Fungal transformation of lignocellulosics as revealed by chemical and ultrastructural analyses.recent advances on fungal treatment of lignocellulosic materials

Abstract

Utrastructural and chemical aspects of lignocellulose transformation by fungi during natural decay and solid-state fermentation are presented.

An extensive fungal transformation of wood by Ganodenna australe, showing both selective and simultaneous degradation patterns, was found in Chilean rain forest, and studied under in vitro conditions. After a preferential decay of lignin and xylans, this fungus produces a partially
delignified and highly digestible material, 'huempe" that is used as cattle feed.

Lignin alteration during wood decay was analyzed by spectroscopic and degradative techniques. The S/G ratios from CuO alkaline degradation of wood were only slightly greater that those obtained from CPMAS 13C-NMR. Very high values were obtained in some of the Chilean woods studied. The composition of the hemicellulose fraction of the latter was also unusual. The CuO degradation gave information on the alteration oflignin by fungi. This was followed by the study of the non-condensed fraction by acidolysis and thioacidolysis.

A decrease in the S/G ratio during fungal degradation was found in both condensed and non-condensed fractions of lignin and, compared with that by other fungal species, an extensive oxidation of lignin was observed during wood decay by G. australe.

Full text

RESEARCH REVIEW
Summary.Wood is the main renewable material on Earth and is largely used as
building material and in paper-pulp manufacturing. This review describes the com-
position of lignocellulosic materials, the different processes by which fungi are
able to alter wood, including decay patterns caused by white, brown, and soft-rot
fungi, and fungal staining of wood. The chemical, enzymatic, and molecular
aspects of the fungal attack of lignin, which represents the key step in wood decay,
are also discussed. Modern analytical techniques to investigate fungal degradation
and modification of the lignin polymer are reviewed, as are the different oxidative
enzymes (oxidoreductases) involved in lignin degradation. These include laccases,
high redox potential ligninolytic peroxidases (lignin peroxidase, manganese perox-
idase, and versatile peroxidase), and oxidases. Special emphasis is given to the
reactions catalyzed, their synergistic action on lignin, and the structural bases for
their unique catalytic properties. Broadening our knowledge of lignocellulose
biodegradation processes should contribute to better control of wood-decaying
fungi, as well as to the development of new biocatalysts of industrial interest based
on these organisms and their enzymes. [Int Microbiol 2005; 8(3):195-204]
Key words:wood-rotting fungi · lignin · analytical pyrolysis · oxidoreductases ·
catalytic mechanisms
Biodegradation of lignocellu-
losics: microbial, chemical,
and enzymatic aspects of
the fungal attack of lignin
Lignocellulosic materials
Forests represent approximately 27% of the world’s land
area, and wood is the predominant commercial product from
forests. Global wood consumption is around 3500 million
m3/year, and has increased over 65% since 1960. More than
half of this consumption is for fuel. The remainder of the
global roundwood consumption is largely for pulp and paper
products, building materials, and other wood in service.
Wood and other lignocellulosic materials are formed by
three main polymeric constituents, cellulose, lignin, and
hemicelluloses [25]. Cellulose is a linear and highly ordered
(often crystalline) polymer of cellobiose (D-glucopyranosyl-
β-1,4-D-glucopyranose) that represents over 50% of wood
weight. By contrast, lignin is a three-dimensional network
built up of dimethoxylated (syringyl, S), monomethoxylated
(guaiacyl, G) and non-methoxylated (p-hydroxyphenyl, H)
phenylpropanoid units, derived from the corresponding p-hy-
droxycinnamyl alcohols, which give rise to a variety of sub-
units including different ether and C—C bonds. Acetylated
lignin units have been recently identified in non-woody plants
using analytical pyrolysis [7].
Lignin is highly resistant towards chemical and biologi-
cal degradation, and confers mechanical resistance to wood.
The highest concentration of this recalcitrant polymer is
found in the middle lamella, where it acts as a cement
between wood fibers, but it is also present in the layers of the
cell wall (especially the secondary cell-wall), forming,
together with hemicelluloses, an amorphous matrix in which
the cellulose fibrils are embedded and protected against
biodegradation [14]. Lignin composition in terms of the
Ángel T. Martínez1*
Mariela Speranza1
Francisco J. Ruiz-Dueñas1
Patricia Ferreira1
Susana Camarero1
Francisco Guillén1,3
María J. Martínez1
Ana Gutiérrez2
José C. del Río2
1Biological Research
Center, CSIC, Madrid, Spain
2Institute of Natural Resources
and Agrobiology of Sevilla,
CSIC, Spain
3Present address: University of
Alcalá, Madrid, Spain
INTERNATIONAL MICROBIOLOGY (2005) 8:195-204
www.im.microbios.org
Received 24 March 2005
Accepted 15 May 2005
*Corresponding author:
A.T. Martínez
Centro de Investigaciones Biológicas, CSIC
Ramiro de Maeztu, 9
28040 Madrid, Spain
Tel. +34-918373112. Fax +34-925360432
E-mail: ATMartinez@cib.csic.es

