A Dual-Focus Workflow for Simultaneously Engineering High Thermostability of Aldo-Keto Reductase for the Degradation of 3-Keto-Deoxynivalenol

-DON, thereby alleviating mycotoxin contamination in food and feed. However, the previously reported AKR13B3 mutant AKR-V3 exhibited high catalytic activity but poor thermostability, restricting its industrial application. Here, guided by molecular dynamics flexibility analysis and consensus sequence design, we engineered the variant M5 with markedly improved thermostability. M5 showed a 38.3-fold longer half-life at 60 °C and retained slightly enhanced catalytic efficiency. Quantum mechanics/molecular mechanics (QM/MM) calculations revealed that M5 possessed a lower activation barrier than AKR-V3, which explained its superior activity retention under high-temperature conditions. Moreover, structural analyses and molecular dynamics (MD) simulations further demonstrated that the enhanced thermostability of M5 stemmed from reinforced hydrogen bonding, increased hydrophobic packing, elevated structural rigidity, and optimized surface electrostatic potential. This work provides an effective strategy for designing thermostable detoxification enzymes to eliminate mycotoxins in industrial applications.