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.

Therecentlyisolatedbacterialstrain80/3representsoneofthemostabundant16SrRNAphylotypesdetectedinthehealthyhumanlargeintestineandbelongstotheRuminococcaceaefamilyofFirmicutes.Thecompletedgenomesequencereportedhereisthefirstforamemberofthisimportantfamilyofbacteriafromthehumancolon.Thegenomecomprisestwolargechromosomesof2.24and0.73Mbp,leADIngustoproposethenameRuminococcusbicirculansforthisnewspecies.Analysisofthecarbohydrateactiveenzymecomplementsuggestsanabilitytoutilizecertainhemicelluloses,especiallyβ-glucansandxyloglucan,forgrowththatwasconfirmedexperimentally.Theenzymaticmachineryenablingthedegradationofcelluloseandxylanbyrelatedcellulolyticruminococciishoweverlackinginthisspecies.Whilethegenomeindicatedthecapacitytosynthesizepurines,pyrimidinesandall20aminoacids,onlygenesforthesynthesisofnicotinate,NAD⁺,NADP⁺andcoenzymeAweredetectedamongtheessentialvitaminsandco-factors,resultinginmultiplegrowthrequirements.Invivo,thesegrowthfactorsmustbesuppliedfromthediet,hostorothergutmicroorganisms.OtherfeaturesofecologicalinterestincludetwotypeIVpilins,multipleextracytoplasmicfunction-sigmafactors,aureaseandabilesalthydrolase.

FourcorewoodtypeswereexaminedfromsaplingtreesoftwoclonesofPinusradiatagrowninaglasshouse.Treesweregrowneitherstraighttoproducenormalcorewood,tiltedat45°fromtheverticaltoproduceoppositecorewoodandcompressioncorewood,orrockedtoproduceflexurecorewood.MeancellulosemicrofibrilangleoftracheidwallswasestimatedbyX-raydiffractionandlongitudinalswellingmeasuredbetweenanovendryandmoisturesaturatedstate.Ligninandacetylcontentsofthewoodsweremeasuredandthemonosaccharidecompositionsofthecell-wallpolysaccharidesdetermined.Finelymilledwoodwasanalysedusingsolution-state2DNMRspectroscopyofgelsfromfinelymilledwoodinDMSO-d₆/pyridine-d₅.Althoughtherewasnosignificantdifferenceincellulosemicrofibrilangleamongthecorewoodtypes,compressioncorewoodhadthehighestlongitudinalswelling.Alignincontent>32%andagalactosylresiduecontent>6%clearlydividedseverecompressioncorewoodfromtheothercorewoodtypes.Relationshipscouldbedrawnbetweenlignincontentandlongitudinalswelling,andbetweengalactosylresiduecontentandlongitudinalswelling.The2DNMRspectrashowedthatthepresenceofH-unitsinligninwasexclusivetocompressioncorewood,whichalsohadahigher(1→4)-β-D-galactancontent,definingauniquecompositionforthatcorewoodtype.

Theimmobilizedendo-andexo-arabinanasesfromCaldicellulosiruptorsaccharolyticusproducedcontinuouslyanaverageof16.5gl^-1L-arabinosefrom20gl^-1sugarbeetarabinanatpH5.0and75°Cfor216h,withaproductivityof9.9gl^-1h^-1andaconversionyieldof83%.

Background:Thedegradationofplantmaterialsbyenzymesisanindustryofincreasingimportance.Forsustainableproductionofsecondgenerationbiofuelsandotherproductsofindustrialbiotechnology,efficientdegradationofnon-edIBLeplantpolysaccharidessuchashemicelluloseisrequired.Foreachtypeofhemicellulose,acomplexmixtureofenzymesisrequiredforcompleteconversiontofermentablemonosaccharides.Inplant-biomassdegradingfungi,theseenzymesareregulatedandreleasedbycomplexregulatorystructures.Inthisstudy,wepresentamethodologyforevaluatingthepotentialofagivenfungusforpolysaccharidedegradation.Results:Throughthecompilationofinformationfrom203articles,wehavesystematizedknowledgeonthestructureanddegradationof16majortypesofplantpolysaccharidestoformagraphicaloverview.Asacaseexample,wehavecombinedthiswithalistof188genescodingforcarbohydrate-activeenzymesfromAspergillusniger,thusformingananalysisframework,whichcanbequeried.Combinationofthisinformationnetworkwithgeneexpressionanalysisonmono-andpolysaccharidesubstrateshasallowedelucidationofconcertedgeneexpressionfromthisorganism.Onesuchexampleistheidentificationofafullsetofextracellularpolysaccharide-actinggenesforthedegradationofoatspeltxylan.Conclusions:Themappingofplantpolysaccharidestructuresalongwiththecorrespondingenzymaticactivitiesisapowerfulframeworkforexpressionanalysisofcarbohydrate-activeenzymes.Applyingthisnetwork-basedapproach,weprovidethefirstgenome-scalecharacterizationofallgenescodingforcarbohydrate-activeenzymesidentifiedinA.niger.

