@-D-1,6-GLUCANASES IN FUNGI'

ELWN T. REESE, FREDERICK W. PARRISH,~ AND ~ / I A R Y MANDELS

Abstract P-D-1,6-Glucans apparently occur rarely in nature, yet many fungi are able

to produce an enzyl1le capable of hydrolyzing them. The P-1,6-glucanases tend to be constitutive enzymes, similar in this respect to the P-1,3-gluca11ases. They act on /3-1,6-gluca11s in a random fashion, producing a series of P-1,6-linked oligosaccharides, including gentiobiose. The P-1,6-glucanases can be separated from other /3-glucanases by zone electrophoresis.

Introduction A systematic survey of the 0-glucanases of fungi (10, 11, 12) is being con-

tinued with this report on P-1,6-glucanases. In the past, occasio~lal incidental references have been made to the hydrolysis of P-1,6-glucans by fungal enzyme preparations, but these have been interpreted as an action of a ltnown glucanase such as cellulase (3 , s ) rather than as the actioil of a separate enzyme. In this paper the P-1,6-glucanases are shown to be of wide occurrence in fungi and to be highly specific enzymes that can be separated readily froin other glucan- ases by means of zone electrophoresis.

Methods Most of the methods used in this study have been described earlier (7,11,12).

The P-1,6-glucan (pustulan), used as a substrate, was isolated from dry Umbilicaria pztsta~lata by the procedure of Lindberg and McPherson (6). I t is a glucan containing only P-1,6 links. The yields of crude glucan were over 10yo of the dry weight of the lichen. This material contained some galactan. The crude product (6 g) was further purified by treatilleilt with 0.33 N sulphuric acid (100 ml) a t 100" C for 15 minutes followed by fractional pre- cipitation with ethanol. Thirteen percent ethanol brought down a black residue that was discarded. Additional ethanol (to the 50y0 level) precipitated the purified pustulan. This product, representing 7540% of the crude pus- tulan, was washed with acetone and dried i n vacuo. On acid hydrolysis ( N H2SO4; 100"; 4 hours), i t gave only glucose (paper chromatogram, isopropanol: acetic acid :water::27 :4:9), and in yields of over 95y0.

For estimation of the P-1,6-glucanase activity, 0 .5 nll of enzyme solution, appropriately diluted with buffer, was added to an equal volume of 0.47, P-1,6-glucan in 0.05 il& citrate buffer (pH 4.5) . After incubation a t 50" C for 1 hour, reducing sugars produced as a result of hydrolysis were estimated with dinitrosalicylic acid. A solution has one P-1,6-glucanase unit per ml (U/ml) if, when assayed as above, i t produces 0.5 mg of reducing sugar as glucose (13). This procedure follows closely that used for the estinlation of the other P-glucanases (11, 12).

'Manuscript received December 14, 1961. Contl-ibution from the Pioneering Research Division, I-IQ Quartermaster Research and - .-

Engineering Commancl. U.S. Army, NaticIi, Massach~~setts. 2Present address: Corn Products Ref. Co., Argo, Illi~lois.

Canadian Journal of Microbiology. Volume 8 (1962)

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328 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 8. 1962

Results A. Search for P-1,6-Glz~canases in Fungi

One hundred of the 150 fungi that were grown on a medium containing glucose and 0-1,6-glucan produced P-1,6-glucanase. After incubation on a shaker a t 29' for a period of 1-3 weeks, the cell-free culture medium was assayed for enzyme activity. The most active fungi (Table I) are in the genus Penicillium. Relatively few of the highly active cellulase-, or P-1,3-glucanase- producing organisins are active P-1,6-glucanase producers.

The 0-1,6-glucanases of fungi, like the P-1,3-glucanases, tend to be con- stitutive. Except for a few fungi, such as Poria cocos, growth on the glucan does not result in the marked increase in yields characteristic of such adaptive enzymes as the P-1,4-glucanases and P-1,2-glucanases. Indeed, the yields on glycerol (Table I) are sometimes higher than those obtained on the glucan.

B. Properties of P-1,6-Glzccanases of Fzcngi The pH optimum for the P-1,6-glucanases of six fungi was 4 . 0 f 0 . 4

(Fig. 1 shows three examples). Enzyme activities are low a t pH 2 .0 and a t pH 7.0. The temperature optimum (pH 4 .1 , 30 minutes' incubation) ranged

TABLE I Effect of substrate on fungal production of P-1,6-glucanase

Substrate

P-1,6-Glucan* Glycerol+

Name QM No. (Enzyme in U/ml of rnedi~~m)

A. ungu i s A. vzrsicolor

Beauveria bnssialza F~tsariunz roseuln

Penic i l l iu ln brefeldialzzlnz P . brefeldia~zz~vz

P . digitalz~nz P . ekrlichii

P. spinz~losz~?lz Poria cocos

Trichoder?ita li,onorzlnt Trichodernta viride

*Organisms grown on ~lucose (0.3 %) + P-1,6-glucan (0.3 %'0) tGrown on glycerol, 0.5 %.

