Vol. 142, No. 2JOURNAL OF BACTERIOLOGY, May 1980, p. 414-4230021-9193/80/05-0414/10$02.00/0

Lyticase: Endoglucanase and Protease Activities That ActTogether in Yeast Cell LysisJANET H. SCOTTt AND RANDY SCHEKMAN*

Biochemistry Department, University of California, Berkeley, California 94720

Yeast lytic activity was purified from the culture supernatant of Oerskoviaxanthineolytica grown on minimal medium with insoluble yeast glucan as thecarbon source. The lytic activity was found to consist of two synergistic enzymeactivities which copurified on carboxymethyl cellulose and Sephadex G-150, butwere resolved on Bio-Gel P-150. The first component was a ,8-1,3-glucanase witha molecular weight of 55,000. The Km for yeast glucan was 0.4 mg/ml; that forlaminarin was 5.9 mg/ml. Hydrolysis of fi-1,3-glucans was endolytic, yielding amixture of products ranging from glucose to oligomers of 10 or more. The sizedistribution of products was pH dependent, smaller oligomers predominating atthe lower pH. The glucanase was unable to lyse yeast cells without 2-mercapto-ethanol or the second lytic component, an alkaline protease. Neither of theseagents had any effect on the glucanase activity on polysaccharide substrates. Theprotease had a molecular weight of 30,000 and hydrolyzed Azocoll and a varietyof denatured proteins. The enzyme was unusual in that it had an affinity forSephadex. Although the activity was insensitive to most protease inhibitors, itwas affected by polysaccharides; yeast mannan was a potent inhibitor. Theenzyme did not have any mannanase activity, however. Neither pronase nortrypsin could substitute for this protease in promoting yeast cell lysis. A partiallypurified fraction of the enzymes, easily obtained with a single purification step,had a high lytic specific activity and was superior to commercial preparations inregard to nuclease, protease, and chitinase contamination. Lyticase has beenapplied in spheroplast, membrane, and nucleic acid isolation, and has proveduseful in yeast transformation procedures.

Enzymes capable of degrading the cell wall ofyeasts have been isolated from the culture su-pernatants of numerous microorganisms (13). Ingeneral, the yeast lytic activity can be attributedto a fi-1,3-glucanase, although lytic ,8-1,6-glucan-ases have been characterized (16). Usually, theglucanase activity will not lyse viable yeast cellswithout either a thiol reducing agent or a secondenzyme activity. For some procedures the prep-aration of yeast spheroplasts requires pure lyticenzymes of known specificity. We have devel-oped a high-yield purification of yeast lytic en-zymes ("lyticase") from the culture supernatantof Oerskovia xanthineolytica and characterizedthe f8-1,3-glucanase and the protease that areresponsible for the lytic activity.

MATERIALS AND METHODSMaterials. The following products were obtained

commercially: pustulan and Azocoll (Calbiochem);laminarin (Pierce Chemical Co. or Calbiochem); poly-ethylene glycol (Carbowax 600 powder, Union CarbideCorp.); CM 52 and DE 52 cellulose (Whatman, Inc.);

t Present address: National Institutes of Health, Bethesda,MD 20205.

Sephadex G-150 (Pharmacia Fine Chemicals, Inc.);Bio-Gel P-150, P-30, and P-2 (Bio-Rad Laboratories);yeast extract and peptone (Difco Laboratories); bulkyeast (Red Star); calf thymus DNA, pancreatic DNaseI, myoglobin, hemoglobin, cytochrome c, lysozyme,and ovalbumin (Sigma Chemical Co.); bovine serumalbumin (Miles Laboratories, Inc.); Zymolyase 5000and 60000 (Kirin Brewery); and Glusulase (Endo Lab-oratories). A premarket sample of Teichozyme Y wasgenerously provided by Worthington BiochemicalCorp. Mannan from Saccharomyces cerevisiae X2180-1B and mnn2 prepared by Fehling's precipitation (12)and the mannan acetolysis fractions were a gift of R.Douglas. Escherichia coli alkaline phosphatase was agift of K. Telander. Rabbit actin was a gift of C. Greer.

Insoluble yeast fi-glucan was prepared from RedStar yeast by repeated alkali extractions done by theprocedure of Cabib and Bowers (2) through step 1only; no acid extraction was performed. After eachextraction, the glucan was collected by centrifugationin a Sorvall GS3 rotor at 9,000 rpm (14,000 x g) for 15mm. Sedimentation in a Sharples centrifuge did notclear the supernatant solution, and the resultant glu-can was not an effective inducer, possibly as a resultof a lack of small glucan oligomers. The glucan wasadjusted to pH 7 with HCl before the final centrifu-gation and then was washed twice with distilled water.The pellet was then dried by washing once with

414

VOL. 142, 1980

ethanol and twice with ether. The yield was about 15g of glucan per lb (454 g) of yeast, so approximately 6lb (2.7 kg) of yeast was required to provide sufficientglucan for a 20-liter culture of 0. xanthineolytica.

