Biotechnolo

Journal of Biotechnology 41 (1995) 1-17

ELSEVIER

Review

Enzymes of white-rot fungi involved in lignin degradation

ecological determinants for wood decay
U. Tuor a,1,K. Winterhalter

b, A. Fiechter

and

a**

a Swiss Federal Institute of Technology, Instirute of Biolechnology, ETH-Hiinggerberg, CH-8093 Ziirich, Switzerland
b Swiss Federal hstitule of Technology, Department of Biochemistry, ETH-Zenlrum, CH-8092 Zikich, Switzerland

Received 12 November 1994; accepted 17 March 1995

Abstract
White-rot fungi preferably degrade wood from deciduous trees, whilst wood decay by brown-rot fungi is
predominant on coniferous substrates. A compilation of recent publications on ligninolytic fungal species and their
substrate preference is presented. These organisms can be classified on the basis of their enzyme systems, but an
unambiguous allocation to specific hosts proved to be difficult. Environmental conditions may be crucial in governing
the selectivity of fungal biodegradation of wood components. Possible mechanisms of primary attack of the wood cell
wall by hemicellulose degradation are discussed. Furthermore, a hypothetical scheme for lignin biodegradation
involving oxidative cleavage of phenol& C,-oxo-substituted
substructures is presented.
Keywords: White-rot fungi; Ecology; Wood biodegradation; Microbial population; Lignin degradation; Hemicellulose

degradation; Ligninase; Manganese peroxidase; Lactase

1. Introduction

The stems of higher plants obtain mechanical

strength and rigidity by the structural elements
cellulose, hemicellulose and lignin, which are synthesized and deposited in the plant cell walls.
Under microgravity conditions aboard the space
shuttle, lignification in pine, oats and mung beans
seedling was decreased by 6-24% (Cowles et al.,
1989). In recent years the enzymatic machinery of

* Corresponding author.
1 Present
address: Helbling Ingenieurunternehmung,
Hohlstr. 610, CH-8048 Ziirich, Switzerland.

selected wood degrading white-rot fungi has been

elucidated to a large extent. The discovery of
ligninase (lignin peroxidase (Lip) Glenn et al.
(1983), Tien and Kirk, 1983) and Mn-peroxidase
(MnP; Kuwahara et al., 1984) from Phanerochaete chrysosporium triggered biochemical research on lignin biodegradation. Here we present
the available information on ecological determinants of white-rot wood decay and relate it to the
present knowledge of the enzymology of white-rot
fungi and other microorganisms. Based on present knowledge, the biochemistry and physiology
of white-rot wood decay can not easily be correlated with environmental conditions because of
the variability of the fungi and their weak speci-

016%1656/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved

SSDZ 0168-1656(95)00042-9

U.

Tuor et al. /Journal

of Biotechnology 41 (1995) 1 -I 7

ficity towards their hosts. As research proceeds,

our perception of lignin degradation is changed
from an oxidative depolymerization
process
caused by a single enzyme to a process of concerted oxidative and reductive conversions in
which different classes of enzymes participate.
The emerging hypothetical schemes also involve
cleavage of lignin-carbohydrate
complexes by
hemicellulases as crucial steps in the overall lignin
biodegradation.

Table 1
Number of functional groups in lignin per 100 C,C, units
Functional group

Spruce lignin

Birch lignin

Methoxyl
Phenolic hydroxyl (free)
Benzyl alcohol
Noncyclic benzyl ether
Carbonyl

92-96
15-30
15-20
7-9
20

139-158
9-13

Their content may vary depending on the origin of the lignin

(e.g., middle lamella or secondary cell wall lignin). The values
are compiled from Adler (1977) and Sarkanen and Ludwig
(1971).

2. Host specificity of wood-rotters

Hardwood and softwood are distinguished by
structural elements building the phenylpropane
backbone of the lignin component. Lignin is a
threedimensional,
optically
inactive phenylpropanoid polymer randomly synthesized from
coniferyl, p-coumaryl and sinapyl alcohol precursors (Sarkanen and Ludwig, 1971; Fig. 1). Softwood lignin is referred to as guaiacyl lignin, containing more than 95% coniferyl alcohol (4-hydroxy-3-methoxy-cinnamyl alcohol) units. A structural model of spruce lignin was proposed by
Adler (1977). The remaining elements are mainly
p-coumaryl (6hydroxycinnamyl)
alcohols with
trace amounts of sinapyl (3,5-dimethoxy-4-hydroxy-cinnamyl alcohol) alcohols. Typical hardwood lignins, also classified as guaiacyl-syringyl
lignins, contain coniferyl- and sinapyl alcohol-derived subunits. The relative amount of the latter

OH

p-coumaryl

alcohol

varies from 26 to 60% (Chang and Sarkanen,

1973). Hardwood lignins typically contain 1.2-1.5
methoxyl
groups
per phenylpropane
unit
(Sarkanen and Hergert, 1971). A structural representation was proposed by Nimz (1974). Although
classified as guaiacyl-syringyl lignin, grass lignin
also contains additional small amounts of pcoumaryl alcohol-derived
subunits. The mentioned differences in chemical composition are
reflected in the frequency of functional groups
(Table 1): spruce (softwood) lignin contains 92-96
methoxyl and 15-20 free phenolic functional
groups per 100 phenylpropane units, whereas the
corresponding
numbers for birch (hardwood)
lignin are 139-158 and 9-13, respectively
(Sarkanen and Ludwig, 1971; Adler, 1977).
According to macroscopic differences of their
substrate utilization, wood-rotters are classified
into three specific decay groups: white-rot,

OH

coniferyl

OH

alcohol

Fig. 1. Precursor alcohols of lignin.

sinapyl alcohol

U. Tuor et al. /Journal

of Biotechnology 41 (1995) I -I 7

brown-rot and soft-rot fungi. Most wood-rotting

fungi are able to degrade both cellulose and
lignin in wood, but have different degradation
rates for cellulose, hemicellulose
and lignin.
Brown-rot fungi have an obvious preference for
coniferous substrates (gymnosperm& A survey of
substrate relationships reported that 19% of
North American polypores are brown-rot fungi,
among which 60 out of 71 (85%) occur primarily
on conifers, and that they are mainly soft-wood
degraders. They develop predominantly in coniferous forest regions as they are more efficient in
gaining energy from wood for growth and reproduction than white-rot fungi, favouring their survival and propagation in colder climates with a
shorter growing season (Gilbertson, 1980). On the
other hand, there are numerous examples of
wood-rotting fungi that have a geographic distribution not corresponding to their most suitable
hosts (Gilbertson, 1980).
In contrast, white-rot fungi predominantly degrade wood from deciduous trees (angiosperms).
In a survey of a total of 65 central European
wood-destroying basidiomycetes, four were reported to only attack coniferous wood, whereas
34 were attacking angiosperms exclusively. 27 of
the fungi attack both (Rypacek, 1977). Table 2
presents a overview of white-rot fungi whose
ligninolytic system has been investigated. Where
known, their preferred host is indicated, and the
dominance of hard-wood substrates is evident.
In general, wood rotting fungi have broad host
ranges and can only be designated soft- or hardwood degraders. Growth on principal monoses of
soft- or hardwood hemicellulose correlates with
the specialization of white-rot and brown-rot
fungi. It was therefore suggested that the ability
of hemicellulose
degradation
may determine
preference for coniferous or deciduous substrates
(Rypacek, 1977).

