Suitableasareplacementforbirchwoodxylanasasubstrateforβ-xylanaseinDNSAreducingsugarassay.
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,reproduc
IBLe,automatableassayforthisenzymethatisbasedontheuseofachemicallydefinedsubstratehasnotbeendescribedtodate.Reportedhereinisanewenzymecoupledassayprocedure,termedtheXylX6assay,thatemploysanovelsubstrate,namely4,6-
O-(3-ketobutylidene)-4-nitrophenyl-β-4
5-
O-glucosyl-xylopentaoside.ThedevelopmentofthesubstrateandassociatedassayisdiscussedhereandtherelationshipbetweentheactivityvaluesobtainedwiththeXylX6assayversustr
ADItionalreducingsugarassaysanditsspecificityandreproducibilitywerethoroughlyinvestigated.
AComparisonofPolysaccharideSubstratesandReducingSugarMethodsfortheMeasurementofendo-1,4-β-Xylanase.
McCleary,B.V.&McGeough,P.(2015).Appl.Biochem.Biotechnol.,177(5),1152-1163.
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Themostcommonlyusedmethodforthemeasurementofthelevelofendo-xylanaseincommercialenzymepreparationsisthe3,5-dinitrosalicylicacid(DNS)reducingsugarmethodwithbirchwoodxylanassubstrate.ItiswellknownthatwiththeDNSmethod,muchhigherenzymeactivityvaluesareobtainedthanwiththeNelson-Somogyi(NS)reducingsugarmethod.Inthispaper,wehavecomparedtheDNSandNSreducingsugarassaysusingarangeofxylan-typesubstratesandaccuratelycomparedthemolarresponsefactorsforxyloseandarangeofxylo-oligosaccharides.Purifiedbeechwoodxylanorwheatar
ABInoxylanisshowntobeasuitablereplacementforbirchwoodxylanwhichisnolongercommerciallyavailable,anditisclearlydemonstratedthattheDNSmethodgrosslyoverestimates
endo-xylanaseactivity.UnliketheDNSassay,theNSassaygavetheequivalentcolourresponsewithequimolaramountsofxylose,xylobiose,xylotrioseandxylotetraosedemonstratingthatitaccuratelymeasuresthequantityofglycosidicbondscleavedbythe
endo-xylanase.TheauthorsstronglyrecommendcessationoftheuseoftheDNSassayformeasurementof
endo-xylanaseduetothefactthatthevaluesobtainedaregrosslyoverestimatedduetosecondaryreactionsincolourdevelopment.
PurificationandCharacterizationofaThermostableβ-mannanasefromBacillussubtilisBE-91:PotentialApplicationinInflammatoryDiseases.
Cheng,L.,Duan,S.,Feng,X.,Zheng,K.,Yang,Q.&Liu,Z.(2016).BioMedResearchInternational,ArticleID6380147.
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β-mannanasehasshowncompelling
BIOLOGicalfunctionsbecauseofitsregulatoryrolesinmetabolism,inflammation,andoxidation.Thisstudyseparatedandpurifiedtheβ-mannanasefrom
BacillussubtilisBE-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-mannanaseactivitywassignificantlyenhancedinthepresenceofMn
+,Cu
2+,Zn
2+,Ca
2+,Mg
2+,andAl
3+andstronglyinhibitedbyBa
2+,andPb
2+.
Kmand
Vmaxvaluesforlocustbeangumwere7.14 mg/mLand107.5 μmol/min/mLversus1.749 mg/mLand33.45 µmol/min/mLforKonjacglucomannan,respectively.Therefore,β-mannanasepurifiedbythisworkshowsstabilityathightemperaturesandinweaklyacidicorneutralenvironments.Basedonsuchdata,theβ-mannanasewillhavepotentialapplicationsasadietarysupplementintreatmentofinflammatoryprocesses.
Immobilizationandstabilizationofcommercialβ-1,4-endoxylanaseDepolTM333MDPbymultipointcovalentattachmentforxylanhydrolysis:Productionofprebiotics(xylo-oligosaccharides).
MartinsdeOliveira,S.,Moreno-Perez,S.,Romero-Fernández,M.,Fernandez-Lorente,G.,Rocha-Martin,J.&Guisan,J.M.(2017).BiocatalysisandBiotransformation,1-10.
