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

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 Umg-1 for arachidonic acid (AA). The molecular mass of the native ALOX3c was 156kDa 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 Umg-1, 1.96s-1μM-1) > docosahexaenoic acid (DHA, 13.6 Umg-1, 0.91s-1μM-1) > eicosapentaenoic acid (EPA, 10.5 Umg-1, 0.65s-1μM-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 0.1mM Zn2+ in the presence of 10mM cysteine. Under these reaction conditions, 6.88 U mL-1 D. rerio 5S-LOX converted 1.0mM of AA, EPA, and DHA to 0.91mM 5S-HETE, 0.72mM 5S-hydroxyeicosapentaenoic acid (5S-HEPE), and 0.68mM 7S-hydroxydocosahexaenoic acid (7S-HDHA) in 25, 30, and 25min, corresponding to molar conversion rates of 91, 72, and 68% and productivities of 2.18, 1.44, and 1.63mMh-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.