JOURNAL OF BACrERIOLOGY, June, 1965Copyright 0 1965 American Society for Microbiology

Vol. 89, No. 6Printed in U.S. A.

Enzymatic Hydrolysis of Yeast Cell WallsI. Isolation of Wall-Decomposing Organisms and Separation and

Purification of Lytic EnzymesHIROSATO TANAKAl AND HERMAN J. PHAFF

Department of Food Science and Technology, University of California, Davis, California

Received for publication 27 January 1965

ABSTRACTTANAKA, HIROSATO (University of California, I)avis), AND HERMAN J. PHAFF. En-

zymatic hydrolysis of yeast cell walls. I. Isolation of wall-decomposing organisms andseparation and purification of lytic enzymes. J. Bacteriol. 89:1570-1580. 1965.-A num-

ber of microorganisms, able to decompose and grow on yeast cell walls, were isolatedfrom soil. These isolates demonstrated various types of attack on yeast walls. A bac-terium, identified as Bacillus circulans, and a species of Streptomyces produced clear,lysed zones when grown on an agar medium containing baker's yeast cell walls. Thestreptomycete formed glucanase, mannanase, and protease, but B. circulans producedonly glucanases. Purified mannan could be prepared from the culture fluid of B. circu-lans grown on baker's yeast cell walls. In a liquid, mineral medium, extracellular lyticenzyme production by B. circulans was optimal after 3 days of aerobic growth at 30 Cwith 0.5% baker's yeast cell walls as the carbon source. Twelve other carbon sources

were ineffective as inducers. Among a number of polysaccharides tested, the crude en-zymes of B. circulans hydrolyzed only 0-1---3 glucan (laminarin) and ,3-1--*6 glucan(pustulan), both by a random mechanism, to a mixture of dimer and glucose. The,B-1--*3 and ,3-1--+6 glucanases were separated from each other by diethylaminoethyl cel-lulose column chromatography. Water-soluble oat glucan, which contains in the linearchain both ,-1--+3 and f-1--4 bonds, was also hydrolyzed by the bacterial 0-1--*3 glu-canase. The products of this reaction indicated that this enzyme hydrolyzes ,B-1--+3 or

,B-1--+4 glucosidic linkages, provided the B-glucopyranosyl units composing these bondsare substituted in the 3 position by another glucose unit.

Present knowledge of the chemistry and finestructure of yeast cell walls is still quite incom-plete, and is largely confined to Saccharomycescerevisiae. Divergent views exist, even on thechemical composition and location of the majorpolysaccharides, glucan and mannan. 'Most ofthe chemical studies of these polysaccharideshave been done after drastically treating eitherthe intact cells or isolated walls with acid oralkali (or both).The literature on t-his subject was recent]y

reviewed by Phaff (1963), Nickerson (1963),Clarke and Stone (1963), and Northcote (1963).To supplement the chemical studies and to

expand our knowledge of the structure of yeastwalls, an enzymatic analysis was thought to bepromising.A number of cell wall-decomnposing micr o-

organisms were isolated fr-om soil. An activestrain of Bacillus circulans was chosen for de-tailed study. In the culture fluiid of this organism,

1 Present address: Department of BiologicalSciences, Purdue University, Lafayette, Ind.

grown on baker's yeast cell walls, an endo-,B-1-*3 and an endo-3-1--6 glucanase were shownto be present. These enzymes were separatedfrom each other, and some of their propertiesare rel)orted.

MATERIALS AND METHODS

Types of cell-wall material. Various substrates,rich in wall materials, were explored for use ascarbon sources in culture media. Autolyzed andwashed baker's yeast (AWY) was used in someparts of this work. Equal amounts by weight ofwater and commercial compressed baker's yeastwere mixed. A small amount of toluene was addedand the suspension was incubated at 55 C for 3days. The insoluble residue was centrifuged, andwas washed with distilled water by repeatedcentrifugations until the supernatant liquid wasfree from cytoplasmic particles. The cell pastewas lyophilized and stored in a desiccator. AWYcells (Fig. la) have distinct cell walls clearlydiscernible with either a phase contrast or a lightmicroscope. The cytoplasmic contents are some-what retracted from the cell walls because ofautolysis.

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FIG. 1(a). Autolyzed and washed baker's yeast cells. (b) Purified baker's yeast cell walls prepared by theuse of a colloid mill.

For the preparation of large quantities of cellwalls, an Eppenbach laboratory colloid mill(model QV-6) was chosen; glass beads with anaverage size of 120 , were used (Garver and Ep-stein, 1959). Use of 0.05 M phosphate buffer (pH6.2) was found to reduce precipitation of proteininside the cell envelope. Cells were usually cut atone place, and the cytoplasmic content seemed tobe almost completely removed after breakage.Unbroken cells, cytoplasmic material, and glassbeads were separated from walls by differentialcentrifugation in an International centrifuge with0.05 M phosphate buffer until the walls were vir-tually free from these extraneous materials.Final washing was done with distilled water.Yield was 12 to 156/a on the dry weight basis.The purified cell walls are shown in Fig. lb.

Substrates. A small quantity of purified lami-narin (no. 2208) was obtained from the Kelco Co.,San DJiego, Calif. Crude soluble laminarin waspurchased from the Institute of Seaweed Research,Inveresk, Midlothian, Scotland. The dark brownmaterial was purified as follows. Crude laminarin(10 g) was dissolved in 100 ml of distilled water,and was dialyzed against distilled water. The solu-tion was then passed through a column of 50 g ofDowex 50 (H+ form) ion-exchange resin. Thecolumn was washed with sufficient water to re-cover all of the laminarin. The eluate was thenpassed through a column of 40 g of hide powder(Frank F. Marshall, Ridgway, Pa.). Owing to theD)owex treatment, almost all of the brown pigmentcould be adsorbed by the hide powder. The fil-trate was freeze-dried; the yield was 30 to 40%1c.

