Chitosanase-catalyzed hydrolysis of 4-methylumbelliferyl beta-chitotrioside

4-Methylumbelliferyl beta-chitotrioside [(GlcN)(3)-UMB] was prepared from 4 -methylumbelliferyl tri-N-acetyl-beta-chitotrioside [(GlcNAc)(3)-UMB] using chitin deacetylase from Colletotrichum lindemuthianum, and hydrolyzed by c hitosanase from Streptomyces sp. N174, The enzymatic deacetylation of (GlcN Ac)(3)-UMB was confirmed by H-1-NMR spectroscopy and mass spectrometry, Whe n the (GlcN)(3)-UMB obtained was incubated with chitosanase, the fluorescen ce intensity at 450 nm obtained by excitation at 360 nm was found to increa se with proportion to the reaction time, The rate of increase in the fluore scence intensity was proportional to the enzyme concentration. This indicat es that chitosanase hydrolyzes the glycosidic linkage between a GlcN residu e and UMB moiety releasing the fluorescent UMB molecule, Since (GlcN), itse lf cannot be hydrolyzed by the chitosanase, (GlcN)(3)-UMB is considered to be a useful low molecular weight substrate for the assay of chitosanase. Th e k(cat) and K-m values obtained for the substrate (GlcN)(3)-UMB were deter mined to be 8.1 x 10(-5) s(-1) and 201 mu M, respectively. From TLC analysi s of the reaction products, the chitosanase was found to hydrolyze not only the linkages between a GlcN residue and UMB moiety, but also the linkages between GlcN residues. Nevertheless, the high sensitivity of the fluorescen ce detection of the UMB molecule would enable a more accurate determination of kinetic constants for chitosanases.