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.
Highlights
Lipoxygenases (LOX, EC. 1.13.11.X) are widely distributed in eukaryotes and were more recently identified in bacteria as well [1]
We present the development of an enzymatic cascade consisting of lipase, LOX and catalase for efficient synthesis of linoleic acid hydroperoxides starting from safflower oil (Scheme 1)
LOX dosing HPODE concentration increased to around 80% after 3 h, even exceeding the control reaction with lipoxygenase 1 from Glycine max (LOX-1) dosage starting from linoleic acid (Figure 4a)
Summary
Lipoxygenases (LOX, EC. 1.13.11.X) are widely distributed in eukaryotes and were more recently identified in bacteria as well [1]. The main biocatalytic application is the production of natural green note aroma compounds for the flavor and fragrance industry by combined action of LOX and hydroperoxide lyase for splitting of the hydroperoxides [8,12,13] This Hock-type rearrangement may be catalyzed chemically leading to volatile C6-aldehydes and the corresponding C12-ω-oxoacids [14,15]. The ω-oxoacids are an interesting class of products and were proposed as bifunctional monomer precursors for green polymer synthesis [16] Another possible LOX application could be the synthesis of plant metabolites like -12-oxophytodienoic acid (12-OPDA) in an enzymatic cascade mimicking the LOX pathway [17]. Solubility of the fatty acid substrates and strong foaming due to molecular oxygen injection under alkaline conditions, which leads to soap formation, are major issues in high-yield production of hydroperoxides with soybean LOX-1.
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