Yeast 14, 1297–1306 (1998)

A New Method for Quantitative Determination ofPolysaccharides in the Yeast Cell Wall. Application tothe Cell Wall Defective Mutants of Saccharomycescerevisiae

NATHALIE DALLIES, JEAN FRANCqOIS* AND VERONIQUE PAQUET

Centre de Bioingenierie Gilbert Durand, UMR-CNRS 5504, LA. INRA, Departement de Genie Biochimique etAlimentaire, Institut National des Sciences Appliquees, Complexe Scientifique de Rangueil,31077 Toulouse Cedex 04, France

A reliable acid hydrolysis method for quantitative determination of the proportion of â-glucan, mannan and chitinin Saccharomyces cerevisiae cell wall is reported together with a simple extraction procedure to quantify within astandard error of less than 2% the proportion of the wall per gram of cell dry mass. This method is an optimizedversion of Saeman’s procedure based on sulfuric acid hydrolysis of complex polysaccharides. It resulted in an almostcomplete release of glucose, mannose and glucosamine residues from cell wall polysaccharides. After completeremoval of sulfate ions by precipitation with barium hydroxide, the liberated monosaccharides were separated andquantified by high performance anion-exchange chromatography with pulsed amperometric detection. Thesuperiority of this method over the hydrolysis in either trifluoroacetic or hydrochloric acid resides in its higherefficiency regarding the release of glucose from â1,6-glucan and of glucosamine from chitin. The sulfuric acid methodwas successfully applied to determine the â-glucan, mannan and chitin contents in cell walls of geneticallywell-characterized yeast mutants defective in cell wall biosynthesis, and in Schizosaccharomyces pombe cell walls. Thesimplicity and reliability of this procedure make it the method of choice for the characterization of cell walls fromS. cerevisiae mutants generated in the EUROFAN programme, as well as for other pharmacological andbiotechnological applications. ? 1998 John Wiley & Sons, Ltd.

— cell wall; chitin; â-glucan; Saccharomyces cerevisiae

*Correspondence to: J. M. Francois, Centre de BioingenierieGilbert Durand, UMR-CNRS 5504, LA. INRA, Departementde Genie Biochimique et Alimentaire, Institut National desSciences Appliquees, Complexe Scientifique de Rangueil, 31077Toulouse Cedex 04, France. Tel: (+33) 561559492; fax: (+33)

INTRODUCTION

The cell wall of Saccharomyces cerevisiae mayaccount for between 20 and 30% of the cell drymass. It is mainly composed of mannoproteins andâ-glucans (85–90% of cell wall dry mass) and asmaller amount of chitin (1–3%) and lipids (2–5%)(Fleet, 1991; Klis, 1994). The proportion of these

561559400; e-mail: fran_jm@insa-tlse.fr

CCC 0749–503X/98/141297–10 $17.50? 1998 John Wiley & Sons, Ltd.

components may vary according to the strains andthe culture conditions (McMurrough and Rose,1967; Catley, 1988).

Methods available for cell wall architectureanalysis are based on the separation of structuralcomponents by either chemical or enzymaticmethods. The chemical fractionation of the cellwall polysaccharides results in three main frac-tions: an alkali-soluble fraction consisting mainlyof â1,3-glucan and mannan and some â1,6-glucan;an alkali-insoluble acid-insoluble fraction, con-taining â1,3-glucan linked to chitin; and an

acid-soluble alkali-insoluble fraction composed of

Received 10 February 1998Accepted 9 May 1998

1298 . .

