THE CELLULASES OF TRICHODERMA VIRIDE
Mode of action and application in biomass conversion
BILUOTH .VVK
LANDEDCTi:(x;v*cuoOL
CENTRALE LANDBOUWCATALOGUS
0000 0174 6383
MoqM
Promotor :dr. W. Pilnik, hoogleraar inde levensmiddelenleer
Co-promotor: dr. ir.A.G.J. Voragen, wetenschappelijkhoofdmedewerker
[^ ^ ;
G. BELDMAN
THECELLULASESOF TRICHODERMA
Mode of action and application inbiomass conversion
Proefschrift
ter verkrijging van degraad van
doctor inde landbouwwetenschappen
opgezagvan de rector magnificus,
dr. C. C. Oosterlee,
in het openbaar te verdedigen
op dinsdag 25november 1986
desnamiddagste vier uur in de aula
van de Landbouwuniversiteit te Wageningen
1986
DRUKKERIJ VAN DENDEREN B.V.
GRONINGEN
\^y U M l
>no\ , i | >
VIRIDE
Abstract
Beetpulpandpotato fibrewere liquefied and saccharified withacombinationof
cellulase fromTrichodermavirideandpectinase fromAspergillus niger.Cellwall
polysaccharides werehydrolysed extensively.Theapplication ofapackedcolumn
reactor,connected withahollow fibreultrafiltrationunitwas investigated for
theenzymatic hydrolysisofbeetpulp.Ahighdegreeofpolysaccharide hydrolysis
of spentgrainby theactionofcellulaseswasobtained afterpretreatmentwith
sulfuric acidorsodiumhydroxyde.Essentially onlymonomeric sugarswerepresent
intheenzymedigestoftheacidpretreated material.Sixendoglucanases,three
exoglucanasesand aB-glucosidase werepurified fromacommercial cellulase
preparation ofT.virideorigin.Theseenzymeswerecharacterized with respectto
theiractivities onseveralcellulosic substratesaswellaschemicalandphysical
properties.The B-glucosidase showednocellobiaseactivity.Randomand less
randomacting endoglucanases weredistinguished.Adsorption,kinetic andsynergisticbehaviours of theseenzymeswere investigated oncrystallinecellulose.It
wasshownthatanoptimal ratioofendoglucanase toexoglucanasewasneeded in
order toobtain amaximal degreeof synergism.Thisratiocouldberelated tothe
adsorptionbehavioursof thecellulases,suggesting that synergismbetweenendoglucanasesandexoglucanases takesplace inveryclosecooperation atthesurface
ofthecelluloseparticle.From activity experimentsonxylan specific andnonspecific glucanasescouldbedemonstrated.However,thenon-specific glucanases
prefertohydrolyse B-1,4-glucosidiclinkages over B-1,4-xylosidicbonds.
Stellingen
1. Het door Ruy e t a l . geconstateerde synergisme tussen
niet-geadsorbeerd endo-B-glucanase en door competitieve
a d s o r p t i e vrijgekomen exo-g-glucanase geeft geen
informatie over het synergisme in de geadsorbeerde f r a c t i e .
Ruy, V.V.V., Kim, C. and Mandeti, M., Biote.ch.nol. Bioeng. 1983, 26,
488-496
2. De c o n s t a t e r i n g van Sprey en Lambert dat het met behulp
van i s o e l e c t r i s c h focusing geisoleerde c e l l u l a s e - x y l a n a s e g-glucosidase bevattende complex van e i w i t t e n homogeen
b l i j k t t e zijn b i j herfocusing i s het intrappen van een
open deur.
SpJizy, B. and LambeAt, C, FEMS UicAobiol.
Lett.
1983, 18, 277-222
3. De op b a s i s van a c t i v i t e i t opgegeven opbrengsten van
gezuiverde c e l l u l a s e s zijn n i e t r e l e v a n t .
Kitt&teineA-EbeAle,
17, 131-142
R., Hofielminn, M. and SchxeleA,
P., food Chem. 1985,
4. De door F u j i i en Shimizu gebruikte endo- en exo-0glucanases zijn v o l s t r e k t onvoldoende gezuiverd voor het
toetsen van het door deze onderzoekers gebruikte model
dat enzymatische hydrolyse van oplosbare c e l l u l o s e derivaten b e s c h r i j f t .
Vuijlt, M. and Shimizu,
M., Biotechnol.
Bixteng. 1986, 28, 878-882
5. Mendienttebeseffendattijdensdeproduktievanbietsuiker,bijonvoldoendecontrole,depHentemperatuur
indediffusietorenvrijweloptimaalkunnenwordenvoor
g-eliminatievepectine-afbraak.
6.DeineenJapanspatentomschreventoepassing van
cyclodextrinevoordebereiding vanpoedervormigewhiskey
moetwordengezienalseenvormvandrankmisbruik.
Jpn. Kokai Tokkyo Koko, J? 57.149.238
7.Erzullenv66rdeingebruikneming vanbepaaldenieuwe
biotechnologische processen internationale maatregelen
moetenwordengenomenomtevoorkomendatderde-wereld
landen,zoalsnade invoeringvanhetisomerase-proces
isgebeurd,weerhetkindvanderekening zullenworden.
8.Hetverdientaanbeveling hetmodelvanWood dateen
verklaring geeftvoor synergismetijdens enzymatische
hydrolysevanonoplosbaarkristallijncellulosete
toetsenopdeafbraakvannatief zetmeel.
Wood,T.M. and McCtae, S.T.,
New Vzlhl,
Vfioc. BZoconveM,. Symp. I.I.T.,
19??, pp 111-141
9.Geziendemilieuvervuilendeaktiviteiten enhetgebruik
vantermenzoalsbio-industrie lijktdeagrarische sector
zich steedsverder teontwikkelen toteen"volwaardige"
industrietak metalledaarbijbehorende negatieve
aspecten.
10.NadekernrampinTsjernobyl iseenstemeerduidelijk
gewordenhoezeer informatieverleningendemocratie
gekoppeldzijn.
11. Indiendevermindering vanhetbegrotingstekort steeds
meerwordtgezocht inbezuinigingenbijhetonderwijs,
kanhetkabinetbinnenkortookhetcellentekortopheffen
door simpelwegtralies teplaatsenvoorderamenvande
danverlaten studentenflats.
Stellingenbehorendebijhetproefschrift "Thecellulasesof
Tn.-ichodz.Kma vinidz. Modeofactionandapplication inbiomass
conversion"doorGerritBeldman.Wageningen 25november1986.
aanmijn ouders,
aan Hannie
The work presented in thisthesiswassupported byagrant from
the European Community (contract number ESE-R-038-NL)
Dankbetuiging
Allen,dieopdeeenofanderewijzehebbenbijgedragentot
detotstandkoraingvanditproefschrift,wilikhartelijkdanken.
Naastalledienstverlenende afdelingenvanhetBiotechnion,ben
ikaanenkelemenseninhetbijzonderdankverschuldigd:
ProfessorDr.WalterPilnik,diemijindegelegenheidheeft
gesteldhetonderzoek teverrichtenendoor zijnzeernuttige
opmerkingenensuggesties,maarookvaakamusanteanekdotes,een
bronvaninspiratieisgeweestenzalblijven.
MarjoSearle-vanLeeuwen,diedoorvoortreffelijkeiwitchromatografiewerkeenzeerwezenlijkebijdrageheeftgeleverd.
Hetwasbijzonderprettigommet jousamentewerken.
FonsVoragen,mijnco-proraotor,waar ikaltijdmetmijn
problementerechtkon,dienauwbetrokkenwasbijhetonderzoek
enmetwieiksamenmetveelplezieropstapgingomdevoortgangsrapportagesvanhetE.G.-project teverzorgen.
FransRombouts,metwieheteengenoegenwasomdediverse
resultatentebediscussierenendietevensnuttigeadviezen
heeftgegevenbetreffendedeteksteninhoudvanhetmanuscript.
JanHennekam,dieaandehoofdstukken2en3eenbijzondere
bijdrageheeftgeleverdenmetwieikopeenkameraadschappelijkemanierhebkunnensamenwerken.
HenkScholsenJanCozijnsen,dieervoorhebbengezorgddat
respectievelijkdeHPLC-enGC-apparatuur bedrijfszeker functioneerden.
HelgaBelling,dieveelvandevoortgangsrapporten heeft
getypt.
MichelGeuskens,CorBakker,JanOldeheuvelenAnsJacobs,
dieinhetkadervanhunhoofdvakonderzoek enkele resultaten
hebbengeboekt,dieindiversevoortgangsrapporten verwerktkondenworden.
Mijnmoeder,diemijnaardeH.B.S.stuurdeendie -hoewel
dattoendertijdnietalsvanzelfsprekend gold-ergeenenkel
bezwaar tegenmaaktedatikgingstuderen.
Maar zonder jou,Hannie,washetallemaalnietgegaan.
Contents
Chapter 1 Introduction
Chapter 2 Applicationofcellulase andpectinasefrom
fungaloriginfortheliquefactionand
saccharificationofbiomass
Chapter 3 Enzymatichydrolysisofbeerbrewers'spent
grainandtheinfluenceofpretreatments
21
Chapter 4 ThecellulaseofTrichodermaviride
Purification,characterization andcomparison
ofalldetectable endoglucanases,exoglucanases
andp-glucosidases
33
Chapter 5 Adsorptionandkineticbehaviours ofpurified
endoglucansesandexoglucanases fromTrichoderma
viride
57
Chapter 6 Synergism incellulosehydrolysisbyendoglucanases andexoglucanasespurified from
Trichodermaviride
75
Chapter 7 Specific andnon-specific glucanasesfrom
Trichodermaviride
87
Summary
104
Samenvatting
106
1 Introduction
Althoughonlyapproximatively0.1-0.3%ofthesolarenergy,
reachingtheearth,isfixedthroughphotosynthesisbygreen
plants (1),theannualproductionoforganicplantsubstance
fromCC>2-fixationisenormousandestimated tobeabout 1()H—
-lO-^tons (2,3).Utilizationofonlyafractionofthese
materialscould,atleastpartially,helptosolvetheproblems
offoodandenergyshortage.
About 40%ofplantbiomassconsistsofcellulose,thelinear
polymerofanhydro-D-glucoseunits,linkedby P(l,4)-glucosidic
bonds (Fig.1).Thedegreeofpolymerization ofnativecellulose
1,'IUCOSI'
|CH20H
ccllobiose
FIGURE 1. Conformation of the cellulose molecule.
isgenerally intheorderof 10,000,whichimpliesamolecular
massof 1.5xl06 andachainlengthofabout 5urn(4).The
hydroxylgroups areinequatorial andthehydrogenatomsinthe
axialpositions.Everyotherchainunitisrotated 180°around
themainaxis,whichresultsinanunstrained linearconfiguration.Bundlesofcellulosemolecules,whichareparallelordered
innativecellulose (5),formelementary fibrilswitha
partiallycrystalline structurecausedbyintra-andintermolecularhydrogenbonds.Aggregates ofthesefibrils (microfibrils)arethebasisoftherigid structureofplantcell
walls,where theyareassociatedwithavarietyofother
polymers,suchaspectin,hemicellulose,ligninandstructural
proteins (6).
Theplantcellwallconsistsofamiddle lamella,primary
wallandsecondarywall.Theamountofpectic substances,which
arepolygalacturonides withnon-uronide carbohydrates covalently
bound toanunbranchedchainof (1,4)a-D-galacturonicacid
units,decreases inthisorder.Thewallsofsoft,nondifferentiated tissues areoftheprimary type,richin
hemicellulose andpectinandcontaining atypeofcellulose
whichhasalowerdegreeofpolymerization andcrystallinity
thaninthesecondarywall(7).
Thehemicelluloses areagroupofpolysaccharides,which
containpentoses (D-xylose,L-arabinose)and/orhexoses (Dgalactose,D-mannose,D-glucose)assugarresidues.Theydo
occurashomoglycansaswellasheteroglycans,containingdifferenttypesofsugar residues (8).Inwoodyplanttissuethe
cellulose fibrilsarecloselyassociatedwith lignin,ahighly
polydispersepolyphenolicmacromoleculeofnine-carbonphenylpropaneunits.
Theabundanceofcellulosehasbeenandstillisachallenge
formanybiotechnologist toconvertthismaterialbytheuseof
biochemical routestoglucose syrups.The literatureonthis
fieldofresearch isextensively reviewed anddealsaboutthe
quality andavailibilityofbiomass (3,9,10),pretreatments
andeconomics (1,2,9-12),processconsiderations (1,9)and
especiallyproductionandmodeofactionofcellulases (1,3,9,
12-21).
Cellulolytic enzymes areproducedbyalargenumberof
micro-organisms,including fungi,actinomycetes,gliding
bacteria (myxobacteria)andtruebacteria (21).Bacterial
cellulasesareoftencell-wallbound.Highlyactivecellulases
arefound intheculturemediaoffungi.Thereforetheseenzymes
arestudied extensively,especiallywithregard totheir ability
todegradehighlyorderedcrystallinecellulose.Thoroughly
investigated arecellulases fromTrichodermaspp. (T_.viride,T.
reeseiandT_.koningii),Fusariumsolani,Penicillium
funiculosum,SporotrichumpulverulentumandTalaramyces
emersonii (forreferences:seereviews 1-3,9, 12-21).
Thedegradationofcelluloserequirestheparticipationof
manyenzymes.The firstconcepttoexplaintheenzymatic
degradation ofcrystalline cellulosewasproposedbyReeseet
al. (22).Anon-hydrolyticC^enzymewasthoughttocause
disaggregation ofcellulosechainsincrystallinecellulose,
whichresults inamodified amorphouscelullose.Inturn,this
cellulosecanbeattackedbyhydrolyticC x enzymes,producing
solubleproducts.
C
1
Crystallinecellulose—»amorphouscellulose
C
x
•solubleproducts
InlaterstudiesitwasfoundthatahighlypurifiedC^
componentisactuallyahydrolyticenzyme,whichreleasesa
cellobiosemolecule fromthenon-reducing chain-end (23, 24).
Thisenzymeisclassifiedasacellobiohydrolase (1,4-p-D-glucan
cellobiohydrolaseEC3.2.1.91),whichactsco-operativelywith
twoothertypesofenzymesinhydrolysisofnativecellulose.
Theseare:endo-1,4-p-glucanase (endo-1,4-p-D-glucanglucanohydrolase,EC3.2.1.4)andp-glucosidaseorcellobiase
((3-D-glucosideglucohydrolase,EC3.2.1.21).Insomecellulase
systemsaglucohydrolase (1,4-p-D-glucanglucohydrolase,EC
3.2.1.74)ispresent.Amodelforthedegradationofnative
cellulosebytheactionoftheseenzymesisproposedbyKlyosov
exoglucosidase
cellulose
endoglueanase
»-G„
cellobiohydrolase
endoglueanase + cellobiohydrolase
cellobiase
»-Ga
»G
T
exoglucosidase
FIGURE 2 . Model according to Klyosov for the breakdown of cellulose. G, glucose.
Cellobiosecan
beproduced fromcellulosebyasequential
attackofendoglueanaseandcellobiohydrolaseviacellooligosaccharidesorbyacombined actionofthesetwoenzymes
whichisthoughttotakeplaceinverycloseco-operationatthe
surfaceofcrystalline areasofthecellulose fibrils.This
latterrouteissuggestedtoberesponsibleforthesynergism,
oftenobservedinmixturesofendoglucanasesandcellobiohydrolases (21).Cellobiasehydrolysescellobiose,whichisastrong
inhibitorofbothendoglueanaseandcellobiohydrolase,totwo
glucoseunits.Direct formationofglucosecan
beestablishedby
exoglucosidase activity.
Inthebeginningof1980theCommissionoftheEuropean
Communities startedtopromotearesearchprogramme concerning
theproductionandthermochemicalandbiologicalconversionof
biomass.Themainpartoftheresearchdescribedinthisthesis
wascarriedoutwithintheframeworkofthisprogramme.Theaim
ofthestudywastoapplypolysaccharidedegradingenzymesin
theconversionofagricultural residuestofermentable sugar
solutions.Inthiscasebioconversionofby-productsinthe
beet-sugar andpotato-starch industries (Chapter 2)aswellas
ofbeerbrewers'spentgrain (Chapter 3)were investigated.
Sincecellulose isthemainconstituentofmostplant
biomass,specialattentionwaspaidtothemodeofactionofthe
cellulasecomplexpresent inacommercialpreparationderived
fromTrichodermaviride.Althoughcellulases fromthisfungus
havebeenstudied extensively,there isstillsomedebateon
themodeofactionoftheseenzymesoncrystalline cellulose (9,
21). Inordertoinvestigate theroleofthedifferent
cellulasespresent inthecommercialpreparation allendoglucanase,exoglucanase and(3-glucosidaseactivitieswere
isolated (Chapter 4).Adsorptionandkineticbehavioursofthese
purified enzymesaswellasthesynergism,observedduring
combined actionofendoglucanase andexoglucanase (cellobiohydrolase)oncrystallinecellulosewerestudied (Chapter 5and
6). Finallythenon-specificmodeofactionofsomethepurified
enzymesonarabino-xylanwasinvestigated (Chapter7 ) .
REFERENCES
1.Tangu,S.K.,ProcessBiochem.1982,Mary/June,36-45
2.Ryu,D.D.Y. andMandels,M.,EnzymeMicrob.Technol.1980, 2,
91-102
3.Bisaria,V.S.andGhose,T.K.,EnzymeMicrob.Technol.1981,
2, 90-104
4.Fan,L.T.,Lee,Y.H.andBeardmore,D.H.,Adv.Biochem.Eng.
1980,14, 101-117
5.Sarko,A.,Tappi 1978,16,59-61
6.Keegstra,K.,Talmadge,K.W.,Brauwer,W.D.andAlbersheim,
P.,PlantPhysiol.1973,51,188-196
7.McNeil,M.,Darvill,A.G.,Fry,S.C.andAlbersheim, P.,
Ann.Rev.Biochem. 1984,53,625-663
8.Aspinall,C O . , inThePolysaccharides,PergamonPress,New
York,1970
9.Ladisch,M.R.,Lin,K.W.,Voloch,M.andTsao,G.T.,Enzyme
Microb.Technol.1983,5,82-102
10.Keenan,J.D.,ProcessBiochem. 1979,May, 9-15
11.Ladisch,M.R.,ProcessBiochem. 1979,Jan.,21-25
12.Mandels,M.,ASMNews 1981,47,174-178
13.Coughlan,M.P.andFolan,M.A.,Int.J.Biochem. 1979,10,
103-108
14.Ghose,T.K. andGhosh,P.,ProcessBiochem. 1979,Nov.,
20-25
15.Mandels,M.andAndreotti,R.E.,ProcessBiochem.,1978,
May, 6-13
16.Gosoyr,J. andEriksen,J. inMicrobialEnzymesandBioconversions (Rose,A.H.ed.), AcademicPress,NewYork,1980,
p.p. 283-332
17.Shewale,J.G., Int.J.Biochem.,1982,14,435-443
18.Woodward,J.andWiseman,A.,EnzymeMicrob.Technol.1982,
4,73-79
19.Montenecourt,B.S.,TrendsinBiotechnol.,1983,1,156-161
20.Lutzen,N.W.,Nielsen,M.H.,Oxenboell,K.M.,Schulein,M.
andStentebjerg-Olesen,B.,Phil.Trans.R.Soc.Lond.,
1983,300,283-291
21.Wood,T.M.,Biochem. Soc.Trans.,1985,13,407-410
22.Reese,E.T.,Siu,T.G. andLevinson,H.S.,J.Bacteriol.
1950,59,485-497
23.Halliwell,G.andGriffin,S.,Biochem.J., 1973,135,587594
24.Wood,T.M.andMcCrae,S.I.,Carbohydr.Res.,1977,57,
117-133
25.Klyosov,A.A.,Sinitsyn,A.P.andRabinowitch,M.L.in
EnzymeEngineering (Weetall,H.H.andRoyer,G.P.ed.)vol.
5,PlenumPress,NewYork,1980,p.p.153-165
2 Applicationofcellulaseandpectinasefromfungal origin
fortheliquefaction andsaccharification of biomass
Published in Enzyme Microb. Technol. 1984,6, 503-507
APPLICATIONOFCELLULASEANDPECTINASEFROMFUNGALORIGINFOR
THELIQUEFACTION ANDSACCHARIFICATION OFBIOMASS
G.Beldman,F.M. Rombouts,A.G.J.VoragenandW.Pilnik
DepartmentofFoodScience,AgriculturalUniversity,Wageningen
Commercialcellulase [seel,4-(l,3;l,4)-|3-D-glucan4-glucanohydrolase,EC3.2.1.4]fromTrichodermavirideandpectinase
[poly (1,4-a-D-galacturonide)glycanohydrolase,EC3.2.1.15]
fromAspergillusnigerhavebeenapplied toproduce fermentation
syrupsfromsugar-beetpulpandpotatofibre.Cellulosic,hemicellulosic andpecticpolysaccharides ofthesesubstrateswere
hydrolysed extensively.Recoveryofenzymeshasbeeninvestigated inapacked-columnreactor,connectedwithahollow-fibre
ultrafiltrationunit.Enzymesappeared tobestableinthistype
ofreactor,althoughpartoftheenzymeactivitywaslost,
especiallybyadsorptionontothesubstrateresidue.
INTRODUCTION
Currentagriculturalsourcesoffermentablecarbohydratesare
sugar-beet,sugar-cane,corn,wheat,rice,potatoesand
Jerusalem artichokes.Inonewayoranother theserawmaterials
mustbeprocessed toobtainfermentable sugars:theintracellularcarbohydrate isextracted fromtheplantcellsand,inthe
caseofstarchandinulin,thepolysaccharides arehydrolysedto
glucoseand fructose,respectively.
Theplantcellsaresurroundedbyastructuraltissue,the
cellwall,which isbuiltupofathinprimarywallanda
thicker secondarywallwiththemiddle lamella locatedbetween
adjacentcells (1).Cellulose,hemicellulose,pectinand lignin
arethemainconstituentsoftheplantcellwall.
Largequantitiesofplantcell-wallmaterialbecomeavailable
asby-products inthebeet-sugar andpotato-starchindustries.
Forinstance,intheNetherlands 4.7 x10 5tons (drymatter)
ofbeetpulpand 0.6 x10 5tons (drymatter)ofpotatofibre
areproduced.
Thepurposeofthepresentworkistoinvestigatethe
enzymatichydrolysisofthesematerials.Suchaprocessis
8
attractive fortwomainreasons:firsttheproductionof
fermentable sugarsfromthepolysaccharides,andsecondthe
disruptionofthecell-wallmatrix,resulting inliquefactionof
thematerialsandthereleaseofintracellularcarbohydrates.
A largespectrumofpolysaccharide-degrading enzymes (cellulases,pectinases,hemicellulases)isneeded forthebioconversionofplantmaterial.Enzymeactionisrestricted bythe
accessibilityofthesubstrate.Thecellulases especiallyare
hindered intheiractiononcellulosebythepresenceofthe
otherpolysaccharides (and lignin).Combinationsofpolysaccharidedegradingenzymesareknowntoactsynergisticallyinthe
degradationofthecell-wallmatrix(2).
Thispaperdealswiththeapplicationofcommercialcellulolyticandpectolytic enzymesof fungalorigintohydrolysisof
beetpulpandpotato fibre.Ahydrolysisprocessofbeetpulp
whichcanbeoperated onacontinuousbasis,withrecoveryof
enzymes,wasinvestigated inacolumnreactorconnected toa
hollow-fibreultrafiltrationunit.
MATERIALSANDMETHODS
Biomass
Pressed andunpressed beetpulp (21.1and 9.8%drymatter
content,respectively)andpotato fibre (15.9%drymatter
content)were fromDutchfactories.
Enzymepreparations
Commercialenzymepreparations,MaxazymeCL [cellulase,1,4(1,3;l,4)-p-D-glucan4-glucanohydrolase,EC3.2.1.4,from
Trichodermaviride],Rapidase C80 [pectinase,poly(l,4-a
-D-galacturonide)glycanohydrolase,EC 3.2.1.15,from
Aspergillusniger]andHazymeRapidase [a-amylase,1,4-a-Dglucanglucanohydrolase,EC 3.2.1.1,fromAspergillus niger]
werekindlyprovidedbyGist-Brocades,Delft,theNetherlands.
Analysismethods
Reducing sugarsweremeasured according toSomogyi (3).The
galacturonidecontentwasestimated usingthemethodofAhmed
andLabavitch (4).Theneutralsugarcompositionwasdetermined
bygaschromatography afterSaemanhydrolysis (5)andderivatizationtoalditolacetates (6). Solubleneutralsugarswere
analysedbyHPLCusingaLichrosorb 10NH2column.Cellulose
determinationwasdoneaccording tothemethodofUpdegraff
(7). TheKjeldahlmethodwasusedforproteinanalyses.Starch
contentwasmeasured usingtheBoehringer enzymekit
(Boehringer-Mannheim,FRG).
Liquefaction
Enzymicliquefactionofbeetpulp (unpressed)andpotatofibre
wasfollowdbymeasurementofconsistencychanges inathermostaticallycontrolled rotatingreactionvesselofaBrabender
Viskograph (Brabender,OHB,Duisburg,FRG),at40°Cand100
revmin -1 , for 4h.Thepulpwasground inameat-grinder.To
fallwithintheresistance rangecapableofbeingmeasuredby
theinstrument,70gwaterhadtobeaddedto100gpulp.
Thepulpwas incubatedwithacombinationofcellulaseand
pectinaseatpH 5.2 and 3.8.Thecontributionofindividual
cellulaseandpectinasepreparations tochangesinconsistency
wasinvestigated atpH3.8.Thecellulaseconcentrationwas 1.08
mgm l - 1 andthepectinaseconcentration 1.35 mg ml - 1 .
Saccharification
Beetpulp (unpressed)andpotatofibreweresaccharified ina
stirredbatchreactorat40°CandpH 4.0 for 24h.Thebeet
pulpwasground andcellulase andpectinasewereaddedtoa
concentrationof 2.0 and2.5mgml"-'-,respectively.
Saccharification ofpotato fibrewascarriedoutaftera
gelatinizationpretreatmentofthestarch (15minat85°C).
Enzymeconcentrationswere (mgml~l): celluase,2.0;pectinase, 2.5;anda-amylase,0.5.TopreventmicrobialgrowthNaN3
wasaddedtoaconcentrationof0.01%.
Packed-column reactor experiments
Saccharificationofpressedbeetpulpandpotatofibrewas
studied inapacked-column reactor,connectedwithahollowfibreultrafiltrationunit (AmiconCH4,H1P5cartridge,5000MW
cut-off).Thissystem (Figure 1)iscapableofcontinuous
operation,withrecoveryandre-useoftheenzyme.Thecolumn
hadavolumeof2.56 litreandcontained 1850gpulpwhichwas
broughttopH4.0with 10%HC1.Anenzyme solution,whichgavea
cellulaseconcentration inthesystemof 5.68mgml"landa
pectinaseconcentrationof7.14mgml~l,waspumpedupinto
thecolumnandrecirculated.Percolationpump1wasoperatedat
1.01 litreh - 1 . Columnandenzymevesselwerekeptat40°C.
