Cellulase Assay Kit (CellG3 Method)

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.

Suitable for auto/analyser formats.

Product Code
180 / 360 assays per kit / 720 (auto-analyser)

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Colourimetric method for the determination of endo-1,4-β-
glucanase (cellulase) in enzyme preparations and fermentation

(1) Benzylidene-G3-β-CNP + H2O → Blocked-GX + G(3-X)-β-CNP

                (thermostable β-glucosidase)
(2) G(3-X)-β-CNP + H2O → D-glucose + CNP

      (alkaline solution)
(3) CNP → phenolate ion (yellow colour)
Note: CNP = 2-Chloro-4-nitrophenol

Kit size:                            180 / 360 assays / 720 (auto-analyser)
Method:                            Spectrophotometric at 400 nm
Total assay time:              ~ 20 min
Detection limit:                0.05 U/mL
Application examples:
Fermentation broths, industrial enzyme preparations and biofuels research
Method recognition:       Novel method
                                The analogous fluorimetric reagent (R-CELLFLR)
                                           is also available with 10-fold greater sensitivity.


  • 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

Novel substrates for the measurement of endo-1,4-β-glucanase (endo-cellulase).

McCleary, B. V., Mangan, D., Daly, R., Fort, S., Ivory, R. & McCormack, N. (2014). Carbohydrate Research, 385, 9-17.

Quantitative fluorometric assay for the measurement of endo-1,4-β-glucanase.

Mangan, D., McCleary, B. V., Liadova, A., Ivory, R. & McCormack, N. (2014). Carbohydrate Research, 395, 47-51.

A novel automatable enzyme-coupled colorimetric assay for endo-1,4-β-glucanase (cellulase).

Mangan, D., Cornaggia, C., McKie, V., Kargelis. T. & V. McCleary, B. V. (2016). Analytical and Bioanalytical Chemistry, 408(15), 4159-4168.

Balanced trafficking between the ER and the Golgi apparatus increases protein secretion in yeast.

Bao, J., Huang, M., Petranovic, D. & Nielsen, J. (2018). AMB Express, 8(1), 37.

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