Autoclaved yeast was prepared by mixing Red Staryeast with an equal volume of distilled water andautoclaving for 20 min. The yeast cell wall residue wascollected by centrifugation and washed twice withdistilled water. The pellet was stored frozen untilneeded.

Strains and growth conditions. Arthrobacterluteus strain 73-14 was obtained from Jeremy Thorner(University of California, Berkeley). This strain wasoriginally obtained from Yashishi Yamamoto (KirinBrewery, Takasaki, Gunma, Japan). This organismhas since been reclassified by Mary Lechevalier (Rut-gers University) as Oerskovia xanthineolytica.

0. xanthineolytica was grown at 30°C on modifiedSistrom's medium (18) designated M63 containing(grams per liter): KH2PO4 (13.6), (NH4)2SO4 (2.0),KOH (4.2), MgSO4.7H20 (0.2), and Fe2(SO4)3.6H20(0.001). This was supplemented with thiamine andbiotin (1 ,tg/ml each). Medium contained one of thefollowing carbon sources: glucose, 0.4%; washed auto-claved yeast, 1.5%; or insoluble yeast glucan, 0.4%.The lytic assay employed haploid S. cerevisiae

X2180-1B as substrate. Cells were grown on YPD (1%yeast extract, 2% Difco peptone, 2% glucose) to anabsorbance at 600 nm (A600; Zeiss PMQII spectropho-tometer, 1-cm cuvette) of 2 or less (they were lesssensitive to lysis if grown to a higher density). Cellgrowth was arrested by the addition of 10 mM sodiumazide. Cells were harvested by centrifugation and wereresuspended in 50 mM potassium phosphate (pH 7.5)plus 10mM azide. Yeast suspensions were used within1 week.

S. cerevisiae 1412-4d (a MAL3 SUC3) was used forthe invertase release experiment.Enzymatic assays. Unless otherwise stated, the

lytic assay incubation mixture contained: 50 mM po-tassium phosphate (pH 7.5), 10 mM 2-mercaptoetha-nol, a yeast cell suspension sufficient to give an Aw* of0.8 to 0.9, and enzyme, in a total volume of 2.0 ml.Enzyme was added last, and the A8w was read beforeand after a 30-min incubation at 30°C in a shakingwater bath. One lytic unit is defined as a 10% decreasein absorbance in 30 min. The assay was sigmoidal withrespect to time and enzyme concentration; activitywas determined from the slope between 20% and 70%reduction in Aso as provided by increasing enzymeconcentrations. Assays for lytic synergy were done intwo ways. The first was a standard assay done oncombined enzyme fractions. Fold synergy is defined asthe activity of the combined fractions divided by thesum of the activity of the two fractions by themselves.The other assay measured lysis in the absence of 2-mercaptoethanol when purified glucanase was mixedwith other fractions.The protease assay mixture contained 10 mg of

Azocoll per ml, 100 mM potassium phosphate (pH7.5), and enzyme, in a total volume of 0.5 ml. After a30-min incubation at 30°C with shaking, the insolubleAzocoll was removed by centrifugation, and the A520of the supematant solution was measured against ablank containing no enzyme. One protease unit is

ENZYMATIC LYSIS OF YEAST 415

defined as one A520 unit solubilized in 30 min.The chitinase assay mixture contained 50 mM po-

tassium phosphate (pH 7.5), 0.5 mg of colloidal chitin(1) per ml, and enzyme, in a total volume of 1 ml. After1 h at 30°C, the reaction was stopped by boiling for 3min. The undegraded chitin was removed by centrif-ugation, and portions of the supematant solution weretreated with Sephadex G-25-ffitered Glusulase (2) ina total volume of 0.5 ml for 30 min at 300C withshaking. (This treatment breaks oligomers of N-ace-tylglucosamine down to monomers.) The reactiontubes were boiled for 3 min and tested for N-acetylglu-cosamine by the Reissig reaction (14). One unit re-leases 1 nmol of N-acetylglucosamine in 1 min.The DNase assay mixture contained 0.5 ml of DNA

solution (0.2 mg of polymerized calf thymus DNA perml, 8 mM CaCl2, 50 mM Tris, pH 7.5), 50 mM buffer(pH 7.5), and enzyme, in a total volume of 1 ml. Theincrease in A2m0 was measured over a few minutes. TheKunitz unit (7) was used.The glucanase assay mixtures contained up to 0.1

ml of enzyme and 0.1 ml of glucan substrate (2.0 mg/ml) or laminarin (10 mg/ml) in 0.1 M potassium phos-phate buffer (pH 7.5). The reactions were incubatedat 300C for 0.5 to 4 h and were stopped by the additionof 0.2 ml of alkaline copper tartrate reagent, used forthe determination of reducing sugar (19). The sub-strates for ,8-1,3- and ,B-1,6-glucanases were laminarinand pustulan, respectively. When yeast glucan wasused as substrate, the insoluble material was removedby centrifugation before the solubilized reducingsugars were assayed. a-Mannanase activity was as-sayed in an analogous fashion except that the pH ofthe phosphate buffer was 7.0. One unit releases 1 ,umolof reducing sugar equivalent per min.

