Endo-Beta-Galactosidasecleavesinternalβ(1-4)galactoselinkagesinunbranched,repeatingpoly-N-acetyllactosaminestructures.Sulfatedstructuressuchaskeratansulfatearealsocleaved.Branchingand/orfucosylationofthesubstratemaydecreaseoreliminatecleavage.
Endo-Beta-Galactosidaseisusefulforidentifyingandremovingpoly-N-acetyllactosaminestructuresonmanyBIOLOGicallyimportantglycoconjugates.
SourceRecombinantfromBacteroidesfragilis
EC3.2.1.103
CAS55072-01-0
SpecificityCleavesinternalß(1-4)galactoselinkagesinunbranched,repeatingpoly-N-acetyllactosamine[GlcNAc-ß(1-3)Gal-ß(1-4)]nstructuresarethepreferredsubstrate.Sulfatedstructuressuchaskeratansulfatearealsocleaved.Branchingand/orfucosylationofthesubstratemaydecreaseoreliminatecleavage.SulfationofC-6ongalactosewillblockcleavage.Oligosaccharidesoftheneo-lactogrouparecleavedatgreatlyeducedratesdependingonthedeviationfromthepreferredsubstrate.Forexample,Gal-ß(1-3)GlcNAc-ß(1-3)Gal-ß(1-4)Glciscleavedat5×10-5therateofkeratansulfate(seeref.4).SpecificityissimilartotheEscherichiafreundiienzyme.SpecificityissimilartotheEscherichiafreundiienzymeexceptthatitislimitedtocleavingN-acetyllactosamineextensionsontetraantennarystructuresoferythropoietin(seeref5).
Contents
60µlaliquotofenzyme(0.9U)in20mMtris-HCl,pH7.5
1vialreactionbuffer-250mMSodiumphosphate,pH5.8
SpecificActivity>140U/mg
Activity>14U/ml
Molecularweight~32,000daltons
OptimumpH5.8
Suggestedusage
Forglycoproteins:
1.Addupto100µgofglycoproteintoatube.
2.Add4ul5Xbufferandwaterto19µl.
3.Add1µlenzyme.
4.Incubateat37˚Cfor2hrs.
Procedureforoligosaccharides:
Sameasaboveexceptincubatefromseveralhourstoseveral
daysdependingonthesubstrate.Addbovineserumalbumento2mg/mltostABIlizetheproteinduringextendedincubations.
SpecificActivity
OneunitofEndo-Beta-Galactosidaseisdefinedastheamountthatwillliberateoneµmoleofreducingsugarperminuteat37˚CandpH5.8frombovinecornealkeratansulfate.
StabilityStableatleast24monthswhenstoredproperly.Severaldaysexposuretoambienttemperaturewillnotreduceactivity.Activeforatleast5daysunderreactionconditions.
PurityEndo-Beta-Galactosidaseistestedforcontaminatingproteaseasfollows:10μgofdenaturedBSAisincubatedfor24hoursat37°Cwith2μLofenzyme.SDS-PAGEanalysisofthetreatedBSAshowsnoevidenceofdegradation.
TheproductionstrainofE.colihasbeenextensivelytestedanddoesnotproduceanydetectableglycosidases.
Endo-Beta-GalactosidaseReferences
1.Scudder,P.,Uemura,K.,Doby,J.,Fukuda,M.N.&Feizi,T.(1983)Isolationandcharacterizationofanendo-b-galactosidasefromBacteroidesfragilisBiochem.J.213,485-494.
2.Scudder,P.,Hanfland,Pl,Uemura,K.&Feizi,T.(1984)Endo-b-galactosidasesofBacteroidesfragilisandEscherichiafreundiihydrolyzelinearbutnotbranchedoligosaccharidedomainsofglycolipidsoftheneolactoseries.J.Biol.Chem.259,6586-6592.
3.Scudder,P.Tang,P.W.,Hounsell,E.F.,Lawson,A.M.,Mehmet,H.&Feizi,T.(1986)Isolationandcharacterizationofsulfatedoligosaccharidesreleasedfrombovinecornealkeratansulphatebytheactionofendo-b-galactosidase.Eur.J.Biochem.157,365-373.
4.Murata,T.,Hattori,T.Amarume,S.Koicki,A.&Usui,T.(2003)https://www.ncbi.nlm.nih.gov/pubmed/?term=Murata%2C+T.%2C+Hattori%2C+T.+Amarume%2C+S.+Koicki%2C+A.+%26amp%3B+Usui%2C+T.+(2003)Eur.J.Biochem270,3709-3719.
5.Hokke,C.H.,Bergwerff,A.A.,VanDedem,D.W.,Kamerling,J.P,andVliegenthart,J.F.(1995)StructuralanalysisoftheN-andO-linkedcarbohydratechainsofrecombinanthumanerythropoietinexpressedinChinesehamsteroveraycells.SialylationpattersandbranchlocationofdimericN-acetyllactosamineunits.Eur.J.Biochem.228,981-1008.
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