Catalase Activity Assay for V. vulnificus

V. vulnificus strains (MO6-24/O, Δfur, Δfur complemented with pRK415-fur, ΔkatG) were cultured overnight in LB medium, washed, and sub-cultured in fresh LB medium. All cells were harvested at exponential phase (A₆₀₀ value of approximately 1.0) and sonicated using Ultrasonic Homogenizer (KUS-650, KBT, Seongnam, Korea) in 10 cycles with 1 s of sonication followed by 2 s of rest for each cycle. After centrifugation, the supernatant was concentrated using Amicon Ultra-0.5 mL Centrifugal Filter Units (10,000 NMWL, UFC501024, Merck-Millipore, Germany). Each of 50 μL sample was mixed with 10 mM hydrogen peroxide (Duksan, Ansan, Korea). The reaction tubes were vortexed, incubated for 2 min at 37°C, and 600 μL of working solution was added. The working solution consisted of 100 mL cobalt (II) solution (20.3 g / 1 L DIW), 100 mL sodium hexametaphosphate solution (10 g / 1 L DIW), 800 mL sodium bicarbonate solution (180 g/ 2 L DIW) (Hadwan, 2018 (link)). The tubes were vortexed for 5 s and then kept at room temperature for 10 min in the dark. After 10 min, the catalase activities were measured at 440 nm using a Multimode Plate Reader (PerkinElmer, Waltham, MA, United States).