Lysis of Yeast Cell Walls

Transcription

1 Eur. J. Biochem. 63, (1976) Lysis of Yeast Cell Walls Lytic P-(l+ 3)-Glucanases from Bacillus circulans WL-12 Frank M. ROMBOUTS and Herman J. PHAFF Department of Food Science and Technology, University of California, Davis, California (Received October 3/December 2, 1975) Bacillus circulans WL-12, when grown in a mineral medium with yeast cell walls or yeast glucan as the sole carbon source, produced five 8-glucanases. Two /?-(1-+3)-glucanases(I and 11), which are lytic to yeast cell walls, were isolated from the culture liquid by batch adsorption on yeast glucan, and separated by chromatography on hydroxylapatite. Lytic /?-( 1 + 3)-glucanase I was further purified by carboxymethylcellulose chromatography. The specific activity of lytic P-( )-glucanase I on laminarin was 4.1 U per mg of protein. The enzyme moved as a single protein with a molecular weight of during sodium dodecylsulfate electrophoresis in slab gels. It was specific for the p-(1+3)-glucosidic bond but the enzyme did not hydrolyze laminaribiose. Hydrolysis of laminarin went through a series of oligosaccharides, and laminaribiose and glucose accumulated till the end of the reaction. A small amount of gentiobiose was also produced from laminarin. Products from yeast cell walls and yeast glucan included laminaripentaose, laminaritriose, laminaribiose, glucose, and gentiobiose, but no laminaritetraose was detected. This glucanase has an optimum ph of 5.5. Laminarin hydrolysis followed Michaelis-Menten kinetics. A K, of mg of laminarin per ml and a V of 5.6 micro-equivalents of glucose released/min per mg enzyme protein were calculated. The enzyme required no metal ions. The lytic p-(1 --f 3)-glucanase I is a powerfully lytic enzyme, completely solubilizing the glucan of yeast cell walls. Synergism with lytic /?-( 1 +6)-glucanase could be observed during yeast glucan hydrolysis. Lytic B-( 1 -+3)-glucanase I1 could be further purified by carboxymethyl-cellulose and diethylamino-ethyl-agarose chromatography. At this stage lytic P-( 1 --f 3)-glucanase I1 was still contaminated with some non-lytic P-(1-+3)-glucanase that could not be removed by any further chromatographic steps. The enzyme has an optimum ph of 6.5 to 7; its properties were not studied further. Bacillus circulans WL-12 was isolated as an organism capable of degrading yeast cell walls [l]. It was found that this bacterium produces a number of P-glucanases when grown on bakers yeast cell walls or on alkali-insoluble bakers yeast glucan [l Fleet and Phaff [2] isolated both a /?-(1+3)- and a P-(1 +6)-glucanase in highly purified form from the culture liquid, and observed that the hydrolytic action of these enzymes on isolated yeast cell walls was negligible. Neither of the enzymes nor the combination of the two could lyse cell walls to an extent that clearing could be observed in a cell wall agar plate. Also, the enzymes were unable to hydrolyze the microfibrillar glucan network present in Succharomyces walls [4]. The enzymes were therefore considered to be non-lytic. Subsequently, Rombouts and Phaff [3] found that lytic enzymes, which were present in the culture liquid of Bacillus circulans WL-12, could be -~ Enzyme. Endo-l,3-/~ -glucanase (EC ). isolated by adsorption onto alkali-insoluble yeast glucan. Fractionation of the lytic preparation on hydroxylapatite revealed that three lytic enzymes were present, viz. one lytic /?-(1+6)-glucanase and two lytic P-(1+3)-glucanases. The isolation of the lytic enzymes from the culture liquid by glucan adsorption, as well as purification and properties of the lytic p-(1+6)-glucanase have been described [3]. The present paper deals with the purification of one lytic P-(1+3)-glucanase and a study of its properties. It also describes the presence and partial purification of a second lytic P-(1+3)-glucanase. MATERIALS AND METHODS Microorganisms and Culture Conditions Bacillus circulans WL-12 [l] was obtained from the culture collection of the Department of Food Science and Technology (University of California,

2 122 Lytic /3-(1+3)-Glucanases from Bucillus circuluns WL-I2 Davis, California). B. circulans WL-12 was grown in a liquid mineral medium with alkali-insoluble bakers yeast glucan as the substrate for induction of P-glucanases [3]. Preparation of Yeast Cell Walls and Yeast Glucun Bakers yeast cell walls and alkali-insoluble bakers yeast glucan were prepared as described by Rombouts and Phaff [3]. Enzyme Assays Standard assays for glucanase activity were performed as described in [3]. One unit of glucanase is defined as that amount of enzyme which releases one micromole of reducing sugar equivalent, expressed as glucose, per min under the standard assay conditions. For monitoring of lytic activity, a previously described turbidimetric assay was used [3]. Lytic activity on cell walls was also checked by observing clearing in an agar plate containing suspended cell walls (cup-plate technique, [3]). Peptidase and phosphatase activities were determined using Azocoll (Calbiochem) and p-nitrophenylphosphate (Calbiochem), respectively, as substrates [3]. Phosphomannanase activity was estimated as described by McLellan et al. [5], using phosphomannan from Hansenula holstii as substrate. Substrates Laminarin (Nutritional Biochemicals Co.) was used for the estimation of p-(l +3)-glucanase activity [3]. This predominantly p-( 1 + 3)-linked glucan from Laminaria hyperborea (L. cloustoni) is known to be composed of a cold-water insoluble unbranched fraction and a soluble branched fraction. The branch of the soluble fraction consists of a single p-(l+6)- linked glucose residue [6]. Pustulan (ex P. papullosa, Calbiochem), a p-( 1 +6)-glucan, was routinely used as a substrate for j?