Synergistic enhancement of activity and stability for haloalcohol dehalogenase in aqueous‐organic biphasic systems

Abstract The haloalcohol dehalogenase‐catalyzed conversion of 1,3‐dichloro‐2‐propanol to epichlorohydrin is a promising biosynthetic route. However, poor stability of substrate and product in aqueous media requires an aqueous‐organic biphasic system, which challenges the enzyme's catalytic performance and conformational robustness. To address these bottlenecks, this study developed a synergistic protein engineering strategy via structural analysis and computer‐aided rational design. Long‐range conformational modulation and direct substrate–enzyme interaction optimization were combined to improve the catalytic efficiency, and a holistic stabilization strategy that coordinates rational reshaping of organic solvent channels and optimization of protein binding free energy was proposed to enhance stability in the biphasic system. The obtained variant (HheC 4M R123A+A159T+L143N+V206A+F244W ) exhibited a 16.1‐fold prolonged half‐life (138.3 h) and 3.5‐fold higher catalytic efficiency ( k cat / K m ) than the parental enzyme. The engineering strategy established in this work not only broadens the application prospects of haloalcohol dehalogenases, but also provides new insights for protein engineering in organic or biphasic systems.