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)isnotsusceptIBLetotransglycosylationandservesamiablyasaroutinequantitativeassaytoolwiththepotentialtorunkineticassaysduetothelowpK_a(∼5.5)ofthechromogenicmoietywhile4,6-O-benzylidene-4-methylumbelliferyl-β-6³-α-D-maltotriosyl-maltotrioside(BzMUG₃G₃,Hexafluor)wasfoundtobesusceptibletotransglycosylationwithLD.ItisanticipatedthatHexafluormayfindextensiveuseinapplicationswherehighsensitivityisrequiredsuchashighthroughputscreeningstudies.

Proceduresforthequantitativeextraction,activation,andassayoflimit-dextrinaseincerealflourshavebeendeveloped.Extractionandactivationrequireincubationinbuffercontaining20mmcysteineforatleast16horwith25mmdithiothreitolfor5h.Activityisassayedwithasoluble,dyedsubstrate(Red-Pullulan)oraninsoluble,dyed,andcross-linkedsubstrate(Azurine-CL-Pullulan)whichisdispensedintabletform(Limit-DextriZymetablets).

Twoproteinshavebeenisolatedfrombarley(Hordeumvulgarecv.Harrington)thatinhibitedlimitdextrinasefromgerminatedbarley.Theseinhibitorswerepurifiedbycarboxymethylcelluloseionexchangechromatographyandchromatofocusing.TheywereshowntohaveapproximateM_rsof15kandisoelectricpointsofapproximately6•7and7•2(measuredbyisoelectricfocusing).

Twoinhibitorsofmaltlimitdextrinasewerepurifiedfromacrudebarleyextract(cv.Harrington)byCMcelluloseionexchangechromatographyandchromatofocusing.Theinhibitorswereheat-stableproteinsofM_rapproximately15kandisoelectricpointsof6•7(lowpIinhibitor)and7•2(highpIinhibitor).BothinhibitorswereactiveoverawidepHrange,andweremosteffectiveatpH5•5to6•5,thepHoptimumofthelimitdextrinaseenzyme.Inactivationofthelimitdextrinaseenzymebyeitherinhibitorcouldbereversedbywarningthecomplexat40°Cinthepresenceofreducingagents.

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.

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.

Limitdextrinase,isanimportantenzymeinthehydrolysisofstarchfromcerealstofermentablesugars.WorkisdescribedwhichdemonstratestheimportanceofthisenzymeintheproductionofScotchwhisky.Thestudyconsiderstheoccurrence,survivalandactionoflimitdextrinase(totalandfree)duringfermentationinmaltandgraindistilleries.Theresultsofbothlaboratoryanddistillerystudiesrevealedthatlimitdextrinasecansurvivetheconditionsencounteredduringmashingandisnotonlypresentinthefermenterbutitsactivitycanincreaseduringfermentation.ThisobservationhasimportantimplicationsfortheproductionofScotchwhisky,sincethefermentationsubstrate(wash)isnotboiled,theenzymeisthereforeavailabletodegradedextrinsintofermentablesugars,andcanpotentiallyincreasetheyieldofalcohol.

Thereisnotastrongcorrelationbetweentheamountofthioredoxininbarleyandthetotalproteincontentofthegrain.Thelevelofdetectablethioredoxindecreasesduringgermination,whereastheamountoflimitdextrinaseincreases,forthemostpartinaboundform.Undernocircumstancescouldareleaseoflimitdextrinaseinanactiveformbedemonstratedthroughtheagencyofthethioredoxinsystem.Bycontrastthethiolagentdithiothreitol,especiallyinthepresenceofcalcium,didpromotetheactivationoflimitdextrinase.Ofmostpronouncedeffect,however,wastheloweringofpH.Three-foldhigherlimitdextrinaseactivitywasachievedwhenthepHwasloweredto4.0.

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.KineticconstantsofLDcatalyzedpullulanhydrolysiswerefoundtoK_m,app=0.16±0.02mg/mLandK_cat,app=79±10s^-1byfittingtheuncompetitivesubstrateinhibitionMichaelis–Mentenequation,whichreflectssignificantsubstrateinhibitionand/ortransglycosylation.Theresultingcatalyticcoefficient,K_cat,app/K_m,app=488±23mL/(mgs)is3.5-foldhigherthanforbarleymaltLD.Surfaceplasmonresonanceanalysisshowedα-,β-,andγ-cyclodextrinbindingtoLDwithK_dof27.2,0.70,and34.7µM,respectively.

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.

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.

Twenty-fourmaltsampleswereassayedforlimitdextrinaseactivityusingachromogenicassaydevelopedrecentlyinourgroup.Theassayutilizesasmallsolublechromogenicsubstratewhichishydrolyzedselectivelybylimitdextrinaseinacoupledassaytoreleasethechromophore2-chloro-4-nitrophenol.Thereleaseofthechromophore,correspondingtotheactivityoflimitdextrinase,canbefollowedbymeasuringtheUVabsorptionat405nm.The24maltsamplesrepresentedawidevariationoflimitdextrinaseactivities,andtheseactivitiescouldbeclearlydifferentiatedbytheassay.Theresultsobtainedwerecomparablewiththeresultsobtainedfromacommerciallyavailableassay,Limit-DextrizymefromMegazymeInternationalIreland.Furthermore,theimprovedassayusesasolublesubstrate.Thatmakesitwellsuitedforhigh-throughputscreeningasitcanbehandledina96-wellplateformat.