Laboratory of Immunopathology Keizo Asami (LIKA), Federal University of Pernambuco-UFPE, Av. Prof. Moraes s/n, 50670-901 Recife, PE, Brazil.
Department of Morphology and Animal Physiology, Federal Rural University of Pernambuco-UFRPE, Av. Dom Manoel de Medeiros s/n, 52171-900 Recife, PE, Brazil.
J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Aug 15;1092:524-529. doi: 10.1016/j.jchromb.2018.04.040. Epub 2018 Apr 24.
A fibrinolytic enzyme was produced by microalga Chlorella vulgaris cultivated in autotrophic and mixotrophic conditions added corn steep liquor, purified by a single chromatographic step, then biochemical characterization and in vitro thrombolytic activity was performed. Maximum cell concentration (1637.45 ± 15 mg L) and productivity (181.93 mg L day) was obtained in mixotrophic culture using 1% corn steep liquor. Enzyme-extracted microalgal biomass was purified by acetone precipitation and DEAE Sephadex anion exchange chromatography up to 2 fold with recovery of 4.0%. After purification, fibrinolytic activity was 1834.6 U mg and 226.86 mm by spectrophotometry and fibrin plate assays, respectively. SDS-PAGE results exhibited a protein band of about 45 kDa and fibrinolytic band was detected by fibrin zymography. Enzyme activity was enhanced in the presence of Fe and inhibited by phenylmethane sulfonyl fluoride (PMSF) and ethylenediamine tetracetic acid (EDTA), which suggest it to be a metal-dependent serine protease. The extract also showed a red blood cell lysis <4% and in vitro thrombolytic activity of 25.6% in 90 min of reaction. These results indicate that the fibrinolytic enzyme from C. vulgaris may have potential applications in the prevention and treatment of thrombosis.
微藻小球藻在添加玉米浆的自养和混合营养条件下培养生产纤溶酶，经单一色谱步骤纯化，然后进行生化特性和体外溶栓活性分析。在混合营养培养中使用 1%玉米浆可获得最大细胞浓度（1637.45±15mg/L）和生产力（181.93mg/L/day）。酶提取的微藻生物质通过丙酮沉淀和 DEAE Sephadex 阴离子交换色谱法纯化，回收率为 4.0%，达到 2 倍。纯化后，分光光度法和纤维蛋白平板测定的纤溶活性分别为 1834.6 U/mg 和 226.86mm。SDS-PAGE 结果显示约 45kDa 的蛋白带，纤维蛋白酶谱检测到纤溶带。酶活性在 Fe 的存在下增强，而被苯甲磺酰氟（PMSF）和乙二胺四乙酸（EDTA）抑制，表明它是一种依赖金属的丝氨酸蛋白酶。该提取物在 90min 的反应时间内还表现出<4%的红细胞裂解和 25.6%的体外溶栓活性。这些结果表明，小球藻的纤溶酶可能在预防和治疗血栓形成方面具有潜在的应用。
