REVIEW ARTICLE

Lignin-degrading enzymes
Loredano Pollegioni1,2, Fabio Tonin1 and Elena Rosini1,2

degli studi dell’Insubria, Varese, Italy
1 Dipartimento di Biotecnologie e Scienze della Vita, Universita
2 The Protein Factory, Centro Interuniversitario di Biotecnologie Proteiche, Politecnico di Milano, ICRM CNR Milano, Universit

a degli Studi
dell’Insubria, Italy

Keywords A main goal of green biotechnology is to reduce our dependence on fossil

biorefinery; green biotechnology; lignin
valorization; ligninolytic enzymes;
lignocellulosic biomass
Correspondence
L. Pollegioni, Dipartimento di Biotecnologie
e Scienze della Vita, Universit
a degli studi
dell’Insubria, via J. H. Dunant 3,
21100 Varese, Italy
Fax: +39 0332421500
Tel: +39 0332421506
E-mail: loredano.pollegioni@uninsubria.it
(Received 16 September 2014, revised 29
December 2014, accepted 30 January 2015)
doi:10.1111/febs.13224
reserves and to increase the use of renewable materials. For this, lignocellu-
lose, which is composed of cellulose, hemicellulose and lignin, represents
the most promising feedstock. The latter is a complex aromatic heteropoly-
mer formed by radical polymerization of guaiacyl, syringyl, and p-hydroxy-
phenyl units linked by b-aryl ether linkages, biphenyl bonds and
heterocyclic linkages. Accordingly, lignin appears to be a potentially valu-
able renewable aromatic chemical, thus representing a main pillar in future
biorefinery. The resistance of lignin to breakdown is the main bottleneck in
this process, although a variety of white-rot fungi, as well as bacteria, have
been reported to degrade lignin by employing different enzymes and cata-
bolic pathways. Here, recent investigations have expanded the range of nat-
ural biocatalysts involved in lignin degradation/modification and significant
progress related to enzyme engineering and recombinant expression has
been made. The present review is focused primarily on recent trends in lig-
ninolytic green biotechnology to suggest the potential (industrial) applica-
tion of ligninolytic enzymes. Future perspectives could include synergy
between natural enzymes from different sources (as well as those obtained
by protein engineering) and other pretreatment methods that may be
required for optimal results in enzyme-based, environmentally friendly,
technologies.

Introduction
The term lignin derives from the Latin word ‘lignum’,
which means wood. It represents one of the three com-
ponents of lignocellulosic biomass, along with cellulose
and hemicellulose. Lignin is the second most abundant
natural substance in nature after cellulose and, annu-
ally, approximately 5 9 106 metric tons of lignin is
produced industrially [1]. Indeed, lignin is the most
abundant renewable source of aromatic polymers on
earth: its degradation is mandatory for carbon recy-
cling.
Chemically, lignin is a cross-linked macromolecular
material derived from oxidative coupling of monolig-
nols, mainly hydroxycinnamyl alcohols: the three main
components are p-coumaryl, coniferyl and synapyl
alcohols. Lignins show plant-specific composition, with
molecular weight and linkage motifs (Fig. 1) varying
according to plant species and environmental factors.
Lignification is obtained by cross-linking reactions
of the lignin monomers or by polymer–polymer cou-
pling via radicals produced by oxidases: the resonance

Abbreviations
ABTS, 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); DyP, dye-decolorizing peroxidases; HBT, 1-hydroxybenzo-triazole; LiP, lignin
peroxidase; LRET, long-range electron transfer; MnP, manganese peroxidase; PDB, Protein Data Bank; VA, veratryl alcohol; VP, versatile
peroxidase.

1190 FEBS Journal 282 (2015) 1190–1213 ª 2015 FEBS

L. Pollegioni et al. Enzymatic degradation of lignin

Fig. 1. (A) Structure of primary monomers

(top) and corresponding structural units in
lignin (bottom) indicated as p-hydrophenyl
(H), guaiacyl (G) and syringyl (S) units. (B)
Schematic representation of a lignin
structure: the most frequent bonds are
indicated. Softwood lignin contains mostly
G units and very low levels of H units;
hardwood lignin contains similar levels of
G and S units (and traces of H units); and
grasses contain all three units (G/S/H ratio
is 96 : traces : 4, 50 : 50 : traces,
70 : 25 : 5, respectively).

delocalization of radicals to couple at different sites
yields an array of units linked by carbon–carbon and
carbon–oxygen (ether) bonds. The most frequent
bonds include b-O-4, b-5, b-b, 5-5, 5-O-4 and b-1 cou-
plings (Fig. 1B).
As a result of its complex structure, almost 98% of
this renewable polymer derived from biomass is
burned as a source of energy in the pulp and paper
industry. Accordingly, a great biotechnological chal-
lenge is to develop sustainable technologies for con-
verting waste biomass to biofuels, chemicals and new
biobased materials (such as bioplastics).
Most biorefineries focus on cellulose and hemicellu-
lose valorization, the so-called ‘sugar-based platform’
[2], whereas lignin is usually considered to be a low-
value product. A major aim of modern biorefineries is
to (efficiently) convert lignocellulosic materials into
valuable products by unraveling the limitations as a

FEBS Journal 282 (2015) 1190–1213 ª 2015 FEBS 1191

Enzymatic degradation of lignin
result of a non-uniform structure, unique chemical
reactivity and the presence of various organic and
inorganic impurities in lignin samples. The controlled
breaking down of C-C and C-O bonds in lignin by a
selective depolymerization process could produce a
whole series of monomeric, aromatic species. Thus, lig-
nin could represent a renewable source of aromatic
fine chemicals.
The main strategies for depolymerizing lignin to
produce valuable chemicals are cracking or hydro-
lysis reactions, catalytic reduction, catalytic oxidation
and enzymatic reactions [3–6]. A detailed description
of available lignin preparations is provided elsewhere
[7].
The present review summarizes the state-of-art in
enzymatic systems active on the main lignin bonds,
with a particular emphasis on heterologous enzyme
expression and protein engineering studies that aim to
improve their practical use. In the 1980s, reactive oxy-
gen species were reported to be involved in cleaving
lignin chains in basidiomyceteous white-rot fungal cul-
tures: the ligninolytic activity in Phanerochaete chry-
sosporium cell extracts was correlated with production
of H2O2 [8]. Over the years, four enzymatic activities
3; 4  dimethoxybenzaldehyde þ 1  ð3; 4
have been reported to depolymerize lignin in decaying
plant cell walls: lignin peroxidases (LiPs), manganese
peroxidases (MnPs), versatile peroxidases (VPs) and
laccases. These enzymes have gained attention as
potential biological catalysts for lignin biodegradation.
Of note, lignocellulolytic enzymes are too large to
penetrate into undecayed wood cell walls; therefore,
reactive oxygen species could be the agents responsible
for local lignin decay. Phenolic compounds related to
recurring lignin phenolic moieties are known to medi-
ate the oxidation of nonphenolic compounds by a
radical mechanism, once oxidized by laccases to phen-
oxyl radicals. Laccases possess relative low redox
potentials (≤ 0.8 V) that restrict their action to the
oxidation of the phenolic lignin components. The oxi-
dation of nonphenolic lignin moieties is performed via
redox mediators: compounds of small molecular
weight act as electron shuttles performing the oxida-
tion of complex polymers (e.g. lignin) that do not
have access to the enzyme’s active site. After the enzy-
matic oxidation and the conversion into stable radi-
cals, mediators diffuse far away, thus enabling the
oxidation of target compounds not considered to be
substrates of laccase, as a result of their high redox
potential and high volume. An ideal redox mediator
would be a small-size molecule capable of generating
stable radicals (in the oxidized form) that do not inac-
tivate the enzyme, and that is also easily recyclable,
environmental-friendly (specially obtained from natu-
1192
L. Pollegioni et al.
ral resources) and available at low cost [9–12]. Indeed,
the process of lignin degradation is enhanced by sev-
eral additional and accessory enzymes, and some of
them are now available. A recent review reporting on
immobilization of LiP, MnP and laccase is also avail-
able [13].
Peroxidases
The extracellular fungal LiP, MnP and VP are all
members of class II peroxidases within the superfamily
of heme peroxidases [14].
LiP
Reaction and properties
LiP (EC 1.11.1.14), a glycoprotein of 38–46 kDa (and
pI of 3.2–4.0) containing 1 mol of iron protoporphyrin
IX per 1 mol of protein, catalyzes the H2O2-dependent
oxidative depolymerization of lignin. The reaction can
be represented as:
1; 2  bisð3; 4  dimethoxyphenylÞpropane  1; 3  diol
þ H2 O2
 dimethoxyphenylÞethane  1; 2  diol þ H2 O
ð1Þ
LiP shows a very high redox potential (E00  1.2 V
versus SHE at pH 3.0) compared to laccases ( 0.8 V
at pH 5.5), horseradish peroxidases (0.95 V at pH 6.3)
and MnP (0.8 V at pH 4.5) [15]: this property enables
LiP to catalyze the oxidation of nonphenolic aromatic
compounds, even in the absence of a mediator. The
reaction catalyzed by LiP is reported in Fig. 2A [7]. In
more detail: 2-electron oxidation of ferric [Fe(III)] LiP
produces compound I intermediate, a oxoferryl iron
porphyrin radical cation [Fe(IV)=O+] with the reduc-
tion of H2O2. Next, two consecutive one-electron
reduction steps of compound I [from an electron
donor substrate, such as veratryl alcohol (VA)], yield-
ing at first compound II [Fe(IV)=O] and VA radical
cation [VA+] and then returning LiP to the ferric oxi-
dation state, completed the catalytic cycle (central loop
in Fig. 2B). Indeed, a single 2-electron reduction can
return compound I to the resting state in some cases
[7]. Oxidation of phenolic compounds is associated
with inactivation of LiP as a result of the accumula-
tion of compound III (Fig. 2B); VA+ appears to
protect LiP from inactivation as a result of the pro-
duction of compound III [16]. Notably, in the presence
of dioxygen, additional oxidative steps take place to
form the quinine form and promote aromatic ring
cleavage.
FEBS Journal 282 (2015) 1190–1213 ª 2015 FEBS
L. Pollegioni et al. Enzymatic degradation of lignin

Fig. 2. Reaction catalyzed by LiP. (A)

Mechanism of the reaction of LiP on a B
nonphenolic arylglycerol b-aryl ether lignin
model compound [31, 124]. (B) Details of
the steps in the catalytic cycle of LiP in
the presence of VA as substrate:
compounds I, II and III are intermediates
with differing heme oxidation states [7].
The lower loop represents the reaction of
LiP compound II with H2O2 at pH 3.0 in
the presence of excess H2O2 and the
absence of a reducing substrate, yielding a
catalytic inactive form of the enzyme,
known as compound III. Compound III is
then converted to the resting enzyme by
spontaneous autoxidation or by oxidation
with a VA radical cation.

LiPs are active on a wide range of aromatic com-
pounds, such as VA, methoxybenzenes and nonphenol-
ic b-O-4 linked arylglycerol b-aryl ethers with a redox
potential up to 1.4 V in the presence of H2O2 [16]
(Table 1). Using a lignin model dimer as the substrate,
the cation radical decays with the spontaneous Ca-Cb
fission of the alkyl side chain, with the products resem-
bling those found when fungi degrade lignin [17]. LiPs
can oxidize phenolic lignin model compounds more
readily than nonphenolic ones [18]. Unfortunately, the
size of the oligomeric lignin model substrates signifi-
cantly affects the catalytic efficiency of LiP (i.e. the
activity of LiP on a b-O-4-linked lignin model trimer
is 25-fold lower than that observed on VA) [19].
The crystal structure of P. chrysosporium LiP is
composed of eight main and eight minor a-helices and
a limited b structure, and it is organized in a proximal
and a distal domain (Fig. 3) [20–23]. The heme is
embedded in a crevice between the domains: two small
channels connect the prosthetic group to the solvent
(see below). The LiP contains four disulfide bridges,
two Ca2+ binding sites (one per each domain, puta-
tively involved in maintaining the topology of the
active site) and two glycosylation sites in the proximal
domain. The heme iron is coordinated with H176 at
the proximal side, and W339 is hydrogen-bonded to
the distal H146. The peroxide binding site is located
on the distal side of the heme; a channel connects this
pocket to the exterior of the protein. The negative
charge(s) that develops in the cleavage of peroxides is
stabilized by R43, which also stabilizes the ferryl oxy-
gen of compound I; the distal H47 acts as a H+ accep-
tor for the bound peroxide. Notably, W171 on the
protein surface shows hydroxylation at Cb (post-trans-
lational modification): this residue plays a main role in
the binding and oxidation of VA through long-range
electron transfer (LRET), as also demonstrated by
site-directed mutagenesis studies [24]. Characterization
of W171A (which is unable to oxidize VA) and F267L
(showing an increased Km) variants of LiP isoenzyme
H8 from Phanerochaete crysosporium demonstrated
that the site including these two residues, rather than
the heme access channel, is the site of VA oxidation
[25].
Expression of LiP
Very recently, a strain of P. crysosporium was geneti-
cally modified to co-express two endogenous genes
encoding for a LiP (lipH8) and a MnP (mnp1) yielding
4.5 kUL1 and 9.1 kUL1, respectively, and a spe-
cific activity of 29.9 U(mg LiP)1 (on VA as sub-

FEBS Journal 282 (2015) 1190–1213 ª 2015 FEBS 1193

Enzymatic degradation of lignin L. Pollegioni et al.