-(l+6)-glucanase assay [3]. Pachyman was a defatted laboratory preparation from Poria cocos. This p-( 3 +3)-glucan of high molecular weight is water-insoluble. Laminaribiose, laminaritriose, oat glucan, pseudonigeran, peroxidized laminarin, cellulose dextrins, bakers yeast mannan and phosphomannan (from Hansenula holstii) were obtained from the laboratory collection [7-91. All other substrates and chemicals were obtained commercially. Analytical Measurements Reducing sugars were determined by the Nelson- Somogyi method [lo]. Glucose was used for the construction of a standard curve. Protein was measured by the method of Lowry et al. [l l]. Chromatography Descending paper chromatography of degradation products of polysaccharides, and column chromatography of enzymes were done by previously described methods [3]. Glucan Adsorption of Enzymes Lytic P-glucanases were separated from the crude culture liquid of B. circulans WL-12 by adsorption on alkali-insoluble yeast glucan as described [3]. Gel Electrophoresis Enzyme molecular weight was determined by slab gel electrophoresis on polyacrylamide gel in the presence of sodium dodecylsulfate (cf. [3]). RESULTS Purification of Lytic p- ( I +3) -Glueanuse I A mixture of extracellular lytic and non-lytic p-( 1 + 3)- and p-( 1 +6)-glucanases is produced when B. circulans WL-12 is grown in a minimal medium with alkali-insoluble yeast glucan as the substrate [3]. After removal of bacterial cells and glucan remnants by centrifugation, the lytic enzymes were separated from the culture liquid by a two-step adsorption onto yeast glucan in a batch operation as described by Rombouts and Phaff [3]. By chromatography on a hydroxylapatite column (Fig.2 in [3]) the mixture of lytic enzymes could be resolved into three fractions, represented by three peaks: (I) lytic activity together with p-(1+6)-glucanase passed through the column during loading and washing with the starting buffer; (11) lytic activity, high p-( 1 + 3)-glucanase activity and some p-( )- glucanase eluted at a sodium phosphate concentration of M; the lytic P-(l+3)-glucanase present in this peak was designated as lytic p-(1-+3)-glucanase 11; (111) highly lytic activity with very low p-(1+3)- glucanase activity eluted at M sodium phosphate; the lytic p-(1+3)-glucanase present in this peak was named lytic fi-( 1 + 3)-glucanase I. The fractions containing lytic p-(1+3)-glucanase I were dialyzed against 0.01 M sodium succinate buffer ph 5.0 and applied to a CM-cellulose column equilibrated with the same buffer. It was found that some residual non-lytic B-(1+ 3)-glucanase and lytic /3-(1+3)-glucanase 11 did not bind to this column (cf: Fig.2). After washing the column with equilibration buffer, elution was done as indicated in Fig. I. The lytic P-(1-+3)-glucanase 1 eluted as a sharp peak at 0.55 M NaCI. Portions of 0.05 ml of this enzyme,

3 F. M. Rombouts and H. J. Phaff 123 applied to wells in a cell wall agar plate produced zones of complete lysis with a sharp outer border (cf. Fig.6, well B). Fractions 43 to 61 (97 ml) were pooled and dialyzed against M sodium succinate ph 6.0. The protein content of the enzyme solution was measured by the Lowry method [Ill using a sample that was concentrated fifteen-fold by freezedrying and reconstitution. The enzyme was dispensed in 3-ml quantities in small tubes and kept frozen at - 25 'C, until used for the determination of its properties. i i Fraction number Fig. 1. CM-cr/h/ose chromatography of lytic fi-(l+jj-ghcanrt.w 1 enriched by Iiydroxylapatite cliromatogruphy. The enzyme solution (230 ml with U per ml of p-(l-r3)-glucanase activity) was applied to a CM-cellulose column (20x 1.3 cm) equilibrated with 0.1 M sodium succinate buffer ph 5.0. Thc column was washed with IlOml (fractions 1 to 22) of the equilibration buffer, and eluted with a linear gradient (fractions 23 to 77) to 1.0 M NaCl ( of 0.01 M sodium succinate buffer ph 5.0 in the mixing chamber and 150 ml of 0.01 M sodium succinate ph 5.0 in 1.0 M NaCl in the reservoir). Fraction volumes of 5 ml each were collected every nine min. Protein (0); fl-(1+3)-glucanase I (0); lytic activity (turbidimetric assay) (+) Table 1 presents a summary of the steps involved in the purification of the lytic p-(1+3)-glucanase 1. The enzyme represented only a very small portion of the total /I-( 1 +3)-glucanase (laminarinase) activity present in the auto-digest of the insoluble glucan. Since the assay for lytic B-(1+3)-glucanase measures overall laminarinase activity (which includes nonlytic /3-(1+3)-glucanase activity) the degree of purification of the lytic enzyme I cannot be calculated from the apparent specific activity values. The purified enzyme protein represented only 0.032% of the protein in the dialyzed culture fluid. Assuming that 75 % of the lytic enzyme was lost during the purification steps, the lytic /I-( 1 + 3)-glucanase I was purified 78O-fold. Puriscation ojlytic /3-(1+3)-Glucanase II The enzyme mixture of peak I1 which eluted from the hydroxylapatite column at a sodium phosphate concentration of M (Fig.2 in [3]) contained lytic activity, high /3-( 1 + 3)-glucanase activity and some ~-(l-t6)-glucanase. A total volume of 154 ml of this enzyme solution was dialyzed against 0.01 M sodium succinate buffer, ph 5.0 and loaded onto a CM-cellulose column, equilibrated with the same buffer. Elution was done as shown in Fig.2. Lytic activity with very high p-(1+3)-glucanase activity passed through the column without binding (fractions 3 to 39). This peak contained 86% of the /I-(I-+ 3)-glucanase applied to the column. Portions of 0.05 ml of various fractions of this enzyme, applied to wells in a cell wall agar plate, produced after 22 h of incubation at 30 C rather large (5 mm) zones of complete lysis with a fuzzy outer border. Another small lytic peak with only a trace of /I-(1+3)- glucanase activity eluted off the column at a calculated salt concentration of 0.50 M (around fraction 80). Fractions of this enzyme peak gave small zones (2 mm) of complete lysis with a sharp outer border. Both the behavior in the cup-plate assay and the molarity of salt at which this enzyme eluted from the CM-cellulose column (cf Fig. 1) suggested that this Table 1. Summary of the steps involved in pur+cation of lylic /I-(l+3)-glucunase I Hydroxylapatite chromatography gives the combined eluates from two column applications. During this step pustulanase and 35.1 U (63%) of /~-(1+3)-giucanase(both non-lytic and lytic ~-(1+3)-glucanase 11) were separated out. Apparent specific activity is sum of lytic and non-lytic fl-(1+3)-glucanase activity Purification step Volume fl-(1+3)-glucanase Protein Apparent specific activity conccntration activity ml U/ml mg/ml U/mg protein Dialyzed culture filtrate Solution after glucan adsorption Hydroxylapatite chromatography CM-cellulose chromatography

4 124 Lytic B-(1+3)-Glucanases from Bacillus circulans WL E rn E -.~ c a' r ;. 0 m J Fraction number 0 Fig. 2. CM-celluhe chromatography oj'lytic ~-(1+3)-glucana.se I1 enriched by hydrosylupatite chromatography. The enzyme solution (154 ml with 0.23 U per ml of [~-(1+3)-glucanase activity) was applied to a CM-cellulose column (14x 1.3 cm) equilibrated against 0.01 M sodium succinate buffer ph 5.0 (fractions 1 to 30). The column was washed with 110 ml of equilibration buffer (fractions 31 to 48). Elution (fractions 49 to 144) was done with a linear gradient to 1.5 M NaCl (250 ml 0.02 M sodium succinate buffer ph 5.0 in the mixing chamber and 250 in1 of 0.01 M sodium succinate ph 5.0 in 1.5 M NaCl in the reservoir). Fraction volumes of 5 ml each were collected every 10 min. Protein (0); ~-(1-3)-glucanase (0); fl-(1+6)-g~ucdnase (A); lytic activity (turbidimetric assay) (+) } 1.5 L ! '3 100 iractior rurnber Fig. 3. DEAE-Bio-Gel A chromatography qf'lytic /~-(l-ts)-glucunase II. The enzyme solution (175 ml with 0.16 U per ml B-(1+3)-glucanase activity) was applied to a DEAE-Bio-Gel A column (15 x 1.3 cm) equilibrated against 0.01 M sodium succinate buffer ph 6.0. The column was washed with 120 ml of equilibration buffer (fractions 1 to 24). Elution (fractions 25 to 103) was done with a linear gradient to 1.0 M NaCl (200 ml 0.01 M sodium succinate buffer ph 6.0 in the mixing chamber and 200 ml 0.01 M sodium succinate buffer ph 6.0 in 1.0 M NaCl in the reservoir). Fraction volumes of 5 ml each were collected every 10 min. Protein (0); fl-(1+3)-glucanase (0); lytic activity (turbidimetric assay) (+) enzyme represented some of the lytic p-( )-glucanase I. At a calculated salt concentration higher than 0.55 M NaCl (fractions 85 and higher) a lytic enzyme with p-(1+6)-glucanase activity eluted off the column, with considerable tailing. This enzyme produced rather large (5 mm) zones of incomplete lysis with a fuzzy outer border. This enzyme probably represented some of the lytic p-(1+6)-glucanase [3], although in this case the enzyme eluted off the CM- cellulose column at a somewhat higher salt concentration. Fractions 3 to 39 (175 ml) from the CM-cellulose column were pooled, dialyzed against 0.01 M sodium succinate buffer ph 6.0 and applied to a DEAE- Bio-Gel A column. Elution was done as shown in Fig (1+3)-Glucanase eluted off the column in two peaks (fractions 40 and 50, respectively) at calculated salt concentrations of 0.20 and 0.33 M.