â1,6-glucan (Manners et al., 1973; Fleet, 1991).Because chemical methods are rather harsh andcan damage the original structure of the polymers,more gentle methods involving enzymes capable ofspecifically digesting one component without alter-ing the others have been devised, as for instancethe use of pronase to isolate mannoproteins fromâ-glucans or Quantazyme, a recombinant â1,3-glucanase, to digest â1,3-glucan without degradingâ1,6-glucan (Hartland et al., 1994; Kapteyn et al.,1996; Lu et al., 1995; Kollar et al., 1997). Comp-lementary to these techniques, Hong et al. (1994b)have developed a cell wall fractionation procedurefor yeast cells incubated with radiolabelled glucosein order to quantify the actual changes in cell wallstructure after the introduction of a specific muta-tion (namely deletion of KNR4). Alternatively,methods for a rapid estimation of the proportionof carbohydrates, proteins and lipids in the yeastcell wall primarily require a separation of the cellwall from other cell components and a procedurefor complete hydrolysis of complex cell wall poly-mers. The liberated monosaccharides can then beeasily separated and quantified by improved high-performance anion-exchange chromatography(HPAEC) using pellicular resins and pulsed-amperometric detection (PAD) (Hardy et al.,1988). With this method, monosaccharides can beaccurately detected without any derivatizationwith a sensitivity of 0·1 nmol per injected amount.

Several procedures, differing in the nature andthe concentration of acids, have been used toachieve complete hydrolysis of cell wall polymers.In their original work leading to the isolation of 63cell-wall-defective mutants of S. cerevisiae basedon Calcofluor white hypersensitivity, Ram andcoworkers (1994) characterized these mutants by achange in the mannose/glucose ratio after hydroly-sis of isolated cell wall in 2 -trifluoroacetic acid(TFA). This method was, however, unable toestimate chitin contents because TFA is not veryefficient in cleaving the â1,4-glucosyl linkages ofthis polymer (Tracey, 1956; Hardy et al., 1988).Conversely, the use of a 72% solution of sulfuricacid (H2SO4) following Saeman’s procedure(described in Selvendran et al., 1974) apparentlyresulted in an almost complete release of mono-saccharides from various fibrous substrates includ-ing xylan, cellulose and wheat bran (Garleb et al.,1989). Other hydrolytic agents such as hydro-chloric acid (HCl) or hydrofluoric acid (HF) havealso been used for hydrolysis of either complexcarbohydrates of plant cell walls (Chambers and

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Clamp, 1971; Ip et al., 1992) or of chitin from yeast(Popolo et al., 1997), but these methods sufferfrom the fact that they cannot be readily adaptedfor quantification of the released monosaccharidesby HPAEC.

Therefore, our purpose was to compare differentacid hydrolysis procedures of cell wall polymers inorder to propose a simple, accurate and reproduc-ible protocol for the quantification of polysaccha-rides from cell walls of yeast mutants generated inthe EUROFAN programme. We report here thatsulfuric acid hydrolysis coupled to HPAEC-PADanalysis is the most complete, accurate, and repro-ducible method for determination of carbohydratecontents of the yeast cell wall.

MATERIALS AND METHODS

MaterialsLaminarin (â1,3-glucan), mannan (á1,2- 1,3-

and 1,6-mannose) and chitin (â1,4-N-acetylglucosamine), chitinase (from Serratiamarcescens) and N-acetylglucosaminidase werepurchased from Sigma Co., and pustulan (â1,6-glucan) from Calbiochem. Other enzymes andbiochemicals were obtained from Boehringer.Growth media were from Difco. Solutions of 50%NaOH used for HPAEC-PAD analysis were fromFisher Scientific. All other chemicals were of thehighest grade available.

Yeast strains and culture conditionsThe yeast strains used in this work are listed in

Table 1. Strain JF291 was used as the referenceS. cerevisiae strain for optimizing cell wall isolationand extraction of polysaccharides. Yeast cells weregrown in shake flasks (filled to 20% of their totalvolume) at 30)C and 200 rpm in YPD medium (10 gyeast extract, 20 g bactopeptone and 20 g glucoseper litre). Unless otherwise stated, yeast cultureswere collected at 2#107 cells/ml (mid-exponentialphase of growth) for cell wall extraction.

Isolation of cell wall

Disruption by hydraulic pressure using the FrenchPress (‘macro-method’) This method was usedfor the accurate determination of the proportion ofthe wall relative to the yeast cell dry mass. Expo-nential or stationary phase cells (1–2 g dry mass)were harvested by centrifugation (5 min, 2000 g)and washed three times with cold deionized water.

