Proteolytic activities in two wood-decaying

Printed in Great Britain
Microbiology (1995), 141, 1575-1 583
Proteolytic activities in two wood-decaying
basidiomycete fungi, Serpula lacrymans and
Coriolus versicolor
Rekha V. Wadekar,’ Michael J. North’ and Sarah C. Watkinson’
Author for correspondence: Sarah C. Watkinson. Tel: +44 1865 275000. Fax: +44 1865 275074.
e-mail : sarah.watkinson @ plant-sciences.oxford.ac.uk
1
Department of Plant
Sciences, University of
Oxford, South Parks Road,
Oxford OX1 3RB, UK
2
Department of Biological
and Molecular Sciences,
University of Stirling,
Stirling FK9 4LA, UK
Proteolytic enzyme activities of the wood-decaying basidiomycetes Serpula
lacrymans and Coriolus versicolor, have been characterized using azocasein as
substrate and by electrophoretic analysis with gelatin-containing
polyacrylamide gels (gelatin-SDS-PAGE). In S. /acryinans#intracellular and
extracellular azocaseinase activity was optimal at pH 5.6 and was inhibited by
pepstatin A. Gelatin-SDS-PAGE revealed two highly active proteinases, 51 and
54 (apparent M, 65000 and 30000, respectively) and two less active enzymes,
S2 and 53 (apparent M, 47000 and 43000, respectively). S1, the predominant
intracellular proteinase, was present at all ages of the mycelium (tested up
t o 3 months). It is active over a broad pH range, with highest activity
around neutral pH. As 51 was partially inhibited by 1,lO-phenanthroline, the
enzyme was considered t o be a metalloproteinase although EDTA and
phosphoramidon had no effect. A proteinase apparently identical t o
S 1 was also detected in the medium of older cultures. 54 is a pepstatinsensitive aspartic proteinase; its activity was highly pH-dependent and it was
inactive in gelatin gels at pH 50 and above. S2 and 53 were identified as
intracellular metalloproteinases, present in relatively young and growing
cultures. They were distinct from S1 as they were inhibited by EDTA and
phosphoramidon. During starvation-induced autolysis of S. lacrymans,
proteinase 51 was the only enzyme present throughout (and the intracellular
azocaseinase activity increased), which suggested a likely role of 51 in intrahyphal protein mobilization. 54 is more likely to play a part in extracellular
digestion of protein. The azocaseinase activities of cultures of C. versicolor
were optimal at pH 7-0 (intracellular) and pH 5 6 (extracellular). Mycelial
extracts gave one major band of proteinase activity in gelatin gels, C1
(apparent M, 62-64000). Since the activity was sensitive to inhibitors of both
serine and metalloproteinases, there may have been overlapping bands due t o
enzymes of both types. Extracellular samples gave a more complex pattern,
(five bands, C2-C6, M, 50000-100000). C2 and C4 are PMSF-sensitive
proteinases, C5 and C6 are probably metalloproteinases, while C3, which was
most active at pH 4.0, was unaffected by any of the inhibitors tested, including
pepstatin A. No aspartic proteinase equivalent t o 5. lacrymans 54 appeared t o
be produced by C. versicolor. From the information gained about the
intracellular or extracellular location of these enzymes, and the conditions
under which they are active, an in vivo role may be tentatively ascribed to
some of them.
Keywords : Serptlla lacr_ymans,Coriolus versicolor, basidiomycetes, proteolytic activity,
wood-decaying fungi
0001-9646 0 1995 SGM
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1575
R. V. W A D E K A R , M. J. N O R T H a n d S. C. W A T K I N S O N
INTRODUCTION
Wood-decaying basidiomycetes are able to develop very
large colonies with wood, a carbon-rich, but nitrogenpoor material, as their sole source of nutrients. Their
growth presumably requires sensitive control of their
nitrogen economy, involving regulation of proteinase
activity both for the extracellular digestion of the protein
in wood, and for the intracellular turnover and spatial
reallocation of nitrogen from mycelial protein. Secretion
of extracellular proteinase from the hyphae could be
necessary for acquiring nitrogen from wood protein, but
represents a potential waste of a colony's limited nitrogen
resources. Both positive and negative feedback controls
of extracellular proteinase secretion are therefore likely.
