Production and characterization of N-acyl-D-glutamate amidohydrolase from Pseudomonas sp. strain 5f-1

N-Acyl-D-glutamate amidohydrolase from Pseudomonas sp. strain 5f-1 was inducibly produced by D isomers of N-acetylglutamate, glutamate, aspartate, and asparagine. The enzyme has been purified to homogeneity by DEAE-cellulose, (NH₄)₂SO₄ fractionation, and chromatofocusing followed by gel filtration on a Sephadex G-100 column. The enzyme was a monomer with molecular weight of 55,000. The enzyme activity was optimal at pH 6.5 to 7.5 and 45°C. The isoelectric point and the pH stability were 8.8 and 9.0, respectively. N-Formyl, N-acetyl, N-butyryl, N-propionyl, N-chloroacetyl derivatives of D-glutamate and glycyl-D-glutamate were substrates for the enzyme. At pH 6.5 in 100 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer at 30°C, a K(m) of 6.67 mM and a V(max) of 662 μmol/min/mg of protein for N-acetyl-D-glutamate were obtained. None of the metal ions stimulated the enzyme activity. Na⁺, K⁺, Mg²⁺, and Ba²⁺ acted as stabilizers. Hg²⁺, Cu²⁺, Zn²⁺, Fe³⁺, and EDTA were strongly inhibitory.