Purification and properties of an endo-α-mannan hydrolase from the mushroom Volvariella volvacea

ARCHIVES

Vol.

OF BIOCHEMISTRY

AND

241, No. 2, September,

Purification

BIOPHYSICS

pp. 533-539,

and Properties of an Endo-wmannan Hydrolase from the Mushroom Vo/variel/a volvacea SUMAN

Inditzn

1985

Institute

KHOWALA

of Chemical

Received

Biology,

December

AND

4 Raja

S. SENGUPTA’

S.C. Mullick

3, 1984, and in revised

Road, form

Calcutta-700

April

032, India

29, 1985

The enzyme, endo-a-mannanase, from culture filtrate of a mushroom Volvariella has been purified 73-fold by acetone precipitation, ion-exchange chromatography (DEAE-Sephadex), and gel-permeation chromatographies on Bio-Gel P-300 and on Sephacryl S-200 columns. The enzyme preparation gave a single protein band on sodium dodecyl sulfate-disc gel electrophoresis at pH 6.8 and has a molecular weight of approx. 56,000. It has no CY-or /3-mannosidase activity and does not act on p-glucoor galactomannan. The enzyme shows maximum activity on baker’s yeast a-mannan at pH 5.0 and at 55”C, and is fairly stable between pH 3 and 6 and temperatures up to 50°C. The Km is 32.25 mg manna&ml. Enzyme activity is inhibited by H$+, sodium azide, iodoacetic acid, EDTA, and Ag+, in decreasing order. o 1985 Academic press, inc. volvacea

Endo-a-mannanase splitting of a-linked mannosidic linkages may be developed into an interesting biochemical tool either to modify or to study different glycopeptides and glycoproteins of biological interests, but no such enzyme has yet been purified from any source. The production and characterization of an inducible endo-a-mannanase in the culture filtr,ate of the mushroom, Volvariella volvacea, has been reported (1). The present paper describes the purification and properties of the enzyme. MATERIALS

AND

pepsin, and fl-lactoglobulin) were purchased from Sigma Chemical Company. Bio-Gel P-300 and Sephacryl S-200 were the products of Bio-Rad Laboratories, and Pharmacia Fine Chemicals, respectively. Iodoacetie acid and EDTA were purchased from E. Merck (Darmstadt, FRG). Other chemicals used were of chemically pure quality. The synthetic medium and other fermentative conditions for the optimum production of enzyme by I? volvacea have been reported earlier (1). Fermentation was conducted in shaker flasks and was continued for 4 days at 30 + 1°C. Enzyme assay. Mannanase activity was assayed and enzyme activity was expressed as described earlier (1). Liberation of reducing groups by the enzyme in a reaction mixture (0.4 ml) containing 25 mg mannan in 0.1 M acetate buffer (pH 5.0) incubated for 4 h at 40°C was measured. Protein and carbohydrate determination. Protein was estimated according to Lowry et al (2) using bovine serum albumin as standard. Carbohydrate was estimated with the orcinol-HaSO, reagent according to Brown and Anderson (3), using yeast mannan as standard. Polyacrylamide gel electrqphoresis. Gel electrophoresis was carried out in 0.02 M phosphate buffer, pH 6.8, using 5 and 10% acrylamide (4, 5). A constant current of 1.5 mA per gel (7 cm) was applied for 6 h at 25°C. Gels were stained overnight with Coomassie brilliant blue and destained with methanol/ acetic acid/water.

METHODS

a-Mannan (bsaker’s yeast), pnitrophenyl-or-n-mannopyranoside, pnitrophenyl-B-D-mannopyranoside, 1-0-methyl-cy-o-glucopyranoside, l-O-methyl-@-Dglucopyranoside, 1-O-methyl-a-D-galactopyranoside, 1-0-methyl-@-rl-galactopyranoside, DEAE-Sephadex (A-50), locust bean gum, guar gum, carboxymethylcellulose (low viscosity), sodium dodecyl sulfate (SDS): Cooma.ssie brilliant blue, N-W-methylenebisacrylamide, acrylamide, and molecular weight marker proteins (bovine serum albumin, ovalbumin,

’ To whom correspondence * Abbreviation used: SDS,

should sodium

be addressed. dodecyl sulfate. 533

0003-9861/85 Copyright All rights

$3.00

Q 1995 by Academic Press, Inc. of reproduction in any form resewed.

