beta-N-Acetylhexosaminidases (EC 3.2.1.52) belong to an enzyme family that hydrolyzes terminal beta-D-N-glucosamine and beta-D-N-galactosamine residues from oligosaccharides. In this report, we purified a novel beta-N-acetylhexosaminidase (Peb-NAHA1) from the marine zoanthid Palythoa caribaeorum by applying ammonium sulfate fractionation, affinity chromatography on a chitin column, followed by two rounds of size exclusion chromatography. SDS-PAGE analysis indicated a single band protein of apparent homogeneity with a molecular mass of 25 kDa. The purified enzyme preferentially hydrolyzed p-nitrophenyl-2-acetoamide-2-deoxyamide-2-deoxy-beta-D-N-acetylglucosami de (pNP-GlcNAc) and to a lesser extent p-nitrophenyl-2-acetoamide-2-deoxyamide-2-deoxy-beta-D-N-acetylgalactosa mide (pNP-GalNAc). Detailed kinetic analysis using pNP-GlcNAc resulted in a specific activity of 57.9 U/mg, a K-m value of 0.53 mM and a V-max value of 88.1 mu mol/h/mg and k(cat) value of 0.61 s(-1). Furthermore, purified Pcb-NAHA1 enzyme activity was decreased by HgCl2 or maltose and stimulated in the presence of Na2SeO4, BaCl2, MgCl2, chondroitin 6-sulfate, and phenylmethylsulfonylfluoride. The optimum activity of Pcb-NAHA1 was observed at pH 5.0 and elevated temperatures (45-60 degrees C). Direct sequencing of proteolytic fragments generated from Pcb-NAHA1 revealed remarkable similarities to plant chitinases, which belong to family 18, although no chitinase activity was detected with Pcb-NAHA1 We conclude that beta-N-acetylhexosaminidases, representing a type of exochitinolytic activity, and endo-chitinases share common functional domains and/or may have evolved from a common ancestor. (c) 2007 Elsevier Inc. All rights reserved.