Abstract Fibrinolytic proteases are a promising alternative in the pharmaceutical industry, they are used in the treatment of cardiovascular diseases, especially thrombosis. Microorganisms are the most interesting source of fibrinolytic proteases. The aim of this study was the production of fibrinolytic protease from Streptomyces parvulus DPUA 1573, the recovery of the protease by aqueous two-phase system and partial biochemical characterization of the enzyme. The aqueous two-phase system was performed according to a 24-full factorial design using polyethylene glycol molar mass, polyethylene glycol concentration, citrate concentration and pH as independent variables. It was analyzed the effect of different ions, surfactants, inhibitors, pH and temperature on enzyme activity. The best conditions for purifying the enzyme were 17.5% polyethylene glycol 8,000, 15% Phosphate and pH 8.0, it was obtained a partition coefficient of 7.33, a yield of 57.49% and a purification factor of 2.10-fold. There was an increase in enzyme activity in the presence of Fe2+ and a decrease in the presence of $\beta$-Mercaptoethanol, phenylmethylsulfonyl fluoride and Iodoacetic acid. The optimum pH was 7.0 and the optimum temperature was 40 ºC. The purified protease exhibited a molecular mass of 41 kDa. The fibrinolytic protease from Streptomyces parvulus proved to be a viable option for the development of a possible drug with fibrinolytic action.

Resumo: A mastite é uma inflamação na glândula mamária que pode acarretar perdas na produção e na qualidade do leite, gerando prejuízos econômicos para a pecuária leiteira. O tratamento é baseado na utilização de antibióticos, sendo, em muitos casos, ineficazes devido à resistência bacteriana já conhecida para esta doença. O objetivo deste trabalho foi selecionar linhagens de Streptomyces spp. produtoras de biocompostos com atividade antimicrobiana frente a isolados do gênero Staphylococcus multirresistentes de búfalas com mastite. Bem como, determinar os melhores parâmetros de produção, e avaliar a produção simultânea de ácido clavulânico. A seleção de Streptomyces spp. com capacidade de produzir compostos com atividade antimicrobiana foi realizada através da técnica bloco de gelose. Dentre as 30 espécies de Streptomyces spp. testadas, o micro-organismo Streptomyces parvulus DPUA 1573 apresentou melhores resultados, sendo capaz de inibir o crescimento de 7 isolados Staphylococcus spp. multirresistentes. Posteriormente, a espécie selecionada Streptomyces parvulus DPUA 1573 foi cultivada em diferentes condições pré-determinadas pelo planejamento fatorial 24, onde as variáveis independentes foram: concentração de soja (0,5; 1,0; 1,5%), glicose (0; 0,5; 1g/L), agitação (150; 200; 250rpm) e temperatura (28; 32; 37°C) e todos os ensaios do planejamento foram monitorados até 120 horas de cultivo. Todas as variáveis independentes influenciaram positivamente no crescimento celular, enquanto que para atividade antimicrobiana apenas as variáveis temperatura e agitação apresentaram efeitos significativos positivos. O líquido metabólito produzido por Streptomyces parvulus DPUA 1573 foi capaz de inibir o crescimento de sete Staphylococcus spp. multirresistentes. As melhores condições de cultivo para a produção de moléculas bioativas por este micro-organismo foi a 37?C, com 250rpm de agitação por período de 72 horas. Nos ensaios que apresentaram atividade antimicrobiana, foi avaliada a produção de ácido clavulânico ao longo do cultivo. A maior concentração de ácido clavulânico foi de 269,84g/L obtidas nas condições de 1,5% de farinha de soja em ausência de glicose no tempo de 96 horas. A linhagem Streptomyces parvulus DPUA 1573 foi eficiente contra Staphylococcus spp. multirresistentes isolados de mastite em búfalas, ainda apresentando concomitantemente produção de ácido clavulânico com o potencial uso farmacêutico.

Abstract: Mastitis is an inflammation in one or more mammary glands which can lead to reduction in production and quality of milk causing economic losses to dairy farming. The use of antibiotics is the key for the treatment of this disease, but in many cases ineffective due to bacterial resistance already known for this condition. The aim of this study was to select strains of Streptomyces spp. producing biomolecules with antimicrobial activity against multidrug-resistant Staphylococcus isolated from buffaloes with mastitis, as well as to determine the best production parameters to the evaluation of simultaneous production of clavulanic acid. Thirty species of Streptomyces spp. were used to selecting the greatest producer spectrum of antimicrobial activity (agar block technique), with selection of Streptomyces parvulus DPUA 1573, and 7 multidrug-resistant Staphylococcus spp. sensitive to its biocompounds. The selected strain of Streptomyces parvulus DPUA 1573 was cultured in different conditions predetermined by the factorial design 24, where the independent variables were: soybean concentration (0.5, 1.0, 1.5%), glucose (0, 0.5, 1g/L), agitation (150, 200, 250rpm) and temperature (28, 32, 37°C); all the tests were monitored up to 120 hours of cultivation. All independent variables influenced positively the cell growth, while for antimicrobial activity only the variables temperature and agitation showed positive effects. The antimicrobial bio compounds showed activity against seven multidrug-resistant Staphylococcus spp under the conditions: temperature 37°C, agitation 250rpm, with 72 hours of production process. In the tests which showed antimicrobial activity, was also assessed the production of clavulanic acid along with the cultivation. The highest concentration of clavulanic acid was 269.84g/L obtained under the conditions of 1.5% of soybean flour and absence of glucose in 96 hours. The strain Streptomyces parvulus DPUA 1573 was effective against multidrug-resistant strains of Staphylococcus spp. of mastitis from buffaloes, still showing concomitantly production of clavulanic acid for pharmaceutical use.