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(6) demonstrates a broad substrate range, along with remarkable thermostability and alkaline stability, exhibiting optimal activity at pH 11.0 and 60 degrees 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 beta-keratin P2450, and hemoglobin bind to this pocket, forming anti-parallel beta-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.