Production, purification, and characterization of an extracellular endo-P-1,3-glucanase from a monokaryon of Schizophyllum commune ATCC 38548 defective in exo-P-1,3-glucanase formation

ANDREAS PROKOP, PETER RAPP,~ AND FRITZ WAGNER Itlstitrttfiir Biochemie rind Biotechnologie, Technische Universitat Bt.aunschweig, 0-3300 Bt.altnschweig, Germany

Received April 14, 1993

Revision received October 14, 1993

Accepted October 15, 1993

PROKOP, A., RAPP, P., and WAGNER, F. 1994. Production, purification, and characterization of an extracellular endo-a-l,3- glucanase from a monokaryon of Schizophyllum commune ATCC 38548 defective in exo-P-1,3-glucanase formation. Can. J. Microbiol. 40: 18-23.

Production of extracellular P-1,3-glucanase activity by a monokaryotic Schizophyllum commrtne strain was monitored and results indicated that the P-glucanase activity consisted of an endo-P-l,3-glucanase activity, besides a negligible amount of P-1,6-glucanase and P-glucosidase activity. Unlike the P-I ,3-glucanase production of the dikaryotic parent strain S. commune ATCC 38548, the P-1.3-glucanase formation of the monokaryon was not regulated by catabolite repression. The endo-P-I ,3-glucanase of the monokaryon was purified from the culture filtrate by lyophilization, anion exchange chromatography on Mono Q, and gel filtration on Sephacryl S-100. It appeared homogeneous on SDS-PAGE with a molecular mass of 35.5 kDa and the isoelectric point was 3.95. The enzyme was only active toward glucans containing P-1,3-linkages, including lichenan, a P-1 ,3-1,4-D-glucan. It attacked laminarin in an endo-like fashion to form laminaribiose, laminaritriose, and high oligosaccharides. While the extracellular P-glucanases from the dikaryotic S. commune ATCC 38548 degraded significant amounts of schizophyllan, the endo-P-l,3-glucanase from the monokaryon showed greatly reduced activity toward this high molecular mass fi-1,3-/P-1,6-glucan. The K, of the endoglucanase, using laminarin as substrate, was 0.28 mg/mL. Optimal pH and temperature were 5.5 and 50°C, respectively. The enzyme was stable between pH 5.5 and 7.0 and at temperatures below 50°C. The enzyme was completely inhibited by 1 mM H ~ ~ + . Growth of the monokaryotic S. commune strain was not affected by its constitutive endo-P- l,3-glucanase formation.

Key words: endo-P-l,3-glucanase, Schizophyllum commune, monokaryon, constitutive endo-P-l,3-glucanase formation.

PROKOP, A,, RAPP, P., W WAGNER, F. 1994. Production, purification, and characterization of an extracellular endo-P- l,3- glucanase from a monokaryon of Schizophyllutn commune ATCC 38548 defective in exo-P- 1,3-glucanase formation. Can. J. Microbiol. 40 : 18-23.

