1999 Volume 125 Issue 4 Pages 760-769
L-Glutamate dehydrogenase (GLDH) independent of NAD (P) and oxygen was first obtained from the psychrotrophic bacterium Aeromonas sp. L 101, originally isolated from the organs of salmon (Oncorhynchus keta). GLDH was purified by a series of chromatography steps on DEAE-Sepharose, Superdex 200 pg, Q-Sepharose, CM-Sepharose, and Phenyl-Sepharose. The purified protein was determined to have a molecular mass of 110 kDa and a pI of 5. 7. Maximum activity was obtained at 55°C and pH 8. 5. The activity of GLDH at 4 and 20°C was 38 and 50%, respectively, of that at 50°C. GLDH was coupled to cytochrome c and several redox dyes including 1-methoxy-5-methylphenazinium methylsulfate (1-Methoxy PMS), 2, 6-dichlorophenylindophenol (DCIP), 9-dimethylaminobenzo[α]phenoxazin-7-ium chloride (meldola's blue), 3, 3'-[3, 3'-dimethoxy-(1, 1'-biphenyl)-4, 4'-diyl]-bis[2-(4-nitrophenyl)-5-phenyl-2 H tetrazolium chloride] (nitroblue tetrazolium; NBT), and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2 H tetrazolium (INT). The presence of NAD (P) and oxygen gave no oxidation activity to GLDH. Spectroscopic profile and ICP data indicated a b-type cytochrome containing iron.
