Malt β-glucanase: 100 assays (manual format) /400 assays (auto-analyser format)Lichenase: 100 / 200 assays (manual format) /330 assays (auto-analyser format)

The MBG4 reagent contains a single substrate, namely 4,6-O-benzylidene-2-chloro-4-nitrophenyl-β-(3¹-β-D-cellotriosyl-glucoside) (BCNPBG4). The benzylidene acetal group prevents any hydrolytic action by exo-acting hydrolytic enzymes such as β-glucosidase or cellobiohydrolase. Mixed linkage β-glucanase (endo-1,3:1,4-β-glucanase) / lichenase (EC 3.2.1.73) acts specifically to release 2-chloro-4-nitrophenol (CNP) from this substrate. The rate of release of CNP is directly related to the β-glucanase/lichenase activity in a sample. The reaction is terminated and the phenolate colour is developed on addition of Tris buffer solution (pH = 10.0).

Note that the substrate is not hydrolysed by β-glucosidase or cellobiohydrolase. The substrate can be hydrolysed by certain endo-cellulases (e.g. Trichoderma sp.) but this does not result in an increase in absorbance.

Data calculators are located in theDocumentation tab.

Novel approaches to the automated assay of β-glucanase and lichenase activity.

Mangan, D., Liadova, A., Ivory, R. & McCleary, B. V. (2016). Carbohydrate Research, 435, 162-172. Link to Article Read 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-3¹-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.

Colourimetric method for the determination ofendo-1,3:1.4-β-glucanase/lichenase in crude malt extracts andenzyme preparations

Principle: (endo-1,3:1,4-β-glucanase/lichenase)(1)Benzylidene-BGTETB-β-CNP + H₂O → Benzymidene-BGTETB+ CNP (tris buffer)(2) CNP→ phenolate ion (yellow colour)Note:CNP = 2-Chloro-4-nitrophenol

Kit size:Malt β-glucanase 100 assays (manual) / 400 (auto-analyser) orLichenase 100 / 200 assays (manual) / 330 (auto-analyser)

Method: Spectrophotometric at 400 nmTotal assay time: Malt β-glucanase 20 min (manual) / 10 min (auto-analyser) orLichenase 10 min (manual) / 10 min (auto-analyser)Detection limit:Malt β-glucanase 4.3 x 10^-4 U/mL orLichenase 9.1 x 10^-5 U/mLApplication examples: Crude malt extracts, industrial enzyme preparationsMethod recognition: Novel method

Advantages

• Very cost effective
• All reagents stable for > 2 years
• Specific forendo-1,3:1,4-β-glucanase/lichenase
• Simple,convenient, rapid assay
• Well suited to automation
• Malt flour standard and lichenase standard included