- Preparation method of L-theanine
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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.
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- Characterization of l -Theanine Excitatory Actions on Hippocampal Neurons: Toward the Generation of Novel N-Methyl- d -aspartate Receptor Modulators Based on Its Backbone
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l-Theanine (or l-γ-N-ethyl-glutamine) is the major amino acid found in Camellia sinensis. It has received much attention because of its pleiotropic physiological and pharmacological activities leading to health benefits in humans, especially. We describe here a new, easy, efficient, and environmentally friendly chemical synthesis of l-theanine and l-γ-N-propyl-Gln and their corresponding d-isomers. l-Theanine, and its derivatives obtained so far, exhibited partial coagonistic action at N-methyl-d-aspartate (NMDA) receptors, with no detectable agonist effect at other glutamate receptors, on cultured hippocampal neurons. This activity was retained on NMDA receptors expressed in Xenopus oocytes. In addition, both GluN2A and GluN2B containing NMDA receptors were equally modulated by l-theanine. The stereochemical change from l-theanine to d-theanine along with the substitution of the ethyl for a propyl moiety in the γ-N position of l- and d-theanine significantly enhanced the biological efficacy, as measured on cultured hippocampal neurons. l-Theanine structure thus represents an interesting backbone to develop novel NMDA receptor modulators.
- Sebih, Fatiha,Rousset, Matthieu,Bellahouel, Salima,Rolland, Marc,De Jesus Ferreira, Marie Celeste,Guiramand, Janique,Cohen-Solal, Catherine,Barbanel, Gérard,Cens, Thierry,Abouazza, Mohammed,Tassou, Adrien,Gratuze, Maud,Meusnier, Céline,Charnet, Pierre,Vignes, Michel,Rolland, Valérie
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p. 1724 - 1734
(2017/08/21)
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- Studies on the Biochemical Formation Pathway of the Amino Acid l -Theanine in Tea (Camellia sinensis) and Other Plants
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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.
- Cheng, Sihua,Fu, Xiumin,Wang, Xiaoqin,Liao, Yinyin,Zeng, Lanting,Dong, Fang,Yang, Ziyin
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p. 7210 - 7216
(2017/08/30)
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- Application of recombinant Bacillus subtilis γ-glutamyltranspeptidase to the production of l-theanine
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l-Theanine, which has seen increasing use in the functional food industry, can be prepared via enzymatic synthesis using γ-glutamyltranspeptidase (GGT; EC 2.3.2.2). In this study, the GGT from Bacillus subtilis 168 was cloned and expressed as a secreted protein using Escherichia coli BL21(DE3). The enzymatic properties of the GGT and the optimal conditions for the enzymatic synthesis of l-theanine were investigated in detail. The activity of the enzyme was optimal at pH 10; the optimal temperature was 50 °C. Desirable pH stability was observed between pH 5 and pH 12, and adequate thermostability was seen at 50 °C. In 5 h at 37 °C, the enzyme converted 200 mM l-glutamine and 2.2 M ethylamine to l-theanine with a final yield of 78%. Yields of l-theanine decreased to 58% when using 500 mM Gln and 45% when using 1 M Gln. The yield of l-theanine obtained at high substrate concentration provides the basis for the industrial-scale production of l-theanine.
