Carboxymethylated(DS~0.4),mediumviscositycellulose.Recommendedsubstratefortheassayofcellulasesbyreducingsugarmethods.Formostendo-cellulases,thisisamuchmoresensitivesubstratethanCMC-7M(DS=0.7).
Afibrolyticpotentialinthehumanileummucosalmicrobiotarevealedbyfunctionalmetagenomics.
Patrascu,O.,Béguet-Crespel,F.,Marinelli,L.,LeChatelier,E.,Abraham,A.,Leclerc,M.,Klopp,C.,Terrapon,N.,Henrissat,B.,Blottière,H.M.,Doré,J.&ChristelBéra-Maillet.(2017).ScientificReports,7,40248.
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Thedigestionofdietaryfibersisamajorfunctionofthehumanintestinalmicrobiota.Sofarthisfunctionhasbeenattributedtothemicroorganismsinhabitingthecolon,andmanystudieshavefocusedonthisdistalpartofthegastrointestinaltractusingeasilyaccessiblefecalmaterial.However,microbialfermentations,supportedbythepresenceofshort-chainfattyacids,aresuspectedtooccurintheuppersmallintestine,particularlyintheileum.Usingafosmidlibraryfromthehumanilealmucosa,wescreened20,000clonesfortheiractivitiesagainstcarboxymethylcelluloseandxylanschosenasmodelsofthemajorplantcellwall(PCW)polysaccharidesfromdietaryfibres.ElevenpositiveclonesrevealedabroadrangeofCAZymeencodinggenesfromBacteroidesandClostridialesspecies,aswellasPolysaccharideUtilizationLoci(PULs).Thefunctionalglycosidehydrolasegeneswereidentified,andoligosaccharidebreak-downproductsexaminedfromdifferentpolysaccharidesincludingmixed-linkageβ-glucans.CAZymesandPULswerealsoexaminedfortheirprevalenceinhumangutmicrobiome.Severalclustersofgenesoflowprevalenceinfecalmicrobiomesuggestedtheybelongtounidentifiedstrainsratherspecificallyestablishedupstreamthecolon,intheileum.Thus,theilealmucosa-associatedmicrobiotaencompassestheenzymaticpotentialforPCWpolysaccharidedegradationinthesmallintestine.
PurificationandCharacterizationofaThermostableβ-mannanasefromBacillussubtilisBE-91:PotentialApplicationinInflammatoryDiseases.
Cheng,L.,Duan,S.,Feng,X.,Zheng,K.,Yang,Q.,&Liu,Z.(2016).BioMedResearchInternational,ID6380147.
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β-mannanasehasshowncompellingbiologicalfunctionsbecauseofitsregulatoryrolesinmetabolism,inflammation,andoxidation.Thisstudyseparatedandpurifiedtheβ-mannanasefromBacillussubtilisBE-91,whichisapowerfulhemicellulose-degradingbacteriumusinga“two-step”methodcomprisingultrafiltrationandgelchromatography.Thepurifiedβ-mannanase(about28.2 kDa)showedhighspecificactivity(79,859.2 IU/mg).TheoptimumtemperatureandpHwere65°Cand6.0,respectively.Moreover,theenzymewashighlystableattemperaturesupto70°CandpH4.5–7.0.Theβ-mannanaseactivitywassignificantlyenhancedinthepresenceofMn2+,Cu2+,Zn2+,Ca2+,Mg2+,andAl3+andstronglyinhibitedbyBa2+andPb2+.KmandVmaxvaluesforlocustbeangumwere7.14 mg/mLand107.5 µmol/min/mLversus1.749 mg/mLand33.45 µmol/min/mLforKonjacglucomannan,respectively.Therefore,β-mannanasepurifiedbythisworkshowsstabilityathightemperaturesandinweaklyacidicorneutralenvironments.Basedonsuchdata,theβ-mannanasewillhavepotentialapplicationsasadietarysupplementintreatmentofinflammatoryprocesses.
Enzymaticpreparationofmushroomdietaryfibre:Acomparisonbetweenanalyticalandindustrialenzymes.
Wong,K.H.&Cheung,P.C.K.(2009).FoodChemistry,115(3),795-800.
