Versatile peroxidase as a valuable tool for generating new biomolecules by homogeneous and heterogeneous cross-linking

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new biomolecules by homogeneous and heterogeneous cross-linking

Davinia Salvachúa, Alicia Prieto, Maija-Liisa Mattinen, Tarja Tamminen, Tiina Liitiä, Martina Lille, Stefan Willför, Angel T Martínez, María Jesús

Martínez, Craig Faulds

To cite this version:

Davinia Salvachúa, Alicia Prieto, Maija-Liisa Mattinen, Tarja Tamminen, Tiina Liitiä, et al.. Ver- satile peroxidase as a valuable tool for generating new biomolecules by homogeneous and hetero- geneous cross-linking. Enzyme and Microbial Technology, Elsevier, 2013, 52 (6-7), pp.303-311.

�10.1016/j.enzmictec.2013.03.010�. �hal-01268160�

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Versatile peroxidase as a valuable tool for generating new

biomolecules by homogeneous and heterogeneous cross-linking

Davinia Salvachúa

, Alicia Prieto

, Maija-Liisa Mattinen

, Tarja Tamminen

, Tiina Liitiä

, Martina Lille

, Stefan Willför

, Angel T. Martínez

, María Jesús Martínez

,

Craig B. Faulds

aCentrodeInvestigacionesBiológicas,CSIC,RamirodeMaeztu9,E-28040Madrid,Spain

bVTTTechnicalResearchCentreofFinland,P.O.Box1000,FI-02044VTT,Finland

cProcessChemistryCentre,ÅboAkademiUniversity,Porthansgatan3,FI-20500Turku,Finland

Keywords:

Enzymaticpolymerization Organicco-solvent Lignan

Peptide

␤-Casein

Feruloylatedarabinoxylan

a b s t r a c t

Themodificationandgenerationofnewbiomoleculesintendedtogivehighermolecular-massspecies forbiotechnologicalpurposes,canbeachievedbyenzymaticcross-linking.Theversatileperoxidase(VP) fromPleurotuseryngiiisahighredox-potentialenzymewithoxidativeactivityonawidevarietyofsub- strates.Inthisstudy,VPwassuccessfullyusedtocatalyzethepolymerizationoflowmolecularmass compounds,suchaslignansandpeptides,aswellaslargermacromolecules,suchasproteinandcomplex polysaccharides.Differentanalytical,spectroscopic,andrheologicaltechniqueswereusedtodetermine structuralchangesand/orvariationsofthephysicochemicalpropertiesofthereactionproducts.The lignanssecoisolariciresinolandhydroxymatairesinolwerecondensedbyVPformingupto8unitpoly- mersinthepresenceoforganicco-solventsandMn²⁺.Moreover,11unitofthepeptidesYIGSRandVYV werehomogeneouslycross-linked.Theheterogeneouscross-linkingofoneunitofthepeptideYIGSR andseverallignanunitswasalsoachieved.VPcouldalsoinducegelationofferuloylatedarabinoxylan andthepolymerizationof␤-casein.TheseresultsdemonstratetheefficacyofVPtocatalyzehomo-and hetero-condensationreactions,andrevealitspotentialexploitationforpolymerizingdifferenttypesof compounds.

© 2013 Elsevier Inc. All rights reserved.

1. Introduction

Biotransformationis a useful way for modifying or produc- ingnovelstructuresandmaterials,whichcanthenbeexploited ina broad rangeof applications.Theenzymatic polymerization andhetero-conjugationofvarioussubstratesisamethodofpri- maryinteresttoreachthatgoal.Biocatalysisisadvantageousover chemicalprocedures,since:(i)it isanenvironmentallyfriendly alternativethatusesmilderandlesscontaminantreactions[1]and (ii)itcanproducemorespecificcross-links,asmanyenzymeshave highchemo-,regio-,andenantioselectivity[2].Theuseofoxidore- ductases,asradical-formingenzymesystems,torenderhomoor hetero-polymersofverydiversemoleculesisanattractiveexam- pleofthis,andtheenzymaticallysynthesizedpolymersmayexhibit

∗Correspondingauthor.Tel.:+34918373112;fax:+34915360432.

E-mailaddress:mjmartinez@cib.csic.es(M.J.Martínez).

1 Current address: Biotechnologie des Champignons Filamenteux, INRA- UniversitéAixMarseille,PolytechMarseille,163avenuedeLuminy,13288Marseille Cedex09,France.

neworimprovedpropertiesincomparisonwiththeirrespective precursors[3].

Versatileperoxidases(VP)areaninterestinggroupofoxidore- ductases(EC1.11.1.16;describedasaReactiveBlack5:hydrogen peroxideoxidoreductase)whoseactivityinthesepolymerization reactionshasnotbeenpreviouslyexploredatthemolecularlevel.

These enzymesare secretedby fungiand includedin theclass IIofthesuperfamilyofplant-fungal-bacterialheme-peroxidases, togetherwithmanganeseperoxidases(MnP),ligninperoxidases (LiP), generic peroxidases (GP)as Coprinopsiscinerea (synonym Coprinuscinereus)peroxidase (CiP) [4]. Todate, these enzymes and their encoding genes have been found and characterized onlyinwhiterot,wood-decayingBasidiomycotabelongingtothe class Agaricomycetes,asdescribed in thecomparativegenomic researchrecentlypublishedbyFloudaset al.[5].VPsconstitute an example of enzyme multifunctionality, combining the cat- alyticpropertiesofMnP,LiP,andlowredox-potentialperoxidases.

Therefore,VPdisplaysawideoxidativeactivityonsubstrateshav- ingdifferentchemicalstructuresandredox-potentials,including compoundsthatcannotbeoxidizedbylowredox-potentialper- oxidases, suchas fungal GP, horseradishperoxidase (HRP) and otherplantperoxidases[6–9].RecentresearcheffortsofVPshave

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Fig.1. Chemicalstructuresofphenolicmodelcompounds:(a)SECO,(b)HMR,(c)MR,(d)CYCLO,(e)7-HSECOlignans,(f)tyrosine,and(g)trans-ferulicacid.

focusedontheunderstandingoftheirreactionmechanismsand structure–functionrelationships[10,11],inthesearchofadequate systemsfortheexpressionofVP[12],andinenzymeimprovement throughdirectedevolution[13].Nevertheless,thepotentialappli- cationsofVPshavenotyetbeenfullyexploited,inspiteofbeinga verypromisinggroupofenzymesfromabiotechnologicalpointof view[14].

