Arangeofα-L-arabinofuranosyl-(1-4)-β-D-xylo-oligosaccharides(AXOS)wereproducedbyhydrolysisofwheatflourarabinoxylan(WAX)andaciddebranchedarabinoxylan(ADWAX),inthepresenceandabsenceofanAXH-d3α-L-arabinofuranosidase,byseveralGH10andGH11β-xylanases.ThestructuresoftheoligosaccharideswerecharacterisedbyGC-MSandNMRandbyhydrolysisbyarangeofα-L-arabinofuranosidasesandβ-xylosidase.TheAXOSwerepurifiedandusedtocharacterisetheactionpatternsofthespecificα-L-arabinofuranosidases.Theseenzymes,incombinationwitheitherCellvibriomixtusorNeocallimastixpatriciarumβ-xylanase,wereusedtoproduceelevatedlevelsofspecificAXOSonhydrolysisofWAX,suchas3²-α-L-Araf-(1-4)-β-D-xylobiose(A³X),2³-α-L-Araf-(1-4)-β-D-xylotriose(A²XX),3³-α-L-Araf-(1-4)-β-D-xylotriose(A³XX),2²-α-L-Araf-(1-4)-β-D-xylotriose(XA²X),3²-α-L-Araf(1-4)-β-D-xylotriose(XA³X),2³-α-L-Araf-(1-4)-β-D-xylotetraose(XA²XX),3³-α-L-Araf-(1-4)-β-D-xylotetraose(XA³XX),2³,3³-di-α-L-Araf-(1-4)-β-D-xylotriose(A²⁺³XX),2³,3³-di-α-L-Araf-(1-4)-β-D-xylotetraose(XA²⁺³XX),2⁴,3⁴-di-α-L-Araf-(1-4)-β-D-xylopentaose(XA²⁺³XXX)and3³,3⁴-di-α-L-Araf-(1-4)-β-D-xylopentaose(XA³A³XX),manyofwhichhavenotpreviouslybeenproducedinsufficientquantitiestoallowtheiruseassubstratesinfurtherenzymicstudies.ForA^2,3XX,yieldsofapproximately16%ofthestartingmaterial(wheatarabinoxylan)havebeenachieved.Mixturesoftheα-L-arabinofuranosidases,withspecificactiononAXOS,havebeencombinedwithβ-xylosidaseandβ-xylanasetoobtainanoptimalmixtureforhydrolysisofarabinoxylantoL-arabinoseandD-xylose.

Plantcellwallsareconstructedfromadiversityofpolysaccharidecomponents.Molecularprobesdirectedtostructuralelementsofthesepolymersarerequiredtoassaypolysaccharidestructuresinsitu,andtodeterminepolymerrolesinthecontextofcellwallBIOLOGy.Here,wereportontheisolationandthecharacterizationofthreeratmonoclonalantibodiesthataredirectedto1,5-linkedarabinansandrelatedpolymers.LM13,LM16andLM17,togetherwithLM6,constituteasetofantibodiesthatcandetectdifferingaspectsofarabinanstructureswithincellwalls.EachoftheseantibodiesbindsstronglytoisolatedsugarbeetarabinansamplesinELISAs.Competitive-inhibitionELISAsindicatetheantibodiesbinddifferentiallytoarabinanswiththebindingofLM6andLM17beingeffectivelyinhibitedbyshortoligoarabinosides.LM13bindspreferentiallytolongeroligoarabinosides,anditsbindingishighlysensitivetoarabinanaseaction,indicatingtherecognitionofalongerlinearizedarabinanepitope.Incontrast,thebindingofLM16tobranchedarabinanandtocellwallsisincreasedbyarabinofuranosidaseaction.Thepresenceofallepitopescanbedifferentiallymodulatedinvitrousingglycosidehydrolasefamily43andfamily51arabinofuranosidases.Inaddition,theLM16epitopeissensitivetotheactionofβ-galactosidase.Immunofluorescencemicroscopyindicatesthattheantibodiescanbeusedtodetectepitopesincellwalls,andthatthefourantibodiesrevealcomplexpatternsofepitopeoccurrencethatvarybetweenorgansandspecies,andrelatebothtotheprobableprocessingofarabinanstructuralelementsandthedifferingmechanicalpropertiesofcellwalls.

