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

Coenen, Anna; Marti, Valentin Gala; Müller, Kira; Sheremetiev, Maria; Finamore, Lorenzo; Schörken, Ulrich

doi:10.1007/s12010-022-04095-0

Cited by 7 publications

(21 citation statements)

References 47 publications

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“…However, the three-enzyme cascade with LOX-1 resulted in a much lower conversion. In our previous study, we showed that the HPL reaction is significantly faster than the LOX reaction (Coenen et al 2022). Hence, the slow formation of 13S-HPODE could be the cause of the lower overall activity observed in the coupled three-enzyme system.…”

Section: Discussionmentioning

confidence: 96%

“…12-Oxo-9( Z )-dodecenoic acid was dissolved in ethanol and frozen at − 80 °C until further use. The amount and purity of 12-oxo-9( Z )-dodecenoic acid were confirmed by GC analysis as described previously (Coenen et al 2022 ). The remaining hexanal concentration was less than 1.5% compared to 12-oxo-9( Z )-dodecenoic acid.…”

Section: Methodsmentioning

confidence: 99%

“…The transaminases TR 2 , TR 3 , and TR 6 , which were cloned into pRhokHi-2 (for TR 2 ) and pBXCH (for TR 3 and TR 6 ), were expressed in E. coli MC1061 as described previously (Coscolín et al 2019). Hydroperoxide lyase from C. papaya (HPL CP-N ) was expressed in E. coli BL21(DE3) using the pET-28a( +) expression vector as described previously (Coenen et al 2022).…”

Section: Cloning and Expression Of Enzymesmentioning

confidence: 99%

“…The eluted protein fractions were concentrated with Pierce™ Protein Concentrators PES, 10 K MWCO from Thermo Fisher Scientific (Waltham, MA, USA). The His6-tagged transaminases TR 2 , TR 3 , and TR 6 were purified by affinity chromatography as described by Coscolín et al (2019), and HPL CP-N was purified as described by Coenen et al (2022). Protein concentrations were measured with Bradford reagent (Bradford 1976), and the purification process was monitored with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli 1970).…”

Section: Protein Purificationmentioning

confidence: 99%

“…For this purpose, lipoxygenase (LOX) and hydroperoxide lyase (HPL) originating from the oxylipin pathway were coupled to a transaminase reaction. In previous work from our group, 12-oxo-9( Z )-dodecenoic acid and hexanal were obtained from safflower oil in an enzyme cascade utilizing lipase, LOX, and HPL (Coenen et al 2022 ). To date, the application of LOX and HPL has mainly focused on the green note synthesis of C6- and C9-aldehydes and their corresponding alcohols (Gigot et al 2012 ; Vincenti et al 2019 ; Stolterfoht et al 2019 ).…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 96%

Section: Methodsmentioning

confidence: 99%

Section: Cloning and Expression Of Enzymesmentioning

confidence: 99%

Section: Protein Purificationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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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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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Heterologous Expression of Toxic White Spot Syndrome Virus (WSSV) Protein in Eengineered Escherichia coli Strains

Chen

Wang

et al. 2023

Appl Biochem Biotechnol

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

Cited by 7 publications

References 47 publications

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

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

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

Heterologous Expression of Toxic White Spot Syndrome Virus (WSSV) Protein in Eengineered Escherichia coli Strains

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