534

KHOWALA

AND

SDS-gel electrophoresis of 2-mercaptoethanoltreated enzyme was done according to Laemmli (6) using both 5 and 10% polyacrylamide gels. A constant current of 1.5 mA per gel (11.5 em) was applied for 5 h at 25°C. Purijkation of a-mannanose. This was carried out at 4°C unless otherwise specified. The filtered broth (4 1) was cooled to 0°C. Three volumes of prechilled (-20°C) acetone was added in a fine stream to lliter hatches of culture filtrate with thorough mixing for precipitation of mannan. The gummy precipitate retaining enzyme protein was quickly recovered, initially by decantation and then by centrifugation of the aqueous acetone mixture. The precipitate was then dissolved in 1 liter of 0.01 M acetate buffer, dialyzed in the same buffer and lyophilized to 50 ml. The enzyme solution was then applied to a DEAEgel column (4.5 X 40 cm), preequilibrated with 0.01 M buffer (pH 5.0) and washed with the same buffer at a flow rate of 20 ml/h. The enzyme fraction (637 ml) eluted from the ionexchange column by application of a buffered NaCl gradient (0-0.8~) was concentrated to 23 ml by lyophilization. The solution was then equilibrated against 0.1 M acetate buffer and applied to a BioGel P-300 column (2.3 X 44 cm). The column was then eluted with the same buffer at a flow rate of 4 ml/h. The active fraction (57.5 ml) eluted from a BioGel P-300 column was concentrated to 8 ml and dialyzed at 25°C against 0.1 M acetate buffer (pH 5.0), containing 1% (w/v) SDS. The dialyzed solution was then subjected to gel chromatography on a Sephacryl S-200 column with 1% (w/v) SDS buffer as eluant. a-Mannanase activity was eluted in two fractions. Excess KC1 was added to enzyme fractions I (96 ml) and II (76 ml) to precipitate SDS as the K salt, and supernatants were exhaustively dialyzed. Studies wz the properties of th,e pw-i~ed enzyme. Fraction II from the Sephacryl S-200 column was used as the source of purified a-mannanase. Temperature and activity profile. Optimum temperature for the hydrolysis of mannan by the purified enzyme was determined by incubating 0.07 pg of purified enzyme with 40 mg mannan in 0.1 M acetate buffer (pH 5.0) at different temperatures. Thermal stability of the enzyme in the same buffer was determined by assaying the residual activity at 40°C of the enzyme solution (0.7 pg/ml) preincubated for 1 h at different temperatures (20~80°C). pH and activity pro&e. Buffers used (0.1 M): KCIHCl (pH l.O-2.0), phthalate-HCl (pH 2.5-4.0), phthalate-NaOH (pH 4.5-6.0), and Tris-HCl (pH 7.0~8.0), to determine optimum pH for the enzyme action on mannan. For determination of pH stability, enzyme was preincubated for 1 h at different pH values and then assayed at pH 5.0. Lktermination of depolymerizing activity. Depoly-

SENGUPTA merizing action of the enzyme on mannan was assayed in an Ostwald viscometer according to Hylin and Sawai (7). The incubation mixture (2.5 ml) contained 0.258 unit of enzyme in 12.5% mannan solution in acetate buffer. Assay of yeast cell wall lytic activity. Cell wall was prepared from baker’s yeast according to Mill (8). Cell wall suspension (-2 mg carbohydrate) was incubated with 0.258 unit of enzyme in 0.25 ml buffer at 40°C for 10 and 40 min. Incubation mixtures were then centrifuged at 10,OOOg and supernatants were assayed for carbohydrate content. RESULTS

