Tese (doutorado)—Universidade de Brasília, Faculdade de Medicina, 2007. / Submitted by Larissa Ferreira dos Angelos (ferreirangelos@gmail.com) on 2010-01-07T22:03:45Z
No. of bitstreams: 1
2007_MonicadeOliveiraSantos.PDF: 948235 bytes, checksum: 8bcc87f3398c3399d83dc39e4e2a3cba (MD5) / Approved for entry into archive by Daniel Ribeiro(daniel@bce.unb.br) on 2010-01-07T22:39:25Z (GMT) No. of bitstreams: 1
2007_MonicadeOliveiraSantos.PDF: 948235 bytes, checksum: 8bcc87f3398c3399d83dc39e4e2a3cba (MD5) / Made available in DSpace on 2010-01-07T22:39:25Z (GMT). No. of bitstreams: 1
2007_MonicadeOliveiraSantos.PDF: 948235 bytes, checksum: 8bcc87f3398c3399d83dc39e4e2a3cba (MD5)
Previous issue date: 2007-12-12 / Um cDNA (Pbnag, nº de acesso GenBank AF 419158) codificante para N-acetil-β-D-glicosaminidase (NAG) de Paracoccidioides brasiliensis, isolado Pb 01 (ATCC MYA826) foi caracterizado e classificado como uma enzima pertencente à família 20 de glicosil hidrolase. PbNAG foi clonado em pGEX-4T3 e expresso em Escherichia coli. A análise em gel apresentou a presença de uma proteína de massa molecular estimada em 64,8 kDA. A proteína expressa foi purificada em resina glutationa-sepharose 4B e utilizada para produção de anticorpo policlonal. O anticorpo produzido anti-PbNAG apresentou reação com a proteína nativa, em extrato total, de micélio e levedura de P. brasiliensis. Na imunocitolocalização o anticorpo anti-PbNAG reagiu com a proteína NAG nativa presente na parede de células leveduriformes P. brasiliensis. A enzima recombinante PbNAG foi caracterizada apresentando afinidade pelo substrato ρ-nitrofenil-β-D-N-acetilglucosamina (ρNP-GlcNAc), com Km de 0.112 mM, Vmax de 0.0017 mM min-1 mg proteina-1, temperatura ótima de 37ºC e pH ótimo de 4.4. A enzima foi pouco inibida por glicose e apresentou inibição por glicosamina, N-acetilglicosamina. Os estudos mostram que PbNAG está presente na parede celular de células leveduriformes de P. brasiliensis, sugerindo seu papel no metabolismo da parede fúngica. ____________________________________________________________________________________ ABSTRACT / The full-length cDNA Pbnag enconding a N-acetyl-β-Dglucosaminidase (NAG) GeneBank accession number AF 419158 of Paracoccidioides brasiliensis was characterized and classified as a NAG belonging to the glycosil hydrolase family 20. The PbNAG was expressed in Escherichia coli and purified to homogeneity by affinity chromatography on glutathione-Sepharose 4B. SDS-PAGE analysis revealed the presence of one protein species with molecular mass estimated of 64.8 kDa. Immunoelectron microscopy demonstrated the presence of the native protein in the cell wall of P. brasiliensis. The recombinant protein was evaluated regarding to its kinetics properties The Km and Vmax values for the enzyme using ρ- nitrophenyl-β-D-N-acetylglucosamine (ρNP-GlcNAc) as substrate, were 0.112 mM and 0.0017 mM, respectively. The pH optimum for the enzyme was 4.4 and maximum activity was obtained at 37 °C. The enzyme activity was poorly affected by glucose and was inhibited by glucosamine, Nacetylglucosaminide. The studies show that PbNAG is present in the cell wall of yeast cells of P. brasiliensis suggesting its potential role in the fungal cell wall additionally.
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.unb.br:10482/3037 |
| Date | 12 December 2007 |
| Creators | Santos, Mônica de Oliveira |
| Contributors | Soares, Célia Maria de Almeida |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Source | reponame:Repositório Institucional da UnB, instname:Universidade de Brasília, instacron:UNB |
| Rights | info:eu-repo/semantics/openAccess |
Page generated in 0.0025 seconds