Impact of the removal of N-terminal non-structured amino acids on activity and stability of xylanases from Orpinomyces sp. PC-2

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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

Impact

of

the

removal

of

N-terminal

non-structured

amino

acids

on

activity

and

stability

of

xylanases

from

Orpinomyces

sp.

PC-2

Rafaela

Zandonade

Ventorim

a

_,

_Tiago

_Antônio

_de

_Oliveira

_Mendes

b

_,

Larissa

Mattos

Trevizano

a

_,

_Ana

_Maria

_dos

_Santos

_Camargos

a

_,

Valéria

Monteze

Guimarães

a,∗

a_BIOAGRO,_Universidade_Federal_de_Vic¸_osa,_Vic¸_osa,_MG_36.570000,_Brazil

b_Departamento_de_Bioquímica_e_Biologia_Molecular,_Universidade_Federal_de_Vic¸_osa,_Vic¸_osa,_MG_36.570000,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received20April2017

Receivedinrevisedform19July2017 Accepted1August2017

Availableonline3August2017

Keywords: Xylanase Orpinomyces Thermostability

Moleculardynamicssimulation

a

b

s

t

r

a

c

t

Xylanasescatalyzetherandomhydrolysisofxylanbackbonefromplantbiomassandthus,theyhave applicationintheproductionofbiofuels,Kraftpulpsbiobleachingandfeedindustry.Here,xylanases derivedfromOrpinomycessp.PC-2wereengineeredguidedbymoleculardynamicsmethodstoobtain morethermostableenzymes.Basedonthesemodels,27aminoacidresiduesfromtheN-terminalwere predictedtoreduceproteinstabilityandtheimpactofthisremovalwasvalidatedtotwoenzyme con-structs:smallxylanaseWild-Type(SWT)obtainedfromWild-Typexylanase(WT)andsmallxylanase Mutant(SM2)generatedfromM2mutantxylanase(V135A,A226T).Thetailremovalpromotedincrease inspeciﬁcactivityofpuriﬁedSWTandSM2,whichachieved5,801.7and5,106.8Umg−1_of_protein,

respec-tively,whiletheWTactivitywas444.1Umg−1_of_protein._WT,_SWT_and_SM2_showed_half-life_values_at

50◦Cof0.8,2.3and29.5h,respectively.Overall,inviewoftheresults,weproposethatthepresenceof non-structuredaminoacidintheN-terminalleadstodestabilizationofthexylanasesandmaypromote lessaccessofthesubstratetotheactivesite.Therefore,itsremovalmaypromoteincreasedstabilityand enzymaticactivity,interestingpropertiesthatmakethemsuitableforbiotechnologicalapplications.

1. Introduction

Xylanases(E.C.3.2.1.8)catalyzetherandomhydrolysisofxylan backbone,amainconstituentofplantbiomass,inanendwise man-nertoyieldxylooligosaccharides[1].Duetothisremarkablerole inthedeconstructionofxylan,theseenzymeshavearousedgreat interestfortheproductionofbiofuels,biobleachingofKraftpulps andinthefeedindustry[2].

Xylanasesareclassiﬁedwithinglycosidehydrolase(GH) fami-lies5,8,10,11,and30accordingtoCarbohydrate-ActiveenZymes (CAZy)database(www.CAZy.org)basedonsimilaritiesofthe cat-alytic domainsequences [3,4]. Comparedto theother families, GH11xylanaseshaveseveralinterestingpropertiesthatmakethem suitableforbiotechnologicalapplications,suchashighsubstrate selectivity,highcatalyticefﬁciency,andactivityinabroadranges ofpHandtemperature[2].

Xylanases from the same GH family show similar three-dimensional structures, active-site geometries and conserved

∗ Correspondingauthor.

E-mailaddress:vmonteze@ufv.br(V.M.Guimarães).

enzymaticmechanism.Thexylanasefamily11presentacompact globularstructurewithasingle␣-helixandtwoextendedpleated ␤-sheetsformingajelly-rollfold[5].Themainfeatureofthis fam-ilyisthepresenceofalongcleftthatspanstheentiremolecule. Thiscleftcontainstheactivesiteincludingtwoglutamateresidues thatdirectlyparticipateofxylanhydrolysis,inwhichoneactingas acid/basecatalystandtheotherasanucleophile[5,6].

GH11 xylanase (XynA) produced by the anaerobic fungus Orpinomycessp.PC-2showed higherspeciﬁc activitycompared with equivalent enzymes from other sources [7]. This native enzymewasmostefﬁcientinthepHrangingfrom5.0to6.5and temperaturesfrom40to50◦C[7,8].However,morestableenzymes arerequiredforbiotechnologicalprocesses,suchaspulpandpaper, animalfeedandbiofuelindustries.Thexylanasesmustbesuitable forthepHandtemperatureconditionsoftheseprocesses, exhibit-inghighthermostabilityandwidepHadaptability[2,9].

