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HighpuritydyedandcrosslinkedLimit-Dextrizymetabletsforthemeasurementofenzymeactivity,forresearch,biochemicalenzymeassaysandinvitrodiagnosticanalysis.
Fortheassayoflimit-dextrinaseandpullulanase.ContainingAZCL-Pullulan.
Newupdatedassayprotocolemployingamyloglucosidaseintosamplepreparation.
Colourimetricandfluorimetricsubstratesfortheassayoflimitdextrinase.
Mangan,D.,McCleary,B.V.,Cornaggia,C.,Ivory,R.,Rooney,E.&McKie,V.(2015).JournalofCerealScience,62,50-57.
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Themeasurementoflimit-dextrinase(LD)(EC3.2.1.142)ingrainsamplessuchasbarley,wheatorricecanbeproblematicforanumberofreasons.TheintrinsicLDactivityinthesesamplesisextremelylowandtheyoftencontainalimit-dextrinaseinhibitorand/orhighlevelsofreducingsugars.LDalsoexhibitstransglycosylationactivitythatcancomplicatethemeasurementofitshydrolyticactivity.AminormodificationtotheindustrialstandardLimit-Dextrizymetablettestissuggestedheretoovercomethistransglycosylationissue.Inaddition,twonewsubstratesaredescribedthatcanbeadoptedforuseinanauto-analyserformat.4,6-O-benzylidene-2-chloro-4-nitrophenyl-β-63-α-D-maltotriosyl-maltotrioside(BzCNPG3G3,Hexachrom)isnotsusceptIBLetotransglycosylationandservesamiablyasaroutinequantitativeassaytoolwiththepotentialtorunkineticassaysduetothelowpKa(∼5.5)ofthechromogenicmoietywhile4,6-O-benzylidene-4-methylumbelliferyl-β-63-α-D-maltotriosyl-maltotrioside(BzMUG3G3,Hexafluor)wasfoundtobesusceptibletotransglycosylationwithLD.ItisanticipatedthatHexafluormayfindextensiveuseinapplicationswherehighsensitivityisrequiredsuchashighthroughputscreeningstudies.
Measurementofthecontentoflimit-dextrinaseincerealflours.
McCleary,B.V.(1992).CarbohydrateResearch,227,257-268.
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Proceduresforthequantitativeextraction,activation,andassayoflimit-dextrinaseincerealflourshavebeendeveloped.Extractionandactivationrequireincubationinbuffercontaining20mmcysteineforatleast16horwith25mmdithiothreitolfor5h.Activityisassayedwithasoluble,dyedsubstrate(Red-Pullulan)oraninsoluble,dyed,andcross-linkedsubstrate(Azurine-CL-Pullulan)whichisdispensedintabletform(Limit-DextriZymetablets).
Detectionofalimitdextrinaseinhibitorinbarley.
Macri,L.J.,MacGregor,A.W.,Schroeder,S.W.&Bazin,S.L.(1993).JournalofCerealScience,18(2),103-106.
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Twoproteinshavebeenisolatedfrombarley(Hordeumvulgarecv.Harrington)thatinhibitedlimitdextrinasefromgerminatedbarley.Theseinhibitorswerepurifiedbycarboxymethylcelluloseionexchangechromatographyandchromatofocusing.TheywereshowntohaveapproximateMrsof15kandisoelectricpointsofapproximately6•7and7•2(measuredbyisoelectricfocusing).
Purificationandcharacterisationoflimitdextrinaseinhibitorsfrombarley.
MacGregor,A.W.,Macri,L.J.,Schroeder,S.W.&Bazin,S.L.(1994).JournalofCerealScience,20(1),33-41.
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Twoinhibitorsofmaltlimitdextrinasewerepurifiedfromacrudebarleyextract(cv.Harrington)byCMcelluloseionexchangechromatographyandchromatofocusing.Theinhibitorswereheat-stableproteinsofMrapproximately15kandisoelectricpointsof6•7(lowpIinhibitor)and7•2(highpIinhibitor).BothinhibitorswereactiveoverawidepHrange,andweremosteffectiveatpH5•5to6•5,thepHoptimumofthelimitdextrinaseenzyme.Inactivationofthelimitdextrinaseenzymebyeitherinhibitorcouldbereversedbywarningthecomplexat40°Cinthepresenceofreducingagents.
Limitdextrinasefromgerminatingbarleyhasendotransglycosylaseactivity,whichexplainsitsactivationbymaltodextrins.
