Purification and biochemical characterization of recombinant alanine dehydrogenase from Thermus caldophilus GK24

Abstract

The recombinant alanine dehydrogenase (ADH) from E. coli containing Thermus caldophilus ADH was purified to homogeneity from a cell-free extract. The enzyme was purified 38-fold with a yield of 68% from the starting cell-free extract. The purified enzyme gave a single band in polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 45 kDa. The pH optimum was 8.0 for reductive amination of pyruvate and 12.0 for oxidative deamination of L-alanine. The enzyme was stable up to 70°C. The activity of the enzyme was inhibited by 1 mM Zn2+, 20% hexane, and 20% CHCl3. However, 10 mM Mg2+ and 40% propanol had no effect on the enzyme activity. The Michaelis constants (Km) for the substrates were 50 μM for NADH, 0.2 mM for pyruvate, 39.4 mM for NH4+, 2.6 mM for L-alanine, and 1.8 mM for NAD+.