Xyloglucaniswidelybelievedtofunctionasatetherbetweencellulosemicrofibrilsintheprimarycellwall,limitingcellenlargementbyrestrictingtheabilityofmicrofibrilstoseparatelaterally.Totestthebiomechanicalpredictionsofthis“tetherednetwork”model,weassessedtheabilityofcucumber(Cucumissativus)hypocotylwallstoundergocreep(long-term,irreversibleextension)inresponsetothreefamily-12endo-β-1,4-glucanasesthatcanspecificallyhydrolyzexyloglucan,cellulose,orboth.Xyloglucan-specificendoglucanase(XEGfromAspergillusaculeatus)failedtoinducecellwallcreep,whereasanendoglucanasethathydrolyzesbothxyloglucanandcellulose(Cel12AfromHypocreajecorina)inducedahighcreeprate.Acellulose-specificendoglucanase(CEGfromAspergillusniger)didnotcausecellwallcreep,eitherbyitselforincombinationwithXEG.Testswithadditionalenzymes,includingafamily-5endoglucanase,confirmedtheconclusionthattocausecreep,endoglucanasesmustcutbothxyloglucanandcellulose.Similarresultswereobtainedwithmeasurementsofelasticandplasticcompliance.BothXEGandCel12Ahydrolyzedxyloglucaninintactwalls,butCel12AcouldhydrolyzeaminorxyloglucancompartmentrecalcitranttoXEGdigestion.XyloglucaninvolvementintheseenzymeresponseswasconfirmedbyexperimentswithArabidopsis(Arabidopsisthaliana)hypocotyls,whereCel12Ainducedcreepinwild-typebutnotinxyloglucan-deficient(xxt1/xxt2)walls.Ourresultsareincompatiblewiththecommondepictionofxyloglucanasaload-bearingtetherspanningthe20-to40-nmspacingbetweencellulosemicrofibrils,buttheydoimplicateaminorxyloglucancomponentinwallmechanics.Thestructurallyimportantxyloglucanmaybelocatedinlimitedregionsoftightcontactbetweenmicrofibrils.

Componentsofmodularcellulases,type-Acellulose-bindingmodules(CBMs)bindtocrystallinecelluloseandenhanceenzymeeffectiveness,butstructuraldetailsoftheinteractionareuncertain.WeanalyzedcellulosebindingbyEXLX1,abacterialexpansinwithabilitytoloosenplantcellwallsandwhosedomainD2hastype-ACBMcharacteristics.EXLX1stronglybindstocrystallinecelluloseviaD2,whereasitsaffinityforsolublecellooligosaccharidesisweak.Calorimetryindicatedcellulosebindingwaslargelyentropicallydriven.WesolvedthecrystalstructuresofEXLX1complexedwithcellulose-likeoligosaccharidestofindthatEXLX1bindstheligandsthroughhydrophobicinteractionsofthreelinearlyarrangedaromaticresiduesinD2.Thecrystalstructuresrevealedauniqueformofligand-mediateddimerization,withtheoligosaccharidesandwichedbetweentwoD2domainsinoppositepolarity.Thisreportclarifiesthemoleculartargetofexpansinandthespecificmolecularinteractionsofatype-ACBMwithcellulose.