Les auteurs ont CtudiC la production d'une activitC P-1,3-glucanase extracellulaire par une souche monocaryote de Schizophyllum cotnmune; les rCsultats ont indiquC que l'activitt P-glucanase est composCe d'une endo-P-l,3-glucanase, en plus d'une quantitC negligeable d'activitC P-1,6-glucanase et P-glucosidase. Contrairement i la production de P-1,3-glucanase de la souche parentale dicaryote de S. commutle ATCC 38548, la synthbse de P-1,3-glucanase du monocaryon n'a pas CtC dCterminCe par la repression d'un catabolite. L'endo-P-1,3-glucanase du monocaryon a CtC purifiCe du filtrat de culture par lyophilisation, chromatographie Cchangeuse d'anion sur Mono Q et filtration sur gel de Sephacryl S-100. L'Clectrophorbse en gel dCnaturant de polyacrylamide (SDS-PAGE) a rCvClt une protCine homogbne de masse molCculaire de 3 5 3 kDa et le point isoClectrique a CtC trouvC i 3,95. L'enzyme fut active seulement envers les glucanes contenant des liaisons P-1,3, incluant la lichenane, une p - 1 3 1 ,4-D-glucane. Elle a attaquC la laminarine selon un modble de type endo pour former le laminaribiose, le laminaritriose et des oligosaccharides plus complexes. Tandis que les P-glucanases de la souche dicaryote de S. commune ATCC 38548 ontdCgradC des quantitCs considCrables de schizophyllane, l'endo- l,3-glucanase du monocaryon a montrC une activitC fortement rCduite envers cette P-1,3-/P-1,6-glucane de poids moltculaire ClevC. Le K, de l'endoglucanase a CtC calculi i 0.28 mg/mL en utilisant la laminarine comme substrat. Le pH et la tempCrature optimums ont CtC respectivement de 5,5 et 50% L'enzyme a CtC stable entre les pH 5,5 et 7,O ainsi qu'aux tempCratures inferieures i 50°C. L'enzyme a CtC complbtement inhibCe par l'ion Hg2+ i 1 mM. La croissance de la souche monocaryote de S. commlrne n'a pas CtC affectCe par la synthbse de son endo-p- l,3-glucanase constitutive.

Mots clis : endo-P-l,3-glucanase, Schizophyllum comtnune, monocaryon, synthbse d'une endo-P-l,3-glucanase constitutive. [Traduit par la rCdaction]

Introduction Schizophyllum commune is a dikaryotic, wood-rotting basid-

iomycete, which secretes several P-glucanases under condi- tions of carbon source limitation. Wessels (1969a) separated an extracellular enzyme preparation of this fungus into an exo-p- 1,3-glucanase, an endo-& l,3-glucanase, a P- 1,6- glucanase, a P-glucosidase, and an R-glucanase. Whereas all glucanases in this enzyme preparation degraded yeast glucan,

'Author to whom all correspondence should be sent at the following address: Gesellschaft fur Biotechnologische Forschung mbH, D-38124 Braunschweig, Germany.

only the latter was able to degrade R-glucan, a highly branched glucan with P-1,3- and P-1,6-linkages, which is chemically linked to chitin and is a portion of the alkali-insoluble inner layer of the hyphal wall (Sietsma and Wessels 1979). The alkali- soluble S-glucan is mainly located at the outer surface of the wall and has an a-1,3-polyglucose structure (Sietsma and Wessels 1977). Apart from these cell-wall polysaccharides, S. commune also produces a high molecular mass water-soluble polysac- charide, termed schizophyllan, which is present at the hyphal surface and in the culture supernatant (Kikumoto et al. 1970; Sietsma and Wessels 1977; Miinzer 1989). It consists of a P-1,3-D-glucan with one 6-linked P-D-glucopyranose group

Printcd in Canada 1 Imprim6 au can ad;^

Can

. J. M

icro

biol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

Uni

vers

ity o

f P.

E.I

. on

11/1

6/14

For

pers

onal

use

onl

y.

PROKOP ET AL. 19

attached to every third residue of the main chain (Kikumoto et al. 1971; Saito et al. 1979; Tabata et al. 198 1).

In the dikaryotic S. commune the incompatibility genes A and B regulate morphogenetic events, such as nuclear migration, nuclear pairing, hook-cell formation, conjugate division, hook- cell septation, and hook-cell fusion (Raper and Hoffman 1974). On the other hand, in monokaryons the formation of hook cells and fruiting bodies is normally repressed, since the incompati- bility genes are in the A-off and B-off status (Raper and Hoffman 1974). During our studies of schizophyllan production by mono- karyons and dikaryons of S. commutle, several monokaryotic strains were obtained by isolating the two types of monokaryotic protoplasts and their reversion to hyphal growth. The two incom- patibility types were termed protoclone A and B, because mating interactions between members of these two groups produced the original dikaryon (Prokop et al. 1992).