Anexo-arabinanase,designatedAbnx,waspurifiedfromaculturefiltrateofPenicilliumchrysogenum31Bbyammoniumsulfateprecipitation,anion-exchangechromatography,andhydrophobicchromatography.Abnxhadanapparentmolecularmassof47kDa.Theenzymereleasedonlyarabinobiosefromthenonreducingterminusofα-1,5-L-arabinanandshowednoactivitytowardsp-nitrophenyl-α-L-arabinofuranosideandα-1,5-L-arabinofuranobiose.Abnxisthefirstenzymewiththismodeofaction.

Thestructureofarabinanandgalactandomainsinassociationwithcellulosemicrofibrilswasinvestigatedusingenzymaticandalkalidegradationprocedures.Sugarbeetandpotatocellwallresidues(called‘natural’composites),richinpecticneutralsugarsidechainsandcellulose,aswellas‘artificial’composites,createdbyinvitroadsorptionofarabinanandgalactansidechainsontoprimarycellwallcellulose,werestudied.Thesecompositesweresequentiallytreatedwithenzymesspecificforpecticsidechainsandhotalkali.Thedegradationapproachusedshowedthatmostofthearabinanandgalactansidechainsareinstronginteractionwithcelluloseandarenothydrolysedbypecticsidechain-degrADIngenzymes.Itseemsunlikelythatisolatedarabinanandgalactanchainsareabletotetheradjacentmicrofibrils.However,cellulosemicrofibrilsmaybetetheredbydifferentpecticsidechainsbelongingtothesamepecticmacromolecule.

Pectinsofvaryingstructuresweretestedfortheirabilitytointeractwithcelluloseincomparisontothewell-knownadsorptionofxyloglucan.Ourresultsrevealthatsugarbeet(Betavulgaris)andpotato(Solanumtuberosum)pectins,whicharerichinneutralsugarsidechains,canbindinvitrotocellulose.Theextentofbindingvarieswithrespecttothenatureandstructureofthesidechains.Additionally,branchedarabinans(Br-Arabinans)ordebranchedarabinans(Deb-Arabinans;isolatedfromsugarbeet)andgalactans(isolatedfrompotato)wereshownbindtocellulosemicrofibrils.TheadsorptionofBr-ArabinanandgalactanwaslowerthanthatofDeb-Arabinan.ThemaximumadsorptionaffinityofDeb-Arabinantocellulosewascomparabletothatofxyloglucan.Thestudyofsugarbeetandpotatoalkali-treatedcellwallssupportsthehypothesisofpectin-celluloseinteraction.Naturalcompositesenrichedinarabinansorgalactansandcellulosewererecovered.Thebindingofpectinstocellulosemicrofibrilsmaybeofconsiderablesignificanceinthemodelingofprimarycellwallsofplantsaswellasintheprocessofcellwallassembly.

Pectinisaclassofcomplexcellwallpolysaccharideswithmultiplerolesduringcelldevelopment.Assigningspecificfunctionstoparticularpolysaccharidesisinitsinfancy,inpart,becauseofthelimitednumberofmutantsandtransformantsavailablewithmodifiedpecticpolymersintheirwalls.Pectinsarealsoimportantpolymerswithdiverseapplicationsinthefoodandpharmaceuticalindustries,whichwouldbenefitfromtechnologyforproducingpectinswithspecificfunctionalproperties.Inthisreport,wedescribethegenerationofpotato(SolanumtuberosumL.cvPosmo)tubertransformantsproducingpecticrhamnogalacturonanI(RGI)withalowlevelofarabinosylation.ThiswasachievedbytheexpressionofaGolgimembrane-anchoredendo-α-1,5-arabinanase.SugarcompositionanalysisofRGIisolatedfromtransformedandwild-typetubersshowedthatthearabinosecontentwasdecreasedbyapproximately70%intransformedcellwallscomparedwithwildtype.ThemodificationoftheRGIwasconfirmedbyimmunolabelingwithanantibodyrecognizingα-1,5-arabinan.Thisisthefirsttime,toourknowledge,thatthebiosynthesisofaplantcellwallpolysaccharidehasbeenmanipulatedthroughtheactionofaglycosylhydrolasetargetedtotheGolgicompartment.

