β-D-galactosidegalactohydrolase,Exo-(1-4)-beta-D-galactanase,
Lactase

BetaGalactosidasefromStreptococcuspneumoniaereleasesonlyβ(1-4)-linked,non-reducingterminalgalactosefromcomplexcarbohydratesandglycoproteins.β(1-4)galactoseisbyfarthemostcommonlinkagefoundinN-linkedoligosaccharides.Forothergalactosidaselinkages,ß(1-3,4,6)-Galactosidase(E-BG02)fromBovinetestesisrecommended.Theenzymeisasactiveontetraantennaryoligosaccharidesasondisaccharidescontainingβ(1-4)-linkedgalactose.FucoselinkedtothepenultimateN-acetylglucosaminewillblockcleavageofthegalactose.Upto100υgofasialofetuincanbecompletelyβ(1-4)-degalactosylatedinlessthan1hourusing3mUofenzyme.

SourcerecombinantStreptococcuspneumoniae<>

EC3.2.1.23<>

Contents
ß-(1-4)Galactosidasein20mMTris-HCl,25mMNaCl(pH7.5).

Includedwith20µLand60µLpacksizes:
5xReactionBuffer6.0(250mMsodiumphosphate,pH6.0).

SpecificActivity>6U/mg
Activity>3U/ml

Molecularweight~250,000daltons

SuggestedUsage
1.Addupto100μgofasialoglycoproteinor1nmolofoligosaccharidetotube.
2.Addwaterto14μL
3.Add4μL5XReactionBuffer
4.Add2μLβ(1-4)Galactosidase
5.Incubateat37°Cfor1hour.

Forglycoproteins,cleavagemaybemonitoredbySDS-PAGEifthesizedifferentialbetweennativeandde-galactosylatedproteinissufficientfordetection.

Note:Theoptimumforcleavageofoligosaccharidesis~pH6.0.

SpecifictityNon-reducingterminalBeta-(1-4)-galactose.Numberofantennaedoesnotaffectcleavagerate.FucoselinkedtothepenultimateN-acetylglucosaminewillblockcleavageofthegalactose.

SpecificActivityAssayOneunitofBetaGalactosidaseisdefinedastheamountofenzymerequiredtoproduce1µmoleofp-nitrophenol(pNP)in1minuteat37˚CpH5fromp-nitrophenyl-beta-D-galactopyranoside.

StorageStoreenzymeat4˚C.

PurityEachlotofBetaGalactosidaseistestedforcontaminatingproteaseasfollows;10μgofdenaturedBSAisincubatedfor24hourswith2μLofenzyme.SDS-PAGEanalysisofthetreatedBSAshowsnoevidenceofdegradation.

Theproductionhoststrainhasbeenextensivelytestedanddoesnotproduceanydetectableglycosidases.

StABIlityStableatleast12monthswhenstoredproperly.Severaldaysexposuretoambienttemperatureswillnotreduceactivity.

BetaGalactosidaseReferences

1.Glasgow,LR.,J.C.PaulsonandR.L.Hill.SystematicpurificationoffiveglycosidasesfromStreptococcuspneumonia.J.BiolChem252:8615-8623(1977).

2.Kobata,A.Useofendo-andexoglycosidasesforstructuralstudiesofglycoconjugates.AnalBiochem100:1-14(1979).

3.Prime,S.J.Dearnley,A.M.Venton,R.B.ParekhandC.J.Edge.Oligosaccharidesequencingbasedonexo-andendoglycosidasedigestionandliquidchromatographicanalysisoftheproducts.JChromatogrA720:263-274(1996)

4.Dwek,R.A.,C.J.Edge,D.J.Harvey,M.R.WormaldandR.B.Parekh.Analysisofglycoprotein-associatedoligosaccharides.AnnRevBiochem62:65-100.