Invertase was assayed by the method of Goldsteinand Lampen (5), a two-step procedure. The A40 wasread, with glucose used as a standard. One unit equals1 ,umol of glucose released per min.

Protein determinations were done as described byLowry et al. (9) on samples which had been precipi-tated with 7% trichloroacetic acid. Bovine serum al-bumin was used as a standard.

Identification of polysaccharide hydrolysisproducts. Bio-Gel P-2 (400 mesh) was used to sepa-rate oligomers. The column was 1 by 53 cm. Fractions(0.5 ml) were collected at a flow rate of 5 ml/h andassayed for carbohydrate by the phenol-sulfuric acidmethod of Dubois (3). Positive identification of thesugars was made by descending paper chromatogra-phy in ethyl acetate-pyridine-water (5:3:2) as the sol-vent. Chromatograms were developed for 24 h, andsugars were located with the silver nitrate stain (21).Oligomers of the laminarin series, obtained from R.Cohen, who had made them by partial acid hydrolysisof laminarin, were used as standards.

Gel electrophoresis. Polyacrylamide gels (12%)were electrophoresed in the presence of SDS (sodiumdodecyl sulfate) as described by Laemmli (8). Gelswere stained for protein with Coomassie blue.Molecular-weight determinations. (i) Subunit

molecular weights were estimated from SDS gels, withbovine serum albumin (68,000), rabbit actin (42,000),pancreatic DNase I (31,000), trypsin (23,800), and ly-sozyme (14,400) as standards. (ii) Molecular weights

416 SCOTT AND SCHEKMAN

in solution were estimated both by gel filtration onBio-Gel P-150, with bacterial alkaline phosphatase(80,000), hemoglobin (68,000), ovalbumin (43,000), andmyoglobin (17,000) as standards, and by sucrose veloc-ity gradient centrifugation in the manner of Martinand Ames (10), with bacterial alkaline phosphatase(80,000), typsin (23,800), myoglobin (17,000), cyto-chrome c (12,400), and lysozyme (14,400) as standards.Enzyme purification: (i) Growth and induc-

tion. 0. xanthineolytica was grown on 20 liters ofglucan or yeast medium for 24 to 36 h, starting with a1% inoculum of a culture grown on glucose medium toan A6o0 of 2 to 3. Cells were grown either in Erlenmeyerflasks using one-fifth the volume of medium or, forlarge-scale preparations, in a New Brunswick fermen-tor (two 10-liter cylinders) with mum aeration at250 rpm. Samples of the culture supernatant weremonitored for lytic activity, and cells and residualsubstrate were removed by centrifugation (20 min at14,000 x g in a Sorvall GS3 rotor) at the estimatedpoint of highest activity. The turbidity of the mediummade the monitoring of growth by absorbance impos-sible. The use of a steam-driven Sharples centrifugewas not satisfactory for clarifying the supernatantsolution. The following procedures were done at 0 to40C.

(ii) Concentration and dialysis. The culture su-pernatant was concentrated to about one-twentieth ofits original volume by dialysis against solid polyethyl-ene glycol. The culture supernatant was placed in 2-in. (5-cm)-diameter dialysis tubing which was thencovered with Carbowax 6000 powder. The viscousliquid which accumulated outside the tubing wasdrained off, and new polyethylene glycol was appliedseveral times during the procedure, which took about24 h. The concentrate (fraction I) was dialyzed against10 mM sodium succinate (pH 5.0) in the same tubingafter collecting the liquid at one end and tying it off.

(iii) Carboxymethyl cellulose chromatogra-phy. CM-52 cellulose was equilibrated with 10 mMsodium succinate (pH 5.0). A 200-ml slurry was mixedwith fraction I and stirred for 1 h. The mixture waspoured into a column (4 by 20 cm) and allowed tosettle. The unabsorbed solution was collected, thecolumn was washed with one column volume of buffer,and the activity was eluted with two volumes of buffercontaining 0.25 M NaCl. Ten-milliliter fractions werecollected, and those containing lytic activity (fractionH) were pooled. Column chromatography with saltgradient elution did not improve the purificationachieved.

(iv) Sephadex G-150 chromatography. A por-tion of fraction II was concentrated 10-fold by dialysisagainst solid polyethylene glycol, and 1 ml was appliedto a 30-ml column (1.5 by 20 cm) of Sephadex G-150equilibrated with 50 mM succinate buffer (pH 5.0).Flow rate was critical; for best separation, a flow rateof s4 ml/h was used. The column was eluted with 120to 150 ml of 50 mM succinate buffer (pH 5.0), and 2-ml fractions were collecte4. The second broad peak ofprotein which contained all the lytic activity (fractionIII) was pooled.

