Purification of an NADH-(dichlorophenol-indophenol) oxidoreductase from Bacillus stearothermophilus

An NADH-(dichlorophenol-indophenol) oxidoreductase was purified 104-fold and in 25% overall yield from the thermophilic bacterium Bacillus stearothermophilus, strain PH24. After solubilization in 2M-NaCl at 70 degrees C, the enzyme was purified by ion-exchange and hydroxyapatite chromatography, followed by affinity chromatography on immobilized Cibacron Blue 3GA. The purified enzyme had a mol.wt. of 43 000 and had an absorption spectrum characteristic of flavoprotein. The enzyme activity was enhanced by FMN and by CN-. The enzyme was inhibited by EDTA and by rho-chloromercuribenzoic acid.