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.

Hydroperoxide lyases (HPLs) catalyze the splitting of 13S-hydroperoxyoctadecadienoic acid (13S-HPODE) into the green note flavor hexanal and 12-oxo-9(Z)-dodecenoic acid, which is not yet used industrially. Here, HPL from Carica papaya (HPLCP) was cloned and functionally expressed in Escherichia coli to investigate synthesis of 12-oxo-9(Z)-dodecenoic acid in detail. To improve the low catalytic activity of full-length HPLCP, the hydrophobic, non-conserved N-terminal sequence was deleted. This enhanced enzyme activity from initial 10 to 40 U/l. With optimization of solubilization buffer, expression media enzyme activity was increased to 2700 U/l. The tetrameric enzyme was produced in a 1.5 l fermenter and enriched by affinity chromatography. The enzyme preparation possesses a slightly acidic pH optimum and a catalytic efficiency (kcat/KM) of 2.73 × 106 s−1·M−1 towards 13S-HPODE. Interestingly, HPLCP-N could be applied for the synthesis of 12-oxo-9(Z)-dodecenoic acid, and 1 mM of 13S-HPODE was transformed in just 10 s with a yield of 90%. At protein concentrations of 10 mg/ml, the slow formation of the 10(E)-isomer traumatin was observed, pointing to a non-enzymatic isomerization process. Bearing this in mind, a one-pot enzyme cascade starting from safflower oil was developed with consecutive addition of Pseudomonas fluorescens lipase, Glycine max lipoxygenase (LOX-1), and HPLCP-N. A yield of 43% was obtained upon fast extraction of the reaction mixtures after 1 min of HPLCP-N reaction. This work provides first insights into an enzyme cascade synthesis of 12-oxo-9(Z)-dodecenoic acid, which may serve as a bifunctional precursor for bio-based polymer synthesis.

Hydroperoxy‐9Z,11E‐octadecadienoic acid (13‐HPODE) can be obtained from safflower oil in an enzyme cascade utilizing lipase, lipoxygenase (LOX), and catalase for in situ oxygen generation. The application of immobilized enzymes may open a new path to a cost‐efficient production of 13‐HPODE, which is used for the synthesis of green note aroma hexanal. Ten immobilization supports are compared for immobilization of lipoxygenase‐1 from Glycine max (LOX‐1) and oxirane‐based Immobead 150 P proves to be best with a maximum LOX‐1 activity of 22 470 U g−1. The immobilizate is successfully recycled in eight consecutive batches and maintains full activity over a period of 16 h using a 3D‐printed column reactor. Catalase from Micrococcus lysodeikticus and LOX‐1 are co‐immobilized on Immobead 150 P allowing a constant production of 13‐HPODE for up to six cycles with a maximum product conversion of 45% and a 13‐regioselectivity of 83% . In this two‐enzyme system with H2O2‐dosage, foam generation is significantly reduced. Co‐immobilization of LOX‐1 and lipaseis possible; however, rapid lipase deactivation occurs. Therefore, a two‐reactor approach with oil hydrolysis in the first reactor is proposed. Immobilized lipases from C. rugosa are suitable for safflower oil hydrolysis and maintain full activity over ten hydrolysis cycles. Practical applications: Linoleic acid hydroperoxide (13‐HPODE) is the starting material for the synthesis of the green note aroma compound hexanal. The byproduct of the hydroperoxide splitting is ω‐oxododecenoic acid, which is currently not employed industrially. The bifunctional oxodocecenoic acid is interesting as precursor for the synthesis of polymer building blocks. Simple one‐step derivatization of the oxo‐group can yield suitable C12 monomers such as dicarboxylic acids, ω‐amino acids, or ω‐hydroxy acids. Cost‐efficiency is a key parameter to incorporate these new biobased building blocks for polymer applications. In this approach, immobilized enzymes are used for the synthesis of 13‐HPODE starting from safflower oil with in situ oxygen generation to prevent excessive foam formation. A two‐reactor concept is designed to circumvent hydroperoxide‐induced lipase deactivation. Direct comparison of both batch and continuous process is performed and provides information for the implementation of the enzyme cascade and the design of an optimized reactor system.