Megazyme/Pullulanase/LimitDextrinase Assay Kit (PullG6 Method)/KPullG6/100 / 200 assays per kit

PullG6assayforthemeasurementofpullulanaseemploysawatersolubledefinedsubstrate,namely4,6-O-benzylidene-4-nitrophenyl-6³-α-D-maltotriosyl-maltotriose(BPNPG3G3),coupledwiththeancillaryenzymesα-glucosidaseandβ-glucosidase.Uponhydrolysisofthesubstrateatthe1,6-α-linkagebypullulanaseorlimit-dextrinase,thereleased4-nitrophenyl-β-maltotriosideisimmediatelyhydrolysedtoglucoseand4-nitrophenolbytheconcertedactionoftheα-glucosidaseandβ-glucosidaseenzymesinthereagentmixture.Thereactionisterminatedandphenolateionsaredevelopedbyadditionofdilutealkali.Theabsorbanceisreadat400nmandthevalueobtainedcorrelatesdirectlywithpullulanaseactivity.

Colourimetricandfluorometricsubstratesformeasurementofpullulanaseactivity.

McCleary,B.V.,Mangan,D.,McKie,V.,Cornaggia,C.,Ivory,R.&Rooney,E.(2014).CarbohydrateResearch,393,60-69.

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Specificandhighlysensitivecolourimetricandfluorometricsubstratemixtureshavebeenpreparedforthemeasurementofpullulanaseandlimit-dextrinaseactivityandassaysemployingthesesubstrateshavebeendeveloped.Thesemixturescomprisethermostableα-andβ-glucosidasesandeither4,6-O-benzylidene-2-chloro-4-nitrophenyl-β-maltotriosyl(1-6)α-maltotrioside(BzCNPG₃G₃,1)asacolourimetricsubstrateor4,6-O-benzylidene-4-methylumbelliferyl-β-maltotriosyl(1-6)α-maltotrioside(BzMUG₃G₃,2)asafluorometricsubstrate.Hydrolysisofsubstrates1and2byexo-actingenzymessuchasamyloglucosidase,β-amylaseandα-glucosidaseispreventedbythepresenceofthe4,6-O-benzylidenegrouponthenon-reducingendD-glucosylresidue.Thesubstratesarenothydrolysedbyanyα-amylasesstudied,(includingthosefromAspergillusnigerandporcinepancreas)andareresistanttohydrolysisbyPseudomonassp.isoamylase.Onhydrolysisbypullulanase,the2-chloro-4-nitrophenyl-β-maltotrioside(3)or4-methylumbelliferyl-β-maltotrioside(4)liberatedisimmediatelyhydrolysedtoD-glucoseand2-chloro-4-nitrophenolor4-methylumbelliferone.Thereactionisterminatedbytheadditionofaweakalkalinesolutionleadingtotheformationofphenolateionsinsolutionwhoseconcentrationcanbedeterminedusingeitherspectrophotometricorfluorometricanalysis.Theassayprocedureissimpletouse,specific,accurate,robustandreadilyadaptedtoautomation.

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

Gluten-freesourcesoffermentableextract:effectoftemperatureandgerminationtimeonqualityattributesofteff[Eragrostistef(zucc.)trotter]maltandwort.

DiGhionno,L.,Marconi,O.,Lee,E.G.,Marconi,O.,Rice,C.J.,Sileoni,V.&Perretti,G.(2017).JournalofAgriculturalandFoodChemistry,65(23),4777-4785.

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ThisstudywasconductedtoevaluatethebehaviorofawhiteteffvarietycalledWitkopduringmaltingbyusingdifferentparameters(germinationtemperatureandduration)andtoidentifythebestmaltingprogram.Sampleswereevaluatedforstandardqualitymaltandwortattributes,pastingcharacteristics,β-glucanandarabinoxylancontent,andsugarprofile.Itwasconcludedthatmaltingteffat24°Cfor6daysproducedacceptablemaltintermsofqualityattributesandsugarprofileforbrewing.Themainattributeswere80.4%extract,80.9%fermentability,1.53mPasviscosity,7.4EBC-Ucolor,129mg/LFAN,and72.1g/Loftotalfermentablesugars.Statisticalanalysisshowedthatpastingcharacteristicsofteffmaltwerenegativelycorrelatedwithsomemaltqualityattributes,suchasextractandfermentability.Witkopteffappearedtobeapromisingrawmaterialformaltingandbrewing.However,thesmallgrainsizemayleadtodifficultiesinhandlingmaltingprocess,andabespokebrewhouseplantshouldbedevelopedfortheproductionatindustrialscale.

Colourimetricmethodforthedeterminationofpullanase
orlimit-dextrinase

Principle:
                          (pullulanase/limit-dextrinase)
(1)Benzylidene-G₃-(α-1,6)-G₃-β-PNP+H₂O→Benzylidene-G₃
                                       +G₃-β-PNP

(thermostableα-glucosidaseandβ-glucosidase)
(2)G₃-β-PNP+H₂O→D-glucose+PNP

  (alkalinesolution)
(3)PNP→phenolateion(yellowcolour)

Note:PNP=4-nitrophenol

Kitsize:                         100/200assays
Method:                         Spectrophotometricat400nm
Totalassaytime:             10minforpullanasepreparations
                           30minformaltextractscontaining
limit-dextrinase
Detectionlimit:                0.18U/mLforpullulanasepreparations
(50-folddilution)
              0.01U/gforlimitdextrinaseinmilledmalt
Applicationexamples:Assayofmicrobialpullulanasepreparations
Measurementoflimit-dextrinasein
maltextracts
Methodrecognition:   Novelmethod

Advantages

Highsensitivity
Suitableformanualandauto-analyserformats
Notransglycosylationinterference
Verycosteffective
Allreagentsstablefor>1yearafterpreparation
Veryspecific
Simpleformat
Standardincluded

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Megazyme/Pullulanase/LimitDextrinase Assay Kit (PullG6 Method)/KPullG6/100 / 200 assays per kit

Colourimetricandfluorometricsubstratesformeasurementofpullulanaseactivity.

Colourimetricandfluorimetricsubstratesfortheassayoflimitdextrinase.

Gluten-freesourcesoffermentableextract:effectoftemperatureandgerminationtimeonqualityattributesofteff[Eragrostistef(zucc.)trotter]maltandwort.