McDougall,G.J.,Ross,H.A.,Swanston,J.S.&Davies,H.V.(2004).Planta,218(4),542-551.
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Limitdextrinase(EC3.2.1.41)fromgerminatingbarley(HordeumvulgareL)canbeactivatedbymillimolarconcentrationsoflinearmaltodextrinswithadegreeofpolymerisation≥2.Theactivationwasassay-dependent;itwasdetectedusingassaysbasedonthesolubilisationofcross-linkeddyedpullulanbutnotinassaysthatdirectlymeasuredcleavageeventssuchastheformationofnewreducingtermini.Thisstronglysuggestedthatmaltodextrinsdidnotincreasethecatalyticrateoflimitdextrinasei.e.thisisnotatrueactivation.Ontheotherhand,considerableactivationwasnotedinassaysthatmeasuredpullulandegradationbyreductioninviscosity.Takentogether,thissuggestedthatmaltodextrinsalteredthemodeofactionoflimitdextrinase,causingmorerapiddecreasesinviscosityorgreatersolubilisationofdye-linkedpullulanfragmentspercleavageevent.Theproposedmechanismofactivationbyalterationinactionpatternwasreminiscentofinitialworkinthediscoveryofxyloglucanendotransglycosylase.Therefore,theABIlityoflimitdextrinasetocatalysetransglycosylationreactionsintopullulanwastestedandconfirmedbyanassaybasedontheincorporationofafluorescentlylabelledmaltotriosederivativeintohigher-molecular-weightproducts.Thetransglycosylationreactionwasdependentonlimitdextrinaseactivityandwasenhancedinmorehighlypurifiedpreparationsoflimitdextrinase.Transglycosylationwasinhibitedbyunlabelledmaltotriose.Howtransglycosylationaccountsfortheapparentactivationoflimitdextrinasebymaltodextrinsandthephysiologicalrelevanceofthisnovelreactionarediscussed.
Efficientsecretoryexpressionoffunctionalbarleylimitdextrinaseinhibitorbyhighcell-densityfermentationofPichiapastoris.
Jensen,J.M.,Vester-Christensen,M.B.,Møller,M.S.,Bønsager,B.C.,Christensen,H.E.M.,Hachem,M.A.&Svensson,B.(2011).ProteinExpressionandPurification,79(2),217-222.
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Thelimitdextrinaseinhibitor(LDI)frombarleyseedsactsspecificallyonlimitdextrinase(LD),anendogenousstarchdebranchingenzyme.LDIisa14kDahydrophobicproteincontainingfourdisulfidebondsandoneunpairedthiolgrouppreviouslyfoundtobeeitherglutathionylatedorcysteinylated.Itisamemberoftheso-calledCM-proteinfamilythatincludesα-amylaseandserineproteaseinhibitors,whichhavebeenextremelychallengingtoproducerecombinantlyinfunctionalformandingoodyields.Here,LDIisproducedinveryhighyieldsbysecretoryexpressionbyPichiapastorisapplyinghighcell-densityfermentationina5Lfed-batchbioreactor.Thusabout200mgofLDI,whichshowedtwofoldhigherinhibitoryactivitytowardsLDthanLDIfrombarleyseeds,waspurifiedfrom1LofculturesupernatantbyHis-tagaffinitychromatographyandgelfiltration.ElectrosprayionizationmassspectrometryverifiedtheidentityoftheproducedglutathionylatedLDI-His6.Ata1:1MratiotherecombinantLDIcompletelyinhibitedhydrolysisofpullulancatalyzedby5–10nMLD.LDIretainedstabilityinthepH2–12rangeandatpH6.5displayedahalf-lifeof53and33minat90and93°C,respectively.TheefficientheterologousproductionofLDIsuggestssecretoryexpressionbyP.pastoristobeapromisingstrategytoobtainotherrecombinantCM-proteins.
ThesurvivaloflimitdextrinaseduringfermentationintheproductionofScotchwhisky.
Walker,J.W.,Bringhurst,T.A.,Broadhead,A.L.,Brosnan,J.M.&Pearson,S.Y.(2001).JournaloftheInstituteofBrewing,107(2),99-106.
