PURIFICATION AND CHARACTERIZATION OF EXTREMELY THERMO-STABLE GLUTAMATE-DEHYDROGENASE FROM A HYPERTHERMOPHILIC ARCHAEON, THERMOCOCCUS-LITORALIS

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 degrees C for 30 min and at pH 7-11 at 80 degrees C for 20 min. The maximum enzyme activity in L-glutamate deamination was obtained around 95 degrees C. Both optimum pHs for L-glutamate deamination and alpha-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 alpha-keto acids such as alpha-ketocaporoate, alpha-ketovalerate and pyruvate were also aminated as well as alpha-ketoglutarate in the presence of NADPH and ammonia. Michaelis constants for the substrates were as follows: NADP, 0.045 mM; L-glutamate, 2.1 mM; alpha-ketoglutarate, 1.0 mM; ammonia, 5.6 mM; and NADPH, 0.044 mM. N-Terminal 24 amino acid sequence of the enzyme was determined to be; VEQDPFEIAVKQLERAAQYMDISE.