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

• Published in: Applied microbiology and biotechnology Vol. 74; no. 6; pp. 1240 - 1247
• Main Authors: Geueke, Birgit, Weckbecker, Andrea, Hummel, Werner
• Format: Journal Article
• Language: English
• Published: Berlin Berlin/Heidelberg : Springer-Verlag 01.04.2007; Springer; Springer Nature B.V
• Subjects: Acidic soils; Amino acid oxidase; Amino Acid Sequence; Amino Acids; Amino Acids - metabolism; Arthrobacter; Arthrobacter - enzymology; Arthrobacter - genetics; Arthrobacter protophormiae; Bacteria; Bacterial Proteins; Bacterial Proteins - chemistry; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Biological and medical sciences; Biotechnology; Cell density; chemistry; D-Amino acids; D-Amino-Acid Oxidase; D-Amino-Acid Oxidase - chemistry; D-Amino-Acid Oxidase - genetics; D-Amino-Acid Oxidase - metabolism; D-methionine; E coli; Electrophoresis, Polyacrylamide Gel; Enantiomers; enzymology; Escherichia coli; Fermentation; Fundamental and applied biological sciences. Psychology; genes; genetics; Gram-positive bacteria; Hydrophobicity; isolation & purification; metabolism; Methionine; Molecular Sequence Data; nucleotide sequences; Recombinant Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; screening; Sequence Homology, Amino Acid; soil sampling; Substrate Specificity; Characterization; D-Amino-acid oxidase; Oxidoreductases; Enzyme
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-006-0776-9

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 x 10⁶ U of d-AAO were obtained from 15 l of fermentation broth.