Recombinant Oleate Hydratase from Lactobacillus rhamnosus ATCC 53103: Enzyme Expression and Design of a Reliable Experimental Procedure for the Stereoselective Hydration of Oleic Acid

• Published in: Catalysts Vol. 10; no. 10; p. 1122
• Main Authors: Castagna, Antonio, De Simeis, Davide, Ferrandi, Erica E., Marzorati, Stefano, Monti, Daniela, Serra, Stefano, Valentino, Mattia
• Format: Journal Article
• Language: English
• Published: Basel MDPI AG 01.10.2020
• Subjects: biocatalysis; Biocatalysts; Biotransformation; Catalysis; Catalysts; Catalytic activity; Chemical reactions; Cloning; Composition; E coli; Enzymes; Escherichia coli; Ethanol; Experiments; Fatty acids; Gene amplification; Gene expression; Genetic aspects; heterologous expression; Hydration; Lactobacilli; Lactobacillus; Lactobacillus rhamnosus; Lactose; Methods; Microorganisms; Molecular weight; Monounsaturated fatty acids; oleate hydratase; Oleic acid; Probiotics; Protein expression; Proteins; Stereoselectivity; Substrate inhibition; Italy
• ISSN: 2073-4344; 2073-4344
• DOI: 10.3390/catal10101122

Different microbial strains are able to transform oleic acid (OA) into 10-hydroxystearic acid (10-HSA) by means of the catalytic activity of the enzymes oleate hydratase (EC 4.2.1.53). Lactobacillus rhamnosus ATCC 53103 performs this biotransformation with very high stereoselectivity, affording enantiopure (R)-10-HSA. In this work, we cloned, in Escherichia coli, the oleate hydratase present in the above-mentioned probiotic strain. Our study demonstrated that the obtained recombinant hydratase retains the catalytic properties of the Lactobacillus strain but that its activity was greatly affected by the expression procedure. According to our findings, we devised a reliable procedure for the hydration of oleic acid using a recombinant E. coli whole-cell catalyst. We established that the optimal reaction conditions were pH 6.6 at 28 °C in phosphate buffer, using glycerol and ethanol as co-solvents. According to our experimental protocol, the biocatalyst does not show significant substrate inhibition as the hydration reaction can be performed at high oleic acid concentration (up to 50 g/L).