Highpuritydyed,solubleAzo-WheatArabinoxylanforthemeasurementofenzymeactivity,forresearch,biochemicalenzymeassaysandinvitrodiagnosticanalysis.
PreparedbydyeingwheatflourarabinoxylanwithRemazolBrilliantBlueRdye.Substratefortheassayofendo-1,4-β-D-xylanase.
Novelsubstratesfortheautomatedandmanualassayofendo-1,4-β-xylanase.
Mangan,D.,Cornaggia,C.,Liadova,A.,McCormack,N.,Ivory,R.,McKie,V.A.,Ormerod,A.&McCleary,D.V.(2017).CarbohydrateResearch,445,14-22.
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endo-1,4-β-Xylanase(EC3.2.1.8)isemployedacrossabroadrangeofindustriesincludinganimalfeed,brewing,baking,biofuels,detergentsandpulp(paper).Despiteitsimportance,arapid,reliable,reproducible,automatableassayforthisenzymethatisbasedontheuseofachemicallydefinedsubstratehasnotbeendescribedtodate.Reportedhereinisanewenzymecoupledassayprocedure,termedtheXylX6assay,thatemploysanovelsubstrate,namely4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-45-O-glucosyl-xylopentaoside.ThedevelopmentofthesubstrateandassociatedassayisdiscussedhereandtherelationshipbetweentheactivityvaluesobtainedwiththeXylX6assayversustraditionalreducingsugarassaysanditsspecificityandreproducibilitywerethoroughlyinvestigated.
EvidencefortemporalregulationofthetwoPseudomonascellulosaxylanasesbelongingtoglycosidehydrolasefamily11.
Emami,K.,Nagy,T.,Fontes,C.M.G.A.,Ferreira,L.M.A.&Gilbert,H.J.(2002).JournalofBacteriology,184(15),4124-4133.
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Pseudomonascellulosaisahighlyefficientxylan-degradingbacterium.Genesencodingfivexylanases,andseveralaccessoryenzymes,whichremovethevarioussidechainsthatdecoratethexylanbackbone,havebeenisolatedfromthepseudomonadandcharacterized.Thexylanasegenesconsistofxyn10A,xyn10B,xyn10C,xyn10D,andxyn11A,whichencodeXyn10A,Xyn10B,Xyn10C,Xyn10D,andXyn11A,respectively.Inthisstudyasixthxylanasegene,xyn11B,wasisolatedwhichencodesa357-residuemodularenzyme,designatedXyn11B,comprisingaglycosidehydrolasefamily11catalyticdomainappendedtoaC-terminalX-14module,ahomologueofwhichbindstoxylan.Localizationstudiesshowedthatthetwoxylanaseswithglycosidehydrolasefamily(GH)11catalyticmodules,Xyn11AandXyn11B,aresecretedintotheculturemedium,whereasXyn10Cismembranebound.xyn10C,xyn10D,xyn11A,andxyn11Bwereallabundantlyexpressedwhenthebacteriumwasculturedonxylanorβ-glucanbutnotonmediumcontainingmannan,whereasglucoserepressedtranscriptionofthesegenes.Althoughallofthexylanasegeneswereinducedbythesamepolysaccharides,temporalregulationofxyn11Aandxyn11Bwasapparentonxylan-containingmedia.Transcriptionofxyn11Aoccurredearlierthantranscriptionofxyn11B,whichisconsistentwiththepredictedmodeofactionoftheencodedenzymes.Xyn11A,butnotXyn11B,exhibitsxylanesteraseactivity,andtheremovalofacetatesidechainsisrequiredforxylanasestohydrolyzethexylanbackbone.AtransposonmutantofP.cellulosainwhichxyn11Aandxyn11Bwereinactivedisplayedgreatlyreducedextracellularbutnormalcell-associatedxylanaseactivity,anditsgrowthrateonmediumcontainingxylanwasindistinguishablefromwild-typeP.cellulosa.Basedonthedatapresentedhere,weproposeamodelforxylandegradationbyP.cellulosainwhichtheGH11enzymesconvertdecoratedxylansintosubstitutedxylooligosaccharides,whicharethenhydrolyzedtotheirconstituentsugarsbythecombinedactionofcell-associatedGH10xylanasesandsidechain-cleavingenzymes.