3. Oxidative enzymes in lignin biodegradation

The extracellular oxidative enzymes ligninase,
manganese peroxidase and lactase may be defined as phenoloxidases. Both ligninase (EC No.
1.11.1.14 Diarylpropane:
oxygen, hydrogen-per-

oxide oxidoreductase) and manganese peroxidase

(EC No. 1.11.1.13 Mn(I1): hydrogen-peroxide oxidoreductase) belong to the class of peroxidases
and oxidize their substrates by two consecutive
one-electron oxidation steps with intermediate
cation radical formation. Due to its high redox
potential, the preferred substrates for LIP are
nonphenolic methoxyl-substituted lignin subunits,
whereas MnP acts exclusively as an phenoloxidase on phenolic substrates using Mn2+/Mn3+ as
an intermediate redox couple. Oxidation of phenolic substrates by ligninase leads to their polymerization (Higuchi, 1986; Odier et al., 1988).
Ring-cleavage of aromatic rings is a key step of
lignin mineralization. Non-phenolic syringyl and
biphenyl model compounds are oxidized by lignin
peroxidase and subsequent ring cleavage. In contrast, oxidation of the corresponding phenolic
compounds by ligninase did not yield ring-opened
products (Hattori and Higuchi, 1991a,b). Alkoxyl
groups activate aromatic rings towards oxidation
by ligninase, partly explaining why syringyl lignin
is easier to degrade than guaiacyl lignin (Eriksson
et al., 1990). Lactase (EC No. 1.10.3.2. (benzenediol: 0, oxidoreductase) is a true phenoloxidase with broad substrate specificity. It oxidizes
phenols and phenolic lignin substructures by
one-electron abstraction with formation of radicals that can repolymerize or lead to depolymerization (Higuchi, 1989). Demethylation reactions
of terminal phenolic units catalyzed by lactase
may therefore be of importance for native lignin
degradation, and the utilization of lactase or laccase-producing white-rot fungi for biopulping has
become a focus of interest recently (Reid and
Paice, 1994; Messner and Srebotnik, 1994). In
view of these findings it seems likely that structural and chemical differences in the very inhomogeneous lignin substrate should lead to a specialization in the respective degrading microorganisms, particularly the oxidative enzymes expressed by each organism. Table 2 gives an
overview of the white-rot fungi investigated and
the oxidative enzyme activities as published in the
literature. They are classified in Table 2 into five
main groups:
I. White-rot fungi expressing Lip, MnP and
lactase. This group contains the best known

Conalus LYrsicolor

I
10.3.2)

(lIttIe)

(15c

1.1i.i.1.3)

Lactase

hosts

(EC

(EC 1.11.1.14)

MnP

UP

Enzymes

fungi, their oxidative enzymes and their preferred

Microorganism

White-rot

Table 2

none

(EC

1.1.3.7)

Atyl alcohol

oxidase

Phenol-

oxidase
activity

Waldnrl-

and

Bourbonnais

et al..

Maltseva et al., 19Y1:

Golovlevatl al., 1993

P&it and Gold, 1991

Leatham and
Stahmann, 1981:
Leatham. 1986:
Forrenter et al., 1990

Petroski et al., 1980;

Lobos ct al.. lr)4

Riittimann
1992a,b:

Paice, 1989;
Boyle et al., 1992

LY87,

Fukurumi.

Sannia et al., 1991

19X7:
rt al , 198X;

Waldnrr,

Hatakka et al.. 1987:

Niku-Paavola et al.. 1988
Sonnia et al., 19%;

Glenn et al., 1983;

Tien and Kirk, 1983:
Leisola et al., 1987
Perez and Jcffrics. 1990:
Riittimann ct al.. 1992~

Fahraeus and
Reinhammar. 1967.
Dodson et al., 1987;
Johansson and
Nyman, 1987;
Waldner, 1987:
Waldner et al., 1988

Ref.

trccb

presumably

deciduous

trees

white-rot
fungus uncertain
classification

white-rot

little iaccasr production

(Andcr ct al.. 1980:
Eriksson cl al, 1983)

Remarks

birch, aspen, spruce, wheat,

barley, grape straw
deciduous trees

deciduous

nd

deciduous tree\
rarely comters
deciduous lreen

deciduous trees

Host

\
2
5
$
%
5
e
R
%

none

Trametes cingulata
x

trees

trees

trees

trees

trees

none = does not produce

na
enzyme; blank = no reports on enzyme activity; na = information

Polysticrus uersicolor

see text. x = enzyme activity reported;

Farmer et al., 1960

Kirk, 1987; Waldner, 1987; na

Waldner et al., 1988
Heoea brasiliensis
Geiger et al., 1986a,b
(rubber tree)
conifers
Mustranta, 1987
na
Guillen et al., 1990

none

none

Kirk, 1987; Waldner,

Waldner et al., 1988
none

et al., 1989

deciduous trees
(beech, alder)
1987; na

deciduous trees
(cherry)
deciduous trees
and conifers

deciduous
(beech)
deciduous

deciduous
na
deciduous
(beech)

na
na

none

Phellinus weirii
Pleurotw eryngii

Pheliinus noxius

nonq

Fames lignosus

Hiittermann

Polyporus oarius

Waldner, 1987;
Hiittermann et al., 1989;
Muheim et al., 1990;
Muheim, 1991;

et al., 1989

For explanations

Hiittermann

none

et al., 1989

Hiittermann

Xylaria polymorpha

et al., 1989
et al., 1989
et al., 1989

Hiittermann
Hiittermann
Hiittermann

et al., 1989

x
x
x

Polyporus brwnalis
Polyporus pinsitus
Ustulina deusta

et al., 1989
et al., 1989

Hiittermann
Hiittermann

na

deciduous

Biswas-Hawkes et al., 1987; deciduous trees

and conifers
Hiittermann et al., 1989

Hilttermann

x
x

Pleurotus florida
Polyport~s platensis

Vares et al., 1993

Phlebia wemellows

beech

Hiittermann

et al., 1989

na

Daedaleopsis
confragosa
Phallus impudicus

Phlebia ochraceofuloa

Hevea brasiliensis
(rubber tree)

Geiger et al., 1986a,b;

Galliano et al., 1991
Waldner, 1987;
Waldner et al., 1988
Dill and Kraepelin, 1988

Oudemansiella
radicata

Bjerkandera ad&a

Ganodenna
applanatum

IV

111 Coriolus pruinosum

Rigidoporus iignosus none

syn. Daedaleopsis

white-rot

white-rot

fungus

not available.

syn. Inonotus weirii

classification white-rot
fungus uncertain
syn. Coriolus uersicolor

presumably
fungus

not white-rot

syn. Hypoxylon deustum

presumably
fungus

syn. Merulius tremel~osus

indirect detection

confragosa
*

U. Tuor et al. /Journal

of Biotechnology 41 (1995) I-1 7

white-rot fungi Coriotus L:ersicolor, Phanerochaete chrysosporium and Phlebia radiata. P.

chrysosporium is listed within this group since
lactase production was reported (Ander et al.,
1980; Eriksson et al., 1983). However, this fungus is generally considered not to produce laccase. All of them usually colonize deciduous
trees, only Phlebia radiata occasionally degrades
conifers.
II. White-rot fungi simultaneously producing
both types of phenoloxidases MnP and lactase,
but reportedly not secreting detectable levels of
lignin peroxidase. Nevertheless, these fungi are
strong lignin degraders. Dichomitus squalens and
the edible fungus Lentinulu edodes belong to
this group.
III. White-rot fungi with LIP and one of either
phenoloxidases.
Lactase is the predominant
phenoloxidase produced, only in the case of
Coriolus pruinosum was MnP production reported. These fungi grow on hardwood. As an
exception, only Phlebia tremellosus also degrades coniferous wood.
IV. Four white-rot fungi secrete LIP without
phenoloxidases. Again with one exception they
are hardwood degraders.
V. The last group probably consists of fungi
which are incompletely characterized. Notably
Fames lignosus and Trametes cingulata are
white-rot degraders, but neither of the oxidative
enzymes was detected.
Furthermore,
references to aryl alcohol oxidase and more general reports on phenoloxidase
activity are given in Table 2. Apparently, white-rot
fungi achieve lignin biodegradation with several
different combinations of peroxidases and oxidases. Lignin degradation by fungi that mainly
produce LiP most likely runs along different
pathways and involves different redox mediators
as opposed to fungi predominantly secreting MnP
and laccases. The fungal species investigated may
also express gene products differently in agitated
or stationary cultures than during growth on solid
substrates in natural environments. Contrary to
liquid culture, P. chrysosporium produces MnP as
major peroxidase when grown on wood (Datta et
al., 1991). C. subvermispora and P. brevispora
tested negative for LIP production in agitated

culture, whereas the use of southern hybridization technique with a cDNA probe from P.
chrysosporium revealed the presence of LiP genes
in both fungi (Riittimann et al., 1992a,b). In the
case of P. brevispora, LIP production in liquid
culture was reported earlier (Perez and Jeffries,
1990).
Rather than pointing to a particular set of
enzymes responsible for lignin breakdown, the
data of Table 2 suggest that neither ligninase,
manganese peroxidase nor lactase is essential for
white-rot wood decay. Work with a ligninasenegative mutant of P. chrysosporium showed no
obligatory relationship between ligninase expression and lignin degradation. This LiP mutant
produced MnP, but no detectable amounts of
Lip, and was still able to mineralize lignin, albeit
at a lower rate than the wild type (Boominathan
et al., 19901. Lignin degradation by other wild-type
white-rot and brown-rot fungi which do not secrete lignin peroxidase was observed (Buswell
and Odier, 1987; Bourbonnais and Paice, 1989).
C. subvermisporu, known not to produce Lip,
does degrade nonphenolic synthetic lignin and
model compounds when grown on its natural
substrate wood, but does not do so in liquid
culture (Srebotnik et al., 1994). In cell free systems, LiP does not mineralize lignin (Kirk and
Farrell, 1987; Hammel et al., 1993). Haemmerli
et al. (1986) observed polymerization of different
lignin preparations when incubated with purified
ligninase. However, partial depolymerization of
synthetic, radiolabeled lignin was achieved using
ligninase (Hammel and Moen, 1991; Hammel et
al., 1993) or MnP (Wariishi et al., 1991), provided
that low hydrogen peroxide concentrations are
maintained. Depolymerization by MnP alone is
limited to phenolic lignin preparations and can
be stopped completely by chemical blocking of
phenolic functions (Hammel et al., 1993). Complete mineralization requires cellular uptake of
depolymerization products and further intracellular catabolism.
3.1. Crystal structure of lignin peroxidase and manganese peroxidase
Communication$ on the tertiary structure of
ligninase at 2.6 A resolution (Edwards et al.,

