential,
ranging from 0.5 to 1.0 V versus NHE, is low enough to allow
electron abstraction by the laccase T1 copper (Giardina et al.,
2010). Moreover, substrates characterized by a high redox
potential (non-phenolic substrates) cannot be directly oxi-
dized by laccase and therefore in order to broaden the range of
those substrates being oxidized, a laccase needs a presence of
a mediator. A mediator is a small molecule that acts as an
electron shuttle and expands the oxidative capacity of laccase
(Giardina et al., 2010). It does so by an ability to form a
strongly oxidizing intermediate (i.e. Medox) upon oxidation.
After a mediator loses an electron for laccase, it diffuses away
from the catalytic pocket of the enzyme and, in turn, carries
out a mono-electronic oxidation of any substrate that, due to
its size or steric hindrance may not directly enter into the
active site of the laccase (Galli & Gentili, 2004). The better
the mediator the more conjugated double-bond system
facilitating electron abstraction it has. One of the examples Figure 9. Schematic representation of the mediator-substrate oxidation
for a mediator having a strong mediating capacity is sinapic of lignin model compound; 4-methoxybenzalcohol, catalyzed by
acid (SA). It can be fast oxidized by laccase and produce a laccases via two different routes: (a) the radical Hydrogen Atom
Transfer (HAT) route, and (b) the Electron Transfer (ET) route. Medox
high concentration of stable free radicals (Camarero et al., represents oxidized mediator and Medred represents a reduced mediator
2008). However, be aware, most compounds described as (schematic representation of the mechanism adapted from Fabbrini et al.,
laccase mediators are not strictly redox mediators since their 2002).
 DOI: 10.3109/07388551.2014.949617
giving the highly reactive N-oxyl radical (4N–O’; Baiocco
et al., 2003). The latter abstracts in turn the benzylic hydrogen
from the non-phenolic substrate of lignin and itself forms
an aldehyde (etc. conversion of 4-methoxybenzylalcohol into
a 4-methoxybenzaldehyde, Figure 9a). The driving force of
this reaction is the enthalpy balance between the dissociated
bond (C–H) in the non-phenolic, target substrate and the
formation of the NO–H bond in the mediator. Therefore,
this type of mechanism requires substrates with relatively weak
C–H bonds and mediators which can form N-oxyl radicals
upon enzymatic oxidation and removal of an electron (Fabbrini
et al., 2002). In addition, the efficiency of catalytic oxidation of
various 4N–OH compounds [expressed as log(kcat/Km)]
depends on the difference between the redox potential of the
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Tennessee on 09/11/14
4N–OH compound and the redox potential of the T1 copper of
laccase (Morozova et al., 2007a,b).
In contrast, mediation by ABTS is suggested to involve the
oxidation of the non-phenolic substrate by an Electron
Transfer (ET) pathway (Figure 9b). In this type of mechanism,
the Medox form of ABTS that is responsible for oxidation of
the non-phenolic substrates of lignin is ABTS++ (rather than
a radical cation – ABTS+’ that can only react with lignin
phenolic groups; Fabbrini et al., 2002; Morozova et al.,
2007a,b). In addition, the initial monoelectronic oxidation of
the benzylic alcohol by the previously oxidized mediator is
followed by a fast C–H deprotonation of the intermediate
radical cation of the alcohol that drives the conversion to
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aldehyde (Baiocco et al., 2003).
Mediators of natural origin
Mediators of natural origin are compounds that are available
in nature and which may be lignin degradation products, plant
phenolics (precursors for lignin biosynthesis) or secondary,
extracellular, fungal metabolites (Cañas & Camarero, 2010).
Among the latter ones, e.g. 4-hydrobenzylic alcohol
(Johannes & Majcherczyk, 2000) and 4-hydroxybenzoic
acid (Murayama et al., 2007), have been proven able to
oxidize benzo[a]pyrene (PAH). In contrast, the first dis-
covered secondary metabolite produced by Pycnoporus
cinnabarinus; 3-hydroxyanthranilic acid (Eggert et al.,
1996), which was believed to mediate oxidation of non-
phenolic substrates and synthetic lignin during laccase
catalysis was shown to generate a cinnabarinic acid during
the oxidative coupling, which was unable to mediate the
oxidation of non-phenolic compounds (Li et al., 2001).
Hence, widely available phenolic substrates that originate
from plants and which are most likely the true mediators of
laccase activity during fungal (white-rots) biodegradation of
lignin have also been confirmed to take part in transformation
of PAH and decolorization of some industrial dyes (Camarero
et al., 2008).
There are few more examples of oxidation of phenolic
substrates with help of natural mediators used in industrial
applications, including acetosyringone, syringaldehyde