Laminaribiose, laminaritriose, laminaritetra-ose, cellotriose, and cellotetraose were kindly

supplied by D. S. Feingold and E. F. Neufeld ofthe University of California, Berkeley. Pustulanwas obtained through the courtesy of E. T. Reeseof the Quartermaster Research and EngineeringCenter, Natick, Mass. Oat glucan, 3-O-f3-D-cello-triosyl-D-glucose and 3-0-f3-D-cellobiosyl-D-glu-cose were gifts of P. A. J. Gorin, and crown gallpolysaccharide produced by Agrobacterium radio-bacter was kindly supplied by J. F. T. Spencer,both of the Prairie Regional Laboratory, Saska-toon, Saskatchewan, Canada. Colloidal chitin wasobtained through the courtesy of D. M. Reynoldsof the University of California, Davis. Cellulosedextrin was prepared according to the methoddescribed by Fuller and Norman (1942).

Stock cuiltur es. Wall-decomposing organismswere stored on agar media containing 0.5% lyophi-lized AWY, 0.1%,X yeast autolysate (Albimi),0.1% glucose, and 1.36% KH2PO4 (pi1 adjusted to6.5 with NaOH). The medium was agitated in thetube before slanting, to insure an even suspensionof AWY.Assay of reducing gi-ou0ps. Liberation of aldehyde

groups during hydrolysis of laminarin by f-1---*3glucanase was followed by a semimicrohypoioditeprocedure (Yemm, 1935); 1 meq of iodine reducedcorresponded to 0.513 mmole of reducing groups.Assay of protein. Protein concentration was

estimated by the method of Lowry et al. (1951).Crystallized bovine serum albumin was used asthe standard.

Protein chromatography. Cellulose IA, N'-diethyl-aminoethyl ether (DEAE cellulose) was an East-man product (no. 7392). The I)EAE cellulosewas equilibrated at pH 8.0 with 1.0 M tris(hydroxy-methyl)aminomethane (Tris)-HCI buffer, fol-

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lowed by five washings with 100-ml portions ofdistilled water per gram of DEAE cellulose, andwas then poured into a chromatography tube.The column was washed with 1 liter of distilledwater per gram of DEAE cellulose. The convexlyincreasing gradient elution was effected by a de-vice described by Palmer (1955).

Detection of lytic activity. A cup-plate method(Dingle et al., 1953) was adapted for detectionand rough estimation of the lytic activity in thefractions collected from the columns. The mediumfor this assay contained 0.1% purified baker'syeast walls, 0.85% Ionagar no. 2 (Oxoid), 0.01%Merthiolate, and 0.1 M phosphate buffer (pH 6.5).The walls were suspended evenly by sonic treat-ment (Son Blaster, series 200, The Narda Ultra-sonics Corp., Mineola, N.Y.). The medium wasatutoclaved for 5 min to melt the agar. Heating,incidentally, made the walls slightly more sus-ceptible to the lytic enzymes. The medium (100ml) was poured into a 9-in. pie plate (22.86 cm indiameter). Holes of 6 mm in diameter were madewith a no. 2 cork borer. After adding 0.05-misamples of enzyme solution to each hole, the platewas incubated at 30 or 37 C and the linear extentof clearing (wall lysis) was measured.

RESULTSIsolation and purification of organisms which

decompose baker's yeast cell walls. Small particlesof various soils were placed on the surface of anagar medium containing 1.0%0 AWY (servingas a source for both carbon and nitrogen), 1.0%K2HPO4, 0.01% MgSO4.7H20, and 2% agar(Difco). The relatively high pH of this medium(8.0) suppressed the normally occurring abun-dant mycelial growth of Mlucor-like molds, andthus facilitated the isolation of lytic bacteria.The plates were incubated at 30 C. Lysed zonesal)l)eared around some of the soil particles.Colonies of lytic organisms were purified on theabove medium. Isolates included three strains ofStreptomyces, six strains of Bacillus, two strainsof small gram-negative motile rods, and severalstrains of molds.

Optimal pH ar.d action of the lytic enzymes on.1 11-Y cells. An agar medium containing 0.5%7,AWY, 0.1 %0 yeast autolysate (Albimi), and1.36I7KH2PO4 was adjusted to pH 5.0, 6.0,6.5, 7.0, 7.5, and 8.0 with NaOH. Growth of thetest organisms and development of lysed zonesaround the colonies were observed. Although theoptimal pH for growth differed among the strains,all of the organisms te4ted formed the mostextensive lysed zones at pH 6.5.

Next, microscopic observations were made toobtain further information on the ability of theorganisms to attack the walls of baker's yeast.A liquid medium (at pH 6.5) was the same asthat listed above but contained 1% lyophilizedAWY. Fresh slant cultures of the cell wall-decomposing organisms were inoculated in 100ml of this medium in 250-ml Erlenmeyer flasks,and were allowed to grow on a shaker at 30 C.The organisms started their growth within 24to 48 hr and, as growth progressed, the AWYcells underwent various conspicuous changescaused by the action of enzymes produced bythe organisms.