Inourpreviouswork,thedirectedevolutionmethodologyusing themutagenictechnique oferror-pronePCRwascarriedoutto improve thethermostability of xylanases derived from Orpino-mycesXynA[9].Thismethodologywaseffectivetoidentifyfour mutantxylanases,whichexhibitedhigherthermostabilityat60◦C comparedtothewild-typeenzyme(WTXynA). However,these

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morethermostablemutantenzymesshoweddrasticreductionof activity.AnexceptionwastheM2mutantxylanase(V135A,A226T) thatexhibitedahalf-lifeof33.2minat60◦C,approximatelyfour timeshigherthanthewild-typeenzyme(7.92min)andmaintained similaractivity.Therefore,theM2mutantxylanaseshowedtobe mostpromisingforfutureindustrialapplicationfromthe biotech-nologicalpointofview.

Severalstudieshavedemonstratedtheimpactofunstructured aminoacids,locatedintheN-terminalregion,inthe thermosta-bilityofxylanases[10,11].TheremovalofN-terminalresiduesby strategiesofenzymerationalengineeringcouldpromote enhance-mentofenzymethermostability.Inthiswork,MolecularDynamics (MD) approaches were taken toevaluate alterations in the N-terminalregionofWTxylanasederivedfromOrpinomycesXynA thatcouldpotentiallyaffectproteinstabilityandactivityinorder togenerateimprovedxylanasesandtwoenzymeconstructswere producedtovalidatethesimulationresults.

2. Materialandmethods 2.1. Strains,vectorsandreagents

E.coliBL21-CodonPlus(DE3)-RIPLcompetentcellswere pur-chasedfromStratagene(La Jolla,CA,USA)and usedforprotein expression. The expression vector pET24(b) and the KOD DNA polymerasekitwerepurchased fromNovagen– EMDMillipore (SanDiego, CA,USA).The T4DNA ligasekit and enzymesNdeI andXhoI werepurchasedfromFermentas –ThermoFisher Sci-entiﬁc(Waltham,MAUSA).TheGelExtractionDNAkitandDNA plasmidialpuriﬁcationMiniprepkitwerepurchasedfromQiagen (Venlo,Germany).TheE.colistrainDH5␣(Stratagene)wasusedfor propagationandmanipulationofplasmids.Beechwoodxylanand xylosewerepurchasedfromSigmaChemicalCo.(St.Louis,MO, USA).

2.2. Predictionofsignalpeptides

TheSignalP4.0server( http://www.cbs.dtu.dk/services/SignalP-4.0/)[12]settledtoEukaryoteswasusedtopredictexport pep-tidesignalandcleavagesitefromOrpinomycesXynAxylanaseand theProtParam tool(http://web.expasy.org/protparam/)[13] was usedtopredictphysicalandchemicalparameters ofwilde-type andmutantproteins.

2.3. Xylanasemoleculardynamics

ThePhyre2 web server(http://www.sbg.bio.ic.ac.uk/phyre2/) [14]wasusedtoobtainthe3DmodeloftheWTXynAcatalytic domainandSWTxylanase.Asinthepreviouswork[9],themodels obtainedwereconstructedbasedonthexylanasefrom Neocallimas-tixpatriciarum(PDBID2C1F,2.1Åresolution)[15]thatshows91% identitywiththecatalyticdomainofOrpinomycesXynA. Consider-ingthisalignment,itwaspossibletoconstructamodelforresidues 13-230ofthewild-typeXynA,withoutsignalpeptide,and2-213of theSWT.Themodelwascheckedfortheirstereochemicaland over-allstructuralqualityusingPhyre2Investigator[14],Procheck[16], Verify3D[17,18],ERRAT[19],RAMPAGE[20],ProSA-web[21]and JPred4[22].Thethree-dimensionalstructureofmodeledprotein wasanalyzedusingthePyMOLMolecularGraphicsSystem(Version 1.8,Schrödinger,LLC).