SecondarysubstratebindingstronglyaffectsactivityandbindingaffinityofBacillussubtilisandAspergillusnigerGH11xylanases.
Cuyvers,S.,Dornez,E.,Rezaei,M.N.,Pollet,A.,Delcour,J.A.&Courtin,C.M.(2011).FEBSJournal,278(7),1098–1111.
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Thesecondarysubstratebindingsite(SBS)ofBacillussubtilisandAspergillusnigerglycosidehydrolasefamily11xylanaseswasstudiedbysite-directedmutagenesisandevaluationofactivityandbindingpropertiesofmutantenzymesondifferentsubstrates.ModificationoftheSBSresultedinanuptothree-folddecreaseintherelativeactivityoftheenzymesonpolymericversusoligomericsubstratesandhighlightedtheimportanceofseveralaminoacidsintheSBSforminghydrogenbondsorhydrophobicstackinginteractionswithsubstrates.WeakeningoftheSBSincreasedKdvaluesbyupto70-foldinbindingaffinitytestsusingnaturalsubstrates.TheimpactthatmodificationsintheSBShavebothonactivityandonbindingaffinitytowardspolymericsubstratesclearlyshowsthatsuchstructuralelementscanincreasetheefficiencyofthesesingledomainenzymesontheirnaturalsubstrates.
Comparisonofprebioticeffectsofarabinoxylanoligosaccharidesandinulininasimulatorofthehumanintestinalmicrobialecosystem.
Grootaert,C.,VandenAbbeele,P.,Marzorati,M.,Broekaert,W.F.,Courtin,C.M.,Delcour,J.A.,Verstraete,W.&VandeWiele,T.(2009).FEMSMicrobiologyEcology,69(2),231–242.
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Inthisstudy,theprebioticpotentialofarabinoxylanoligosaccharides(AXOS)wascomparedwithinulinintwosimulatorsofthehumanintestinalmicrobialecosystem.Microbialbreakdownofbotholigosaccharidesandshort-chainfattyacidproductionwascoloncompartmentspecific,withascendingandtransversecolonbeingthepredominantsiteofinulinandAXOSdegradation,respectively.Lactatelevels(+5.5mM)increasedintheascendingcolonduringAXOSsupplementation,whilepropionatelevels(+5.1mM)increasedinthetransversecolon.Theconcomitantdecreaseinlactateinthetransversecolonsuggeststhatpropionatewaspartiallyformedovertheacrylatepathway.Furthermore,AXOSsupplementationstronglydecreasedbutyrateintheascendingcolon,thisinparallelwithadecreaseinRoseburiaspp.andBacteroides/Prevotella/Porphyromonas(−1.4and−2.0logCFU)levels.Inulintreatmenthadmoderateeffectsonlactate,propionateandbutyratelevels.Denaturinggradientgelelectrophoresisanalysisrevealedthatinulinchangedmicrobialmetabolismbymodulatingthemicrobialcommunitycomposition.Incontrast,AXOSprimarilyaffectedmicrobialmetabolismby‘switchingon’AXOS-degradingenzymes(xylanase,arabinofuranosidaseandxylosidase),withoutsignificantlyaffectingmicrobialcommunitycomposition.OurresultsdemonstratethatAXOShasahigherpotencythaninulintoshiftpartofthesugarfermentationtowardthedistalcolonparts.Furthermore,duetoitsstrongerpropionate-stimulatingeffect,AXOSisacandidateprebioticcapableofloweringcholesterolandbeneficiallyaffectingfatmetabolismofthehost.
Arabinoxylan‐oligosaccharides(AXOS)affecttheprotein/carbohydratefermentationbalanceandmicrobialpopulationdynamicsofthesimulatorofhumanintestinalmicrobialecosystem.
Sanchez,J.I.,Marzorati,M.,Grootaert,C.,Baran,M.,VanCraeyveld,V.,Courtin,C.M.,Broekaet,W.F.Declour,J.A.,Verstraete,W.&VandeWiele,T.(2009).MicrobialBiotechnology,2(1),101-113.