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The cellular pellet was resuspended in 20 ml coldTris–HCl, 10 m, pH 8·0 at a concentration ofabout 50 mg cell dry mass/ml. Disruption wascarried out by several passages in a French Press ata pressure of 18,000 psi (1450 kg/cm2) and fol-lowed by microscopic examination. The breakagewas stopped when the cell suspension showedmore than 95% of sheared and broken cells. Theclassical method of blue methylene staining wasnot used since it also stained apparently intactcells. In general, four passages were enough forwild-type strains, but additional passages werenecessary when the chitin content in the cell wallswas higher. After disruption, the cell suspensionwas brought to 20 ml with water, and an aliquot of5 ml was used for determination of cell dry mass(see below). The cell walls were pelleted at 3800 gfor 5 min and washed several times with colddeionized water until the supernatant becameclear. The pellet was finally resuspended in 5 ml ofdeionized water and 3 ml of this suspension wasdried to a constant mass in an oven at 110)C todetermine the proportion of cell wall relative tocell dry mass. The remaining part was used forcarbohydrate analysis.

Disruption with glass beads (‘micro method’) Thismethod was used for rapid carbohydrate analysisin yeast cell walls and in particular to quantify

the mannose/glucose ratio. About 20 mg (cell dry

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mass) of exponential or stationary phase cells wereharvested and washed three times with cold deion-ized water. Yeast cells were disrupted in 0·5 ml ofTris–Cl 10 m, pH 8, in the presence of 0·5 g ofglass beads (0·45–0·55 mm in diameter) for fourcycles of 20 s each, using a mini-Bead Beater(Biospect Products, Bartlesville, OK, USA) with20-s intervals on ice. Alternatively, disruption canbe carried out in glass tubes vortexing the cellsuspension on a bench-top vortex set at full speedfor several periods of 1 min, with intervals of 1 minon ice. The percentage of cell breakage was esti-mated as described above and the cycles werestopped when more than 95% of the cells werebroken. The cell suspension was collected and theglass beads were extensively washed with coldTris–Cl buffer. The supernatant and washings werepooled and centrifuged again at 3800 g for 5 min.The pellet, containing the cell walls, was washedseveral times with cold deionized water until thesupernatant became clear, and stored at "20)Cuntil use.

Hydrolysis procedures

Table 1. Yeast strains used in this study.

Strain Genotype Source or reference

S. cerevisiaeJF291 MATa leu2 ura3-52 his3 This studyAR27 MATá ura3-52 Ram et al. (1994)cwh53 MATá ura3-52 cwh53/fks1 Ram et al. (1994)cwh4 AR27 cwh4 Ram et al. (1994)cwh9 AR27 cwh9 Ram et al. (1994)cwh43-1 AR27 cwh43-1 Ram et al. (1994)X2180-1A MATa mal mel gal2CUP1MNN9 ATCCmnn9* MATa mal mel gal2CUP1mnn9 ATCCJY102 MATá leu2-3,112 lys2-801 ura3-52KNR4 Hong et al. (1994a)ZH401C† MATá leu2-3,112 lys2-801 ura3-52 knr4::LEU2 Hong et al. (1994a)YDK5-1C MATa leu2 his3 can1 H. BusseyTR98‡ MATá kre6::HIS3 leu2 his3 can1 H. Bussey

S. pombeSPO25 his+ ade6-M216 leu1-32 ura4-Ä18his3-Ä2 C. Gancedo

*Isogenic to X2180-1A; †isogenic to JY102; ‡isogenic to YDK5-1C.

