alpha-L-fucosidefucohydrolase,alpha-L-fucosidase,alpha-(1-6)fucosidase

Removesnon-reducing,terminalbranchedfucosewhenlinkedalpha-(1-6)tothecoreGlcNAcofaGlcNAc-GlcNAcdisaccharidestructurethathasbeenlabeledwithafluorescentmolecule.Todate,thefluorescentlabelsANTS,ANDSAand2-AA(LT-KAA-A2)havebeendemonstratedtofunctionwiththefucosidase.

Thereisnocleavageofunlabeledoligosaccharides.Theoneexceptionisthataterminalunbranchedα(1-3)orα(1-4)fucoseiscleavedintheabsenceofanyreportermolecule.

α(1-6)Fucosidaseisusefulfordeterminingcorefucosylation.

SourcerecombinantElizabethkingiameningosepticumexpressedinE.Coli

EC3.2.1.51

Contents
Alpha-(1-6)fucosidasein20mMTris-HCl,25mMNaCl,(pH7.5).

Includedwith20µLand60µLpacksizes:
200µl5xReactionBuffer5.0(250mMsodiumphosphate,pH5.0)

SpecificActivity>1.5U/mg
Activity>1.0U/ml

Molecularweight~55,000daltons

FormulationTheenzymeisprovidedasasterile-filteredsolutionin20mMTris-HCl,25mMNaClpH7.5.

Suggestedusage
1.Addupto1nmoleoflabeledoligosaccharidetoatube.
2.Addde-ionizedwatertoatotalof15µl.
3.Add4µlof5xReactionBuffer5.0.
4.Add1µlofAlpha-(1-6)-Fucosidase.
5.Incubatefor3hoursat37˚C.

Specificityα(1-6)linkedcorefucosewhencovalentlyattachedtoareportermoleculeatthereducingterminalGlcNAc.Theoneexceptionisthataterminalunbranchedα(1-3))orα(1-4)fucoseiscleavedintheabsenceofanyreportermolecule.Reportermoleculesknowntosupportcleavageareamino-napthalenedisulfonicandtri-sulfonicacidsand2-aminobenzoicacid(2AA).However,2-aminobenzamide(2-AB)
willnotsupportcleavage.Shorteroligosaccharidessuchastrimannosylchitobiosearemorecompletelydigestedthanlongerderivativeswhichmayrequirelongerincubationtimes.

SpecificActivityAssayOneunitofFucosidaseactivityisdefinedastheamountofenzymerequiredtocleave1µmoleoffucosefrom4-methylumbelliferyl-alpha-L-fucosidein1minuteat37˚CandpH5.0.

StorageStoreenzymeat4˚C.

PurityEachlotofα(1-6)Fucosidaseistestedforcontaminatingproteaseasfollows:10μgofdenaturedBSAisincubatedfor24hourswith2μLofenzyme.AnalysisoftheBSAbandafterSDS-PAGEshouldshownoevidenceofdegradation.

Theproductionhoststrainhasbeenextensivelytestedanddoesnotproduceanydetectableglycosidases.

StABIlityStableatleast12monthswhenstoredproperly.Severaldaysexposuretoambienttemperatureswillnotreduceactivity.

Alpha-(1-6)FucosidaseReferences

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