Fibrinolytic enzymes with a direct mechanism of action and safer properties are currently requested for thrombolytic therapy. This paper reports on a new enzyme capable of degrading blood clots directly without impairing blood coagulation. This enzyme is also non-cytotoxic and constitutes an alternative to other thrombolytic enzymes known to cause undesired side effects. Twenty-four Bacillus isolates were screened for production of fibrinolytic enzymes using a fibrin agar plate. Based on produced activity, isolate S127e was selected and identified as B. subtilis using the 16S rDNA gene sequence. This strain is of biotechnological interest for producing high fibrinolytic yield and consequently has potential in the industrial field. The purified fibrinolytic enzyme has a molecular mass of 27.3 kDa, a predicted pI of 6.6 and a maximal affinity for Ala-Ala-Pro-Phe. This enzyme was almost completely inhibited by chymostatin with optimal activity at 48°C and pH 7. Specific subtilisin features were found in the gene sequence, indicating that this enzyme belongs to the BPN group of the S8 subtilisin family and was assigned as AprE127. This subtilisin increased thromboplastin time by 3.7% (37.6 to 39 s) and prothrombin time by 3.2% (12.6 to 13 s), both within normal ranges. In a whole blood euglobulin assay, this enzyme did not impair coagulation but reduced lysis time significantly. Moreover, in an in vitro assay, AprE127 completely dissolved a thrombus of about 1 cc within 50 min and, in vivo, reduced a thrombus prompted in a rat-tail by 11.4% in 24 h compared to non-treated animals.

中文翻译：

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由枯草芽孢杆菌新菌株产生的溶栓酶的纯化和表征。

目前需要具有直接作用机制和更安全特性的纤维蛋白溶解酶用于溶栓治疗。本文报道了一种能够直接降解血块而不影响血液凝固的新型酶。这种酶也是非细胞毒性的，可以替代其他已知会引起不良副作用的血栓溶解酶。使用纤维蛋白琼脂平板筛选24株芽孢杆菌分离物以产生纤维蛋白溶解酶。根据产生的活性，选择分离株 S127e 并将其鉴定为枯草芽孢杆菌使用 16S rDNA 基因序列。该菌株在产生高纤维蛋白溶解产率方面具有生物技术意义，因此在工业领域具有潜力。纯化的纤维蛋白溶解酶的分子量为 27.3 kDa，预测 pI 为 6.6，对 Ala-Ala-Pro-Phe 具有最大亲和力。该酶几乎完全被 chymostatin 抑制，在 48°C 和 pH 7 时具有最佳活性。在基因序列中发现了特定的枯草杆菌蛋白酶特征，表明该酶属于 S8 枯草杆菌蛋白酶家族的 BPN 组，并被指定为 AprE127。这种枯草杆菌蛋白酶使凝血活酶时间增加了 3.7%（37.6 至 39 秒），使凝血酶原时间增加了 3.2%（12.6 至 13 秒），均在正常范围内。在全血优球蛋白测定中，这种酶不会影响凝血，但会显着缩短裂解时间。此外，在一个在体外试验中，AprE127 在 50 分钟内完全溶解了约 1 cc 的血栓，并且在体内，与未处理的动物相比，在 24 小时内，鼠尾中的血栓减少了 11.4%。