Nitrogen derived from wood is accumulated in the
mycelium to levels well above those in surrounding
wood, for example Serpzlla lacymans mycelium utilizing
wood as a sole nutrient source contains 3.7% nitrogen
compared with 0.07 % in the wood substrate (Watkinson
e t al., 1981). Nitrogen is accumulated in the mycelium
as free amino acids and an ethanol-insoluble protein
fraction yielding amino acids on hydrolysis. In 1-week-old
mycelium of S. lacymans grown in a synthetic culture
medium with a nitrogen content comparable to that of
wood, the total free amino acid was equivalent to 74 pM
nitrogen, and the amino acid released by hydrolysis of the
ethanol insoluble component of the mycelium to 2054 pM
nitrogen, per g dried mycelium (Venables & Watkinson,
1989a). The mycelium in established parts of the colony
can thus be regarded as a rich source of nitrogen, most of
which is initially in the form of protein. Intracellular
proteinases which hydrolyse mycelial protein and make it
available for re-use, are likely to play a key role in the
nitrogen economy of the whole colony.
Regulation of proteinases of wood-decay basidiomycetes
is likely to be a part of the physiological processes of
morphogenesis, because of the close relationship between
fungal nutrition and morphogenesis. The composition
and spatial arrangment of the nutrient substrate affects not
only the rate of growth of the colony but also the
differentiation of structures such as mycelial strands. Such
developmental responses probably result from evolutionary optimization of foraging. Fungi such as S. lacymans
grow on discontinuous nutrient resources, the fungal
colony behaving like a foraging individual, with new
growth localized in parts of the colony where new
resources are most abundant (Rayner etal., 1985;Dowson
e t al., 1988). As their colonies grow, different parts of the
same mycelium may be growing into freshly-encountered
wood, or extending out from it supported by translocation
of nutrients from the food base, or senescing and
autolysing. The advance of the mycelium from one food
resource to another has been shown by 15Nlabelling to be
accompanied by a transfer of nitrogen into the new
resource (Watkinson, 1984), a transfer which is expected
because of the high carbon:nitrogen ratio of wood. It
therefore seems likely that intracellular proteolysis to
mobilize protein stores is induced by a requirement for
nitrogen, and that sites of induction and proteolysis could
be at separate points in the colony. Hedlund e t al. (1991)
1576
have shown that insect grazing can induce proteolysis at a
distance in Mortierella isabellina colonies.
Very little is known about the proteolytic enzymes of
timber-decaying fungi. Before their role and regulation
can be understood, it is necessary to establish the
characteristic enzymes. In this study, two species have
been used. S. lacymans, the wood dry rot fungus, was
chosen for its economic significance and because it
develops mycelial strands for exploitation of its resources.
Corioltls versicalar was chosen as a timber-decaying basidiomycete with a very different physiology; it is a white rot
and does not normally form strands.
METHODS
Organisms and culture conditions. The isolates of S.lacymans
(Wulf. Fr.) Schroet (culture no. 12C) and C. versicolor (L. ex
Fr) (culture no. 28A) were supplied by the Forest Products
laboratory of the Building Research Establishment, UK. They
were subcultured and maintained on 2 YOmalt agar [2 % (w/v)
malt extract and 1-5YO(w/v) Oxoid no. 3 agar].
Static liquid cultures were grown on peptone medium (20 ml),
containing 1 YO (w/v) bactopeptone, 0.3 YOyeast extract and
0.2% D-glucose in 50 ml conical flasks, with each flask
inoculated with a 10 mm disc cut submarginally from a 5-10-dold fungal colony growing on 2 YO(w/v) malt agar. The flasks
were incubated at 22 "C and harvested at various ages as
indicated.
For autolysis experiments, cultures were grown initially on
peptone medium. Two-week-old healthy cultures were then
transferred on day 15 to salts only 'starvation' medium (gl-':
KH,PO,, 1; MgSO,. 7H,O, 0.5; FeSO,. 7H,O, 0.01 ; KC1, 1).
Cultures were harvested at the time of transfer to 'starvation'
medium and periodically after transfer. Parallel sets of cultures
were used for preparing samples for azocaseinase assays and
gelatin-SDS-PAGE analysis.
Azocasein hydrolysis. Samples used to measure proteinase
activity with azocasein as substrate were prepared as follows.
After washing with deionized water, all the mycelium from a
single flask was ground in a mortar with purified sand (1 g) and
5 ml extraction buffer. The extraction buffer was 0.2 M acetate,
pH 5.6, except when the pH dependence was to be determined
when the concentration of the buffer was 0.05 M. The resultant
homogenate was spun for 10 min at 1990g in an Econospin
bench top centrifuge and the supernatant was used to analyse
the intracellular activity. Extracellular samples were collected
by separating culture fluid from mycelium by filtration. Freshly
prepared samples were used directly for azocaseinase assays.
Occasionally the extracellular samples were stored in a refrigerator at 4 "C, never for longer than 24 h, but storage had no
effect on proteinase activity.
The assay procedure was based on the method originally
described by Prestidge e t al. (1971), and differed only in respect
of the azocasein concentration used (0.5 YOinstead of 2 YO,
w/v). Azocasein hydrolysis was measured at 25 "C in reaction
mixture containing 0.1 ml buffer,0.1 ml deionized water, 0.3 ml
intracellular sample or 0.05 ml extracellular sample, 0-5 ml
0.5% azocasein (Sigma) and water to a total volume of 1 ml.
