Thermostable alanine dehydrogenase from thermophilic Bacillus sphaericus DSM 462

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Abstract
Alanine dehydrogenase (L‐alanine:NAD+ oxidoreductase, deaminating) was simply purified to homogeneity from a thermophile, Bacillus sphaericus DSM 462, by ammonium sulfate fractionation. red‐Sepharose 4B chromatography and preparative slab gel electrophoresis. The enzyme had a molecular mass of about 230 kDa and consisted of six subunits with an identical molecular mass of 38 kDa. The enzyme was much more thermostable than that from a mesophile, B. sphaericus, and retained its full activity upon heating at 75 °C for at least 60 min and with incubation in pH 5.5–9.5 at 75 °C for 10 min. The enzyme can be stored without loss of its activity in a frozen state (– 20 °C, at pH 7.2) for over 5 months. The optimum pH for the L‐alanine deamination and pyruvate amination were around 10.5 and 8.2, respectively. The enzyme exclusively catalyzed the oxidative deamination of L‐alanine in the presence of NAD+, but showed low amino acceptor specificity; hydroxypyruvate, oxaloacetate, 2‐oxobutyrate and 3‐fluoropyruvate are also aminated as well as pyruvate in the presence of NADH and ammonia. Initial velocity and product inhibition studies showed that the reductive amination proceeded through a sequential mechanism containing partially random binding. NADH binds first to the enzyme, and then pyruvate and ammonia bind in a random fashion. The products are sequentially released from the enzyme in the order L‐alanine then NAD+. A dead‐end inhibition by the formation of an abortive ternary complex which consists of the enzyme, NAD+ and pyruyate was included in the reaction. A possible role of the dead‐end inhibition is to prevent the enzyme from functioning in the L‐alanine synthesis. The Michaelis constants for the substrates were as follows: NADH, 0.10 mM; pyruvate, 0.50 mM: ammonia. 38.0 mM; L‐alanine, 10.5 mM and NAD+, 0.26 mM.