Stability and Activity Collaborative Improvement of Carbonyl Reductase Based on the Modification Strategy for Transition Zone of the Flexible and Rigid Regions: An Application for Vibegron Chiral Intermediate Synthesis in High Efficiency

Carbonyl reductases (EC 1.1.1.148, CRs) make up a group of oxidoreductases that catalyze the asymmetric reduction of prochiral ketones or aldehydes to produce the corresponding chiral alcohols, which are widely used in pharmaceutical and fine chemical industries. However, challenges in improving the enzymatic activity and stability continue to hinder the broader industrial application of the biocatalysts. In this study, a novel strategy was developed to target the transition region between the rigid and flexible domains of EaSDR6 from Exiguobacterium sp. s126. B-factor analysis was used to guide the introduction of mutations (K36D/T75K) at the domain interface. The resulting variant, EaSDR6^K36D/T75K, exhibited nearly a 5-fold increase in catalytic efficiency (k_cat/K_m = 1.53 mM^-1·s^-1) and an improvement of 10.4 °C in thermal stability (T_m = 54.8 °C). This transition zone engineering enhanced NADPH binding and stabilized the active site, effectively overcoming the activity-stability limitation. In a BuOAc-H₂O biphasic system, EaSDR6^K36D/T75K was employed to catalyze the asymmetric synthesis of (2S,3R)-2-((tert-butoxycarbonyl) amino)-3-hydroxy-3-phenylpropanoate, a key intermediate for vibegron synthesis. A conversion of 97% was achieved within 36 h at a total substrate concentration of 200 g/L, accompanied by >99% enantiomeric excess (e.e) and >99% diastereomeric excess (d.e). This study presents a practical approach for enhancing enzyme performance and advancing green chiral alcohol synthesis.