Figure 2a shows AWY cells in the 3-day culturefluid of the Streptomyces strain WL-6. The cellwalls as well as their evtoplasmic contents werealmost completely digested, leaving only somehighly refractile, spherical bodies, indicatingthat these were very riesistant to enzymaticaction. This type of decomposition was probablydue to the combined action of cell wall-decom-posing enzymes (polysaccharidases) and proteo-lytic enzymes. The latter are presumed to attackthe cytoplasmic contents.

Figure 2b shows AWY cells attacked byBacillus strain WL-12. In comparing this figurewith that of unattacked AWY cells (Fig. la),it is evident that the cell wall is completelyabsent in the former, but the inner cytoplasmiccontents are left practically intact. This indi-cates that proteolytic activity was either absentor very weak in the culture fluid of BacillusWL-12.

Figure 2c shows a type of attack exhibited byBacillus strain WL-5, which produced an in-completely clear zone on the cell-wall plate.Affected AWY cells were very refractile, and thesurface of the cells was irregular.

Figures 2d and 2e are two illustrations ofAWY cells affected by the enzymes of a strainof Aspergillus. Cytoplasmic material was almostcompletely riemoved, and the cell walls werevery thin. What appear to be bud scars and abirth scar became accentuated and visible owingto the action of enzymes of this organism.

Lysis of purified cell walls. As shown in Fig.la, AW-Y cells contain residual cytoplasmicmaterial, not removed by autolysis. Therefore,the ability to digest purified cell walls was testednext. The medium contained 0.25% purifiedcell walls, 0.1%lo yeast autolysate (Albimi), and

FIG. 2. Various types of lysis of autolyzed washed baker's yeast (AWY) cells by enzymes produced dur-ing growth of the organisms listed. The medium contained 1% AWY, 0.jco yeast autolysate (Albimi), and1.360co KH2PO4; pH adjusted to 6.5. Photographs were taken after 3 days of incubation at 30 C on a shaker(phase photomicrographs). (a) AWY cells attacked by Streptomyces strain WL-6. (b) AWY cells attacked byBacillus WL-12. (c) AWY cells attacked by Bacillus WL-5. (d) AWY cells attacked by enzymes of Asper-gillus WL-20. (e) Same as d.

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1.36% KH2PO4, and the pH was adjusted to 6.5with NaOH. The various isolates were inoculatedby touching the surface of the agar mediumlightly with the tip of an inoculating needle.Various degrees of clearing of the cell wallsaround the colonies were observed, rangingfrom none to very distinct.

Streptomyces strain WL-6 and Bacillus strainWL-12 showed the most distinct lysed zones;the clearing by Bacillus WL-5 was incomplete.The water-clear lysed zone produced by BacillusWL-12 is illustrated in Fig. 3.

Action of the lytic enzymes produced by threeselected organisms on cell ualls of baker's yeast.Based on the results just described, three strainswere chosen for more detailed study. Table 1summarizes the particular features of the strainsselected.To obtain extracellular lytic enzymes, the

strains were inoculated into a medium containing

FIG. 3. Growth of Bacillus strain WL-12 after 2days on agar medium containing 0.25% baker'syeast cell walls, 0.1% yeast autolysate, and 0.1 M

phosphate buffer (pH 6.5). Note the zone of lysedcell walls around the bacterial growth.

0.1% purified cell walls, 0.1% yeast autolysate(Albimi), 1.36% KH2PO4, and the pH was ad-justed to 6.5 with NaOH. Portions of 100 mlof the medium were dispensed in 250-mI flasksand allowed to grow on a shaker at 30 C. After40 hr, the walls had disappeared from the mediuminoculated with Bacillus WL-12. After 48 hr,the same happened with the medium inoculatedwith Streptomyces WL-6. In the culture fluidinoculated with Bacillus WL-5, the cell wallsbecame much thinner than those in the controlbut remained recognizable after 3 days of in-cubation. The culture fluids were clarified bycentrifugation, dialvzed against 0.01 M phosphatebuffer (pH 6.5), and tested on baker's yeast cellwalls.

Purified baker's yeast cell walls (2 mg) were

suspended by sonic treatment in 6 ml of thedialyzed culture fluids. Reduction of the tur-bidity was followed in a Klett-Summersonphotoelectric colorimeter with a red filter (no.62). The results are shown in Fig. 4.

Next, the products of the above reactionswere analyzed by paper chromatography. When

no further decrease in turbidity occurred, thereaction mixtures were dried under vacuum,

dissolved in a small quantity of water, and ap-

plied in various concentrations on Whatmanno. 1 filter paper. Descending chromatographywas done with isopropanol-ethyl alcohol-water(7:2:1).In the reaction mixture of Streptomyces WL-6,

glucose and mannose were clearly present, inaddition to small amounts of oligosaccharides.This indicated that this streptomycete producedboth mannanase and glucanase.From the reaction mixture of Bacillus WL-12,

glucose and two oligosaccharides were detectedon paper. Mannose was absent, indicating thatthis strain did not produce mannanase. Thechromatogram of the reaction mixture of strainWL-5 showed only higher oligosaccharides.

Fate of mannan during grou th of BacillusWVL-12 on baker's yeast cell walls. If, as shown

above, strain WL-12 forms no mannanase, thewater-soluble mannan should still be present in

TABLE 1. Lytic action during growth of three selected strains on two cell-wall materialsincorporated in solid or liquid media

Degree of clearing or digestionCulture designation

AWY agar Cell-wall agar AWY in liquid

Streptomyces WL-6.......... Complete Complete CompleteBacillus WL-12.............. Incomplete Complete Only cell walls digestedBacillus WL-5............... Incomplete Incomplete Cell walls affected partially

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the centrifuged culture fluid. Bacillus strainWL-12 was grown in the same medium as usedabove, except that 0.5% purified cell walls wereused as the carbon source. An equal volume ofcold Fehling solution was added to the centri-fuged, clear, supernatant culture fluid. Thebluish-white precipitate was purified accordingto the method described by Haworth et al. (1937)for the purification of mannan. The white, water-soluble polysaccharide was hydrolyzed by acid,and, when analyzed by paper chromatography,it contained only mannose.