II Tuor et L /Journal

of Biotechnology 41 (1995) l-17

19931, 2.5 A resolution (Piontek et al., 1993) and

2.0 A (~oulos et al., 1993) have appeared rzcently. The chainfold of the enzyme comprises

343 amino acids with three ordered sugar moieties and one heme molecule. A view of the
crystallographically determined structure is given

Fig. 2. A view of the crystallographically determined structure of lignin peroxidase (Piontek et al., 1993). a-Helices are shown in
blue, while p-strands are drawn as yellow arrows. The catalytic heme molecule together with the proximal and distal histidine are
shown as ball and stick models, as well as three glycosylation sites and the eight cysteine residues forming four disulfide bridges.
Two calcium ions are represented as enlarged, violet spheres. The plot was generated using the program MOLSCRIPT.

U. Tuor et ul. /Journal

of Biotechnology

in Fig. 2. Noteworthy features are the mostly

helical fold with separate domains on either side
of the heme. LIP strikingly resembles cytochrome
c peroxidase (CCP) with a similar spatial arrangement of 11 helical segments, despite the fact that
the amino acid sequence identity of the two enzymes is below 20% (Piontek et al., 1993: Poulos
et al., 1993). LiP contains four disulfide bridges
formed by eight cysteine residues which are absent in CCP (Piontek et al., 1993; Poulos et al..
1993).
Comparisons of the active centers of LiP and
CCP show differences which in part account for
the reactivity differences observed. The smaller
heme pocket in LiP is separated from the solvent
and probably provides the binding site for small
aromatic molecules, e.g., veratryl alcohol. In addition, the active site of LIP seems to be stabilized by two structural calcium ions which have
not been observed in CCP (Poulos et al., 1993).
The enzyme contains one N-acetylglycosamine at
Asn-257 and two mannose residues, supposedly
originating from an 0-glycosylation site (Piontek
et al.. 1993: Poulos et al.. 1993).
The porphyrin system consists0 of one pentacoordinate iron placed 0.15-0.27 A above the saddle-shaped heme plane. It coordinates with a
distal and a proximal histidine and a distal arginine. Unique features of LIP like a low pH optimum near 3.0 (Tien et al., 1986) and the capacity
to oxidize nonphenolic substrates with high redox
potential (Schoemaker. 1990; Gold et al., 1989;
Kirk and Farrell, 1987) can now be explained on
a molecular level. pH-dependent protonation of
carboxylates which are present in the heme pocket
favour substrate binding and electron transfer
(Poulos et al., 1993). Weaker ligation of the ligninase

heme

iron

with the proximal

histidine

re-

sults in a higher electron deficit and thus higher

redox potential than in CCP (Piontek et al., 1993).
Since LIP and MnP exhibit good sequence homology, LIP electron density maps were also applied for the refinement of crystallographic data
obtained from MnP crystals. Preliminary crystallographic high-resolution
data has been presented, but a detailed structural description of
MnP and its binding site is not available as yet
(Sundaramoorthy et al., 1994).

41 (1995)

I - 17

3.2. Enzyme multiplicity

Ligninolytic activity in P. chrysosporium and
other white-rot fungi is associated with multiple
isoenzymes. At least 21 heme peroxidases are
produced in liquid cultures of P. chrysosporium
(Leisola et al., 1987; Tien, 1987). The physical
and kinetic characteristics of the isozymes are
very similar, but differences in stability, quantity
and catalytic properties have been described
(Farrell et al., 1989; Giumoff et al., 1990). They
may be the consequence of three different structural genes or post-translational
modifications.
Coriolus versicolor (Morohoshi,
1991), Panus
tigrinus (Maltseva et al., 1991), Rigidoporus lignosus (Geiger et al., 1986b), and Ceriporiopsis subcermispora (Lobes et al., 1994) secrete lactase
isoenzymes. The significance of the multiplicity of
ligninolytic enzymes to lignin degradation remains to be explained; it was suggested that they
possess different importance in the delignification process, or that they may act synergistically
(Farrell et al., 1989). The isozymes may also reflect the inhomogeneity of the substrate and intermediate products at different stages of wood
decay and among plant species.

4. Importance of ecological conditions

All white-rot basidiomycetes degrade the three
wood polymers cellulose, hemicellulose and lignin,
albeit at different rates and extent. Based on
these criteria white-rot fungi have been divided
into classes of simultaneous degraders of lignin
along with wood polysaccharides or selective
lignin degraders (Eriksson et al., 1990; Blanchette,
1991). Classification according to these groups
proved to be difficult since many white-rot fungi
show both types of decay while acting on the
same substrate, or they attack lignin and polysaccharides sequentially (Eriksson et al., 1990). Environmental conditions such as temperature, humidity, microclimate, nitrogen content of the substrate and compartimentalization may also govern
the selectivity of lignin biodegradation in vivo.
The gaseous regimes within the wooden substrate
influence enzyme activies. At the hyphal level,

U. Tuor et al. /Journal

of Biotechnology 41 (1995) 1 -I 7

elevated carbon dioxide levels (> lo-20%) with

concomitant depressed oxygen levels ( < 1%) were
measured (Thacker and Good, 1952), suggesting
that in vivo oxidative lignin degradation may occur under microaerobic conditions. P. chrysosporium efficiently degraded dioxane/HCl-extracted
straw lignin even under an atmosphere of 30%
CO, and 10% 0, (Leisola et al., 1984). There is
experimental evidence that a pure oxygen atmosphere is toxic to the fungus (Leisola et al., 1984;
Reid and Seifert, 1980).
A special case of selective lignin removal in
hardwoods is caused by Ganoderma applanatum
in evergreen rainforests of southern Chile and
known as palo podrido. Comparing palo podrido
with lignin degradation by G. applanatum in temperate climate it was concluded that low nitrogen
concentration, high carbon dioxide/low oxygen
partial pressure, high humidity and low temperature are the most important environmental factors for selective lignin degradation (Dill and
Kraepelin, 1988). Palo podrido is a result of the
rainforest climate, whereas in Europe this type of
wood decay is very atypical. It will only appear if
a similar microclimate can build up in a secluded
compartment of a tree or stem. The same report
states that the substrate of palo podrido contains
lower amounts of nitrogen of 0.037-0.073% dry
weight as opposed to amounts > 0.07% in European tree species. Under these conditions microbial cellulose consumption is inhibited. Since approximatly 50% of the total nitrogen in wood is
bound to lignin (Dill et al., 19841, lignin degradation with simultaneous nitrogen utilization would
result in mycelial growth.
Lignin degradation occurs at low pH. Its optimum differs between species. P. chrysosporium
degrades lignin best at pH 4.0 with decreasing
activity towards lower pH values (Kirk et al.,
1978). In another study, lignin degradation by P.
chrysosporium and P. sajor-caju increased with
decreasing pH down to pH 3.0 (Boyle et al.,
1992). Accordingly, the enzymes purifed from P.
chrysosporium have their activity optimum at pH
3.0 (Lip; Tien et al., 1986) and pH 4.5 (MnP;
Glenn and Gold, 1985). The pH optimum of
lactase from Phellinus noxius is at pH 4.6 (Geiger
et al., 1986b). The pH of the natural substrate

varies between 4 and 6 for both hardwoods and

softwoods (Gray, 1958) and corresponds to the
growth optimum for most wood rotting fungi.
Furthermore, fungi are able to maintain a low pH
environment at the hyphal level by secreting a
polysaccharide slime layer (Buchala and Leisola,
1987).