Theultrafiltrationunitwasexposed toroomtemperature.
Enzymeinactivationwasstudied inthe column/hollow-fibre
reactor inthepresenceandabsenceofbeetpulp.Sampleswere
assayed fortheirenzymeactivitiesafterremovalofthesugars
withAmiconmembranecones,typeCF25.
10
H20
sample
pump 2
column
reactor
hollow fiber
ultrafiltration unit
enzyme
vessel
• product
pumpl
Fig. 1.Experimental set-up forsaccharificationofbeetpulpin
acolumn/hollow-fibremembranereactor.
Enzymeacitivityassays
Exo-l,4-|3-D-glucanaseactivity inthecommercialenzymepreparationswasmeasuredbytheincreaseinreducingsugarsfrom
Whatmanno.1filterpaper (filterpaperactivity,FPA).Pieces
of 2x2cm (33.2mg)wereincubatedwithdifferentamountsof
enzymein6ml 0.1 Msodiumacetatebuffer,pH4.0 at40°C.
Activityoftheamountofenzymewhichgave10%hydrolysisafter
2hincubationwasdeterminedusingtheNelson-Somogyimethod.
Endo-l,4-p-glucanase activitywasmeasuredviscometrically
withcarboxymethyl cellulose (typeAkucellAF0305,Akzo,
Arnhem,theNetherlands;carboxymethylcellulase,CMCaseactivity)at30°Caccording toAlminandEriksson (8),usinga0.25%
substrateconcentration.
Activityonpectinwasdetermined bythesameviscometric
method,using 0.25%pectinwithadegreeofesterificationof
38% (ObipectinLtd,Bischoszell,Switzerland;pectinase
activity),atpH4.0 and30°C.
Residualcellulase activity inthepacked-columnreactorwas
measuredonAvicel (TypeSF,Serva,Heidelberg,FRG;avicelase
activity),for 20h,at30°Cin0.05 Msodiumacetatebuffer,
pH5.0,usinga0.5%substratesuspension.After centrifugation
thesupernatantwasanalysed forreducingsugars.
Polygalacturonase,pectinlyase,pectinesterase and(3-D-glucosidaseweremeasured accordingtoVoragenetal. (2)All
activitieswereexpressed inInternationalUnits (U)pergramof
enzymepreparation,withtheexceptionofCMCaseandpectinase
activities,whichareexpressed asrateofchangeinspecific
fluidity, A* S pmin~l.
11
RESULTS
Beetpulpandpotato fibrewere analysed fortheircarbohydrate compositions togiveanindicationofthekindsof
enzymestouse,aswellastogiveabasisforassessing changes
insugarcomposition afterenzymatic hydrolysis.Thecommercial
cellulase andpectinasepreparationswere screened fortheir
differentenzymeactivities.Optimum conditionswithrespectto
pHandenzymecomposition fortheliquefaction ofbeetpulpand
potatofibreweremeasured viscometrically.Pectinaseandcellulasewereused separately,aswellasincombination.
Polysaccharide hydrolysis limitsandsugaryieldswere
determined inastirredbatchreactorandcompared tothe
resultsobtained inacolumnhollow-fibre reactor,whichwas
applied torecoverenzymes.
Analysisofbiomass
Table 1givesthechemicalcompositionofthebiomassusedin
thepresentexperiments.Themainpolysaccharides inbeetpulp
arecellulose,pectinandahemicellulosefractionconsisting
predominantly ofarabinose.Thereissomeresidualsucroseleft
inthepulp,especially intheunpressedsample.
Potatofibrehasasimilarcomposition.L-Arabinose andDgalactosearethemainhemicellulosicsugars.Thefibrecontains
aconsiderable amountofstarch.
Theamountof ligninwasnotdetermined,butisknowntobe
low (2-4%)insimilarmaterials(9).
Table 1. Chemical composition of beet pulp and potato fibre.
(%
Cellulose
Pectin
Hemicellulose
Araban
Xylan
Mannan
Galactan
Sucrose
Starch
Protein
Ash
12
Beet pulp
of dry ma tter)
Unpressed
Pressed
Potato fibre
(% of dry
matter)
18.7
11.7
18.7
15.0
26.0
15.3
14.4
13.2
0.8
0.0
2.9
1.2
0.4
2.8
5.8
0.0
6.0
3.9
1.8
0.3
7.9
0.0
17.3
0.0
5.1
9.0
11.8
17.1
14.9
5.-2
Table 2.Proteinandenzyme levelsincommercialcellulasefrom
Trichodermavirideandpectinase fromAspergillusniger.
Enzymepreparation
(Ug"1)
FPase
CMCasea
P-D-Glucosidase
Pectinlyase
Pectinesterase
Polygalacturonase
Proteinb
Cellulase
Pectinase
39.3
750.0
32.4
0.0
0.0
0.0
337.0
0.0
344.0
120.0
50.4
767.0
421.0
180.0
a
Arbitraryunits (min"1)
"ragg"lenzymepreparation
Activitiesofcommercialenzymepreparations
Exo-l,4-|3-D-glucanase (FPase)activity (FPA)wasonlyfound
inthecellulasepreparation (Table 2 ) .Nopectinaseconcentrationwashighenoughtoobtain10%hydrolysisoffilterpaper
within 2h.Endo-l,4-p-D-glucanase (CMCase)andfi-D-glucosidase
activitieswere found inbothpreparations,withthepectinase
preparationhavingamuchhigher fi-D-glucosidase level.No
pectolytic activitywasfound inthecellulasepreparation.
Liquefaction
Treatmentofbeetpulpwithacombinationofcellulaseand
pectinase atpH 5.2 showedanintermittentdelayinthe
viscositydecrease (Figure 2 ) .After 2htheprocessof
liquefactionaccelerated,however,andasimultaneouspHshift
frompH5.2 to3.8wasobserved (datanot shown). Thedecrease
inpHcanbeexplainedbyassuming ade-esterification ofpectin
bypectinesterase action.ThepHeffectwasconfirmedbythe
fastinitialdecrease inviscosityofpulp,whichwasbroughtto
pH 3.8priortoenzymeaddition.
Incubationofpulpwithcellulase andpectinase separately
showedthatthepectinasepreparationcausesadramatic
viscosity loss (Figure 2)andthatoptimumactionoccursinan
acidicpH (Figure3).
Essentially thesameresultswereobtained from liquefaction
experimentswithpotatofibre:thecombinationofcellulaseand
pectinase inducedthefastestdecrease inviscosity,butthe
pectinasepreparationwaslargely responsible fortheeffect
(resultsnot shown).
13
Incubation time (h)
Fig.2.Decrease ofviscosityof sugar-beet pulp,treatedwith
pectinase (1.35mg ml"-'-),cellulase (1.08ragml"-'-)anda
combinationof theseenzymes,atdifferentpHvalues.• ,
Cellulase +pectinase,pH 5.2;• , cellulase,pH 3.8;A ,
pectinasepH 3.8;X, cellulase +pectinase,pH3.8.
Fig.3.OptimumpH for liquefactionofbeetpulp,measuredwith
viscograph andoptimum pHforpolygalacturonase.X , Activityon
beetpulp;•,activityonpolygalacturonic acid.
Forthesereasonsallother liquefactionandsaccharification
experimentswerecarried outatpH4.0.
Saccharification
Figure 4showstheincrease inreducing sugarsfromunpressed
14
1§
25"
25
Hydrolysis time :h)
Fig. 4.Reducing sugar released frombeetpulpandpotatofibre
during saccharificationwithcellulase (2.0mgml" 1 )and
pectinase (2.5mg m l " 1 ) .X , Beetpulp;O,potatofibre.
beetpulpandpotato fibreasafunctionofhydrolysistime.
Almost 80%ofthetotalsolidsofbeetpulpwerebroughtinto
solutionasreducing sugars.Potato fibregave 60%conversion
oftotalsolidstoreducingsugars.
Polysaccharidehydrolysis limitswerecalculated fromthe
carbohydrate analysisoftheresidues.Morethan90%ofbeet
pulppolysaccharideswerehydrolysed (Table 3).Polysaccharide
hydrolysis limitsofpotatofibrewere lowerforcelluloseand
pectin.However,starchandthehemicellulosepolymersofLarabinoseandD-galactosewerealsoextensively hydrolysed.
Table 3.Enzymehydrolysis limitsofmainpolysaccharidesof
beetpulpandpotatofibre.
(%
Substrate
hydrolysis;
Polysaccharide
Beet pulp
Potatofibre
Cellulose
Pectin
Hemicellulose
Araban
Galactan
Starch
91.0
91.0
75.0
58.0
93.0
90.0
0.0
93.0
96.0
99.0
15
Table 4.Sugarcompositionofenzymedigestafterincubationof
beetpulpandpotatofibrewithcellulase andpectinase.
Substrate
(g/gdryweightof substrate)
Sugar
Beetpulp
Potatofibre
D-Glucose
D-Fructose
L-Arabinose
D-Galactose
Uronicacid
0.38
0.16
0.17
0.02
0.15
0.45
0.00
0.04
0.06
0.11
HPLC-analysisshowed thatalmostallreleased sugarswerein
monomericform;nocellobiosecouldbedetected.Sucrosefrom
beetpulpwasinverted toD-glucoseandD-fructose.Thefinal
producthadasugarcompositionasgiveninTable4.D-Glucose,
D-fructose,L-arabinose andD-galactosearethemainsugarmonomers fromneutralpolysaccharides.Uronic acid isthehydrolysis
productfrompectinmolecules.
Packed-column reactorexperiments.
Thecumulative sugarproductionfrompressedbeetpulpduring
30
A0
50
Hydrolysis time ( h )
Fig. 5.Cumulative sugarproduction frompressedbeetpulp
duringenzymatic saccharificationinthecolumn/hollow-fibre
membrane reactor,A, D-Glucose; V,uronic acid;X, L-arabinose;
°,D-fructose.
16
enzymictreatment inacolumn/hollow-fibremembranereactoris
showninFigure 5.Hydrolysiswasfollowed for48h.Sugarswere
measured attheultrafiltrationoutlet.Hydrolysis limitsfor
celluloseandpectin inthecolumnreactorwerecalculated from
thepolysaccharide contentoftheresidueandfoundtobe
similar tothoseinthebatchreactor:cellulosewas 89%hydrolysedandpectin95%.
Enzymeinactivation inthistypeofreactorwasstudiedin
thepresenceandabsenceofbeetpulp (Table5).Residualenzyme
acitvitymeasured intheabsenceofbeetpulpgivesanindicationoftheamountofenzymewhich isinactivated by temperature
andshear.Inthepresenceofbeetpulptheadditionalamountof
enzyme,lostbyadsorpiontothesubstrate,isalsomeasured.
Cellulaseactivity (avicelase,CMCase)wasstabletoshearand
heat.Somewhatmorepectinaseactivitywaslostunderthese
circumstances.Theadditional lossesofavicelase andpectinase
activitiesinthepresenceofsubstratewere 31and24%,
respectivelywhile p-D-glucosidaseactivity seemed tobe
stabilized inthepresenceofsubstrate.
Treatmentofpotato fibreinthecolumnreactorgavepoor
results.Thesoftstructureofthefibrecausedbadflow
characteristics ofthecolumnwhentheenzymesolutionwas
pumpedthrough.
Table 5.Residualenzyme activity incolumn/hollow-fibrereactor
after 48hofoperationwithorwithoutbeetpulp.
Residual activity
(% ofor iginal activity)
Enzyme
Absenceof
substrate
Presenceof
substrate
Avicelase
CMCase
p-D-Glucos idase
Pectinase
82.0
80.0
84.6
66.7
50.8
72.0
98.0
42.9
DISCUSSION
Theresultspresented inthispaper indicatethatcommercial
polysaccharide-degrading enzymescanbeusedtoconvertsome
solidplantbiomasstofermentable sugar solutions.Acombinationofpectolytic andcellulolyticenzymeswasabletoproduce
monomeric sugarsolutionsfrombeetpulpandpotatofibre.
Cell-wallpolysaccharides frombeetpulpwereextensively
hydrolysed:morethan90%conversionwasobtained.Celluloseand
17
especiallypectin frompotato fibrewere somewhatmore resistant
toenzymatichydrolysis.Thebasisforthisresistanceofpotato
fibreisnotclear.Ligninmayplayaslightrole,thoughits
influencemustbelimitedbecauseitisaminorcomponent.
Cellobiose isknowntobeastrong inhibitor ofcellulase.Since
Trichoderma cellulasepreparations aremostlydeficientin
cellobiase activity,additionofAspergilluscellobiase reduces
cellobiose levelsandincreasesglucosecontent inthedigest
(10).Thisisconfirmedbytheexperimentsdescribed inthis
paper:bytheuseofacombinationofTrichodermacellulaseand
Aspergilluspectinasewithahighp-D-glucosidaselevel,no
significant amountsofcellobioseaccumulatedduringhydrolysis
ofbeetpulpandpotatofibre.Therefore,thelowerextentof
cellulosehydrolysis inpotatofibrewasapparentlynotcaused
byproductinhibition.Probablythecellulosefibrilswerestill
shieldedbythepectinmoleculeswhichwerenotdegradedbythe
pecticenzymes.The lowdegreeofhydrolysisofthispectinmay
becausedbyitshighdegreeofacetylation (unpublished
results).
There isasynergistic actionofcellulases andpectinasesin
plantbiomassconversion.Consistencymeasurementsofbeetpulp
andpotatofibre showed thatthefirststepoftheprocess,the
disintegrationoftheplantcellwalls,resultsinliquefaction
ofthematerial.Thisprocess isoptimalatpH3.5-4.0andis
mainlycausedbythepolygalacturonase fromthepectolytic
enzymewhichhasitsoptimum inthesameregion,whilepectin
lyasehasitsoptimum atpH6 (11).Inasecond step,theextensivehydrolysisofthecellwallpolysaccharides,acellulase
preparationwithhighactivityoncrystalline cellulose (FPA)
wasneeded.
Theuseofbeetpulpandpotatofibre,substrateswithlow
lignincontents,gavehighyields incellulosehydrolysis(7590%). Itisknownthatinmaterialwithahighlignincontent
much lowercelluloseconversionsareobtained.Expensivetreatments suchasballmilling (12),attritormilling (13),compressionmilling (14),acidandalkalinepretreatment(15),
steamexplosion (16)ortreatmentwithorganic solvents (17)
havetobeusedtoraisetheyield.
Resultsofthehydrolysisofbeetpulpinapacked-column/
hollow-fibre reactor showed thepossibilities foracontinuous
process.Theenzymesarekeptinthereactor,theproduced
sugars leavethereactor throughtheultrafiltrationmembrane
andsubstrate isfedcontinuouslye.g.byacascadeofreactors.
18
Simpleoperationofthistypeofreactor isposiblebecauseno
stirringdevice isneededandahighsolid/liquid ratiocanbe
obtained.Recoveryofenzymesbyultrafiltration appeared tobe
attractivebecause theenzymeswereratherresistantto
inactivationunder thesecircumstances.However,partofthe
enzymeactivity,especially avicelaseactivity,waslostby
adsorptionontheresidue.Ourresults (Chapter 4)andthoseof
othersindicatethatadsorbed cellulasecanberecoveredbyapH
shift (18)tothealkaline region (pH10)orbytheuseof
surfactant(19).
REFERENCES
1.Keegstra,K.,Talmadge,K.W.,Bauer,w.D.andAlbersheim,
P.,PlantPhysiol.1973,51,188-196
2.Voragen,A.G.J.,Heutink,R.andPilnik,W.,J.Apll.
Biochem.1980, 2, 452-468
3.Somogyi,M.,J.Biol.Chem.1952,195,19-23
4.Ahmed,A.E.R.andLabavitch,J.M.,J.FoodBiochem.1977,
1,361-365
5.Saeman,J.F.,Moore,W.E.andMillet,M.A.,MethodsCarbohydr.Chem. 1963,3,54-69
6.Jones,T.M. andAlbersheim,P.,PlantPhysiol.1972,49,
926-936
7.Updegraff,D.M., Anal.Biochem. 1969,32,420-424
8.Almin,K.E.andEriksson,K.E.Biochim.Biophys.Acta1967,
139,238-247
9.Voragen,F.G.J.,Timmers,J.P.J.,Linssen,J.P.H.,Schols,
H.A. andPilnik,W.,Z.Lebensm.Unters.Forsch.1983,177,
251-256
10.Ruy,D.D.Y.andMandels,M.,EnzymeMicrob.Technol.1980,
2,91-102
11.Rombouts,F.M. andPilnik,W.inMicrobialEnzymesandBioconversions (Rose,A.H.ed.), AcademicPress,NewYork,
1980,pp.227-281
12.Mandels,M.,Hontz,L.andNystrom,J., Biotechnol.Bioeng.
1974, 1&_,1471-1493
13.Ryu,S.K. andLee,J.M., Biotechnol.Bioeng,1983,25,53-65
14.Ryu,D.D.Y.,Lee,S.B.,TassinariT.andMacy,C , Biotechnol.Bioeng.1982, 22, 1047-1067
15.Detroy,R.W.,Lindenfelsen,L.A., Sommer,S.andOrton,W.L.
Biotechnol.Bioeng.1981,23,1527-1535
16.Buchholz,K.,Puis.J., Godelmann,B.andDietrichs,H.H.,
ProcessBiochem.1981,Dec./Jan.,37-43
17.Ladisch,M.R.,Ladisch,C M . andTsao,G.T.,Science1978,
201,743-745
18.Reese,E.T.,ProcessBiochem. 1982,May/June,2-6
19.Castanon,M.andWilke,C.R.,Biotechnol.Bioeng.1981,23,
1365-1372
19
3 Enzymatic hydrolysis ofbeer brewers'spent grain and
theinfluence of pretreatment
Submitted for publication in Biotechnol. Bioeng.
21
ENZYMATICHYDROLYSISOFBEERBREWERS'SPENTGRAINANDTHE
INFLUENCEOFPRETREATMENTS
G.Beldman,J.HennekamandA.G.J.Voragen
DepartmentofFoodScience,AgriculturalUniversity,Wageningen
Abatchofspentgrainwasfoundtocontain 15.1%cellulose,
2.0%starch,24.8%hemicelluloseand 2.3%uronicacidondry
matter.Foruntreated spentgrain,approximately 47%ofthese
polysaccharideswerehydrolysed byamixtureoftwocommercial
cellulases.
Hammermilling andUltraTurraxblending improved enzymatic
hydrolysisby 10%;extrusionat170°Cinasingle screw
extruderyielded 18%moresugars.
Incubationfor4hoursat90°Cwith0.5NNaOHor0.5N
H S0
2 4 9 r e a t lyimprovedenzymatic hydrolysis.About80%of
thepolysaccharideswerehydrolysed byacombinationofchemical
pretreatmentandenzymatichydrolysis.Thecharacter ofthe
producedpolysaccharide fragmentsvariedconsiderably andwas
dependentonwhether alkalioracidwasusedduringchemical
pretreatment.
INTRODUCTION
Theenzymatic saccharificationofplantmaterialhasbeen
showntobeofinterest invarious fields,suchastheproductionoffruitjuices (1,2)andtheutilizationofbiomass(3).
Acombinationofcellulase,pectinaseandhemicellulasesis
usuallyused,becauseofthechemicalcompositionofthematrix
ofplantcellwalls.
Forapples,beetpulpandpotatofibrealmostacomplete
hydrolysisofpolysaccharides isobtainedbycombining cellulase
andpectinase.Fornon-parenchymatic tissuethesituationis
somewhatdifferent:pectinisaminorcomponentandthehemicellulosecontentismuchhigher.Enzymeactionisrestrictedby
theligninbarrier andbythehighcrystallinityofcellulosein
thismaterial.Forsuchmaterialsmechanical,thermalorchemicalpretreatmentsarenecessarytoachieveefficienthydrolysis
(4,5).
22
Thispaperdescribesvariousenzymatictreatmentsand
chemicalandphysicalpretreatment,usingbrewers'spentgrain
assubstrate.Spentgrainistheresidueofmaltandgrainwhich
remains inthemash-kettle after the liguefied and saccharified
starchhasbeenremovedbyfiltration.
MATERIALSANDMETHODS
Samplesofspentgrainwereobtained fromtheHeineken
Brewery atZoeterwoude (theNetherlands).Sub-sampleswere
freeze-dried andhammermilled toparticles smaller than0.5 mm
(dried Asls), inorder todeterminetheircomposition.From
other sub-samples,AlcoholInsoluble Solids (AIS)wereprepared
by4successiveextractionswith96%ethanol,at50°Cand
Water InsolubleSolids (WIS)by4successiveextractionswith
distilledwaterat50°C .Thesesampleswerealsosubjected to
thesametreatmentandanalysisofthoseused fordriedAsls.
Analysismethods
Moistureandashweredetermined bystandardmethods.
Cellulosewasdetermined according toUpdegraff (6),uronic
acidswithm-hydroxydiphenylusing themethodofAhmed-Labavitch
(7), starchwithanenzymatic testofBoehringer, (Boehringer,
Mannheim,FRG),nitrogenaccording toKjeldahl (8).Sugar
compositionwasdeterminedbyGC-analysisofthe alditolacetate
derivatesofthesugars (9)(glasscolumn,3mx 2mm i.d., of
3%OV275onChromosorbWAWat190°C)whichwereobtainedbya
modified Saemanhydrolysis (10)for4hoursat100°Cin2.0N
H 2 S0 4 .
Enzymaticallyorchemically solubilized sugarswereassayed
aftercentrifugationof insoluble solids,usingtheNelsonSomogyimethod forreducingendgroups (11)andtheDubois
method fortotalsugars (12).Glucosewasused asstandard.
Theinsoluble solidcontent aftervarioustreatmentswas
determinedbywashing,centrifugingandfreeze-drying the
residuesandexpressed aspercentage oftheoriginalamountof
drymatter.
SolubleproductswereanalysedbyHPLC (SpectraPhysicsSP
8000,eguipedwithaWatersR401refractive indexdetector),
usingtheAminexHPX 87Pcolumn (300x7.8mm,Bio-Rad Labs,
Richmond CA,USA).Amixedbed ion-exchanger,containing equal
amountsoftheion-exchange resinsAG 50W-X4 (H+)andAG3-X4A
(OH~)(Bio-RadLabs)wasusedasprecolumn.
23
Pretreatments
Physicaltreatmentsappliedwerehammermilling (0.5mm
sieve),UltraTurraxblending (for5minutes)andextrusion ina
Battenfield singlescrewextruder at170°C,usingaspeedof
45-105rpmand a1:3 screwcompression ratio.Forchemical
treatments 2.5 gfreeze-dried spentgrainwassuspended in20ml
solutionswithvariousconcentrations ofNaOHorH2SO4
(0.01-0.50 N ) , incubated at90°Cduring 4hoursand
subsequentlyneutralised topH4.85.
Enzymatic treatments
Severalcommercialenzymepreparationsweretested fortheir
ability tohydrolyse spentgrainpolysaccharides (TableI ) .
Suspensions ofspentgrain (10%w/w,ondryweight)in0.1M
succinatebufferpH4.85wereincubatedwithenzymes for70
hoursat40°C.Toinhibitmicrobialgrowth 0.01%sodiumazide
and0.2%sodium sulphitewereaddedaspreservatives.Physical
pretreated sampleswereincubatedunder similarconditions,
using 1% (w/w)Rbhmcellulase,supplementedwith1%Rohmhemicellulase (afterhammermillingandUltraTurraxblending)or1%
BioconBiocellulase supplementedwith 1%Rohmcellulase (after
extrusion).This lattercombinationofenzymeswasalsousedfor
biodegradation ofchemicallypretreated samples,usingthesame
reactionconditions.
Table I . Commercial enzyme preparations used for enzymatic
hydrolysis of spent grain.
Enzymes
Supplier
Maxazyme CL 2000
Gist Brocades N.V., Delft, the
Netherlands
Cellulase "2230A"
Rohm GmbH, Darmstadt, W-Germany
Hemicellulase "EL129-79"
"
"
"
Biocellulase A.C.
Biocon Ltd., Cork, Ireland
Cellobiase "SP188"
Novo I n d u s t r i A/S, Bagsvaerd, Denmark
Pectinase K29
Rapidase, Seclin, France
Cellulase "9108"
"
"
RESULTS AND DISCUSSION
Composition of s p e n t g r a i n
The wet s p e n t g r a i n c o n s i s t e d of 20.4% d r y m a t t e r , 19.6% WIS
and 17.6% AIS. The c o m p o s i t i o n s of t h e d r i e d s p e n t g r a i n i s
24
Table II.Composition ofspentgrain (g/100gdrymatter), after
drying (Asls),extractionwithethanol (AIS)andextractionwith
H 2 0 (WIS).
Constituent
Asls
AIS
WIS
Ash
Protein
Uronic Acids
Cellulose
Hemicellulose
Starch
SolubleSugars
Fat+Lignin
(bydifference)
3
23
2
15
24
2
3
25
3.8
26.5
2.5
17.9
30.4
2.3
0.0
16.6
3.1
N.D
2.5
16.9
31.1
1.7
0.0
N.D
5
8
3
1
8
0
5
0
N.D. =notdetermined
Table III.Sugarcomposition of spentgrains (g/100gdry
matter), after 2.0 NH2SO4hydrolysis.Spentgrain samples
wereobtained after drying (Asls),extractionwithethanol (AIS)
andextractionwithH2O (WIS).Values areexpressed asthe
amountofanhydro-sugar.
Anhydro-Sugar
Asls
AIS
Rhamnose/Fucose
Arabinose
Xylose
Mannose
Galactose
Glucose
0.0
7.6
15.5
0.5
1.2
18.8
0.0
8.9
19.8
0.6
1.2
22.5
WIS
0.0
8.4
20.0
0.4
1.2
22.6
presented inTable II.Hemicellulose andcellulose arethemain
polysaccharides.Theamountofpectic substances,expressedas
uronicacids,waslow.Starchwasonlypresent inminimal
amounts.Table IIIshowsthesugarcompositionsofthedifferent
samplesaftercompletehydrolysiswith2NH2SO4.Ifthesum
ofstarchandcellulosecontentpresented inTable IIis
comparedwiththeamountofglucose foundbyGC-analysis (Table
III), itcanbeseenthatdifferentmethodsofanalysisgave
verysimilarresults.
Enzymatichydrolysisofuntreated spentgrain
TableIVshowsthesolubilizationandhydrolyisofpolysaccharides after incubationofspentgrain (Asls)withseveral
enzymesorcombinationsofenzymes.Differences inpolysaccharidehydrolysisbythevariousenzymepreparationsare
small.Increasing theenzymeconcentration from 1%to2%(w/w,
25
Table IV.Amount ofreducing sugars (RS)andtotalsugars(TS),
released from spentgrainbytreatmentwithvariousenzyme
preparations for 70hoursat40°CandpH4.85,aswellas
ratioofreducing sugarstototalsugarsanddegreeofpolysaccharidehydrolysis.