Identification of Bacillus strain WL-12. As a

result of these exploratory experiments, BacillusWL-12 was selected for further study because(i) the culture fluiid had high lytic activity, (ii)no mannanase was produced, and (iii) the cul-ture fluid appeared to have negligible proteolyticactivity. Hence the lytic enzyme(s) produced bythis organism was limited to that which hy-drolyzes the glucan component of the wall, andit was decided to study this system first.The strain properties subjected to taxonomic

analysis were based on the traits listed by Smith,Gordon, and Clark (1946) and those listed inBergey's Manual. The morphological and physio-logical characteristics agreed well with those ofBacillus circulans Jordan 1890 emend. Ford 1916.

100> _ _ ~~~~Bacillus Str. WL S

C, ~~~~~~~Streptomyces Sir. WL6

50

Bocillus Str. WL 12

HOURS

FIG. 4. Decrease in turbitdiy of baker's yeastcell wall suspensions by the action of extracellularenzymes produced by three selected strains. Reactionmixtures contained 2 mg of cell walls in 6 ml ofculture fluid which had been dialyzed against 0.01 Mphosphate buffer (pH 6.6). Merthiolate (0.01%),which did not affect the lytic activity, was added as a

preservative. Temperature, 30 C.

TABLE 2. Action of a crude lytic enzymepreparation* on various polysaccharides

at pH 6.6

Substrate Hydrol- Predominantysis glucosidic bond

Cellulose (Whatman pow-der) ................... -

Cellodextrin.............. -Laminarin................ +Postulan ................. +Crown gall polysaccharide. -Colloidal chitint.......... -14

(N-acetyl-glucosa-mine)

Soluble starch............ - c-1a-i-4 (linearchains)

* The enzyme preparation was concentratedculture fluid of Bacillus circulans grown on baker'syeast walls and dialyzed against distilled water.

t Chitin was included because it is one of thecell-wall components. Its decomposition wasstudied by turbidimetric analysis as well as bychromatography.

The observed characteristics of our strain werereported in detail by Tanaka (1963).

Inducibility of the lytic enzyme. Various carboncompounds were tested for their suitability assubstrates for lytic enzyme production.The media contained 0.1% yeast autolysate,

1.36% KH2PO4, and 0.5% of test compound;the pH was adjusted to 6.5 with NaOH. Thecompounds tested were: L-arabinose, D-xylose,D-glucose, D-fructose, D-galactose, maltose, su-crose, lactose. cellobiose, cellodextrin, cellulose,starch, D-mannitol, and cell walls of baker'syeast. The flasks were inoculated and placed ona shaker at 30 C. Samples were taken after 24and 36 hr of incubation. The culture fluids werecentrifuged, and lytic activity was estimatedby the cup-plate method after allowing 24 hr at30 C for enzyme action. The cells grew well withall of the compounds, except cellobiose, cello-dextrin, and cellulose. Only the culture fluid ofthe medium containing baker's yeast cell wallsformed a distinct lysed zone on the test plate.Very weak lysis was caused by the culture fluidsof the media containing L-arabinose and D-xylose.

Substrate specificity of the crude enzyme. Theculture fluid of B. circulans grown in the cell-wall medium was concentrated under vacuum,dialyzed against distilled water, filtered througha Millipore filter, and tested for its polysac-charidase activity on the substrates listed inTable 2.

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Substrate (2 mg in 0.1 ml of distilled water)was dispensed into a small test tube which wasthen placed in a larger screw-capped tube. Afterautoclaving, 0.1 ml of sterile 0.1 M phosphatebuffer (pH 6.5) was added. Biefore the additionof enzyme, 10 pliters of the mixture were spottedon paper as a control. Then, 0.2 ml of enzymesolution was added. After 5 hr and again after48 hr, 20 pliters of the reaction mixture werespotted on Whatman no. 1 papel and developedwith n-butanol-acetic acid-water (4:1:5). 'Mono-and oligosaccharides were detected by sprayingthe paper with aniline hydrogen phthalate. Theresults (Table 2) indicate that, on the basis of thesubstrates tested, only a f-1i3 and a 0-1-*6glucanase were present.

Assay of fl-i-3 glucanase. Routine assays offl-i13 glucanase were done by measuring thelinear rate of reducing-group production at 30 Cwith 0.4% soluble laminarin in 0.05 M succinatebuffer (pH 5.8). The initial rate was a linearfunction of enzyme concentration in the rangein which assays were made. Based on a number ofassays at different pH values, optimal activitywas found at pH 5.8. This is somewhat belowthe value of 6.5, which was optimal for the rateof optical density decrease of cell-wall suspensionsas the substrate.One unit of l-1 3 glucanase was defined as

that amount of enzyme which liberated 1 ,umoleof aldehyde groups per hr at 30 C and pH 5.8.

