Honda Y, Kirihata M, Fukamizo T, Kaneko S, Tokuyasu K, Brzezinski R
Laboratory of Bioorganic Chemistry, College of Agriculture, Osaka Prefecture University, Sakai, Osaka, 599-8531, Japan.
J Biochem. 1999 Sep;126(3):470-4. doi: 10.1093/oxfordjournals.jbchem.a022475.
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 chitosanase from Streptomyces sp. N174. The enzymatic deacetylation of (GlcNAc)(3)-UMB was confirmed by (1)H-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 k(cat) and K(m) values obtained for the substrate (GlcN)(3)-UMB were determined to be 8.1 x 10(-5) s(-1) and 201 microM, 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.
4-甲基伞形酮基β-壳三糖[（GlcN）₃-UMB]由4-甲基伞形酮基三-N-乙酰-β-壳三糖[（GlcNAc）₃-UMB]使用来自菜豆炭疽菌的几丁质脱乙酰酶制备，并由链霉菌属N174的壳聚糖酶水解。（GlcNAc）₃-UMB的酶促脱乙酰作用通过¹H-NMR光谱和质谱得到证实。当将得到的（GlcN）₃-UMB与壳聚糖酶一起温育时，发现通过在360nm激发获得的450nm处的荧光强度随反应时间成比例增加。荧光强度的增加速率与酶浓度成正比。这表明壳聚糖酶水解GlcN残基和UMB部分之间的糖苷键，释放出荧光性的UMB分子。由于（GlcN）₃本身不能被壳聚糖酶水解，因此（GlcN）₃-UMB被认为是用于壳聚糖酶测定的一种有用的低分子量底物。底物（GlcN）₃-UMB的kcat和Km值分别测定为8.1×10⁻⁵ s⁻¹和201μM。从反应产物的TLC分析发现，壳聚糖酶不仅水解GlcN残基和UMB部分之间的键，还水解GlcN残基之间的键。然而，UMB分子荧光检测的高灵敏度将能够更准确地测定壳聚糖酶的动力学常数。