5. Importance of hemicellulose degradation and

succession of microbial populations
The twisted arrangement of cellulose, hemicellulose and lignin was described in a model by
Kerr and Goring (1975). Lignin is chemically
bound to the hemicellulose moiety in the S2
layer, forming a matrix encrusting the cellulose
microfibrils. Xylans and lignin are closely associated and covalently bound in wood. Lignincarbohydrate complexes (LCCs) have been isolated from wood meal extracts after removal of
lignin or in the lignin fraction directly, and they
have been recently reviewed (Koshijima et al.,
1989; Jeffries, 1990).
Lignin-carbohydrate bonds are formed by ester
or ether bonds, linking the cu-position of the
lignin phenylpropane unit to either carboxyl or
free hydroxyl groups of hemicelluloses, respectively. They occur in low frequency. The sugarlignin linkage of a pine wood sample consists of
benzyl ether bonds predominantly at C-6 in hexoses and mostly at C-3 and to a lesser extent at
C-2 in pentoses (Koshijima et al., 1989). o-Xylose
and o-mannose are the major LCC components.
Evidence for glycosylic lignin-carbohydrate bonds
was not reported.
5.1. Accessability of the substrate
Lignin is a high-molecular, hydrophobic polymer and thus not soluble in aqueous solution,
raising the question about its accessability by
extracellular oxidative enzymes and cofactors. The
situation is complicated by the fact that lignin
does not serve as a sole growth substrate for
white-rot fungi (Ander and Eriksson, 1975; Kirk
et al., 1976). It was therefore suggested that the
purpose of lignin removal is to expose cellulose

10

U. Tuor et al. /Journal

of Biotechnology 41 (1995) l-1 7

and hemicellulose fibers for consumption and

further fungal growth (Kirk and Fenn, 1982). On
the other hand, the hemicelluloses also physically
restrict the access of enzymes to lignin. The close
association of lignin and hemicelluloses suggests
that the primary attack of the wood cell wall
requires enzymatic degradation of the hemicelluloses prior to lignin degradation. The covalent
bonds of hemicelluloses to lignin may be hydrolyzed, resulting in lignin-carbohydrate complexes
of lower molecular weight capable of diffusing
from the matrix. Alternatively, the hemicellulases
could strip their substrates off the cell wall, rendering the residual lignin exposed to attack by
LiP or MnP. Sound wood cells cannot be invaded
by wood decay enzymes (Srebotnik et al., 1988;
Blanchette et al., 1989). Hydrolyzation and depolymerization of the hemicellulose component
would render lignin accessible for LiP and MnP.
5.2. Degradation of hemicellulose
Hemicellulose degradation could happen in
two ways: either the wood-rotting fungi secrete
hemicellulases, or the fungal lignin degradation is
preceeded by hemicellulolytic activity of bacterial
Endo-xylanases,
@xylosidases and
consortia.
endo-mannanases have been purified from a large
number of fungi and bacteria, and they are produced by most wood-degrading fungi (for an
overview see Eriksson et al., 1990). At an early
stage of wood inoculation, P. chrysosporium expresses xylanases which require close contact with
their substrates as shown by immunolabeling experiments (Joseleau and Ruel, 1992). Xylanases
and hemicellulases together with peroxidases appear during incipient stages of decay in the hyphal cell wall of P. chrysosporium and subsequently move towards the secondary cell wall
(Blanchette et al., 1989). Immunolabeling showed
that xylanases do not diffuse into degraded wood
cell walls, whereas LiP and MnP penetrate degraded sections of the cell wall. MnP exhibited a
pronounced preference for the highly lignified
middle lamella and cell corners (Joseleau and
Ruel, 1992). Although white-rot fungi are investigated for their ability to selectively remove lignin,
one should not overlook their ability to degrade

hemicelluloses during the process. Hemicellulases, primarily xylanases and mannanases of fungal origin, are currently used in the treatment of
pulp on a technical scale as a biological pretreatment for delignification in paper manufacture
(Buchert et al., 1992; Viikari et al., 1992,1994).
5.3. Bacterial wood biodegradation
Bacteria also attack softwood and hardwood
cell walls. They have been described as primary
wood colonizers (Liese and Greaves, 1975) and
may inhibit or synergistically promote growth of
wood degrading fungi. Cell wall decay by mixed
bacterial cultures in natural environments as well
as in vitro was classified in degradation by erosion, cavitation and tunneling (Daniel et al., 1987).
Fungal lignin degradation results in the formation of low molecular weight, mostly aromatic
carboxylic acids, which may be further metabolized by bacteria. Bacterial cultures mineralize
intermediates from model compound degradation
by white-rot fungi with little concomitant label
incorporation into bacterial biomass Wicuna et
al., 1993).

6. The role of cr-carbonyl groups in lignin degradation

Structural analysis of spruce lignin determined
a frequency of 15-30 free phenolic hydroxyl
groups and 20 carbonyl groups per 100 phenylpropane units (Table 1; Sarkanen and Ludwig,
1971). Frequent occurrence of phenolic, C,-oxosubstituted substructures in native lignin and in
partially degraded lignin can therefore be assumed. Furthermore,
formation of a-carbonyl
groups is prominent during lignin biodegradation
(Kirk and Chang, 1975; Chen and Chang, 1985)
and during metabolism of a dimeric P-0-Cmodel
compound (Fenn and Kirk, 1984). Destroying chiral centers by C,-oxidation not alone at C,, but
also via enolization at C,, was suggested to be
important in decreasing the steric complexity of
the polymer (Shimada, 1980).
Enhanced depolymerization of spruce lignins
in ligninolytic cultures of P. chrysosporium was

U. Tuor et al. /Journal

of Biotechnology

observed after selective chemical C,-oxidation,

whereas C,-oxidation actually retarded degradation of an alkylated P-0-4-model
compound
(Fenn and Kirk, 1984). In a more recent study
with dimeric P-0-Cmodel
compounds, homogeneous MnP oxidized a phenolic C,-carbonyl
model compound, leading to alkyl-phenyl cleavage and C,-C,
cleavage (Tuor et al., 1992).
Based on these results, the earlier observation
could be explained by oxidative C,-C, cleavage
and alkyl-phenyl cleavage in the lignin polymer
which was initiated by MnP. The findings strongly
suggest that the MnP-system can directly attack
phenolic lignin substructures
with cr-carbonyl
groups, leading to oxidative degradation of the
lignin polymer via C,-C,
cleavage or alkylphenyl cleavage. The free diffusion of the
Mn/organic acid complex would favour such a
depolymerization
pathway.
The
observed
degradative pathway for the C,-carbonyl compound by the MnP system suggests an important,
hitherto unreported pathway for the degradation
of polymeric lignin.

7. Hypothetical schemes for Iignin biodegradation

It is evident that mineralization of the complex
lignin substrate by a microorganism requires two
key processes: breakdown of the polymer and
ring cleavage of the aromatic nuclei. Model compound studies in vivo and with ligninolytic enzymes yielded a wealth of knowledge on oxidative
cleavage reactions promoting these degradative
reactions (reviewed in: Higuchi, 1986; Kirk and
Farrell, 1987; Gold et al., 1989; Schoemaker, 1990;
Umezawa, 1988). Analysis of degraded lignin
preparations and lignin model compounds suggested that lignin biodegradation is an oxidative
process. This view was confirmed with the discovery of Iignin peroxidase and manganese peroxidase and the elucidation of the mechanistic action of these enzymes. However, we must be
extremely careful in extrapolating the already
complex results obtained from model compound
degradation studies to lignin biodegradation. Initial expectations that complete in vitro lignin
mineralization could be achieved by LiP and/or

