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Документ Exopolysaccharide Production and Peculiarities of C6-Metabolism in Acinetobacter sp. Grown on Carbohydrate Substrates(2002) Pirog, Tatiana; Kovalenko, M.; Kuzminska, Yu.An Acinetobacter sp. strain grown on carbohydrate substrates (mono- and disaccharides, molasses, starch) was shown to synthesize exopolysaccharides (EPS). Glucose catabolism proved to proceed via the Embden–Meyerhof–Parnas and Entner–Doudoroff pathways. Pyruvate entered the tricarboxylic acid cycle due to pyruvate dehydrogenase activity. Pyruvate carboxylation by pyruvate carboxylase was the anaplerotic reaction providing for the synthesis of intermediates for the constructive metabolism of Acinetobacter sp. grown on C6-substrates. The C6-metabolism in Acinetobacter sp. was limited by coenzyme A. Irrespective of the carbohydrate growth substrate (glucose, ethanol), the activities of the key enzymes of both C2- and C6-metabolism was high, except for the isocitrate lyase activity in glucose-grown bacteria. Isocitrate lyase activity was induced by C2-compounds (ethanol or acetate). After their addition to glucose-containing medium, both substrates were utilized simultaneously, and an increase was observed in the EPS synthesis, as well as in the EPS yield relative to biomass. The mechanisms responsible for enhancing the EPS synthesis in Acinetobacter sp. grown on a mixture of C2- and C6-substrates are discussed.Документ Peculiarities of ethanol metabolism in an Acinetobacter sp. Mutant strain defective in exopolysaccharide synthesis(2002) Pirog, Tatiana; Sokolov, I.; Kuzminska, Yu.; Malashenko, YuriActivities of the key enzymes of ethanol metabolism were assayed in ethanol-grown cells of an Acinetobacter sp. mutant strain unable to synthesize exopolysaccharides (EPS). The original EPS-producing strain could not be used for enzyme analysis because its cells could not to be separated from the extremely viscous EPS with a high molecular weight. In Acinetobacter sp., ethanol oxidation to acetaldehyde proved to be catalyzed by the NAD+-dependent alcohol dehydrogenase (EC 1.1.1.1.). Both NAD+ and NADP+ could be electron accepters in the acetaldehyde dehydrogenase reaction. Acetate is implicated in the Acinetobacter sp. metabolism via the reaction catalyzed by acetyl-CoA-synthetase (EC 6.2.1.1.). Isocitrate lyase (EC 4.1.3.1.) activity was also detected, indicating that the glyoxylate cycle is the anaplerotic mechanism that replenishes the pool of C4-dicarboxylic acids in Acinetobacter sp. cells. In ethanol metabolism by Acinetobacter sp., the reactions involving acetate are the bottleneck, as evidenced by the inhibitory effect of sodium ions on both acetate oxidation in the intact cells and on acetyl-CoA-synthetase activity in the cell-free extracts, as well as by the limitation of the C2-metabolism by coenzyme A. The results obtained may be helpful in developing a new biotechnological procedure for obtaining ethanol-derived exopolysaccharide ethapolan. В клетках выращенного на этаноле мутантного штамма Acinetobacter sp., не образующего экзополисахариды (ЭПС), определены активности ключевых ферментов метаболизма этанола. Клетки исходного ЭПС-образующего штамма не могли быть использованы для проведения энзимологических исследований ввиду невозможности их отделения от высоковязкого ЭПС с высокой молекулярной массой. Установлено, что окисление этанола до ацетальдегида у Acinetobacter sp. катализируется НАД+-зависимой алкогольдегидрогеназой (КФ 1.1.1.1.). Акцепторами электронов в ацетальдегиддегидрогеназной реакции являются НАД+ и НАДФ+. Ацетат вовлекается в метаболизм Acinetobacter sp. при участии ацетил-КоА-синтетазы (КФ 6.2.1.1.). Наличие изоцитратлиазы (КФ 4.1.3.1.) свидетельствует о том, что анаплеротической последовательностью реакций, восполняющих пул С4-дикарбоновых кислот в клетках Acinetobacter sp., является глиоксилатный цикл. «Узким» местом метаболизма этанола у бактерий Acinetobacter sp. является вовлечение ацетата в метаболизм, о чем свидетельствует ингибирование окисления ацетата в интактных клетках и активности ацетил-КоА-синтетазы в бесклеточном экстракте ионами натрия, а также лимитирование С2- метаболизма коэнзимом А. Полученные данные являются основой для разработки новой технологии получения экзополисахарида этаполана на основе этанола.Документ Regulation of Acetate Metabolism in a Strain of Acinetobacter sp. Growing on Ethanol(2003) Pirog, Tatiana; Kuzminska, Yu.Ethanol metabolism in Acinetobacter sp. is shown to be limited by the rate of acetate assimilation,a reaction catalyzed by acetyl-CoA synthetase (EC 6.2.1.1). Effects of ions (sodium, potassium, and magnesium), by-products of ethanol and acetaldehyde oxidation (NADH and NADPH), and pantothenic acid on this enzyme are studied (sodium, NADH, and NADPH inhibit acetyl-CoA synthetase; pantothenic acid, potassium, and magnesium act as enzyme activators). Conditions of culturing were developed under which ethanol, acetaldehyde, and acetate in Acinetobacter cells were oxidized at the same rates, producing a threefold increase in the activity of acetyl-CoA synthetase in the cell-free extract. The results of studies of acetyl-CoA synthetase regulation in a mutant strain of Acinetobacter sp., which is incapable of forming exopolysaccharides, provide a basis for refining the technology of ethapolan production involving the use of C2 substrates.