TheD-Glucuronic/D-Galacturonictestkitisa simple,reliableandaccuratemethodforthemeasurementandanalysisofD-hexuronicacids(specificallyD-glucuronicacidandD-galacturonicacid)inplantextracts,culturemedia/supernatantsandothermaterials.Suitableformanual,auto-analyserandmicroplateformats.Identificationoffeaturesassociatedwithplantcellwallrecalcitrancetopretreatmentbyalkalinehydrogenperoxideindiversebioenergyfeedstocksusingglycomeprofiling.Li,M.,Pattathil,S.,Hahn,M.G.&Hodge,D.B.(2014).RSCAdvances,4(33),17282-17292.LinktoArticleReadAbstractAwoodydicot(hybridpoplar),anherbaceousdicot(goldenrod),andagraminaceousmonocot(cornstover)weresubjectedtoalkalinehydrogenperoxide(AHP)pretreatmentandsubsequentenzymatichydrolysisinordertoassesshowtaxonomicallyandstructurallydiversebiomassfeedstocksrespondtoamildalkalineoxidativepretreatmentandhowdifferingfeaturesofthecellwallmatrixcontributetoitsrecalcitrance.Usingglycomeprofiling,wedeterminedchangesintheextractABIlityofnon-cellulosicglucansfollowingpretreatmentbyscreeningextractsofthepretreatedwallswithapanelof155cellwallglycan-directedmonoclonalantibodiestodeterminedifferencesintheabundanceanddistributionofnon-cellulosicglycanepitopesintheseextractsandassesspretreatment-inducedchangesinthestructuralintegrityofthecellwall.Twotaxonomically-dependentoutcomesofpretreatmentwereidentifiedthatbothimprovedthesubsequentenzymatichydrolysisyieldsbutdifferedintheirimpactsoncellwallstructuralintegrity.Specifically,itwasrevealedthatgoldenrodwallsexhibiteddecreasesinallclassesofalkali-extractableglycansindicatingtheirsolubilizationduringpretreatment,whichwasaccompaniedbyanimprovementinthesubsequentextractabilityoftheremainingcellwallglycans.Thecornstoverwallsdidnotshowthesamedecreasesinglycanabundanceinextractsfollowingpretreatment,butrathermildincreasesinallclassesofcellwallglycans,indicatingoverallweakerassociationsbetweencellwallpolymersandimprovedextractability.Thehybridpoplarwallswererelativelyunaffectedbypretreatmentintermsofcomposition,enzymatichydrolysis,andtheextractabilityofcellwallglycansduepresumablytotheirhigherlignincontentanddenservascularstructure.CorrelatingligninstructuralfeaturestophasepartitioningbehaviorinanovelaqueousfractionationofsoftwoodKraftblackliquor.Stoklosa,R.J.,Velez,J.,Kelkar,S.,Saffron,C.M.,Thies,M.C.&Hodge,D.B.(2013).GreenChemistry,15(10),2904-2912.LinktoArticleReadAbstractInthiswork,asetofsoftwoodligninswererecoveredfromaKraftblackliquorusinganovelpH-basedfractionationprocessinvolvingsequentialCO2acidificationandseparationofthesolvatedaqueousligninfraction.TheserecoveredligninsfractionswerecharacterizedwithrespecttopropertiesthatmayberesponsIBLefortheirphasepartitioningbehavioraswellaspropertiesthatmayrendertheligninsmoresuitableformaterialsapplications.LigninfractionswererecoveredbetweenapHrangeof12.8and9.5withthebulkofthelignin(90%)recoveredbetweenapHof11.1and10.0.Whileallthefractionswerefoundtoconsistprimarilyofligninasvalidatedbysamplemethoxylcontent,thefirstfractionstophaseseparatedwerefoundtobeespeciallyenrichedinaliphaticextractivesandpolysaccharides.FromthebulkoftheligninthatwasrecoveredbetweenapHof11.1and10.0anumberofnoteworthytrendswerediscerniblefromthedata.Specifically,thephenolichydroxylcontentwasfoundtoexhibitastrongnegativecorrelationtothefractionationpHandexhibitedanearly50%increasewithrecoveryatdecreasingpH,whiletheGPC-estimatedmolecularweightsand13CNMR-estimatedβ-O-4contentshowedstrongpositivecorrelationstothepHatrecovery.Thealiphatichydroxylcontentexhibitedminimaldifferencesbetweenrecoveryconditions.Overall,theseresultssuggestthatthisfractionationapproachcangenerateligninfractionsenrichedinselectphysicalorstructuralpropertiesthatmaybeimportantfortheirapplicationasfeedstocksforrenewablechemicalsormaterials.DietarySupplementationwithSolublePlantainNon-StarchPolysaccharidesInhibitsIntestinalInvasionofSalmonellaTyphimuriumintheChicken.Parsons,B.N.,Wigley,P.,Simpson,H.L.,Williams,J.M.,Humphrey,S.,Salisbury,A.M.,Watson,A.J.,Fry,S.C.,O"Brien,D.,Roberts,C.L.,O"Kennedy,N.,Keita,Å.V.,Söderholm,J.D.,Rhodes,J.M.&Campbell,B.J.(2014).PloSOne,9(2),e87658.LinktoArticleReadAbstractSolublefibres(non-starchpolysaccharides,NSP)fromedibleplantsbutparticularlyplantainbanana(Musaspp.),havebeenshowninvitroandexvivotopreventvariousentericpathogensfromadheringto,ortranslocatingacross,thehumanintestinalepithelium,apropertythatwehavetermedcontrabiotic.HerewereportthatdietaryplantainfibrepreventsinvasionofthechickenintestinalmucosabySalmonella.InvivoexperimentswereperformedwithchicksfedfromhatchonapelletdietcontainingsolubleplantainNSP(0to200mg/d)andorallyinfectedwithS.Typhimurium4/74at8dofage.Birdsweresacrificed3,6and10dpost-infection.Bacteriawereenumeratedfromliver,spleenandcaecalcontents.InvitrostudieswereperformedusingchickencaecalcryptsandporcineintestinalepithelialcellsinfectedwithSalmonellaentericaSEROvarsfollowingpre-treatmentseparatelywithsolubleplantainNSPandacidicorneutralpolysaccharidefractionsofplantainNSP,eachcomparedwithsalinevehicle.Bacterialadherenceandinvasionwereassessedbygentamicinprotectionassay.InvivodietarysupplementationwithplantainNSP50mg/dreducedinvasionbyS.Typhimurium,asreflectedbyviablebacterialcountsfromsplenictissue,by98.9%(95%CI,98.1–99.7;PInvitrostudiesconfirmedthatplantainNSP(5–10mg/ml)inhibitedadhesionofS.Typhimurium4/74toaporcineepithelialcell-line(73%meaninhibition(95%CI,64–81);PPex-vivohumanilealmucosashowedthatthiseffectwasassociatedwithincreasedshortcircuitcurrentbutnochangeinelectricalresistance.TheinhibitoryactivityofplantainNSPlaymainlywithintheacidic/pectic(homogalacturonan-rich)component.SupplementationofchickfeedwithplantainNSPwaswelltoleratedandshowspromiseasasimpleapproachforreducinginvasivesalmonellosis.Optimizationofaformic/aceticaciddelignificationtreatmentonbeechwoodanditsinfluenceonthestructuralcharacteristicsoftheextractedlignins.Simon,M.,Brostaux,Y.,Vanderghem,C.,Jourez,B.,Paquot,M.&Richel,A.