α-D-galactosidegalactohydrolase,melibiase.

AlphaGalactosidasefromE.colicleavesα(1-3)-andα(1-6)-linked,non-reducingterminalgalactosefromcomplexcarbohydratesandglycoproteins.Thereisnoactivityonα(1-4)linkedgalactose.Itisparticularlyefficientforremovingα-linkedgalactoseunderconditionswherethepHmustbeneutralorabove,forexample,withlivingcells.

ForcleavingBetaGalactosidasewerecommendß-(1-4)Galactosidase(E-BG07),recombinantfromStreptococcuspneumoniaeorß-(1,3,4,6)Galactosidase(E-BG02),purifiedfrombovinetestes.

SourcerecombinantfromE.coliinE.coli

EC3.2.1.22

Contents
AlphaGalactosidaseenzymein50mMsodiumphosphate,pH7.5

Includedwith20µLand60µLpacksizes:
5xReactionBuffer–250mMsodiumphosphate,pH6.5

SpecificActivity>30U/mg
Activity80U/ml

Molecularweight~80,000daltons

SuggestedUsage
1.Addupto100μgofglycoproteinor1nmolofoligosaccharidetotube.
2.Addwaterto13μL
3.Add4μL5XReactionBuffer.
4.Add2μLα(1-3,6)galactosidase.
5.Incubateat37°Cfor1hour.

NOTE:Longerincubationsarenecessaryiffucoseispresentonthepenultimatesugar.

SpecifictityCleavesAlpha-(1-3)-andAlpha-(1-6)-linked,non-reducingterminalgalactosefromcomplexcarbohydratesandglycoproteins.

SpecificActivityOneunitofAlpha-(1-3,6)-Galactosidaseisdefinedastheamountofenzymerequiredtoproduce1µmoleofp-nitrophenol(pNP)in1minuteat25°CpH6.5fromp-nitrophenyl-alpha-D-galactopyranoside.

StorageStoreenzymeat4˚C.

PurityEachlotofAlphaGalactosidaseistestedforcontaminatingproteaseasfollows;10μgofdenaturedBSAisincubatedfor24hourswith2μLofenzyme.SDS-PAGEanalysisofthetreatedBSAshowsnoevidenceofdegradation.

Theproductionhoststrainhasbeenextensivelytestedanddoesnotproduceanydetectableglycosidases.

StABIlityStableatleast12monthswhenstoredproperly.Severaldaysexposuretoambienttemperatureswillnotreduceactivity.

AlphaGalactosidaseReferences

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

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

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

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