Quantitative fluorometric analysis of plant and microbial chitosanases

Abstract

A quantitative fluorometric assay for chitosanase activity in bacterial and plant tissues was developed. The assay can be conducted with either finely milled preparations of chitosan in suspension or dissolved chitosan; activity is based on measurements of glucosamine (GlcN) or oligomers of GlcN. GlcN is detected fluorometrically after reaction with fluorescamine with detection in the nanomole range. Fluorescence measurements of chitosanase activity and radioassay of chitinase in commercial preparations of chitinase from Streptomyces griseus revealed that both activities were present. Specific activities for the S. griseus chitosanase using suspended and soluble chitosans were respectively 1.24 and 6.4 μmol GlcN·min −1·mg protein −1. Specific activity of the S. griseus chitinase was 0.98 μmol GlcN·min −1·mg protein −1. Sweet orange callus tissue was tested for chitosanase and chitinase activity. It was necessary to remove small amine-containing molecules from the callus preparations before chitosanase activity could be assayed. The specific activity for chitinase and chitosanase in desalted extracts of nonembryogenic Valencia sweet orange callus tissue was determined to be 18.6 and 89.4 nmol GlcN·min −1·mg protein −1, respectively.