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

• LIU Jian

  Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University

• ASANO Yu

  Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University

• IKOMA Keiko

  Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University

• YAMASHITA Satoshi

  Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University

• HIROSE Yoshihiko

  Amano Enzyme Inc.

• SHIMOYAMA Takefumi

  Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University

• TAKAHASHI Seiji

  Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University

• NAKAYAMA Toru

  Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University

• NISHINO Tokuzo

  Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University

抄録

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℃ and pH 8.5, respectively. The k_<cat> and K_m values for NADF were 41 s^<-1> and 2.5 mM at 37℃ 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.

収録刊行物

• Journal of bioscience and bioengineering

  Journal of bioscience and bioengineering 114 (4), 391-397, 2012-10-25

  公益社団法人日本生物工学会

キーワード

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

詳細情報

• CRID: 1574231877586014720
• NII論文ID: 110009543722
• NII書誌ID: AA11307678
• ISSN: 13891723
• 本文言語コード: en
• データソース種別:
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