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ThecommercialenzymeDepolTM333MDP(D333MDP)wasimmobilizedbymultipointcovalentattachmentonto10%cross-linkedagarosebeadssupporthighlyactivatedwithaldehydegroups.Theenzymeimmobilizationprocesswasveryefficient,retaining86%ofitsinitialcatalyticactivity.ThermalstabilityoftheimmobilizedD333MDPbiocatalystsvariedaccordingtotheincubationtimeoftheenzyme-support.Theoptimalimmobilizedbiocatalystwasproducedafter24 hofincubationunderalkalineconditionsandlongerincubationtimesresultedinalossofstability.Theoptimalimmobilizedbiocatalystwas60-and50-foldmorestableatpH5.5andpH7at50°Cthanthesolubleenzyme,respectively.ActivityandstabilityatpH5.5wereenhancedwhentheoptimalimmobilizedbiocatalystwasmodifiedbychemicalaminationoftheenzymesurface.Thechemicalaminationoftheimmobilizedenzymesurfacewas5-foldmorestableatpH5.5and50°Ccomparedwiththeunmodifiedimmobilizedbiocatalyst.Thebestimmobilizedbiocatalysts(containing100UI/gofsupport)wereevaluatedinthebeechwoodxylanhydrolysisreactionat50°CandpH5.5.80%ofthereducingsugarswerereleasedafter6 hofhydrolysiswiththeaminatedbiocatalyst.Xylanhydrolysisreactionwiththeaminatedbiocatalystwas80%fasterthanwiththenon-aminatedone.Thefinalcompositionofthexylooligosaccharides(XOS)obtainedwasidentifiedandquantifiedbyHPAEC-PADwhichshoweditwascomposedof90%ofxylobioseand5%ofxylotrioseandxylose.Theaminatedimmobilized-stabilizedbiocatalystwasusedforfourcyclesofhydrolysiswithnolossofcatalyticactivity,resultinginhighlyactiveandstablederivativesuitableforindustrialprocesses.
Improvementofthecatalyticcharacteristicsofasalt-tolerantGH10xylanasefromStreptomyceRocheiL10904.
Li,Q.,Sun,B.,Li,X.,Xiong,K.,Xu,Y.,Yang,R.,Hou,J.&Teng,C.(2017).InternationalJournalofBiologicalMacromolecules,InPress.
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AGH10xylanaseSrxyn10fromStreptomycerocheiL10904,anditstruncatedderivative,Srxyn10M,wereinvestigated.Bothdisplayedgreatsalt-tolerantability,retainingmorethan95%and91%activityafterincubationat37°Cfor1hin3.0Mand5.0MNaCl,respectively.Theyexhibitedaspecialhydrolyticpropertyofformingxylobioseasthemajorproductandproducedfewerxylosecompoundswhencombinedwithareportedxylanasewhiledigestingcorncobxylans.Themutant,Srxyn10M,wasconstructedfromSrxyn10bydeletingtheC-terminalcarbohydrate-bindingmodule.Itpossesseda3.26-foldhigherspecificactivityonbeechwoodxylanthanSrxyn10.Moreover,Srxyn10MshowedgreatersubstrateaffinityandcatalyticefficiencythanSrxyn10whenbeechwoodxylan,birchwoodxylan,andoat-speltxylanwereusedassubstrates.Thethermostabilitywasalsogreatlyimproved.Therefore,theapplicationpotentialwasmarkedlyenhancedbytheimprovementoftheseproperties.
Influenceofviscosityonthegrowthofhumangutmicrobiota.
Tamargo,A.,Cueva,C.,Álvarez,M.D.,Herranz,B.,Bartolomé,B.,Moreno-Arribas,M.V.&Laguna,L.(2017).FoodHydrocolloids,InPress.
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Numerousstudiessupportthebeneficialeffectsofdietaryfibre.Itiswellknownthatfibreincreasesviscosityatintestinallevel.Therefore,theeffectsoffibreongutmicrobiotacouldbeduenotonlybyitsintestinalbacteriafermentationbutalsototheincreaseinviscositybyitself.Theaimofthisstudywastoevaluatetheeffectofviscosityonthegrowthofgutmicrobiotaatphysiologicalconditions.Forthispurpose,fourcompartmentsfromagastrointestinalsimulator(simgi®)werefilledwithGutNutrientMedium(GNM)plusdifferentagarconcentrations(0,0.30,0.45and0.60%),inoculatedwithfaecalmicrobiota,andincubated48hunderanaerobicconditions.Sampleswerecollectedatthreetimepoints(0,24hand48h)forrepresentativeintestinalbacterialenumerationandrheologicalcharacterization.IncubationofGNMgelswithfaecalmicrobiotachangedthemediumviscosityovertime,evenwithconstantconditions(temperatureandpH).Insuchwaythat,inabsenceofagar(lowviscosity),viscosityslightlyincreasedovertime;however,inviscousmediums,viscositydecreasedovertime.Inrelationtothegrowthofgutmicrobiota,resultsshowedthatviscosityfavouredthegrowthoftotalanaerobesandClostridiumspp.;incontrast,totalnumberofaerobesandmembersofthegenusEnterococcuscorrelatednegativelywithviscosityincrement.Inconclusion,changesinintestinalviscosityseemtoselectivelymodifymicrobiotacomposition.Thisisapioneerworktounderstandtheeffectoffoodviscosityinthegastrointestinalsystem,showingthatviscosityisanimportantfactoritselftoconditionthegrowthofdifferentbacteria’sgroups.