Enzyme
RS
TS
(mg/100 mgd.m.
RS
-TS
polysaccharide
hydrolysis
)
(%)
l%*ROhmcellulase
2%Rb'hmcellulase
1%BioconBiocellulase
1%Maxazyme
1%Rapidase cellulase
9.7
10.4
12.2
8.4
11.2
19.8
21.2
19.8
15.8
20.9
0.49
0.49
0.62
0.53
0.54
41
44
41
33
44
1%Rb'hmcellulase+
1%Rb'hmhemicellulase
1%Novocellobiase
1%Biocon Biocellulase
12.0
10.3
12.3
21.0
20.8
22.4
0.57
0.50
0.55
44
43
47
1%BioconBiocellulase+
1%Rb'hmhemicellulase
1%Rapidase pectinase
13.1
13.7
22.3
19.8
0.59
0.69
46
41
*w/wbasedondrymatter
**Total sugarsreleased,as%ofpolysaccharides presentin
spentgrain (i.e.48mg/100mgtotal solids).
ondrymatter)resulted inonlyslightlymore solubilized
sugars.Mixingthecellulasewithotherenzymepreparations,includingothercellulases (toovercomepossibleincompleteenzyme
systems)hadonlyminoreffect.Onthebasisoftheamountof
totalsugars,whichweremeasured inthesolublefraction,47%
ofthepolysaccharideswerehydrolysedbyamixtureofR6hm
cellulaseandBioconBiocellulase.
HPLCanalysisshowed that,besidesofsomeoligomers,glucose
wasthemainmonomeric sugarfoundinthedigestofthis
combinationofenzymes (Fig.1A).Re-incubationoftheresidue
ofenzymetreated spentgrain (afterremovalofthesoluble
sugars)withfreshenzymeresulted inonlyminimaladditional
polysaccharidehydrolysis.Only 5%ofthepentosansand19%of
theglucansofthisresiduewerehydrolysed.Theseresults
indicatethattheincompletehydrolysis ismainlycausedbythe
structuralfeaturesoftheunattackedcellwallfragmentsand
notbyproductinhibition.
Physicalpretreatment
Comparedwithuntreated spentgrain,bothhammermillingand
26
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27
UltraTurraxblending,whichtransform themacro-structureof
spentgraintosmallfragments,yielded about10%moretotal
sugarsafterenzymictreatment.Thesesugarswerederived
equally fromcellulose andhemicellulosehydrolysis.This
indicatesthattheoverallsusceptibility toenzymatichydrolysiswassomewhat improvedbythesetreatmentswhich,however,
didnotaffectthemicro-structural features suchas
crystallinity andspecific surfaceareaofcellulose,knownto
havemoreinfluence onthedegradibility (13,14).Themicrostructurecanbeaffectedbytheefficientbutexpensiveball
millingtreatment,preferablywithsimultaneous enzymatic
hydrolysis(15).
Extrusion resulted in18%additional solublesugars,mainly
becausecellulosehydrolysiswasincreased (resultsnot shown).
Chemicalpretreatment
AscanbeseenfomTableV,NaOH treatment resulted inthe
releaseof lesssoluble sugarsfromspentgrain (35%for 0.5 N
NaOH)thantheH 2 S0 4 treatment (66%for 0.5 NH S0 4 ). IncomparisonwiththeHSO treatment,NaOHonlyslightlyhydrolysed the
extractedpolysaccharides,resulting inalowratioofreducing
sugarstototalsugars.Solubilizationofnon-sugar components
wassubstantial inthe sampletreatedwithNaOH.Fromthe
materialtreatedwith 0.5 NNaOHonly 38%oftheoriginaltotal
solidsremained asinsoluble.However,atNaOHorH-SO.
TableV.Amountofreducing sugars (RS)tototal sugars (TS)
released from spentgrainbyNaOHandH2SO4treatment,as
wellastheratioofreducing sugarstototalsugars,amountof
insoluble residualdrymatter anddegreeof polysaccharide
hydrolysis.
Treatment
RS
TS
(mg/100 mgd
H20
RS
TS
Polysaccharide
hydr olysis
(%)
0.4
2.0
96.5
0.0
0.50 N
0.10 N
0.01 N
1.5
0.4
0.4
17.0
6.4
2.0
38.4
87.6
94.2
0.09
0.06
0.20
35
13
4
H 2 S0 4 0.50 N
0.10 N
0.01 N
14.6
4.8
0.6
31.0
13.9
2.9
59.2
79.5
89.0
0.46
0.35
0.21
66
29
6
NaOH
calculated asinTableIV
28
Insolubleresidue
.m.)
0
concentrationsbelow 0.1 Nscarcelyanysolubilizationwas
effected,neitherofsugarnorofnon-sugarcomponents.
HPLC-analysisofsugarsobtained afterH 2 S0 4 treatment showed
thatarabinosyl linkagesofspentgrainarehighly susceptible
toacidhydrolysis,becauseatlowerH2SO4concentrationmainly
arabinosewasreleased asmonomer (Fig. IB). AthigherH 2 S0 4
concentration nexttoarabinose alsoxylose and several
oligomerswereproduced (Fig.1C).Under theseconditions almost
noglucosewasreleased.Thissuggeststhatcellulosewasnot
hydrolysed bythisacidtreatment.
Inthesoluble fractionofthealkalitreatedmaterialonly
largeroligomersandnomonomerswerefound (resultsnot shown).
Enzymatichydrolysis ofchemicallypretreated samples
EnzymatichydrolysisofNaOHpretreated spentgrainresulted
inanamountofsolubilized totalsugars,whichwasaboutequal
totheamountoftotalsugarsfound inthedigestoftheHSO
pretreatedmaterial (TableVI). Comparedwiththeresults
obtainedwhennotreatmentwasapplied,bothchemicalpretreatmentsdoubledenzymatic hydrolysis ofpolysaccharides.However,
thenatureofthesolubilized sugarsdifferedconsiderably.The
amountofreducing sugarsobtained fromtheacidpretreated
spentgrainwasmuchhigherthanthevalue foundafterenzymic
conversionoftheNaOHpretreatedmaterial,indicating thatthe
hydrolysisproductsfromthefirsttreatmentwerealmost
completely inmonomericform.
TableVI.Amountofreducing sugars (RS)andtotalsugars (TS),
released fromchemicallypretreated spentgrainbyacombination
ofBiocellulase andRbhmcellulase,aswellastheratioof
reducing sugarstototal sugars,amountof insoluble residual
drymatter anddegreeofpolysaccharidehydrolysis.
Treatment
RS
TS
(mg/100 mgd
Insoluble
residue
m. )
RS
TS
Polysaccharide
hydrolysis
(%)
H 2 0+enzyme
12.4
22.0
76.6
0.56
46
NaOH0.5ON + enzyme
0.10N
0.01N
20.0
15.8
13.4
37.8
26.8
22.6
35.4
61.5
74.8
0.52
0.59
0.59
79
56
47
H2SO40.50N +enzyme
0.10N
0.01N
34.2
20.2
12.8
40.4
28.0
22.4
47.7
68.0
70.1
0.85
0.72
0.57
85
58
47
calculated asinTableIV.
29
Thiswasconfirmed byHPLCanalysisofthesolubleproducts
inbothdigests.Actuallyonlythemonomersglucose,xyloseand
arabinose andalmostnooligomerswereobtained ashydrolysis
productsoftheacidpretreated sample (Fig. ID). Essentiallyno
cellobiosewasdetected.Thisimpliesthatthecombinationof
cellulaseswhichwereusedarenotdeficient incellobiase
activityandpossibleproductinhibitionbycellobiosecouldbe
prevented. Intheenzymicproductofthe0.5 NNaOHpretreated
materialonly 51%ofthetotalpeakareawasfoundtobemonomer
and 29%aslarger oligomers,appearing inthe "void"ofthe
chromatogram (Fig.1E).Itisnotclearwhytheselatter
oligomersarenotfurtherhydrolysed bytheenzymestosmaller
products. Itmaybethattheseoligomers areheteroglycanswhich
areonlydegraded byaspecialenzyme,forinstancean
arabinofuranosidase,lacking intheenzymepreparationswhich
wereused.Presumably theseoligomersare (atleastpartially)
hydrolysedduringH2SO4pretreatmentandarethereforenot
detected intheenzymedigestofthismaterial.The relatively
small "void"peak inthechromatogram oftheproductof 0.5 N
H2SO4treatmentalone (Fig.1C)confirmsthispossibility.
REFERENCES
1.Voragen,A.G.J.,Heutink,R.andPilnik.W.,J.Appl.
Biochem.1980,2,452-468
2.Voragen.A.G.J,andPilnik.w.,FlussigesObst 1981,48,
261-264
3.Beldman,G.,Voragen,A.G.J.,Rombouts,F.M.andPilnik,W.,
EnzymeMicrob.Technol.1984,6, 503-507
4.Fan,L.T.,Lee,Y.H.andBeardmore,D.H., 2 n d Int.
Symposium onBioconversion andBiochemical Engineering,
Delhi,India,3-6 March1980
5.Gharpuray,M.M.,Lee,Y.H.andFanL.T.,Biotechnol.
Bioeng.1983,25,157-172
6.Updegraff,D.M., Anal.Biochem.,1969,32,420-424
7.Ahmed,A.E.R.andLabavitch,J.M., J.FoodBiochem. 1977, 1,
361-365
8.Roozen,J.P.andOuwehand,R.,Voedingsmiddelentechnol.
1978,22,1-3
9.Jones,T.M. andAlbersheim,P.,PlantPhysiol.1972,49,
926-936
10. Selvendran,R.R.,March,J.F.,Ring,S.G., Anal.Biochem.
1979,96,282-292
11.Nelson,N.,J. Biol.Chem.1944,153,375-380
12.Dubois,M.,Gilles,K.A.,Hamilton,J.K., Rebers,F.A. and
Smith,F.,Anal.Chem. 1956,28,350-356
30
13. Lee,S.B., Shin,H.S.,Ryu,D.D.Y.andMandels,M.,
Biotechnol.Bioeng. 1982,24,2137-2153
14. Lee,S.B., Kim,I.H., Ryu,D.D.Y.andTaguchi,H.,
Biotechnol.Bioeng. 1983,25,33-51
15.Kelsey,R.G. and Shafizadeh,F.,Biotechnol.Bioeng.1980,
22, 1025-1037
31
4 Thecellulase of Trichoderma viride
Purification, characterization andcomparison ofalldetectable endoglucanases,
exoglucanases and fi-glucosidases
Published in Eur. J. Biochem. 1985, 146,301-308
33
THECELLULASEOFTRICHODERMAVIRIDE
Purification,characterization andcomparisonofalldetectable
endoglucanases,exoglucanasesandp-glucosidases
G.Beldman,M.F.Searle-vanLeeuwen,F.M.RomboutsandA.G.J.
Voragen
DepartmentofFoodScience,AgriculturalUniversity,Wageningen
Sixendoglucanases (EndoI;II;III;IV;V;VI),threeexoglucanases (ExoI;II;III)anda(3-glucosidase (p-glucI)wereisolated fromacommercialcellulasepreparationderivedfrom
Trichodermaviride,usinggelfiltrationonBio-Gel,anionexchangeonDEAE-Bio-GelA,cationexchangeonSE-Sephadexand
affinitychromatography oncrystallinecellulose.Molecular
massesweredetermined bypolyacrylamidegelelectrophoresis.
Onegroupofendoglucanases (EndoI,EndoIIandEndoIV)with
M r of 50000,45000and23500weremorerandom intheir attack
oncarboxymethylcellulosethananothergroup (EndoIII,EndoV
andEndoVI)showingM r of 58000,57000and 53000respectively.EndoIIIwasidentified asanewtypeofendoglucanasewith
relativelyhighactivityoncrystalline cellulose andmoderate
activtityoncarboxymethylcellulose.ExoIIandExoIIIwith
M r of 60500and62000respectively showeddistinct adsorption
affinitiesonacolumnofcrystallinecellulose andcouldbe
elutedbyapHgradienttoalkalineregions.Theseenzymeswere
cellobiohydrolases asjudgedbyhigh-pressure liquidchromatographyoftheproductsobtained fromincubationwithH3PO4swollencellulose.Itwasconcluded thattheseexoglucanasesare
primarilyactiveonnewlygenerated chainends.ExoIwasessentiallyanothertypeofexoglucanasewhich inthefirst instance
wasabletosplitoffacellobiosemoleculefromachainendand
thenhydrolysethismolecule inasecond steptotwoglucose
units.p-GlucIwasanewtypeofaryl-p-D-glucosidasewhichhad
noactivityoncellobiose.TheenzymehadaM r of76000and
wasmoderately activeonCM-cellulose,crystalline celluloseand
xylanandhighly activeonp-nitrophenyl-p-D-glucoseandpnitrophenyl-p-D-xylose.
Abbreviations.HPLC,high-pressure liquid chromatography;
SDS,sodiumdodecylsulphate.
34
Enzymes.1,4-p-D-Glucanglucanohydrolase (EC3.2.1.4);1,4-pD-glucancellobiohydrolase (EC3.2.1.91);(3-D-glucosideglucohydrolaseor(3-glucosidase (EC3.2.1.21); 1,4-p-D-glucanglucohydrolase (EC3.2.1.74).
INTRODUCTION
Muchattention ispaidtotheenzymatic hydrolysisofcellulosebecause itisarenewablecarbonsourceandavailablein
largequantities.Cellulases fromfungaloriginareknowntobe
mostpowerfulincellulosehydrolysis.Cellulase preparations
arederived,amongother,fromTrichoderma reesei (1, 2 ) ,
Trichodermaviride (3),Trichodermakoningii (4)andSporotrichumpulverulentum (5).Thesemicroorganismsproduceamulticomponentenzymesystem,including the1,4-fJ-D-glucanglucanohydrolase (endoglucanase;EC 3.2.1.4),1,4-p-D-glucancellobiohydrolase (exoglucanase;EC3.2.1.91)andp-D-glucosideglucohydrolase p-glucosidase;EC 3.2.21).Acombinationofthese
threetypesofenzymes isnecessary forthecompletehydrolysis
ofcrystalline cellulose.Endoglucanase andexoglucanaseare
knowntoactsynergistically incellulosehydrolysis (6),while
p-glucosidaseisneeded forremovalofcellobiose,astrong
inhibitor ofbothendoglucanase andexoglucanase (7).However,
thedebateonthemechanism ofcellulosehydrolysis still
continues.Thediversityofendoglucanases,exoglucanasesand
p-glucosidasesfromfungaloriginisemphasizedbymanyreview
reportsonthissubject (7-13).Comparative studiesare
difficult andsometimesconfusing sinceseveralspecieswith
different strainsareusedbyvarious investigatorsand
characterization isdoneusingdifferentcriteria.
Thefirststepinastudyonthemodeofactionofcellulases
istheisolationofsubstantial amountsofallenzymesofthe
complex.Thepurposeofthepresentworkwastheintegral
fractionationofacommercialcellulasepreparationderived from
Trichodermavirideandthecharacterization ofallendoglucanases,exoglucanases andp-glucosidaseswhichcouldbedetected
andpurified,withrespecttomolecularmass,isoelectricpoint,
glycoproteinnature,temperatureoptimum,degreeofrandomness
inCM-cellulosehydrolysis andspecific activityonseveral
typesofcellulose.
Anewtypeofendoglucanasewithrelativelyhighactivityon
crystallinecellulosewasisolated.Wepurifiedanaryl-p-D-
35
glucosidase,notreportedbefore intheliterature,whichis
abletoactonp-nitrophenyl-(3-D-glucosidebut lackstheability
tohydrolysecellobiose.
Affinitychromatography oncrystalline cellulosewasusedfor
theseparationoftwodifferentcellobiohydrolases.Hydrolysis
productsoftheseexoglucanaseswereanalysedbyHPLCand
compared tothehydrolysisproductsofanother typeofexoglucanase,isolated fromthesameenzymepreparation.Themode
ofactionofthecellulasecomplex,withrespecttotheroleof
theseexoglucanases,isdiscussed.
MATERIALSANDMETHODS
Enzymepreparation
Maxazyme CI,acommercialcellulasepreparation fromTrichodermavirideorigin,waskindlyprovidedbyGist-Brocades
(Delft,TheNetherlands).
Enzymeassays
Enzymeactivitiesweremeasured towardsAvicel (typeSF,
Serva,Heidelberg,FRG;Avicelase activity),CM-cellulose (type
AkucellAF 0305,Akzo,Arnhem,TheNetherlands;CM-cellulase
activity),p-nitrophenyl-p-D-glucopyranoside (Koch-Light,
Colnbrook,Bucks,England;p-glucosidaseactivity),cellobiose
(BDHChemicalsLtd,Poole,England)and^PC^-swollencellulosewhichwasobtainedbythemethoddescribed byWood(14).
Allactivitieswereexpressed inInternationalUnits (U)ml"l.
Avicelase activity.Thereactionmixture forAvicelase
activitymeasurementconsisted of0.5ml 1%Avicelsuspensionin
0.05 Msodiumacetatebuffer,pH 5.0 and0.5mlofenzyme
solution.Incubationtookplaceinashaking incubator at30°C
for 20h.Aftercentrifugation0.5mlwastakenandanalysed for
reducing sugarsbythemethod ofNelsonSomogyi (15).Specific
activitesweremeasured ataproteinconcentrationof 25ug/ml.
CM-cellulase activity.CM-cellulase activitywasmeasured
inamixturecontaining 0.4mlofa0.5%CM-cellulose solution
in0.05 M sodium acetate,pH 5.0 and 0.1mlofenzymesolution.
After incubationat30°Cfor 1h,thereactionwasstoppedand
reducing sugarsweremeasured byadditionoftheNelsonSomogyi
reagent.Sampleswerecentrifugedbeforereadingabsorbanceat
520nm.CM-cellulase activitywasalsomeasured viscometrically
at30°Caccording toAlminandEriksson (16),usinga0.25%
substrateconcentration.Activitywasexpressed asrateof
36
changeinspecific fluidity, A$ S p(min _1 ). Aproteinconcentrationof 1ug/mlwasused forspecific activitymeasurements.
P-Glucosidaseactivity.The(3-glucosidaseassaywasperformedbyadditionof0.1mlofenzymesolutionto0.9mlof0.1%
p-nitrophenyl-p-D-glucopyranosidein0.05 Msodiumacetate
buffer,pH 5.0 andincubated for 1hat30°C.Thereactionwas
stoppedbyadditionof 1ml0.5 Mglycinebuffer,pH 9.0,containing0.002MEDTA.Theconcentrationofp-nitrophenolwas
measured at400nm,usinganabsorptioncoefficient of13700
lyr^-cm--1-.Specific activitiesweremeasured usingaprotein
concentrationof5ug/ml.
Cellobiaseactivity.Enzymefractions,containing(3-glucosidaseactivity,were screened forcellobiaseactivityby
incubationof0.4mlenzymesolution in0.1 Msodium acetatepH
5.0,containing 20u.gproteinwith0.1ml 0.2%cellobiose inthe
samebuffer.After incubationat30°Cfor 1h,theenzymes
wereinactivated byplacingthetubesinaboilingwaterbath
for 10min.Glucoseconcentrationwasmeasured bythehexokinase
method (Boehringerenzymekit,Boehringer,Mannheim,FRG).
ActivitytowardsH-^PC^-swollencellulose.Activity
towardsI^PC^-swollencellulosewasmeasuredbyadditionof
0.1 mlenzymesolutionto0.9ml 0.55%H3P04-swollencellulosein0.05 MsodiumacetatepH 5.0.Incubationtookplacefor
20hat30°C.Aftercentrifugation 0.5 mlofthe supernatant
wasanalysed forreducing sugars.Specificactivitieswere
measured ataproteinconcentrationof1u-g/ml.
Columnchromatography
ColumnchromatographywascarriedoutonBio-GelP10 (100-200
mesh),Bio-GelP100 (100-200mesh),DEAE-Bio-GelA (Bio-Rad
Laboratories,Richmond,USA)SE-SephadexC50 (PharmaciaFine
Chemicals,Uppsala,Sweden)andAvicelcrystallinecellulose.
Eluted fractionswereanalysed forAvicelase,CM-cellulaseand
(3-glucosidaseacitivities.Proteinwasdetected at280nm.
Proteinconcentrationsweremeasuredusing theFolinreagent
(17)withbovine serumalbuminasthestandard. Chromatography
experimentswerecarriedoutat4°C.Allbufferscontained
0.01%NaN3 aspreserving agent.Detailsonthechromatographic
stepsaregivenunderResults.
Sodiumdodecylsuplhate/polyacrylamide-gelelectrophoresis
Slab-gelelectrophoresis onan11%polyacrylamidegel,using
thediscontinuousbuffer systemofLaemmli (18)wasappliedto
analyseenzymepurity.Sampleswere3.5 timesdilutedwith
37
samplebuffer,consistingof 0.125 MTris/HCl,pH 6.8,2.5%
(w/v)SDS,2.5% (v/v)2-mercaptoethanol,25%(v/v)glyceroland
0.0025%bromphenolbleuandsubsequentlyboiled for 2min.Gels
were stained forproteinwithCoomassiebrilliantbleuandfor
carbohydrate,using theperiodicacid/Schiff reagent(19).
Bovine serumalbumin (Mr 68000),ovalbumin (Mr 45000),and
chymotrypsinogen (Mr 25000)wereusedasstandards.
Isoelectric focusing
Analytical thin-layergelisoelectric focusingwasperformed
inthepHrange3.5-9.5onteLKB2117Multiphor (LKB-Produkter
AB,Stockholm,Sweden)usingtheapplicationnoteprovidedby
themanufacturer.Theprocedurewasslightlymodified: 0.4ml
0.004%riboflavinwasreplacedby0.4ml 1%ammonium persulphate
and 50ulN,N,N',N',-tetramethylethylethylendiamine (TEMED).
Afterelectrofocusing thegelwasfixedwithasolutioncontaining 30%methanol/10%trichloroacetic acid/3.5%sulphosalicylic
acid for 1handthenwithasolutioncontaining 30%methanol/
12% trichloroacetic acid for 2h.Beforestainingallthe
ampholineswereremovedbywashingwith 40%methanol/10%acetic
acid foratleast 36h.
Temperature optimum
Temperatreoptimaofallpurifiedenzymesweredeterminedby
measuring theformationofreducing sugarsfromCM-celluloseat
temperatures ranging from10°to90°C.Additionally,
temperature optimaofenzymes showingfi-glucosidaseactivity
weredeterminedwithp-nitrophenyl-p-D-glucopyranosideand
enzymes showingmainlyAvicelase activitywereanalysedwith
Avicelassubstrate.
ProductanalysisonHPLC
InordertoidentifypurifiedenzymesshowinghighAvicelase
and lowCM-cellulaseactivity,theseproteinswere incubatedat
a concentrationof 10ug/mlwith 1%f^PC^-swollencellulose
at30°C in0.05 MsodiumacetatepH 5.0,for 2,6and 21h.
Aftercentrifugationofresidualcellulose (10min,3000xg)
theenzymes inthesupernatantwere inactivatedbyplacingthe
tubesinaboilingwaterbathfor 5min.Productswereanalysed
byHPLC (SpectraPhysicsSP8000)usinganAminexHPX 87Pcolumn
andwater astheeluent.
38
DEAE Bio-Gel A
NgCI - qrodient
I—1Bio-Gel P10C
|
• EAE Bio-Gel A
pH gradient
|
DEAE Bio-Gel A
I pH qrodient
m.
|SE-Sepondey]
1
JsE Sephode* |
|BIO- G&l P1001
|BIO-G&[ P1QO|
Isio-Get PIOol
I
Avicet
I
Fig.1.Flowsheetofthepurificationofendoglucanases,
exoglucanasesand(3-glucosidasesfromacommercial enzyme
preparation.
RESULTS
Purification studies
Theprocedurewhichwasappliedforfractionationofthe
cellulasecomplex intoitsendoglucanase,exoglucanaseand (3glucosidase componentsisshowninFig.1.Anamountof10g
cellulasepreparationwasdissolvedin30ml0.01Msodium
acetate,pH5.0,centrifuged 20minat50000xgtoremove
solidsanddesaltedonaBio-GelP10column (30x950mm)
equilibrated inthesamebuffer.Allofthecellulase activity
appearedasonepeakinthevoidvolume (resultsnotshown).
ThispeakwaspooledandappliedtoaDEAE-Bio-GelAcolumn,
equilibrated in0.01Msodium acetatepH5.0.Thecolumnwas
washedwithbufferandthenelutedwithasodiumchloride
gradientinthesamebuffer (Fig. 2 ) .
Peak H i , containingmostofthep-glucosidaseandCMcellulase activitywaspooled,lyophilized,dissolved inasmall
volumeandappliedtoaBio-GelP100column,equilibratedin
0.02MsodiumcitratepH3.5.Afterelutionwiththesamebuffer
threepeakswereobtainedofwhichtwopeaksshowedcellulase
activity (Fig. 3 ) .
PeakIIIiw a s dialysedandconcentrated in0.01Msodium
citratepH3.5inanultrafiltrationcell (Diaflotype50,
Amicon,MA,USA)usingthePM10membrane.Thisconcentrated
materialwasappliedtoaSE-SephadexC50column,equilibrated
in0.01MsodiumcitratepH3.5.Thecolumnwaselutedwitha
39
flatandthenasteepsodiumcitrategradient (Fig.4 ) .
ExtensivepurificationofEndoIIandEndo IIIwasobtained
after rechromatographyonthesameSE-Sephadexcolumn.Allthe
fourpeaks,EndoI,EndoII,EndoIIIandExoI,containing
cellulaseand(3-glucosidaseactivity,weredialysed andconcen-
150
200
Fraction number
Fig.2.DEAE-Bio-GelAchromatographyofcrudecellulasepreparationafterdesaltingonBio-GelP10.Fractions fromtheBioGelcolumncontainingcellulase activitywerepooled (140ml)
andappliedtotheDEAE-Bio-GelAcolumn (30x200mm).The
columnwaswashedwith 300ml0.01 Msodium acetatepH5.0and
thenelutedwithalineargradientofsodiumchlorideinthe
samebuffer.The flowratewas25ml/handfractionvolumewas
5.5ml. (O)Absorbanceat280nm; (X)Avicelase; (Q)CM-cellulase;(A) p-glucosidase;(—)sodiumchlorideconcentration.
J \v"H
20
•A-J$=HlS&p}
40
60
Fraction number
r^,^
Fig.3.Bio-GelP100chromatographyofpool IIj. Pool 11^
from theDEAE-Bio-GelAcolumnwasconcentratedtoasmall
volumeandappliedtotheBio-GelP100column (20x900mm).The
columnwaselutedwith 0.02MsodiumcitratepH3.5withaflow
rateof25ml/h.Thefractionvolumewas5ml. (O)Absorbanceat
280nm; (X)Avicelase; (a)CM-cellulase; (A)p-glucosidase.