41 (1995) 1-I 7

11

MnP alone have not been fulfilled. There is growing experimental evidence that reductive processes play a pivotal roIe in lignin biodegradation
and are also part of the ligninolytic system. A
range of monomeric and dimeric aromatic aldehydes and acids are reduced to the corresponding
alcohols in ligninolytic cultures of P. chrysosporium, and the responsible enzymes have been
purified and characterized (Ander et al., 1980;
Enoki et al., 1981; Leisola and Fiechter, 1985;
Muheim et al., 1990). In addition, the enzymes
cellobiose:quinone
oxidoreductase
(Westermark
and Eriksson, 1975) and NADH:quinone oxidoreductase (Constam et al., 1991; Muheim, 1991),
which are involved in quinone reduction, have
been isolated and purified from the same fungus.
Hypothetical schemes of lignin biodegradation
taking these conversions into account have been
published by Eriksson et al. (19901, Schoemaker
(19901, and Schoemaker et al. (1991). The degradation scheme by the latter authors emphasizes
that lignin degradation by P. chrysosporium must
involve both oxidative and reductive conversions.
Oxidative fragmentation and cleavage reactions
are required for chemical breakdown of the polymer, whereas extracellular and intracellular stages
of degradation are distinguished: given the size
and the insoluble nature of lignin, its breakdown
must take place outside the cell. This is followed
by cellular uptake of the soluble, monomeric and
small oligomeric fragments for further processing.
In general, H,O,-requiring
oxidations occur extracellularly
and intracellularly,
whereas the
NAD(P)H-dependent
reductions take place in the
cell. Lignin depolymerization
therefore can be
considered as an oxidative process, whereas the
metabolism of lignin fragments involves a combination of oxidations and reductions. The compartmentalization of oxidative and reductive conversions calls for a powerful transport mechanism
through the cell wall. Evidence for this was presented with the rapid removal of quinone reductase substrates in liquid culture of P. chrysosporium, and the observation of subsequent secretion
of the reduction products by the cells shortly
after (Tuor et al., 1993).
The cleavage of phenylpropane side-chains in
the course of the degradation yields C,, C, and

C, fragments,
which can be considered
as subfor the enzyme glyoxal oxidase and thus
supply extracellular
hydrogen peroxide. Aromatic
aldehydes formed by C,,-C,
cleavage in the depolymerization
process can be reduced
to the
corresponding
benzyl alcohols by the enzyme aryl
alcohol dehydrogenase.
This makes these compounds susceptible
to attack by the peroxidases
of the ligninolytic
system, generating
quinones
and ring-opened
products.
The importance of C,-oxidation
in the primary
attack of the lignin polymer and subsequent
polymer degradation
was already outlined.
Its role in
the depolymerization
of the polymer is shown in
Fig. 3 (Tuor, 1992). The initial steps of lignin
degradation
by P. chrysosporiunz seem to involve
a combination
of oxidative conversions
catalyzed
by MnP and LIP, possibly in conjunction
with the
action of Mn ions, veratryl alcohol. or other
compounds,
as redox mediators. This primary attack would lead to C,,-oxidation
in the lignin
strates

polymer or to C,-C,
cleavage and alkyl-phenyl
cleavage under release of small lignin fragments
(Fig. 3). The C,-carbonyl
groups aIready present
or formed in the process above are linked to both
phenolic
and non-phenolic
aromatic
rings. For
further degradation,
the latter have to undergo
demethylation
to yield phenols or can be separated from the polymer by oxidation of an adjacent B-ring by LIP, followed by Cp-ether
bond
cleavage (Kirk et al., 1986).
Phenolic
lignin
substructures
with a C,carbonyl function, both native or stemming from
oxidation
of phenolic
substructures
by MnP or
oxidation of non-phenolic
structures
by LIP and
subsequent
demethylation,
could in contrast be
attacked by MnP. This process would lead to the
liberation
of lignin fragments
from the polymer
via C,-C,
cleavage and alkyl-phenyl
cleavage.
Importantly,
these results suggest that no further
enzymes capable of cleaving C,-0x0 substructures
need be contemplated.
Since MnP production
by

Ihi/ 1%0-4-cleavage

phenolic
lr-c=O

C,,-Clc-cleavage
q

=;)alkyl-phenylcleavage

11-I/(+0-4-cleavage

Fig. 3. Hypothetical
scheme leading to oxidative depolymerization
involving mediation by veratryl alcohol or Mn. respectively.
=
ing reactions without structural
breakdown.

of the lignin polymer under

. potentially depolymerizative

the action of LiP and MnP. possibly

reaction pathways:
+ . lignin-alter-

U. Tuor et al. /Journal

of Biotechnology 41 (1995) l-l 7

P. chrysosporium reaches its maximum about 2 d

before LiP production (Leisola, 1988), it is conceivable that the oxidation of C,-carbonyl
residues by MnP is key to the primary attack of
the lignin polymer.
Direct degradation of the non-phenolic lignin
substructures can occur only under the action of
LiP via C,-C, cleavage or alkyl-phenyl cleavage
according to Fig. 3. Recent results with cultures
of C. subvermispora (Table 21, however, suggest
that an efficient in vivo depolymerization pathway of nonphenolic lignin exists that does not
depend on the secretion of Lip. The presence of
the natural substrate wood triggered the formation of C,-C, cleavage products, which were not
detected in liquid medium, and subsequent mineralization by an hitherto unknown mechanism
(Srebotnik et al., 1994).

8. Concluding remarks
The ubiquitous presence of the substrate wood
has always been a trigger for the investigation of
lignin biodegradation. During the last decade the
focus of applied research moved from the utilization of a single purified enzyme preparation to
the selective use of different enymes or whole
organisms. The main fields of application are
biobleaching and biopulping in paper manufacture and biodegradation of xenobiotics, especially
chlorinated phenols and dioxins. Remarkably,
fungal hemicelluloses could be faster developed
into application on an industrial scale than the
lignin degrading enzymes. Despite the progress in
our understanding
of catalytic function and
mechanism of the latter enzymes, the knowledge
of their attack on the polymeric lignin in the
natural substrate is too poor for being applied
today.
More recent research work indicates that
white-rot fungi produce a great diversity of enzyme combinations to accomplish a very complicated task generally termed lignin degradation.
Environmental conditions like temperature, moisture, as well as pH and nitrogen and oxygen
levels at the location of enzyme action, are governing factors. The growing knowledge will make

13

it possible to exploit metabolic pathways specifically and allow access to the tremendous biotechnological potential of white-rot fungi.

Acknowledgements
The authors thank Dr. 0. Petrini (Institute of
Microbiology, Swiss Federal Institute of Technology, Zurich) for his competent advice in taxonomy.

References
Adler, E. (1977) Lignin chemistry - past, present and future.
Wood Sci. Technol. 11, 169-218.
Ander, P. and Eriksson, K.-E. (1975) Influence of carbohydrates on lignin degradation by the white-rot fungus
Sporotrichum pulverulentum. Svensk Paperstidn. 78, 643652.
Ander, P., Hatakka, A. and Eriksson, K.-E. (1980) Vanillic
acid metabolism by the white-rot fungus Sporotrichum
pulverulentum. Arch. Microbial. 125, 189-202.
Biswas-Hawkes, D., Dodson, A.P.J., Harvey, P. and Palmer,
J.M. (1987) Ligninases from white-rot fungi. In: Odier, E.
(Ed.) Lignin Enzymic and Microbial Degradation. Symposium Intern. INRA Paris, pp. 125-130.
Blanchette, R.A., Abad, A.R., Farrell, R.L. and Leathers,
R.L. (1989) Detection of lignin peroxidase and xylanase by
immunocytochemical labeling in wood decayed by basidiomycetes. Appl. Environ. Microbial. 55, 1457-1465.
Blanchette, R.A. (1991) Delignification by wood-decay fungi.
Ann. Rev. Phytopathol. 29, 381-398.
Boominathan, K., Balachandra Dass, S., Randall, T.A., Kelley, R.L. and Reddy, A. (1990) Lignin peroxidase-negative
mutant of the white-rot basidiomycete Phanerochaete
chrysosporium. J. Bacterial. 172, 260-265.
Bourbonnais, R. and Paice, M.G. (1989) Oxidative enzymes
from the lignin-degrading fungus Pleurotus sajor-caju. In:
Lewis, N.G. and Paice, M.G. (Eds.), Biogenesis and
Biodegradation of Plant Cell Polymers, ACS Symposium
Series 399, pp. 472-481.
Boyle, CD., Kropp, B.R. and Reid, I.D. (1992) Solubilization
and mineralization of lignin by white rot fungi. Appl.
Environ. Microbial. 58, 3217-3224.
Buchala, A.J. and Leisola, M.S.A. (1987) Structure of the
P-glucan secreted by Phanerochaete chrysosporium in continuous culture. Carbohydr. Res. 165, 146-149.
Buchert, J., Kantelinen, A., Ratto M., Siika-aho, M., Ranua,
M. and Viikari, L. (1992) Xylanases and mannanases in
the treatment of pulp. In: Kuwahara, M. and Shimada, M.
(Eds.) Biotechnology in the Pulp and Paper Industry, Uni
Publishers Co., Tokyo, pp. 139-144.

