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Degradation of isolated barley endosperm cell walls by purified endo-(1 → 3)(1 → 4)-β-D -glucanases and malt extracts
Journal of Cereal Science7 (1988) 153-168
Degradation of Isolated Barley Endosperm Cell Walls by Purified Endo-(1--+ 3)(1--+4)-p-n-glucanasesand Malt Extracts*t PAMELA BRUNSWICK, DAVID J. MANNERS and J. ROGER STARKt
Heriot- Watt University, DepartmentofBrewing and Biological Sciences, Edinburgh EHl lHX, u.K. Receivedin revisedform 7 December1987
Thestructureof isolatedbarleyendospermcell wall fragmentswasstudiedemploying The endomalt extracts and two purified endo-(I-;..3)(1-;..4)-~-D-glucanases. (1-;.. 3)(1 -;.. 4)-~-D-glucanases werecharacterisedby their activity onvarioussubstrates and possessedmolecular weights and isoelectricpoints of 27 kD, 33 kD and pI 8·5 and ~ 10·8 respectively.Theseisoenzymeseffected a limited degradationof barley endospermcell wall polysaccharide,releasingglucosein addition to the expected oligosaccharideproducts.Thereleaseof glucosesuggestedthat ~-glucosidases may be associatedwith the isolatedcell wall fragments.The degreeof cell wall carbohydrate degradationwas increasedby the presenceof a mixture of basic ~-glucanases; whereascrude enzymeextracts from barley malt, containing proteolytic activity, releasedup to 72 % of the total carbohydrate,including arabinoxylanbreakdown products. The pattern of the cell wall digestions was, however, not altered by pretreatmentof the cell wall fragments with a-amylaseand a general proteinase, forwarding the view that proteolysis is not a pre-requisitefor endo-~-glucanase attack. Examination of digested endosperm cell wall fragments by electron microscopyindicatedthat the enzymesof the basicproteinfraction from barleymalt clearly attackedthe inner pitted surfaceof the walls, althoughsolubilisationof the outerscalysurface,evenin the presenceof crudeenzymeextracts,waslessapparent. The observationsdid not confirm the proposalthat the sequentialappearanceof proteinasesandcarbohydrases during germinationreflecttheir role in endospermcell wall breakdown.
Introduction The endospermcell walls of barleyaredegradedenzymically during germinationof the grain, with the concomitantsolubilisationof the starchyendosperm.C ell walls are thus regardedas potentially important sites for the regulation of endospermmodification during malting and of carbohydraterecoveryduring the mashingstageof brewing1 • .. Studieson ~-Glucanases, PartVIII. For PartVII, seeBrunswick,P., Manners,D. J., Stark, J. R. J. Ins/. Brew. 93 (1987) 181-186. t Dedicatedto ProfessorLuis F. Leloir on the occasionof his 80th birthday. t To whom correspondenceshouldbe addressed. Abbreviations used: -CBZ = carbobenzoxy-;eM- = carboxymethyl-;pCMB = p-chloromercuribenzoic acid; DEAE- = diethylaminoethyl-; DDT = DL-dithiothreitol; PMSF = phenylmethylsulphonylfluoride; TNBS = 2,4,6,-trinitrobenzeneI-sulphonicacid. 0733-5210/88/020153 + 16 $03.00/0
© 1988 AcademicPressLimited
154
PAMELA BRUNSWICK ET AL.
In barley, the endospermcell walls are composedmainly of a mixed linkage This matrix glucanconstitutes70-72% by weight of the cell wall, the remainderbeingcomposedof matrix and microfibrillar arabinoxylan,protein and cellulose or glucomannan2-4.The high ~-glucan to cellulose ratio provides a flexible, easily degradablestructure. Although the composition of the endospermcell wall has been established,the physicalarrangemento f thecomponentsis lesswell-defined.The glucansandpentosans canbe classedaccordingto their waterand alkali solubility, indicating that their degree of polymerizationor binding within the walls canvary5. Of interestto the presentstudy is the suggestedbilayeredstructureof endospermcell walls, with the inner pitted surface of the wall composedof the more water-solublepolysaccharides 2. Furthermore,it hasbeenproposedthat the sequentiala ppearanceof enzymesduring germination could give an indication of the structural arrangementof the cell wall barrier6 • The suggestionthat ~-glucan 'solubilase',a carboxypeptidase,catalysesthe first stagesof cell wall degradation6 was supportedby earlierpublicationswhich showed that proteolysis,prior to hot water extraction,could removeover 96 % of the ~-glucan presenta ndcausestructuralcollapseof thecell waIF. In fact, a peptide-glucancomplex, isolatedfrom barley endosperm,could be degradedto a molecularsize similar to that derivedfrom greenmalt by digestionwith thermolysin or by hydrazinolysis7 • However, contraryto the proposalof preliminary proteolysis,it hasbeendemonstratedthat both an endo-(l-+3)-~-glucanase from malted barley and a fungal endo-(l-+4)-~-glucanase can extensivelydegradeisolatedcell wall fragments4 • The controversyover whetherproteolysisis a pre-requisitefor cell wall degradation by ~-glucanases is discussedin the presentstudy.Partiallypurified enzymeextractsfrom barley malt and purified, characterisedendo-(l-+3)(1-+ 4)-~-D-glucanase isoenzymes were preparedand employed to study the cell wall structure and its solubilisation. Breakdown of the cell wall was observedby the releaseof soluble oligosaccharide productsand by electronmicroscopy. ~-(1~3)(1-+4)-glucan2-4.
Experimental Preparation of dehuskedkilned barley malt
Barley grain (GoldenPromise,1980 harvest)wasdehuskedin 50 % (v Iv) sulphuricacid solution for 3 h at room temperature.Themixture was neutralisedwith sodiumcarbonateand the grain was thoroughly rinsed by light rubbing in water. After air-drying overnight, the grain was subjectedto a steepingprogramof 8 h steep,16 h air rest, 8 h steep,12 h air rest, 0'5 h steepand drain. The grain was germinatedat 16°C for 16 h. Germinated'green'barleygrain was stored at -15°C or subjectedto kilning at 60°C for 16 h and placedin dry storage. Isoenzymeextraction and separationprocedure
Endo-(I-+3)(1-+4)-~-D-glucanases wereextractedandpartially purified8 • The dehuskedmalt was extractedat 0-4 °C in a solution (2 l/kg of grain) of 0·2 MNa acetatebuffer, pH 4'8, containing 10 mM sodiumazide, 10 mM EDTA, 3 mM PMSF and 3 mM DTT. Following centrifugationand
BARLEY CELL WALL DEGRADATION
155
dialysis, the enzyme extract was subjected to DEAE-cellulose chromatographyin 20 mM citrate-phosphate buffer, pH 8. Proteineluting at the void volumeof the DEAE-cellulosepadwas pooled and dialysedwith 0·1 M acetatebuffer, pH 4·8. A portion of the extract, containing86·6mg protein, loadedonto a column of CM-cellulose (40 x 1·8 em) and elutedwith 200 ml startingbuffer, followed by a linear gradient,550 ml, from 0·1 to 1'0 M acetate,pH 4'8, producedthe elution profiles shown in Fig. I (4'2 ml fractions). Endo-(1.....3)(1 .....4)-B-o-glucanaseisoenzymesfor the study of endospermcell walls were prepared by an equivalent bulk separationof 409 mg protein on a 17 x 6 cm CM-cellulose column.
Isoenzymepurification Endo-(1.....3)(1 .....4)-B-o-glucanaseisoenzymes,labelled I and II accordingto their consecutive elution from the CM-cellulosecolumn, were chromatofocusedon columnsof PBE 74 (32'5 x 1'0 cm) and PBE 118 (22'0 x 1·0 cm) Pharmaciaresinsrespectively.The PBE 74 was equilibratedin 25 mM ethanolamine-HCIbuffer, pH 9-4 andelutedwith dilute (1 : 10) Polybuffer96-HCI, pH 7'5. PBE 118 resin was equilibriated with 25 mM triethylamine-HCl, pH 10·8 and the column was eluted with dilute (I :45) Pharmalyte-HCI,pH 8. The pH elution profile was measuredin the collectedeluantfractions(2 ml), in additionto assayingfor enzymicactivity on varioussubstrates. Assaysinvolving reducingpower determinationsin the presenceof Polybuffer were centrifuged prior to absorbancereading. Further purification of the individual isoenzymeswas achievedby molecular sieve chromatographyon a calibratedcolumn(86'0x 2'2 cm) of SephadexG-75 (Pharmacia).Theequilibration and elution buffer employed was 50 mM Na acetate buffer, pH 4·8, containing 50 mM KCI. Fractionsof theeluant(1'7 ml) wereassayedfor enzymicactivity andthe pooledfractionsdialysed against0·1 MNa acetatebuffer, pH 4,8, beforestorageat -IS DC.
Assayof carbohydraseactivities Enzymic activities on carbohydratesubstrateswere determinedaccordingto the reducingpower and glucoseoxidase(cellobiaseactivity only) methodsof Brunswick et alB. In addition to the substratescellobiose, lichtenin, laminarin and soluble starch, the polysaccharidepullulan was included at a concentrationof O' I % (w Iv) and 2 h incubation. Enzymic activity is reportedas equivalent I!g of o-glucose released,measuredwith reducing power reagentsat 600 nm, per 0·1 ml sampleper assaytime. Endo-B-glucanaseand arabinoxylanaseactivities were determined more specifically by viscometry 8 and paper chromatography.T he substratesemployed to measureendo-(1-.;.3)-I3glucanase, endo-(1.....4)-[3-glucanase, endo-(1..... 3)(1 .....4)-I3-glucanases and arabinoxylanase activities were CM-pachyman(0,8 % (w Iv», CM-cellulose(0'13% (w Iv», 40 DC water-extracted barley B-glucan (0'7 % (w Iv» and rye arabinoxylan(0'2% (w Iv» respectively.Viscometry was performedat 30 DC in a M4-type V-tube viscometer,in the presenceof 6 mM Na acetatebuffer, pH 5 and 0·3 mM DTT. The 'Units' of enzymeactivity areexpressedasthe rateof increasein the reciprocalof specific dynamicviscosity with time8 • Paperchromatographyof oligosaccharideproductsemployed the following solvent systems (proportions by voL); ethyl acetate-pyridine-water(10:4: 3), ethyl acetate-aceticacid-formic acid-water (18:3: I :4) and n-propanol--ethyl acetate-water(14:2: 7). Reducing sugars were . detectedby alkaline silver nitrate reagents9•
Assayof carboxypeptidaseactivity Carboxypeptidaseactivity was determinedby the methodof Baxter10, basedon that reportedby Satakeet alY, and employing 0·4 mM CBZ-L-phenylalanylalanineas substrate.
