Functional identification of a feather-degrading thermitase-like S8 serine protease from Fervidobacterium islandicum AW-1

Abstract
Thermophilic species of Fervidobacterium are well known for their ability to degrade native chicken feathers, yet the specific proteases responsible for this activity at the single-enzyme level have remained unresolved because previously identified enzymes primarily acted on synthetic substrates or required synergistic activity in crude extracts. Here, we identified and characterized Fervitase, a feather-degrading thermitase-like S8 serine protease from Fervidobacterium islandicum AW-1. Recombinant Fervitase efficiently hydrolyzed native chicken feathers as a purified enzyme in the presence of dithiothreitol and exhibited maximal activity at 80 °C and pH 8.0. LC–MS/MS analysis demonstrated broad cleavage specificity with preferential recognition of threonine, leucine, and serine residues at the P1 position. Notably, Fervitase selectively degraded insoluble feather keratin while exhibiting minimal activity toward soluble protein substrates, suggesting evolutionary specialization for keratin degradation. Structural and mutational analyses further demonstrated that Fervitase-specific structural elements are essential for maturation, catalytic activity and thermostability. Together, this study resolves a long-standing ambiguity in Fervidobacterium keratinolysis and expands current understanding of functional adaptation in thermophilic S8 proteases.