Materials

Chemicals used for enzymatic reactions and FOX assay were obtained from the following sources: linoleic acid (LA; C18:2 Δ9Z,12Z) from Nu-Chek Prep, Inc., Minnesota, USA; fatty acid hydroperoxide standards (13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13-HPODE), 13(S)-hydroperoxy-9Z,11E,15Z-octadecatrienoic acid (13-HPOTE), 15(S)-hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-HPETE), 12(S)-hydroperoxy-5Z,8Z,10E,14Z,17Z-eicosapentaenoic acid (12-HPEPE), and 17(S)-hydroperoxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid (17-HPDHE)) from Larodan, Solna, Sweden; xylenol orange tetrasodium salt, iron(II) sulfate heptahydrate, cumene hydroperoxide (CuHP), hydrogen peroxide (HP), and perchloric acid from Sigma-Aldrich, Missouri, USA.

The gene of Burkholderia thailandensis lipoxygenase (Bt-LOX) (An et al. 2015) (NCBI ABC36974.1) with codons optimized for expression in Escherichia coli (Table S1) was purchased from GenScript Biotech, The Netherlands. The gene was obtained in a pET-19b plasmid (Novagen, USA), inserted between the NdeI and BlpI restriction sites. A 10 × His-tag was present at the N terminus of the enzyme and was used for protein purification. Materials used for the enzyme production and purification were obtained from the following sources: Escherichia coli BL21(DE3) competent cells from Invitrogen, California, USA; Luria Bertani medium, pepstatin A, and ampicillin sodium salt from Sigma-Aldrich, Missouri, USA; isopropyl β-d-1-thiogalactopyranoside (IPTG) from Duchefa Biochemie B.V., Haarlem, The Netherlands; BugBuster master mix and Ni–NTA His-bind resin from Millipore-Merck, Darmstadt, Germany; cOmplete mini EDTA-free protease inhibitor cocktail from Roche, Mannheim, Germany; VivaSpin spin filters from GE Healthcare, Buckinghamshire, UK.

Protein expression and purification

Recombinant E. coli BL21(DE3) transformed with the pET-19b_Bt-LOX plasmid was cultivated in Luria Bertani medium at 37 °C with shaking at 250 rpm. When the optical density of the bacterial culture at 600 nm (OD600) reached 0.6 − 0.8, 0.5 mM IPTG was added and the culture was further incubated at 16 °C with shaking at 150 rpm for 48 h. Then, the cells were harvested by centrifugation at 7000 × g for 15 min at 4 °C and stored at − 20 °C until protein purification.

To purify the Bt-LOX enzyme, a lysis solution was first added to the frozen cell pellet obtained from 200 mL culture medium. The lysis solution consisted of one Mini EDTA-free cOmplete protease inhibitor cocktail tablet and 1 µM pepstatin A dissolved in 10 mL of BugBuster Master Mix. Centrifugation at 16,000 × g for 20 min at 4 °C was carried out to remove cell debris, and the resulting supernatant was filtered using a 0.22-μm membrane filter. Subsequently, purification was conducted using a gravity flow column containing 1 mL of Ni–NTA His-bind resin. Prior to sample application, the column was equilibrated with 10 column volumes (CV) of an equilibration buffer consisting of 50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole adjusted to pH 7.0 with a 1 M HCl solution. The filtered supernatant was then applied to the column, which was subsequently washed with 2 CV each of four washing buffers pH 7, containing 50 mM NaH2PO4, 300 mM NaCl, and increasing concentrations of imidazole of 20, 50, 100, and 150 mM, respectively. Elution of the purified enzyme was achieved using 4 CV of elution buffer pH 7, containing 50 mM NaH2PO4, 300 mM NaCl, and 250 mM imidazole. The desalting of the elution fractions was performed using a VivaSpin spin filter with a molecular weight cut-off of 10 kDa. The purified enzyme was stored in 100 mM phosphate buffer pH 7, its concentration was determined using the BCA assay, and its size was confirmed by SDS-PAGE, showing a single band at approximately 75 kDa.

Absorption spectral analysis of the ferric-xylenol orange complex

The FOX assay was carried out with some modifications of the previously described method (Gay and Gebicki 2002; Pinto et al. 2007). The xylenol orange reagent containing 2.0 mM ferrous sulfate, 0.29 mM xylenol orange tetrasodium salt, and 440 mM perchloric acid in methanol/water (9:1) was freshly prepared. To find the optimum absorbance at which the concentration of the ferric-xylenol orange (Fe3+–XO) complex can be determined, 30 µL of cumene hydroperoxide (0–10.52 mM dissolved in water) and 150 µL xylenol orange reagent were mixed well. The mixtures were then incubated for 15 min at room temperature (~ 20 °C) and the absorption spectra of the mixtures were measured in the wavelength range of 400–650 nm using a Spectramax ID3 multi-detection microplate reader (Molecular Devices, LLC, San Jose, California, USA). When the absorbance of the samples exceeded 2.0, the samples were diluted in 75% methanol in water. The actual absorbance of the sample was then calculated, taking the dilution factor into account.