156
PAMELA BRUNSWICK ET AL.
SDSelectrophoresis SDS disc-electrophoresiswas based upon the method of Weber and Osborn12 with minor modifications.
Preparation ofbarley endospermcell walls Fragmentsof endospermcell walls were isolated from dehuskedbarley by the methods of Fincher2 , Ballance4 and Ballanceand Manners5 • The cell walls were stored in 70% ethanolat 0-4 °C. The preparationcontained 17 % (w Iw) protein, of which one-fifth was water-soluble following homogenisation.
Cell wall pre-treatments Cell wall fragmentswere digestedwith filter-sterilised salivary ex-amylasein aqueoussolution containing0·02% (w Iv) Na azide,for 16 h at 30°C. In addition, the fragmentswere subjectedto digestion,at 37 °C, by ProteinaseK, 8 x 10-3 AnsonUnits/ml (BDH Chemicals,u.K.) in 20 mM Na phosphatebuffer, pH 7, also containingazide.During thesedigestionprocessesthe cell walls were kept suspendedby gentleinversionand the digestionswere terminatedby washingthe walls free of enzyme.
Cell wall digestionby carbohydrases Suspendedendospermcell wall fragmentswereincubatedwith variousenzymepreparationsin a volume of 3 ml containing3 mM Na azide,3 mM DTT, and 0,1 M Na acetatebuffer, pH 4,8. Zero time sampleswere takenfrom the supernatantafter the addition of enzymeat 0-4 °C, whereas total carbohydratewasmeasuredin thesuspensionmixture.Timedintervalsamplesof supernatant were taken during incubation of the digest at 30°C. Control digestionscontainedno added enzyme.The sampleswere centrifuged and the supernatantassayedfor the presenceof total carbohydrateby the phenol-sulphuricacid method13. Cell wall carbohydratedigestionis reported asthe releaseof equivalentI!g D-glucoseper total digestvolumeaftercorrectionfor the changein digestvolume and loss of carbohydratedue to sampling.
Scanningelectron microscopy Cell wall fragmentswerewashedin successivelyhigherconcentrationsof ethanolandfinally with ether. The air-dried fragments were coated with gold to a thickness of 30 nm before being examinedundera CambridgeStereoscanelectronmicroscopeat an acceleratingvoltageof27 kY.
Results and Discussion
Purity and characterisationof fJ-glucanasesemployedin cell wall digestion
Two endo-(l-+3)(1 -+ 4)-~-D-glucanases werepurified from dehuskedbarleyasoutlined in the Experimentalsectionand previouslyB,14. The purification at eachstageis shown in TableI. An initial 3'4-fold purificationoverDEAE-cellulose(TableI) wascomparable to that observed by Manners and Wilson15 for endo-(1-+3)(1 -+ 4)-~-glucanase extractionfrom barley malt. A l-8-fold purification was reportedby Woodward and Fincher16 for DEAE-cellulosepurification, althoughwhen a salting out and desalting
BARLEY CELL WALL DEGRADATION
,157
TABLE I. Purification of endo-(l -+ 3)(1 ->- 4)-p-n-glucanases from dehusked,germinated, kilned barley
Fractionation procedure Crude (combined1+11) Step 1; DEAE-cellulose (combined1+11) Step 2: eM-cellulose IsoenzymeI IsoenzymeII Step 3: SephadexG-75 IsoenzymeI IsoenzymeII Alternative step 3; Chromatofocusing and SephadexG-75 IsoenzymeI IsoenzymeII
Total protein (mg)
Total activity (units)
Specific activity (units/mgx 102)
Purification factors Eachstep
Overall
20820
204
0·98
6988
216
3·1
3'4
3-4
839 489
37 61
4·4 12,5
1'4 4'0
12·8
42 49
15 24
36 49
8'2 4'0
37 50
145 23
4'0 0·5
148 23
4·2
3-7
6'0 0·8
4'5
Valuesgiven at eachstepare the means(per kg of dehuskedgrain) of two or more repeatedexperiments. Recoveriesof> 100% are attributableto interferenceby additi,:esin the crudeextract.
stepwasincluded,the overallpurification reportedwas 1O·9-fold16 • It was notedthat the activity extractedfrom germinated total protein and endo-(1-+ 3)(1 -+ 4)-~-glucanase barley by the latter authors16 was much higher than that observedin the presentstudy of germinated'green'barleyextracts(TableII). Thediscrepancyin proteinrecoverywas probablyattributableto the useof 1kg grain in the presentstudy as comparedto 10 kg by the other authors16, Differences in actual values of reported enzyme activities measuredby viscometry are inevitable becauseof the effect of initial substrate concentration,incubationconditions and variation betweenthe polymeric substrates ~-glucan substrateconcentration employed.In thepresentstudythelaboratory-prepared was 0'71 % (wjv), as compared to 0·18% (wjv) employed by Woodward and Fincher16• 17 ; thus, decreasesin the viscosity of the substratesolution by enzymic hydrolysis produceddiffering rangesof valuesfor total and specificactivity, It was of interestto note that, whereasthe presenceof the additivesDDT, PMSF, EDTA and azide improved the initial endo-(l-+3)(1-+4)-p-glucanaseactivity from activity germinatedbarley by almost 100%, the recoveryof endo-(l-+3)-~-glucanase was little affected (Table II). During theseproceduresit was also observedthat the presenceof DDT was required in order to maintain endo-(l-+3)(1-+ 4)-~-glucanase activity during dilution or cold storage.
PAMELA BRUNSWICK ET AL.
158
TABLE II. Purification of endo-~-glucanases from germinatedor kilned dehuskedbarley by DEAE-cellulosechromatography Endo-(l-e".3)-I3-glucanase
Endo-(1-+ 3)(1 - 4)-p-glucanase Extraction and fractionation procedure
Total protein (mg)
Total activity (units)
Germinatedbarley; no additives Crude 352 18895 DEAB-cellulose 7375 135 Germinatedbarley; plus additives Crude 21379 280 DEAE-cellulose 5595 260 Kilned barley; plus additives Crude 20820 204 DEAE-cellulose 6988 216
Specific activity (units/ mg)
Purification factor
Total activity (units)
Specific activity (units/ mg)
Purification factor
1·9 l·g
0·98
805 672
4·3 9-1
2'1
1-3 4·6
3-5
715 617
3·3 11·0
3-3
0·98 3·1
3-4
569 709
2·7 10·2
3·7
Valuesgiven per kg of dehuskedgrain. Recoveriesof> 100 % are attributableto interferenceby additives (seeExperimental)in the crudeextract.
TABLE III. Estimationof the molecularweights of enzymesextractedfrom kilned germinated barley malt
V.(ml)
SephadexG-75 chromatography Endo-(l --+ 3)(1--+4)-I3-g1ucanaseI Endo-(1-+ 3)(1 -+ 4)-j3-g1ucanaseII J3-Amylase Cellobiase SOS electrophoresis Endo-(I_3)(l-+4)-j3-g1ucanase I Endo-(1 --+ 3)(1 --+ 4)-j3-g1ucanaseII
kD/mobility
194·9
0'3139
180'7 170·5
0·2574 0·2167 0·1837
162·2
0·610 0'575
loglO mol. wt.
mol. wt. (kD)
4·403 4'527 4·616 4·687
33'7±0'7 48·7±0·9
4'465 4'513
29·2±1·5 32'6±0'8
25'3±1·6 41'3±1-6
Standardproteins employedwere: bovine serum albumin, 68 kD; ovalbumin, 43 kD; ~-lactoglobulin, 36'5 kD; chymotrypsinogenA, 25·7kD; ~-lactoglobulin monomer, 18'2kD; equine myoglobin, 17-6 kD; cytochromeC, 11'7 kD.
In the presentstudy, two endo-(l-+3)(1- 4)-f3-glucanaseswere separatedby eMcellulose chromatographyand identified, by hydrolysis of the substratelichenin and viscometrically,as the peakslabelled'!' and'II' in Fig. 1. The purification factor for the two isoenzymesfrom kilned barleymalt was 1·4-fold and4-fold (overa1l4'5-foldand l2'B-fold) respectively(Table I). This correspondsto the 8-fold purification of a single mixed-linked type enzymefrom barley malt reportedby Mannersand Wilson15• The purification of two equivalent isoenzymesfrom 'unkilned' germinated barley was
BARLEY CELL WALL DEGRADATION
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FIGURE I. eM-Cellulosecolumnelutionprofilesof extractsfrom 3-daygerminated, dehuskedbarley. Enzymic activities measuredby the releaseof reducing oligosaccharidesor glucose from (a) lichenin, (b) cellobiose, (c) laminarin and (d) soluble starch. See Experimental for details. Linear acetategradient D· 1-1'0 M shown as- - - .