196 INT. MICROBIOL. Vol. 8, 2005
H:G:S ratio varies between different vascular plant groups.
Woody gymnosperms (softwoods) have the highest lignin
content, and their lignin is made up mostly of G units. By
contrast, lignin of woody angiosperms (hardwoods) consists
of S and G units, and that from non-woody angiosperms con-
tains also H units. Lignin composition between the different
wood tissues and cell-wall layers also varies. For example,
middle-lamella lignin typically has a lower S/G ratio than
lignin from the secondary wall.
The third structural component, hemicelluloses (poly-
oses), has an intermediate degree of complexity and is made
up of different pentose and hexose residues, which are often
acetylated, and generally form branched chains. Typically,
hemicelluloses in softwood are glucomannans, whereas those
in hardwoods are mainly xylans together with variable percen-
tages of galactose, arabinose, rhamnose and methylglucuronic
acid units, and acetyl groups. Other non-structural components
of wood include compounds extractable with organic solvents
(the so-called extractives) which can be either polar (e.g. phe-
nols and tannins) or apolar (e.g. fats and sterols), water-soluble
compounds (e.g. sugars and starch), as well as proteins and
ashes. These components together generally represent less
than 5% of the dry weight of wood but can reach 20% in some
softwoods (e.g. in some Cupressaceae)[14].
The above constituents form the three main types of wood
tissue elements, namely fibers, vessels, and parenchyma cells
(Fig. 1A,B). In gymnosperms, wood tissues have a relatively
simpler structure than in angiosperms; they consist of 90–95%
tracheid cells (softwood fibers) and low amounts of parenchy-
ma, which includes the specialized resin channels in conifers.
Parenchymatic rays, which contain phenolic and lipophilic
extractives and water-soluble compounds as storage material,
have a radial arrangement in wood. Vessels are large cells with
a longitudinal arrangement, and they are responsible for the
transport of water and nutrients along the plant stem. Finally,
fibers, which are also longitudinally arranged, represent most
of the wood volume and are characterized by their thick cell
walls, which provide support to the tree.
Wood biodegradation
Lignocellulose degradation is a central step for carbon recy-
cling in land ecosystems. Moreover, fungal decay of wood in
MARTÍNEZ ETAL.
Int. Microbiol.
Fig. 1. Wood anatomy and fungal degradation.
In vitro degradation of Eucalyptus globulus
wood by Inocutis jamaicensis (using the soil-
block test). Images obtained using low-temper-
ature scanning-electron microscopy. (A,B):
Transversal and tangential sections, respective-
ly, of a hardwood showing large vessels (v),
parenchymatic rays (r) and fibers (f) (initial
degradation of wood with hyphae inside ves-
sels in shown in B). (C): Simultaneous white-
rot decay characterized by strong degradation
of all cell-wall components, including abun-
dant mycelium (with extracellular mucilage)
inside a vessel. (D): White-rot selective delig-
nification characterized by preferential degra-
dation of lignin and separation of fibers due to
destruction of the middle lamella. Black arrow-
heads in B indicate hyphae inside vessels, as
shown in C. White arrowheads in C and D
show cell-wall degradation and fiber separa-
tion, respectively. Bars: 500 µm in A and B, 50 µm
in C, and 20 µm in D.