Optimal conditions for synthesis of A-1--*3glucanase. Elaboration of f-1-*3 glucanase byB. circulans in shaking and in standing cultureswas followed for a period of 5 days. Cultureswere agitated on a model V rotary action shaker(New Brunswick Scientific Co., New Brunswick,N.J.) operated at approximately 200 oscillationsper minute. The medium contained 0.5% cellwalls, 0.67%o Yeast Nitrogen Base (Difco), and1.36% KH2PO4 in 1 liter of medium, and thepH was adjusted to 6.7 with NaOH. Seed culturewas prepared in the same medium, and 10 ml ofactively growing culture were inoculated into 1liter of fresh medium in a 3-liter Fernbach flask.Enzyme production in shaking culture started

TABLE 3. Effect of cell-wall concentration on theproduction of ,3-i- glucanase in cultures

grown for 4 days at 30 C

Cell wall concn M . .(tS, w/v) ,3-l-13 (lucanase activity

units/mi0.25 3.90.5 9.61.0 13.32.0 5.0

without lag. Enzyme concentration in the mediumincreased very rapidly for 2 days, increasedslightly during the next 2 days, and leveled off,reaching 10 units/ml, after 5 days.

In the standing culture, both growth andenzyme production were very poor, comparedwith the shaking culture.To test the effect of substrate concentration,

the same medium was used except that the cell-wall concentration was varied (Table 3). Enzymeconcentration was maximal in the medium with1 o cell walls, and there was a marked decreasewhen the cell-wall concentration was increasedto 2%. The yield of enzyme per unit weight ofcell walls utilized was highest at 0.5%.

Separation and purification of bacterial fi-glucanases. Evidence in Table 2 shows that thelytic enzyme preparation from B. circulanscatalyzed the hydrolysis of l-1-3 and fBl--*6polyglucosides. An attempt was therefore madeto separate these two activities.

Preliminary experiments indicated poor sepa-ration on DEAE cellulose columns. A chanceobservation indicated that this difficulty couldbe overcome if the centrifuged and water-dialyzed culture fluid was first treated withprotamine sulfate for the purpose of removingnucleic acids. A voluminous precipitate of nicleicacid-protamine sulfate complex appeared if a5% aqueous solution of protamine sulfate wasadded to the dialyzed culture fluid. To avoidpassing large volumes of fluid over DEAEcolumns, it was found advantageous to concen-trate the centrifuged culture liquid under vacuumin a rotary flash evaporator to approximately10% of the original volume. Concentration wasfollowed by dialysis against distilled water andtreatment with protamine sulfate (optimalconcentration approximately 4 ml of 5% pro-tamine sulfate per 100 ml of concentrated culturefluid). Concentration and dialysis against dis-tilled water caused no loss in activity of f-1-3glucanase. Its activity per milliliter slightlyincreased after removal of nucleic acids.The lytic enzymes were adsorbed on DEAE

cellulose after dialyzing the protamine sulfate-treated culture fluid against 0.005 M Tris-HClbuffer (pH 8.0). When cold Fehling solution wasadded to the first effluent, a voluminous precipi-tate was formed, indicating that the nonutilizedmannan of the culture fluid was not adsorbed onsuch columns. Thus, the column was washedwith distilled water, and the adsorbed enzymeswere subjected to gradient elution. Representa-tive fractions were tested for lytic activity bythe cup-plate method.Two types of lytic activities were encountered.

Early fractions contained an enzyme which

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formed rapidly spreading zones of large diameterwhich contained incompletely lysed residual wallmaterial. Later fractions contained an enzymewhich formed sharp, clear zones of small diameteron the test plate. The early fractions whichformed partially lysed zones hydrolyzed f-1i-*6glucan (pustulan) but did not hydrolyze f-1i-3glucan (laminarin). On the other hand, fractionswhich formed sharp clear zones hydrolyzed,-B-] 3 glucan but did not hydrolyze fli-*6glucan.A complete separation of the two activities

was achieved by the following method. Proteinchromatography was done with 0.1 M phosphatebuffer (pH 7.2) as the upper limit of gradient.First, several large inert protein peaks appeared(Fig. 5), accompanied by a considerable amountof yellowish pigment, which was present in theculture fluid. With a calculated buffer concentra-tion between 0.04 and 0.06 M, f-I -*6 glucanase(forming the large, opaque, lysed zones on thetest plate) was eluted. To insure completeelution of f-i -*6 glucanase, elution was generallycontinued until 750 ml of effluent were collected.To elute the f-i -*3 glucanase, a second gradient

elution was conducted, with the upper limit ofgradient at 0.5 M acetate buffer (pH 5.0). Figure6 shows the cup-plate assay of the fractions con-taining fl-1-*3 glucanase. The largest amount ofthe enzyme was eluted beginning at 0.04 Mbuffer strength. Fractions containing up to 0.3M acetate showed lysed zones on the plate. Nosignificant peak in absorbancy at 280 m,u wasobserved, indicating a very low concentration ofprotein in the purified f-1 3 glucanase frac-

40

co

FIG. 5. Protein chromatography of protaniine-treated dialyzed culture fluid (2,700 ml of originalculture fluid) with 3 g of DEAE cellulose. Upperlimit of gradient, 0.1 Mii phosphate buffer (pH 7.2).Protein concentration was expressed as opticaldensity at 280 mjy. Diameters of lysed zones due to,1-i-*6 glucanase action on the cell-wall plate wasbest measured at 8 hr of incubation and is indicatedby dashed straight lines. Fractions were 7.5 ml each.