Sulfuric acid hydrolysis Commercial polysaccha-rides or cell walls (1 mg) were wetted with 75 ìl of72% (w/w) H2SO4 and left at room temperature for3 h. The slurry was diluted to 1 ml in the presence

of 0·3 mg/ml galactose (used as internal standard)

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to a final concentration of 2 -H2SO4 and heatedin sealed tubes for 4 h at 100)C. The tubes werecooled on ice and the hydrolysate was diluted to9 ml with MilliQ water. Sulfate ions were precipi-tated by drop-wise addition of saturated Ba(OH)2until neutral pH was reached (checked with pHpaper). The volume was adjusted to 25 ml and theBaSO4 precipitate was pelleted at 3800 g for 5 min.The supernatant was removed very carefully andleft at 4)C overnight to allow precipitation of theremaining sulfate ions which were removed by asecond centrifugation. The supernatant was readyto be used for monosaccharide analysis withHPAEC-PAD. It was also verified that loss ofsugars by adsorption on the BaSO4 precipitates didnot exceed 3%.

Trifluoroacetic acid hydrolysis Hydrolysis of0·5 mg cell walls or commercial polysaccharideswas carried out in 1 ml of 2 -TFA in the presenceof 0·02 mg of galactose as internal standard. Thissuspension was heated in sealed tubes at 100)C for1–10 h. The tubes were cooled on ice and TFA wasevaporated using a Buchi rotary evaporator. Thedry residue was resuspended in 1 ml of MilliQwater and the liberated carbohydrates wereanalysed with HPAEC-PAD as described below.Other conditions of temperature (95–120)C) andacid concentration (4 and 6 ) were also tested.

HCl hydrolysis Commercial polysaccharides orcell walls (1 mg) were resuspended in 3 ml of2 -HCl (or 6 -HCl for chitin) and heated insealed tubes at 100)C for 1–10 h. The tubes werecooled on ice and the hydrolysate was neutralizedwith 2 -NaOH. The final volume was adjusted to25 ml with MilliQ water. Liberated monosaccha-rides were measured enzymatically (see below).

Quantification of sugars by HPAECChromatography of samples was performed us-

ing a Dionex Bio-LC system (Sunnyvale, CA). Themonosaccharides liberated by acid hydrolysis (glu-cose, mannose, glucosamine and galactose) wereseparated on a CarboPac PA1 anion-exchangecolumn (4#250 mm), equipped with a CarboPacPA guard column. Elution was performed at roomtemperature (22–25)C) at a flow rate of 1 ml/minwith 18 m-NaOH. After 30 min of isocratic elu-tion, the column was rinsed for 10 min with200 m-NaOH to prevent clogging of the columnwith residual non-hydrolysed polysaccharides.

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Detection of sugars was performed with a pulsedamperometric detector PAD equipped with a goldelectrode, according to the manufacturer’s manual.A solution of NaOH was prepared from a commer-cial 50% solution of NaOH with ultra-pure MilliQwater, degassed by flushing helium in the solutionand kept pressurized with the eluant degas moduleof Dionex. Quantification of sugars was performedusing the response factors calculated from the peakareas of the reference monosaccharides pre-treatedwith the corresponding acid solution.

Colorimetric and enzymatic determination ofsugars

Total sugars per g dry material from commerciallaminarin, pustulan and mannans were determinedby the anthrone method (Trevelyan and Harrison,1956) using glucose as a standard for the two firstpolysaccharides and mannose for the third one.Unless otherwise stated, the total carbohydratecontent of the cell walls was measured by thephenol-sulphuric acid method according to Duboiset al. (1956). Since the colorimetric response isdependent on the nature of the sugar, a mannose/glucose ratio of 1·0 was used as a standard, as thisratio was considered from literature data (Ramet al., 1994) as a good indicator of the proportionof â-glucans and mannans in the yeast cell walls.Glucose and mannose were also determined enzy-matically using conventional spectrophotometricmethods as described in Bergmeyer (1982). Chitinhydrolysis and assay of released glucosamine wasdone according to Popolo et al. (1997).

