Purification and characterization of a chitinase from Amycolatopsis orientalis with N-acetyllactosamine-repeating unit releasing activity

We report a novel enzyme from the culture filtrate of Amycolatopsis orientalis, that endoglycosidically releases an N-acetyllactosamine-repeating unit (Galβ1,4GlcNAcβ1,3Galβ1,4GlcNAc, LN2) from a synthetic chromogenic substrate Galβ1,4GlcNAcβ1,3Galβ1,4GlcNAcβ-pNP (1). The enzyme activity was purified by 80% saturated ammonium sulfate precipitation followed by gel filtration and affinity chromatography. The enzyme splits 1, Galβ1,4GlcNAcβ-pNP (2), GlcNAcβ1,3Galβ1,4GlcNAcβ-pNP (3), and GlcNAcβ1,4GlcNAcβ-pNP (4) into the corresponding oligosaccharides and p-nitrophenol. The catalytic efficiencies (kcat/Km) for compounds 1, 2, and 4 were 0.6, 0.05, and 13, respectively. Compound 4 acts as a fairly good substrate for the enzyme, and LN2-releasing activity was inhibited by 4 and GlcNAcβ1,4GlcNAcβ1,4GlcNAcβ-pNP (7), indicating that this enzyme activity is derived from a kind of chitinase. The enzyme hydrolyzed 1 by a mechanism leading to retention of the anomeric configuration. This is the first report of a N-acetyllactosamine-repeating unit releasing enzyme.