40
Fraction number
Fig.4.SE-Sephadexchromatographyofpool III^.Thedialysed
andconcentrated poolIII^ fromtheBio-GelP100columnwas
appliedtotheSE-Sephadexcolumn (25x300mm)equilibratedin
0.01Msodiumcitrate,pH3.5.Thecolumnwaswashedwith 200ml
ofthesamebufferandthenelutedwithalineargradient from
0.01Mto0.025Msodiumcitrate,pH3.5.From fraction 150to
205thecolumnwaswashedwith275ml0.025MsodiumcitratepH
3.5andthenelutionwascontinuedwithalineargradientto0.1
MsodiumcitratepH3.5.Theflowratewas6ml/handthefractionvolumewas5ml.(o)Absorbance at280nm; (X)Avicelase;
(D)CM-cellulase; (A)p-glucosidase; (—) sodium citrate
concentration.
40
60
Fraction number
Fig. 5.SE-Sephadex chromatography ofpool III2- TheconcentratedpoolI H 2 fromtheBio-GelP100columnwasappliedto
the SE-Sephadexcolumn (20x200mm)equilibrated in0.02M
sodiumcitratepH3.5.Thecolumnwaswashedwith475mlofthe
samebufferandthenelutedwithalineargradient to0.1M
sodiumcitratepH3.5.Theflowratewas20ml/handfraction
volumewas10ml.(O)Absorbanceat280nm;(X)Avicelase;(D)
CM-cellulase; (—) sodiumcitrateconcentration.
41
tratedbyultrafiltrationin0.05 Msodium acetatepH5.0.These
proteinsappeared asasinglebandonSDS/polyacrylamidegel
electrophoresis (Fig.13).ThefirsttwopeaksofFig.4,
containingcellulaseactivity (EndoIandEndoII)were typical
endoglucanases showinghighspecificactivitytowardsCM-celluloseand lowactivity towardsAvicel (Table1 ) .
Thepeakreferred toasEndoIIIhad lowspecific activity
towardsCM-celluloseandamuchhigheractivitytowardsAvicel.
Thisenzymewasabletoproducecellotriose,cellobioseand
glucosefromr^PO^j-swollencellulose (Fig.14d)andthereforeidentified asanendoglucanase.Therewasnoevidencefor
cellotrioseproduction fromtransglucosidase activityofthis
enzyme,because therelativepeakareasof G3,G2andG^
remainedconstant inthecourseoftheincubation (analysed
after 2,6and 21h;resultsnot shown).
ThelastpeakoftheelutionprofileofFig.4 (ExoI)showed
higharyl-p-glucosidaseactivity,andalsohighactivity towards
cellobiose,H3P04-swollencellulose andAvicel.HPLCanalysisoftheproductsobtainedafter incubationwithH3PO4swollencellulose showed thatthisenzymewasanexoglucanase,
releasingcellobiosemoleculeswhichwerehydrolysed bythesame
Table 1.Specific activitiesofendoglucanases,exoglucanases
and p-glucosidasepurified fromcommercial cellulasederived
fromTrichodermaviride.
Specific activitiesweremeasured at 30°CandpH 5.0 towards
CM-cellulose,Avicel,t^PC^-swollencellulose,p-nitrophenyl
(3-D-Glucopyranoside (pNp-Glc)andcellobiose assubstrates.For
experimentaldetails seethetext.Abbreviations:n.d.,not
detected; -,notdetermined.
Enzyme
Specific activity towards
CMcellulose
Avicel
H 3 P0 4 swollen
cellulose
p-Np-Glc
cellobiose
0.0130
0.0068
0.0156
0.0026
0.0073
0.0044
0.0160
0.0064
0.0078
0.0018
0.64
0.46
0.45
0.33
0.61
0.36
0.63
0.053
0.030
0.45
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.79
n.d.
n.d.
0.40
-
U/mg
EndoI
EndoII
EndoIII
EndoIV
EndoV
EndoVI
ExoI
ExoII
ExoIII
0-GlucI
42
13.1
20.1
3.2
9.6
14.7
15.8
1.8
0.26
0.033
0.29
0.19
n.d.
enzymetotwoglucoseunits (Fig.14a,b,c ) .
PoolIII2fromtheBio-GelP100column (Fig.3)wasconcentratedbyultrafiltrationandapplied toaSE-Sephadexcolumn
(Fig.5).Thecolumnwaswashedwith 0.02 Msodiumcitrateand
elutedwithasodiumcitrategradient.Thepeakcontaining
CM-cellulaseactivitywaspooled anddialysed and concentrated
in0.05MsodiumacetatepH5.0 byultrafiltration.Theprotein
waspureasjudgedbySDS/polyacrylamidegel electrophoresis
(Fig.13).Thisenzymewasidentified asatypical endoglucanase
(Endo IV), havinghighactivity towardsCM-celluloseandlow
activitytowardsAvicel (Table1 ) .
PeakII2fromtheDEAE-Bio-GelAcolumn (Fig.2.)was
rechromatographed onthesamecolumn,butthistimeusingapH
gradient (Fig.6).Thepeak III3,containing CM-cellulase
activity,wasconcentratedbylyophilizationandapplied toa
Bio-GelP100column,equilibrated in0.05 MsodiumcitratepH
4.0 (Fig.7 ) .Thepeakcontaining CM-cellulase activity (EndoV)
wasdialysedandconcentrated in0.05 Msodiumacetate,pH 5.0
byultrafiltration. SDS/polyacrylamide gelelectrophoresisof
thisprotein showed asingleband (Fig.13).Theenzymewas
identified asanendoglucanase,withhighactivity towardsCMcellulose and lowactivitytowardsAvicel (Table1 ) .
ThesamepHgradientpurification steponDEAE-Bio-GelAwas
used for furtherpurificationofproteins inpoolII3fromthe
40
Fraction number
60
Fig. 6.DEAE-Bio-GelAchromatography ofpool112- Pool II 2
fromthefirstDEAE-Bio-GelAcolumnwasdilutedwithwaterto
the sameconductivity asthe startingbuffer (0.02Msodium
acetatepH5.6)and applied totheDEAE-Bio-GelAcolumn (30x
200mm)equilibrated inthesamebuffer.Thecolumnwaseluted
withapHgradient in0.02 Msodiumacetate frompH 5.6 topH
3.8.Theflowratewas 25ml/handfractionvolumewas 10ml.
(O)Absorbance at280nm; (X)Avicelase; (o)CM-cellulase.
43
<
0.15
Bio - G e l P100
EndoV
-t>^-*efc
20
W
Fraction number
Fig. 7.Bio-Gel P100chromatography ofpool III3.The
lyophilizedpool III3 from theDEAE-Bio-GelAcolumnwas
dissolved inasmallvolumeand applied totheBio-GelP100
column (20x900mm)equilibrated in0.05 MsodiumcitratepH
4.0.Thecolumnwaselutedwith thesamebuffer.Theflowrate
was 25ml/hand fractionvolumewas 5ml. (o)Absorbance at280
nm; (x)Avicelase; (Q) CM-cellulase.
firstDEAE-Bio-Gelcolumn.TwoCM-cellulase peaksandonep-glucosidasepeakwereobtained after thispurification step (Fig.
8). Thefistpeakwasidentified asthesamematerialas
fraction III3 (Fig.6;resultsnot shown).Bio-GelP100chromatographywasused for furtherpurificationofpoolIII4,
containing CM-cellulase activity (Fig.9 ) ,andpool III5,containing p-glucosidase activity (Fig.10).
Thepeak inFig.9containing CM-cellulase activity (EndoVI)
wasconcentrated byultrafiltrationandidentified asapure
endoglucanase.ApartoftheCM-cellulase activityofEndoVI
appeared asasecondpeak inthepurificationofthep-glucosidase (Fig.10).Thisproteinwasidentified aspureEndoVI.
Thep-glucosidase peakofFig.10 (p-glucI)consisted ofa
pureenzyme (Fig.13)withhighactivity towardsp-nitrophenylp-D-glucopyranoside.However,thisenzymeshowednoactivity
towardscellobiose andonlymoderate activity againstAvicel,
H3P04-swollencellulose andCM-cellulose (Table 1).The
enzymewasalsoactive towardsxylanandthep-nitrophenyl
derivatives ofp-D-xyloseanda-L-arabinose (resultsnot shown).
Peak II 4 fromthefirstfractionation ontheDEAE-Bio-GelA
column (Fig.2 ) ,containingessentiallyonlyAvicelase activity
wasconcentrated by lyophilization andapplied totheBio-Gel
P100column (Fig.11).Thepeak Illg,containingmostofthe
Avicelaseactivitywassubjected toaffinitychromatography on
44
Avicelcellulose.Twopeaks (ExoIIandExo III), containing
Avicelase activity,wereeluted fromthecolumnbyapHgradient
of 0.1Mpotassiumphosphate,frompH6.0 topH11.8 (Fig.12).
Thefractionscontainingproteinwere immediatelyneutralized to
pH7byadditionof 2.5ml0.2MKH2PO4,dialyzedandcanOEAE Bio-GelA
't
008-
60
Fraction number
Fig. 8.DEAE-Bio-GelAchromatography ofpoolII3.PoolII3
fromthefirstDEAE-Bio-GelAcolumnwasdilutedwithwaterto
thesameconductivity asthestartingbuffer,0.02Msodium
acetatepH 5.6,andapplied totheDEAE-Bio-GelAcolumn (30x
200mm)equilibrated inthesamebuffer.Thecolumnwaseluted
with apHgradient in0.02Msodium acetate frompH 5.6 topH
3.8. The flowratewas25ml/hand fractionvolumewas 10ml.
(O)Absorbanceat280nm; (x)Avicelase; (•) CM-cellulase; (A)
p-glucosidase.
EndojZI
<
010-
A,
\
aW.
40
60
Fraction number
80
Fig. 9.Bio-GelP100chromatography ofpoolIII 4 .The
lyophilized poolIII 4 from theDEAE-Bio-GelAcolumnwas
dissolved inasmallvolumeand applied totheBio-GelP100
column (20x900mm)equilibrated in0.05 MsodiumcitratepH
4.0.Thecolumnwaselutedwith thesamebuffer.Theflowrate
was 25ml/handfractionvolumewas 5ml. (o)Absorbance at280
nm; (x)Avicelase; (a)CM-cellulase; (£)B-glucosidase.
45
centrated in0.05 Msodiumacetatebyultrafiltration.ExoII
waseluted fromthecolumnatpH 10.3andExoIIIatpH11.8.
BothExoIIandExoIIIgave singlebandsongelelectrophoresis.Theseenzymeswere identified asexoglucanases,havinghigh
specificactivity towardsAvicelandvery lowactivity towards
0 glue.1
60
Fraction number
100
Fig. 10.Bio-Gel P100chromatography ofpool III5.The
lyophilizedpoolIII5fromtheDEAE-Bio-GelAcolumnwas
dissolved inasmallvolumeand applied totheBio-GelP100
column (20x900mm)equilibrated in0.05 M sodiumcitratepH
4.0.Thecolumnwaselutedwiththesamebuffer.Theflowrate
was 25ml/hand fractionvolumewas 5ml. (o)Absorbance at280
nm; (•) CM-cellulase; (A)fi-glucosidase.
Fraction number
Fig. 11.Bio-GelP100chromatography ofpoolII4.The
lyophilized pool II 4 from thefirstDEAE-Bio-GelAcolumnwas
dissolved inasmallvolume andapplied totheBio-GelP100
column (20x900mm)equilibrated in0.05 MsodiumcitratepH
4.0.Thecolumnwaselutedwiththe samebuffer.Theflowrate
was 25ml/hand fractionvolumewas 5ml. (o)Absorbance at280
nm; (x)Avicelase; (D)CM-cellulase; (A)3-glucosidase.
46
CM-celluloseandH3P04-swollencellulose (Table 1). These
enzymesreleasedcellobioseastheonlyproductduringincubationwithH3PC>4-swollencellulose (Fig.14e,f).
Degreeofrandomnessof endoglucanases
TheratiooftheactivityofendoglucanasesonCM-cellulose,
measuredviscometrically,totheactivityonCM-cellulose,measuredbytheincreaseofreducingsugars (i.e.A$sp/reducing
200
250
Fraction number
Fig. 12.Avicelchromatography ofpool I H g . PoolH l g was
dialysedbyultrafiltrationto0.1 Mpotassium/sodium phosphate
pH6.0 andappliedtotheAvicelcolum (22x 100mm),equilibrated inthesamebuffer..Thecolumnwaswashedwith 500ml
buffer and thenelutedwithapHgradient in0.1 Mpotassium/sodiumphosphatefrompH6.0 topH11.8.Theflowratewas50
ml/handfractionvolumewas 5ml. (O)Absorbance at280ran;(X)
Avicelase; (O)CM-cellulase.
Table 2.Degreeof randomness inattackonCM-celluloseofendoglucanases isolated fromTrichodermaviride.
ThespecificactivityonCM-celluloseoftheendoglucanases,
measured viscometrically,wasdivided bytheactivityfound from
measurements ofreducingpower.
Enzyme
A<tS p / r e d u c i n g power
EndoI
EndoII
EndoIII
EndoIV
EndoV
EndoVI
1.00
0.77
0.40
0.88
0.50
0.49
47
Endo Endo Endo
I
II
III
Exo
Endo
I
IV
Endo Endo (Jgluc Exo Exo
V
VI
I
II
III
-6B000
-4S0OO
-25C---
Fig. 13.SDS/polyacrylamidegelelectrophoresisofendoglucanases,exoglucanases and p-glucosidasepurified fromcommercial
cellulasederived fromTrichodermaviride.Thegelhadafinal
acrylamideconcentrationof 11%.Proteinswerestainedwith
Coomassiebrilliantblue.Standards:bovine serum albumin (Mr
68000),ovalbumin (Mr 45000)andchymotrypsinogen (Mr
25000).
10
15
20
Elution time (mini
10
1'5
26
Elution time (min)
Fig. 14.HPLCanalysisofproducts released fromH3PO4swollencellulose.Incubation tookplace in0.05 Msodium
acetatepH 5.0 at 30°Cwith:Exo Ifor 2h (a),6h(b),21h
(c); EndoIII for 21h (d);ExoIIfor 21h (e);Exo IIIfor21
G2,cellobiose;G3,cellotriose;RI,
h (f).G^,glucose;
refractiveindex.
48
power),isanindicationofthedegreeofrandomness incellulosehydrolysis.Essentially twotypesofendoglucanases could
bedistinguished (Table 2).EndoI,EndoIIandEndoIVgavea
highquotient andthereforewereidentified asrandomly acting
endoglucanases.EndoIII,EndoVandEndoVIwerealess-random
typeendoglucanases.
Chemicalandphysicalproperties
Molecularmassesofthepurifiedendoglucanases,exoglucanasesand(3-glucosidasescouldbeestimated fromtheresultsof
theSDS/polyacrylamidegelelectrophoresis (Fig.13).Mostof
thepurified enzymeshadM r intherange 45000to76000 (Table
3). EndoIVappeared tobeanexceptionally smallprotein.The
specialnatureofthisenzymewasemphasized byitsrelatively
highisoelectricpoint.Moreover,EndoIVwastheonlyenzymeof
allthepurifiedenzymespresented inthispaperwhichcouldnot
bestainedbytheperiodic acid/Schiff reagent.Alltheother
endoglucanases,exoglucanases and(3-glucosidaseswere identified
asglycoproteins.
TheglucanasesEndoI,EndoIIandEndoIIIandtheglucanase
ExoI,whichwereobtainedbypurificationonthefirstSESephadexcolumnandwhichthereforehad arelativelyhighisoelectricpoint (pi5.3-6.9)-,showedatemperature optimumfor
hydrolysis ofCM-celluloseat60°C.Thistemperatreoptimum
wasabout 10°Chigher thantheoptimaltemperatureofallthe
otherendoglucanases,exoglucanases and p-glucosidases,which
wereisolated intheotherpurification stepsandhavealower
isoelectric point (pi2.8 -4.4).Againthereisanexception
forEndoIV,whichhadapiof7.7 andatemperature optimumof
48°C.Thetemperature optimaoftheenzymes showing(3-glucosidaseactivitywerealsomeasured onp-nitrophenylp-D-glucoside.
Therewasnosignificantdifference ofoptimaltemperaturewhen
thissubstratewasusedinsteadofCM-cellulose.Replacementof
CM-cellulosebyAvicelhadalmostnoinfluenceonthe
temperature optimaofExoIIandExoIII,althoughtheincubationtimewith the latter substratewas 20hinstead of 1hwith
CM-cellulose.Although there isnoexplanation forthecorrelationbetweenhightemperatureoptimaandhigh isoelectric
points,theendoglucanase isolated fromthe thermophilic
bacterium Clostridium thermocellumalsohasthecombinationof
thesetwoproperties (Nget al. [20]).
49
Table 3.Chemicalandphysicalpropertiesofendoglucanases,
exoglucanases and0-glucosidasepurified fromcommercialcellulasederived fromTrichodermaviride.
Forexperimentaldetails seethetext.Abbreviations:P,
positive,andN,negativeonstainingwithperiodic acid/Schiff
reagent; -,notdetermined;pi, isoelectricpoint;pNp-Glc,
p-nitrophenylp-D-glucoside.
Enzyme
Mr-
CMcellulose
EndoI
EndoII
EndoIII
EndoIV
EndoV
EndoIV
ExoI
ExoII
ExoIII
(3-GlucI
50000
45000
58500
23500
57000
52000
53000
60500
62000
76000
5
6
6
7
4
3
5
3
3
3
Glycoprotein
Temp,optimumtowards
Pi
3
9
5
7
4
5
3
5
8
9
60
60
60
48
50
50
60
52
52
52
Avicel
pNp-Glc
62
50
50
49
DISCUSSION
EndoI,EndoII,EndoIV,EndoVandEndoVIwere identified
astypicalendoglucanases (1,4-p-D-Glucanglucanohydrolases),
showinghighactivitytowardsCM-cellulose.ThespecificactivitiesoftheseenzymesonAvicelwereatleast10 3timeslower
Thesetwoproperties aregenerallyaccepted todistinguishendo-
50
glucanases fromexoglucanases.Schoemakeretal.[21,22]describe
a low-molecular-mass endoglucanase IIfromPancellaseSS,acommercialcellulasefromTrichodermaviride,whichclearlyresemblesEndoIVwithrespecttomolecularmassandlowcarbohydrate
content.ThesameenzymewasalsopurifiedbyFagerstametal.
[23]fromT.virideQM9414andidentified asaproteinwitha
highisoelectricpoint (pi7.5),which isconsistentwithour
observations.
Schoemaker reportstwootherendoglucanaseswithdifferent
degreesofrandomness intheiractiononCM-cellulose:endoglucanaseIIIandendoglucanase IV [21,22].Theless-random type
ofenzyme,referredtobytheseauthorsasendoglucanase IIIwas
verysimilartoEndoV,reported inthispaper.EndoV isa
less-random typeofhydrolase thanEndoIsinceitgaveless
decreaseofviscositycompared totheincreaseofreducing
sugarsduringincubationwithCM-cellulose.Forthisreasonand
because ithasthesamemolecularmass,EndoIwasidentified as
thesameendoglucanase (endoglucanase IV),isolatedbySchoemaker [21, 22].
EndoVIisaglucanasewiththesameproperties asEndoV,
withrespecttotemperatureoptimum,carbohydratecontentand
degreeofrandomness inhydrolysisofCM-cellulose.Its
molecularmass issomewhat lower.EndoVIwasnotdescribedby
Schoemaker [21,22].However,Okada [24]purified a less-random
typeofendoglucanase (cellulaseIl-b)fromMeicellase,acommercialcellulase fromT.viridewhich issimilar toEndoVI.
Untilnowthreedifferenttypesofp-glucosidaseisolated
fromT.viridehavebeenreported.Twooftheseenzymesare
extracellular andhaveM r of47000 [25]and76000 [26]respectively.Another,intracellular p-glucosidasewitha M rof
98000,wasisolatedbyInglinetal.[27].Alloftheseenzymes
showedaryl-p-D-glucosidaseaswellascellobiaseactivity.Here
wedescribeanewtypeofp-glucosidase (p-glucI)whichis
activetowardsp-nitrophenylp-D-glucosidebutisunableto
hydrolysecellobiose.Theenzymeshowed abroad substrate
specifcity:itwasmoderately activetowardsCM-cellulose,
Avicelandxylanandhighly activetowards thep-nitrophenyl
derivativesofp-D-glucoseandp-D-xylose.
Fagerstam etal.[23]isolated threecellobiohydrolases:a
majorcomponentwithapiof 3.95 andtwominorcomponentswith
pi 3.80 and 4.15 respectively.ExoIII,described inthispaper,
resembles themajorexoglucanase component,whileExoIIiscomparable totheminorcomponentofpi 3.80.ExoIIandExoIII
51
showeddifferentadsorptionaffinitiestocrystalline cellulose
andcouldeasilybeseparated byspecificdesorptionwithapH
gradientwithout significant lossofenzymeactivity.ThediversityofthecellobiohydrolaseswasconfirmedbyGumet al.[28],
who isolated threeexoglucanases fromMeicelasePandonefrom
T_.virideQM9123.Theseauthorsconcluded thatthecarbohydrate
content istheprincipal factorwhichdifferentiatesthecellobiohydrolase enzymes.However,Nakayamaetal. [29]concluded
that limitedproteolysismaypartiallyberesponsible forthe
multiplicity ofendoglucanases fromT_.viride.Ourresultsgave
noevidence foranysignificantproteolysisduringenzyme
purification:although theperiodoftimebetween twosubsequent
chromatographic stepsinseparate isolationexperimentswas
varied,nodistinctdifferences inelutionprofileswere
obtained.
ExoIIandespecially ExoIIIacted synergisticallywiththe
endoglucanasesdescribed inthispaper (resultstobepublished;
Chapter 6). Anewtypeofsynergismwasdemonstratedby
Fagerstametal.[30].Theirresults showsynergistic action
betweenthecellobiohydrolase (CBHI)described in [23]and
anothercellobiohydrolase (CBH II), activeagainst crystalline
celluloseandwhichcouldberesolved intotwoiso-enzymeswith
pivaluesof 5.0 and 5.6 respectively.Theseenzymesproduce
cellobiose asthemainproductwhenactingoncystalline
cellulose.However,significant amountsofglucosewerealso
measured.Althoughtheseauthorsgivenofurther informationon
molecularmassesandspecific activities towardsthesubstrates
whichwereused inthiswork,theresultsstrongly suggestthat
theCBHIIisnotonlyacellobiohydrolase butanexoglucanase
identicaltoExoI,described inthispaper.ExoIisan
enzymewiththesamepivalueandproducesnotonlycellobiose
fromcellulosebutalsoglucose sincethisenzymehascellobiase
activityaswell.Moreover,theexoglucanase showedastrong
synergistic actionwithExoIIandExoIII (resulttobe
published;Chapter 6).Similarexoglucanaseswereisolatedby
Shikataetal. [31]fromCellulase-Onozuka derived fromT.
virideandbyWoodetal.[32]fromPenicilliumfuniculosum.The
latter found littleaccumulation ofcellobiose after an18-h
incubationwith^PC^-swollencelluloseand identified this
enzymeasa1,4-p-D-glucanglucohydrolase (EC3.2.1.74).
EndoIIIshowed lowactivity towardsCM-cellulosebutfrom
allpurified endoglucanases thisenzymehadthehighest specific
activity againstAvicel.Anenzymewiththesepropertiesis
52
usuallycalled anexoglucanase.However,fromseverallinesof
evidencepresented inthispaperwesuggest thatthisenzymeis
clearly anendoglucanase.Firstly,becauseEndoIIIreleased
cellotriose,cellobioseandglucose fromH3PC>4-swollen
cellulose,which isanindication foritsendo-typeattack.
Secondly,thisendoglucanase showed asynergistic actionin
combinationwithExoIII.Thirdly,nosynergismwasobserved
whenEndoIIIwasworking inconcertwithanother endoglucanase
(resultstobepublished;Chapter 6). Asfarasweknow,this
enzymehasnotbeendescribedbefore intheliterature,anda
furthercharacterizationwillbepublished inasubsequent
paper.
Comparisonofthethreepurifiedexoglucanaseswithrespect
totheiractivitiestowardsseveralsubstrates showssomeinterestingphenomena.TheactivityofExoIagainstAvicelcelluloseisabouttwiceashigh,compared totheactivitiesofExo
IIandExoIII,towardsthissubstrate.However,usingH3PO4
-swollencellulose asthesubstrateExoIshowed 10-20times
moreactivity thanExoIIandExoIII.Thesetwocellobiohydrolasesarenotabletodegradethissoluble,non-crystalline
cellulosealthoughtherearesufficientchainendsavailable,
sinceExoIishighlyactivetowardsthissubstrate.
ExoIIandExoIIIhaveapparently amuchhigher affinity
towardscrystalline cellulosethantowardsamorphouscellulose
(CM-cellulose;H3PC>4-swollencellulose).Thelowaffinity
towardsthesesubstratesisneitherdetermined bytheavailabilityofchainends,whereExoIIandExoIIIaresupposed tobind
andexerttheircatalytic action,norbycharge interactions
betweenenzymeandsubstrate,becauseendoglucanaseswith
different isoelectricpoints (intherangefrompi 3.5 to 6.9)
allshowhighaffinity tothesesubstratesundertheexperimentalconditions (pH5.0; 30°C).WhydoExoIIandExoIIIonly
havesuchhighaffinityforcrystalline areas,whileinthese
veryregionstheenzymesareunabletohydrolyse?OneexplanationcouldbethatExoIIandExoIIIalsohaveanon-hydrolytic
activityduringdegradationofcellulose:theinductionof
structuralchanges inthecrystalline regions,resultingin
betteraccessibilityofthecellulosechainstoendoglucanases.
Thisisthewell-knownC^,C xconceptofReese[33],recently
supportedbytheelectronmicroscopic studyofChanzyetal.[34]
Anotherexplanation,which ismoreinaccordancewiththe
hydrolytic natureofExoIIandExoIII,couldbethatthese
cellobiohydrolases adsorbonthecrystalline regionsandonly
53
become activewhenanendoglucanase generatesanewchainend.
Theresults suggest thatthecellobiohydrolase isespecially
activeonsuchnewlygenerated chainends,sincesubstrateswith
accessiblechainends (CM-cellulose,I^PC^-swollencellulose)arescarcely attacked.Thismeansthatthisenzymewould
havetowork inveryclosecooperationwith theendoglucanase,
maybe asanenzyme-enzyme complexonthesurfaceofthe
cellulosechains,assuggestedbyWood [6]. Adetailed studyon
theadsorptionkineticsoftheseexoglucanases andendoglucanaseswillgivemoreinsight intothistheoryandwillbe
published inasubsequentpaper.
PartofthisworkwassupportedbyagrantfromtheEuropean
Community (contractnumberESE-R-038-NL).
