CLONING, EXPRESSION, AND NUCLEOTIDE-SEQUENCE OF THE N-ACYL-D-ASPARTATE AMIDOHYDROLASE GENE FROM ALCALIGENES XYLOSOXYDANS SUBSP XYLOSOXYDANSA-6

The gene (termed daa) encoding N-acyl-D-aspartate (D-Asp) amidohydrola se (D-AAase) from the Alcaligenes xylosoxydans subsp. xylosoxydans (Al caligenes A-6) was cloned in Escherichia coli (E. cell) JM109. The daa gene consists of 1,494 nucleotides and encodes 498 amino acid residue s. The molecular weight of D-AAase was calculated to be 53,581. The N- terminal amino acid sequence (NH2-TDRSTLDDAP-) predicted by the nucleo tide sequence matched exactly those of the purified D-AAase from both Alcaligenes A-6 and cloned E. coil, 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 fro m Alcaligenes A-6 showed high overall homology (56%), D-AAase from Alc aligenes A-6 showed 25 similar to 29% homology with Bacillus stearothe rmophilus, porcine, and human L-aminoacylases. The daa was highly expr essed in E. coli, and the recombinant enzyme was purified to homogenei ty with 17.8% yield.