Purity~90%.Highviscosity.Ara:Xyl=38:62.Contaminants:Othersugars~3%.
Preparationofarabinoxylobiosefromryexylanusingfamily10Aspergillusaculeatusendo-1,4-β-D-xylanase.
Rantanen,H.,Virkki,L.,Tuomainen,P.,Kabel,M.,Schols,H.&Tenkanen,M.(2007).CarbohydratePolymers,68(2),350-359.
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CommercialxylanasepreparationShearzyme®,whichcontainstheglycosidehydrolasefamily10endo-1,4-β-D-xylanasefromAspergillusaculeatus,wasusedtoprepareshort-chainarabinoxylo-oligosaccharides(AXOS)fromryearabinoxylan(AX).AmajorAXOSwasformedasahydrolysisproduct.LongerAXOSwerealsoproducedasminorproducts.ThepureGH10xylanasefromA.aculeatuswasusedasacomparisontoensurethattheformedAXOSwereconsequenceoftheendoxylanase‘sfunctioninsteadofsomesideenzymespresentinShearzyme.ThemajorAXOSwaspurifiedandthestructureconfirmedwithvariousanalysismethods(TLC,HPAEC-PAD,MALDI-TOF-MS,andone-andtwo-dimensionalNMRspectroscopywithnano-probe)asα-L-Araf-(1→3)-β-D-Xylp-(1→4)-D-Xylp(arabinoxylobiose).Thisisthefirstreporton13CNMRdataofpurearabinoxylobiose.Theyieldofarabinoxylobiosewas12%fromthequantifiedhydrolysisproducts.Inconclusion,GH10endoxylanasefromA.aculeatusisthusabletocutefficientlythexylosidiclinkagenexttothearabinofuranosyl-substitutedxyloseunitwhichisnottypicalforalltheGH10endoxylanases.Interestingly,pureA.aculeatusxylanaseshowednotablyactivitytowardsp-nitrophenyl-β-Dxylopyranose.InpreviouslystudieslongerAXOShavebeenproducedwithShearzymebuttheformationofshort-chainAXOSbyA.aculeatusGH10xylanasehasnotbeenstudiedbefore.
XylooligosaccharidesfromhardwoodandcerealxylansproducedbyathermostablexylanaseascarbonsourcesforLactobacillusbrevisandBifidobacteriumadolescentis.
Falck,P.,Precha-Atsawanan,S.,Grey,C.,Immerzeel,P.,Stalbrand,H.,Adlercreutz,P.,&NordbergKarlsson,E.(2013).JournalofAgriculturalandFoodChemistry,61(30),7333-7340.
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Tocomparexylansfromforestrywithagriculturalorigins,hardwoodxylan(birch)andcerealarabinoxylan(rye)werehydrolyzedusingtwovariantsofthexylanaseRmXyn10A,full-lengthenzymeandcatalyticmoduleonly,fromRhodothermusmarinus.Cultivationsoffourselectedbacterialspecies,usingthexylooligosaccharide(XOS)containinghydrolysatesascarbonsource,showedselectivegrowthofLactobacillusbrevisDSMZ1264andBifidobacteriumadolescentisATCC15703.BothstrainswereconfirmedtoutilizetheXOSfraction(DP2–5),whereasputativearabinoxylooligosaccharidesfromtheryearabinoxylanhydrolysatewereutilizedbyonlyB.adolescentis.Escherichiacolididnotgrow,despiteitscapabilitytogrowonthemonosaccharidesarabinoseandxylose.ItwasalsoshownthatPediococcusparvulusstrain2.6utilizedneitherxylosenorXOSforgrowth.Insummary,RmXyn10Aoritscatalyticmoduleprovedsuitableforhigh-temperaturehydrolysisofhardwoodxylanandcerealarabinoxylan,producingXOSthatcouldqualifyasprebioticsforuseinfunctionalfoodproducts.
Understandinghownoncatalyticcarbohydratebindingmodulescandisplayspecificityforxyloglucan.
Luís,A.S.,Venditto,I.,Temple,M.J.,Rogowski,A.,Baslé,A.,Xue,J.,Knox,J.P.,Prates,J.A.M.,Ferreira,L.M.A.,Fontes,C.M.G.A.,Najmudin,S.&Gilbert,H.J.(2013).JournalofBiologicalChemistry,288(7),4799-4809.