(2014).JournalofChemicalTechnologyandBiotechnology,89(1),128-136.LinktoArticleReadAbstractBackground:InordertoreplacepetRochemicalsbybio-basedligninproductsinhighvalue-addedapplications,aformic/aceticacidtreatmentwasadaptedtobeechwood(FagussylvaticaL.)forligninextraction.Results:Beechwoodparticlesweredelignifiedatatmosphericpressurebyaformicacid/aceticacid/watermixture.Cookingtimeandtemperaturewereoptimizedfordelignification,pulpyieldand2-furfuralconcentration.Responsesurfacedesignanalysisrevealedthatdelignificationyieldincreasedwithcookingtimeandtemperature.Conclusion:Themulti-criteriaoptimizationofdelignificationwasusedtofindtheidealcookingconditions(5h07min,104.2°C)tomaximizedelignification(70.5%)andpulpyield(58.7%)and,toalesserextent,minimize2-furfuralproduction.Treatmentconditionswerefoundtoinfluencethechemicalstructureofextractedlignins.Cookingtimeandtemperatureinverselyinfluencedligninmolecularweights.Fractionationandimprovedenzymaticdeconstructionofhardwoodswithalkalinedelignification.Stoklosa,R.J.&Hodge,D.B.(2015).BioEnergyResearch,8(3),1224-1234.LinktoArticleReadAbstractInthiswork,analkalinedelignificationwasinvestigatedforseveralindustriallyrelevanthardwoodstounderstandthekineticsofxylansolubilizationanddegradationandtheroleofresiduallignincontentinsettingcellwallrecalcitrancetoenzymatichydrolysis.Between34and50 %ofthexylanwassolubilizedduringtheheat-upstageofthepretreatmentandundergoesdegradation,depolymerization,aswellassubstantialdisappearanceoftheglucuronicacidsubstitutionsonthexylanduringthebulkdelignificationphase.Animportantfindingisthatsubstantialxylanisstillpresentintheliquorwithoutdegradation.Cellulosehydrolysisyieldsintherangeof80to90 %wereachievablewithin24–48 hforthediversehardwoodssubjectedtodelignificationbyalkaliatmodestenzymeloADIngs.Itwasfoundthatsubstantialdelignificationwasnotnecessarytoachievethesehighhydrolysisyieldsandthathybridpoplarsubjectedtopretreatmentremovingonly46 %oftheligninwascapableofreachingyieldscomparabletohybridpoplarpretreatedto67or86 %ligninremoval.Decreasingthelignincontentwasfoundtoincreasetheinitialrateofcellulosehydrolysistoglucosewhilelignincontentsunderapproximately70 mg/goriginalbiomasswerefoundtoslightlydecreasethemaximumextentofhydrolysis,presumablyduetodrying-inducedcelluloseaggregationandporecollapse.Pretreatmentswereperformedonwoodchips,whichnecessitateda“disintegration”stepfollowingpretreatment.Thisallowedtheeffectofcomminutionmethodtobeinvestigatedforthethreehardwoodssubjectedtothehighestlevelofdelignification.Itwasfoundthatadditionalknife-millingfollowingdistintegrationdidnotimpacteithertherateorextentofglucanandxylanhydrolysis.KineticpropertiesofRhizopusoryzaeRPG1endo-polygalacturonasehydrolyzinggalacturonicacidoligomers.Mertens,J.A.&Bowman,M.J.