- Chen, Xingyi,Su, Lingqia,Wu, Dan,Wu, Jing
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p. 1429 - 1439
(2014/11/07)
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- Synthesis of amino acid using a flow-type microreactor containing enzyme-mesoporous silica microsphere composites
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A flow-type microreactor containing composite materials of a theanine synthetase (glutaminase) and mesoporous silica with 23.6 nm pore diameter (SBA-15 microsphere) was developed for the continuous synthesis of l-theanine, a unique amino acid. Enzyme-immobilisation ability and enzymatic activity in the SBA-15 microsphere with large mesopores were higher than those of SBA-15 with a 5.4 nm pore diameter. Moreover, the glutaminase-SBA-15 microsphere composites displayed higher selectivity in theanine production than the free enzyme did in a batch experiment. A direct visualization of composites of fluorescently labelled glutaminase and SBA-15 microsphere immobilised in the flow channel of the microreactor by a combination of differential interference contrast and fluorescence microscopy revealed that the enzymes were uniformly dispersed throughout the mesoporous silica particles, because of the successful encapsulation of the enzyme. The enzyme-encapsulated microreactor exhibited a high conversion of l-glutamine to l-theanine with local control of the reaction temperature. In addition to this advantage of the microreaction system, the microreactor enabled the on-off regulation of enzymatic activity during continuous theanine synthesis by controlling the reaction temperature or the pH of the substrate solution.
- Matsuura, Shun-Ichi,Chiba, Manami,Tomon, Emiko,Tsunoda, Tatsuo
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p. 9021 - 9030
(2014/03/21)
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- Enzymatic synthesis of theanine from glutamic acid γ-methyl ester and ethylamine by immobilized Escherichia coli cells with γ- glutamyltranspeptidase activity
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Theanine (γ-glutamylethylamide) is the main amino acid component in green tea. The demand for theanine in the food and pharmaceutical industries continues to increase because of its special flavour and multiple physiological effects. In this research, an improved method for enzymatic theanine synthesis is reported. An economical substrate, glutamic acid γ-methyl ester, was used in the synthesis catalyzed by immobilized Escherichia coli cells with γ-glutamyltranspeptidase (GGT) activity. The results show that GGT activity with glutamic acid γ-methyl ester as substrate was about 1.2-folds higher than that with glutamine as substrate. Reaction conditions were optimized by using 300 mmol/l glutamic acid γ-methyl ester, 3,000 mmol/l ethylamine, and 0.1 g/ml of immobilized GGT cells at pH 10 and 50°C. Under these conditions, the immobilized cells were continuously used ten times, yielding an average glutamic acid γ-methyl ester to theanine conversion rate of 69.3%. Bead activity did not change significantly the first six times they were used, and the average conversion rate during the first six instances was 87.2%. The immobilized cells exhibited favourable operational stability.
- Zhang, Fei,Zheng, Qing-Zhong,Jiao, Qing-Cai,Liu, Jun-Zhong,Zhao, Gen-Hai
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experimental part
p. 1177 - 1182
(2011/09/16)
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- Molecular cloning and characterization of γ-Glutamyltranspeptidase from pseudomonas nitroreducens IFO12694
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y-Glutamyltranspeptidase from Pseudomonas nitroreducens IFO12694 (PnGGT) exhibited higher hydro-lytic activity than transfer activity, as compared with other y-glutamyltranspeptidases (GGTs). PnGGT showed little activity towards most of L-amino acids and towards glycyl-glycine, which is often used as a standard y-glutamyl accepter in GGT transfer reactions. The preferred substrates for PnGGT as a y-glutamyl accepter were amines such as methylamine, ethylamine, and isopropylamine.
- Imaoka, Masashi,Yano, Shigekazu,Okumura, Masashi,Hibi, Takao,Wakayama, Mamoru
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p. 1936 - 1939
(2011/06/11)
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- A novel catalytic ability of γ-glutamylcysteine synthetase of Escherichia coli and its application in theanine production
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γ-Glutamylcysteine synthetase (γGCS, EC 6.3.2.2) catalyzes the formation of γ-glutamylcysteine from L-glutamic acid (Glu) and L-cysteine (Cys) in an ATP-dependent manner. While γGCS can use various amino acids as substrate, little is known about whether it can use non-amino acid compounds in place of Cys. We determined that γGCS from Escherichia coli has the ability to combine Glu and amines to form γ-glutamylamides. The reaction rate depended on the length of the methylene chain of the amines in the following order: n-propylamine > butylamine > ethylamine methylamine. The optimal pH for the reaction was narrower and more alkaline than for the reaction with an amino acid. The newly found catalytic ability of γGCS was used in the production of theanine (γ-glutamylethylamine). The resting cells of E. coli expressing γGCS, in which ATP was regenerated through glycolysis, synthesized 12.1 mm theanine (18 h) from 429 mm ethylamine.
