Enzyme activities of monokaryotic and dikaryotic strains of the marine Basidiomycete Nia vibrissa

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

Mono- and dikaryotic 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), carbohy- drate metabolism (amylase, cellulase, chitinase, B-glucosidase, laminarinase, pec- tate transeliminase, xylanase), fat metabolism (lipase), nitrogen metabolism (caseinase, gelatinase, nitrate reductase, urease). A l l experiments were con- ducted at 22 °C on agar plates or test tubes with media containing synthetic seawater or deionized water. Most of the strains showed an identical enzyme pattern. Amylase, caseinase, cellulase, gelatinase, laminarinase, lipase, nitrate reductase, peroxidase and xylanase were found in all 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 Gasteromycete. Previous investigations of Nla vibrissa dealt primarily with mor- phology and taxonomy ( D O G U E T 1967, K O H L M E Y E R 1963), the few physiologi- c a l investigations were carried 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 pattern 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 from 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

Biological material: The origin and suppliers of Nia vibrissa strains are given in the first 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 microfluorimetry 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).

Media, 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 extract (0.1 g/1) medium made up with synthetic seawater (GPYS) 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 sea- water was prepared with R I L A M A R I N E MIX (RILA P R O D U C T S , Teaneck, N.J., USA). The incubation temperature was 22 °C.

Enzyme tests: For the determination of laccase with syringaldazine ( H A R K I N and OBST 1973), cellulase with carboxymethylcellulose ( T E A T H E R and WOOD 1982), cellulase with avicel coupled with R B B R (Remazol-brillant-blue-R) (NG and Z E I K U S 1980, SMITH 1977, C O L L E T T 1984) and B-glucosidase with 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 after T H I E M et a l . 1977) by staining the non-degraded laminarin with congo red ( T E A T H E R and W O O D 1982; as modified 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 02 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 precipitating the residual xylan by ethanol ( F L A N N I G A N 1980). For the remaining enzyme tests (laccase with a - naphthol, laccase with guajacol, laccase with benzidin, peroxidase with benzidin, tyrosinase with p-cresol and glycin, amylase with soluble starch, chitinase with colloidal chitin, laminarinase with insoluble laminarin, pectate transeliminase with pectin from apples, lipase with tween 80 and C a C l2, caseinase with skim milk powder, gelatinase with gelatin and nitrate reductase with N a N 0³ 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).

A l l direct enzyme tests (for explanation see Table 1) and the growth determina- tion were conducted in triplicate; for the indirect enzyme tests 5 plates were used for each enzyme/strain combination. Growth determination and enzyme tests were carried 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 car- bohydrate metabolism, both on synthetic (S) and deionized water (I) media, are given in arbitrary units for the monokaryotic and dikaryotic strains of Nia vibrissa in Table 1. From Table 1 the following results can be summarized:

Amylase, caseinase, cellulase, gelatinase, laminarinase, lipase, nitrate reductase, peroxidase and xylanase are produced by all strains in at least one test on I- and/or S-medium.

Chitinase, 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 activity investigated using several tests (cellulase, laccase and laminarinase), it is evident that for cellulase the results differ only quanti- tatively, whereas for laccase the results depend on the strain and enzyme tests used. When tested with soluble laminarin, laminarinase was found in 100 % of the strains, when tested with insoluble laminarin, laminarinase was not found in any strain.

No general picture could be formed of the e f f e c t of synthetic or deionized water medium on enzyme activity, since some enzymes are produced by a high percentage of strains on seawater medium (chitinase, g-glucosidase, peroxidase, urease), laccase was produced preferentially on deionized water medium (lac- case), whereas lipase was found exclusively on deionized water medium. A m y - lase, caseinase, cellulase, gelatinase, nitrate reductase and xylanase were pro- duced by all strains on both media.

Neither 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 (MOLITORIS 1976 and M O L I T O - RIS 1979) laccase being specific of the white rot type of wood degradation and responsible for the linkage of lignin with cellulose degradation (MOLITORIS 1979). Owing to methodological difficulties, laccase was investigated using dif- ferent tests. Taking the results together, all strains are able to produce laccase, preferentially on I-medium. This agrees well with the fact that Nla vibrissa is a wood-degrading fungus of the white rot type, confirming also the results of L E I G H T L E Y and E A T O N (1979). Laccase production was also specifically 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 propa- gation (MOLITORIS 1976).