(v) Bio-Gel P-150 chromatography. Fractionmwas concentrated 10-fold by dialysis against polyethyl-ene glycol and applied to a 70-ml column (1.5 by 40

cm) of Bio-Gel P-150. The column was equilibratedand eluted with 50 mM sodium succinate buffer (pH5.0). Fractions of 2 ml were collected at a flow rate of4 ml/h. The two protein peaks (fraction IVa and b)were pooled separately.

(vi) DEAE-celiulose chromatography. FractionIVb was dialyzed against 10mM potassium borate (pH8.5) and applied to a 20-ml column (1.5 by 10 cm) ofDE-52 equilibrated with the same buffer. The unab-sorbed material (fraction V) was pooled. The remain-ing protein was eluted with borate buffer containing0.1 M NaCl.

RESULTSInduction and purification. When 0. xan-

thineolytica was grown with either autoclavedyeast or purified yeast glucan as a carbon source,a large number of hydrolytic enzymes were se-creted into the culture medium. These included,8-1,3-glucanases, chitinase, and proteases. In-duction of lytic and protease activities on yeastand glucan media was compared (Fig. 1). Lyticactivity was induced to a comparable level oneither carbon source, although induction took 12to 24 h longer with the glucan medium. Theinduction of protease, however, was reduced by80% when glucan medium was used, so theseconditions were chosen for purification of thelytic activity.For large-scale (20-liter) fermentor prepara-

tions, it was necessary to concentrate the culturesupernatant. This was accomplished by dialysisagainst solid polyethylene glycol. This concen-trate was absorbed onto carboxymethyl celluloseand eluted with 0.25M NaCl, effecting a 2.7-foldpurification.

D E,800- 4

0~~~~~~~~~~~~~~0

Hours After Inoculation

FIG. 1. Lyticase and protease induction. Cultures(200 ml) ofautoclaved yeast (open symbols) or glucanmedium (closed symbols) were inoculated with 2 mlofa glucose-grown culture. Samples (5 ml) were takenat intervals, the cells were removed by centrifugation,and the supernatant solution was assayed for lytic(circles) andprotease (triangles) activities.

J. BACTERIOL.

ENZYMATIC LYSIS OF YEAST 417

Early attempts at gel filtration with Sephadexresulted in an apparent total loss of lytic activity.However, it was found that several column vol-umes of buffer served to elute the activity, sug-gesting that the lytic enzymes were selectivelyretained by the dextran matrix. This affinitystep completely removed chitinase activity (Fig.2). The retardation of the lytic activity wasunaffected by either salt concentration or pH,but was abolished if the elution buffer contained1 M glucose.The lytic activity did not absorb to Bio-Gel P-

150. Gel permeation chromatography on thismaterial yielded two peaks, one containing lyticand glucanase activities and the other containingonly protease activity (Fig. 3). There was ashoulder of lytic activity where the two peaksoverlapped. The material in the first peak fromthis column, fraction IVa, cochromatographed,with the lytic and glucanase activities and theprotein eluting together. The lytic specific activ-ity was constant across the peak. This fractiondisplayed a single major band on SDS-polyacryl-amide gels with a few light bands attributable toproteolysis of the major band (Fig. 4).When fraction IVa and fraction IVb (the sec-

ond peak from the Bio-Gel P-150 column) weremixed, pronounced synergy (two- to five fold)for lysis was observed. Fraction IVb containedresidual glucanase activity which was separatedfrom the protease activity by DEAE-cellulosechromatography. The protease activity flowedthrough the column, whereas the glucanase waseluted with 0.1 M NaCl. The protease activitychromatographed as a single species on Bio-GelP-30 and showed a single major band on SDS-polyacrylamide gels (Fig. 4F), although proteo-

~-2-

DI -

O ~~~~~~~~~~~~60000-

0.2-

4000

0.1

2000

Froction Number

FIG. 2. Sephadex G-150 chromatography. A con-

centrated sample offraction H (1 ml, 7mg ofprotein)was applied to a 30-ml Sephadex G-150 column (1.5by 19 cm) equilibrated with 50 mM succinate buffer,pH 5.0. The column was eluted with the same buffer,and 2-mi fractions were collected. Open circles, AM;closed circles, lytic activity; crossed bars, chitinaseactivity. Excluded volume is fraction 5; included vol-ume is fraction 16.

N ~~~~~~~~~~~~~~~~~~~~~N,

22.0-1

4)~~~~~~~~~~~0

15 20 25 30 35

Fraction Number

FIG. 3. Bio-Gel P-150 chromatography. FractionIII was concentrated against polyethylene glycol toc1.5 ml and was applied to a 70-mi Bio-Gel column(1.5 by 40 cm) equilibrated with 50 mM sodium suC-cinate buffer, pH 5.0. The column was developed withthe same buffer, and 2-mi fractions were collected.The flow rate was 5 ml/h. Symbols 0, A2w; *, Iyticactivity; A, protease activity; A, ,B-1,3-glucanase ac-tivity. Excluded volume is fraction 11; included vol-ume is fraction 35.

