Novel approaches to the automated assay of β-glucanase and lichenase activity. (29th November 2016)

• Record Type: Journal Article
• Authors: Mangan, D.; Liadova, A.; Ivory, R.; McCleary, B.V.
• Is Part Of: Carbohydrate research. Volume 435(2016)
• Page Start: 162
• Page End: 172
• Publication Date: 2016-11-29
• Subjects: β-glucanase -- Lichenase -- Assay procedure -- 4, 6-O-benzylidene-2-chloro-4-nitrophenyl-β-31-cellotriosyl-β-glucopyranoside -- MBG4 -- Colourimetric -- Oligosaccharides; Carbohydrates -- Periodicals; Chemistry, Organic -- Periodicals; Biochemistry -- Periodicals; Carbohydrates -- Periodicals; Chimie organique -- Périodiques; Glucides -- Périodiques; Biochemistry; Carbohydrates; Chemistry, Organic; Periodicals; Electronic journals; 507.78
• DOI: 10.1016/j.carres.2016.10.006
• Languages: English
• ISSNs: 0008-6215

Abstract

Abstract: We report herein the development of a novel assay procedure for the measurement of β-glucanase and lichenase (EC 3.2.1.73) in crude enzyme extracts. Two assay formats based on a) a direct cleavage or b) an enzyme coupled substrate were initially investigated. The 'direct cleavage' substrate, namely 4, 6- O -benzylidene-2-chloro-4-nitrophenyl-β-3 1 -cellotriosyl-β-glucopyranoside (MBG4 ), was found to be the more generally applicable reagent. This substrate was fully characterised using a crude malt β-glucanase extract, a bacterial lichenase ( Bacillus sp.) and a non-specific endo -1, 3(4)-β-glucanase from Clostridium thermocellum (EC 3.2.1.6). Standard curves were derived that allow the assay absorbance response to be directly converted to β-glucanase/lichenase activity on barley β-glucan. The specificity of MBG4 was confirmed by analysing the action of competing glycosyl hydrolases that are typically found in malt on the substrate. Manual and automated assay formats were developed for the analysis of a) β-glucanase in malt flour and b) lichenase enzyme extracts and the repeatability of these assays was fully investigated. Graphical abstract: Highlights: The rational design of colourimetric substrates for β-glucanase is investigated. Characterisation of 2 novel substrates performed using lichenase/β-glucanase enzymes. Manual and automated assay formats are developed using the lead substrate. The repeatability/reproducibility of all assay formats is thoroughly investigated.