ABSTRACT
Nattokinase is an enzyme produced by Bacillus subtilis subsp. natto that contains strong fibrinolytic activity. It has potential to treat cardiovascular diseases. In silico analysis revealed that nattokinase is considered as an antigen, thus hindering its application for injectable therapeutic protein. Various web servers were used to predict B-cell epitopes of nattokinase both continuously and discontinuously to determine which amino acid residues had been responsible for the immunogenicity. With the exclusion of the predicted conserved amino acids, four amino acids such as S18, Q19, T242, and Q245 were allowed for mutation. Substitution mutation was done to lower the immunogenicity of native nattokinase. Through the stability of the mutated protein with the help of Gibbs free energy difference, the proposed mutein was S18D, Q19I, T242Y, and Q245W. The 3D model of the mutated nattokinase was modeled and validated with various tools. Physicochemical properties and stability analysis of the protein indicated that the mutation brought higher stability without causing any changes in the catalytic site of nattokinase. Molecular dynamics simulation implied that the mutation indicated similar stability, conformation, and behavior compared to the native nattokinase. These results are highly likely to contribute to the wet lab experiment to develop safer nattokinase.