reportedto be O'7-fold and 6'4-fold (overall 7-fold and 64-fold) respectively,after a similar procedurebut employingelution buffer pH changesratherthana concentration gradient16,1 7. At this stagethe two endo-(1-+3)(1-+4)-p-glucanases were contaminatedwith endo(1-.+ 3)-p-glucanase,cellobiaseand J3-amylaseactivities, as shownin Fig. 1. No activity on pullulan or CBZ-phenylalanylalaninewas observedin the eM-cellulosecolumn fractions. The isoenzymeswere further purified by gel filtration on SephadexG-75, or a combination of chromatofocusing and gel filtration (Table I; Figs 2, 3). A gel filtration stepalonegavea further purification of isoenzymesI and II of 8'2-fold and4fold (overall 37-fold and 50-fold) respectively(Table I). Combinedwith a chromatofocusingstep,thesepurificationswere alteredto 4-fold (overall 148-fold) for isoenzyme I, but deterioratedto O'S-fold (overall 23-fold) for isoenzymeII (Table I). This latter deteriorationcould be attributedto the instability of isoenzymeII at the high alkaline pH valuesemployed. By comparison,the presentedpurificationsover SephadexG-75 arein a similar range to the publishedvalues of 105-fold15 and 3Q-200-fold18 by other workers employing
160
PAMELA BRUNSWICK ET AL. 9·5
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FIGURE 2. Chromatofocusingon PBE 74 resin of endo-(l-+3)(1-+ 4)-~-glucanase isoenzymeI. Enzymicactivitiesmeasuredby the releaseof reducingoligosaccharides or glucosefrom (a)- - , lichenin:---, pH gradient9·0-7·5.(b) - - , cellobiose;---, solublestarch.(c) - - , laminarin. seeExperimentalfor details.
commercialkilned barley.WoodwardandFincherreportedpurificationsof 67-fold and 4'6-fold by combined chromatographyover CM-Sepharoseand two columns of SephadexG-75 (giving overall 467-fold and 294-fold) for two similar isoenzymesfrom germinatedbarley16·17.The presentresultsappearedto bein line with thoseof previous workers. The data suggeststhat the yield and purification of theseisoenzymescould undoubtedlybe improvedabovevaluespreviouslyreportedl 5-18 by including an initial salting out step(as employedby previousworkers)16.17in the presentprocedure. werecharacterisedby their isoelectricpoints Thetwo endo-(l-+ 3)(1 -+ 4)-~-glucanases (chromatofocusingdata, Figs 2, 3), molecularweights(calibratedSephadexG-75 data and SDS electrophoresis,Table III) and by their profiles of hydrolysis of various carbohydrates(Table IV). Results were consistentwith those for two endo-(l-+3) (1-+ 4)-J3-glucanase isoenzymespurified from unkilned germinatedbarleyby Woodward and Fincher16.17, exceptthat no activity towardsCM-pachymanwas observedin the presentpreparations(Table IV).
BARLEY CELL WALL DEGRADATION
161
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FIGURE 3. Chromatofocusingon PBE 118 resin of endo-(l--*3)(I--*4)-p-glucanase isoenzymeII. ---, pH gradient10'8-8,0;- - , hydrolysis of lichenin measuredby reducingpower. No othercarbohydraseactivity observedexceptfor tracecellobiose in pooledlicheninasepeak. TABLE IV. Substratespecificity of purified endo-(l -+ 3)(I -+ 4)-p-glucanaseisoenzymesI and II from dehuskedkilned germinatedbarley Hydrolytic activity Substratetested Barley p-glucan 40°C water extract 90 °C water extract Lichenin Low mol. wt p-glucanoligosaccharides CM-pachyman Laminarin CM-cellulose Cellobiose Rye arabinoxylan Larchwooddyed CM-xylan
IsoenzymeI
IsoenzymeII
+++ +++ +++
+++ +++ +++
+
+ +
See 'Experimental' for details of techniquesand incubation conditions. Dyed CM-xylan hydrolysis determinedby thereleaseof solubledyedcarbohydrate.+ + +, hydrolytic activity observed;- , no hydrolysis observed;+, tracehydrolytic activity observed.
Isoenzyme I, pI8·4-8·5 and mol. wt. 27,250 (± 1600) (Table III), appeared chromatographicallypure by SDS electrophoresis.Any remaining contaminating (pI < 7'5, 41 kD) and cellobiase (pI's,..., 8'3 and ,..., 8'6, mol. wt. 49 kD), ~-amylase laminarinase(pI's> 9'0. 8'0-8'3, 8·0 and 7-8) activities observedduring chromatofocusing (Fig. 2) were removed by gel filtration (Table III). The void volume laminarinasepeak (pI> 9) of chromatofocusingcan be attributed to a previously althoughmalt extractsareknown to containtwo ~reportedendo-(l-+3)-~-glucanase19.
162
PAMELA BRUNSWICK ET AL.
glucan 'solubilases' with pI values acidic and 9-352 °. Laminarinase actlVlty that overlapped with the endo-(1--+ 3)(1 4)-p-glucanaseduring chromatofocusingalso exhibiteda high affinity towardslaminaribiose,the remaininglaminarinasepeaksbeing inferred to be due to the presenceof p-glucosidaseisoenzymes. IsoenzymeII, pI > 10·8 (Fig. 3) andmol. wt. 33,125(± 750) (TableIII), appearedto be separatedfrom all contaminatingenzymeactivities by the chromatofocusingstep. The observedvalues for mol. wt. and pI of the two isoenzymeswere close to those reportedfor two equivalentisoenzymesfrom unkilnedmaltI6 • 1? It seemedcoincidental that two out of four carboxypeptidases presentin a crudemalt extractwerereportedto possessisoelectricpoints of 8·4 and> 1021. SDS electrophoresisof isoenzymeII showedtraces of at least one contaminating protein. This was inferred to be a cellobiaseenzymesince trace amountsof cellobiase activity could not be removedfrom the preparation(Table IV). In addition to this contaminant,b othof thepurifiedisoenzymesexhibitedtraceactivity on rye arabinoxylan but no other contaminatingpolysaccharidedegradingactivities were observed(Table IV). Contaminatingarabinoxylanaseactivity, and, in addition, hydrolysis of eMpachyman,wereobservedin theendo-(l --+ 3)(1--+4)-p-glucanaseisoenzymepreparations of Woodward and FincherI6 • 17 . The arabinoxylanaseactivity may be attributed to contamination of the pentosan substrateswith traces of glucan. Alternatively, arabinoxylanmoleculesmaybesusceptibleto endo-(I...."..3)(1-;.4)-p-glucanasehydrolysis by imitating the conformationalstructureof a mixed-linkagef)-glucan. The physiologicalsignificanceof two endo-(1----3)(1----4)-p-glucanaseisoenzymesin germinating grain is important in the context of their role in the breakdown of endospermcell walls. Recentpublicationssuggestthat these isoenzymesare derived from different tissuesandareunderseparategeneticcontrop2.Theequivalentisoenzyme I is reportedly synthesisedpredominantly in the scutellum, while isoenzyme II is synthesisedexclusivelyin the aleurone22. A third, putativeisoenzymehasbeendetected in imbibed barley22, Of interest, also, is the dynamic nature of cell wall p-glucanin Graminaceouscoleoptilesand its role in coleoptileelongation23. Endospermcell wall studies
Barley endospermcell walls were examinedfor the releaseof solublecarbohydrateand by electronmicroscopyfollowing intervals of digestionwith barley malt crudeenzyme extract, DEAE-cellulosepartially purified extract and purified endo-(1-;.3)(1"""" 4)-f)glucanases. After a 24 h incubationwith endo-(1...."..3)(1----4)-p-glucanaseisoenzymesI or II, at 2'09 x 10-2 U/ digest, the endospermcell walls remainedstructurally intact, the two isoenzymesreleasingequivalentand additive amountsof solublecarbohydrate(Table V). The comparative instability of isoenzyme lover that of isoenzyme II was demonstratedby employingreducedconcentrationsof enzyme;in this casethe release of solublecarbohydratereached11 % and 22 % respectively,with a crudemalt enzyme extractreleasing58 % by comparison(Fig. 4), Interferenceby the releaseof endogenouscell wall carbohydrateand contaminating starchgranuleswas removedby treatmentof the walls with a-amylaseand dialysis at
163
BARLEY CELL WALL DEGRADATION
TABLE V. Digestionof barley endospermcell wall fragmentsby purified endo-(1-+3)(1-+4)~-glucanase isoenzymesI and II Releaseof solublecarbohydratefrom cell walls ( = Ilg D-glucose/3ml of digestinterval shown-1)
Content
24h
48 h
72h
96 h
IsoenzymeIR IsoenzymeIIR IsoenzymeI plus isoenzymeII b
441 450 439
34 7 0
8
0 0 0
0 0
• Purified isoenzymesI and II were employedat an initial concentrationof2'09U/3ml of digest;a further 1·050U were addedafter 3 days. b 1·045U of eachisoenzymewas usedinitially; a further 0'525 U of eachwas addedafter 3 days.
