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Título: | Purification and characterization of an extracellular trypsin-like protease of Fusarium oxysporum var. lin. |
Autor(es): | Barata, Ricardo Andrade Andrade, Milton Hércules Guerra de Rodrigues, Roberta Dias Castro, Ieso de Miranda |
Palavras-chave: | Trypsin-like protease Fusarium Inhibitor effects |
Data do documento: | 2002 |
Referência: | BARATA, R. A. et al. Purification and characterization of an extracellular trypsin-like protease of Fusarium oxysporum var. lin. Journal of Bioscience and Bioengineering USA, v. 94, n. 4, p. 304-308, 2002. Disponível em: <http://www.sciencedirect.com/science/article/pii/S1389172302801682>. Acesso: 10 jan. 2017. |
Resumo: | An alkaline serineprotease, capable of hydrolyzing Nu-benzoyl-DL arginine p-nitroanilide, was secreted by Fusurium oxysporum var. hi grown in the presence of gelatin as the sole nitrogen and carbon source. The protease was purified 65-fold to electrophoretic homogenity from the culture supernatant in a three-step procedure comprising QSepharose chromatography, aMnity chromatography, and FPLC on a MonoQ column. SDS-PAGE analysis of the purified protein indicated an estimated molecular mass of 41 kDa. The protease had optimum activity at a reaction temperature of 45OC and showed a rapid decrease of activity at 48OC. The optimum pH was around 8.0. Characterization of the protease showed that Ca*+ and MgZ+ cations increased the activity, which was not inhibited by EDTA or l,lO-phenanthroline. The enzyme activity on Nubenzoyl-DL arginine p-nitroanilide was inhibited by 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride,p-aminobenzamidine dihydrochloride, aprotinin, 3-4 dichloroisocoumarin, and IVtosyl-L-lysine chloromethyl ketone. The enzyme is also inhibited by substrate concentrations higher than 2.5x lo-4 M. The protease had a Michaelis-Menten constant of 0.16 mM and a V,, of 0.60 pm01 released product .min-‘.mg’ enzyme when assayed in a non-inhibiting substrate concentration. The activity on Nu-benzoyl-DL arginine p-nitroanilide was competitively inhibited by p-aminobenzamidine dihydrochoride. A Ki value of 0.04 mM was obtained. |
URI: | http://www.repositorio.ufop.br/handle/123456789/8915 |
Link para o artigo: | http://www.sciencedirect.com/science/article/pii/S1389172302801682 |
DOI: | https://doi.org/10.1016/S1389-1723(02)80168-2 |
ISSN: | 1389-1723 |
Licença: | O periódico Journal of Bioscience and Bioengineering concede permissão para depósito deste artigo no Repositório Institucional da UFOP. Número da licença: 3266010491564. |
Aparece nas coleções: | DEFAR - Artigos publicados em periódicos |
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ARTIGO_PurificationCharacterizationExtracellular.pdf | 590,37 kB | Adobe PDF | Visualizar/Abrir |
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