Intravascular thrombosis is a prime cause of cardiac complications worldwide. Microbial fibrinolytic proteases are of clinical significance in thrombosis treatment. The present study discusses the purification and characterization of a protease from Bacillus cereus S46, ascertaining its in vitro thrombolytic activity against a blood clot. By the three-step purification involving precipitation, dialysis, and diethylaminoethyl-cellulose ion-exchange chromatography, a 12.37-fold purification of the enzyme to homogeneity was achieved. The apparent molecular mass of the protease was 30 kDa, as found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum activity of the enzyme was observed at pH 8.0 and 40°C. The enzyme retained an 82.19% residual activity at pH 8.0 and 40°C for 1 h. The Km and Vmax values of the protease with casein were 0.0027 mM and 9.712 µmol/min, respectively. In an in vitro assay, the purified protease resulted in 97.02% lysis of the blood clot. The fibrinolytic potential of the enzyme, together with its characteristics of being active and stable under near-physiological conditions, may suggest its application as a therapeutic agent.

中文翻译：

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蜡样芽孢杆菌 S46 的纤溶蛋白酶：其体外溶栓潜力的纯化、表征和评价

血管内血栓形成是全世界心脏并发症的主要原因。微生物纤溶蛋白酶在血栓形成治疗中具有临床意义。本研究讨论了蜡样芽孢杆菌 S46 蛋白酶的纯化和表征，确定其体外抗血栓的溶栓活性。通过涉及沉淀、透析和二乙氨基乙基纤维素离子交换色谱的三步纯化，实现了酶的 12.37 倍纯化至均一性。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳发现蛋白酶的表观分子量为30 kDa。在 pH 8.0 和 40°C 下观察到酶的最佳活性。该酶在 pH 8.0 和 40°C 下保持 82.19% 的残留活性 1 小时。含有酪蛋白的蛋白酶的 Km 和 Vmax 值分别为 0.0027 mM 和 9.712 µmol/min。在体外试验中，纯化的蛋白酶导致血凝块的溶解率为 97.02%。该酶的纤维蛋白溶解潜力及其在接近生理条件下的活性和稳定特性，可能表明其可用作治疗剂。