FEMS MicrobiologyLetters66 (1990)15-22
Published by Elsemer
15
FEMSLE 03810
Effect of aromatic monomers on production of
carbohydrate-degrading enzymes by white-rot and brown-rot fungi
T e r r y L. H i g h l e y a n d
Jessie A. Mtc a le s
U S Department of Agriculture. Forest Service Forest Products Laboratory t One Glfford P|nchot Drwe Madison,
WI, U S A 53705-2398
RecetveA18 July 198Q
Rewslon receivedand accepted 16 August 1989
Key words: Brown-rot; Whate-rot; Cellulose, Xylanase; Enzyme inhibition; Wood decay; Phenohes
1 SUMMARY
The effects were studied of 12 monomerlc
aromatic compounds on the production of six
carbohydrate-degradmg enzymes of two brown-rot
fungi. Postta placenta and Gloeophyllum trabeum.
and one white-rot fungus, Cortolus verstcolor Most
compoundswere inhibitory to growth of the decay
fungi at concentrations of 0 05c~. At lower concenlrattons, the phenohcs often stimulated growth
Relatively lugh concentranons ( > 0 1%) of aromatic monomers were required to inhibit xylanase
of P. placenta m sltu The effects of the monomers
incorporated m hqmd medm on enzyme produc-
Correspondence to T L Highley, U S Department of AsHculture, Forest Service,Forest Products Laboratory. I Gffford
Pmcbot Drive, Madison, WI 53705.2398.U S A
1 The Forest Products Laboratory is maintained in cooperation with the University of Wisconsin This article was
wraten and prepared by U S Government employees on
officialtime, and iI is therefore in Ihe pubhc domain and not
subject to copyright,
The use of trade names In Ihls artncl¢does not imply endorsement by the U S Department of Agricultureof any product or
service
t*on varied depending on compound and funsal
species. Some compounds were quite inhibitory to
production of enzyme activities at very low concentratlons. For example, eateehol and vamlhn (50
ppm) caused 100% inhibition of xylanase and
fl-l,4-endoglueanase production by P placenta
However, no aromatic monomer was strongly inhibitory to production of all enzyme activities of
all fungL
2 INTRODUCTION
Decay fungi are exposed to aromatic menumerle degradation products or extracttves during
degradation of wood. The low molecular weight
aromatic hgmn breakdown products include compounds such as vamlhn, vanJlhc acid. gualacol.
veratrlc acid, p-hydroxybenzotc acid. and synngte
a o d [1] These compounds may sumulate or Inhibit the growth of mlero-orgamsms [2.31. Aromatic compounds may also inhibit enzyme activity
[4-61. Highley [71 found that cellulases from wood
decay fungi generally were not i,',hlbtted in SltU by
phenols but that several were inhibited upon
oxidation The effect of aromatic compounds on
0378-1097/89/$0350 1989Federaaon of European MicrobzologlcalSocleues
16
productmn of degradatlve enzymes by wood decay
fungi is virtually unknown. In thts study, we examined the effects of aromattc monomers m culture medta on the productmn of glycosJdases,
xylanase, and cellulase (fl-l,4 endoglucanase) of
brown-rot and white-rot fungi. Compounds that
mhibtt productton of these enzymes may point to
new methods of controlhng decay.
3. MATERIALS A N D M E T H O D S
3 1 Fnngt
Two brown-rot fnugl, Posua placema (Ft.) M.
Lars. et Lomb. (MAD-698) and Gloeophyllum
trabeum (Pers.Fr.) Murr. (MAD-617), and one
white-rot fungus, Corwlus verswolor (L. Fr ) Quel.
(MAD-697), were used in most experiments.
Phanerochaete chrysospormm Burds. (ME-461) was
included in growth studies.
The strains were stored at 4 ° C on two malt
agar slants.
