Characterization and biotechnological application of recombinant xylanases from Aspergillus nidulans

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ContentslistsavailableatScienceDirect

International

Journal

of

Biological

Macromolecules

jo u r n al h om ep age :w w w . e l s e v i e r . c o m / l o c a t e / i j b i o m a c

Characterization

and

biotechnological

application

of

recombinant

xylanases

from

Aspergillus

nidulans

Gabriela

P. Maitan-Alfenas

a,b,∗

_,

_Mariana

_B.

_Oliveira

a,c

_,

_Ronaldo

_A.P.

_Nagem

c

_,

Ronald

P. de

Vries

b

_,

_Valéria

_M.

_Guimarães

a

a_Department_of_Biochemistry_and_Molecular_Biology,_Universidade_Federal_de_Vic¸_osa,_MG,_Brazil

b_Fungal_Physiology,_CBS-KNAW_Fungal_Biodiversity_Centre_&_Fungal_Molecular_Physiology,_Utrecht_University,_Uppsalalaan_8,₃₅₈₄_CT,_Utrecht,_The

Netherlands

c_Department_of_Biochemistry_and_Immunology,_Universidade_Federal_de_Minas_Gerais,_Belo_Horizonte,_MG,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received29February2016

Receivedinrevisedform14April2016 Accepted17May2016

Availableonline25May2016 Keywords: Xylanase Aspergillusnidulans Pichiapastoris Sacchariﬁcation Sugarcanebagasse

a

b

s

t

r

a

c

t

TwoxylanasesfromAspergillusnidulans,XlnBandXlnC,wereexpressedinPichiapastoris,puriﬁedand characterized.XlnBandXlnCachievedmaximalactivitiesat60◦_C_and_pH_7.5_and_at₅₀◦_C_and_pH_6.0,

respectively.XlnBshowedtobeverythermostablebymaintaining50%ofitsoriginalactivityafter49h incubatedat50◦C.XlnBhaditshighestactivityagainstwheatarabinoxylanwhileXlnChadthebest activityagainstbeechwoodxylan.BothenzymeswerecompletelyinhibitedbySDSandHgCl2.Xylotriose

at1mg/mlalsototallyinibitedXlnBactivity.TLCanalysisshowedthatthemainproductofbeechwood xylanhydrolysisbyXlnBandXlnCwasxylotetraose.AnadditiveeffectwasshownbetweenXlnBand XlnCandthexylanasesoftwotestedcommercialcocktails.Sugarcanebagassesacchariﬁcationresults showedthatthesetwocommercialenzymaticcocktailswereabletoreleasemoreglucoseandxylose aftersupplementationwithXlnBandXlnC.

1. Introduction

Hemicelluloseisthesecondmostabundantorganicmaterial

intheworldanditpresentsaverycomplexstructurecomposed

of various residues [1,2]. Xylan, the major hemicellulose

poly-mer,ishydrolyzedby␤-1,4-endoxylanase,␤-1,4-xylosidaseand

accessoryenzymes[3–5].␤-1,4-Endoxylanases (E.C.3.2.1.8) are

glycosidehydrolasesthatcatalyzethehydrolysisof␤-1,4-xylosidic

linkagesofthexylanbackbone[6].Theyareproducedbymany

organismsbuttheirmaincommercialsourcesareasmallnumber

ofﬁlamentousascomycetefungi[7].

␤-1,4-Endoxylanasesfromfungibelongtotwoglycoside

hydro-lasefamilies:GH10andGH11,andthesetwofamiliesdifferintheir

substratespeciﬁcity[7,8].Bothfamiliescatalyzethehydrolysisof

xylanthroughtheretentionoftheanomericconﬁguration,andtwo

glutamateresidueshavebeenimplicatedintheircatalytic

mecha-nism[9].However,GH10endoxylanaseshaveabroadersubstrate

speciﬁcityandarethereforeveryimportantforcomplete

degra-dationofsubstitutedxylans[8,10].Theyalsodifferwithrespect

∗ Correspondingauthorat:DepartmentofBiochemistryandMolecularBiology, UniversidadeFederaldeVic¸osa,MG,Brazil.

E-mailaddress:gabmaitan@yahoo.com.br(G.P.Maitan-Alfenas).

totheircatalyticdomains,whichinGH10endoxylanasespresent

an␣/␤8TIM-barreltopology, while GH11endoxylanaseshave a

jelly-rollstructure[2,11].

Xylanasescanbeusedforvariousbiotechnologicalapplications

suchasbioconversionoflignocellulose intofermentablesugars,

clariﬁcationofjuices,preparationofanimalfeedandpulp

bleach-ing[12,13].However,forindustrialapplications,xylanasesneedto

havedesirablepropertiessuchasstabilityinawidepHand

tem-peraturerange,highspeciﬁcactivitiesandlowproductioncosts

[14,15].Therefore,moleculartechniqueshavebeenusedtoobtain

xylanaseswithidealcharacteristicsforcommercialpurposes[7].

Heterologousexpressioninyeasthasalotofadvantagesamong

whichtheproductionofsolubleandcorrectlyfoldedrecombinant

proteinsthathaveundergonepost-translationalmodiﬁcations,the

capacityofgrowthathighcelldensitiesandtheabilitytosecrete

reasonableamountsofprotein[16].Pichiapastorishasbecomea

welldescribedand widelyappliedexpressionhostforxylanase

productionmainlybecausethisyeastgiveshighexpressionunder

its own promoters [17]. Furthermore, there is no secretion of

endogenouslignocelluloliticenzymesinsigniﬁcantamountsbyP.

pastorisandtherecombinantproteinsarealmostpureheterologous

enzymepreparations[18].Inaddition,proteinscanbeobtainedby

inexpensivelarge-scalecultivationofP.pastoriswhichreducecosts

oftheprocess[19].

http://dx.doi.org/10.1016/j.ijbiomac.2016.05.065

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TwoxylanasesfromAspergillusnidulanswerepuriﬁedand

char-acterized[20–22]andthegeneswhich expressthesexylanases,

AN1818(xlnB)andAN3613(xlnC),wereclonedinP.pastoris[23,24].