Ageneencodinganα-L-arabinofuranosidase,designatedSaAraf43A,wasclonedfromStreptomycesavermitilis.ThededucedaminoacidsequenceimpliesamodularstructureconsistingofanN-terminalglycosidehydrolasefamily43moduleandaC-terminalfamily42carbohydrate-bindingmodule(CBM42).TherecombinantenzymeshowedoptimalactivityatpH6.0and45°CandwasstableoverthepHrangeof5.0–6.5at30°C.Theenzymehydrolysedp-nitrophenol(PNP)-α-L-arabinofuranosidebutdidnothydrolyzePNP-α-L-arabinopyranoside,PNP-β-D-xylopyranoside,orPNP-β-D-galactopyranoside.Debranched1,5-arabinanwashydrolyzedbytheenzymebutarabinoxylan,arabinogalactan,gumarabic,andarabinanwerenot.Amongthesyntheticregioisomersofarabinofuranobiosides,onlymethyl5-O-α-L-arabinofuranosyl-α-L-arabinofuranosidewashydrolyzedbytheenzyme,whilemethyl2-O-α-L-arabinofuranosyl-α-L-arabinofuranosideandmethyl3-O-α-L-arabinofuranosyl-α-L-arabinofuranosidewerenot.Thesedatasuggestedthattheenzymeonlycleavesα-1,5-linkedarabinofuranosyllinkages.Theanalysisofthehydrolysisproductofarabinofuranopentaosesuggestedthattheenzymereleasesarabinoseinexo-actingmanner.Theseresultsindicatethattheenzymeisdefinitelyanexo-1,5-α-L-arabinofuranosidase.TheC-terminalCBM42didnotshowanyaffinityforarabinogalactananddebranchedarabinan,althoughitboundarabinanandarabinoxylan,suggestingthattheCBM42boundtobranchedarabinofuranosylresidues.Removalofthemoduledecreasedtheactivityoftheenzymewithregardtodebranchedarabinan.TheCBM42playsaroleinenhancingthedebranchedarabinanhydrolyticactionofthecatalyticmoduleinspiteofitspreferenceforbindingarabinofuranosylsidechains.

ThesoftrotfungusPenicilliumpurpurogenumsecretesawidevarietyofxylanolyticenzymestothemedium,amongthemthreeα-L-arabinofuranosidases.Thisworkreferstoarabinofuranosidase2(ABF2).Thisenzymewaspurifiedtohomogeneityandcharacterized;itisaglycosylatedmonomerwithamolecularweightof70 000andanisoelectricpointof5.3.Whenassayedwithp-nitrophenylα-L-arabinofuranoside(pNPAra)theenzymefollowedMichaelis–MentenkineticswithaK_Mof0.098mm.TheoptimumpHis5andtheoptimaltemperature60°C.ABF2showedweakactivityonnaturalpolymericsubstrates,suchassugarbeetarabinan,debranchedarabinan,andarabinoxylan.Theseresults,togetherwithitslowK_M(pNPAra)anditsactivitytowardsshortarabinooligosaccharides,suggestthattheenzymebelongstotheexoα-L-arabinosylhydrolasesnotactiveonpolymers.Theabf2geneanditsCDNAweresequenced,andthegenewasfoundtopossesssevenintrons.Thematureproteinis618aminoacidslongwithacalculatedmolecularweightof67 212.Aminoacidsequencealignmentsshowthattheenzymebelongstofamily51oftheglycosylhydrolases,althoughitdiffersinsomepropertiesfromotherenzymesofthisfamily.

Bacillussubtilisisabletoutilizearabinopolysaccharidesderivedfromplantbiomass.Here,bycombininggeneticandphysiologicalanalyseswecharacterizetheAraNPQimporterandidentifyprimaryandsecondarytransportersofB.subtilisinvolvedintheuptakeofarabinosaccharides.WeshowthattheABC-typeimporterAraNPQisinvolvedintheuptakeofα-1,5-arabinooligosaccharides,atleastuptofourL-arabinosylunits.Althoughthissystemisthekeytransporterforα-1,5-arabinotrioseandα-1,5-arabinotetraose,theresultsindicatethatα-1,5-arabinobiosealsoistranslocatedbythesecondarytransporterAraE.Thisbroad-specificityprotonsymporteristhemajortransporterforarabinoseandalsoisaccountablefortheuptakeofxyloseandgalactose.Inaddition,MsmXisshowntobetheATPasethatenergizestheincompleteAraNPQimporter.FurThermore,theresultssuggesttheexistenceofatleastonemoreunidentifiedMsmX-dependentABCimporterresponsIBLefortheuptakeofnonlinearα-1,2-andα-1,3-arabinooligosaccharides.ThisstudyassignsMsmXasamultipurposeB.subtilisATPaserequiredtoenergizedifferentsaccharidetransporters,thearabinooligosaccharide-specificAraNPQ-MsmXsystem,aputativeMsmX-dependentABCtransporterspecificfornonlineararabinooligosaccharides,andthepreviouslycharacterizedmaltodextrin-specificMdxEFG-MsmXsystem.

