Biotransformation of Polyunsaturated Fatty Acids to Trioxilins by Lipoxygenase from Pleurotus sajor-caju

• Published in: Chembiochem : a European journal of chemical biology Vol. 24; no. 23; p. e202300556
• Main Authors: Seo, Min-Ju, Lee, Tae-Eui, Yeom, Soo-Jin, Oh, Deok-Kun, Shin, Kyung-Chul
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 01.12.2023
• Subjects: Arachidonic acid; Biotransformation; Chromatography; Chromatography, Liquid; Docosahexaenoic acid; Docosahexaenoic Acids - metabolism; E coli; Eicosapentaenoic acid; Enzymes; Fatty acids; Fatty Acids, Unsaturated; High performance liquid chromatography; Lipoxygenase; Lipoxygenase - metabolism; Liquid chromatography; Liquid oxygen; Mass spectrometry; Mass spectroscopy; Pleurotus; Polyunsaturated fatty acids; Substrate specificity; Substrates; Tandem Mass Spectrometry; Pleurotus sajor-caju; trioxilin; fatty acid; lipoxygenase; lipid mediator
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.202300556

A lipoxygenase from Pleurotus sajor-caju (PsLOX) was cloned, expressed in Escherichia coli, and purified as a soluble protein with a specific activity of 629 μmol/min/mg for arachidonic acid (AA). The native PsLOX exhibited a molecular mass of 146 kDa, including a 73-kDa homodimer, as estimated by gel-filtration chromatography. The major products converted from polyunsaturated fatty acids (PUFAs), including AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), were identified as trioxilins (TrXs), namely 13,14,15-TrXB , 13,14,15-TrXB , and 15,16,17-TrXB , respectively, through high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. The enzyme displayed its maximum activity at pH 8.0 and 20 °C. Under these conditions, the specific activity and catalytic efficiency of PsLOX for PUFAs exhibited the following order: AA>EPA>DHA. Based on HPLC analysis and substrate specificity, PsLOX was identified as an arachidonate 15-LOX. PsLOX efficiently converted 10 mM of AA, EPA, and DHA to 8.7 mM of 13,14,15-TrXB (conversion rate: 87 %), 7.9 mM of 13,14,15-TrXB (79 %), and 7.2 mM of 15,16,17-TrXB (72 %) in 15, 20, and 20 min, respectively, marking the highest conversion rates reported to date. Collectively, our results demonstrate that PsLOX is an efficient TrXs-producing enzyme.