产品资料

Megazyme/Cellulase Assay Kit (CellG5 Method)/K-CellG5-2V/60 / 120 assays (manual) / 240 (auto-analyser)

TheCellG5assayreagentforthemeasurementofendo-cellulase(endo-1,4-β-glucanase)containstwocomponents;
1)4,6-O-(3-Ketobutylidene)-4-nitrophenyl-β-D-cellopentaoside(BPNPG5)and2)Thermostableβ-glucosidase.Theketoneblockinggrouppreventsanyhydrolyticactionbytheβ-glucosidaseonBPNPG5. Incubationwithanendo-cellulasegeneratesanon-blockedcolourimetricoligosaccharidethatisrapidlyhydrolysedbytheancillaryβ-glucosidase.Therateofformationof4-nitrophenolisthereforedirectlyrelatedtothehydrolysisofBPNPG5bytheendo-cellulase.ThereactionisterminatedandthephenolatecolourisdevelopedonadditionofTrisbuffersolution(pH9.0).
TheCellG5assayrepresentsahugestepforwardinthemethodologyforthemeasurementofcellulasethattrADItionallyreliedonsubstratessuchasCM-cellulose,Avicel,cellooligosaccharides,filterpaperordyedpolysaccharidesincludingCMCCongoredorcelluloseazure.

Novelsubstratesforthemeasurementofendo-1,4-β-glucanase(endo-cellulase).

McCleary,B.V.,Mangan,D.,Daly,R.,Fort,S.,Ivory,R.&McCormack,N.(2014).CarbohydrateResearch,385,9-17.

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Aspecificandsensitivesubstratefortheassayofendo-1,4-β-glucanase(cellulase)hasbeenprepared.Thesubstratemixturecomprisesbenzylideneend-blocked2-chloro-4-nitrophenyl-β-cellotrioside(BzCNPG3)inthepresenceofthermostableβ-glucosidase.Hydrolysisbyexo-actingenzymessuchasβ-glucosidaseandexo-β-glucanaseispreventedbythepresenceofthebenzylidenegrouponthenon-reducingendD-glucosylresidue.Onhydrolysisbycellulase,the2-chloro-4-nitrophenyl-β-glycosideisimmediatelyhydrolysedto2-chloro-4-nitrophenolandfreeD-glucosebytheβ-glucosidaseinthesubstratemixture.Thereactionisterminatedandcolourdevelopedbytheadditionofaweakalkalinesolution.Theassayprocedureissimpletouse,specific,accurate,robustandreadilyadaptedtoautomation.Thisprocedureshouldfindwidespreadapplicationsinbiomassenzymologyandinthespecificassayofendo-1,4-β-glucanaseingeneral.

Quantitativefluorometricassayforthemeasurementofendo-1,4-β-glucanase.

Mangan,D.,McCleary,B.V.,Liadova,A.,Ivory,R.&McCormack,N.(2014).CarbohydrateResearch,395,47-51.

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Thereisagrowingdemandforresearchtoolstoaidthescientificcommunityinthesearchforimprovedcellulaseenzymesforthebiofuelindustry.Inthiswork,wedescribeanovelfluorometricassayforcellulase(endo-1,4-β-glucanase)whichisbasedontheuseof4,6-O-benzylidene-4-methylumbelliferyl-β-cellotrioside(BzMUG3)inthepresenceofanancillaryβ-glucosidase.Thisassaycanbeusedquantitativelyoverareasonablelinearrange,orqualitativelyasasolutionscreeningtoolwhichmayfindextensiveuseintheareaofmetagenomics.

Anovelautomatableenzyme-coupledcolorimetricassayforendo-1,4-β-glucanase(cellulase).

Mangan,D.,Cornaggia,C.,McKie,V.,Kargelis.T.&V.McCleary,B.V.(2016).AnalyticalandBioanalyticalChemistry,408(15),4159-4168.

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endo-1,4-β-Glucanase(endo-cellulase,EC3.2.1.4)isoneofthemostwidelyusedenzymesinindustry.Despiteitsimportance,improvedmethodsfortherapid,selective,quantitativeassayofthisenzymehavebeenslowtoemerge.In2014,anovelenzyme-coupledassaythataddressedmanyofthelimitationsoftheexistingassaymethodologywasreported.Thisinvolvedtheuseofabifunctionalsubstratechemicallyderivedfromcellotriose.Reportedhereinisamuchimprovedversionofthisassayemployinganovelsubstrate,namely4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-D-cellopentaoside.

CompletegenomesequenceofBacillussp.275,producingextracellularcellulolytic,xylanolyticandligninolyticenzymes.

Gong,G.,Kim,S.,Lee,S.M.,Woo,H.M.,Park,T.H.,&Um,Y.(2017).JournalofBiotechnology,254, 59-62.

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Technologiesfordegradationofthreemajorcomponentsoflignocellulose(e.g.cellulose,hemicelluloseandlignin)areneededtoefficientlyutilizelignocellulose.Here,wereportBacillussp.275isolatedfromamudflatexhibitingvariouslignocellulolyticactivitiesincludingcellulase,xylanase,laccaseandperoxidaseinthecellculturesupernatant.ThecompletegenomeofBacillussp.275straincontains3832proteincordingsequencesandanaverageG+Ccontentof46.32%ononechromosome(4045,581bp)andoneplasmid(6389bp).Thegenesencodingenzymesrelatedtothedegradationofcellulose,xylanandligninweredetectedintheBacillussp.275genome.Inaddition,thegenesencodingglucosidasesthathydrolyzestarch,mannan,galactosideandarABInanwerealsofoundinthegenome,implyingthatBacillussp.275haspotentiallyawiderangeofusesinthedegradationofpolysaccharideinlignocellulosicbiomasses.

Colourimetricmethodforthedeterminationof
endo-1,4-β-glucanase(cellulase)inenzymepreparationsandfermentationproducts

Principle:
                    (endo-1,4-β-glucanase)
(1)3-Ketobutylidene-G₅-β-PNP+H₂O→Blocked-G_X +G_(5-X)-β-PNP

        (thermostableβ-glucosidase)
(2)G_(5-X)-β-PNP+H₂O→D-glucose+PNP

   (alkalinesolution)
(3)PNP→phenolateion(yellowcolour)
Note: PNP=4-nitrophenol

Kitsize:
K-CellG5-4V120/240assays(manual)/480(auto-analyser)
or
K-CellG5-2V60/120assays(manual)/240(auto-analyser)

Method:                     Spectrophotometricat400nm
Totalassaytime:         10min
Detectionlimit:              3.5x10^-4U/mL
Applicationexamples:
Fermentationbroths,industrialenzymepreparationsandbiofuelsresearch
Methodrecognition:   Novelmethod

Advantages

Verycosteffective
Allreagentsstablefor>4years
Completelyspecificforcellulase(endo-1,4-glucanase)
Generallyapplicableandhighlysensitive
Simpleformat.Wellsuitedtoautomation
Standardincluded

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Megazyme/Cellulase Assay Kit (CellG5 Method)/K-CellG5-2V/60 / 120 assays (manual) / 240 (auto-analyser)

Novelsubstratesforthemeasurementofendo-1,4-β-glucanase(endo-cellulase).

Quantitativefluorometricassayforthemeasurementofendo-1,4-β-glucanase.

Anovelautomatableenzyme-coupledcolorimetricassayforendo-1,4-β-glucanase(cellulase).

CompletegenomesequenceofBacillussp.275,producingextracellularcellulolytic,xylanolyticandligninolyticenzymes.