lytic breakdown products were detected afterstorage for prolonged periods at 0°C or afterincubation at elevated temperatures. This wasprobably due to action of the protease on itself.The protease displayed virtually no lytic activityby itself but stimulated the lytic activity of theglucanase. This was the source of the synergyseen earlier.The summary of the purification achieved in

a typical preparation starting with culture su-pernatant from cells grown on yeast glucan isshown in Table 1.Properties of the glucanase: (i) Electro-

phoretic properties and molecular weight.When the glucanase was electrophoresed onSDS-polyacrylamide gels, only a single majorband appeared, with an apparent molecularweight of 55,000 (Fig. 4D and H). A few lightbands with slightly higher mobilities were seenwhen 5 to 10 ,ug of protein was applied to thegel, but these bands were seen across the entireBio-Gel P-150 peak and could be due to cleavageby a contaminating protease in trace amounts.The low-molecular-weight species were en-hanced by treatment of the glucanase with pu-rified protease. The molecular weight of theglucanase as calculated from its elution on Bio-Gel P-150 was 65,000, indicating a monomericstructure.

(ii) pH optimum. The glucanase activitymeasured with laminarin had a pH optimum of6.0 (Fig. 5). The lytic activity, however, showeda pH optimum at 8.0 with a steep drop betweenpH 7.5 and 7.0. At pH 9 and above, yeast cells

VOL. 142, 1980

418 SCOTT AND SCHEKMAN

A B C D E F G H

-|_m _ 41b f:t..oin" MO q

FIG. 4. SDS-polyacrylamide gel electrophoresis. Samples were applied to a 12% polyacrylamide gel. (A)Dialyzed concentrate of culture fluid (fraction I), 15 pg; (B) carboxymethyl cellulose pool (fraction II), 10 pg;(C) Sephadex G-150pool (fraction III), 10pg; (D) Bio-Gel P-150peak I (fraction IVa), 10 pg; (E) Bio-Gel P-150peak HI (fraction IV), lOpg; (1F) DEAE-cellulose, unabsorbed material (fraction 19, 5pg; (G) DEAE-cellulose,salt wash, 10 pg; (H) fraction IVa rechromatographed on Bio-Gel P-150, 10 pg.

TABLE 1. Purification of lytic activityLytic activity Protease activity

Fraction~ ~ /mg Fold pu~rifi- Yil % /g Fold purifi- il %U/g cation Yil /g cation Yil

Culture supernatant .11,600 1 100 9.9 1 100FractionI..11,400 0.99 85 18.7 1.91 163bFraction II................25,000 2.7 57 26.3 2.67 57F'raction III ...............48,000 4.2 44 18.1 1.84 19bFraction IVa...............42,O00 3.6 25 - - -

Fraction IVb .................24,000 2.07 5 79.9 8.1 20Fraction V....<. _._. - 98.9 10.0 11

a The greater than 100% Yield may have been due to the instability of the protease in the culture supematant.b The low yield of protease at this step was due to the separation from another nonlytic protease.'The specific activity goes down as a result of separation from the synergistic protease.

d Some lysis was seen with large amounts of pure protease.

became sensitive to lysis in the absence of en-zyme, and the assay was invalid.

(iii) Substrate specificity and action pat-tern. The glucanase was active on both lami-narin and insoluble yeast glucan, both predom-inantly ,B-1,3-linked glucose polymers, butshowed no activity on pustulan, a 8-1,6-linkedglucan, or mannan. The enzyme released nomore than 5% of the potential reducing ends of

either laminarin or yeast glucan. Approximately50% of the glucan as measured by the phenol-sulfuric acid assay was converted to soluble oli-gomers after exhaustive hydrolysis; the additionof new enzyme and buffer to the insoluble resi-due did not result in the release of any morecarbohydrate to the supernatant solution. All ofthe laminarin was converted to small oligomersafter exhaustive hydrolysis.

J. BACTERIOL.

ENZYMATIC LYSIS OF YEAST 419

(0-)

5 6 7 8 9 10

pHFIG. 5. pH optima. Assays were performed in the

following buffers: pH 5.0 to 6.5, sodium succinate;pH6.5 to 8.0, potassium phosphate; pH 8.0 to 9.0, potas-sium borate; pH 9.0 to 10.5, sodium carbonate. Sym-bols: A, laminarinase activity ofpurified glucanase(fraction IVa); 0, lytic activity ofpure glucanase; A,proteolytic activity ofpure protease (fraction V9.

The products of exhaustive glucanase diges-tion of laminarin are shown in Fig. 6. The en-zyme cleaved endolytically and released a vari-ety of oligomers. The final products were pHdependent. At pH 6.0, the pH optimum for lam-inarinase activity, smaller oligomers predomi-nated, with major amounts of laminaritriose,laminaribiose, and glucose. At pH 7.5, the opti-mal pH for the lytic reaction, larger oligomers,pentasaccharides or higher, predominated. Thehydrolysis of yeast glucan showed a similar pHdependence, with trisaccharides and pentasac-charides the major product at pH 6.0 and 7.5,respectively.

