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Megazyme/Cellulase Assay Kit (CellG3 Method)/K-CellG3/180 / 360 assays per kit / 720 (auto-analyser)
TheCellG3assayreagentforthemeasurementofendo-cellulase(endo-1,4-β-glucanase)containstwocomponents;
1)4,6-O-benzylidene-2-chloro-4-nitrophenyl-β-D-cellotrioside(BCNPG3)and2)Thermostableβ-glucosidase.Thebenzylideneblockinggrouppreventsanyhydrolyticactionbytheβ-glucosidaseonBCNPG3. Incubationwithanendo-cellulasegeneratesanon-blockedcolourimetricoligosaccharidethatisrapidlyhydrolysedbytheancillary β-glucosidase. Therateofformationof2-chloro-4-nitrophenolisthereforedirectlyrelatedtothehydrolysisofBCNPG3bytheendo-cellulase. ThereactionisterminatedandthephenolatecolourisdevelopedonadditionofTrisbuffersolution(pH9.0).
Pleasenotethatanewassaykit(K-CellG5)isnowavailableforthemeasurementofendo-cellulase. TheCellG5reagentcontainsacellopentaosecoreandexhibitsvastlyimprovedsensitivityforsomecellulases. Inaddition,theexchangeofthebenzylideneblockinggroupinCellG3for3-keto-butylideneinCellG5improvesthesubstrate’swatersolubilitysignificantly,allowingforareductionintheconcentrationofDMSOrequiredintheassay. AsDMSOisknowntoinhibitcertaincellulases,thisisanotherbenefitinusingCellG5. MegazymenowrecommendstheuseofK-CellG5forallassaysforthemeasurementofendo-cellulase.
Suitableforauto/analyserformats.
Novelsubstratesforthemeasurementofendo-1,4-β-glucanase(endo-cellulase).
Quantitativefluorometricassayforthemeasurementofendo-1,4-β-glucanase.
Anovelautomatableenzyme-coupledcolorimetricassayforendo-1,4-β-glucanase(cellulase).
Colourimetricmethodforthedeterminationofendo-1,4-β-
glucanase(cellulase)inenzymepreparationsandfermentation
products
Principle:
(endo-1,4-β-glucanase)
(1)Benzylidene-G3-β-CNP+H2O→Blocked-GX+G(3-X)-β-CNP
(thermostableβ-glucosidase)
(2)G(3-X)-β-CNP+H2O→D-glucose+CNP
(alkalinesolution)
(3)CNP→phenolateion(yellowcolour)
Note:CNP=2-Chloro-4-nitrophenol
Kitsize: 180/360assays/720(auto-analyser)
Method: Spectrophotometricat400nm
Totalassaytime: ~20min
Detectionlimit: 0.05U/mL
Applicationexamples:
Fermentationbroths,industrialenzymepreparationsandbiofuelsresearch
Methodrecognition: Novelmethod
Note: Theanalogousfluorimetricreagent(R-CELLFLR)
isalsoavailablewith10-foldgreatersensitivity.
Advantages
- Verycosteffective
- Allreagentsstablefor>2yearsafterpreparation
- Completelyspecificforcellulase(endo-1,4-glucanase).Thesubstrateisnothydrolysedbyβ-glucosidase,cellbiohyrolaseoranyotherenzymestested
- KineticassayspossIBLeduetosignificantphenolateionpresence(andUVabsorbance)atpH5-6
- Simpleformat.Wellsuitedtoautomation
- Standardincluded