Purification, characterization, and primary structure of a novel N-acyl-d-amino acid amidohydrolase from Microbacterium natoriense TNJL143-2

Jian Liu, Yu Asano, Keiko Ikoma, Satoshi Yamashita, Yoshihiko Hirose, Takefumi Shimoyama, Seiji Takahashi, Toru Nakayama, Tokuzo Nishino

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)


A novel N-acyl-d-amino acid amidohydrolase (DAA) was purified from the cells of a novel species of the genus Microbacterium. The purified enzyme, termed AcyM, was a monomeric protein with an apparent molecular weight of 56,000. It acted on N-acylated hydrophobic d-amino acids with the highest preference for N-acetyl-d-phenylalanine (NADF). Optimum temperature and pH for the hydrolysis of NADF were 45°C and pH 8.5, respectively. The kcat and Km values for NADF were 41 s-1 and 2.5 mM at 37°C and pH 8.0, although the enzyme activity was inhibited by high concentrations of NADF. Although many known DAAs are inhibited by 1 mM EDTA, AcyM displayed a 65% level of its full activity even in the presence of 20 mM EDTA. Based on partial amino acid sequences of the purified enzyme, the full-length AcyM gene was cloned and sequenced. It encoded a protein of 495 amino acids with a relatively low sequence similarity to a DAA from Alcaligenes faecalis DA1 (termed AFD), a binuclear zinc enzyme of the α/β-barrel amidohydrolase superfamily. The unique cysteine residue that serves as a ligand to the active-site zinc ions in AFD and other DAAs was not conserved in AcyM and was replaced by alanine. AcyM was the most closely related to a DAA of Gluconobacter oxydans (termed Gox1177) and phylogenetically distant from AFD and all other DAAs that have been biochemically characterized thus far. AcyM, along with Gox1177, appears to represent a new phylogenetic subcluster of DAAs.

Original languageEnglish
Pages (from-to)391-397
Number of pages7
JournalJournal of Bioscience and Bioengineering
Issue number4
Publication statusPublished - 2012 Oct


  • D-Amino acids
  • D-Aminoacylase
  • D-Phenylalanine
  • Microbacterium natoriense
  • N-Acyl-d-amino acid amidohydrolase
  • Optical resolution
  • α/β-Barrel amidohydrolase superfamily

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology


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