FEMS Microbiology Reviews 13 (1994) 235-240
© 1994 Federation of European Microbiological Societies 0168-6445/94/$26.00
Published by Elsevier
235
FEMSRE 00349
Enzymes and small molecular mass agents involved
with lignocellulose degradation
C.S. Evans *, M.V. Dutton, F. Guill4n and R.G. Veness
School of Biological Sciences, University of Westminster, 115 New Cavendish St., London W1M RIS, UK
Abstract: Electron microscopic and biochemical studies of lignocellulose degradation by wood-rotting fungi have shown that
enzymes such as lignin peroxidases, manganese-dependent peroxidases, laccases and cellulases are too large to penetrate
undegraded secondary wood cell walls. Degradation occurs by surface interaction between cell wall and enzymes, but initiation of
decay at a distance from the fungal hyphae must involve diffusible low-molecular mass agents. The roles of hydrogen peroxide,
veratryl alcohol, oxalate, Fe2+-Fe 3+ and Mn2+-Mn 3+, as such agents in lignocellulose degradation are discussed.
Key words: Lignocellulose degradation; Hydrogen peroxide; Oxalate; White-rot fungi; Brown-rot fungi
Introduction
The use of immunogold-cytochemical labelling
techniques with electron microscopy of wood infected by basidiomycetes has assisted in the elucidation of the localisation of enzymes which degrade lignocellulose [1]. The precise location of
enzyme molecules in situ has enabled a correlation to be made with the biochemical data available and the physico-chemical observations of
degraded wood.
Most of the biochemical studies on lignin
degradation have applied to the effect of isolated
enzymes on lignin-model compounds, with relatively few observations of degradation of 'typical'
polymeric lignins. The major enzymes found to
affect lignin and lignin-model compounds are laccases (polyphenol oxidases), lignin-peroxidases
* Corresponding author.
SSDI 0 1 6 8 - 6 4 4 5 ( 9 3 ) E 0 0 7 5 - U
(LIP) and manganese-dependent peroxidases
(MnP). Although some reports of lignin degradation have suggested that polymeric lignin is degraded, most work has shown that these enzymes
will mineralise lignin-model compounds and fragments of lignin rather than degrade polymeric
lignin. In fact, there is some scepticism that the
studies on lignin-model compounds have revealed
very little about lignin degradation in situ [2].
Enzymic digestion of cellulose has been more
clearly demonstrated, with the identification and
characterisation of endoglucanases, cellobiohydrolases and /3-glucosidases [3]. However, biochemical evidence is still needed to explain the
differences in wood decay caused by brown-rot
and white-rot fungi with respect to cellulose and
hemicellulose degradation. Brown-rot fungi appear to degrade wood cell walls by inducing a
general thinning of the secondary wall at some
distance from the location of the hyphae in contrast to white-rot fungi which, in addition, frequently cause localised troughs in the wall close
236
to the hyphae [4]. Cellulases and hemicellulases
have been isolated from both white- and brownrot fungi though it has been thought that a nonenzymic mechanism operates in brown-rot decay
involving Fe 2+ and hydrogen peroxide. Studies
on the molecular size distribution of cellulose
following attack by white-rot and brown-rot fungi
showed that brown-rots cleaved completely
through the amorphous regions of the cellulose
fibrils whilst white-rots caused a progressive decay by attacking the surfaces of the microfibrils
[5].
Enzyme localisation with electron microscopic
study of infected wood has shown that lignocellulolytic enzymes cannot penetrate into the wood
structure except where the wood cell wall is already partially decayed. Most of the enzymes
localised by immunogold-cytochemical labelling
techniques were located at the cell wall surfaces
and once the wood cell wall was partially eroded
the enzymes began to penetrate into the wall.
This pattern of enzyme distribution was typical of
•
ligninases
U
cellul~s~
O
B--glucosidase
LiP, laccase, endo-l,4-/3-glucanase and 1,4-fl-Dglucan cellobiohydrolase 1, but cellobiase (1,4-/3glucosidase) was always located within the extracellular polysaccharide sheath surrounding the
hyphae [1].
These data are supported by measurements of
pore sizes in wood which show that large
molecules such as enzymes would be unable to
penetrate into the cell wall [6]. We have presented a hypothesis that there is regulation of the
relative sequence of lignocellulolytic enzymes
defining the spatial arrangement between hyphae
and the wood cell wall with initiation of decay by
low-molecular mass mediators. Fig. 1 presents
this hypothesis which has led us to investigate the
potential low-molecular mass compounds which
may play a role in lignocellulose degradation.