(iv) Kinetics. Glucanase activity was meas-ured at different substrate concentrations ofboth yeast glucan and laminarin. The resultswere markedly different. The apparent Km forglucan was 0.4 mg/ml, whereas the apparent Kmfor laminarin was 5.9 mg/ml, revealing a 10-foldhigher affinity of the enzyme for its naturalsubstrate. The maximum velocity was calculatedto be 1.05 nanoequivalents per min for glucanand 5.63 nanoequivalents per min for laminarin.The turnover numbers calculated were 120min-' molecule-' for glucan and 620 min-' mol-ecule-' for laminarin.

Stability. The purified glucanase was quitestable. Fifty percent of the activity remainedafter 3 weeks at room temperature. Seventypercent of the activity remained after 3 weeks at2 to 40C or frozen at -18°C, with or without 20%glycerol. For long-term storage, freezing wasused, but for periods of less than 1 month, stor-age at 2 to 4°C in the presence of sodium azidewas satisfactory.

Fraction Number

FIG. 6. Glucanase digestion products of lami-narin. Digestion mixtures (1-ml total volume) con-tained 20 mg of laminarin, 10 mM sodium succinatebuffer (pH 6.0) or 10 mM potassium phosphate (pH7.5), and 6 U ofpurified glucanase (as measured onlaminarin atpH 6.0). Digestions were carried out for24 h at 30°C. Samples were loaded on a Bio-Gel P2column and analyzed as described in Materials andMethods. Symbols: 0, pH 7.5; 0, pH 6.0. Excludedvolume is fraction 29; included volume is fraction 65.

(vi) Activation and inhibition. As men-tioned above, the Bio-Gel P-150 chromatogra-phy yielded two peaks of activity, one of glucan-ase and one of protease, which, when assayedtogether, gave up to five times the expectedactivity. Further purification of the second peakled to the separation of the protease from resid-ual glucanase activity. The synergy was due tothe protease activity. When 2-mercaptoethanolwas removed from the lytic assay mixture, theglucanase lost its lytic activity entirely (Fig. 7A).The addition of either protease or 2-mercapto-ethanol restored the lytic activity (Fig. 7B). Nei-ther the protease nor the 2-mercaptoethanol hadany effect on the glucanase activity on polysac-charide substrates.

Inhibition of the lytic activity of the glucanaseby various sugars was also examined. Glucanwas a potent inhibitor (50% inhibition at 0.03mg/ml); laminarin inhibited at 10 times the con-centration for glucan. Glucose was inhibitory atlow concentrations (50% at 0.8 mg/ml), but man-nan inhibited only at high concentrations (50%at 3.1 mg/ml).

(vii) Invertase release. The extent of solu-bilization of invertase, a cell wall mannoprotein,was used as a measure of lytic enzyme digestionof the cell wall. After a 1-h preincubation with2-mercaptoethanol, either the glucanase or theprotease or both released essentially all the in-vertase, even at low concentrations of enzyme(Fig. 8A). Preincubation was required because

VOL. 142, 1980

420 SCOTT AND SCHEKMAN

0

-J

0

-J

._

p-Mercaptoethanol, mM

4C

20

0.05 0.10

Protease, UG15

FIG. 7. Stimulation of the lytic activity of the glu-canase by the protease and 2-mercaptoethanol. (A)Standard lytic assays wereperformed except that theamount of2-mercaptoethanol was varied from 0 to 40mM. The glucanase (fraction IVa) was held constantat 0.8 U per assay. Symbols: 0, 2-mercaptoethanoland glucanase; 0, 2-mercaptoethanol alone. (B) Asabove except that no 2-mercaptoethanol was addedand protease (fraction V) was added in amountsranging from 0.01 to 0.15 U per assay. Symbols: 0,

glucanase andprotease; 0, protease alone.

2-mercaptoethanol interfered with the invertaseassay. In the absence of the 2-mercaptoethanolpretreatment (Fig. 8B), the glucanase did notrelease any invertase; the protease alone re-leased a maxium of 30%, but the combinationreleased up to 70% of the invertase to the super-natant solution. Invertase activity was unaf-fected by these levels of protease.Properties of the protease. The synergistic

protease and glucanase activities copurifiedthrough the Sephadex G-150 step. Separationwas achieved on Bio-Gel P-150, and completepurification was accomplished with DEAE ion-exchange chromatography. This protease dis-played several unique properties. First, it re-stored lytic activity to the glucanase in the ab-sence of 2-mercaptoethanol. Neither trypsin norpronase was able to substitute for the nativeprotease, even when added at up to 20,000 timesthe proteolytic activity as measured on Azocoll.Second, the protease was retarded on Sephadexand eluted after the first column volume. Thisretention, also seen with the purified protease,indicated that the protease had an affinity forthe matrix itself and was not indirectly boundthrough the glucanase. Third, the protease,

40

enC 10c

B. 80 _

60

40-

20

0 20 30 40 50

kLI/A600FIG. 8. Invertase release by the lytic enzymes. (A)