In contrast to the dikaryotic strains, which produced con- siderable amounts of schizophyllan, the monokaryons produced quantities slightly above detection limits. To know whether this production of trace amounts of schizophyllan by the mono- karyons is due to a constitutive P- 1,3-glucanase, a monokaryon of S. commune ATCC 38548 was selected and its P-glucanase production was studied. Moreover, the kind and number of p- 1,3-glucanases produced by this monokaryon were examined.

Materials and methods Maiel.inls

Mono Q HR 10110, Sephacryl S-100 HR, and Superose 12 HR 10130 were obtained from Pharmacia-LKB (Freiburg, Germany). Laminarin, lichenan, xylan from birchwood, laminaribiose, and gentiobiose were purchased from Sigma (Deisenhofen, Germany). Pustulan was obtained from Calbiochem (Bad Soden, Germany); sodium carboxymethyl- cellulose and amylose from potato were from Serva (Heidelberg, Germany). Schizophyllan was isolated and purified according to Schulz and Rapp (1 99 1). Periodate-oxidized laminarin was prepared according to Goldstein et al. (1965). All other reagents were analytical grade from

I

-

A A A

commercial sources. Time (h)

Growth ofnlicroolga~lisnrs The dikaryotic strain S . coniniline ATCC 38548 and its parental

monokaryons, protoclones A No. 9 and B No. 5, were cultivated on chemically defined medium as previously described (Prokop et al. 1992). Growth was determined according to Prokop et a]. (1992).

Enzyme assays p- 1,3-Glucanase activity was routinely assayed by incubating 0.5 mL

0.5% (wlv) laminarin in 50 mM sodium phosphate buffer (pH 5.5) with 0.5 mLenzyme solution at 50°C for 10 min, and the amount of reducing sugars produced was determined by the method of Somogyi-Nelson (Somogyi 1952). One unit (U) of P-1,3-glucanase activity was defined as the amount of enzyme that produced 1 pmol of reducing equivalentlmin under the given conditions.

Plateirz determirlniion Concentrations of soluble protein were estimated by the absorbance

at 280 nm or by the colorimetric method of Bradford (1976), using bovine serum albumin as the standard.

Enzyme purification Shake flask cultures of protoclone B No. 5 grown for 96 h according

to Prokop et al. (1992) were filtered through nylon cloth (120 pm). Indigo granules were removed by consecutive membrane filtration with Sartorius SM 13400 (Gottingen, Germany), Millipore HAWP 047, and Millipore GS (Eschborn, Germany) at a pressure of 2 bar (1 bar = 100 kPa). The resultant culture supernatant was lyophilized, and the residue was dissolved in 50 mL of 50 mM sodium phosphate buffer (pH 5.5) and dialyzed against the same buffer for 6 h at 4°C. The dialyzed sample was centrifuged and the supernatant was applied to a Mono Q HR 10110

FIG. 1. Growth and production of extracellular P- 1,3-glucanase activity of the monokaryons of S . conlm~rne ATCC 38548, protoclones A No. 9 (A) and B No. 5 (B), and of the dikaryotic parent strain S. commune ATCC 38548 (C). Cultures were grown in 250 rnL chemically defined medium in I-L Erlenmeyer flasks on a rotary shaker (100 rpm) at 27°C. 0 , mycelial dry mass; m, P-1,3-glucanase activity; 0, residual glucose in culture supernatant.