14

U. Tuor et al. /Journal of Biotechnology 41 (1995) I-1 7

Buswell, J.A. and Odier, E. (1987) Lignin biodegradation.

CRC Crit. Rev. Biotechnol. 6, l-60.
Chang, H.-M. and Sarkanen, K.V. (1973) Species variation in
lignin: effect of species on rate of kraft delignification.
Tappi 56, 132-134.
Chen, C.L. and Chang, H.-m. (1985) Chemistry of lignin
biodegradation. In: Higuchi, T. (Ed.), Biosynthesis and
Biodegradation of Wood Components, Academic Press,
San Diego, CA, pp. 535-556.
Constam, D., Muheim, A., Zimmermann, W. and Fiechter, A.
(1991) Purification and characterization of an intracellular
NADH:quinone
oxidoreductase
from Phanerochaete
chrysosporium. J. Gen. Microbial. 137, 2209-2214.
Cowles, J.R., LeMay, R., Jahns, G., Scheld, W.H. and Peterson, C. (1989) Lignification in young plant seedlings grown
on earth and aboard the space shuttle. In: Lewis, N.G. and
Paice, M.G. (Eds.), Biogenesis and Biodegradation of Plant
Cell Polymers, ACS Symposium Series 399, pp. 203-213.
Daniel, G.F., Nilsson, T. and Singh, A.P. (1987) Degradation
of lignocellulosics by unique tunnel-forming bacteria. Can.
J. Microbial. 33, 943-948.
Datta, A., Bettermann, A. and Kirk, T.K. (1991) Identification
of a specific manganese peroxidase among ligninolytic
enzymes secreted by Phanerochaete chrysosporium. Appl.
Environ. Microbial. 57, 1453-1460.
Dill, I., Salnikow,
J. and Kraepelin,
G. (1984)
Hydroxyproline-rich protein material in wood and lignin of
Fagus sylvatica. Appl. Environ. Microbial. 48, 1259-1261.
Dill, I. and Kraepelin, G. (1988) Der Abbau von Lignin/Cellulose durch Weissfiiule-Pilze:
Einfluss spezifischer
Gkologischer Faktoren. Forum Mikrobiologie 11/88, 484489.
Dodson, P.J., Evans, C.S., Harvey, P.J. and Palmer, J.M.
(1987) Production and properties of an extracellular peroxidase from Coriolus versicolor which catalyzes C,-C,
cleavage in a lignin model compound. FEMS Microbial.
Lett. 42, 17-22.
Edwards, S.L., Raag, R., Wariishi, H., Gold, M.H. and Poulos, T.L. (1993) Crystal structure of lignin peroxidase.
Proc. Natl. Acad. Sci. USA 90, 750-754.
Enoki, A., Goldsby, G.P. and Gold, M.H. (1981) P-Ether
cleavage of the lignin model compound 4-ethoxy3methoxyphenylglyceroI_P-guaiacyl ether and derivatives by
Phanerochaete chrysosporium. Arch. Microbial. 129, 14114.5.
Eriksson, K.-E., Johnsrud, SC. and Vallander, L. (1983)
Degradation of lignin and lignin model compounds by
various mutants of the white-rot fungus Sporotrichum puluerulentum. Arch. Microbial. 135, 161-168.
Eriksson, K.-E, Blanchette, R.A. and Ander, P. (1990) Microbial and enzymatic degradation of wood and wood components. Springer Series in Wood Science, Springer Verlag,
Berlin.
Fahraeus, G. and Reinhammar, B. (1967) Large-scale production and purification of lactase from cultures of Polyporus
versicolor and some properties of lactase, A. Acta Chem.
Stand. 21, 2367-2378.

Farmer, V.C., Henderson, M.E.K. and Russel, J.D. (1960)

Aromatic alcohol oxidase activity in the growth medium of
Polystictus uersicolor. Biochemistry 74, 257-262.
Farrell, R.L., Murtagh, K.E., Tien, M., Mozuch, M.D. and
Kirk, T.K. (1989) Physical and enzymatic properties of
lignin peroxidase
isoenzymes
from Phanerochaete
chrysosporium. Enzyme Microb. Technol. 11, 322-328.
Fenn, P. and Kirk, T.K. (1984) Effects of C,-oxidation in the
fungal metabolism of lignin. J. Wood Chem. Technol. 4,
131-148.
Forrester, IT., Grabski, A.C., Mishra, C., Kelley, B.D., Strickland, W.N, Leatham, G.F. and Burgess, R.R. (1990) Characteristics and N-terminal amino acid sequence of a manganese peroxidase purified from Lentinula edodes cultures
grown on a commercial wood substrate. Appl. Microbial.
Biotechnol. 33, 359-365.
Fukuzumi, T. (1987) Ligninolytic enzymes of Pleurotus sajorcaju. In: Odier, E. (Ed.), Lignin Enzymic and Microbial
Degradation, Symposium Intern. INRA Paris, pp. 137-142.
Galliano, H., Gas, G. and Boudet, A.M. (1991) Lignin degradation by Rigidoporus lignosus involves synergistic action
of two oxidizing enzymes-Mn peroxidase and lactase. Enzyme Microb. Technol. 13, 478-482.
Geiger, J.P., Huguenin, B., Nicole, M. and Nandris, D. (1986al
Laccases of Rigidoporus lignosus and Phellinus noxius II.
Effect of R. lignosus lactase Ll on thioglycolic lignin of
hevea. Appl. Biochem. Biotechnol. 13, 97-l 10.
Geiger, J.P., Rio, B., Nandris, D. and Nicole, M. (1986bl
Laccases of Rigidosporus lignosus and Phellinus noxius I.
Purification and some physicochemical properties. Appl.
Biochem. Biotechnol. 12, 121-133.
Gilbertson, R.L. (1980) Wood-rotting fungi of north America.
Mycologia 72, 1-49.
Glenn, J.K., Morgan, M.A., Mayfield, M.B., Kuwahara, M.
and Gold, M.H. (1983) An extracellular HzOa-requiring
enzyme preparation involved in the lignin biodegradation
by the white rot basidiomycete Phanerochaete chrysosporium. Biochem. Biophys. Res. Commun. 114, 1077-1083.
Glenn, J.K. and Gold, M.H. (1985) Purification and characterization of an extracellular Mn(II)-dependent peroxidase
from the lignin-degrading basidiomycete, Phanerochaete
chrysosporium. Arch. Biochem. Biophys. 242, 329-341.
Glumoff, T., Harvey, P.J., Molinari, S., Goble, M., Frank, G.,
Palmer, J.M., Smit, J.D. and Leisola, M.S.A. (1990) Lignin
peroxidase from Phanerochaete chrysosporium. Eur. J.
Biochem. 187, 515-520.
Gold, M.H., Wariishi, H. and Valli, K. (1989) Extracellular
peroxidases involved in lignin degradation by the white rot
basidiomycete Phanerochaete chrysosporium. In: Whitaker,
J.R. and Sonnet, P.E. (Eds.), Biocatalysis in Agricultural
Biotechnology, ACS Symposium series 389, Chapter 9,
American Chemical Society, Washington, DC, pp. 127140.
Golovieva, L.A., Leontievsky, A.A., Maltseva, O.V. and Myasoedova, N.M. (1993) Ligninolytic enzymes of the fungus
Punus tigrinus 8/l& Biosynthesis, purification and properties. J. Biotechnol. 30, 71-77.

