Production and Preparation of Glutamate Synthase from Brevibacterium flavum

概要

論文の詳細を見る
A procedure was developed for preparation of glutamate synthase (GOGAT) from Brevibaterium flavum (ATCC 14067). Glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were obtained as by-products. The productivity of GOGAT was increased about 45-fold by aerobic culture in a specific medium with 0.3% yeast extract, 0.01% EDTA-Fe and others. Glutamate synthase (GOGAT) was isolated as homogeneous protein (350-fold purification, 6% yield) by ammonium phosphate fractionation and column chromatographies on DEAE-cellulose, Sepharose 6B, hydroxyapatite, and Sephadex G-200. Ammonium phosphate precipitation was effective in protecing the enzyme from inactivatio, and hydroxyapatite column chromatography was effective for removal of glutamate dehydrogenase (GDH). The enzyme (molecular weight 260,000) consisted of two different subunits with molecular weights of 190,000 and 60,000. It contained 8 moles of non-heme iron, 2 moles of FMN, and 2 moles of FAD, and showed adsorption mixima at 280,380-390 and 440 nm. No enzyme activity was lost after 2 months at -20℃ in 0.02 M potassium phosphate buffer (pH 6.5) with 50% glycerol and 2 mM α-ketoglutarate, and 75% of the initial activity remained after 7 months. some properties of glutamate synthase (GOGAT) and glutamine synthetase (GS) are also decribed.
社団法人日本生物工学会の論文