column coupled to a Pharmacia fast performance liquid chromatography system (FPLC). Unbound protein was washed from the column with 50 mM sodium phosphate buffer (pH 5.5). Bound protein was eluted by using a linear gradient of 0 to 1 M NaCl in 50 mM sodium phosphate buffer (pH 5.5). Elution was monitored for protein at 280 nm. The flow rate was 1 mL/min and fractions of 3 mL were collected. Fractions containing P-1,3-glucanase activity were pooled and applied to a Sephacryl S-100 HR column (2.5 x 80 cm). Proteins were eluted isocratically with 50 mM sodium phosphate buffer (pH 5.5) containing 0.3 M sodium chloride, at a flow rate of 1 mL/min. Fractions of 5 rnL were collected and analyzed for protein and P-1,3-glucanase activity. Fractions containing P-1,3-glucanase activity were dialyzed against 20 mM Tris-HC1 buffer (pH 7.2) for 6 h and applied to a Mono Q HR 10/10 column coupled to a Pharmacia FPLC. Unbound protein was eluted with 20 mM Tris-HC1 buffer (pH 7.2) and the P-1,3- glucanase activity was eluted by using a linear gradient of 0 to 1 M NaCl in20 rnM Tris-HC1 buffer (pH 7.2) at a flow rate of I mL/min. Fractions of 1 mL were collected and the purified P-1,3-glucanase was stored at -15°C in 50 mL of 50 mM sodium phosphate buffer (pH 5.5).

Can

. J. M

icro

biol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

Uni

vers

ity o

f P.

E.I

. on

11/1

6/14

For

pers

onal

use

onl

y.

CAN. J. MICROBIOL. VOL. 40. 1994

Fraction number

FIG. 2. FPLC chromatogram of P-1,3-glucanase elution from a Mono Q HR I O / I O column with 50 m M sodium phosphate buffer (pH 5.5) and a linear NaCl gradient. For other conditions see Materials and methods. 0 , P-1,3-glucanase activity; broken line, A280; diagonal solid line, linear NaCl gradient.

SDS-PAGE ancl isoe1ectr.i~ foc~tsing Purity and apparent molecular mass of the P-1,3-glucanase were

examined by SDS-PAGE using the automatic electrophoresis system from Pharmacia (Phast system) (Freiburg, Germany). Apolyacrylamide gel with a 10-15% gradient was used. Protein bands were located by silver staining according to the manufacturer's instructions. The following SDS molecular mass markers (Pharmacia) were used: phos- porylase 11 (94 kDa), bovine albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), and a-lactalbumin (14.4 kDa).

Isoelectric point was determined in a Phast Gel IEF 3-9 from Pharmacia with the Phast system, using a canier ampholyte in the pH range of 3-10. Protein bands were visualized by silver staining according to the manufacturer's instructions and the calibration kit contained the following marker proteins: amyloglucosidase ( 3 . 9 , soybean trypsin inhibitor (4.55). P-lactoglobulin A (5.2), bovine carbonic anhydrase B (5.85), human carbonic anhydrase B (6.55), horse myoglobin (6.85), horse myoglobin (7 .33, lentil lectin (8.15), lentil lectin (8.45). lentil lectin (8.65), and trypsinogen (9.3).

Determirlation of K,,, The rate of laminarin hydrolysis was measured for a series of

concentrations of laminarin from 0.002 to 0.8 mg/mL. The K,,, value was estimated by a Lineweaver-Burk plot of 1/S vs. I/V.

Irii~ihitiorl The purified enzyme ( 1 ~ g ) was incubated in 2 mL 50 mM sodium

phosphate buffer (pH 5.5) with each potential inhibitor (1 and 5 inM) at room temperature. After 60 min the activity toward laminarin was assayed under standard conditions.

Analysis of hydrolysis products The purified glucanase (0.5 ~ g ) was incubated with 5 mg laminarin

in 1 mL 50 mM sodium phosphate buffer (pH 5.5) at 50°C. Samples were removed after 1 h and hydrolysis was stopped by heating the samples in a boiling water bath for 10 min. The products were detected by thin-layer chromatography (TLC) (Kottutz and Rapp 1990).

