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Concentrated cell-extract of Pseudomonas taetrolens Y-30, isolated as a methylamine-assimilating organism, formed gamma-glutamylethylamide (theanine) from glutamic acid and ethylamine in a mixture containing the alcoholic fermentation system of baker's yeast for ATP-regeneration. Glutamine synthetase (GS), probably responsible for theanine formation, was isolated from the extract of the organism grown on a medium containing 1% methylamine, 1% glycerol, 0.5% yeast extract, and 0.2% polypepton as carbon and nitrogen sources. The molecular mass was estimated to be 660 kDa by gel filtration and 55 kDa by SDS-polyacrylamide gel electrophoresis, suggesting that Ps. taetrolens Y-30 GS consists of 12 identical subunits. The enzyme required Mg2+ or Mn2+ for its activity. Under the standard reaction condition for glutamine formation (pH 8.0 with 30 mM Mg2+), GS showed 7% and 1% reactivity toward methylamine and ethylamine respectively of that to ammonia. Reactivity to the alkylamines varied with optimum pH of the reaction in response to divalent cation in the mixture: pH 11.0 was the optimum for the Mg2+ -dependent reaction with ethylamine, and pH 8.5 was the optimum for the Mn2+ -dependent reaction. In a mixture of an optimum reaction condition with 1000 mM ethylamine (at pH 8.5 with 3 mM Mn2+), reactivity increased up to 7% of the reactivity to ammonia in the standard reaction condition. The isolated GS formed theanine in the mixture with the yeast fermentation system.

bioscience, biotechnology, and biochemistry

Purification and Characterization of Glutamine Synthetase of<i>Pseudomonas taetrolens</i>Y-30: An Enzyme Usable for Production of Theanine by Coupling with the Alcoholic Fermentation System of Baker’s Yeast

Purification and Characterization of Glutamine Synthetase ofPseudomonas taetrolensY-30: An Enzyme Usable for Production of Theanine by Coupling with the Alcoholic Fermentation System of Baker’s Yeast