Cloning, expression, and nucleotide sequence of the N-acyl-d-aspartate amidohydrolase gene from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

The gene (termed daa) encoding N-acyl-d-aspartate (d-Asp) amidohydrolase (d-AAase) from the Alcaligenes xylosoxydans subsp. xylosoxydans (Alcaligenes A-6) was cloned in Escherichia coli (E. coli) JM109. The daa gene consists of 1,494 nucleotides and encodes 498 amino acid residues. The molecular weight of d-AAase was calculated to be 53,581. The N-terminal amino acid sequence (NH₂-TDRSTLDDAP-) predicted by the nucleotide sequence matched exactly those of the purified d-AAase from both Alcaligenes A-6 and cloned E. coli, with the exception of the removal of the N-terminal methionine processed after translation. A comparison of the amino acid sequence of d-AAase with that of d-aminoacylase from Alcaligenes A-6 showed high overall homology (56%). d-AAase from Alcaligenes A-6 showed 25∼29% homology with Bacillus stearothermophilus, porcine, and human l-aminoacylases. The daa was highly expressed in E. coli, and the recombinant enzyme was purified to homogeneity with 17.8% yield.