197
INT. MICROBIOL. Vol. 8, 2005
service results in billion-euro losses. Basidiomycetes are the
main wood rotters due to their ability to degrade or modify
lignin, an enzymatic process that originated in the Upper
Devonian period in parallel with the evolution of vascular plants
[12]. Wood-rotting basidiomycetes are classified as white-rot
and brown-rot fungi based mainly on macroscopic aspects
[42,49]. Table 1 summarizes the characteristics of wood attack
by several types of fungi. Basidiomycetes can overcome diffi-
culties in wood decay, including the low nitrogen content of
wood and the presence of toxic and antibiotic compounds.
Extracellular oxidative enzymes (oxidoreductases) secreted by
fungi are involved in degradation of cell-wall components (see
below). White-rot basidiomycetes, the most frequent wood-rot-
ting organisms, are characterized by their ability to degrade
lignin, hemicelluloses, and cellulose, often giving rise to a cellu-
lose-enriched white material. Due to the ability of white-rot
basidiomycetes to degrade lignin selectively or simultaneously
with cellulose, two white-rot patterns have been described in dif-
ferent types of wood, namely selective delignification, also
called sequential decay, and simultaneous rot [35] (Fig. 1C,D).
BIODEGRADATION OF LIGNOCELLULOSICS
Table 1.Anatomical, chemical features of different types of wood decaying and staining fungi*
White rot Brown rot Soft rot Stain fungi
Decay aspect and
consistency Bleached appearance, lighter in color than
sound wood, moist, soft, spongy, strength
loss after advanced decay.
Brown, dry, crumbly, pow-
dery, brittle consistency,
breaks up like cubes, drastic
loss of strength at initial
stage of decay. Very uni-
form ontogeny of wood
decay.
Soft consistency in wet
environments. Brown and
crumbly in dry environ-
ments. Generally uniform
ontogeny of wood decay.
Discoloration sapwood areas
(specks, spots and patches),
blue (softwood), black (hard-
wood), red, or other colors.
Discoloration due to colored
hypha, or physiological res-
ponse of tree against damage.
Host (wood-type) Simultaneous rot Selective deligni-
fication
Hardwood, rarely
softwood Hardwod and
softwood
Softwoods; seldom hard-
woods. Forest ecosystems
and wood in service.
Generally hardwoods (soft-
woods very slightly degra-
ded). Forest ecosystems,
waterlogged woods, histo-
ric archaeological wood,
utility poles.
Both softwoods and hardwo-
ods in forest ecosystems, and
during transport and storage
of timber.
Cell-wall constituents
degraded Cellulose, lignin
and hemicellulose.
Brittle fracture.
Initial attack selec-
tive for hemicellu-
loses and lignin,
later cellulose also.
Fibrous feature.
Cellulose, hemicelluloses.
Lignin slightly modified.
In some cases, extended
degradation of hardwood
(including middle lamella).
Cellulose and hemicellulo-
ses, lignin slightly altered. Wood extractives and water-
soluble compounds (sugars
and starch).
Anatomical features Cell wall attacked
progressively from
lumen. Erosion
furrows associated
with hyphae.
Lignin degradation
in middle lamella
and secondary wall.
Middle lamella dis-
solved by diffusion
mechanism (not in
contact with
hyphae), radial cavi-
ties in cell wall.
Degradation at a great dis-
tance from hyphae (diffu-
sion mechanism). Entire
cell wall attacked rapidly
with cracks and clefts.
Cell wall attack in the pro-
ximity of hyphae starts
from cell lumen.
Logitudinal biconical
cylindrical cavities in
secondary wall (Type 1).
Secondary wall erosions
from cell lumen (Type 2).
Facultative soft-rot decay
by some basidiomicetes.
Colonization primarily affec-
ting ray parenchyma and resin
channels (produced through
pits).
Causal agents Basidiomycetes
(e.g. T. versicolor,
Irpex lacteus,
P. chrysosporium
and Heteroba-
sidium annosum)
and some Ascomy-
cetes (e.g. Xylaria
hypoxylon).
Basidiomycetes (e.g.
Ganoderma australe,
Phlebia tremellosa,
C. subvermispora,
Pleurotus spp. and
Phellinus pini).
Basidiomycetes exclusi-
vely (e.g. C. puteana,
Gloeophyllum trabeum,
Laetiporus sulphureus,
Piptoporus betulinus,
Postia placenta and
Serpula lacrimans).
Ascomycetes (Chaetomium
globosum, Ustulina deusta)
and Deuteromycetes
(Alternaria alternata,
Thielavia terrestris,
Paecilomyces spp.), and
some bacteria. Some white
(Inonotus hispidus) and
brown-rot (Rigidoporus
crocatus) basidiomycetes
cause facultative soft-rot
decay.
Ascomycetes (e.g. Ophios-
toma and Ceratocystis spp.)
and Deuteromycetes (e.g.
Aureobasidium pullulans,
Phialophora spp. and
Trichoderma spp.)
*Based on Eriksson et al [12], Schwarze et al. [42], and Zabel and Morrell [49].

