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.

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

Principle:
(endo-1,4-β-glucanase)
(1) 3-Ketobutylidene-G₅-β-PNP + H₂O → Blocked-G_X + G_(5-X)-β-PNP

(thermostable β-glucosidase)
(2) G_(5-X)-β-PNP + H₂O → D-glucose + PNP

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