[delignification of paper pulps (Camarero et al., 2007), dye
decolorization (Murugesan et al., 2009), dehalogenation of
pesticides (Torres-Duarte et al., 2009)], vanillin, acetovanil-
line, ferulic acid and p-coumaric acid [anthracene
and benzo[a]pyrene transformation (Cañas et al., 2007)].
Structure, functionality and tuning up of laccases 11
Natural mediators typically follow the Hydrogen Atom
Transfer (HAT) oxidation mechanism (Figure 9a; d’Acunzo
& Galli, 2003), and are regarded as the most efficient non-
phenolic degraders in laccase catalyzed reactions. The term
‘‘natural’’ not only refers to the natural origin of these
compounds, but also defines their role in nature, as being the
most likely true mediators during laccase catalyzed biodeg-
radation of lignin polymers (Cañas & Camarero, 2010).
What is a good laccase? Desirable features of an
industrial oxidoreductase
Given the different applications, the required features may not
be fulfilled by a single good laccase discovered in nature or
developed by a focused molecular evolution strategy.
Laccases described in recent publications (Moreno et al.,
2013; Ryu et al., 2013), are proposed to assist in the
pretreatment of biomass by catalyzing the removal of
inhibitors in lignocellulosic slurries, but the suggested
applications of laccases range from paper and pulp bleaching
(Galli et al., 2011), detergent and textile applications
(bleaching purposes; Janssen et al., 2004), gelation and
emulsion stabilization of food systems (Zaidel et al., 2012,
2013), bioremediation (Galli et al., 2011) and even biomed-
ical applications have been proposed in connection with
development of a laccase tolerant to human blood components
(Maté et al., 2013). Moreover, as described already 20–25
years ago, laccases can increase water-soluble degradation
products by catalysis of ring opening of low molecular weight
phenolic lignin model compounds (Kawai et al., 1988) as well
as catalysis of C–C bond cleavage of ‘‘synthetic lignin’’
substances (Iimura et al., 1995).
Resistance of laccase to inactivation by the free
radicals
The presently known laccases have a number of drawbacks
with respect to robustness, redox potential and dependency
of mediators. Although the mediators stimulate the catalysis,
the laccase may be sensitive to the free radicals of the
mediator, which subsequently may inactivate the laccase and
reduce the rate of oxidation of the non-phenolic lignin
structures.
For example, the mediator, violuric acid inactivates the
laccases from Trametes villosa, Pycnoporus cinnabarinus,
Botrytis cinerea and Myceliophthora thermophila at a much
higher rate than that of N-hydroxybenzotriazole (HBT).
If violuric acid free radicals generated by the laccases could
be consumed by the substrate fast enough, then inactivation of
the laccases may be avoided and oxidation of the substrate
would mainly depend on the kcat of violuric acid (Li et al.,
1999).
High kcat for an effective laccase mediator system with
high redox potential
The kcat is dependent upon the electron transfer from a
substrate to the T1 copper, which in turn is dependent on the
difference in the redox potential between the laccase and a
substrate, and the affinity between the laccase and the
substrate.
 12 A. K. Sitarz et al.
Increase of the redox potential value of T1Cu of
laccases of more than 0.7 V
It has been experimentally shown that the laccase from the
thermophilic mould Myceliophthora thermophila has a
redox potential which is too low to efficiently oxidize
1 -(3,4-dimethoxyphenyl)-2 -(2-methoxyphenoxy)propan-1,3-
diol in the presence of 1-HBT or violuric acid. Since an
effective laccase mediator system (LMS) must have a redox
potential high enough to oxidize non-phenolic lignin, then
laccase should also have redox potential high enough to make
the oxidation of an effective laccase mediator kinetically
possible (Li et al., 1999). Xu et al. (1996) suggested a number
of factors that may influence the redox potential of the T1Cu
site in fungal laccases. The first is the solvent accessibility
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Tennessee on 09/11/14
previously reported for the iron–sulfur proteins (Langen et al.,
1992). The second may be an internal hydrogen bonding
to the ligands at the T1Cu site, which could change
the electron density at the copper center. The third factor
may be contributed by the dialectric anisotropy around the
T1Cu site.
Laccase optimization by molecular evolution
Several novel laccases have been discovered in diversity
screenings, but none with superior performance on biomass
delignification. Further optimization by directed evolution
techniques is therefore necessary to meet industrial needs.
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One of the main targets for improvement is the T1Cu site,
which defines the redox potential of the laccases. A higher