AlthoughthenativelyproducedenzymesfromA.nidulanswere

previouslystudied,thefunctionalpropertiesoftheseheterologous

xylanasesandtheirpotentialforlignocellulosicbiomasshydrolysis

remainedlargelyunexplored.

Inthiswork,xlnBandxlnCwereproducedbyP.pastorisandthe

correspondingxylanases,XlnB(GH10)andXlnC(GH11),

respec-tively,werepuriﬁedandbiochemicallycharacterized.Theirability

tosupplementcommercialenzymaticcocktailsforuseinsugarcane

bagassehydrolysiswasevaluated.

2. Materialsandmethods

2.1. Materials

Substrates including p-nitrophenyl-␤-d-glucopyranoside

(pNP␤Glc), p-nitrophenyl-␤-d-xylopyranoside (pNP␤Xyl),

␳-nitrophenyl-␤-d-mannopyranoside (pNP␤Man),

␳-nitro-phenyl-␣-d-mannopyranoside (pNP␣Man), p-nitrophenyl-

␤-d-galactopyranoside (pNP␤Gal), p-nitrophenyl-

␣-d-galactopy-ranoside (pNP␣Gal), p-nitrophenyl-␣-d-arabinofuranoside

(pNP␣Ara), p-nitrophenyl-␤-d-cellobioside (pNP␤Cel),

car-boxymethylcellulose(CMC),Avicel®,xylanfrombirchwood,xylan

frombeechwood,oatspeltxylan,dinitrosalicylicacid(DNS)and

methanol were purchased from Sigma Chemical Co. (St. Louis,

MO,USA).Arabinoxylan(wheatﬂour,lowviscosity−8cSt)was

obtainedfromMegazyme(Wicklow,Ireland).Yeastextractwas

purchasedfromHimediaLaboratoriesCo.(Mumbai,Maharashtra,

India).ThechemicalreagentsNaOH,H2SO4andpotassiumsodium

tartratewereobtainedfromVetecFineChemical(DuquedeCaxias,

RJ,Brazil).ThecommercialenzymaticmixturesMultifect®CLand

Accellerase® 1500werepurchasedfromGenencorInternational

Inc.(Rochester,NY,USA).Sugarcanebagassewaskindlydonated

by Jatiboca Sugar and Ethanol Plant, Urucânia, MG, Brazil. All

othersreagentsusedinthisstudywereofanalyticalgrade.

2.2. Pichiapastorisstrainsandcultivationconditions

The P. pastoris strains used in this study were previously

described[23,24].Thestrainsweregrownin25mlofbuffered

com-plexglycerolmedium(BMGY)in250mlshakeﬂasksat28◦Cand

250rpmfor16h.AliquotcellsweredilutedtoanOD1.0(600nm)

with25mlofbufferedcomplexmethanolmedium(BMMY)and

incubatedfor72hwithdailyadditionof150␮lof100%methanol.

Theculturewascentrifugedat5000rpmfor10minaccordingto

thePichiaExpressionKitManual(Invitrogen).

2.3. Proteinanalysis

Proteinconcentrationin theenzymatic extract of P.pastoris

andinthecommercialenzymaticmixtureswasdeterminedbythe

CoomassieBluebindingmethodusingbovineserumalbumin(BSA)

asastandard[25].

2.4. Xylanaseassay

Allenzymaticassayswerecarriedoutin100mMsodiumacetate

buffer,pH5,at50◦C.Theywereperformedintriplicateandthe

meanvalueswerecalculated.Relativestandarddeviationsofthe

measurementswerebelow5%.Xylanaseactivitywasdetermined

usingxylanfrombeechwood(1%w/vatﬁnalconcentration)as

sub-strate.Theenzymaticreactionswereinitiatedbytheadditionof

100␮loftheappropriatelydilutedenzymesolutionto400␮lof

thepolysaccharidesubstratesolutionpreparedinbuffer.The

reac-tionmixtureswereincubatedfor15minand thetotal reducing

sugarcontentreleasedwasdeterminedat540nmbyDNSmethod

[26]usingxyloseasstandard.Oneenzymeunit(U)wasdeﬁned

astheamountofenzymenecessarytoproduce1␮molofxylose

equivalentperminute.

2.5. Xylanasepuriﬁcation

ThecrudeextractsfromP.pastoriswerecentrifugedat15,000g

for30minat4◦C.Thehistidinetagswereusedforthe

puriﬁca-tionoftherecombinantproteinsbynickelafﬁnitychromatography.

The xylanasepuriﬁcationwascarried out atroomtemperature

undernativecondition,accordingtoprotocolsdescribed inThe

QIAExpressionistTM_Mannual_(Fifth_Edition,_March_2001).