(a)
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time
(h)
FIGURE 4. Digestiqn of barley endospermcell walls by enzyme extracts from dehuskedkilned barleymalt. (a) and(b) Cell walls digestedwith endo-(I -+ 3)(1 -+ 4)-~glucanaseisoenzymesI and II respectively. (c) Cell walls digested with both isoenzymesin a ratio of I: 1. (d) Cell walls digestedwith crudeenzymeextractfrom dehuskedkilned barley.Digests(a), (b) and(d) initial endo-(I -? 3)(1-+4)-~-glucanase activity of 5·23x 1O-3U/3ml; (c) initial activity of 10-5 x 10-3U/3 m!. Q-4 °C. Following this additionalstep,endo-(1-73)(1-74)-p-glucanases I andII released
14 % and 29 % of the cell wall carbohydraterespectively(Fig. 5). Thesefigures could only be increasedto a maximumof 20 and 31 % respectivelyby the further addition of freshenzyme(Fig. 5). This incompletesolubilisationof cell wall carbohydratesuggested that, providedthat productinhibition wasnot occurringin both cases,a portion of the p-glucanwas unavailablefor hydrolysis. Comparisonof the presentresults with those of previous workers highlights the importanceof enzymespecificity and concentration.Solubilisation of endospermcell
164
PAMELA BRUNSWICK ET AL. 220 200
(a)
(b)
(c)
(d)
FIGURE 5. Digestion of a-amylasetreatedbarley endospermcell walls by enzyme extractsfrom dehuskedkilned barleymalt. (a) and(b) Cell walls digestedwith endo(1 ->- 3)(1-;. 4)-p-glucanaseisoenzymesI and II respectively.(d) and (c) Cell walls digestedwith dialysedcrudeand DEAE-cellulosepartially purified enzymeextracts from dehuskedkilned barley respectively.All digests containedan initial endoactivity of 5'23 x 10-3 U/3 ml. A further 1·96 x 10-3 U/3 ml (I ->- 3)(1-;. 4)-p-glucanase and 5'23 x 10-3 U/3 ml were addedto digests(a) and (b) respectivelyafter 9 daysof incubation.Shadedarearepresentsthe carbohydratereleasedeach 1'5 h/3 ml. Total area representsthe total carbohydratereleased,(a) 14 and 20 % after further digestion,(b) 29 and 31 % after further digestion,(c) 43 % after further digestionand (d) 59 % after further digestion. walls by 60-70% over 24 hand75-80 % after 5 days,hasbeenachievedemployinghigh concentrationsof a fungal 'licheninase'(endo-(1-+4)-~-glucanase5) or a purified malt endo-(l-+3)-~-glucanase4. It wasnotedon review, however,that the publisheddatadid not appearto be correctedfor carbohydrateleachingfrom the cell wall or comparedto the actual total carbohydratepresent. Neverthelessthe figures did suggest a total solubilisation of the p-glucan matrix4. In contrast, other authors have reported the collapseof cell wall structureafter the extractionof most of the p-glucan7 • In the presentstudy the productsof endo-(1->-3)(1->-4)-p-glucanasedigestionof cell walls, surprisingly, included glucose, identified by paper chromatography. This conflicted with the evidenceobtainedby prolongedhydrolysisof p-glucansubstrateby the purified isoenzymes,in which the products were the expected tri- and tetrasaccharides.Although tracecellobiaseactivity could not be removedfrom isoenzymeII (TableIV), contaminationcould not explainthe releaseof glucosecausedby isoenzyme 1. The anomaly could be attributed to the presenceof p-glucosidases,k nown to be presenti n ungerminatedbarleygrain, which maybecomeattachedto thecell wall during the isolation processand be reactivatedunderthe incubationconditions.In supportof this suggestionis evidencethat Graminaceouscoleoptile cell walls, isolatedin glycerol and organicsolvent,containedfirmly boundautolytic activity which releasedreducing sugars,including glucose,andmixed linkageglucansfrom the walls during incubation24. By comparison, DEAE-cellulose partially purified enzyme extract from malt
BARLEY CELL WALL DEGRADATION
165
(containingequivalentendo-(1~ 3)(1 ~ 4)-~-glucanase activity) hydrolysed43 % of the cell wall carbohydrate(Fig. 5). This extract contained cellobiase, ~-amylase and laminarinase(endo-(l ~ 3)(~-glucanase and P-glucosidases),in addition to endo-(l -+ 3) (1 -+ 4)-p-glucanase,activities (see purification and characterisationsection). No CBZphenylalanylalanineor pullulan hydrolysing activity was present. Of the enzymes presenti t appearedthat theendo-(I ~ 3)( I ~ 4)-p-glucanaseisoenzymeswereresponsible for the major part of cell wall breakdown.However, productsfrom this digestion now included trace amounts of arabinose and xylose, indicating that breakdown of arabinoxylanmay playa significant additional role. Since arabinoxylan is presentin both the matrix and microfibrillar parts of the cell walls2 - 4 (total ~ 23 %), and no collapseof cell wall structurewas observedin any of the presentdigestions,it may be inferred that the more accessiblematrix materialwas the site of pentosansolubilisation. Incubation of the cell walls with a crude enzymeextract from barley malt (again containingequivalentendo-(l ~ 3)(1 --7 4)-p-glucanaseactivity) released59 % of the total carbohydratepresent(Figs 4, 5). In addition to the abovementionedenzymicactivities, this preparationincludes the reportedheat-stablep-glucan 'solubilase'carboxypeptidase6 activity. Clearly the presenceof this and otheracidic proteinsprofoundlyaffected the degreeof endospermcell wall breakdown,althoughthe major degradativeprocess
FIGURE 6. Electron micrographsof barley endospermcell wall fragmentsisolated in 70 % ethanol;showing(a) the inner pitted surface x 1200, and (b) the outerscaly surface x 1320.
166
PAMELA BRUNSWICK ET AL.
still appearedto be the action of endo-(l-+3)(1 -+ 4)-~-glucanases. It must also be emphasised,however,that if oneconsidersthat the solublecarbohydratepresentin the •zero time' sampleto bedueentirely to a rapid initial digestion,ratherthan,asassumed, due to the presenceof interfering contaminantsin the crudeenzymepreparation,then the solubilisationfactor comescloseto 72 %, the latter value beingequivalentto a total solubilisation.In this casethe acidic enzymeswould be consideredto cell wall ~-glucan playa major role in cell wall breakdown. The controversyover whetheracidic carboxypeptidaseaction on a proteinjJ3-glucan complex constitutesthe first obligatory step in endospermcell wall degradation19• 2o , could not be clarified by the above results. Furthermore, evidence showing that proteolysisin combinationwith hot waterextractioncould releaseover 96 % of the cell 7 wall ~-glucan , promptedthe study of cell wall solubilisationby endo-(l-+ 3)( I -+ 4)-13glucanasesfollowing proteolysis.Preliminary proteolysisof the cell wall fragmentsby 'proteinase K', however, did not alter the observed solubilisation of cell wall carbohydrateshownin Fig. 5. In conclusionit appearedthat proteolysiswas not a prerequisiteoccurrenceprior to endospermcell wall degradationby endo-(l-+3)(1-+ 4)-~-
FIGURE 7. Eleclron micrographsof barley endospermcell wall fragmentsafter isoenzymeI or II, inner digestionwith: (a) purified endo-(1-+ 3)(1 -->- 4)-~-glucanase wall x 1200,(b) DEAE-cellulosepartially purified enzymeextract,innerwall x 2400, (c) DEAE-cellulosepartially purified enzymeextract,outerwall x 1800. Incubation conditionsas in Fig. 5.
BARLEY CELL WALL DEGRADATION
167
glucanases,althoughthe presentresultsmay not necessarilybe applicablein situ. Since isolation of cell wall fragmentsmay have exposedcomponentsof the cell wall which would normally be protected from direct enzyme attack in situ, a further visual examinationof digestedendospermcell walls was included in the presentstudy. Subsequentelectronmicroscopystudieson the endospermcell wall fragmentsafter endo-(1-,.3)(1--+4)-p-glucanasedigestion were unableto confirm the order of enzyme attack.The isoenzymesexhibited a limited attackwhich was observedonly as a rough appearanceof tl:W cell wall surfaces(Figs 6, 7(a)). The disruptionof cell wall characteristicswas moredistinct following digestionwith crude and DEAE-cellulosepartially purified enzymeextractsfrom malt (Figs 6, 7(b), 7(c». Theenzymesstronglyattackedthe innerpitted surface(Fig. 7(b», but degradation of the outer scalysurfacewas less obvious (Fig. 7(c». Consideringthe in situ sourceof theseenzymes,from the scutellumand aleurone22, their apparentpreferentialattackon the inner wall surfacewas surprising.The datadid, however,supportthe proposalof a bilayeredcell wall structurewith the more water solubleproteinson the inner surface2 In summary,it is concludedthat, despitethe presentedenzymic evidence,a visual examinationof endospermcell walls suggestedthat endo-(l--+3)(1--+4)-p-glucanasesin situ may be unable to hydrolyse the outer exposedcell wall surface.This does not confirm the view that proteolysis, and, more specifically, carboxypeptidaseactivity, precedesor acts in combinationwith glucanasesin the breakdownof endospermcell walls during germination.Contraryevidenceto this view is presentedby the inability of a generalproteinaseto stimulatecell wall degradationby l3-glucanases. Furthermore,the role of arabinoxylan-degrading enzymesremainsin questionsince it was noted that kilned malt extracts solubilised the majority of the cell wall carbohydratewithout a significant degradationof the pentosan. It appearsrelevantto note that the first immediatebarrier to the releaseof enzymes from the aleuronecells into the endospermwould be the ontogenicallyrelatedaleurone walls. Thesebilayered walls contain protein and various polysaccharides,including a high contentof pentosan,mixed-linkagep-glucanand only low amountsof (1 --+ 3)-13glucan at the endosperminterface25 • Although the aleurone walls possessplasmodesmata,it cannot be disregardedthat the sequentialappearanceof enzymesduring germinationmay reflect their role initially in aleuroneratherthan endospermcell wall breakdown.
The authorsare indebtedto Dr G. H. Palmerfor helpful discussions.
References 1. 2. 3. 4. 5. 6.
Thompson,R. G. and LaBerge,D. E. MBAA Tech. Q. 14 (1977) 238-243. Fincher, G. B. J. Inst. Brew. 81 (1975) 116-122. Palmer,G. H. Proc. Am. Soc. Brew. Chem. 33 (1975) 174-180. Ballance,G. M. Ph.D. Thesis,Heriot-Watt University, Edinburgh(1976). Ballance,G.M. and Manners,D.J. Cal'bohyd. Res. 61 (1978) 107-118. Bamforth, C. W. Eur. Brew. COllv. Congress(1981) 335-346.
168 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
PAMELA BRUNSWICK ET AL. Forrest, I. S. and Wainwright, T. J. Inst. Brew. 83, (1977) 279-286. Brunswick, P., Manners,D. J. and Stark, J. R. J. Inst. Brew. 93 (1987) 181-186. Trevelyan,W. E., Procter,D. P. and Harrison,J. S. Nature 166 (1950) 444-445. Baxter, E. D. J. Inst. Brew. 84 (1978) 271-275. Satake,K., Okuyama,T., Ohashi,M. and Shinoda,T. J. Biochern. 47 (1960) 654-660. Weber, K. and Osborn,M. B. J. Bioi. Chern. 244 (1969) 4406-4412. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. and Smith, F. Anal. Chern. 28 (1956) 350--356. Brunswick, P. Ph.D. Thesis,Heriot-Watt University, Edinburgh (1982). Manners,D. J. and Wilson, G. Carbohyd. Res.48 (1976) 255-264. Woodward,J. R. and Fincher,G. B. Eur. J. Biochern. 121 (1982) 663--669. Woodward, J. R. and Fincher,G. B. Carbohyd. Res.106 (1982) 111-122. Luchsinger,W. W. and Richards,A. W. Arch. Biochem.Biophys.106 (1964) 65--70. Ballance,G. M. and Meredith, W. O. S. J. Inst. Brew. 82 (1976) 64--67. Bamforth, C. W., Martin, H. L. and Wainwright, T. J. Inst. Brew. 85 (1979) 334-338. Bamforth, C. W. and Martin, H. L. J. Inst. Brew. 87 (1981) 81-84. Stuart,I. M., Loi, L. and Fincher, G. B. Plant Physiol. 80 (1986) 310--314. Luttenegger,D. D. and Nevins, D. J. Plant Physiol. 77 (1985) 175--178. Lamport, D. T. A. Ann. Rev. Plant Physiol. 21 (1970) 235-270. Bacic, A. and Stone,B. A. Aust. J. Plant Physiol. 8 (1981) 475-495.
