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Overproduction and characterization of a recombinant D-amino acid oxidase from Arthrobacter protophormiae


A screening of soil samples for d-amino acid oxidase (d-AAO) activity led to the isolation and identification of the gram-positive bacterium Arthrobacter protophormiae. After purification of the wild-type d-AAO, the gene sequence was determined and designated dao. An alignment of the deduced primary structure with eukaryotic d-AAOs and d-aspartate oxidases showed that the d-AAO from A. protophormiae contains five of six conserved regions; the C-terminal type 1 peroxisomal targeting signal that is typical for d-AAOs from eukaryotic origin is missing. The dao gene was cloned and expressed in Escherichia coli. The purified recombinant d-AAO had a specific activity of 180 U mg protein−1 for d-methionine and was slightly inhibited in the presence of l-methionine. Mainly, basic and hydrophobic d-amino acids were oxidized by the strictly enantioselective enzyme. After a high cell density fermentation, 2.29 × 106 U of d-AAO were obtained from 15 l of fermentation broth.

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This project was financially supported by the Degussa AG and the Deutsche Bundesstiftung Umwelt (Förderschwerpunkt Biotechnologie, ICBio, Az 13094). We thank Truc Pham for technical assistance and Holger Gieren for expert help with the fermentation.

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Correspondence to Birgit Geueke.

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Geueke, B., Weckbecker, A. & Hummel, W. Overproduction and characterization of a recombinant D-amino acid oxidase from Arthrobacter protophormiae . Appl Microbiol Biotechnol 74, 1240–1247 (2007).

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