Targeted Adherence and Enhanced Degradation of Feather Keratins by a Novel Prepeptidase C-Terminal Domain-Fused Keratinase

Abstract

Keratinases are valuable enzymes for converting feather keratin waste into bioactive products but often suffer from poor substrate specificity and low catalytic efficiency. This study reported the creating of a novel keratinase with targeted adherence and specific degradation on feather keratins by fusing prepeptidase C-Terminal (PPC) domain. A PPC domain of metalloprotease E423 specifically adsorbed feather keratins by hydrogen bonds and hydrophobic interactions in a time- and temperature-dependent manner. Stepwise N-/C-terminal truncations disclosed the essential core sequence composed of 21 amino acid residues determining the keratin-targeted adherence. Fusion of the core fragment with a flexible linker (GGGGS)₁ achieved the optimal secretion, and improved the catalytic efficiency of a representative keratinase 4-3Ker-MAV by 0.97-fold. Moreover, the feather degradation rate increased from 65 to 82%, representing the highest reported performance for a keratinase. This PPC-fusion strategy opens new horizons in enzyme engineering, promising not only to revolutionize keratin waste valorization but also to inspire the design of substrate-specific biocatalysts across diverse industrial applications.