Enhanced Thermostability of Nattokinase by Computation-Based Rational Redesign of Flexible Regions

作者：Liangqi Chen, Kongfang Yu, Aixia Ma, Wenhui Zhu, Hong Wang, Xiyu Tang, Yaolei Tang, Yuan Li, Jinyao Li · 发表于：Journal of Agricultural and Food Chemistry · 年份：2024 · DOI：10.1021/acs.jafc.4c02335 · 被引用次数：16 · 研究领域：Enzyme Production and Characterization、Advanced Proteomics Techniques and Applications、Protein Hydrolysis and Bioactive Peptides

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.