2.6. SDS–PAGEandzymogramanalysisforxylanolyticactivities

SDS–PAGEwasperformedusinga12%(w/v)polyacrylamidegel

witha5%stacking geland theMini-ProteanIIsystem(BioRad)

accordingtothemethodpreviouslydescribed withsome

modi-ﬁcations[27].Thesamplepreparationforzymogramanalysiswas

performedbymixing0.5Uofenzymewith5␮lofloadingbuffer

containingSDS2%(w/v).ThemixturewasappliedinaSDS-PAGE

containing1%ofbirchwoodxylanand,afterrunning,thegelwas

dividedintotwoparts.Onepart,containingthemolecularmarker

obtainedfrom BioRad(BioRad Precision PlusProteinUnstained

Standard),wasstainedwithComassieBrilliantBlue.Theotherpart

ofthegelcontainingjustenzymaticsampleswaswashedtwicefor

30minin20%isopropanol(v/v)toremoveSDSandallowrefolding

oftheproteinsinthegel.Thegelwaswashedagainfor30minin

100mMacetatebuffer,pH5,toremove2-propanoland

immedi-atelyincubatedatsamebufferfor15minat50◦Cfordevelopment

of xylanase activity.Afterthat, the gelwas submergedin 0.1%

(w/v)Congoredsolutionfor30minanddestainedwith1MNaCl

untilpale-redhydrolysiszonesappearedagainstaredbackground.

Aceticacidat0.5%concentrationwasaddedtoexposethebands

andthegelturnedtoadarkbluecolor.

2.7. Proteindigestionandidentiﬁcationbymassspectrometry

(MS)

Proteinspotswereremovedmanuallyfromthegels,reducedby

DTT(Dithiothreitol)andalkylatedbyiodoacetamide.Thedigestion

wascarriedoutwithtrypsinina50mMammoniumbicarbonate

buffer, pH7.8, containing 20ng/␮l ofsequencing grade trypsin

(Promega)at37◦Covernight.Peptideswereextractedfromthe

spotswith30␮lof50mMammoniumbicarbonatesolution,

fol-lowedbyincubationfor10minwithoccasionalvortexmixing.After

that,thesupernatantwascollectedandtransferredtoa0.5ml

plas-ticmicrocentrifugetube.Thisextractionwasperformedtwomore

times.Thevolumeoftheextractwascompletelydriedby

evap-orationinaspeed-vac.Trypticpeptidesweresolubilizedin30␮l

ofMSgradewater(Sigma-Aldrich)containingtriﬂuoroaceticacid

0.1%(v/v).

Forproteinidentiﬁcationbymassspectrometry,thetryptic

pep-tideswereanalyzedusingaMALDI-TOF/TOFmassspectrometer

modelUltraﬂexIII(BrukerDaltonics).Thesamplesofthetryptic

peptidesweremixedwith␣-cyano-4-hydroxylcinnamicacidina

proportionof1:1.Themassspectraobtainedwereprocessedusing

FlexAnalysissoftware(BrukerDaltonics)andapeaklist(mgf

for-mat)wasusedforidentiﬁcationoftheproteinsbyMSionssearch

using the Mascot software against theNCBI protein databases.

Forthesearch,thefollowingparameterswereconsidered:amass

toleranceof75ppmfortheparentalion,ﬁxedmodiﬁcationfor

(3)

foroxidationofthemethionineresidues.Thesequencesobtained

wereconﬁrmedbydenovosequencingmanuallyandthetrypic

cleavagepartnersoftheproteinswereanalyzedbyinterpretation

ofthespectrausingtheﬂexAnalysissoftware.

2.8. Enzymaticcharacterization

2.8.1. EffectsofpHandtemperature

TheinﬂuenceofpHandtemperatureonxylanaseactivitieswas

determinedusingthestandardenzymaticassay,exceptthatthepH

valuesweremodiﬁedtoarangeof2.0–14.0,usingdifferentbuffer

solutions,andthetemperaturerangedfrom25to70◦C.

ThepHstabilityofxylanaseswasdeterminedbypre-incubating

enzymesolutionsinthepHrangeof2.0–14.0for1h,onice.After

pre-incubation, the mixturewas used for determining residual

activity, according to standard assay, using xylan from

beech-wood as the substrate. Thermal stability was investigated by

pre-incubatingtheenzymaticsolutionsin100mMsodiumacetate

buffer,pH5.0,attemperaturesof50and60◦C,forperiodsas

indi-catedinthetext.Aliquotsoftheenzymeswerecollectedatspeciﬁc

timesandsubmittedtothestandardassay,measuringthe

remain-ingactivity.Therelative activitieswerecalculatedinrelationto

xylanaseactivitywithoutpre-incubation,whichwasconsideredto

be100%.Resultsoftheanalysesarepresentedasmean±SD for

threemeasurements.

2.8.2. Substratespeciﬁcity

Enzymaticassayswereperformedwithvarioussynthetic,

natu-ralandpolymericsubstrates.FPaseandendoglucanaseactivities

were determined using Whatman No. 1 ﬁlter paper (1×6cm,

50mg)and1%CMC/1%Avicel®assubstrates,respectively,

accord-ing topreviously described standard conditions [28].The total

reducingsugarsliberatedduringtheenzymaticassayswere

quan-tiﬁedbythedinitrosalicylicacid(DNS)method[26]usingglucose

asthestandard. Xylanaseactivityusingxylanfrombeechwood,

oatspeltxylan andwheatarabinoxylan,allat1%(w/v)

concen-tration, was determined as the standard assay. ␤-Glucosidase,

␤-xylosidase,␣-and␤-mannosidase,␣-and␤-galactosidase,

␣-arabinofuranosidase and ␤-cellobiase activities were measured

using␳PN␤Glc,␳NP␤Xyl,␳NP␣/␤Man,␳NP␣/␤Gal,␳NP␣Araand

␳NP␤Celassubstrates,respectively.Thereaction mixtures

con-tained100␮loftheappropriatelydilutedenzymesolution,125␮l

ofthesyntheticsubstratesolution(1mMatﬁnalconcentration)

and275␮lof100mMsodiumacetatebufferpH5.0.Thereaction

mixturewasincubatedfor30minanditwasstoppedbyaddition

of0.5mlofa 0.5Msodiumcarbonatesolution.Absorbancewas

measuredat410nmand theamountof ␳-nitrophenolreleased

wasestimatedusingastandardcurve.Thedatapresentedforall

enzymeactivitydeterminationsaremeanvalues±SDofthree

mea-surements.