Table 1. Structures of most common substrates for the different Table 1. (Continued).
lignin-degrading enzymes reported in the present review.
Substrate Structure
Substrate Structure
Sinapyl alcohol
2,6-Dimeth-
oxyphenol

4-Methoxyphenol Syringyl alcohol

ABTS

Veratryl alcohol

Cathecol

strate) and 11.7 U(mg MnP)1 (as Mn3+-malonate

Coniferyl alcohol complex formation) [26].
Concerning recombinant expression, an engineered
cDNA from P. crysosporium encoding the LiP isoen-
zyme H2 has been successfully overexpressed in Esc-
herichia coli [27]. It was produced in an inactive and
Guaiacol insoluble form in inclusion bodies: the active enzyme
was produced by refolding with a buffer containing
glutathione, urea, Ca2+ and heme (final yield of
3.4 mg enzymeL1 of culture). The turnover number
of purified recombinant LiP (at pH 2.5) was 39 s1,
Methoxybenzene measured as the oxidation of VA; this was similar to
the value obtained when the enzyme was isolated from
the same source (30 s1). Subsequently, the recombi-
nant enzyme was reported to possess a MnP activity,
with a turnover number of 14 s1.
The LiP isoenzyme H8 from P. chrysosporium was
Methylhydr-
also overexpressed in E. coli: the recombinant pro-
oquinone
tein was recovered from inclusion bodies with a
folding efficiency of 1% in terms of active enzyme
[24]. Indeed, this enzyme was homologously overex-
pressed under the control of the glyceraldehyde 3-
phosphate dehydrogenase promoter, reaching approx-
Phenol red
imately 2 mgL1 of pure enzyme in 4 days of
growth [28].
P. chrysosporium LiP was also expressed in Pichi-
a methanolica cells under alcohol oxidase promoter
control and contained the LiP a-factor signal pep-
tide from P. chrysosporium or Saccharomyces cerevisi-
ae; the extracellular activity was 932 UL1 and
1933 UL1, respectively [29].

1194 FEBS Journal 282 (2015) 1190–1213 ª 2015 FEBS

L. Pollegioni et al. Enzymatic degradation of lignin

A B

Fig. 3. Crystal structure of

P. chrysosporium LiP [Protein Data Bank
(PDB) code: 1LGA]. (A) Tertiary structure
of LiP: blue, proximal, C-terminal domain;
green, distal, N-terminal domain; heme is
indicated as yellow ball sticks. (B) Details
of the heme environment. The proximal
H176 forms hydrogen-bonds with D238,
which facilitates stabilization of Fe(IV)-O
intermediate in compound I (the Asp-His-
Fe triad is a conserved feature of plant
peroxidase superfamily).

In more recent work, the cDNA encoding a MnP
(mnp1) and that encoding for a LiP (clg5) were fused and
the chimeric protein was expressed in E. coli cells [30].
Following protein refolding and activation (in the pres-
ence of Ca2+ and heme) the MnP activity reached
27 UL1, with a specific activity of 0.3 U(mg protein)1,
whereas the LiP activity was below detection levels.
Drawbacks in using LiP for oxidative depolymerization
of lignin
A purified isoenzyme from the fungus P. chrysospori-
um in aqueous/organic solvent mixtures at low lignin
concentrations (~ 4 mgL1) was able to cleave the
Ca-Cb bond of the propyl side chain of synthetic
lignins to give benzylic aldehydes at the Ca position
(i.e. soluble lower molecular weight products similar
to those obtained from P. chrysosporium cultures
incubated with lignin for 6–24 h) [31]. The main
drawback was the polymerization reaction that
occurred because the small phenolic products were
not removed. The preference of LiP for phenolic lig-
nin units explains the propensity to polymerize rather
than depolymerize lignin samples under in vitro condi-
2Mn(III) þ 2H2 O ð2Þ
tions [32]. To minimize phenoxy radical coupling, lim-
iting low concentrations of the substrate enabled the
depolymerization and re-polymerization of a synthetic
lignin obtained from coniferyl alcohol to a similar
extent [31].
Lignin isolated samples are very polydisperse: the
measurement of the molecular size range of lignin, as
well as the changes that can be caused by the process
of lignin degradation, is a challenging task. For deter-
mination of the molecular size of lignin samples, the
most advantageous methods are gel permeation chro-
matography, light scattering and vapor pressure
osmometry (the use of ultrafiltration and MS was
also reported) [33,34]. Details concerning lignin char-
acterization using direct (based on unmodified lignin
samples) and indirect (via chemical modification of
the samples) methods have been reported previously
[12].
MnP
Reaction and properties
MnP (EC 1.11.1.13) is a glycosylated heme protein
with molecular mass of ~ 40–50 kDa; it represents
the most common lignin-modifying peroxidase pro-
duced and secreted by almost all wood-colonizing
basidiomycetes, including white-rot and various soil-
colonizing fungi. This enzymatic activity was detected
in P. chrysosporium almost 30 years ago [35,36]: the
46-kDa acidic (pI 4.5) glycoprotein was demonstrated
to depolymerize and (re)polymerize the synthetic lig-
nin molecules produced starting from coniferyl and/or
sinapyl alcohol in the presence of 0.2 mM Mn2+, at
pH 4.5 and at 37 °C [37]. MnP catalyzes the reac-
tion:
2Mn(II) þ 2Hþ þ H2 O2
Indeed, MnP is the only heme peroxidase showing a
one-electron Mn2+ oxidation reaction mechanism
(Fig. 4). The Mn3+ dissociates from the enzyme and is
complexed by carboxylic acids, in particular oxalate
and malate: the Mn3+-chelator complex works as a
diffusible oxidant of phenolic compounds, giving a
phenoxy radical intermediate that yields various degra-
dation products. The Mn3+ form oxidizes phenolic
compounds (such as 2,6-dimethoxyphenol, guaiacol, 4-
methoxyphenol and phenolic lignin residues), whereas
it is inactive on VA or nonphenolic substrates
(Table 1) [38].

FEBS Journal 282 (2015) 1190–1213 ª 2015 FEBS 1195

Enzymatic degradation of lignin
Fig. 4. The catalytic cycle of the MnP resembles that of LiP
(Fig. 2B). The conversion of compound I into compound II can be
achieved by using alternative electron donors (i.e. ferrocyanide and
a variety of phenolic compounds) [125].
LiP and MnP from P. chrysosporium show a similar
tertiary structure based on a helical topography: they
are globular proteins formed by 11–12 a-helices pre-
dominantly in two domains, with a central cavity con-
taining the heme group (Fig. 5A) [20,39,40]. The
structural similarity points to divergent evolution. Pro-
tein stability is strongly based on disulfide bridges: LiP
contains four disulfide bridges, whereas MnP contains
an additional bridge (in its C-terminal region) and two
Ca2+ binding sites that are located in a cation-binding
site on the protein surface; the site can accommodate a
number of different metal ions. Concerning the heme
prosthetic group, it is stabilized by the two Ca2+ ions:
one tightly bound on the proximal side and the other
bound on the distal side. Mn2+ binding is largely a
result of R177 whose orientation is dependent on a
salt-bridge with E35 [41]. The extracellular MnP from
P. chrysosporium, for which the crystal structure was
 resolution, showed the presence of
solved at 1.45 A
divalent Cd2+ bound at the Mn-binding site: this rep-
resented the first report of MnP-inhibitor complex
structure [42]. Cd2+ also binds to a putative second
weak metal-binding site (constituted by the carboxy-
terminal oxygen, the side chain of D84 and two sol-
vent molecules) displaying tetrahedral geometry. In the
open conformation of E39, a network of hydrogen
bonds connect E39 and D84 (i.e. the ligands in the
two metal sites) through D85 and water. This second
site is exposed to the solvent and could accommodate
a Mn2+ ion with a bound chelator.
1196
L. Pollegioni et al.
Recombinant expression of MnP from P. chrysosporium
P. chrysosporium possesses at least six different MnP.
The recombinant MnP from P. chrysosporium (isoen-
zyme H4) has been expressed in E. coli [43]: the inac-
tive and insoluble protein was refolded in the presence
of 2 M urea, CaCl2, heme and oxidized glutathione.
The recombinant, active MnP showed the same spec-
tral properties as the native fungal enzyme, a kcat of
450 s1 on Mn2+, and Km values for H2O2 and Mn2+
of 96 lM and 52 lM, respectively (Table 2).
P. chrysosporium MnP was also successfully
expressed in Pichia pastoris cells [44]: active MnP
(containing the a-factor secretion signal) was secreted
in broth culture, reaching a maximum of 120 UL1.
The kcat value for H2O2 was lower (168 s1), whereas
the Km value for Mn2+ was similar to that of native
enzyme (Table 2). The expression level of the active
MnP was increased by adding heme to the fermenta-
tion broth: a logarithmic relationship between heme
concentration and MnP activity was observed
[45]. The scale-up to a 2-L fed-batch bioreactor, in
the presence of 0.1 gL1 of heme, increased the
MnP activity to 2500 UL1, corresponding to
15.6 mgL1.
Protein engineering of MnP
The interconversion of structure/function between
MnP and LiP was made apparent by site-directed
mutagenesis studies. The S168W variant of P. chrysog-
enum MnP was demonstrated to be a hybrid enzyme
because it retained the manganese oxidase activity
(kcat  260 s1 at pH 4.3) (Table 2) and acquired the
ability to oxidize a multitude of LiP substrates (i.e. a
kcat value of 11 s1 was observed in the presence of
VA) [46,47]. S168 in MnP corresponds to the W171
residue located on the surface of LiP (i.e. the VA oxi-
dation site).
As noted above, MnP purified from P. chrysospori-
um is susceptible to thermal inactivation as a result of
a loss of the Ca2+ ion [48], and the recombinant
enzyme, lacking glycosylation, was also more suscepti-
ble [49]. The double substitution A48C and A63C
introduced a disulfide bond in proximity to the distal
calcium-binding site of MnP [50]. This MnP variant
showed activity and spectral properties resembling
those of wild-type, glycosylated MnP but proved to be
more stable: the half-life of inactivation at 42 °C
increased from 2 min for the wild-type to > 1 h for
the enzyme variant. Indeed, the enzyme variant was
also more stable at higher pH values (i.e. at pH 6.0).
Furthermore, the M273L and N81S substitutions
FEBS Journal 282 (2015) 1190–1213 ª 2015 FEBS
L. Pollegioni et al. Enzymatic degradation of lignin

A B

Fig. 5. Structure of MnP and VP. (A)

Schematic representation of the 3D
structure of MnP from P. chrysosporium
(PDB code: 1YYD) [40]: blue) proximal,
C-terminal domain; green) distal, N-
terminal domain; heme is indicated as
yellow ball sticks. (B) 3D structure of
P. eryngii VP (PDB code: 2BOQ) [55]. (C)
Axial view of the heme region, including
the putative Mn2+ binding site in the
crystal structure of P. eryngii VP [54]. The
putative LRET pathways from H232 and
from W164 (pathways I and II) are
indicated as well as pathway III from P76,
absent in vpl and envisaged from the
homology model of isoenzyme VSP1 [55].