Results Fol.mation of P-1,3-glucatzase nctivity

In a previous study, several inonokaryotic strains were obtained from the dikaryotic S. commune ATCC 38548 (Prokop et al. 1992). Figure 1 shows the formation of extracellular

p-1,3-glucanase activity during cultivation in shake flasks of two selected monokaryotic strains, the so-called protoclone A No. 9 and protoclone B No. 5, and their dikaryotic parent strain S. conzmune ATCC 38548. A few hours after inoculation of protoclone A No. 9 and protoclone B No. 5, P-1,3-glucanase activity could be measured in the culture supernatants. It increased simultaneously with growth and reached (after 96 h) maximal values of 0.89 and 1.28 UImL for protoclone A No. 9 and protoclone B No. 5, respectively (Figs. 1Aand 1B). By com- parison, the dikaryotic S. conznzune ATCC 38548 began to secrete very small amounts of extracellular p-1,3-glucanase activity only after 144 h cultivation when the culture medium was depleted of glucose (Fig. 1C). The culture of the dikaryotic strain reached maximal values of 0.8 U/mL p-glucosidase activity, 0.3 U/mL p-1,3-glucanase activity, and 0.05 U/mL p-1,6-glucanase activity after 720 h of cultivation in shake flasks with 3% (w/v) glucose as carbon source (data not shown). Both monokaryons secreted indigo into the culture medium but produced only trace amounts of schizophyllan, in contrast to the dikaryotic parent strain S. comnzutze ATCC 38548, which secreted approximately 10 g/L, of this polysaccharide and no detectable indigo after 96 h of growth (Prokop et al. 1992). Growth of both monokaryons was not impaired by the con- stitutive P-1,3-glucanase foimation, since the two monokaryotic strains foimed (after 120 h) the same amount of mycelial dry mass as the dikaryotic parent strain ATCC 38548 (Fig. 1).

Enzyme pur$cation The supernatant of a shake flask culture of the protoclone B

No. 5 grown for 96 h and liberated from indigo granules by successive membrane filtration was lyophilized and dialyzed. Anion exchange chromatography on Mono Q HR 10/10 resulted in a large peak of P-1,3-glucanase activity (Fig. 2). Fractions from this peak were pooled and applied to a Sephacryl S-100 HR column. A single peak of P-1,3-glucanase activity was obtained and its dialyzed fractions were applied a second time to a Mono Q HR 10/10 column. SDS-PAGE of the fractions from the peak obtained revealed one protein band (Fig. 3). By all these pro- cedures an apparently pure p-1,3-glucanase was obtained with a

Can

. J. M

icro

biol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

Uni

vers

ity o

f P.

E.I

. on

11/1

6/14

For

pers

onal

use

onl

y.

PROKOP ET AL. 2 1

FIG. 3. SDS-PAGE of the purified endo-P-l,3-glucanase. For con- ditions see Materials and methods. The band in the first two lanes represents the purified enzyme. The positions of the molecular mass markers are indicated.

TABLE I . Summary of steps used in the purification of endo-P-1,3- glucanase

Total Specific protein activity Purification Yield

(mg) (Ul~ng) (-fold) (%)

Culture supematant 65.3 Lyophilization

and dialysis 32.9 Mono Q HR 1011 0

(PH 5.5) 7.7 Sephacryl S- 100 HR 2.4 Mono Q HR 10/10

(pH 7.2) 0.19

specific activity of 139.4 U/mg, a purification factor of 28.4, and a yield of 8.2% (Table 1).

Properties ofthe enzyme SDS-PAGE of the purified P-1,3-glucanase suggested a

molecular mass of 35.5 kDa (Fig. 3). By gel filtration, using a Superose 12 HR 10130 column coupled to FPLC, the molecular mass of the enzyme was estimated to be 33.0 kDa (data not shown). The isoelectric point was found to be 3.95 (Fig. 4).

The optimal pH for enzyme activity was 5.5 and the maximal rate of laminarin hydrolysis was reached at 50°C. The enzyme was stable between pH 5.5 and 7.0 during 16 h incubation at room temperature. It maintained its full activity during incuba- tion for 1 h at temperatures below 50°C.

FIG. 4. Isoelectric focusing gel of the purified endo-P- l,3-glucanase. For conditions see Materials and methods. The band in the first three lanes represents the purified enzyme. The positions of the isoelectric point markers are indicated.

