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doi:10.1016/j.pep.2007.10.024 
Copyright © 2007 Elsevier Inc. All rights reserved.
Identification of a novel β-N-acetylhexosaminidase (Pcb-NAHA1) from marine Zoanthid Palythoa caribaeorum (Cnidaria, Anthozoa, Zoanthidea)
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Djair S.L. Souzaa, b, c, Maria F. Grossi-de-Saa, d, 
, 
, , Luciano P. Silvaa, Octavio L. Francod, José E. Gomes-Juniora, b, Gustavo R. Oliveiraa, e, Thales L. Rochaa, Cláudio P. Magalhãesa, Brener M. Marraa, b, Maíra Grossi-de-Saa, Eduardo Romanoa, César Martins de Sáb, Erich Kombrinkf, Arnubio V. Jiméneza, b, g and Luiz R.D. Abreuc
Received 7 October 2007;
Abstract
β-N-Acetylhexosaminidases (EC 3.2.1.52) belong to an enzyme family that hydrolyzes terminal β-d-N-glucosamine and β-d-N-galactosamine residues from oligosaccharides. In this report, we purified a novel β-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-β-d-N-acetylglucosamide (pNP-GlcNAc) and to a lesser extent p-nitrophenyl-2-acetoamide-2-deoxyamide-2-deoxy-β-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 μmol/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 β-N-acetylhexosaminidases, representing a type of exochitinolytic activity, and endo-chitinases share common functional domains and/or may have evolved from a common ancestor.
Keywords: β-N-Acetylhexosaminidase; Palythoa caribaeorum; Chitin-active-enzymes; Inactivated chitinases
Article Outline
- Materials and methods
- Materials
- Extraction and purification of the β-N-acetylhexosaminidase
- β-N-Acetylhexosaminidase assays
- Chitinolytic assays
- Mass spectrometry and de novo sequencing
- Immunoblotting
- β-N-acetylhexosaminidase biochemical characterization
- Results
- Purification of β-N-acetylhexosaminidase
- Substrate specificity
- Determination of Km and Vmax
- Effect of pH and temperature
- Effect of chemical compounds on enzyme activity
- Sequence and Western blot analysis of Pcb-NAHA1
- Discussion
- Purification of β-N-acetylhexosaminidase
- Substrate specificity
- Determination of Km and Vmax
- Effects of pH and temperature
- Effect of chemical compounds on enzyme activity
- Sequence and Western blot analysis of Pcb-NAHA1
- Acknowledgements
- References
