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-alpha-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-degrees-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 alpha-H- and alpha-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 (k(cat)/K(m) ratio) was measured With DL-alpha-allyl alanine amide, DL-alpha-methyl phenylalanine amide, and DL-alpha-methyl leucine amide.