title: Purification and Characterization of an l-Amino Amidase from Mycobacterium neoaurum ATCC 25795.
abstract: 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.
authors.affiliation: Bio-Organic Chemistry Section, DSM Research, 6160 MD Geleen, The Netherlands.
authors.forename: H F
authors.initials: HF
authors.lastname: Hermes
authors.forename: R F
authors.initials: RF
authors.lastname: Tandler
authors.forename: T
authors.initials: T
authors.lastname: Sonke
authors.forename: L
authors.initials: L
authors.lastname: Dijkhuizen
authors.forename: E M
authors.initials: EM
authors.lastname: Meijer