Other analytical proceduresThe amounts of proteins in cell wall extracts

were measured by the Bradford method (Bradford,1972) using bovine serum albumin as a standard.Glycogen and trehalose in whole cells and cell wallswere determined as described previously (Parrouand Francois, 1997). The presence of nucleic acidsin cell walls was estimated by the absorbance at260 nm as described by Catley (1988). The lipidcontent in cell walls was determined by extractionwith chloroform/methanol following the proceduredescribed by Kaneko and Itoh (1976).

RESULTS AND DISCUSSION

Cell breakage and isolation of pure cell wallsThe first aim of this study was to develop an

easy method to isolate clean cell walls and to

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quantify their mass as a proportion of total yeastcell mass. A number of methods for disruptingyeast cells have been extensively reviewed byCatley (1988). It has been reported that isolationof cell walls could also be obtained after auto-claving lyophilized cells (Kasahara et al., 1994).However, this apparently simple and fast methodwas not reliable since yeast cells were only partiallylysed and not broken. Though more tedious andtime-consuming, the mechanical breakage of yeastcells with the French press was found to be themost reliable procedure to isolate cell walls fromother cell components. Almost 100% cell breakagewas obtained after five passages in the press, set ata pressure of 18,000 psi (21450 kg/cm2) with anoptimal concentration of 50 mg/ml of cell drymass. After low-speed centrifugation and extensivewashing with cold Tris–Cl buffer, the pellet waschecked for the lack of any unbroken cells and forthe absence of nucleic acids and glycogen. Thisfraction was considered to consist of pure cell wallsand accounted for 24·7% of the cell dry mass forour control strain, JF291, with a standard devia-tion of less than 1% from three samples of thesame culture. This deviation increased to about 3%with samples harvested from six independent yeastcultures harvested at the same growth phase. Acloser analysis of the cell walls indicated that theycontained 93·7% of total carbohydrates as deter-mined by the colorimetric method of Dubois et al.(1956), 5·7% of proteins, about 2% of lipids andless than 1% of glycogen and nucleic acids. Theseresults were in agreement with earlier observations(Fleet, 1991; Catley, 1988), and the low amount oflipids was an indication of the purity of cell wallsand the absence of membranes. Interestingly, theproportion of cell wall mass in yeast was about thesame whether the cell walls were prepared fromexponential or stationary phase yeast cells grownon glucose. Further work should be done to verifywhether other culture media and conditions affectthis proportion.

For a semi-quantitative method aiming at arapid determination of the glucose, mannose andglucosamine proportion in the cell walls, we devel-oped a small-scale method of cell wall isolationbased on cell disruption with glass beads. Almostcomplete cell breakage was obtained with about10 mg dry mass in either a mini-bead beater, afterthree to five runs of 20 s each, or after four times of1 min vortexing on a bench-top vortex. Extensivewashings of the beads were, however, needed torecover all cellular materials from the glass beads

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before subsequent centrifugation steps. Analysis ofthe pellet indicated that it was mostly composed ofcell wall carbohydrates (about 95% of dry mass).

Table 2. Monosaccharide recoveries (%) from stan-dard mixtures after treatment under three different acidhydrolysis conditions.

Sugars TFA H2SO4

HCl2

Glucosamine 95·6&0·2 96&1·0 97·5&0·5Galactose 97·7&0·3 93·9&0·6 ndGlucose 98·8&0·4 98·0&0·5 97·3&0·6Mannose 97·8&0·3 91·2&1·0 95·3&0·5

The monosaccharide solutions contained 20 mg/l of glucose,galactose and mannose and 5 mg/l of glucosamine. Hydrolysiswas carried out as follows: trifluoroacetic acid (TFA): 2 -TFAfor 4 h at 100)C; H2SO4 hydrolysis: contact with 72% H2SO4

for 3 h at room temperature followed by 4 h at 100)C in2 -H2SO4; HCl: hydrolysis for 2 h at 100)C. Sugars weredetermined as described in Materials and Methods. Valuesgiven are the average&standard errors of four independentexperiments; nd, not determined.