The buffers used were : 0-5 M citrate/phosphate (pH 4*0),0.2 M
sodium acetate/acetic acid (pH 4-0-5.6) and 0.15 M sodium/
potassium phosphate (pH 5-6-7.0). The reaction was stopped by
adding 2 ml of 7 Yo (v/v) perchloric acid. The tubes were spun
at 1990g for 10 min and then 2.6 ml of supernatant was mixed
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Proteolytic activities of wood-decaying fungi
with 0.4 ml 10 M NaOH. In controls, the perchloric acid was
added immediately before the azocasein. Activity was measured
One A unit was equivalent to the hydrolysis
as change in A436.
of 1.33 mg azocasein. Activity was expressed as specific activity
[mg azocasein hydrolysed h-' (unit dry weight fungus)-'] or
as total activity (mg azocasein hydrolysed per h per flask,
irrespective of mycelial weight).
0-07
Gelatin-SDSPAGE. Samples were prepared as follows. The
protein in mycelial extracts or culture filtrates obtained as
described above was concentrated by precipitation with ammonium sulphate. The sample was taken to 80% saturation by
addition of solid ammonium sulphate and then centrifuged at
1990g for 30 min. Supernatant fluid was discarded and the
pellet was resuspended in extraction buffer. The ammonium
sulphate was removed by Amicon mini-concentrators
(Centricon 3 or 10 ultrafilter units, cut off 3000 or 10000,
respectively), by spinning for approximately 30 min at
5000 r.p.m. The exact time varied and depended on the starting
volume of the samples.
PH
Electrophoresis was carried out as described by Lockwood e t al.
(1987). Samples were mixed with an equal volume of electrophoresis buffer containing 0.0625 M Tris/HCl (pH 6-8), 2 YO
(w/v) SDS, 5 YO(w/v) 2-mercaptoethanol, 20 YO(v/v) glycerol
and 0.002 YObromophenol blue. They were then electrophoresed
in 10 Yo (w/v) polyacrylamide gels containing 0.2 'YO gelatin
using the SDS-discontinuous buffer system described by Hames
(1981). After loading the samples (10-20 ml, approximately
12-25 mg protein) onto the stacking gel, they were electrophoresed at a constant current of 15 mA for about 45 min per
gel. After electrophoresis the gels were treated with 2.5 % (v/v)
Triton X-100 for 30 min. The proteinase bands were developed
by incubating gels overnight in an appropriate incubation buffer
in a shaking incubator at 25 "C. The gels were then stained with
either 0.1 YOCoomassie Blue in 40 YO(v/v) methanol and 10 YO
(v/v) acetic acid or 0.1 YOamido black in 7 % (v/v) acetic acid.
They were destained with acetic acid. The apparent M, of the
proteinases were estimated from their mobility in relation to
that of standard molecular marker proteins from Sigma.
-
RESULTS
T w o methods were selected for analysing the proteinase
activities of S.lacymans a n d C. versicolor. Azocasein was
used as a substrate t o measure relative proteinase activity
0
4
3.0
Protein determination. Protein concentration was determined
by the method of Bradford (1976), using BSA as a standard.
Determination of inhibitor sensitivities. The effect of
inhibitors on azocaseinase activity was determined by incubating samples with inhibitor at room temperature for 1 h prior
to assay. Control samples containing an equivalent volume of
water or organic solvent were pre-incubated identically. For
gelatin-SDS-PAGE analysis, samples were incubated with
inhibitors, water, or organic solvent as above before loading.
After electrophoresis the individual lanes were cut out into
strips, treated with Triton X-100 for 30 min and incubated
overnight ( 15-18 h) in the appropriate incubation buffer
containing the same inhibitor or solvent. Stock solutions of
EDTA, leupeptin, iodoacetic acid and phosphoramidon were
prepared in water, pepstatin and PMSF were dissolved in
ethanol, and 1,lo-phenanthroline solutions were prepared in
either DMSO or methanol. Final concentrations of inhibitors
were chosen as recommended in the literature (North, 1989;
Salvesen & Nagase, 1989).
All inhibitors were obtained from Sigma except for phosphoramidon which was supplied by Scientific Marketing Associates.
~
4.0
5-0
6.0
7.0
8.0
PH
...............................................................................................................,.,..................................,,....
