title: Colourimetric and fluorometric substrates for measurement of pullulanase activity.
abstract: Specific and highly sensitive colourimetric and fluorometric substrate mixtures have been prepared for the measurement of pullulanase and limit-dextrinase activity and assays employing these substrates have been developed. These mixtures comprise thermostable α- and β-glucosidases and either 4,6-O-benzylidene-2-chloro-4-nitrophenyl-β-maltotriosyl (1-6) α-maltotrioside (BzCNPG3G3, 1) as a colourimetric substrate or 4,6-O-benzylidene-4-methylumbelliferyl-β-maltotriosyl (1-6) α-maltotrioside (BzMUG3G3, 2) as a fluorometric substrate. Hydrolysis of substrates 1 and 2 by exo-acting enzymes such as amyloglucosidase, β-amylase and α-glucosidase is prevented by the presence of the 4,6-O-benzylidene group on the non-reducing end D-glucosyl residue. The substrates are not hydrolysed by any α-amylases studied, (including those from Aspergillus niger and porcine pancreas) and are resistant to hydrolysis by Pseudomonas sp. isoamylase. On hydrolysis by pullulanase, the 2-chloro-4-nitrophenyl-β-maltotrioside (3) or 4-methylumbelliferyl-β-maltotrioside (4) liberated is immediately hydrolysed to D-glucose and 2-chloro-4-nitrophenol or 4-methylumbelliferone. The reaction is terminated by the addition of a weak alkaline solution leading to the formation of phenolate ions in solution whose concentration can be determined using either spectrophotometric or fluorometric analysis. The assay procedure is simple to use, specific, accurate, robust and readily adapted to automation.
authors.affiliation: Megazyme International Ireland, Bray Business Park, Southern Cross Road, Bray, County Wicklow, Ireland. Electronic address: barry@megazyme.com.
authors.forename: Barry V
authors.initials: BV
authors.lastname: McCleary
authors.affiliation: Megazyme International Ireland, Bray Business Park, Southern Cross Road, Bray, County Wicklow, Ireland. Electronic address: david@megazyme.com.
authors.forename: David
authors.initials: D
authors.lastname: Mangan
authors.affiliation: Megazyme International Ireland, Bray Business Park, Southern Cross Road, Bray, County Wicklow, Ireland.
authors.forename: Vincent
authors.initials: V
authors.lastname: McKie
authors.affiliation: Megazyme International Ireland, Bray Business Park, Southern Cross Road, Bray, County Wicklow, Ireland.
authors.forename: Claudio
authors.initials: C
authors.lastname: Cornaggia
authors.affiliation: Megazyme International Ireland, Bray Business Park, Southern Cross Road, Bray, County Wicklow, Ireland.
authors.forename: Ruth
authors.initials: R
authors.lastname: Ivory
authors.affiliation: Megazyme International Ireland, Bray Business Park, Southern Cross Road, Bray, County Wicklow, Ireland.
authors.forename: Edward
authors.initials: E
authors.lastname: Rooney
mesh_terms: D003124:Colorimetry; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry; D005470:Fluorometry; D006026:Glycoside Hydrolases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure
chemical_list: D005456:Fluorescent Dyes; D006026:Glycoside Hydrolases; C100181:pullulanase
keywords: 4,6-O-Benzylidene-2-chloro-4-nitrophenyl-β-maltotriosyl (1-6)-α-d-maltotrioside (BzCNPG(3)G(3)); 4,6-O-Benzylidene-methylumbelliferyl-β-maltotriosyl (1-6)-α-d-maltotrioside (BzMUG(3)G(3)); Assay procedure; Limit dextrinase; Pullulanase