The absence of tyrosinase in the p-cresol test agrees with 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 ( R O H R - M A N N and MOLITORIS 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 vi- brissa is regularity found on wood and was shown to produce laccase, which par- ticipates in wood degradation, it was to be expected that cellulases and other enzymes participating in the use of cellulose and other carbohydrates as energy source would be found.

Furthermore, all 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-Glucosi- dase (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 latter enzyme, however, only in small amounts. Chitinase (EC 3.2.1.14.), the enzyme degrading chitin, a substrate on which Nia vibrissa is not found, was produced to a much lesser degree. Cellulase 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 similar set of enzymes as described here for

Table 1 (Part 1). Enzyme activities of 3 monokaryotic and 8 dikaryotic strains of Nia vibrissa on synthetic seawater and deionized water media.

Enzyme a c t i v i t y 1) Enzyme

(Substrate)

M2> Dikaryons Monokaryons % o f3)

Enzyme (Substrate)

M154 M167 M168 M169 M170 M171 M175 M21 M172 M173 M174 s t r a i n REDOX METABOLISM

L a c c a s e / d4) ( -Naphthol)

I +++ + +1 0 +++ ( + ) +++ + 0 ++ +++ 82%

L a c c a s e / d4) ( -Naphthol)

S ++ ( + ) * 0 0 0 0 0 0 0

-

Laccase/d (Guajacol)

I +++ +1

- -

- - -

Laccase/d (Guajacol)

s ⁺⁺

- - -

- -

-

L a c c a s e / i5^ (Benzidine)

I ++

-

- - - -

L a c c a s e / i5^ (Benzidine)

s + 9%

L a c c a s e / i ( S y r i n g - a l d a z i n )

I ++

- - -

- -

L a c c a s e / i ( S y r i n g -

a l d a z i n ) s 0%

P e r o x i d a s e / i (Benzidin)

I +++

- -

- -

-

P e r o x i d a s e / i (Benzidin)

s ^{+++ + +} (+)1 +1 + ++1 +1 ++ ++1 +1 100%

T y r o s i n a s e / i (p-Cresol)

I 0%

T y r o s i n a s e / i (p-Cresol)

s 0%

CARBOHYDRATE METABOLISM Amylase/d

( S o l u b l e Starch)

I + ++ + + + + ++ + ++ ++ + 100%

Amylase/d ( S o l u b l e

Starch) S ++ ++ +++ +++ ++ ++ +++ ++ +++ +++ ++ 100%

C e l l u l a s e / i (CMC6 ))

I ++ +++ +++ +++ ++ +++ ++ ++ ++ ++ ++ 100%

C e l l u l a s e / i (CMC6 ))

s ^{++ ++ ++} ++ ++ ++ ++ ++ ++ ++ ++ 100%

C e l l u l a s e / d ( A v i c e l l +RBBR)

I ++ ++ ++ ++ ++ +1 +++ ++ +++ ++ ++ 100%

C e l l u l a s e / d ( A v i c e l l

+RBBR) s ^{++ ++ ++ ++} +++ ++ +++ + ++ +1 ++ 100%

C h i t i n a s e / d ( C o l l o i d a l C h i t i n )

I ++1 ++1 18%

C h i t i n a s e / d ( C o l l o i d a l

C h i t i n ) s ^{+ +} (+) ++

- -

-

B-Glucosidase /d (Arbutin)

I

-

- -

-

B-Glucosidase /d (Arbutin)

s + + 1 ++ +++ ++ ++ +1 ++ +1

-

Laminarinase I 0%

/CL ( i n s o i u o i e

Laminarin) s

- - - - - - - - - -

Laminarinase / i ( S o l u b l e Laminarin)

I ++ ++ ++ ++ + ++ + ++ ++ ++ + 100%

Laminarinase / i ( S o l u b l e

Laminarin) s ^{+ ++} ++ ++ ++ +++ + +

-

Table 1 (Part 2).

Enzyme a c t i v i t y 1) Enzyme

(Substrate)

M2^ Dikaryons Monokaryons % o f3)

Enzyme (Substrate)

M154 M167 M168 M169 M170 M171 M175 M21 M172 M173 M174 • a c t i v e s t r a i n CARBOHYDRATE METABOLISM

P e c t a t e7) t r a n s e l i m i n . / i ( P e k t i n A)

I (+)1 (+) ( + ) + (+)

-

P e c t a t e7) t r a n s e l i m i n .

