Screening, production and biochemical characterization of a new fibrinolytic enzyme produced by Streptomyces sp. (Streptomycetaceae) isolated from Amazonian lichens

Thrombosis is a pathophysiological disorder caused by accumulation of fibrin in the blood. Fibrinolytic proteases with potent thrombolytic activity have been produced by diverse microbial sources. Considering the microbial biodiversity of the Amazon region, this study aimed at the screening, production and biochemical characterization of a fibrinolytic enzyme produced by Streptomyces sp. isolated from Amazonian lichens. The strain Streptomyces DPUA1576 showed the highest fibrinolytic activity, which was 283 mm2. Three variables at two levels were used to assess their effects on the fibrinolytic production. The parameters studied were agitation (0.28 - 1.12 g), temperature (28 - 36 ºC) and pH (6.0 - 8.0); all of them had significant effects on the fibrinolytic production. The maximum fibrinolytic activity (304 mm2) was observed at 1.12 g, 28 ºC, and pH of 8.0. The crude extract of the fermentation broth was used to assess the biochemical properties of the enzyme. Protease and fibrinolytic activities were stable during 6 h, at a pH ranging from 6.8 to 8.4 and 5.8 to 9.2, respectively. Optimum temperature for protease activity ranged between 35 and 55 °C, while the highest fibrinolytic activity was observed at 45 ºC. Proteolytic activity was inhibited by Cu2+ and Co2+ ions, phenylmethylsulfonyl fluoride (PMSF) and pepstatin A, which suggests that the enzyme is a serine protease. Enzymatic extract cleaved fibrinogen at the subunits A-chain, A-chain, and -chain. The results indicated that Streptomyces sp. DPUA 1576 produces enzymes with fibrinolytic and fibrinogenolytic activity, enzymes with an important application in the pharmaceutical industry.The authors grateful acknowledge the financial support of Fundação de Amparo a Pesquisa do Estado de Pernambuco (FACEPE, Pernambuco, Brazil, N. 0158-2.12/11), CNPq/ RENORBIO (National Counsel of Technological and Scientific Development, N.55146/2010-3) and National Council for the Improvement of Higher Education (CAPES, Brazil) for the scholarship. The author thanks editor and reviewers for their review and comments.info:eu-repo/semantics/publishedVersio

Response surface analysis on the effect of temperature and pH on Growth and proteolytic activity of thermophilic Bacillus sp

Proteolytic activity and cell biomass of thermophilic Bacillus sp. strain were evaluated at various levels of initial pH and temperature by applying response surface methodology. The mineral medium containing yeast extract (0.01%) and starch (1%) was used throughout the experiment. The results of statistical analysis revealed the polynomial model with high coefficient of determination (R² = 0.8) for the biomass and total proteolytic activity of the strain studied. This model showed a satisfactory adjustment of the statistic model with the experimental data. The p values showed that the temperature and pH had significant effect on biomass and proteolytic activity (P<0.05) of strain tested. The highest proteolytic activity (2.333 U/ml/h) of the Bacillus sp. was predicted at 41º C and pH 4.8. The high biomass values were observed at broad range of temperature and pH.<br>Atividade proteolítica total e biomassa de uma linhagem de Bacillus sp. termofílico foram analisados em vários níveis de pH inicial e temperatura utilizando a metodologia de superfície de resposta. O meio mineral com extrato de levedura (0.01%) e amido (1%) foi utilizado no experimento. Os resultados de análise estatística da metodologia de resposta de superfície definiram um modelo polinomial para a biomassa e atividade proteolítica da linhagem de Bacillus sp. com alto coeficiente de determinação (R² = 0.8), mostrando um ajuste satisfatório do modelo estatístico obtido com os dados experimentais. Os valores de p mostraram que a temperatura e pH tiveram efeito significante em biomassa e atividade total (P <0.05) da linhagem testada. A atividade proteolítica mais alta (2.368 U/ml/h) da linhagem de Bacilus sp. foi prevista pelas curvas de superfície de resposta em temperatura a 41º C e pH igual a 4.8. Os valores de biomassa altos foram previstos para ampla faixa de temperatura e pH