Fig. 1. pH-dependence of azocaseinase activity of intracellular
(closed symbols) and extracellular (open symbols) samples from
(a) cultures of 3-week-old 5. lacrymans and (b) 2-week-old C.
versicolor. Buffers: 0.5 M citratelphosphate
0.2 M acetate
(H,
0.15 M phosphate (V,V). Each point is the mean of
four t o six samples from three replicate cultures assayed by the
sampling out method.
a),
i n unfractionated samples, while gelatin-SDS-PAGE was
used in a n attempt to identify t h e individual enzymes
which contribute to the overall activity.
Azocaseinase activity in S. lacrymans
Azocaseinase activity could be detected in samples derived
from mycelia a n d from culture medium (Fig. la). In both,
activity was optimal a t pH 5.6 (Fig. l a ) which confirmed
earlier findings for extracellular activity (Venables, 1987).
A s azocasein tends t o precipitate at l o w pH it was n o t
possible to use it for comparable assays below pH 4.
Both intracellular a n d extracellular sample activity,
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1577
R. V. W A D E K A R , M. J. N O R T H a n d S. C. W A T K I N S O N
Table 7. Effect of inhibitors and assay pH o n azocaseinase activity in 3-week-old
S. lacrymans
The pH values for inhibition assays were selected so as to optimize the activities of those enzymes
previously found to be susceptible to the inhibitor being assayed. The values given are means f s of~
4 to 6 readings of samples from two or more replicate cultures.
____
Inhibitor
Concn
(mM)
Pepstatin A
PMSF
EDTA
10-Phenanthroline
Iodoacetic acid
Pepstatin A plus
1,lo-phenanthroline
0.036
0.036
1
1
5
5
10
t
Intracellular
Assay
Extracellular
Inhibition
P*
Inhibition
(Yo)
87( f1.4)
23( f11.8)
1
1
0
8*0(& 5.1)’
0
100
11(f8.7)
5.6
7.0
6.2
6.2
6.2
7.0
6.2
5.6
7.0
86( f0.7)
45( f11.9)
5
0
25( f1.6)
15( f8*9)*
0
96( f 1.1)
24( & 23.8)
2
3
PH
(W
5-6
7.0
5.6
5.6
5.6
7.0
5.6
5.6
7.0
Assay
* Activity enhanced.
t0.036 mM Pepstatin A and 5 mM phenanthroline.
(a)
1
(b)
2
kDa
65 47 43 -
1
4
5
6
7
8
9
10
kDa
- 51
- s2
- 53
65 -
- 51
30 -
- 54
pH 4.0
pH 5.0
pH 5.6
pH 6.2
pH 7.0
Fig. 2. Band patterns in gelatin-containing gels following electrophoretic separation o f proteinases from cultures o f 5.
lacrymans. (a) Bands produced when samples were taken from a 2-week-old culture. Lanes: 1, intracellular sample; 2,
extracellular sample (for details of sample preparation see Methods). Gels were incubated a t pH 6.2. (b) Bands from
intracellular samples (lanes 1, 3, 5, 7 and 9) and extracellular samples (lanes 2, 4, 6, 8 and 10) incubated a t a range of pHs
after separation. Cultures were 12 weeks old. M, values were estimated by comparison with standards (Sigma kits
MW-SDS-70L and MW-SDS-200) run in parallel (not shown).
measured at pH 5.6, were reduced by more than 85% by
pepstatin A, a specific inhibitor of aspartic proteinases
(Table 1). For the extracellular activity, measured at
pH 6.2, 1,lO-phenanthroline caused a reduction of
approximately 25 %, suggesting a contribution from
metalloproteinases. At pH 7.0, 1,lo-phenanthroline
enhanced activity, and pepstatin had less effect than at
pH 5-6, for both intracellular and extracellular samples.
None of the other inhibitors tested had a significant effect.
1578
Gelatin-SDSPAGE of S. lacrymans proteinases
Analysis of mycelial extracts and culture filtrates showed
a relatively simple proteinase pattern for both (Fig. 2a).
Samples were examined from cultures of different ages
from 10 d to 3 months old. In 10-d-old cultures, three to
four faint intracellular proteinase bands were detected,
with hardly any activity being found in extracellular
samples (data not shown). Analysis of older cultures
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Proteolytic activities of wood-decaying fungi
Table 2. Effect of inhibitors on azocaseinase activity in 2-week-old C. versicolor
...........................................................................................................................................................................................................
........................................
See legend to Table 1 for details.
Inhibitor
Pepstatin A
PMSF
EDTA
1,lo-Phenanthroline
Iodoacetic acid
PMSF plus 1,lOphenanthrolinet
Concn
(mM)
Intracellular
Assay
0.072
2
5
5
10
Inhibition
PH
(%I
5-6
7.0
7-0
7-0
7-0
7-0
0
27( f1.4)
56( f1.87)
62( f0.53)
0
78( & 2.23)
Extracellular*
Inhibition (%)
0
14( f1.8)
66( f0.67)
74( f066)
0
74( f2.53
* All extracellular assays were at pH 5.6.
t 2 mM PMSF and 5 mM 1,lO-phenanthroline.
