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HighpuritydyedandcrosslinkedAmylazymeRedtabletsforthemeasurementofenzymeactivity,forresearch,biochemicalenzymeassaysandinvitrodiagnosticanalysis.
Ahighlysensitivesubstrateforthemeasurementofα-amylase.
Newchromogenicsubstratesfortheassayofalpha-amylaseand(1-4)-β-D-glucanase.
McCleary,B.V.(1980).CarbohydrateResearch,86(1),97-104.
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Newchromogenicsubstrateshavebeendevelopedforthequantitativeassayofalpha-amylaseand(1→4)-β-D-glucanase.Thesewerepreparedbychemicallymodifyingamyloseorcellulosebeforedyeing,toincreasesolubility.Afterdyeing,thesubstrateswereeithersolubleorcouldbereADIlydispersedtoformfine,gelatinoussUSPensions.Assaysbasedontheuseofthesesubstratesaresensitiveandhighlyspecificforeitheralpha-amylaseor(1→4)-β-D-glucanase.Themethodofpreparationcanalsobeappliedtoobtainsubstratesforotherendo-hydrolases.
Comparisonofendolytichydrolasesthatdepolymerise1,4-β-D-mannan,1,5-α-L-arABInanand1,4-β-D-galactan.
McCleary,B.V.(1991).“EnzymesinBiomassConversion”,(M.E.HimmelandG.F.Leatham,Eds.),ACSSymposiumSeries460,Chapter34,pp.437-449.AmericanChemicalSociety,Washington.
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Hydrolysisofmannan-typepolysaccharidesbyβ-mannanaseisdependentonsubstitutiononandwithinthemain-chainaswellasthesourceoftheβ-mannanaseemployed.Characterisationofreactionproductscanbeusedtodefinethesub-sitebindingrequirementsoftheenzymesaswellasthefine-structuresofthepolysaccharides.Actionofendo-arabinanaseandendo-galactanaseonarabinansandarabinogalactansisdescribed.Specificassaysforendo-arabinanaseandarabinan(infruit-juiceconcentrates)arereported.
Measurementofpolysaccharidedegradingenzymesusingchromogenicandcolorimetricsubstrates.
McCleary,B.V.(1991).ChemistryinAustralia,58,398-401.
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Enzymicdegradationofcarbohydratesisofmajorsignificanceintheindustrialprocessingofcerealsandfruits.Intheproductionofbeer,barleyisgerminatedunderwelldefinedconditions(malting)toinducemaximumenzymesynthesiswithminimumrespirationofreservecarbohydrates.Thegrainsaredriedandthenextractedwithwaterundercontrolledconditions.Theamylolyticenzymessynthesizedduringmalting,aswellasthosepresentintheoriginalbarley,convertthestarchreservestofermentablesugars.Otherenzymesactonthecellwallpolysaccharides,mixed-linkageβ-glucanandarabinoxylan,reducingtheviscosityandthusaidingfiltration,andreducingthepossibilityofsubsequentprecipitationofpolymericmaterial.Inbaking,β-amylaseandα-amylasegivecontrolleddegradationofstarchtofermentablesugarssoastosustainyeastgrowthandgasproduction.Excessquantitiesofα-amylaseintheflourresultinexcessivedegradationofstarchduringbakingwhichinturngivesastickycrumbtextureandsubsequentproblemswithbreadslicing.Juiceyieldfromfruitpulpissignificantlyimprovedifcell-walldegradingenzymesareusedtodestroythethree-dimensionalstructureandwaterbindingcapacityofthepecticpolysaccharidecomponentsofthecellwalls.Problemsofroutineandreliableassayofcarbohydratedegradingenzymesinthepresenceofhighlevelsofsugarcompoundsareexperiencedwithsuchindustrialprocess.
Measurementofα-amylaseincereal,foodandfermentationproducts.
McCleary,B.V.&Sturgeon,R.(2002).CerealFoodsWorld,47(7),299-310.
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InGeneral,thedevelopmentofmethodsformeasuringα-amylaseispioneeredintheclinicalchemistryfieldandthentranslatedtootherindustries,suchasthecerealsandfermentationindustries.Inmanyinstances,thistransferoftechnologyhasbeendifficultorimpossIBLetoachieveduetothepresenceofinterferingenzymesorsugarsandtodifferencesinthepropertiesoftheenzymesbeinganalysed.Thisarticledescribesmanyofthecommonlyusedmethodsformeasuringα-amylaseinthecereals,food,andfermentationindustriesanddiscussessomeoftheadvantagesandlimitationsofeach.
Comparisonofshort-wavelengthinfrared(SWIR)hyperspectralimagingsystemwithanFT-NIRspectrophotometerforpredictingalpha-amylaseactivitiesinindividualCanadianWesternRedSpring(CWRS)wheatkernels.
Xing,J.,Symons,S.,Hatcher,D.&Shahin,M.(2011).BiosystemsEngineering,108(4),303-310.
