A unique polysaccharide- and glycoside-degrading enzyme complex from the wood-decay fungus Poria placenta

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 97, No. 4, 1980

Pages 1499-1504

December 31, 1980

A UNIQUE POLYSACCHARIDE-

AND GLYCOSIDE-DEGRADING

COMPLEX FROM THE WOOD-DECAY

ENZYME

P o ~ a placenta

FUNGUS

KARL E. WOLTER, TERRY L. HIGHLEY, and FAYE J. EVANS, Forest Products Laboratory, USDA Forest Service, Box 5130, Madison, Wisconsin 53705.

Received November 12, 1980 SUMMARY A multifunctional enzyme, active on both polysaccharides and glycosides, has been isolated from the wood-decay fungus P. placenta. A molecular weight of 185,000 Dalton was determined with lower molecular weight subunits characterized on dodecyl sulfate-polyacrylamide gel electrophoresis. An unusual isoelectric point of 1.8 was established for this protein. INTRODUCTION The objective of this research was to identify the properties cellular polysaccharidewood-decay

fungus,

Poria placenta.

carboxymethylcellulose and B-D-xyloside.

and glycoside-degrading

of the extra-

enzymes produced by brown-rot

The fungus can hydrolyze xylan, mannan,

(CMC), ~- and B-D-glucoside,

In a previous paper

~- and B-D-galactoside,

(i), the polysaccharide-degrading

enzyme

activities present in culture filtrates of the fungus were studied and the effects of culture conditions

on their production determined.

These enzymes of

P. placenta differ from those of most fungi in that their production is not repressed by simple sugars.

MATERIALS AND METHODS Culture and Preparation of Crude Extracellular Enzymes

Poria placenta (Fr.) Cke. (Madison 698) was grown in stationary culture on a previously described basal salts medium (2) containing 0.1% galactomannan (Sigma), 0.5 % Solka-Floc (Brown), 0.5% glucose, and 0.5% milled hemlock wood [Tsuga hereto The Laboratory Wisconsin.

is maintained in cooperation with the University of

The U.S. Government's right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged.

0006-291X/80/241499-06501.00/0 1499

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BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

phylla (Raf.) Sarg.]~/.

After sterilization at 121 ° C for 15 minutes, flasks with medium were inoculated with washed mycelial suspension precultured on the basal medium containing 1% glucose and incubated at 27 ° C in the dark for 30 days. Mycelial mats were separated by suction filtration through glass filter paper and eluted in 0.1M, pH 5.0 acetate buffer. This solution was concentrated by hollow fiber P-10 (Amicon) ultrafiltration and further concentrated and dialyzed with Schleicher and Schuell membranes--lO,000, 25,000, or 75,000 molecular weight (MW) cutoff. All manipulations were carried out at room temperature: enzyme activities were not affected by ambient conditions or by repeated freezing and thawing. Enzyme Assays Polysaccharidase activities were assayed by increase in reducing groups at 40 ° C (3) as described previously (i) (Table i). Substrates used were xylan (NBC), CMC (Hercules), galactomannan (Sigma), and glucomannan [from loblolly pine, Pinus tadea L., isolated by R. Scott, FPL (unpublished)]. A unit of enzyme activity was defined as the amount needed to liberate reducing power equivalent to 1 micromole (~M) of glucose per hour at 40 ° C. ~-D-galactosidase, B-D-galactosidase, ~-D-glucosidase, B-D-glucosidase, and B-D-xylosidase activities were assayed by determining the liberation of p-nitrophenol from respective p-nitrophenol substrate as previously described (3). A unit of enzyme activity was defined as the amount liberating 1 ~M of p-nitrophenol per hour at 40 ° C. Total protein was determined by the method of Lowry (4). Thin-Layer

Isoelectric Focusing

(TLIEF)

Thin-layer isoelectric focusing was performed with a Desaga Double Chamber apparatus. Preparative (prep) plates were prepared with Sephadex G-200 (Pharmacia). Carrier ampholytes (LKB) incorporated were in the 3-5 pH range. Lower ranges of 2-4 pH were initially obtained from Brinkmann, while subsequent runs contained ampholytes from Serva and LKB. Purified enzymes from prep plates were further defined on a Sephadex G-75 gel plate containing appropriate ampholytes. A low temperature circulator maintained the chamber at 5 ° C during focusing. Samples were focused for approximately 20 hours at a constant voltage of 250V with additional focusing at 500V for 2 hours. Protein bands were located with 2% Coomassie blue R 250. The pH of the protein bands was determined directly on the gel plates with a microcombination pH probe (Microelectrodes Inc.). Gels containing the protein bands were removed from the plates and the protein eluted with 0.1M, pH 5 acetate buffer. Ampholyte contaminants from this procedure were removed via collodion filtration (75,000 MW cutoff) before determinations for activity were made° Molecular Weight Determinations Molecular weight of the TLIEF-purified protein was determined by gel chromatography according to the procedure suggested by Pharmacia Fine Chem. Co. The protein solution was applied to a 43 x 1.5 cm equilibrated column of Ultragel AcA 34 and eluted with degassed phosphate buffer, pH 6. Fractions were collected and assayed for enzyme activity, and molecular weight was determined by comparison of elution volumes with those of standard proteins. ~/ The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the U.S. Department of Agriculture of any product or service to the exclusion of others which may be suitable.