5 F. M. Rombouts and H. J. Phaff 125 The fl-(l-+3)-glucanaseluting at 0.20 M NaCl (fractions 35 to 44, containing 36 % of the p-(1-+3)-glucanase activity applied to the column) was non-lytic; it was thought to be the non-lytic p-(1-+ 3)-glucanase described by Fleet and Phaff [2]. Only a small part of this enzyme present in the original culture fluid adsorbed to the glucan during glucan adsorption of the lytic enzymes. The j-(1+3)-glucanase peak eluting at 0.33 M salt (fractions 45 to 55, containing 31 '%, of the j-( 1 -+3)-glucanase activity applied to the column) was lytic (p-(1+3)-glucanase II), as shown by both the turbidimetric and by the cup-plate assays. In the cup-plate assay after 20 h of incubation at 30 "C, the peak fractions of the lytic peak still showed a zone of 5 mm of complete lysis with a fuzzy outer border. However, as is evident from Fig. 3 there was considerable overlap of the two peaks. In other experiments attempts were made to separate these two enzymes on DEAE-cellulose ; however the lytic /3-( 1 + 3)-glucanase I1 adsorbed almost completely and irreversibly to the column material. Thus DEAE-Bio-Gel A proved to be superior for the separation (even though only partial) of the two glucanases. Attempts to further eliminate the non-lytic 8- (1 -+3)-glucanase from the lytic /3-(1+3)-glucanase I1 by rechromatography of fractions 45 to 55 on DEAE- Bio-Gel A, under the same conditions as those described in Fig. 3, were unsuccessful. The enzymes eluted as a single peak. Chromatography of this portion of lytic j-(1-+3)-glucanase I1 on a hydroxylapatite column equilibrated against M sodium phosphate buffer ph 6.5 gave no further separation into non-lytic /3-(1-+3)-glucanase and lytic fl-(1-+3)- glucanase 11. On a Sephadex G-100 column the two enzymes eluted in peaks with great overlap. Separation of the enzymes by gel filtration on Bio-Gel P-100 was not attempted. A chromatography step by which the non-lytic p-(1+3)-glucanase contaminant could be removed quantitatively from the lytic j- (1 -+ 3)-glucanase I1 was not found. The storage stability of this mixed enzyme preparation was rather poor. A 50 % activity loss was measured after one week of storage at 2 "C. The j-(lh3)- glucanase activity and lytic activity decreased simultaneously. The lytic /3-(1+3)-glucanase I1 showed an optimum ph of 6.5 to 7 on laminarin in a series of Tris/succinate buffers, but the curve showed a shoulder at ph 5.0, which is known to be the optimum value for non-lytic j-(l-+ 3)-glucanase [2]. Other properties of the enzyme were not determined. PROPERTIES OF THE LYTIC B-(l+3)-GLUCANASE I Electrophoretic Properties; Molecular Weight Only a limited amount (175 micrograms) of purified enzyme protein was available. The enzyme was concentrated 15-fold by freeze-drying followed by reconstitution; quantities of 0.75 microgram of protein were applied to single wells of a slab gel for sodium dodecylsulfate electrophoresis. Only a single weak band of protein was observed. With this technique, approximately 0.2 microgram of protein is visible in a single band [12]. On the basis of its mobility in the gel, the enzyme was assigned a molecular weight of The possibility that the enzyme dissociated into two subunits of equal molecular weight of during dodecylsulfate electrophoresis was not ruled out, in view of its relative elution volume on a Sephadex G-100 column (not shown). Suhstsate Specijicity and Action Pattern As seen from Table 2 the enzyme specifically hydrolyzed molecules containing the /3-( )-glucosidic bond. However, laminaribiose was not hydrolyzed. Since in oat glucan pairs of /?-(I +4)-glucosidic bonds alternate with single /3-(1-+3)-bonds [23], the enzyme presumably cleaves /3-( 1 +4)-bonds also, provided one of the fl-glucopyranosyl units composing these bonds is substituted in the 3-position by another glucose unit [l], The enzyme contained neither p-dglucosidase, c?-d-glucosidase, phosphatase nor peptidase activities. For the determination of the action pattern, laminarin, pustulan, pachymdn, bakers' yeast cell walls and bakers' yeast glucan (final concentration 5 mg per ml), were incubated at 30 "C in the presence of U per ml of enzyme and 0.01 M sodium succinate buffer ph 6.0. Samples were withdrawn after 2, 8, 32, and 120 h. After inactivation by boiling for 5 min, the samples were desalted and chromatogrdphed on Whatman no. 1 paper [3]. A mixture of lamina- ribiose, laminaritriose, laminaritetraose and laminaripentaose as well as authentic gentiobiose ( RGIL values with the ethyl acetate/pyridine/water solvent of 0.80, 0.58, 0.40, 0.29 and 0.52, respectively) were used as standards. The hydrolysis of laminarin