A new spectrophotometric method of assay for chitosanase based on Calcofluor white dye binding.

A new spectrophotometric method for the assay of chitosanase based on complex
formation of the substrate chitosan with Calcofluor white dye is described. The
absorption maximum for the chitosan--Calcofluor complex is determined to be
406 nm. The apparent minimum size of chitosan for complex formation is 5-7 kDa.
Therefore, those enzymes that do not generate glucosamine or reducing groups as
products of hydrolysis at levels not measurable by the available methods of assay
can be assayed by the present method. In the standardized procedure 2OOpg of
chitosan in acetate buffer pH 4.5 with the enzyme in a reaction volume of 1 S ml is
incubated at 45°C for 1 h, after which 1.5 ml of Calcofluor white (0.05%) is added,
kept for 1 h and absorbance at 406nm measured by a spcctrophotometer. The
chitosanase unit is arbitrarily defined as the reduction in absorbance by O.Ol/min.