Degradation of Isolated Barley Endosperm Cell Walls by Purified Endo-(1--+ 3)(1--+4)-p-n-glucanasesand Malt Extracts*t PAMELA BRUNSWICK, DAVID J. MANNERS and J. ROGER STARKt
Heriot- Watt University, DepartmentofBrewing and Biological Sciences, Edinburgh EHl lHX, u.K. Receivedin revisedform 7 December1987
Thestructureof isolatedbarleyendospermcell wall fragmentswasstudiedemploying The endomalt extracts and two purified endo-(I-;..3)(1-;..4)-~-D-glucanases. (1-;.. 3)(1 -;.. 4)-~-D-glucanases werecharacterisedby their activity onvarioussubstrates and possessedmolecular weights and isoelectricpoints of 27 kD, 33 kD and pI 8·5 and ~ 10·8 respectively.Theseisoenzymeseffected a limited degradationof barley endospermcell wall polysaccharide,releasingglucosein addition to the expected oligosaccharideproducts.Thereleaseof glucosesuggestedthat ~-glucosidases may be associatedwith the isolatedcell wall fragments.The degreeof cell wall carbohydrate degradationwas increasedby the presenceof a mixture of basic ~-glucanases; whereascrude enzymeextracts from barley malt, containing proteolytic activity, releasedup to 72 % of the total carbohydrate,including arabinoxylanbreakdown products. The pattern of the cell wall digestions was, however, not altered by pretreatmentof the cell wall fragments with a-amylaseand a general proteinase, forwarding the view that proteolysis is not a pre-requisitefor endo-~-glucanase attack. Examination of digested endosperm cell wall fragments by electron microscopyindicatedthat the enzymesof the basicproteinfraction from barleymalt clearly attackedthe inner pitted surfaceof the walls, althoughsolubilisationof the outerscalysurface,evenin the presenceof crudeenzymeextracts,waslessapparent. The observationsdid not confirm the proposalthat the sequentialappearanceof proteinasesandcarbohydrases during germinationreflecttheir role in endospermcell wall breakdown.
Introduction The endospermcell walls of barleyaredegradedenzymically during germinationof the grain, with the concomitantsolubilisationof the starchyendosperm.C ell walls are thus regardedas potentially important sites for the regulation of endospermmodification during malting and of carbohydraterecoveryduring the mashingstageof brewing1 • .. Studieson ~-Glucanases, PartVIII. For PartVII, seeBrunswick,P., Manners,D. J., Stark, J. R. J. Ins/. Brew. 93 (1987) 181-186. t Dedicatedto ProfessorLuis F. Leloir on the occasionof his 80th birthday. t To whom correspondenceshouldbe addressed. Abbreviations used: -CBZ = carbobenzoxy-;eM- = carboxymethyl-;pCMB = p-chloromercuribenzoic acid; DEAE- = diethylaminoethyl-; DDT = DL-dithiothreitol; PMSF = phenylmethylsulphonylfluoride; TNBS = 2,4,6,-trinitrobenzeneI-sulphonicacid. 0733-5210/88/020153 + 16 $03.00/0
© 1988 AcademicPressLimited
154
PAMELA BRUNSWICK ET AL.
In barley, the endospermcell walls are composedmainly of a mixed linkage This matrix glucanconstitutes70-72% by weight of the cell wall, the remainderbeingcomposedof matrix and microfibrillar arabinoxylan,protein and cellulose or glucomannan2-4.The high ~-glucan to cellulose ratio provides a flexible, easily degradablestructure. Although the composition of the endospermcell wall has been established,the physicalarrangemento f thecomponentsis lesswell-defined.The glucansandpentosans canbe classedaccordingto their waterand alkali solubility, indicating that their degree of polymerizationor binding within the walls canvary5. Of interestto the presentstudy is the suggestedbilayeredstructureof endospermcell walls, with the inner pitted surface of the wall composedof the more water-solublepolysaccharides 2. Furthermore,it hasbeenproposedthat the sequentiala ppearanceof enzymesduring germination could give an indication of the structural arrangementof the cell wall barrier6 • The suggestionthat ~-glucan 'solubilase',a carboxypeptidase,catalysesthe first stagesof cell wall degradation6 was supportedby earlierpublicationswhich showed that proteolysis,prior to hot water extraction,could removeover 96 % of the ~-glucan presenta ndcausestructuralcollapseof thecell waIF. In fact, a peptide-glucancomplex, isolatedfrom barley endosperm,could be degradedto a molecularsize similar to that derivedfrom greenmalt by digestionwith thermolysin or by hydrazinolysis7 • However, contraryto the proposalof preliminary proteolysis,it hasbeendemonstratedthat both an endo-(l-+3)-~-glucanase from malted barley and a fungal endo-(l-+4)-~-glucanase can extensivelydegradeisolatedcell wall fragments4 • The controversyover whetherproteolysisis a pre-requisitefor cell wall degradation by ~-glucanases is discussedin the presentstudy.Partiallypurified enzymeextractsfrom barley malt and purified, characterisedendo-(l-+3)(1-+ 4)-~-D-glucanase isoenzymes were preparedand employed to study the cell wall structure and its solubilisation. Breakdown of the cell wall was observedby the releaseof soluble oligosaccharide productsand by electronmicroscopy. ~-(1~3)(1-+4)-glucan2-4.
Experimental Preparation of dehuskedkilned barley malt
Barley grain (GoldenPromise,1980 harvest)wasdehuskedin 50 % (v Iv) sulphuricacid solution for 3 h at room temperature.Themixture was neutralisedwith sodiumcarbonateand the grain was thoroughly rinsed by light rubbing in water. After air-drying overnight, the grain was subjectedto a steepingprogramof 8 h steep,16 h air rest, 8 h steep,12 h air rest, 0'5 h steepand drain. The grain was germinatedat 16°C for 16 h. Germinated'green'barleygrain was stored at -15°C or subjectedto kilning at 60°C for 16 h and placedin dry storage. Isoenzymeextraction and separationprocedure
Endo-(I-+3)(1-+4)-~-D-glucanases wereextractedandpartially purified8 • The dehuskedmalt was extractedat 0-4 °C in a solution (2 l/kg of grain) of 0·2 MNa acetatebuffer, pH 4'8, containing 10 mM sodiumazide, 10 mM EDTA, 3 mM PMSF and 3 mM DTT. Following centrifugationand
BARLEY CELL WALL DEGRADATION
155
dialysis, the enzyme extract was subjected to DEAE-cellulose chromatographyin 20 mM citrate-phosphate buffer, pH 8. Proteineluting at the void volumeof the DEAE-cellulosepadwas pooled and dialysedwith 0·1 M acetatebuffer, pH 4·8. A portion of the extract, containing86·6mg protein, loadedonto a column of CM-cellulose (40 x 1·8 em) and elutedwith 200 ml startingbuffer, followed by a linear gradient,550 ml, from 0·1 to 1'0 M acetate,pH 4'8, producedthe elution profiles shown in Fig. I (4'2 ml fractions). Endo-(1.....3)(1 .....4)-B-o-glucanaseisoenzymesfor the study of endospermcell walls were prepared by an equivalent bulk separationof 409 mg protein on a 17 x 6 cm CM-cellulose column.
Isoenzymepurification Endo-(1.....3)(1 .....4)-B-o-glucanaseisoenzymes,labelled I and II accordingto their consecutive elution from the CM-cellulosecolumn, were chromatofocusedon columnsof PBE 74 (32'5 x 1'0 cm) and PBE 118 (22'0 x 1·0 cm) Pharmaciaresinsrespectively.The PBE 74 was equilibratedin 25 mM ethanolamine-HCIbuffer, pH 9-4 andelutedwith dilute (1 : 10) Polybuffer96-HCI, pH 7'5. PBE 118 resin was equilibriated with 25 mM triethylamine-HCl, pH 10·8 and the column was eluted with dilute (I :45) Pharmalyte-HCI,pH 8. The pH elution profile was measuredin the collectedeluantfractions(2 ml), in additionto assayingfor enzymicactivity on varioussubstrates. Assaysinvolving reducingpower determinationsin the presenceof Polybuffer were centrifuged prior to absorbancereading. Further purification of the individual isoenzymeswas achievedby molecular sieve chromatographyon a calibratedcolumn(86'0x 2'2 cm) of SephadexG-75 (Pharmacia).Theequilibration and elution buffer employed was 50 mM Na acetate buffer, pH 4·8, containing 50 mM KCI. Fractionsof theeluant(1'7 ml) wereassayedfor enzymicactivity andthe pooledfractionsdialysed against0·1 MNa acetatebuffer, pH 4,8, beforestorageat -IS DC.
