Antioxidant activity assay by the Oxford cup method

To test whether MpmMn-SOD has antioxidant activity, the tolerance of the Trx-His-MpmMn-SOD-overexpressed E. coli cells to hydroperoxide was determined by the Oxford cup method (Liu et al., 2018). The bacteria BL21(pET-32a-MpmMn-SOD) and BL21(pET-32a) were grown overnight at 37 °C in LB broth containing ampicillin (Amp⁺) (50 mg/L), and then diluted 1: 100 in LB medium. The diluted cells were further incubated at 37 °C until a final optical density of 0.4∼0.6 at 595 nm. These cells were induced with 0.3 mM IPTG at 25 °C for 10 h. Then, 100 µL BL21(pET-32a-MpmMn-SOD) and BL21(pET-32a) were, respectively, added to fresh LB (Amp⁺) solid medium in plates. After the medium is solidified, five Oxford cups were placed on the plate, then 100 µL of different concentrations (100, 75, 50, 25 and 0 mmol/L) of H₂O₂ were respectively added to the top of the cups. The plates were incubated overnight at 37 °C with three replicates per treatment group. BL21(pET-32a) cells were used as the control. The agent diffused into the surrounding area through the cup to form a decreasing concentration gradient. Observe the zone of inhibition formed around the cup and record the diameter of the zone. The inhibition zones were measured as described by Burmeister et al. (2008).