Moleculardynamicssimulationswerecarriedoutonacomputer clusterequippedwith2IntelXeonprocessorsX5650,2.66GHzand 48GBofRAMonaLinuxPlatform,usingNAMDsoftware(Version 2.11)[23]andperformedwithCHARMMforceﬁeld(Version1.9.2) [24].VMDinterface[25]wasusedtopreparethexylanasemodelsas

wellastomakevisualizationsandanalysisoftrajectories.The sys-temsizewaschosentodistancetheproteinatomsapproximately 5.0Åoftheedgeofthesimulationwaterbox.Thesystemswere minimizedat313or323Kfor1000stepstoequilibratethe posi-tionalrestraintsontheproteinswithsolventmolecules.Theﬁnal productionofsimulationsweredonefor10nswiththetimestepof 2fsat313and323K.PressurewascontrolledwithaLangevin pis-ton(1atm)andtemperaturebyLangevinthermostat.TheParticle MeshEwald(PME)algorithm[26]wasusedtomodelthe electro-staticinteractions.Trajectoriesweresavedatevery5000steps. 2.4. PrimersconstructionandPCRampliﬁcations

A pair of oligonucleotides, XF1

(5GGCAATTCCATATGGGTCAAAGATTAAGCGTTGG3) and XR1 (5GCCGCTCGAGTTAACGAGGAGCAGAACCTTGTTT3),was synthe-sized toperforme ampliﬁcation of genesencoding the selected xylanases (SWTand SM2).XF1matchedwiththenucleotide85 andXR1matchedwithantisensenucleotides745-765ofthegene ofxylanaseAfromOrpinomycesPC-2.Theunderlinedand double-underlined nucleotides represent the NdeI and XhoI restriction sites,respectively. TheplasmidpET24(b)withthenon-mutated andmutatedxynAinsertsservedasatemplate.

PCR wasperformed in a 50␮Lvolume containing 5␮L 10X buffer,5␮Mofeachprimer,2.0mMofeachdNTP,1.5unitKOD polymerase, 2.5mM MgCl2, and 2ng plasmid DNA. PCR reac-tionswerecarriedoutinaMastercyclerThermocycler(Eppendorf, Germany)withthefollowingconditions:initialheatingat94◦Cfor 3min,then3cyclesof95◦Cfor20s,37◦Cfor30sand72◦Cfor 60s,then30cyclesof95◦Cfor20s,65◦Cfor30sand72◦Cfor45s, followedbya72◦Cincubationperiodfor10min.

2.5. Cloningandsequenceanalysis

ThePCRproductwasvisualizedona0.8%(w/v)agarosegel, puri-fiedusingtheGelExtractionkit,digestedwithNdeIandXhoIand clonedintotheexpressionvectorspET24b(+)usingtheT4DNA lig-asekit.TheligatedproductwasusedtotransformE.coliDH5␣.The recombinantplasmidialDNAwaspurifiedfromisolatedcolonies andverifiedbyDNAsequencingwithanautomaticMacrogenPCR sequencer(Gasan-dong,Geumchun-gu,SeoulKorea).

Inordertodetermineifmutationpatternsweremaintained,the DNAsequencesweretranslatedintotheirproteinsequencesand comparedtotheinitialsequencesusingtheCLUSTALW(Version 1.81) alignment program (http://www.ebi.ac.uk/clustalw). Plas-midialDNAswerethenextractedfromE.coliDH5␣andtransferred bythermalshockprotocoltoE.coliBL21-CodonPlus(DE3)-RIPL. 2.6. Xylanaseexpression

E.colicultures(1mL)wereinitiallyinoculatedin40mLofLB/Kan medium (triptone 1%, NaCl 1%, yeast extract 0.5%) and shaken overnightat250rpmand37◦C(NewBrunswick,ScientiﬁcCo., Edi-son,NJ,USA).Atotalof40mLofthecultureswereinoculatedin 1LofSOB/Kanmedia(triptone2%,yeastextract0.5%,NaCl0.05%, KCl2.5mM,MgSO40.01M,NaOH0.5mM)andshakenat250rpm and37◦CuntiltheOD600reached1.0.Afterthat,2.5mLofIPTG

solution(100mM)wereaddedto1Lcultures(ﬁnalconcentration of0.25mM)andshakenat37◦C,250rpmfor4htoinducexylanase expression.

Afterinduction,thecultureswereincubatedonicefor30min andthencentrifuged(15,000×g)at4◦Cfor30min.Thepelletwas resuspendedin25mLofsodiumphosphatebuffer,100mM,pH6.5. Theintracellularxylanasepreparationswereobtainedafter soni-cationcyclesofthirtytimesfor10swithabreakof10sbetween eachsonication.Thissuspensionwasthencentrifugedat4◦Cand

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15,000×gfor20min,andthesupernatantwasusedasancrude enzymeextract.

