Synthesis of Linoleic Acid 13-Hydroperoxides from Safflower Oil Utilizing Lipoxygenase in a Coupled Enzyme System with In-Situ Oxygen Generation

Abstract

Linoleic acid hydroperoxides are versatile intermediates for the production of green note aroma compounds and bifunctional ω-oxo-acids. An enzyme cascade consisting of lipoxygenase, lipase and catalase was developed for one-pot synthesis of 13-hydroperoxyoctadecadienoic acid starting from safflower oil. Reaction conditions were optimized for hydroperoxidation using lipoxygenase 1 from Glycine max (LOX-1) in a solvent-free system. The addition of green surfactant Triton CG-110 improved the reaction more than two-fold and yields of >50% were obtained at linoleic acid concentrations up to 100 mM. To combine hydroperoxidation and oil hydrolysis, 12 lipases were screened for safflower oil hydrolysis under the reaction conditions optimized for LOX-1. Lipases from Candida rugosa and Pseudomonas fluorescens were able to hydrolyze safflower oil to >75% within 5 h at a pH of 8.0. In contrast to C. rugosa lipase, the enzyme from P. fluorescens did not exhibit a lag phase. Combination of P. fluorescens lipase and LOX-1 worked well upon LOX-1 dosage and a synergistic effect was observed leading to >80% of hydroperoxides. Catalase from Micrococcus lysodeikticus was used for in-situ oxygen production with continuous H2O2 dosage in the LOX-1/lipase reaction system. Foam generation was significantly reduced in the 3-enzyme cascade in comparison to the aerated reaction system. Safflower oil concentration was increased up to 300 mM linoleic acid equivalent and 13-hydroperoxides could be produced in a yield of 70 g/L and a regioselectivity of 90% within 7 h.