Puti&cation of mnnanase. Table I represents the purification of the enzyme from the culture filtrate of the mushroom V: volvacea. The enzyme could not be recovered by ammonium sulfate precipitation, as an insoluble precipitate was always formed above 30% saturation of the salt. Although enzyme activity was present in the suspended precipitate, it could not be released in the supernatant by the alteration of pH and ionic strength of the buffer. However, mannanase was coprecipitated with mannan present in the culture filtrate by acetone, and 50% enzyme activity was recovered in this step with 4-fold purification. The enzyme was further purified 19-fold by ion-exchange chromatography on DEAE-Sephadex gel using a NaCl gradient. As the enzyme fraction, on molecular seiving on Bio-Gel P-300 column, attained slightly higher specific activity, it was assumed that the eluted fraction probably contains little or no nonenzyme protein. The Bio-Gel fraction showed a single band in gel electrophoresis at pH 6.8 (Fig. 2a), but resolved into five bands on SDS-gel electrophoresis at pH 8.3 (Fig. 2b). In addition, the fraction has hydrolytic activity on a number of unrelated polysaccharides (Table III). Thus, it was assumed that the enzyme has probably been eluted from Bio-Gel P300 as a single mannan-protein complex containing other enzyme proteins. The complex appears homogeneous in gel electrophoresis but dissociates only under denaturing conditions. As 1% SDS in the incubation mixture was found not to be inhibitory for mannanase activity, gel fil-

ENDO-o-MANNAN

HYDROLASE

FROM

TABLE

VolvatieZlu

535

volwaceu

I

PURIFICATIONOF MANNANASEFROMTHE CULTURE FILTRATEOF V volvacea

Enzyme Step

sample

Protein (w)

Total (units

Specific activity (units per mg protein)

activity X lo-*)

Recovery yield

1

Culture (4000

filtrate ml)

955

49.00

Step 2

Acetone (1000

precipitate ml)

115.5

23.906

20.69

48.78

Step 3

DEAE-Sephadex (A-50) fractions (75-165) (637 ml)

16.28

16.10

98.89

32.85

19.27

Step 4

Bio-Gel (P-300) fractions (36-58) (57.5 ml)

8.0

9.85

123.125

20.12

23.99

Step 5

Seph,acryl fraction (96 ml)

4.2

5.55

132.14

11.32

25.94

0.636

2.37

372.64

4.83

72.628

S-200 I (13-36)

Seph,acryl S-200 Fraction II (4260) (76 ml)

tration of the enzyme complex in SDS buffer on Sephacryl S-200 was tried. It is evident from Fig. 1 that mannanase activity was resolved into high- (I) and lowmolecular-weight (II) fractions. Mannanase (I) was found to be identical with the Bio-Gel P-300 fraction having similar electrophoretic mobility (Fig. 2b) and activity profile (Table III). On the other hand, there was about a 3-fold increase in specific activity of the enzyme eluted in the mannanase (II) fraction. This fraction gave a single band on gel electrophoresis at pH 6.8 (Fig. 2~) and also under denaturing lconditions (Fig. 2d). No mobility of the enzyme protein was observed at pH 4.5 or 8.3. Physicochemical properties of the purijkd enzyme. The enzyme exhibits optimum hydrolytic activity on yeast mannan in 0.1 M acetate buffer (pH 5.0) at temperatures around 55°C. About 30 and 60% of the maximum activity were observed at 10 and 80°C under the same experimental conditions. The enzyme is relatively thermostable, not losing any activity by 1 h preincubation at 50°C in the same buffer.

5.1308

Purification (fold)

100.0

1.0

4.032

But, activity is completely lost when temperature is increased from 60 to ‘70°C. However, the enzyme is stable at room

30 -

- 0.3

0 IO

20 Frooion

30 number

40 l4ml

50

60

70

/tube1

FIG. 1. Sephacryl S-200 gel filtration of a-mannanase: ---, mannanase activity; -, protein as determined from Am. The concentrated and dialyzed BioGel,P-300 fraction (8 ml) was loaded on the column (1.8 X 4.8 cm) of Sephacryl S-200 equilibrated with 0.1 M acetate buffer, pH 5.0, containing 1% (w/v) SDS. Elution was accomplished with the same buffer at a flow rate of 40 ml/h.

536

a

KHOWALA

b

AND

d

FIG. 2. Polyacrylamide gel electrophoresis of (Ymannanase. (a) Electrophoretogram of the enzyme from Bio-Gel P-300 chromatography on 5% polyacrylamide gel. (b) Electrophoretogram of the reduced and denatured Bio-Gel P-300 enzyme on 5% polyacrylamide gel with 0.1% SDS. (c) Electrophoretogram of the purified enzyme (fraction II from Sephacryl S-200 chromatography) on 10% polyacrylamide gel. (d) Electrophoretogram of the reduced and denatured enzyme (fraction II from Sephacryl S-200 chromatography) on 10% polyacrylamide gel with 0.1% SDS.