2.7. Intracellularxylanasespuriﬁcation

TheintracellularxylanaseswereloadedintoaQ-Sepharoseion exchangecolumn(1.6×2.5cm)(GEHealthcareLifeSciences, Upp-sala,Sweden)previouslyequilibratedwith25mMsodiumborate buffer, pH8.5. The sample wasalsoequilibrated and eluted in thesamebuffer, followedbyalinearsalt gradientconsistingof 25mMsodiumboratebuffer,pH8.5andthesamebuffercontaining 1MNaCl.TheprocesswasperformedbyFastProteinLiquid Chro-matography(ÄKTAProteinPuriﬁcationSystem–GEHealthcareLife Sciences,Uppsala,Sweden)withaﬂowrateof4mL/min.Active fractionswerepooledandanalyzedforpuritybySDS-PAGEusing 12.5%(w/v)acrylamidegel[27].Theproteinbandswerevisualized bysilverstaining[28].

Theproteinconcentrationintheenzymaticextractwas deter-minedbytheCoomassieBluebindingmethodusingbovineserum albumin(BSA)asthestandard[29].

2.8. Massspectrometry

Mass spectrometric analysesof theWT and SWT xylanases werecarried out by matrix-assistedlaser desorption-ionization time-of-flightmassspectrometry(MALDI-TOF-MS)method. Puri-fiedxylanasesweresubjectedtodigestionwithsequencinggrade modifiedtrypsin(Promega,Madison,MI,USA)accordingtotheAn In-SolutionDigestionProtocolofKinterandShermanet[30].The peptidefragmentswereanalyzedonABSCIEXMALDITOF/TOFTM 5800System(AppliedBiosystems,FosterCity,CA,USA).Afterdata acquisition, a list of peaks was obtainedfrom the raw MS/MS datausingthe4000SeriesExploreSoftware(AppliedBiosystems). Thepeptidesequenceswereassociatedtom/zpeaksthroughthe theoreticaldigestionand ionizationpredictedbyMS-digesttool implementedintheProteinProspectorwebserver(Version5.18.1) [31].

2.9. Xylanaseassay

Xylanaseactivitywasdeterminedbymeasuringthereleaseof reducingsugarsfrombeechwoodxylanusingthedinitrosalicylic acidreagent(DNS) [32].The xylanase standard assaywas per-formedin500␮Lreaction volumescontaining400␮L (1%w/v) beechwoodxylandilutedin100mMsodiumphosphatebufferpH 6.5and0–100␮Lofappropriatelydilutedenzymepreparations. Thereactionwascarriedoutfor30minat40◦Candstoppedby theadditionof0.5mLoftheDNSreagent.Testtubeswereboiled for5minandthenchilledonicewaterfor10min.Theamountof reducingsugarsreleasedwasdeterminedat540nm.D-xylosewas usedasthestandard.Oneunitofxylanaseactivitywasdeﬁnedas theamountofenzymethatreleased1␮molofxyloseperminunder standardassayconditions.

Thedataofxylanaseactivitywerepresentedasmeanvalues oftriplicateassaysinwhichthestandarddeviationswerealways lessthan10%andtheresultswereexpressedasmeans±standard deviations.

2.10. EffectofpH,temperatureandthermostabilityonxylanase activity

TheinﬂuenceofpHonxylanaseactivitywasdeterminedunder standardassayconditions,exceptthatpHrangewasfrom2.0to 11.0.Thebuffersatﬁnalconcentrationof100mMwere:sodium phosphatebuffer(pHvaluesof2,3,7and8),sodiumcitratebuffer (pH4–6)andsodiumcarbonatebuffer(pH9–11).

Fig.1. Homologymodelofmaturexylanase(WT)catalyticdomain.Thetailthat couldaffectxylanasestabilityintheN-terminalregionisshowninyellowandthe twocatalyticglutamatesareshowninbluesticks.(Forinterpretationofthe refer-encestocolorinthisﬁgurelegend,thereaderisreferredtothewebversionofthis article.)

Theeffectoftemperatureonenzymeactivitywasdetermined understandardassayconditions,exceptthattemperatureranged from20to80◦C.

Thermalstabilitywasdeterminedby incubatingthepuriﬁed dilutedenzymeat50◦Cin100mMsodiumphosphatebuffer,pH 6.5. Afterincubation at differenttimes, aliquots wereremoved andtheresidualxylanaseactivitiesweremeasuredbythe stan-dardassay.Half-lifevalueoftheenzymeswascalculatedusingthe CurveExpertprogram(Version1.4)[33]fromthedataofresidual activitydeterminedat50◦C.