3 1 1 Cultwatlon conditions: For determlmng
the effect of the aromatic monomers on enzyme
production, the fungi were cultivated on a previously described basal medmm [8] with the addition of 1.0% mterocrystalhne cellulose (Avteel)
and 0 1% cdlobmsc as carbon sources. For growth
studies, the fungi were grown on the basal medium
wRh 1% eellobms¢ as carbon source. Twenty-five
mllhhters of medium were added to 250-ml
Erlenmeyer flasks, and the flasks were stenhzed at
121°C for 20 nun. After cooling, falter-sterilized
coumenc actd, 2,6-dlmethoxyphenol, catechol, feruhc acid, galhc acid, guamcol, protocatachmc acid,
pyrogailol, tannic acid, vandlm, veratrol, or pentachlorophenol was added to the medium. The
flasks were inoculated wtth agar plugs and incubated m the dark for 3 weeks in stauonary
culture at 2 7 ° C and 70% relattve humtdlty. The
culture were harvested by fdtratton on 25-mm-d~ameter glass-fibcred paper (Whatman). The filters
were washed with dtstdled water and dried at
5 0 ° C for blomass determmatton. The filtrate ohtamed was used to determine enzyme activities.
All expenments were repeated at least twice with
three rephcat~ons
3 1 2. Enzyme actlvr',te~ The activity of endo-fl1.4-xylanase and endo-fl-lA-glucanase (eellulasc)
was detcnmned by spectroscopic measurement of
the increase in reducing groups at 40 o C after 24 h
[9]. The substrate for xylanase was larchwood
xylan (Sigma), and the substrate for cellulase (.81,4-endogluanase) was carboxymethylcellulose
(Hercules). The activity of a-O-galactosldase, ,8D-galactosldase, fl-D-xylosidas¢, and ,8-D-glucostdase were deternuncd by spectroscopic measuremerit at 425 n m of the hberatlon of a-mtrophenol
from the respectwe p-mtrophenyl derlvatlzed substrafes [10].
3 2. StabJhty of xylanase to phenols
Crude culture filtrate from P. placenta grown
in 1% eelloblose and basal salts for 3 weeks was
dmlyzed for 48 h m 0 ! M citrate buffer, pH 5.0,
to remove egcess reducing sugars. This crude enzyme mixture was then added to dilutions of
catechoL vamlhn, and tanntc acid for final pbenohc concentrations selected from among the followrag: 0.01%, 0.05%, 0.10%, 0.50%, 1.0%, and 10.0%.
The higher concentrations of tanmc acid and
catechol could no;. be used, because they interfered with the reducing-sugar assay. Stock solutions of phenohcs were made in absolute ethanol
and adjusted to pH 5.0 with 0.01 M HCI or
NaOH. The enzyme-phenol mtxtures were incubated in the dark at 2 5 ° C for 2 h. The increase
in reducing sugars was then determined after 24 h
at 4 0 ° C
4. RESULTS A N D DISCUSSION
In vitro, cellulase from wood decay fungt has
been found to be quite stable to mcubatton with
pentachlorophenol (penta). Sharp [11] found that
1% penta did not affect cellulase and amylase
activities of several fungi, including wood decay
fungi. Highley [7] did not fred stgmflcant inhibition of cellulase from several wood decay fungi
below a concentration of 1.5%. In the present
study (Table 1), pentachlorophenol at a very low
concentration (5 ppm) m culture medmm was
extremely mhlbaory to production of carbohy-
17
Table 1
Effect of pentachlorophcnol a on enzymeproduction by wood
decay fungi
Enzyme
/I.glucostdase
h-galactomdase
~-xylomdase
,O.galacto~dase
Xylanase
C©llulase
Enzymeacuwty (percentageof control)
P placenta G trabeurn
C verswolor
50
5
50
5 50 5
0
24 ltlO lO0 0
143
0
26
65
74 0
109
0
8
57
66 0
1130
0
16
72 1(.10 4
30
20 100
82
73 0
153
25
25
70 100 0
100
Pentachlorophenol concentratmns of 50 and 5 ppm
drate-degradmg enzymes from P. placenta and C
verslcolor but not G. trabeu:n. '.~hu~., ~cnl~chlorophenol does not act by inhibiting these particular
enzymes in sttu, but must affect their release in au
active form into culture m~dlum.
