Identification of a novel B-N-acetylhexosaminidase (Pcb-NAHA1) from marine Zoanthid Palythoa caribaeorum (Cnidaria, Anthozoa, Zoanthidea)

• Autor(a) principal: Souza, Djair S.L.
• Data de Publicação: 2008
• Outros Autores: Grossi-de-Sá, Maria Fátima, Silva, Luciano P., Franco, Octavio L., Gomes-Júnior, José E., Oliveira, Gustavo R., Rocha, Thales L., Magalhães, Cláudio P., Marra, Brener M., Grossi-de-Sá, Maíra, Romano, Eduardo, Martins de Sá, César, Kombrink, Erich, Jiménez, Arnubio V., Abreu, Luiz R.D.
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Repositório Institucional da UCB
• Texto Completo: http://twingo.ucb.br:8080/jspui/handle/10869/443; https://repositorio.ucb.br:9443/jspui/handle/123456789/7696
• topic: b-N-Acetylhexosaminidase; Palythoa caribaeorum; Chitin-active-enzymes; Inactivated chitinases

B-N-Acetylhexosaminidases (EC 3.2.1.52) belong to an enzyme family that hydrolyzes terminal b-D-N-glucosamine and b-D-N-galactosamine residues from oligosaccharides. In this report, we purified a novel b-N- acetylhexosaminidase (Pcb-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-b-D-Nacetylglucosamide (pNP-GlcNAc) and to a lesser extent p-nitrophenyl-2-acetoamide-2-deoxyamide-2-deoxy-b-D-N-acetylgalactosamide (pNP-GalNAc). Detailed kinetic analysis using pNP-GlcNAc resulted in a specific activity of 57.9 U/mg, a Km value of 0.53 mM and a Vmax value of 88.1 lmol/h/mg and kcat 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 _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 b-N-acetylhexosaminidases, representing a type of exochitinolytic activity, and endo-chitinases share common functional domains and/or may have evolved from a common ancestor.