2.11. Kineticproperties

Thekineticparameters(k_M andkcat)ofthepuriﬁedxylanases weredeterminedbynonlinearcurveﬁttingusingthe Michaelis-MentenplotandtheCurveExpertprogram(Version1.4)[33].The substratebeechwoodxylanwasusedatconcentrationsbetween 0.25and10mg/mLin100mMsodiumphosphatebuffer,pH6.5 andtheassayswerecarriedoutat40◦Cfor30min.

3. Resultsanddiscussion

3.1. Moleculardynamicssimulationsandsmallxylanase evaluation

Predictionanalyzesoftheexportsignalpeptideandthecleavage sitefromOrpinomycesXynAxylanaseidentiﬁedaprocessingsite betweenaminoacidresidues18and 19togeneratethemature enzyme.

ThemoleculardynamicsanalysisofthematureWTxylanase revealedthattheﬁrsttenresiduesintheN-terminalregionhaveno deﬁnedsecondarystructure,weaklytointeractwiththeremaining globalstructureandhighmobility,reachingaminoacidsnearthe catalyticsite.Therefore,wesuggestthatremovalofthisN-terminal tailcouldimprovethestabilityofthemolecule.Fig.1showsthe matureWT xylanase3D structure, emphasizingthe N-terminal destabilizingregion.

Inordertoverifyifremovalofthisregionwouldcompromise thexylanaseactivity,aMDtrajectoryof10nswasevaluatedto investigatetheaverageRMSﬂuctuationsofaminoacids(Fig.2).It isseenthatthemajorityoftheresiduesofthesmallxylanase(SWT –maturexylanasewithouttheﬁrsttenaminoacids)showing

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dis-Fig.2.AverageRMSﬂuctuationvalues(Å)asafunctionofaminoacidsequenceupto10nssimulationtrajectoryat313K(A)and323K(B).MaturexylanaseWT(black); smallxylanaseSWT,withouttheN-terminaltail(gray).

Fig.3. Distance(Å)betweenthecatalyticglutamateresiduesofthexylanasesalongamoleculardynamictrajectoryof10nsat313K(A)and323K(B).MaturexylanaseWT (black);smallxylanaseSWT,withouttheN-terminaltail(gray).

Fig.4.Radiusofgyration(Å)ofthexylanasesalongamoleculardynamictrajectoryof10nsat313K(A)and323K(B).MaturexylanaseWT(black);smallxylanaseSWT, withouttheN-terminaltail(gray).

placementslessthan9.0Åat313Kandlessthan5.0Åat323K. Ontheotherhand,theRMSﬂuctuationsobservedforaminoacids locatedinthemoststableconformationsof␤-sheetsand␣-helix ofmaturexilanase(WT)werehigherthantheobserved displace-mentsforallresiduesofSWT.Thisresultindicatedthatremoval oftheN-terminaltailcouldpromotethestabilizationofindividual residuesandofthewholemolecule.

Thedistancebetweenthecatalyticglutamateresidues(E115 and E203)of theSWTand WT(E124 and E212)wasevaluated (Fig.3).Positioningofthecatalyticresiduesontheactivesite, dur-ingsimulationsatbothevaluatedtemperatures,suggeststhatthe tailremovalpromotedconsiderablestructuralchangesintheactive site,resultinginagainofﬂexibilityintheSWT,whichprobably ensuresthecatalyticperformanceofthisenzyme.

Theradiusofgyration(Rg)ofthematurexylanaseWTandsmall xylanaseSWTwerepresentedinFig.4.TheRgisaparameterthat describestheequilibriumconformationofatotalsystem,indicating proteinstructurecompactness[34].ThelowerRgvalue,presented bytheSWTstructureat313K,indicatesthatthesmallenzymehasa

morecompactstructurewhichmayhaveimplicationsonthe over-allproteinstability,whileat323Kbothxylanasesacquiredsimilar Rgvalues.

The information obtained from molecular dynamics analy-sesindicatedthatremovaloftheN-terminaltailcouldpromote increasedenzymestability,sincethisN-terminalregionhaveno definedsecondarystructurethatappearshighlyflexible.Besides that,theN-terminaltaillocationnearcatalyticaminoacidsinthe 3Dstructure ofthexylanasemolecule(Fig.1), suggestthatthis tailcanalsoinfluencetheproperpositioningofthesubstrateinthe cavityofthecatalyticsite,whichcouldresultinreducedenzymatic activity.

3.2. N-terminalevaluationbymassspectrometry

InordertoconﬁrmthedifferencesintheN-terminalregionof thexylanases,thegenesencodingtheWTandSWTwerecloned intotheexpressionvectorpET24(b)andusedtotransformE.coli BL21(DE3)RIPL.Theexpressedenzymeswerepuriﬁed,digestedand

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Fig.5. N-terminalregionoftheWTandSWTxylanaseswiththerespectivesignal peptideprocessingsitespredictedbySignalPServerandbyMS.

