Enhancing the thermostability of the zearalenone lactonase ZENM through an integrated computer-aided rational design...

Zearalenone (ZEN) lactonases offer an environmentally benign route for mycotoxin detoxification, yet their industrial application is often limited by insufficient thermostability. Here, we employed an integrated computational-experimental workflow to enhance the thermal resilience of ZENM, a ZEN lactonase from Monosporascus sp. GIB2. By combining FireProt- and PROSS-guided design with ΔΔG-based screening and structure-based filtering, we identified 33 candidate single-point substitutions and experimentally validated their effects. The most promising variant, A59P, located within a solvent-exposed flexible loop (residues 57-71), retained wild-type catalytic activity while exhibiting markedly improved thermostability. At 55 °C and pH 9.0, A59P showed a 9.8-fold longer half-life (27.84 min vs. 2.85 min for the wild type) and a 7.6 °C higher half-inactivation temperature (T₅₀ = 62.5 °C vs. 54.9 °C). Differential scanning fluorimetry further showed that the apparent melting temperature increased from 59.17 ± 1.17 °C for WT to 82.37 ± 1.34 °C for A59P, corresponding to a ΔT_m of +23.20 °C. All-atom molecular dynamics simulations at 323-343 K further suggested that A59P attenuates heat-induced fluctuations in the loop (residues 57-71), limits temperature-driven global conformational expansion, and reduces the structural sensitivity of ZENM to elevated temperature, while maintaining overall comparable intramolecular hydrogen-bond levels to WT. Collectively, these results identify A59P as an effective thermostabilizing substitution in ZENM and support local loop rigidification as a key contributor to the improved thermal resilience of the enzyme.