Figure 2

The presence of the covalent FAD cofactor in GOOX. (A): An SDS-PAGE of GOOX and mutant variants stained by Coomassie blue (upper) and under 254 nm transillumination (lower), which shows intrinsic fluorescence of the covalently-bound FAD upon acidification; protein samples were overloaded to facilitate the detection of FAD intrinsic fluorescence. (B): UV–VIS scanning of GOOX, showing two automatically-determined maxima of 375 and 440 nm, which are similar to the absorbance (Abs) peaks of GOOX-T1 at 380 and 444 nm, respectively [10]; the 440-nm peak disappeared when the enzyme was reduced by 50 mM sodium hydrosulfite (dotted solid line) or by 200 mM cellobiose (dash line).