Purification and Some Properties of Glutaminase from Pseudomonas nitroreducens IFO 12694

抄録

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 hydrolyzed glutamine optimally at pH 9, and its K_m was 6.5mM. D-Glutamine, γ-glutamyl p-nitroanilide, γ-glutamylmethylamide, γ-glutamylethylamide (theanine), and glutathione showed respectively 107, 85, 78, 74, and 82% reactivity of glutamine. Zn²⁺, Ni²⁺, Cd²⁺, Co²⁺, Fe²⁺, and Cu²⁺ 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 K_m for glutamine and hydroxylamine were 4mM 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.