BacillussubtilissynthesizesatleastonearabinanaseencodedbytheabnAgenethatisabletodegradethepolysaccharidearabinan.Here,wereporttheexpressioninEscherichiacoliofthefull-lengthabnAcodingregionwithaHis₆-tagfusedtotheC-terminus.TherecombinantproteinwassecretedtotheperiplasmicspaceandcorrectlyprocessedbytheE.colisignalpeptidase.ThesubstratespecificityofpurifiedAbnA,thephysico-chemicalpropertiesandkineticparametersweredetermined.FunctionalanalysisstudiesrevealedGlu215asakeyresidueforAbnAhydrolyticactivityandindicatedthatinadditiontoAbnAB.subtilissecretesotherenzyme(s)abletodegradelinear1,5-α-L-arabinan.

Astrainofathermophilicbacterium,tentativelydesignatedBacillusthermodenitrificansTS-3,witharabinan-degradingactivitywasisolated.Itproducedanendo-arabinase(ABN)(EC3.2.1.99)andtwoarabinofuranosidases(EC3.2.1.55)extracellularlywhengrownat60°Conamediumcontainingsugarbeetarabinan.TheABN(tentativelycalledanABN-TS)waspurified7,417-foldbyanion-exchange,hydrophobic,sizeexclusion,andhydroxyapatitechromatographies.ThemolecularmassofABN-TSwas35kDaasdeterminedbysodiumdodecylsulfate-polyacrylamidegelelectrophoresis,andtheisoelectricpointwaspH4.5.TheenzymewasobservedtobemorethermostablethanknownABNs;ithadahalf-lifeof4hat75°C.Theenzymehadoptimalactivityat70°CandpH6.0.TheenzymehadapparentK_mvaluesof8.5and45mg/mlandapparentV_maxvaluesof1.6and1.1mmol/min/mgofproteinagainstdebranchedarabinan(α-1,5-arabinan)andarabinan,respectively.Theenzymehadnopectin-releasingactivity(protopectinaseactivity)fromsugarbeetprotopectin,differingfromanABN(protopectinase-C)frommesophilicBacillussubtilisIFO3134.ThepatternofdegradationofdebranchedarabinanbyABN-TSindicatedthattheenzymewasanendo-actingenzymeandthemainendproductswerearabinobioseandarabinose.TheresultsofpreliminaryexperimentsindicatedthattheculturefiltrateofstrainTS-3issuitableforL-arabinoseproductionfromsugarbeetpulpathightemperature.

Thegenesoftwoα-L-arabinofuranosidases(AbfI andII)fromfamilyGH62havebeenidentifiedinthegenomeofAspergillusfumigatuswmo.BothgeneshavebeenexpressedinPichiapastorisandtheenzymeshavebeenpurifiedandcharacterized.AbfIiscomposedof999 bp,doesnotcontainintronsandcodesforaprotein(ABFI)of332aminoacidresidues.abfIIhas1246 bp,includinganintronof51 bp;theproteinABFIIhas396aminoacidresidues;itincludesafamily1carbohydrate-bindingmodule(CBM)intheN-terminalregion,followedbyacatalyticmodule.ThesequenceofABFIandthecatalyticmoduleofABFIIshowa79 %identity.Bothenzymesareactiveonp-nitrophenylα-L-arabinofuranoside(pNPAra)withK_Mof94.2and3.9 mMforABFIandII,respectively.OptimaltemperatureforABFIis37°CandforABFII42°C,whilethepHoptimumisabout4.5to5forbothenzymes.ABFIIshowsahigherthermostability.Whenassayedusingnaturalsubstrates,bothshowhigheractivityoverryearabinoxylanascomparedtowheatarabinoxylan.ABFIIonlyisactiveonsugarbeetpulparabinanandbothareinactivetowardsdebranchedarabinan.Thehigherthermostability,higheraffinityforpNPAraandwideractivityovernaturalsubstratesshownbyABFIImayberelatedtothepresenceofaCBM.Theavailabilityoftherecombinantenzymesmaybeusefulinbiotechnologicalapplicationsfortheproductionofarabinose.