(2-10 weeks old) revealed a proteinase S1, with an
apparent M, of 65 000, in intracellular samples of all ages,
and an apparently identical enzyme in the medium. The
extracellular form was at higher levels at later stages and
was presumably released into the medium as the culture
aged. Proteinase S1 was most active around neutral p H
and was affected by only one of the proteinase inhibitors
tested, 1,lo-phenanthroline, by which it was partially
inhibited (Table 3). Some loss of S1 activity was observed
with ethanol and methanol when used in controls. An
attempt to concentrate samples by acetone precipitation
resulted in inactivation. Two other intracellular
proteinases, S2 and S3 (apparent Mr 47000 and 43000,
respectively), were detected in younger cultures up to
4 weeks old (Fig. 2a) but were absent from mycelium of
older cultures. Their activity was totally inhibited by all
three of the metalloproteinase inhibitors tested, namely
1,lO-phenanthroline and, in contrast to S1, both EDTA
and phosphoramidon.
The other major proteinase of 5’.lacymans was S4 which
had a lower apparent M, (about 30000). It was found
predominantly in the medium and was not detected in the
youngest cultures (10 d old), although it was detectable
from 2 weeks onwards. Within the pH range tested, it was
most active at pH 4.0 and was not detected on gels
incubated at pH 5.0 and above (Fig. 2b). This proteinase
was almost completely inhibited by pepstatin A (Table 3),
identifying it as an aspartic proteinase. Proteinase S4
appeared to be modified, presumably by limited proteolysis, during storage at -20 OC, as additional bands
were apparent after 4 weeks’ storage, but since the new
bands were detectable on substrate-containing gels, these
altered forms had clearly retained activity (data not
shown).
A comparison of the results of gelatin-SDS-PAGE
analysis with those of azocaseinase assays suggested that
proteinases S1, S2 and S3 could have been responsible for
only a small proportion of the azocaseinase activity at
pH 5.6. These intracellular metalloproteinases were
inhibited by 1,lo-phenanthroline but this inhibitor did
not significantly affect the intracellular azocaseinase
activity. The results are, however, consistent with proteinase S4 being responsible for most of the extracellular
azocaseinase activity at low pH since both were inhibited
by pepstatin. At pH 7.0 pepstatin inhibits less than half
the azocaseinase activity in both intracellular and extracellular samples, so S4 presumably contributes less to
overall activity at neutral pH. Phenanthroline enhanced
azocaseinase activity at pH 7.0 rather than inhibiting it,
and reduced the inhibition caused by pepstatin when the
two inhibitors were used together (Table 1). The reason
for this enhancement of azocaseinase activity at neutral
pH is not clear.
Proteinase S4 was not found in the gelatin-SDS-PAGE
analysis of intracellular samples, and no pepstatin-sensitive proteinase was present to account for the pepstatinsensitive azocaseinase activity. A possible explanation for
this was that the intracellular samples contained a different
aspartic proteinase with very little activity towards
gelatin. However, analysis of samples by SDS-PAGE
using gels in which azocasein was substituted for gelatin
failed to reveal any additional proteinases which could
have been responsible for the intracellular azocaseinase. A
second possibility considered was that an intracellular
proteinase may have been inactivated during electrophoresis. As extracellular S4 was not inactivated under
these conditions this would have required the presence
of either a different, less stable enzyme, or an
inactivating factor(s) in the mycelium. The latter possibility was tested as follows. Concentrated samples of
mycelial extract, medium and a mixture of the two were
compared by gelatin-SDS-PAGE. A significant amount
of S4 activity was detected in both extracellular and mixed
samples, but not the intracellular sample (data not shown).
Thus, if an inhibitor or an inactivating factor had been
present it could have been there only in limited amounts.
All mixtures of intracellular and extracellular samples
gave the expected levels of azocaseinase activity indicating
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1579
R. V. W A D E K A R , M. J. N O R T H a n d S. C. W A T K I N S O N
1
kDa
100 -
c150
-
2
3
4
- c2
- c3
-c4
- c5
- C6
10
Fig. 3. Patterns o f bands in gelatin-containing gels following
electrophoretic separation o f proteinases from cultures o f C.
versicolor. lntracellular (lanes 1 and 3) and extracellular (lanes 2
and 4) samples were incubated a t pH 4.0 (1 and 2) and pH 6-2 (3
and 4). Mrvalues were estimated by reference t o markers as in
Fig. 2.
that cell extracts contained neither an inhibitor nor an
activator of azocaseinase activity. The nature of the
enzyme responsible for the pepstatin-sensitive azocaseinase of S. lacrymans mycelium therefore remains to be
elucidated.
