Previously, the crude extracts of recombinant Nattokinase (NK) variants i.e. NatTK and NatOC and one wild type Douchi Fibrinolytic Enzyme (DFE) from Indonesian traditional fermented foods has been shown to demonstrate fibrinolytic activity. Both NKs contain substitutions of D41N, V192A and 252-RLQHTLEALSTM-263 but NatOC has additional V4F. In the present study, the effects of amino acid substitutions in NK variants and G169A in DFE on their enzyme characteristics were evaluated. Pure proteins were obtained using two sequential steps chromatography using ion exchange and a gel filtration columns. Their activities were determined with fibrin plate, fibrin zymography, fibrinogen hydrolysis, and chromogenic assays. The fibrinogen degradation profile of the wild type NK (NatWT) was different to the NK variants but similar to DFEs. Optimum activity of all the NKs and DFEs was achieved at 50 °C while the optimum pH for NatWT/DFEs and NK variants were 8 and 7, respectively. DFE_G169A exhibited higher fibrinogen degradation rate and fibrin specific activity than DFE. PMSF inhibited all the NKs and DFEs while SDS and EDTA caused lower activity. The NK variants were more resistant towards Na⁺ and Ca²⁺ but more sensitive to K⁺. The amino acid substitutions in NK variants alter their fibrinogen degradation profile, optimum working pH, working pH range, and resistance to some ions. Substitutions in NK variants likely promote structural changes, particularly with the binding mode of the calcium ion cofactor. The results provide a beneficial basis for future development of fibrino(gen)olytic proteins with improved properties for cardiovascular diseases therapy.

中文翻译：

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印尼传统发酵食品中细菌纤溶蛋白的分子表征。

以前，重组纳豆激酶（NK）变体（即NatTK和NatOC）和一种来自印度尼西亚传统发酵食品的野生型豆FE纤溶酶（DFE）的粗提物已显示出纤溶活性。两个NK均包含D41N，V192A和252-RLQHTLEALSTM-263的取代，但NatOC具有附加的V4F。在本研究中，评估了NK变体中的氨基酸取代和DFE中的G169A对其酶特性的影响。通过使用离子交换和凝胶过滤柱的两个连续步骤色谱法获得纯蛋白。它们的活性用血纤蛋白板，血纤蛋白酶谱，血纤蛋白原水解和生色测定法确定。野生型NK（NatWT）的纤维蛋白原降解曲线与NK变体不同，但与DFE相似。所有NK和DFE的最佳活性均在50°C下实现，而NatWT / DFE和NK变体的最佳pH分别为8和7。DFE与DFE相比，_G169A表现出更高的纤维蛋白原降解率和纤维蛋白比活性。PMSF抑制所有NK和DFE，而SDS和EDTA引起较低的活性。NK变体对Na ⁺和Ca ^2+的抵抗力更强，但对K ^+的敏感性更高。NK变体中的氨基酸取代改变了它们的纤维蛋白原降解特性，最佳工作pH，工作pH范围以及对某些离子的抵抗力。NK变体中的取代可能促进结构变化，尤其是钙离子辅因子的结合模式。该结果为纤维蛋白水解酶蛋白的进一步开发提供了有益的基础，该蛋白具有改善心血管疾病治疗的性能。