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Colourimetric and fluorometric substrates for measurement of pullulanase activity.
- Source :
-
Carbohydrate research [Carbohydr Res] 2014 Jul 01; Vol. 393, pp. 60-9. Date of Electronic Publication: 2014 Apr 28. - Publication Year :
- 2014
-
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.<br /> (Copyright © 2014 Elsevier Ltd. All rights reserved.)
Details
- Language :
- English
- ISSN :
- 1873-426X
- Volume :
- 393
- Database :
- MEDLINE
- Journal :
- Carbohydrate research
- Publication Type :
- Academic Journal
- Accession number :
- 24938640
- Full Text :
- https://doi.org/10.1016/j.carres.2014.04.014