Effect of starvation-induced autolysis on the
proteinase activities of S. lacrymans
In fungi such as S. lacrymans, the breakdown of intracellular protein during starvation-induced autolysis may
be of major importance for the relocation of nitrogen
sources within the colony. T o examine the possible
involvement in this process of the proteinases revealed
here, cultures of S. lacvmans were starved by transferring
them to salts-only medium, and proteinase activity was
measured using azocasein and gelatin-SDS-PAGE.
30
50
Incubation time (d)
70
culture medium before transfer
mx
6-
EP
:z;
4-
vEa LJ
g2
- 2
Rise in activity in fresh medium
immediatelyafter transfer
10
30
50
1
..........................................................................................................................................................
Fig. 4. Azocaseinase activity in 5. lacrymans after transfer t o
'starvation' medium. Cultures were incubated for 2 weeks o n
peptone medium before transfer and harvested periodically
(48 h and 2, 4 and 7 weeks) after transfer. (a) lntracellular total
(V)and specific (m) activity a t pH 6.2; (b) extracellular total
activity a t pH 4.0. Each point represents the mean o f six samples
from t w o replicate cultures.
When cultures were starved, the intracellular azocaseinase
specific activity and total activity, measured at p H 6.2,
showed a sharp increase during the 48 h after transfer
(Fig. 4a). This increase accompanied the reduction in
biomass and in total mycelial protein due to autolysis (Fig.
5). During subsequent incubation the total intracellular
azocaseinase activity decreased. Extracellular total activity, measured at pH 4-0, accumulated significantly by
the second week after transfer (Fig. 4b). Proteolytic
activity was first detected in the medium 2 h after transfer.
Of the individual proteinases detected by gelatin-SDSPAGE, the activity of the EDTA-sensitive proteinases S2
and S3 progressively decreased in activity after transfer,
and were almost absent by week four (Fig. 6). The other
metalloproteinase, S1, was still active within mycelium at
weeks two and four, and also gradually appeared in the
medium. Pepstatin-sensitive extracellular proteinase S4
was detected as a faint band on gels incubated at pH 4.0
with samples harvested at 2 and 4 weeks after transfer. Its
activity was considerably less than that found in extracellular samples taken prior to transfer (data not shown).
During starvation the pH of the culture medium changed.
In the first 48 h after transfer, the pH dropped from 4.5 to
1580
0
Incubation time (d)
600
n
400
m
.-C
:
aJ
CI
m
.200
20
40
Incubation time (d)
60
f8
.,
..........................................................................................................................................................
Fig. 5. Changes in dry weight and total protein content o f the
fungal mycelium per flask during starvation o f 5. lacrymans.
Dry weight, mean reading o f three replicate cultures; 0 ,
mycelial protein, based on the samples prepared from t w o
replicate cultures for PAGE analysis.
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Proteolytic activities of wood-decaying fungi
Table 3. Effect of inhibitors on 5. lacrymans and C. versicolor proteinases detected by
gelatin-PAGE
....................................... .
.................
Inhibition of bands was detected at optimal pH conditions by incubating all gel portions at pH 4 0 and
6.2, chosen on the basis of the different observed enzymic pH optima in SDS-PAGE.
.........................................................................................................
I
.....I......
.....................
Inhibition of bands*
Inhibitor
Concn
(mM)
Pepstatin A
0.036
PMSF
1
Leupeptin
2
EDTA
1
1,lO5
Phenanthroline
Phosphoramidon 0.01
10
Iodoacetic acid
S. Zacrymans
Intracellular
Extracellular
NE
S4JJ
NE
NE
NE
NE
S2JL s3.14
SlJ, S2JJ,
S34J
s244, s344
NE
SI(extra)J
NE
NE
NE
Concn
(mM)
0.072
2
2
5
5
ND
10
C.versicoZor
Intracellular
NE
c14
Extracellular
NE
( 3 . 1s4.1J.
NE
NE
c1
c1
C5$, C6$J
C5$, C644
ND
ND
NE
NE
Not determined.
* Effects observed: 4,partial inhibition; $4,complete inhibition; NE, no effect.
ND,
3.9, but subsequently increased again (Table 4). Despite
the reduced pH, the enzyme that would be most active
under these conditions, the aspartic proteinase S4, was
only detected as a faint band.
Because no aspartic proteinases could be detected in
intracellular samples using gelatin-SDS-PAGE, the contribution of these enzymes to the increased activity during
autolysis was assessed by measuring the extent to which
pepstatin inhibited the azocaseinase activity present before
and 48 h after transfer of mycelia to starvation medium.
