A new bacterial l-amino acid oxidase with a broad substrate specificity: purification and characterization

Abstract

The Gram-positive bacterium Rhodococcus opacus DSM 43250 produces an l-amino acid oxidase ( l-AAO) with a very broad substrate specificity. This enzyme has been purified to homogeneity and a detailed biochemical characterization was carried out. The complete nucleotide sequence of the l-AAO gene was determined and the primary structure of l-AAO was deduced. The molecular mass of the native enzyme was 99 kDa determined by gel filtration, 54.2/108.5 kDa measured by MALDI-TOF/MS, 53.2 kDa for the subunit calculated after SDS/PAGE. The coenzyme-binding motif G–X–G–X–X–G which is known for all l-AAOs was found very close to the N-terminus of the protein. l-AAO oxidized 39 out of 43 tested l-amino acids. The kinetic data for 16 of these l-amino acids were determined revealing K m -values in the range of 15–30 μM for substrates like l-phenylalanine, l-leucine, l-citrulline and l-lysine. The stability of l-AAO can be increased by storage or incubation of the enzyme in glycine/NaOH buffer. The protein has a pI of 4.8 and a slightly basic pH-optimum at pH 8–9 measured for l-alanine, l-phenylalanine and l-leucine as substrates. The ability for resolution of racemic mixtures was investigated and d-amino acids with an enantiomeric excess of >99% were obtained.