Table 2. Kinetic parameters of different MnPs.

Km (lM)
Activity
Enzyme Host (s1) H2O2 Mn2+ References

P. chrysosporium 410 107 42 [43]

Wild-type E. coli 450 96 52 [43]
S168W variant E. coli 260 110 110 [47]
Wild-type P. pastoris 168 58 [44]
P. eryngii MnPL2 118 (165 Umg1) 4 19 [128]
B. adusta MnP1 132 (180 Umg1) 10 17 [128]

yielded MnP variants highly resistant to 1 mM H2O2
[51].
After MnP activity was identified in the white-rot fun-
gus P. chrysosporium, the enzyme was also discovered in
most of the wood lignin and litter-decaying basidiomy-
cetes. Phlebia radiata secretes several lignin heme-
containing peroxidases in the extracellular broth,
including three isoenzymes of MnP. Engineering of the
MnP3 isoenzyme produced five enzyme variants that
were overexpressed in E. coli [52]; after the inclusion
bodies refolded, 1.4 mgL1 fermentation broth of
active MnP3 was purified (~ 7% of refolding efficiency).

FEBS Journal 282 (2015) 1190–1213 ª 2015 FEBS 1197

Enzymatic degradation of lignin
VP
Reaction and properties
VP (EC 1.11.1.16) is characterized by a broad sub-
strate preference and by sharing typical features of the
MnP and LiP fungal peroxidase families. For example,
both VP and LiP oxidize VA (the latter showing
higher affinity and efficiency) but VP only oxidizes
Reactive Black 5 and other dyes. The VP activity has
been identified in the white-rot fungal genera Pleurotus
(i.e. Pleurotus eryngii and Pleurotus ostreatus) and
Bjerkandera (i.e. Bjerkandera adusta) [53].
The crystal structure of VP from P. eryngii (heterol-
ogously expressed in E. coli W311O) has been solved
to 1.3 A and includes 11–12 helices, four disulfide
bridges, two structural Ca2+ sites, a heme pocket con-
taining the distal His47 and proximal His169 residues,
and a Mn2+ binding site (Fig. 5B) [54,55]. The Mn2+
binding site is formed by one of the heme propionates
and the carboxylates of Glu36, Glu40 and Asp175 resi-
dues (Fig. 5C) and resembles that observed in the
MnP. The substrate promiscuity of VP is the result of
a high redox potential (E00 > +1.4 V versus SHE) and
the presence of different catalytic sites connected to
the heme pocket for low- and high-redox potential
substrate oxidation [53]. Three putative long-range
electron-transfer pathways for oxidating high redox
potential aromatic substrates have been identified,
comprising the residues: (a) H232–D231 and the proxi-
mal H169; (b) W164, L165 and the methyl-group C of
the heme (the only pathway involved in VA oxida-
tion); and (c) P76, A77, D78 and the distal H47
(Fig. 5C). Site-directed mutagenesis studies demon-
strated that only the W164 pathway is involved in oxi-
dation of VA and Reactive Black 5 dye [55].
The catalytic cycle of VP resembles the one reported
for LiP (Fig. 2B): the enzyme catalyzes electron trans-
fer from the substrate to form compound I and II
intermediates. The oxidation of Mn2+ and aromatic
substrates by direct electron transfer to the heme and
Table 3. Expression yield of VP from different sources.
Enzyme Host
P. ostreatus
P. ostreatus (modified strain)
P. eryngii wild-type P. crysosporium
P. eryngii wild-type E. nidulans
P. eryngii wild-type S. cerevisiae
R4 variant S. cerevisiae
P. eryngii vpl2 E. coli
1198
L. Pollegioni et al.
LRET from W164 in VP is similar to that observed in
P. chrysosporium LiP, with the main exception that
W164 exists in the form of a neutral radical [55]. The
basic VP catalytic cycle proposed previously [56] was
then implemented to account for VA oxidation. This
model implies the presence of two enzyme forms,
termed IB and IIB (in equilibrium with the classical
compounds I and II), characterized because one oxida-
tion equivalent would be in the form of a tryptophan
radical, whose presence is required during oxidation of
high-redox potential substrates.
The catalytic efficiency of VP from P. eryngii on
Mn2+ is similar to that of MnP: in contrast, VP is 10-
fold less efficient than LiP in oxidizing VA [55,57] and
is active on both phenolic and nonphenolic b-O-4 lig-
nin model dimers. P. eryngii VP degraded a 0.25 mM
guaiacylglycerol-b-guaiacyl ether and veratrylglycerol-
b-guaiacyl ether solution with a 65% and 20% yield,
respectively, in the presence of 0.1 mM H2O2 [58].
Indeed, VP from P. ostreatus induced the oxidative
degradation of a nonphenolic b-O-4 lignin model
dimer [59] and a significant depolymerization of a syn-
thetic lignin was obtained in the presence of VA only.
Expression of P. eryngii VP
A genetically modified P. ostreatus strain with high VP
productivity, reaching at most 230 UL1 by using a
synthetic medium, was isolated [60]. By optimizing the
culture conditions, 21 mgL1 of VP (7300 UL1)
could be produced (Table 3): the kinetic parameters
for Mn2+, H2O2 and VA of P. ostreatus VP are
reported in Table 4 [61]. Co-expression in P. crysospo-
rium of a MnP and LiP (from the same microorgan-
ism) and of VP vpl2 from P. eryngii enabled a high
level of extracellular production of this VP: 21 kUL1
culture on 2,6-dimethoxy-phenol as substrate [26].
The proper folding and expression of an active form
of recombinant VP is strongly hampered by its com-
plex structure (i.e. by the presence of four disulfide
Expression level
(UL1) (mgL1) References
230 0.6 [60]
7300 21 [61]
21 000 342 [26]
0.4 [56]
21 [64]
57 000 [64]
12.5 [63]
FEBS Journal 282 (2015) 1190–1213 ª 2015 FEBS
L. Pollegioni et al. Enzymatic degradation of lignin

Table 4. Kinetic parameters of different VPs.

VA Mn2+ ABTS H2O2

1 1 1
Enzyme Host Activity (s ) Km (lM) Activity (s ) Km (lM) Activity (s ) Km (lM) Activity (s1) Km (lM) References

P. eryngii 12.9 3000 118 19 [55]

Wild-type E. coli 8.0 2750 298 189 [55]
Wild-type E. coli (0.25 mUmg1)a (27.5 mUmg1)a [62]
Wild-type S. cerevisiae 54 45 220 540 135 51 [64]
R4 variant S. cerevisiae 75 120 365 56 490 200 [64]
2-1B variant S. cerevisiae 98 4300 850 34 1720 800 [64]
vpl2b E. coli 6.4 138 78 5.4 0.7 [63]
P. ostreatus 3.2 (4.4 Umg1) 31 (43.1 Umg1) 36 (50.8 Umg1) [69]
39 (54 Umg1) 207 (289 Umg1) 411 (574 Umg1) [59]
E. coli 13 (18 Umg1) 186 (259 Umg1) 142 (198 Umg1) [59]
Amycolatopsis E. coli 24 210 [69]
sp. 75iv2
(DyP2)

a
Specific activity.
b
After removing thioredoxin tag.

bridges, two Ca2+ ions and the heme prosthetic
group). An enzyme expression level resembling that
achieved for the homologous VP from P. eryngii was
obtained by employing Emericella nidulans or Aspergil-
lus niger as hosts (~ 0.4 mgL1) (Table 3) [56]. Unfor-
tunately, the heterologous expression in E. coli cells
resulted in the formation of inclusion bodies and low
enzymatic activity (Table 4) [62]. Most recently, vpl2
gene from P. eryngii was successfully overexpressed in
E. coli BL21(DE3) cells as a chimeric protein fused to
thioredoxin and by inducing protein expression by
IPTG in the presence of 0.1 gL1 of hemin [63]. Over-
expressed VP-thioredoxin was soluble and was pro-
duced with an overall yield of 12.5 mg pure
proteinL1 of fermentation broth. Specific activity
increased > 10-fold after removing the tag: the main
kinetic parameters are reported in Table 4. VP from
P. eryngii was more efficiently produced as a soluble
and active enzyme in S. cerevisiae, reaching a secretion
level of ~ 21 mgL1 (Table 3) [64].
Protein engineering of P. eryngii VP
To obtain a highly stable enzyme against temperature,
alkaline pH and H2O2, P. eryngii VP was subjected to
six rounds of directed evolution using the yeast S. ce-
revisiae as expression host [64]. Among the selected
enzyme variants, the R4 VP showed an improved
affinity for 2,20 -azino-bis(3-ethylbenzothiazoline-6-sul-
phonic acid) (ABTS) (10-fold lower Km value than the
wild-type VP) and activity (i.e. kcat = 365 s1), thus
enhancing its kcat/Km ratio (16-fold compared to the
wild-type VP) (Table 4). A similar behavior was
observed for the thermostable 2-1B variant on ABTS;
however, Mn2+ binding was negatively affected
(yielding a 94-fold enhancement of the Km value for
Mn2+). Indeed, these evolved VP variants can operate
at higher H2O2 concentrations than the wild-type
enzyme, with a significant improvement in activity
(from 135 s1 to 1720 s1 for the wild-type enzyme
and the 2-1B variant, respectively) (Table 4). At satu-
rating H2O2 concentrations, the R4 and 2-1B variants
(containing four and seven point substitutions, respec-
tively) showed specific activities of 3530 Umg1 and
11.3 kUmg1, respectively, on ABTS. Under these
conditions, the expression of R4 VP reached
~ 57 kUL1 of culture broth (ABTS oxidation activ-
ity) (Table 3), the highest value reported so far for a
peroxidase [64].
To improve resistance to H2O2, VP from P. eryngii
was recently the object of a rational design investiga-
tion based on site-directed mutagenesis of residues
which are easily oxidized (e.g. methionine) and close
to heme and the H2O2-binding site. A number of sin-
gle point variants, as well as double and triple point
variants, with improved stability over time in the pres-
ence of 4 mM H2O2 have been isolated [65].
Dye-decolorizing peroxidases (DyPs)
Among the machinery involved in degrading lignin in
the soil bacterium Rhodococcus jostii RHA1, three
DyPs have been identified recently [66]. DyPs, existing
in a wide range of fungi and bacteria, contain a heme
prosthetic group, possess a ferredoxin-like fold (resem-
bling that of chlorite dismutase) and have been classi-
fied into four subfamilies (A–D). DyPs exhibit
peroxidase activity toward anthraquinones, ABTS, car-
otenoids, methoxylated aromatics (i.e. VA) and lignin
model compounds: indeed, RHA1 degrades lignin and

