Purification, fibrinolytic activity and substrate binding of nattokinase from Bacillus subtilis: A rapid and sensitive detection for fibrinolytic activity of nattokinase

Abstract

Nattokinase (NK, EC 3.4.21.62) is an alkaline serine protease secreted by B. subtilis, which has a strong fibrinolytic activity in vitro and in vivo. Here, we fermented NK using a B. subtilis strain and purified the protein, designed a peptide segment derived from fibrin as a substrate of NK. Based on fluorescence resonance energy transfer (FRET), we found that NK exhibits a high hydrolytic activity towards the peptide, with Km of 9.33 ± 1.28 μM, kcat of 0.20 ± 0.01 s−1, and kcat/Km of 21,436.23 s−1 M−1, demonstrating that the peptide is a substrate for NK. Furthermore, we investigated the binding of the substrate with NK by tryptophan fluorescence quenching, molecular docking and dynamics simulation. Fluorescence quenching showed that the substrate binds to NK mainly through hydrogen bonding and van der Waals forces, with dissociation constants KD of 13.73 and 21.24 μM at 25 and 35 °C, respectively. Molecular docking and dynamics analysis revealed that the substrate recognizes the active site of NK, and provides new information on the characteristics of the binding of the substrate with NK. Our study demonstrated a rapid and sensitive method for the quantitative measurement of fibrinolytic activity of NK based on the substrate, which is very reproducible and rapid with virtually complete results in approximately 20 min.