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Table l.--Polysaccharidase and $1ycosidase activities in crude~ collodion~ and TLIEF eluate

Specific enzyme activity (units/mg proteir~I/) Substrate

Enzyme

Crude Collodion TLIEF extract concentrate eluate dialyzed .............................................................................. Xylan

Xylanase

4.4

7.3

3.5

Glucomannan

Mannanase

2.5

6.0

1.8

Carboxymethylcellulose

Cellulase

1.4

2.1

0.8

~-D-Glucoside

B-D-Glucosidase

7.2

13.1

~-D-Glucoside

~-D-Glucosidase

1.3

5.8

B-D-Galactoside

B-D-Galactosidase

18.7

61.9

29.6

e-D-Galactoside

~-D-Galactosidase

25.7

61.9

35.1

B-D-Xyloside

B-D-Xylosidase

1.9

7.6

0.26 0.095

0.17

!/One unit of enzyme activity is the amount which will release i ~M of p-nitrophenol or reducing equivalents to 1 ~M of glucose per hour at 40°C.

Additional molecular weight of the TLIEF-purified protein was determined by an alternate method of sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis by comparing the relative electrophoretic mobility (Rf) with those of protein standards of known MW's (5). Low MW (14,300 to 94,000 D) and high MW (34,000 to 200,000 D) natural proteins were from Bio-Rad. Purified enzyme and standards were denatured in a 0.025M Tris buffer (pH 7.4) with 1% mercaptoethanol and heated for 5 minutes at i00 ° C. Standard and sample aliquots (I00 ~i) were applied to a vertical (EC) discontinuous polyacrylamide slab of Cyanagum 41 (95% acrylamide and 5% bisacrylamide) (6,7). The gel consisted of 4% spacer gel (0.1M, pH 6.7) and a 10% running gel (0.5M, pH 8.9). The buffer was Tris/glycine (O.04M, pH 8.3). Gels were run at 200 milliamperes (mA) until samples'were stacked and then 3 hours at 300 mA, which effected adequate separation. Gels were stained with a 2% Coomassie blue R 250 stain in a methanol-water-acetic acid solution (4.5:4.5:1.0 v:v:v) and destained with water-methanol-acetic acid (6.75:2.5:0.75 v:v:v) until protein bands were clear. RESULTS AND DISCUSSION Liquid column chromatography Sephadex 200, hydroxylapitite,

separations with Sepharose 6-B, Sephadex I00, DEAE Bio-Gel A, and Ultragel AcA 34 were un-

successful in separating ~-D-galactosidase, ~-D-glucosidase,

B-D-xylosidase,

xylanase,

procedures were equally unsuccessful.

B-D-galactosidase,

CMCase, or mannanase.

Therefore,

purification.

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~-D-glucosidase, Other standard

TLIEF was used in subsequent

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Prep samples initially were run on wide-range ampholytes which showed all the enzyme activities to be in one or two bands at the low end of the pH gradient (about 3.0).

Thus, pH 2-4 and 3.5-5 ampholytes were used in subsequent TLIEF

experiments. Protein samples from prep plates were spotted on analytical gels. enzyme activities assayed

All the

(Table I) were localized in one protein band.

When

pH 2-4 ampholytes were used, the band was in the same location for all replications at a pl of approximately complex from knowledge,

1.8.

This very low pl for the carbohydrolase

Poria placenta appears to be quite unusual.

To the authors'

such a low pl has never been reported for any other fungal enzyme

prior to this study, although a nonenzyme acid glycoprotein of pl 1.8 has been reported from human serum and pepsin has been documented at pl 2.2 from pig (8). Enzyme activities

(Table i) indicate that all activities sought were in the

crude dialyzed extract.

Purification

membrane indicated substantially

and concentration

on a 75,000 MW collodion

increased activity for all enz~nes.

Further

purification with TLIEF should have increased specific activity on a protein basis; however,

this was not the case.

Even though there was a decided reduction

in activity of TLIEF, the enzyme was still present.

