Peroxidase assay kit

The mycelia were harvested from liquid CM after a two-day inoculation, and transferred into new liquid CM added with 2 mM Mn²⁺. The two-day inoculation mycelia were frozen by liquid nitrogen. The H₂O₂ content, activities of SOD, CAT and POD, were determined according to the technical bulletins about fluorometric hydrogen peroxide assay kit (Sigma, St. Louis, MO, USA), SOD assay kit (Sigma, St. Louis, MO, USA), catalase assay kit (Sigma, St. Louis, MO, USA), and peroxidase assay kit (Sigma, St. Louis, MO, USA). The fungal biomass was determined by the protein content according to previous protocols [107 (link)]. These experiments were performed three independent times.

We used the Peroxidase Assay Kit (Sigma Aldrich, MAK311). This assay utilizes the chromogenic Fe³⁺−xylenol orange reaction in the mixture. A purple complex is formed when Fe²⁺ is oxidized to Fe³⁺ by peroxides present in the sample that can be measured at 585 nm, and the absorbance is proportional to the peroxide level in the sample. The optimized formulation reduces interference by substances in the raw samples. A concentration of 1 μM H₂O₂ equals 34 ng/mL, with a H₂O₂ range detection between 0.2–30 μM (7–1020 ng/mL). Samples (50 mg) were ground with liquid nitrogen, and 0.5 mL of ultrapure water were added. Samples were mixed for 1 min and centrifuged at 14,000 rpm for 15 min at 4 °C. The supernatant was recovered for the assay, and a 40 µL sample was added to 200 µL of detection reagent in a 96-well plate. After 30 min of incubation, the absorbance was measured as before. The H₂O₂ contents in the samples were calculated from a standard curve (0, 3, 6, 9, 12, 18, 24, and 30 μM).