Purification and Some Properties of Glutaminase from Pseudomonas nitroreducens IFO 12694.
Glutaminase (EC 3.5.1.2) was isolated from Pseudomonas nitroreducens IFO 12694 grown on 0.6% sodium glutamate as a nitrogen source (325-fold purification, 13% yield). The molecular weight of the enzyme was estimated to be 40,000 by gel filtration and SDS-gel electrophoresis. The enzyme hydro-lyzed glutamine optimally at pH 9, and its Km was 6.5 mm. d-Glutamine, γ-glutamyl p-nitroanilide, γ-glutamylmethylamide, γ-glutamylethylamide (theanine), and glutathione showed respectively 107, 85, 78, 74, and 82% reactivity of glutamine. Zn(2+), Ni(2+), Cd(2+), Co(2+), Fe(2+), and Cu(2+) repressed the enzyme activity strongly. Glutaminase formed γ-glutamylhydroxamate in the reaction mixture containing glutamine and hydroxylamine (transferring reaction). The optimum pH of the transferring reaction was 7-8, and the Km for glutamine and hydroxylamine were 4 mm and 120 mm, respectively. γ-Glutamyl derivatives hydrolyzable by glutaminase showed reactivity for the transferring reaction. Methylamine or ethylamine was replaceable for hydroxylamine with 3 or 8% reactivity. The effect of divalent cations was not so striking as in the hydrolyzing reaction.
a Department of Bioscience and Biotechnology, Faculty of Science and Engineering , Ritsumeikan University, Noji-cho 1916 , Kusatsu 525 , Japan.
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Takashi
a Department of Bioscience and Biotechnology, Faculty of Science and Engineering , Ritsumeikan University, Noji-cho 1916 , Kusatsu 525 , Japan.
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Takeshi
a Department of Bioscience and Biotechnology, Faculty of Science and Engineering , Ritsumeikan University, Noji-cho 1916 , Kusatsu 525 , Japan.
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Masashi
a Department of Bioscience and Biotechnology, Faculty of Science and Engineering , Ritsumeikan University, Noji-cho 1916 , Kusatsu 525 , Japan.
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Yasuki
a Department of Bioscience and Biotechnology, Faculty of Science and Engineering , Ritsumeikan University, Noji-cho 1916 , Kusatsu 525 , Japan.
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Nobuo
a Department of Bioscience and Biotechnology, Faculty of Science and Engineering , Ritsumeikan University, Noji-cho 1916 , Kusatsu 525 , Japan.
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Yu-Ichi
a Department of Bioscience and Biotechnology, Faculty of Science and Engineering , Ritsumeikan University, Noji-cho 1916 , Kusatsu 525 , Japan.
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Ju-Ichi
Pseudomonas nitroreducens; glutaminase; γ-glutamyl transferring reaction