Megazyme/Amylazyme Red Tablets/TAMZRD200T/200 Tablets

Newchromogenicsubstrateshavebeendevelopedforthequantitativeassayofalpha-amylaseand(1→4)-β-D-glucanase.Thesewerepreparedbychemicallymodifyingamyloseorcellulosebeforedyeing,toincreasesolubility.Afterdyeing,thesubstrateswereeithersolubleorcouldbereadilydispersedtoformfine,gelatinoussuspensions.Assaysbasedontheuseofthesesubstratesaresensitiveandhighlyspecificforeitheralpha-amylaseor(1→4)-β-D-glucanase.Themethodofpreparationcanalsobeappliedtoobtainsubstratesforotherendo-hydrolases.

Hydrolysisofmannan-typepolysaccharidesbyβ-mannanaseisdependentonsubstitutiononandwithinthemain-chainaswellasthesourceoftheβ-mannanaseemployed.Characterisationofreactionproductscanbeusedtodefinethesub-sitebindingrequirementsoftheenzymesaswellasthefine-structuresofthepolysaccharides.Actionofendo-arabinanaseandendo-galactanaseonarabinansandarabinogalactansisdescribed.Specificassaysforendo-arabinanaseandarabinan(infruit-juiceconcentrates)arereported.

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.

InGeneral,thedevelopmentofmethodsformeasuringα-amylaseispioneeredintheclinicalchemistryfieldandthentranslatedtootherindustries,suchasthecerealsandfermentationindustries.Inmanyinstances,thistransferoftechnologyhasbeendifficultorimpossibletoachieveduetothepresenceofinterferingenzymesorsugarsandtodifferencesinthepropertiesoftheenzymesbeinganalysed.Thisarticledescribesmanyofthecommonlyusedmethodsformeasuringα-amylaseinthecereals,food,andfermentationindustriesanddiscussessomeoftheadvantagesandlimitationsofeach.

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.

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.

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.

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.