SENGUPTA

lose, CM-cellulose, dextran, polyglucuronic. acid, arabinogalactan, glucomannan (guar gum), and galactomannan (locust bean gum) are not attacked by the enzyme (Table III). The purified enzyme has neither mannan depolymerizing nor yeast cell wall lytic activity. No appreciable reduction in the viscosity of mannan solution was noted by the action of enzyme until 10 h of incubation. Similarly, no carbohydrate was liberated by the action of enzyme on yeast cell wall. The rate of liberating reducing groups from yeast mannan by the enzyme was found to be more or less exponential up to 6 h of incubation. Beyond this period the rate of hydrolysis is lowered gradually and stops after 8 h of incubation. Km and I’,,,,, values determined from LineweaverBurk plots (Fig. 4) were found to be 32.25 mg mannan/ml and 21.31 X lo-’ pmol mannose min-l pg enzyme-l, respectively. The developed paper chromatogram (Fig. 5) of the incubation mixtures (4-48 h) indicates that mannose is not present in any mixture. The number of smaller oligosaccharides in the hydrolysate inTABLE

II

EFFECTOF SOME METAL IONSAND INHIBITORSON MANNANASE ACTIVITY Residual

temperature (30°C) and also toward lyophilization, freezing and thawing. a-Mannanase exhibits hydrolytic activity on mannan in the acid pH range only, being maximal at pH 5.0 (Fig. 3), and also could retain 55% activity even at pH 2.0. The enzyme is also fairly stable over a pH range of 3 to 6. H2+ and Ag+ were found to be potent inhibitors whereas Cu2+, Zn2+, Ca2+, and Mgz+ have no effect on enzyme activity. NaN3, EDTA, and CH21COOH inhibit enzyme activity at 20 IYIM concentrations (Table II). The enzyme has no CY-or @-mannosidic, glucosidic, or galactosidic activity. Cellu-

Chemicals

Hg2+ &+ Caz+ cl?+ Znz+ Mg’+ NaN3 CHJCOOH EDTA

2mM

5 75.5 100 100 100 100 9.5 35.0 36.5

activity

20mM 0 2.0 70.9 76.5 80.0 67.5 0 0 0

Note. Activity was assayed by incubating 0.0258 unit of enzyme in 0.4 ml (final volume) containing 0.1 M acetate buffer (pH 5.0), 25 mg mannan, and the chemicals at 40°C for 4 h and measuring the reducing group liberated following the method already described (1).

ENDO-a-MANNAN TABLE SPECIFIC

HYDROLASE

FROM

Volvarielh

537

volvacea

III

ACTIVITIES OF I! volvacea MANNANASE DIFFERENT SUBSTRATES

ON

Specific activity of (Ymannanase from

Substrate

Bio-Gel P-300 fraction

(25 mg/ml)

Sephacryl s-zoo fraction

I -I/K,

Carboxymethylcellulose Dextran Polyglucuronic ,acid Arabinogalactan Mannan

50.3 20.2 35.35 15.2 123.125

0 0 0 0 372.64

Note. The enzyme shows no hydrolytic activity with 1-0-methyl-ol-D-glucopyranoside, l-o-methylfl-D-glucopyranoside, 1-O-methyl-cu-D-galactopyranoside, l-O-methyl-fi-D-galactopyranoside, *pnitrophenyl-a-D-mannopyranoside, glucomannan, galactomannan, or *p-nitrophenyl-fl-D-mannopyranoside. Different glycosides and carbohydrates were incubated with 0.07 ,ug of enzyme protein in 0.1 M acetate buffer, pH 5.0, lper incubation mixture for 4 h, and activity was assayed in the usual way. Specific activity is expressed in terms of mannose equivalents produced per milligram of enzyme protein per hour. *Mannosidic ac:tivity was assayed using pnitrophenyl-a-D-mannopyranoside and p-nitrophenyl-flD-mannopyranoside as substrates according to methods described earlier (1).

creases with time, but two spots just following mannose were found to be common in all enzymatic digests. Carbohy-

0 1% 3

5 Pf-

7

9

FIG. 3. pH optima for a-mannanase action. The stock enzyme was diluted with respective buffers of different pH. Activity was assayed by using 0.07 pg of enzyme protein under standard assay conditions.

