180 / 360 assays per kit / 720 (auto-analyser)
Prices exclude VAT
Available for shipping
|Content:||180 / 360 assays per kit / 720 assays (auto-analyser)|
Short term stability: 2-8oC,
Long term stability: See individual component labels
|Stability:||> 2 years under recommended storage conditions|
|Assay Format:||Spectrophotometer, Auto-analyser|
|Limit of Detection:||0.05 U/mL|
|Reproducibility (%):||~ 3%|
|Total Assay Time:||~ 20 min|
|Application examples:||Fermentation broths, industrial enzyme preparations and biofuels research.|
|Method recognition:||Novel method. Note: The analogous fluorimetric reagent (R-CELLFLR) is also available with 10-fold greater sensitivity|
The CellG3 assay reagent for the measurement of endo-cellulase (endo-1,4-β-glucanase) contains two components;
1) 4,6-O-benzylidene-2-chloro-4-nitrophenyl-β-D-cellotrioside (BCNPG3) and 2) thermostable β-glucosidase. The benzylidene blocking group prevents any hydrolytic action by the β-glucosidase on BCNPG3. Incubation with an endo-cellulase generates a non-blocked colourimetric oligosaccharide that is rapidly hydrolysed by the ancillary β-glucosidase. The rate of formation of 2-chloro-4-nitrophenol is therefore directly related to the hydrolysis of BCNPG3 by the endo-cellulase. The reaction is terminated and the phenolate colour is developed on addition of Tris buffer solution (pH 9.0).
Please note that a new assay kit (K-CellG5) is now available for the measurement of endo-cellulase. The CellG5 reagent contains a cellopentaose core and exhibits vastly improved sensitivity for some cellulases. In addition, the exchange of the benzylidene blocking group in CellG3 for 3-keto-butylidene in CellG5 improves the substrate’s water solubility significantly, allowing for a reduction in the concentration of DMSO required in the assay. As DMSO is known to inhibit certain cellulases, this is another benefit in using CellG5. Megazyme now recommends the use of K-CellG5 for all assays for the measurement of endo-cellulase.
- Very cost effective
- All reagents stable for > 2 years after preparation
- Completely specific for cellulase (endo-1,4-glucanase). The substrate is not hydrolysed by β-glucosidase, cellbiohyrolase or any other enzymes tested
- Kinetic assays possible due to significant phenolate ion presence (and UV absorbance) at pH 5-6
- Simple format. Well suited to automation
- Standard included
(Aspergillus niger) E-CELBA - Cellulase (endo-1,4-β-D-glucanase)
(Bacillus amyloliquefaciens) E-CELTE - Cellulase (endo-1,4-β-D-glucanase)
(Talaromyces emersonii) E-CELTH - Cellulase (endo-1,4-β-D-glucanase)
(Thermobifida halotolerans) E-CELTR - Cellulase (endo-1,4-β-D-glucanase)
(Trichoderma longibrachiatum) E-CELTM - Cellulase (endo-1,4-β-D-glucanase)
Mangan, D., Cornaggia, C., McKie, V., Kargelis. T. & V. McCleary, B. V. (2016). Analytical and Bioanalytical Chemistry, 408(15), 4159-4168.
endo-1,4-β-Glucanase (endo-cellulase, EC 188.8.131.52) is one of the most widely used enzymes in industry. Despite its importance, improved methods for the rapid, selective, quantitative assay of this enzyme have been slow to emerge. In 2014, a novel enzyme-coupled assay that addressed many of the limitations of the existing assay methodology was reported. This involved the use of a bifunctional substrate chemically derived from cellotriose. Reported herein is a much improved version of this assay employing a novel substrate, namely 4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-D-cellopentaoside.Hide Abstract
McCleary, B. V., Mangan, D., Daly, R., Fort, S., Ivory, R. & McCormack, N. (2014). Carbohydrate Research, 385, 9-17.
A specific and sensitive substrate for the assay of endo-1,4-β-glucanase (cellulase) has been prepared. The substrate mixture comprises benzylidene end-blocked 2-chloro-4-nitrophenyl-β-cellotrioside (BzCNPG3) in the presence of thermostable β-glucosidase. Hydrolysis by exo-acting enzymes such as β-glucosidase and exo-β-glucanase is prevented by the presence of the benzylidene group on the non-reducing end D-glucosyl residue. On hydrolysis by cellulase, the 2-chloro-4-nitrophenyl-β-glycoside is immediately hydrolysed to 2-chloro-4-nitrophenol and free D-glucose by the β-glucosidase in the substrate mixture. The reaction is terminated and colour developed by the addition of a weak alkaline solution. The assay procedure is simple to use, specific, accurate, robust and readily adapted to automation. This procedure should find widespread applications in biomass enzymology and in the specific assay of endo-1,4-β-glucanase in general.Hide Abstract
Mangan, D., McCleary, B. V., Liadova, A., Ivory, R. & McCormack, N. (2014). Carbohydrate Research, 395, 47-51.
There is a growing demand for research tools to aid the scientific community in the search for improved cellulase enzymes for the biofuel industry. In this work, we describe a novel fluorometric assay for cellulase (endo-1,4-β-glucanase) which is based on the use of 4,6-O-benzylidene-4-methylumbelliferyl-β-cellotrioside (BzMUG3) in the presence of an ancillary β-glucosidase. This assay can be used quantitatively over a reasonable linear range, or qualitatively as a solution screening tool which may find extensive use in the area of metagenomics.Hide Abstract