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.
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.
Yamamoto
Sachiko
S
Department of Bioscience and Biotechnology, Faculty of Science and Technology, Ritsumeikan University, Shiga, Japan.
Uchimura
Kousuke
K
Wakayama
Mamoru
M
Tachiki
Takashi
T
D004735:Energy Transfer; D005285:Fermentation; D019649:Food Industry; D005974:Glutamate-Ammonia Ligase / Q000302:isolation & purification* / Q000378:metabolism; D005971:Glutamates / Q000096:biosynthesis*; D011549:Pseudomonas / Q000201:enzymology* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D005971:Glutamates; C026166:theanine; D005974:Glutamate-Ammonia Ligase