Bacteroidesisadominantgenuswithintheintestinalmicrobiotaofhealthyhumans.KeyadaptationsoftheBacteroidestothedynamicintestinalecosystemincludeadiverserepertoireofgenesinvolvedinsensingandprocessingnumerousdiet-andhost-derivedpolysaccharides.Onesuchadaptationisthecarbohydrate-sensinghybridtwo-componentsystem(HTCS)familyofsignallingsensors,whichhasbeenwidelyexpandedwithintheBacteroides.UsingBacteroidesthetaiotaomicronasamodel,wehavecreatedachimericHTCSconsistingofthewell-characterizedsensingdomainofoneHTCS,BT1754,andtheregulatorydomainofanotherHTCS,BT0366,toexploretheregulatorycapabilitiesofthesemolecules.WefoundthattheBT0366regulatoryregiondirectlybindstoandmediatesinductionoftheadjacentpolysaccharideutilizationlocus(PUL)usingwhole-genometranscriptionalprofilingafterinducingsignallingthroughourchimericprotein.WealsofoundthatBT0366activationsimultaneouslyleadstorepressionofdistalPULsinvolvedinmucuscarbohydrateconsumption.TheseresultssuggestanovelmechanismbywhichanHTCSenforcesanutrienthierarchywithintheBacteroidesviainductionandrepressionofmultiplePULs.Thus,hybridtwo-componentsystemsprovideamechanismforprioritizingconsumptionofcarbohydratesthroughsimultaneousbindingandregulationofmultiplepolysaccharideutilizationloci.

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

AfterdegradationofsugarbeetcellwallswithDriselase®andfractionationofthesolubilisedproductsbyhydrophobicinteractionchromatography,adehydrodiferuloylatedoligoarabinanwasisolated.Itsstructurewasassignedtotwodimersof(1→5)-linkedarabinoseunitsesterifiedbyacentral8-O-4′ferulicdimer.Theseresultsprovidethefirstdirectevidencethatpecticarabinansinsugarbeetcellwallsmaybecovalentlycross-linkedthroughdehydrodiferulates.

(1,3)(1,4)-β-D-Glucan(mixed-linkageglucanorMLG),acharacteristichemicelluloseinprimarycellwallsofgrasses,wasinvestigatedtodeterminebothitsroleincellwallsanditsinteractionwithcelluloseandothercellwallpolysaccharidesinvitro.BindingisothermsshowedthatMLGadsorptionontomicrocrystallinecelluloseisslow,irreversible,andtemperature-dependent.MeasurementsusingquartzcrystalmicrobalancewithdissipationmonitoringshowedthatMLGadsorbedirreversiblyontoamorphousregeneratedcellulose,formingathickhydrogel.Oligosaccharideprofilingusingendo-(1,3)(1,4)-β-glucanaseindicatedthattherewasnodifferenceinthefrequencyanddistributionof(1,3)and(1,4)linksinboundandunboundMLG.ThebindingofMLGtocellulosewasreducedifthecellulosesampleswerefirsttreatedwithcertaincellwallpolysaccharides,suchasxyloglucanandglucuronoarabinoxylan.ThetetheringfunctionofMLGincellwallswastestedbyapplyingendo-(1,3)(1,4)-β-glucanasetowallsamplesinaconstantforceextensometer.Cellwallextensionwasnotinduced,whichindicatesthatenzyme-accessibleMLGdoesnottethercellulosefibrilsintoaload-bearingnetwork.

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.

AnenzymaticmixtureofcellulasesandxylanaseswasproducedbyPleurotusostreatususingmicrocrystallinecelluloseasinducer,partiallycharacterizedandtestedinthestatisticalanalysisofArundodonaxbioconversion.ThePlackett-BurmanscreeningdesignwasappliedtoidentifythemostsignificantparametersfortheenzymatichydrolysisofpretreatedA.donax.AsthemostsignificantinfluenceduringtheenzymatichydrolysisofA.donaxwasexercisedbythetemperature(°C),pH,andtime,thecombinedeffectofthesefactorsinthebioconversionbyP.ostreatuscellulaseandxylanasewasanalyzedbya3³factorialexperimentaldesign.Itisworthnotingthatthebestresultof480.10 mgofsugars/gds,obtainedat45°C,pH3.5,and96hoursofincubation,wassignificantalsowhencomparedwiththeresultspreviouslyreachedbyprocessoptimizationwithcommercialenzymes.