Plantcellwallismainlycomposedbycellulose,hemicelluloseandlignin.Theheterogeneousstructureandcompositionofthehemicellulosearekeyimpedimentstoitsdepolymerizationandsubsequentuseinfermentationprocesses.Thus,thisstudyaimedtoperformascreeningofthermophilicandthermotolerantfilamentousfungicollectedfromdifferentregionsoftheSãoPaulostate,andanalyzetheproductionofβ-xylosidaseandarabinanaseatdifferenttemperatures.Theseenzymesareimportanttocellwalldegradationandsynthesisofendproductsasxyloseandarabinose,respectively,whicharesignificantsugarstofermentationandethanolproduction.Atotalof12fungalspecieswereanalyzedand9ofthemgrewat45°C,suggestingathermophilicorthermotolerantcharacter.AdditionallyAspergillusthermomutatusanamorphofNeosartoryaandA.parasiticusgrewat50°C.AspergillusnigerandAspergillusthermomutatuswerethefilamentousfungiwiththemostexpressiveproductionofβ-xylosidaseandarabinanase,respectively.Ingeneralformostofthetestedmicroorganisms,β-xylosidaseandarabinanaseactivitiesfrommycelialextract(intracellularform)werehigherinculturesgrownathightemperatures(35-40°C),whilethecorrespondentextracellularactivitieswerefavorablysecretedfromculturesat30°C.ThisstudycontributestocatalogueisolatedfungiofthestateofSãoPaulo,andthesefindingscouldbepromisingsourcesforthermophilicandthermotolerantmicroorganisms,whichareindustriallyimportantduetotheirenzymes.

Arabinanisacomponentofpectin,whichisoneofthepolysaccharidespresentinlignocelluose.Theenzymesdegradingthemainchainofarabinanaretheendo-(EC3.2.1.99)andexo-arabinanases(3.2.1.-).Onlythreeexo-arabinanaseshavebeenbiochemicallycharacterized;theybelongtoglycosylhydrolasefamily93.Inthiswork,thecDNAofanexo-arabinanase(Arap2)fromPenicilliumpurpurogenumhasbeenheterologouslyexpressedinPichiapastoris.Thegeneis1310 bplong,hasthreeintronsandcodesforaproteinof380aminoacidresidues;thematureproteinhasacalculatedmolecularmassof39 823 Da.TheheterologouslyexpressedArap2hasamolecularmassintherangeof60-80 kDaduetoheterogeneousglycosylation.TheenzymeisactiveondebranchedarabinanwithoptimumpHof4-5.5andoptimaltemperatureof40°C,andhasanexo-typeactionmode,releasingarabinobiosefromitssubstrates.Theexpressionprofileofarap2incorncobandsugarbeetpulpfollowsadifferentpatternandisnotrelatedtothepresenceofarabinan.Thisisthefirstexo-arabinanasestudiedfromP.purpurogenumandthefirstexpressedinyeast.TheavailabilityofheterologousArap2maybeusefulforbiotechnologicalapplicationsrequiringacidicconditions.

Lignocellulosicbiomassfromvarioustypesofwoodhasbecomearenewableresourceforproductionofbiofuelsandbiobasedchemicals.Becausexylanisthemajorcomponentofwoodhemicelluloses,highlyefficientenzymestoenhancexylanhydrolysiscanimprovetheuseoflignocellulosicbiomass.Inthisstudy,axylanolyticgeneclusterwasidentifiedfromthecrudeoil-degradingthermophilicstrainGeobacillusthermodenitrificansNG80-2.Theenzymesinvolvedinxylanhydrolysis,whichincludetwoxylanases(XynA1,XynA2),threeβ-xylosidases(XynB1,XynB2,XynB3),andoneα-L-arabinofuranosidase(AbfA),havemanyuniquefeatures,suchashighpHtolerance,highthermostability,andabroadsubstraterange.Thethreeβ-xylosidaseswerehighlyresistanttoinhibitionbyproduct(xylose)accumulation.Moreover,thecombinationofxylanase,β-xylosidase,andα-L-arabinofuranosidaseexhibitedthelargestsynergisticactiononxylandegradation(XynA2,XynB1,andAbfAonoatspeltorbeechwoodxylan;XynA2,XynB3,andAbfAonbirchwoodxylan).Wehavedemonstratedthattheproposedenzymaticcocktailalmostcompletelyconvertscomplexxylantoxyloseandarabinofuranoseandhasgreatpotentialforuseintheconversionofplantbiomassintobiofuelsandbiochemicals.