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Acomparativestudyonpreparingdietaryfibres(DFs)fromthreemushroomsclerotia,namely,Pleurotustuber-regium(PTR),Polyporusrhinocerus(PR)andWolfiporiacocos(WC),usinganalyticalorindustrialenzymes(includingα-amylase,proteaseandamyloglucosidase),wasconducted.Apartfromenzymeactivityandpurity,theireffectsontheyieldofsclerotialDFaswellasitsmajorcomponents,suchasβ-glucans,chitinandresistantglycogen(RG),wereinvestigatedandcompared.Theactivitiesofallindustrialenzymesweresignificantlylowerthanthoseoftheircorrespondinganalyticalones,exceptfortheFungamyl®SuperMA,whichhadthehighestα-amylaseactivity(6395U/g).However,thisfungalα-amylasewaslessabletodigestthethreesclerotialglycogenswhencomparedwiththebacterialalternatives.Amongstalltestedenzymes,onlyanalyticalandindustrialamyloglucosidaseswerefoundtohavesignificantamountofcontaminatingcellulase(7.05–7.07U/ml)andlichenase(4.62–4.67U/ml)activities,whichwouldcauseendo-depolymerizationoftheβ-glucan-typecellwallcomponents(3.39%reductioninglucoseresidueafterRGcorrection)ofthePTR,leadingtoamarkedα-amylasehydrolysisofitsotherwisephysically-inaccessiblecytoplasmicglycogen(20.3%reductioninRGcontent).CommercialproductionofthethreenovelsclerotialDFs,usingtheindustrialenzymes,wouldbefeasiblesince,inadditiontotheireconomicadvantage,boththeyield(PTR:81.2%;PR:86.5%;WC:96.2%ofsampleDM)andtotalnon-starchpolysaccharidecontents(PTR:88.0%;PR:92.5%;WC:91.1%DF-richmaterialsofDM)oftheirresultingsclerotialDFswerecomparabletothelevelsofthosepreparedusinganalyticalenzymes.
CharacterizationofafunctionalsolubleformofaBrassicanapusmembrane-anchoredendo-1,4-β-glucanaseheterologouslyexpressedinPichiapastoris.
Mølhøj,M.,Ulvskov,P.&DalDegan,F.(2001).PlantPhysiology,127(2),674-684.
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TheBrassicanapusgene,Cel16,encodesamembrane-anchoredendo-1,4-β-glucanasewithadeducedmolecularmassof69kD.Asforothermembrane-anchoredendo-1,4-β-glucanases,Cel16consistsofapredictedintracellular,chargedNterminus(methionine1-lysine70),ahydrophobictransmembranedomain(isoleucine71-valine93),andaperiplasmiccatalyticcore(lysine94-proline621).Here,wereportthefunctionalanalysisofΔ1-90Cel16,theNterminallytruncatedCel16,missingresidues1through90andcomprisingthecatalyticdomainofCel16expressedrecombinantlyinthemethylotrophicyeastPichiapastorisasasolubleprotein.Atwo-steppurificationprotocolyieldedΔ1-90Cel16inapureform.ThemolecularmassofΔ1-90Cel16,whendeterminedbysodiumdodecylsulfate-polyacrylamidegelelectrophoresis,wasabout130kDandabout60kDafterenzymaticremovalofN-glycans,fittingtheexpectedmolecularmassof59kD.Δ1-90Cel16washighlyNglycosylatedascomparedwiththenativeB.napusCel16protein.Δ1-90Cel16hadapHoptimumof6.0.TheactivityofΔ1-90Cel16wasinhibitedbyEDTAandexhibitedastrongdependenceoncalcium.Δ1-90Cel16showedsubstratespecificityforlowsubstitutedcarboxymethyl-celluloseandamorphouscellulose.Itdidnothydrolyzecrystallinecellulose,xyloglycan,xylan,(1→3),(1→4)-β-D-glucan,thehighlysubstitutedhydroxyethylcellulose,ortheoligosaccharidescellotriose,cellotetraose,cellopentaose,orxylopentaose.SizeexclusionanalysisofΔ1-90Cel16-hydrolyzedcarboxymethylcelluloseshowedthatΔ1-90Cel16isatrueendo-actingglucanase.
Propertiesoffamily79β-glucuronidasesthathydrolyzeβ-glucuronosyland4-O-methyl-β-glucuronosylresiduesofarabinogalactan-protein.
Konishi,T.,Kotake,T.,Soraya,D.,Matsuoka,K.,Koyama,T.,Kaneko,S.,Igarashi,K.,Samejima,M.&Tsumuraya,Y.(2008).CarbohydrateResearch,343(7),1191-1201.