Togetherwithfungi,saprophyticbacteriaarecentraltothedecompositionandrecyclingofbiomassinforestenvironments.TheBacteroidetesphylumisabundantindiversehabitats,andseveralspecieshavebeenshowntobeabletodeconstructawidevarietyofcomplexcarbohydrates.ThegenusChitinophagaisoftenenrichedinhotspotsofplantandmicrobialbiomassdegradation.WepresentaproteomicassessmentoftheabilityofChitinophagapinensistogrowonanddegrademannanpolysaccharides,usinganagaroseplate-basedmethodofproteincollectiontominimisecontaminationwithexopolysaccharidesandproteinsfromlysedcells,andtoreflecttherealisticsettingofgrowthonasolidsurface.WeshowthatselectPolysaccharideUtilisationLoci(PULs)areexpressedindifferentgrowthconditions,andidentifyenzymesthatmaybeinvolvedinmannandegradation.Bycomparingproteomicandenzymaticprofiles,weshowevidencefortheinducedexpressionofenzymesandPULsincellsgrownonmannanpolysaccharidescomparedwithcellsgrownonglucose.Inaddition,weshowthatthesecretionofputativebiomass-degradingenzymesduringgrowthonglucosecomprisesasystemfornutrientscavenging,whichemploysconstitutivelyproducedenzymes.Significanceofthisstudy:Chitinophagapinensisbelongstoabacterialgenuswhichisprominentinmicrobialcommunitiesinagriculturalandforestenvironments,whereplantandfungalbiomassisintensivelydegraded.Suchdegradationishugelysignificantintherecyclingofcarboninthenaturalenvironment,andtheenzymesresponsibleareofbiotechnologicalrelevanceinemergingtechnologiesinvolvingthedeconstructionofplantcellwallmaterial.Thebacteriumhasacomparativelylargegenome,whichincludesmanyuncharacterisedcarbohydrate-activeenzymes.Wepresentthefirstproteomicassessmentofthebiomass-degradingmachineryofthisspecies,focusingonmannan,anabundantplantcellwallhemicellulose.Ourfindingsincludetheidentificationofseveralnovelenzymes,whicharepromisingtargetsforfuturebiochemicalcharacterisation.Inaddition,thedataindicatetheexpressionofspecificPolysaccharideUtilisationLoci,inducedinthepresenceofdifferentgrowthsubstrates.Wealsohighlighthowaconstitutivesecretionofenzymeswhichdeconstructmicrobialbiomasslikelyformspartofanutrientscavengingprocess.

Background:Pectinisanabundantcomponentinmanyfruitandvegetablewastesandcouldthereforebeanexcellentresourceforbiorefinery,butiscurrentlyunderutilized.Fungalpectinasesalreadyplayacrucialroleforindustrialpurposes,suchasforfoodstuffprocessing.However,theregulationofpectinasegeneexpressionisstillpoorlyunderstood.Foranoptimalutilizationofplantbiomassforbiorefineryandbiofuelproduction,adetailedanalysisoftheunderlyingregulatorymechanismsiswarranted.Inthisstudy,weappliedthegeneticresourcesofthefilamentousascomycetespeciesNeurosporacrassatoscreenfortranscriptionfactorsthatplayamajorroleinpectinaseinduction.Results:Thepectindegradationregulator-1(PDR-1)wasidentifiedthroughatranscriptionfactormutantscreeninN.crassa.TheΔpdr-1mutantexhibitedaseveregrowthdefectonpectinandalltestedpectin-relatedpoly-andmonosaccharides.BiochemicalaswellastranscriptionalanalysesofWTandtheΔpdr-1mutantrevealedthatwhilePDR-1-mediatedgeneinductionwasdependentonthepresenceofL-rhamnose,italsostronglyaffectedthedegradationofthehomogalacturonanbackbone.Theexpressionoftheendo-polygalacturonasegh28-1wasgreatlyreducedintheΔpdr-1mutant,whiletheexpressionlevelsofallpectatelyasegenesincreased.Moreover,apdr-1overexpressionstraindisplayedsubstantiallyincreasedpectinaseproduction.PromoteranalysisofthePDR-1regulonallowedrefinementoftheputativePDR-1DNA-bindingmotif.Conclusions:PDR-1ishighlyconservedinfilamentousascomycetefungiandispresentinmanypathogenicandindustriallyimportantfungi.OurdatademonstratethatthefunctionofPDR-1inN.crassacombinesfeaturesoftworecentlydescribedtranscriptionfactorsinAspergillusniger(RhaR)andBotrytiscinerea(GaaR).Theresultspresentedinthisstudycontributetoabroaderunderstandingofhowpectindegradationisorchestratedinfilamentousfungiandhowitcouldbemanipulatedforoptimizedpectinaseproduction.