The effect of the aromatic compounds on
growth of the white- and brown-rot fungi with
cellobtose as the carbon source is presented in
Table 2. At a concentration of 0.05%, most compounds were inhibitory to growth. None of the
fungi exhibited growth with feruhc acid and pyrogallol at 0.05%. At concentrations of 0.005% and
0.0005%. the phenolics were not inlubttory and in
many cases stimulated growth of both white- and
brown-rot fungi. Stimulation of growth of wluterot fungi by aromatic compounds has been previously noted [21.
Aromatic monomers that may be formed during the synthesis and degradation of hgnln in
wood tissues may be inhibitory to enzymes Their
toxicity is based on their ability to bind and
inactivate proteins [12]. Relatively high concentrations of the three phenoltcs tested in this study
were required to inhibit the activity of xylanase
from P. placenta (Table 3).
Catechol was the most toxic substance inhibitory to xylanase in sigH. A catechol concentration
of 0.1~0 was required for total mhtbltton. The
enzyme was most resistant to v~udhn; a collcentratlon of 1% resulted in 40% mhlbmon These
data correspond with those of Sharma et al. [6].
who found that most phenohcs, with the exception
of quatacol and caffeic acid. did not affect the
xylanase activity of Aspergdlus Japomcus at concentrattons of 0 1%; catechol was not included in
their study. HJghley [8] reported that only the
oxidized forms of catechol and vandlm inhibited
the soluble cellulase of brown-sot fungi; nonoxidized compounds did not affect celhilase activity. The growth of P placenta was inhibited by
several phenoltcs at lower concentrations than
inhibited xylanase (Table 1). The compounds exadently interfere with some other aspect of
metabolism. The carbohydrate moiety associated
with the xylanase may be protecting the enzyme
from the phenolic material. For example, glycoproteins present on the spores of Colletotr~chum
gramtmcola have a high affinity for purified condensed tannins and may protect the fungus from
the toxic phenohcs present in plant tissue [13]. The
presence of phenohcs in low concentrations appears to increase the activity of the xylanase,
mdlcatmg that certain glycoprotem-phenohc interactions may actually be beneficial for the
fungus.
The effects of the aromatic compounds incorporated into liquid culture on xylanase, cellulase,
and glycostdase production depended on compound and fungal species (Tables 4-- 6). With the
white-rot fungus. Co verswolor (Table 4). gualacol
was the most inhibitory to production of all enzyme actwmes. Veratrol and 2,6-dtmethoxyphenol
also inhibited formation of all enzymes. Catechol
inhibited production of cellulase and xylanase but
not the clycosldases. Except for mhibRlon of glycosldases by feruhc acid (50 ppmL the remaining
phenohcs were not particularly inhibitory In fact,
In several instances, enzyme activities were increased. Muller et al [2]. studying the effect of
phenohcs on regulation of cellulase production by
the whRe-rotter Trametes verstcolor, also found
stimulation by iigmn-related phenols. They suggested that stimulation ts not directly related to
effects by phenols but IS redirect. The phenol ~s
oxidized by laccase to qmnone. The quinone is
then reduced by the enzyme cellobtose quinoneoxldoreductase, while celloblono-lactone is formed
from cellohiose. Formatmn of cellobtono-lactone
prevents catabohe repression of cdlulase by cellobiose, resulting in increased celhilase production.
0
33
0
0
48
58
0
14
30
66
0
55
60
42
53
71
58
62
60
56
60
52
56
58
53
53
52
62
59
59
58
58
0
0
12
0
18
41
12
33
0
32
32
0
73
70
56
75
65
69
70
50
63
85
65
0005
005
00005
005
0005
p z.hrr~ovpormnl
C ~r$1co[or
Whlte-rol mycehal mass (rag)
Concentrauon levels of 0 05%. 0 005~. a n d 0.0005~.