Different substrates previously used in enzymatic polymer- ization reactions [15–18], representing low-molecular mass, oligomeric, and polymeric substrates, were chosen toevaluate thepolymerizingabilityofVP.Lignansarediphenoliccompounds foundinthecellwallofhigherplants,formedby␤–␤coupling of two cinnamyl precursors [19], and their chemical structure dependsespeciallyontheplantspeciesfromwhichtheyareiso- lated.Thesecompoundscanappearinside-streamsfromindustrial processingoflignocellulosicmaterial,e.g.mechanicalpulpingand paperprocessing, and shouldbe removedto avoid undesirable effects,suchasinterferenceswithprocesschemicals[20].Poly- merizationoflignansintolargermoleculesisonewaytoeliminate theseunwantedeffects.Lignansmayalsoserveasprecursorsforthe enzymaticproductionofvalue-addedpolymersormaterialswith improvedfunctionalproperties[21].Thesesyntheticreactionsare challenging,sincemostofthemmayonlybeperformedinthepres- enceoforganicsolvents,jeopardizingthestabilityoftheenzyme catalyst.Ontheotherhand,theenzymaticpolymerizationofbioac- tivepeptides,proteinsas␤-casein,orferuloylatedarabinoxylans (FAX),whichhavewell-knownfunctionalproperties[19,22–24], couldleadtotailoredproductswithimproved/modifiedorganolep- ticorfunctionalpropertiessuchasreducedfatcontent,texture, solubility,mouthfeel,betterdigestibility,emulsification,viscosity, gelling,orresistancetoheatorproteolyticattackduringenzyme digestion[25,26].

Therefore,theaimofthepresentstudywastodetermineifthe VPfromPleurotuseryngiiisabletocatalyzethecovalenthomo- geneousand/orheterogeneouscross-linkingofselectedsmalland largemolecules,inthepresenceandabsenceoforganicsolvents, thusproducingnovelbiocompounds.Theextentofthecondensa- tionreactionswasalsoevaluated.

2. Materialsandmethods 2.1. Substrates

Thelignansusedinthisstudy (Fig.1),namelysecoisolariciresinol(SECO), hydroxymatairesinol(HMR),matairesinol(MR),cyclolariciresinol(CYCLO),and7- hydroxy-secoisolariciresinol(7-HSECO)werepreparedasdescribedearlier[27–29].

Thebioactivepeptides EPPGGSKVILF,RKRSRKE,VEPIPY,YIGSR,andVYV were obtainedfromSigma(St.Louis,MO,USA).YSTwasboughtfromBiokemis(Saint Petersburg, Russia). GLY was obtained from Fluka (Buchs, Switzerland). The bovine␤-caseinprotein(24kDa,85%purity)waspurchasedfromSigma–Aldrich (Taufkirchen,Germany).FAXfrommaizebran,containinganalkali-extractablefer- ulicacidcontentof6.2mgg⁻¹,waskindlygiventoCBFbyCambridgeBiopolymers Ltd.(Cambridge,UK).

2.2. Enzymeactivity

VPwasisolatedandpurifiedfromP.eryngiiculturesaspreviouslydescribed [30].ItsMn²⁺-oxidizingactivitywasdeterminedspectrophotometricallyat238nm throughtheformationoftheMn³⁺·tartratecomplex(ε238=6500M⁻¹cm⁻¹)ina

reactionmixturecontaining0.1mMMnSO4(Mn²⁺)in25mMsodiumtartratebuffer (pH5.0),withtheadditionof0.1mMH2O2tostartthereaction.Theeffectof twoorganicsolventsonVPactivitywasalsocheckedthroughtheevaluationof Mn²⁺-independentactivitiesfollowingtheoxidationof1mM2,6-dimethoxyphenol (DMP)todimericcoerulignone(ε469=55,000M⁻¹cm⁻¹)and1mM2,2-azino-bis(3- ethylbenzothiazoline-6-sulphonicacid)(ABTS)toABTS⁺(ε436=29,300M⁻¹cm⁻¹)in 25mMsodiumtartratebuffer(pH5.0).Solutionscontainingfrom0%to50%(v/v)of eitherethanolor1,2-propanediolin25mMsodiumtartratebuffer(pH5.0),inthe presenceorabsenceof0.1mMMn²⁺and0.1mMH2O2,werepreparedassolvents forthesubstrates.TheVPstabilityduring24h-reactionswasalsocheckedin25mM sodiumtartratebuffer(pH5.0),20%,and50%ethanol(dissolvedinthesamebuffer atequalfinalconcentration),withandwithoutMn²⁺.Thepercentageofresidual activitywascalculatedat0,0.5,2,and24husingMn^2+/H2O2assubstratesandtak- ingtheinitialactivityinbufferas100%.Measurementswerecarriedoutintriplicate atroomtemperature.Oneunitofactivity(1U)isdefinedastheamountofenzyme releasing1␮molofproductperminuteunderthedefinedreactionconditions.

2.3. Substratesolutions

The lignanSECO (3mM), all peptides (3mM), ␤-casein (1mgmL⁻¹), and FAX(30mgmL⁻¹)werepreparedin25mMsodiumtartratebuffer(pH5.0).The remaininglignansweredissolvedin20%ethanolinthesamebufferata3mMfinal concentration,except7-HSECO,whichwasdissolvedin50%ethanolbuffer.Allsolu- tionswereleftstandingforatleast30mintobestabilizedbeforecommencingthe enzymatictreatments.

