Institute of Materia Medica, College of Pharmacy, Xinjiang University, Urumqi 830017, China.
Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830017, China.
J Agric Food Chem. 2024 Jun 26;72(25):14241-14254. doi: 10.1021/acs.jafc.4c02335. Epub 2024 Jun 12.
Nattokinase is a nutrient in healthy food natto that has the function of preventing and treating blood thrombus. However, its low thermostability and fibrinolytic activity limit its application in food and pharmaceuticals. In this study, we used bioinformatics analysis to identify two loops (loop10 and loop12) in the flexible region of nattokinase rAprY. Using this basis, we screened the G131S-S161T variant, which showed a 2.38-fold increase in half-life at 55 °C, and the M3 variant, which showed a 2.01-fold increase in activity, by using a thermostability prediction algorithm. Bioinformatics analysis revealed that the enhanced thermostability of the G131S-S161T variant was due to the increased rigidity and structural shrinkage of the overall structure. Additionally, the increased rigidity of the local region surrounding the active center and its mutated sites helps maintain its normal conformation in high-temperature environments. The increased catalytic activity of the M3 variant may be due to its more efficient substrate binding mechanism. We investigated strategies to improve the thermostability and fibrinolytic activity of nattokinase, and the resulting variants show promise for industrial production and application.
纳豆激酶是一种存在于健康食品纳豆中的营养物质，具有预防和治疗血栓的功能。然而，其低热稳定性和纤维蛋白溶解活性限制了其在食品和制药领域的应用。在本研究中，我们使用生物信息学分析鉴定了纳豆激酶 rAprY 柔性区域中的两个环（loop10 和 loop12）。在此基础上，我们使用热稳定性预测算法筛选出半衰期在 55°C 下提高了 2.38 倍的 G131S-S161T 变体，以及活性提高了 2.01 倍的 M3 变体。生物信息学分析表明，G131S-S161T 变体的热稳定性增强是由于整体结构的刚性增加和结构收缩。此外，活性中心及其突变位点周围局部区域的刚性增加有助于在高温环境下保持其正常构象。M3 变体催化活性的增加可能与其更有效的底物结合机制有关。我们研究了提高纳豆激酶热稳定性和纤维蛋白溶解活性的策略，所得变体有望用于工业生产和应用。