Fig.6. SDS-PAGE(12.5%)oftheE.coliintracellularcrudeextractandpurified xylanases.1–molecularweightmarker,2–proteinprofilefromexpressionofWT 3–proteinprofilefromexpressionofSWT,4–proteinprofilefromexpressionof SM2,5–molecularweightmarker,6–purifiedWT,7–purifiedSWT,8–purifiedSM2. Proteingelwasstainedwithsilvernitrate.

analyzedbymassspectrometry(MALDI-TOF-MS).Theanalysesof theidentifiedpeptidessuggestthatprocessingofsignalpeptideby E.colioccurredatacleavagesitedifferentfromthatpredictedby SignalP(Fig.5).BasedonthepredictionsofSignalP,thesize dif-ferencebetweenWTandSWTxylanaseswouldbeequivalentto nineaminoacidresidues,which couldformasmallloopatthe N-terminalregionofWT.Ontheotherhand,theanalysesofthe identifiedpeptidesshowedthattheseenzymesexpressedinE.coli exhibitedadifferenceof25aminoacidresidues.Thissequenceof25 aminoacidresiduescouldformanunstructuredloop,whichcould negativelyaffectthepositioningofthesubstrateinthecatalytic cavity, reducing the enzymatic activity of WT.TheMS analyses showedthepeptidesof697.909m/zand807.139m/z,whichwere obtainedfromWTandSWTfragmentation,respectively(Fig.S1). Thesepeptideswereincompatiblewiththeexpectedproductsfrom trypticcleavageofthesexylanases.The697.909m/zwas compat-iblewiththe AITTVAKfragment,which is partof theexpected FLFALAITTVAKpeptide.Similarly,the807.139m/zwasequivalent totheGGQNQHKfragment,whichispartoftheLSVGGGQNQHK peptide.Theseresultsindicatethattheprocessedsignalpeptideof WTcorrespondedtothefirsttenaminoacidresidues,whilethe pro-cessedsignalpeptideofSWTwascorrespondingtothefirsteight aminoacidresidues(Fig.5).Therefore,itispossibletoassumethat thedifferenceinmassbetweentheseenzymeswouldbeequivalent to25aminoacidresidues.ThisvariationinmolecularmassofWT andSWTxylanaseswasconfirmedbySDS-PAGE(Fig.6).The pro-teinbandcorrespondingtopurifiedWTxylanase(lane6)presented highermolecularmasscomparedtopurifiedSWTband(lane7). TheseobservationsareinagreementwiththeWTandSWT molec-ularmassesestimatedbyProtParam,thatwere26,640.48Daand 24,300.89Da,respectively.

3.3. Theeffectoftailremovalonxylanaseproperties

Thespecificactivitydeterminedfromcrudeenzymeextractsof WTand SWTxylanasesweresignificantlydifferent.Thespecific activityofSWTwas3,752.1Umg−1ofprotein,whileatthesame conditionsthespecificactivityofWTwas231.7Umg−1ofprotein. TheproteinprofileoftheintracellularcrudeextractsfromE.coli expressingWTorSWT(Fig.6)showedthatSWTwasthemost

abun-dantproteininthecrudeextract(lane3),whiletheamountofWT wasrelativelylowandsimilartoseveralotherproteinsexpressed fromE.coli(lane2).Inthisway,thehigherconcentrationofSWTin theactiveproteinextractcouldcontributetoitssuperiorspeciﬁc activity,howeverotherfactorsareinvolved,whichcouldexplain thedifferenceinthespeciﬁcactivityofthesexylanases.

VariousstudieshaveshownthattheN-terminalregionofGH11 xylanasesisrelatedtoenzymestability[11]andrecentmolecular dynamicinformationhasindicatedthatproteinunfoldinginitiates there[35].E.colihastheN-endrulepathway,whichrelatesthe invivohalf-lifeofaproteintotheidentityofitsamino-terminal residue.ItwasshownthatunstableproteinscontainedArg,Lys, Phe,Leu,TrpandTyrattheaminoterminus[36,37].Inourﬁndings, thepresenceofdestabilizingaminoacidresiduesinthexylanase N-terminalcouldreducetheproteinstability.Infact,theN-terminal region of WT is highly rich in Arg, Lys, Phe and Leu residues (AAD04194).DegradationofWTxylanasecouldoccurpriorto sig-nalpeptideprocessingintheperiplasm,asobservedinexpression systemsofrecombinantproteinsbyE.coli[37,38].Thisamino ter-minusregioncouldpotentiallycontributetolowerstabilityand reduced speciﬁcactivityof theWT, howeverother factorsthat affecttheexpressionoftheenzymesmaybeinvolved[11,37].