REFERENCES
1.Mandels,M.,Meideiros,J.E.,Andreotti,R.E.&Bisset,F.H.
Biotechnol.Bioeng.1981,23,2009-2026
2.Tangu,S.K.,Blanch,H.W. &Wilke,C.R., Biotechnol.Bioeng.
1981,23,1837-1849.
3.Shin,S.B.Kitakawa,Y.,Suga,K.I.&Ichikawa,K.,
J. Ferment.Technol.1978,56,396-402
4.Wood,T.M.,Biochem.J. 1968,109,217-227
5.Eriksson,K.E.&Pettersson,B.,Eur.J.Biochem. 1975,51,
193-206
6.Wood,T.M.&McCrae,S.I.,Biochem.J. 1978,r7_l,61-72
7.Woodward,J. &Wiseman,A.,EnzymeMicrob.Technol.1982,4,
73-79
8.Ladisch,M.R.,Lin,K.W.,Voloch,M.&Tsao,G.T.,Enzyme
Microb.Technol.1983,5,82-102
9.Goksoyr,J. &Eriksen,J. inMicrobialEnzymesandBioconversions (Rose,A.H.,ed.)1980,pp.283-329,Academic
Press,NewYork
10.Ruy,D.D.Y. &Mandels,M.,EnzymeMicrob.Technol.1980,2,
91-102
11.Lee,Y.H.&Fan,L.T.,Adv.Biochem.Eng.1980,17,101-129
12.Bisaria,V.S.&Ghose,T.K., EnzymeMicrob.Technol.1981,
3,90-104
13. Shewale,J.G.,Int.J.Biochem. 1982,1_4,435-443
14.Wood,T.M., BiochemJ. 1971,121,353-362
15. Somogyi,M.,J.Biol.Chem.1952,195,19-23
16.Almin,K.E.&Eriksson,K.E.,Biochim.Biophys.Acta,1967,
139,238-247
17.Lowry,O.H.,Rosebrough,N.J.,Farr,A.L.&Randall,R.J.,
J.Biol.Chem.1951,193,265-275
18.Laemmli,U.K.,Nature (Lond.)1970,227,680-685
19.Colowick,S.P.,Kaplan,N.D.,MethodsEnzymol.1972,28,
54-62
20.Ng,T.K. &Zeikus,J.G., Biochem.J. 1981,199,341-350
21.Schoemaker,S.P. &Brown,R.D.,Biochim.Biophys.Acta,1978,
523,133-146
22.Schoemaker,S.P. &Brown,R.D.,Biochim.Biophys.Acta,1978,
523,147-161
54
23.Fagerstam,L.,Hakansson,U.,Pettersson,G. &Andersson,L.
Proc.Bioconversion Syrup.IITDelhi,1972,165-178
24.Okada,G.,J.Biochem (Tokyo)1975,77,33-42
25.Berghem,L.E.R.&Pettersson,L.G.,Eur.J.Biochem.1974,
46, 295-305
26.Gong,C.S.,Ladisch,M.R.&Tsao,G.T.,Biotechnol.Bioeng.
1977,19,959-981
27. Inglin,M.,Feinberg,B.A. &Loewenberg,J.R., Biochem. J.
1980,185,515-519
28.Gum,E.K.&Brown,R.D.,Biochim.Biophys.Acta 1977,492,
225-231
29.Nakayama,M.,Tomita,Y.,Suzuki,H.&Nisizawa,K.,J.Biochem. (Tokyo)1976,79,955-966
30.Fagerstam,L.G. &Pettersson,L.G.,FEBSLett.1980,153,
113-118
31. Shikita,S.&Nisizawa,K.,J.Biochem. (Tokyo)1975,78,
499-512
32.Wood,T.M.&McCrae,S.I.,Carbohydr.Res.1982,110,291303
33.Reese,E.T.,Rec.Adv.Phytochem. 1976,11,311-367
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Lett. 1983,153,113-118
55
5 Adsorption and kineticbehaviours of purified
endoglucanases and exoglucanases from
Trichodermaviride
Accepted for publication in Biotcchnol. Bioeng.
57
ADSORPTIONANDKINETICBEHAVIOURSOFPURIFIEDENDOGLUCANASESAND
EXOGLUCANASES FROMTRICHODERMAVIRIDE.
G.Beldman,A.G.J.Voragen,F.M. Rombouts,M.F.Searle-van
LeeuwenandW.Pilnik.
DepartmentofFoodScience,AgriculturalUniversity,Wageningen
Adsorptiononcrystallinecelluloseof sixendoglucanases (Endo
I,II,III,IV,V andVI;1,4-p-D-glucanglucanohydrolase,EC
3.2.1.4)andtwoexoglucanases (ExoIIand III; 1,4-p-D-glucan
cellobiohydrolase,EC 3.2.1.92),purified fromacommercial
cellulasepreparationofTrichodermavirideorigin,was studied.
EndoI,IIIandVadsorbed stronglyonAvicelcellulose,while
adsorptionofEndoII,IVandVIwasmuch lower.Also,thetwo
exoglucanasescouldbedivided intooneenzyme (ExoIII)which
hadahighadsorptionaffinity andanotherenzyme (ExoII)which
adsorbedonlymoderately.Adsorptiondatafitted theLangmuirtypeadsorption isotherm.However,adsorptionwasonlypartially
reversiblewithrespecttodilution.Norelationcouldbefound
betweenadsorption affinityanddegreeofrandomnessincellulosehydrolysis,measured asthediversityof released
hydrolytic products.Kineticmeasurements indicated thatonly
partoftheadsorbed enzymemoleculesarehydrolyticallyactive.
INTRODUCTION
Cellulose isarenewablecarbohydrate sourceandavailablein
largequantities.Manyattemptshavebeenmadetoutilizethis
enormousamountofcarbohydrate throughenzymatic hydrolysis
intoglucose.Cellulases,capableofhydrolysisofcrystalline
cellulosearederived fromseveralfungi (1-5).Theextracellular
multi-enzyme systemofthesemicro-organisms ischaracterizedby
threemaincomponents:the1,4-p-D-glucan glucanohydrolaseor
endoglucanase (EC3.2.1.4),the1,4-p-D-glucan cellobiohydrolase
orexoglucanase (EC3.2.1.91)andthep-D-glucosideglucohydrolaseor6-glucosidase (EC3.2.1.21).Themultiplicityof
theenzymecomplexandthesynergistic actionbetween
endoglucanase andexoglucanase (6)causemanydifficultiesin
58
studying themodeofactionofthecomplex.
Thesubstrate-enzyme system isheterogeneous andconsistsof
thewater insolublecelluloseandthewater solubleenzymes.
This impliesthatthefirststepincellulolysis isbindingof
thecellulaseenzymesonthesurfaceofthecellulosefibrils.
Afterbindingoftheenzymemolecules theactualcatalytic
action,i.e.thehydrolysisofthesusceptible glucosidicbonds,
takesplace.Thephysicochemicalheterogeneity ofthecellulose
introducesanothermultiplicity,whichhas large influenceon
theadsorptioncharacteristics ofthecellulaseenzymes,aswell
asontheextentofhydrolysis.
Crystallinityofcellulosewasshowntobethemainparameter
whichdetermines itsbiodegradability (7,8 ) .Besidesofthe
enzymeandcelluloseconcentration,theamountofadsorbed
cellulaseperunitmassofcellulose,dependsonthecrystallinityindexandthespecific surfacearea.Theadsorption
equilibriumconstant,obtained fromtheLangmuir-typeadsoption
isotherm,usingaconstantcelluloseconcentration,decreases
withdecreasingcrystallinity indexandthemaximumprotein
adsorptionconstant increaseswithincreasing specific surface
area (9,10).Atpresentalltheresearchcarriedouton
adsorptionofcellulaseoncellulosic substrateshasbeendone
withthecompleteculture filtrate (9-14), commercialenzyme
preparations (15)orpartiallypurified and characterized
endoglucanase andexoglucanase fractions (16).Informationon
theadsorptionofvariousendoglucanase andexoglucanase
componentsofthesepreparationswasobtained fromactivity
measurementsoncarboxymethylcellulose (endoglucanase)and
filterpaper (exoglucanase) (11-14). However,ithasbeenproven
thatsuchassaysareunsuitable forcalculationsofenzyme
quantities (17, 18).
Theworkpresented inthispaperavoids suchdrawbacksbythe
useofhighlypurifiedenzymes.Theadsorption characteristics
and thereactionkineticparametersof sixendoglucanasesand
twoexoglucanases,purified fromacommercialcellulasepreparation,derived fromTrichodermaviride (19)weredeterminedon
crystallineAvicelcellulose.Theadsorptionbehaviourofendoglucanaseswerecompared totheirmodeofactionincellulose
hydrolysis,especiallywithrespecttothereleaseofsoluble
products.
59
MATERIALSANDMETHODS
Enzymes
Sixendoglucanases (EndoI,II,III,IV,VandVI)andtwo
exoglucanases (ExoIIandIII)were isolated fromthecommercial
cellulasepreparationMaxazymeCL (Gist-Brocades,Delft,the
Netherlands)asdescribed before (19).Ifnecessarytheenzyme
solutionswereconcentrated tothedesiredprotein concentration
(1-2mg/ml)byultrafiltration (DiafloType 50,Amicon,Mass.,
USA)using thePM10membrane.
Adsorption studies
Variousamountsofthepurifiedendoglucanases,orexoglucanasesandappropriate amountsof0.05 Msodium acetatebufferpH
5.0weremixedwith afixedamountofAvicelcellulose (TypeSF,
Serva,Heidelberg,F.R.G.)togiveafinalvolumeof 0.5ml.The
enzymeproteinconcentrationvariedbetween 0.1 and 1.0mg/ml.
Thefinalcelluloseconcentrationwas20mg/ml.Incubationtook
place for 1hour at30°C in0.05Msodium acetatepH5.0with
continuousmixingonarollerdrum (NewBrunswickScientific,
Edison,USA).Aftercentrifugation ofthecellulose (10min,
3000x g ) , thesupernatantwasanalysedonnon-adsorbed protein
usingtheFolinreagent (20).Cellulase,adsorbedoncellulose
wasestimatedby substracting theamountofdissolvedprotein
fromthevaluesobtained inasimilarexperimentwithout
cellulose.
Reversibility oftheadsorptionwasstudiedunder conditions
ofabout50%saturation.Thecellulosewascentrifugedoff,
followedbyreplacementoftwo-third ofthetotalvolumeby
freshbuffer.Afterresuspending thecellulose,furtherincubationtookplaceduring 1and 8hours,usingthesameconditions.
Thesampleswerecentrifuged againandthefreeprotein
concentrationwasdetermined.
Kinetic studies
Thereactionmixtures forkinetic studiesonAvicelcellulose
consistedofvariousconcentrations of substrate (11.43to90.91
mg/ml)andafixedenzymeconcentration (25ug/ml)in0.05M
sodium acetatebufferpH 5.0.Thefinalvolumewas 1ml.
Incubationtookplaceonarollerdrumat30°Cfor 20hours.
After centrifugation aliquotsofthereactionmixtureswere
takenandanalysed forreducing sugarsbythemethodofNelson
andSomogyi(21).
60
Hydrolysisproducts analysis
Thesixendoglucanaseswereincubatedwith2%Avicelcelluloseinafinalvolumeof0.5 mlat30°Cin0.05Msodium
acetatebuffer for 6hours.Theenzymeconcentrationwas60
y.g/ml.Aftercentrifugationofthecellulose,aliquotsof 0.4
mlweretakenfromthesupernatant,theenzymeswere inactivated
byplacingthetubesinaboilingwater-bath for5min.and
evaporatedwithdryair.Theresiduewasdissolved in75ul 0.1
MPb(N03)2andcentrifugedtoremovesolids.Products inthe
supernatantwereanalysedbyHPLC (SpectraPhysicsSP8000)
usingtheAminexHPX 87Pcolumn (300x7.8mm,Bio-RadLabs,
Richmond CA,USA)coupledwithaguardcolumn,containingequal
amountsoftheion-exchange resinsAG 50W-X4 (H+)andAG3-X4A
(OH~)(BioRad labs). Thecolumnwasoperatedwithwateras
eluentat85°Candaflowrateof0.5ml/min.Detectiontook
placewithanERMA-ERL7510refractive indexdetector,
thermostatedat40°C.
RESULTS
Adsorptionstudies
After incubationofvariousamountsofpurified endoglucanase
orexoglucanasewithafixed amountofAvicelcellulose thefree
proteinconcentration P (mg/ml)wasdetermined.Thequantityof
adsorbedenzymewascalculated andP a d s wasdefined asthe
amountofadsorbedproteindivided throughtheamountofcellulose (mgprotein/mgcellulose).Fromthesedatatheadsorption
isotherms (PadsvsP)wereconstructed fortheendoglucanases
(Fig.1)andexoglucanases (Fig.2).
Onthebasisoftheseisothermstwogroupsof endoglucanases
couldbedistinguished.Onegroup,consistingofEndo I,Endo
IIIandEndoV adsorbed stronglyoncrystallinecellulose,while
anothergroupofendoglucanases,EndoII,EndoIVandEndoVI,
adsorbedmoderately (EndoII)oronlyveryslightly.Fromthe
twoexoglucanases ExoIIIappearedtoadsorbmuch stronger than
ExoII.
Adsorptiondataof thecompletecellulasecomplexareknown
toobeytheLangmuir-typeadsorptionisotherm (10,15,22, 24):
K
p
p* pads,m
ads=
P
1+
[1]
K p .P
61
wherePisthefreeproteinconcentration (mg/ml); P a( j si s t h e
amountofadsorbedprotein (mgadsorbedprotein/mg cellulose).
p
adsmi s t h e maximalamountofadsorbedprotein (mgadsorbed
protein/mgcellulose)andKpistheadsorption equilibrium
constant (mg/ml) -1 .
0A
0.6
1.0
P(mg/ml)
Fig. 1.Adsorption isothermsofpurified endoglucanaseson
Avicelcrystalline cellulose (20rag/ml),o , EndoI;»,EndoII;
V,EndoIII;D, EndoIV;X, EndoV;&, EndoVI.
0.2
Hydrolysisproducts analysis
Thesixendoglucanaseswere incubatedwith2%Avicelcelluloseinafinalvolumeof 0.5mlat30°Cin0.05 Msodium
acetatebuffer for6hours.Theenzymeconcentrationwas60
ug/ml.Aftercentrifugationofthecellulose,aliguotsof 0.4
mlweretakenfromthesupernatant,theenzymeswere inactivated
byplacingthetubesinaboilingwater-bath for 5min.and
evaporatedwithdryair.Theresiduewasdissolved in75y.10.1
MPb(N03>2andcentrifugedtoremove solids.Productsinthe
supernatantwereanalysedbyHPLC (SpectraPhysicsSP8000)
usingtheAminexHPX 87Pcolumn (300x7.8 mm,Bio-Rad Labs,
Richmond CA,USA)coupledwithaguardcolumn,containing equal
amountsoftheion-exchange resinsAG 50W-X4 (H+)andAG3-X4A
(OH")(BioRad labs). Thecolumnwasoperatedwithwateras
eluentat85°Candaflowrateof0.5ml/min.Detectiontook
placewithanERMA-ERL7510refractiveindexdetector,
thermostatedat40°C.
RESULTS
Adsorption studies
Afterincubationofvariousamountsofpurified endoglucanase
orexoglucanasewithafixed amountofAvicelcellulose thefree
proteinconcentration P (mg/ml)wasdetermined.Thequantityof
adsorbedenzymewascalculated andP , wasdefined asthe
amountofadsorbedproteindividedthroughtheamountofcellulose (mgprotein/mgcellulose).Fromthesedatathe adsorption
isotherms (Padsv s p^ wereconstructed fortheendoglucanases
(Fig.1)andexoglucanases (Fig.2).
Onthebasisoftheseisothermstwogroupsof endoglucanases
couldbedistinguished.Onegroup,consistingofEndoI,Endo
IIIandEndoV adsorbed stronglyoncrystallinecellulose,while
anothergroupofendoglucanases,EndoII,EndoIVandEndoVI,
adsorbedmoderately (EndoII)oronlyveryslightly.Fromthe
twoexoglucanasesExoIIIappearedtoadsorbmuch stronger than
ExoII.
Adsorptiondataof thecompletecellulasecomplexareknown
toobeytheLangmuir-typeadsorption isotherm (10,15,22, 24):
K
p
ads=
p- pads,m
1
P
[1]
+K p .P
61
where P i s t h e free p r o t e i n concentration (mg/ml); P a ds i s t n e
amount of adsorbed p r o t e i n (mg adsorbed protein/mg c e l l u l o s e ) .
P a d s m i s t h e maximal amount of adsorbed p r o t e i n (mg adsorbed
protein/mg c e l l u l o s e ) andKp i s t h e adsorption equilibrium
constant (mg/ml) - 1
2 30
/
-
rfX'
.^
V^*
iff
.
y,*
—&
0.2
1.0
0A
0.6
P (mg/ml)
Fig. 1. Adsorption isotherms of purified endoglucanases on
Avicel c r y s t a l l i n e cellulose (20 mg/ml). o , Endo I ; • , Endo I I ;
V, Endo I I I ; Q , Endo IV; X, Endo V; A, Endo VI.
'0
0.2
0.3
0.4
P( mg/ml)
Fig. 2. Adsorption isotherms of purified exoglucanases on Avicel
c r y s t a l l i n e cellulose (20 mg/ml). O, Exo I I ; X, Exo I I I .
62
0.1
Rearrangement ofeq.[l]gives:
P
1
[2]
•ads
K
p
p* ads,m
p
ads,m
AplotofP/PadsversusPwillgiveastraight line,from
whichtheadsorptionparametersPads,ma n d K p c a n b e
calculated.Usingeq. [2]fortheexperimentaldataofadsorptionofthepurifiedendoglucanasesandexoglucanases,straight
lineswereobtained (Fig.3,4,5 ) ,indicating thatthesedata
fittheLangmuir-typeadsorption isotherm indeed.Adsorption
parametrers,obtained fromtheselinesarepresented inTable
I.
Reversibilityofadsorption isoneoftheconditionswhich
havetobeobeyed toattachanyphysicalsignificance tothe
adsorptionparameters obtained fromtheLangmuireq.[1].
Therefore reversibilitywasinvestigatedbythecentrifugation
ofcelluloseandreplacementofalargepartofthesupernatant,
containingnon-adsorbedprotein,byfreshbuffer.After
0.2
0.3
P(mg/ml)
Fig. 3.Langmuir-type adsorptionplotsofEndoI (O), EndoIII
(V)andEndoV (x)•
63
resuspendingthecellulose andfurther incubation,theamountof
releasedprotein (AP ads )wasmeasured andexpressedas
percentageoftheamountofprotein,whichtheoreticallycould
desorbatfullreversibility,usingtheLangmuir-type isotherm
(TableII).
0.4
0.6
P(mg/ml)
Fig. 4.Langmuir-type adsorptionplotsofEndo II (•),EndoIV
OandEndoVI <£)•
0.2
0.3
P(mg/ml)
Fig. 5.Langmuir-type adsorptionplotsofExoII (O)andExoIII
(X).
0
64
0.1
Table I.Adsorptionparametersandreactionkinetic parameters
ofpurified endo-andexoglucanases fromTrichodermaviride.
Enzyme
EndoI
EndoII
EndoIII
EndoIV
EndoV
EndoVI
Exo II
Exo III
p
R
K
v
ads,m
m
max
(mg.protein/
(umol/
(mg/ml)-1 mgcellulose) (mg/ml)~1(mg/ml) mg.min)
K,
0.88
0.28
11.67
2.50
0.89
3.44
4.96
6.92
0.126
0.090
0.026
0.0028
0.105
0.0041
0.0066
0.063
0.111
0.025
0.303
0.007
0.094
0.014
0.037
0.436
46.3
90.6
14.4
130.7
64.3
122.9
44.1
12.0
0.196
0.099
0.085
0.152
0.133
0.122
0.060
0.019
TableII.Degreeofreversibility ofadsorption.Amountof
desorbed enzymeprotein (APa(js)a t 1n°urdesorptionand
calculated values atfullreversible reaction;n.d.,not
determined.
Enzyme
Endo1
EndoII
EndoIII
EndoIV
EndoV
EndoVI
Exo II
Exo III
AP
% reversibility
ads
measured
calculated from
Langmuir i sotherm
1.10
2.19
1.82
n.d.
0.91
n.d.
n.d.
10.39
4.10
5.06
3.15
n.d.
1.99
n.d.
n.d.
13.81
27
43
58
n.d.
46
n.d.
n.d.
75
AdsorptionofEndoI,II,III,VandExoIIIwasonly
partiallyreversible.Evenalongerdesorptiontimeupto8
hoursdidnotincrease theamountofreleasedprotein (datanot
shown).ExoIIIgavethehighest levelofreversibility: 75%of
theamountofprotein,whichcould theoreticallydesorbatfull
reversible reaction,wasreleased.DataondesorptionofEndo
IV,VIandExoIIarenotgivenbecause theseenzymes adsorb
onlyslightly.Thereforedetectionofthesmallamountsofthese
enzymeswhichcoulddesorbwasnotpossible,using themethods
described.
65
Because adsorptionofatleastsomeoftheendoglucanasesand
exoglucanases isnotcompletelyreversible,nophysical
significance canbeattributed totheLangmuirconstants,
calculated fromeq.[2]andpresented inTable I.ThereforeKp
cannotinterpreted asanaffinityconstantofadsorption.Asa
parameterwhichdescribesthedegreeofinteractionbetweenthe
enzymesandthecellulose,Rwasintroduced anddefined asthe
slopeoftheisothermatitsorigin (P=0),whereno interaction
betweentheenzymemoleculestakesplace.Thisslopeequalsthe
inverseinterceptofthelinesobtained fromeq.[2] (Fig.3,4,
5), so:
R=K
p •pads,m
[3]
Rhasthedimensionof (mgcellulose/ml) '.FromTable 1it
canbeseenthatthestrongest interactionbetweenthe
endoglucanases andcellulosewasfoundforEndoIII.AlsoEndoI
andEndoV showed arelatively largeR-value.These latter
enzymeshave,incomparisonwithEndoIII,alarger P ac } Sm'
whichmeansthatmoreadsorption sitesonthecellulosechains
areavailable.However,inthecaseofEndoIandEndoV,the
affinityofenzymetosubstrate islowandhighinitial
concentrationsoftheseenzymesareneeded toreachsaturation.
SmallvaluesforRwereobtained forEndoII,IVandVI.
Although interactionbetweenEndoIIandAvicelisratherpoor,
relativelymanyadsoptionsitesforthisenzymearepresenton
thistypeofcellulose (highP a( j sm )•ForEndoIVandVI,
however,theamountofcellulosewhichwasused inthese
experimentswasalready saturatedbytheseenzymesatalow
proteinconcentration.ExoIIIisthemostabundant exoglucanase
inthecommercialcellulasepreparation fromwhichitwas
purified (19)andshowedaninteractionwithAvicelwhichwas
much stronger thanforExoII.Alsothesaturation levelof
adsorption forExoIIIwasabouttentimeshigherthanthevalue
found forExoII.
Kinetic studies
Hydrolysiskineticsbytheendo-andexoglucanaseswas
studiedusingAvicelcellulose asthesubstrate.Thekinetic
parametersK,^andV m a x wereobtained fromtheLineweaverBurkplotsoftheexperimentaldata (Fig.6,7)andpresented in
TableI.Forthedifferentendoglucanases thelowestK m values
were found forthoseenzymeswhichhaveahighinitialrateof
adsorption,i.e.EndoI,IIIandV (Table I ) .Thesameobserva-
66
tionwasmadeforthetwoexoglucanases.The lowerK m ofExo
IIIisattendedwiththehighervalue forR.
-0.06 -0.04
-0.02
0.08
0
0.02 0.04 0.06
1/S (mg/ml)
Fig.6.Lineweaver-Burkplotsofpurifiedendoglucanasesusing
Avicelcrystallinecellulose asthesubstrate,o,EndoI;
•,EndoII;V , EndoIII;D, EndoIV;x, EndoV;A , EndoVI.
X
120-
c 100E
en
E
^
yy
80-
B
>
y
/
*
S
A
/
/
/
y3
y
60
/
X
0
A>
/ 4 0 o
/
-0.08
20-
-0.04
0.04
1/S(mg/ml
0.12
i-l
Fig.7.Lineweaver-Burk plotsofpurifiedexoglucanases.
O,ExoII;X, ExoIII.
67
Hydrolysisproducts analysis
Hydrolysisproducts fromincubationsoftheendoglucanases
withAvicelcellulosewereanalysedonHPLCandpresentedin
Figure 8.EndoI,II,IIIandIVproduced amixtureofglucose,
cellobioseandcellotriose.Smallamountsofcellotetraosewere
found inthereactionproductsofEndoIVafteraveryshort
incubationtime (1hour;datanot shown). Onlyglucoseand
cellobiosewereobtained fromincubationswithEndoV andEndo
VI.Thepresenceoftransglucosidase activitycouldbeexcluded,
becauserelativepeakareasafter 2and 6hoursofincubation
werecomparable (datanot shown).
Thedifferenceswhichwereobserved inadsorptionbehaviours
oftheendoglucanases (Fig.1)didnotparallelthediversityin
hydrolytic actionoftheseenzymes.Fromtheendoglucanases I,
IIIandVwhichdoadsorbtoagreatextent,EndoIandIII
produce G2,G3andG4,whileEndoVproducesonlyG2and
G^.Ontheotherhand,fromtheendoglucanaseswhichboth
produceonlyG 2 andG^duringcellulosehydrolysis,one
(EndoV)adsorbsstronglyonitssubstrate,whiletheother
enzyme (EndoVI)doesnot.
Thedifferentdegreesofrandom attackduringcellulose
hydrolysisoftheseendoglucanases results inreactionmixtures
G,
10
200
10
200
10
20
elutiontime(min)
Fig. 8. HPLC analysis of products released from Avicel crystalline cellulose by Endo I ( a ) ,Endo II ( b ) ,Endo III ( c ) ,Endo IV
(d), Endo V (e) and Endo VI (f).G^, glucose; G 2 , cellobiose; G3, cellotriose.
68
withamoreorlessdiversecomposition (G4,G3,G2and
G-^)orinamorehomogeneous reactionproductcontainingonly
G2and G]_.However,itappearsthatthedegreeof randomness
isnotdetermined bytheadsorptioncharacteristics.
DISCUSSION
Severalinvestigators showed thatadsorptionofcellulaseon
crystallinecellulose isarelatively fastprocess.Ooshimaet
al. (15)stated thattheequilibrium ofadsorptionof
TrichodermaviridecellulaseonpureAvicelcellulosewas
reachedwithin 30minutesattemperaturesbetween 5-50°C.A
contacttimeof 15minuteswasfoundtobesufficienttoattain
a steady stateofadsorption inamixtureofTalaromvces
emersonii cellulase andanumberofcellulosic substrateswith
differentcompositions (12).Fortheexperimentsdescribedhere,
acontact timeof 1hourwasusedtoestablish equilibrium.
