Molecular Cloning of an Extremely Thermostable Alanine Racemase from Aquifex pyrophilus and Enzymatic Characterization of the Expressed Protein

A homologous gene to alanine racemase was cloned from a hyperthermophilic bacterium, Aquifex pyrophilus. The cloned gene encodes a protein of 341 amino acids, which has a significant homology to alanine racemase of Bacillus stearothermophilus, Lactobacillus brevis, and E. coli. When the gene was expressed in Escherichia coli, it produced a 40 kDa protein. The purified protein contains one mole pyridoxal 5-phosphate per one mole of protein, which is essential for catalytic activity of alanine racemase. The purified protein catalyzed racemization of L-alanine to D-alanine, or vice versa, indicating that the cloned gene encoded alanine racemase. It also showed significant racemization activity against L-serine and ${\alpha}-aminobutylic$ acid. The A. pyrophilus alanine racemase showed strong thermostability, and it maintained catalytic activity in the presence of organic solvents.