FIG. 6. Gradient elution of bacterial endo-fl-1-*3glucanase with the upper limit of gradient, 0.5 Macetate buffer (pH 5.0). Arrow indicates the firstfraction. Fractions were applied counterclockwisefrom the outer toward inner circles. Note the clearzones produced in fractions containing ,3-1*glucanase (36 hr of incubation at 30 C).

tions. The activity of f-i1 3 glucanase decreasedconsiderably if the second elution was not doneimmediately after the first elution. Later it wasfound more convenient to elute f-1 3 glucanaserapidly with 1 M acetate buffer (pH 5.0) and tofollow this by prompt dialysis against 0.03 Mphosphate buffer (pH 6.5).

Table 4 shows the yield and specific activityof fl-i-*3 glucanase obtained by the last-men-tioned procedure. The results show that 89%of the activity was recovered, and the overallspecific activity increased 32-fold.A flow sheet of the separation and purification

of f-i1 -*3 and f-i -*6 glucanases is shown in Fig. 7.Action pattern ofl-i-1 3 glucanase on laminarin

and related oligosaccharides. Formation of reac-tion products during the hydrolysis of laminarinby f-1i3 glucanase was followed chromato-graphically (Table 5).

Oligosaccharides with a (legree of polymeriza-tion (DP) higher than 5 had disappeared after 1hr. However, in some experiments with weakerenzyme solutions. as many as seven sugars couldbe detected during the early stages of the reac-tion. The chromatographic mobility of the threefastest moving sugars was the same as that ofauthentic glucose, laminaribiose, and laminari-triose. Furthermore, the logarithms of the ratiosof movement of the various oligosaccharides to

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TABLE 4. Purification of J0-1-I3 glucanase by theuse of a column of 2 g of DEAE cellulose

SpecificPrepn fS-1-3 Total Total activityGlucanase units protein (units/mg

of protein)

units/ml mg

Initial* .... 80 10,800 326.7t 33Eluate . . 23 9,660 9.24 1,045

* A protamine sulfate-treated concentratedculture fluid; 03-1--+6 glucanase was eluted as in-dicated in the text.

t Total protein based on the concentrateddialyzed culture fluid before protamine sulfatetreatment.

Centrifuged culture fluid (2 liters)

Vacuum concentrated to 200 ml, dialyzedagainst distilled water

Dialyzed concentrated culture fluid

Add 8 ml of 5% protamine sulfate solutionand centrifuge. Discard precipitate.

Supernatant fluid

Dialyze against 0.005 M Tris-HCl bufferpH 8.0

Deposit on DEAE cellulose column (2 g) previ-ously equilibrated with Tris buffer at pH 8.0and washed.

Gradient elution with 0.1 M phosphate buffer(pH 7.2) as the upper limit of gradient.

Wash column with 200 ml of water ,3-1-*6glucanase

Elute with 1 M acetate buffer, pH 5.0 (300 ml)

Dialyze against distilled water or buffe

,6-1-*3 glucanaseFIG. 7. Steps in the separation and purification

of j3-l3 and l-1-6 glucanases.

that of glucose (RGI) were found to bear a linearrelationship to the presumed DP of the inter-mediates (Jeanes, Wise, and Dimler, 1951).This relation is further proof that the reactionproducts detected on paper were members of ahomologous polymeric series, since more than90% of the linkages in laminarin are known to beof the 3-1 --3 type (Peat, Whelan, and Lawley,1958).The disappearance of higher oligosaccharides

and the gradual appearance of lower oligosac-charides and glucose clearly indicate a randommechanism of cleavage of laminarin by p3-1 -+3glucanase.

Action-pattern analysis of laminaritetraoseshowed that this tetrasaccharide was preferen-tially hydrolyzed to trimer and monomer, withtraces of dimer being formed also during the earlystages of the reaction. With the same enzymeconcentration, laminaritriose was hydrolyzedmuch more slowly to dimer and monomer.Laminaribiose was not hydrolyzed by the en-zvme. Because laminarin is hydrolyzed in arandom pattern, the enzyme may be termedendo-fl-1 -3 glucanase.

Action of bacterial endo-fl-1 -cl glucanase onoat glucan. Recently Parrish, Perlin, and Reese(1960) showed that laminarinase from Rhizopusarrhizus hydrolyzes either ,B-1 --3 or l-1i-+4glucosidic linkages in polysaccharides contain-ing both of these bonds, provided either one ofthese linkages is adjacent to a fli-+3 glucosidiclinkage.To compare bacterial f-1--3 glucanase with

the fungal enzyme, oat glucan was subjected tohydrolysis by our bacterial enzyme, and thereaction products were analyzed paper chro-matographically.As markers, glucose, cellobiose, cellotriose,

cellotetraose, laminaribiose, laminaritriose, 3-0-fl-D-cellotriosyl-D-glucose and 3-O-0-D-cellobiosyl-D-glucose were used. The epiphase of ethylacetate-acetic acid water (3:1:3) and of ethylacetate-pyridine-water (10:4:3) were found toseparate all of the products and markers satis-factorily on Whatman no. 1 paper. In both ofthe solvent systems, the major reaction productsshowed the same chromatographic mobilities as

TABLE 5. Chromatographic analsyis of the productsof hydrolysis cf 0.5% laminarin (SS 2208) by

concentrated culture liquid of Bacilluscirculans at pH 6.5*

'rime of incubation (hr)Product

0 1 3 5 10

Laminaripen-taose. - ++++ (+)t - -

Laminaritetra-ose .......... - ++ ++± (+)

Laminaritriose. - + + +++ ++++Laminarihiose.. - + + ++ +++Glucose... - + ++ ++ +++

* The solvent system used was n-butanol-acetic acid-water (4:1:5) on Whatman no. 1filter paper.

t Weak spots.