0

0.02

0.04

0.06

0.08

I/CSl(m~‘xml)

FIG. 4. Lineweaver-Burk plot. Variable amounts (5-80 mg) of mannan were incubated for 4 h with 0.0258 unit of enzyme, and liberation of reducing groups was measured.

drate spots could not be identified as authentic oligosaccharide samples were not available. Molecular weight and subunits of mannanase. The molecular weight of a-man-

nanase as determined from the relative mobility of different standard proteins on SDS-electrophoresis was approximated to be 56,000. The enzyme gave a single protein band (Fig. Zd), indicating that it contains similar or no subunits. DISCUSSION

Endoglycosidases active on different glycoproteins have been studied (g-14), but no endo-a-mannanase was available for the purpose. An extracellular inducible endo-cY-mannanase was characterized in the culture filtrate of the mushroom, F/: volvacea (1). During the process of purification of enzyme from the culture filtrate, ammonium sulfate precipitation of the enzyme was not successful, but coprecipitation of the enzyme with mannan by acetone recovered about 50% of culture filtrate activity. Yeast invertase, which is similarly precipitated with mannan by acetone from cell lysate, is believed to be covalently linked with mannan (4). Recovery of mannanase with mannan may be accounted for its attachment with substrate or due to its coprecipitation. The enzyme purified by ion-exchange and Bio-Gel P-300 chro-

538

KHOWALA

Monnose --r;-:-:

48h

24h

a E? 0

c

Line Of opplicolion

-

fr0n1

4h

AND

n

u G

0 v 0 0 0 0 0 0 u SOlVeIll

FIG. 5. Descending paper chromatogram of the enzyme-hydrolyzed products of mannan (baker’s yeast) by V volvacea a-mannanase. Hydrolysis was carried out by incubating 2.58 units of enzyme in 2 ml of 0.1 M acetate buffer, 150 mg mannan, at 40°C. Undigested mannan was precipitated with 3 vol absolute ethanol, and clear supernatants were lyophilized. The solvent system used was ethyl acetate/ pyridine/water (5/3/2, v/v) (24). Spots of carbohydrates were detected by the silver nitrate-sodium hydroxide reagent (25), using mannose as a reference sugar.

matography appears to be a high-molecular-weight protein which is incapable of migrating in both 10 and 7.5% polyacrylamide gels under disc electrophoresis at acid (4.5) and alkaline (8.3) pII’s. However, this fraction gave a single band in a 5% gel at pH 6.8, but under denaturing conditions resolved into five bands (Fig. Zb). The fraction also showed hydrolytic activities on a number of unrelated polysaccharides (Table III). So, it is supposed that the fraction is a mannan-protein complex containing different enzymes but not an oligomeric single enzyme. We tried to liberate mannanase from the complex under partial denaturing condition not

SENGUPTA

affecting enzyme activity. The complex resolved into high- and low-molecularweight mannanases under Sephacryl S200 gel chromatography in the presence of 1% SDS. The low-molecular-weight mannanase was characterized as a single polypeptide of approximately M, 56,000, and is devoid of other carbohydrase activities as found in the Bio-Gel fraction (Table III). Thus, it appears that yeast mannan in the medium forms a heterogenous mannan-protein complex with different enzyme proteins, including mannanase, and that the complex does not resolve even under electrophoretic conditions. It is to be pointed out that a number of enzymes, viz., a-glucosidase (15), alkaline phosphatase (15), and invertase (16) isolated from yeast lysate were reported to be mannoproteins, and invertase was specifically postulated to be a glycosidically mannan-linked enzyme (4). However, according to Nickerson et al. there is some evidence that mannan-protein complex is present in yeast cell wall, but it is not established that they form a covalently linked unit (17). The enzyme purified has relatively high Km and low V, values with yeast mannan as substrate. It may be assumed that either the enzyme is not equally active on different mannosidic linkages present in native mannan or its action is being hindered by the highly ramified structure of the polysaccharide. An endo c~(l6)mannanase which is inactive on native mannan has been reported (18). Yeast mannan may also possess some blocked structure within the molecule which is inaccessible to enzyme action, similar to that reported in konjac mannan for pmannanase action (19). The enzyme does not have lytic action similar to other cell wall lytic endoglycosidases (20-22). It is incapable of depolymerizing mannan or of liberating carbohydrate from intact yeast cell wall, as reported for phosphomannanase from Bacillus circulars (23). However, the enzyme liberates manno-oligodextrins from native