2.8.3. Effectofionsandreducingagents

Theeffectsofionsandreducingagentsonxylanaseactivities

wereassayedbythestandardmethod.Reactionmixturescontained

100␮loftheappropriatelydilutedenzymesolutionwithan

ade-quateamountoftheionorreducingagentforaﬁnalconcentration

of2and10mM(exceptfor␤-mercapthoethanolthatpresenteda

ﬁnalconcentrationof1mM)and400␮lofxylanasesolution

pre-paredinbuffer. Thereaction wasincubated for15minat50◦C

andthereducingsugarsweremeasuredat540nm,asdescribed

before.Thedatapresentedforallenzymeactivityassaysaremean

values±SDofmeasurementsperformedintriplicate.

2.8.4. Productseffect

Thexylooligosaccharideseffectonxylanaseactivities(XlnBand

XlnC)wasevaluatedthroughtheadditionofthesecompoundsto

thereactionmixdescribedonSection2.4.Xylotriosewasusedin

theconcentrationsof0.1and1mg/mlandxylotetraosewasused

at0.1mg/ml.

2.8.5. Kineticcharacterization

ThekineticparametersKmandVmaxofthexylanasesXlnBand

XlnCwereestimatedusingSigmaPlot®10.0.Theenzymaticactivity

assayswereperformedasdescribedonSection2.4withincreasing

concentrations of the substrates beechwood xylan, wheat

ara-binoxylanand oatspeltxylan. Thecatalytic constant (kcat)was

calculateddividingVmaxbytheenzymeconcentrationintheassay.

2.8.6. Compositionalanalysisofbeechwoodxylanhydrolysis

products

Thecomposition ofhydrolysis productsfrom1%beechwood

xylanbythexylanasesXlnBandXlnCandthecommercial

cock-tailsAccellerase®1500andMultifectCL® wasanalyzedonsilica

gelplate(Sigma)byThinLayerChromatography(TLC).Thereaction

mixturescontained150␮lofpuriﬁedenzymesorthecommercial

cocktailsand300␮lof1%beechwoodxylanin0.1Msodiumacetate

bufferpH5.0.Bufferwasusedforthenegativecontrol.Thereaction

wasincubatedby20hat50◦Cand,afterthisperiod,25_␮lofthe

mixwasspottedonsilicaplate.Theglassbowl(15×15cm)was

saturatedwiththemixturepropanol:aceticacid:waterintheratio

1:1:0.1(v/v).Afterelution,thehydrolysisproductswere

visual-izedwiththeapplicationofthedevelopingsolution(1%␣-naphthol

and10%phosphoricacidinethanol)andheatingoftheplateat

120◦Cduring10min.Xylose,xylotrioseandxylotetraosesolutions

at5mg/mlconcentrationwereusedasstandards.

2.9. Additiveeffectsofthexylanaseactivities

To investigate the presence of additive effect between the

xylanases XlnB andXlnC and thexylanase activitiespresent in

thecommercialcocktails(Multifect® CLandAccellerase® 1500),

xylanaseassayswereperformedforthefollowingmixtures:10FPU

Multifect®CL+15UXlnB;10FPUMultifect®CL+15UXlnC;10FPU

Multifect® CL+7.5UXlnB+7.5UXun3613; 10 FPU Accellerase®

1500+15UXlnB;10FPUAccellerase®1500+15UXlnCand10FPU

Accellerase®1500+7.5UXlnB+7.5UXlnCandthemeasured

val-ueswereobtained. Thetheoreticalactivitieswerecalculatedby

thesumofthemeasuredactivitiesintheassayscontainingonly

thecommercialcocktailandonlythexylanase(individualor

mix-tureforms).Thetheoreticalvalueswerecomparedtothemeasured

activitiesandtheadditiveeffectwasexpressedasapercentageof

thetheoreticalactivity.

2.10. Sugarcanebagassepretreatment

Sugarcanebagassewaswashedanddriedinanovenat70◦C

untilreachingaconstantmass,afterwhichitwasfurthermilled

(particlesizelessthan1mm)andsubmittedtoalkaline

pretreat-ment prior tobeing employed in sacchariﬁcation experiments.

Sodiumhydroxide at1.0%(w/v)concentrationwasusedto

pre-treatthemilledsugarcanebagassesamplesatasolidloadingof10%

(w/v).Thepretreatmentwasperformedinanautoclaveat120◦C

for60min.Pretreatedbagassewasseparatedintosolidandliquid

fractionsusingaBuchnerfunnelﬁttedwithﬁlterpaper.Thesolid

fractionwaswashedthoroughlywithdistilledwater,sealedina

hermeticvesseltoretainmoistureandstoredat−20◦_C.

2.11. Sugarcanebagassesacchariﬁcation

Thepuriﬁedxylanasesandthecommercialcocktailscontaining

cellulases(Multifect® CLandAccellerase® 1500)wereappliedin

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Fig.1. (A)and(B)SDS-PAGEforxylanasesextractsafteraffinitychromatography.(C)Zymogramofxylanases(SDS-PAGE12%containing1%ofbirchwoodxylan).M− molecularmassmarkerstainedwithComassieBrilliantBlue.1−PurifiedextractcontainingXlnB.2−PurifiedextractcontainingXlnC.3−CrudeextractcontainingXlnB.4 −CrudeextractcontainingXlnC.

ofalkali-pretreatedsugarcanebagassewasperformedin125ml

Erlenmeyerﬂaskswith20mlworkingvolume,ataninitialsolid

concentrationof8%drymatter(w/v)in100mMsodiumacetate

bufferatpH5.0.Thesacchariﬁcationassayscontained:the

indi-vidualcommercialcocktails(10FPaseunits(FPU)/gofbiomass);

ortheindividualcommercialcocktails(10FPU/g biomass)

sup-plementedwithXlnBorXlnC(15U/gbiomass);ortheindividual

commercialcocktails(10FPU/gbiomass)supplementedwithXlnB

andXlnC(7.5U/gbiomassforeachenzyme).Sodiumazide(10mM)

andtetracycline(40␮g/ml)wereaddedtothereactionmixtureto

inhibitmicrobialcontamination.Thereactionswerecarriedoutin

duplicateinanorbitalshakerat250rpmand50◦Cfor72h.

