Purification and Some Properties of Glutamate Synthase from Gluconobacter suboxydans grown on Glutamate as a Nitrogen Source

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Glutamate synthase (L-glutamine : 2-oxoglutarate aminotransferase, NADPH oxidizing; EC 2.6.1.53) was purified 450-fold with a 3% yield from Gluconobacter suboxydans IFO 3172 grown on glutamate as a nitrogen source. The preparation was homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was calculated to be about 500,000 by gel filtration on Sepharose 6B. The enzyme exhibited absorption maxima at 280,370-380 and 420-440 nm. The 420-440 nm peak decreased on addition of sodium hydrosultite or NADPH and reappeared on further addition of α-ketoglutarate(α-KGA) and glutamine.The optimum pH of the reaction was 8.5. The enzyme lost 70-80% of the initial activity after 10 min incubation at 55℃ in 0.1 M potassium phosphate buffer (pH 6.8).The presence of α-KGA, glutamine and EDTA was effective in protecting the activity.The enzyme specifically required glutemine, α-KGA and NADPH as substrates and coenzyme. Their K_m values were 0.59mM, 8.6μM and 14μM, respectively. The activity was inhibited by many substances, among which methionine, methionine sulfoximine and NADP were potent inhibitors.
社団法人日本生物工学会の論文
1983-04-25