Purification and Characterization of Extremely Thermo-Stable Glutamate Dehydrogenase from a Hyperthermophilic Archaeon,Thermococcus Litoralis

Abstract

Glutamate dehydrogenase (L-glutamate: NADP oxidoreductase, deaminating, EC 1.4.1.4) from a hyperthermophilic archaeon, Thermococcus litoralis DSM 5473 was purified to homogeneity from the crude extract. The enzyme had a molecular mass of about 300 kDa and consisted of six subunits with identical molecular masses of 37 kDa. The enzyme was extremely themostable; the activity was not lost after incubation at 95°C for 30 min and at pH 7–11 at 80°C for 20 min. The maximum enzyme activity in L-glutamate deamination was obtained around 95°C. Both optimum pHs for L-glutamate deamination and a-ketoglutarate amination were around 7.8. The enzyme exclusively catalyzed the oxidative deamination of L-glutamate in the presence of NADP but showed low amino acceptor specificity; several α-keto acids such as α-ketocaporoate, α-ketovalerate and pyruvate were also aminated as well as α-ketoglutarate in the presence of NADPH and ammonia. Michaelis constants for the substrates were as follows: NADP, 0.045 mM; L-glutamate, 2.1...