Small molecules would be capable of diffusing
into the wood cell wall structure to initiate decay,
so opening up the pore size in wood to allow
enzymes to penetrate to complete the degradative process.
ML
S~
Fe
Mn
OXALATE
S~
VERATRYL ALCOHOL
CATION
mucilage
Fig. 1. Distribution of lignocellulolytic enzymes and potential small molecular size mediators during white-rot decay of wood.
237
Small molecular m a s s molecules
EXTRACELLULAR MEDIUM
Hydrogen peroxide
Much research has centred on the role of
hydrogen peroxide in both cellulose and lignin
degradation. Cellulose degradation by brown-rot
fungi was considered to be less enzyme-dependent than with white-rot fungi. The involvement
of Fenton's reaction, Fe 2+ and H202 to produce
hydroxyl radicals which were active in depolymerising cellulose, in addition to enzymes, was
thought to be a reason for the complete extraction of cellulose from the lignocellulosic matrix of
wood by brown-rots [7]. However, white-rots also
produce H202 from the action of enzymes such
as glyoxal oxidase, glucose oxidase and aryl alcohol oxidase, so it would be expected that the
depolymerisation of cellulose would occur in a
similar manner to the brown-rots. Although Fe 2÷
has been detected in some quantity in specific
woods [21], the presence of hydrogen peroxide in
wood has not been shown. Hydrogen peroxide
has been measured in culture media of fungi
under specific conditions [7,10,11]. The Fenton's
reaction mechanism has not been proven to be
the primary mode of cellulose degradation in
wood, mainly because the life time of extracellular H202 in the environment is unknown and its
diffusion into the wood cell wall has not been
demonstrated [8].
Hydrogen peroxide is necessary for the action
of the ligninolytic enzymes LiP and Mn-peroxidase. Its logical site of production would be in
close association with them in the mucilage surrounding the hyphae to enable specific interaction. Glyoxal oxidase is thought to be a major
source of extracellular H202, and is produced in
culture under growth conditions that are identical
for the production of LiP [9]. In addition, aryl
alcohol oxidases are also responsible for hydrogen peroxide production by ligninolytic fungi [10].
Study of Pleurotus eryngii has shown the existence of a cyclic system of H202 production
involving aryl-alcohol oxidase and two dehydrogenases (Fig. 2). This cycle, which has been demonstrated with benzyl and veratryl compounds, is
probably operating with a high number of aromatic alcohols, aldehydes and acids derived from
H=O=
T
Alcohol
H202
)
T
Aldehyde,
oxld&le
Alcohol
(
oxldlse
Aldehyde
Aryl-llcohol
dlhydrogenlle
) Acid
(
Acid
Ar yl-lldehyde
dehydrogenale
MYCELIUM
Fig. 2. Cycle of hydrogen peroxide production by aryl-alcohoI
oxidase and dehydrogenase activities in Pleurotus eryngii.
fungal metabolism and lignin degradation, due to
the wide substrate specificity of the enzymes implicated. In this multi-enzymatic system, H202 is
produced extracellularly through the action of
aryl-alcohol oxidase on aromatic alcohols and
aldehydes, with the alcohols being the best substrates. The oxidised products of aryi-alcohol oxidase are recycled by the intracellular dehydrogenases. An equilibrium between the production
and a rapid system of reduction of H202 seems
to be attained to keep H 2 0 z at constant levels.
Similarly, the activities of aryl alcohol oxidase
and dehydrogenases seem to regulate the concentrations of aromatic compounds [11].
Veratryl alcohol
Veratryl alcohol is produced by many white-rot
species as a secondary metabolite. It has been
postulated that a mediator molecule such as the
cation radical of veratryl alcohol, produced by
interaction with LiP, could be involved as an
agent in lignin depolymerisation [12]. It would
have the ability to diffuse away from the enzymes
secreted by the hyphae, although whether the
life-time of the radical would be sufficient to
allow it to permeate the wood cell wall can be
questioned.
