Purification and characterization of novel N-acyl-D-aspartate amidohydrolase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

• Published in: Bioscience, biotechnology, and biochemistry Vol. 57; no. 7; pp. 1145 - 1148
• Main Authors: Moriguchi, M. (Oita Univ. (Japan)), Sakai, K, Katsuno, Y, Maki, T, Wakayama, M
• Format: Journal Article
• Language: English
• Published: Tokyo Taylor & Francis 1993; Japan Society for Bioscience Biotechnology and Agrochemistry; Oxford University Press
• Subjects: ACIDE ASPARTIQUE; ACIDO ASPARTICO; ALCALIGENES; Alcaligenes - enzymology; AMIDE HYDROLASE; AMIDE HYDROLASES; AMIDO HIDROLASA; Amidohydrolases - chemistry; Amidohydrolases - isolation & purification; Amidohydrolases - metabolism; Amino Acid Sequence; Amino Acids - analysis; ASPARTIC ACID; Aspartic Acid - analogs & derivatives; Aspartic Acid - metabolism; Bacteriology; Biological and medical sciences; Biotechnology; Cations, Divalent - pharmacology; CHEMICAL STRUCTURE; CHEMICOPHYSICAL PROPERTIES; Electrophoresis, Polyacrylamide Gel; ESTRUCTURA QUIMICA; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Hydrolysis; Isoelectric Point; Metabolism. Enzymes; Microbiology; Molecular Sequence Data; Molecular Weight; OPTICAL PROPERTIES; PROPIEDADES FISICO-QUIMICAS; PROPIEDADES OPTICAS; PROPRIETE OPTIQUE; PROPRIETE PHYSICOCHIMIQUE; PURIFICACION; PURIFICATION; Stereoisomerism; STRUCTURE CHIMIQUE; Substrate Specificity; Sulfhydryl Reagents - pharmacology; Purification; Stability; Enzymatic activity; Molecular weight determination; Alcaligenes; N terminal-Sequence; Enzyme inhibitor; Bacteria; Hydrolases; Physicochemical properties
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.57.1145

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 (K m =12.5 mM), N-acetyl (K m =2.52 mM), N-propionyl (K m =0.194 mM), N-butyryl (K m =0.033 mM), and N-glycyl (K m =1.11 mM) 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 (Hg 2+ , Ni 2+ , Cu 2+ ) and thiol reagents (N-ethylmaleimide, iodoacetic acid, dithiothreitol, and p-chloromercuribenzoic acid). The N-terminal amino acid sequence and amino acid composition were analyzed.