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REESE ET AL.: GLUCANASES IN FUNGI

FIG. 1. Effect of p H and temperature on activity of /3-1,6-gluca1~ases. 0 - - - 0 Sporotricltt~m prz~i~tosunt Q M 826. 0 - - - 0 Penicilliunz ocl~ro-cl~loron Q M 477. A- A Penicilliz~m brefeldinnum Q M 1873.

pH:M/20 citrate buffer; 50"; 30 min; p~lstulan 2 mg/ml. Temp.: M/20 citrate buffer, p H 4 .1 ; 30 ~ n i n ; pustulan 2 mg/ml.

from 50" to 60" C (Fig. 1). All but one of the enzymes were inactivated in less than 10 minutes a t 70" C. The enzyme from Sporotrichum pruinosum, however, remained highly active over a 2-hour period a t this temperature.

Enzyme activity increased with substrate concentration up to 6-8 mg of glucan per ml of reaction mixture. The Michaelis constants (K,,), estimated from a plot of the reciprocal of the velocity vs. the reciprocal of the substrate concentration, are between 0 . 4 and 1 . 3 mg/inl for the five enzymes tested (Penicillium brefeldianz~nz, Penicillium parvz~m, Penicilliz~m ehrlichii, Peni- cillium oclzro-chloron, Sporotrichzsm pruinos~~m).

Enzyine concentration curves, and time curves, under optimal conditions (PI-I 4 . 0 ; 50" C; glucail = 5 ing/ml) were linear until the extent of l~yclrolysis, as measured by reducing value, exceeded 0. S ing/inl.

C. Presence of other Carbohydrases in P-1,6-Gbcanase Preparations P-1,6- and P-1,3-glucanases, since they are constitutive in fungi, are usually

present in fungal eilzyine preparations regardless of the substrate on which the organism is grown. P-1,2- and P-1,4-glucanases, since they are adaptive, are much less common. Some of the inore active P-1,6-glucanase preparations are listed (Table 11). Most of thein also contain high P-1,3-glucanase activity. The preparations froin Penicilliunz melinii and one of the P. parvum pre- parations have high 0-1,2-glucanase activity because they were grown on the P-1,2-glucan. The Sporotrichum pruinosum preparation has high cellulase activity because it was grown on cellulose.

P-Glucosidase (cellobiase), which also hydrolyzes laminaribiose and gentio- biose, is present in appreciable quantities in most of the /3-1,6-glucanase pre- parations (Table 11). Amylase and xylanase, constitutive enzymes in most fungi, are also undoubtedly present in most of these.

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330 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 8. 1962

TABLE I1 Presence of other P-glucanases and of P-glucosidase in P-1,6-glucanase preparations

Glucanase, U/mgt Cello- Enzyme QM biase,

preparation of: No. P-1,6- P-1,3- P-1,2- P-1,4- U/mg

Aspergillz~s qz~adricinctus 6874 3 .4 14.0 <0 .1 <0 .1 1 . 7 Cellulase, Miles* 2 .0 4 .3 0 .2 10.0 10.0 Penicilliunr brefeldianum 1873 14.0 4 .2 0 .2 0 0.9

*Commercial sample from Takamine Division. 10.000 of their units per mg. Sa~nple redissolved and precipitated with acetone.

tiill other salnples are acetone precipitates of culture solutions.

FIG. 2. Separation of P-1,6-glucanase from other P-glucanases by zone electrophoresis. Trichoderma viride QM 6a; Penicilliunz spinz~losz~,n QM 7643.

Ordinate = relative enzyme activity; abscissa = distance moved (in cm) of each enzy~nic component after 20 hours a t 8 v/cm. - = cathode; + = anode.

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R E E E ET AL.: GLUCANASES IN FUNGI 33 1

D. Separation of 0-1,6-Glz~canases from Related Enzymes by Zone Electro- phoresis

0-1,6-Glucanase preparations (2.5-20 mg) from 15 fungi were subjected to starch zone electrophoresis as previously described (7, 12). After electro- phoresis, the starch block (100 X 3 . 3 X0 .3 cm) was cut into one hundred 1-cm pieces; each piece was extracted, ancl the eluates were analyzed for the various enzymes lcnown to be present. In the four cxperiinents shown (Figs. 2 and 3 ) , the 0-1,6-glucanases separated nicely froin P-1,3- and P-1,4-glucanases. These preparations contain no ,8-1,2-glucanase activity. In previous worlc (1 2), it was shown that P-1,6-glucanases move separately from 0-1 ,2-glucanases. Each of these four 0-glucanases moves independently, and each acts only on i ts own substrate. Even \vl~ere components overlap, it is obvious from position and shape of the pealts that these enzymes are clistinct. Likewise, the P- gluca~lases do not act on salicin, i.e., they are not 0-glucosidases (Fig. 3). The 0-1,6-glucanases of most fungi (15 tested) lnove as a single component. In Penicilli~lm sl~inz~loszim (Fig. 2) and two other fungi, there are two com- ponents, and in three fungi there are three active components.

