Protein engineering approaches to enhance nattokinase activity and stability structure function relationship

Abstract

This study focused on expressing recombinant NK (rNK) in Escherichia coli and addressing issues of protein insolubility. Initial expression in E. coli C41 led to inclusion body formation and enzymatic inactivity, but co-expression with the pGro7 chaperone system using the pET32a vector significantly enhanced solubility and yield. The recombinant enzyme exhibited optimal activity at 37°C and pH 8.0, with a specific activity of 4188 IU/mg and kinetic parameters indicating high catalytic efficiency. Molecular docking identified key fibrin-binding residues, Ser300, Leu302, and Asp303, critical for stable enzyme-substrate interaction. Site-directed mutagenesis revealed that substitutions at these positions significantly impaired fibrin binding without affecting basic proteolytic activity. Among engineered variants, NK(E262D) exhibited the most promising performance, with enhanced catalytic efficiency (2.2-fold increase in kcat/Km), improved substrate affinity, and the highest fibrinolytic activity (2414 U/mg). In contrast, NK(A259T) showed severely reduced activity and stability due to structural disruptions in the fibrin-binding pocket. Thermal and pH stability assays confirmed broader activity ranges for the engineered enzymes, with NK(E262D) maintaining significant activity at elevated temperatures and acidic pH. NK(E262D) represents a superior variant with enhanced therapeutic potential for fibrinolytic therapy, laying the foundation for future development of safer and more effective thrombolytic biopharmaceuticals. newline