S. cerevisiae 1412-4D was grown on yeast extract-peptone-maltose (2%) overnight to an Aeo of 2. Cellswere centrifuged, washed, and resuspended in 50mMpotassium phosphate (pH 7.5). 2-Mercaptoethanolwas added to give a final concentration of 10 mM,and the cells were incubated at 30°C for 1 h. Thecells were again centrifuged, washed, and resus-pended in phosphate buffer. To 0.5 ml of cells at anA6 of 2, 5 to 50 iLl of enzyme(s) was added, andincubation was conducted for 30 min at 300C. Cellswere centrifuged and resuspended in 0.5 ml ofphos-phate buffer. Samples of both the supernatant solu-tion and the cell suspension were assayed for inver-tase activity. Percent release is the amount in thesupernatant solution divided by the sum of theamounts in both the supernatant solution and the cellsuspension. (B) Same as the above except that theincubation with 2-mercaptoethanol was omitted.Symbols: A, glucanase alone (stock solution, 1,000 Ulml); A, protease alone (stock solution, 10 U/ml); 0,

both enzymes.

though unaffected by most protease inhibitors,was inhibited by polysaccharides, in particularby bulk mannan (Table 2). The protease did not,however, display any a-mannanase activity. Amolecular weight of 30,000 for the protease wasdetermined by SDS-gel electrophoresis and oneof 25,000 was determined by sucrose velocitysedimentation, suggesting a monomeric struc-ture. The protease displayed an alkaline pHoptimum (Fig. 5). The purified protease wasstable at -20°C at pH 8.5 (potassium boratebuffer, 10 mM). The addition of glycerol desta-bilized the enzyme. Approximately 50% of the

60 A

40

20

20 10 20 30 40

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B

ENZYMATIC LYSIS OF YEAST 421

activity was lost in 1 week at room temperature.Comparison to commercial preparations.

Fraction II and the purified glucanase were com-pared with several commercial lytic enzymepreparations: Glusulase, Zymolyase 5000 and60,000, and Teichozyme Y. Glusulase is a con-centrate of snail digestive juice; Zymolyase andTeichozyme Y are lyophilized powders from cul-ture supernatants ofA. luteus and 0. xanthineo-lytica, respectively. Both the Zymolyase 5000and Teichozyme Y powders contained onlyabout 10% protein by weight. Zymolyase 60,000is a more purified form of Zymolyase 5000. Thecontent of lytic, chitinase, DNase, and proteaseactivities indicated considerable contaminationof the commercial preparations (Table 3). Frac-tion II was significantly freer from the contami-nating activities and had a high lytic specificactivity. Contaminating activities were not de-tected in the purified glucanase.

Fraction II, our standard laboratory reagentfor making spheroplasts, had a pH optimum of7.5, required reducing reagent for full activity,and was inhibited significantly by glucose. The

TABLE 2. Protease inhibition by carbohydrates andglycoproteins

Concn (mg/Compound ~~~ml) givingCompound 50% inhibi-

tion

Mannan, X2180-1B .......... ......... 0.18Mannan, mnn2 ....................... 0.24Acetolysate Ia ........................ 2.0Acetolysate IIa .................... 5.0Mannotetraose ....................... >10.0L.Aminarin ........................... >10.0Mannose ............................ 200Glucose ............................. 200Ovalbumin .......................... 25Bovine serum albumin ........ ........ >100

a Acetolysates I and II are mannan acetolysis prod-ucts with an average size of 100 and 10 mannoseresidues, respectively.

enzyme preparation was very stable. At 2 to 40Cno loss of activity was detected over severalmonths when stored with sodium azide as an

antimicrobial agent. At 220C the enzyme de-cayed 50% in 1 week, but could be stabilized bysaturated ammonium sulfate or 20% glycerol.The preparation lost 50% of its activity whenfrozen.

DISCUSSIONThe purification of lytic activity described in

this paper has two critical steps. First, the use ofpurified glucan as the carbon source during en-

zyme induction results in a much lower induc-tion of protease. A partially purified fraction isstable and highly effective in the formation ofspheroplasts. The second critical step is theSephadex affinity column. Lytic enzymes bindto the column and are separated from otherhydrolytic enzymes which elute in the columnvolume. This step is similar to the glucan ab-sorption step employed by both Rombouts andPhaff (15) and Jeifries (T. Jeffries, Ph.D. thesis,Rutgers University, New Brunswick, N.J., 1975).In our hands, the Sephadex affinity step was

more reliable.The yeast lytic enzyme system ("lyticase")

produced by 0. xanthineolytica consists of a ,8-1,3-glucanase and an alkaline protease. The glu-canase attacks ,8-1,3-glucans in a random endo-lytic fashion, releasing oligosaccharides, in a pH-dependent fashion. At pH 7.5, the optimum forcell lysis, oligomers of five or more predomi-nated, whereas at pH 6.0, the optimum for lam-inarinase activity, smaller oligomers and glucosepredominated. This effect, which was seen withboth yeast glucan and laminarin, probably is dueto a pH effect on the enzyme. This action pattemwas different from that seen for other lytic glu-canases, with which laminaripentaose is the ma-jor or sole hydrolysis product (6, 15, 23). Theenzyme showed a 10-fold higher affinity for glu-can than for laminarin. Consistent with this was