Theresultsclearlyshowthatthegroupofpurified
endoglucanases,aswellasthetwoexoglucanases,canbedivided
intotwosubclassesofenzymes:thoseenzymeswhichdoadsorb
strongly (EndoI,III,V,andExoIII)andthosewhichabsorb
onlyslightlyormoderately oncrystalinecellulose (EndoII,
IV,VIandExoII).Theexistenceofanon-adsorbingendoglucanasecomponent inacellulasecomplexofT.reeseiwasalso
observedbyRyuetal.(16).Theresults inthispaper indicate
thatsuchanon-adsorbing endoglucanasecomponentconsists
primarilyofdistinct,specificendoglucanases.
Oneoftheconsequencesofsuchaspecificadsorption forthe
recoveryofthecompletecellulasecomplexafter (incomplete)
cellulosehydrolysis,isthattheenzymecompositionofthe
adsorbed fractioncanbecompletelydifferent fromthecompositionoftheoriginalcomplex.Thisshouldbeconsideredwhen
recoveryofenzymes isdonebyadsorptiononfreshsubstrateas
suggestedbysomeauthors(11).
Alsothereversibilityoftheadsorptionwasexamined.It
appeared thatatleastapartoftheenzymemoleculeswas
irreversibly adsorbedonthecellulosechains,evenafterprolongeddesorptiontime.However,theexperimentaldataofthe
adsorptionexperiments,fortuitously fitted theLangmuir-type
adsorption isotherm (eq.[l]),whichmakes thisequationuseful
forcalculationsoftheamountsofadsorbedcellulaseprotein.
69
atthecelluloseconcentration (20mg/ml)used inthese
experiments.
Thisfindingwasusedtoevalutatethereactionkinetic
experiments.Itcanbeassumed thatataveryhighcellulose
concentration (S-»°°)alloftheavailableenzyme (P av )is
adsorbed tothesubstrate andthemaximalreactionrate (V max )
isachieved.Itisreasonable tosupposethat,going fromlowto
highsubstrateconcentration anincreaseofreactionrate
parallels anincreaseoftheamountofadsorbedenzyme.Leeet
al. (10)showed thatthiswastrueforadsorptionofthe
completecellulasecomplexofT_.reesei.Alsoassumingthatthe
distributionoftheactiveandpossible inactiveenzymes remains
unchangedduringadsoption,onecanexpectthat,atacertain
celluloseconcentration,thepartoftotalavailableenzyme
which isadsorbed,expressed asPacis/Pav'equalstheratio
ofactualreactionrateandmaximalreactionrate(i.e.
V/V m a x )atthatspecificcelluloseconcentration.This latter
ratiocanbeobtaineddirectly fromthekineticexperiments,
whileeqs.[l]makesitpossibletocalculate theratioof
adsorbed andavailableenzymeproteinatasubstrate
concentrationof20mg/ml.
Itcanbeconcluded fromTable IIIthatformostofthe
purifiedendoglucanases andexoglucanases theseratiosarenot
equivalent.Especially forthoseenzymeswhichdoadsorbtoa
greatextent i.e.EndoI,III,ivandExoIII,itcanbe
estimated thatalargepartoftheavailableenzymeisalready
adsorbed atthiscelluloseconcentration,whilethereaction
ratefortheseenzymesundertheseconditionswasonlyamuch
smallerpartoftheirV m a x . Forinstance fromEndoI69%of
theavailableenzymeproteinwasadsorbed,butonly 31%ofthe
Vmaxw a s achieved.
Table III.Partoftotalavaiblableenzymewhich isadsorbedand
partofmaximalreaction rateatacelluloseconcentration of20
mg/ml.
Enzyme
EndoI
EndoII
EndoIII
EndoIV
EndoV
EndoVI
Exo II
Exo III
70
P a d s / p av
0.69
0.33
0.85
0.12
0.65
0.21
0.38
0.90
v
/ v max
0.31
0.18
0.58
0.13
0.24
0.14
0.31
0.63
Anexplanation forthisdiscrepancymaybethatapartofthe
adsorbed enzymemoleculesarenotbound attheir catalyticsite.
Anotherexplanation isthattheseenzymesdo indeed adsorbat
theircatalytic site,butarenotabletoexpresstheirhydrolyticaction.Itmaybepossible thattheactivityofthese
enzymes -for instance anendoglucanase -willonlyfind
expression ifanotherenzyme,inthiscaseanexoglucanase,is
alsopresent.Ontheotherhandpartoftheexoglucanase
activitywillonlybemeasured ifanendoglucanasemolecule
adsorbsatthesamespotandcreatesanewchain-end.This
phenomenonmayexplainpartially thesynergismbetweenendo-and
exoglucanases.
Inaprevious study (Chapter 4)weshowed thatdifferent
degreesofrandomness intheattackofcellulosechainsby
endoglucanases exist (19).Thisisexpressed astheratioofthe
liquefying activity tothesaccharifying activity.Thevalueof
thisratiowasshowntobethehighest forEndoI,EndoIIand
EndoIV.Theresults inthispaper showthatthesecellulases
indeed arethemostrandomtypesofthesixendoglucanaseswhich
werepurified fromTrichodermaviride,because aconsiderable
amountofcellotriosewasproducesbytheseenzymes.Low
liquefying/saccharifying valueswereobtained forEndoVand
EndoVI,which isconsistent tothefactthattheseenzymesgave
onlycellobioseandglucoseashydrolysisproduct.Onecould
expectthatanenzymewhich istightlyadsorbed atitssolid
substrate islessmobile andtherefore lessrandom inhydrolytic
actionthananenzymewhichadsorbsonlyslightly.However,no
suchrelationship couldbeobserved forthe endoglucanases
investigated inthisstudy.Randomness isratherdetermined by
thefacthowtheenzymestructurally bindstoitssubstrate,
thanbyhowstrongly itbinds.Theextremely lowdegreeof
randomness incellulose attackbyEndoIII (19)isincontrast
with theheterogeneouscomposition of itshydrolysisproduct
(G3,G2and G^). Possiblythiscanbeexplainedbya
singlechainmultiple attackmechanism ofthisenzyme,contrary
tothemultiplechainattackoftheotherendoglucanases.
71
NOMENCLATURE
P
p
ads
Pav
p
adsm
Kp
R
V
v
max
Km
freeenzyme (mg/ml)
adsorbedenzyme (mg/mgcellulose)
availableenzyme (mg/mgcellulose)
maximum adsorbedenzyme (mg/mgcellulose)
adsorptionequilibrium constant (mg/ml) -1
initialrateofadsorption (mgcellulose/ m l ) - 1
actualreactionrate (umol/mg.min)
maximalreactionrate (umol/mg.min)
Michaelis-Menten constant (mg/ml)
REFERENCES
1.Mandels,M.,Meideiros,J.E.,Andereotti,R.E.andBisset,
F.H.,Biotechnol.Bioeng.1981,23,2009-2026
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Bioeng.1981,23,1837-1849
3.Shin,S.B.,Kitakawa,Y.,Suga,K.I.andIchikawa,K.,
J. Ferment.Technol.1978,56_,396-402
4.Wood,T.M.,Biochem.J. 1968,109_,217-227
5.Eriksson,K.E.andPettersson,B.,Eur.J.Biochem.1975,
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8.Ohmine,K.,Ooshima,H.andHarano,Y.,Biotechn.Bioeng.
1983,25,2041-2053
9.Lee,Y.H.andFan,L.T.,paperpresented attheSixthInternationalFermentation Symposium,London,Ontario,Canada,
1980
10.Lee,S.B.,Shin,H.S.andRyu,D.D.Y.Biotechnol.
Bioeng.1982,24. 2137-2153
11.Castanon,M.,andWilke,C.R. Biotechnol.Bioeng.1980,22,
1037-1053
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271-280
13.Goel,S.C.andRamachandran,K.B.J.Ferment.Technol.1983,
61,281-286
14.Deshpande,v.,Mishra,c , Ghadge,G.D.,Secta,R.andRao,
M.,Biotechnol.Lett.1983,5,391-394
15.Ooshima,H.,Sakata,MandHarano,Y.,Biotechnol.Bioeng.
1983,25,3103-3114
16.Ryu,D.D.Y.,Kim,C.andMandels,M.,Biotechnol.Bioeng.
1984,26,488-496
17. Lindner,W.A.,Dennison,C.andQuicke,G.V.Biotechnol.
Bioeng.1983,25,377-385
18.Canevascini,G.andGattlen,C.Biotechnol.Bioeng.1981,
23,1573-1590
19.Beldman,G.,Searle-vanLeeuwen,M.F.,Rombouts,F.M. and
Voragen,A.G.J.,Eur.J.Biochem. 1985,146,301-308
72
20.Lowry,O.H.Rosebrough,N.J.,Farr,A.L.andRandell,R.J.,
J.Biol.Chem.1951,193,262-275
21. Somogyi,M.,J.Biol.Chem. 1952,195,19-23
22. Peitersen,N.,Medeiros,J. andMandels,M.,Biotechnol.
Bioeng. 1977,19,1091-1094
23.Reese,E.T.,Process Biochemistry May/June,2-6 (1982).
24. Stuart,J.Y. andRistroph,D.L.,Biotechnol.Bioeng.1985,
27, 1056-1059
73
6 Synergismincellulose hydrolysis byendoglucanases and
exoglucanasespurified from Trichoderma viride
Submitted for publication in Biotechnol. Bioeng.
SYNERGISM INCELLULOSEHYDROLYSISBYENDOGLUCANASESANDEXOGLUCANASESPURIFIEDFROMTRICHODERMAVIRIDE
G.Beldman,A.G.J.Voragen,F.M. RomboutsandW.Pilnik.
Department ofFoodScience,AgriculturalUniversity,Wageningen.
Synergistic effectsincellulosehydrolysisbetween sixendoglucanases (EndoI,II,III,IV,VandVI)andtwoexoglucanases
(cellobiohydrolases ExoIIandExo III),whichwerepurified
fromacommercialcellulasepreparation,derived from
Trichodermaviridewere investigated.Especially ExoIIIacted
strongly synergisticallywithallendoglucanases.Thedegreeof
synergistic effectwas largelydependentupontheinitialratio
ofendoglucanase toexoglucanase andmaximalataspecific
value,whichcouldqualitativelyberelated totheadsorption
behavioursoftheendoglucanases.Thoseendoglucanaseswhich
adsorbstronglyoncrystallinecellulose (EndoI,IIIandVI)
needed tobeused insmalleramounts toreachmaximaldegreeof
synergism,thanEndoII,IVandVIwhichwehavefoundareto
adsorb less.Theseresults indicate thatonlythose
endoglucanase andexoglucanasemoleculeswhichareadsorbed on
thecellulosechainsasacomplexwithaspecific ratioofboth
enzymesareinvolved insynergistic effectsoninsoluble
crystallinecellulose.
Probablybecauseofcellobiase activity,another exoglucanase
(ExoI)showed synergistic actionwhenworking inconcertwith
ExoIIandExoIII.
INTRODUCTION
Oneofthemostintriguingphenomena incellulosehydrolysis
bythecellulasesofseveralfungiisthesynergistic action
betweentheindividualcomponentsoftheseenzymemixtures.The
enzymeswhichplayamainroleinsynergism aretheendoglucanase [1,4-p-D-glucanglucanohydrolase (EC3.2.1.4)]andthe
exoglucanase [1,4-p-D-glucancellobiohydrolase (EC3.2.1 .91.)].
Bothtypesofenzymeswerefoundtoexistinmultiple formsin
a.o.theculture filtratesofTrichodermaviride (1-3),
TrichodermaKoningii (4,5 ) ,Sporotrichumpulverulentum(6),
76
Penicillium funiculosum (7, 8), Fusariumsolani (9)and
Talaromycesemersonii(10).
Synergism incellulosehydrolysisbythecellulasecomplexof
severalofthesefungihasbeendemonstrated by recombination
experimentsofthepurifiedendo-andexoglucanases (4,5).Also
"cross-synergism"hasbeenobservedbetweentheendo-and
exo-actingenzymes fromdifferentmicrobialorigins (7).
Synergism intheenzymaticdegradation ofcrystalline cellulose
isnotsimplyexplained bythesequential attackofendoglucanasesandexoglucanases.Forinstance itwasfoundbyWoodet
al.(5)thatoneofthefourendoglucanases fromT_.Koningii,
whichwasthemostrandomactingenzyme incellulose attack,and
thereforeexpected tocreaterelativelymanychain-ends foran
exo-glucanasetoacton,showedonlyvery little synergistic
effectwhenworking togetherwiththecellobiohydrolase from
this fungus.Theseauthorsstatethattheformationofacomplex
ofendoglucanase andcellobiohydrolase onthesurfaceofthe
cellulasechain isessential for synergism.
Fromthemechanisticmodeldescribed byOkazakiet al. (11)
itwasconcluded thatthedegreeofsynergistic effect (DSE),
which isdefined astheratiooftheobserved activityofthe
combined endo-andexoglucanase enzymes,tothesumofthe
observed individual activities,largelydependsontheratioof
endoglucanase andexoglucanase,aswellasonthedegreeof
polymerizationofthesubstrate.Themodelwasused topredict
synergisticeffects,usingkineticdataobtained from
incubationswithsoluble substrates.Thevalueofthismodelto
predictadegreeofsynergistic effect (DSE)during hydrolysis
ofinsolublecrystalline cellulose isquestionable.
Anexplanation forsynergism fromanotherpointofviewwas
givenbyRyuetal. (12 ),who studied thesequential adsorption
oftheendoglucanase andexoglucanase componentsofT.reesei
and foundthatsynergismwasgradually "introduced" intothe
non-adsorbed enzymefractionbycompetitive adsorptionofoneof
theglucanases.Theseinvestigators statethatsynergism ofthe
cellulasecomplex remainsundisturbed duringadsorption,but
shownodataonsynergismbetweencellulases intheadsorbed
fraction.Theworkwasdonewithpartiallypurifiedenzymes,
which stillcontained severaliso-enzymes.Intwoprevious
paperswedescribed thepurification andbiochemicalcharacterizationofallendoglucanases andexoglucanases ofacommercial
enzymepreparation fromT_.viride (1),aswellastheadsorption
andkineticbehaviours oftheseenzymes (13).Thispaper
77
presentstheresultsoftherecombinationexperimentswiththese
purifiedenzymes.Thesynergistic effectswhichwereobserved
couldpartiallybeexplained inthelightofthereported
adsorptionbehavioursofthesecellulases.
MATERIALSANDMETHODS
Enzymepreparations
MaxazymeCIisacommercialcellulasepreparation from
TrichodermavirideoriginandwasprovidedbyGist-Brocades,
Delft,theNetherlands.Fromthispreparation sixendoglucanases
(EndoI,IIIII,IV,V andVI)andthreeexoglucanases (ExoI,
ExoIIandIII)were isolated asdescribed inapreviouspaper
(1).
Recombination experiments
Endoglucanases incombinationwithExoIIIonAvicel
cellulose.Fromtheendoglucanasepreparations (EndoI,II,
III,IV,V,VI)afixed amountof90ugenzymewascombinedwith
anamountofExoIIIranging from6to200ugprotein.Incubation
with 0.5%Avicelcellulose (TypeSF,Serva,Heidelberg,F.R.G.)
tookplaceundercontinuouslymixingonarollerdrumduring20
hoursat30°Cin0.05 M sodiumacetatebuffer.Thefinal
volumewas1ml.Inaparallelexperiment theendoglucanasesand
ExoIIIwereincubated separatelyusingthesameconditionsas
inthecombinationexperiment.After incubationthecellulose
wascentrifuged off (10min.,3000xg)andthesupernatantwas
analysed onreleased reducing sugarsbythemethodofNelson
Somogyi (14)aswellasontotalsugarsbythemethodofDubois
et al.(15).
EndoIVincombinationwithExoIIIonH^PC^-swollen
cellulose.RecombinationofEndoIVwithExoIIIwascarried
outasdescribed above,using 0.5%^PC^-swollencellulose
insteadofAvicel.The^PC^-swollencellulosewasprepared
bythemethod ofWood(16).
FixedamountsofendoglucanasesorExoIincombination
withExoIIandExoIIIonAvicelcellulose.Endoglucanases
orExoI (25ug)werecombinedwith 25ugofrespectively ExoII
orExoIII.Incubationofthecombinations aswellasofthe
separateenzymestookplaceduring 20hoursat30°Cina
volumeof1ml0.05MsodiumacetatepH5.0with0.5%Avicel
cellulose.Afterremovalofresidualcellulose,theamountof
78
reducing sugarsinthesupernatantwasdetermined.
EndoIIIincombinationwithEndoIonAvicelcellulose.
EndoIIIwasrecombinedwithEndoI,using90ugofboth
enzymes,andincubated at30°Cwith0.5%Avicelduring20
hours.Thefinalvolumewas1ml0.05Msodium acetatepH5.0.
The sameincubationwascarriedoutwiththeseparateenzymes.
Productswereanalysedasdescribedabove.
EndoglucanasesorExoIIIincombinationwithMaxazymeCl
onAvicelcellulose.MaxazymeCl,inafinalconcentrationof
500ug/ml,wascombinedwiththeindividualendoglucanases,
respectivelyExoIII,andincubatedat30°Cin0.05Msodium
acetatebufferpH5.0.Thepurifiedglucanaseswereusedina
concentrationof77ug/ml.After incubationduring 20hoursthe
cellulosewascentrifuged andtheamountofreducing sugarsin
thesupernatantwasdeterminedandcomparedtothevalues
obtained fromseparate incubationsofMaxazymeClandpurified
cellulases,usingthesameconditions.
RESULTS
Endoglucanases incombinationwithExoIII
Recombinationofthepurified exoglucanaseExoIIIand
endoglucanases EndoI,II,III,IV,VandVIresulted inall
casesinasynergistic effectafterincubationwithAvicel
crystalline cellulose.Thedegreeofsynergistic effect(DSE)
couldbecalculatedbycomparingtheactivityofthe
combinationstothesumoftheactivitiesoftheindividual
enzymes.Byusingafixedconcentrationoftheendoglucanases
andavaryingconcentrationofExoIIIitwasfound
thattheDSEdependsupontheendoglucanase toexoglucanase
ratio.
Figure1showsthatforallcombinationsofendoglucanase
withExoIIIamaximumDSEwasobservedataspecific ratioof
theseenzyms.ThemaximalvalueofDSEwasdependentuponthe
methodsofanalysisofthehydrolysisproducts.Analysisof
released reducing sugars,whichgivesavalueforevery
glucosidic bondwhichisenzymaticallycleaved,resultedin
maximalDSEvaluesvaryingbetween1.60 (Fig.1C)and3.45(Fig.
IE).
DeterminationoftotalsugarsbythemethodofDubois
representsasolubilizationeffectoftheenzymatic degradation
ofcelluloseandgives,withtheexceptionofthecombinationof
79
B
12.5-
ty
^
^Y—X
a
2.0-
/
-
\
X
/
XV
\
1.5-
1.0-1
2.5-
2.5
2.0-
2.0
1.5-
'x^
\
\ 1-5-
4
"A.
1.0-1
2.5
4.0-'
A/-A„
A—&
/
3.0A'
X
'ASA
2.0
/
*Nt
^
2.0-
1.5X\
1.0J
1.0
10.0
1.0
10.0
Endo/Exo ( g / g !
Fig. 1.Degreeof synergistic effect (DSE),obtained after
recombinationofExoIIIwithvarious amountsofEndoI(A),
EndoII (B),EndoIII (C),Endo IV (D),EndoV (E)andEndoVI
(F),duringhydrolysisofAvicel (A-F)andH3PO.<}-swollen
cellulose (D).Productsweremeasured asreducingsugars (RS)as
T
well astotalsugars (TS).A , Avicel,RS; y.,Avicel,
S ;O,
H3P04-swollencellulose,RS;D #H3P04-swollencellulose,TS
80
EndoIIIwithExoIII (Fig.1C)lowermaximalDSEvaluesbetween
1.75 and 2.45.Theweight-ratioofendoglucanase toexoglucanase
wheremaximalDSEoccurs,isgiveninTableI.Takeninto
accountthemolecularmassesoftheseenzymes (1),alsothe
molarratioscouldbecalculated. Inourpreviousstudyon
adsorptionofpurified endoglucanases andexoglucanases,we
showed thatEndoI,IIIandVadsorbstronglyonAvicel
cellulose.Especially theseenzymes showtheirmaximalDSEata
much lowerendoglucanase toexoglucanase ratiothanEndoII,IV
andVIwhenworking inconcertwithExoIII.
Synergistic effects inhydrolysisofamorphousH3PO4swollencellulosewasinvestigated forthecombinationofEndo
IVandExo III.FromFigure IDitcanbeseenthatthecombinationoftheseenzymesresults inDSEvalues,whicharemuch
lowerthanthosevaluesobtained inhydrolysisofAvicel
cellulose.Alsoamuchbroadermaximumwasobserved,atalower
Table I.Initialendoglucanase toExo IIIratios,atwhich
maximalDSEwasobserved under theexperimentalconditions.
ExoIIIin
with
EndoI
EndoII
Endo III*
EndoIV
EndoV*
EndoVI
combination
Endo/Exo III
mass ratio
molar ratio
1.5
3.75
2.50
5.0
2.0
2.75
1.9
5.2
2.7
13.2
2.2
3.3
Adsorb stronglyonAvicelcellulose(13).
Table II.DSE-valuesobtained forthecombinationofendoglucanasesandExoIwithExoIIandExoIIIusinganenzyme
mass ratioof1.
Combinationwith
Enzyme
EndoI
EndoII
EndoIII
EndoIV
EndoV
EndoVI
Exo I
ExoII
ExoIII
0.9
1.4
1.0
1.2
1.3
1.3
1.4
1.5
2.1
1.2
1.7
2.0
1.6
3.5
81
endoglucanase toexoglucanaseratio.
Endoglucanases andExoIincombinationwithExoIIandExo
III.Thetwocellobiohydrolases ExoIIandExoIII,whichwere
isolated fromMaxazyme CI,werecomparedwith respecttotheir
capacities toactsynergisticallywhenworking togetherwiththe
endoglucanases andExoIonAvicelcellulose (TableII).
DSE-valuesmeasured forthecombinations ofExoIIwiththese
enzymeswere foundtobelowerthanthevaluesobtained ina
similarexperimentusingExoIII.Actuallynosynergismwas
found forthecombinationofExoIIwithEndoIandEndoIII.
Theenzymeconcentrationused intheseexperimentswas25ug/ml
foreachenzyme (Endo/Exo= 1). Atthisproteinconcentrationa
combination ofanendoglucanasewithExoIIIexhibited alower
DSE-valuethanobserved inthepreviousexperiment inwhicha
proteinconcentrationof 90ug/mlwasused forbothenzymes.Exo
I,whichwe foundtobeanenzymeabletorelease cellobiose
fromcellulose andsubsequently hydrolyse thisproductto
glucose,worked strongly synergisticallywithExoIII.
EndoIIIincombinationwithEndoI.Inapreviouspaper
(1;Chapter 4)somedoubtsremainedontheendo/exoglucanase
natureofEndo III.Inorder toinvestigatewhether ornotEndo
IIIisatrueendoglucanase,theabilityofthisenzymetoact
synergisticallywithEndoIwasstudied.EndoIwasshowntobe
a trueendoglucanase (1).ADSE-valueof 0.97wasfound,indicating thatthesetwoenzymes arenotabletoact synergistically
andEndoIIIcannotbeclassified asanexoglucanase.
Endoglucanases andExoIIIincombinationwithMaxazyme CI.
Thesynergistic effectswhichcouldbeobtainedwithacombinaDSE
Exom
EndoI
EndoU
EndoIII
EndoE
EndoY
EndoH
0.6
Fig. 2.Degreeofsynergistic effect (DSE),obtained after
recombinationoftheoriginalcellulasepreparationMaxazymecl
withpurified endoglucanases andExoIII.
82
tionoftwopurified cellulases,wasalsostudied inmuchmore
complexmixtureofthesetypesofenzymes.Maxazyme Cl,the
originalcellulasepreparation fromwhich theendo-andexoglucanaseswerepurified,wassupplementedwiththeindividual
enzymesandinvestigated onitsAvicelhydrolysing capacity,in
comparisonwiththeactivitiesobtained inseparateincubations.
Figure 2showsthatendoglucanases II,III,IV,VandVIare
abletoincreasetheactivityoftheoriginalcellulase
preparation,byacting synergisticallywith it.TheDSE-values
obtainedvariedbetween 1.06 foracombinationwithEndoIIto
1.24whenEndoIIIwascombinedwithMaxazymeCL.EndoIandExo
III,however,gaveanegative synergistic effectwhen
supplemented totheoriginalcellulasepreparation.
DISCUSSION
Itisshownthatallsixendoglucanases (EndoI,II,III,IV,
V,VI)whichwere isolated fromthecommercial cellulase
preparationMaxazyme Cl,canactsynergistically incellulose
hydrolysiswiththecellobiohydrolase ExoIII,obtained fromthe
samepreparation.Thedegreeofsynergistic effect (DSE)fora
specificcombinationofendo-andexoglucanase,isatleast
dependentupontwomain factors:firstlytheratioinwhichboth
enzymes arecombined andsecondlythenatureofthesubstrate
whichisused (intheseexperimentscrystallineAvicelor
amorphousH3PC>4-swollencellulose).
ThefactthattheDSEobtainsamaximalvalueatonespecific
endoglucanase toexoglucanase ratioisingoodagreementwith
thetheoreticalmodelofOkazaki (11),who stated thatenzymatic
cellulolysis isstronglyaffectedbythisratio,aswellasby
thedegreeofpolymerizationofthesubstrate.However,these
authorsusedtheirmodelonthebasisofsolublesubstrates,
assuminghomogeneous reactionconditions.When insoluble
crystalline cellulose isused,thereaction isheterogeneous and
another factor isintroduced: theselective adsorption
characteristics oftheindividualendo-andexoglucanases.
Theresults,presented inthispaper,showthatfor
hydrolysisofAvicelcellulosetheoptimal initial endoglucanase
toexoglucanase ratio isgreatlydependentupontheadsorption
behavioursoftheseenzymes.EndoI,EndoIIIandEndoV,which
enzymesareknowntoadsorbstronglyonAvicel (13),exhibited
83
theirmaximalsynergistic effectwithExoIII (whichalso
adsorbs stronglyonAvicel)atamuch lowerinitialendo-to
exoglucanase ratiothaninthose incubationswhereEndoII,Endo
IVandEndoVIwereused. Itwasshowninourpreviousworkthat
these latterendoglucanases adsorbonly slightlyonAvicel.The
observationscanbeexplained byassumingthatonlythose
cellulaseswhichareadsorbed ontheinsolublecellulosechains,
participate intheformationofacomplexofendo-andexoglucanase,which isresponsible forthesynergisticeffect.In
agreementtothefindingsofWoodetal. (4),theactivityofan
exoglucanase inthiscomplexcanonlyfindexpressionafterthe
formationofanewchain-end bytheendoglucanase.Ontheother
handtheactionofanendoglucanase canonlybeexpressed ifit
isimmediately followedbytheremovalofacellobiosemolecule,
astheresultoftheactionofanexoglucanase,inorderto
preventthereformationofthefirstbrokenbond.