redox potential at the primary electron acceptor site is
desirable to oxidize a range of recalcitrant polymers in the
biomass. Generally, different regions of the laccase polypep-
tide chain have been subjected to specific modifications
(Table 2). They involve:
(1) Replacement of aromatic amino acids residues with non-
aromatic side chains to make laccases less vulnerable to
free-radicals and to prevent their inactivation (Li et al.,
1999).
(2) Improvements of the substrate-binding site to accommo-
date a better docking of the reducing substrate.
Based on predicted modeling, specific amino acid residues
involved in hydrophobic protein–ligand interactions were
selected and substituted by mutation (Table 2; Galli et al.,
2011; Mohamad et al., 2008). By this method, the
T. versicolor laccase became more active towards bulky
phenolic substrates. The modeling also revealed that PHE162
was a critical residue located in the T1Cu site, and that
substitution with an apolar alanine residue resulted in an
increased oxidation of a bulky substrate such as bisphenol A
(BPA; Table 2). Moreover, a double mutation in positions
PHE162ALA and PHE332ALA produced a cooperative
effect, resulting in 98% oxidation of BPA in only 5 h.
Optimization of the T1Cu redox potential can, in part, be
correlated to the axial ligands hydrophobicity, which is
presumed to increase the redox potential of the primary
electron acceptor site. This hypothesis is true for laccase from
Bacillus subtilis (CotA; Durão et al., 2006), E. coli (CuO;
Sakurai & Kataoka, 2007) and Pseudomonas aeuroginosa’s
azurin (Karlsson et al., 1989; Pascher et al., 1993), where
mutation of an axial Met ! Leu resulted in increase in redox
Crit Rev Biotechnol, Early Online: 1–17
potential by as much as 100 mV, and 60 mV for the latter two,
respectively.
Alteration of the C-terminus tail
A C-terminal protruding tail is 13–14 amino acids long in
the amino acid sequence of laccases from ascomycetes
M. albomyces (Hakulinen et al., 2002), M. thermophila
(Bulter et al., 2003; Zumárraga et al., 2008) and up to 16
amino acids long in the laccase from basidiomycete Pleurotus
ostreatus (Autore et al., 2009). In ascomycetes laccases, this
tail is generally cleaved by proteolysis at a conserved cleavage
site [Asp-Ser-Gly-(Leu/Val/Ile)] to produce an active form of
the enzyme. Analysis of the 3D structure of the laccase from
Melanocarpus albomyces has shown this C-terminal exten-
sion as a plug that blocks the solvent channel, thus leading to
the hypothesis that its cleavage is required to favor the
entrance of oxygen and the subsequent exit of water
molecules (Hakulinen et al., 2002). Moreover, creating a
C-truncated version of ascomycete Mycelophthora thermo-
phila laccase, by introducing a stop codon at the processing
site, leads to a 10-fold decrease in catalytic efficiency of the
enzyme (Table 2). It seems that the C-terminal tail exerts a
strong influence during processing steps, which in turn affects
the mature protein activity. According to this theory, the
C-terminal tail forms a loop interacting with the active site to
prevent binding of copper ions during processing. It is also
assumed that a change of two consecutive amino acids
(GlyGlu to AspLys) in M. thermophila C-terminal tail might
contribute to a higher grade of tightness between the
C-terminus and the main enzymatic core, which would
affect the protein folding and the final mature protein
(Zumárraga et al., 2008). In any case, closure of the tunnel,
leading to the trinuclear copper site, certainly affects the
function of the trinuclear copper site. Whether this feature is
possible among basidiomycete laccases, which have different
C-terminal residues, is not known.
Festa et al. (2008) obtained a more stable and substrate
specific laccase from basidiomycete by mutagenesis of
C-terminal tail of laccase in position PRO494THR
(Table 2), simultaneously allowing a higher accessibility of
water molecules to the T1Cu site by increasing mobility of
loops that form the reducing substrate-binding site, and
possibly leading to an increased activity of laccase. Another
confirmation of C-terminal end significance on performance
and stability of laccase from basidiomycetes was shown in
experiments of Autore et al. (2009), who decisively ruled out
that four C-terminal amino acids act as a plug that blocks the
access of oxygen and water to the trinuclear T2/T3 copper
cluster. Experiments were performed on a laccase mutant of
Pleurotus ostreatus, designed according to the results of
Zumárraga et al. (2008). In case of P. ostreatus, the presence
of a C-terminal tail had a positive effect on the specific
activity to KM ratio towards phenolic substrates such as
2,6-dimethoxyphenol (DMP) and syringaldazine (SGZ), but
not ABTS.
Improved activity and temperature regimes
The first study on directed evolution of laccase was carried
out by Butler et al. (2003), who performed functional

