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

Souza DS, Grossi-de-Sa MF, Silva LP, Franco OL, Gomes-Junior JE, Oliveira GR, Rocha TL, Magalhães CP, Marra BM, Grossi-de-Sa M, Romano E, de Sá CM, Kombrink E, Jiménez AV, Abreu LR

Embrapa Recursos Genéticos e Biotecnologia, PqEB-Final W5 Norte-Cp02372, Brasilia, DF, Brazil.

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.

Published 1 February 2008 in Protein Expr Purif, 58(1): 61-9.
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