FEBS Journal 282 (2015) 1190–1213 ª 2015 FEBS 1199

Enzymatic degradation of lignin
lignocellulose, producing a number of monocyclic phe-
nolic compounds [67]. R. jostii RHA1 contains two
DyPs: DypA, which is predicted to be in the periplasm
and is putatively involved in iron uptake, and DypB,
which is responsible for lignin degradation. DypB
showed the greatest kinetic efficiency for ABTS (kcat/
Km = 2000 M1s1) and also oxidized Mn2+ (kcat/
Km = 25.1 M1s1), a reactivity that could enlarge its
ability to degrade lignin-based compounds. Engineer-
ing of the Mn-binding pocket enabled a 14-fold
improvement in the MnP activity of DyPB [68].
The strain Amylocolatopsis sp. 75iv2 also demon-
strated high capacity to modify lignin to produce acid-
precipitable polymeric lignin (as well as extracellular
phenol oxidation activity): DyP2, a member of C-type
subfamily, was isolated and characterized [69]. DyP2
has novel function for this family, with high activity
both as a peroxidase and MnP (kcat/Km in the range
105 to 106 M1s1): MnP activity is close the same
order of magnitude as canonical MnPs and VPs which
are involved in fungal lignin breakdown. As a peroxi-
dase, it demonstrates high activity against a broad
range of peroxidase substrates, including RB5. DyP2
also demonstrates an oxidase reactivity, dependent on
Mn2+, with a Km value similar to that for MnP activ-
ity, and broadens its substrate scope to include the
degradation of nonphenolic lignin model compounds
[69]. Crystallographic studies show that a distinct Mn-
binding site exists, located 15 A from the heme site.
DyP2 was reported to rapidly break down lignin
model dimers containing the b-O-4 linkage in the
absence of redox mediators: the presence of a low
redox potential phenolic site (DE = 0.6–0.8 V) in the
substrate is mandatory for this activity [69].
Laccase
Reaction and properties
Laccase (EC 1.10.3.2, benzenediol:oxygen oxidoreduc-
tase) is a polyphenol oxidase that catalyzes the overall
reaction:
4 benzenediol þ O2
4 benzosemiquinone þ 2H2 O ture of nitrite reductases more than that of large lac-
ð3Þ
A recent and comprehensive review of laccases is
provided elsewhere [70]. Fungal laccases are produced
as intracellular and extracellular isoenzymes differing
in oligomeric state (monomeric or dimeric) and glyco-
sylation level (10–45%) [71]. Laccase contains four
copper ions of three different types: type 1 copper in
the T1 site shows a strong absorption band at around
600 nm, thus giving laccase its characteristic blue
1200
L. Pollegioni et al.
color; one type 2 and two type 3 copper ions form a
trinuclear cluster (in the T2 and T3 sites) coordinated
by a highly conserved pattern of four histidine resi-
dues. In the resting enzyme form, the four copper ions
are in the +2 oxidation state [24].
The structure of laccase from Trametes versicolor
containing a full complement of coppers, the complete
polypeptide chain and seven carbohydrate moieties
was solved previously [72] (Fig. 6A). All known fungal
laccases show a similar structure, consisting of three b-
barrel sequential domains related to small blue copper
proteins. The T1 site is located in the third domain
and the substrate binding site is located in a small,
negatively-charged cavity close to the T1 site, whereas
the T2–T3 site is placed between the first and the third
domain (Fig. 6B). A detailed observation shows the
presence of two channels: one for dioxygen diffusion
to reach the two type 3 copper ions, and the second
for release of water from the type 2 copper ion
(Fig. 6B). Of note, the coordination distances in the
T1 site were proposed to affect the redox potentials.
The structure of T. versicolor laccase was solved at
2.4 A resolution in complex with 2,5-xylidine, a weak
reducing substrate and the compound used as inducer
in the fungus culture [73]. The cavity accommodating
2,5-xylidine is wide (10 9 10 9 20 A)  and can host
substrates of various sizes. Several residues (F162,
L164, F265, F332 and F337) make hydrophobic inter-
actions with the aromatic ring of the ligand and two
charged/polar residues interact with its amino group:
the conserved aspartate at position 206 and H458.
The latter also coordinates the copper Cu1 that func-
tions as the primary electron acceptor: H458 repre-
sents the entrance door of this electron during its
transfer from the substrate to Cu1. The structure of
laccase–2,5-xylidine complex has been considered a
suitable model for engineering the efficiency and spec-
ificity of laccases.
On the other hand, bacterial laccase from Strepto-
myces coelicolor A3(2) significantly differs from all lac-
cases studied so far: it consists of two domains and
forms trimers, thus resembling the quaternary struc-
cases (Fig. 6C) [74]. To produce a geometry of the
active site similar to that of large laccases in S. coeli-
color laccase, three trinuclear copper clusters are
placed between domains 1 and 2 of each pair of neigh-
bor chains.
Laccase possesses moderately low redox potentials
(0.42–0.79 V) and can oxidize phenolic compounds
(i.e. polyphenols, methoxy-substituted phenols and
aromatic diamines, with Km values in the range 1–
10 mM) to the corresponding radicals [70]. The log
FEBS Journal 282 (2015) 1190–1213 ª 2015 FEBS
L. Pollegioni et al. Enzymatic degradation of lignin

A B

Fig. 6. (A) Ribbon diagram of laccase from

T. versicolor showing two channels
leading to the T2/T3 cluster (PDB code:
1GYC) [72]. (B) Structural details of the
laccase active site: the T1, T2 and T3 sites
are indicated. The T1 copper is
coordinated with Nd1 of an His and a
thiolate sulfur of a Cys as equatorial
ligands; the T2 copper is coordinated to
two His-Ne2 and one OH– yielding a
trigonal coplanar configuration; each T3 C
copper coordinates three His residues and
a bridging oxygen atom and shows a
distorted tetrahedral geometry. The arrows
mark the flow of substrates, electrons and
O2. Further details are provided elsewhere
[70]. (C) Comparison of the ribbon diagram
of laccase from T. versicolor organized in
three sequentially arranged domains (left)
and of trimeric bacterial laccase from
S. coelicolor A3(2) organized in two
domains A1 and B2 (PDB code: 3CG8)
(right) [74].

(kcat/Km) value increases with the redox potentials
difference between the T1 site and the substrate donor
[75]. The substrate acceptance of laccases can be
enlarged using mediator compounds: laccase slowly
oxidizes ABTS, thus allowing the degradation of non-
phenolic lignin substrates (see below) [9] and the indi-
rect oxidation of large molecules. Indeed, laccase does
not use hydrogen peroxide.
Regarding the catalytic cycle, the enzyme catalyzes a
four single-electron substrate oxidation reaction and
the enzyme is fully reduced by O2 through two consec-
utive two-electron steps (Fig. 7) [7,70]. An important
issue for laccase activity is the pH-dependence of the
activity. For phenolic substrates, the pH-dependence is
bell-shaped: the activity increase with the pH is attrib-
uted to the pH-dependence decrease in redox poten-
tials of the phenol groups, whereas the decrease in
activity with pH has been ascribed to hydroxide inhibi-
tion of the trinuclear cluster [76]. The maximum of this
bell-shaped curve occurs at acidic pH values, although
a high activity at alkaline pH is a desired feature for
industrial applications.
Importantly, laccases subtract one electron from
phenolic-OH groups of phenolic lignin model com-
pounds forming phenoxy radicals, which often
undergo polymerization via radical coupling. This
reaction is also accompanied by demethylation, qui-
none formation and ring cleavage [77]. Phenolic b-1
lignin models are degraded by laccase as a result of
phenoxy radical formation, which leads to Ca oxida-
tion, alkyl-aryl cleavage and Ca-Cb cleavage (Fig. 8A)
[77,78]. Indeed, laccases also oxidize nonphenolic
model compounds and b-1 lignin dimers in the
presence of a mediator such as ABTS, 1-hydroxybenzo-
triazole (HBT) and the natural compound 3-hydroxy-
anthranilic acid [9]. The oxidation of a nonphenolic
b-O-4 model compound by employing laccase coupled
to HBT is reported in Fig. 8B, promoting aromatic ring
cleavage, Ca-Cb cleavage and Ca oxidation.
Interestingly, laccases are widely distributed in
higher plants (i.e. Rhus vernicifera, where laccase is
involved in the xylem tissue lignifications) [79], in fungi
(i.e. Pleurotus), and in a few bacteria (including Bacil-
lus). Atypical laccases include those possessing two
domains (i.e. from Botrytis cinerea and Tricholo-
ma giganteum), those showing a homodimeric (i.e.
Thapsia villosa, Phellinus ribis, Rhizoctonia solani, etc.)
or a tetrameric structure (i.e. Agaricus bisporus D621,
Podospora anserina, Aspergillus nidulans, etc.) and
those with unusual spectral properties (i.e. white or
yellow laccases; see below) [70].
The constitutive, extracellular production of lac-
cases in basidiomycete fungi is low [80], although it
is significantly stimulated by specific inducers: for
example, metals (Cu2+, Ca2+, Ag+ and Mn2+); aro-
matic or phenolic compounds related to lignin or lig-
nin derivatives; ethanol (laccase production from
T. versicolor employing a medium containing

FEBS Journal 282 (2015) 1190–1213 ª 2015 FEBS 1201

Enzymatic degradation of lignin
40 gL1 of ethanol was 2.6 kUL1, a 20-fold
increase compared to the basal level); and copper (it
is important for the proper enzyme folding). Notably,
when growing T. pubescens using glucose as the main
carbon source, a significant laccase production
started when the sugar was consumed from the med-
ium; in a medium containing 40 gL1 of glucose,
10 gL1 of peptone and 2 mM Cu2+, 330 kU of lac-
caseL1 of fermentation broth was obtained. A
review of laccase gene transcriptional regulation is
provided elsewhere [70].
Recombinant expression
The cDNA coding for the lac1 gene isolated from Pyc-
noporus cinnabarinus was expressed in the methylo-
trophic yeast P. pastoris: after 10 days of cultivation,
laccase activity in the culture medium reached a value
1202
L. Pollegioni et al.
Fig. 7. Proposed mechanism for the
reduction and reoxidation of the copper
sites in the catalytic cycle of laccase
[7,126]. The cycle starts with oxidation of
the substrate by the T1 copper
(Cu2+ ? Cu+), the primary electron
acceptor (the T1 site shows a relatively
high redox potential, ~ 700–800 mV). This
electron is transferred to the T2/T3
trinuclear site through a conserved His-
Cys-His motif (the rate-limiting step in
catalysis): a successive four-electron
oxidation (from different substrate
molecules) produced the fully reduced
enzyme form (step 1). The molecular
oxygen binds to the T2/T3 site and
converts to a peroxide intermediate with
transfer of two electrons from the T3
coppers (step 2). The peroxide
intermediate decays to an oxy radical
facilitated by electron transfer from the T2
copper and accelerated by pH decrease:
by 2-electron reductive cleavage of the
O-O bond a water molecule is released
(steps 3 and 4).
of 20 UL1 (8 mgL1) and the enzyme was hypergly-
cosylated [81].
The cDNA encoding for an extracellular laccase
from Streptomyces ipomoea CECT 3341 was cloned
and the protein overexpressed in E. coli [82]: it pos-
sessed a good stability at high pH (the enzyme
retained its initial activity after 36 h of incubation at
pH values between 5.0 and 9.0) and at high tempera-
ture, as well as resistance to high NaCl concentrations
and to several laccase inhibitors. The substrate prefer-
ence of this laccase was pH-dependent: at alkaline pH,
a wide range of phenolic compounds were oxidized,
including the syringyl and guaiacil moieties derived
from lignin. At optimal pH, a Km of 0.4 mM and kcat
of 10 s1 were determined in the presence of ABTS.
T. versicolor, a typical white-rot fungus, secretes sev-
eral laccase isoenzymes. The LccB and LccC isoen-
zymes were recently expressed in P. pastoris GS115,
FEBS Journal 282 (2015) 1190–1213 ª 2015 FEBS
L. Pollegioni et al. Enzymatic degradation of lignin

Fig. 8. (A) Three different pathways of

oxidation of phenolic b-1 lignin model
compounds by laccase [77,78]. (B) Three
different pathways of oxidation of
nonphenolic b-O-4 lignin model
compounds by laccase in the presence of
the HBT mediator [127].

reaching an expression level of 34 kUL1 [83]. The
two isoenzymes show similar kinetic properties: the
specific activity was 51 Umg1 and 63 Umg1 and
the Km for ABTS was 0.43 mM and 0.29 mM for LccB
and LccC, respectively. On the other hand, production
levels for P. pastoris X33 expressing the LccA isozyme
were lower (18 kUL1) [84]. Indeed, the lcc1 gene was
cloned into the genome of Yarrowia lipolytica, reach-
ing an expression level of 250 UL1 of culture med-
ium [85].