went through a series of oligosaccharides. Laminarihexaose to laminaritriose, very little laminaribiose, some glucose and some gentiobiose were the products after 2 h of incubation. Laminarihexaose to laminaritetraose were still present in low concentrations after five days of reaction. while the amount of laminaribiose and glucose increased greatly throughout the incubation period. The concentration of laminaritriose increased over the first 32 h and then decreased slightly. The gentiobiose level increased during the first 32 h and then remained constant. The production of gentiobiose from laminarin showed the enzyme to cleave the two P-(l+3)-bonds adjacent to a glucose unit with a j-( 1 -+6)-glucosyl branch. The action pattern on laminarin, and also the activity on periodate oxidized

6 ~ ~~ ~ ~~ ~~ 126 Lytic fi-(1+3)-glucanases from Bacillus circulans WL-12 I o'6 Table 2. Substrate speciji'city of lytic /L(l+3)-glucanase I from B. circulans WL-12 Substrates (4 mg per nil; ph 6) were incubated with enzyme (0,0015 U per ml, final concentration) at 30 "C for 12 h, after which time formation of reducing groups was measured. +, reducing groups formed; -, 170 reducing groups formed Substrate Main linkage Hydrolysis type Laminarin 8-1,3 + Laminaritriose " - Laminaribiose 8-1,3 Glucan (S. cerevisiae) p-1,3; 8-1,6 Cell walls (S. crrevisiae) p-1,3;,8-1,6 Periodate oxidized laminarin 8-3,3 Pachyman 8-1,3 Oat glucd11 8-1,4; 8-1,3 + Cellulose dextrins 8-1,4 - Pseudonigeran a-1,3 - Dextran a-i,6 - Amylose a-i,4 - Yeast mannan ~-1,6; ~-1,3; ~-1,2 - Phosphomannan (H.holstii) (mannose) a-1,2; a-1,3 - (mannose) Sucrose - Meth yl-8-d-glucoside - Phen yl-8-o-ghcoside - p-nitrophenyl-8-d-glucoside - p-nitrophenyl-a-d-glucoside - p-nitrophenylphosphate - Azocoll A._.- > c " m 0' I PH Fig.4. OptimumpH of lytic 8-(1+3/-glucanase I. Reaction mixtures (0.5 ml) were composed of 0.1 ml of enzyme ( U per ml), 0.2 ml of laminarin (10 mg per ml) and 0.2 ml of a series of Tris/ succinate buffers (0.2 M tris-(hydroxymethy1)-aminomethanc brought to the desired ph with 0.2 M succinic acid). Incubation, 4 hat 30 "C walls; its concentration did not increase significantly in later samples. a Determined by paper chromatography. See Materials and Methods for phosphatase and peptidase assays. laminarin (Table 2) proved the lytic p-( 1 +3)-glucanase I to be an endoenzyme. No products were observed from pustulan during any stage of the incubation period. Laminaritriose, glucose and a trace of laminaribiose were the products detected during the early stage of pachyman hydrolysis. Glucose and laminaribiose increased steadily over the entire incubation period, while laminaritriose increased over the first eight hours of incubation and then decreased considerably. Bakers' yeast cell walls and alkali-insoluble yeast glucan showed very similar degradation patterns, although glucan was slightly more susceptible to degradation than were cell walls. The products observed after 2 h of incubation were laminaritriose, glucose and a trace of laminaribiose. Laminaribiose and glucose accumulated throughout the incubation period. The concentration of laminaritriose increased up to 32 h of incubation and then decreased. No laminaritetraose was ever detected. Laminaripentaose was present in low concentrations after 8 and 32 h of incubation, but only a trace of this compound remained after 5 days. A gentiobiose spot was found in the eight-hour sample from yeast glucan and in the 32-h sample from cell Storage and Stability The lytic p-(1+3)-glucanase I was moderately stable at 2 "C in the ph range of 5 to 7 in buffers of to 1.0 M. About 25% of the activity was lost during one week of storage under these conditions. The enzyme was insensitive to 0.01 % sodium azide. For longer periods, the enzyme was stored in M sodium succinate buffer ph 6.0, in 3-ml quantities in small tubes, frozen at - 25 "C. Under these storage conditions activity loss over a period of two months was less than 10%. Kinetics The enzyme had no metal requirements, since the presence of 0.01 M ethylenediamine tetraacetic acid had no noticeable effect. Laminarin hydrolysis rate was linear for the first 3.5 h (composition of reaction mixtures, 0.2 mg per ml of substrate, U per ml of enzyme, 0.08 M sodium succinate buffer ph 6.0). If the assumption is made that the reducing groups of the oligosaccharide products of laminarin contribute equally as much as does glucose to the absorbance values found in the Nelson-Somogyi test, the average degree of polymerization of the products was estimated to be 5.5 (corresponding to 18% hydrolysis) at the point where the hydrolysis rate was no longer linear.