TABLE 2. Substrate specificity of endo-P-l,3-glucanase

Relative Linkage activity (%)

Laminarin Periodate-oxidized laminarin Lichenan Pustulan Schizophyllan Sodium carboxymethylcellulose Amylose Xylan Cellobiose l~-Nitrophenyl-~-~-glucopyranoside"

Nors: Reaction ~nixture containing I pg purified cnzyrne and 2.5 nig substrate in 1 rnL 50 n1M sodium phosphate buFfer (pH 5.5) was incubated at 50°C for 10 min.

"Reaction mixture was incubetc(1 at 40°C for 10 min.

The enzyme had an apparent K,, of 0.28 mg/mL for laminarin as substrate.

The effect of various potential inhibitors on the P-1,3- glucanase activity of the purified enzyme was studied and the results indicate that 1 and 5 mM Cu2+ reduced the activity to 41 and 5%, respectively, whereas 1 mM Hg" completely inhibited the 0-1,3-glucanase activity. Concentrations of 1 and 5 mMPb2+, Zn", urea, sodium iodoacetate, and L-cysteine did not affect the endo-P- l,3-glucanase activity.

Can

. J. M

icro

biol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

Uni

vers

ity o

f P.

E.I

. on

11/1

6/14

For

pers

onal

use

onl

y.

22 CAN. 1. MICROBII

The substrate specificity of the enzyme is shown in Table 2. It was characterized to be an endo-P-l,3-glucanase by its ability to hydrolyze periodate-oxidized laminarin (Table 2) and by its action pattern on laminarin. After 1 h hydrolysis of laminarin under standard assay conditions the product distribution con- sisted of 20% laminaribiose, 30% laminaritriose, and 50% higher oligosaccharides. In addition, Table 2 shows that the enzyme was only active toward glucans with P-1,3-linkages. It exhibited a high activity toward lichenan (a P-1,3- 1 ,4-D-glucan) and greatly reduced activity toward schizophyllan, which was 3% of that toward laminarin. On the other hand, the activity toward schizophyllan by the extracellular endo- and exo-P-1,3- glucanase-containing preparation of the dikaryotic parent strain ATCC 38548 was 15% of that toward laminarin under the same conditions. The endoglucanase from the monokaryon did not attack pustulan, amylose, cellulose, xylan, cellobiose, or p-nitrophenyl-P-D-glucopyranoside.

Discussion During our studies to improve schizophyllan production by

finding new strains of S. commurze with a higher capacity for schizophyllan synthesis, several monokaryons from S. comnzu17e ATCC 38548 were isolated (Prokop et al. 1992). Comparing the properties of two monokaryotic strains with those of the dikaryotic parent strain S. commutze ATCC 38548, it became evident that they differ in the secretion of P-1,3-glucanase activ- ity, schizophyllan, and indigo, but not in growth. The dikaryotic parent strain produced considerable amounts of schizophyllan, but no indigo, and the P-glucanase formation was under cata- bolite repression. In contrast, the monokaryons constitutively produced significant amounts of extracellular P- 1,3- glucanase activity and secreted indigo but only trace amounts of schizo- phyllan. To determine whether the constitutively produced P-1,3-glucanase activity was responsible for the deficiency in schizophyllan, this enzyme activity was analyzed. It was found that besides trace amounts of P- 1,6-glucanase and P-glucosidase activity only an endo-P-l,3-glucanase was present in the culture supernatant of the monokaryotic protoclone B No. 5. On the other hand, when the culture was deprived of glucose, the dikaryotic parent strain S. commune ATCC 38548 secreted at least one endo- and one exo-p-1,3-glucanase, a P-glucosidase, and p-1,6-glucanase activity (Munzer 1989). The same spectrum of enzyme activities was measured in culture supernatants from the dikaryotic S. commune strain K8 (Wessels 1969~) . Wessels et al. (1972) showed additionally that schizophyllan was prefer- entially hydrolyzed by exo-p-1,3-glucanase. This result is sup- ported by the finding that the endo-P-l,3-glucanase of the monokaryon exhibited a very small activity toward this high molecular mass p- 1,3-/P- 1,6-glucan compared with that of the exo- and endo-l,3-P-glucanase-containing enzyme preparation of the dikaryotic parent strain S. commutze ATCC 38548. There- fore, we conclude that the deficient schizophyllan production by the monokaryon is not due to this strain's constitutive endo-P- 1,3-glucanase formation. It is rather suggested that the indigo production, the lack of schizophyllan secretion, and the absence of catabolite repression of extracellular P-1,3-glucanase for- mation by both types of monokaryons as well as the deficiency of exo-p-1,3-glucanase secretion by the protoclone B No. 5 are a result of the A-off and B-off status of the incompatibility genes of the monokaryons (Wessels 1969h; Raper and Hoffman 1974).