Oxalate
It has been known for many years that brownrot fungi secrete large amounts of oxalate into
238
their environment, although evidence of accumulation of oxalate by white-rots had not been reported [13]. The absence of oxalate in cultures of
white-rots was accounted for by the fact that
white-rots but not brown-rots produce oxalate
decarboxylase, an enzyme found intracellularly in
hyphae. Our studies have shown that many
white-rots also secrete and accumulate oxalate,
sometimes of the same order as in brown-rot
cultures, e.g. Trametes (Coriolus) cersicolor produces up to 10 mM in culture media compared
with 3 mM by Coniophora puteana and 28 mM by
Poria ~'aporaria [14]. The timing of secretion by
cultures differs between the two types of fungi,
with brown-rots accumulating oxalate throughout
growth, whereas white-rots accumulate oxalate in
secondary metabolic phase only. In primary
growth phase, oxalate decarboxylase is produced,
both intra- and extracellularly by white-rot cultures, but the enzyme is not detectable when
cultures reach secondary metabolism. This correlates with the accumulation of oxalate in secondary growth phase but not in primary phase for
white-rot species. The role of oxalate secretion is
still unclear, but evidence is gradually accumulating to indicate its involvement in the lignocellulolytic process.
Oxalate has been shown to chelate cations
such as Ca 2+, Fe 2+, NH~- from its environment,
frequently forming crystals of insoluble calcium
oxalate, which has led to speculation that this is a
means of environmental detoxification [14]. Calcium, however, is an important constituent in
plant cell walls. It can be withdrawn from calcium
pectate and sequestered by oxalate. In conjunction with pectinases secreted by wood-rotting
fungi, this can lead to significant changes in the
cell wall structure [15]. The pore size within the
cell wall would be enlarged by the removal of
calcium ions and may permit access by enzyme
molecules which were previously excluded. In addition it has been demonstrated in vitro that
oxalate will breakdown hemicellulose and depolymerise cellulose [16].
Another role for oxalate may be to enable the
Fenton's reaction to cycle by reducing Fe ~+ to
Fe 2+, with the concomitant production of active
hydronium ions H3 O+, regenerating Fe 2+ for
reaction with H202. The exact concentrations of
ions may be important in this reaction as the
Fenton reaction has been shown to be inhibited
by t mM oxalic acid [17]. Recent studies have
shown that oxalate can inhibit LiP-catalysed oxidations of non-phenolic substrates [18], but this
was very. dependent on the-ionic state of oxalate,
with oxalic acid being ineffective while the dicarboxylate ion was more effective than the monocarboxylate ion.
The observed properties of oxalate suggest
there must be a controlling mechanism in vivo if a
synchronised process of lignocellulose degradation is to occur. This would be more important in
white-rot decay, which has ligninolytic capacity,
than with brown-rot decay, which is essentially
cellulosic and hemicellulosic and not ligninolytic.
The enzyme oxalate decarboxylase has been described as an intracellular enzyme in white-rots,
but we have shown that it is also secreted by the
hyphae and capable of being induced oxalic acid
by to produce up to an 100-fold increase in the
extracellular medium. The protein has similar
properties to that of Collybia L~elutipes with respect to molecular mass of 64 kDa, pH optimum
at 3.0, and very acidic p I values [19]. Other
properties of this enzyme are currently under
investigation.
Manganese
The potential for Mn 3+ to be a mobile agent
responsible for initial degradation has also been
proposed. The production of Mn 3+ through the
action of Mn-peroxidases which degrade phenolic
lignins has been well recorded [2]. Manganese has
been shown to accumulate in softwoods decayed
by a wide range of white-rot fungi in structural
studies using scanning electron microscopy with
atomic emission spectrometry and X-ray microanalysis [20]. A system for recycling Mn 2+ to
Mn 3+ would be necessary to continue the
degradative action.
Conclusions
Although many possible low-molecular mass
molecules have been suggested as candidates for
239
a m o b i l e f a c t o r to p e r m e a t e w o o d cell walls a n d
initiate decay, n o n e has b e e n conclusively p r o v e n
as such. H o w e v e r , it is likely that m a n y such
agents are involved to a d d r e s s th e d e g r a d a t i o n of
the c o m p l e x s t r u c t u r e of the w o o d cell wall. T h e
i n t e r a c t i o n b e t w e e n l o w - m o l e c u l a r mass m e d i a tors an d large e n z y m e s will be d e t e r m i n e d by th e
specific e n z y m e m e c h a n i s m s involved, a n d the
availability o f the m e d i a t o r m o l e c u l e s . S o m e of
these, such as veratryl a lc o h o l and oxalate, are
p r o d u c e d as a result o f fungal m e t a b o l i s m and
t he i r s e c r e t i o n has e n a b l e d the fungi to c o l o n i s e
and d e g r a d e t h e w o o d cell wall s t r u c t u r e m o r e
effectively than o t h e r organisms. F u r t h e r study
will ai m at e l u c i d a t i n g this i n t r i g u i n g p r o b l e m .
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10
11
12
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Acknowledgements
W e are g r a t e f u l to A F R C ,
f u n d i n g this work.
8
U K and E C for
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