Directed evolution of a keratinase BLk from Bacillus licheniformis to enhance the solvent tolerance

Abstract

Abstract Keratinase, an important protein used in hair degradation, requires stability enhancement in industrial applications due to the harsh reaction environment for keratin hydrolysis. Previous studies have focused on improving the thermostability of keratinase. In this study, directed evolution was applied to enhance the organic solvent stability of keratinase BLk from Bacillus licheniformis. Three excellent mutants were screened and exhibited significantly improved stability in various solvents, although similar results were not observed in terms of thermostability. The identified mutations were located on the enzyme's surface. The half-life of the D41A, A24E, and A24Q mutants increased by 47-, 63-, and 61-fold, respectively, in the presence of 50% (v/v) acetonitrile compared to the wild-type. Similarly, in the presence of 50% (v/v) acetone, the half-life of these mutants increased by 22-, 27-, and 27-fold compared to the wild-type. Importantly, the proteolytic activity of all selected mutants was similar to that of the parent keratinase BLk. Furthermore, molecular dynamic simulation was employed to analyze the possible reasons for the enhanced solvent stability. The results suggest that increased intramolecular interactions, such as hydrogen bonds and hydrophobic interactions, may contribute to the improved solvent tolerance. The mutants obtained in this study hold significant potential for industrial applications.