A Comparative Biochemical Study of Oleate Hydratases

In the search for more efficient chemical processes to valorize renewable feedstocks, oleate hydratases (Ohys) represent promising biocatalysts. These enzymes can convert cis-Δ⁹ fatty acids like oleic acid into their hydroxy fatty acid counterparts using only water as co-reagent and with unparallelled selectivity. A multitude of Ohys has been described, however, direct comparison is hard due to the use of different assays. For this work, we characterized and compared four Ohys across different homologous families (HFams): those from Elizabethkingia meningoseptica (EmOhy, HFam11), Stenotrophomonas nitritireducens (SnOhy, HFam11), Rhodococcus erythropolis (ReOhy, HFam3), and Rhodococcus pyridinivorans (RpOhy, HFam2). All enzymes, especially SnOhy, expressed well in Escherichia coli (28-99 mg/L_culture). FAD occupancy after purification was 51% or lower for all Ohys. ReOhy had lost the flavin altogether, making supplementation a necessity for its activity. Reduction of the cofactor to FADH₂ clearly showed a favorable effect on the activity of all Ohys, with product yields increasing from modest to 12-fold. Although SnOhy showed the highest initial reaction rates, EmOhy reached the highest 10-hydroxystearic acid (10-HSA) yields at higher oleic acid loading (10 mM) and prolonged reaction times. Thermostability was analyzed by thermal shift assays, with EmOhy appearing as most thermostable (T_m ^app = 54 °C).