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Optimization of production, biochemical characterization and in vitro evaluation of the therapeutic potential of fibrinolytic enzymes from a new Bacillus amyloliquefaciens

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Abstract

The capacity of fibrinolytic enzymes to degrade blood clots makes them of high relevance in medicine and in the pharmaceutical industry. In this work, forty-three microorganisms of the genus Bacillus were evaluated for their potential to produce fibrinolytic proteases. Thirty bacteria were confirmed as producers of fibrinolytic enzymes, the best results obtained for the strain Bacillus amyloliquefaciens UFPEDA 485. The optimization of the enzyme production conditions was done by a central composite design (CCD) star 23 that allowed to define the optimal conditions for soybean flour and glucose concentrations and agitation rate. The highest fibrinolytic activity (FA) of 813 U mL–1 and a degradation of blood clot in vitro of 62% were obtained in a medium with 2% (w/v) of soybean flour and 1% (w/v) glucose at 200 rpm after 48 h of cultivation, at pH 7.2 and 37 °C. The obtained fibrinolytic enzyme was characterized biochemically. Fibrinolytic activity was inhibited by PMSF (fluoride methylphenylsulfonyl - C7H7FO2S) 91.52% and EDTA (ethylenediaminetetraacetic acid - C10H16N2O8) 89.4%, confirming to be a serine-metallo protease. The optimum pH and temperature were 7.0 and 37 oC, respectively, and the enzyme was stable for 12 h. The fibrinolytic activity at physiological conditions of this enzyme produced by Bacillus amyloliquefaciens UFPEDA 485, as well as its long term stability, demonstrate that it has suitable characteristics for human and veterinary applications, and promises to be a powerful drug for the treatment of vascular diseases.

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Authors and Affiliations

  1. Department of Morphology and Animal Physiology, Federal Rural University of Pernambuco, 52171-900, Recife, PE, Brazil

    Fabiana América Silva Dantas de Souza, Amanda Emmanuelle Sales, Raquel Pedrosa Bezerra & Ana Lúcia Figueiredo Porto

  2. Department of Natural Sciences, University of Pernambuco (Campus Mata Norte), 55.800-000, Nazaré da Mata, PE, Brazil

    Fabiana América Silva Dantas de Souza

  3. Laboratory of Immunopathology Keizo Asami, Federal University of Pernambuco, 50670-901, Recife, PE, Brazil

    Pablo Eugênio Costa e Silva & Ana Lúcia Figueiredo Porto

  4. Department of Nanotechnology, Center of Strategic Technologies of the Northeast, 50740-540, Recife, PE, Brazil

    Germana Michelle de Medeiros e Silva

  5. Department of Antibiotics, Federal University of Pernambuco, 50670-901, Recife, PE, Brazil

    Janete Magali de Araújo

  6. Center for Research in Environmental Sciences, Catholic University of Pernambuco, 50050-900, Recife, PE, Brazil

    Galba Maria de Campos Takaki

  7. Academic Unit of Garanhuns, Federal Rural University of Pernambuco, 55296-901, Garanhuns, PE, Brazil

    Tatiana Souza Porto

  8. Centre of Biological Engineering, University of Minho, Campus Gualtar, 4710-057, Braga, Portugal

    José António Couto Teixeira

Authors
  1. Fabiana América Silva Dantas de Souza
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  2. Amanda Emmanuelle Sales
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  3. Pablo Eugênio Costa e Silva
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  6. Janete Magali de Araújo
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  7. Galba Maria de Campos Takaki
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  8. Tatiana Souza Porto
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  10. Ana Lúcia Figueiredo Porto
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Correspondence to Fabiana América Silva Dantas de Souza.

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de Souza, F.A.S.D., Sales, A.E., Costa e Silva, P.E. et al. Optimization of production, biochemical characterization and in vitro evaluation of the therapeutic potential of fibrinolytic enzymes from a new Bacillus amyloliquefaciens . Macromol. Res. 24, 587–595 (2016). https://doi.org/10.1007/s13233-016-4089-2

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  • DOI: https://doi.org/10.1007/s13233-016-4089-2

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