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

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.

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.

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.