Sam-ples(0.5ml)weretakenfromthereactionmixtureatdifferenttime

intervalsforprocessmonitoring.Thesesampleswereimmediately

heatedto100◦Ctodenaturetheenzymes,cooledandthen

cen-trifugedat15,000gfor5min.

2.12. Analysisofhydrolysisproducts

Productsofthesacchariﬁcationassayswereanalyzedbyhigh

performance liquid chromatography (HPLC) using a Shimadzu

series10Achromatograph.TheHPLCwasequippedwithanAminex

HPX–87P column(300×7.8mm)and refractive indexdetector.

Thecolumnwaselutedwithwaterataﬂowrateof0.6ml/minand

itoperatedat80◦C.

ThetotalreducingsugarsweremeasuredbytheDNSmethod

[26]usingamixofglucoseandxyloseasstandard.

3. Resultsanddiscussion

3.1. PuriﬁcationandbiochemicalcharacterizationofXlnBand

XlnC

After72hcultivationofP.pastoris,xylanaseactivitiesinthe

crudeextractwere80.45U/mland35.64U/mlforXlnBandXlnC,

respectively.Expressedintermsoftheproteincontent,thespeciﬁc

activitieswere311.82U/mgofproteinand105.13U/mgofprotein

forXlnBandXlnC,respectively.Afterpuriﬁcation,thexylanases

exhibitedasinglebandinSDS-PAGE,withmolecularmassvalues

of34kDaforXlnB(Fig.1A)and24kDaforXlnC(Fig.1B).These

val-uesareinagreementwiththemolecularmassexpectedforthese

proteins.ThezymographyshowedthatXlnBandXlnCwereactive

puriﬁedxylanases(Fig.1C).TheidentityofXlnBandXlnCwere

con-ﬁrmedbymassspectrometryanalysis(Fig.2).Denovosequencing

ofionm/z2222.01(Fig.2B)presentinMS1ofXlnBpuriﬁedextract

(Fig.2A) revealedthe sequence IMHWDVVNEI/LFNEDGTFRthat

correspondtoapeptideproductoftryptichydrolysisofXlnB.Also,

denovosequencingofionm/z2222.01(Fig.2D)presentinMS1of

XlnCpuriﬁedextract(Fig.2C)revealedthesequences

RVSWFQEF-TATGEI/LandYNAPSI/LEGTAthatcorrespondtopeptidesproduct

oftryptichydrolysisofXlnC.

XlnB exhibited substantialactivity againstbeechwood xylan

withinapHrangeof5.0-9.0andtemperaturerangeof50–60◦C,

whileXlnCshowedthehighestactivitiesinthepHrange5.0-8.0and

thesametemperaturerange.TheoptimalpHandtemperaturewere

7.5and60◦CforXlnB,whileXlnCachievedmaximalactivityatpH

6.0and50◦C(Fig.3A–D).SinceXlnBandXlnCbothretainedmore

than75%oftheiractivitiesatpH5.0,thestandardactivityassay

wasperformedatthispHvalue.Thesedataareinaccordancewith

previousstudieswhichreportedthatrecombinantxylanasesfrom

Aspergillusterreus,Plectosphaerellacucumerina,andAlternariasp.

HB186,alsoexpressedinP.pastoris,showedmaximalactivityat

pH5.0and60◦C[29],pH6.0and40◦C[30]andpH6.0and50◦C,

respectively[31].Inpreviousstudies,A.nidulansxylanasesXlnB

(X34)and(XlnC)(X24),puriﬁedfromA.nidulanshadoptimalpHand

temperaturevaluesof6.0/56◦Cand5.5/52◦C,respectively[20,21].

ComparisonofoptimapHandtemperaturevaluesforthenative

andheterologousxylanases,showedthatexpressioninP.pastoris

resultedinasigniﬁcantincreaseinthepHvaluesatwhichthese

enzymesexhibitedmaximumactivity,especiallyforXlnBwhich

haditsoptimumpHchangedfrom6to7.5.

XlnBandXlnCshowedsigniﬁcantstabilityoverawidepHrange

(Fig.3A–B).Afterpre-incubationfor30mininpHvaluesbelow4.0

andabove10.0,XlnBrecoveredover65%and90%ofitsinitial

activ-ity,respectively,whenthisenzymewasreturnedtopH5.0.The

resultsshowthatXlnBwasabletorefoldandretrieveits

activ-ityafterpre-incubationinextremepHranges.Similarresultswere

observedforXlnCthatrecoveredover90%ofitsinitialactivityafter

30minpre-incubationinpHvalueslowerthan4.0andhigherthan

9.0.