/ i ( P e k t i n A) S n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

X y l a n a s e / i (Xylan)

I (+)e ++ ++ + + ++ + + + + 100%

X y l a n a s e / i (Xylan)

s (+) ++ + + + + (+) + + + + 100%

FAT METABOLISM L i p a s e / d

(Tween 80)

I +1 ++ + + +1 +1 ++ ++ ++ +++1 ++1 100%

L i p a s e / d (Tween 80)

s 0%

NITROGEN METABOLISM Caseinase/d

(Skim-milk -powder)

I +++ ++ ++ ++ +++ +++ +++ +++ +++ +++ +++ 100%

Caseinase/d (Skim-milk

-powder) s ⁺⁺ ++1 ++1 ++1 ++ ++ + ++ (+)1 ++ +++ 100%

G e l a t i n a s e / i ( G e l a t i n )

I ++ +++ ++ ++ ++ ++ ++ ++ ++ ++ + 100%

G e l a t i n a s e / i ( G e l a t i n )

s ^{++ ++ +++ +++ ++ +++ +++ +++ ++ +++} 100%

N i t r a t e r e d u c t a s e / i

(NaN03)

I + (+) + (+) (+) (+) (+) (+) + (+) (+)1 100%

N i t r a t e r e d u c t a s e / i

(NaN03) s (+) (+)1 (+) (+) (+)1 (+) (+) (+)1 (+) (+) (+)1 100%

Urease/d I +++ (+)1

- - - -

-

{urea/ s ⁺⁺⁺ (+)1 (+)1 (+)1

-

-

1) Enzyme a c t i v i t y g i v e n as h i g h e s t a c t i v i t y w i t h i n t h e t e s t p e r i o d , i n a r b i - t r a r y u n i t s : -; (+); +; ++; +++; n.d. « n o t 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 o f no growth; e = e a r l y a c t i v i t y , o n l y i n t h e f i r s t incuba- t i o n week; 1 = l a t e a c t i v i t y a f t e r the 3rd i n c u b a t i o n week;

2) M = Medium; I = D e i o n i z e d w a t e r ; 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 s h o w i n g 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 b e f o r e 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 a n d / o r d e t e c t i n g r e a g e n t i s added a f t e r t h e i n c u b a t i o n p e r i o d ;

6) CMC * C a r b o x y m e t h y l c e l l u l o s e sodium;

7) P e c t a t e t r a n s e l i m . = P e c t a t e 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 vil- losa ( R O H R M A N N and M O L I T O R I S 1986).

F a t metabolism

Lipase (EC 3.1.1.3.) enables the fungus to use fat from various substrates as its energy source. This enzyme was found (at least on I-medium) in all strains tested. This corresponds with the results of N E R U D et a l . (1982) who found lipase in 15 of the 16 wood-degrading terrestrial Basidiomycetes and also with G E S S N E R (1979) who found it in 20 higher marine fungi from 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 Basidiomycete Halocyphina villosa.

Nitrogen metabolism

Protein-hydrolysing enzymes (caseinase (EC 3.4.) and gelatinase (EC 3.4.)) were found in large amounts in all Nia vibrissa strains and on all media tested, which corresponds with the results 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 - phina villosa and the results of P I S A N O et al. (1964), who found gelatinase in 13 out of 14 marine fungi.

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 enzy- me of systematic significance in terrestrial 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 pre- sent in all 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 nitrate reductase was discussed (MO- LITORIS and S C H A U M A N N 1986) as a positive selective advantage to f u l f i l l the nitrogen requirement by using the nitrogen of the nitrate present in seawater.

This view is confirmed by the results of R A U and MOLITORIS (1991). The pre- sence of nitrate reductase in all strains of Nia vibrissa and in both media in the present investigation fits well into this picture.

Summing up the results, it may be stated that generally the enzymes found in Nia vibrissa agree well with expectations correlated with the natural substrates

and living conditions of this marine Basidiomycete.

Acknowledgements

The authors are grateful to E.B.G. Jones, J . Kohlmeyer, A. Nakagiri and K.

Schaumann, for kindly providing fungal cultures, to S. Rohrmann, for helpful dis- cussions in enzyme methodology and to R. Owen, for c r i t i c a l l y reading the Eng- lish text.

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