Purification and Characterization of an L-Amino Amidase from Mycobacterium neoaurum ATCC 25795

An L-amino amidase from Mycobacterium neoaurum ATCC 25795 responsible for the enantioselective resolution of DL-α-methyl valine amide was purified and characterized. The purification procedure included ammonium sulfate fractionation, gel filtration, and anion-exchange chromatography, which resulted in a homogeneous preparation of the enzyme with a native molecular mass of 136 kDa and a subunit molecular mass of 40 kDa. The purified enzyme displayed the highest activity at 50°C and at pH 8.0 and 9.5. The enzyme was strongly inhibited by the metal-chelating agent 1,10-phenanthroline, the disulfide-reducing agent dithiothreitol, and the cysteine proteinase inhibitor iodoacetamide. The purified amino amidase showed a unique L-enantioselective activity towards a broad range of both α-H- and α-alkyl-substituted amino acid amides, with the highest activity towards the cyclic amino acid amide DL-proline amide. No activity was measured with DL-mandelic acid amide nor with the dipeptide L-phenylalanine-L-leucine. The highest catalytic efficiency (kcat/Km ratio) was measured with DL-α-allyl alanine amide, DL-α-methyl phenylalanine amide, and DL-α-methyl leucine amide.