The purified endo-P-l,3-glucanase from the monokaryon protoclone B No. 5 was active only toward glucans containing

p- 1,3-linkages. The high activity of the enzyme toward lichenan, a P-D-glucan containing 1,3- and 1,4-bonds, raises the question whether this enzyme should better be considered as a 8-1,3-1,4- glucanase. But unlike a true lichenase (EC 3.2.1.73), which is unable to hydrolyze P-D-glucans containing only P-1,3-bonds, the endoglucanase from the monokaryotic protoclone B No. 5 exhibited higher activity toward laminarin than toward lichenan. The endo-P-l,3-glucanase partially purified by Wessels (1 969a) also exhibited activity not only toward laminarin but also toward lichenan. This suggests that the endoglucanase from the rnono- karyon B No. 5 and that from the dikaryotic S. commune strain K8 used by Wessels (1969~) are probably identical. An extracellular endo-P-1,3-glucanase with similar substrate spec- ificity and even with a comparable molecular mass (32.1 kDa) and K,,, for laminarin (0.3 14 mg/mL) was isolated by Clark et al. (1978) from cultures of Rhizopus u1.r-hizus QM 1032. However, the isoelectric points of the enzymes were substantially different. The optimal pH and temperature for enzyme activity as well as the pH and temperature stability of the endo-P-1,3-glucanase from the monokaryon protoclone B No. 5 were within the range of other endo-P-1,3-glucanases from filamentous fungi (Clark et al. 1978; Iwamuro et al. 1985; Totsuka and Usui 1986; Tangarone et al. 1989). The complete inhibition of the endo-P- 1,3-glucanase of the monokaryon protoclone B No. 5 by 1 mM Hg" suggests the requirement of sulfhydryl groups for the enzymic hydrolysis of laminarin. Finally, the monokaryotic S. commuize strain protoclone B No. 5 is one of the rare rnicro- organisms that constitutively secretes only an endo- and no exo-P- 1,3-glucanase.

Acknowledgment This work was supported by a grant (03-8807) from the

Bundesminister fur Forschung und Technologie, Germany.

Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein- dye binding. Anal. Biochem. 72: 248-254.

Clark, D.R., Johnson, J., Jr., Chung, K.H., and Kirkwood, S. 1978. Purification, characterization, and action-pattern studies on the endo-1,3-0-D-glucanase from Rhizoprts ar-r-hizus QM 1032. Carbohydr. Res. 61: 457-477.

Goldstein, I.J., Hay, G.W., Lewis, B.A., and Smith, F. 1965. Controlled degradation of polysaccharides by periodate oxidation, reduction, and hydrolysis. Methods Carbohydr. Chem. 5: 361-369.

Iwamuro, Y., Aoki, M., and Mikami, Y. 1985. Purification and some properties of an endo-0- l,3-glucanase from Por.odisc~rlus pendulus. J. Ferment. Technol. 63: 61-66.

Kikumoto, S., Miyajima, T., Yoshizumi, S., Fujimoto, S., and Kimura, K. 1970. Polysaccharide produced by Schizophyllunz commune. Part I . Formation and some properties of an extracellular polysaccharide. Nippon Nogei Kagaku Kaishi, 44: 337-342.

