Kiel 1991
Enzyme activities of monokaryotic and dikaryotic strains of the
marine Basidiomycete Nia vibrissa
G. Schimpfhauser and H. P. Molitoris
Botanisches Institut an der Universitat Regensburg
Postfach 397, D-8400 Regensburg, Germany
Abstract
M o n o - and d i k a r y o t i c isolates of the marine wood-inhabiting Basidiomycete Nla
vibrissa
from various marine zones were investigated in semiquantitative tests
for 15 enzymes in redox metabolism (laccase, peroxidase, tyrosinase), carbohydrate metabolism (amylase, cellulase, chitinase, B-glucosidase, laminarinase, p e c tate transeliminase, xylanase), fat metabolism (lipase), nitrogen metabolism
(caseinase, gelatinase, n i t r a t e reductase, urease). A l l experiments were c o n ducted at 22 ° C on agar plates or test tubes w i t h media containing synthetic
seawater or deionized water. Most of the strains showed an identical enzyme
pattern. A m y l a s e , caseinase, cellulase, gelatinase, laminarinase, lipase, n i t r a t e
reductase, peroxidase and xylanase were found in a l l strains, tyrosinase in none.
The production of enzymes did not show any significant differences as regards
the nuclear status (mono- or dikaryon) or the biogeographical origin of the
strains.
Introduction
Nla vibrissa M O O R E and M E Y E R S (1959), is the only wood degrading marine
G a s t e r o m y c e t e . Previous investigations of Nla vibrissa dealt p r i m a r i l y w i t h morphology and taxonomy ( D O G U E T 1967, K O H L M E Y E R 1963), the few physiologic a l investigations were c a r r i e d out usually using only one isolate ( D O G U E T 1968,
1969a, 1969b; for further references see also S C H I M P F H A U S E R 1990). In their
search for the enzyme p a t t e r n of the fungus, L E I G H T L E Y and E A T O N (1979)
tested for enzymes involved in both lignin degradation (laccase and tyrosinase)
and carbohydrate metabolism (cellulase, mannanase and xylanase). The presence
of laccase indicated a white rot type of wood degradation.
In this paper several monokaryotic and dikaryotic strains f r o m several marine
biogeographical^ zones ( H U G H E S 1974, K O H L M E Y E R 1983) were compared for
the production of 15 enzymes on synthetical seawater and deionized water
media.
Material and methods
B i o l o g i c a l m a t e r i a l : The origin and suppliers of Nia vibrissa strains are given in
the f i r s t paper in this series ( S C H I M P F H A U S E R and M O L I T O R I S 1991). Three of
the 11 strains were shown by m i c r o f l u o r i m e t r y to be monokaryotic (see
S C H I M P F H A U S E R and M O L I T O R I S 1991).
M e d i a , incubation, growth and evaluation: The strains were transferred to 9 cm
d petri dishes and grown on a glucose (1 g/1) - peptone (0.5 g/1) - yeast e x t r a c t
(0.1 g/1) medium made up w i t h synthetic seawater ( G P Y S ) or deionized water
(GPYI), as described in M O L I T O R I S and S C H A U M A N N (1986). The synthetic seaw a t e r was prepared w i t h R I L A M A R I N E MIX ( R I L A P R O D U C T S , Teaneck, N.J.,
U S A ) . The incubation temperature was 22 °C.
E n z y m e tests: F o r the determination of laccase w i t h syringaldazine ( H A R K I N
and OBST 1973), cellulase w i t h carboxymethylcellulose ( T E A T H E R and W O O D
1982), cellulase w i t h a v i c e l coupled w i t h R B B R ( R e m a z o l - b r i l l a n t - b l u e - R ) (NG
and Z E I K U S 1980, S M I T H 1977, C O L L E T T 1984) and B-glucosidase w i t h arbutin
( K R E I S E L and S C H A U E R 1987), see R O H R M A N N and M O L I T O R I S (1991). The
presence of laminarinase was determined with soluble laminarin in G P Y - m e d i u m
containing 5 % soluble laminarin (prepared a f t e r T H I E M et a l . 1977) by staining
the non-degraded l a m i n a r i n with congo red ( T E A T H E R and W O O D 1982; as
m o d i f i e d by R O H R M A N N , personal communication). Urease a c t i v i t y (splitting of
urea into C 0
and ammonia) results in a pH increase which is demonstrated by
turning red the pH indicator phenol-red ( C H R I S T E N S E N 1946 in K R E I S E L and
S C H A U E R 1987). Xylanase a c t i v i t y was shown by p r e c i p i t a t i n g the residual xylan
by ethanol ( F L A N N I G A N 1980). F o r the remaining enzyme tests (laccase w i t h a naphthol, laccase w i t h guajacol, laccase with benzidin, peroxidase w i t h benzidin,
tyrosinase w i t h p-cresol and g l y c i n , amylase w i t h soluble starch, chitinase w i t h
c o l l o i d a l c h i t i n , laminarinase w i t h insoluble laminarin, pectate transeliminase
w i t h p e c t i n from apples, lipase with tween 80 and C a C l , caseinase w i t h skim
milk powder, gelatinase w i t h gelatin and n i t r a t e reductase w i t h N a N 0 and Gries
Ilosvaye reagent) see M O L I T O R I S and S C H A U M A N N (1986).
