Engineering Acid Adaptability of Zearalenone Lactonase Guided by an Integrated Structural and Computational Strategy

Abstract Zearalenone (ZEN) lactonases are promising biocatalysts for detoxifying ZEN-contaminated food and feed. However, their poor activity and stability under acidic conditions limit practical applications. Here, we establish an integrated computationally/structurally guided hydrophobic microenvironment engineering as a paradigm for improving the acid adaptability of ZEN lactonases. Constant pH molecular dynamics (CpHMD) simulations revealed that acidic conditions suppress proton abstraction by altering catalytic-residue protonation and destabilizing key structural regions. Structure-guided engineering introduced Pro and Phe residues to stabilize acid-sensitive regions, while hydrophobic microenvironment engineering promoted substrate repositioning and the formation of near-attack conformations. The engineered M4 mutant exhibited a 111.7-fold enhancement in acid stability. Validation with 5Z-7-oxozeaenol supported a hydrophobic gating mechanism, in which the hydrophobic microenvironment governs substrate positioning, aligns catalytic residues, and stabilizes reactive conformations. M4 degraded approximately 70% of ZEN under simulated gastrointestinal pH conditions, demonstrating its potential for ZEN detoxification.