7 F. M. Rombouts and H. J. Phaff 121 1/[Sl (ml/mg) Fig. 5. Lineweaver-Burk plot of larninarin hydrolysis with lytic /I-( I +3)-g/ucanuse I. Reaction mixtures contained (final concentrations) to 0.20 mg per ml of laminarin, U per ml of enzyme, and 0.08 M sodium succinate buffer ph 6.0. Reducing sugars were measured after 2 h of incubation at 30 "C. o is expressed as change in absorbance at 520 nm Fig. 6. Cup-plate test ofthe Iytic /l-glucana.se.y ofb. circulans WL-12. Portions of 0.05 ml of the various enzyme solutions were applied to wells made with a No. 2 cork borer in a cell wall agar plate. Enzymes applied to different wells were: well A, U of lytic fi-(1+6)-glucanase; well B, U of lytic /l-(i- 3)-glucanase I; well C, U of lytic fi-(1-.3)-glucanase 11. Incubation, 15 h at 30 ' C The optimum ph as determined with a series of Tris/succinate buffers was 5.5 (Fig.4). At ph 6.0 the measured activity was approximately the same in 0.08 M sodium phosphate as in sodium succinate buffers, but it was 15 % lower in Tris/succinate buffer. Enzyme activities were measured at different substrate concentrations (Fig. 5). A K, of mg laminarin per ml and a V of 5.6 microequivalents of glucose released/min per mg of enzyme protein were determined from the figure. When the data of Fig. 5 were used in a log [S] versus log u/(v - v) plot, a straight line was obtained from which a K, of mg laminarin per ml was calculated. The slope of the line was 1.04, a value close to 1, which is typical of enzymes following Michaelis-Menten kinetics [14]. Action of the Various Lytic p-glucanases on Bakers' Yeast Cell Walls and on Bakers' Yeast Glucan All three of the lytic enzymes of B. circulans WL-12 were applied to wells in a cell wall agar plate (Fig. 6). The lytic p-(1+3)-glucanase I (Fig. 6, well B) produced a ring of complete lysis with a very sharp outer border. Under a phase contrast microscope no remnants of cell walls could be detected in the lysed zone. The enzyme was powerfully lytic in very low concentrations. The lytic p-(1+6)-glucanase [3] showed rapidly spreading but incomplete lysis (Fig. 6, well A). The outer border of the lysed zone was rather vague. Under a phase contrast microscope the cell walls still appeared to be present in the lysed zone but they showed considerably less contrast. The lytic p-(1+3)- glucanase 11, purified as far as possible by the technique applied, showed a moderately spreading zone of lysis with a fuzzy outer border (Fig. 6, well C). Lysis slowly proceeded to completion, and in the zone immediately around the well, no remnants of cell walls could be detected. It is evident from Fig. 1 and 3 that, based on their p-( 1 + 3)-glucanase (laminarinase) activity, the lytic fl-(1+3)-glucanase I is much more strongly lytic than the lytic fl-(1+3)-glucanase 11. Bakers' yeast glucan was hydrolyzed with lytic fl-(1+3)-glucanase I, with lytic fl-(1+6)-glucanase and with a combination of the two lytic enzymes (Fig. 7). With lytic fl-(1+3)-glucanase I the rate of release of reducing groups from glucan was constant for the first 2 h, corresponding to 1.4:! hydrolysis of the substrate. and then decreased slightly. During the linear period, the rate of release of reducing groups from the insoluble glucan was 55'x as compared to that obtained with soluble laminarin under the same reaction conditions. In contrast it could be calculated from data given by Fleet and Phaff [2], that the initial activity of the non-lytic fl4-3)- glucanase of B. circulans WL-12 was times lower on cell walls than on laminarin (these authors did not use yeast glucan as substrate). In

8 128 Lytic ~-(1+3)-Glucanases from Bacillus circulms WL Time (h) Fig. I. Hydrolysis of alkali-insoluble bakers' yeast glucan with lyric ~-(l-+3)-glucanase I, Iytic b-(l+6/-glucanase and a comhinutian o/ the t~;o enzymes. Reaction mixtures contained (final concentrations) 1 mg of glucan per ml, 0.08 M sodium succinate buffer ph 6.0 and either U of lytic b-(l-+3)-glucanase I per ml (curve A) or 0.01 U of lytic ~-(3+6)-glucanase per ml (curve B) or U of lytic P-(l+3)-glucanase 1 plus U of lytic /3-(1+6)-g~ucanase per ml (curve C). At time intervals of 30 min. samples were withdrawn and analyzed for reducing Sugars addition, the total release of reducing groups from glucan by the lytic P-(1+3)-glucanase I was much greater than