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Plantbiomassiscentraltothecarboncycleandtoenvironmentallysustainableindustriesexemplifiedbythebiofuelsector.Plantcellwalldegradingenzymesgenerallycontainnoncatalyticcarbohydratebindingmodules(CBMs)thatfulfilatargetingfunction,whichenhancescatalysis.CBMsthatbindβ-glucanchainsoftendisplaybroadspecificityrecognizingβ1,4-glucans(cellulose),β1,3-β1,4-mixedlinkedglucansandxyloglucan,aβ1,4-glucandecoratedwithα-1,6-xyloseresidues,bytargetingstructurescommontothethreepolysaccharides.Thus,CBMsthatrecognizexyloglucantargettheβ1,4-glucanbackboneandonlyaccommodatethexylosedecorations.HereweshowthattwocloselyrelatedCBMs,CBM65AandCBM65B,derivedfromEcCel5A,aEubacteriumcellulosolvensendoglucanase,bindtoarangeofβ-glucansbut,uniquely,displaysignificantpreferenceforxyloglucan.ThestructuresofthetwoCBMsrevealaβ-sandwichfold.Theligandbindingsitecomprisestheβ-sheetthatformstheconcavesurfaceoftheproteins.Bindingtothebackbonechainsofβ-glucansismediatedprimarilybyfivearomaticresiduesthatalsomakehydrophobicinteractionswiththexylosesidechainsofxyloglucan,conferringthedistinctivespecificityoftheCBMsforthedecoratedpolysaccharide.Significantly,andincontrasttootherCBMsthatrecognizeβ-glucans,CBM65Autilizesdifferentpolarresiduestobindcelluloseandmixedlinkedglucans.Thus,Gln106iscentraltocelluloserecognition,butisnotrequiredforbindingtomixedlinkedglucans.Thisreportrevealsthemechanismbywhichβ-glucan-specificCBMscandistinguishbetweenlinearandmixedlinkedglucans,andshowhowtheseCBMscanexploitanextensivehydrophobicplatformtotargetthesidechainsofdecoratedβ-glucans.
Peroxidase-mediatedoxidativecross-linkinganditspotentialtomodifymechanicalpropertiesinwater-solublepolysaccharideextractsandcerealgrainresidues.
Robertson,J.A.,Faulds,C.B.,Smith,A.C.&Waldron,K.W.(2008).JournalofAgriculturalandFoodChemistry,56(5),1720-1726.
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Analysisofwheatbranandspentgrainshowsthatconcentrationsofferulateanddiferulatesofferconsiderablescopetomodifythecross-linkingofferuloylatedpolysaccharidesandhencethemechanicalpropertiesoftheseresidues.Insolutionferulicacid(FA)canbereadilypolymerizedbyhorseradishperoxidase,butwhenesterifiedtoapolysaccharide,theprofileofdiferulatesbecomesrestricted.ThissituationalsoexistsinmuroandsuggestsstructuralconstraintsmaylimittheavailabilityofFAforcross-linking.Atrelativelylowpolysaccharideconcentration,(~3%),stericrestrictionswereapparentingelspreparedusingisolatedpolysaccharides.Mechanicalpropertiessuchasswellingalsoappeartobefixedattheserelativelylowpolysaccharideconcentrations.Thislimitsthepotentialtomodifymechanicalpropertiesinmurothroughoxidoreductaseactivity.Tomodifymechanicalpropertiessuchtreatmentswillneedtofocusonincreasingthe“flexibility”ofthecellwallmatrixandtheaccessibilityofenzymestothecellwallmatrix.
RestorationofmatureetiolatedcucumberhypocotylcellwallsusceptibilitytoexpansinbypretreatmentwithfungalpectinasesandEGTAinvitro.
Zhao,Q.,Yuan,S.,Wang,X.,Zhang,Y.,Zhu,H.&Lu,C.(2008).PlantPhysiology,147(4),1874-1885.