Concerningthermalstability(Fig.3E–F),XlnBwasverystable

at50◦Candretainedabout70%ofitsoriginalactivityafter2hof

pre-incubationatthistemperatureandthisresidualactivitywas

maintaineduntil6hofpre-incubation.Thisenzymestillretained,

at50◦C,morethan30%ofitsoriginalactivityafter100hof

(5)

Fig.2.Proteinidentiﬁcationbymassspectrometry(MS).MS1spectraoftrypticpeptidesofXlnB(A)andXlnC(C),respectively.Denovosequencingoftheionswithm/z 2222.01(B)and2202.91(D),whichwereobtainedinMS1oftrypticpeptidesofXlnBandXlnC,respectively.

Fig.3.EffectofpHandtemperatureonthexylanasesfromA.nidulans.(A)EffectofpHonactivity(ⵦ)andstability(䊉)ofXlnB.(B)EffectofpHonactivity(ⵦ)andstability (䊉)ofXlnC.(C)EffectoftemperatureonXlnB.RelativeactivitieswerecalculatedinrelationtoactivitiesdeterminedatoptimapHandtemperature.(D)Effectoftemperature onXlnC.RelativeactivitieswerecalculatedinrelationtoactivitiesdeterminedatoptimapHandtemperature.(E)Thermostabilityat50(䊉)and60◦C(ⵦ)forXlnB.(F) Thermostabilityat50(䊉)and60◦_C_(ⵦ)_for_XlnC.

at60◦C,XlnBlostactivityafter9hofpre-incubation.XlnCshowed

lowerthermalstabilitycomparedtoXlnB,sincethisenzyme

main-tainedonly28%ofitsinitialactivityafter6hofincubationat50◦C

andtheactivitywaszeroafter30minat60◦C.XlnBandXlnC

pre-sentedhalf-lifevaluesof49h20minand1h10min,respectively,at

50◦C.Thehalf-lifevaluesofXlnBandXlnCat60◦Cweredrastically

reducedto20minand5.8min,respectively.Thehigher

thermosta-bilityat50◦Cand60◦CexhibitedbyXlnB,whichbelongstoGH10,

comparedtoXlnC,a GH11 xylanase,couldbeexplainedbythe

structuraldifferencesbetweenGH10andGH11xylanases.GH10

Xylanasestendtoshowhigher thermalstability sincethe␣/␤8

structureofGH10xylanasesiscomposedbypropellersandsheets

and(␣/␤)istherepetitiveunit[32].FortheGH11xylanases,the

jelly-rollstructureiscomposedofmanysheetsandonlyone

pro-pellerandonesheetistherepetitiveunit.Thepropellerstructures

foldbetterthanthesheetstructures,whichcouldprovidehigher

stabilityoftheGH10enzymes.

TheanalysisofXlnBandXlnCspeciﬁcitiesagainstseveral

sub-strates showed that these enzymes didnot hydrolyze the aryl

syntheticsubstratescontainingxyloseordifferent

monosaccha-rides(Suppl.Data,Table1).Ontheotherhand,theywereableto

hydrolyzedifferentxylans.Thisresultconﬁrmsthattheseenzymes

exhibitendoxylanaseactivity,asexpected,sincetheclonedgenes

correspondtoGH10andGH11xylanases.Inrelationtothenatural

substrates,XlnBshowedhighactivityagainstwheatarabinoxylan,

whichisacomplexstructureofxylan,containingmany

arabino-furanosedecorations[33].Incontrast,XlnChadthebestactivity

(6)

Table1

RelativeactivityofXlnBandXlnCinthepresenceofdifferentionsandreducing agents.Relativeactivitieswerecalculatedinrelationtothexylanaseactivitywithout pre-incubationwhichwasconsideredtobe100%.

XlnB XlnC

Ion/ReducingAgent Concentration (mM) RelativeActivity (%)±DP HgCl2 10 0 0 2 0 0 ZnCl2 10 56.12±0.01 73.65±0.02 2 58.07±0.00 98.24±0.02 NaCl 10 97.88±0.00 93.24±0.02 2 92.76±0.00 114.44±0.04 MgCl2 10 86.79±0.03 129.97±0.02 2 83.65±0.03 113.59±0.01 EDTA 10 57.62±0.01 76.68±0.03 2 64.34±0.01 90.17±0.01 SDS 10 0.00 0.00 2 0.00 0.00 MnCl2 10 93.18±0.10 119.70±0.02 2 101.58±0.04 116.52±0.02 CaCl2 10 90.28±0.06 95.44±0.01 2 90.14±0.03 113.21±0.04 CuSO4 10 0 0 2 0 93.66±0.01 ␤-mercapthoethanol 1 87.64±0.01 122.66±0.01

xylan,containing95%ofxylose[13].Similarresultsweredescribed

forthenativelyproducedenzymessinceinthatstudyXlnBdidnot

showactivityagainstCMC,␳NP␤Xil,␳NP␤Gli,␳NP␣Ara,␳NP␤Gal

and␳NP␣ManandXlnCpresentedhigheractivityagainst

birch-woodxylan,followedbyarabinoxylanandinsolubleoatspeltxylan

[20,21].

Theenzymesshoweddistinctsensitivitiestoionsandreducing

agents(Table1).XlnBandXlnCwerecompletelyinhibitedbySDS

andHgCl2.CuSO4completelyinhibitedXlnBbuttotalinactivation

ofXlnCwasachievedonlyat10mMconcentration.Thedenaturing

actionofSDSprobablyaffectedtheintegrityoftheenzyme

tridi-mensionalstructurewhichisfundamentalforitscatalyticactivity

[34].TheionCu2+_is_known_to_be_a_strong_inhibitor_of_xylanases

[29,30].XlnCwasactivatedbyMnCl2andMgCl2,butMgCl2reduced

theXlnBactivity.␤-Mercaptoethanol,areducingagent,promoted

theactivationofXlnCwhichcanbeexplainedbythefactthatsome

reducedchemicalligationsintheenzymestructurearefavorable

forthecatalyticactivity[34].Theactivitiesofnativexylanaseswere

differentlyaffectedbythepresenceofions[20].ThenativeXlnB

wascompletelyinhibitedby1mMofHg2+_and_Cu2+_,_while_the_same

concentrationoftheseionsreducedtheactivityofnativeXlnC.The

activityofthesenativeenzymeswerereducedbyMn2+_[20]_.