Theseenzymeswerepurifiedusingionexchange chromatog-raphyandtheelectrophoreticprofileofthesepurifiedxylanases confirmedthepresenceofasingleproteinbandofWTandSWT (Fig. 6). The specific activities of purified SWT and WT were 5,801.7Umg−1 of protein and 444.1Umg−1 of protein, respec-tively. This higher specific activity of SWT indicates that the removalofresiduesin theN-terminalregioncouldpromotean improvement inxylanaseactivity.In sum, itseemsthat the N-terminaltailremovalpositivelyaffectedtheexpressionandactivity ofSWT.

Basedonthis information,wedecidedtousetheN-terminal region removal approach to evaluate its effect on a mutant xylanase,alsoderivedfromOrpinomycesXynA.

3.4. Productionofamutantsmallxylanase

The mutant xylanase M2 (V135A, A226T) was previously obtained by directed evolution [9]. The M2 enzyme presented improvedthermostabilityat50and60◦C,butitsactivityinthe E.coliintracellularcrudeextractwas34.4Umg−1 ofprotein[9]. Inordertoincreasetheexpressionandactivityofamorestable xylanase,theN-terminalregionoftheM2xylanasewasremoved, generatingthesmallxylanaseSM2.Afterintroductionofa methi-onineresidue(startcodon)toensuresuccessfulproteinsynthesis, genesequencingindicatedthatthemutationpatternintroduced previouslywasmaintained.Thenewsmallenzymewasexpressed inE.coliBL21(DE3)RIPLandtheSM2activityintheintracellular crudeextractreached2,846.8Umg−1ofprotein.Afterpuriﬁcation, theSM2enzymeshowedspeciﬁcactivityof5,106.8Umg−1at40◦C, pH6.5.

3.5. Characterizationofxylanases

ThepuriﬁedWT,SWTandSM2xylanaseshadanoptimum tem-perature at60◦Cand showedsimilarbehavior at temperatures from20to80◦C(Fig.7A).Thisvalueofoptimaltemperaturewasthe sameasthatexhibitedbyM2xylanase[9],andbyBacillussubtilis xylanase[39],andhigherthanseveralfungixylanasesthatpresent optimalactivitiesattemperaturesbelow45◦C[40,41].

TheWT,SWTandSM2showedoptimumpHwithintherange of5–8(Fig.7B).ItisinterestingtohighlightthattheSWTshowed considerableresidualactivitywhenassayedinpH9,about50%, whiletheresidualactivityofWTandSM2werearound10%and 30%,respectively.SM2,WTandSWTenzymesshowedconsiderable

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Fig.7. A–EffectoftemperatureonWT,SWTandSM2activityatpH6.5.Thexylanaseactivitywasmeasuredundertemperaturesrangingfrom20to80◦_C._B_–_Effect_of_pH

onWT,SWTandSM2activityat40◦_C._Xylanase_activity_was_measured_under_pH_values_ranging_from_2.0_to_11.0._C_–_{Thermostability}_at₅₀◦_C._Relative_activity_is_expressed

asapercentageofthemaximumenzymeactivityunderstandardassayconditions.Alldataplottedareaverageoftriplicates.WT(),SWT(䊉),SM2().

Table1

Propertiesofxylanases.Thevaluesofhalf-life(t1/2),kMandkcatconstantsweredeterminedfromactivitystandardassay,usingpuriﬁedxylanasesandbeechwoodxylanas

substrate.

Enzymes t1/2at50◦C(h) kM(mgmL−1) kcat(min−1) kcat/kM(mL/min−1mg−1)

WT 0.77 1.05 8.0×106 _8.0_×₁₀6

SWT 2.27 0.73 2.3×109 _3.3_×₁₀9

SM2 29.46 0.62 1.8×1010 _2.9_×₁₀10

residualactivitywhenassayedinawidepHrangefromacidicto

alkalinepHvalues,similartoxylanasesfromothersources[40,41].

TheSM2retainedapproximately45%ofitsmaximalxylanase activ-ityatpH3(Fig.7B).Thesexylanasescouldbemoresuitablefor biotechnologicalapplicationsthatrequireacidic andneutralpH conditions. For industrial applications,it is interesting that the xylanasesexhibitgreateractivityinawidepHrangeandhigh tem-perature.However,naturalxylanasesthatarethermostable and acidoralkalitolerantarelimited[42].

