@article{CoenenMartiM{\"u}lleretal.2022,
  author    = {Coenen, Anna and Marti, Valentin Gala and M{\"u}ller, Kira and Sheremetiev, Maria and Finamore, Lorenzo and Sch{\"o}rken, Ulrich},
  title     = {Synthesis of Polymer Precursor 12-Oxododecenoic Acid Utilizing Recombinant Papaya Hydroperoxide Lyase in an Enzyme Cascade},
  journal   = {Applied Biochemistry and Biotechnology},
  volume    = {194},
  number    = {12},
  issn      = {0273-2289},
  doi       = {10.1007/s12010-022-04095-0},
  institution = {Angewandte Naturwissenschaften (F11)},
  pages     = {6194 -- 6212},
  year      = {2022},
  abstract  = {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.},
  language  = {en}
}