2
2
3
A l l d i r e c t enzyme tests (for explanation see Table 1) and the growth d e t e r m i n a tion were conducted in t r i p l i c a t e ; for the indirect enzyme tests 5 plates were
used for each enzyme/strain combination. G r o w t h determination and enzyme
tests were c a r r i e d out at weekly intervals for at least 7 weeks.
Results and discussion
The results of the enzyme tests for 15 enzymes in redox, fat, nitrogen and c a r bohydrate metabolism, both on synthetic (S) and deionized water (I) media, are
given in arbitrary units for the monokaryotic and d i k a r y o t i c strains of Nia
vibrissa in Table 1. F r o m Table 1 the following results can be summarized:
A m y l a s e , caseinase, cellulase, gelatinase, laminarinase, lipase, n i t r a t e reductase,
peroxidase and xylanase are produced by a l l strains in at least one test on Iand/or S-medium.
C h i t i n a s e , 3-glucosidase, laccase, pectate transeliminase and urease are found
for about 2/3 of the strains in at least one test on I- and/or S-medium.
Tyrosinase is not produced by any of the strains tested.
Regarding the enzyme a c t i v i t y investigated using several tests (cellulase, laccase
and laminarinase), it is evident that for cellulase the results d i f f e r only quantit a t i v e l y , whereas for laccase the results depend on the strain and enzyme tests
used. When tested w i t h soluble l a m i n a r i n , laminarinase was found in 100 % of
the strains, when tested w i t h insoluble laminarin, laminarinase was not found in
any strain.
No general p i c t u r e could be formed of the e f f e c t of synthetic or deionized
water medium on enzyme a c t i v i t y , since some enzymes are produced by a high
percentage of strains on seawater medium (chitinase, g-glucosidase, peroxidase,
urease), laccase was produced p r e f e r e n t i a l l y on deionized water medium (laccase), whereas lipase was found exclusively on deionized water medium. A m y lase, caseinase, cellulase, gelatinase, nitrate reductase and xylanase were produced by a l l strains on both media.
N e i t h e r nuclear status nor the biogeographical origin of the strains appeared to
have a significant influence on enzyme production.
Redox metabolism
Peroxidase (EC 1.11.1.7.), tyrosinase (EC 1.14.18.1.) and laccase (EC 1.10.3.2.)
are involved in wood synthesis and degradation ( M O L I T O R I S 1976 and M O L I T O RIS 1979) laccase being s p e c i f i c of the white rot type of wood degradation and
responsible for the linkage of lignin w i t h cellulose degradation ( M O L I T O R I S
1979). Owing to methodological d i f f i c u l t i e s , laccase was investigated using d i f ferent tests. Taking the results together, a l l strains are able to produce laccase,
p r e f e r e n t i a l l y on I-medium. This agrees w e l l w i t h the f a c t that Nla vibrissa is a
wood-degrading fungus of the white rot type, c o n f i r m i n g also the results of
L E I G H T L E Y and E A T O N (1979). L a c c a s e production was also s p e c i f i c a l l y found
in the peridia of Nla vibrissa fruitbodies in the guajacol test (this paper) pointing
to the often-discussed role of phenoloxidases in fungal morphogenesis and propagation ( M O L I T O R I S 1976).
The absence of tyrosinase in the p-cresol test agrees w i t h previous results by
L E I G H T L E Y and E A T O N (1979) for Nia vibrissa. This enzyme was not found in
the other wood-degrading marine Basidiomycete, Halocyphina
villosa
(ROHRM A N N and M O L I T O R I S 1986) either, whereas peroxidase was found in our tests
in Nia vibrissa and previously for Halocyphina
villosa ( R O H R M A N N and M O L I TORIS 1986).
Carbohydrate metabolism
Since wood is frequently found as a substrate for marine fungi, and since Nia vibrissa is regularity found on wood and was shown to produce laccase, which part i c i p a t e s in wood degradation, it was to be expected that cellulases and other
enzymes p a r t i c i p a t i n g in the use of cellulose and other carbohydrates as energy
source would be found.
