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

• Specific, fluorometric enzyme-coupled assay for the measurement of cellulase is reported.
• The synthesis of the fluorometric substrate is described.
• Benzylidene acetal functionalisation prevents action of the exo-glycosidase present.
• 4-Methylumbelliferyl functionalisation provides fluorogen for simple quantitative detection.
• Substrate can also be employed as a highly sensitive probe for metagenomic cellulase screening.

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.

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