title: Identification of a novel beta-N-acetylhexosaminidase (Pcb-NAHA1) from marine Zoanthid Palythoa caribaeorum (Cnidaria, Anthozoa, Zoanthidea).
abstract: 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 (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 25kDa. The purified enzyme preferentially hydrolyzed p-nitrophenyl-2-acetoamide-2-deoxyamide-2-deoxy-beta-d-N-acetylglucosamide (pNP-GlcNAc) and to a lesser extent p-nitrophenyl-2-acetoamide-2-deoxyamide-2-deoxy-beta-d-N-acetylgalactosamide (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 micromol/h/mg and k(cat) value of 0.61s(-1). Furthermore, purified Pcb-NAHA1 enzyme activity was decreased by Hg Cl(2) or maltose and stimulated in the presence of Na(2)SeO(4,) BaCl(2), MgCl(2,) 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.
authors.affiliation: Embrapa Recursos Genéticos e Biotecnologia, PqEB-Final W5 Norte-Cp02372, Brasilia, DF, Brazil.
authors.forename: Djair S L
authors.initials: DS
authors.lastname: Souza
authors.forename: Maria F
authors.initials: MF
authors.lastname: Grossi-de-Sa
authors.forename: Luciano P
authors.initials: LP
authors.lastname: Silva
authors.forename: Octavio L
authors.initials: OL
authors.lastname: Franco
authors.forename: José E
authors.initials: JE
authors.lastname: Gomes-Junior
authors.forename: Gustavo R
authors.initials: GR
authors.lastname: Oliveira
authors.forename: Thales L
authors.initials: TL
authors.lastname: Rocha
authors.forename: Cláudio P
authors.initials: CP
authors.lastname: Magalhães
authors.forename: Brener M
authors.initials: BM
authors.lastname: Marra
authors.forename: Maíra
authors.initials: M
authors.lastname: Grossi-de-Sa
authors.forename: Eduardo
authors.initials: E
authors.lastname: Romano
authors.forename: César Martins
authors.initials: CM
authors.lastname: de Sá
authors.forename: Erich
authors.initials: E
authors.lastname: Kombrink
authors.forename: Arnubio V
authors.initials: AV
authors.lastname: Jiménez
authors.forename: Luiz R D
authors.initials: LR
authors.lastname: Abreu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D037421:Anthozoa / Q000201:enzymology* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013379:Substrate Specificity; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D001619:beta-N-Acetylhexosaminidases