TheXylX6assayreagentforthemeasurementofendo-xylanase(endo-1,4-β-xylanase)containstwocomponents;1)4,6-O-(3-Ketobutylidene)-4-nitrophenyl-β-D-45-glucosyl-xylopentaosideand2)β-xylosidase.Theketoneblockinggrouppreventsanyhydrolyticactionbytheβ-xylosidaseorotherexo-actingglycosidasesontheXylX6substrate.Incubationwithanendo-xylanasegeneratesanon-blockedcolourimetricoligosaccharidethatisrapidlyhydrolysedbytheancillaryβ-xylosidase.Therateofformationof4-nitrophenolisthereforedirectlyrelatedtothehydrolysisofXylX6bytheendo-xylanase.ThereactionisterminatedandthephenolatecolourisdevelopedonadditionofTrisbuffersolution(pH=10.0).
NotethatstandardcurvesrelatingtheabsorbanceobtainedusingtheXylX6assaytoendo-xylanaseactivityonthenativesubstrates,wheatarABInoxylanandbeechwoodxylan,areprovidedintheSupportingInformationfileundertheDocumentationtab.
Novelsubstratesfortheautomatedandmanualassayofendo-1,4-β-xylanase.
AComparisonofPolysaccharideSubstratesandReducingSugarMethodsfortheMeasurementofendo-1,4-β-Xylanase
Colourimetricmethodforthedeterminationof endo-1,4-β-xylanase inany
sampletypeincludingcrudeenzymeextracts,fermentationbrothsor
commercialenzymepreparations
Principle:
(endo-1,4-β-xylanase)
(1) 3-Ketobutylidene-GX5-β-PNP+H2O→Blocked-GXY +X(5-Y)-β-PNP
(β-glucosidase)
(2)X(5-Y)-β-PNP+H2O→D-xylose+PNP
(alkalinesolution)
(3)PNP→phenolateion(yellowcolour)
Note: PNP=4-nitrophenol
Kitsize:
K-XylX6-1V100assays(manual)/200(auto-analyser)
or
K-XylX6-2V200assays(manual)/400(auto-analyser)Method: Spectrophotometricat400/405 nm
Totalassaytime: 10min
Detectionlimit: 5.3x10-4U/mL
Applicationexamples:
Fermentationbroths,industrialenzymepreparations,animalfeed,biofuelsresearch,barley
maltanalysis
Methodrecognition: Novelmethod
Advantages
- Verycosteffective
- Allreagentsstablefor>4years
- Completelyspecificforendo-1,4-β-xylanase
- Generallyapplicableandhighlysensitive
- Simpleformat.Wellsuitedtoautomation
- Excellentreproducibility
- Standardincluded