Feruhc
Vamlhn
Pyrogallol
Coumarlc acid
Veralrol
Gualacol
Catachol
T a n m c acid
Galhc acid
2,6- Dimelhoxyphenol
Control
Compound
Mycehal mass produced in the presence of aromatt¢ monomers"
Table 2
005
0
16
0
20
74
48
12
17
55
29
0
00005
68
70
60
73
65
66
67
71
69
74
0
P placeata
53
66
28
53
65
65
57
55
72
38
38
0005
51
46
37
44
67
74
07
55
70
53
0
00005
Brown-rot mycehal mass (rag)
0
18
0
O
48
58
O
0
23
23
0
005
49
52
33
44
58
52
59
51
41
41
31
OG05
G rrabe:tm
45
42
dO
41
54
54
51
48
44
51
0
00~05
Table 3
Inl~b]tloa of xylanase m posltaplacenta bY phenohcs a
Compound
Concentratton
Percentage of control
Vamlhn
O
O.Ol
0.10
l O0
tO00
0
0.0!
005
0 l0
0 50
0
0.01
0 05
010
0 50
100
140
Catechol
Tanmc actd
160
60
0
100
125
lO0
o
0
100
100
150
125
0
f u n g u s ( T a b l e s 5 a n d 6). In general, p r o d u c t i o n o f
xyl a na s e a n d cellulase m the l i q u i d t, ultures w a s
n o t greatly l n h t b l t e d b y the p h e n o b c s . V a m l l m (50
p p m ) a n d catechol (50 p p m ) were the m o s t i n h i b i tory to these e n z y m e s m P. placenta, c a u s i n g
c o m p l e t e mMbltlOn. T h e o n l y p h e n o l i c c o m p l e t e l y
Inhibitory t o x y l a n a s e a n d cellulase p r o d u c t i o n
f r o m G. trabeum w a s 2 . 6 - d l m e t h o x y p h e n o l .
C a t e c h o l w a s qui t e i n h i b i t o r y to glycosidase p r o d u c t i o n o f b o t h P. placenta a n d G. trabeum. Bxylosldase p r o d u c a o n b y b o t h fungi w a s sensitive
to the p h e n o h c s Seven p h e n o l s c a u s e d g r e a t e r
t h a n 50% m l u b m o n o f fl-xylostdase p r o d u c t i o n b y
P placenta, a n d eight p h e n o l s caused g r e a t e r t h a n
50% m h l b m o n of fl-xylosidase p r o d u c t i o n b y G.
trabeum.
A s w i t h the white-rotter, the a r o m a t i c c o m p o u n d s oft e n s u m u l a t e J e n z y m e acttvlties w i t h the
t
t w o b r o w n - r o t t e r s . H o s e v e r . i n v o l v e m e n t o f laccase a n d cellobtono-l.~ctone, as p r o p o s e d for
s t l m u l a t t o n of a whtte-rOtter c a r b o h y d r a t e - d e g r a d i n g e n z y m e , lS p r o b a b l y n o t revolved w i t h the
b r o w n - r o t t e r s , because d e g r a d a t l v e e n z y m e s f r o m
a A¥cfage of [hrec reptlCallOnS
E n z y m e f o r m a t i o n b y the t w o b r o w n - r o t fungi
i n r e s p o n s e to the a r o m a t i c c o m p o u n d s differed
f r o m each o t h e r a n d d if f er ed f r o m the w h i t e - r o t
Table 4
Effect of aromatic monomers on enzyme production by Corwtus verstco[or
Compound
Feruhc actcl
l~rogallo|
Galh¢ acid
Countenc acid
Tanmc actd
Vamlhn
Calechol
Veratrol
Gumacol
2,6-Dimelhoxyphenol
Concentratton a
Glycostdase (percentage of connol)
fl-glucoa-galacto/]-~tytcsldase
~ldase
sldaSe
50
5
50
5
50
S
rio
5
50
5
50
5
50
5
50
5
50
5
50
5
3
166{,
70
67
2256
130
713
180
1080
210
43
510
100
140
2
4
4
4
52
10
a Concentranons of 50 and 5 ppm
0
837
24
42
708
160
800