F u r t h e r m o r e , a l l strains tested produce amylase (EC 3.2.1.1.), cellulase (mainly
E C 3.2.1.4. and E C 3.2.1.91) and xylanase (EC 3.2.1.8.) on both media. B-Glucosidase (EC 3.2.1.21), laminarinase (EC 3.2.1.6.) and pectate transeliminase (EC
4.2.2.2.) were found in most of the strains and media tested, the l a t t e r enzyme,
however, only in small amounts. Chitinase (EC 3.2.1.14.), the enzyme degrading
c h i t i n , a substrate on which Nia vibrissa is not found, was produced to a much
lesser degree. C e l l u l a s e and xylanase were also found by L E I G H T L E Y and
E A T O N (1979) in Nia vibrissa. A s i m i l a r set of enzymes as described here for
Table 1 (Part 1). E n z y m e a c t i v i t i e s of 3 monokaryotic and 8 d i k a r y o t i c strains
of Nia vibrissa on synthetic seawater and deionized water media.
Enzyme a c t i v i t y 1)
Enzyme
(Substrate)
2
M>
Dikaryons
Monokaryons
M154 M167 M168 M169 M170 M171 M175 M21
3
% of )
M172 M173 M174 s t r a i n
REDOX METABOLISM
4
Laccase/d )
( -Naphthol)
Laccase/d
(Guajacol)
5
Laccase/i ^
(Benzidine)
Laccase/i
(Syringaldazin)
Peroxidase/i
(Benzidin)
Tyrosinase/i
(p-Cresol)
I
+++
+
0
+++
( + ) +++
+
0
S
++
( + )* 0
0
0
0
0
0
0
I
+++
+1
++
+
++
I
++
++
+++1 ++
-
-
++
-
++
s
-
++
-
+1
+++
-
+
-
++
-
s
82%
++
27%
+++
54%
++
45%
+++
54%
+
++
-
-
-
++
+++
I
+++
-
-
(+)e
-
-
( + ) ( + )1 ( + ) ( + )
s
+++
+
+
(+)1 +1
+
++1
I
+++
+++
(+)
-
-
9%
++
54%
-
54%
s
0%
+1
++
++1
+1
100%
I
0%
s
0%
CARBOHYDRATE METABOLISM
Amylase/d
(Soluble
Starch)
Cellulase/i
(CMC )
I
+
++
+
+
+
+
++
+
++
++
+
100%
100%
S
++
++
+++
+++
++
++
+++
++
+++
+++
++
I
++
+++
+++
+++
++
+++
++
++
++
++
++
100%
s
++
++
++
++
++
++
++
++
++
++
++
100%
I
++
++
++
++
++
+1
+++
++
+++
++
++
100%
s
++
++
++
++
+++
++
+++
+
++
+1
++
100%
++1
++1
18%
+++
+++
72%
6)
Cellulase/d
(Avicell
+RBBR)
Chitinase/d
(Colloidal
Chitin)
I
s
-
+++
( + )1 +++
++
+
+1
++
+++
++
Laminarinase
I
/CL
Laminarin)
s
Laminarinase
/ i (Soluble
Laminarin)
I
++
++
s
+
++
(insoiuoie
-
+
B-Glucosidase I
/d (Arbutin)
s
-
+
(+)
++
++
+
-
-
-
++
+1
++
+
+1
-
+++
64%
+++
91%
0%
-
-
-
-
-
-
-
-
-
-
++
++
+
++
+
++
++
++
+
++
++
++
+++
+
+
-
+
++
0%
100%
91%
Table 1 (Part 2).
Enzyme a c t i v i t y 1)
Enzyme
(Substrate)
Dikaryons
2
M^
Monokaryons
M154 M167 M168 M169 M170 M171 M175 M21
3
% of )
• active
M172 M173 M174
strain
CARBOHYDRATE METABOLISM
7
Pectate )
I
transelimin.
/ i (Pektin A) S
Xylanase/i
(Xylan)
FAT
(+)1
(+)
n.d.
n.d.
(+) +
(+)
-
(+) (+) (+) (+) (+)
n.d.
n.d.
n.d.
n.d.
n.d.
++
+
+
++
+
++
+
+
+
+
(+)
++
+
+
+1
+1
++
I
(+)e ++
s
(+)
I
+1
n.d.
91%
n.d.
n.d.
n.d.
+
+
+
100%
+
+
+
+
100%
++
++
+++1 ++1
n.d.