198 INT. MICROBIOL. Vol. 8, 2005
Brown-rot fungi, which grow mainly on softwoods, represent
only 7% of wood-rotting basidiomycetes. This group of basi-
diomycetes can degrade wood polysaccharides after only a partial
modification of lignin, resulting in a brown material consisting of
oxidized lignin, which represents a potential source of aromatic
compounds for the stable organic matter fraction in forest soils.
Although only white-rot and brown-rot basidiomycetes
can degrade wood extensively, some ascomycetes and their
asexual states, the so-called deuteromycetes, can colonize
wood in contact with soil. This results in a decrease in the
mechanical properties of wood, giving rise to so-called soft-
rot, a process that often involves bacteria (Table 1). Soft-rot
fungi can degrade wood under extreme environmental condi-
tions (high or low water potential) that prohibit the activity of
other fungi. Moreover, some basidiomycetes also cause a soft-
rot-type decay pattern. Finally, a limited number of ascomy-
cetous fungi, called stain fungi, can colonize wood through
parenchymatic rays and resin channels causing discoloration of
softwood tissues but a very limited degradation, which mainly
affects extractives and water-soluble materials.
Chemical analysis of degraded wood
The main wood degradation patterns at advanced stages of
decay can be identified macroscopically and microscopically,
as described above. General changes in the chemical compo-
sition of wood can be also observed after fungal-induced decay
(Table 1). However, a precise analysis of the degradation type
requires chemical analysis of cellulose and lignin contents and
of the modifications in the decayed wood. Lignin in wood is
traditionally estimated by the Klason method, which is based
on total acid hydrolysis of polysaccharides and gravimetric
estimation of the lignin content (after deducing ashes and
protein). This method, however, is time-consuming, and
requires a considerable sample volume. In addition, its appli-
cation to samples containing modified lignin (as in rotted
wood samples) is problematic because it has been developed
for lignin in sound (undecayed) wood. Estimation of Klason
lignin is often combined with analysis of wood polysaccha-
ride composition by gas chromatography (GC) of the mono-
saccharides present in the acid hydrolysate.
Several modern spectroscopic and degradative methods
have been used to analyze lignin or polysaccharides in wood
[30]. Among them, pyrolysis coupled to gas chromatogra-
phy-mass spectrometry (Py-GC/MS) has several great
advantages as a technique enabling rapid analysis of small
samples, yielding a precise identification of the proportion of
H, G, and S lignin units [8]. Figure 2 shows a Py-GC/MS
analysis of the fungal decay of a hardwood (Eucalyptus glo-
bulus). Two regions, corresponding to the polysaccharide-
and lignin-derived compounds, can be distinguished in the
pyrograms. Due to the higher stability of the degradation
products of lignin (the main ones shown in Fig. 2D) than of
polysaccharides, which yield several compounds that cannot
be chromatographied, Py-GC/MS analyses result in overesti-
mation of lignin. However, this method can be used to com-
pare the changes in the relative proportion of lignin and poly-
saccharides in decaying wood. Moreover, the amounts of G-
lignin and S-lignin (no H-lignin has been detected in eucalypt
wood) can be separately estimated by Py-GC/MS analysis.
Figure 2A shows wood decay by the white-rot basid-
iomycete Ceriporiopsis subvermispora, which results in a
relative decrease in the lignin peaks and a relative increase in
the carbohydrate peaks. By contrast, the soft-rot deuteromy-
cete Paecilomyces sp. decreases cellulose content to a larger
extent than lignin content (Fig. 2B). Figure 3 shows wood
degradation patterns by the basidiomycetes Bjerkandera
adusta, C. subvermispora, Coniophora puteana, Crepidotus
variabilis, Funalia trogii, Melanotus hepatochrous, Phanero-
chaete chrysosporium, Phlebia radiata, and Pleurotus pul-
monarius (Fig. 3A), the ascomycetes Mollisia sp, Ophios-
toma piliferum (including an inoculum commercialized as
Cartapip) and Ophiostoma valdivianum (Fig. 3B), and the
deuteromycetes Fusarium oxysporum, Kirramyces eucalypti
and Paecilomyces sp. (Fig. 3C), which can be differentiated
based on their lignin/carbohydrate and S/G Py-GC/MS
ratios. The highest cellulose enrichments are produced by the
basidiomycetes C. subvermispora and C. variabilis, the for-
mer also causing the highest modification of lignin, as
revealed by the S/G ratio. In addition, Paecilomyces sp. and
other deuteromycetes produce an increase in the relative
lignin content in wood (due to preferential removal of poly-
saccharides), whereas ascomycetes slightly modify wood
composition, as revealed by the Py-GC/MS analysis.
Enzymatic aspects
Lignin degradation and/or modification by basidiomycetes is
the key step in lignocellulose decay. Therefore, the enzymes
and mechanisms involved in lignin attack are described
below. For a discussion of the subsequent steps in the degra-
dation of wood polysaccharides, we recommend other
reviews [36,43].
Laccases have been known for many years in plants,
fungi, and insects, where they play a variety of roles, includ-
ing synthesis of pigments, fruit-body morphogenesis, and
detoxification [34]. Their production in fungal plate cultures
was considered to be a characteristic unique to white-rot
MARTÍNEZ ETAL.