TABLE 3. Comparison of activities found in various lytic enzyme preparationsActivity (U/mg) Ratio

Lytic enzyme souce Chiti-Lytic/prooLyticehi-Lytic Protease ruenase tease tinase Lytic/DNase

Zymolyase 500 3,200 3,200 120 1.9 4,900 28 1,700 0.66Zymolyase 60000 ...... 4,700 50 0.4 14,000 94 12,000 0.34Glusulase ..............is5a 0.2 1.2 _b 66 12 __bTeichozyme Y 240 240 30 6.1 51,000 8 38 4.6 x 10-3Fraction II .32,00 32,000 29 22 <100 1,100 1,400 >320Fraction III .22,00 22,000 13 1.4 <100 1,800 16,000 >220Fraction IVa ........... 31,000 <1 X 10-2 <0.5 <100 >3 x 10W >62,000 >310

a The published pH optimum of this preparation (6.0) gave an even lower value.b Intrinsic absorbance of glusulase made the assay impossible.

VOL. 142, 1980

422 SCOTT AND SCHEKMAN

the observation that 10 times as much laminarinas glucan was required to inhibit the lytic activ-ity. A high affinity for yeast glucan may accountfor the lytic capacity of the glucanase. Glucan-ase-mediated lysis required either 2-mercapto-ethanol or the aLkaline protease, neither ofwhichaffects the activity on laminarin or glucan. Thereducing agent and the protease probably acteddirectly on the yeast cell surface.The alkaline protease was quite specific. Al-

though the proteolytic activity measured on theAzocoll substrates was low, small amounts wereeffective in lytic synergy with the glucanase.Neither trypsin nor pronase (or other proteasespresent in the culture fluid) could be substitutedfor the protease in the lytic reaction. The pro-tease was unaffected by a variety of proteaseinhibitors, but it was strongly inhibited by man-nan. An affinity for polysaccharides may explainthe retention of the protease by the Sephadexmatrix. The purified protease was free from de-tectable glucanase and a-mannanase activity;thus, lysis was promoted by attack on the pro-tein portion of the mannoprotein, not on thecarbohydrate portion. Mercaptoethanol maysubstitute for the protease by reducing disulfidebonds in the mannoprotein.

Villanueva (22) has proposed that the glucanlayer of the cell wall, which is responsible forstructural integrity and rigidity, is covered by alayer of mannoprotein which must be modifiedin some way before a lytic glucanase can reachits substrate and promote cell lysis. This theoryis supported by the synergistic effect of glucan-ase and protease activities in cell lysis and ininvertase solubilization. The results of the latterexperiment suggest that some invertase is ex-posed at the cell surface and some is sequesteredbeneath the glucan layer, accounting for theextra invertase released by the combination ofboth enzymes.The enzyme system described here is similar

to that isolated from Arthrobacter GJM byVrsanka et al. (23, 24). Here also, cell lysis re-quires the combined action of a ,8-1,3-glucanaseand an alkaline protease or 2-mercaptoethanol.The characteristics of the individual proteins,however, are different. The GJM glucanase issmaller (20,000 daltons) and specifically releaseslaminaripentaose. Pronase substitutes for thealkaline protease in their system, but this couldbe due to a difference in the cell wall of thesubstrate yeast, CCY 21-4-13. Although Zy-molyase is the only ,B-1,3-glucanase which isclaimed to be active against yeast cells by itself,the recent isolation from crude Zymolyase of aprotease which promotes yeast cell lysis by theglucanase (4) suggests that the glucanase may

not be pure. Assays which use glucan or cellwalls as substrate (such as the cup-plate assayemployed by Rombouts and Phaff [15, 16] forthe Bacillus circulans enzymes) show no re-quirement for thiols or protease in the assay butdo not establish the effectiveness of the enzymeagainst yeast cells.The partially purified fraction II lyticase was

easily obtained in large quantities with a singlepurification step. It had a high lytic specificactivity and was purer than any commercialpreparation with regard to nuclease, protease,and chitinase contamination. This fraction hasbeen used successfully for spheroplast formation(11), membrane isolation (17), and nucleic acidisolation (20), and has proved useful in yeasttransformation procedures.

ACKNOWLEDGMENTSWe thank Jeremy Thorner and Patrick Gerstenberger for

providing the Oerskovia strain, for communicating their un-published results, and for the idea of replacing autoclavedyeast with extracted glucan as a carbon source.

This work was supported in part by grants from the Uni-versity of California Cancer Research Committee, the Univer-sity of California Biomedical Research Support Fund, theCalifornia Division of the American Cancer Society, and theNational Science Foundation.

ADDENDUM IN PROOFWe found recently that one alkaline extraction of

yeast cells (instead ofthree) produces a glucan fractionthat is satisfactory in the induction of lyticase withlow protease content.

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