Cloning and Sequencing of a Gene Encoding D-Aminoacylase from Alcaligenes xylosoxydans subsp. Xylosoxydans A-6 and Expression of the Gene in Escherichia Coli

The gene encoding the D-aminoacylase of Alcaligenes xylosoxydans subsp. xylosoxydans A-6(Alcaligenes A-6) was cloned and its complete nucleotide sequence was identified. The D-aminoacylase structural gene consists of 1452 nucleotides and encodes 484 amino acid residues. The molecular weight of D-aminoacylase was calculated to be 51, 918. This value agreed well with the apparent molecular weight of 52, 000 found for the purified enzyme from Alcaligenes A-6 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The N-terminal amino acid sequence (NH₂-SQSDSQPFDLLRAG-) predicted by the nucleotide sequence exactly matched those of the purified D-aminoacylase both from Alcaligenes A-6 and from cloned Escherichia coli (E. coli), with the exception of the removal of the N-terminal methionine processed after translation. The purified recombinant enzyme showed almost the same enzymatic properties as the native enzyme from Alcaligenes A-6. Alcaligenes A-6 D-aminoacylase showed 25-29% homology with L-aminoacylases from Bacillus stearothermophilus, porcine, and humans.