199
INT. MICROBIOL. Vol. 8, 2005
BIODEGRADATION OF LIGNOCELLULOSICS
Int. Microbiol.
Fig. 2. Py-GC/MS analysis of the
same hardwood degraded in vitro by
the white-rot basidomycete C. sub-
vermispora (A) and the soft-rot
deuteromycete Paecilomyces sp.
(B), and the corresponding control
(C) showing carbohydrate derived
(a–m) and lignin derived (1–26)
compounds. (D) Chemical structure
of the main G-type and S-type
lignin marker compounds. E. glob-
ulus wood chips were treated with
the different fungi under solid-state-
fermentation conditions. Adapted
from del Río et al. [8]. Peak identifi-
cation: a, butanedial; b, 1,4-pentadi-
ene-3-one; c, unknown; d, 3-hydro-
xypropanal; e, 3-furaldehyde; f, (2H)-
furan-2-one; g, 2-furaldehyde; h,
hydroxymethylfuran; i, (5H)-furan-2-
one; j, 2,5-dihydro-5-methylfuran-2-
one; k, 2,3-dihydro-5-methylfuran-2-
one; l, 4-hydroxy-5,6-dihydro-(2H)-
pyran-2-one; m, 2-hydroxy-3-methyl-
2-cyclopenten-1-one; 1, guaiacol; 2,
2,3,4-dihydroxybenzaldehyde; 3, 4-
methylguaiacol; 4, 4-ethylguaiacol; 5,
4-vinylguaiacol; 6, eugenol; 7, syrin-
gol; 8, cis-isoeugenol; 9, vanillin; 10,
trans-isoeugenol; 11, 4-methylsy-
ringol; 12, homovanillin; 13, ace-
toguaiacone; 14, 4-ethylsyringol; 15,
guaiacylacetone; 16, 4-vinylsyringol;
17, propiovanillone; 18, 4-allylsy-
ringol; 19, 4-propylsyringol; 20, cis-
4-(prop-2-enyl)syringol; 21, syringal-
dehyde; 22, trans-4-(prop-2-enyl)sy-
ringol; 23, homosyringaldehyde; 24,
acetosyringone; 25, syringylacetone;
26, propiosyringone.
Fig. 3. Hardwood biodegradation patterns: Py-GC/MS analysis of decayed
wood (Fig. 2) permits the definition of degradation clusters corresponding to
basidiomycetes (A), ascomycetes (B), and deuteromycetes (C) (an untreated
control is also included). Adapted from del Río et al. [8].
Int. Microbiol.

200 INT. MICROBIOL. Vol. 8, 2005
basidiomycetes [27], although some brown-rot fungi produce
laccase in liquid cultures [29]. These phenoloxidases have a
low redox potential that allows direct oxidation only of pheno-
lic lignin units, which often comprise less than 10% of the total
polymer. The interest in laccases for biotechnological applica-
tions increased with the discovery of their ability to oxidize
high redox potential substrates in the presence of synthetic
mediators [3], which allows the degradation of xenobiotic com-
pounds [40] and chlorine-free bleaching of paper pulp [4].
Natural mediators involved in lignin biodegradation remain to
be identified, although some lignin-derived phenols could act
as efficient laccase mediators [5].
Lignin peroxidase (LiP) and manganese peroxidase (MnP)
were discovered in the mid-1980s in P. chrysosporium and
described as true ligninases because of their high redox potential
[16,31]. LiP degrades non-phenolic lignin units (up to 90% of the
polymer), whereas MnP generates Mn3+, which acts as a diffu-
sible oxidizer on phenolic or non-phenolic lignin units via lipid
peroxidation reactions [26]. More recently, versatile peroxidase
(VP) has been described in Pleurotus [32,41] and other fungi as
a third type of ligninolytic peroxidase that combines the catalyt-
ic properties of LiP, MnP, and plant/microbial peroxidases oxi-
dizing phenolic compounds [24].
Other extracellular enzymes involved in wood lignin
degradation are oxidases generating H2O2, and mycelium-
associated dehydrogenases that reduce lignin-derived com-
pounds. The former include the aryl-alcohol oxidase (AAO)
described in Pleurotus eryngii [18] and other fungi, and P.
chrysosporium glyoxal oxidase [28]. Fungal aryl-alcohol
dehydrogenases (AAD) and quinone reductases (QR) are
also involved in lignin degradation [20,21].
As shown in Fig. 4, laccases or ligninolytic peroxidases
(LiP, MnP, and VP) produced by white-rot fungi oxidize the
lignin polymer, thereby generating aromatic radicals (a) [12].
These evolve in different non-enzymatic reactions, including
C4-ether breakdown (b), aromatic ring cleavage (c), Cα-Cβ
breakdown (d), and demethoxylation (e) [22]. The aromatic
aldehydes released from Cα-Cβbreakdown of lignin, or syn-
thesized de novo by fungi (f, g) [21] are the substrate for
H2O2generation by AAO in cyclic redox reactions involving
also AAD [19]. Phenoxy radicals from C4-ether breakdown
(b) can repolymerize on the lignin polymer (h) if they are not
first reduced by oxidases to phenolic compounds (i), as
reported for AAO [33]. The phenolic compounds formed can
be again reoxidized by laccases or peroxidases (j). Phenoxy
radicals can also be subjected to Cα-Cβbreakdown (k), yielding
MARTÍNEZ ETAL.
Int. Microbiol.
Fig. 4. A scheme for lignin biodegradation including
enzymatic reactions and oxygen activation, (for expla-
nation see text). Updated from Gutiérrez and Martínez
[22].

