Megazyme/Arabinoxylan (Wheat Flour; for Reducing Sugar Assays)/PWAXYRS/10 grams

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.

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.