ENDO-a-MANNAN

HYDROLASE

yeast mannan. Thus, the enzyme may be considered as an endo-a-mannanase first purified from any source. ACKNOWLEDGMENTS The authors sre grateful to Professor B. K. Bachhawat, Director of the Institute, for his kind interest and valuable suggestions during the progress of the work. A fellowship grant to SK. by the Council of Scientific and Industrial Resarch (India) is gratefully acknowledged. REFERENCES 1. KHOWALA, S,, AND SENGUPTA, S. (1984) Canad J. Microbial. 30, 657-662. 2. LOWRY, 0. II., ROSEBROUGH, N. J., FARR, A. L., AND RANIDALL, R. J. (1951) J. Biol Chem 193, 265-275. 3. BROWN,

W., AND ANDERSON, 0. (1971) J. Chre matogr. 57,255-263. 4. NEUMANN, N. P., AND LAMPEN, J. 0. (196’7) Bb chemistrg 6.468-475. 5. GASCON, S., NEUMANN, N. P., AND LAMPEN, J. 0. (1968) J. Biol. Chem 243, 1573-1577. 6. LAEMMLI, U. K. (1970) Nature &md,on) 227,680685. 7. HYLIN,

8. 9. 10. 11.

J. W., AND SAWAI, K. (1964) J. Biol. Chem. 239, 990-!)92. MILL, P. J. (1966) J. Gen Microbial. 44, 329-341. TAKASAKI, S., AND KOBATA, A. (1976) J. BioL Chem 2511, 3610-3615. YAMASHITA, K., TACHIBANA, Y., TAKASAKI, S., AND KOB~TA, A. (1976) Nature 262, 702-703. MURAMATSU, T., KOIDE, N., CECCARINI, G., AND

FROM

Volvariella

ATKINSON,

539

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P. H.

(1976)

J. Biol.

Chem

251,

4673-4679.

12. ITO, S., YAMASHITA, K., SPIRO, R. G., AND KOBATA, A. (1977) J. BiocAem 81. 1621-1626. 13. TAI, T., YAMASHITA, K., ITO, S., AND KOBATA, A. (1977) J. BioL Chem, 252, 6687-6698. 14. TAI, T., ITO, S., YAMASHITA, K., MURAMATSU, T., AND KOBATA, A., (1975) B&hem. Biophys. Res. Commun 65, 968-974. 15. LAMPEN, J. 0. (1968) Antonie van leeuwenhock 34, 1-18. 16. LAMPEN, J. 0. (1971) in The Enzymes (Boyer, P. D., ed.), Vol. 5, pp. 291-305, Academic Press, New York. 17. NICKERSON, W. J., FALCONE, G., AND KESSLER, G. (1961) Symp. Sot. Gen Physiol. 205, 18. 18. NAKAJIMA, T., AND BALLOU, C. E. (1974) in Advances in Enzymology (Meister, A., ed.), Vol. 40,

pp. 262-264.

19. SUGIYAMA, N., SHIMAHARA, H., ANDOH, T., AND TAKEMOTO, M. (1973) Agric Biol CRAWL (Tokyo) 37, 9-17. 20. TORRIANI, A., AND PAPPENHEIMER, A. M. (1962) J. Biol Chem 237, 3-13. 21. BENDER, W., MOLBERT, F. E., KNIFFERMANN, H., RUDOLPH, C., THUROW, H., AND STIRM, S. (1973) Virology 56, 134-149. 22. THUROW, H., NIEMANN, H., AND STIRM, S. (1975) Carbohydr. Res. 41,257-271. 23. MCLELLAN, W. L., JR., MCDANIEL, L. E., AND LAMPEN, J. 0. (1970) J. Bacttiol 102, 261269. 24. JONES, G. H., AND BALLOU, C. E. (1969) J. Biol Chem. 244, 1943-1951. 25. ANET, E. F. L. J., AND REYNOLDS, T. M. (1954) Nature (London) 174, 930.