Chitosanase-Catalyzed Hydrolysis of 4-Methylumbelliferyl -Chitotr ioside

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Abstract
4-Methylumbelliferyl β-chitotrioside [(GlcN)3-UMB] was prepared from 4-methylumbel-liferyl tri-N-acetyl-β-chitotrioside [(GlcN Ac)3-UMB] using chitin deacetylase from Col-letotrichum lindemuthianum, and hydrolyzed by chitosanase from Streptomyces sp. N174. The enzymatic deacetylation of (GlcNAc)3-UMB was confirmed by 1H-NMR spectroscopy and mass spectrometry. When the (GlcN)3-UMB obtained was incubated with chitosanase, the fluorescence intensity at 450 nm obtained by excitation at 360 nm was found to increase with proportion to the reaction time. The rate of increase in the fluorescence intensity was proportional to the enzyme concentration. This indicates that chitosanase hydrolyzes the glycosidic linkage between a GlcN residue and UMB moiety releasing the fluorescent UMB molecule. Since (GlcN)3 itself cannot be hydrolyzed by the chitosanase, (GlcN)3-UMB is considered to be a useful low molecular weight substrate for the assay of chitosanase. The kat and Km values obtained for the substrate (GlcN)3-UMB were determined to be 8.1 ×10−5 s−1 and 201 μM, respectively. From TLC analysis 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 fluorescence detection of the UMB molecule would enable a more accurate determination of kinetic constants for chitosanases.

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