2.4. Cross-linkingassays

Theenzymereactionsdetailedbelowwereinitiatedbyadditionof0.1mMH2O2, supplementingwithaseconddosageafter1hofincubationandbrieflyagitatedafter eachH2O2supplementation.Asanexception,theFAXassayswereperformedwith asingledosageof0.2mMH2O2atthebeginningofthetreatment.Unlessother- wisestated,thesetreatmentswereperformedinthepresenceofMn²⁺(0.1mM), induplicate,atroomtemperature.Negativecontrolsconsistedofreactionslacking VPorH2O2incubatedunderthesameconditionsthanthetestreactions(withand withoutMn²⁺).

2.4.1. Homogeneouscross-linkingoflignansandpeptides

Thepolymerizationofdifferentsubstrateswasperformedin1.5mLEppendorf tubeswitha1.5UmL⁻¹VPinafinalvolumeof200␮L.Aliquots(3␮L)ofeach reactionmixturewereremovedafter0.5and2hofincubationforsubsequentanaly- sisbymatrix-assistedlaserdesorption/ionization-timeofflight-massspectroscopy (MALDI-TOFMS).Lignantreatmentswerealsoperformedduring24h,intheabsence ofMn²⁺.Tostopthesereactions0.05%(w/v)NaN3wasaddedandsamplesfromlig- nantreatmentswerelyophilizedforfurthersizeexclusionchromatography(SEC) analysis.Theeffectonthepolymerizationefficiencyofalowerenzymaticdose (0.15UmL⁻¹)andahigherH2O2concentration(0.5mM),during0.5,2and24h, wasstudiedandseparatelyassayedusingHMRlignanassubstrate.

2.4.2. Heterogeneouscross-linkingoflignanswithpeptides

Reactionscontainingequalvolumes(85␮L)ofthe3mMsolutionsoftheSECOor HMRlignansandofthetyrosine-containingpeptidesweremixedwith1.5UmL⁻¹ VP,inafinalvolumeof200␮L.Aliquots(3␮L)werewithdrawnafter0.5and2h reactionandanalyzedbyMALDI-TOFMS.

2.4.3. Homogeneouscross-linkingofˇ-casein

ThreeVPdoses(0.015,0.15,and1.5UmL⁻¹)wereassayedin1mLreactions for␤-caseinpolymerization,incubatingseparatelyfor2,6,and24hwithcontinu- ousstirringat300rpm.Aliquots(30␮L)fromeachtreatmentwereseparatedand immediatelymixedwithloadingbuffer(10␮L),boiledfor10minandanalyzed bysodiumdodecylsulphate-polyacrylamidegelelectrophoresis(SDS-PAGE).The remainingenzymaticreactionswerestoppedwithNaN3(0.05%,w/v)andsamples werelyophilizedforfurthertransmissionelectronmicroscopy(TEM)analysis.

2.4.4. Homogeneouscross-linkingofFAX

TheabilityofVPtocross-linkFAX,inducinggelformation,wasinvestigatedin reactionscontainingVPdosesof0.015,0.15,1.5UmL⁻¹inatotalvolumeof1.5mL.

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Mixtureswerebrieflyvortexedand1.3mLwasimmediatelyremovedandplaced ontherheometerplate.Rheologicalanalysisstarted3minafterH2O2activation.

Forswellingexperiments,reactionswerepreparedwiththesameenzymedoses,in 2mLsyringes(diameter1.5cm),inafinalvolumeof1mL.Reactionswereallowed togelfor15hbeforeanalyzingtheswellingdegree.

2.5. MALDI-TOFMSanalyses

MALDI-TOFMSspectraofVP-treatedlignansandpeptideswererecordedona BrukerAutoflexIIinstrumentequippedwithaN2-laser(337nm,100␮J)andprevi- ouslycalibratedwithpeptideandproteinstandardsolutionsfromsamedistributor (Bremen,Germany).Fortheanalyses,3␮Lofthereactionsolutionweremixed1:1 (v:v)withsaturated␣-cyano-4-hydroxycinnamicacidmatrixfromSigma–Aldrich (St.Louis, MO,USA)dissolvedin 0.1%trifluoroaceticacidfrom Fluka(Buchs, Switzerland)containing50%acetonitrile.1␮Lofthesample-matrixsolutionwas spottedontothestainlesssteeltargetplateandallowedtodryatroomtemperature.

Positiveionmassspectrawererecordedinreflectormode(m/zrange500–3500)and linearmode(m/zrange3500–10000).FlexAnalysis(version2.4)wasusedfordata analysis(Bruker,Bremen,Germany).Lignansweredetectedastheirsodiumadducts.

2.6. SECanalyses

Samplesfromlignantreatmentsweredissolvedin0.1MNaOHandanalyzed byhighperformanceSECelutingwiththesamesolvent(0.5mLmin⁻¹flowrate) at25^◦C,inMCX1000and100,000 ˚Acolumnsconnectedintandemandcoupled toaprecolumn(allfromPSSMainz,Germany).Theelutionprofileswerefollowed at280nmwithaWatersUVdetector.Themolarmassdistributions(MMD),and weightaveragemolarmasses(Mw)werecalculatedagainstpolystyrenesulphonate (Na-PSS)standards,usingtheWatersEmpower2software.

2.7. SDS-PAGEanalysis

SDS-PAGEwasusedtoanalyzetheformationof␤-caseinpolymerssmaller than250kDa.SampleswereloadedontoCriterionTGXStain-Free^TMprecastgels (4–20%)andvisualizedontheCriterionStainFree^TMImagerSystem.PrecisionPlus Protein^TMStandards(10–250kDa)wereusedformolecularweightestimations.All instrumentsandreagentswerepurchasedtoBio-Rad(Hercules,CA,USA).

2.8. Microscopyanalysis

Lyophilized␤-caseinsamples,subjectedornottoa24hVPtreatment,were dissolvedindistilledwateror6Mureatoobservenon-enzymaticaggregatesand enzymaticcross-links,respectively.Glow-dischargedcarbon-coatedformvargrids wereplacedface-downoveradropletofsample.After1min,thegridwasremoved, blottedbrieflywithfilterpaperandnegativelystainedwith2%uranylacetatefor 40s,blottedquicklyandair-dried.SampleswereobservedbyTEMinaJEOL1230 instrument(Tokyo,Japan)operatedat100kV.

