Characterization and Substrate Specificity of a Recombinant Acetyl Xylan Esterase from Halalkalibacterium halodurans through a Dual-Strategy Rational Design

Acetylxylan esterases (AXEs) hydrolyze acetyl groups in xylan but are limited by their narrow substrate specificity. To improve their industrial applications, we engineered a CE7 family HhAXE from Halobacillus halodurans . The purified enzyme exhibited optimal activity at pH 8.5 and 40 °C toward ρ-nitrophenyl acetate (ρNPA), with stability across pH 8.0–9.0 and 30–45 °C. Fe 3+ and Mn 2+ (10 mM) enhanced activity by 184.94 and 195.13%, respectively, and 20% DMSO increased activity by 105.56%. Using dual computational strategies─PROSS-based thermostabilization and machine learning-guided optimization─we generated mutant L260R, which showed a 136.16% increase in ρNPA activity and improved affinity for longer-chain substrates (ρNPB, ρNPO). The mutant also achieved 140.99, 132.68, and 127.98% higher deacetylations of beechwood, arabinoxylan, and corncob, respectively. Molecular docking and dynamics simulations revealed structural changes that enhanced the substrate-binding and catalytic efficiency. The engineered HhAXE-L260R shows potential as a biocatalyst for lignocellulose valorization and offers a computational approach to AXE optimization.