Oleate Hydratase from <i>Lactobacillus rhamnosus</i> ATCC 53103: A FADH₂-Dependent Enzyme with Remarkable Industrial Potential

• Main Authors: Stefano Serra, Davide De Simeis, Stefano Marzorati, Mattia Valentino
• Format: Article
• Language: English
• Published: MDPI AG 2021-08-01
• Series: Catalysts
• Subjects: oleate hydratase; <i>Lactobacillus rhamnosus</i> ATCC 53103; heterologous expression; oleic acid; 10-hydroxystearic acid; biocatalysis
• Online Access: https://www.mdpi.com/2073-4344/11/9/1051

Recently, we described the preparation of the recombinant oleate hydratase from <i>Lactobacillus rhamnosus</i> ATCC 53103. We observed that the purified C-terminal His-tagged enzyme was completely inactive and the catalytic activity was partially restored only in presence of a large amount of flavin adenine dinucleotide (FAD). In the present work, we assess that this hydratase in the presence of the reduced form of flavin adenine dinucleotide (FADH₂) is at least one hundred times as active as in the presence of the same concentration of FAD. By means of two different biochemical processes, we demonstrated unambiguously that oleate hydratase from <i>Lactobacillus rhamnosus</i> ATCC 53103 is a FADH₂-dependent enzyme. As a first relevant application of this discovery, we devised a preparative procedure for the stereoselective synthesis of (<i>R</i>)-10-hydroxystearic acid. Accordingly, the hydration of oleic acid (up to 50 g/L) is performed on a multigram scale using the recombinant hydratase and FADH₂ generated in situ as cofactor. The produced (<i>R</i>)-10-hydroxystearic acid (ee > 97%) precipitates from the reaction solvent (water/glycerol/ethanol) and is conveniently recovered by simple filtration (>90% yield).