Purification and Characterization of Novel N-Acyl-D-aspartate Amidohydrolase from Alcaligenes xylosoxydans subsp. Xylosoxydans A-6

Mitsuaki Moriguchi, Kenji Sakai, Yutaka Katsuno, Tetsuyoshi Maki, Mamoru Wakayama

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) produced N-acyl-D-aspartate amidohydrolase (D-AAase) in the presence of N-acetyl-D-aspartate as an inducer. The enzyme was purified to homogeneity. The enzyme had a molecular mass of 56 kDa and was shown by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) to be a monomer. The isoelectric point was 4.8. The enzyme had maximal activity at pH 7.5 to 8.0 and 50°C, and was stable at pH 8.0 and up to 45°C. N-Formyl (Km=12.5mM), N-acetyl (Km=2.52mM), N-propionyl (Km=0.194mM), N-butyryl (Km=0.033mM), and N-glycyl (Km=1.11mM) derivatives of D-aspartate were hydrolyzed, but N-carbobenzoyl-D-aspartate, N-acetyl-L-aspartate, and N-acetyl-D-glutamate were not substrates. The enzyme was inhibited by both divalent cations (Hg2+, Ni2+, Cu2+) and thiol reagents (N-ethylmaleimide, iodoacetic acid, dithiothreitol, and p-chloromercuribenzoic acid). The N-terminal amino acid sequence and amino acid composition were analyzed.

Original languageEnglish
Pages (from-to)1145-1148
Number of pages4
JournalBioscience, biotechnology, and biochemistry
Volume57
Issue number7
DOIs
Publication statusPublished - Jan 1 1993

Fingerprint

Alcaligenes
D-Aspartic Acid
Purification
Enzymes
p-Chloromercuribenzoic Acid
Iodoacetic Acid
Amino Acids
Sulfhydryl Reagents
Ethylmaleimide
Dithiothreitol
Divalent Cations
Isoelectric Point
Molecular mass
Electrophoresis
Sodium Dodecyl Sulfate
Polyacrylamide Gel Electrophoresis
Glutamic Acid
Amino Acid Sequence
Monomers
Derivatives

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Analytical Chemistry
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology
  • Organic Chemistry

Cite this

Purification and Characterization of Novel N-Acyl-D-aspartate Amidohydrolase from Alcaligenes xylosoxydans subsp. Xylosoxydans A-6. / Moriguchi, Mitsuaki; Sakai, Kenji; Katsuno, Yutaka; Maki, Tetsuyoshi; Wakayama, Mamoru.

In: Bioscience, biotechnology, and biochemistry, Vol. 57, No. 7, 01.01.1993, p. 1145-1148.

Research output: Contribution to journalArticle

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abstract = "Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) produced N-acyl-D-aspartate amidohydrolase (D-AAase) in the presence of N-acetyl-D-aspartate as an inducer. The enzyme was purified to homogeneity. The enzyme had a molecular mass of 56 kDa and was shown by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) to be a monomer. The isoelectric point was 4.8. The enzyme had maximal activity at pH 7.5 to 8.0 and 50°C, and was stable at pH 8.0 and up to 45°C. N-Formyl (Km=12.5mM), N-acetyl (Km=2.52mM), N-propionyl (Km=0.194mM), N-butyryl (Km=0.033mM), and N-glycyl (Km=1.11mM) derivatives of D-aspartate were hydrolyzed, but N-carbobenzoyl-D-aspartate, N-acetyl-L-aspartate, and N-acetyl-D-glutamate were not substrates. The enzyme was inhibited by both divalent cations (Hg2+, Ni2+, Cu2+) and thiol reagents (N-ethylmaleimide, iodoacetic acid, dithiothreitol, and p-chloromercuribenzoic acid). The N-terminal amino acid sequence and amino acid composition were analyzed.",
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