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Arabinoxylan‐oligosaccharides(AXOS)arearecentlynewlydiscoveredclassofcandidateprebioticsas–dependingontheirstructure–theyarefermentedindifferentregionsofgastrointestinaltract.Thiscanhaveanimpactontheprotein/carbohydratefermentationbalanceinthelargeintestineand,thus,affectthegenerationofpotentiallytoxicmetabolitesinthecolonoriginatingfromproteolyticactivity.Inthisstudy,wescreeneddifferentAXOSpreparationsfortheirimpactonthe
invitrointestinalfermentationactivityandmicrobialcommunitystructure.Short‐termfermentationexperimentswithAXOSwithanaveragedegreeofpolymerization(avDP)of29allowedpartoftheoligosaccharidestoreachthedistalcolon,anddecreasedtheconcentrationofproteolytic
Markers,whereasAXOSwithloweravDPwereprimarilyfermentedintheproximalcolon.Additionally,prolongedsupplementationofAXOSwithavDP29totheSimulatorofHumanIntestinalMicrobialEcosystem(SHIME)reactordecreasedlevelsofthetoxicproteolyticmarkersphenolandp‐cresolinthetwodistalcoloncompartmentsandincreasedconcentrationsofbeneficialshort‐chainfattyacids(SCFA)inallcolonvessels(25–48%).Denaturantgradientgelelectrophoresis(DGGE)analysisindicatedthatAXOSsupplementationonlyslightlymodifiedthetotalmicrobialcommunity,implyingthattheobservedeffectsonfermentationmarkersaremainlycausedbychangesinfermentationactivity.Finally,specificquantitativePCR(qPCR)analysisshowedthatAXOSsupplementationsignificantlyincreasedtheamountofhealth‐promotinglactobacilliaswellasof
Bacteroides–Prevotellaand
Clostridiumcoccoides–Eubacteriumrectalegroups.ThesedataallowconcludingthatAXOSarepromisingcandidatestomodulatethemicrobialmetabolisminthedistalcolon.
Microbialcommunitydevelopmentinadynamicgutmodelisreproducible,colonregionspecific,andselectiveforbacteroidetesandclostridiumclusterIX.
VandenAbbeele,P.,Grootaert,C.,Marzorati,M.,Possemiers,S.,Verstraete,W.,Gérard,P.,Rabot,S.,Bruneau,A.,ElAidy,S.,Derrien,M.,Zoetendal,E.,Kleerebezem,M.,Smidt,H.&VandeWiele,T.(2010).AppliedandEnvironmentalMicrobiology,76(15),5237-5246.
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Dynamic,multicompartmentinvitrogastrointestinalsimulatorsareoftenusedtomonitorgutmicrobialdynamicsandactivity.Thesereactorsneedtoharboramicrobialcommunitythatisstableuponinoculation,colonregionspecific,andrelevanttoinvivoconditions.Togetherwiththereproducibilityofthecolonizationprocess,thesecriteriaareoftenoverlookedwhenthemodulatorypropertiesfromdifferenttreatmentsarecompared.Wethereforeinvestigatedthemicrobialcolonizationprocessintwoidenticalsimulatorsofthehumanintestinalmicrobialecosystem(SHIME),simultaneouslyinoculatedwiththesamehumanfecalmicrobiotawithahigh-resolutionphylogeneticmicroarray:thehumanintestinaltractchip(HITChip).Followinginoculationoftheinvitrocoloncompartments,microbialcommunitycompositionreachedsteadystateafter2weeks,whereas3weekswererequiredtoreachfunctionalstability.ThisdynamiccolonizationprocesswasreproducibleinbothSHIMEunitsandresultedinhighlydiversemicrobialcommunitieswhichwerecolonregionspecific,withtheproximalregionsharboringsaccharolyticmicrobes(e.g.,Bacteroidesspp.andEubacteriumspp.)andthedistalregionsharboringmucin-degradingmicrobes(e.g.,Akkermansiaspp.).Importantly,theshiftfromaninvivotoaninvitroenvironmentresultedinanincreasedBacteroidetes/Firmicutesratio,whereasClostridiumclusterIX(propionateproducers)wasenrichedcomparedtoclustersIVandXIVa(butyrateproducers).Thiswassupportedbyproportionallyhigherinvitropropionateconcentrations.Inconclusion,high-resolutionanalysisofinvitro-culturedgutmicrobiotaoffersnewinsightonthemicrobialcolonizationprocessandindicatestheimportanceofdigestiveparametersthatmaybecrucialinthedevelopmentofnewinvitromodels.
