Versatile ferrous oxidation–xylenol orange assay for high‑throughput screening of lipoxygenase activity

Chrisnasari, Ruth and Ewing, Tom A. and Hennebelle, Marie and van Berkel, Willem J. H. and Vincken, Jean-Paul (2024) Versatile ferrous oxidation–xylenol orange assay for high‑throughput screening of lipoxygenase activity. Applied Microbiology and Biotechnology, 108 (266).

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Lipoxygenases (LOXs) catalyze dioxygenation of polyunsaturated fatty acids (PUFAs) into fatty acid hydroperoxides (FAHPs), which can be further transformed into a number of value-added compounds. LOXs have garnered interest as bio�catalysts for various industrial applications. Therefore, a high-throughput LOX activity assay is essential to evaluate their performance under diferent conditions. This study aimed to enhance the suitability of the ferrous-oxidized xylenol orange (FOX) assay for screening LOX activity across a wide pH range with diferent PUFAs. The narrow linear detection range of the standard FOX assay restricts its utility in screening LOX activity. To address this, the concentration of perchloric acid in the xylenol orange reagent was adjusted. The modifed assay exhibited a fvefold expansion in the linear detection range for hydroperoxides and accommodated samples with pH values ranging from 3 to 10. The assay could quantify vari�ous hydroperoxide species, indicating its applicability in assessing LOX substrate preferences. Due to sensitivity to pH, bufer types, and hydroperoxide species, the assay required calibration using the respective standard compound diluted in the same bufer as the measured sample. The use of correction factors is suggested when fnancial constraints limit the use of FAHP standard compounds in routine LOX substrate preference analysis. FAHP quantifcation by the modifed FOX assay aligned well with results obtained using the commonly used conjugated diene method, while ofering a quicker and broader sample pH range assessment. Thus, the modifed FOX assay can be used as a reliable high-throughput screening method for determining LOX activity.

Item Type: Article
Subjects: Q Science > QD Chemistry
Divisions: Faculty of Technobiology > Department of Biology
Depositing User: RUTH CHRISNASARI - 210001
Date Deposited: 19 Mar 2024 03:03
Last Modified: 19 Mar 2024 03:03

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