Federal Rural University of Pernambuco-UFRPE, Department of Animal Morphology and Physiology, Dom Manoel de Medeiros Ave., s/n, Dois irmãos, 52171-900 Recife, PE, Brazil.
Federal University of Pernambuco-UFPE, Laboratory of Immunopathology Keizo Asami (LIKA), Prof. Moraes Ave., s/n, Várzea, 50670-901 Recife, PE, Brazil.
An Acad Bras Cienc. 2023 Aug 14;95(suppl 1):e20220552. doi: 10.1590/0001-3765202320220552. eCollection 2023.
A fibrinolytic enzyme from the microalga Dunaliella tertiolecta was produced under mixotrophic conditions using different corn steep liquor (CSL) concentrations ( 0 ≤ CLS ≤ 0.75%), purified using a combination of salting out and ion-exchange chromatography, and then biochemical characterized. Cultivation of this microalga using 0.5% CSL led to the highest maximum cell concentration (1.960±0.010 mg L-1) and cell productivity (0.140g L-1 day-1), besides a high fibrinolytic activity of the extract obtained by the homogenization method (102 ±1 U mL-1). The enzyme extracted from the microalgal biomass was 5-fold purified with a 20% yield and was found to have a specific activity of 670 U mg-1. The enzyme, whose molecular weight determined by fibrin zymography was 10 kDa, was shown to be stable at pH 3.0-9.0 and up to 70°C with optimal pH and temperature values of 8.0 and 50°C, respectively. When compared to other fibrinolytic enzymes, this protease stood out for its high fibrinolytic activity, which was enhanced by Fe2+, inhibited by Zn2+, Cu2+, Mg2+, and Ca2+, and strongly inhibited by phenylmethylsulfonyl fluoride, suggesting that it belongs to the serine metalloprotease family. Moreover, thanks to its thermal stability, the enzyme may be easily preserved and activated under high-temperature conditions.
从杜氏盐藻中生产的纤维蛋白溶酶在混养条件下使用不同浓度的玉米浆（CSL）（0≤CLS≤0.75%）产生，通过盐析和离子交换层析的组合进行纯化，然后进行生化特性分析。在 0.5%CSL 下培养这种微藻可获得最高的最大细胞浓度（1.960±0.010mgL-1）和细胞生产率（0.140gL-1day-1），此外通过匀浆法获得的提取物具有高的纤维蛋白溶酶活性（102±1UmL-1）。从微藻生物质中提取的酶经过 5 倍纯化，收率为 20%，比活为 670Umg-1。通过纤维蛋白凝胶电泳测定的酶分子量为 10kDa，在 pH3.0-9.0 和 70°C 下稳定，最适 pH 和温度分别为 8.0 和 50°C。与其他纤维蛋白溶酶相比，该蛋白酶的纤维蛋白溶酶活性较高，Fe2+可增强其活性，Zn2+、Cu2+、Mg2+和 Ca2+可抑制其活性，苯甲基磺酰氟可强烈抑制其活性，表明其属于丝氨酸金属蛋白酶家族。此外，由于其热稳定性，该酶可在高温条件下容易保存和激活。
