NADP-SPECIFIC GLUTAMATE-DEHYDROGENASE FROM ALKALIPHILIC BACILLUS SP KSM-635 - PURIFICATION AND ENZYMATIC-PROPERTIES

An NADP-specific glutamate dehydrogenase [L-glutamate: NADP(+) oxidore ductase (deaminating), EC 1.4.1.4] from alkaliphilic Bacillus sp, KSM- 635 was purified 5840-fold to homogeneity by a several-step procedure involving Red-Toyopearl affinity chromatography. The native protein, w ith an isoelectric point of pH 4.87, had a molecular mass of approxima tely 315 kDa consisting of six identical subunits each with a molecula r mass of 52 kDa. The pH optima for the aminating and deaminating reac tions were 7.5 and 8.5, respectively. The optimum temperature was arou nd 60 degrees C for both. The purified enzyme had a specific activity of 416 units/mg protein for the aminating reaction, being over 20-fold greater than that for deaminating reaction, at the respective pH opti ma and at 30 degrees C. The enzyme was specific for NADPH (K-m 44 mu M ), 2-oxoglutarate (K-m 3.13 mM), NADP(+) (K-m 29 mu M), and L-glutamat e (K-m 6.06 mM). The K-m for NH4Cl was 5.96 mM. The enzyme could be st ored without appreciable loss of enzyme activity at 5 degrees C for ha lf a year in phosphate buffer (pH 7.0) containing 2 mM 2-mercaptoethan ol, although the enzyme activity was abolished within 20 h by freezing at -20 degrees C.