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Limitdextrinase,isanimportantenzymeinthehydrolysisofstarchfromcerealstofermentablesugars.WorkisdescribedwhichdemonstratestheimportanceofthisenzymeintheproductionofScotchwhisky.Thestudyconsiderstheoccurrence,survivalandactionoflimitdextrinase(totalandfree)duringfermentationinmaltandgraindistilleries.Theresultsofbothlaboratoryanddistillerystudiesrevealedthatlimitdextrinasecansurvivetheconditionsencounteredduringmashingandisnotonlypresentinthefermenterbutitsactivitycanincreaseduringfermentation.ThisobservationhasimportantimplicationsfortheproductionofScotchwhisky,sincethefermentationsubstrate(wash)isnotboiled,theenzymeisthereforeavailabletodegradedextrinsintofermentablesugars,andcanpotentiallyincreasetheyieldofalcohol.
Thioredoxininbarley:couldithavearoleinreleasinglimitdextrinaseinbrewerymashes?
Heisner,C.B.&Bamforth,C.W.(2008).JournaloftheInstituteofBrewing,114(2),122-126.
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Thereisnotastrongcorrelationbetweentheamountofthioredoxininbarleyandthetotalproteincontentofthegrain.Thelevelofdetectablethioredoxindecreasesduringgermination,whereastheamountoflimitdextrinaseincreases,forthemostpartinaboundform.Undernocircumstancescouldareleaseoflimitdextrinaseinanactiveformbedemonstratedthroughtheagencyofthethioredoxinsystem.Bycontrastthethiolagentdithiothreitol,especiallyinthepresenceofcalcium,didpromotetheactivationoflimitdextrinase.Ofmostpronouncedeffect,however,wastheloweringofpH.Three-foldhigherlimitdextrinaseactivitywasachievedwhenthepHwasloweredto4.0.
SecretoryexpressionoffunctionalbarleylimitdextrinasebyPichiapastorisusinghighcell-densityfermentation.
Vester-Christensen,M.B.,Hachem,M.A.,Naested,H.&Svensson,B.(2010).ProteinExpressionandPurification,69(1),112-119.
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Heterologousproductionoflargemultidomainproteinsfromhigherplantsisoftencumbersome.Barleylimitdextrinase(LD),a98kDamultidomainstarchandα-limitdextrindebranchingenzyme,playsamajorroleinstarchmobilizationduringseedgerminationandispossiblyinvolvedinstarchbiosynthesisbytrimmingofintermediatebranchedα-glucanstructures.HighlyactivebarleyLDisobtainedbysecretoryexpressionduringhighcell-densityfermentationofPichiapastoris.TheLDencodinggenefragmentwithoutsignalpeptidewassubclonedin-framewiththeSaccharomycescerevisiaeα-factorsecretionsignaloftheP.pastorisvectorpPIC9Kundercontrolofthealcoholoxidase1promoter.Optimizationofafed-batchfermentationprocedureenabledefficientproductionofLDina5-Lbioreactor,whichcombinedwithaffinitychromatographyonβ-cyclodextrin–SepharosefollowedbyHiloadSuperdex200gelfiltrationyielded34mghomogenousLD(84%recovery).TheidentityoftherecombinantLDwasverifiedbyN-terminalsequencingandbymassspectrometricpeptidemapping.Amolecularmassof98kDawasestimatedbySDS–PAGEinexcellentagreementwiththetheoreticalvalueof97419Da.KineticconstantsofLDcatalyzedpullulanhydrolysiswerefoundtoKm,app=0.16±0.02mg/mLandKcat,app=79±10s-1byfittingtheuncompetitivesubstrateinhibitionMichaelis–Mentenequation,whichreflectssignificantsubstrateinhibitionand/ortransglycosylation.Theresultingcatalyticcoefficient,Kcat,app/Km,app=488±23mL/(mgs)is3.5-foldhigherthanforbarleymaltLD.Surfaceplasmonresonanceanalysisshowedα-,β-,andγ-cyclodextrinbindingtoLDwithKdof27.2,0.70,and34.7µM,respectively.
Theadvantagesofusingnaturalsubstrate‐basedmethodsinassessingtherolesandsynergisticandcompetitiveinteractionsofbarleymaltstarch‐degrADIngenzymes.
Osman,A.M.(2002).JournaloftheInstituteofBrewing,108(2),204-214.
