Enzyme activity quantification in Vibrio alginolyticus

The determination of pyruvate dehydrogenase (PDH), alpha-ketoglutarate dehydrogenase (KGDH), succinate dehydrogenase (SDH), and malate dehydrogenase (MDH) activity was carried out as previously described (Cheng et al., 2017 (link)). The activity of phosphoenolpyruvate carboxykinase (PEPCK) enzymatic activity was performed through the PEPCK Assay Kit according to the manufacturer’s manual (Suzhou Comin Biotechnology, Suzhou, China). V. alginolyticus cultured in medium with OFX was collected and washed three times with sterile saline. The bacterial cells were suspended in × 1 PBS (pH 7.0) to OD600 = 1.0. Samples of 30 ml were collected by centrifugation at 8,000 rpm for 5 min. Pellets were resuspended in phosphate-buffered saline (PBS), and the cells were lysed by sonication for 10 min (200 W total power with 35% output, 2 s pulse, 3 s pause over ice). The solution was then centrifuged with 12,000 rpm at 4°C for 10 min to remove insoluble materials. The protein concentration of the supernatant was quantified by the BCA protein concentration determination kit (Beyotime, P0009). Then, 200 μg proteins were used for the determination of enzyme activity.