Computational simulation to improve the activity of a heterologous feruloyl esterase secreted by Bacillus subtilis.

Feruloyl esterases (FAEs) release ferulic acid from plant cell walls but suffer from low efficiency and poor stability. A putative FAE (FAE-BSaf) from Bacillus safensis was expressed in Bacillus subtilis via a non-classical pathway, exhibiting optimal activity at 55 °C and pH 8.5. Using AlphaFold3 modeling and ΔΔG predictions, variant L13W showed 1.5-fold higher specific activity than the wild type, while G72P enhanced ferulic acid release from wheat bran only when expressed in B. subtilis 166D4. Alanine scanning confirmed His144 and His274 as essential catalytic residues, indicating that targeted modification of the substrate-binding pocket is a viable strategy to improve FAEs for lignocellulose bioconversion.