Identification of a novel B-N-acetylhexosaminidase (Pcb-NAHA1) from marine Zoanthid Palythoa caribaeorum (Cnidaria, Anthozoa, Zoanthidea)
| Autor: | Souza, Djair S.L., 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. |
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| Jazyk: | angličtina |
| Rok vydání: | 2008 |
| Předmět: | |
| Zdroj: | Repositório Institucional da UCB Universidade Católica de Brasília (UCB) instacron:UCB |
| Popis: | Made available in DSpace on 2016-10-10T03:52:23Z (GMT). No. of bitstreams: 5 Identification of a novel B N acetylhexosaminidase_Pcb_NAHA1.pdf: 298920 bytes, checksum: b6ceb77cf2800c60845c35248addc653 (MD5) license_url: 52 bytes, checksum: 2f32edb9c19a57e928372a33fd08dba5 (MD5) license_text: 24259 bytes, checksum: f1f24f769b03eb8f9cd3f53c1090841c (MD5) license_rdf: 24658 bytes, checksum: 9d3847733d3c0b59c7c89a1d40d3d240 (MD5) license.txt: 1887 bytes, checksum: 445d1980f282ec865917de35a4c622f6 (MD5) Previous issue date: 2008 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. Sim Publicado |
| Databáze: | OpenAIRE |
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