Table 2. Laccase modifications and their potential industrial applications.
Organism
Laccase tuned by directed evolution and rational design
Myceliophthora thermophila
Myceliophthora thermophila
Basidimycete PM1(HRPL)
Pycnoporus cinnabarinus
Myceliophthora thermophila
Basidiomycete PM1 (HRPL)
Improvements in the reduction potential of T1 copper center
Trametes versicolor
Trametes versicolor
Alterations in the C-terminus of laccases
Pleurotus ostreatus
Pleurotus ostreatus
Melanocarpus albomyces
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Tennessee on 09/11/14
Mutant Modification
MtL T2 A(n20)P, S3I, E86G,
A108V, N303S, F351L,
T366M, Y403H, S450P,
N454K, L489L, L536F,
Y552N, H(c2)R
MtL 6C9 A(n20)P, P(n20)H, S3I,
E86G, A108V, N303S,
F351L, T366M, Y403H,
S450P, N454K, S462S,
L476V, L489L, L536F,
Y552N, H(c2)R,
OB-1 V[a10]D, N[a23]K,
A[a87], V162A, H208Y,
S224G, A239P, D281E,
S426N, A461T
a*-3PO A[a9]D, F[a48]S, S[a58]G,
G[a62]R, E[a86]G,
N208S,R280H, N331D,
D341N,P394H
MtL-R2!IG88 N109S, D530E (E38G in
pro-leader sequence)
OB-1!ChU-B E[a27]K, I[a66]M, F396I,
V452I, F454E
F162A/F332A F162A, F332A
F162A F162A
3M7C L112F, P494T (C-terminal
loop)
POXA1bD4 truncation of 4 amino acids
on the C-terminus
529PLKA533
Tr(L559G) and truncation of 4 amino acids
Tr(delDSGL559) on the C-terminus
556DSGL559, or L559G
For personal use only.
Expression host Desired characteristics Industrial application Ref.
S. cerevisiae Robust at high temp. 170-fold decontamination, pulp Bulter et al. (2003),
(BJ5465) increase in activity 22-fold bleaching, bioremedi- Zumárraga et al. (2007)
increase in kcat ation (PAH)
S. cerevisiae 3.5-fold increase in stability in organic chemistry Alcalde et al. (2005)
DOI: 10.3109/07388551.2014.949617
(BJ5465) 20% acetonitrile and 30% etha-
nol mixture
S. cerevisiae 34 000-fold increase in activity, pulp biobleaching, food Sayut & Sun (2010),
highly robust to temp., organic processing, textile treat- Maté et al. (2010)
solvents, and acidity ment (and more)
S. cerevisiae 8100-fold increase in activity, (biomedical, plant biomass Camarero et al. (2012)
(BJ5465) improved secretion. Shift in pH upgrading)
activity to neutral
S. cerevisiae pH activity optimum shifted Not specified/evaluated Torres-Salas et al. (2013)
(BJ5465) towards neutral (opt. from pH
4 to pH 6)
S. cerevisiae 41 800-fold increase in activity, pH Blood tolerance (biomed- Mate et al. (2013)
shift to more neutral, Cl- and ical nanotechnol)
blood tolerance
Yarrowia lipolytica 98% consumption of bisphenol A bioremediation, biofuel Galli et al. (2011)
(Po1g) (BPA) in 5 h cells, paper and pulp
bleaching
Yarrowia lipolytica 99, 63, 78 and 45% consumption of bioremediation, biofuel Galli et al. (2011)
(Po1g) 2-t-Bu-phenol, 3,5-di- t-Bu- cells, paper and pulp
phenol, BPA, and trimeric sub- bleaching
strate, respectively
S. cerevisiae higher activity, and increase in immobilization (stain Festa et al. (2008)
(W303-1 A) stability removal)
S. cerevisiae higher specific activity to KM ratio acidic bio-processes (xeno- Autore et al. (2009)
(W303-1 A) towards DMP, and syringalda- biotic transform.),
zine but not ABTS organic synthesis
Trichoderma reseei 20-fold lower expression levels, Not specified/evaluated. Andberg et al. (2009)
RutC-30 unstable protein, reduced or no
activity on ABTS and syringal-
dazine for Tr(L559G) and
Tr(delDSGL559), respectively
(continued )
Structure, functionality and tuning up of laccases
13
 14 A. K. Sitarz et al. Crit Rev Biotechnol, Early Online: 1–17