U. Tuor et al. /foumal

of Biotechnology 41 (1995) l-l 7

Gray, V.R. (1958) The acidity of wood. J. Inst. Wood Sci. 1,

58-64.
Guillen, F., Martinez, A.T. and Martinez, M.J. (1990) Production of hydrogen peroxide by aryl-alcohol oxidase from the
ligninolytic fungus Pleurotus eryngii. Appl Microbial.
Biotechnol. 32, 465-469.
Haemmerli, SD., Leisola, M.S.A. and Fiechter, A. (1986)
Polymerization of lignins by ligninases from Phanerochaete
chrysosporium. FEMS Microbial. Lett. 35, 33-36.
Hammei, ICE. and Moen, M.A. (1991) Depolymerization of a
synthetic lignin in vitro by lignin peroxidase. Enzyme
Microbial. Technol. 13, 15-18.
Hammel, KE., Jensen, K.A., Mozuch, M.D., Landucci, L.L.,
Tien, M. and Pease, E.A. (1993) Ligninolysis by a purified
lignin peroxidase. J. Biol. Chem. 268, 12274-12281.
Hatakka, A,, Kantelinen, A., TervilZ-Wile, A. and Viikari, L.
(1987) Production of ligninases by Phlebia radiafa in agitated cultures. In: Odier, E. (Ed.), Lignin Enzymic and
Microbial Degradation. Symp. Intern. INRA Paris, pp.
185-189.
Hattori, T. and Higuchi, T. (1991a) Degradation of phenolic
and nonphenolic syringyl and biphenyl lignin model compounds by lignin peroxidase. Mokuzai Gakkaishi 37, 542547.
Hattori, T. and Higuchi, T. (1991b) Degradation of lignin
substructure model compounds in the presence of veratryl
alcohol or veratryl-@-D-xyloside by lignin peroxidase.
Mokuzai Gakkaishi 37, 548-554.
Higuchi, T. (1986) Catabolic pathways and role of ligninases
for the degradation of lignin substructure models by whiterot fungi. Wood Res. 73, 58-81.
Higuchi, T. (1989) Mechanisms of lignin degradation by lignin
peroxidase and lactase of white-rot fungi. In: Lewis, N.G.
and Paice, M.G. (Eds.), Biogenesis and Biodegradation of
Plant Cell Polymers. ACS Symposium Series 399, pp.
482-502.
Hiittermann, A., Milstein, O., Nicklas, B., Trojanowski, J.,
Haars, A. and Kharazipour, A. (1989) Enzymatic modification of lignin for technical use. In: Glasser, W.G. and
Sarkanen, S. (Eds.), Lignin-Properties and Materials. ACS
Symposium Series Vol. 397, pp. 361-370.
Jeffries, T.W. (1990) Biodegradation of lignin-carbohydrate
complexes. Biodegradation 1, 163-176.
Johansson, T. and Nyman, P.O. (1987) A manganese(II)-dependent extracellular peroxidase from the white-rot fungus Trametes versicolor. Acta Chem. Stand. B41, 762-765.
Joseleau, J.P. and Ruel, K. (1992) Ultrastructural examination
of lignin and polysaccharide degradation in wood by whiterot fungi. In: Kuwahara, M. and Shimada, M. (Eds.),
Biotechnology in the Pulp and Paper Industry. Uni Publishers Ltd., Tokio, pp. 195-202.
Kerr, A.J. and Goring, D.A.I. (1975) The ultrastructural arrangement of the wood cell wall. Cellul. Chem. Technol. 9,
563-573.
Kirk, T.K (1987) Lignin-degrading enzymes. Philos Trans. R.
Sot. London A 321, 461-474.
Kirk, T.K. and Chang, H.-m. (1975) Decomposition of lignin

15

by white-rot fungi II. Characterization of heavily degraded

lignins from decayed spruce. Holzforschung 29, 56-64.
Kirk, T.K., Connors, W.J. and Zeikus, J.G. (1976) Requirement for a growth substrate during lignin decomposition
by two wood-rotting fungi. Appl. Environ. Microbial. 32,
192-194.
Kirk, T.K., Schultz, E., Connors, W.J., Lorenz, L.F. and
Z&us, J.G. (1978) Influence of culture parameters on
lignin metabolism by Phanerochaete chrysosporium. Arch.
Microbial. 117, 277-285.
Kirk, T.K. and Fenn, P. (1982) Formation and action of the
ligninolytic system in basidiomycetes. In: Swift, M.J., Frankland, J. and Hedger, J.N. (Eds.1, Decomposer basidiomycetes Br. Mycol. Sot. Symp. 4. Cambridge University
Press, Cambridge, pp. 67-90.
Kirk, T.K., Tien, M., Kersten, P.J., Mozuch, M.D. and
Kalyanamaran, B. (1986) Ligninase of Phanerochuete
chrysosporium. Biochem. J. 236, 279-287.
Kirk, T.K. and Farrell, R.L. (1987) Enzymatic combustion:
The microbial degradation of lignin. Annu. Rev. Microbio1. 41, 465-505.
Koshijima, T., Watanabe, T. and Yaku, F. (1989) Structure
and properties of the lignin-carbohydrate polymer as an
amphipathic substance. In: Glasser, W.G. and Sarkanen,
S. (Ed%), Lignin-Properties and Materials. ACS Symposium Series Vol. 397, pp. 11-28.
Kuwahara, M., Glenn, J.K., Morgan, M.A. and Gold, M.H.
(1984) Separation and characterization of two extracellular
HzO,-dependent
oxidases from ligninolytic cultures of
Phanerochaete chrysospvtium. FEBS Lett. 169, 247-250.
Leatham, G.F. (1986) The ligninolytic properties of Lentinus
edodes and Phanerochaete chrysosporium. Appl. Microbial.
Biotechnol. 24, 51-58.
Leatham, G.F. and Stahmann, M.A. (1981) Studies on the
lactase of Lentinus edodes: specificity, localization and
association with the development of fruiting bodies. J.
Gen. Microbial 125, 147-157.
Leisola, M.S.A., Ulmer, D.C. and Fiechter, A. (1984) Factors
affecting lignin degradation in lignocellulose by Phanerochaete chrysosporium. Arch. Microbial. 137, 171-175.
Leisola, M.S.A. and Fiechter, A. (1985) New trends in lignin
biodegradation. In: Mizrahi, A. and van Wezel, A.L. (Eds.),
Advances in Biotechnological Processes. Alan R. Liss Inc.,
New York, pp. 59-89.
Leisola, M.S.A., Kozulic, B., Meussdoerffer, F. and Fiechter,
A. (1987) Homology among multiple extracellular peroxidases from Phanerochaete chrysosporium. J. Biol. Chem.
262, 419-424.
Leisola, M.S.A. (1988) White-rot Lignin Degradation. Habilitationsschrift, ETH Ziirich.
Liese, W. and Greaves, H. (1975) Micromorphology of bacterial attack. In: Liese, W. (Ed.), Biological Transformantion
of Wood by Microorganisms. Springer, New York, pp.
74-88.
L.obos, S., Larrain, J., Salas, L., Cullen, D. and Vicuna, R.
(1994) Isozymes of manganese-dependent
peroxidase and
lactase produced by the lignin-degrading basidiomycete

16

U. Tuor et al. /Journal

of Biotechnology 41 (1995) l-l 7

Ceriporiopsis subuermispora. .I. Gen. Microbial. 140, 26912698.

Maltseva, O.V., Niku-Paavola, M.-L., Leontievsky, A.A., Myasoedova, N.M. and Golovleva, L.A. (1991) Ligninolytic
enzymes of the white-rot fungus Panus tigrinus. Biotechnol. Appl. Biochem. 13, 291-302.
Messner, K. and Srebotnik, E. (1994) Biopulping: An overview
of developments in an environmentally safe paper-making
technology. FEMS Microbial. Rev. 13, 351-364.
Morohoshi, N. (1991) Laccases from the ligninolytic fungus
Coriolus uersicolor. In: Leatham. G., and Himmel, M.E.
(Eds.), Enzymes in Biomass Conversion. ACS Symposium
Series 460, pp. 204-207.
Muheim, A., Waldner, R., Leisola, M.S.A. and Fiechter, A.
(1990) An extracellular aryl-alcohol oxidase from the
white-rot fungus Bjerkandera adusta. Enzyme Microb.
Technol. l&204-209.
Muheim, A. (1991) Novel ligninolytic enzymes from white-rot
fungi. PhD Thesis, Diss. ETH No. 9501, Ziirich.
Mustranta, A. (1987) Production of peroxidase by Inonotus
weirii. Appl. Microbial. Technol. 27, 21-26.
Niku-Paavola, M.L., Karhunen, E., Salola, P. and Raunio, V.
(1988) Ligninolytic enzymes of the white-rot fungus Phlebia
radiata. Biochem. J. 254, 877-884.
Nimz, H.H. (1974) Beech lignin-proposal of a constitutional
scheme. Angew. Chem. Intl. Ed. 13, 313-321.
Odier, E., Mozuch, M.D., Kalyanaraman, B. and Kirk, T.K.
(1988) Ligninase-mediated phenoxy radical formation and
polymerization unaffected by cellobiose:quinone oxidoreductase. Biochimie 70, 847-852.
Perez, J. and Jeffries, T.W. (1990) Mineralization of 14C-ringlabeled synthetic lignin correlates with the production of
lignin peroxidase, not of manganese peroxidase or lactase.
Appl. Environ. Microbial. 56, 1806-1812.
P&i&, F. and Gold, M.H. (1991) Manganese regulation of
manganese peroxidase expression and lignin degradation
by the white-rot fungus Dichomitus squa!ens. Appl. Environ. Microbial. 57, 2240-2245.
Petroski, R.J., Peczynska-Czech, W. and Rosazza, J.P. (1980)
Analysis, production and isolation of extracellular lactase
from Polyporus anceps. Appl. Environ. Microbial. 40,
1003-1006.
Piontek, K., Glumoff, T. and Winterhalter, K. (1993) Low pH
crystal structure of glycosylated lignin peroxidase from
Phanerochaete chrysosporium at 2.5 A resolution. FEBS
Lett. 315, 119-124.
Poulos, T.L., Edwards, S.L., Wariishi, H. and Gold, M.H.
(1993) Crystallographic refinement of lignin peroxidase at
2 A. J. Biol. Chem. 268,4429-4440.
Reid, I.D. and Seifert, K.A. (1980) Lignin degradation by P.
chrysosporium in hyperbaric oxygen. Can. J. Microbial. 26.
1168-1171..
Reid, I.D. and Paice, M.G. (1994) Biological bleaching of
kraft pulps by white-rot fungi and their enzymes. FEMS
Microbial. Rev. 13, 369-376.
Riittimann, C., Schwember, E., Salas, L., Cullen, D. and

