Megazyme/Xylan (Beechwood; purified)/PXYLNBE10G/10 grams

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.

Themostcommonlyusedmethodforthemeasurementofthelevelofendo-xylanaseincommercialenzymepreparationsisthe3,5-dinitrosalicylicacid(DNS)reducingsugarmethodwithbirchwoodxylanassubstrate.ItiswellknownthatwiththeDNSmethod,muchhigherenzymeactivityvaluesareobtainedthanwiththeNelson-Somogyi(NS)reducingsugarmethod.Inthispaper,wehavecomparedtheDNSandNSreducingsugarassaysusingarangeofxylan-typesubstratesandaccuratelycomparedthemolarresponsefactorsforxyloseandarangeofxylo-oligosaccharides.Purifiedbeechwoodxylanorwheatarabinoxylanisshowntobeasuitablereplacementforbirchwoodxylanwhichisnolongercommerciallyavailable,anditisclearlydemonstratedthattheDNSmethodgrosslyoverestimatesendo-xylanaseactivity.UnliketheDNSassay,theNSassaygavetheequivalentcolourresponsewithequimolaramountsofxylose,xylobiose,xylotrioseandxylotetraosedemonstratingthatitaccuratelymeasuresthequantityofglycosidicbondscleavedbytheendo-xylanase.TheauthorsstronglyrecommendcessationoftheuseoftheDNSassayformeasurementofendo-xylanaseduetothefactthatthevaluesobtainedaregrosslyoverestimatedduetosecondaryreactionsincolourdevelopment.

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-mannanaseactivitywassignificantlyenhancedinthepresenceofMn⁺,Cu²⁺,Zn²⁺,Ca²⁺,Mg²⁺,andAl³⁺andstronglyinhibitedbyBa²⁺,andPb²⁺.K_mandV_maxvaluesforlocustbeangumwere7.14 mg/mLand107.5 μmol/min/mLversus1.749 mg/mLand33.45 µmol/min/mLforKonjacglucomannan,respectively.Therefore,β-mannanasepurifiedbythisworkshowsstabilityathightemperaturesandinweaklyacidicorneutralenvironments.Basedonsuchdata,theβ-mannanasewillhavepotentialapplicationsasadietarysupplementintreatmentofinflammatoryprocesses.

ThecommercialenzymeDepol^TM333MDP(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.

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.

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.