that from cell walls with non-lytic p-( 1 + 3)-glucanase after prolonged incubation. For example, after 4.5 h the lytic j-(1+3)-glucanase I ( U per ml, 1 mg glucan per id) had split 2.6% of the glucosidic bonds present in the substrate. Calculated from data of Fleet and Phaff [2], the non-lytic fl-(l+ 3)-glucanase liberated only 0.4 of the reducing groups of cell walls (0.5 U of enzyme per ml, 2.5 mg of cell walls per ml) during 45 h of incubation at 30 "C. As shown for the lytic and nonlytic p-(1+6)-glucanases of B. civculans WL-12 [3], the lytic p-(1+ 3)-glucanase I hydrolyzes insoluble yeast glucan much more rapidly and to a greater extent than the non-lytic p-( 1 +3)-glucanase. As seen from Fig.7, there was no synergism between lytic j3-(1-+3)-glucanase I and lytic fl-(l+6)- glucanase during the first half hour of the reaction, since during that stage of the reaction curve C runs in between curves A and B, at equal distances from these curves. After 1 h of reaction, synergism was obvious and it became more pronounced as the reaction proceeded. From the action patterns of the enzymes on yeast glucan it was evident that synergism can not be explained in terms of relief of product inhibition. It was thought more likely that synergism is due to increased accessibility to the substrates through the simultaneous action of the enzymes. DISCUSSION The P-Glucanases of B. circulans WL-12 This bacillus produces a number of 8-glucanases when grown on yeast cell walls or on yeast glucan. One non-lytic p-( 1 +6)-glucanase [2], one lytic p- (1 +6)-glucanase [3] and one non-lytic p-( )- glucanase [2] have already been fully purified and described. In this paper, the purification and properties of lytic B-(l-t3)-glucanase I and the partial purification of lytic p-(1+3)-glucanase I1 are described. Recently Kobayashi et al. [15] and Tanaka et al. [16] also explored the production of multiple 8-glucanases by B. circulans WL-12. Their activity profile, as shown by polyacrylamide gel electrophoresis of the enzyme mixture produced on S. cerevisiae cells, revealed four different p-glucanases active on soluble laminarin. These enzymes have as yet not been purified and studied further, but most likely they will prove to be identical with the three fl-(1-+3)-glucanases mentioned above and the lytic p-( 1 +6)-glucanase, which is known to release gentiobiose from soluble laminarin [3]. The bacterium was found to produce six laminarinase activities when Pyricularia oryzae mycelium was used as the substrate [16]; one of these, which was not produced in significant concentration on S. cerevisiae cells, was further purified by electrophoresis and some of its properties have been determined [17]. This enzyme had a molecular weight of Its properties appeared generally similar to those described for the lytic P-(l-+3)-glucanase I. In view of the results of these authors [16] it may be possible to further separate contaminating non-lytic fi-( )-glucanase from lytic p-(1+3)-glucanase I1 of Bacillus circulaizs by polyacrylamide gel electrophoresis. Properties of p-(l-+3)-glucunases and Their Action on Yeast Cell Walls The three p-(1+3)-glucanases of B. circulans WL-12 differ strikingly in their action on yeast cell walls and on insoluble yeast glucan. The non-lytic p-( 1 + 3)-glucanase gives an almost negligible release of reducing sugars from yeast cell walls [2], whereas the highly lytic p-(1+3)-glucanase I produces reducing sugars from insoluble yeast glucan at an initial rate which is 55% of that from soluble laminarin at the same enzyme concentration. The lytic p-(1-+3)- glucanase 11 occupies an intermediate position, since, relative to its laminarinase activity, its lytic activity on cell walls was much lower than that of lytic p-(1+ 3)-glucanase I. Glucanase components with high and low lytic activity are also known to be produced by Arrhrobacter [18]. The substrate specificity and the endo-action pattern on laminarin of the lytic p-( )-glucanase I were generally similar to those described for the non-lytic p-( 1 -+3)-glucanase

