Optimizing reaction parameters for the enzymatic synthesis of epoxidized oleic acid with oat seed peroxygenase

Peroxygenase is a plant enzyme that catalyzes the oxidation of a double bon d to an epoxide in a stereospecific and enantiofacially selective manner. A microsomal fraction containing peroxygenase was prepared from oat (Avena s ativa) seeds and the enzyme immobilized onto a hydrophobic membrane. The en zymatic activity of the immobilized preparation was assayed in 1 h by measu ring epoxidation of sodium oleate (5 mg) in buffer-surfactant mixtures. The pH optimum of the reaction was 7.5 when t-butyl hydroperoxide was the oxid ant and 5.5 when hydrogen peroxide was the oxidant. With t-butyl hydroperox ide as oxidant the immobilized enzyme showed increasing activity to 65 degr eesC. The temperature profile with hydrogen peroxide was flatter, although activity was also retained to 65 degreesC. In 1 h reactions at 25 degreesC at their respective optimal pH values, t-butyl hydroperoxide and hydrogen p eroxide promoted epoxide formation at the same rate. larger-scale reactions were conducted using a 20-fold increase in sodium oleate (to 100 mg). Reac tion time was lengthened to 24 h. At optimized levels of t-butyl hydroperox ide 80% conversion to epoxide was achieved. With hydrogen peroxide only a 3 3% yield of epoxide was obtained, which indicates that hydrogen peroxide ma y deactivate peroxygenase.