expression of a thermophilic laccase in S. cerevisiae. The

Janssen et al. (2004)

laccase gene was subjected to ten rounds of directed evolution

Andberg et al. (2009)

Aehle et al. (2002),

and screening. The laccase activity was hereby increased
Ref.
170-fold and in addition also had a higher temperature
tolerance (Table 2).

Improved tolerance to organic solvents

Another directed evolution study was carried out on the
tomato stains), detergent laccase from Myceliophthora thermophila. The designed
and textile applications
Industrial application
Not specified/evaluated

bleaching (paprika and

laccase was tolerant to high concentrations of organic solvents

such as methanol, ethanol (30%), acetonitrile (20%) and
dimethylosulfoxide (Alcalde et al., 2005; Bulter et al., 2003).
Specific mutants, such as GLU182LYS gave better stability in
organic media, or increased the redox potential and resistance
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Tennessee on 09/11/14

against denaturation under external factors (mutation

SER280ASN), or modified the channel leading to the
trinuclear center, which might affect the transit of O2 to the
bleaching ability of paprika and
11-fold increase in Ki of NaN2

activity, 2 - to 3-fold increased

decreased thermal stability for
inactive Sc(delDSGL559) mutant,

active pocket (mutation ASN552HIS), or enhanced substrate

on ABTS, narrower pH opt.,

4 - to 6-fold increased specific

binding proximal to the T1Cu and increased the tolerance to

Desired characteristics

co-solvents (mutation LEU429VAL).

Sc(delDSGL559)

Improved catalysis and superb stability

tomato stains

The PM1 laccase from basidiomycetes has recently been

improved by directed evolution by Alcalde’s research group
(Table 2; Maté et al., 2010). The most efficient laccase
contained 15 different mutations; five in the yeast’s a-factor
For personal use only.

Table 2. Continued

signal sequence and 10 in the mature protein. The laccase

activity was improved 32 000-fold. The superior laccase was
Expression host

Aspergillus niger

also very tolerant to temperature, pH range and organic

S. cerevisiae

solvents. Mutations in the signal sequence (yeast a-factor)

enhanced functional expression, whereas the mutations in the
mature protein improved its catalytic capacities by altering
interactions with the surrounding residues (Maté et al., 2010;
truncation of 4 amino acids

556DSGL559, or L559G

Sayut & Sun, 2010). More recently, further evolution of the

ing peptides YGYLPSR,
SLLNATK, KASAPAL,
end by carotenoid-bind-
elongation of C-terminus

mutant obtained (‘‘OB-1’’) has resulted in the development of

on the C-terminus

a ‘‘blood tolerant’’ laccase with tolerance to above neutral

Modification

IERSATAPPP,
CKASAPALC

pH, and chloride ions. Notably, mutations in the region close

to the T1 copper site (the F396I and F454E mutations;
Table 2) were within 7.5 Å from the T1 copper site
(Maté et al., 2013). This indicates the significance of this
structural region (Figure 5) for halide inhibition as well as
overall laccase activity.
Sc(delDSGL559)
Sc(L559G) and

M254F/E346V,

Conclusions and perspectives

Mutant

E348Q

Detailed biochemical studies of the function of the trinuclear

cluster in the copper-catalyzed oxidoreduction mechanism of
laccases have now produced an improved understanding of
the remarkable catalytic mechanism of laccases. Protein
structural information has contributed to our understanding of
some of the structural features of fungal laccases and has
given insight into the role of individual amino acids in the
Melanocarpus albomyces

Stachybotrys chartarum

catalysis. The T1Cu site plays an important role in defining

the redox potential of the enzyme. One of the examples of a
troublesome region of the laccase may be the C-terminal tail.
Depending on the organism, the activity of the enzyme can be
Organism

enhanced or can decrease dramatically when this region is

modified, indicating that subtle modifications may have large
effects on laccase activity. No currently known laccase
 DOI: 10.3109/07388551.2014.949617
combines the desired attributes of being active on a broad
range of substrates, at a wide pH range, while being robust at
high temperature. To decrease the cost of operations and
develop sustainable processes, it is important to search for
new laccases and use directed evolution to engineer improved
laccases. Discovery of better laccase mediators of natural
origin that are environmentally friendly will also be important
when considering laccases for new applications. Overall, the
laccases have a wide range of applications, and they can be
tuned for enhanced functions.
Declaration of interest
The authors report no declarations of interest.
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Tennessee on 09/11/14
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