COMPARISON OF 4 METHODS FOR MEASURING CHITINASE ACTIVITY AND THE APPLICATION OF THE 4-MUF ASSAY IN AQUATIC ENVIRONMENTS

Four methods were compared for measuring chitinase activity in natural aquatic environments and microcosms. The four methods included: (1) the enumeration of chitinoclastic microorganisms; (2) the use of chitin azure as a substrate; (3) the use of fluorogenic [4-methylumbelliferyl (4-MUF)] chitin derivatives as substrates; and (4) the use of H-3-chitin as a substrate. The 4-MUF assay required the least amount of preparation, the shortest time to complete, and was one of the most sensitive and highly reproducible assays. Optimum assay conditions included: (1) concentration of the trimer (4-MUF-beta-D-N,N',N" triacetylchitotriose) at 5-mu-M.ml-1; (2) pH at 7.4; (3) temperature 35-degrees-C; (4) time at 24 h; and (5) final pH after incubation adjusted to 10-11 (to enhance fluorescence). Using this method, chitinase activity was determined to be highest in the aerobic region of aquatic sediments, slightly lower in the anaerobic zone, and lowest in the water column (5% of the total activity). The majority of enzymatic activity in sediments was associated with organic particles (95%) while 5% was associated with inorganic matter (sand). The results suggest that the 4-MUF assay is a sensitive, reliable, easily applied method for measuring chitinase activity in aquatic environments.