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

Marti, Valentin Gala; Coenen, Anna; Schörken, Ulrich

doi:10.3390/catal11091119

Cited by 10 publications

(31 citation statements)

References 41 publications

(54 reference statements)

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“…The development of enzyme cascades possesses significant advantages over successive reactions including a shift of equilibria without the need for isolation of intermediates [ 48 , 49 ]. In our previous work, we developed a cascade reaction for 13S-HPODE synthesis from safflower oil utilizing lipase from P. fluorescens , LOX-1 from soybeans, and catalase for in situ oxygen generation [ 11 ]. Now we wanted to couple lipase and LOX-1 reaction with HPL CP-N to prove the concept of a one-pot synthesis of 12-oxo-9(Z)-dodecenoic acid and hexanal from safflower oil.…”

Section: Resultsmentioning

confidence: 99%

“…Safflower oil was purchased from Gefro (Germany) and had a fatty acid composition of 77.2% linoleic acid, 13.3% oleic acid, 2.4% stearic acid, 6.7% palmitic acid, and 0.4% of other fatty acids [ 11 ]. 13S-HPODE, 12-oxo-9(Z)-dodecenoic acid, and 12-oxo-10(E)-dodecenoic acid standards were from Larodan (Sweden).…”

Section: Methodsmentioning

confidence: 99%

“…Linoleic acid was prepared from safflower oil by alkaline hydrolysis and enrichment by urea crystallization as described before [ 11 ]. Linoleic acid was then diluted in 2.5 l of cold 50 mM sodium borate buffer (pH 9.5) to a final concentration of 1 mM.…”

Section: Methodsmentioning

confidence: 99%

“…The organic phase was separated and washed with water before evaporating residual solvent under vacuum. Final HPODE content and regioisomeric ratio were determined photometrically at 234 nm and by HPLC analysis as described before [ 11 ].…”

Section: Methodsmentioning

confidence: 99%

“…Recently, the combination of lipase and lipoxygenase (LOX) with green surfactant and in situ oxygen generation was developed by our group for high-yield synthesis of 13S-HPODE from safflower oil [ 11 ]. The subsequent HPL cleavage of 13S-HPODE leads to 12-oxododecenoic acid and hexanal.…”

Section: Introductionmentioning

confidence: 99%

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Synthesis of Polymer Precursor 12-Oxododecenoic Acid Utilizing Recombinant Papaya Hydroperoxide Lyase in an Enzyme Cascade

Coenen

Marti

Müller

et al. 2022

Appl Biochem Biotechnol

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Synthesis of Polymer Precursor 12-Oxododecenoic Acid Utilizing Recombinant Papaya Hydroperoxide Lyase in an Enzyme Cascade

Coenen

Marti

Müller

et al. 2022

Appl Biochem Biotechnol

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Immobilization of Lipoxygenase, Catalase, and Lipase for a Reactor Design Targeting Linoleic Acid Hydroperoxidation

Marti

Müller

Spektor

et al. 2022

Euro J Lipid Sci & Tech

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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.

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Synthesis of 12-aminododecenoic acid by coupling transaminase to oxylipin pathway enzymes

Coenen

Ferrer²,

Jaeger

et al. 2023

Appl Microbiol Biotechnol

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Biobased polymers derived from plant oils are sustainable alternatives to petro based polymers. In recent years, multienzyme cascades have been developed for the synthesis of biobased ω-aminocarboxylic acids, which serve as building blocks for polyamides. In this work, we have developed a novel enzyme cascade for the synthesis of 12-aminododeceneoic acid, a precursor for nylon-12, starting from linoleic acid. Seven bacterial ω-transaminases (ω-TAs) were cloned, expressed in Escherichia coli and successfully purified by affinity chromatography. Activity towards the oxylipin pathway intermediates hexanal and 12-oxododecenoic acid in their 9(Z) and 10(E) isoforms was demonstrated for all seven transaminases in a coupled photometric enzyme assay. The highest specific activities were obtained with ω-TA from Aquitalea denitrificans (TRAD), with 0.62 U mg−1 for 12-oxo-9(Z)-dodecenoic acid, 0.52 U mg−1 for 12-oxo-10(E)-dodecenoic acid and 1.17 U mg−1 for hexanal. A one-pot enzyme cascade was established with TRAD and papaya hydroperoxide lyase (HPLCP-N), reaching conversions of 59% according to LC-ELSD quantification. Starting from linoleic acid, up to 12% conversion to 12-aminododecenoic acid was achieved with a 3-enzyme cascade comprising soybean lipoxygenase (LOX-1), HPLCP-N and TRAD. Higher product concentrations were achieved by the consecutive addition of enzymes compared to simultaneous addition at the beginning. Key points • Seven ω-transaminases converted 12-oxododecenoic acid into its corresponding amine. • A three-enzyme cascade with lipoxygenase, hydroperoxide lyase, and ω-transaminase was established for the first time. • A one-pot transformation of linoleic acid to 12-aminododecenoic acid, a precursor of nylon-12 was achieved. Graphical Abstract

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

Cited by 10 publications

References 41 publications

Synthesis of Polymer Precursor 12-Oxododecenoic Acid Utilizing Recombinant Papaya Hydroperoxide Lyase in an Enzyme Cascade

Synthesis of Polymer Precursor 12-Oxododecenoic Acid Utilizing Recombinant Papaya Hydroperoxide Lyase in an Enzyme Cascade

Immobilization of Lipoxygenase, Catalase, and Lipase for a Reactor Design Targeting Linoleic Acid Hydroperoxidation

Synthesis of 12-aminododecenoic acid by coupling transaminase to oxylipin pathway enzymes

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