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Existingmethodsofassayofmaltstarch-degradingenzymeswerecriticallyappraised.Newmethodsbasedonnaturalsubstrates,namelystarchanditsnaturalintermediate-derivative,weredevelopedforalltheenzymes,exceptlimitdextrinaseforwhichpullulanwasused.Thermostability,optimaltemperaturesandpHswereestablished.α-Amylaseandlimitdextrinasewerethemostthermostableandβ-amylase,α-glucosidaseandmaltaseweretheleaststablewhilediastaseoccupiedanintermediateposition.Theoptimaltemperatureswerecongruentwiththermostability,β-amylasehavingthelowest(50°C)andα-amylasethehighest(65°C)withtheremainingenzymes,includingdiastase,fallinginbetween.Incontrast,α-amylasehasthelowestoptimalpH(pH4.5)andβamylasethehighest(pH5.5)whiletheothershavepHsinbetweenthetwovalues.Therolesoftheenzymeswereevaluatedtakingintoaccountthelevelofactivity,thermostability,optimumpH,thenatureoftheproduct(s),andtherelevancetobrewing.β-Amylaseproductionofmaltosewassynergisticallyenhanced,mostlybyα-amylasebutalsolimitdextrinase.α-Glucosidaseandmaltaseareunimportantforbrewing,becauseoftheirlowactivityandthenegativeimpactonβ-amylaseactivityandthenegativeeffectofglucoseonmaltoseuptakebyyeast.Thestarch-degradingenzymes(diastase)inagramofmaltwereabletodegrademorethan8gboiledstarchintoreducingsugarsin10minat65°C.Thelatter,suggeststhatitwillbepossibletogelatinisemostofthemaltstarchatahighertemperatureandensureitshydrolysistofermentablesugarsbymixingwithsmallerportionsofmaltandmashingatlowertemperaturese.g.50–60°C.
Oligosaccharideandsubstratebindinginthestarchdebranchingenzymebarleylimitdextrinase.
Møller,M.S.,Windahl,M.S.,Sim,L.,Bøjstrup,M.,Hachem,M.A.,Hindsgaul,O.,Palcic,M.,Svensson,B.&Henriksen,A.(2015).JournalofMolecularBIOLOGy,427(6),1263-1277.
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Completehydrolyticdegradationofstarchrequireshydrolysisofboththeα-1,4-andα-1,6-glucosidicbondsinamylopectin.Limitdextrinase(LD)istheonlyendogenousbarleyenzymecapableofhydrolyzingtheα-1,6-glucosidicbondduringseedgermination,andimpairedLDactivityinevitablyreducesthemaltoseandglucoseyieldsfromstarchdegradation.CrystalstructuresofbarleyLDandactive-sitemutantswithnaturalsubstrates,productsandsubstrateanaloguesweresoughttobetterunderstandthefacetsofLD–substrateinteractionsthatconfinehighactivityofLDtobranchedmaltooligosaccharides.Forthefirsttime,anintactα-1,6-glucosidicallylinkedsubstratespanningtheactivesiteofaLDorpullulanasehasbeentrappedandcharacterizedbycrystallography.Thecrystalstructurerevealsboththebranchandmain-chainbindingsitesandisusedtosuggestamechanismfornucleophilicityenhancementintheactivesite.Thesubstrate,productandanaloguecomplexeswerefurtherusedtooutlinesubstratebindingsubsitesandsubstratebindingrestraintsandtosuggestamechanismforavoidanceofdualα-1,6-andα-1,4-hydrolyticactivitylikelytobeabiologicalnecessityduringstarchsynthesis.
Achromogenicassaysuitableforhigh-throughputdeterminationoflimitdextrinaseactivityinbarleymaltextracts.
Bøjstrup,M.,Marri,L.,Lok,F.&Hindsgaul,O.(2015).JournalofagriculturalandFoodChemistry,63(50),10873-10878.
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Twenty-fourmaltsampleswereassayedforlimitdextrinaseactivityusingachromogenicassaydevelopedrecentlyinourgroup.Theassayutilizesasmallsolublechromogenicsubstratewhichishydrolyzedselectivelybylimitdextrinaseinacoupledassaytoreleasethechromophore2-chloro-4-nitrophenol.Thereleaseofthechromophore,correspondingtotheactivityoflimitdextrinase,canbefollowedbymeasuringtheUVabsorptionat405nm.The24maltsamplesrepresentedawidevariationoflimitdextrinaseactivities,andtheseactivitiescouldbeclearlydifferentiatedbytheassay.Theresultsobtainedwerecomparablewiththeresultsobtainedfromacommerciallyavailableassay,Limit-DextrizymefromMegazymeInternationalIreland.Furthermore,theimprovedassayusesasolublesubstrate.Thatmakesitwellsuitedforhigh-throughputscreeningasitcanbehandledina96-wellplateformat.
ValidationofMethods

RACIStandardMethod