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).
Colourimetric method for the determination of
endo-1,4-β-glucanase (cellulase) in enzyme preparations and fermentation products
Principle:
(endo-1,4-β-glucanase)
(1) 3-Ketobutylidene-G5-β-PNP + H2O → Blocked-GX + 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
Kit size:
K-CELLG5-4V 120 / 240 assays (manual) / 480 (auto-analyser)
or
K-CELLG5-2V 60 / 120 assays (manual) / 240 (auto-analyser)
Method: Spectrophotometric at 400 nm
Total assay time: 10 min
Detection limit: 3.5 x 10-4 U/mL
Application examples:
Fermentation broths, industrial enzyme preparations, biofuels research
Method recognition: Novel method
Advantages
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