Crystallization and Properties of N-Benzoylglycine Amidohydrolase from Pseudomonas putida

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N-Benzoylglycine amidohydrolase (hippurate hydrolase EC 3.5.1.32), which catalyzes the hydrolysis of hippuric acid to benzoic acid and glycine, was found in a cell-free extract of Pseudomonas putida C692-3 grown on a medium containing hippuric acid. The enzyme was purified from the extract by ammonium sulfate fractionation and column chromatographies on DEAE-cellulose, DEAE-Sephadex A-50, hydroxyapatite, and Sepharose CL-6B. The enzyme was finally crystallized. The crystalline enzyme was almost homogeneous on electrophoresis. The enzyme had a molecular weight of about 170, 000 and consisted of four subunits identical in molecular weight (approximately 42, 000). The enzyme hydrolyzed N-benzoylglycine most rapidly, and N-benzoyl-L-alanine and N-benzoyl-L-aminobutyric acid. The Km value for these substrates were 0.72mM, 0.87 mM, and 0.87mM, respectively. The optimum pH of the enzyme reaction was 7.0 to 8.0 and the enzyme was stable from pH 6.0 to 8.0.
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