E. Enzynzatic f1ydrolysis P~odz~cts of /3-1,6-Glucans Our 0-1,6-glucanases act on P-1,6-glucans in a ranclonl fashion to produce

a series of 0-1,6-linlced oligosaccharides (Fig. 4). Glucose is generally present even when the enzyme preparation, or fraction, is free of P-glucosidase.

SCLEROTIUM ROLFSll A. NIGER

FIG. 3. Separation of P-glucosidase lroin P-glucanases by zone electrophoresis. Aspergi l lus n iger Q M 877; Sclerobiztvz rolfsi i Q M 7740. P-Glucosidase = "Salicinase".

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332 CANADIAN JOURNAL O F MICROBIOLOGY. VOL. 8, 1962

When the P-1,6-glucanase preparation contains an appreciable amount of 0-glucosidase (as in the Aspergillus niger enzyme, Fig. 3), the intermediates are hydrolyzed as rapidly as they are produced, and glucose is the only pro- duct. Hydrolysis by the P-1,6-glucanase freed of P-glucosidase (by zone electrophoresis) gave the typical series of P-1,6-linlted oligosaccharides, including glucose. This purified P-1,6-glucanase did not hydrolyze gentiobiose. The glucose produced from the enzymatic hydrolysis of the glucail must have come from the longer inembers of the 0-1,6-oligosaccharide series.

Pustulan (2.0 g) was hydrolyzed by the P-1,6-glucanase of Penicilliurn digitatztm, QM 518, a preparation low in 0-glucosidase. The hydrolyzate was fractionated on a carbon column. The following fractions were obtained:

Gentiotetraose, and longer chains 838 mg Gentiotriose (plus traces of gentiobiose and glucose) 286 ing Gentiobiose (plus trace of glucose) 419 mg Glucose (approxin~ately) 100 mg

1643

The disaccharide product has the same RG values as authentic gentiobiose in two solvent systeins used (isopropanol :acetic acid :water, 27 : 4 : 9 ; and ethyl acetate: pyridine: water, 10: 4: 3). The acetylated disaccharide recrystallized from ethanol has the properties of 0-gentiobiose octaacetate (m.p. 196. 5-197O C ; [a] = -5.3", 6 5 . 5 , CHCl,).

When the log (1-Rj)/Rj is plottecl against the number of glucose units in the oligosaccharide, a straight line is obtained. This irldicates (4) that the

FIG. 4. Chromatogram of hydrolyzates of P-1,6-glucans by enzymes. P-1,6-Glucanases of Pen ic i l l i l ~?n brefeldiarrtlm QM 1873 (El) and of P. elzrlichii QM 1874 (E?). Glucans: pustnlan (P); lutean from Dr. B. A. Stone (Ls); lutean of P. kelicwil QM 1852 (La) .

Hydrolysis conditions: 1 hour; 50" C ; 1% ghican; pH 4.5. C = control of glucose (G) and gentiobiose (GB). Cross hatching is ~lsed to indicate the intensity of color.

distance moved by component Ro = distance moved by glucose

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REESE ET AL.: GLUCAN.4SES I N FUNGI 333

observed hydrolysis products all belong to the same P-1,6 series. Conversely, the absence of spots t h a t do not fall on the line indicates the absence of other linkages. These observations confirln those of Lindberg and McPherson (6), and of Dralte (3).

A lutean preparation (a gift from B. A. Stone), and a lutean fro111 Penicillizlm hel icz~m QM 1852 (prepared b y us) were also subjected to enzyme hydrolysis (Fig. 4). T h e same products were formed from lutean as from pustulan, thus establishing all as P-1,6-glucans.

Discussion P-1,6-Glucanases are found in over two-thirds of the fungi tested. Ye t

in nature, the substrate, P-1,6-glucan, is apparently of rare occurrence. I t has been reported in the lichen, Umbilicaria pz~stztlata (3, 6). I t is produced by members of the Penic i l l i~ lm luteum group in the form of a malonic acid ester (1). T h e P-1,6 linltage also occurs admixed with P-1,3 linkages in glucans of yeast (8), of Candida albicans (2), and of marine algae (9). All of these examples are from a rather restricted section of the plant kingdom, the thallo- phyta. As with the P-1,3-glucans, the infrequent occurrence of the P-1,6- glucans in nature may be more apparent than real since there are so few investigators loolting for glucans. Perhaps the P-1,6-glucans (in pure or mixed form) are comnlon reserve foodstuffs in fungi, hence the presence in these of a constitutive enzyme required for hydrolysis of the P-1,6-glucan. This is con- sistent with the fact t h a t a constitutive amylase is produced in fungi where it is apparently required for the hydrolysis of the reserve glucan, glycogen.

