Oleate hydratase activity assay.

The ability of isolates to convert LA (Sigma-Aldrich, Dublin, Ireland) and RA (Sigma-Aldrich, Dublin, Ireland) to CLA was performed as follows. Enzymatic reactions were performed in triplicate. The standard reaction conditions consisted of 1 ml of reaction mixture (50 mM potassium phosphate buffer, pH 6.0) containing 0.75 mg/ml purified OleH, 20 μM FAD, 10 mM individual unsaturated fatty acid, and 2% (vol/vol) ethanol. The reactions were carried out for 15, 30, 45, and 60 min anaerobically at 37°C. Two negative controls were used consisting of all the components except substrates and all the components except the purified protein.