(2016).BiocatalysisandAgriculturalBiotechnology,5,11-16.LinktoArticleReadAbstractThekineticcharacteristicsofRhizopusoryzaeendo-polygalacturonase,RPG1,hydrolyzinggalacturonicacidoligomers(GalpA)nweredetermined.RPG1generates(GalpA)3asadominantproductofpolygalacturonicacidand(GalpA)4-6hydrolysis.Theenzymecanhydrolyze(GalpA)3,buthydrolysisoccursatasignificantlylowerraterelativetooligomerswithahigherdegreeofpolymerization.Hydrolysisoftheα-1,4glycosidicbondbyRPG1isanendothermicprocesswithaδHapp,of1.03±0.04 kcal/mol.Determinationofkineticconstantsbyisothermaltitrationcalorimetryshowedthatforoligomers(GalpA)3-6,theKmdecreasedandtheKcatincreasedasthelengthofthe(GalpA)oligomerincreased.FixedtimepointassaysfollowedbychromatographicanalysisprovidedapparentKcatvaluessimilartothosefoundusingisothermaltitrationcalorimetry.Assaystodeterminetowhatextenttheenzymeissubjecttoproductinhibitiondemonstratedthattheenzymeiscompetitivelyinhibitedby(GalpA)2whenusing(GalpA)4assubstrate.TheapparentKiof767µMissignificantlyhigherthantheKmvaluesobtainedfortheseriesofgalacturonicacidoligomers.Characterisationofthreefungalglucuronoylesterasesonglucuronicacidestermodelcompounds.Hüttner,S.,Klaubauf,S.,deVries,R.P.&Olsson,L.(2017).AppliedMicroBIOLOGyandBiotechnology,1-11.LinktoArticleReadAbstractTheglucuronoylesterases(GEs)thathavebeenidentifiedsofarbelongtofamily15ofthecarbohydrateesterasesintheCAZyclassificationsystemandarepresumedtotargetesterbondsbetweenligninalcoholsand(4-O-methyl-)D-glucuronicacidresiduesofxylan.FewGEshavebeencloned,expressedandcharacterisedtodate.Characterisationhasbeendoneonavarietyofsyntheticsubstrates;however,thenumberofcommerciallyavailablesubstratesisverylimited.WeidentifiednovelputativeGEsfromawidetaxonomicrangeoffungiandexpressedtheenzymesoriginatingfromAcremoniumalcalophilumandWolfiporiacocosaswellasthepreviouslydescribedPcGE1fromPhanerochaetechrysosporium.AllthreefungalGEswereactiveonthecommerciallyavailablecompoundsbenzylglucuronicacid(BnGlcA),allylglucuronicacid(allylGlcA)andtoalowerdegreeonmethylglucuronicacid(MeGlcA).TheenzymesshowedpHstabilityoverawidepHrangeandtolerated6-hincubationsofupto50°C.KineticparametersweredeterminedforBnGlcA.ThisstudyshowsthesuitabilityofthecommerciallyavailablemodelcompoundsBnGlcA,MeGlcAandallylGlcAinGEactivityscreeningandcharacterisationexperiments.WeenrichedthespectrumofcharacterisedGEswithtwonewmembersofarelativelyyoungenzymefamily.Duetoitsbiotechnologicalsignificance,thisfamilydeservestobemoreextensivelystudied.Thepresentedenzymesarepromisingcandidatesasauxiliaryenzymestoimprovesaccharificationofplantbiomass.ActionofaGH115α-glucuronidasefromAmphibacillusxylanusatalkalineconditionpromotesreleaseof4-O-methylglucopyranosyluronicacidfromglucuronoxylanandarabinoglucuronoxylan.Yan,R.,Vuong,T.V.,Wang,W.&Master,E.R.(2017).EnzymeandMicrobialTechnology,104, 22-28.LinktoArticleReadAbstractGlucuronicacidand/or4-O-methyl-glucuronicacid(GlcA/MeGlcA)aresubstituentsofthemainxylanspresentinhardwoods,conifers,andmanycerealgrains.α-GlucuronidasesfromglycosidehydrolasefamilyGH115cantargetGlcA/MeGlcAfrombothinternallyandterminallysubstitutedregionsofxylans.ThecurrentstudydescribesthefirstGH115α-glucuronidase,AxyAgu115A,fromthealkaliphilicorganismAmphilbacillusxylanus.AxyAgu115AwasactiveinawidepHrange,anddemonstratedbetterperformanceinalkalineconditioncomparedtoothercharacterizedGH115α-glucuronidases,whichgenerallyshowoptimalactivityinacidicconditions.Specifically,itsrelativeactivitybetweenpH5.0andpH8.5wasabove