T h e P-1,6-glucanases are endoenzymes acting on P-1,6-glucans by random hydrolysis t o yield gentiobiose and 11ighe1- members of this oligosaccharide series. Relatively little glucose is produced unless the preparation also contains P-glucosiclase. In this, the P-1,6-glucanases resemble the other P-glucanases which act b \ ~ random splitting. Exo-P-glucrunases,acting from the chain end, are found only among the P-1,3-glucanases iirllere they appear t o be more common than the endoenzymc. Rcports of cxocellulases are basecl mainly on the pro- duction of cellobiose as the major product of hydrolysis. This is insufficient evidence.

'The cnzymcs 1q.cl1-olyzing P-1,6-glucans show no activity 011 P-glucosides or on other P-glucans. Since fungal enzl-lne preparations are always mixtures, this specificity can be shown only when the 0-1,6-glucanase is obtained free of other enzynlcs by techniques such a s clcctropl~orcsis. P-1,6-Glucanases usually movc as a single enzyme component, bu t a few have two or three active components. T h e presence of multiple enzl-mes is known also for P-1,2-, P-1,3-, and P-1,4-glucanases.

For the most part , the P-1,6-glucanases appear t o be constitutive enzymes. In this, they reseinble the P-1,3-glucanases, and differ from the adaptive P-1,2-, and P-1,4-glucanases. As a result, i t is possible t o predict the complexity of P-glucanases in a culture filtrate from the n a t ~ ~ r e of the substrate on which the fungus is grown. T h e absolute quantities of the glucanases vary, of course, with the enzyme-producing capacity of the different organisms.

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334 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 8, 1962

Acknowledgments \Ve wish to thank B. Lindberg (Swedish Forest Products Laboratory,

Stocltholn~) for supplying Umbilicaria pz~stz~lata and B. A. Stone (University of Melbourne, Australia) for a sample of lutean. Miss Dorothy Fennel1 ltindly supplied most of the fungal cultures.

References 1. ANDERSON, C. G., I-IA\VORTH, W. N., RAISTRICIC, I-I., and STACEY, M. Molecular con-

stitution of luleose. Riochem. J. 33, 272-279 (1939). 2. BISHOP, C. T., BL:\NK, F., and GARDNER, P. E. Cell-wall polysaccharicles of Candida

albicans: glucan, Inannan, and chitin. Can. J . Chem. 38, 869-881 (1960). 3. DRAI~E, B. Uber einige polysaccharide der Flechten, vornehmlich das Lichenin und das

neuentclec1;te Pust~llin. Biocheln. Z. 313, 388-399 (1943). 4. FRENCII, D. ancl WILD, G. M. Correlation of carbohydrate structure with papergrarn

mobility. J. Am. Chern. Soc. 75, 2612-2616 (1953). 5. I<OOIMAN, P. Some properties of cellulase of M y i o t l ~ e c i ~ ~ ~ ~ ~ uerrucaria and some other

fungi. 11. Enzyrnologia, 18, 371-384 (1957). 6. LINDBERG, B. and NICPHERSON, J. Chemistry of lichens. VI. Pustulan. Acta Chern. Scand.

8, 985-988 (1954). 7. MANDELS, M., MILLER, G. L., and SLATER, R. Separation of fungal carbohydrases by

starch bloclc electrophoresis. Arch. Biochem. Riophys. 93, 115-121 (1961). 8. PEAT, S., Wa~r,:zx, W. J., and ED\\T:\RDS, T. E. Polysaccharides of baker's yeast.

11. Yeast glucan. J. Chern. Soc. 3862-3868 (1958). 9. PEAT, S., ~ V A E L A N , W. J., and LA\VLEY, H. G. The structure of laminarin. 11. iVIinor

s t r u c t ~ ~ m l features. J. Chem. Soc. 729-737 (1958). 10. RERSE, E. T. a ? d LEVINSON, H. S. Comparative s t~rdy of the brealido\vn of cellulose by

microorganrsms. Physiol. Plantarum, 5, 345-366 (1952). 11. Rsasr:, E. 'T. and MANDELS, M. P-~-1,3-Gluca11ases in fungi. Can. J. Microbiol. 5, 173-185

(1959). 12. REESE, E. T., PARRISII, F. W., and N~ANDELS, M. P-~-1,2-Glucanases i n fungi. Can. J.

Microbiol. 7, 309-317 (1961). 13. SUMXER, J. R. and Soare~ts, G. I;. 12aborntory esperilne~lts in bioIogical chemistry.

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