GlycosideHydrolasesfromatargetedCompostMetagenome,activity-screeningandfunctionalcharacterization.
Dougherty,M.J.,D’haeseleerP.,Hazen,T.C.,Simmons,B.A.,Adams,P.D.&Hadi,M.Z.(2012).BMCBiotechnology,12,38.
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Background:Metagenomicsapproachesprovideaccesstoenvironmentalgeneticdiversityforbiotechnologyapplications,enablingthediscoveryofnewenzymesandpathwaysfornumerouscatalyticprocesses.Discoveryofnewglycosidehydrolaseswithimprovedbiocatalyticpropertiesfortheefficientconversionoflignocellulosicmaterialtobiofuelsisacriticalchallengeinthedevelopmentofeconomicallyviableroutesfrombiomasstofuelsandchemicals.Results:Twenty-twoputativeORFs(openreadingframes)wereidentifiedfromaswitchgrass-adaptedcompostcommunitybasedonsequencehomologytorelatedgenefamilies.TheseORFswereexpressedinE.coliandassayedforpredictedactivities.SevenoftheORFsweredemonstratedtoencodeactiveenzymes,encompassingfiveclassesofhemicellulases.Fourenzymeswereoverexpressedinvivo,purifiedtohomogeneityandsubjectedtodetailedbiochemicalcharacterization.TheirpHoptimarangedbetween5.5-7.5andtheyexhibitmoderatethermostabilityupto~60-70°C.Conclusions:SevenactiveenzymeswereidentifiedfromthissetofORFscomprisingfivedifferenthemicelluloseactivities.Theseenzymeshavebeenshowntohaveusefulproperties,suchasmoderatethermalstabilityandbroadpHoptima,andmayserveasthestartingpointsforfutureproteinengineeringtowardsthegoalofdevelopingefficientenzymecocktailsforbiomassdegradationunderdiverseprocessconditions.
HemicellulaseproductioninChrysosporiumlucknowenseC1.
Hinz,S.W.A.,Pouvreau,L.,Joosten,R.,Bartels,J.,Jonathan,M.C.,Wery,J.&Schols,H.A.(2009).JournalofCerealScience,50(3),318-323.
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Filamentousfungiarewidelyusedforenzymeproductionforthebiofuelindustry.TheascomycetousfungusChrysosporiumlucknowenseC1wasisolatedasanaturalproducerofneutralcellulases.ItisatpresentanattractivealternativetowellknownfungilikeAspergillussp.andTrichodermareeseiforproteinproductiononacommercialscale.Besidesmanycellulases,alargenumberofhemicellulases(particularlyxylanasesandarabinofuranosidases)andesterases(acetylxylanesterasesandferulicacidesterases)encodinggeneshavealsobeenidentifiedintheC1genome.ManyoftheseextracellularenzymeshavebeenselectivelyexpressedinC1andthenpurifiedandcharacterized.Fourarabinofuranosidases,twoacetylxylanesterases,twoferulicacidesterases,anα-glucuronidaseandfourxylanaseshavebeenpurifiedandcharacterized.Alltheseenzymeswerefoundtobeactivetowardsarabinoxylans,demonstratingthehighpotentialofC1asaproducerofhemicellulolyticenzymes.
Impactofcellwall‐degradingenzymesonwater‐holdingcapacityandsolubilityofdietaryfibreinryeandwheatbran.
Petersson,K.,Nordlund,E.,Tornberg,E.,Eliasson,A.C.,&Buchert,J.(2013).JournaloftheScienceofFoodandAgriculture,93(4),882-889.