Assayof carbohydraseactivities Enzymic activities on carbohydratesubstrateswere determinedaccordingto the reducingpower and glucoseoxidase(cellobiaseactivity only) methodsof Brunswick et alB. In addition to the substratescellobiose, lichtenin, laminarin and soluble starch, the polysaccharidepullulan was included at a concentrationof O' I % (w Iv) and 2 h incubation. Enzymic activity is reportedas equivalent I!g of o-glucose released,measuredwith reducing power reagentsat 600 nm, per 0·1 ml sampleper assaytime. Endo-B-glucanaseand arabinoxylanaseactivities were determined more specifically by viscometry 8 and paper chromatography.T he substratesemployed to measureendo-(1-.;.3)-I3glucanase, endo-(1.....4)-[3-glucanase, endo-(1..... 3)(1 .....4)-I3-glucanases and arabinoxylanase activities were CM-pachyman(0,8 % (w Iv», CM-cellulose(0'13% (w Iv», 40 DC water-extracted barley B-glucan (0'7 % (w Iv» and rye arabinoxylan(0'2% (w Iv» respectively.Viscometry was performedat 30 DC in a M4-type V-tube viscometer,in the presenceof 6 mM Na acetatebuffer, pH 5 and 0·3 mM DTT. The 'Units' of enzymeactivity areexpressedasthe rateof increasein the reciprocalof specific dynamicviscosity with time8 • Paperchromatographyof oligosaccharideproductsemployed the following solvent systems (proportions by voL); ethyl acetate-pyridine-water(10:4: 3), ethyl acetate-aceticacid-formic acid-water (18:3: I :4) and n-propanol--ethyl acetate-water(14:2: 7). Reducing sugars were . detectedby alkaline silver nitrate reagents9•
Assayof carboxypeptidaseactivity Carboxypeptidaseactivity was determinedby the methodof Baxter10, basedon that reportedby Satakeet alY, and employing 0·4 mM CBZ-L-phenylalanylalanineas substrate.
156
PAMELA BRUNSWICK ET AL.
SDSelectrophoresis SDS disc-electrophoresiswas based upon the method of Weber and Osborn12 with minor modifications.
Preparation ofbarley endospermcell walls Fragmentsof endospermcell walls were isolated from dehuskedbarley by the methods of Fincher2 , Ballance4 and Ballanceand Manners5 • The cell walls were stored in 70% ethanolat 0-4 °C. The preparationcontained 17 % (w Iw) protein, of which one-fifth was water-soluble following homogenisation.
Cell wall pre-treatments Cell wall fragmentswere digestedwith filter-sterilised salivary ex-amylasein aqueoussolution containing0·02% (w Iv) Na azide,for 16 h at 30°C. In addition, the fragmentswere subjectedto digestion,at 37 °C, by ProteinaseK, 8 x 10-3 AnsonUnits/ml (BDH Chemicals,u.K.) in 20 mM Na phosphatebuffer, pH 7, also containingazide.During thesedigestionprocessesthe cell walls were kept suspendedby gentleinversionand the digestionswere terminatedby washingthe walls free of enzyme.
Cell wall digestionby carbohydrases Suspendedendospermcell wall fragmentswereincubatedwith variousenzymepreparationsin a volume of 3 ml containing3 mM Na azide,3 mM DTT, and 0,1 M Na acetatebuffer, pH 4,8. Zero time sampleswere takenfrom the supernatantafter the addition of enzymeat 0-4 °C, whereas total carbohydratewasmeasuredin thesuspensionmixture.Timedintervalsamplesof supernatant were taken during incubation of the digest at 30°C. Control digestionscontainedno added enzyme.The sampleswere centrifuged and the supernatantassayedfor the presenceof total carbohydrateby the phenol-sulphuricacid method13. Cell wall carbohydratedigestionis reported asthe releaseof equivalentI!g D-glucoseper total digestvolumeaftercorrectionfor the changein digestvolume and loss of carbohydratedue to sampling.
Scanningelectron microscopy Cell wall fragmentswerewashedin successivelyhigherconcentrationsof ethanolandfinally with ether. The air-dried fragments were coated with gold to a thickness of 30 nm before being examinedundera CambridgeStereoscanelectronmicroscopeat an acceleratingvoltageof27 kY.
Results and Discussion
Purity and characterisationof fJ-glucanasesemployedin cell wall digestion
Two endo-(l-+3)(1 -+ 4)-~-D-glucanases werepurified from dehuskedbarleyasoutlined in the Experimentalsectionand previouslyB,14. The purification at eachstageis shown in TableI. An initial 3'4-fold purificationoverDEAE-cellulose(TableI) wascomparable to that observed by Manners and Wilson15 for endo-(1-+3)(1 -+ 4)-~-glucanase extractionfrom barley malt. A l-8-fold purification was reportedby Woodward and Fincher16 for DEAE-cellulosepurification, althoughwhen a salting out and desalting
BARLEY CELL WALL DEGRADATION
,157
TABLE I. Purification of endo-(l -+ 3)(1 ->- 4)-p-n-glucanases from dehusked,germinated, kilned barley
Fractionation procedure Crude (combined1+11) Step 1; DEAE-cellulose (combined1+11) Step 2: eM-cellulose IsoenzymeI IsoenzymeII Step 3: SephadexG-75 IsoenzymeI IsoenzymeII Alternative step 3; Chromatofocusing and SephadexG-75 IsoenzymeI IsoenzymeII
Total protein (mg)
Total activity (units)
Specific activity (units/mgx 102)
Purification factors Eachstep
Overall
20820
204
0·98
6988
216
3·1
3'4
3-4
839 489
37 61
4·4 12,5
1'4 4'0
12·8
42 49
15 24
36 49
8'2 4'0
37 50
145 23
4'0 0·5
148 23
4·2
3-7
6'0 0·8
4'5
Valuesgiven at eachstepare the means(per kg of dehuskedgrain) of two or more repeatedexperiments. Recoveriesof> 100% are attributableto interferenceby additi,:esin the crudeextract.
stepwasincluded,the overallpurification reportedwas 1O·9-fold16 • It was notedthat the activity extractedfrom germinated total protein and endo-(1-+ 3)(1 -+ 4)-~-glucanase barley by the latter authors16 was much higher than that observedin the presentstudy of germinated'green'barleyextracts(TableII). Thediscrepancyin proteinrecoverywas probablyattributableto the useof 1kg grain in the presentstudy as comparedto 10 kg by the other authors16, Differences in actual values of reported enzyme activities measuredby viscometry are inevitable becauseof the effect of initial substrate concentration,incubationconditions and variation betweenthe polymeric substrates ~-glucan substrateconcentration employed.In thepresentstudythelaboratory-prepared was 0'71 % (wjv), as compared to 0·18% (wjv) employed by Woodward and Fincher16• 17 ; thus, decreasesin the viscosity of the substratesolution by enzymic hydrolysis produceddiffering rangesof valuesfor total and specificactivity, It was of interestto note that, whereasthe presenceof the additivesDDT, PMSF, EDTA and azide improved the initial endo-(l-+3)(1-+4)-p-glucanaseactivity from activity germinatedbarley by almost 100%, the recoveryof endo-(l-+3)-~-glucanase was little affected (Table II). During theseproceduresit was also observedthat the presenceof DDT was required in order to maintain endo-(l-+3)(1-+ 4)-~-glucanase activity during dilution or cold storage.
PAMELA BRUNSWICK ET AL.
158
TABLE II. Purification of endo-~-glucanases from germinatedor kilned dehuskedbarley by DEAE-cellulosechromatography Endo-(l-e".3)-I3-glucanase
Endo-(1-+ 3)(1 - 4)-p-glucanase Extraction and fractionation procedure
Total protein (mg)
Total activity (units)
Germinatedbarley; no additives Crude 352 18895 DEAB-cellulose 7375 135 Germinatedbarley; plus additives Crude 21379 280 DEAE-cellulose 5595 260 Kilned barley; plus additives Crude 20820 204 DEAE-cellulose 6988 216
Specific activity (units/ mg)
Purification factor
Total activity (units)
Specific activity (units/ mg)
Purification factor
1·9 l·g
0·98
805 672
4·3 9-1
2'1
1-3 4·6
3-5
715 617
3·3 11·0
3-3
0·98 3·1
3-4
569 709
2·7 10·2
3·7
Valuesgiven per kg of dehuskedgrain. Recoveriesof> 100 % are attributableto interferenceby additives (seeExperimental)in the crudeextract.
TABLE III. Estimationof the molecularweights of enzymesextractedfrom kilned germinated barley malt
V.(ml)
SephadexG-75 chromatography Endo-(l --+ 3)(1--+4)-I3-g1ucanaseI Endo-(1-+ 3)(1 -+ 4)-j3-g1ucanaseII J3-Amylase Cellobiase SOS electrophoresis Endo-(I_3)(l-+4)-j3-g1ucanase I Endo-(1 --+ 3)(1 --+ 4)-j3-g1ucanaseII
kD/mobility
194·9
0'3139
180'7 170·5
0·2574 0·2167 0·1837
162·2
0·610 0'575
loglO mol. wt.
mol. wt. (kD)
4·403 4'527 4·616 4·687
33'7±0'7 48·7±0·9
4'465 4'513
29·2±1·5 32'6±0'8
25'3±1·6 41'3±1-6
Standardproteins employedwere: bovine serum albumin, 68 kD; ovalbumin, 43 kD; ~-lactoglobulin, 36'5 kD; chymotrypsinogenA, 25·7kD; ~-lactoglobulin monomer, 18'2kD; equine myoglobin, 17-6 kD; cytochromeC, 11'7 kD.