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ENZYMOLYSIS OF YEAST CELL WALLS

3-0-3-cellotriosyl-D-glucose and 3-0-,B-cellobiosyl-D-glucose. Traces of laminaribiose and glucosewere also present. This pattern of hydrolysis,which was similar to that of the mold enzyme(Parrish et al., 1960), indicated that the mech-nism determining the specificity of f-1-*3glucanase of fungal origin also seems to apply tothe bacterial enzyme.

Action pattern of bacterial /3-1-6 glucanase onpustulan. By paper chromatographic analysis,pustulan, a /3-1-*6 linked polyglucoside, wasfound to be hydrolyzed also by a random mecha-nism. Table 6 shows the pattern of hydrolysisof endo-f-1-*6 glucanase.Of the fl-1-*6 oligosaceharides, only gentio-

biose was available as an authentic compound.Four sugars were present in the 1-hr reactionmixture. With weaker enzyme solutions, higheroligosaccharides also appeared during early stagesof hydrolysis.

In two solvent systems tested, epiphase ofn-butanol-acetic acid-water (4:1:5) and ethylacetate-pyridine-water (10:5:6), logio(RGI X 10)showed a linear relationship to the presumedDP of the reaction products.From these results, together with the fact that

pustulan contains primarily l-1i-+6 linkages, thefour sugars which moved most rapidly duringchromatography were considered to be glucose,gentiobiose, gentiotriose, and gentiotetraose.The final reaction products were gentiobiose andglucose. It was established in a separate experi-ment that gentiobiose was not hydrolyzed bythe enzyme solution.

DISCUSSIONThe main objective of the present study was

to explore the possibility of introducing enzy-matic techniques for comparative studies of thecell-wall composition of different yeasts. It wasfelt that microbial enzymes would be the pre-ferred too]. Although the digestive fluid of snailsis very effective in digesting yeast cell walls, it isnot only very complex in composition (Holdenand Tracey, 1950), but is also difficult to obtainin large quantities. An enzymatic analysis cansupplement chemical investigations on the kindsand frequency of various glycosidic bonds inpolysaccharides (Reese and Mandels, 1959)and throw additional light on the fine structure ofyeast walls.The present paper is primarily concerned with

the nature of the lytic enzymes of a strain ofB. circulans, although this work and that of others(Phaff, 1963). has shown that the ability ofmicroorganisms to elaborate wall-lytic enzymesis quite widespread. B. circulans was chosen forstudy because it caused lysis of baker's yeast cellwalls by a relatively simple extracellular enzyme

TABLE 6. Chromatographic analysis of the productsof hydrolysis of 0.6% pustulan by endo-0-1--+6

glucanase in the concentrated culture fluidof Bacillus circulans (0.05 M phosphate

buffer, pH 6.5

Time of incubation (hr)Product

0 1 6 24

Gentiotetraose.... Trace ++ ()Gentiotriose ...... Trace +++ + (+)Gentiobiose. .... Trace ++ +++ ++++Glucose .......... Trace (+) + ++

* Weak spots.

system, one limited to the hydrolysis of the glucanportion of the cell wall. The culture fluid had noaction on the mannan component and virtuallyno proteolytic activity. Earlier, Horikoshi andSakaguchi (1958) isolated another strain of B.circulans, which, when grown on Aspergillusmycelium, elaborated a strong lytic activitytowards walls of certain fungi. The optimal pHof their partially purified enzyme system wasapproximately 6.5, the same value found optimalfor yeast-wall lysis in our study. A heated suspen-sion of Saccharomyces sake underwent a loweringin turbidity when treated with their enzymepreparation. In a later study (Horikoshi et al.,1963), the A-1--->3 glucanase of their strain ofB. circulans was subjected to purification andshown to hydrolyze laminarin by a random-actionpattern to the dimer stage with a pH optimumat 5.8. However, absence of f-1---6 glucanaseactivity in either the crude or purified enzymewas not demonstrated.Our own work has shown that the inducible

glucanase system of B. circulans WL-12 couldbe purified and resolved into two components,a f-1-*3 glucanase and a f-1i6 glucanase. Thefirst component had a much more pronouncedlytic activity on purified baker's yeast wallsthan did the second enzyme. Both enzymes wereshown to possess randomly splitting properties(endo-glucanases). fl-1i-3 Glucanase hydrolyzedlaminarin to laminaribiose plus glucose, andf-1i6 glucanase hydrolyzed pustulan to gentic-biose plus glucose. These two enzymes aretherefore additional memberis of a group ofrandomly splitting enzymes whieh hydrolyzelinear polvsaccharides to a mixture of dimer andmonomer (Phaff, 1959).

In spite of some uncertainties in the structure ofyeast glucan, it is clear that this polymer containsa great majority of f-1 3 bonds, whereas thebalance is probably limited to fl-1-*6 bonds(Peat, Turvey, and Evans, 1958). If enzymes areto be used for bond analysis in polysaccharides

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TANAKA AND PHAFF

with mixed linkages, reliance has to be placed onthe stereospecificity of the enzyme in question.In this connection the recent findings of Parrishand Perlin (1960) and Parrish et al. (1960) areof great significance. Their work has shown thatthe susceptibility of bonds in a :-linked glucanwith mixed linkages appears to be determined bythe position of substitution in the fl-glucopy-ranosyl unit which is to be cleaved at carbon 1,rather than by the 1 -* n linkage of this unit.Thus, in a situation where we have -G 1-*3G1 -4G- in a polymer chain which is treated with,B-1i-*3 glucanase (laminarinase), it is the 1-34bond which will be hydrolyzed, because thecentral glucose unit is substituted in the 3 posi-tion. Laminaribiose is not hydrolyzed becausethe 3-glucopyranosyl unit of the disaccharide isnot substituted in the 3 position. To test whetherthe f-1-*3 glucanase of B. circulans would behavein the same manner as the Rhizopus enzyme usedby Parrish and co-workers, soluble oat glucanwas included among the substrates tested. Ourresults show that the action pattern of the bac-terial enzyme was similar to that of the fungallaminarinase, including its inability to hydrolyzelaminaribiose.

