Colourimetric and fluorimetric substrates for the assay of limit dextrinase.
Mangan, D., McCleary, B. V., Cornaggia, C., Ivory, R., Rooney, E. & McKie, V. (2015). Journal of Cereal Science, 62, 50-57.
The measurement of limit-dextrinase (LD) (EC 220.127.116.11) in grain samples such as barley, wheat or rice can be problematic for a number of reasons. The intrinsic LD activity in these samples is extremely low and they often contain a limit-dextrinase inhibitor and/or high levels of reducing sugars. LD also exhibits transglycosylation activity that can complicate the measurement of its hydrolytic activity. A minor modification to the industrial standard Limit-Dextrizyme tablet test is suggested here to overcome this transglycosylation issue.
In addition, two new substrates are described that can be adopted for use in an auto-analyser format. 4,6-O-benzylidene-2-chloro-4-nitrophenyl-β-63-α-D-maltotriosyl-maltotrioside (BzCNPG3G3, Hexachrom) is not susceptible to transglycosylation and serves amiably as a routine quantitative assay tool with the potential to run kinetic assays due to the low pKa (∼5.5) of the chromogenic moiety while 4,6-O-benzylidene-4-methylumbelliferyl-β-63-α-D-maltotriosyl-maltotrioside (BzMUG3G3, Hexafluor) was found to be susceptible to transglycosylation with LD. It is anticipated that Hexafluor may find extensive use in applications where high sensitivity is required such as high throughput screening studies.
Colourimetric and fluorometric substrates for measurement of pullulanase activity.
McCleary, B. V., Mangan, D., McKie, V., Cornaggia, C., Ivory, R. & Rooney, E. (2014). Carbohydrate Research, 393, 60-69.
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.
Characterization of the human β-glucan receptor and its alternatively spliced isoforms.
Willment, J. A., Gordon, S. & Brown, G. D. (2001). Journal of Biological Chemistry, 276(47), 43818-43823.
β-1,3-D-Glucans are biological response modifiers with potent effects on the immune system. A number of receptors are thought to play a role in mediating these responses, including murine Dectin-1, which we recently identified as a β-glucan receptor. In this study we describe the characterization of the human homologue of this receptor and show that it is structurally and functionally similar to the mouse receptor. The human β-glucan receptor is a type II transmembrane receptor with a single extracellular carbohydrate recognition domain and an immunoreceptor tyrosine activation motif in its cytoplasmic tail. The human β-glucan receptor is widely expressed and functions as a pattern recognition receptor, recognizing a variety of β-1,3- and/or β-1,6-linked glucans as well as intact yeast. In contrast to the murine receptor, the human receptor mRNA is alternatively spliced, resulting in two major (A and B) and six minor isoforms. The two major isoforms differ by the presence of a stalk region separating the carbohydrate recognition domain from the transmembrane region and are the only isoforms that are functional for β-glucan binding. The human receptor also binds T-lymphocytes at a site distinct from the β-glucan binding site, indicating that this receptor can recognize both endogenous and exogenous ligands.