60 / 120 assays (manual) / 240 assays (auto-analyser)
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60 / 120 assays (manual) / 240 assays (auto-analyser) or
120 / 240 assays (manual) / 480 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:||1.2 x 10-3 U/mL|
|Reproducibility (%):||~ 3%|
|Total Assay Time:||10 min|
|Application examples:||Fermentation broths, industrial enzyme preparations and biofuels research.|
|Method recognition:||Novel method|
The CellG5 assay reagent for the measurement of endo-cellulase (endo-1,4-β-glucanase) contains two components;
1) 4,6-O-(3-Ketobutylidene)-4-nitrophenyl-β-D-cellopentaoside (BPNPG5) and 2) thermostable β-glucosidase. The ketone blocking group prevents any hydrolytic action by the β-glucosidase on BPNPG5. 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 4-nitrophenol is therefore directly related to the hydrolysis of BPNPG5 by the endo-cellulase. The reaction is terminated and the phenolate colour is developed on addition of Tris buffer solution (pH 9.0).
The CellG5 assay represents a huge step forward in the methodology for the measurement of cellulase that traditionally relied on substrates such as CM-cellulose, Avicel, cellooligosaccharides, filter paper or dyed polysaccharides including CMC Congo red or cellulose azure.
- Very cost effective
- All reagents stable for > 4 years
- Completely specific for cellulase (endo-1,4-glucanase)
- Generally applicable and highly sensitive
- 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)
Prediction of potential malt extract and beer filterability using conventional and novel malt assays.
Cornaggia, C., Evans, D. E., Draga, A., Mangan, D. & McCleary, B. V. (2019). Journal of Institute of Brewing, In Press.
Colourimetric assays were used to measure the activities of six key hydrolases endogenous to barley: β‐glucanase, xylanase, cellulase, α-amylase, beta‐amylase and limit dextrinase. The analysed barley malt samples were previously characterised by 27 conventional malt quality descriptors. Correlations between enzymatic activities and brewing parameters such as extract yield, fermentability, viscosity and filterability were investigated. A single extraction protocol for all six hydrolases was optimised and used for multi‐enzyme analysis using fully automatable assay formats. A regression analysis between malt parameters was undertaken to produce a relationship matrix linking enzyme activities and conventional malt quality descriptors. This regression analysis was used to inform a multi‐linear regression approach to create predictive models for extract yield, apparent attenuation limit, viscosity and filterability using the Small‐scale Wort rapId Filtration Test (SWIFT) and two different mashing protocols – Congress and a modified infusion mash at 65oC (MIM 65oC). It was observed that malt enzyme activities displayed significant correlations with the analysed brewing parameters. Both starch hydrolases and cell wall hydrolase activities together with modification parameters (i.e. Kolbach index) were found to be highly correlated with extract yield and apparent attenuation limit. Interestingly, it was observed that xylanase activity in malts was an important predictor for wort viscosity and filterability. It is envisaged that the automatable measurement of enzyme activity could find use in plant breeding progeny selection and for routine assessment of the functional brewing performance of malt batches. This analytical approach would also contribute to brewing process consistency, product quality and reduced processing times.Hide Abstract
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 22.214.171.124) 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
Gong, G., Kim, S., Lee, S. M., Woo, H. M., Park, T. H., & Um, Y. (2017). Journal of Biotechnology, 254, 59-62.
Technologies for degradation of three major components of lignocellulose (e.g. cellulose, hemicellulose and lignin) are needed to efficiently utilize lignocellulose. Here, we report Bacillus sp. 275 isolated from a mudflat exhibiting various lignocellulolytic activities including cellulase, xylanase, laccase and peroxidase in the cell culture supernatant. The complete genome of Bacillus sp. 275 strain contains 3832 protein cording sequences and an average G + C content of 46.32% on one chromosome (4045,581bp) and one plasmid (6389bp). The genes encoding enzymes related to the degradation of cellulose, xylan and lignin were detected in the Bacillus sp. 275 genome. In addition, the genes encoding glucosidases that hydrolyze starch, mannan, galactoside and arabinan were also found in the genome, implying that Bacillus sp. 275 has potentially a wide range of uses in the degradation of polysaccharide in lignocellulosic biomasses.Hide Abstract