Stability of the monosaccharides under threedifferent hydrolysis conditions

Since acid hydrolysis of glucan, mannan andchitin leads to the release of glucose, mannose andglucosamine (as N-acetyl-glucosamine is convertedinto glucosamine under acid hydrolysis; Hardyet al., 1988), it was necessary to establish thedegree of stability of these monosaccharides, in-cluding the galactose used as an internal standard,under the three different acid conditions. In agree-ment with previous work (Hardy et al., 1988; Ramet al., 1994), acid hydrolysis in 2-TFA at 100)Cfor 4 h gave rise to a recovery of sugars close to95% (Table 2). Longer incubation times or ahigher temperature did not alter these recoveries.In contrast, increasing the TFA concentration to4 and 6 considerably promoted the destructionof the monosaccharides. Treatment with sulfuricacid, according to Saeman’s procedure (as de-scribed in Selvendran et al., 1979), resulted inrecoveries between 93–96% relative to the un-treated sugars (Table 2). However, a prolongedincubation at 100)C for 8–18 h caused a significantloss of material, as already noticed by Selvendranet al. (1979). Treatment with 2 -HCl at 100)C forincubation periods between 0·5 to 5 h was not

destructive for these monosaccharides.

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Table 3. Monosaccharide recoveries from commercial polymers hydrolysed in threedifferent acid conditions.

AcidLaminarin(% glucose)

Pustulan(% glucose)

Mannan(% mannose)

Chitin(% glucosamine)

TFA 100 73 77 10H2SO4 98 95 89 80HCl 52·7 59 86 11

Hydrolysis of commercial polysaccharides and sugar analysis were as in Table 2.Values are given as % of liberated monosaccharides by acid hydrolysis relative to total sugars whichwere determined either by the anthone method for laminarin, pustulan, and mannan or enzymaticallyfor chitin as described in Materials and Methods.Data presented are the means of four independent experiments with a standard deviation of 3%.

Comparison of three acid hydrolysis procedures oncommercial polysaccharides and on isolated cellwalls

Considering the carbohydrate composition ofyeast cell walls, we initially investigated the effi-ciency of the three acid hydrolysis conditionsin releasing monosaccharides from commercialâ1,3-glucan (laminarum), â1,6-glucan (pustulan),mannan and chitin. The purity of these polysac-charides, as determined by the chemical reactionwith phenol-sulfuric or anthrone, was found tobe close to 95% for laminarin, 86% for pustulan,95% for mannan, and 96% for chitin, as estimatedby enzymatic hydrolysis using chitinase andN-acetylglucosaminidase. These values were usedto calculate the recoveries of the monosaccharidesliberated by acid treatments of the commercialpolymers. The results of this experiment are sum-marized in Table 3. It can be seen that when theH2SO4 hydrolysis method was used, the yields ofglucose released from laminarin and pustulan, andthat of mannose from mannan were generallyhigher when TFA or HCl was used. The higherefficiency of sulfuric acid treatment was mainly dueto an almost complete release of glucose frompustulan (â1,6-glucan), while this polymer wasonly hydrolysed by 73% and 59% in TFA and HCl,respectively. Furthermore, an 80% hydrolysis ofchitin into glucosamine was reached with thesulfuric acid procedure, whereas only 10% ofhydrolysis could be obtained in 2 -TFA andHCl. We also confirmed previous reports (see forinstance, Popolo et al., 1997) that treatment ofchitin in 6 -HCl at 100)C for 4 h led to almostcomplete hydrolysis of this polymer into gluco-samine residues. However, this condition resulted

in 50–70% destruction of glucose and mannose

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(not shown). With the sulfuric acid method, weconfirmed the recommendation statement in theprocedure of Saeman (see Selvendran et al., 1979)that the preincubation step in 72% H2SO4 at roomtemperature before further incubation in 2 of thesame acid at 100)C, was a critical step to get amaximal recovery of sugars.