80%,andwas35%ofmaximumatpH10.5;althoughtheenzymelost30%and80%relativeresidualactivityafter24-hpre-incubationatpH9andpH10,respectively.AxyAgu115Awasalsosimilarlyactivetowardsglucuronoxylanaswellascomparativelycomplexxylanssuchassprucearabinoglucurunoxylan.AccommodationofcomplexxylanswassupportedbydockinganalysesthatpredictedaccessibilityofAxyAgu115Atobranchedxylo-oligosaccharides.MeGlcAreleasebyAxyAgu115Afromeachxylansamplewasincreasedbyupto30%byperformingthereactionatpH11.0ratherthanpH4.0,revealingappliedbenefitsofAxyAgu115Aforxylanrecoveryandprocessing.Fungalglucuronoylesterases:genomeminingbasedenzymediscoveryandbiochemicalcharacterization.Dilokpimol,A.,Mäkelä,M.R.,Cerullo,G.,Zhou,M.,Varriale,S.,Gidijala,L.,Brás,J.L.A.,Jütten,P.,Piechot,A.,Verhaert,R.,Faraco,V.,Hilden,K.S.&deVries,R.P.(2017).NewBiotechnology,InPress.LinktoArticleReadAbstract4-O-Methyl-D-glucuronicacid(MeGlcA)isaside-residueofglucuronoarabinoxylanandcanformesterlinkagestolignin,contributingsignificantlytothestrengthandrigidityoftheplantcellwall.Glucuronoylesterases(4-O-methyl-glucuronoylmethylesterases,GEs)cancleavethisesterbond,andthereforemayplayasignificantroleasauxiliaryenzymesinbiomasssaccharificationfortheproductionofbiofuelsandbiochemicals.GEsbelongtoarelativelynewfamilyofcarbohydrateesterases(CE15)intheCAZydatabase(www.cazy.org),andsofararoundtenfungalGEshavebeencharacterized.ToexploreadditionalGEenzymes,weusedagenomeminingstrategy.BLASTanalysiswithcharacterizedGEsagainstapproximately250publiclyaccessiblefungalgenomesidentifiedmorethan150putativefungalGEs,whichwereclassifiedintoeightphylogeneticsub-groups.Tovalidatethegenomeminingstrategy,21selectedGEsfrombothascomyceteandbasidiomycetefungiwereheterologouslyproducedinPichiapastoris.Oftheseenzymes,18wereactiveagainstbenzylD-glucuronatedemonstratingthesuitabilityofourgenomeminingstrategyforenzymediscovery.UV-methodforthedeterminationofD-GlucuronicAcidorD-GalacturonicAcidinhydrolysatesofplantmaterialandpolysaccharidesandothermaterialsPrinciple:                    (Uronatedehydrogenase;UDH)(1)D-Glucuronicacid+NAD++H2O→D-glucarate+NADH+H+                     (Uronatedehydrogenase;UDH)(2)D-Galacturonicacid+NAD++H2O→D-galactarate+NADH+H+Kitsize:                         *100assays(manual)/1000(microplate)                                       /1000(auto-analyser)* Thenumberofmanualtestsperkitcanbedoubledifallvolumesarehalved. ThiscanbereadilyaccommodatedusingtheMegaQuantTM WaveSpectrophotometer(D-MQWAVE).Method:                          Spectrophotometricat340nmReactiontime:                 ~10minat25°Cor~5minat37°CDetectionlimit:                ~17mg/LApplicationexamples:HydrolysatesofplantmaterialandpolysaccharidesandothermaterialsMethodrecognition:   NovelmethodAdvantagesVerycosteffective Allreagentsstablefor>2yearsduringuse Onlytestkitavailable Simpleformat Mega-Calc™softwaretoolisavailablefromourwebsiteforhassle-freerawdataprocessing Standardincluded Suitableformanual,microplateandauto-analyserformats

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