When endoxylanases act on xylan, the products are

xylooligosaccharides that can inhibit the enzyme activity by

a retro-inhibition mechanism [7]. The effects of the products

xylotriose and xylotetraose on the activities of XlnB and XlnC

showedthatxylotriosedidnotinibitthesexylanasesat0.1mg/ml,

butat1mg/mlXlnBactivitywasfullyinhibitedandXlnCactivity

was reduced to 66.7% of its original activity. Xylotetraose at

0.1mg/mlconcentrationdidnotaffecttheactivityofXlnBandonly

slightlyreducedtheXlnCactivityto93.3%ofitsoriginalactivity.

ThisresultindicatesthatXlnBismoresensitivetoxylotriose,while

XlnCwasaffectedbyxylotetraose.

ThecalculatedvaluesofKm,kcatandkcat/Kmforthexylanases

XlnBandXlnCagainstthedifferentxylansareshowninTable2.

XlnB and XlnC had similar catalytic efﬁciency values (kcat/Km)

againstthetestedsubstrates,exceptthatXlnCwasmoreefﬁcient

tocatalyzethehydrolysisofoatspeltxylan,whileXlnBhydrolyzed

wheatarabinoxylanlessefﬁciently.Thekineticconstantvaluesof

nativeXlnBandXlnCagainstthedifferentxylans[21],were

com-paredtothevaluesobtainedfortheheterologousenzymes.The

nativeXlnBshowedKmvaluesagainstbirchwoodxylan,

arabinoxy-lanandoatspeltsxylanof1.78,1.5and4.15mgml−1,respectively,

whilethesevaluesforXlnCwere4.37,12.14and12.43mgml−1,

respectively.Theseresultssuggestthattheheterologousenzymes

displaysimilarkineticpropertiesasthenativexylanases.

ThehydrolysisproductsofbeechwoodxylanbyXlnBandXlnC

wereanalyzedinTLCandcomparedwiththeproductsobtained

afterbeechwoodxylanhydrolysisusingthecommercialenzymatic

mixtures Accellerase® 1500andMultifectCL® (Suppl.Data, Fig.

1A).ThemainproductofxylanhydrolysisbyXlnBandXlnCwas

xylotetraose,whilexylotrioseappearedinlessconcentration.The

occurrenceofthexylooligossacharidesxylotrioseandxylotetraose

andtheabsenceofxyloseastheﬁnalproductconﬁrmsthe

activ-ityendoxylanolyticofXlnBandXlnC.Theproductsobtainedafter

hydrolysisbyAccellerase®1500andMultifectCL®werexylotriose,

xylotetraoseand oligossacharideswitha polymerizationdegree

higherthanfour.Theabsenceofstandardswithhigher

polymeriza-tiondegreethanxylotetraoseanddecoratedstandardsprevented

theidentiﬁcationofotherxylanhydrolysisproductsbythetested

commercialcocktails.

3.2. EffectsofXlnBandXlnCincommercialenzymaticmixtures

XlnBandXlnCwereaddedtothecommercialenzymatic

mix-turesMultifectCL® andAccellerase®1500andtheﬁnalxylanase

activitieswerehigherthanthetheoreticalactivities(Suppl.Data,

Fig.1B).Thecommercialcocktailsalreadyhaveseveralenzymes

that are able to hydrolyze xylan, such as endoxylanases,

␣-arabinofuranosidases,_{␤-xylosidases,}andothers[35].Still,XlnBand

XlnChadpositiveeffectontheactionofthexylanasespresentinthe

commercialcocktails.However,thiseffectvarieddependingonthe

commercialmixtureandtheuseofXlnBorXlnC,probablyduethe

mechanismsofactionofXlnB(GH10)andXlnC(GH11)andthe

dif-ferentxylanasespresentinMultifectCL® andAccellerase® 1500.

Thehighestadditiveeffectwasobservedfor MultifectCL®

sup-plementedwithXlnBorwithbothenzymes(XlnB+XlnC),which

were86.53%and86.09%,respectively.However,XlnCwasmore

efﬁcientfor Accellerase® 1500supplementation.These additive

effectsobservedinbeechwoodxylanhydrolysisindicatethatthe

additionofXlnBandXlnCinthecommercialenzymaticmixtures

usedforthehydrolysisofsugarcanebagassecouldimprovethe

overallxylanolyticactivity.

Table2

MichaelisMentenconstant(Km),maximalvelocity(Vmax),catalyticconstant(kcat)andcatalyticefﬁciency(kcat/Km)forXlnBandXlnC,usingdifferentxylans.

Enzyme Substrate Km(mgml−1) Vmax

(␮mol/minml−1₎ kcat(min

−1₎ _kcat/Km

(mlmg−1_min−1₎

XlnB Beechwoodxylan 1.66 193.27 5.56106 _3.53₁₀6

Wheatarabinoxylan 6.67 626.55 1.90107 _2.85₁₀6

Oatspeltxylan 7.45 151.50 4.59106 _6.16₁₀5

XlnC Beechwoodxylan 4.23 435.82 1.69107 _3,99₁₀6

Wheatarabinoxylan 10.27 934.03 1.108 _1.07₁₀7

(7)

Fig.4. Sacchariﬁcationassaysofalkali-pretreatedsugarcanebagasse.(A)Glucoseand(B)xylosereleaseusingMultifect®_CL_as_the_cellulase_source._(C)_Glucose_and_(D)_xylose

releaseusingAccellerase®₁₅₀₀_as_the_cellulase_source.

