Fig. 5

Azoreductase activity of AzoA and EF0404 and their cofactor preferences. The proteins used were purified without their prosthetic group, that is purified using the denaturation/renaturation protocol. For the enzymes presenting azoreductase activity, methyl red reduction is monitored by fluorescence intensity at 250/395 nm during 60 min in presence 10 mg.l⁻¹ of indicated enzyme. a To test the importance of FMN for these enzymes, methyl red (100 μmol.l⁻¹) reduction is followed without addition of FMN (−) and with 5 μmol.l⁻¹ of FMN (− •). NADPH is added at 500 μmol.l⁻¹. b Methyl red (100 μmol.l⁻¹) reduction is followed in presence of 500 μmol.l⁻¹. NADPH (• • •) or NADH (− −) to evaluate cofactors preference. FMN is added at 5 μmol.l⁻¹. The control wells contained the reaction buffer (methyl red, FMN, NAD (P) H) without enzyme