We then applied the sulfuric acid hydrolysis onpure yeast cell walls obtained from the controlstrain JF291, and compared it with TFA and HClhydrolysis. The time of acid treatment was the sameas in Table 1, and the yields of glucose, mannoseand glucosamine are reported in Table 4. The totalamount of sugars liberated by TFA hydrolysis wasonly 61% of cell wall dry mass, while it was closeto 93% when determined by anthrone or phenol-sulfuric acid. Hydrolysis with HCl was also notsatisfactory as the amount of sugars released wasonly 73% of cell wall dry mass. The best yields wereobtained using the sulfuric acid procedure. Thismethod liberated twice as much glucose and gluco-samine than TFA, and the total sugars releasedunder this condition amounted to 92% of cell walldry mass. While any modifications of this pro-cedure with respect to the time of incubation, tem-perature or even acid concentration gave no betteryields, we again confirmed that a preliminary con-tact of cell wall material with 72% H2SO4 at roomtemperature was critically important for an effi-cient subsequent hydrolysis of the polysaccharidesin hot 2 -H2SO4, because a direct treatment in2 -acid lowered sugar recovery and caused incom-plete digestion of chitin. These observations en-tirely agreed with previous experiments carried outon vegetable fibers (Selvendran et al., 1979).

As a consequence of a more effective hydrolysis

of â-glucan by H2SO4 treatment, the value of the

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mannose/glucose ratio of 1·0 for wild-type strainsdetermined with TFA hydrolysis (Ram et al., 1994)must be corrected to 0·6–0·7. Using this ratio as astandard, a rapid estimation of the total sugars inyeast cell walls can be obtained with the anthroneor phenol sulfuric acid method. However, thesecolorimetric methods are highly approximativesince the sugar ratio does not represent the in vivocarbohydrate composition of cell walls from yeastaffected by either environmental conditions orgenetic modifications, and furthermore they do notgive any information about the chitin content.Therefore, quantitative and reliable data on carbo-hydrate composition of yeast cell walls could onlybe obtained with the sulfuric acid hydrolysis of cellwalls followed by HPAEC-PAD analysis.

Table 4. Yields (% of cell wall material) of glucose, mannose and glucosamine released from cell walls ofS. cerevisiae wild-type strain by three different acid hydrolysis conditions.

Acid

Carbohydrate composition (% cell wall dry mass&S.E.)Total sugars

(% of cell dry mass)Glucose Mannose Glucosamine

TFA 27·1&0·20 32·6&1·0 1·3&0·05 61HCl 39·1&1·20 32·3&0·70 0·9&0·05 73H2SO4 52·8&0·25 36·9&0·25 2·3&0·05 92

The cell walls were obtained from exponentially growing cells of JF291 cultivated on glucose (collected at 2#107 cells/ml).Hydrolysis conditions and sugar analysis were as in Table 2. The values are the means&standard deviation of four independentexperiments.

Carbohydrate composition of isolated cell wallfrom S. cerevisiae cell wall mutants andSchizosaccharomyces pombe using the ‘micromethod’

We validated the H2SO4 hydrolysis method byreinvestigating the cell wall carbohydrate compos-ition of some well-characterized cell wall mutantsand we compared our results with those obtainedby the TFA hydrolysis method. These results arereported in Figure 1 and Table 5. Control strainsused in this work, namely JF291, AR27, X2180,JY102 and SEY 6210 all exhibited about the samecontent of mannan and â-glucan, giving rise to amannose/glucose ratio of 0·6–0·7, while chitin con-tent was found to be slightly higher than usuallyreported (2·1&0·2% instead of 1%). The cwh53mutant known to be deficient in â(1,3) glucansynthase (Ram et al., 1995) showed a 50% reduc-tion of â-glucans as reported previously with theTFA method (Ram et al., 1994) while the mnn9

mutant defective in N-linked outer chain syn-

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thesis (Ballou, 1990) contained 80% less mannoseresidues than the wild type. However, becausehydrolysis with sulfuric acid allowed a better quan-titative liberation of glucosamine from chitin, itcould be shown that both mutations resulted in a10-fold increase in chitin content which amountedto about 20–22% of total sugars in cell walls.

