Obtainment and characterization of digestive aspartic proteases from the fish Caranx hippos (Linnaeus, 1766)

Abstract This work aimed to obtain aspartic proteases of industrial and biotechnological interest from the stomach of the crevalle jack fish (Caranx hippos). In order to do so, a crude extract (CE) of the stomach was obtained and subjected to a partial purification by salting-out, which resulted in the enzyme extract (EE) obtainment. EE proteases were characterized physicochemically and by means of zymogram. In addition, the effect of chemical agents on their activity was also assessed. By means of salting-out it was possible to obtain a purification of 1.6 times with a yield of 49.4%. Two acid proteases present in the EE were observed in zymogram. The optimum temperature and thermal stability for EE acidic proteases were 55 ºC and 45 °C, respectively. The optimum pH and pH stability found for these enzymes were pH 1.5 and 7.0, respectively. Total inhibition of EE acid proteolytic activity was observed in the presence of pepstatin A. dithiothreitol (DTT) and Ca2+ did not promote a significant effect on enzyme activity. In the presence of heavy metals, such as Al3+, Cd2+ and Hg2+, EE acidic proteases showed more than 70% of their enzymatic activity. The results show that it is possible to obtain, from the stomach of C. hippos, aspartic proteases with high proteolytic activity and characteristics that demonstrate potential for industrial and biotechnological applications.

摘要 本研究旨在从大西洋鲹（Caranx hippos）的胃部获取具有工业及生物技术应用价值的天冬氨酸蛋白酶（aspartic proteases）。为此，研究人员制备了该鱼类胃部的粗提取物（CE），并通过盐析法对其进行部分纯化，最终得到酶提取物（EE）。随后，对酶提取物中的蛋白酶开展了理化性质表征及酶谱（zymogram）分析。此外，还评估了化学试剂对该类蛋白酶活性的影响。通过盐析法可获得1.6倍的纯化效果，回收率达49.4%。酶谱分析显示，酶提取物中存在两种酸性蛋白酶。该酸性蛋白酶的最适温度与热稳定性分别为55℃与45℃；最适pH值与pH稳定性分别为pH 1.5与pH 7.0。在胃蛋白酶抑制剂A（pepstatin A）存在时，酶提取物的酸性蛋白水解活性被完全抑制。二硫苏糖醇（DTT）与Ca²⁺对该酶活性无显著影响。在Al³⁺、Cd²⁺、Hg²⁺等重金属环境中，该酸性蛋白酶仍保留超过70%的酶活性。研究结果表明，从大西洋鲹（Caranx hippos）胃部可获取具有高蛋白水解活性的天冬氨酸蛋白酶，其特性显示出在工业及生物技术领域的应用潜力。