PUBLICATION

Characterization of Arachidonate 5S-Lipoxygenase from Danio rerio with High Activity for the Production of 5S- and 7S-Hydroxy Polyunsaturated Fatty Acids

Authors
Shin, K.C., Lee, J., Oh, D.K.
ID
ZDB-PUB-221018-100
Date
2022
Source
Applied Biochemistry and Biotechnology   195(2): 958-972 (Journal)
Registered Authors
Keywords
5S-Hydroxyeicosatetraenoic acid, Arachidonate 5S-lipoxygenase, Arachidonic acid, Danio rerio, Enzyme characterization, Zebrafish
MeSH Terms
  • Docosahexaenoic Acids
  • Arachidonate Lipoxygenases*
  • Fatty Acids, Unsaturated
  • Arachidonate 15-Lipoxygenase
  • Chromatography, Liquid
  • Hydroxyeicosatetraenoic Acids/metabolism
  • Arachidonic Acid/metabolism
  • Zebrafish*/metabolism
  • Tandem Mass Spectrometry
  • Animals
(all 10)
PubMed
36251113 Full text @ Appl. Biochem. Biotechnol.
Abstract
A recombinant putative lipoxygenase (LOX) from Danio rerio (zebrafish), ALOX3c protein with 6-histidine tag, was purified using affinity chromatography, with a specific activity of 17.2 U mg-1 for arachidonic acid (AA). The molecular mass of the native ALOX3c was 156 kDa composed of a 78-kDa dimer by gel-filtration chromatography. The product obtained from the conversion of AA was identified as 5S-hydroxyeicosatetraenoic acid (5S-HETE) by HPLC and LC-MS/MS analyses. The specific activity and catalytic efficiency of the LOX from D. rerio for polyunsaturated fatty acids (PUFAs) followed the order AA (17.2 U mg-1, 1.96 s-1 μM-1) > docosahexaenoic acid (DHA, 13.6 U mg-1, 0.91 s-1 μM-1) > eicosapentaenoic acid (EPA, 10.5 U mg-1, 0.65 s-1 μM-1) and these values for AA were the highest among the 5S-LOXs reported to date. Based on identified products and substrate specificity, the enzyme is an AA 5S-LOX. The enzyme exhibited the maximal activity at pH 8.0 and 20 °C with 0.1 mM Zn2+ in the presence of 10 mM cysteine. Under these reaction conditions, 6.88 U mL-1 D. rerio 5S-LOX converted 1.0 mM of AA, EPA, and DHA to 0.91 mM 5S-HETE, 0.72 mM 5S-hydroxyeicosapentaenoic acid (5S-HEPE), and 0.68 mM 7S-hydroxydocosahexaenoic acid (7S-HDHA) in 25, 30, and 25 min, corresponding to molar conversion rates of 91, 72, and 68% and productivities of 2.18, 1.44, and 1.63 mM h-1, respectively. To the best of our knowledge, this study is the first to describe the bioconversion into 5S-HETE, 5S-HEPE, and 7S-HDHA for the application of biotechnological production.
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