Data obtained from at least six samples from two
replicate cultures showed that while total azocaseinase
activity (measured at pH 5.6) rose from 1-45kO.1 to
2-05kO-035mg azocasein hydrolysed per h per flask, the
amount of activity inhibited by 0.044 mM pepstatin did
not change significantly (0.885 & 0,025 mg azocasein
hydrolysed per h per flask before transfer and
0-925& 0.085 mg azocasein hydrolysed per h per flask 48 h
after transfer). Thus the increase in intracellular activity
was not due to aspartic proteinases. However, most of the
extracellular activity recovered 48 h after transfer was
pepstatin-sensitive.
The results suggest that it was likely to be proteinase S1
which was mainly responsible for the increase in intracellular azocaseinase activity, while the activity released
immediately into the medium was due to the pepstatinsensitive aspartic proteinase, S4. The release of the latter
probably resulted from autolysis rather than any increased
production of enzyme.
Azocaseinase activity in C. versicolor
The intracellular activity of C. versicolor azocaseinase was
optimal at neutral pH while the extracellular activity was
highest at pH 5.6 (Fig. lb). These latter results can be
compared with those reported by Staszczak & Nowak
Table 4. Changes in pH of culture filtrates recovered a t
each harvest during autolysis of 5. lacrymans
Values are based on the readings obtained from two replicate
cultures.
Culture mediumlharvest
PH
Original peptone medium
Peptone medium after 2 weeks’
mycelial growth
Original ‘starvation ’ medium
48 h*
2 weeks*
4 weeks*
7 weeks*
6.65
3-23
4-48
3.88
4.42
5.56
5.30
* Period after transfer to starvation medium.
(1984) who determined two p H optima of 7.0 and 5.0-5-4,
respectively, for both intracellular and extracellular activities of C. versicolor (strain ATCC 44308) using haemoglobin as substrate. The azocaseinase activity in C.
versicolor was inactivated by a different set of inhibitors
from those effective in 5’. lacrymans samples, suggesting
that other classes of proteinases were involved (Table 2).
The intracellular activity at p H 6-2 was reduced by 27 %O
by PMSF suggesting that a serine proteinase contributed.
As activity was inhibited by more than 50% by EDTA
and 1,lO-phenanthroline, a metalloproteinase was also
present. Similar inhibitory effects were observed on the
extracellular activity. A combination of PMSF and 1,lOphenanthroline gave slightly greater inhibition than that
with 1,lo-phenanthroline alone. This was not apparent
with the extracellular activity.
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1581
R. V. W A D E K A R , M. J. N O R T H a n d S. C. W A T K I N S O N
1,lo-phenanthroline suggesting contributions from both
serine and metalloproteinases (Table 2). Proteinase C3
was not affected by any of the inhibitors tested, including
pepstatin. Proteinase C2 was completely inhibited by
PMSF suggesting that it was a serine proteinase, although
one that was not affected by leupeptin. The effects of
inhibitors on the other proteinases were more difficult to
discern. C4 appeared similar to C2 in being sensitive to
PMSF, while proteinases C5 and C6 were most affected by
1,lo-phenanthroline, although E D T A was much less
inhibitory. A comparison with the data for C. versicolor
azocaseinase activity suggests that the latter could be
accounted for largely by the proteinases revealed by
gelatin-SDS-PAGE. The intracellular C1 band was
affected by the same agents as those which inhibited the
intracellular azocaseinase activity, namely PMSF, EDTA
and 1,lo-phenanthroline. Metalloproteinases were the
major contributors to the extracellular azocaseinase activity, and so proteinases C5 and C6 were most likely
involved.
2
51 s2 53 -
2
- 51 (extra)
DISCUSSION
Fig. 6. Proteinase patterns o f S. lacrymans during starvation.
Samples were obtained from the cultures harvested at (a)
2 weeks and (b) 4weeks after transfer o n t o 'starvation'
medium. Lanes: 1, Intracellular; 2, extracellular. Gels were
incubated a t pH 6.2. The pattern for the culture a t the time of
transfer is shown in Fig. 2(a). Samples loaded: (a) lane 1, 8 p g
protein (equivalent to 160 pl); lane 2, equivalent to 530 PI; (b)
lane 1, 6.2 pg protein (equivalent t o 320 PI);lane 2, equivalent
t o 400 pI. Fig. 2(a): lane 1, 12 pg protein (equivalent t o 80 pl);
lane 2, equivalent t o 160 pI.
