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

• Novel, selective, enzyme-coupled assay for the measurement of cellulase is reported.
• The synthesis of a range of colourimetric cellulase substrates is described.
• Benzylidene acetal functionalisation prevents action of the exo-glycosidase present.
• Nitrophenyl functionalisation provides a chromophore for simple quantitative detection.
• Analysis of various cellulase enzymes acting on these substrates is performed.

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.

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