Whenpresentedwithnutrientmixtures,severalhumangutBacteroidesspeciesexhibithierarchicalutilizationofglycansthroughaphenomenonthatresemblescataboliterepression.However,itisunclearhowcloselytheseobservedphysiologicalchanges,oftenmeasuredbyalteredtranscriptionofglycanutilizationgenes,mirroractualglycandepletion.Tounderstandtheglycanprioritizationstrategiesoftwocloselyrelatedhumangutsymbionts,BacteroidesovatusandBacteroidesthetaiotaomicron,weperformedaseriesoftimecourseassaysinwhichbothspecieswereindividuallygrowninamediumwithsixdifferentglycansthatbothspeciescandegrade.Disappearanceofthesubstratesandtranscriptionofthecorrespondingpolysaccharideutilizationloci(PULs)weremeasured.Eachspeciesutilizedsomeglycansbeforeothers,butwithdifferentprioritiesperspecies,providinginsightintospecies-specifichierarchicalpreferences.Ingeneral,thepresenceofhighlyprioritizedglycansrepressedtranscriptionofgenesinvolvedinutilizinglower-prioritynutrients.However,transcriptionalsensitivitytosomeglycansvariedrelativetotheresidualconcentrationinthemedium,withsomePULsthattargethigh-prioritysubstratesremaininghighlyexpressedevenaftertheirtargetglycanhadbeenmostlydepleted.Coculturingoftheseorganismsinthesamemixtureshowedthatthehierarchicalordersgenerallyremainedthesame,promotingstablecoexistence.Polymerlengthwasfoundtobeacontributingfactorforglycanutilization,therebyaffectingitsplaceinthehierarchy.OurfindingsnotonlyelucidatehowB.ovatusandB.thetaiotaomicronstrategicallyaccessglycanstomaintaincoexistencebutalsosupporttheprioritizationofcarbohydrateutilizationbasedoncarbohydratestructure,advancingourunderstandingoftherelationshipsbetweendietandthegutmicrobiome.

Inordertocharacterizetwoα-L-arabinofuranosidases(α-L-AFases),Abf1Geo12andAbf2Geo12,producedbyGeobacillusstearothermophilusstrain12,thegenes(abf1andabf2)codingfortheseenzymeswereclonedandsequenced.Basedontheproteinsequencesimilarities,approximately57kDatwoα-L-AFaseswereassignedtotheglycosidehydrolasefamily51.Toobtainpureenzymes,theabf1andabf2geneswereclonedintopET28a+expressionvectorandrecombinantα-L-AFaseswereproducedinE.coliBL21(DE3):pLysS.Characterizationofrecombinantα-L-AFasesrevealedthatAbf1Geo12andAbf2Geo12wereactiveinabroadtemperaturerangefrom50to85°Candfrom40to80°C,respectively.Also,theAbf1Geo12wasactiveinabroadpHrangefrom5.0to9.0.TheoptimumpHandtemperatureforAbf1Geo12weredeterminedaspH6.0and65°C,respectively,whereastheoptimumpHandtemperatureforAbf2Geo12weredeterminedaspH5.5and60°C,respectively.Basedoncharacterizationstudies,itwasdeterminedthattheAbf1Geo12wasmorestablethanAbf2Geo12andpreviouslyidentifiedα-L-AFasesfromG.stearothermophilus.Usingp-nitrophenylα-L-arabinofuranosideasasubstrate,theK_mandV_maxvaluesforAbf1Geo12andAbf2Geo12weredeterminedas0.31mMand290U/mgfortheformerenzymeand0.19mMand213.2U/mgforthelatterenzyme,respectively.TheactivitiesofAbf1Geo12andAbf2Geo12werestronglyinhibitedby1mMHg²⁺.Interestingly,Cu²⁺andCo²⁺stimulatedtheactivityofAbf1Geo12,buttheyreducedtheactivityofAbf2Geo12.TherecombinantenzymesreleasedL-arabinosefromsugarbeetarabinan,arabinobiose,arabinotriose,arabinotetraoseandarabinopentaose.Consequently,thesecharacterizedtwoenzymesmaybeusedinindustrialfieldssincetheyarestableathightemperatures.