Biochemical characterization and molecular docking of a novel alkaline-stable keratinase from Amycolatopsis sp. BJA-103.

Amycolatopsis sp. BJA-103 was isolated for its exceptional feather-degradation capability, leading to the purification, cloning, and heterologous expression of the keratinase enzyme, KER0199. Sequence analysis places KER0199 within the S8 protease family, revealing <60 % sequence similarity to known proteases. The recombinant KER0199-His demonstrates a broad substrate range, along with remarkable thermostability and alkaline stability, exhibiting optimal activity at pH 11.0 and 60 °C, despite the absence of cysteine residues essential for disulfide bonding. Structural modeling reveals a predominantly negatively charged surface and a flat, low-electrostatic-potential substrate-binding pocket. Substrate-binding models, predicted using AlphaFold3 and molecular dynamics simulations, indicate that substrates such as casein, chicken feather β-keratin P2450, and hemoglobin bind to this pocket, forming anti-parallel β-sheets with residues G97 to G99 and establishing extensive hydrogen bonds with key residues near the enzyme's active site. These findings suggest that AlphaFold-based substrate binding predictions, combined with an analysis of intermolecular forces, provide a valuable tool for assisting in the elucidation of enzyme specificity and substrate recognition. KER0199, the first characterized S8 family keratinase from the Amycolatopsis genus, shows great potential for industrial applications.