2.9. Rheologicalmeasurementsandgelswellinganalysis

Thegelationofthecross-linkedFAXwasmonitoredbyusinganAR-G2rheome- ter(TAInstruments,Crawley,UK)inoscillatorymodeataconstanttemperatureof 22^◦C.Aplate–plategeometrywithadiameterof40mmandagapof1mmwasused forthemeasurements,withasolventtraptopreventsampledryingduringanalysis.

Gelformationwasfollowedduring4h,bymonitoringthestoragemodulus(G),the lossmodulus(G)andthephaseangleataconstantfrequencyof0.1Hzandastrain of0.01%.

Toevaluatethegelswellingproperties,cross-linkedFAXswereallowedtoswell in10mLofa0.02%(w/v)NaN3solution.After32h,sampleswereblotted,weighted, andsubsequentlyaddedtonewNaN3solutionsatroomtemperature.Theequilib- riumswellingwasreachedwhentheweightofthesamplesdidnotchangemorethan 3%.Theswellingratio(q)iscalculatedaccordingtotheequation:q=(Ws−Wi)/Wi, whereWsistheweightoftheswollengelateachmeasuredtimeandWiisthe weightofthegelbeforeswelling.

3. Resultsanddiscussion

3.1. Influenceoforganicco-solventsandMn²⁺onVPactivityand stability

Thelignansincludedinthepresentstudywereselectedfortheir differentstructuresandthedegreeofsolubilityinaqueoussolu- tionsororganicco-solvents.Enzymescanbeseverelyaffectedby thepresence oforganicsolvents,which generallycauseasharp activity drop due to modification of the protein conformation [31].Forthisreason,VPactivitywasfirstestablishedinthepres- enceofdifferentconcentrationsofethanoland1,2-propanediol,

Fig.2.Schemeoftheversatileperoxidase(VP)catalyticcycleadaptedfromRuiz- Due ˜nasetal.[10].Asshown,VPcanoxidize,amongothers:(i)phenolicsubstrates (PhOH),suchaslignansandtyrosine,tothecorrespondingphenoxyradicals(PhO·) whichareabletopolymerize;and(ii)Mn²⁺toMn³⁺,thelatteractingasadiffusible oxidizerofdifferentcompoundsincludingphenols.

two solventschosenfor theirability tosolubilizesoftwoodand hardwoodligninsand forbeingcompletely water-miscible.Ini- tialactivitiesinaqueousbuffersweredeterminedtobe15UmL⁻¹ forMn²⁺,1UmL⁻¹withABTS,and0.4UmL⁻¹forDMP.VP-activity againstABTSandDMPincreasedinthepresenceofMn²⁺,reach- ing1.6UmL⁻¹and3.6UmL⁻¹,respectively.Thisenhancedactivity onphenolsanddyesinMn²⁺-containingreactionshasbeenprevi- ouslyreported[30],anditisrelatedtothecatalyticversatilityof VP,oxidizingtheminbothMn²⁺-independentandMn²⁺-mediated reactions(Fig.2).

Fig. 3 shows the effect of the two solvents on Mn²⁺, DMP, andABTSoxidation. As1,2-propanediolconcentrationincreased (Fig.3a),aconcurrentdecreaseinVPresidualactivitywasobserved.

ThedecreasewassignificantlylowerinreactionswithMn²⁺,espe- ciallyathighsolventconcentrations,althoughsomeactivitywas retainedinallreactionsevenwith50%ofthisorganicsolvent.On theotherhand,theeffectofethanolonVPactivity(Fig.3b)was diverse.WithMn²⁺asthesubstrate,VPwasquitestableinupto 40%ethanol,withonlya30%decreaseinactivity.Theoxidation ofDMPinthepresenceofMn²⁺appearednottobeaffectedbythe useofethanolconcentrations≤20%.TheactivityofaB.adustaVPat lowethanolconcentrationshasalsobeendescribed[32]although, accordingtoourdata,theVPfromP.eryngiiseemstobemoreresis- tant.However,ahighactivitylosswasobservedinDMPreactions withoutMn²⁺thatcouldbeexplainedbyreducedaccessibilityof ethanolthroughthenarrowMn-oxidationchannel,comparedwith themainhemeaccess-channelwhereDMPisoxidized[33].From theaboveresults,andtakingintoaccountthatmostofthelignans usedinthisstudyaresolubleatlowethanolconcentrations(20%), thissolventwasselectedforfurtherenzymestabilityassays.The enzymeinbuffer,aswellasinthepresenceorabsenceofMn²⁺, retainedtheinitialactivityduringthe24hofincubation.With20%

ethanolintheabsenceofMn²⁺,theresidualactivitywas84and43%

at0.5and24h,respectively,whileinthepresenceoftheionwas 87and82%(with50%ethanoltheenzymelostitsactivityquickly, retaining3.4%and2.1%activityafter0.5hwithandwithoutMn²⁺, respectively).ItcanthusbeconcludedthatthepresenceofMn²⁺

exertsastimulatingandstabilizingeffectontheoxidationreactions andVP respectively.Theformer effectisduetoMn³⁺-mediated

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Fig.3.Effectof(a)1,2-propanedioland(b)ethanolonVPactivityagainstthreedif- ferentsubstrates(Mn²⁺,ABTSandDMP).ABTSandDMPreactionswerefollowedin thepresenceandabsenceof0.1mMMn²⁺.Allreactionswereinitiatedwith0.1mM H2O2.100%correspondedtotheenzymaticactivityintheabsenceofthoseorganic solvents,andvaluesareexpressedasthepercentageofresidualactivity.Standard deviationsfromtriplicateswerelessthan5%oftheaveragevalueinallcases.

oxidationofDMPandABTS,whilethestabilizingeffectindicates lowerinhibitionwhentheVPMn²⁺-oxidationsiteisoccupiedby theionduringincubationwithethanol.