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Thecarbohydratemoietiesofarabinogalactan-proteins(AGPs),whicharemainlycomposedofGal,L-Ara,GlcA,and4-Me-GlcAresidues,areessentialforthephysiologicalfunctionsoftheseproteoglycansinhigherplants.Forthisstudy,wehaveidentifiedtwogenesencodingfamily79β-glucuronidases,designatedAnGlcAaseandNcGlcAase,inAspergillusnigerandNeurosporacrassa,respectively,basedontheaminoacidsequenceofanativeβ-glucuronidasepurifiedfromacommercialpectolyticenzymepreparationfromA.niger.AlthoughthededucedproteinsequencesofAnGlcAaseandNcGlcAasewerehighlysimilar,therecombinantenzymesexpressedinPichiapastorisexhibiteddistinctsubstratespecificitytoward4-Me-GlcAresiduesofAGPs:recombinantAnGlcAase(rAnGlcAase)substantiallyliberatedbothGlcAand4-Me-GlcAresiduesfromradishAGPs,whereasrecombinantNcGlcAase(rNcGlcAase)activityonthe4-Me-GlcAresiduesofAGPswasverylow.MaximumactivityofrAnGlcAasehydrolyzingPNPβ-GlcAoccurredatpH3.0–4.0,whereasthemaximumrNcGlcAaseactivitywasatpH6.0.TheapparentKmvaluesofrAnGlcAasewere30.4µMforPNPβ-GlcAand422µMforβ-GlcA-(1→6)-Gal,andthoseofrNcGlcAasewere38.3µMand378µM,respectively.Similartothenativeenzyme,rAnGlcAasewasabletocatalyzethetransglycosylationofGlcAresiduesfromPNPβ-GlcAtovariousmonosaccharideacceptorssuchasGlc,Gal,andXyl.WeproposethatbothAnGlcAaseandNcGlcAaseareinstancesofanoveltypeofβ-glucuronidasewiththecapacitytohydrolyzeβ-GlcAand4-Me-β-GlcAresiduesofAGPs,althoughtheydiffersignificantlyintheirpreferences.
Axyloglucan-specificfamily12glycosylhydrolasefromAspergillusniger:recombinantexpression,purificationandcharacterization.
Master,E.R.,Zheng,Y.,Storms,R.,Tsang,A.&Powlowski,J.(2008).Biochem.J,411,161-170.
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AnewGH12(glycosylhydrolase12)familyXEG[xyloglucan-specificendo-β-1,4-glucanase(EC3.2.1.151)]fromAspergillusniger,AnXEG12A,wasoverexpressed,purifiedandcharacterized.WhereassevenxyloglucanasesfromGH74andtwoxyloglucanasesfromGH5havebeencharacterizedpreviously,thisisonlythethirdcharacterizedexampleofaGH12familyxyloglucanase.GH12enzymesarestructurallyandmechanisticallydistinctfromGH74enzymes.Althoughover100GH12sequencesarenowavailable,littleisknownaboutthestructuralandbiochemicalbasesofxyloglucanbindingandhydrolysisbyGH12enzymes.ComparisonoftheAnXEG12AcDNAsequencewiththegenomesequenceofA.nigershowedthepresenceoftwointrons,oneinthecodingregionandthesecondoneinthe333-nt-long3´-untranslatedregionofthetranscript.TheenzymewasexpressedrecombinantlyinA.nigerandwasreadilypurifiedfromtheculturesupernatant.Theisolatedenzymeappearedtohavebeenprocessedbyakexin-typeprotease,whichremovedashortprosequence.Thesubstratespecificitywasrestrictedtoxyloglucan,withcleavageatunbranchedglucoseinthebackbone.Theapparentkineticparametersweresimilartothosereportedforotherxyloglucan-degradingendoglucanases.ThepHoptimum(5.0)andtemperatureresultinginhighestenzymeactivity(50–60°C)werehigherthanthosereportedforaGH12familyxyloglucanasefromAspergillusaculeatus,butsimilartothoseofcellulose-specificendoglucanasesfromtheGH12family.Phylogenetic,sequenceandstructuralcomparisonsofGH12familyendoglucanaseshelpedtodelineatefeaturesthatappeartobecorrelatedtoxyloglucanspecificity.
Degradationofcarbohydratemoietiesofarabinogalactan-proteinsbyglycosidehydrolasesfromNeurosporacrassa.
Takata,R.,Tokita,K.,Mori,S.,Shimoda,R.,Harada,N.,Ichinose,H.,Kaneko,A.,Igarashi,k.,Samejima,M.,Tsumuraya,Y.&Kotake,T.(2010).CarbohydrateResearch,345(17),2516-2522.