FEBS Journal 282 (2015) 1190–1213 ª 2015 FEBS 1203

Enzymatic degradation of lignin
Protein engineering of laccases from different sources
A directed evolution approach enabled the isolation of
three variants of Lcc1 laccase that yielded a four-fold
increase in enzyme productivity (i.e. 1000 UL1) [85].
The better variant (L185P/Q214K, named rM-4A)
showed a 2.4-fold increase in the catalytic efficiency
toward ABTS (with a kcat and Km value of 210 s1
and 120 lM, respectively). The recombinant Lcc1 lac-
case was used in dye decolorization.
By employing a combined approach of directed evo-
lution and rational design, the thermostable laccase
from the basidiomycete PM1 (showing a moderate
high redox potential, i.e. > 0.7 V) was engineered by
Mate et al. [86]. The original signal sequence was
replaced by the a-factor preproleader, and the protein
was expressed in S. cerevisiae. The OB-1 variant iso-
lated after eight rounds of random mutagenesis
showed high activity and stability in terms of tempera-
ture, pH range and organic solvents; a 34 000-fold
enhancement in expression of the OB-1 activity
resulted from improvement of maximal activity and of
the expression level (~ 8 mgL1). A turnover rate of
200 s1 and a Km value of 6.3 lM was apparent with
the substrate ABTS.
Subsequently, an additional high redox potential lac-
case isolated from P. cinnabarinus (named PcL), and
whose original signal sequence was replaced by the a-
factor preproleader for expression in S. cerevisiae, was
the object of protein engineering studies by the same
research group [87]. After six rounds of directed evolu-
tion (by error-prone PCR and DNA shuffling), the
best variant obtained (a-3PO) showed a 13.7-fold
higher kcat for ABTS than the wild-type (483 s1 ver-
sus 38 s1, respectively), improved kinetic parameters
for various substrates (i.e. the catalytic efficiency for
sinapic acid and 2,6-dimethoxyphenol increased 5.4-
and 1.6-fold, respectively), an enzyme production of
300 UL1 and a secretion level of ~ 2 mgL1. More-
over, the 7A9 enzyme variant was overexpressed in
A. niger: to facilitate the secretion process, the a-factor
signal was replaced by the glucoamylase sequence from
A. niger. A production level of 23 mgL1 was
achieved and the recombinant enzyme displayed bio-
chemical features similar to those of the variant
expressed in S. cerevisiae.
White laccases
In recent years, it has been reported that atypical ‘yel-
low or white’ laccases (i.e. those lacking the peak at
around 600 nm in the absorption spectrum and in
which the A280/A610 ratio is ~ 20) from the white-rot
1204
L. Pollegioni et al.
fungi Panus tigrinus and P. radiata catalyze the oxida-
tion of nonphenolic b-1 lignin model compounds in
the absence of a mediator [88,89]. The first white lac-
case was purified from the basidiomycete P. ostreatus
by Palmieri et al. [90]: this enzyme contains only one
copper, two zinc atoms, and one iron atom in each
protein molecule. The laccase from Trametes hirsuta
contains copper and manganese in a 3 : 1 ratio.
A white laccase was recently purified from the deu-
teromycete fungus Myrothecium verrucaria NF-05, a
strain producing up to 40 kU of laccaseL1 after
13 days of cultivation [91]. The highest activity was
apparent at pH 4.0 and 30 °C and was significantly
enhanced by adding Na+, Mn2+, Cu2+ and Zn2+ (it
was instead inhibited by dithiothreitol, NaN3 and hal-
ogen anions). The enzyme showed a higher affinity for
ABTS (Km = 86 lM) than for other tested aromatic
substrates (i.e. cathechol) and a turnover rate of
270 s1. This enzyme could be effectively used for dye
decolorization.
Bacterial laccases
Currently, only fungal laccases are used in biotechno-
logical processes and, as stated above, most of the
characterized enzymes have been isolated from fungi.
To date, few bacterial laccases have been studied,
although rapid progress in genome analysis suggests
that these enzymes are widespread in bacteria. Here,
the most representative enzyme is the CotA laccase
from Bacillus subtilis, an endospore coat protein show-
ing a high thermostability [92,93] and for which the
crystal structure has been determined [94].
The 65-kDa CotA protein was purified to homoge-
neity from an overproducing E. coli strain [93]. The
optimal enzymatic activity was found at pH 3.0 for
ABTS and at pH 7.0 for syringaldazine. Remarkably,
the purified enzyme showed an inactivation half-life of
~ 2 h at 80 °C. The copper content of recombinant
CotA from B. subtilis when produced in E. coli cells
was strongly dependent on the presence of copper and
oxygen in the culture medium: in a copper-supple-
mented medium, the switch from aerobic to microaero-
bic conditions produced an 80-fold increase in copper
protein incorporation and 20-fold enhancement in the
kcat value (Table 5) [95].
A CotA laccase from Bacillus licheniformis was also
expressed in E. coli [96]. As shown in Table 5, the
kinetic constants Km and kcat for ABTS were 6.5 lM
and 83 s1. The highest activity was observed at
85 °C; however, it is as thermostable as CotA from
B. subtilis. Because the application of this laccase has
been hampered by expression levels (~ 26 mgL1), the
FEBS Journal 282 (2015) 1190–1213 ª 2015 FEBS
L. Pollegioni et al. Enzymatic degradation of lignin

Table 5. Kinetic parameters of different CotA bacterial laccases on ABTS as substrate.

Source Host kcat (s1) Km (lM) kcat/Km Reference

B. subtilis E. coli 17 106 0.16 [93]

B. subtilis (microaerobic) E. coli 322 124 2.60 [95]
B. licheniformis E. coli 83 6.5 12.80 [96]
B. pumilus E. coli 290 80 3.63 [98]

CotA double variant K316N/D500G was produced Cleaving b-O-4 ether bond
that reached an 11.4-fold higher level of expression
An extracellular b-etherase capable of cleaving the
than the wild-type enzyme [97]. Indeed, this laccase
phenolic b-O-4 lignin model dimer (i.e. the guaiacyl-
variant is also more efficient than the wild-type
glycerol b-guaiacyl ether) has been isolated from the
enzyme in converting ferulic acid (21% conversion ver-
fermentation broth of an ascomycete of the genus
sus 14%) and in decolorizing a range of industrial
Chaetomium, in particular, by the newly isolated fun-
dyes.
gus 2BW-1 (approximately 250 UL1) [102]. This 65-
Recently, a CotA laccase from Bacillus pumilus was
kDa enzyme catalyzes the addition of two molecules
also expressed and purified from E. coli cells [98]. The
of H2O at Ca and Cb positions and the cleavage of
recombinant enzyme showed a high activity (kcat for
the b-aryl ether bond of the model compound. The
ABTS was 290 s1) (Table 5), a pH optimum of 4.0, a
proposed reaction mechanism might proceed through
temperature optimum at around 70 °C and a wide
an intermediate quinine methide in which b-aryl ether
substrate specificity.
bond scission occurs, as shown in Fig. 9A. This is con-
The laccase from S. coelicolor shows also interesting
sistent with the observation that the b-aryl ether cleav-
features since it is relatively small in size (~ 32 kDa)
age enzyme requires a hydroxyl group and a Ca
and possesses a high activity at alkaline pH values
alcohol structure. A water molecule attacks the Ca
[99]: it is representative of a new enzyme family, the
position in the quinonemethide, the b-aryl ether link-
two-domain laccases, lacking the second domain
age is cleaved, and an additional water molecule
involved in the substrate binding (Fig. 6C). The
attacks the Ca position. As a result, the guaiacylglyc-
recombinant enzyme expressed in E. coli was highly
erol b-O-guaiacyl is converted to guaiacylglycerol and
stable (it retained activity as dimer in denaturating gels
guaiacol.
after boiling and SDS treatment), showed an optimal
Subsequently, an additional b-O-4 aryl-ether cleaving
kinetic efficiency on 2,6-dimethoxyphenol at pH 9.4
enzyme system from the protobacterium Sphingobium
(Km of ~ 0.5 mM and kcat of 10 s1), and was quite
sp. SYK-6 was identified and characterized [103]. This
resistant under various conditions (i.e. in the presence
reaction is part of a three-step process (shown in
of 1 mM NaN3). Laccase from S. ipomoea was also
Fig. 9B): the three proteins involved, LigD (a Ca-dehy-
overexpressed in E. coli BL21(DE3) as host and used
drogenase), LigF (a b-etherase) and LigG (a glutathione
for bleaching of kraft pulps [100]. Interestingly, an effi-
lyase belonging to the omega class of glutathione trans-
cient expression system was set up in Streptomyces livi-
ferase class and whose structure was solved) [104], were
dans (homologous expression host of this bacterial
successfully expressed and purified in E. coli [105]. This
laccase), reaching an expression level of 350 mgL1,
enzymatic system was demonstrated to specifically
the highest production yield reported so far for a bac-
cleave the b-aryl ethers of a model lignin substrate, the 1-
terial laccase [101]. The secreted enzyme showed an
(4-hydroxy-3-methoxy-phenyl)-2-(2-methoxyphenolxy)-
oxidative activity on a wide pH range, depending on
1,3-propanediol: LigD catalyzes the NAD+-dependent
the tested substrate.
oxidation of Ca of lignin substrate, LigF cleaves the
intermediate with attachment of glutathione at the Cb
Additional enzymatic delignification activities position; glutathione is then oxidized by LigG and the
product is released. Altogether, the enzyme cascade Lig-
In the past decade, several new enzymatic approaches
DFG catalyzes the reductive cleavage of the ether bond
for delignification have been reported, based on the
and the oxidation of the secondary alcohol via reduction
dermination of a number of novel biochemical path-
of NAD+ and oxidation of two molecules of reduced
ways. It has been suggested that these accessory
glutathione. Here, the co-substrates NAD+ and
enzymes enhance lignin degradation as a result of the
glutathione can be regenerated by introducing the
well-known lignolytic enzymes (see above).