3.3. Sacchariﬁcationexperiments

Alkali-pretreatedsugarcanebagassewashydrolyzedby

com-mercialcocktails(Multifect® CLand Accellerase® 1500)withor

withoutsupplementationbyXlnBand/orXlnC(Fig.4).

Concerningsugarcane bagasse sacchariﬁcationbyMultifect®

CL,ahigherreleaseofglucose(6.94g/l)wasachievedwhenthe

commercialcocktailwassupplementedwithbothXlnBandXlnC.

Xylanases are not able to produce glucose directly but these

enzymescouldstimulate cellulose hydrolysis since theyacton

thehemicellulosefractionandfacilitatetheaccessofcellulolytic

enzymestocellulose[36,37].Multifect®CLwithout

supplemen-tation and withboth XlnB and XlnC resulted in similarxylose

release(3.36and 3.37g/l,respectively).Therefore thexylanases

didnotaffectthereleaseofxylosefromoligosaccharidesbythe

␤-xylosidasepresentinthecommercialcocktails,butlikelymainly

affectedthexylandepolymerization.

Whensugarcanebagassesacchariﬁcationwasperformedusing

Accellerase® 1500,thesupplemented mixtures alsoresulted in

higherglucoserelease.Accellerase®1500withoutsupplementation

released6.66g/lofthissugar,whileAccellerase®1500withXlnC

released8.3g/lofglucose.SupplementationwithXlnCalso

pro-motedthebestreleaseofxylose(4.54g/l).Theseresultsshowthat

XlnC(GH11)hasabetterpotentialforsugarcanebagasse

hydroly-siswhenusedasasupplementfortheAccellerase®1500.Indeed,

GH11xylanaseshaveforalongtimebeenusedas

biotechnologi-caltoolsinvariousindustrialapplicationsduetotheirinteresting

properties suchas highsubstrate selectivity and high catalytic

efﬁciency,smallsize(around20kDa)andvarietyofpHand

tem-perature optimum [11]. The sacchariﬁcation assays performed

usingAccellerase®1500resultedinhigherreleaseofglucoseand

xylosethanthoseperformedusingMultifect® CL.Thisindicates

thatAccellerase® 1500leadstohighersugarreleasefrom

alkali-pretreatedsugarcanebagasse,withorwithoutsupplementation.

Table3

Totalreducingsugars(theresultsaremeanvalues±SDofthreemeasurements) andpercentageofglucanandxylanconversionsafter72hofsugarcanebagasse sacchariﬁcationbythedifferentenzymemixtures.

EnzymaticMixture ReducingSugars±SD SacchariﬁcationYield −Conversion (␮mol/ml) Glucan(%) Xylan(%) MultifectCL 68.90±0.03 14.03 14.45 MultifectCL+XlnB 68.36±0.05 14.38 12.94 MultifectCL+XlnC 71.84±0.08 14.73 12.73 MultifectCL+XlnB+XlnC 72.30±0.12 16.25 14.45 Accellerase 74.93±0.06 15.55 16.82 Accellerase+XlnB 79.19±0.05 17.53 17.25 Accellerase+XlnC 80.33±0.03 19.40 19.63 Accellerase+XlnB+XlnC 69.61±0.01 18.00 17.04

Totalreducingsugarsweremeasuredafter72hof

sacchariﬁ-cationexperiments(Table3).Theseresultsconﬁrmedthatwhen

Multifect®CLwasusedforsugarcanebagassehydrolysis,thebest

yieldwasachievedbysupplementationwithbothXlnBandXlnC

and that for Accellerase® 1500 the maximal release of sugars

occurredwhenitwassupplementedwithXlnC.

Complementing crude enzyme extracts shows signiﬁcant

promisesinceitcanresultinsynergisticeffectsthatimprovethe

efficiency of biomass saccharification [38]. The saccharification

yieldsinourexperimentswerelowerthan20%whichcanbe

asso-ciatedwiththehighsolidconcentrationusedfortheexperiments

andalsotothemildconditionofalkalipretreatment.Althoughhigh

solidconcentrationisknowntodecreaseconversionrates,itcan

reducetheamountofwaterintheprocessandgenerateamore

concentratedproduct,whichcanresultinadecreaseinprocess

costs[39].Moreover,themildconditionsareimportanttoreduce

inhibitorformationand environmentalimpacts buttheycanbe

(8)

4. Conclusion

TwoxylanasesfromA.nidulansexpressedinP.pastoris,XlnB

(GH10)andXlnC(GH11),werestudiedandtheirabilitytodegrade

differentxylanswasdemonstrated.Theenzymeswerestablein

a wide pH range and reasonable temperatures and were able

toact synergisticallywithenzymes present in commercial

cel-lulase cocktails. They also demonstrated satisfactory results as

supplementsofcommercialenzymecocktailsforsacchariﬁcation

ofalkali-pretreatedsugarcanebagasse.Therefore,XlnBandXlnC

havea great potentialfor biotechnologicalconversions,as

sup-plementsofcommercialcocktailsforbiomasssacchariﬁcationin

secondgenerationethanolproductionprocesses.

Conﬂictofinterest

Theauthorsdeclarenoﬁnancialorcommercialconﬂictof

inter-est.

Acknowledgements

WeacknowledgetheBrazilianinstitutionsCAPESforthe

schol-arshipgrantedtotheﬁrstandsecondauthorsandFAPEMIGand

CNPqfortheresourcesprovidedtocompletethisexperiment

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,

intheonlineversion,athttp://dx.doi.org/10.1016/j.ijbiomac.2016.

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