Kikumoto, S., Miyajima, T., Kimura, K., Okubo, S., and Komatsu, N. 1971. Polysaccharide produced by Scl~i-.o~~hyllnm conmzutle. Part 11. Chemical structure of an extracellular polysaccharide. Nippon Nogei Kagaku Kaishi, 45: 162-168.

Kottutz, E., and Rapp, P. 1990. 1,3-0-Glucan synthase in cell-free extracls from mycelium and protoplasts of Sclerotium ghlcar~icun~. J. Gen. Microbial. 136: 1517-1523.

Miinzer, S. 1989. Produktion and Charakterisierung eines von Schizo- pl~yllum cornmutle ATCC 38548 gebildeten extracellularen 0-1,3- Glucans. Ph.D. thesis, Institut fur Biochemie und Biotechnologie Technische Universitat Braunschweig, Braunschweig, Germany.

Prokop, A., Rapp, P., and Wagner, F. 1992. Production of extracellula~ P-1,3-/j3-1,6-glucan by mono- and dikaryons of Schizophyll~tn commztne. Exp. Mycol. 16: 197-206.

Can

. J. M

icro

biol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

Uni

vers

ity o

f P.

E.I

. on

11/1

6/14

For

pers

onal

use

onl

y.

PROKOP ET AL. 23

Raper, J.R., and Hoffman, R.M. 1974. Schizophyll~rrn commune. In Handbook of genetics. Edited by R.C. King. Plenum Press, New York, London. pp. 597-626.

Saito, H., Ohki, T., and Sasaki, T. 1979. A llC-nuclear magnetic resonance study of polysaccharide gels. Molecular architecture in the gels consisting of fungal, branched 1,3-P-D-glucans (lentinan and schizophyllan) as manifested by conformational changes induced by sodium hydroxide. Carbohydr. Res. 74: 227-240.

Schulz, D., and Rapp, P. 1991. Properties of the polyalcohol prepared from the P-~-glucan schizophyllan by periodate oxidation and borohydride reduction. Carbohydr. Res. 222: 223-23 1.

Sietsma, J.H., and Wessels, J.G.H. 1977. Chemical analysis of the hyphal wall of Sch~zophyllurn comnzune. Biochim. Biophys. Acta, 496: 225-239.

Sietsma, J.H., and Wessels, J.G.H. 1979. Evidence for covalent linkages between chitin and P-glucan in a fungal wall. J. Gen. Microbiol. 114: 99-108.

Somogyi, M. 1952. Notes on sugar determination. J. Biol. Chem. 195: 19-28.

Tabata, K., Ito, W., and Kojima, T. 1981. Ultrasonic degradation of schizophyllan, an antitumor polysaccharide produced by Schizo- phyllum commune Fries. Carbohydr. Res. 89: 121-135.

Tangarone, B., Royer, J.C., and Nakas, J.P. 1989. Purification and characterization of an endo-1,3-P-D-glucanase from Trichoderma longihrachiaturn. Appl. Environ. Microbiol. 55: 177-1 84.

Totsuka, A,, and Usui, T. 1986. Separation and characterization of the endo-P- l ,3-D-glucanase from Rhizoctot~ia solani. Agric. Biol. Chem. 50: 543-550.

Wessels, J.G.H. 1969a. A P- 1,6-glucan glucanohydrolase involved in hydrolysis of cell-wall glucan in Schizophyll~rm commune. Biochim. Biophys. Acta, 178: 191-193.

Wessels, J.G.H. 1969h. Biochemistry of sexual morphogenesis in Schizophyllum commune: effect of mutations affecting the incornpati- bility system on cell-wall metabolism. J. Bacterial. 98: 697-704.

Wessels, J.G.H., Kreger, D.R., Marchant, R., Regensburg, B.A., and De Vries, O.M.H. 1972. Chemical and morphological characteri- zation of the hyphal wall surface of the basidiomycete Schizophyllum commune. Biochim. Biophys. Acta, 273: 346-358.

Can

. J. M

icro

biol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

Uni

vers

ity o

f P.

E.I

. on

11/1

6/14

For

pers

onal

use

onl

y.