de Souza Thayana Cruz, Schwarz Marcos Gustavo Araujo, da Silva Daniela Marinho, Maia Carolina Rabelo, de Araújo Cláudia Patrícia Mendes, Balieiro Antônio Alcirley da Silva, de Oliveira Luiz Antonio, Degrave Wim Maurits Sylvain, Fernandes Ormezinda Celeste Cristo, Mendonça-Lima Leila
Leônidas and Maria Deane Institute, ILMD/Fiocruz, Rua Teresina, 476, Adrianópolis, Manaus, Amazonas 69057-070, Brazil.
Oswaldo Cruz Institute, Fiocruz, Av. Brasil, 4365, Manguinhos, Rio de Janeiro, State of Rio de Janeiro 21040-360, Brazil.
Int J Microbiol. 2024 Oct 29;2024:5306083. doi: 10.1155/2024/5306083. eCollection 2024.
Fibrinolytic agents are essential in treating thrombosis, playing a critical role in improving survival rates in cardiovascular diseases. Microbial fibrinolytic proteases have emerged as promising alternatives due to their affordability, specificity, lower toxicity, and reduced side effects. Consequently, the search for microorganisms capable of producing these enzymes has gained significant economic importance in the pharmaceutical industry. This study reports and characterizes a novel fibrinolytic enzyme produced by CFAM 521, a strain isolated from the Amazon region. The enzyme was purified using a polyethylene glycol (PEG)-phosphate salt aqueous two-phase system (ATPS). The effects of PEG molecular weight, PEG concentration, and phosphate concentration on the protease partition coefficient (K) were evaluated through a 2 full factorial design. The enzyme exhibited both fibrinolytic and fibrinogenolytic activities. After partitioning in a two-phase system with 10% (w/w) PEG and 15% (w/w) sodium phosphate, the fibrinolytic proteases were predominantly retained in the salt-rich bottom phase ( = 0.33). The enzyme has a molecular weight of 34 kDa, with optimal pH and temperature at 9°C and 37°C, respectively. Inhibitory analysis confirmed that it is a serine protease, and its activity was enhanced by the addition of Mn. Notably, the enzyme exhibited no hemolytic activity. Therefore, CFAM 521 represents a novel source of fibrinolytic enzymes, highlighting its potential as an alternative for the development of thrombolytic agents.
纤维蛋白溶解剂在治疗血栓形成中至关重要，在提高心血管疾病的生存率方面发挥着关键作用。微生物纤维蛋白溶解蛋白酶因其价格低廉、特异性强、毒性较低和副作用减少而成为有前景的替代品。因此，寻找能够产生这些酶的微生物在制药行业具有重要的经济意义。本研究报告并表征了一种由从亚马逊地区分离的CFAM 521菌株产生的新型纤维蛋白溶解酶。该酶使用聚乙二醇（PEG）-磷酸盐盐水双相系统（ATPS）进行纯化。通过二因素全因子设计评估了PEG分子量、PEG浓度和磷酸盐浓度对蛋白酶分配系数（K）的影响。该酶表现出纤维蛋白溶解和纤维蛋白原溶解活性。在含有10%（w/w）PEG和15%（w/w）磷酸钠的双相系统中分配后，纤维蛋白溶解蛋白酶主要保留在富含盐的下层相中（=0.33）。该酶的分子量为34 kDa，最佳pH和温度分别为9°C和37°C。抑制分析证实它是一种丝氨酸蛋白酶，并通过添加锰增强其活性。值得注意的是，该酶没有溶血活性。因此，CFAM 521代表了一种新型的纤维蛋白溶解酶来源，突出了其作为溶栓剂开发替代品的潜力。