Endoglucanases suchasEndoII,EndoIVandEndoVIneedto
beaddedinarelativelyhighconcentration tocreatea"driving
force"which isstrongenoughfortheadsorpionofatleasta
partoftheavailableenzymemoleculesandtheformationofthe
endoglucanase-cellobiase complex.Theresultsindicatethat
maximalDSEoccursataspecificoptimalratioofadsorbedendoglucanasesandexoglucanases.Itisvery likelythattheendoandexoglucanase needtobepresent insuchacomplex ina1 :1
ratio,inordertoobtainamaximalDSE.
Itisclearthatthisoptimumdependsmoreupontheadsorptioncharacteristics,thanuponthehydrolytic nature (specific
activity,degreeofrandomness,oligosaccharide compositionof
theproduct)oftheindividualenzymes.These latterproperties
oftheenzymesdetermine a.o.thedifferentDSE-valuesat
optimalratioofthevariouscombinations aswellasthe
differencesbetweenDSE-values obtainedbymeasurementofthe
productasreducing sugars (bond-breaking activity)ortotal
sugars (solubilization activity)foronespecificcombination.
The factthatEndoIII,incombinationwithExoIIIgivesa
relatively lowDSE-valueattheoptimalratio,mayberelatedto
thelowdegreeofrandomness incellulose attackofthisenzyme
(1).Wesuggested thatthisenzymeactsaccording toasingle
chainmutipleattackmechanism (13),which isinaccordanceto
thefactthattheDSE-value,measured asbond-breaking activity
andassolubilization activity,differonlyslightly forthe
combinationofthisenzymewithExo III.Thismaybeexplained
bythefactthatEndoIIIismoreor lessrestricted tothesame
84
cellulosechaininacrystallinematrix and solubilization
parallelshydrolytic activity. Incontrast,theactionofthe
otherendoglucanases (EndoI,II,IV,V,VI)incontrary,which
jumpfromchaintochain (multiple-chain attack),willnot
always leadtosolubilization,because thechainisfixed ina
crystallinematrix.However,itcanresult inanewchain-end
foranexoglucanase toacton,creating newmeasurable reducing
end-groups.
ThefactthatsynergismbetweenEndoIVandExoIIIon
amorphous^PC^-swollencellulose islessstrikingthanon
Avicelisingoodagreementwithdatafromtheliterature (12).
WealsofoundthatDSEonthistypeofamorphouscellulosewas
toalesserextentdetermined bytheratioofbothenzymes,
which isexpressed inabroadermaximum (Fig. ID). Probablymore
encounters ofoneoftheseenzymesandthistypeofmoreorless
soluble substrate lead tothereleaseofameasurableproduct.
Thiseffect isonlyslightly increasedbytheadditionofthe
complementaryenzyme.Thistypeofsynergism takesplacemore
ofteninsolution (solublesynergism)while synergism onAvicel
especially takesplaceonthecrystalline surface (insoluble
synergism).
Theendoglucanase natureofEndoIIIwasdiscussed inour
previous study (1)sincethisenzymeexhibited arelatively low
activity towardsCM-celluloseandahighactivitytowards
Avicel,comparedwiththeotherendoglucanases.BytheobservationthatsynergismwaspositivewithExoIIIandnegativein
combinationwithEndoI,weconfirm itsendoglucanasenature.
Thesynergistical actionofExoIwiththecellobiohydrolyses
ExoIIandExoIIIcanatleastpartiallybeexplainedbythe
factthatcellobioseproducedbythese latterenzymesis
hydrolysedbyExoItotwoglucoseunitsgivingmore reducing
sugarsandeliminatingpossibleproduct inhibition.However,it
maybepossiblethatsynergism between theseexoglucanasesis
similar tothesynergistic actionoftwo cellobiohydrolases
describedbyFagerstamandPettersson (17),althoughthese
authorsalsofoundglucose intheenzymedigest,suggesting
similarities toourobservations.
Synergism canalsobeobservedafter additionofpurified
cellulases toamorecomplexmixtureoftheseenzymes.
SupplementingMaxazymeCIwiththeendoglucanases II,III,IV,V
andVIresulted inahigher activitythanthesumof activities
obtained intheseparate incubations.Onecanconcludethat
MaxazymeCIisdeficient intheseendoglucanases.However,this
85
effectcannotbegeneralized sinceadditionofEndoIgavea
negative synergistic effect,indicating againthatsynergismis
morethansimplesuccessive actionofendoglucanase and
exoglucanase,butrather related tootherproperties,suchas
adsorptionnature,oftheseenzymes.Duringhydrolysisof
cellulosebycomplexmixturesofcellulases,suchasculture
filtratesandcommercialenzymepreparations,therewillbea
competitionbetween theindividualenzymestooccupythe
available adsorptionsitesandtoformtheendoglucanase/
exoglucanase complex,mentioned above.Taking intoaccountthat,
inthecaseofT_.viride,atleasteightenzymeswithdifferent
adsoptionparameters areinvolved,onecanconclude thatitis
verydifficulttopredicttheoptimalcompositionofanenzyme
mixture inorder toobtainmaximalsynergism.
REFERENCES
1.Beldman,G.,Searle-vanLeeuwen,M.F., Rombouts, F.M. and
Voragen,A.G.J.,Eur.J.Biochem.1985,146,301-308
2.Schoemaker,S.P. andBrown, R.D., Biochem. Biophys. Acta
1978,523,133-146
3.Schoemaker,S.P. andBrown, R.D., Biochem. Biophys. Acta
1978,523,147-161
4.Wood,T.M.andMcCrae,S.I.,Proc.Bioconvers.Symp.I.I.T.,
NewDelhi,1977,pp111-141.
5.Wood,T.M. andMcCrae,S.I.,Biochem.J. 1978,171,61-72
6.Eriksson,K.E.andPettersson,B.,Eur.J.Biochem.1975,
51,193-206
7.Wood,T.M.,McCrae,S.I.andMacfarlane,C.C., Biochem. J.
1980,189,51-65
8.Wood,T.M. andMcCrae,S.I.,Carbohydr.Res.1982,110,291303
9.Wood,T.M. andMcCrae,S.I.,Carbohydr.Res.1977,57,117133
10.McHale,A.andCoughlan,M.P.,FEBS-Lett.1980,117,319-322
11.Okazaki,M.andMoo-Young,M.,Biotechn.Bioeng.1978,20,
637-663
12.Ryu,D.D.Y.,Kim,C.andMandels,M.,Biotechn.Bioeng.
1984,26^,488-496
13.Beldman,G.,Voragen, A.G.J., Rombouts, F.M., Searle-van
Leeuwen,M.F.andPilnik,W.,accepted forpublicationin
Biotechn.Bioeng.1986
14. Somogyi,M.,J.Biol.Chem.1952,195,19-23
15.Dubois,M.,Gilles,K.A.,Hamiltion,J.K., Rebers,F.A. and
Smith,F.,Anal.Chem. 1956,28,350-356
16.Wood,T.M., Biochem.J. 1971,121,353-362
17. Fagerstam,L.G. andPettersson,L.G., FEBSLett.1980,153,
113-118
86
7 Specific and non-specific glucanasesfrom Trichoderma
viride
Submitted for publication in Biotechnol. Bioeng.
87
SPECIFICANDNON-SPECIFIC GLUCANASESFROMTRICHODERMAVIRIDE
G.Beldman,A.G.J.Voragen,F.M. Rombouts,M.F.Searle-van
LeeuwenandW.Pilnik.
Department ofFoodScience,AgriculturalUniversity,Wageningen
Sixendoglucanases (EndoI,II,III,IV,Vand VI), threeexoglucanases (ExoI,IIandIII)anda(3-glucosidase (3-glucI)
whichwereisolatedfromacommercialcellulasepreparationof
Trichodermavirideorigin,wereexamined astotheir activities
onxylanexoatspelts.EndoI,IIandIII,aswellasExoII
and IIIshowednoactivitytowardsxylanandwereclassified as
specificglucanases.Lessspecificitywasfound fortheendoglucanasesEndoIV,VandVI,ExoIand[3-glucIwhichenzymes
wereabletohydrolysexylan.Withrespect toproduct formation
thesexylanolytic cellulasesfittheclassificationofxylanases
generally accepted intheliterature.Kineticexperimentwith
xylan,CM-celluloseandp-nitrophenyl-p-D-glucosiderevealed
thatEndoIV,VanVIandExoIprefer tohydrolyse(3-1,4-Dglucosidic linkages,p-glucIshowednoclear substratepreference.
INTRODUCTION
Manymicro-organisms areabletoproduceawidevarietyof
polysaccharide degrading enzymes.Importantgroupsofthese
typesofenzymesarecellulasesandxylanases,becausetheycan
potentiallybeemployed toutilize twoofthemostabundant
polysaccharides,respectivelycellulose (1-3)andxylan (4-6).
Hydrolases fromfungaloriginhavebeenstudied extensively.
Manyauthorsreported onthepurification and characterization
ofcellulases (7-14)andxylanases (4,5,15-19)fromthese
sources.Substratecross-specificity ofcellulases andxylanases
isoftenobserved.Forinstance smallamountsofcellulase
activitywerefoundinhomogeneousxylanases,purified from
crudeenzymemixturesobtained fromAspergillus niger (4,18,
19), Trichodermaviride (15)andOxiporussp. (15).Ontheother
handhomogeneous cellulases fromcommercialenzymepreparations
derived fromIrpexlacteus (9)orTrichodermaviride (8)
88
exhibited somexylanaseactivity.Isoelectric focussingofa
mixtureofendo-l,4-p-glucanasesandendo-l,4-|3-xylanasesfrom
Trichodermareesei showed thepossibility todistinguishbetween
specificxylanases,specificglucanases andnon-specific
glucanases,applyingazymogrammethodwithdyed substrates(5).
Thereissomedebateonthenatureofthenon-specific
glucanases found intheculture filtratesofTrichoderma.Sprey
andLambert (20)wereabletosplitacellulasecomplex,which
washomogeneous onisoelectric focussing,intoseveralproteins
withdifferent isoelectricpointsanddistinctactivitieson
CM-cellulose,xylanand 4-methylumbelliferyl-p-D-glucoside.A
mixtureof6Mureaand 1%octylglucosidewasneededtosplitthe
complex.Theseresults indicate thatthefungusreleasesa
tightlyboundcomplexofseveralhydrolases inordertoattacka
multi-componentsubstrateofplantcell-wallpolysaccharides.
ShikataandNisizawa (8)alsofoundacellulasewhichshowed
xylanase and(3-glucosidaseactivity.However,these
investigatorsconcluded fromacompetitionexperiment,using
CM-cellulose,xylanandp-nitrophenyl-|3-D-glucoside,thatthese
threesubstratesreactatthesameactive siteoftheenzyme.
Thisconclusion issupportedbythefactthatboth substrates
haveananalogous structure.Themainchainofxylanconsistsof
p-D-xylopyranosylunits,whichare1,4-fS-linked,comparableto
thep-1,4-linkedglucose residuesofcellulose.However,xylan
isamoreheterogeneouspolysaccharide andusuallybranched(5).
Dependentonthesourcefromwhich itisextracted,xylan
containesbrancheswithvariousamountsofL-arabinofuranosyl
andD-xylo-,D-gluco-,andD-galactopyranosylunits.Also
D-glucuronicand4-0-methyl-D-glucuronicacidresiduesare
linkedtothemainchain.Moreover,xylans fromseveralkindsof
woodareesterifiedwithaceticacid.
Inprevious reportswedescribed thepurificationand
characterization ofsixendoglucanases,threeexoglucanasesand
a p-glucosidasefrom Trichodermaviride (7).Alsothe
adsorptionandreactionkinetics (21),aswellasthe
synergisticbehaviours (22)oftheseenzymesweredescribed.The
aimofthepresentworkwastoinvestigate thespecificor
non-specific natureoftheseglucanases,bydeterminationof
theiractivitiestowardsxylanandseveralxylo-andarabinoxylo-oligosaccharides.
89
MATERIALSANDMETHODS
Enzymepreparations
Sixendoglucanases (EndoI,II,III,IV,VandVI), three
exoglucanases (ExoI,IIandIII)anda3-glucosidase ((3-glucI)
werepurified fromMaxazymeCL (Gist-Brocades,Delft,the
Netherlands),acommercialcellulasepreparation from
Trichodermavirideorigin,aspreviously reported(7).
Substrates
CM-cellulose,typeAkucelAF 0305wasfromAkzo,Arnhem,the
Netherlands.Xylanexoatspeltswas fromKoch-Light,Colnbrook,
Bucks,England.Thismaterialwascharacterized bymethylation
analysisasdescribed byTalmadgeetal. (23)andhada
compositionaspresented inTableI.Almost 90%ofthesugar
residuesare(3-D-(1,4)-linkedxyloseunits,ofwhichabout13%
have singleordoublebranchpoints.
Enzyme incubationswithxylan
Theendoglucanases,exoglucanases and(3-glucosidasewere
incubatedat30°Cin0.1 MsodiumacetatebufferpH 5.0with1
mg/mlxylan.Theenzymeconcentrationwas60ug/mlinafinal
volumeof1ml.After anincubationtimeof 3and 21hoursthe
reactionwasstoppedbyadditionof0.1ml 1.0M leadnitrate.
Theprecipitatewhichwasformed at4-6°Cwascentrifuged.The
sampleswhichwere incubatedduring 3hourswere concentrated
Table I.Typesof linkagespresent inxylanexoatspelts (mole
percentages).
90
Sugar residue
Typesof
D-Xylose
0-1
0-1,
0-1,
0-1,
0-1,
linkages
0-4
0-3, 0-4
0-2, 0-4
0-2, 0-3, 0-4
mole%
4.0
75.7
9.9
1.5
0.6
L-Arabinose
0-1
0-1, 0-2
0-1, 0-3
0-1, 0-5
5.1
1.8
0.2
0.5
D-Glucose
0-1, 0-4
0.6
D-Galactose
0-1, 0-3, 0-6
0.1
threetimesbyevaporationandcentrifuged againtoremove
additionalprecipitate.Theclear supernatantwasanalysedby
HPLC,using aSpectraPhysicsmodelSP 8000,eguipedwithan
AminexHPX 87Pcolumn (300x7.8mm,Bio-Rad Labs,Richmond LA,
USA)asdescribed earlier (21;Chapter5).
Preparationofoligomersfromxylan
Xylo-andarabinoxylo-oligosaccharides wereprepared ona
larger scalefortheuseofpeakidentificationinthe
HPLCanalysisandasasubstrate forfurtherenzymeincubation.
Cellulases,exhibitingxylanase activity,wereincubatedwith
xylaninavolumeof 10ml,usingtheconditions asmentioned
above.After 21hoursofincubationtheenzymeswere inactivated
byplacingthetubesinaboilingwaterbathfor5min.
Insoluble residueswere removedbycentrifugation andthesupernatantwasconcentrated tentimesbyevaporationundervacuum.
Theconcentratewasfractionated onaBio-GelP2 (200-400mesh,
Bio-RadLabs)column (100x1.0 cm)usingdegassed distilled
wateraseluent.Fractionsof0.52mlwerecollectedand
analysed forsugarsbythephenol-sulphuric acidassay(24).
Peakswereanalysed fortheir sugarcompositionsbygaschromatographyafterhydrolysis in2Ntri-fluoracetic acidand
derivatization toalditolacetates (25).Thechromatografic
behavioursofthemono-andoligosaccharide fractionsobtained
fromthegelpermeationchromatographywere alsodeterminedby
HPLC.
Enzymeincubationswith oligosaccharides
Purifiedoligosaccharides obtained fromthedegradation
productofxylanwithEndoIVwerere-incubatedwiththose
cellulases,whichwere foundtobeactiveonxylan.Incubation
tookplacewithanoligosaccharide concentrationof1.3mg/ml
andanenzymeconcentrationof 30ug/ml,during 20hoursat
30°Cin0.1 Msodium acetatebufferpH 5.0.Inactivation,
samplepreparation andHPLCanalysiswascarriedoutas
describedabove.
Kinetic experiments
Reactionkineticparameters ofthepurifiedcellulaseswere
determined onCM-cellulose forEndoI,II,III,IV,VandVIand
onxylanandp-nitrophenyl-p-D-glucoside (pNp-Gluc)forthose
enzymes,whichwere showntobeactiveonthesetypesofsubstrates.TheincubationwithCM-cellulose tookplaceat30°C
91
in0.05 Msodium acetatebufferpH 5.0 using several substrate
concentrationsbetween1.3and8.0mg/mlandanenzymeconcentrationof 1ug/ml.Thereactionmixtures forkinetic studies
onxylanconsisted ofvariousconcentrations ofsubstrate,
ranging from 1.9 to15.4mg/mlandanenzymeconcentrationof2
Ug/mlin0.1 Msodium acetatepH 5.0.Theincubationtookplace
at30°C.After thedesired reactiontimes,aliguotsofthe
reactionmixturesweretakenandanalysed forreducing sugarsby
themethod ofNelsonandSomogyi (26).Thesampleswerecentrifugedbeforereadingabsorbanceat520nm.Incubationwith
pNp-Glucwasdoneunder thesameconditionswithasubstrate
concentration between 0.03 and 30mg/ml.Theconcentrationof
p-nitrophenolwasmeasured after 1hourat400nm,usingthe
absorptioncoefficient of13700W-'-cm--'-.
RESULTS
Enzyme incubationswithxylan
Fromthetenglucanases,purifiedbyusfromacommercial
cellulasepreparationderived fromTrichodermaviride (7)only
fiveenzymes showed activity towardsxylan (TableII).Even
afterprolonged incubation (21hours)withEndoI,EndoII,Endo
III,ExoIIandExoIIInodegradationproducts fromxylancould
beobservedbyHPLC.FromFigure 1itcanbeseenthatEndoIV
releasedmainlyxylobiose (X2)andxylotriose (X3)fromxylan.
Theratiooftheseoligosaccharides remained aboutequalaftera
longer incubationperiod.
Theidentificationoftheoligomer peakswasdonewith
standards,obtained fromgelpermeationchromatographyon
Bio-GelP2 (seenextparagraph).Morediversityofhydrolysis
productswasobserved afterincubationofxylanwithEndoV.
AlthoughX2wasthemainproduct,alsoxylose (X-|),X3 and
xylotetraose (X4)weredetected.The largeroligomerwasnot
foundafter 21hoursof incubation.OnshorttermEndoVI
produced amixtureofX1,X2,X3 andX4,however thisenzymewas
alsoabletodegradetheseoligomerstomonomericxylose,which
wasfoundastheonlyproductafterprolonged incubation.Since
xylanexoatspeltscontainsalsoasmallamountof p-D-(l,4)linkedglucose (Table I), whichcanbecleavedoffbythese
enzymes,someglucose (G-j)wasfound inalmostallhydrolysates.
BesidesG1 andXJ1anamountofarabinose (A1)wasreleased
92
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Table II.Activityofcellulases fromTrichodermavirideon
xylan.P,positive;N,negative.
Enzyme
Endo I
EndoII
EndoIII
EndoIV
EndoV
EndoVI
Exo I
Exo II
Exo III
p-glucI
Activity
N
N
N
P
P
P
P
N
N
P
fromthepolymeric substratebyExoI.Withtheexceptionofa
smallamountofG1,X1 andX 2 ,(3-glucIproducedxylose asthe
onlyhydrolysisproduct.
Preparationofoligomers fromxylan
GelpermeationchromatographyonBio-GelP2wasusedto
preparexylo-andarabinoxylo-oligomersfromxylan.These
oligomerswereproduced fortworeasons:firstlyasstandards
forpeak identification inHPLCanalysisandsecondlyas
substrates forfurtherenzymatic conversion.Figure 2showsthe
chromatogramsofthedigestsofxylanswiththosecellulases
whichwereshowntobeactiveonthissubstrate,i.e.EndoIV,
EndoV,EndoVI,ExoIandp-gluc I.Theelutionprofilesarein
goodagreementwiththeresultspresented inFigure 1,however,
sinceBio-GelP2separatesthesesaccharidesonlyonthebasis
ofmolecularmassdifferences andbecausexylan isaheterogeneous substrate,thepeaksarenotnecessarilyhomogeneous.
Therefore samplesofthepeakswerefurther characterized
withrespecttothesugarcompositions aswellasbyHPLC
analysisoftheoligomers.Theresultsaresummarized inTable
IIIandsometypicalchromatograms aregiveninFigure 3.The
dimerandtrimerfractionsobtained fromthedigestionofxylan
withEndoIV,EndoV,EndoVIandExoI,consisted ofpure
xyloseandgavesinglepeaksbyHPLCanalysisatretentiontimes
ofrespectively 15.05 (Figs.3A2 and 3B2)and 13.35min.
(Figs.3A3 and 3B 3 ). Thereforethesepeakswereidentified asX2
andX,.ThepentamerpeaksfromtheBio-GelP2chromatographyof
xylandigestswithEndoIV,EndoV andEndoVIconsistedof
xyloseandarabinose inaratioof 4 :1andgavesinglepeaks
ataretentiontimeof 10.73min.,whensubjected toHPLC
94
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E R , T, Tr D, M„
M i l l
0.5
50
LO-
70
90
fraction number
OS-
50
70
90
70 90
fractionnumber
50
Fig. 2. Bio-Gel P2 chromatography of products released from
xylan by Endo IV ( A ) ,Endo V ( B ) ,Endo VI ( C ) ,Exo I (D) and
(3-gluc I (E).M o ,monomer; Di, dimer; Tr, trimer; Te, tetramer;
Pe, pentamer; Hx, hexamer.
Table III. Sugar composition and identification of oligosaccharides from P2-gelpermeatlon chromatochraphy, obtained
after incubation of xylan with several cellulases, purified from
Trichoderma viride. SC, Sugar composition of the peaks from
P2-column; X, 100% xylose; X:A, ratio of xylose to arabinose
residues; RT, Retention time on HPLC analysis; OC, Oligosaccharide composition of the peaks from P2-column.
Pentamer
Hexamer
X
13.35
X:A=4:1
10.62
x3
X4A1
X:A=5:1
10.15
X5A1
X
13.35
X:A=4:1
10.73
X:A=5:1
10.15
x3
X4A1
X5A1
X:A=4:1
10.73
X:A=5:1
10.20
X5A,
Enzyme
Dimer
Trimer
E n d o IV
SC X
RT 15 05
OC x2
SC X
RT 15 05
OC *2
Endo V
E n d o VI
Exo I
p-gluc I
Tetramer
SC X
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OC x 2
X4A1
SC X
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13.42
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12.07 13.35
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12 07 1 1 . 3 2
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10.73
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10.26
X:A=5:1
10.90
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10.08
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analysis (Fig.3A5).Thishetero-oligosaccharide couldbe
identifiedas X^A-^. Thecorresponding hexamer fractionsofthese
digestsgaveaxylosetoarabinoseratioof 5onsugar analysis
andsinglepeaksataretention timeof 10.15min. afterHPLC
(Fig.3Ag)•Thesepeakswerethereforeclassified asX5A-].
Although thepenta-andhexamerpeaks foundafter separation
oftheExoIdigestontheP2-columngavealsosinglepeaks
after rechromatographyontheHPLCcolumn (Figs.3B5 and 3Bg),
thesugarcompositionwasanindicationofheterogeneity.A
xylosetoarabinose ratioof 3was found inthepentamerpeak
showingthat inadditiontoX4A-1,acertainamountofacompound
withahigherarabinosecontent,probablyX3A2waspresentin
thisfraction.Thehexamerpeakgaveaxylose toarabinose ratio
of 4,whichmadeustoconcludethatitwasamixtureofX5A1
andX4A2.TheresolutionoftheAminexHPX 87Pcolumn,however,
maynotbegoodenoughtoseparatethesesaccharides.
Different oligosaccharideswithegualmolecular masses could
alsobedemonstrated inthefJ-glucIhydrolysate.Thedimer
fractionconsisted ofpurexylose andcould beseparated into
twopeaksontheHPLCcolumn (Fig. 3C 2 )• Oneofthesepeaks
correspondedwiththe"normal"(3-D-(1,4)-linkedxylobiose,
while theotherpeakatretentiontimeof 10.26min.probably
represented the fJ-D-(l,3)-or(3-D-(1,2)-linkedisomer.The
xylosetoarabinoseratioof 6inthetrimerfractionandthe
twopeaksintheHPLCchromatogramshowed thatthisfraction
consistedofX 3 andX2A1.Fromsimilarpointsofevidencethe
conclusioncouldbedrawnthatalsothetetramer andpentamer
fractionswereheterogeneous andconsisted of respectively
X3A1+X4 andX4Ai+X5.
Enzymeincubationswith oligosaccharides
Oligosaccharides X2,X3,X4A1 andX5A1,whichwere isolated
fromthexylandigestofEndoIVwerereincubatedwiththe
xylanolytic cellulases.Thecompositionsofthedegradation
productsarepresented inFigure 4.Ascouldbeexpected these
oligosaccharideswerenotfurtherdegradedbyEndoIV.EndoV
wasnotactiveonX 2 .However,X3 wasconverted toawiderange
ofxylo-oligosaccharides,demonstrating transferase activityof
thisenzyme.TheX4A-)andX5A-1 saccharideswerenot,oronly
veryscarcelyattackedbythisenzyme.EndoVIappeared tobe
activeontheshort-chain saccharides:X2 andX3 were largely
degraded tothemonomer.ThisenzymewasnotactiveonX4Aj,but
fromX^A-i somexylosewasreleased.ThemainpeakofX5A^ witha
97
X5A,
Xi,Ai
~i
1
r
-i
1
1—•
r
B2
X^A,
X
X
5A1
3
X,
X,
-l
1
r
-4 X5A1+
X^A,
X,
i
J
3
i
i
-i
r
1
r
0,
X^A,
XsA,
X,
n
1
r
*
I
'
X3A,
\X 2 A 1 ? X 1
10 ' 20 ' 1b ' 20 ' 1b ' z'o'
X5V
X4A1
16 ' 2b
elution time (min)
F i g . 4 . HPLC a n a l y s i s of p r o d u c t s o b t a i n e d from i n c u b a t i o n s of
X2, X3, X4A-] and X5A-| w i t h Endo IV (A1-A4), Endo V (B1-B4), Endo
VI (C1-C4), Exo I (D1-D4) and p - g l u c I (E-|- E4 ). P r o d u c t s from
X2 : A1-E1; X3 : A 2 - E 2 ; X4A-j : A3-E3 and X5A-, : A4- E 4 .
