We report a novel enzyme from the culture filtrate of Amycolatopsis orientalis, that endoglycosidically releases an N-acetyl-lactosamine-repeating unit (Gal beta 1,4GIcNAc beta 1,3Gal beta 1,4GlcNAc, LN2) from a synthetic chromogenic substrate Gal beta 1,4GIcNAc beta 1,3Gal beta 1,4GlcNAc beta-pNP (1). The enzyme activity was purified by 80% saturated ammonium sulfate precipitation followed by gel filtration and affinity chromatography. The enzyme splits 1, Gal beta 1,4GlcNAcl beta-pNP (2), GlcNAc beta 1,3Gal beta 1,4G1cNAc beta-pNP (3), and GIcNAc beta 1,4GlcNAc beta-pNP (4) into the corresponding oligosaccharides and p-nitrophenol. The catalytic efficiencies (k(cat)/K-m) 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 GlcNAc beta 1,4GlcNAc beta 1,4GlcNAc beta-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. (c) 2005 Elsevier Inc. All rights reserved.