Gelatin-SDSPAGE of C. versicolor proteinases
Gelatin-SDS-PAGE analysis of C. versicolor revealed a
more complex proteolytic system than that of S. lacrymans
(Fig. 3). In intracellular samples derived from 10 to 15-dold cultures there was a single proteinase band, C1,
corresponding to an apparent M,of 62-64000. Its activity
was detected over a wide range of pH but was highest at
pH 6.2. The band was always diffuse even when different
volumes and concentrations of sample were loaded, and it
seems likely that more than one proteinase was responsible. Five bands in the M , range 50-100000 were
detectable in extracellular samples. Of these, proteinase
C3 represented the most active enzyme. Its activity was
highest at pH 4.0, declined with increasing pH and was
difficult to detect at pH 7.0. Three of the other proteinases
(C2, C4 and C5) were most active around neutral pH but
gave less defined bands. Proteinase C6 could be detected
over the whole pH range tested (pH 4-0-7.0). A proteinase
with similar mobility to C6 was sometimes apparent in
intracellular samples. None of the extracellular proteinases
exactly corresponded to intracellular proteinase C1. The
activity of the C1 band was reduced by PMSF, EDTA and
1582
At present very little is known about the proteinases of
wood-decay fungi. This analysis has shown that the two
species studied here each produce a number of different
proteinases typical of fungi (North, 1982), although the
two species do not have similar enzymes. Four
proteinases, S1, S2, S3 and S4, were the main proteolytic
enzymes of 5.lacrymans. In many ways these were typical
of fungal proteinases, being aspartic (S4) and metalloproteinases (Sl, S2 and S3). Proteinase S1, a metalloproteinase, is intracellular and appeared to be a 'leaking
out' enzyme of the type described by Schanel e t al. (1971),
where an intracellular enzyme is secreted into culture
medium without any change to its molecular properties.
S2 and S3 are apparently strictly intracellular enzymes and
S4 is an extracellular aspartic proteinase. The pepstatinsensitive intracellular activity has not yet been matched to
an enzyme detectable by gelatin-SDS-PAGE, and there is
a probability that a second intracellular aspartic proteinase
is present.
The proteinase levels of S. lacrJmans changed with the
physiological state of the culture. The intracellular
proteinase S1, present in the samples of all ages of the
culture, increased under starved conditions and gradually
accumulated in the medium. Transfer of mycelium to
starvation conditions resulted in the immediate release of
a small amount of proteinase activity, mainly S4, but
release was not sustained, and is more likely to have been
due to mechanical breakage of cells than to specific
induction.
The data suggest possible physiological functions for the
enzymes. S4 could be a digestive enzyme, solubilizing
wood protein under acid conditions during growth. The
hyphae of S.lacrymans grow actively on medium of high
carbon:nitrogen ratio where pH as low as 2.8 can
develop in culture, when the fall in pH during growth is
proportional to the carbon: nitrogen ratio (Watkinson,
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Proteolytic activities of wood-decaying fungi
1775), being usually attributed to the secretion of oxalic
acid (Goksoyr, 1957). Wood itself has a high carbon:
nitrogen ratio, typically approaching 500 : 1. The metalloproteinase S1 could be active in protein turnover, as it is
intracellular and remained active during starvation.
The proteinases seen in C. versicolor were significantly
different, suggesting that the organization of proteolysis
in vivo is different in these two fungi. In C. versicolor only
one intracellular proteinase band (Cl) was found, which
appeared to be a mixture of serine and metalloproteinases.
This band was sensitive to PMSF, and the intracellular
azocaseinase activity from C. versicolor had a broad pH
optimum [features also found in the intracellular proteinase from Agariczls bisporzls stipes (Burton e t al., 1973)].
A complex of extracellular enzymes was found including
serine proteinase, metalloproteinase, and a pepstatininsensitive acid proteinase. In the analysis of the proteinase of C. versicolor, Staszczak & Novak (1784)
found seven distinct intracellular proteinase bands compared with the single one detected here. However, direct
comparisons cannot be made because the culture conditions were different and the proteinases were analysed
using different substrate and electrophoresis conditions.
The extracellular bands found by Staszczak & Novak
(1784) did resemble those described here, and were
possibly equivalent.
The location of extracellular proteinase released from
colonies on solid agar medium was previously found
to differ in S. lacrymans and C. versicolor (Venables &
Watkinson, 1787b). In C. versicolor, extracellular proteolysis was found mainly in the region of growing hyphal
tips, with much less in agar medium under older parts of
the colony. With S. lacymans more activity was found
beneath the central older part of the colony. In nature the
two fungi differ both in physiology and morphology. S.
lacr_ymans causes brown rot, degrading cellulose but not
completely degrading lignin, while C. versicolor decomposes cellulose and lignin, causing white rot. S. lacrymans
is able to grow long distances over non-nutrient surfaces,
supported by nutrients transported through the hyphae
and mycelial cords from a colonized food source. C.
versicolor does not show this behaviour. The results of the
analysis of the proteolytic enzymes support the view that
intracellular protein mobilization plays a more important
role in S. lacrymans than in C. versicolor.
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Received 24 November 1994; revised 14 February 1995; accepted
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1583