In the presentstudy, two endo-(l-+3)(1- 4)-f3-glucanaseswere separatedby eMcellulose chromatographyand identified, by hydrolysis of the substratelichenin and viscometrically,as the peakslabelled'!' and'II' in Fig. 1. The purification factor for the two isoenzymesfrom kilned barleymalt was 1·4-fold and4-fold (overa1l4'5-foldand l2'B-fold) respectively(Table I). This correspondsto the 8-fold purification of a single mixed-linked type enzymefrom barley malt reportedby Mannersand Wilson15• The purification of two equivalent isoenzymesfrom 'unkilned' germinated barley was
BARLEY CELL WALL DEGRADATION
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reportedto be O'7-fold and 6'4-fold (overall 7-fold and 64-fold) respectively,after a similar procedurebut employingelution buffer pH changesratherthana concentration gradient16,1 7. At this stagethe two endo-(1-+3)(1-+4)-p-glucanases were contaminatedwith endo(1-.+ 3)-p-glucanase,cellobiaseand J3-amylaseactivities, as shownin Fig. 1. No activity on pullulan or CBZ-phenylalanylalaninewas observedin the eM-cellulosecolumn fractions. The isoenzymeswere further purified by gel filtration on SephadexG-75, or a combination of chromatofocusing and gel filtration (Table I; Figs 2, 3). A gel filtration stepalonegavea further purification of isoenzymesI and II of 8'2-fold and4fold (overall 37-fold and 50-fold) respectively(Table I). Combinedwith a chromatofocusingstep,thesepurificationswere alteredto 4-fold (overall 148-fold) for isoenzyme I, but deterioratedto O'S-fold (overall 23-fold) for isoenzymeII (Table I). This latter deteriorationcould be attributedto the instability of isoenzymeII at the high alkaline pH valuesemployed. By comparison,the presentedpurificationsover SephadexG-75 arein a similar range to the publishedvalues of 105-fold15 and 3Q-200-fold18 by other workers employing
160
PAMELA BRUNSWICK ET AL. 9·5
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FIGURE 2. Chromatofocusingon PBE 74 resin of endo-(l-+3)(1-+ 4)-~-glucanase isoenzymeI. Enzymicactivitiesmeasuredby the releaseof reducingoligosaccharides or glucosefrom (a)- - , lichenin:---, pH gradient9·0-7·5.(b) - - , cellobiose;---, solublestarch.(c) - - , laminarin. seeExperimentalfor details.
commercialkilned barley.WoodwardandFincherreportedpurificationsof 67-fold and 4'6-fold by combined chromatographyover CM-Sepharoseand two columns of SephadexG-75 (giving overall 467-fold and 294-fold) for two similar isoenzymesfrom germinatedbarley16·17.The presentresultsappearedto bein line with thoseof previous workers. The data suggeststhat the yield and purification of theseisoenzymescould undoubtedlybe improvedabovevaluespreviouslyreportedl 5-18 by including an initial salting out step(as employedby previousworkers)16.17in the presentprocedure. werecharacterisedby their isoelectricpoints Thetwo endo-(l-+ 3)(1 -+ 4)-~-glucanases (chromatofocusingdata, Figs 2, 3), molecularweights(calibratedSephadexG-75 data and SDS electrophoresis,Table III) and by their profiles of hydrolysis of various carbohydrates(Table IV). Results were consistentwith those for two endo-(l-+3) (1-+ 4)-J3-glucanase isoenzymespurified from unkilned germinatedbarleyby Woodward and Fincher16.17, exceptthat no activity towardsCM-pachymanwas observedin the presentpreparations(Table IV).
BARLEY CELL WALL DEGRADATION
161
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FIGURE 3. Chromatofocusingon PBE 118 resin of endo-(l--*3)(I--*4)-p-glucanase isoenzymeII. ---, pH gradient10'8-8,0;- - , hydrolysis of lichenin measuredby reducingpower. No othercarbohydraseactivity observedexceptfor tracecellobiose in pooledlicheninasepeak. TABLE IV. Substratespecificity of purified endo-(l -+ 3)(I -+ 4)-p-glucanaseisoenzymesI and II from dehuskedkilned germinatedbarley Hydrolytic activity Substratetested Barley p-glucan 40°C water extract 90 °C water extract Lichenin Low mol. wt p-glucanoligosaccharides CM-pachyman Laminarin CM-cellulose Cellobiose Rye arabinoxylan Larchwooddyed CM-xylan
IsoenzymeI
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Isoenzyme I, pI8·4-8·5 and mol. wt. 27,250 (± 1600) (Table III), appeared chromatographicallypure by SDS electrophoresis.Any remaining contaminating (pI < 7'5, 41 kD) and cellobiase (pI's,..., 8'3 and ,..., 8'6, mol. wt. 49 kD), ~-amylase laminarinase(pI's> 9'0. 8'0-8'3, 8·0 and 7-8) activities observedduring chromatofocusing (Fig. 2) were removed by gel filtration (Table III). The void volume laminarinasepeak (pI> 9) of chromatofocusingcan be attributed to a previously althoughmalt extractsareknown to containtwo ~reportedendo-(l-+3)-~-glucanase19.
162
PAMELA BRUNSWICK ET AL.
glucan 'solubilases' with pI values acidic and 9-352 °. Laminarinase actlVlty that overlapped with the endo-(1--+ 3)(1 4)-p-glucanaseduring chromatofocusingalso exhibiteda high affinity towardslaminaribiose,the remaininglaminarinasepeaksbeing inferred to be due to the presenceof p-glucosidaseisoenzymes. IsoenzymeII, pI > 10·8 (Fig. 3) andmol. wt. 33,125(± 750) (TableIII), appearedto be separatedfrom all contaminatingenzymeactivities by the chromatofocusingstep. The observedvalues for mol. wt. and pI of the two isoenzymeswere close to those reportedfor two equivalentisoenzymesfrom unkilnedmaltI6 • 1? It seemedcoincidental that two out of four carboxypeptidases presentin a crudemalt extractwerereportedto possessisoelectricpoints of 8·4 and> 1021. SDS electrophoresisof isoenzymeII showedtraces of at least one contaminating protein. This was inferred to be a cellobiaseenzymesince trace amountsof cellobiase activity could not be removedfrom the preparation(Table IV). In addition to this contaminant,b othof thepurifiedisoenzymesexhibitedtraceactivity on rye arabinoxylan but no other contaminatingpolysaccharidedegradingactivities were observed(Table IV). Contaminatingarabinoxylanaseactivity, and, in addition, hydrolysis of eMpachyman,wereobservedin theendo-(l --+ 3)(1--+4)-p-glucanaseisoenzymepreparations of Woodward and FincherI6 • 17 . The arabinoxylanaseactivity may be attributed to contamination of the pentosan substrateswith traces of glucan. Alternatively, arabinoxylanmoleculesmaybesusceptibleto endo-(I...."..3)(1-;.4)-p-glucanasehydrolysis by imitating the conformationalstructureof a mixed-linkagef)-glucan. The physiologicalsignificanceof two endo-(1----3)(1----4)-p-glucanaseisoenzymesin germinating grain is important in the context of their role in the breakdown of endospermcell walls. Recentpublicationssuggestthat these isoenzymesare derived from different tissuesandareunderseparategeneticcontrop2.Theequivalentisoenzyme I is reportedly synthesisedpredominantly in the scutellum, while isoenzyme II is synthesisedexclusivelyin the aleurone22. A third, putativeisoenzymehasbeendetected in imbibed barley22, Of interest, also, is the dynamic nature of cell wall p-glucanin Graminaceouscoleoptilesand its role in coleoptileelongation23. Endospermcell wall studies
Barley endospermcell walls were examinedfor the releaseof solublecarbohydrateand by electronmicroscopyfollowing intervals of digestionwith barley malt crudeenzyme extract, DEAE-cellulosepartially purified extract and purified endo-(1-;.3)(1"""" 4)-f)glucanases. After a 24 h incubationwith endo-(1...."..3)(1----4)-p-glucanaseisoenzymesI or II, at 2'09 x 10-2 U/ digest, the endospermcell walls remainedstructurally intact, the two isoenzymesreleasingequivalentand additive amountsof solublecarbohydrate(Table V). The comparative instability of isoenzyme lover that of isoenzyme II was demonstratedby employingreducedconcentrationsof enzyme;in this casethe release of solublecarbohydratereached11 % and 22 % respectively,with a crudemalt enzyme extractreleasing58 % by comparison(Fig. 4), Interferenceby the releaseof endogenouscell wall carbohydrateand contaminating starchgranuleswas removedby treatmentof the walls with a-amylaseand dialysis at
163
BARLEY CELL WALL DEGRADATION
TABLE V. Digestionof barley endospermcell wall fragmentsby purified endo-(1-+3)(1-+4)~-glucanase isoenzymesI and II Releaseof solublecarbohydratefrom cell walls ( = Ilg D-glucose/3ml of digestinterval shown-1)
Content
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FIGURE 4. Digestiqn of barley endospermcell walls by enzyme extracts from dehuskedkilned barleymalt. (a) and(b) Cell walls digestedwith endo-(I -+ 3)(1 -+ 4)-~glucanaseisoenzymesI and II respectively. (c) Cell walls digested with both isoenzymesin a ratio of I: 1. (d) Cell walls digestedwith crudeenzymeextractfrom dehuskedkilned barley.Digests(a), (b) and(d) initial endo-(I -? 3)(1-+4)-~-glucanase activity of 5·23x 1O-3U/3ml; (c) initial activity of 10-5 x 10-3U/3 m!. Q-4 °C. Following this additionalstep,endo-(1-73)(1-74)-p-glucanases I andII released
14 % and 29 % of the cell wall carbohydraterespectively(Fig. 5). Thesefigures could only be increasedto a maximumof 20 and 31 % respectivelyby the further addition of freshenzyme(Fig. 5). This incompletesolubilisationof cell wall carbohydratesuggested that, providedthat productinhibition wasnot occurringin both cases,a portion of the p-glucanwas unavailablefor hydrolysis. Comparisonof the presentresults with those of previous workers highlights the importanceof enzymespecificity and concentration.Solubilisation of endospermcell
164
PAMELA BRUNSWICK ET AL. 220 200
(a)
(b)
(c)
(d)
