Department of Chemistry, Faculty of Science and Arts, Sakarya University, Serdivan-Sakarya 54187, Turkey.
Int J Biol Macromol. 2018 Jul 15;114:1295-1304. doi: 10.1016/j.ijbiomac.2018.03.165. Epub 2018 Mar 27.
In this study, a protease enzyme was purified from strawberry by using Sepharose-4B-l-tyrosine-p-amino benzoic acid affinity chromatography. The molecular weight of pure protease was determined 65.8 kDa by SDS-PAGE. The single band observed on the gel showed that the enzyme had a single polypeptide chain and was successfully purified. Purification of the protease by the chromatographic method resulted in a 395.6-fold increase in specific activity (3600 U/mg). Optimum pH and temperature for the enzyme were 6 and 40 °C, respectively. The protease was stable at a wide temperature range of 40 to 70 °C and a pH range of 3.0 to 9.0. Co ions stimulated protease activity very strongly. Cu, Hg, Cd and Mn ions significantly inhibited protease activity. While 2-propanol completely inhibited the enzyme, the enzyme maintained its activity better in the presence of ethanol and methanol. The strawberry protease showed the highest specificity towards hemoglobin among all the natural substrates tested. The specificity of the enzyme towards synthetic substrates was also investigated and it was concluded that it has broad substrate specificity. The obtained results indicated that this purified protease was highly-likely a serine protease and its activity was significantly affected by the presence of metal ions.
在这项研究中，通过 Sepharose-4B-l-酪氨酸-p-氨基苯甲酸亲和层析从草莓中纯化了一种蛋白酶。SDS-PAGE 测定纯蛋白酶的分子量为 65.8 kDa。凝胶上观察到的单带表明该酶具有单条多肽链，并且已成功纯化。通过色谱法纯化蛋白酶可使比活（3600 U/mg）提高 395.6 倍。该酶的最适 pH 和温度分别为 6 和 40°C。该蛋白酶在 40 至 70°C 的宽温度范围和 3.0 至 9.0 的 pH 范围内稳定。辅助离子强烈刺激蛋白酶活性。Cu、Hg、Cd 和 Mn 离子显著抑制蛋白酶活性。而 2-丙醇完全抑制了酶的活性，但在存在乙醇和甲醇的情况下，酶的活性保持得更好。草莓蛋白酶在所有测试的天然底物中对血红蛋白表现出最高的特异性。还研究了该酶对合成底物的特异性，结论是它具有广泛的底物特异性。研究结果表明，这种纯化的蛋白酶很可能是一种丝氨酸蛋白酶，其活性受到金属离子的显著影响。
