Purification and characterization of novel N-acyl-D-aspartate amidohydrolase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6.
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 degrees C, and was stable at pH 8.0 and up to 45 degrees C. N-Formyl (Km = 12.5 mM), N-acetyl (Km = 2.52 mM), N-propionyl (Km = 0.194 mM), N-butyryl (Km = 0.033 mM), and N-glycyl (Km = 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 (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.
Moriguchi
M
M
Department of Applied Chemistry, Faculty of Engineering, Oita University, Japan.
Sakai
K
K
Katsuno
Y
Y
Maki
T
T
Wakayama
M
M
D000421:Alcaligenes / Q000201:enzymology*; D000581:Amidohydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013237:Stereoisomerism; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000596:Amino Acids; D002413:Cations, Divalent; D013439:Sulfhydryl Reagents; D001224:Aspartic Acid; C000179:N-acetylaspartate; D000581:Amidohydrolases; C019751:aspartoacylase