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) pro duced N-acyl-D-aspartate amidohydrolase (D-AAase) in the presence of N -acetyl-D-aspartate as an inducer. The enzyme was purified to homogene ity. 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 acti vity at pH 7.5 to 8.0 and 50-degrees-C, and was stable at pH 8.0 and u p to 45-degrees-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-g lycyl (K(m) = 1.11 mM) derivatives Of D-aspartate were hydrolyzed, but N-carbobenzoyl-D-aspartate, N-acetyl-L-aspartate, and N-acetyl-D-glut amate were not substrates. The enzyme was inhibited by both divalent c ations (Hg2+, Ni2+,Cu2+) and thiol reagents (N-ethylmaleimide, iodoace tic acid, dithiothreitol, and p-chloromercuribenzoic acid). The N-term inal amino acid sequence and amino acid composition were analyzed.