3.2. Smallmoleculescross-linkingbyVP 3.2.1. Lignancross-linkinganalysis

Severalparameters,suchasthedegreeofpolymerization(DP), themolecularmass(MM),andmolarmassdistribution(MMD)of thepolymersenzymatically synthesized,shouldbe analyzedto comparetheefficiencyofVPinlignanspolymerizationwiththat reportedforotheroxidativeenzymes[34].Thepolymerizationof thesubstrates(DPandMM)wasfollowedbyMALDI-TOFMS.Con- troltreatments,inthepresenceandabsenceofMn²⁺,containing lignansandH₂O₂butlackingenzymewereconductedtocheckfor substratesself-polymerization,revealingthepresenceofdimers andtrimersoflignans(Table1).Thesenon-enzymaticcross-links couldarisefromoxidationreactions,involvingO₂orH₂O₂,medi- atedbytracesofmetalions[35,36].Despiteoftheseunspecific

links,theVP/H₂O₂systemwascapableofsynthesizingmolecules ofmuchhigherDPcomparedtothecontrols.Table1showsthat mostcross-linkingoccurredduringthefirst30minofreactionand thatthepresenceofMn²⁺enhancedthepolymerizationefficiency, raisingboththereactionrateandthemaximumDPoftheprod- uctsoverthereactiontimeatallsolventconcentrations.Table2 depictsthepredictedandexperimentalMM(accuracy≤±1Da)of thelongestlignanhomopolymerssynthesizedbyVP,andthespec- traofthereactionproductsarerepresentedinFig.4.Theassembly oflignanmonomersisproducedthroughanetherorcarbon–carbon linkagethatcausestheelimination oftwo hydrogenatomsper cross-link[17].Thetheoreticalmassoftheproductscanbecal- culatedaccordingtotheequation[nMM−(n−1)2H+Na⁺],where nisthenumberofmonomersandMMisthemolecularmassof lignan.Thewater-solubleSECOwasthemostefficientlypolyme- rizedsubstrate,formingnonamersandoctamersinthepresence andabsenceofMn²⁺,respectively,after24hincubation.Thisvalue wassimilarorslightly higherthanthosereportedforthecross- linkingofSECOcatalyzedbyfungal[21]orbacteriallaccases[37].

Overthefirst2hofreaction,polymerizationofbothSECOandHMR progressedatsimilarrates,butafter24hthemaximumDPofthe laterdecreased inoneunitwhiletheSECOpolymerreachedits highestlength(Table1).Buchertetal.[20],reportedthesynthesis ofHMRoligomersafter2hincubationwithafungallaccase,butthe productswere3–4unitssmallerthanthosefoundinthepresent studyatthesamereactiontime.Regarding7-HSECO,thelignan dissolvedin50%ethanol,itisremarkablethattheadditionofMn⁺² producedtwo-foldlargeroligomersascomparedtothereaction withoutthecation,whichcouldbeduetoanincreasedVPcatalytic efficiencyandstability,asdescribedintheprevioussection.

SECanalysisoftheVP-untreatedandtreatedlignans,inthepres- enceofMn²⁺,showedthereductionofthelowMMpeakfromthe substratesalongtheincubationtime,andaparallelincreaseofthe Mwoftheproducts.Fig.5illustratestheMMDprofilesofSECOand HMR,thetwolignansthatreachedthehighestDP.Asinglepeak, correspondingtosubstrates,wasobservedafterSECofcontrolsam- pleswithoutperoxidase.Theappearanceofoligomersalongthe reactiontimeisdetectedasnewshouldersorpeaksattheright ofthesubstratespeak.ThesmallincreaseinthelowMMfraction from24hHMRreactionscouldbeexplainedbythecoexistenceof polymerizationanddegradationactivitiesinthereactionmixture, aspreviouslyreportedforsoluble-ligninsamplestreatedwithVP [38]andMnP[39].Thisfindingisinaccordancewiththefadingof thesignalfromHMRoctamersdetectedbyMALDI-TOFanalysisof thissample(Table1).However,theglobalM_wofthereactioncom- ponentsraisedapproximately100Dafrom2hto24h,suggesting thatpolymerizationpredominatedoverdepolymerizationduring thisperiod.

Asinthecaseforallperoxidases,H₂O₂isessentialforVPactiv- ity and its eventual depletionstops thereaction. The effect of differentVP/H2O2 ratiosonHMRcross-linking wasassayedfor 24htreatments,eitherincreasing5-foldtheperoxideconcentra- tionordecreasing10-foldVPdosage(from1.5to0.15UmL⁻¹),and sampleswereanalyzed atdifferentreactiontimes.Enzyme-free controlsdemonstratedthatHMRtreatmentwith0.5mMH₂O₂did notinduceanyadditionalpolymerization.Incontrast,HMRpoly- mersonly oneDP largerin molecularsizethan those detected inlow-peroxidereactionsweredetectedintheVPdosedwith5- foldmoreH₂O₂system.TheMwvaluesincreasedaround200and 300Dain2hand24h-reactions,comparedtotheequivalentprod- uctsobtainedwith0.1mM H₂O₂.SEC profiles(Fig.5b)showed thatthelowMMpeakdecreasedaround50%inheight,whilea high-MMfractionofalmost thesameintensityappeared. Thus, higherdosesofperoxidedidnotcauseinhibitionofVP,butrather improveditspolymerizationcapacity.Ontheotherhand,areduced VPdosageresultedin theproductionofHMRoligomersof 5,6

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Table1

Effectof0.1mMMn²⁺andincubationtimeontheVP-catalyzedpolymerizationoffivedifferentlignans.Thecontrol(CTL)wasaVP-untreatedsampleincubatedduring24h, addingtwodosagesof0.1mMH2O2atthebeginningofthereactionandafter1h,similarlytotheVP-treatments.Analyseswereperformedonduplicatesoftwoindependent reactions,showingidenticalspectra.