Similar studies could not be conducted withthe f-1-6 glucanase fraction of B. circulans,but, because of its inability to hydrolyze gentio-biose, its stereospecificity may be governed bysimilar criteria.

It may therefore be inferred, on the basis ofanalogy, that in the hydrolysis of yeast glucanby the f-1-3 glucanase of B. circulans, 1-*6bonds might be hydrolyzed in addition to 1 -*3bonds. Similar considerations apply to the bac-terial f-1-6 glucanase. These possibilities willbe discussed in more detail in a following publica-tion.

LITERATIJRE CITED

CLARKE, A. E., AND B. A. STONE. 1963. Chemistryand biochemistry of ,3-1,3-glucans. Rev. PureAppl. Chem. 13:134-156.

DINGLE, J., W. W. REID, AND G. L. SOLOMONS.1953. Application of the 'cup-plate' assay to theestimation of enizymes. J. Sci. Food Agr. 3:149--155.

FULLER, W. H., AND A. G. NORMAN. 1942. A cellu-lose-dextrin mediuni for identifying celluloseorganisms in soil. Proc. Soil Sci. Soc. Am.7:243-246.

GARVER, J. C., AND R. L. EPSTEIN. 1959. Methodfor rupturing large quantities of microorgan-isms. Appl. Microbiol. 7:318-319.

HAWORTH, W. N., E. L. HIRST, AND F. A. ISHER-WOOD. 1937. Polysaccharides. Part XXIV. Yeastmannan. J. Chem. Soc., p. 784-791.

HOLDEN, M., AND M. V. TRACEY. 1950. A study of

enzymes that can break down tobacco-leafcomponents. 2. Digestive juice of Helix on de-fined substrates. Biochem. J. 47:407-414.

HORIKOSHI, K., H. KOFFLER, AND K. ARIMA. 1963.Purification and properties of ,-1-*3 glucanasefrom the "lytic enzyme" of Bacillus circulans.Biochim. Biophys. Acta 73:267-275.

HORIKOSHI, K., AND K. SAKAGUCHI. 1958. Studieson autolysis of Aspergillus oryzae. The lyticphenomenon of Aspergillus oryzae caused byBacillus circulans. J. Gen. Appl. Microbiol. 4:1-11.

JEANES, A., C. S. WISE, AND R. J. DIMLER. 1951.Improved techniques in paper chromatographyof carbohydrates. Anal. Chem. 23:415-420.

LOWRY, 0. H., N. J. ROSEBROUTGH, A. L. FARR,AND R. J. RANDALL. 1951. Protein measurementwith the Folin phenol reagent. J. Biol. Chem.193:265-275.

NICKERSON, W. J. 1963. Symposiumi on biochemicalbases of morphogenesis in fungi. IV. Molecularbases of form in yeasts. Bacteriol. Rev. 27:305-324.

NORTHCOTE, D. H. 1963. The structure and organi-zation of the polysaccharides of yeast. PureAppl. Chem. 7:669-675.

PALMER, J. K. 1955. Chemiical investigations ofthe tobacco plant. X. Determination of organicacids by ion exchange chromatography. Conn.Agr. Expt. Sta. Bull. 589:31.

PARRISH, F. W., AND A. S. PERLIN. 1960. Stericfactors affecting the specificity of polygly-cosidases. Nature 187:1110-1111.

PARRISH, F. W., A. S. PERLIN, AND E. T. REESE.1960. Selective enzymolysis of poly-f3-D-glu-cans, and the structure of the polymers. Can. J.Chemn. 38:2094-2104.

PEAT, S., J. R. TURVEY, AND J. M. EVANS. 1958.Polysaccharides of baker's yeast. Part III. Thepresence of 1:6-linkages in yeast glucan. J.Chem. Soc., p. 3868-3870.

PEAT, S., W. J. WHELAN, AND H. G. LAWLEY.1958. The structure of laminarin. Part 1. Themain polymeric linkage. J. Chem. Soc., p.724-728.

PHAFF, H. J. 1959. The production of certain ex-tracellular enzynmes by microorganisms, p. 76-116. In W. Ruhland [ed.], Encyclopedia of plantphysiology, vol. 11. Springer Verlag, Berlin.

PHAFF, H. J. 1963. Cell wall of yeasts. Ann. Rev.1Iicrobiol. 17:15-30.

REESE, E. T., AND MI. MANDELS. 1959. Use ofenzymes in isolation and analysis of polysac-charides. Appl. Microbiol. 7:378-387.

SMITH, N. R., R. E. GORDON, AND F. E. CLARK.1946. Aerobic mesophilic spore-forming bac-teria. U.S. Dept. Agr. Misc. Publ. No. 559.

TANAKA, H. 1963. Hydrolysis of yeast cell walls bymicrobial enzymes. Ph.D. Thesis, University ofCalifornia, Davis.

YEMM, E. W. 1935. The respiration of barleyplants. I. Methods for the determination ofcarbohydrates in leaves. Proc. Roy. Soc. (Lon-don) Ser. B 117:483-504.

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