Wang Jinyu, Liu Xiaolan, Jing Yan, Zheng Xiqun
College of Food Engineering, Harbin University of Commerce, Harbin 150076, China.
Key Laboratory of Corn Deep Processing Theory and Technology of Heilongjiang Province, College of Food and Bioengineering, Qiqihar University, Qiqihar 161006, China.
Foods. 2024 Apr 23;13(9):1292. doi: 10.3390/foods13091292.
A novel fibrinolytic enzyme was produced by the liquid fermentation of . The enzyme was purified from the culture supernatant by hydrophobic interactions, gel filtration, and ion exchange chromatographies. It was purified by 241.02-fold, with a specific activity of 3619 U/mg and a final yield of 10.02%. SDS-PAGE analysis confirmed the purity of the enzyme, showing a single band with a molecular weight of 19.5 kDa. The first nine amino acids of the N-terminal of the purified enzyme were A-T-Y-T-G-G-S-Q-T. The enzyme exhibited optimal activity at a temperature of 42 °C and pH 7.6. Its activity was significantly improved by Zn, K, Ca, Mn, and Mg while being inhibited by Fe, Fe, Al, and Ba. The activity of the enzyme was completely inhibited by ethylenediamine tetraacetic acid (EDTA), and it was also dose-dependently inhibited by phenylmethylsulfonyl fluoride (PMSF) and soy trypsin inhibitor (SBTI). However, inhibitors such as N-α-tosyl-L-phenylalanine chloromethyl ketone (TPCK), aprotinin, and pepstatin did not significantly affect its activity, suggesting that the enzyme was a serine-like metalloproteinase. The enzyme acted as both a plasmin-like fibrinolytic enzyme and a plasminogen activator, and it also exhibited the capability to hydrolyze fibrinogen and fibrin. In vitro, it demonstrated the ability to dissolve blood clots and exhibit anticoagulant properties. Furthermore, it was found that the enzyme prolonged activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT), and reduced the levels of fibrinogen (FIB) and prothrombin activity (PA). Based on these studies, the enzyme has great potential to be developed as a natural agent for the prevention and treatment of thrombotic diseases.
通过[具体菌种名称]的液体发酵产生了一种新型纤溶酶。该酶通过疏水相互作用、凝胶过滤和离子交换色谱从培养上清液中纯化得到。纯化倍数为241.02倍，比活性为3619 U/mg，最终产率为10.02%。SDS-PAGE分析证实了该酶的纯度，显示出一条分子量为19.5 kDa的单一蛋白条带。纯化酶N端的前九个氨基酸为A-T-Y-T-G-G-S-Q-T。该酶在42℃和pH 7.6时表现出最佳活性。锌、钾、钙、锰和镁显著提高其活性，而铁、铝和钡则抑制其活性。乙二胺四乙酸（EDTA）完全抑制该酶的活性，苯甲基磺酰氟（PMSF）和大豆胰蛋白酶抑制剂（SBTI）也呈剂量依赖性抑制其活性。然而，N-α-对甲苯磺酰-L-苯丙氨酸氯甲基酮（TPCK）、抑肽酶和胃蛋白酶抑制剂等抑制剂对其活性没有显著影响，表明该酶是一种丝氨酸样金属蛋白酶。该酶兼具纤溶酶样纤溶酶和纤溶酶原激活剂的作用，还具有水解纤维蛋白原和纤维蛋白的能力。在体外，它表现出溶解血栓的能力并具有抗凝特性。此外，发现该酶延长活化部分凝血活酶时间（APTT）、凝血酶原时间（PT）和凝血酶时间（TT），并降低纤维蛋白原（FIB）水平和凝血酶原活性（PA）。基于这些研究，该酶具有作为预防和治疗血栓性疾病的天然药物开发的巨大潜力。
