From Computational Prediction to Stable Co-Immobilization: Engineering Aldo-Keto Reductase–Glucose Dehydrogenase Cascade on Amino Resin for Efficient Green Synthesis of ( R )-Ethyl-8-Chloro-6-Hydroxyoctanoate

Multienzyme cascade catalysis is promising for chiral drug synthesis, but the poor stability and difficult recovery of free enzymes limit industrial applications. Conventional immobilization relies on inefficient trial-and-error screening. Here, we report a rational, computationally guided strategy to systematically investigate the co-immobilization compatibility of aldo-keto reductase (AKR) and glucose dehydrogenase (GDH). Using multiscale simulations (bioinformatics, homology modeling, substrate channel prediction, molecular docking, and molecular dynamics), we analyzed active sites, surface properties, and enzyme–support interactions. Amino resin LXTE-704 was identified as the optimal support, enabling efficient dual-enzyme co-immobilization via synergistic covalent and electrostatic adsorption. Molecular dynamics revealed that the resin constrains disordered conformational fluctuations and enhances structural stability of both enzymes. Experimentally, AKR-GDH@LXTE-704 showed markedly improved stability over free enzymes. In the green synthesis of (R)-ethyl-8-chloro-6-hydroxyoctanoate ((R)-ECHO), the co-immobilized system achieved complete substrate conversion within 6 h, afforded the product with an enantiomeric excess (ee) of >99.5%, and retained >90% substrate conversion in the 20th reuse cycle. This rational immobilization strategy provides a new paradigm for developing robust multienzyme cascades for sustainable industrial applications.