98
retention timeof10.73min.shiftedto10.55min.,demonstratingtheformationofX4A-1.ExoIshowed axylanase activity
similartoEndo V:X 2 wasnotdegradedbutX3couldbeattacked
bythisenzyme.The formationofsomeX4displayeditstransferaseactivity.AlthoughExoIliberatedA-jfrompolymericxylan,
surprisingly nosuchactivitywasfoundtowardstheoligomers
X4A1 andX5A1. (3-glucIwashighly activetowards,thexylo-as
wellasarabinoxylo-oligosaccharides, liberatingxyloseas
hydrolysisproduct.Becauseresolutionoftheseparationof
oligomerswasnothighenough,theshoulder onthe XJA-J peak
(Fig.4E3)couldnotunambiguously beidentified asX2A-1.
Kinetic experiments
Inordertoinvestigate thespecificity ofthexylanolytic
cellulases forxylanhydrolysis,thekineticparametersofthese
enzymesweredetermined usingxylan,aswellasCM-celluloseas
substrates.This latter substratewaschoosenbecause itis,
likexylan,moreor lesssoluble.SinceCM-cellulose isan
unsuitable substrate forexoglucanase and|3-glucosidaseactivity
measurements,p-nitrophenyl-(3-D-glucopyranoside (pNp-Gluc)was
usedtodeterminethekineticvaluesofExoIand(3-glucI.The
valueofKjnwasexpressed asthe (anhydro)-glycosidic
substrateconcentration (mM).K c a t wascalculated from V m a x ,
using themolecularmassesoftheenzymesasdetermined
previously (7).TableIVshowsthatEndoIV,VandVI strongly
preferCM-cellulose overxylan.ComparedwithCM-cellulose,a
muchhigherconcentrationofxylan isneeded inorderto
saturatetheseenzymestoattainmaximalreactionrates.EndoIV
was70timesmore specific forCM-cellulose thanforxylan,
mainlydeterminedbydifferences inK c a t .EndoVandEndoVI
wererespectively 20and 17timesmorespecificforCM-cellulose.
ComparisonofkineticvaluesofExoIand(3-glucIonxylan
andpNp-Gluc aredifficult tomake,becauseofthedifferent
naturesofthesesubstrates.However,ExoIhasavery low1^
valueforpNp-Gluc andwas665timesmorespecific forthis
lattersubstratethanforxylan,indicating itspreference for
(3-D-glucosidiclinkages.(3-glucIdidnotshowsuchastrong
substratepreference.Therelativelymuch largerK,,,of(3-gluc
I forpNp-Gluc,aswellasitsactivitytowardscelluloseand
xylandemonstrate thenon-specific natureofthisenzyme.
99
TabelIV.Kineticparameters% (mMglycosidic bonds), V m a x
(vunolrag-min"1),K c a t (sec-1)andK c a t /K m (mM-1 sec"1)
ofpurified cellulases fromTrichodermaviride.pNp-Gluc,
p-nitrophenylp-D-glucopyranoside;-,notactive;P,Positive
but notdetermined.
Enzyme
Substrate
Parameter
CM-cellulose
Endo I
Km
v
max
Kcat
Kcat/Km
Endo I I Km
v
max
Kcat
Kcat/Km
Endo I I I Km
Vmax
Kcat
K
cat/Km
Endo IV Km
v
max
Kcat
Kcat/Km
Endo V
Km
Vmax
Kcat
K
cat/Km
Endo VI Km
v
max
Kcat
Kcat/Km
Exo I
Xylan
pNp-Gluc
106.1
0.69
0.3
0.003
153.0
4.63
4.4
0.029
149.2
5.41
4.7
0.032
15.2
0.49
0.4
0.026
0.11
2.17
1.9
17.3
25.7
0.58
0.7
0.027
21.7
6.79
8.6
0.4
23.7
22.8
19.0
0.8C
45.3
42.6
32.0
0.71
2.8
3 ..6
3 ..5
1 ..25
2 5 ..2
13.5
5.3
0.21
46.1
28.8
27.4
0.
51.
31.
27.
0.
P
59
0
6
4
54
v
"max
Kcat
Kcat/Km
Exo I I
Exo I I I
(3-gluc I Km
^max
Kcat
Kcat/Km
DISCUSSION
The c e l l u l a s e complex of Trichoderma v i r i d e , which was
p r e v i o u s l y shown t o c o n t a i n a t l e a s t s i x e n d o g l u c a n a s e s , t h r e e
e x o g l u c a n a s e s and a p - g l u c o s i d a s e (7) c o u l d be s u b d i v i d e d i n t o
s p e c i f i c g l u c a n a s e s and n o n - s p e c i f i c g l u c a n a s e s . Endo I , Endo I I
100
andEndoIII,aswellasExoIIandExoIIIwerenotactiveon
xylan,evenafteraprolonged incubationtime.Thereforethese
cellulaseswereclassified asspecificglucanases.Thenatureof
theotherendoglucanases (EndoIV,EndoVandEndoVI),
exoglucanase (ExoI)andp-glucosidase ((3-glucI)wereless
specificbecausetheseenzymes showed alsoactivity towards
xylan.
According toseveralauthors (4,5,27)thegroupofxylan
degradingenzymescanbedivided into(3-xylosidases,which
producexylosefromshortxylooligosaccharides,exo-p-xylanases,
whichareactiveonxylanand longerxylo-oligosaccharidesand
liberatexylosefromthenon-reducingendandendo-p-xylanases,
whichattackxylanmoreor lessatrandom.Thislastgroupcan
besubdivided intoendoxylanaseswhichattackbothlongand
shortxylo-oligosaccharides andthosewhichonlyhydrolyse
longerchains.Also severalhighlypurified xylanasepreparationsappeartobecapableofcleaving (1,3)-a-L-arabinofuranosylbranchpoints fromxylan.Theresultspresented in
thispaper showthatthepurifiedxylanolytic cellulasesfit
thisclassification.EndoIVproducedmainlyX 2,X3,X4A-|and
X5A-|fromxylanandwasnotabletoreleaseL-arabinose.Small
xylo-oligosaccharides werenotattacked.Thisenzymeacted
endo-wiseandpreferred xylanor longxylo-oligosaccharides.
EndoVandEndoVIwerealsoendoenzymeswithrespectto
theirxylanaseactivityproducing awidevarietyofoligosaccharidesfromxylan.However,theseenzymeswerealsoableto
attack smallerxylo-oligosaccharides.Thesmallestoligosaccharidewhichcould serveassubstrateforEndoVwasX3.
Thisendo-acting enzymeshowedtransferase activitywithX3as
substrate,which resulted intheformationof oligosaccharides
ranging fromX-|toX 5 .Similartrans-xylosidaseactivitywas
alsofoundforanendoxylanase isolated fromBacilluspumilus
(28).EndoVIcouldhydrolysexylobioseasthesmallest
saccharide,however itsactivitywasonlymoderatecomparedwith
theactivityof(3-glucItowardsthissubstrate.Withrespectto
itsxylan-degradingproperties,thislatterenzyme istherefore
classified asa(3-xylosidase.
Xylose,butnoL-arabinosewas liberated fromX4A1 andX5A1,
indicating thatatleastaconsiderablepartoftheL-arabinosyl
branchesintheseoligosaccharideswereattached tothenonterminalD-xylopyranosylresidues.Oneoftheuncommondegradationproductsofxylanafter incubationwithp-glucIwas
(3-D-(l,-3)or(3-0-(1,2)-linked xylobiose.Theappearanceofthis
101
oligosaccharidemakes itlikelythatbesidesofL-arabinosyl
branchesalsoD-xylosylbranchesexistinxylanexoatspelts.
Thisresult isinagreementwiththemethylationanalysisof
thesubstrate,presented inTableI.
ExoIwasfound tobetheonlyenzymepurifiedbyus,which
wasabletoreleaseL-arabinose fromxylan.Thisenzyme
hydrolysed xylanbyanendo-wisemechanism comparable toEndoV.
Xylotriosewasthesmallestoligosaccharide onwhichactivity
wasobserved.Xylotetraosewasformedastheresultof
trans-xylosidaseactivityofthisenzyme.Surprisinglyno
L-arabinose was liberated fromX4A^ orX5A-1byExoI.Probably
theenzymeneedsalonger sequenceofD-xylopyranosylresidues
tobring thecatalytic siteforarabinofuranosidase activity
intothevicinityofthebranchpoint.
Theresults fromkineticmeasurements showed thatthenonspecificglucanases EndoIV,EndoV,EndoVIandExoIstrongly
prefer (3-1,4-D-glucosidicbondsover(3-1,4-D-xylosidiclinkages.
Itmaybepossible thattheseenzymesaresimilartothe
non-specific cellulase isolatedbyShikata andNisizawa (8)who
showed thatCM-cellulose,xylanandp-nitrophenyl 0-D-glucoside
arehydrolysed atthesameactivesiteoftheenzyme.The
productsofcellulosehydrolysisbyEndoIVareglucose (G-]),
cellobiose (G2)andcellotriose (G3) (21),whileX3andX 2 are
themainproducts fromxylose.Similarproductdifferenceswere
observed forEndoV,EndoVIandExoI.TheenzymesproduceG 2
andG^fromcellulosewhileX3,X 2 andX-|arefound inthexylan
hydrolysatesoftheseenzymes.Ifhydrolysisofboth substrates
takesplaceatthesamecatalytic site,formationofdifferent
oligomerscanonlybeexplained bydifferent interactionsof
xylanandcellulosewith thebinding subsitesoftheenzyme,
causedbystructuraldifferencesofthesesubstrates.
Thenatureofp-glucIisnotquitedefined,becauseit
showed noclear substratepreference.Itmaybepossible that
|3-glucI,althoughelectrophoretically homogeneous,isacomplex
ofsaccharidases,whichcanonlybeseparated under strong
denaturating conditions,asdescribed bySpreyandLambert(20).
Itsfairlyhighmolecularmassof 76000,asdeterminedbySDSgelelectrophoresis (7)could allowforthispossibility.
NOMENCLATURE
KJJ,
max
Kcat
v
102
Michaelis-Menten constant (mM)
maximal reactionrate (limol/mg.min)
molaractivity (sec-1)
REFERENCES
1.Linko,M.,inAdvances inBiochemicalEngineering (Ghose,
T.K. andBlakebrough,N.eds)1977,5.,25-48
2.Ryu,D.D.Y.andMandels,M.,EnzymeMicrob.Technol.1980,
2,91-102
3.Bisaria,V.S.andGhose,T.K., EnzymeMicrob.Technol.1981,
3,90-104
4.Frederick,M.M.,Kiang,C.H.,Frederick,J.R. andReilly,
P.J., Biotechnol.Bioeng.1985,27,525-532
5.Biely,P.,TrendsBiotechnol.1985, 3, 286-290
6.Jeffries,T.W., TrendsBiotechnol.1985,3,208-212
7.Beldman,G.Searle-vanLeeuwen,M.F.,Rombouts,F.M. and
Voragen,A.G.J.,Eur.J.Biochem.1985,146,301-308
8.Shikata,S.andNisizawa,K.J.,Biochem. 1975,78^,499-512
9.Kubo,K.andNisizawa,K.J.,Ferment.Technol.1983,61,
383-389
10. Schoemaker,S.P. andBrown,R.D.,Biochem.Biophys.Acta
1978,523,133-146
11.Wood,T.M., McCrae,S.I.andMacfarlane,C.C.,Biochem. J.
1980,189,51-65
12. Eriksson,K.E.andPettersson,B.,Eur.J.Biochem.1975,
51,193-206
13.Wood,T.M. andMcCrae,S.I.,Biochem.J. 1978,171,61-72
14.McHale,A.andCoughlan,M.P.,FEBSLett.1980,117,
319-322
15.Sinner,M.andDietrichs,H.H.,Holzforschung 1975,29,
168-177
16.Biely,P.,Vrsanka,M.andGorbacheva,I.V., Biochem.
Biophys.Acta 1983,743,155-161
17.Frederick,M.M.,Frederick,J.R., Fratzke,A.R.andReilly,
P.J., Carbohydr.Res.1981,9_7,87-103
18.Shei,J.C.,Fratzje,A.R.,Frederucj,M.M.,Frederick, J.R.
andReilly,P.J., Biotechnol.Bioeng.1985,27,533-538
19.Fournier.R.,Frederick,M.M.Frederick,J.R. andReilly,
P.J., Biotechnol.Bioeng.1985,27^,539-546
20.Sprey,B.andLambert,C.,FEMSMicrobiol.Lett.1983,18,
217-222
21.Beldman,G.Voragen,A.G.J.,Rombouts,F.M. Searle-van
Leeuwen,M.F.andPilnik,W.,accepted forpublicationin
Biotechn.Bioeng.1986
22.Beldman,G.,Voragen,A.G.J.,Rombouts,F.M. andPilnik,W.,
submitted forpublication inBiotechn.Bioeng.1986
23.Talmadge,K.W.,Keegstra,K.,Bauer,W.D.and
Albersheim,P.,PlantPhysiol.1973,51,158-173
24.Dubois,M.,Gilles,K.A.,Hamilton,J.K., Rebers,F.A. and
Smith,F.,Anal.Chem.1956,28,350-356
25.Jones,T.M. andAlbersheim,P.,PlantPhysiol.1972,49,
926-936
26. Somogyi,M.J.,Biol.Chem. 1952,195,19-23
27.Dekker,R.F.H.andRichards,G.N.,Adv.Carbohydr.Chem.
Biochem. 1976,32,277-352
28.Panbangred,W.,Shinmyo,A.,Kinoshita,S.andOkada,H.,
Agric.Biol.Chem. 1983,47,957-963
103
Summary
Theaimoftheworkpresented inthisthesiswastoapply
cellulasesfromTrichodermavirideforthebioconversionof
agricultrual residues,aswellastostudythemodeofactionof
thecellulasecomplexofthisfungus.Theseaimsarespecified
inChapter1.
Chapter 2describestheresultsobtained after treatmentof
beetpulpandpotatofibrewithcellulases fromT_.virideand
pectinasederived fromAspergillusniger.Itappeared thatpolygalacturonase fromthepectinasepreparationwas largely
responsible forthedisintegration (liquefaction)ofthese
materials.Additionofcellulasesincreased therateofliquefaction.Acombinationoftheseenzymeswasabletohydrolyse
thecellwallpolysaccharides ofthese substratesextensively.
Enzymatic hydrolysisofbeetpulpcouldconvientlybecarried
outinapacked-column reactorwhichwaspercolatedwithan
enzymesolutionandfromwhichtheproductswerecollected
throughanultrafiltrationmembrane.Theenzymesappeared tobe
stableinthistypeofreactor andcouldessentiallybereused.
Partoftheenzymeactivitywaslostbyadsorptionontothe
substrateresidue.
Therelativelyhighamountof lignin,present inspentgrain
(theresidueofmalt,after liquefaction andsaccharificationof
thestarch)makesthepolysaccharides inthismaterial resistant
toattackofhydrolases.InChapter 3theeffectofsome
physicalandchemicalpretreatmentsontheenzymatic hydrolysis
of spentgrainisdescribed.Physicalpretreatmentswhich
transform onlythemacro-structure ofthesubstrate improvedthe
enzymatichydrolysisonlyslightly.However,pretreatmentwith
sulphuric acidorsodiumhydroxide (0.5N)doubledthedegreeof
polysaccharide degradationafterenzymic treatment.Themonoandoligosaccharide compositionofthedigestwasdepedenton
thepretreatmentmethod.Predominantlymonosaccharideswere
detected intheenzymicproductoftheacidpretreated spent
grain.Ontheotherhandalargeamountofoligosaccharideswas
present inthedigestofthealkalipretreatedmaterial.
Inordertoinvestigate themodeofactionofthecellulase
complex,alldetectable endoglucanase,exoglucanase and(3-glucosidaseactivitieswere isolated fromacommercialcellulase
104
preparation,derived fromT.viride (Chapter 4).Six
endoglucanases (EndoI;II;III;IV;V;VI),threeexoglucanases
(ExoI;II;III)anda(3-glucosidase (p-glucI)were isolated
andcharacterizedwithrespecttotheiractivitiesonseveral
substratesandsomechemicalandphysicalproperties.TheendoglucanasesEndoI,IIandIVweremorerandom intheirattackon
carboxymethylcellulosethantheotherendoglucanases.Theexoglucanasesproducedcellobiose fromI^PC^-swollencellulose,
however,onlyExoIwasalsoabletohydrolysethisproductto
glucose.p-GlucIwashighly activetowardsp-nitrophenyl-(3-Dglucosebutnottowardscellobiose.
Theadsorptionbehaviours ofthepurifiedendoglucanases and
exoglucanasesoncrystalline cellulose aredescribed inChapter
5.EndoI,IIIandVadsorbedmuch strongeronthistypeof
cellulose thantheotherendoglucanases.Withrespecttothis
behaviour alsotheexoglucanasesweresubdivided:ExoIII
adsorbed stronglyandExoIIonlymoderately.Adsorptionwas
onlypartially reversible.Kineticmeasurements indicated thata
partoftheadsorbedenzymemoleculesarenotactive.Itis
supposed thattheseenzymes (forinstance anendoglucanase)
expresstheirhydrolytic activityonlywhenanotherenzyme
(i.e.anexoglucanase) isalsopresent.Thismightexplainthe
synergism,oftenobservedduringcooperativehydrolysisof
crystalline cellulosebyendoglucanases andexoglucanases.
Amoredetailed investigationonsynergism isdescribed in
Chapter 6.Itappeared thatthedegreeofsynergisticeffectwas
largelydependentontheinitialratioofendoglucanaseto
exoglucanase andmaximalataspecificvalue.Thestrongeran
endoglucanase adsorbsatitssubstrate,thelessisneededof
thisenzymetoreachamaximaldegreeof synergism,whenworking
inconcertwithanexoglucanase.
Thespecificandnon-specific natureofthepurifiedglucanaseswithregard totheiractivityonxylanandcellulosic
substrates ispresented inChapter 7.EndoI,IIandIIIaswell
asExoIIandIIIcouldbeclassified asspecificglucanases.
EndoIV,V,VIandExoIwereabletodegradexylan,however
theseenzymesclearlypreferred tohydrolysefi-1,4-glucosidic
linages.(3-GlucIshowednoclear substratepreference.
105
Samenvatting
Koolhydratenkomenopdewereld inovervloedvoorenkunnen
gebruiktwordenvoordeproductievanlevensmiddelenofals
grondstof voordechemischeoffermentatie-industrie.Helaasis
denatieveverschijningsvormvandemeestevandezestoffenniet
zodanigdatgebruikdirectmogelijkis.Zomoetbijvoorbeeldzetmeel (hetpolymeervana-D-glucose),datineensemi-kristallijne
vormalskorrels indenatuur aanwezigis,eerstdooreenhittebehandelingwordenontslotenenvervolgensmetenzymenworden
vervloeid enversuikerd omeenfermenteerbaresuikeroplossing te
verkrijgen.Ongeveeer 40%vandetotaleplantaardigebiomassaop
aardebestaatuiteenanderpolymeer vanglucose,namelijk
cellulose.Echter indezestof zijndeglucose-eenheden viaeen
(3-(l,4)-glucosidischeverbinding gekoppeld,waardoordeketens
eenstructuur krijgendiesterkeonderlinge interactie,
kristallijnvanaard,mogelijkmaakt.Metbehulpvancellulases
zoucellulosebenutkunnenworden,dooromzettingtotglucose.
Hetwerkbeschreven inditproefschrifthadtotdoelcommerciele
cellulases,afkomstigvandeschimmelTrichodermaviridete
gebruikenvoordebiologische omzettingvanenkeleplantaardige
residuenendaarnaasthetwerkingsmechanismevanhetgebruikte
cellulase-complextebestuderen.Dezedoelstellingen zijnnader
uitgewerkt inhoofdstuk1.
Hoofdstuk2beschrijftderesultatendiewerdenverkregenbij
behandelingvanbietenpulpenaardappelvezelmetcellulase
afkomstigvan T_. virideenpectinasegeproduceerd doorAspergillusniger.Hetbleekdathetenzymepolygalacturonase,aanwezig
inhetpectinasepreparaat,verantwoordelijkwasvooreensnelle
desintegratie (vervloeiing)vanhetmateriaal.Diteffectkon
wordenversterktdoor toevoegingvancellulases.Eencombinatie
vanbeideenzympreparatenwasinstaatde celwandpolysachariden
vandegenoemdesubstratenverregaand tehydrolyseren.
Bietenpulpkonopeeneenvoudigemanierenzymatischomgezetwordendoorgebruik temakenvaneenmet substraatgepaktekolom,
waardoor eenenzymoplossingwerdgepomptenwaaruithetproduct
dooreenultrafiltratiemembraanwerdverwijderd.Deenzymen
bleven stabiel ineendergelijk systeemenzoudeninprincipe
opnieuwgebruiktkunnenworden.Eengedeeltevandeactiviteit
gingverlorendooradsorptie aansubstraatresidue.
106
Bierbostel,hetresiduevanmoutnavervloeiing enversuikeringvanhet zetmeel,bevatrelatiefveellignine,dateen
barrierevormtvoordepolysacharide-afbrekende enzymen.In
hoofdstuk 3wordtbeschrevenwatheteffectvanenkele
mechanischeenchemischevoorbehandelingen isopdeenzymatische
afbreekbaarheid vanbierbostel.Mechanischevoorbehandelingen,
diealleendemacro-structuurveranderenblekendeenzymatische
polysacharide-afbraak slechtsingeringematepositief te
beinvloeden.Daarentegenkondoorvoorbehandelingmet zwavelzuur
ofnatronloog ennabehandelingmeteencombinatievancellulases
depolysacharide-afbraak wordenverdubbeld.Demono-enoligosacharidesamenstellingvanhetverkregenproductwassterkafhankelijkvandegebruiktevoorbehandeling. Inhetenzymatische
productvanhetmet zuurvoorbehandeldemateriaalwerdenvoornamelijkmonosacharidengevonden,terwijlnaalkalischevoorbehandelingnogveeloligosacharidenwerdenaangetoond.
Omhetwerkingsmechanismevanhetcellulasecomplex naderte
kunnenbestuderen isheteeneerstevereistedebeschikking te
hebbenovergezuiverdeengoedomschrevencellulases.Een
procedureomallemeetbareendoglucanase-,exoglucanase-en
p-glucosidase-activiteitentezuiverenuiteenmengselvan
cellulases,aanwezig ineencommercieelenzympreparaatafkomstig
van_T_.viride,staatbeschreven inhoofdstuk 4.Erkondenzes
endoglucanases (EndoI;II;III;IV;V;VI),drie exoglucanases
(ExoI;II;III)eneen(3-glucosidase (p-glucI)wordengeisoleerd.Dezeenzymenwerdennadergekarakteriseerdmetbetrekking
tothunactiviteitopdiverse substratenenenkelechemischeen
fysischeeigenschappen.Demanierwaaropdeendoglucanases Endo
I,IIenIVcarboxymethylcellulosehydrolyseren israeerwillekeurigvanaarddandeoverigeendoglucanases.Dedrieexoglucanasesblekencellobioseteproducerenvan fosforzuur-gezwollen
cellulose,maaralleenExoIwasinstaatditproductverderte
hydrolyseren tottweeglucoseeenheden.p-GlucI,datzeer
actiefwasopp-nitrophenyl-p-D-glucoside,wasdaarentegenniet
actiefopcellobiose.Indithoofdstukwordttevensingegaanop
deroldiedecellobiohydrolases (ExoIIenIII)spelentijdens
deafbraakvankristallijncellulose.Deresultaten suggereren
datdecellobiohydrolasesmetnameactief zijnopnieuwgegenereerdeketenuiteinden.
Hetadsorptiegedrag vandediversegezuiverde endoglucanases
enexoglucanases tenopzichtevankristallijncellulosewas
verschillendvanaard.Uitderesultaten,vermeldinhoofdstuk
5,bleekdatdeendoglucanasesEndoI,IIIenVsterk
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adsorbeerden aandittypecellulose,integenstelling toteen
anderegroependoglucanases,bestaandeuitEndoII,IVenVI,
dieslechtszeermatigadsorbeerden.Eenzelfdeonderscheidkon
gemaaktwordenvoorcellobiohydrolases ExoIIenExoII,waarvan
ExoIIIduidelijk sterker adsorbeerde.Deadsorptiewas slechts
gedeeltelijk reversibel.Kinetischeexperimentengaveneen
aanwijzingdateengedeeltevandegeadsorbeerdeenzymmolekulen
nietinstaat zijnhunhydrolytische eigenshaptotexpressiete
brengen.Mogelijkerwijskomtdeactiviteitvandezeenzymen
(bijvoorbeeld eenendoglucanase)alleendantotuitingalseen
anderenzym (inditgevaleenexoglucanases)ookaanwezig isin
dedirecteomgeving.Ditzoueenverklaringkunnen zijnvoorhet
synergistischeffectdatoverhetalgemeenoptreedtalseen
endoglucanase enexoglucanase gezamenlijkinwerkenop
kristallijncellulose.
Naderonderzoeknaarhetsynergismetussenendoglucanasesen
exoglucanases staatbeschreven inhoofdstuk 6.Dematevan
synergistischeffectbleeksterkaftehangenvande initiele
verhoudingwaarinbeideenzymengebruiktwerdenenwasmaximaal
bijeenbepaaldewaarde.Naarmateeenendoglucanasebeter
adsorbeertbleekerrelatiefmindervanditenzym toegevoegd
hoeventewordenomhetmaximalesynergistische effectmeteen
exoglucanase tebereiken.
Inhoofdstuk 7wordtdeactiviteitvandegezuiverdecellulasestenopzichtevanxylaanbeschreven.EndoI,IIenIII,maar
ookExoIIenIIIvertoondengeenactiviteitopxylaanenwerden
geclassificeerdalsspecifiekeglucanases.EndoIV,VenVI
alsmedeExoIen(3-glucIblekenminder specifiekvanaarden
waren instaatxylaantehydrolyserenvolgenseenpatroondat
overeenkomtmethethydrolytische karakter vanspecifiekexylanases.Kinetischeexperimentenmetverschillende substraten
toondenaandatdeniet-specifiekeendoglucanasesenexoglucanaseExoIbijvoorkeur (3-1,4-glucosidischeverbindingen
hydrolyseren.Hetgeisoleerde p-glucosidasehadeenminder
specifieke substraatvoorkeur.
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Curriculumvitae
GerritBeldmanwerdop2juni 1955teZutphengeboren.Nahet
behalenvanzijndiplomaHBS-B,studeerdehijscheikundegedurendedeperiodevan 1972tot1980aandeRijksUniversiteitte
Groningen.Nazijnkandidaatsexamenwerdalshoofdvakbiochemie
enalsbijvakradiobiologiegedaan.Hetpromotie-onderzoek vond
plaatsvan 1980tot 1984ophetLaboratoriumvoor Levensmiddelen
chemieen-raicrobiologievandeLandbouwHogeschool teWageningen.Nadezeperiodewashijopditzelfde laboratoriumenige
tijdwerkzaamalstijdelijkwetenschappelijk medewerker.Sinds
begin1985ishijwerkzaambijhetNIKO-TNOteGroningen.
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