FIGURE 5. Digestion of a-amylasetreatedbarley endospermcell walls by enzyme extractsfrom dehuskedkilned barleymalt. (a) and(b) Cell walls digestedwith endo(1 ->- 3)(1-;. 4)-p-glucanaseisoenzymesI and II respectively.(d) and (c) Cell walls digestedwith dialysedcrudeand DEAE-cellulosepartially purified enzymeextracts from dehuskedkilned barley respectively.All digests containedan initial endoactivity of 5'23 x 10-3 U/3 ml. A further 1·96 x 10-3 U/3 ml (I ->- 3)(1-;. 4)-p-glucanase and 5'23 x 10-3 U/3 ml were addedto digests(a) and (b) respectivelyafter 9 daysof incubation.Shadedarearepresentsthe carbohydratereleasedeach 1'5 h/3 ml. Total area representsthe total carbohydratereleased,(a) 14 and 20 % after further digestion,(b) 29 and 31 % after further digestion,(c) 43 % after further digestionand (d) 59 % after further digestion. walls by 60-70% over 24 hand75-80 % after 5 days,hasbeenachievedemployinghigh concentrationsof a fungal 'licheninase'(endo-(1-+4)-~-glucanase5) or a purified malt endo-(l-+3)-~-glucanase4. It wasnotedon review, however,that the publisheddatadid not appearto be correctedfor carbohydrateleachingfrom the cell wall or comparedto the actual total carbohydratepresent. Neverthelessthe figures did suggest a total solubilisation of the p-glucan matrix4. In contrast, other authors have reported the collapseof cell wall structureafter the extractionof most of the p-glucan7 • In the presentstudy the productsof endo-(1->-3)(1->-4)-p-glucanasedigestionof cell walls, surprisingly, included glucose, identified by paper chromatography. This conflicted with the evidenceobtainedby prolongedhydrolysisof p-glucansubstrateby the purified isoenzymes,in which the products were the expected tri- and tetrasaccharides.Although tracecellobiaseactivity could not be removedfrom isoenzymeII (TableIV), contaminationcould not explainthe releaseof glucosecausedby isoenzyme 1. The anomaly could be attributed to the presenceof p-glucosidases,k nown to be presenti n ungerminatedbarleygrain, which maybecomeattachedto thecell wall during the isolation processand be reactivatedunderthe incubationconditions.In supportof this suggestionis evidencethat Graminaceouscoleoptile cell walls, isolatedin glycerol and organicsolvent,containedfirmly boundautolytic activity which releasedreducing sugars,including glucose,andmixed linkageglucansfrom the walls during incubation24. By comparison, DEAE-cellulose partially purified enzyme extract from malt
BARLEY CELL WALL DEGRADATION
165
(containingequivalentendo-(1~ 3)(1 ~ 4)-~-glucanase activity) hydrolysed43 % of the cell wall carbohydrate(Fig. 5). This extract contained cellobiase, ~-amylase and laminarinase(endo-(l ~ 3)(~-glucanase and P-glucosidases),in addition to endo-(l -+ 3) (1 -+ 4)-p-glucanase,activities (see purification and characterisationsection). No CBZphenylalanylalanineor pullulan hydrolysing activity was present. Of the enzymes presenti t appearedthat theendo-(I ~ 3)( I ~ 4)-p-glucanaseisoenzymeswereresponsible for the major part of cell wall breakdown.However, productsfrom this digestion now included trace amounts of arabinose and xylose, indicating that breakdown of arabinoxylanmay playa significant additional role. Since arabinoxylan is presentin both the matrix and microfibrillar parts of the cell walls2 - 4 (total ~ 23 %), and no collapseof cell wall structurewas observedin any of the presentdigestions,it may be inferred that the more accessiblematrix materialwas the site of pentosansolubilisation. Incubation of the cell walls with a crude enzymeextract from barley malt (again containingequivalentendo-(l ~ 3)(1 --7 4)-p-glucanaseactivity) released59 % of the total carbohydratepresent(Figs 4, 5). In addition to the abovementionedenzymicactivities, this preparationincludes the reportedheat-stablep-glucan 'solubilase'carboxypeptidase6 activity. Clearly the presenceof this and otheracidic proteinsprofoundlyaffected the degreeof endospermcell wall breakdown,althoughthe major degradativeprocess
FIGURE 6. Electron micrographsof barley endospermcell wall fragmentsisolated in 70 % ethanol;showing(a) the inner pitted surface x 1200, and (b) the outerscaly surface x 1320.
166
PAMELA BRUNSWICK ET AL.
still appearedto be the action of endo-(l-+3)(1 -+ 4)-~-glucanases. It must also be emphasised,however,that if oneconsidersthat the solublecarbohydratepresentin the •zero time' sampleto bedueentirely to a rapid initial digestion,ratherthan,asassumed, due to the presenceof interfering contaminantsin the crudeenzymepreparation,then the solubilisationfactor comescloseto 72 %, the latter value beingequivalentto a total solubilisation.In this casethe acidic enzymeswould be consideredto cell wall ~-glucan playa major role in cell wall breakdown. The controversyover whetheracidic carboxypeptidaseaction on a proteinjJ3-glucan complex constitutesthe first obligatory step in endospermcell wall degradation19• 2o , could not be clarified by the above results. Furthermore, evidence showing that proteolysisin combinationwith hot waterextractioncould releaseover 96 % of the cell 7 wall ~-glucan , promptedthe study of cell wall solubilisationby endo-(l-+ 3)( I -+ 4)-13glucanasesfollowing proteolysis.Preliminary proteolysisof the cell wall fragmentsby 'proteinase K', however, did not alter the observed solubilisation of cell wall carbohydrateshownin Fig. 5. In conclusionit appearedthat proteolysiswas not a prerequisiteoccurrenceprior to endospermcell wall degradationby endo-(l-+3)(1-+ 4)-~-
FIGURE 7. Eleclron micrographsof barley endospermcell wall fragmentsafter isoenzymeI or II, inner digestionwith: (a) purified endo-(1-+ 3)(1 -->- 4)-~-glucanase wall x 1200,(b) DEAE-cellulosepartially purified enzymeextract,innerwall x 2400, (c) DEAE-cellulosepartially purified enzymeextract,outerwall x 1800. Incubation conditionsas in Fig. 5.
BARLEY CELL WALL DEGRADATION
167
glucanases,althoughthe presentresultsmay not necessarilybe applicablein situ. Since isolation of cell wall fragmentsmay have exposedcomponentsof the cell wall which would normally be protected from direct enzyme attack in situ, a further visual examinationof digestedendospermcell walls was included in the presentstudy. Subsequentelectronmicroscopystudieson the endospermcell wall fragmentsafter endo-(1-,.3)(1--+4)-p-glucanasedigestion were unableto confirm the order of enzyme attack.The isoenzymesexhibited a limited attackwhich was observedonly as a rough appearanceof tl:W cell wall surfaces(Figs 6, 7(a)). The disruptionof cell wall characteristicswas moredistinct following digestionwith crude and DEAE-cellulosepartially purified enzymeextractsfrom malt (Figs 6, 7(b), 7(c». Theenzymesstronglyattackedthe innerpitted surface(Fig. 7(b», but degradation of the outer scalysurfacewas less obvious (Fig. 7(c». Consideringthe in situ sourceof theseenzymes,from the scutellumand aleurone22, their apparentpreferentialattackon the inner wall surfacewas surprising.The datadid, however,supportthe proposalof a bilayeredcell wall structurewith the more water solubleproteinson the inner surface2 In summary,it is concludedthat, despitethe presentedenzymic evidence,a visual examinationof endospermcell walls suggestedthat endo-(l--+3)(1--+4)-p-glucanasesin situ may be unable to hydrolyse the outer exposedcell wall surface.This does not confirm the view that proteolysis, and, more specifically, carboxypeptidaseactivity, precedesor acts in combinationwith glucanasesin the breakdownof endospermcell walls during germination.Contraryevidenceto this view is presentedby the inability of a generalproteinaseto stimulatecell wall degradationby l3-glucanases. Furthermore,the role of arabinoxylan-degrading enzymesremainsin questionsince it was noted that kilned malt extracts solubilised the majority of the cell wall carbohydratewithout a significant degradationof the pentosan. It appearsrelevantto note that the first immediatebarrier to the releaseof enzymes from the aleuronecells into the endospermwould be the ontogenicallyrelatedaleurone walls. Thesebilayered walls contain protein and various polysaccharides,including a high contentof pentosan,mixed-linkagep-glucanand only low amountsof (1 --+ 3)-13glucan at the endosperminterface25 • Although the aleurone walls possessplasmodesmata,it cannot be disregardedthat the sequentialappearanceof enzymesduring germinationmay reflect their role initially in aleuroneratherthan endospermcell wall breakdown.
The authorsare indebtedto Dr G. H. Palmerfor helpful discussions.
References 1. 2. 3. 4. 5. 6.
Thompson,R. G. and LaBerge,D. E. MBAA Tech. Q. 14 (1977) 238-243. Fincher, G. B. J. Inst. Brew. 81 (1975) 116-122. Palmer,G. H. Proc. Am. Soc. Brew. Chem. 33 (1975) 174-180. Ballance,G. M. Ph.D. Thesis,Heriot-Watt University, Edinburgh(1976). Ballance,G.M. and Manners,D.J. Cal'bohyd. Res. 61 (1978) 107-118. Bamforth, C. W. Eur. Brew. COllv. Congress(1981) 335-346.
168 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
PAMELA BRUNSWICK ET AL. Forrest, I. S. and Wainwright, T. J. Inst. Brew. 83, (1977) 279-286. Brunswick, P., Manners,D. J. and Stark, J. R. J. Inst. Brew. 93 (1987) 181-186. Trevelyan,W. E., Procter,D. P. and Harrison,J. S. Nature 166 (1950) 444-445. Baxter, E. D. J. Inst. Brew. 84 (1978) 271-275. Satake,K., Okuyama,T., Ohashi,M. and Shinoda,T. J. Biochern. 47 (1960) 654-660. Weber, K. and Osborn,M. B. J. Bioi. Chern. 244 (1969) 4406-4412. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. and Smith, F. Anal. Chern. 28 (1956) 350--356. Brunswick, P. Ph.D. Thesis,Heriot-Watt University, Edinburgh (1982). Manners,D. J. and Wilson, G. Carbohyd. Res.48 (1976) 255-264. Woodward,J. R. and Fincher,G. B. Eur. J. Biochern. 121 (1982) 663--669. Woodward, J. R. and Fincher,G. B. Carbohyd. Res.106 (1982) 111-122. Luchsinger,W. W. and Richards,A. W. Arch. Biochem.Biophys.106 (1964) 65--70. Ballance,G. M. and Meredith, W. O. S. J. Inst. Brew. 82 (1976) 64--67. Bamforth, C. W., Martin, H. L. and Wainwright, T. J. Inst. Brew. 85 (1979) 334-338. Bamforth, C. W. and Martin, H. L. J. Inst. Brew. 87 (1981) 81-84. Stuart,I. M., Loi, L. and Fincher, G. B. Plant Physiol. 80 (1986) 310--314. Luttenegger,D. D. and Nevins, D. J. Plant Physiol. 77 (1985) 175--178. Lamport, D. T. A. Ann. Rev. Plant Physiol. 21 (1970) 235-270. Bacic, A. and Stone,B. A. Aust. J. Plant Physiol. 8 (1981) 475-495.