Oleate hydratases (Ohys, EC 4.2.1.53) are a class of enzymes capable of selective water addition reactions to a broad range of unsaturated fatty acids leading to the respective chiral alcohols. Much research was dedicated to improving the applications of existing Ohys as well as to the identification of undescribed Ohys with potentially novel properties. This study focuses on the latter by exploring the genus Rhodococcus for its plenitude of oleate hydratases. Three different Rhodococcus clades showed the presence of oleate hydratases whereby each clade was represented by a specific oleate hydratase family (HFam). Phylogenetic and sequence analyses revealed HFam-specific patterns amongst conserved amino acids. Oleate hydratases from two Rhodococcus strains (HFam 2 and 3) were heterologously expressed in Escherichia coli and their substrate scope investigated. Here, both enzymes showed a complementary behaviour towards sterically demanding and multiple unsaturated fatty acids. Furthermore, this study includes the characterisation of the newly discovered Rhodococcus pyridinivorans Ohy. The steady-state kinetics of R. pyridinivorans Ohy was measured using a novel coupled assay based on the alcohol dehydrogenase and NAD⁺-dependent oxidation of 10-hydroxystearic acid.

The implementation of a stereoselective Michael addition with water as substrate is still a major challenge by classical, chemical means. Inspired by nature's ability to carry out this attractive reaction with both high selectivity and efficiency, the interest in hydratases (EC 4.2.1.x) to accomplish a selective water addition is steadily rising. The gram-positive bacterial genus Rhodococcus is known as biocatalytic powerhouse and has been reported to hydrate various Michael acceptors leading to chiral alcohols. This study aimed at the in-depth re-investigation of the hydration potential of Rhodococcus whole-cells towards Michael acceptors. Here, two concurrent effects responsible for the hydration reaction were found: while the majority of substrates was hydrated in an oxygen-independent manner by amino-acid catalysis, an enzyme-catalysed water addition to (E)-4-hydroxy-3-methylbut-2-enoic acid was proven to be oxygen-dependent. ¹⁸O₂-labelling studies showed that no ¹⁸O₂ was incorporated in the product. Therefore, a novel O₂-dependent hydratase distinct from all characterised hydratases so far was found.

The C12 specific oxidation of hydroxysteroids is an essential reaction required for the preparation of pharmaceutical ingredients like ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA), which can be synthesized by Wolff-Kishner reduction of the obtained 12-oxo-hydroxysteroids. 12α-hydroxysteroid dehydrogenases (12α-HSDHs) have been shown to perform this reaction with high yields, under mild conditions and without the need of protection and deprotection steps, required in chemical synthesis. Here, the recombinant expression and biochemical characterization of the nicotinamide adenine dinucleotide (NAD ⁺ )-dependent HSDH from Eggerthella lenta (El12α-HSDH) are reported. This enzyme shows comparable properties with the well-known nicotinamide adenine dinucleotide phosphate (NADP ⁺ )-dependent enzyme from Clostridium sp. 48–50. In order to perform a viable and atom efficient enzymatic hydroxysteroid oxidation, NAD(P)H oxidase (NOX) was employed as cofactor regeneration system: NOX uses oxygen (O ₂ ) as sacrificial substrate and produces only water as side product. 10 mM of cholic acid was fully and selectively converted to 12-oxo-CDCA in 24 h. The possibility to employ this system on UCA and 7-oxo-deoxycholic acid (7-oxo-DCA) as substrates was additionally investigated. The performance of the El12α-HSDH was evaluated also in combination with a “classical” regeneration system (oxaloacetate/malate dehydrogenase) showing full conversion in 4 h. Finally, the feasibility of a catalytic aerobic-NAD ⁺ -dependent enzymatic oxidation was shown on a preparative scale (oxidation of CA to 12-oxo-CDCA) employing the El12α-HSDH-NOX system in a segmented-flow-reactor. (Figure presented.).