Production, purification, and characterization of D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6.
The best inducers for D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) were a poor substrate, N-acetyl-gamma-methyl-D-leucine, and an inhibitor, N-acetyl-D-alloisoleucine. The enzyme has been homogeneously purified. The molecular weight of the native enzyme was estimated to be 58,000 by gel filtration. A subunit molecular weight of 52,000 was measured by SDS-PAGE, indicating that the native protein is a monomer. The isoelectric point was 5.2. The enzyme was specific to the D-isomer and hydrolyzed N-acetyl derivatives of D-leucine, D-phenylalanine, D-norleucine, D-methionine, and D-valine, and also N-formyl, N-butyryl, and N-propionyl derivatives of D-leucine. The Km for N-acetyl-D-leucine was 9.8 mM. The optimum pH and temperature were 7.0 and 50 degrees C, respectively. The stabilities of pH and temperature were 8.1 and 40 degrees C. D-Aminoacylases from three species of the genus Alcaligenes differ in inducer and substrate specificities, but are similar with respect to molecular weight and N-terminal amino acid sequence.
Moriguchi
M
M
Department of Applied Chemistry, Faculty of Engineering, Oita University, Japan.
Sakai
K
K
Miyamoto
Y
Y
Wakayama
M
M
D000421:Alcaligenes / Q000201:enzymology*; D000581:Amidohydrolases / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
D000596:Amino Acids; D000581:Amidohydrolases; C019672:aminoacylase I