Molecular dynamics studies of disulfide bonds for enhancing the stability of serine protease PB92

The stability of a protease is crucial for its application in biological processes and various industries. This study aims to improve the stability of serine protease PB92 by introducing disulfide bonds. Using the Disulfide by Design 2 online server, we predicted appropriate cysteine pairs and introduced three pairs of amino acid mutations, resulting in their conversion to cysteines and subsequent formation of disulfide bonds. Additionally, to comprehensively assess the effects of combined mutations, three additional mutations were generated by combining the aforementioned three pairs of mutations. Molecular Dynamics (MD) simulations were performed to evaluate the influence of the introduced mutations. The results demonstrated that the mutant with disulfide residues A71C-A86C and T249C-L261C exhibited higher stability compared to both the wild type and other mutants. Docking results indicated that the introduced mutations did not affect the binding performance of the protease. Employing innovative disulfide bond engineering and theoretical simulation, we introduced two bonds into PB92, enhancing thermal stability. The graphic illustrates these bonds, showcasing their crucial role in stabilizing the proteinase structure.