Purification and characterization of cold-active L-glutamate dehydrogenaseindependent of NAD(P) and oxygen

L-Glutamate dehydrogenase (GLDH) independent of NAD(P) and oxygen was first obtained from the psychrotrophic bacterium Aeromonas sp, L101, originally isolated from the organs of salmon (Oncorhynchus keta). GLDH was purified b y a series of chromatography steps on DEAE-Sepharose, Superdex 200pg, Q-Sep harose, CM-Sepharose, and Phenyl-Sepharose. The purified protein was determ ined to have a molecular mass of 110 kDa and a pi of 5.7. Maximum activity was obtained at 55 degrees C and pH 8.5. The activity of GLDH at 4 and 20 d egrees C was 38 and 50%, respectively, of that at 50 degrees C. GLDH was co upled to cytochrome c and several redox dyes including 1-methoxy-5-methylph enazinium methylsulfate (l-Methoxy PMS), 2,6-dichlorophenylindophenol (DCIP ), 9-dimethylaminobenzo [alpha]phenoxazin-7-ium chloride (meldola's blue), 3,3'- [3,3'-dimethoxy-(1, 1'-biphenyl) -4,4'-diyl]-bis [2-(4-nitrophenyl)-5 -phenyl-2H tetrazolium chloride] (nitroblue tetrazolium; NET), and 2-(4-iod ophenyl) -3-(4-nitrophenyl) -5-phenyl-2H tetrazolium (INT). The presence of NAD(P) and oxygen gave no oxidation activity to GLDH. Spectroscopic profil e and ICP data indicated ab-type cytochrome containing iron.