METABOLISM
Lipase/d
(Tween 80)
NITROGEN
s
0%
METABOLISM
Caseinase/d
(Skim-milk
-powder)
Gelatinase/i
(Gelatin)
I
+++
++
++
++
+++
+++
+++
+++
+++
+++
100%
s
++
++1
++1
++1
++
++
+
++
(+)1 ++
+++
100%
I
++
+++
++
++
++
++
++
++
++
++
+
100%
s
++
++
+++
+++
++
+++
+++
+++
++
+++
(+)
+
(+)
+
(+)
(+)1
Nitrate
reductase/i
(NaN0 )
I
+
s
(+)
Urease/d
I
+++
{urea/
s
+++
3
100%
(+)
(+)
(+)
(+)1
(+)1
(+)1
(+)1
(+)
(+)
(+)
(+)1
(+)
(+)
(+)1
-
-
-
+++
(+)1
-
+++
100%
(+)1
100%
100%
(+)
(+)
(+)1
++
+++
-
54%
+1
++
+1
82%
1) Enzyme a c t i v i t y given as highest a c t i v i t y w i t h i n the t e s t period, i n a r b i t r a r y u n i t s : -; (+); +; ++; +++; n.d. « not d e t e r m i n e d ; 0 = enzyme a c t i v i t y
l a c k i n g because of no growth; e = e a r l y a c t i v i t y , only i n the f i r s t incubat i o n week; 1 = l a t e a c t i v i t y a f t e r the 3rd incubation week;
2) M = Medium; I = D e i o n i z e d water; S = s y n t h e t i c s e a w a t e r ;
3) Percentage o f s t r a i n s showing t h e r e s p e c t i v e enzyme a c t i v i t y i n a t l e a s t
one o f t h e t e s t s ;
4) d « d i r e c t t e s t ; s u b s t r a t e and d e t e c t i n g r e a g e n t c o n t a i n e d i n t h e medium
before i n o c u l a t i o n ;
5) i - i n d i r e c t t e s t ; s u b s t r a t e and/or d e t e c t i n g r e a g e n t i s added a f t e r the
incubation period;
6) CMC * Carboxymethylcellulose sodium;
7) Pectate t r a n s e l i m . = Pectate t r a n s e l i m i n a s e .
Nla vibrissa could be shown for the carbohydrate metabolism of Halocyphina
losa ( R O H R M A N N and M O L I T O R I S 1986).
vil-
Fat metabolism
Lipase ( E C 3.1.1.3.) enables the fungus to use fat f r o m various substrates as its
energy source. This enzyme was found (at least on I-medium) in a l l strains
tested. This corresponds w i t h the results of N E R U D et a l . (1982) who found
lipase in 15 of the 16 wood-degrading t e r r e s t r i a l Basidiomycetes and also w i t h
G E S S N E R (1979) who found it in 20 higher marine fungi f r o m salt marshes.
R O H R M A N N and M O L I T O R I S (1986) showed the presence of lipase also in the
wood-degrading marine B a s i d i o m y c e t e Halocyphina
villosa.
N i t r o g e n metabolism
Protein-hydrolysing enzymes
found in large amounts in a l l
corresponds w i t h the results
phina villosa and the results
out of 14 marine fungi.
(caseinase (EC 3.4.) and gelatinase ( E C 3.4.)) were
Nia vibrissa strains and on a l l media tested, which
of R O H R M A N N and M O L I T O R I S (1986) for H a i o c y of P I S A N O et a l . (1964), who found gelatinase in 13
N i t r a t e reductase was found ( B R E S I N S K Y and S C H N E I D E R 1975) to be an e n z y me of systematic s i g n i f i c a n c e in t e r r e s t r i a l Basidiomycetes. F o r marine fungi it
was shown by M O L I T O R I S and S C H A U M A N N (1986) that this enzyme was p r e sent in a l l marine fungi tested so far and might therefore be a t y p i c a l c h a r a c t e r i s t i c for the marine group of fungi. Since nitrogen often constitutes a l i m i t i n g
f a c t o r in fungal growth, the production of n i t r a t e reductase was discussed ( M O L I T O R I S and S C H A U M A N N 1986) as a positive s e l e c t i v e advantage to f u l f i l l the
nitrogen requirement by using the nitrogen of the n i t r a t e present in seawater.
This view is c o n f i r m e d by the results of R A U and M O L I T O R I S (1991). The p r e sence of n i t r a t e reductase in a l l strains of Nia vibrissa and in both media in the
present investigation f i t s w e l l into this p i c t u r e .
Summing up the results, it may be stated that generally the enzymes found in
Nia vibrissa agree well w i t h expectations c o r r e l a t e d w i t h the natural substrates
and living conditions of this marine Basidiomycete.
Acknowledgements
The authors are g r a t e f u l to E.B.G. Jones, J . K o h l m e y e r , A. N a k a g i r i and K.
Schaumann, for kindly providing fungal cultures, to S. Rohrmann, for helpful discussions in enzyme methodology and to R. Owen, for c r i t i c a l l y reading the E n g lish text.
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