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.

油酸水合酶（Ohys，EC 4.2.1.53）是一类酶，其能够选择性地与多种不饱和脂肪酸加水反应，生成各自的手性醇。大量研究致力于改善现有Ohys的应用，以及鉴定具有潜在新特性的未描述Ohys。这项研究通过探索红球菌属中油酸水合酶的丰富性来研究后者。三个不同的红球菌分支表明存在油酸水合酶，每个分支均由特定的油酸酯水合酶家族（HFam）表示。系统发生和序列分析揭示了保守氨基酸之间的HFam特异性模式。来自两个红球菌的油酸水合酶菌株（HFam 2和3）在大肠杆菌中异源表达，并研究了其底物范围。在此，两种酶均显示出对空间需求和多种不饱和脂肪酸的互补行为。此外，这项研究还包括对新发现的吡咯菌Rhodococcus pyridinivorans Ohy的鉴定。使用基于醇脱氢酶和10-羟基硬脂酸的NAD ⁺依赖性氧化的新型偶联测定法，测定了吡啶并霉R. Ohy的稳态动力学。