FEBS Journal 282 (2015) 1190–1213 ª 2015 FEBS 1205

Enzymatic degradation of lignin
Fig. 9. Cleavage of b-O-4 ether bond. (A) The mechanism of catalysis of the b-aryl ether cleavage enzyme from the fungus Chaetomium
2BW-1 as proposed previously [102]. An asterisk indicates 18O labeling in water and reaction products. (B) Reaction pathway of the LigDFG
enzyme system [105].
NAD+-dependent glutathione reductase from Allochro-
matium vinosum, allowing for a self-sufficient balanced
enzymatic process. Approximately 35% of a model
compound was cleaved after 7 days of reaction [103].
The ability of this enzymatic cascade to release lignin
monomers from complex lignin structures (from differ-
ently prepared real lignin substrates) was also reported
[105]. Notably, LigE and LigP glutathione reductase
catalyzes a reaction similar to that of LigF on a different
isomeric guaiacylglycerol-b-guaiacyl ether intermediate
produced by LigD in the same degradation pathway
[106].
Biphenyl bond cleavage enzyme systems
In addition to b-O-4 aryl-ether linkage, the biphenyl
linkage is also a main component of lignin structure,
commonly occurring between two guaiacyl units
(Fig. 1). Oxidative cleavage of the C3 side chain
attached to the biphenyl group would generate 2,20 -di-
hydroxy-3,30 -dimethoxy-5,50 -dicarboxy-biphenyl, which
can be utilized for the growth of Sphingomonas pauc-
imobilis SYK-6 [107,108]. As shown in Fig. 10, deme-
thylation of one methoxy group was reported to be
catalyzed by the non-heme, iron-dependent demethy-
lase enzyme LigX; the catecholic product of LigX is
the substrate for oxidative cleavage by the extradiol
dioxygenase LigZ, and the ring fission product is
1206
L. Pollegioni et al.
subsequently cleaved by the C-C hydrolase LigY, to
form 5-carboxyvanillic acid and 4-carboxy-2-hydroxy-
pentadienoic acid. Two decarboxylase enzymes (named
LigW and LigW2) have been identified that subse-
quently convert 5-carboxyvanillic acid into the valu-
able compound vanillic acid.
O-Demethylation enzyme systems
S. paucimobilis SYK-6 was reported to grow using the
lignin-derived compounds vanillate and syringate as
the sole source of carbon and energy [109]. In detail,
vanillate and syringate are converted to protocatechu-
ate and 3-O-methylgallate by the O-demethylases
LigM and DesA, respectively [110]. These tetrahydrof-
olate-dependent enzymes were expressed in E. coli,
showing that vanillate and 3-O-methylgallate were also
substrates for DesA (but with a very low relative activ-
ity compared to syringate) and that vanillate and 3-O-
methylgallate were converted to protocatechuate and
gallate, respectively, by LigM enzyme with similar,
specific activities (whereas syringate was not a sub-
strate for LigM) [111].
General oxidative activities
Among the fungal extracellular enzymes proposed to
be involved in lignin degradation, we can include
FEBS Journal 282 (2015) 1190–1213 ª 2015 FEBS
L. Pollegioni et al.
Fig. 10. The pathway for the degradation of the lignin biphenyl component in S. paucimobilis by LigX, LigZ and LigY [108].
general oxidases generating H2O2, which provide the
hydrogen peroxide required by peroxidases, and myce-
lium-associated dehydrogenases [112]. The H2O2-gener-
ating oxidases include aryl-alcohol oxidase
(EC 1.1.3.7) (found in various fungi; e.g. P. eryngii)
and glyoxal oxidase (identified in P. chrysosporium). In
addition, quinone reductases (EC 1.6.5.5) are also
involved in lignin degradation by fungi [113]. More-
over, cellobiose dehydrogenase (EC 1.1.99.18), an
enzyme produced by many different fungi under cellu-
lolytic conditions, is also involved in lignin degrada-
tion in the presence of H2O2 and chelated iron ions
[114]. It was proposed to act on the reduction of qui-
nones, which can be used by ligninolytic enzymes or in
support of a MnP reaction [115].
The I.2.A extradiol dioxygenase subfamily appears
to be of particular importance for breaking down
monocyclic aromatic compounds [116]. Regarding the
catechol 2,3-dioxygenase (EC 1.13.11.2) subfamily, dif-
ferences in kinetic properties have been reported for
variants differing in a few residues [117] (e.g. in the
residue at position 218). The catechol 2,3-dioxygenase
variant possessing His218 showed the highest activity
on catechol, whereas the one possessing Tyr218
showed the highest activity on 4-methylcatechol (and
3-methylcatechol) and a low Km for all catecholic sub-
strates [118].
A recently identified type of hydrolase, the so-called
perhydrolase, has been proposed as useful biocatalyst
to produce peroxycarboxylic acids in situ (by using
H2O2 and carboxylic acids in aqueous media) and sub-
sequently employed for delignification [119,120]. Other
hydrolases, mostly lipases (EC 3.1.1.3), can be used as
excellent biocatalysts for in situ peracid formation in
nonaqueous media, starting from carboxylic acids and
diluted H2O2. Indeed, an immobilized lipase B from
Candida antartica was successfully employed in a per-
acid-mediated lignocellulosic delignification process in
a nonaqueous solvent: here, dimethylcarbonate was
used both as a solvent and as an acyl-donor reagent
for the lipase-mediated oxidation [121]. After 9 h of
incubation of beech wood lignin at 80 °C, the immobi-
FEBS Journal 282 (2015) 1190–1213 ª 2015 FEBS
Enzymatic degradation of lignin
lized enzyme was separated and the remnant wood
was obtained as a yellowish oil after dimethylcarbon-
ate evaporation, suggesting a successful delignification
process. ESI-MS analysis demonstrated the production
of small oligomers from both beech wood and conife-
ryl alcohol-derived synthetic lignin.
Treating unbleached kraft pulp with xylanase
(EC 3.2.1.8) did not release significant amounts of lig-
nin; however, the enzyme facilitated extraction of the
lignin fraction because it removed the linkage between
xylan and lignin [122].
Conclusions
Lignin represents a feedstock of enormous potential
for producing low-molecular-weight chemicals. Bec-
ause of its complex structure, considerable effort has
been devoted to understanding the major natural path-
ways and enzymes involved in lignin degradation. It is
now clear that, in nature, lignin degradation is a
multi-enzymatic process involving an array of acces-
sory enzymes in addition to the four major ligninolytic
oxidases. To reach such an ambitious goal, further
investigations will be aimed at: (a) defining the role
and interaction of enzyme combinations in lignin deg-
radation; (b) determining the role of small molecule
mediators (both natural and synthetic) in the catalytic
mechanism of the ligninolytic enzymes, as well as in
the overall process of lignin degradation; (c) reconsti-
tuting in vitro fungal/bacterial ligninolytic systems by
means of purified enzyme components; and (d) com-
bining enzyme treatment with other pretreatment
methods (the ‘combined’ approach).
Of note, ligninolytic enzymes find applications in a
number of industrial processes other than biorefinery
(e.g. laccase is used for clarifying musts and wines,
waste water treatment, biostaining of denim jeans,
assaying the total antioxidant amount in blood sam-
ples, and as biosensors, etc.). A recent review discuss-
ing the application of laccase as a potential
pretreatment strategy for biofuels production is pro-
vided elsewhere [123]. Similarly, the development of
1207
Enzymatic degradation of lignin
novel ligninolytic enzymes will pave the way for addi-
tional, unrelated industrial uses.
Acknowledgements
This work was supported by grants from FP7 project
ValorPlus (Project number 613802) and Fondazione
Cariplo – Regione Lombardia project Biorefill (Project
ID 42611813) to LP. FT is a student of the PhD
school in ‘Biotechnology, Biosciences and Surgical
Technologies’ at Universit