Asexpected,theSM2wastheenzymemorethermostableat 50◦C, followedbySWTandWTxylanase(Fig.7C).Half-life val-uesoftheSM2,SWTandWTat50◦Cwere1,767.6min,136.4min and46.0min,respectively.After20minofincubationat50◦Cthe WTmaintained62.8%ofitsactivity,andafter2hofincubationthis enzymeexhibitedabout26.2%ofitsoriginalactivity.Ontheother hand,theSWTretained88.8%ofitsactivityafter20minof incuba-tionatthesameconditions,andafter2htheresidualactivitywas stillapproximately43.3%.AlreadythexylanaseSM2maintained 90%activityevenafter4hincubation.TheN-terminaltailremoval couldpromote structuralchanges inthesemoleculesthat could increasethethermalstabilityat50◦C.

Chenetal.[8]purifiedandcharacterizedthenativexylanase fromOrpinomycessp.PC-2.Thecrudeculturefluidpresenteda spe-cificactivityof11.2Umg−1ofproteinandthespecificactivityofthe purifiedenzymewas1,560.0Umg−1 ofprotein.Theenzymewas mostactiveattemperaturesof40–50◦CandpHfrom5.0to6.6.So, theexpressionofthexylanasesderived fromOrpinomycesXynA

inE.colicombinedwiththeN-terminaltailremovalpromotedan increaseintheproductionofxylanaseswithgainofactivityand stability.

The kM values of puriﬁed xilanases were calculated by the

Michaelis-Menten plotusing beechwoodxylan asthesubstrate (Table1).ThekMvaluesfortheSWTandSM2wereclosetoeach

other(0.73and0.62mgmL−1,respectively)andbothwerelower thanthekMvalueofWTxylanase(1.05mgmL−1),indicatingthat

smallxylanasespresentedhigherafﬁnityforthesubstrate.Values ofkcat/kM,aparameterthatevaluatesthecatalyticefﬁciencyofthe

enzymes,were8.0×106_for_WT,_3.3_×₁₀9_for_SWT_and_2.9_×₁₀10

forSM2.Theseresultshighlightthattailremovalgreatlyenhanced thecatalyticefﬁciencyoftheenzymestocatalyzethehydrolysisof beechwoodxylan.

Several studies have focused on changes in the N-terminal regiontoimprovethethermostabilityofxylanases[11,43,44].Yin etal.[43]replacedtheN-terminalsegmentofamesophilicGH11 xylanase fromAspergillus oryzae (AoXyn11) by the correspond-ingregionofahyperthermotolerantGH11xylanase(EvXyn11TS) andthemodiﬁedxylanasewashighlyimprovedandshowed opti-mumtemperatureat75◦Candhighthermostabilityat65◦C.Song etal.[44]obtainedamoreactivexylanasebytheadditionof17 aminoacidstotheN-terminalofGH11xylanasefrom Thermobacil-lusxylanilyticus.Ontheotherhand,theremovalofun-structured residuesintheN-terminalregionresultedinthereductionofthe optimumtemperatureandthermostabilityofanAspergillusniger GH11xylanase[11].Thus,removalofthisN-terminaltailproved

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to be advantageous, since it promoted an increase in enzyme stability, catalytic efﬁciency as well as marked an increase in xylanaseproduction,achievingconsiderablelevelsofexpression. Theseimprovedxylanaseswouldhavepotentialfor biotechnolog-icalapplications,especiallyinprocessesthatrequiremoreactive andstableenzymes.

4. Conclusions

Molecular dynamics simulations contributed to the identi-fication and evaluation of a destabilizing N-terminal tail on OrpinomycesWTxylanase.TwoxylanasescodifiedbyxynAgene without these N-terminal destabilizing residues, one mutated (SM2)andtheothernon-mutated(SWT),wereexpressedathigh levelsinE.coli.Theseenzymesweremoreactiveandstablewhen comparedtoWTxylanaseinabroaderrangeofpHand temper-ature.The positive effect oftail removalon xylanases catalytic propertiesreproducedthepredictionsperformedbytheMD ana-lyzes. The properties of these improved xylanases suggest the potentialofapplyinginseveralbiotechnologicalapplications. Conflictofinterest

Theauthorsreportnoconﬂictsofinterest. Acknowledgments

This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de Minas Gerais-FAPEMIG, the CoordenaçãodeAperfeiçoamentodePessoalde Nível Superior-CAPES and Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq,Brazil.TheauthorsthankDr.DouglasB.Jordan fromtheUSDA,ARS,NCAUR,Peoria,IL,andDr.Xin-LiangLifortheir technicalsupport.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,athttp://dx.doi.org/10.1016/j.ijbiomac.2017. 08.015.

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