17010-37-6Relevant articles and documents
Characterization of theanine-forming enzyme from Methylovorus mays No. 9 in respect to utilization of theanine production
Yamamoto, Sachiko,Wakayama, Mamoru,Tachiki, Takashi
, p. 545 - 552 (2007)
For development of theanine production from glutamic acid and ethylamine by coupling yeast sugar fermentation as an ATP-regenerating system, several strains were selected from among about 200 methylamine- and/or methanol-assimilating bacteria depending on the theanine-forming activity of their permeated cells. The amount of theanine formed by the cells of the selected strains was much larger than that by the cells of Escherichia coli AD494 (DE3) expressing Pseudomonas taetrolens Y-30 glutamine synthetase (GS), which has been found to be a usable enzyme for theanine production. A GS-like enzyme responsible for the theanine-forming reaction was obtained from an obligate methylotroph isolate, Methylovorus mays No. 9. The enzyme was induced by methylamine in the culture medium. A molecular mass of 410-470 kDa was obtained by gel filtration of the enzyme, and 51 kDa by SDS-PAGE analysis. The enzyme showed high activity toward methylamine rather than ammonia, which indicates that it is similar to known γ-glutamylmethylamide synthetase. The isolated enzyme also had high reactivity to ethylamine in a neutral pH range, and formed theanine from glutamic acid and ethylamine in a reaction mixture containing a yeast sugar fermentation system for ATP-regeneration.
Studies on the Biochemical Formation Pathway of the Amino Acid l -Theanine in Tea (Camellia sinensis) and Other Plants
Cheng, Sihua,Fu, Xiumin,Wang, Xiaoqin,Liao, Yinyin,Zeng, Lanting,Dong, Fang,Yang, Ziyin
, p. 7210 - 7216 (2017)
Tea (Camellia sinensis) is the most widely consumed beverage aside from water. The flavor of tea is conferred by certain metabolites, especially l-theanine, in C. sinensis. To determine why more l-theanine accumulates in C. sinensis than in other plants, we compare l-theanine contents between C. sinensis and other plant species (Camellia nitidissima, Camellia japonica, Zea mays, Arabidopsis thaliana, and Solanum lycopersicum) and use a stable isotope labeling approach to elucidate its biosynthetic route. We quantify relevant intermediates and metabolites by mass spectrometry. l-Glutamic acid, a precursor of l-theanine, is present in most plants, while ethylamine, another precursor of l-theanine, specifically accumulates in Camellia species, especially C. sinensis. Most plants contain the enzyme/gene catalyzing the conversion of ethylamine and l-glutamic acid to l-theanine. After supplementation with [2H5]ethylamine, all the plants produce [2H5]l-theanine, which suggests that ethylamine availability is the reason for the difference in l-theanine accumulation between C. sinensis and other plants.
Theanine production by coupled fermentation with energy transfer using γ-glutamylmethylamide synthetase of Methylovorus mays No. 9
Yamamoto, Sachiko,Morihara, Yosuke,Wakayama, Mamoru,Tachiki, Takashi
, p. 1206 - 1211 (2008)
γ-Glutamylmetylamide synthetase (GMAS) of Methylovorus mays No. 9, produced by Eschericia coli AD494 (DE3) harboring pET21aGM, formed theanine from glutamic acid and ethylamine with coupling of the reaction with sugar fermentation of baker's yeast cells as an ATP-regeneration system. Theanine formation was stimulated by the addition of Mn2+ to the reaction mixture, whereas Mg2+ was less effective. Increases to a certain level in the concentrations of GMAS and the substrates in the mixture were effective in increasing theanine formation, but high concentrations of ethylamine (900 mM or more) inhibited yeast sugar fermentation, and eventually decreased theanine formation. The inhibitory effect of ethylamine was restored by increasing the concentration of potassium phosphate buffer in the mixture. Approximately 600 mM (110 mg/ml) theanine was formed in 48 h in an improved reaction mixture containing 600 mM sodium glutamate, 600 mM ethylamine· HCl, 300 mM glucose, 200 mM potassium phosphate buffer (pH 7.0), 30 mM MgCl 2, 5 mM MnCl2, 5 mM AMP, 30 units/ml of GMAS, and 40 mg/ml of yeast cells. The yield of theanine was 100% on the substrates (glutamic acid and ethylamine) and also on the energy source (glucose consumed).
Theanine production by coupled fermentation with energy transfer employing Pseudomonas taetrolens Y-30 glutamine synthetase and baker's yeast cells
Yamamoto, Sachiko,Wakayama, Mamoru,Tachiki, Takashi
, p. 784 - 789 (2005)
Theanine was formed from glutamic acid and ethylamine by coupling the reaction of glutamine synthetase (GS) of Pseudomonas taetrolens Y-30 with sugar fermentation of baker's yeast cells as an ATP-regeneration system. Theanine formation was stimulated by the addition of Mn2+ to the mixture for the coupling. The addition of Mg2+ was less effective. In a mixture containing a larger amount of yeast cells with a fixed level of GS, glucose (the energy source) was consumed rapidly, resulting in a decrease in the final yield of theanine. On the other hand, an increase in GS amounts increased theanine formation in a mixture with a fixed amount of yeast cells. High concentrations of ethylamine enhanced theanine formation whereas inhibited yeast fermentation of sugar and the two contrary effects of ethylamine caused a high yield of theanine based on glucose consumed. In an improved reaction mixture containing 200 mM sodium glutamate, 1,200 mM ethylamine, 300 mM glucose, 50 mM potassium phosphate buffer (pH 7.0), 5 mM MnCl2, 5 mM AMP, 100 units/ml GS, and 60 mg/ml yeast cells, approximately 170 mM theanine was formed in 48 h.
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
Yamamoto, Sachiko,Uchimura, Kousuke,Wakayama, Mamoru,Tachiki, Takashi
, p. 1888 - 1897 (2004)
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 3o 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 Mg 2+-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.
Preparation method of L-theanine
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, (2020/04/02)
The invention relates to the technical field of organic chemical synthesis, in particular to a preparation method of L-theanine. The preparation method of the L-theanine comprises the following steps:(a) reacting L-glutamic acid with a copper salt in a solvent to obtain a chelate A; (b) carrying out an esterification reaction on the chelate A and methanol to obtain a compound B; and (c) after a reaction of the compound B with an aqueous ethylamine solution is finished, adding a decoppering reagent, carrying out stirring for a reaction, and removing a solvent to obtain a crude L-theanine product. According to the preparation method of the L-theanine, the L-glutamic acid is used as an initial raw material and is easy to obtain; in addition, residues in amino acid are protected by adopting acopper chelate manner, so the reaction process is green and environment-friendly. In the step (c), the safer aqueous ethylamine solution is used for the reaction, so the safety of the reaction is improved, requirements on equipment are reduced, production cost is greatly saved, and industrial production is easier.