Lignan Solvent Maximaldegreeofpolymerization^a

−Mn²⁺ +Mn²⁺

CTL 30min 2h 24h CTL 30min 2h 24h

SECO Buffer 2 5 7 8 2 8 8 9

HMR 20%Ethanol 3 5 7 6 3 8 8 7

MR 20%Ethanol 2 4 5 5 2 5 6 7

CYCLO 20%Ethanol 2 5 6 6 2 6 7 7

7-HSECO 50%Ethanol 2 3 3 3 2 6 7 7

aValuesrepresentthemaximumnumberoflignanunitscross-linkedasdetectedbyMALDI-TOFMS.

and7monomersafter0.5,2,and24h,respectively,demonstrat- ingthattheamountofenzymeaffectedonlytheinitialreaction rate,reachingthesameDPvaluesobtainedathigherVPdosesin 24h(Table1).

3.2.2. Peptidecross-linkinganalysis

ThepositiveeffectofMn²⁺inVP-catalyzedpolymerizationreac- tionshasalreadybeendeducedfromthepreviousexperiments.

Thus,theVPtreatmentofpeptides,ofdifferentlengthandamino acidsequence,wasperformedinthepresenceofthiscation.No peptidecross-linkingoccurredintheabsenceofVPorH2O2,aswell asinpeptideslackingtyrosineresidues(Table2)suchasRKRSRKE and EPPGGSKVILF.In contrast,theVP/H₂O₂ system washighly efficientpolymerizingtyrosine-containingpeptides,beingasprevi- ouslydescribedforotheroxidoreductases[15,40,41].Polypeptides wereformedthroughthelossoftwohydrogenatoms,probably duetotheformationofdityrosineorisodityrosinebonds,resulting inmonoisotopicmasseswhosetheoreticalvaluecanbecalculated accordingtotheequation:[nMM−(n−1)2H],wherenisthenum- berofmonomersandMMisthemolecularmassofeachpeptide.

Asanexception,thepolymersfromVEPIPYweredetectedastheir sodiumadduct.Table2showstheexperimentalandpredictedMM fromthehighestDPmoleculessynthesizeduponVP-treatment,and Fig.4depictsthespectraofthereactionproducts.

Thepeptidelengthandthepositionoftyrosineinthesequence hadnoeffectontheDPoftheproductsformedbytheactionofVP.

Thisfindingcontrastswiththeresultsobtainedinsimilarreactions performedwithHRP[41]orCiP[42],inwhichastrongeffectof thesetwoparametersoncross-linkinghasbeenreported.VPhas severalcatalyticsites,onefor(lowefficiency)oxidationofphenols anddyesatthemainhemeaccesschannel,asecondoneforMn²⁺

oxidationata(small)secondhemeaccesschannel,andthethird sitefor(highefficiency)oxidationofphenolicandnonphenolicaro- maticsubstrates,locatedattheproteinsurface[11].Thesefeatures, whicharenotobservedinotherperoxidases(suchasHRP)thatonly presentthe“classical”oxidationsiteatthemainhemechannel,can facilitatesubstrateoxidationevenifthereactivemoiety,suchas thetyrosine,isplacedinthemiddleofthesequence.Therefore,the cross-linkingoftyrosine-containingpeptidesbyHRPmaybevery restrictedbysterichindrancesandenzymeinhibition[41].Incon- trast,VPcanalsooxidizeitssubstratesatasecondexposedcatalytic siteandviaMn²⁺diffusion(Fig.2),bypassingtheseproblems.

Inaddition,VP-induced polymerizationofpeptides wasvery fast,reachingthemaximumDPafter30mininallcases.Regardless thereactiontime,thepredominantsignalsinmostspectracorre- spondedtodimers,althoughtetramersandtrimerswerethemain peaksdetectedafter2hofreactionusingGLYorVYVasthesub- strates,respectively(Fig.4).TheMMDoftheproducts,derivedfrom MALDI-TOFMSspectra,changedwhilethereactionproceededdue

Table2

Assignmentofmasses(m/z)ofthelongestlignanandpeptidepolymersdetectedbyMALDI-TOFMSinVP-catalyzedreactions.Homopolymerizationandheteropolymerization experimentswereperformedinthepresenceof0.1mMMn²⁺,addingtwodosagesof0.1mMH2O2atthebeginningofthereactionandafter1h.Allreactionswereincubated during2hexcludingthehomopolymerizationoflignanswhichwascarriedoutfor24h.HMRpolymerizationwasaccomplishedwith0.5mMH2O2during2h.

Substrate Monomer DP^a Predictedm/z Experimental^bm/z

(Da) (Da) (Da)

Homopolymers Lignans

SECO 362.1 9 3264.9 3264.1(−0.8)

HMR 374.1 9 3372.9 3372.8(−0.1)

MR 358.1 7 2516.7 2517.6(0.9)

CYCLO 360.1 7 2530.7 2531.5(0.8)

7-HSECO 378.1 7 2656.7 2656.4(−0.3)

Peptides

RKRSRKE 959.1 1 959.1 959.6(0.5)

EPPGGSKVILF 1125.3 1 1125.3 1125.5(0.2)

GLY 351.4 8 2797.2 2796.9(−0.3)

YST 369.2 8 2939.0 2939.6(0.6)

VYV 379.5 11 4154.5 4155.0(0.5)

YIGSR 594.7 11 6521.7 6522.6(0.9)

VEPIPY 716.8 7 5027.6 5028.2(0.6)

Heteropolymers

YIGSR/SECO 594.7/362.1 1/5 2394.2 2393.7(−0.5)

YIGSR/HMR 594.7/374.1 1/4 2082.1 2082.6(0.5)

aDP=maximaldegreeofpolymerization.

bThem/zdifferencesbetweenthetheoreticalandthedetectedmasses,showninparentheses,arewithintheexperimentalerrorofthetechnique.