a degli studi dell’Insubria.
Author contributions
ER and FT researched bibliography; ER and FT drew
figures; ER and LP wrote the paper; LP reviewed the
manuscript. All the authors gave final approval.
References
1 Mai C, Majcherczyk A & Huttermann A (2000)
Chemo-enzymatic synthesis and characterization of
graft copolymers from lignin and acrylic compounds.
Enzyme Microb Technol 27, 167–175.
2 FitzPatrick M, Champagne P, Cunningham MF &
Whitney RA (2010) A biorefinery processing
perspective: treatment of lignocellulosic materials for
the production of value-added products. Biores
Technol 101, 8915–8922.
3 Pandey MP & Kim CS (2011) Lignin depolymerization
and conversion: a review of thermochemical methods.
Chem Eng Technol 34, 29–41.
4 Zhou CH, Xia X, Lin CX, Tong DS & Beltramini J
(2011) Catalytic conversion of lignocellulosic biomass
to fine chemicals and fuels. Chem Soc Rev 40,
5588–5617.
5 Nanayakkara S, Patti AF & Saito K (2014)
Chemical depolymerization of lignin involving the
redistribution mechanism with phenols and
repolymerization of depolymerized products. Green
Chem 16, 1897–1903.
6 Dashtban M, Schraft H, Syed TA & Qin W (2010)
Fungal biodegradation and enzymatic modification of
lignin. Int J Biochem Mol Biol 1, 36–50.
7 Wong DWS (2009) Structure and action mechanism of
ligninolytic enzymes. Appl Biochem Biotech 157,
174–209.
8 Forney LJ, Reddy CA, Tien M & Aust SD (1982) The
involvement of hydroxyl radical derived from hydrogen
peroxide in lignin degradation by the white rot fungus
Phanerochaete chrysosporium. J Biol Chem 257,
11455–11462.
9 Bourbonnais R, Leech D & Paice MG (1998)
Electrochemical analysis of the interactions of laccase
1208
L. Pollegioni et al.
mediators with lignin model compounds. Biochim
Biophys Acta 1379, 381–390.
10 Crestini C, Jurasek L & Argyropoulos DS (2003) On
the mechanism of the laccase – mediator system in the
oxidation of lignin. Chem Eur J 9, 5371–5378.
11 Ca~nas AI & Camarero S (2010) Laccases and their
natural mediators: biotechnological tools for
sustainable eco-friendly processes. Biotechnol Adv 28,
694–705.
12 Lange H, Decina S & Crestini C (2013) Oxidative
upgrade of lignin – recent routes reviewed. Eur Polym
J 49, 1151–1173.
13 Asgher M, Shahid M, Kamal S & Iqbal HMN (2014)
Recent trends and valorization of immobilization
strategies and ligninolytic enzymes by industrial
biotechnology. J Mol Catal B-Enzym 101, 56–66.
14 Welinder KG, Mauro JM & Norskov-Lauritsen L
(1992) Structure of plant and fungal peroxidases.
Biochem Soc Trans 20, 337–340.
15 Wang W & Wen X (2009) Expression of lignin
peroxidase H2 from Phanerochaete chrysosporium by
multi-copy recombinant Pichia strain. J Environ Sci
21, 218–222.
16 Valli K, Wariishi H & Gold MH (1990) Oxidation of
monomethoxylated aromatic compounds by lignin
peroxidase: role of veratryl alcohol in lignin
biodegradation. Biochemistry 29, 8535–8539.
17 Hammel KE, Tien M, Kalyanaraman B & Kirk TK
(1985) Mechanism of oxidative C alpha-C beta cleavage
of a lignin model dimer by Phanerochaete chrysosporium
ligninase. Stoichiometry and involvement of free
radicals. J Biol Chem 260, 8348–8353.
18 Lundell T, Wever R, Floris R, Harvey P, Hatakka A,
Brunow G & Schoemaker H (1993) Lignin peroxidase
L3 from Phlebia radiata. Pre-steady-state and steady-
state studies with veratryl alcohol and a non-phenolic
lignin model compound 1-(3,4-dimethoxyphenyl)-2-(2-
methoxyphenoxy)propane-1,3-diol. Eur J Biochem 211,
391–402.
19 Baciocchi E, Fabbri C & Lanzalunga O (2003) Lignin
peroxidase-catalyzed oxidation of nonphenolic trimeric
lignin model compounds: fragmentation reactions in
the intermediate radical cations. J Org Chem 68,
9061–9069.
20 Choinowski T, Blodig W, Winterhalter KH & Piontek
K (1999) The crystal structure of lignin peroxidase at
1.70 A resolution reveals a hydroxy group on the
cbeta of tryptophan 171: a novel radical site formed
during the redox cycle. J Mol Biol 286, 809–827.
21 Edwards SL, Raag R, Wariishi H, Gold MH & Poulos
TL (1993) Crystal structure of lignin peroxidase.
PNAS 90, 750–754.
22 Poulos TL, Edwards SL, Wariishi H & Gold MH
(1993) Crystallographic refinement of lignin peroxidase
at 2 A. J Biol Chem 268, 4429–4440.
FEBS Journal 282 (2015) 1190–1213 ª 2015 FEBS
L. Pollegioni et al.
23 Blodig W, Smith AT, Doyle WA & Piontek K (2001)
Crystal structures of pristine and oxidatively processed
lignin peroxidase expressed in Escherichia coli and of
the W171F variant that eliminates the redox active
tryptophan 171. Implications for the reaction
mechanism. J Mol Biol 305, 851–861.
24 Doyle WA, Blodig W, Veitch NC, Piontek K & Smith
AT (1998) Two substrate interaction sites in lignin
peroxidase revealed by site-directed mutagenesis.
Biochemistry 37, 15097–150105.
25 Gelpke MDS, Lee J & Gold MH (2002) Lignin
peroxidase oxidation of veratryl alcohol: Effects of the
mutants H82A, Q222A, W171A, and F267L.
Biochemistry 41, 3498–3506.
26 Coconi-Linares N, Maga~ na-Ortız D, Guzman-Ortiz
DA, Fernandez F, Loske AM & G omez-Lim MA
(2014) High-yield production of manganese peroxidase,
lignin peroxidase, and versatile peroxidase in
Phanerochaete chrysosporium. Appl Microbiol
Biotechnol 98, 9283–9294.
27 Nie G, Reading NS & Aust SD (1998) Expression of
the lignin peroxidase H2 gene from Phanerochaete
chrysosporium in Escherichia coli. Biochem Biophys Res
Commun 249, 146–150.
28 Gelpke MDS, Mayfield-Gambill M, Cereghino GPL &
Gold MH (1999) Homologous expression of
recombinant lignin peroxidase in Phanerochaete
chrysosporium. Appl Environ Microbiol 65, 1670–1674.
29 Wang HK, Lu FP, Sun WF & Du LX (2004)
Heterologous expression of lignin peroxidase of
Phanerochaete chrysosporium in Pichia methanolica.
Biotechnol Lett 26, 1569–1573.
30 Xie J, Feng L, Xu N, Zhu GH, Yang J, Xu XL & Fu
SY (2007) Studies on the fusion of ligninolytic enzyme
cDNAs and their expression. Bioresources 2, 598–604.
31 Hammel KE, Jensen KA Jr, Mozuch MD, Landucci
LL, Tien M & Pease EA (1993) Ligninolysis by a
purified lignin peroxidase. J Biol Chem 268,
12274–12281.
32 Haemmerli SD, Leisola MSA & Fiechter A (1986)
Polymerisation of lignins by ligninases from
Phanerochaete chrysosporium. FEMS Microbiol Lett
35, 33–36.
33 Brunow G (2005) Methods to reveal the structure of
lignin. In Lignin, Humic Substances and Coal.
Biopolymers Online (Hofrichter M & Steinb€ uchel A,
eds), pp. 89–116. Wiley-VHC, Weinheim.
34 Ghaffar SH & Fan M (2013) Structural analysis for
lignin characteristics in biomass straw. Biomass
Bioenergy 57, 264–279.
35 Glenn JK & Gold MH (1985) Purification and
characterization of an extracellular Mn(II)-dependent
peroxidase from the lignin-degrading basidiomycete,
Phanerochaete chrysosporium. Arch Biochem Biophys
242, 329–341.
FEBS Journal 282 (2015) 1190–1213 ª 2015 FEBS
Enzymatic degradation of lignin
36 Paszczynski A, Huynh VB & Crawford R (1986)
Comparison of ligninase-I and peroxidase-M2 from
the white-rot fungus Phanerochaete chrysosporium.
Arch Biochem Biophys 244, 750–765.
37 Wariishi H, Valli K & Gold MH (1991) In vitro
depolymerization of lignin by manganese peroxidase of
Phanerochaete chrysosporium. Biochem Biophys Res
Commun 176, 269–275.
38 Hammel KE & Cullen D (2008) Role of fungal
peroxidases in biological ligninolysis. Curr Opin Plant
Biol 11, 349–355.
39 Sundaramoorthy M, Kishi K, Gold MH & Poulos TL
(1997) Crystal structures of substrate binding site
mutants of manganese peroxidase. J Biol Chem 272,
17574–17580.
40 Sundaramoorthy M, Kishi K, Gold MH & Poulos TL
(1994) The crystal structure of manganese peroxidase
from Phanerochaete chrysosporium at 2.06-A
resolution. J Biol Chem 269, 32759–32767.
41 Gelpke MD, Youngs HL & Gold MH (2000) Role
of arginine 177 in the MnII binding site of
manganese peroxidase. Studies with R177D, R177E,
R177N, and R177Q mutants. Eur J Biochem 267,
7038–7045.
42 Sundaramoorthy M, Youngs HL, Gold MH & Poulos
TL (2005) High-resolution crystal structure of
manganese peroxidase: substrate and inhibitor
complexes. Biochemistry 44, 6463–6470.
43 Whitwam R & Tien M (1996) Heterologous expression
and reconstitution of fungal Mn peroxidase. Arch
Biochem Biophys 333, 439–446.
44 Gu L, Lajoie C & Kelly C (2003) Expression of a
Phanerochaete chrysosporium manganese peroxidase
gene in the yeast Pichia pastoris. Biotechnol Progr 19,
1403–1409.
45 Jiang F, Kongsaeree P, Charron R, Lajoie C, Xu H,
Scott G & Kelly C (2008) Production and separation
of manganese peroxidase from heme amended yeast
cultures. Biotechnol Bioeng 99, 540–549.
46 Timofeevski SL, Nie G, Reading NS & Aust SD
(1999) Addition of veratryl alcohol oxidase activity to
manganese peroxidase by site-directed mutagenesis.
Biochem Biophys Res Commun 256, 500–504.
47 Timofeevski SL, Nie G, Reading NS & Aust SD
(2000) Substrate specificity of lignin peroxidase and a
S168W variant of manganese peroxidase. Arch
Biochem Biophys 373, 147–153.
48 Sutherland GR & Aust SD (1996) The effects of
calcium on the thermal stability and activity of
manganese peroxidase. Arch Biochem Biophys 332,
128–134.
49 Nie G, Reading NS & Aust SD (1999) Relative
stability of recombinant versus native peroxidases from
Phanerochaete chrysosporium. Arch Biochem Biophys
365, 328–334.
1209
Enzymatic degradation of lignin
50 Reading NS & Aust SD (2000) Engineering a disulfide
bond in recombinant manganese peroxidase results in
increased thermostability. Biotechnol Progr 16,
326–333.
51 Miyazaki C & Takahashi H (2001) Engineering of the
H2O2-binding pocket region of a recombinant
manganese peroxidase to be resistant to H2O2. FEBS
Lett 509, 111–114.
52 Ufot UF & Akpanabiatu MI (2012) An engineered
Phlebia radiata manganese peroxidase: expression,
refolding, purification and preliminary
characterization. Am J Mol Biol 2, 359–370.
53 Hofrichter M, Ullrich R, Pecyna MJ, Liers C &
Lundell T (2010) New and classic families of secreted
fungal heme peroxidases. Appl Microbiol Biotechnol 87,
871–897.
54 Ruiz-Duenas FJ, Morales M, Perez-Boada M,
Choinowski T, Martinez MJ, Piontek K & Martinez
AT (2007) Manganese oxidation site in Pleurotus
eryngii versatile peroxidase: a site-directed mutagenesis,
kinetic, and crystallographic study. Biochemistry 46,
66–77.
55 Perez-Boada M, Ruiz-Duenas FJ, Pogni R, Basosi R,
Choinowski T, Martinez MJ, Piontek K & Martinez
AT (2005) Versatile peroxidase oxidation of high
redox potential aromatic compounds: site-directed
mutagenesis, spectroscopic and crystallographic
investigation of three long-range electron transfer
pathways. J Mol Biol 354, 385–402.
56 Ruiz-Duenas FJ, Martinez MJ & Martinez AT (1999)
Heterologous expression of Pleurotus eryngii
peroxidase confirms its ability to oxidize Mn(2+) and
different aromatic substrates. Appl Environ Microbiol
65, 4705–4707.
57 Tien M, Kirk TK, Bull C & Fee JA (1986) Steady-
state and transient-state kinetic studies on the
oxidation of 3,4-dimethoxybenzyl alcohol catalyzed by
the ligninase of Phanerocheate chrysosporium Burds. J
Biol Chem 261, 1687–1693.
58 Caramelo L, Martinez MJ & Martinez AT (1999) A
search for ligninolytic peroxidases in the fungus
Pleurotus eryngii involving a-keto-c-thiomethylbutyric
acid and lignin model dimers. Appl Environ Microbiol
65, 916–922.
59 Fernandez-Fueyo E, Ruiz-Duenas FJ, Martinez MJ,
Romero A, Hammel KE, Medrano FJ & Martinez AT
(2014) Ligninolytic peroxidase genes in the oyster
mushroom genome: heterologous expression, molecular
structure, catalytic and stability properties, and lignin-
degrading ability. Biotechnol Biofuels 7, 2.
60 Tsukihara T, Honda Y & Watanabe T (2006)
Molecular breeding of white rot fungus Pleurotus
ostreatus by homologous expression of its versatile
peroxidase MnP2. Appl Microbiol Biotechnol 71,
114–120.
1210
L. Pollegioni et al.
61 Tsukihara T, Honda Y, Sakai R & Watanabe T (2006)
Exclusive overproduction of recombinant versatile
peroxidase MnP2 by genetically modified white rot
fungus, Pleurotus ostreatus. J Biotechnol 126, 431–439.
62 Mohorcic M, Bencina M, Friedrich J & Jerala R
(2009) Expression of soluble versatile peroxidase of
Bjerkandera adusta in Escherichia coli. Bioresour
Technol 100, 851–858.
63 Bao X, Liu A, Lu X & Li JJ (2012) Direct over-
expression, characterization and H2O2 stability study
of active Pleurotus eryngii versatile peroxidase in
Escherichia coli. Biotechnol Lett 34, 1537–1543.
64 Garcia-Ruiz E, Gonzalez-Perez D, Ruiz-Duenas FJ,
Martinez AT & Alcalde M (2012) Directed evolution
of a temperature-, peroxide- and alkaline pH-tolerant
versatile peroxidase. Biochem J 441, 487–498.
65 Bao X, Huang X, Lu X & Li JJ (2014) Improvement
of hydrogen peroxide stability of Pleurotus eryngii
versatile ligninolytic peroxidase by rational protein
engineering. Enz Microb Tech 54, 51–58.
66 Roberts JN, Singh R, Grigg JC, Murphy MEP, Bugg
TDH & Eltis LD (2011) Characterization of dye-
decolorizing peroxidases from Rhodococcus jostii
RHA1. Biochemistry 50, 5108–5119.
67 Ahmad M, Taylor CR, Pink D, Burton K, Eastwood
D, Bending GD & Bugg TD (2010) Development of
novel assays for lignin degradation: comparative
analysis of bacterial and fungal lignin degraders. Mol