397
803
94
14
104
28
39
0
23
0
0
13
0
0
500
466
90
666
100
300
100
400
95
l0
tO0
6(}0
92
0
36
0
0
0
0
Glycanas¢ (percentage of control)
fl-galactostdase
Xylanase
Cellulase
5
292
77
31
106
153
lfl0
100
1046
140
154
738
161
892
62
46
77
100
23
3
II
6
7
27
I00
106
132
63
72
15
65
0
13
29
12
O
0
0
1080
100
180
100
7{]0
720
280
580
tO0
300
100
8
0
tO0
0
0
25
10
0
O
57
0
20
Table 5
Effect of aromahc monomers on enzyme production by Postla fllaCenla
Compound
Feruhe acid
Pyrogallol
Protocatechmc
Galhe acid
Coumenc acid
Tannic acid
Vandhn
Catechol
Veratrol
Gumacol
2,6-Dtmethoxyphenol
Concen-
GlycosJdase (percentage of control)
tratlon a
/~-glucosldase
a-galactosldaSe
~-xylosldaSe
fl-galactoSldaSe
Xylanase
Glycanase (percentage of control)
Cellulase
50
5
50
5
50
5
50
5
50
5
50
5
50
5
50
5
50
5
50
5
38
208
59
88
10
81
89
2405
30
54
59
92
70
54
49
49
238d
265
769
615
7
82
88
38
39
285
6
7
15
87
5
11
38
100
4
72
184
177
135
135
I1
49
12
27
20
675
8
18
7
35
2
21
9
38
18
25
567
567
324
297
10
153
14
67
24
154
32
80
54
53
52
100
93
73
11
4
537
487
237
262
43
202
83
168
90
146
23
128
40
142
69
202
0
49
0
100
214
192
38
500
3(J
49
85
153
115
207
44
110
92
100
28
100
0
138
0
83
162
287
75
562
50
5
231
363
94
146
62
188
154
181
283
450
66
33
a Coneentrauons of 50 and 5 p p m
Table 6
Effect of aromaue monomers on enzyme production by Gloeophyllum trabeum
Compound
Feruhc acid
Pyrogallol
Ga)h¢ actd
Coumenc aeol
Tanmc acid
Vamlhn
Catechol
Veratml
Guata¢ol
2,6-DImethoxyphenol
Concert-
Glycosldas¢ (percentage of control)
tratton a
~-gluC~~ldase
50
5
50
5
50
5
50
5
50
$
50
5
50
37
120
480
520
I00
122
100
100
560
220
244
480
0
a-galactoslda~e
Glyc,anaze (percentage of control)
/I-xylostdas¢
~-galactosldase
Xylanase
Cellulase
57
52
78
65
44
65
50
35
70
83
70
70
0
55
46
18
100
43
43
56
34
92
66
74
58
0
60
60
58
130
42
56
72
58
130
100
100
82
0
74
74
59
67
68
~3
45
51
39
7]
107
89
92
55
100
52
83
64
100
55
52
74
76
69
76
76
5
0
0
0
0
55
48
50
5
50
5
50
5
40
58
300
0
l0
11
100
54
142
21
19
25
0
0
0
0
0
0
42
0
218
0
0
0
100
119
73
69
0
0
20
71
66
13
64
0
Conc~n~rauons of 50 and 5 p p m
m a n y brown-rot fungi are not subject to catabohc
repression [8]. Perhaps when enzyme stimulatton
does occur m the brown-rot fungi, the aromattc
c o m p o u n d s may be a c t m g as a cofactor for the
enzymes [14].
T h e aromattc compounds clearly have dtffermg
effects on the productton of extracellular degradattve enzymes by the white- and brown-rot fungi m
liqmd culture. N o c o m p o u n d stood out as being
strongly mbabttory to productton of all enzyme
activities of all three fungi exanuned. Lack o f a
c o m m o n reactmn to the phenohc compounds may
be related to the diverse physiologles o f white- and
brown-rot fungi Thus. it ts not simple to predtct
possible ways to mhtbJt the degradattve eapabihty
of the wood decay fungi wtth a r o m a u c comp o u n d s as a means of controlhng tbelr attack on
wood.
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