Overcoming the concentration-enantioselectivity trade-off in haloalcohol dehalogenase for high-titer...

The biocatalytic production of enantiopure (R)-epichlorohydrin (ECH) using haloalcohol dehalogenases (HHDHs) is often limited by a concentration-dependent erosion of enantioselectivity, a phenomenon primarily driven by mutual inhibition between substrate enantiomers. This work reports the engineering of the C-type HHDH from Agrobacterium radiobacter (HheC) through structure-guided rational modification to overcome this limitation. A lead triple mutant, P84A/N176Q/F186Y, was identified, showing a 9.6-fold increase in catalytic efficiency (k_cat/K_m) toward (S)-ECH and a 2.44-fold increase in the Michaelis constant (K_m) for (R)-ECH, effectively raising the threshold for enantiomeric inhibition and improving kinetic resolution at elevated substrate titers. The mechanistic basis for the enhanced performance was further elucidated by MD simulations, which revealed an optimized binding landscape and a higher frequency of productive binding orientations. These effects were primarily mediated by the structural contraction between the catalytic triad and the substrate. To facilitate practical implementation, a fed-batch biotransformation strategy was developed under optimized pH and temperature conditions and the triple mutant facilitated the synthesis of (R)-ECH with >99% enantiomeric excess (ee) at a 200 mM substrate loading. This process achieved an enantioselectivity factor (E-value) of 20.66, representing a 9.98-fold improvement over that of the wild-type enzyme.