BioSyst 6, 815–821.
68 Singh R, Grigg JC, Qin W, Kadla JF, Murphy ME &
Eltis LD (2013) Improved manganese-oxidizing activity
of DypB, a peroxidase from a lignolytic bacterium.
ACS Chem Biol 8, 700–706.
69 Brown ME, Barros T & Chang MC (2012)
Identification and characterization of a multifunctional
dye peroxidase from a lignin-reactive bacterium. ACS
Chem Biol 7, 2074–2081.
70 Giardina P, Faraco V, Pezzella C, Piscitelli A,
Vanhulle S & Sannia G (2010) Laccases: a never-
ending story. Cell Mol Life Sci 67, 369–385.
71 Thurston CF (1994) The structure and function of
fungal laccases. Microbiol-Sgm 140, 19–26.
72 Piontek K, Antorini M & Choinowski T (2002)
Crystal structure of a laccase from the fungus
Trametes versicolor at 1.90-A resolution containing a
full complement of coppers. J Biol Chem 277,
37663–37669.
73 Bertrand T, Jolivalt C, Briozzo P, Caminade E, Joly
N, Madzak C & Mougin C (2002) Crystal structure of
a four-copper laccase complexed with an arylamine:
insights into substrate recognition and correlation with
kinetics. Biochemistry 41, 7325–7333.
74 Skalova T, Dohnalek J, Ostergaard LH, Ostergaard
PR, Kolenko P, Duskova J, Stepankova A & Hasek J
(2009) The structure of the small laccase from
FEBS Journal 282 (2015) 1190–1213 ª 2015 FEBS
L. Pollegioni et al.
Streptomyces coelicolor reveals a link between laccases
and nitrite reductases. J Mol Biol 385, 1165–1178.
75 Xu F (1996) Catalysis of novel enzymatic iodide
oxidation by fungal laccase. Appl Biochem Biotech 59,
221–230.
76 Xu F (1997) Effects of redox potential and hydroxide
inhibition on the pH activity profile of fungal laccases.
J Biol Chem 272, 924–928.
77 Kawai S, Nakagawa M & Ohashi H (1999) Aromatic
ring cleavage of a non-phenolic beta-O-4 lignin model
dimer by laccase of Trametes versicolor in the presence
of 1-hydroxybenzotriazole. FEBS Lett 446, 355–358.
78 Kawai S, Asukai M, Ohya N, Okita K, Ito T &
Ohashi H (1999) Degradation of a non-phenolic beta-
O-4 substructure and of polymeric lignin model
compounds by laccase of Coriolus versicolor in the
presence of 1-hydroxybenzotriazole. FEMS Microbiol
Lett 170, 51–57.
79 LaFayette PR, Eriksson KE & Dean JF (1999)
Characterization and heterologous expression of
laccase cDNAs from xylem tissues of yellow-poplar
(Liriodendron tulipifera). Plant Mol Biol 40, 23–35.
80 Bollag JM & Leonowicz A (1984) Comparative studies
of extracellular fungal laccases. Appl Environ Microbiol
48, 849–854.
81 Otterbein L, Record E, Longhi S, Asther M &
Moukha S (2000) Molecular cloning of the cDNA
encoding laccase from Pycnoporus cinnabarinus I-937
and expression in Pichia pastoris. Eur J Biochem 267,
1619–1625.
82 Molina-Guijarro JM, Perez J, Munoz-Dorado J,
Guillen F, Moya R, Hernandez M & Arias ME (2009)
Detoxification of azo dyes by a novel pH-versatile,
salt-resistant laccase from Streptomyces ipomoea. Int
Microbiol 12, 13–21.
83 Li Q, Ge L, Cai J, Pei J, Xie J & Zhao L (2014)
Comparison of two laccases from Trametes versicolor
for application in the decolorization of dyes.
J Microbiol Biotechnol 24, 545–555.
84 Li Q, Pei J, Zhao L, Xie J, Cao F & Wang G (2014)
Overexpression and characterization of laccase from
Trametes versicolor in Pichia pastoris. Appl Biochem
Microbiol 50, 140–147.
85 Theerachat M, Emond S, Cambon E, Bordes F, Marty
A, Nicaud JM, Chulalaksananukul W, Guieysse D,
Remaud-Simeon M & Morel S (2012) Engineering and
production of laccase from Trametes versicolor in the
yeast Yarrowia lipolytica. Bioresour Technol 125,
267–274.
86 Mate D, Garcia-Burgos C, Garcia-Ruiz E, Ballesteros
AO, Camarero S & Alcalde M (2010) Laboratory
evolution of high-redox potential laccases. Chem Biol
17, 1030–1041.
87 Camarero S, Pardo I, Canas AI, Molina P, Record E,
Martinez AT, Martinez MJ & Alcalde M (2012)
FEBS Journal 282 (2015) 1190–1213 ª 2015 FEBS
Enzymatic degradation of lignin
Engineering platforms for directed evolution of laccase
from Pycnoporus cinnabarinus. Appl Environ Microbiol
78, 1370–1384.
88 Leontievsky A, Myasoedova N, Pozdnyakova N &
Golovleva L (1997) ‘Yellow’ laccase of Panus tigrinus
oxidizes non-phenolic substrates without electron-
transfer mediators. FEBS Lett 413, 446–448.
89 Leontievsky AA, Vares T, Lankinen P, Shergill JK,
Pozdnyakova NN, Myasoedova NM, Kalkkinen N,
Golovleva LA, Cammack R, Thurston CF et al.
(1997) Blue and yellow laccases of ligninolytic fungi.
FEMS Microbiol Lett 156, 9–14.
90 Palmieri G, Giardina P, Bianco C, Scaloni A, Capasso
A & Sannia G (1997) A novel white laccase from
Pleurotus ostreatus. J Biol Chem 272, 31301–31307.
91 Zhao D, Zhang X, Cui D & Zhao M (2012)
Characterisation of a novel white laccase from the
deuteromycete fungus Myrothecium verrucaria NF-05
and its decolourisation of dyes. PLoS ONE 7, e38817.
92 Hullo MF, Moszer I, Danchin A & Martin-Verstraete
I (2001) CotA of Bacillus subtilis is a copper-dependent
laccase. J Bacteriol 183, 5426–5430.
93 Martins LO, Soares CM, Pereira MM, Teixeira M,
Costa T, Jones GH & Henriques AO (2002) Molecular
and biochemical characterization of a highly stable
bacterial laccase that occurs as a structural component
of the Bacillus subtilis endospore coat. J Biol Chem
277, 18849–18859.
94 Enguita FJ, Martins LO, Henriques AO & Carrondo
MA (2003) Crystal structure of a bacterial endospore
coat component. A laccase with enhanced
thermostability properties. J Biol Chem 278,
19416–19425.
95 Durao P, Chen Z, Fernandes AT, Hildebrandt P,
Murgida DH, Todorovic S, Pereira MM, Melo EP &
Martins LO (2008) Copper incorporation into
recombinant CotA laccase from Bacillus subtilis:
characterization of fully copper loaded enzymes. J Biol
Inorg Chem 13, 183–193.
96 Koschorreck K, Richter SM, Ene AB, Roduner E,
Schmid RD & Urlacher VB (2008) Cloning and
characterization of a new laccase from Bacillus
licheniformis catalyzing dimerization of phenolic acids.
Appl Microbiol Biotechnol 79, 217–224.
97 Koschorreck K, Schmid RD & Urlacher VB (2009)
Improving the functional expression of a Bacillus
licheniformis laccase by random and site-directed
mutagenesis. BMC Biotechnol 9, 12.
98 Reiss R, Ihssen J & Thony-Meyer L (2011) Bacillus
pumilus laccase: a heat stable enzyme with a wide
substrate spectrum. BMC Biotechnol 11, 9.
99 Machczynski MC, Vijgenboom E, Samyn B & Canters
GW (2004) Characterization of SLAC: a small laccase
from Streptomyces coelicolor with unprecedented
activity. Protein Sci 13, 2388–2397.
1211
Enzymatic degradation of lignin
100 Eugenio ME, Hernandez M, Moya R, Martin-
Sampedro R, Villar JC & Arias ME (2011) Evaluation
of a new laccase produced by Streptomyces Ipomoea
on biobleaching and ageing of Kraft pulps.
Bioresources 6, 3231–3241.
101 Dube E, Shareck F, Hurtubise Y, Daneault C &
Beauregard M (2008) Homologous cloning,
expression, and characterisation of a laccase from
Streptomyces coelicolor and enzymatic decolourisation
of an indigo dye. Appl Microbiol Biotechnol 79,
597–603.
102 Otsuka Y, Sonoki T, Ikeda S, Kajita S, Nakamura M
& Katayama Y (2003) Detection and characterization
of a novel extracellular fungal enzyme that catalyzes
the specific and hydrolytic cleavage of lignin
guaiacylglycerol beta-aryl ether linkages. Eur J
Biochem 270, 2353–2362.
103 Sato Y, Moriuchi H, Hishiyama S, Otsuka Y, Oshima
K, Kasai D, Nakamura M, Ohara S, Katayama Y,
Fukuda M et al. (2009) Identification of three alcohol
dehydrogenase genes involved in the stereospecific
catabolism of arylglycerol-beta-aryl ether by
Sphingobium sp. strain SYK-6. Appl Environ Microbiol
75, 5195–5201.
104 Meux E, Prosper P, Masai E, Mulliert G, Dumarcay
S, Morel M, Didierjean C, Gelhaye E & Favier F
(2012) Sphingobium sp SYK-6 LigG involved in lignin
degradation is structurally and biochemically related
to the glutathione transferase omega class. FEBS Lett
586, 3944–3950.
105 Reiter J, Strittmatter H, Wiemann LO, Schieder D &
Sieber V (2013) Enzymatic cleavage of lignin beta-O-4
aryl ether bonds via net internal hydrogen transfer.
Green Chem 15, 1373–1381.
106 Tanamura K, Abe T, Kamimura N, Kasai D,
Hishiyama S, Otsuka Y, Nakamura M, Kajita S,
Katayama Y, Fukuda M et al. (2011) Characterization
of the third glutathione S-transferase gene involved in
enantioselective cleavage of the beta-aryl ether by
Sphingobium sp. strain SYK-6. Biosci Biotechnol
Biochem 75, 2404–2407.
107 Sonoki T, Masai E, Sato K, Kajita S & Katayama Y
(2009) Methoxyl groups of lignin are essential carbon
donors in C1 metabolism of Sphingobium sp. SYK-6.
J Basic Microbiol 49 (Suppl. 1), S98–S102.
108 Bugg TD, Ahmad M, Hardiman EM & Rahmanpour
R (2011) Pathways for degradation of lignin in
bacteria and fungi. Nat Prod Rep 28, 1883–1896.
109 Masai E, Katayama Y, Nishikawa S & Fukuda M
(1999) Characterization of Sphingomonas paucimobilis
SYK-6 genes involved in degradation of lignin-related
compounds. J Ind Microbiol Biotechnol 23, 364–373.
110 Kasai D, Masai E, Miyauchi K, Katayama Y &
Fukuda M (2005) Characterization of the gallate
dioxygenase gene: three distinct ring cleavage
1212
L. Pollegioni et al.
dioxygenases are involved in syringate degradation by
Sphingomonas paucimobilis SYK-6. J Bacteriol 187,
5067–5074.
111 Masai E, Sasaki M, Minakawa Y, Abe T, Sonoki T,
Miyauchi K, Katayama Y & Fukuda M (2004) A
novel tetrahydrofolate-dependent O-demethylase gene
is essential for growth of Sphingomonas paucimobilis
SYK-6 with syringate. J Bacteriol 186, 2757–2765.
112 Martinez AT, Speranza M, Ruiz-Duenas FJ, Ferreira
P, Camarero S, Guillen F, Martinez MJ, Gutierrez A
& del Rio JC (2005) Biodegradation of
lignocellulosics: microbial, chemical, and enzymatic
aspects of the fungal attack of lignin. Int Microbiol 8,
195–204.
113 Guillen F, Martinez MJ, Munoz C & Martinez AT
(1997) Quinone redox cycling in the ligninolytic fungus
Pleurotus eryngii leading to extracellular production of
superoxide anion radical. Arch Biochem Biophys 339,
190–199.
114 Henriksson G, Ander P, Pettersson B & Pettersson G
(1995) Cellobiose dehydrogenase (Cellobiose oxidase)
from Phanerochaete chrysosporium as a wood
degrading enzyme – studies on cellulose, xylan and
synthetic lignin. Appl Microbiol Biotechnol 42,
790–796.
115 Henriksson G, Johansson G & Pettersson G (2000) A
critical review of cellobiose dehydrogenases. J Biotechnol
78, 93–113.
116 Eltis LD & Bolin JT (1996) Evolutionary relationships
among extradiol dioxygenases. J Bacteriol 178,
5930–5937.
117 Williams PA, Assinder SJ & Shaw LE (1990)
Construction of hybrid xylE genes between the two
duplicate homologous genes from TOL plasmid
pWW53: comparison of the kinetic properties of the
gene products. J Gen Microbiol 136, 1583–1589.
118 Junca H, Plumeier I, Hecht HJ & Pieper DH (2004)
Difference in kinetic behaviour of catechol 2,3-
dioxygenase variants from a polluted environment.
Microbiology 150, 4181–4187.
119 Carboni-Oerlemans C, Dominguez de Maria P, Tuin
B, Bargeman G, van der Meer A & van Gemert R
(2006) Hydrolase-catalysed synthesis of
peroxycarboxylic acids: biocatalytic promiscuity for
practical applications. J Biotechnol 126, 140–151.
120 Duncan S, Jing Q, Katona A, Kazlauskas RJ,
Schilling J, Tschirner U & Aldajani WW (2010)
Increased saccharification yields from aspen biomass
upon treatment with enzymatically generated peracetic
acid. Appl Biochem Biotechnol 160, 1637–1652.
121 Wiermans L, Perez-Sanchez M & Dominguez de
Maria P (2013) Lipase-mediated oxidative
delignification in non-aqueous media: formation of
de-aromatized lignin-oil and cellulase-accessible
polysaccharides. ChemSusChem 6, 251–255.
FEBS Journal 282 (2015) 1190–1213 ª 2015 FEBS
L. Pollegioni et al.
122 Varnai A, Huikko L, Pere J, Siika-Aho M & Viikari L
(2011) Synergistic action of xylanase and mannanase
improves the total hydrolysis of softwood. Biores
Technol 102, 9096–9104.
123 Kudanga T & Le Roes-Hill M (2014) Laccase
applications in biofuels production: current status and
future prospects. Appl Microbiol Biotechnol 98,
6525–6542.
124 Chen YR, Sarkanen S & Wang YY (2012) Lignin-
degrading enzyme activities. Methods Mol Biol 908,
251–268.
125 Wariishi H, Akileswaran L & Gold MH (1988)
Manganese peroxidase from the basidiomycete
Phanerochaete chrysosporium: spectral characterization
of the oxidized states and the catalytic cycle.
Biochemistry 27, 5365–5370.
FEBS Journal 282 (2015) 1190–1213 ª 2015 FEBS
Enzymatic degradation of lignin
126 Solomon EI, Penfield KW, Gewirth AA, Lowery MD,
Shadle SE, Guckert JA & LaCroix LB (1996)
Electronic structure of the oxidized and reduced blue
copper sites: contributions to the electron transfer
pathway, reduction potential, and geometry. Inorg
Chim Acta 243, 67–78.
127 Kawai S, Nakagawa M & Ohashi H (2002)
Degradation mechanisms of a nonphenolic beta-O-4
lignin model dimer by Trametes versicolor laccase in
the presence of 1-hydroxybenzotriazole. Enzy Microb
Tech 30, 482–489.
128 Heinfling A, Ruiz-Duenas FJ, Martinez MJ,
Bergbauer M, Szewzyk U & Martinez AT (1998) A
study on reducing substrates of manganese-oxidizing
peroxidases from Pleurotus eryngii and Bjerkandera
adusta. FEBS Lett 428, 141–146.
1213