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BACKGROUND:Ryeandwheatbranweretreatedwithseveralxylanasesandendoglucanases,andtheeffectsonphysicochemicalpropertiessuchassolubility,viscosity,water-holdingcapacityandparticlesizeaswellasthechemicalcompositionofthesolubleandinsolublefractionsofthebranwerestudied.Alargenumberofenzymeswithwell-definedactivitieswereused.Thisenabledacomparisonbetweenenzymesofdifferentoriginsandwithdifferentactivitiesaswellasacomparisonbetweentheeffectsoftheenzymesonryeandwheatbran.RESULTS:ThexylanasesderivedfromBacillussubtiliswerethemosteffectiveinsolubilisingdietaryfibrefromwheatandryebran.Therewasatendencyforahigherdegreeofdegradationofthesolubleorsolubiliseddietaryfibreinryebranthaninwheatbranwhentreatedwithmostoftheenzymes.CONCLUSION:Noneoftheenzymesincreasedthewater-holdingcapacityofthebranortheviscosityoftheaqueousphase.Thecontentofinsolublematerialdecreasedasthedietaryfibrewassolubilisedbytheenzymes.Theamountofmaterialthatmayformanetworktoretainwaterinthesystemwastherebydecreased.
RoleofN-linkedglycosylationintheenzymaticpropertiesofathermophilicGH10xylanasefromAspergillusfumigatusexpressedinPichiapastoris.
Chang,X.,Xu,B.,Bai,Y.,Luo,H.,Ma,R.,Shi,P.&Yao,B.(2017).PloSOne,12(2),e0171111.
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N-Glycosylationisaposttranslationalmodificationcommonlyoccurredinfungiandplaysrolesinavarietyofenzymefunctions.Inthisstudy,axylanase(Af-XYNA)ofglycosidehydrolase(GH)family10fromAspergillusfumigatusharboringthreepotentialN-glycosylationsites(N87,N124andN335)washeterologouslyproducedinPichiapastoris.TheN-glycosylatedAf-XYNA(WT)exhibitedfavorabletemperatureandpHoptima(75°CandpH5.0)andgoodthermostability(maintainingstableat60°C).TorevealtheroleofN-glycosylationonAf-XYNA,theenzymewasdeglycosylatedbyendo-β-N-acetylglucosaminidaseH(DE)ormodifiedbysite-directedmutagenesisatN124(N124T).ThedeglycosylatedDEandmutantN124TshowednarrowerpHadaptationrange,lowerspecificactivity,andworsepHandthermalstability.FurtherthermodynamicanalysisrevealedthattheenzymewithhigherN-glycosylationdegreewasmorethermostable.Thisstudydemonstratedthattheeffectsofglycosylationatdifferentdegreesandsiteswerediverse,inwhichtheglycanlinkedtoN124playedakeyroleinpHandthermalstabilityofAf-XYNA.
Managementofenzymediversityinhigh-performancecellulolyticcocktails.
Reyes-Sosa,F.M.,Morales,M.L.,Gómez,A.I.P.,Crespo,N.V.,Zamorano,L.S.,Rocha-Martín,J.,Molina-Heredia,F.P.&García,B.D.(2017).BiotechnologyforBiofuels,10(1),156.
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Background:Modernbiorefineriesrequireenzymaticcocktailsofimprovedefficiencytogeneratefermentablesugarsfromlignocellulosicbiomass.Cellulolyticfungi,amongothermicroorganisms,havedemonstratedthehighestpotentialintermsofenzymaticproductivity,complexityandefficiency.Ontheotherhand,undercellulolytic-inducingconditions,theyoftenproduceaconsiderablediversityofcarbohydrate-activeenzymeswhichallowthemtoadapttochangingenvironmentalconditions.However,industrialconditionsarefixedandadjustedtotheoptimumofthewholecocktail,resultinginunderperformanceofindividualenzymes.Results:OneofthesecellulolyticcocktailsfromMyceliophthorathermophilahasbeenanalyzedherebymeansofLC–MS/MS.PureGH6familymembersdetectedhavebeencharacterized,confirmingpreviousstudies,andaddedtowholecocktailstocomparetheircontributioninthehydrolysisofindustrialsubstrates.Finally,independentdeletionsoftwoGH6familymembers,asanexampleoftheenzymaticdiversitymanagement,ledtothedevelopmentofastrainproducingamoreefficientcellulolyticcocktail.Conclusions:Thesedataindicatethatthedeletionofnoncontributivecellulases(hereEGVI)canincreasethecellulolyticefficiencyofthecocktail,validatingthemanagementofcellulasediversityasastrategytoobtainimprovedfungalcellulolyticcocktails.