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Matureplantcellwallslosetheirabilitytoexpandandbecomeunresponsivetoexpansin.Thisphenomenonisbelievedtobeduetocross-linkingofhemicellulose,pectin,orphenolicgroupsinthewall.Byscreeningvarioushydrolyticenzymes,wefoundthatpretreatmentofnongrowing,heat-inactivated,basalcucumber(Cucumissativus)hypocotylswithpectinlyase(Pel1)fromAspergillusjaponicuscouldrestorereconstitutedexogenousexpansin-inducedextensioninmaturecellwallsinvitro.RecombinantpectatelyaseA(PelA)andpolygalacturonase(PG)fromAspergillusspp.exhibitedsimilarcapacitytoPel1.Pel1,PelA,andPGalsoenhancedthereconstitutedexpansin-inducedextensionoftheapical(elongating)segmentsofcucumberhypocotyls.However,theeffectiveconcentrationsofPelAandPGforenhancingthereconstitutedexpansin-inducedextensionweregreaterintheapicalsegmentsthaninthebasalsegments,whereasPel1behavedintheoppositemanner.Thesedataareconsistentwithdistributionofmoremethyl-esterifiedpectinincellwallsoftheapicalsegmentsandlessesterifiedpectininthebasalsegments.Associatedwiththedegreeofesterificationofpectin,morecalciumwasfoundincellwallsofbasalsegmentscomparedtoapicalsegments.PretreatmentofthecalciumchelatorEGTAcouldalsorestorematurecellwallssusceptibilitytoexpansinbyremovingcalciumfrommaturecellwalls.Becauserecombinantpectinasesdonothydrolyzeotherwallpolysaccharides,andendoglucanase,xylanase,andproteasecannotrestorethematurewallsextensibility,wecanconcludethatthepectinnetwork,especiallycalcium-pectatebridges,maybetheprimaryfactorthatdeterminescucumberhypocotylmaturecellwallsunresponsivenesstoexpansin.
CharacterizationofXyn30AandAxh43AofBacilluslicheniformisSVD1identifiedbyitsgenomicanalysis.
Sakka,M.,Tachino,S.,Katsuzaki,H.,vanDyk,J.S.,Pletschke,B.I.,Kimura,T.&Sakka,K.(2012).EnzymeandMicrobialTechnology,51(4),193-199.
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ThegenomesequenceofBacilluslicheniformisSVD1,thatproducesacellulolyticandhemi-cellulolyticmultienzymecomplex,waspartiallydetermined,indicatingthattheglycosidehydrolasesystemofthisstrainishighlysimilartothatofB.licheniformisATCC14580.Allofthefifty-sixgenesencodingglycosidehydrolasesidentifiedinB.licheniformisATCC14580wereconservedinstrainSVD1.Inaddition,twonewgenes,xyn30Aandaxh43A,wereidentifiedintheB.licheniformisSVD1genome.Thexyn30AgenewashighlysimilartoBacillussubtilissubsp.Subtilis168xynCencodingforaglucuronoarabinoxylanendo-1,4-β-xylanase.Xyn30A,producedbyarecombinantEscherichiacoli,hadhighactivitytoward4-O-methyl-D-glucurono-D-xylanbutshoweddefiniteactivitytowardoat-speltxylanandunsubstitutedxylooligosaccharides.RecombinantAxh43A,consistingofafamily-43catalyticmoduleoftheglycosidehydrolasesandafamily-6carbohydrate-bindingmodule(CBM),wasanarabinoxylanarabinofuranohydrolase(α-L-arabinofuranosidase)classifiedasAXH-m23andcapableofreleasingarabinosylresidues,whicharelinkedtotheC-2orC-3positionofsinglysubstitutedxyloseresiduesinarabinoxylanorarabinoxylanoligomers.TheisolatedCBMpolypeptidehadanaffinityforsolubleandinsolublexylansandremovaloftheCBMfromAxh43Aabolishedthecatalyticactivityoftheenzyme,indicatingthattheCBMplaysanessentialroleinhydrolysisofarabinoxylan.
Family42carbohydrate-bindingmodulesdisplaymultiplearabinoxylan-bindinginterfacespresentingdifferentligandaffinities.
Ribeiro,T.,Santos-Silva,T.,Alves,V.D.,Dias,F.M.V.,Luís,A.S.,Prates,J.A.M.,Ferraira,L.M.A.,Romao,M.J.&Fontes,C.M.G.A.(2010).BiochimicaetBiophysicaActa(BBA)-ProteinsandProteomics,1804(10),2054-2062.