The reduction of specific

activity of TLIEF has been reported by Richards and Shambe five-fold loss on repeated isoelectric

(9), who found a

focusing of glucosidases,

and stated that

the enzyme is "extensively deactivated most probably during the time spent at its isoelectric point," presumably due to conformational

In preliminary

changes.

separation for molecular size, ultrafiltration

of the crude

protein indicated that all the various enzyme activities were in a fraction greater than 100,000 D.

Further determinations

via comparisons with standard proteins, 185,000

(~ 5,000) D.

of the purified TLIEF protein,

indicated one active peak at approximately

This peak contained all the active proteins

found in the

initial crude extract.

SDS-polyacrylamide

runs indicated that at least five protein bands of lower

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

molecular weight were present in the purified sample.

Apparently

the SDS disrupted

the enzyme complex from P. placenta to small subunits of different sizes. Breakdown of large molecular weight enzymes into subunits of identical size is not uncommon

(10,11,12).

We are aware, however,

of one recent report by Ait et al.

system from culture filtrates of Clostridiwn

(13) in which the cellulolytic

thermocellwn, when partially purified by preparative electrophoresis, single band equal to 125,000 D on analytical polyacrylamide However,

the SDS-polyacrylamide

gel electrophoresis

proteins of lower molecular weight, filtrates

from P. placenta.

the SDS-polyacrylamide

showed a

gel electrophoresis.

indicated at least five

results similar to the ones obtained in our

Ait et al.

(13) also indicated that only one band on

gel was active.

Thus, the carbohydrate-degrading

complex isolated from P. placenta apparently is

also composed of an aggregate of polypeptides,

which under the right conditions,

breaks down into small subunits.

The purified enzyme complex from P. placenta was active on macromolecules well as on glycosides

(Table i.)

To the authors'

action by one carbohydrolase has not been reported. shown multiple enzyme activities after extensive purification galacturonase

knowledge, However,

as

such multiple recent work has

associated with a single enzyme fraction even

(14,15).

Cooper et al. found two isozymes of poly-

and pectin lyase of Ve~ticilliwn albo-atrum which retained almost

identical activity profiles

after wide- and narrow-range

isoelectric

focusing.

Urbanik et al., working with Phoma hibernica,

found one band after isoelectric

focusing with activities

cellulose,

glucomannan,

toward CMC-insoluble

and galacto-glucomannan.

were hydrolyzed

slowly.

Nitrophenyl

xylan, galactomannan,

derivatives of carbohydrates

In both of these cases, it was suggested that the

purified enzyme preparation is a complex most probably composed of different enzyme activities.

The large size of the purified enzyme from P. placenta also

suggests that multifunctional This information

activities may he derived from such a complex.

can be used to manipulate

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the degradative

activities of these

VOI. 97, No. 4, 1980

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

fungi, specifically in wood preservation to inhibit decay or in bioconversion to enhance degradation.

The fact that the production of polysaccharide-degrading

enzymes by P. placenta is not repressed by simple sugars is of special interest because catabolic repression of polysaccharides by degradative products (e.g., from Trichode~a viride) is a serious problem in bioconversion.

Such a non-

specific enzyme as that described here may be particularly useful in converting wood biomass.

REFERENCES

i.

Nighley, T. L. (1976) Mater. Org. ii(i), 33-46.

2.

Highley, T. L. (%973) Wood Fiber i, 50-58.

3.

Nelson, N. (1944) J. Biol. Chem. 153, 375-380.

4.

Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275.

5.

Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412.

6.

Gordon, J. C. (1971) Plant Physiol. 47, 595-599.

7.

Wolter, K. E., and Gordon, J. C. (1975) Physiol. Plant. 33, 219-223.

8.

Malamud, D., and Drysdale, J. W. (1978) Anal. Biochem. 86, 620-647.

9.

Richards, G. N., and Shambe, T. (1976) Carbohydr. Res. 49, 371-381.

i0.

Kemp, J. O., Sutton, D. W., and Hack, E. (1979) Plant Physiol. 63(5), 58.

ii.

Nakos, G., and Mortenson, L. (1971) Biochemistry I0, 455-458.

12.

Umezurike, G. M. (1971) Biochim. Biophys. Acta 227, 419-428.

13.

Ait, N., Creuzert, N., and Forget, P. (1979) J. Gen. Microbiol. 113, 339-402.

14.

Cooper, R. M., Rankin, B., and Wood, R. K. S. (1978) Physiol. Plant Pathol. 15, 101-134.

15.

Urbanik, H., Zalewska-Solrzak, J., and Borowinska, A. (1978) Arch. Microbiol. 118(3), 265-269.

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