Vicuna, R. (1992a) Ligninolytic enzymes of the white-rot

basidiomycetes Phlebia brevispora andceriporiopsis subuermispora. Biotechnol. Appl. B&hem. 16, 64-76.
Riittimann, C., Salas, L. and Vicuna, R. (1992b) Studies on
the ligninolytic system of the white-rot fungus Ceriporiopsis subcermispora. In: Kuwahara, M. and Shimada, M.
(Eds.), Biotechnology in the Pulp and Paper Industry. Uni
Publishers Ltd., Tokio, pp. 243-248.
Rypacek, V. (1977) Chemical composition of hemicelIuloses
as a factor participating in the substrate specificity of
wood-destroying fungi. Wood Sci. Techn. 11, 59-67.
Sannia, G., Giardina, P., Luna, M., Rossi, M. and Buonocore,
V. (1986) Lactase from Pleurotus ostreatus. Biotechnol.
Lett. 8, 797-800.
Sannia, G., Limongi, P., Cocca, E., Buonocore, F., Nitti, G.
and Giardina, P. (1991) Purification and characterization
of a veratryl alcohol oxidase enzyme from the lignin degrading basidiomycete Pleurotus ostreatus. Biochim. Biophys. Acta 1073, 114-119.
Sarkanen, K.V. and Ludwig, C.H. (1971) Definition and
Nomenclature. In: Sarkanen, K.V. and Ludwig, C.H.
(Eds.), Lignins: Occurrence, Formation, Structure and Reactions. John Wiley & Sons, New York, pp. 1-18.
Sarkanen, K.V. and Hergert, H.L. (1971) Classification and
distribution. In: Sarkanen, K.V. and Ludwig, C.H. (Eds.),
Lignins: Occurrence, Formation, Structure and Reactions,
John Wiley & Sons, New York, pp. 43-94.
Schoemaker, H.E. (1990) On the chemistry of lignin biodegradation. Rec. Trav. Chim. Pays-Bas 109, 255-272.
Schoemaker, H.E., Tuor, U., Muheim, A., Schmidt, H.W.H.
and Leisola, M.S.A. (1991) The chemistry of lignin
biodegradation. Catabolism of veratryl alcohol and its
methyl ether. In: Paucht, U.K. and Alderweireldt, F.C.,
(Eds.), Bioorganic Chemistry in Health Care and Technology, Proceedings NATO Advanced Research Workshop,
Plenum Press, New York, pp. 69-86.
Shimada, M. (1980) Stereobiochemical approach to lignin
biodegradation. In: Kirk, T.K., Higuchi, T. and Chang,
H.-m. (Eds.), Lignin Biodegradation: Microbiology, Chemistry and Potential Applications, CRC, Boca Raton, FL,
pp. 195-213.
Srebotnik, E., Messner, K. and Foisner, R. (1988) Penetrability of white-rot degraded pine wood by the lignin peroxidase of Phanerochaete chrysosporium. Appl. Environ. Microbiol. 54, 2608-2614.
Srebotnik, E., Jensen, K.A. and Hammel, K.E. (1994) Fungal
degradation of recalcitrant nonphenolic structures without
lignin peroxidase. Proc. Natl. Acad. Sci. USA 91, 1279412797.
Sundaramoorthy, S., Kishi, K., Gold, M.H. and Poulos, T.L.
(1994) Preliminary crystallographic analysis of manganese
peroxidase from Phanerochaete chrysosporium. J. Mol.
Biol. 238, 845-848.
Thacker, D.G. and Good, H.M. (1952) The composition of air
in trunks of sugar maple in relation to decay. Can. J. Bot.
30, 475-485.

U. Tuor et al. /Journal

of Biotechnology

Tien, M. and Kirk, T.K. (19831 Lignin-degrading enzyme from

the hymenomycete P. chrysosporium burds. Science 221,
661-663.
Tien, M., Kirk, T.K., Bull, C. and Fee, J.A. (1986) Steady-state
and transient-state kinetic studies on the oxidation of
3,4-dimethoxybenzyl alcohol catalyzed by the ligninase of
P. chrysosporium burds. J. Biol. Chem. 261, 1687-1693.
Tien, M. (1987) Properties of ligninase from P. chrysosporium
and their possible applications. CRC Crit. Rev. Microbial.
15, 141-168.
Tuor, U. (1992) Formation and metabolism of quinones in the
biodegradation of lignin model compounds by Phanerochaete chrysosporium. PhD Thesis, Diss ETH No. 9806,
ETH Zurich.
Tuor, U., Wariishi, H., Schoemaker, H.W.H. and Gold, M.H.
(1992) Oxidation of phenolic arylglycerol /&aryl ether lignin
model compounds by manganese peroxidase from Phanerochaete chrysosporium:
Gxidative cleavage of an ~1carbonyl model compound. Biochemistry 31, 4986-4995.
Tuor, U., Schoemaker, H.E., Leisola, M.S.A. and Schmidt,
H.W.H. (1993) Isolation and characterization of substituted 4-hydroxy-cyclohex-2-enones as metabolites of 3,4dimethoxybenzylalcohol and its methyl ether in ligninolytic
cultures of Phanerochaete chrysosporium. Appl. Microbial.
Biotechnol. 38, 674-680.
Umezawa, T. (19881 Mechanism for chemical reactions involved in lignin biodegradation
by Phanerochaete
chrysosporium. Wood Res. 75, 21-79.
Vares, T., Lundell, T.K and Hattakka, AI. (1993) Production

41 (1995) 1-I 7

17

of multiple lignin peroxidases by the white-rot fungus

Enzyme Microb. Technol. 15, 664669.
Vicuna, R., Gonzalez, B., Seelenfreund, D., Riittimann, C.
and Salas, L. (19931 Ability of natural bacterial isolates to
metabolize high and low molecular weight lignin-derived
molecules. J. Biotechnol. 30, 9-13.
Viikari, L., Tenkanan, M., RSttij M., Buchert, J., Kantelinen,
A., Bailey, M., Sundquist, J. and Linko, M. (1992) Important properties of xylanases for use in the pulp and paper
industry. In: Kuwahara, M. and Shimada, M. (Eds.1,
Biotechnology in the Pulp and Paper Industry. Uni Publishers, Tokyo, pp. 101-106.
Viikari, L., Kantelinen, A., Sundquist, J. and Linko, M. (19941
Xylanases in bleaching: From an idea to the industry.
FEMS Microbial. Rev. 13, 335-350.
Waldner, R. (1987) Ligninolytic systems of white-rot fungi.
PhD Thesis, Diss ETH No. 8343, ETH Zurich.
Waldner, R., Leisola, M.S.A. and Fiechter, A. (19881 Comparison of ligninolytic activities of selected white-rot fungi.
Appl. Microbial. Biotechnol. 29, 400-407.
Wariishi, H., Valli, K. and Gold, M.H. (1991) In vitro depolymerization of lignin by manganese peroxidase of Phanerochaete chrysosporium. Biochem. Biophys. Res. Commun.
176, 269-275.
Westermark, U. and Eriksson, K.-E. (1975) Purification and
properties of cellobiose:quinone
oxidoreductase
from
Sporotrichum pulverulentum. Acta Chem. Stand. Ser. B 29,
419-424.
Phlebia ochraceofulva.