TheaimofthisworkwastoidentifyenzymesthatparticipateinthedegradationoftransitorystarchinArabidopsis.Amutantlinewasisolatedbyscreeningleavesattheendofthenightforthepresenceofstarch.Themutanthadahigherstarchcontentthanthewild-typethroughoutthediurnalcycle.Thisaccumulationwasduetoareductioninstarchbreakdown,leADIngtoanimbalancebetweentheratesofsynthesisanddegradation.Noreductionintheactivityofendo-amylase(α-amylase),β-amylase,starchphosphorylase,maltase,pullulanaseorD-enzymecouldbedetectedincrudeextractsofleavesofthemutant.However,nativePAGEingelscontainingamylopectinrevealedthatastarch-hydrolysingactivity,putativelyidentifiedasanendo-amylaseandpresentinwild-typechloroplasts,wasabsentorappreciablyreducedinthemutant.Thisisthefirsttimethataspecificenzymerequiredforstarchdegradationhasbeenidentifiedinleaves.

TypeIpullulanasesareenzymesthatspecificallyhydrolyseα-1,6linkagesinpolysaccharides.ThisstudyreportstheanalysesofanoveltypeIpullulanase(PulASK)fromAnoxybacillussp.SK3-4.PurifiedPulASK(molecularmassof80 kDa)wasstableatpH 5.0-6.0andwasmostactiveatpH 6.0.TheoptimumtemperatureforPulASKwas60°C,andtheenzymewasreasonablystableatthistemperature.PullulanwasthepreferredsubstrateforPulASK,with89.90 %adsorbanceefficiency(variousotherstarches,56.26–72.93 %efficiency).SimilartoothertypeIpullulanases,maltotriosewasformedondigestionofpullulanbyPulASK.PulASKalsoreactedwithβ-limitdextrin,asugarrichinshortbranches,andformedmaltotriose,maltotetraoseandmaltopentaose.Nevertheless,PulASKwasfoundtopreferablydebranchlongbranchesatα-1,6glycosidicbondsofstarch,producingamylose,linearorbranchedoligosaccharides,butwasnonreactiveagainstshortbranches;thus,noreducingsugarsweredetected.ThisissurprisingasallcurrentlyknowntypeIpullulanasesproducereducingsugars(predominantlymaltotriose)ondigestingstarch.TheclosesthomologueofPulASK(95 %identity)isatypeIpullulanasefromAnoxybacillussp.LM14-2(Pul-LM14-2),whichiscapableofformingreducingsugarsfromstarch.Withrationaldesign,aminoacids362-370ofPulASKwerereplacedwiththecorrespondingsequenceofPul-LM14-2.Themutantenzymeformedreducingsugarsondigestingstarch.Thus,weidentifiedanovelmotifinvolvedinsubstratespecificityintypeIpullulanases.Ourcharacterizationmaypavethewayfortheindustrialapplicationofthisuniqueenzyme.

TherootextractofPaederiafoetidaLinn.hasbeentraditionallyutilizedfortheimprovementoftasteandtextureofvariousfoodsinThailand.Toidentifyafactorfortheimprovement,weperformedbiochemicalanalyses.Enzymeassayandzymographicmethodrevealedthatthereisamylolyticactivityintherootextract.ThisandSDS-polyacrylamidegelelectrophoresis(PAGE)analysessuggestthatanenzymeexhibitingamylolyticactivityisadominantproteinintheextracts.ByusingDEAE-columnchromatography,theamylasewaspurifiedtohomogeneity,havingamolecularmassonSDS-PAGEof60 kDa.ThepurifiedenzymeshowedpH-andtemperature-optimumactivitiesat7.0andat50°C,respectively.TheenzymeactivitywasfoundtobestableinthepHandtemperaturerangesof6.0–7.5andof30–60°C,respectively,andwasinhibitedcompletelybytheadditionofHg²⁺andCu²⁺andpartiallybyFe³⁺.Theamylasewasactiveonstarch>dextrin>amylopectin>glycogen>β-limitdextrin,butwasinactiveonpullulanandstarchazure.HPLCanalysisofstarchhydrolysatebytheenzymeshowedmaltoseasamainproductwithnodetectableglucose.TheK_mvalueforstarchofthepurifiedenzymewasdeterminedtobe2.7±0.24 mg ml^-1.Takentogether,itissuggestedthatanenzymeresponsIBLeforamylolyticactivityintherootextractsofPaederiafoetidaLinn.isβ-amylase.

Fortygrassfedbeefsteersclosetoslaughterweight(500kg)wereusedtostudytheeffectsofseason(oneexperimentwascarriedoutinautumnandoneinsummer,samefarm,samedesign),supplementation(grass-fedonly=controlorflakedcornsupplemented=supplduringfourweeksbeforeslaughter)andfastingduringlairage(0hor24hfasting).Thesupplementationwithflakedcornstartedwith0.5kganimal^-1day¹,fedindividuallyandincreasingupto1%ofbodyweight(approximately5kganimal^-1day¹)duringthefirstweek;thisamountwaskeptconstantforthreemoreweeks.Theconcentrationsofmuscleglycogen(MGC),glucose-6-phosphate+glucose(G6P+Gluc)andlactate(LA),glycolyticpotential(GPot),activityofAMP-activatedproteinkinase(AMPK),glycogenphosphorylase(GP)andglycogendebranchingenzyme(GDE)weredeterminedinM.Longissimuslumborum(LL);pHandpostmortemtemperatureat0.5hand24hweremeasured.BiopsiesfromtheLLweretakenfromeachsteeratthebeginningofeachexperiment(B1),at0.5h(B2)and24hpostmortem(B3).Foreachmetabolicsubstrate/productmeasuredinthemusclesamplesalinearmixedeffectmodelwasfitted.GPot,MGCandGPwerehigherandGDEwaslower(P<0.05)inautumnthaninsummer.Carcasstemperatureat0.5hand24hpostmortemwaslowerinautumnthaninsummerandnon-fastedsteershadalowerfinalcarcasstemperaturethanthosefasted(P<0.05).Supplementationandnofastingweresignificant(P<0.05)factorsthathelpedmaintainingahigherMGCandGPotinthesteersbetweenB1(onfarmbiopsy)andB2(atslaughter);nofastingalsohelpedinincreasingGDEactivitypostmortem(betweenB2andB3).TheeffectsoftreatmentsonglycogenreservesandontheactivitiesoftheglycolyticenzymesincludedwerenotreflectedintheultimatepHofthecarcasses,becausenodifferencesintermsofmeanpHduetoanyofthefactorsstudiedwerefound(P>0.05).Perhapsothersubstrates/enzymesthattakepartinmuscleglycolysis/glycogenolysisnotincludedinthisstudyshouldbeanalyzedinfuturestudies;consideringthehighindividualvariabilityobserved,intrinsicfactorsofcattle,likegenetics,shouldbetakenintoconsideration.