- Miyake, Koichiro,Kakita, Shingo
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experimental part
p. 2677 - 2683
(2010/09/11)
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- Theanine production by coupled fermentation with energy transfer using γ-glutamylmethylamide synthetase of Methylovorus mays No. 9
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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).
- Yamamoto, Sachiko,Morihara, Yosuke,Wakayama, Mamoru,Tachiki, Takashi
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p. 1206 - 1211
(2008/12/21)
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- Process for purifying theanine
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The present invention provides a process for purifying theanine from an aqueous solution having a dominant cation and comprising impurities. The process comprises the steps of: introducing the aqueous solution into a column in an amount of from 2 to 20% of the column volume, wherein the column is packed with a cation exchange resin of the same cation type as the dominant cation; then introducing an aqueous mobile phase having a pH of from 2.5 to 8.5 into the column thereby to elute a zone enriched in the impurities followed by a zone enriched in theanine; and then recovering at least one fraction of the zone enriched in theanine.
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Page/Page column 3-4
(2009/01/20)
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- ALCOHOL METABOLISM ACCELERATING COMPOSITION, AND FOOD OR DRINK CONTAINING THE COMPOSITION
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An alcohol-metabolism enhancing composition contains theanine. The composition may be contained in ingesta. Theanine has an effect of quickly reducing blood alcohol concentration, thereby easing or improving troubles due to alcoholic ingestion such as hangover or alcoholic hepatic insufficiency.
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Page/Page column 5
(2008/06/13)
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- SLEEP-IMPROVING COMPOSITION
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[ABSTRACT] An object of this invention is to provide a sleep improvement composition, which contains theanine and other components and can be used safely by anybody on a daily basis without side effects. This object is attained by a sleep improvement composition comprising: (a) the anine; and (b) at least one component selected from the group consisting of chamomile, lavender, Saint John's wort, kawakawa, valerian, passion flower, tryptophan, γ-aminobutyric acid, serotonin, melatonin, and cedrol.
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Page/Page column 8-9
(2008/06/13)
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- Characterization of theanine-forming enzyme from Methylovorus mays No. 9 in respect to utilization of theanine production
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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.
- Yamamoto, Sachiko,Wakayama, Mamoru,Tachiki, Takashi
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p. 545 - 552
(2008/03/27)
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- PROCESS FOR THE PREPARATION OF N(5)-ETHYLGLUTAMINE
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Disclosed relates to a process for preparing N (5)- ethylglutamines economically without a specific purification process via a simplified and safe process, in which glutamic acid derivatives, represented by formula 1, protected by phthaloyl groups react with ethylamine to cause an amidation and a deprotection reaction in turn under the same reaction condition, thus preparing N (5) -ethylglutamines .
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Page/Page column 15
(2008/06/13)
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- Compositions to improve tic disorders
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Tic disorders can be improved by administering theanine, an amino acid that crosses the blood-brain barrier, or a dipeptide consisting of theanine and a different amino acid. In addition, these compositions can be added to foods and drinks or pharmaceuticals.
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Page/Page column 4
(2008/06/13)
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- METHOD FOR PRODUCING THEANINE
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Disclosed is a method for producing theanine characterized in that a glutamic acid alkyl ester represented by the general formula (1) below is reacted with a ketone represented by the general formula (2) below in the presence of t-butyl amine, a secondary amine or a tertiary amine; the thus-obtained compound represented by the general formula (3) below is reacted with ethylamine and then heated in the presence of ethylamine or reacted with a fatty acid. (In the formula (1), R1 represents an alkyl group.) (In the formula (2), R2 represents a hydrogen atom and R3 represents a lower alkanoyl group or a benzoyl group, or alternatively R2 and R3 may form a cycloalkanone ring together with an adjacent carbon atom.) (In the formula (3), R1, R2 and R3 are as defined above.)
