Signal peptide engineering of a novel M4 family keratinase and the action mechanism in promoting pepper tolerance to salt stress through KerJY23-hydrolyzed feather waste

Microbial keratinase has attracted significant attention for its ability to convert keratin waste into high-value products with substantial application potential in the agricultural field. However, the low yield and potential pathogenicity of native keratinase producers have significantly hindered their large-scale industrial application. In this study, the novel M4 family keratinase KerJY-23 was heterologous expressed in Bacillus subtilis , and a systematic signal peptide engineering was performed. The optimal signal peptide AprE was identified through screening of the signal peptide library, resulting in an extracellular protease activity of 185.37 ± 6.72 U/mg protein, which was 2.17 times that of the native signal peptide. Comparative analysis revealed that signal peptides with amino acid residues ≤5 and a positive charge ≤3 in the N-region, high hydrophobicity in the center of H-region, and a conserved A-X-A motif in the C-region are conducive to the secretory expression of KerJY-23. To further explore the potential agricultural applications of KerJY23-hydrolyzed feather waste, we investigated its effects on salt stress response in pepper ( Capsicum annuum L.), a commercially significant horticultural crop. Our findings indicated that the exogenous application of feather hydrolysates for 24 h significantly enhanced salt tolerance in pepper plants. Transcriptomic analysis revealed that feather hydrolysates maintain ROS homeostasis and regulate gene expression associated with salt-responsive transcription factors, phospholipid biosynthesis, and plant hormones, ultimately improving pepper tolerance to salt stress. Our work collectively provides a theoretical basis for the utilization of agro-industrial waste in agricultural practices, thereby facilitating its valorization and contributing to sustainable agricultural development.