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Fig.4.MALDI-TOFmassspectra(reflectormode)ofpolymerizedlignansandpeptides.VP-treatedlignanswereincubatedduring24hwith0.1mMMn²⁺andtwodosagesof 0.1mMH2O2at0and1h(excludingHMRwhoseincubationwascarriedoutduring2hwith0.1mMMn²⁺andtwodosagesof0.5mMH2O2at0and1h):(a)HMR,(b)SECO, (c)MR,(d)CYCLO,and(e)7-HSECO.Peptideswereincubatedfor2hwith0.1mMMn²⁺andtwodosagesof0.1mMH2O2at0and1h:(f)GLY,(g)YST,(h)VYV,(i)YIGSR, and(j)VEPIPY.Thenumberofmonomers(n)ofeachlignanorpeptideisshownabovethecorrespondingpeak.Anenlargementofm/z2200–3400regionofallsubstrates isframedexcludingVEPIPY,VYV,andYIGSRwhoseenlargementscorrespondtothem/z3500–7000Da(linealmode)sincetheyformoligomerslargerthan3500Da.Allthe analyseswereperformedonduplicate(twoindependentreactions),showingidenticalspectra.

totheproductionoflargerspecies.Theproductionofpentamers ofGLY,usingalaccasefromTrameteshirsuta[40]andhexamers ofVYVwiththeC.cinereaperoxidase[15]in24h,arepoorval- ueswhencomparedtothenonamersofGLYandtheundecamers ofVYVobtainedinVP-reactionsof30min(Table2).Theresults suggestthatbeyondacertainDPrange,peptidespolymerizationis notfavoredunderthereactionconditionsusedinthisstudyorby otherstericfactors.Despitethislimitation,ourresultsshowthat, evenatshortreactiontimes,thepolymerizationdegreewashigher usingVP,eventhoughthisenzymehasamorerestrictedactivity comparedtolaccases,especiallyduetothelowH2O2concentration testedinthisstudy.

3.2.3. Heterogeneouscross-linkingoflignanswithpeptides

VPreactionscontainingSECOorHMRandeachofthetyrosine- containing peptides were incubated for 30min and 2h and analyzedtotesttheabilityoftheenzymetocatalyzethesynthesis

ofheteropolymers.TheMALDI-TOFspectraofthereactionprod- uctsforeachpeptide-containingreactionshowedthatinmostcases onlylignanhomopolymerswereformed,detectinguptononamers of SECOand octamersof HMR. Steffensenand co-workers [43]

suggestedthatmonolignolscanhaveantioxidanteffects,avoiding tyrosineoxidationinpeptideswhentheyareaddedtothereaction atthesameconcentration.Moreover,althoughthemolarconcen- trationofbothsubstratesinthereactionmixturewasthesameand theMMofthetripeptides(GLY,VYV,YST)andthelignansisalso similar,thelatercontaintwo-foldmorereactivesitesthanthepep- tides(twophenolsperonetyrosine,respectively).Theprobability forheterocomplexformationappearstoincreasewiththenumber ofreactivesitesinthebiopolymer.Thispropositioncontrastswith theresultsobtainedwithVEPIPY.Inthislastcase,andtakinginto accountthegravimetricamountofthecompoundinthefinalreac- tionmixture,polymerizationwasnotobservedeventhoughthis peptideandthelignanscontainedasimilarnumberofreactivesites.

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Fig.5. MolarmassincreaseafterVPtreatmentinthepresenceof0.1mMMn²⁺of:

(a)SECOand(b)HMR.ControlscorrespondtoVP-untreatedSECOorHMRwith 0.1mMH2O2at24hofincubation.ControlscorrespondingtoVP-untreatedHMR with0.5mMH2O2weresimilartothepreviouscontrolandarenotrepresentedin (b).

Hetero-oligomerswereonlyformedinreactionscontainingthe peptideYIGSRwithbothlignans(Fig.6),althoughlignanhomo- oligomerswerealsodetected.Theseheterogeneouscross-linking reactions took place through the elimination of two hydrogen atoms according to theequation: [nMM(YIGSR)+[nMM(lignan)- (n−1)2H⁺]-2H⁺].Peakscorrespondingtopeptidehomo-oligomers werenotfound,contrastingwiththehighDPoftheYIGSRunits produced when incubated alone with VP (Table2).YIGSR is a peptidederivedfromlamininanditisconsideredasanadhesive ligand,whatcanpossiblyfacilitateitsattachmentandlinkingto othermolecules[44].Theseresultsindicatethat,inthepresence ofbothsubstrates,VPhasapreferenceforlignansandifahetero- crosslinkingoccurs,onlythelignanchainisfurtherelongated.

3.3. Largemoleculescross-linkingbyVP 3.3.1. ˇ-Caseincross-linking

As VP was shown to efficiently cross-link small tyrosine- containingpeptides,themilkprotein␤-casein,consistingof209 aminoacids,fourofwhicharetyrosines[45],wasselectedtodeter- mine if VP couldcross-link largerproteins. The products were analyzedunderdissociatingandreducingconditionsbySDS-PAGE, confirmingthatthecomplexesobservedwereduetotheformation of covalentbondsand notby molecularaggregation.Large-size molecularspecies(approximately150kDa)appearedevenatthe lowestenzymedosageandthebandcorrespondingtothe␤-casein monomerslightlydecreasedoverthereactiontime,suggestingthat alowpercentageofproteinwasmodified(Fig.7,lane6).Incon- trast, mediumandhighdosesof VPresultedin analmosttotal fadingofthe␤-caseinmonomerafter24h(Fig.7,lanes7and10).

Moreover,bandshigherthan250kDawereobservedat thetop oftherunninggel,correspondingtopolymersofatleast10–11

␤-caseinmonomers.Thepolymerizationofcaseinhasbeenprevi- ouslyreportedusingHRP[18]andHRPplusferulicacid[46]with similarresultstothosereportedinthepresentstudy.Bandswith alowermassthan␤-caseinappearedafterlongincubationtimes andinreactionswithhighVPdoses.Aspreviouslydiscussed,the peroxidasecansimultaneouslycatalyzepolymerizationanddegra- dationatextendedreactiontimes(Fig.7,lanes7–10).After24hof incubation,aslightaggregationwasdetectedinallsamples,which couldbeproducedbytheintra-andintermoleculartransference ofradicalsformedfromproteinsduringthereaction[47,48].TEM images(Fig.7)fromwatersolutionsofVP-treatedcaseinallowed the observationof theprotein as fibers, representing a typical

Fig.6.MALDI-TOFmassspectrafromVP-heteropolymerizationofYIGSRpeptidewith(a)SECOlignanand(b)HMRlignanduring2hinthepresenceof0.1mMMn²⁺and H2O2.Aseconddosageof0.1mMperoxidewasaddedafter1hincubation.Theunits’numberofthehomo-andthehetero-polymersisshownabovethecorrespondingpeak.