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Page/Page column 6-7
(2008/06/13)
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- Composition for repressing transformation growth factor beta
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A composition for repressing transformation growth factor β contains theanine as an active substance. The composition is useful for preventing or treating chronic glomerulonephritis, renal interstitial fibrosis, hepatic fibrosis, hepatic cirrhosis, idiopathic interstitial pneumonia, keloid, hidebound disease, arterial sclerosis, myocardial infarction, cardiac fibrosis, restenosis, acute megakaryoblastic leukemia, adult T-cell leukemia, chronic fatigue syndrome or ordinary fatigue.
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Page/Page column 3
(2008/06/13)
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- Theanine production by coupled fermentation with energy transfer employing Pseudomonas taetrolens Y-30 glutamine synthetase and baker's yeast cells
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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.
- Yamamoto, Sachiko,Wakayama, Mamoru,Tachiki, Takashi
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p. 784 - 789
(2008/02/03)
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- 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
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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.
- Yamamoto, Sachiko,Uchimura, Kousuke,Wakayama, Mamoru,Tachiki, Takashi
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p. 1888 - 1897
(2007/10/03)
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- γ-Glutamyl Transfer Reactions by Glutaminase from Pseudomonas nitroreducens IFO 12694 and Their Application for the Syntheses of Theanine and γ-Glutamylmethylamide
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In a mixture containing γ-glutamyl donor (donor) and γ-glutamyl acceptor (acceptor), the glutaminase of Pseudomonas nitroreducens IFO 12694 simultaneously catalyzed a γ-glutamyl transfer reaction and hydrolysis of the donor. The variation of the activities responding to the concentration of glutathione and glycylglycine indicated that the enzyme might be classified in a group of glutaminases that shows hydrolysis prior to transfer reaction. On the other hand, the results with glutamine and ethylamine or methylamine indicated that the enzyme was active in the transfer reaction with suppressed hydrolysis of glutamine, and suggested the possibility of using the reaction for producing γ-glutamylethylamide (theanine) or γ-glutamylmethylamide (γ-GMA). In fact, in a mixture containing high concentrations of substrates (0.7 M glutamine, 1.5 M ethylamine or methylamine) and 0.5 unit/ml glutaminase (borate buffer pH 11), 270 mM (47 g/L) theanine or 250 mM (38 g/L) γ-GMA was formed in 7 h of incubation at 30°C.
- Tachiki, Takashi,Yamada, Takeshi,Mizuno, Katsushige,Ueda, Masashi,Shiode, Ju-Ichi,Fukami, Hiroshi
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p. 1279 - 1283
(2007/10/03)
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- Purification and Characterization of γ-Glutamylmethylamide Synthetase from Methylophaga sp. AA-30
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γ-Glutamylmethylamide synthetase was purified about 70-fold from a cell-free extract of Methylophaga sp.AA-30 by ammonium sulfate fractionation, Octyl-Sepharose column chromatography, and Sephacryl S-300 gel filtration.Only a single protein band was detected after SDS-polyacrylamide gel electrophoresis of the purified preparation; the band was at a position corresponding to a molecular weight of 56,000.The molecular weight of the enzyme was calculated to be 440,000 by Superose 6HR gel filtration, so we suggest that the enzyme is an octomer of identical subunits.The enzyme had maximum activity at pH 7.5 and 40 deg C.It could use ethylamine and propylamine instead of methylamine as the substrate, but it could not use D-glutamate or L-glutamine instead of L-glutamate.
- Kimura, Toshio,Sugahara, Isao,Hanai, Katsuyuki,Tonomura, Yuuko
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p. 708 - 711
(2007/10/02)
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