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Alpha-amylaseactivityinindividualCanadianWesternRedSpring(CWRS)wheatkernelswaspredictedusingspectralinformationacrossthewavelengthregion1235–2450nm.ReflectancespectrawerecollectedfromanSWIR(short-wavelengthinfrared)hyperspectralimagingsystemandabsorbancespectrawererecordedfromanFouriertransformnear-infrared(FT-NIR)spectrometeronthesamekernels.Thepartialleastsquares(PLS)regressiontechniquewasusedtomodelthealpha-amylaseenzymeactivitylevelstothespectralinformation.Thepredictionaccuracyvariedwiththepre-processingmethodsappliedtotheregressorandregressand.Thehighestcoefficientofdetermination(r2)valueobtainedfromtheSWIRhyperspectralimagingsystemwas0.88and0.82fromtheFT-NIRinstrument.Theimagingapproachwasmoresuccessfulbecauseitalsohadtheadvantageofbeingabletolocalisetheregionwherespectrawereextractedfrom.
Theobjectivemeasurementofalpha-amylaseinwheatkernelsusingspectralimaging.
Symons,S.,Xing,J.,Shahin,M.&Hatcher,D.(2010,September).WorldAutomationCongress(WAC),257-262.
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Whenwheatkernelsarewettedintheheadpriortoharvest,thegerminationprocessesareinitiated.Thesymptomsrangefromnoobviousvisiblesignsofenzymeactivationtogrosskerneldisfiguration.Alpha-amylase,astarchdegradingenzymeisthemostprevalentoftheactivatedenzymesintheearlystagesofgerminationandmaycausesignificantend-productqualityloss.Currentanalyticaltechniquesdonotprovidearapidsystemforestimatingindividualkernelsproutdamage.Wehavedevelopedanobjectiveapproachusingnear-infraredspectra(1100-2400nm)fromahyperspectralcameratopredictα-amylaselevelsofindividualkernelsintwoclassesofCanadianwheat.Multivariatemodelinggave,anR2ofupto0.69forpredictingindividualkernelα-amylaselevels.Usingthehyperspectraldata,amultispectralmodelpredictedα-amylaseactivitylevelsofgreaterthan1SKUunit/gwithabetterthan90%accuracy.Atthislevel,thereisnovisiblesignofkernelsprouting.
UsingaShortWavelengthInfrared(SWIR)hyperspectralimagingsystemtopredictalphaamylaseactivityinindividualCanadianwesternwheatkernels.
Xing,J.,VanHung,P.,Symons,S.,Shahin,M.&Hatcher,D.(2009).SensingandInstrumentationforFoodQualityandSafety,3(4),211-218.
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Sproutdamage(pre-harvestgermination)inwheatresultsinhighlydeleteriouseffectsonend-productquality.Alpha-amylase,thepre-dominantenzymeintheearlystageofsproutinghasthemostdamagingeffect.ThispaperintroducesanewmethodusingaSWIRhyperspectralimagingsystem(1000–2500nm)topredicttheα-amylaseactivityofindividualwheatkernels.TwoclassesofCanadianwheat,CanadaWesternRedSpring(CWRS)andCanadaWesternAmberDurum(CWAD),withsamplesofdifferingdegreesofsproutdamagewereinvestigated.Individualkernelswerefirstimagedwiththehyperspectralimagingsystemandthentheα-amylaseactivityofeachkernelwasdeterminedanalytically.Individualkernelα-amylaseactivitypredictionwassignificant(R20.54and0.73)forCWADandCWRS,respectivelyusingPartialLeastSquareregressiononthehyperspectraldata.AclassificationmethodisproposedtoseparateCWRSkernelswithhighα-amylaseactivitylevelfromthosewithlowα-amylaseactivitygivinganaccuracyofabove80%.Thisworkshowsthathyper/multi-spectralimagingtechniquescanbeusedforrapidlypredictingtheα-amylaseactivityofindividualkernels,detectingsproutingatearlystage.
Anovelα-amylasegeneistransientlyupregulatedduringlowtemperatureexposureinapplefruit.
Wegrzyn,T.,Reilly,K.,Cipriani,G.,Murphy,P.,Newcomb,R.,Gardner,R.&MacRae,E.(2000).EuropeanJournalofBiochemistry,267(5),1313-1322.
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Anα-amylasegeneproductwasisolatedfromapplefruitbyreverse-transcriptasePCRusingredundantprimers,followedby5′and3′RACE.Thegeneisamemberofasmallgenefamily.Itencodesaputative46.9kDaproteinthatismostsimilartoanα-amylasegenefrompotato(GenBankaccessionM79328).Inapplefruitthisnewgenewasexpressedatlowlevels,asdetectedbyreverse-transcriptasePCR,inanumberofplanttissuesandduringfruitdevelopment.HighestlevelsofmRNAforthistranscriptwereobserved3to9daysafterplacingapplefruitat0.5°C.Phylogeneticanalysisofaminoacidsequenceplacesthepotatoandappleproteinsasadistinctandseparatenewsubgroupwithintheplantα-amylases,whichappearstohavedivergedpriortothesplitbetweenmonocotyledonousanddicotyledonousplants.Thesetwodivergentα-amylaseslackthestandardsignalpeptidestructuresfoundinallotherplantα-amylases,andhavesequencedifferenceswithintheB-domainandC-domain.However,comparisonswithstructuresofknownstarchhydrolasessuggestthatthesedifferencesareunlikelytoaffecttheenzymaticα-1,4-amylasefunctionoftheprotein.Thisisthefirstreportofupregulationofadicotyledonousα-amylaseinresponsetolowtemperature,andconfirmsthepresenceofanewfamilyofα-amylasesinplants.
