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