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Arabinogalactan-proteins(AGPs)areafamilyofplantproteoglycanshavinglargecarbohydratemoietiesattachedtocore-proteins.ThecarbohydratemoietiesofAGPscommonlyhaveβ-(1→3)(1→6)-galactanasthebackbone,towhichotherauxiliarysugarssuchasL-AraandGlcAareattached.Forthepresentstudy,anα-L-arabinofuranosidasebelongingtoglycosidehydrolasefamily(GHF)54,NcAraf1,andanendo-β-(1→6)-galactanaseofGHF5,Nc6GAL,wereidentifiedinNeurosporacrassa.RecombinantNcAraf1(rNcAraf1)expressedinPichiapastorishydrolyzedradishAGPsaswellasarabinanandarabinoxylan,showingrelativelybroadsubstratespecificitytowardpolysaccharidescontainingα-L-arabinofuranosylresidues.RecombinantNc6GAL(rNc6GAL)expressedinP.pastorisspecificallyactedonβ-(1→6)-galactosylresidues.WhereasAGPfromradishrootswashardlyhydrolyzedbyrNc6GALalone,β-(1→6)-galactansidechainswerereducedtooneortwogalactanresiduesbyacombinationofrNcAraf1andrNc6GAL.TheseresultssuggestthatthecarbohydratemoietiesofAGPsaredegradedbytheconcertedactionofNcAraf1andNc6GALsecretedfromN.crassa.
MolecularcloningandexpressioninEscherichiacoliofaTrichodermavirideendo-beta-(1→6)-galactanasegene.
Kotake,T.,Kaneko,S.,Kubomoto,A.,Haque,M.,Kobayashi,H.&Tsumuraya,Y.(2004).Biochem.J,377(3),749-755.
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Ageneencodingendo-β-(1→6)-galactanasefromTrichodermaviridewasclonedbyreversetranscriptase–PCRandexpressedinEscherichiacoli.Thegenecontainedanopenreadingframeconsistingof1437bp(479aminoacids).Thededucedaminoacidsequenceoftheproteinshowedlittlesimilaritywithotherknownglycosidehydrolases.Asignalsequence(20aminoacids)wasfoundattheN-terminalregionoftheproteinandthemolecularmassofthematureformwascalculatedtobe50.488kDa.ThegeneproductexpressedinE.coliasarecombinantproteinfusedwiththioredoxinandHis6tagshadalmostthesamesubstratespecificityandmodeofactionasnativeenzymepurifiedfromacommercialcellulasepreparationofT.viride,i.e.recombinantenzymeendo-hydrolysedβ-(1→6)-galacto-oligomerswithaDP(degreeofpolymerization)higherthan3,anditcouldalsohydrolyseα-L-arabinofuranosidase-treatedarabinogalactanproteinfromradish.Itproducedβ-(1→6)-galacto-oligomersrangingfromDP2toatleast8attheinitialhydrolysisstageandgalactoseandβ-(1→6)-galactobioseasthemajorproductsatthefinalreactionstage.Theseresultsindicatethattheclonedgeneencodesanendo-β-(1→6)-galactanase.Asfarasweknow,thisisthefirsttimeanendo-β-(1→6)-galactanasehasbeencloned.
CrystalstructuresofClostridiumthermocellumxyloglucanase,XGH74A,revealthestructuralbasisforxyloglucanrecognitionanddegradation.
Martinez-Fleites,C.,Guerreiro,C.I.P.D.,Baumann,M.J.,Taylor,E.J.,Prates,J.A.,Ferreira,L.M.A.,CarlosM.G.A.,Fontes,C.M.G.A.,Brumer,H.&Davies,G.J.(2006).JournalofBiologicalChemistry,281(34),24922-24933.
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Theenzymaticdegradationoftheplantcellwalliscentralbothtothenaturalcarboncycleand,increasingly,toenvironmentallyfriendlyroutestobiomassconversion,includingtheproductionofbiofuels.Theplantcellwallisacomplexcompositeofcellulosemicrofibrilsembeddedindiversepolysaccharidescollectivelytermedhemicelluloses.Xyloglucanisonesuchpolysaccharidewhosehydrolysisiscatalyzedbydiversexyloglucanases.HerewepresentthestructureoftheClostridiumthermocellumxyloglucanaseXgh74Ainbothapoandligand-complexedforms.Thestructures,incombinationwithmutagenesisdataonthecatalyticresiduesandthekineticsandspecificityofxyloglucanhydrolysisrevealacomplexsubsitespecificityaccommodatingseventeenmonosaccharidemoietiesofthemultibranchedsubstrateinanopensubstratebindingterrain.
