RESUMEN
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.
RESUMEN
An l-aminopeptidase of Pseudomonas putida, used in an industrial process for the hydrolysis of d,l-amino acid amide racemates, was purified to homogeneity. The highly l-enantioselective enzyme resembled thiol reagent-sensitive alkaline serine proteinases and was strongly activated by divalent cations. It possessed a high substrate specificity for dipeptides and alpha-H amino acid amides, e.g., l-phenylglycine amide.
RESUMEN
Amino acids play an important role in biochemistry and chemistry. They are the building blocks of proteins and play an essential role in the regulation of the metabolism of living organisms. In general, it can be stated that microbial processes (fermentation) are the industrial production methods of choice for large-scale production of naturally occurring proteinogenic L-alpha-H-amino acids, while for the production of synthetic D- and/or L-alpha-H-amino acids, several other methods are highly competitive. At DSM, several routes, i.e., (chemoenzymatic) synthesis, towards L-alpha-H and D-alpha-H-amino acids have been elaborated since the midseventies. A general process for the synthesis of natural as well as synthetic optically pure amino acids has been developed, using an enzymatic kinetic resolution step on racemic amino acid amides as the key step. In this case, both enantiomers of the alpha-H-amino acids are prepared in one single step. This process has been commercialized since 1988. More recent developments using L- or D-amino peptidases in combination with amino acid amide racemases and an asymmetric transformation concept are discussed.