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Enzymesthatdegradeplantcellwallpolysaccharidesdisplayamodulararchitecturecomprisingacatalyticdomainboundtooneormorenon-catalyticcarbohydrate-bindingmodules(CBMs).CBMsdisplayconsiderablevariationinprimarystructureandaregroupedinto59sequence-basedfamiliesorganizedintheCarbohydrate-ActiveenZYme(CAZy)database.HerewereportthecrystalstructureofCtCBM42Atogetherwiththebiochemicalcharacterizationoftwoothermembersoffamily42CBMsfromClostridiumthermocellum.CtCBM42A,CtCBM42BandCtCBM42Cbindspecificallytothearabinoseside-chainsofarabinoxylansandarabinan,suggestingthatvariouscellulosomalcomponentsaretargetedtotheseregionsoftheplantcellwall.ThestructureofCtCBM42Adisplaysabeta-trefoilfold,whichcomprises3sub-domainsdesignatedasα,βandγ.Eachoneofthethreesub-domainspresentsaputativecarbohydrate-bindingpocketwhereanaspartateresiduelocatedinacentralpositiondominatesligandrecognition.Intriguingly,theγsub-domainofCtCBM42Aispivotalforarabinoxylanbinding,whiletheconcertedactionofβandγsub-domainsofCtCBM42BandCtCBM42Cisapparentlyrequiredforligandsequestration.Thus,thisworkrevealsthatthebindingmechanismofCBM42membersisincontrastwiththatofhomologousCBM13swhererecognitionofcomplexpolysaccharidesresultsfromthecooperativeactionofthreeproteinsub-domainspresentingsimilaraffinities.
CharacterizationofRuminiclostridiumjosuiarabinoxylanarabinofuranohydrolase,RjAxh43B,andRjAxh43B-containingxylanolyticcomplex.
Orita,T.,Sakka,M.,Kimura,T.&Sakka,K.(2017).EnzymeandMicrobialTechnology,104,37-43.
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Anovelgene(axh43B)fromRuminiclostridiumjosuiencodingacellulosomalenzymeconsistingofacatalyticmoduleofsubfamilyGH43_10,afamily-6carbohydrate-bindingmodule,andadockerinmodule,wasexpressedusingEscherichiacoli.RjAxh43Breleasedonlyarabinosefromarabinoxylanand23,33-di-α-L-arabinofuranosylxylotriose,butnot32-α-L-arabinofuranosylxylobioseor23-α-L-arabinofuranosylxylotriose,stronglysuggestingthatRjAxh43Bisanarabinoxylanα-L-1,3-arabinofuranohydrolasecapableofcleavingα-1,3-linkedarabinoseresiduesofdoublyarabinosylatedxylan.WhenAxh43BwasmixedwiththerecombinantscaffoldingproteinRjCipAofR.josuiatamolarratioof6:1,theactivityoftheRjAxh43B-RjCipAcomplex(6:1)towardinsolublewheatarabinoxylanwassimilartothatofRjAxh43Balone,suggestingthatRjAxh43Bdoesnotshowaproximityeffect,whichisdefinedasanactivityenhancementeffectcausedbythepresenceofpluralcatalyticsubunitsadjoiningeachother.WhenRjAxh43AwasmixedwithxylanaseRjXyn10C,theyactedsynergisticallytowardinsolublewheatarabinoxylanandricestrawpowderintheabsenceofRjCipA.Furthermore,theRjAxh43B-RjXyn10C-RjCipA(3:3:3)complexhadhigheractivitytowardinsolublewheatarabinoxylanthanamixtureofRjAxh43BandRjXyn10CwithoutRjCipA,suggestingthatincorporationofaxylanaseandanα-L-arabinofuranosidaseintoacellulosomeisbeneficialformoreefficientlydegradingarabinoxylan.
Arabinoxylanasefromglycosidehydrolasefamily5isaselectiveenzymeforproductionofspecificarabinoxylooligosaccharides.
Falck,P.,Linares-Pastén,J.A.,Karlsson,E.N.&Adlercreutz,P.(2017).FoodChemistry,242,579-584.
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Anarabinosespecificxylanasefromglycosidehydrolasefamily5(GH5)wasusedtohydrolysewheatandryearabinoxylan,andtheproductprofileshowedthatitproducedarabinosesubstitutedoligosaccharides(AXOS)having2-10xyloseresiduesinthemainchainbutnounsubstitutedxylooligosaccharides(XOS).Molecularmodellingshowedthattheactivesitehasanopenstructureandthatthehydroxylgroupsofallxyloseresiduesintheactivesitearesolventexposed,indicatingthatarabinosesubstituentscanbeaccommodatedintheglyconeaswellastheaglyconesubsites.ThearabinoxylanhydrolysatesobtainedwiththeGH5enzymestimulatedgrowthofBifidobacteriumadolescentisbutnotofLactobacillusbrevis.Thisarabinoxylanaseisthusagoodtoolfortheproductionofselectiveprebiotics.