201
INT. MICROBIOL. Vol. 8, 2005
p-quinones. Quinones from gand/or kcontribute to oxygen
activation in redox cycling reactions involving QR, laccases,
and peroxidases (l, m) [20]. This results in reduction of the fer-
ric iron present in wood (n), either by superoxide cation radical
or directly by the semiquinone radicals, and its reoxidation with
concomitant reduction of H2O2to hydroxyl free radical (OH·)
(o) [17]. The latter is a very strong oxidizer that can initiate the
attack on lignin (p) in the initial stages of wood decay, when the
small size of pores in the still-intact cell wall prevents the pen-
etration of ligninolytic enzymes [13]. Then, lignin degradation
proceeds by oxidative attack of the enzymes described above.
In the final steps, simple products from lignin degradation enter
the fungal hyphae and are incorporated into intracellular cata-
bolic routes.
Molecular aspects
Due to their potential use as industrial biocatalysts, the catalyt-
ic mechanisms of lignin-degrading oxidoreductases (including
peroxidases, oxidases, and laccases) have been extensively
investigated and their molecular structures have been described
(Fig. 5).
LiP and MnP were the second and third peroxidases
whose crystal structure was solved [38,39,44], just 10 years
after their discovery in P. chrysosporium. These peroxidases
catalyze the oxidation of the recalcitrant non-phenolic lignin
units by H2O2. This is possible because of the formation of a
high redox potential oxo-ferryl intermediate during the reac-
tion of the heme cofactor with H2O2. This two-electron reac-
tion allows the activated enzyme to oxidize two substrate
units, being reduced to the peroxidase resting state (which
reacts again with peroxide). The catalytic cycle, consisting of
the resting peroxidase and compounds I (two-electron oxi-
dized form) and II (one-electron oxidized form), is common
to other peroxidases. However, two aspects in their molecu-
lar structure provide ligninolytic peroxidases their unique
catalytic properties: (i) a heme environment, conferring high
redox potential to the oxo-ferryl complex; and (ii) the exis-
tence of specific binding sites (and mechanisms) for oxida-
tion of their characteristic substrates, including non-phenolic
aromatics in the cases of LiP, manganous iron in the case of
MnP, and both types of compounds in the case of the new VP.
Similar heme environments in the above three peroxi-
dases (located at the central region of the protein, Fig. 5A)
have been evidenced by 1H-NMR, which allows the signals
BIODEGRADATION OF LIGNOCELLULOSICS
Fig. 5. Molecular structures of enzymes acting synergisti-
cally for lignin biodegradation. (A) Crystal structure of ver-
satile peroxidase at 1.13 Å resolution including heme
cofactor and both Mn2+ (right) and aromatic substrate (left)
oxidation sites. (B) Homology molecular model of AAO, a
flavoenzyme providing H2O2to ligninolytic peroxidases
[46]. (C) Crystal structure of active laccase at 1.90 Å reso-
lution with its three catalytic coppers.
Int. Microbiol.

