Directed evolution modification of the keratinase KerD21 and application in feather powder fermented feed

Abstract BACKGROUND Keratinase, a protease specific for keratin, has the potential to improve the utilization of feathers, a byproduct of poultry farming. However, the activity of wild‐type keratinases often requires optimization to meet the demands of practical applications. RESULTS A high‐throughput single‐cell screening method was developed based on the expression of Pseudomonas aeruginosa PAD21 keratinase KerD21 in Bacillus subtilis . Initial site‐directed mutagenesis targeting the active site significantly improved acid tolerance but failed to enhance its activity. Subsequent directed evolution yielded four active mutant proteins, with KerD21‐D124N A126E exhibiting the highest activity (130.93 ± 3.52 U mL −1 ), representing a 180.17 ± 4.24% increase compared to the wild‐type enzyme. Further evaluation of the optimized KerD21‐D124N A126E keratinase in a lactic acid bacteria‐fermented feed system demonstrated a significant increase in keratin degradation. Specifically, the small peptide content in the feather meal increased from 3.35 ± 0.22% to 15.82 ± 0.07% after 7 days of fermentation. CONCLUSION We report the development of a high‐throughput single‐cell screening method for engineering Pseudomonas aeruginosa keratinase KerD21. This method yielded a mutant protein with significantly enhanced keratinase activity, offering a potentially valuable approach for improving feather utilization. © 2025 Society of Chemical Industry.