We also confirmed the data of Hong et al.(1994a) that the deletion of KNR4 led to a four-fold increase in chitin levels and to a 35% reduc-tion of â-glucan. Deletion of KRE6, a geneproposed to be specifically involved in the elonga-tion of â1,6-glucan chains (Roemer and Bussey,1991) also caused a five-fold increase in chitin leveland only a moderate 16% decrease in â-glucan.Other cwh mutants isolated by Ram et al. (1994)all exhibited enhanced chitin levels accompanyinga reduction in either â-glucan or mannan. Takentogether, these results strongly reinforce the evi-dence that the increased amounts of chitin and ofchitin-bound â1,6-glucosylated proteins are part ofa rescued mechanism responsive to cell weakeninginduced by environmental conditions or geneticmodifications (Kapteyn et al., 1997; Popolo et al.,1997; Kollar et al., 1996; Daran et al., 1997).

This procedure can be extended to other yeastspecies. In particular, we have determined the cellwall carbohydrate composition of a wild-typeS. pombe strain grown on glucose-rich medium. Ahigh amount of glucose was liberated by sulfuricacid treatment (about 80% of cell wall dry mass).Acid hydrolysis also released 10% of both galac-tose and mannose, most likely from the galacto-mannan structure present in this yeast species(Ribas et al., 1991), while only a tiny amount ofglucosamine (less than 0·5%) could be detected, inagreement with previous data (Bush et al., 1974;

Sietsma and Wessels, 1990).

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Figure 1. Percentages of liberated monosaccharides from cell walls isolated from wild-type and cell-wall-defective mutantsof Saccharomyces cerevisiae by treatment with sulfuric acid. Cell walls were isolated by the ‘micro-method’ from yeast cellsharvested at the exponential phase of growth on glucose. The values reported were from a single experiment which wasrepeated two times with a standard deviation of less than 5%. The control values are the average from values obtained withwild-type strains, JF2912, X2180-1A, AR27, JY102 and SEY6210 which differed by less than 5%.

Table 5. Mannose/glucose (Man/Glc) ratios and yields of glucosamine (% of cell wall material) as obtained by TFAand H2SO4 hydrolysis methods from different yeast strains.

Genotype

H2SO4 TFA

Man/Glc ratioGlucosamine(% dry mass) Man/Glc ratio

Glucosamine(% dry mass)

Wild type 0·65–0·70* 2·1 0·90–1·10 0·9–1·10cwh53 2·03 21·5 3·92† 3·3†cwh5 0·39 5·60 0·4† 2·7†cwh9 0·30 4·90 0·47† 2·2†mnn9 0·07 20·3 0·24 4·6knr4 1·6 8·0 nd ndkre6 0·8 10·6 1·9† 3·3†

The cell walls were obtained from exponentially growing cells (harvesting at 2#107 cells/ml) on glucose. Hydrolysis conditions andsugar analysis were as in Table 2. The values are the means of four independent experiments. A standard deviation of 5% wascalculated from the released monosaccharides in four independent experiments.*Values are from the analysis of all the different wild-type strains, JF291, AR27, X2180, JY102 and SEY6210.†Values are from Ram et al. (1994); nd, not determined.

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Summarizing, the H2SO4 hydrolysis methodappears to be much more reliable than hydrolysisin TFA, and it should be useful for the quantita-tive determination of carbohydrate compositionfrom cell wall of yeast mutants generated in theEUROFAN programme, as well as for other bio-technological and pharmacological applications.

ACKNOWLEDGEMENTS

We are grateful to Dr Frans M. Klis for provisionof strains and critical reading of the manuscript.We thank Dr C. Gancedo for gift of S. pombestrain and H. Martin for the construction of theknr4Ä mutant strain. Miss C. Tchilinguirian isacknowledged for her initial role in this work. Thiswork was supported in part by grant 960956 fromthe Region Midi-Pyrenees, by the Commissionof European Union (EUROFAN I programme,BIO4-CT95-0080) and by Lesaffre-Developpement(Marcq-en-Baroeul, France).

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