Laboratório de Biotecnologia e Hemoderivados, Departamento de Ciências Farmacêuticas, Universidade Federal de Pernambuco, Avenida Artur de Sá, 50740-520 Recife, PE, Brazil.
Laboratório de Produtos Bioativos e Tecnológicos, Departamento de Morfologia Animal, Universidade Federal Rural de Pernambuco, Avenida Dom Manuel de Medeiros, 52171-900 Recife, PE, Brazil.
An Acad Bras Cienc. 2021 Dec 10;93(suppl 4):e20210335. doi: 10.1590/0001-3765202120210335. eCollection 2021.
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.
纤维蛋白溶酶蛋白酶在制药工业中是一种很有前途的替代品，它们被用于治疗心血管疾病，特别是血栓。微生物是纤维蛋白溶酶蛋白酶最有趣的来源。本研究的目的是从链霉菌属中生产纤维蛋白溶酶蛋白酶 parvulus DPUA 1573，通过双水相系统回收蛋白酶，并对酶进行部分生化特性分析。双水相系统是根据使用聚乙二醇分子量、聚乙二醇浓度、柠檬酸浓度和 pH 值作为独立变量的 24 全因子设计进行的。分析了不同离子、表面活性剂、抑制剂、pH 值和温度对酶活性的影响。纯化酶的最佳条件是 17.5%聚乙二醇 8,000、15%磷酸盐和 pH 值 8.0，此时分配系数为 7.33，产率为 57.49%，纯化倍数为 2.10 倍。在存在 Fe2+的情况下，酶活性增加，而在存在$\beta$-巯基乙醇、苯甲基磺酰氟和碘乙酸的情况下，酶活性降低。最适 pH 值为 7.0，最适温度为 40 ºC。纯化的蛋白酶表现出 41 kDa 的分子量。链霉菌属 parvulus 的纤维蛋白溶酶蛋白酶被证明是开发具有纤维蛋白溶解作用的潜在药物的可行选择。
