Determination of SsDesB extinction coefficient

SsDesB (156 μM) was heated in triplicate at 95°C for 15 min and clarified by centrifugation at 13,000 rpm for 10 min at room temperature. After the supernatant was removed and reheated again, its absorbance was measured over a range of 200–800 nm. Using Beer’s Law (A = εcl) and the extinction coefficient of FAD (11,300 M^-1cm^-1), the extinction coefficient of DesB was determined, as the concentration of the enzyme should be equivalent to the concentration of free FAD after denaturation. Furthermore, the percent of FAD bound was also determined using Eq (1).