Abstract
Alanine dehydrogenase [L-alanine : NAD+ oxidoreductase (deaminating) EC. 1. 4. 1. 1.] from Bacillus natto KMD 1126 was purified 160-fold by ammonium sulfate fractionation, diethylaminoethyl-cellulose chromatography, Sephadex G-200 gel filtration, and adenosine-5'-phosphate Sepharose 4B affinity chromatography. The purified enzyme preparation showed a single band on polyacrylamide gel disc electrophoresis. Specific activity of the purified enzyme was 21 u/mg for oxidative deamination of L-alanine and lower than that of Bacillus subtilis (1350 u/mg). The molecular weight of the enzyme was 280000 daltons as determined by gel filtration on Sephadex G-200. Optimum pH for oxidative deamination of L-alanine was 10.4-10.7, whereas it was 8.2-8.4 for reductive amination of pyruvate. The enzyme was completely inhibited by Hg2+ and p-chloromercuribenzoate at 10-3 M. Nicotinamide adenine dinucleotide and its reduced form were essential as coenzymes and could not be replaced by nicotinamide adenine dinucleotide phosphate and its reduced form. L-Alanine was oxidatively deaminated by the enzyme and L-sereine was also deaminated at the rate of 0.5% of L-alanine, but D-alanine and other amino acids were not deaminated. That is, the enzyme has relatively high substrate specificity.