Optimization of perchloric acid concentration and its effect on the linear detection range of hydroperoxides

To study the effect of perchloric acid concentration on the linear detection range of the FOX assay, xylenol orange reagent was freshly prepared using different concentrations of perchloric acid. The xylenol orange reagent contained 2.0 mM ferrous sulfate, 0.29 mM xylenol orange tetrasodium salt, and perchloric acid (110, 220, 440, or 660 mM) diluted in methanol/water (9:1). The assay was carried out by mixing 30 μL of the sample and 150 μL xylenol orange reagent in a 96-well microplate. The mixture was then incubated for 15 min at room temperature. The absorbance of the samples was read at 570 nm using the Spectramax ID3 multi detection microplate reader. The concentration of perchloric acid which offers a broader linear detection range while maintaining sufficient sensitivity for the measurement (440 mM) was then selected for further experiments.

Evaluation of the modified FOX assay on different sample pHs and different hydroperoxide species

The effect of the sample pH on the modified FOX assay was evaluated by performing the assay on CuHP calibration curves (0–175.2 µM) diluted in different buffers. The buffers used were 100 mM citrate pH 3.0, 4.0, and 5.0, 100 mM Bis–Tris pH 6.0 and 7.0, and 100 mM Tris–HCl pH 8.0, 9.0, and 10.0. The effect of different hydroperoxide species on the response of the modified FOX assay was assessed by measuring various concentrations of the hydroperoxide species, i.e., CuHP, HP, 13-HPODE, 13-HPOTE, 15-HPETE, 12-HPEPE, and 17-HPDHE. For the sake of stability, all the hydroperoxide standard compounds were dissolved in methanol. When measuring different hydroperoxide species, the methanol/water ratio in the xylenol orange reagent was adjusted to 7:1. This adjustment ensured that the final methanol concentration in the reaction mixture remained consistent with the standard protocol. The molar absorption coefficient (Ɛ) of the Fe3+–XO complex for each hydroperoxide was calculated using Eq. (1). The absorbance of the Fe3+–XO complex at 570 nm (A) per concentration of the hydroperoxide (c) was determined from the slope of the linear part (R2 > 0.99) of the calibration curve. The length of the light path through the solution (l) was determined by calculating the height of the sample solution in the 96-well microplate.

$$\varepsilon =\frac$$

(1)

Determination of Fe3+/hydroperoxide ratio

The number of ferric ions generated by each –OOH group from different hydroperoxide species was expressed as the Fe3+/hydroperoxide ratio. The calculation involved dividing the molar absorption coefficient of the Fe3+–XO complex generated by the –OOH group in each hydroperoxide species by the molar absorption coefficient of the complex generated by ferric ions in the same reagent (Gay et al. 1999b, 1999a). The molar absorption coefficient of the Fe3+–XO complex generated by ferric ions was determined by making a calibration curve of FeCl3 from 0 to 120 µM.

Preparation of solubilized fatty acids

Solubilized LA was freshly prepared according to a previously described protocol with slight modifications (Axelrod et al. 1981). In a 10-mL volumetric flask, 13.5 μL LA was mixed with 12.5 μL of Tween-20 in 4 mL milli-Q water. After adding 0.55 mL of 0.5 M NaOH, the solution became clear, and milli-Q water was added to adjust the volume to 10 mL, resulting in a final LA concentration of 4.3 mM.

Comparison between FOX assay and conjugated diene method

To validate the accuracy of the modified FOX assay, fatty acid hydroperoxide produced from LA by Bt-LOX was measured using two different methods, i.e., FOX assay and conjugated diene method. Solutions containing 13.25 nM Bt-LOX, LA in the range of 4.3–139 µM, and 100 mM buffer pH 6 were used as the enzymatic reaction mixtures. Bis–Tris buffer was used for the FOX assay, while phosphate buffer was used for the conjugated diene method to prevent any interference from absorption by the buffer. The reaction mixtures were homogenized and incubated for 5 min at 25 °C. LOX activity measured based on the FOX assay was performed as described above; 13-HPODE (LA-derived hydroperoxide) was used as the standard for the calibration curve. Concentration of fatty acid hydroperoxide based on the conjugated diene method was determined by measuring the absorbance at 234 nm during 5-min incubation of the enzyme with LA using Jasco V-730 UV–vis/NIR spectrophotometer (Jasco, Easton, Maryland, USA). The concentration of fatty acid hydroperoxide formed was calculated using a molar absorption coefficient of 25,000 M−1 cm−1 (Axelrod et al. 1981).