9 F. M. Rombouts and H. J. Phaff 129 of B. circuluns WL The key difference between the two enzymes lies in their K,,, values for laminarin: 0.55 mg per ml and mg per ml for non-lytic p-( 1 --t 3)-glucanase and lytic p-( )-glucanase I, respectively. A difference in affinity must also exist for insoluble yeast glucan, since the lytic B-( 1 +3)-glucanases could be preferentially adsorbed on glucan and thereby separated from most of the non-lytic p-(1-+3)- glucanase which remains in the culture liquid [3]. The greater affinity of the lytic B-(1+3)-glucanase I for insoluble substrates may be at least one factor responsible for its powerfully lytic properties on yeast cell walls. The ability to split glucosidic bonds other than p-(1+3)-bonds (as in oat glucan [l]) or, /3- (1 -+ 3)-bonds adjacent to glucose units substituted in the 6 position (as in laminarin and yeast cell wall glucan) are thought to be other prerequisites for lytic ability. The latter property would indicate ability to debranch the two types of glucan present in the alkaliinsoluble fraction of yeast cell walls (cf. [3]). Reliance on K,,, values with soluble laminarin as the substrate to explain lytic activity is likely to be of limited value. Neither is specific activity per mg of protein on this substrate an index for this property, which is ten times as high for the non-lytic B-( 1 + 3)-glucanase [2] as for lytic P-(1+3)-glucanase I. In recent years several other lytic p-( 1 --f 3)-glucanases have been reported [ The lytic /?-(1-+3)- glucanase separated by Horikoshi et al. [20] from the culture liquid of another B. circuluns strain grown on Aspergillus oryzae mycelium has properties generally similar to those of the /3-(1+3)-glucanases from B. circuluns WL-12. The enzyme hydrolyzes laminarin to glucose and laminaribiose, via laminaritriose and higher oligosaccharides. Another group of lytic p-(1-+3)-glucanases produces as end products from yeast glucan, pachyman, and laminarin mainly oligosaccharides having five or more glucose residues. A highly lytic representative of this group was isolated from the culture liquid of Cytoplzuga johnsonii [22]. Other examples are the lytic p-( 1 + 3)-glucanase zymolyase from Arthrobacter luteus [23,24] and from another Arthrohacter strain [18, Also, fungal enzymes of this type have been reported [ In contrast to the group of lytic B-(1-+3)-glucanases, such as that from B. circuluns WL-12, most of these enzymes require very long chain substrates, which are degraded to higher oligosaccharides only. These higher oligosaccharides, in turn, may act as inhibitors. A third type of lytic P-(1+3)-glucanase is represented by the exo-p-( 1 + 3)-glucanase of a species of Basidiomycetes QM The constitutive enzyme, which is produced in large amounts by this fungus, liberates only glucose from laminarin [31], although later [35] gentiobiose was found to be produced also from bakers yeast cell walls. Its ability to bypass P-(1+6)-linked glucos$ side chains [36] must be an important aspect of its lytic potential. It would seem that different types of lytic p-(1+3)- glucanases exist, which, according to their properties, can be placed in any of at least three groups. An explanation of the effectiveness of the lytic enzymes would require more knowledge of their specificity than is currently available. In addition, the lytic properties of an enzyme must be affected by the chemical composition of various yeast and mold cell walls, in as much as a highly lytic enzyme is not equally effective on the glucan of every yeast or mold species [37,38]. It is felt that the highly lytic P-(1+3)-glucanase I described in this paper represents a valuable tool in yeast cell wall glucan analysis, since it degrades cell wall material very extensively and to products of low molecular weight. F. M. Rombouts received a NATO Science Fellowship through the Netherlands Organization for the Advancement of Pure Research. REFERENCES I. Tdnaka, H. & Phaff, H. J. (2965) J. Bucteriol. 89, Fleet, G. H. & Phaff, H. J. (1974) J. Bucteriol. IIY, Rombouts, F. M. & Phaff, H. J. (1976) Eur. J. Biochem. 63, Kopecka, M., Phaff, H. J. & Fleet, G. H. (1974) 1. Crff Bid. 62, McLcllan, W. L., McDaniel, L. E. & Lampen, J. 0. (1970) 1. Bucleriol. 102, Nelson, T. E. & Lewis, B. A. (1974) Curhohydr. Res. 33, Fleet, G. H. & Phaff, H. J. (1974) J. Biol. C hem. 249, Abd-El-Al, A. T. H. & Phafl, H. J. (1968) Biochwn. J. IOY, Abd-El-Al, A. T. H. & Phaff, H. J. (1969) Can. J. Microhiol. 15, Spiro, R. G. (1966) Methods Enzymol. X, Lowry, 0. H., Rosebrough, N. J., Farr, A. 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