A rapid and sensitive assay for chitinase using tritiated chitin

Radioactive chitin, prepared by acetylation of chitosan with tritiated acetic anhydride, was used as substrate in a rapid and extremely sensitive assay for chitinase. The procedure is based on the insolubility of chitin and the solubility in water of the reaction product, diacetylchitobiose. The course of the chitinase reaction is nonlinear, a result that cannot be attributed to an artifact of the method, to inhibition by product, or to instability of the enzyme. Some evidence points to structural heterogeneity of the substrate as a cause for this behavior. Reacetylated chitosan was also used as an adsorbent in the purification of chitinase with better results than with the previously used colloidal chitin.