Cloning and characterization of a new oleate hydratase from Acinetobacter ursingii for asymmetric hydration of unactivated alkenes

Objectives To expand the availability of promiscuous oleate hydratases (OAHs) for the asymmetric hydration of unactivated alkenes via sequence-based genome mining combined with targeted amino acid substitution. Results From 100 screened OAHs, 13 candidates were chosen, all exhibiting hydration activity toward oleic acid. These enzymes also showed significant activity with 1-decene (2 mM), with AuOAH, CkOAH, AiOAH, and CeaOAH producing ( S )-2-decanol at concentrations of 822, 603, 495, and 461 μM, respectively. AuOAH, CeaOAH, and CkOAH further demonstrated notable activity with short-chain 1-heptene (2 mM), generating ( S )-2-heptanol concentrations of 156, 115, and 133 μM, respectively. AuOAH, sourced from Acinetobacter ursingii for its relatively high activity and broad substrate range, was purified and characterized, showing turnover rates of 0.43–3.21 nmol min ^-1 mg ^-1 for aliphatic alkenes (C7–C13). The optimization of reaction conditions for whole-cell asymmetric hydration of 1-decene in recombinant E. coli (AuOAH) demonstrated that exogenous illumination with 561.5 nm light (9.4 µmol m ^-2 s ^-1 ) increased 1-decene conversion by approximately 1.5-fold. Similar light-induced enhancements (1.3–2.2-fold) were observed in OAHs from various sources. Under optimized conditions, recombinant E. coli (AuOAH) achieved 13.2–78.7% conversion for various unactivated alkenes (C7–C13) in an aqueous/organic two-phase system, with ee values ≥98%. Conclusions This study significantly enriches the enzymatic toolbox for asymmetric alkene hydration and illustrates the beneficial effect of light illumination on OAH-catalyzed hydration.