QA-Bio/O-Glycosidase/E-G001-200

Specifictity
O-glycosidaseleavesonlyunsubstitutedGal-Beta(1-3)GalNAc-alphadisaccharidesattachedtotheserineorthreonineresiduesofglycoproteinsorglycopeptides.

Substitutionssuchassialicacid,galactose,fucoseorN-acetylglucosaminemustfirstberemovedwiththeappropriateexoglycosidasepriortotreatmentwithEndo-O-Glycosidase.Atminimum,asialadasesuchasSialadaseAu(alpha-2-3,6,8,9),partnumberE-S001,isalmostalwaysrequiredtoremovesialicacids

SourceRecombinantStreptococcuspneumoniaeinE.Coli

EC3.2.1.97

CAS9032-92-2

Contents
60µlaliquotofenzyme(75mU)in50mMsodiumphosphate,pH7.5.
Includedwith20µLand60µLpacksizes:
5xReactionBuffer5.0(250mMsodiumphosphate,pH5.0)

SpecificActivity>12U/mg

Activity>1.25U/ml

Molecularweight180,000daltons

pHrange5-7,optimum5.0

Suggestedusage
1.Addupto100µgofglycoproteintoatube.
2.Addde-ionizedwatertoatotalof13µl.
3.Add4µlof5xReactionBuffer5.0.
4.Add1µlofSialadaseAu(alpha2-,3,6,8,9),partnumberE-S001.
5.Add2µlofO-Glycosidase.
6.Incubatefor1hourat37˚C.

SpecificActivityDefinedastheamountofenzymerequiredtoproduce1µmoleofp-nitrophenol(pNP)in1minuteat37˚C,pH5.0fromp-nitrophenyl-2-acetamido-2-deoxy-3-O-(Beta-D-galactopyranosyl)-alpha-D-galactopyranoside.

StorageStoreenzymeat4˚C.

StABIlityStableatleast12monthswhenstoredproperly.Severaldaysexposuretoambienttemperaturewillnotreduceactivity.Activeforatlease5daysunderreactionconditions.

PurityO-Glycosidaseistestedforcontaminatingproteaseasfollows;10μgofdenaturedBSAisincubatedfor24hoursat37°Cwith2μLofenzyme.SDS-PAGEanalysisofthetreatedBSAshowsnoevidenceofdegradation.

O-GlycosidaseReferences

Bhavanandan,V.P.,J.UmemotoandE.A.Davidson.Characterizationofanendo-alpha-N-acetylgalactosaminidasefromDiplococcuspneumoniae.BiochemBiophys70:738-745(1976).

Fan,J.Q.,K.Yamamoto,H.KumagaiandT.Tochikura.Inductionandefficientpurificationofendo-alpha-N-acetyl-D-galactosaminidasefromAlcaligenessp.AgricBiolChem54:233-234(1990).

Glasgow,LR.,J.C.PaulsonandR.L.Hill.SystematicpurificationoffiveglycosidasesfromStreptococcuspneumoniae.JBiolChem252:8615-8623(1977).

Iwase,H.andK.Hotta.ReleaseofO-linkedglycoproteinglycansbyendo-alpha-N-acetyl-D-galactosaminidase.MethMolBiol14:151-159(1993).

Unemoto,J.,V.P.BhavanandanandE.A.Davidson.Purificationandpropertiesofanendo-alpha-N-acetyl-D-galactosaminidasefromDiplococcuspneumoniae.JBiolChem252:8609-8614(1977).