202 INT. MICROBIOL. Vol. 8, 2005
of both the heme cofactor protons and several amino acid
residues forming the heme pocket to be identified [1]. This is
possible due to the paramagnetic effect caused by the cofac-
tor iron, which displaces the signals of neighbor protons out-
side the region where most protein protons overlap. One of
the main differences observed among peroxidases is the posi-
tion of a protein iron ligand, the Nεof the side-chain of a his-
tidine residue (the so-called proximal histidine). In ligni-
nolytic peroxidases, this residue is displaced away from the
heme iron, increasing its electron deficiency and increasing
the redox potential of the oxo-ferryl complex [31].
In addition, recent studies have contributed to identifica-
tion of the substrate binding sites in ligninolytic peroxidases.
The aromatic substrate binding site and the manganese binding
site were first identified in LiP and MnP [9,16,45], and then
confirmed in the crystal structure of VP solved at atomic reso-
lution (unpublished). These studies revealed that the novel cat-
alytic properties of VP are due to its hybrid molecular architec-
ture, as suggested several years before [6,41]. Mn2+ oxidation
is produced at a binding site near the cofactor, where this
cation is bound by the carboxylates of three acidic residues,
which enables direct electron transfer to one of the heme pro-
pionates (Fig. 5A right). By contrast, veratryl alcohol (and
other lignin model substrates) are oxidized at the surface of the
protein by a long-range electron transfer mechanism that initi-
ates at an exposed tryptophan residue (Fig. 5A left). The
rationale of the existence of this electron transfer mechanism
is related to the fact that many LiP/VP aromatic substrates,
including the lignin polymer, cannot penetrate inside the pro-
tein to transfer electrons directly to the cofactor. Therefore,
these substrates are oxidized at the enzyme surface, and elec-
trons are transferred to the heme by a protein pathway.
The H2O2responsible for oxidative degradation of lignin is
generated by extracellular fungal oxidases, which can reduce
dioxygen to peroxide in a catalytic reaction. Flavin cofactors
are generally involved in this reaction, as in the Pleurotus
flavoenzyme AAO, although glyoxal oxidase from P. chrysos-
porium is a copper-containing oxidase [47]. Among flavoen-
zyme oxidases, fungal glucose oxidase has been crystallized
[48], but this is an intracellular enzyme that is not involved in
lignin degradation. However, its crystal structure has been
used as a template to predict the molecular structure of AAO
(shown in Fig. 5B, including the FAD cofactor) [46]. It has
been shown that AAO is a unique oxidase due both to its spec-
troscopic characteristics (flavin intermediates and reactivity)
and to the wide range of aromatic and aliphatic polyunsaturat-
ed primary alcohols (and even aldehydes) that it is able to oxi-
dize [15]. The molecular structure of AAO includes two cat-
alytically active histidines near the N5 of the flavin ring, which
might help electron transfer to/from the cofactor by acting as
bases in the oxidation of aromatic alcohols (which would pro-
ceed via a hydride transfer mechanism) and as acids in the
reduction of oxygen to H2O2.
As noted above, laccases were the first ligninolytic
enzymes to be investigated, and had been known in plants for
many years. Nevertheless, the first molecular structure of a
complete fungal laccase was published only in 2002. That
year, the crystal structures of the laccases from the basid-
iomycete T. versicolor, and the ascomycete Melanocarpus
albomyces were reported [2,23,37] (Fig. 5C). The first struc-
ture of a bacterial laccase was published one year later [11].
A previously reported laccase structure corresponded to an
inactive form due to the loss of a copper ion during deglyco-
sylation to obtain suitable crystals for X-ray diffraction [10].
The active site of laccases includes four copper ions. Type-I
copper (right sphere in Fig. 5C) acts as electron acceptor
from substituted phenols or amines (the typical laccase sub-
strates); and type-II copper, which transfers the electrons to
the final acceptor, dioxygen, which is reduced to water. The
two type-III coppers act as intermediates in the electron
transfer pathway that also includes one cysteine and two his-
tidine protein residues. The molecular environment of lac-
case type-I copper seems to regulate the redox potential of
the enzyme [37]. The fact that laccase can use atmospheric
oxygen as the final electron acceptor represents a consider-
able advantage for industrial and environmental applications
compared with peroxidases, which require a continuous sup-
ply of H2O2. Taking into account that the advantage of perox-
idases is their higher redox potential, engineering the active
site of laccases to obtain high redox potential variants would
be of considerable biotechnological interest.
Most enzymes involved in wood lignin degradation (a
multienzymatic process that includes, among others, peroxi-
dases, oxidases, and laccases acting synergistically) have
been identified, and the mechanisms of action of several of
them have been established at a considerably precise level.
These enzymes, however, cannot penetrate the compact
structure of sound wood tissues due to their comparatively
large molecular size. Therefore, small chemical oxidizers,
including activated oxygen species and enzyme mediators,
are probably involved in the initial steps of wood decay.
Acknowledgements. These studies have been partially sup-
ported by ENCE (Spain), by Spanish projects AGL2002-393 and BIO2002-
1166, by EU projects QLK5-99-1357 and QLK3-99-590, and by an
EUFORES(ENCE)-PDT/MEC(Uruguay) grant. Carmen Ascaso (CCMA,
CSIC, Madrid) is acknowledged for low-temperature scanning-electron
microscopy facilities. Klaus Piontek (ETH, Zurich) is acknowledged for solv-
ing the VP crystal structure. Lina Bettucci (Universidad de la República,
Montevideo) is acknowledged for an Inocutis jamaicensis strain. M.S
acknowledges MEC for a Postdoctoral Fellowship. F.J.R.-D. thanks CSIC for
an I3P contract. A.G. and S.C. thank MEC for their “Ramón y Cajal” contracts.
MARTÍNEZ ETAL.

