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

Abstract

Concentrated cell-extract of Pseudomonas taetrolens Y-30, isolated as a methylamine-assimilating organism, formed γ-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.