Analyseswereperformedonduplicatesoftwoindependentreactions,showingidenticalspectra.

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Fig.7. Cross-linkingof␤-caseinanalyzedbySDS-PAGEandTEM.(a)SDS-PAGEof

␤-casein(Lane1);␤-caseinplusH2O2(Lane2);␤-caseinplusVP(Lane3);VP/H2O2- treated␤-caseinwith0.015UmL⁻¹ofVP(Lanes4–6);VP/H2O2-treated␤-casein with0.15UmL⁻¹(Lanes7–9);VP/H2O2-treated␤-caseinwith1.5UmL⁻¹(Lanes 10–12).ArrowsaresignalingtheVPband(∼43kDa).The␤-caseinmonomeris framedalongthegel.(b)TEMphotomicrographsofuntreated␤-casein(1),and VP-treated␤-caseinduring24hofincubationwithanenzymedosageof1.5UmL⁻¹ dissolvedinwater(2)orinurea6M(3).Arrowsaresignaling␤-caseinmonomers.

product structure upon molecular aggregation [49]. When VP- treated ␤-casein was dissolved in urea to disrupt aggregates, polymerswereobservedasirregularandcompactstructureswith abroadrangeofsizes.

3.3.2. FAXcross-linking

Smalldeformationoscillatoryrheologywasusedtofollowgel formationcausedbytheoxidativecross-linkingofferuloylatedara- binoxylan.ControltreatmentslackingVPorH₂O₂didnotproduce gelsduring4hreactions,andthesameresultwasobtainedwith thelowestVPdosage(0.015UmL⁻¹).Neithertheuseof1.5UmL⁻¹ ofVPwasusefulforthismonitoringsinceitproducedanimmedi- ategelationafterH₂O₂addition.Adosageof0.15UmL⁻¹VPwas selectedforfurtherexperiments.Theprofileobtainedwith3%FAX fittedthetypicalkineticbehaviordisplayedinenzymaticsystems containinglaccases[50]or otherperoxidases[16].Fig.8 shows aninitialincreaseofbothG’andG”followedbyaplateauregion.

ThatplateaureachedG valuesofaround90PaandG valuesof 0.5Pain10and7minrespectively,indicatingthatthesamplehad gel-likeproperties.Thefinalphaseanglewasverylow(below0.5 degrees),highlightingthehighelasticityofthegel.Thegelation point,calculatedfromthecrossoverpointofGandG,tookplacein about2min.Apartfromthediferuliccovalentbonds,non-covalent linksbetweenarabinoxylansmightalsooccur[22,51]andtherefore themeasuredrheologicalpropertiesdependonthearabinoxylan structuralcharacteristicsandtheferulicacidconcentration.

Gelsweregeneratedonlyinreactionswith0.15and1.5UmL⁻¹ VP,reaching theswelling equilibrium between4 and 6h, with swellingratiovalues(q)of53.4±0.4and65.4±1.1gwater/gFAX, respectively.Thesevalueswerehigherthanthosereportedinother enzyme-catalyzedFAXgelationstudies[52].Thefastreactionrate achievedwithVPcouldcausethetrappingofuncrosslinkedFAX moleculesinsidethegelstructure.Thesemoleculeswouldexpand

Fig.8. TheeffectsofVP-treatmentontherheologicalpropertiesofFAXgels.Samples wereanalyzedinduplicate,showingcoefficientsofvariationlowerthan5%.

quicklyincontactwithwater,leadingtoitsincreasedintakeinthe resultantFAXnetworks[16].

4. Conclusions

Themodificationbyenzymatic cross-linkingofbiomolecules usingtheVPfromP.eryngiihasbeenachieved.Thereactioncon- ditionsduringVPtreatmentshadagreatinfluenceinthereaction yields.Ingeneral,Mn²⁺seemedtoimprovetheVPstabilityand/or itscatalyticefficiencyeveninthepresenceoforganicco-solvents, whichareessentialinmostreactionsinvolvinglignans.Onlypep- tidescontainingtyrosineresidues,regardlessoftheirpositionin thesequence,arecapableofformingacovalentbondthroughthis kindofreactions,andheteropolymerizationoflignanswithapep- tideresultedtobefeasible.Moreover,VP-catalyzedcross-linking producedhighmassmacromoleculesfrom␤-caseinandFAX.In viewoftheseresults,theapplicationofVPforefficientpolymer- izationofoxidizablecompoundsissuggested.Furtherscreening ofotherpotentialsubstratesforVPandstudiesontheoptimiza- tionofthepolymerizationreactionwillbedesignedinthefuture.

TheP.eryngiiVPusedinthisworkiscurrentlybeingsubjectedto structural-functionalstudiesenablingrationaldesign,aswellasto directedmolecularevolution,toimproveitsresistancetopHand H₂O₂,twoimportantchallengesforitsapplicationinbiotechnolo- gicalprocesses.

Conflictofintereststatement

Theauthorsdeclarethattherearenoconflictsofinterest.

Acknowledgements

D.SalvachúathankstheSpanishMinistryofEconomyandCom- petitivenessforaFPUfellowship,P.MatikainenandB.Hillebrandt fortechnicalassistanceandDr.K.Viljanenforthephenolicacids analysis(VTT,Espoo, Finland). We alsothank FernandoEscolar foritshelpinTEMandJ.Gil(CIB,Madrid,Spain).Thisworkhas beencarriedoutwithfundingfromtheEUFP7project“Peroxicats”

(KBBE-2010-4-265397),theSpanishMinistryofScienceandInno- vation (BIO2009-08446, PRI-PIBAR-2011-1402), and the project

“LigninFibre”financedbytheAcademyofFinland(Grantnumber 133091).

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