203
INT. MICROBIOL. Vol. 8, 2005
BIODEGRADATION OF LIGNOCELLULOSICS
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Biodegradación de la lignocelulosa: aspectos
microbiológicos, químicos y enzimáticos del
ataque fúngico a la lignina
Resumen. La madera es el principal material renovable en la Tierra y es
utilizada en gran parte como material de construcción y en la fabricación de
celulosa. Esta revisión describe la composición de los materiales lignocelu-
lósicos y diversos procesos de alteración de la madera por parte de hongos,
como el deterioro causado por los llamados hongos de podredumbre blanca,
de podredumbre parda y de podredumbre blanda y por los hongos cromóge-
nos o manchadores de la madera. También se tratan los aspectos químicos,
enzimáticos y moleculares del ataque a la lignina por los hongos, que es
clave en el deterioro de la madera. Se describen las técnicas analíticas
modernas para investigar la degradación y la modificación del polímero de
la lignina causadas por hongos, así como las diversas enzimas oxidativas
(oxidoreductasas) que intervienen en la degradación de la lignina. Entre
éstas se encuentran lacasas, peroxidasas ligninolíticas de alto potencial
redox (lignina-peroxidasa, manganeso-peroxidasa y peroxidasas versátiles)
y las oxidasas. Se destacan las reacciones catalizadas, su acción sinérgica
sobre la lignina, y las bases estructurales de sus exclusivas propiedades cata-
líticas. Un mejor conocimiento de los procesos de biodegradación de la
lignocelulosa debería contribuir a un mejor control de los hongos descompo-
nedores de la madera, así como al desarrollo de nuevos biocatalizadores de
interés industrial basados en estos organismos y sus enzimas. [Int Microbiol
2005; 8(3):195-204]
Palabras clave: hongos descomponedores de madera · lignina · pirólisis
analítica · oxidoreductasas · mecanismos catalíticos
Biodegradação da lignocelulose: aspectos
microbiológicos, químicos e enzimáticos do
ataque fúngico à lignina
Resumo. A madeira é o principal material renovável na Terra e é utilizada
em grande parte como material de construção e na fabricação de celulose.
Esta revisão descreve a composição dos materiais lignocelulósicos e
diversos processos de alteração da madeira por parte de fungos, como a
deterioração causada pelos chamados fungos de podridão branca, de
podridão parda e de podridão mole e pelos fungos emboloradores ou
manchadores da madeira. Também se tratam os aspectos químicos,
enzimáticos e moleculares do ataque à lignina pelos fungos, que é chave na
deterioração da madeira. Se descrevem as técnicas analíticas modernas para
investigar a degradação e a modificação do polímero da lignina causadas por
fungos, assim como as diversas enzimas oxidativas (oxidoreductasas) que
intervêm na degradação da lignina. Entre estas se encontram lacasas,
peroxidasas ligninolíticas de alto potencial redox (lignina-peroxidasa,
manganeso-peroxidasa e peroxidasas versáteis) e as oxidasas. Se destacam
as reações catalisadas, sua ação sinérgica sobre a lignina, e as bases
estruturais de suas exclusivas propriedades catalíticas. Um melhor conhe-
cimento dos processos de biodegradação da lignocelulosa deveria
contribuir para um melhor controle dos fungos descomponedores da
madeira, assim como ao desenvolvimento de novos biocatalisadores de
interesse industrial baseados nestes organismos e suas enzimas. [Int
Microbiol 2005; 8(3):195-204]
Palavras chave: fungos descomponedores de madeira · lignina· pirólise
analítica · oxidoreductasas · mecanismos catalíticos