theanine and ethylamine

l-Theanine (l-Th) was synthesized by simply mixing the reactants (l-glutamine and ethylamine in water) at 25 °C and Bacillus subtilis γ-glutamyl transferase (BsGGT) covalently immobilized on glyoxyl-agarose according to a methodology previously reported by our research group; neither buffers, nor other additives were needed. Ratio of l-glutamine (donor) to ethylamine (acceptor), pH, enzymatic units (IU), and reaction time were optimized (molar ratio of donor/acceptor=1 : 8, pH 11.6, 1 IU mL

Topics: Bacillus subtilis; Catalysis; Enzymes, Immobilized; Ethylamines; gamma-Glutamyltransferase; Glutamine

l-Theanine is a nonproteinogenic amino acid present almost exclusively in tea plants and is beneficial for human health. For industrial production, l-theanine is enzymatically or chemically synthesized from glutamine/glutamate (or a glutamine/glutamate derivative) and ethylamine. Ethylamine is extremely flammable and toxic, which complicates and increases the cost of operational procedures. To solve these problems, we developed an artificial biosynthetic pathway to produce l-theanine in the absence of supplemental ethylamine. For this purpose, we identified and selected a novel transaminase (NCBI:protein accession number AAN70747) from

Topics: Biosynthetic Pathways; Escherichia coli; Ethylamines; Fermentation; Glutamates

Topics: Adenosine Triphosphate; Carbon-Nitrogen Ligases; Corynebacterium glutamicum; Ethylamines; Fermentation; Glutamates; Glutamic Acid; Industrial Microbiology; Metabolic Engineering

γ-Glutamylcysteine synthetase (γ-GCS) from Escherichia coli, which catalyzes the formation of L-glutamylcysteine from L-glutamic acid and L-cysteine, was engineered into an L-theanine synthase using L-glutamic acid and ethylamine as substrates. A high-throughput screening method using a 96-well plate was developed to evaluate the L-theanine synthesis reaction. Both site-saturation mutagenesis and random mutagenesis were applied. After three rounds of directed evolution, 13B6, the best-performing mutant enzyme, exhibited 14.6- and 17.0-fold improvements in L-theanine production and catalytic efficiency for ethylamine, respectively, compared with the wild-type enzyme. In addition, the specific activity of 13B6 for the original substrate, L-cysteine, decreased to approximately 14.6% of that of the wild-type enzyme. Thus, the γ-GCS enzyme was successfully switched to a specific L-theanine synthase by directed evolution. Furthermore, an ATP-regeneration system was introduced based on polyphosphate kinases catalyzing the transfer of phosphates from polyphosphate to ADP, thus lowering the level of ATP consumption and the cost of L-theanine synthesis. The final L-theanine production by mutant 13B6 reached 30.4 ± 0.3 g/L in 2 h, with a conversion rate of 87.1%, which has great potential for industrial applications.

Topics: Adenosine Triphosphate; Amide Synthases; Catalysis; Directed Molecular Evolution; Escherichia coli; Ethylamines; Glutamate-Cysteine Ligase; Glutamates; Glutamic Acid; High-Throughput Screening Assays; Industrial Microbiology; Protein Engineering

Functional foods have created an open environment for the development of new solutions to health-related issues. In celiac disease, there is still no therapeutic alternative other than the observance of a gluten-free diet. In this context, we developed a wheat flour enriched in l-theanine aimed to be a potential alternative to the gluten-free diet. Through microbial transglutaminase-catalysed transamidation of gluten proteins using ethylamine as amine nucleophile, substantial amounts of glutamine residues were converted in theanine residues. Furthermore, using T-cell lines generated from intestinal biopsy specimens of celiac disease patients, this treatment showed the potential to strongly reduce the ability of gluten proteins to stimulate a T-cell-mediated immune response. From a rheological point of view, the functionality of gluten was retained. Considering L-theanine's evidence-based health benefits, a novel functional food is presented here and for celiac disease can be a path towards the development of an alternative to the gluten-free diet.

Topics: Celiac Disease; Diet, Gluten-Free; Dietary Supplements; Elasticity; Ethylamines; Flour; Functional Food; Glutamates; Glutens; Humans; Intestines; T-Lymphocytes; Transglutaminases; Triticum

l-Theanine has a significant role in the taste of tea (

Topics: Amino Acid Sequence; Camellia sinensis; Ethylamines; Glutamates; Glutamic Acid; Hydrolases; Plant Leaves; Plant Proteins; Protein Transport; Sequence Alignment

Theanine is the most abundant non-protein amino acid in

Topics: Bacteria; Camellia sinensis; Endophytes; Ethylamines; Glutamates; Glutamine; Plant Leaves

l-Theanine is a specialized metabolite in tea (Camellia sinensis) leaves that contributes to tea function and quality. Yellow tea leaves (albino) generally have higher l-theanine contents than green tea leaves (normal), but the reason is unknown. The objective of this study was to investigate why l-theanine is accumulated in yellow tea leaves. We compared original normal leaves (green) and light-sensitive albino leaves (yellow) of cv. Yinghong No. 9. The l-theanine content was significantly higher in yellow leaves than in green leaves (p ≤ 0.01). After supplementation with [

Topics: Camellia sinensis; Ethylamines; Glutamates; Glutamic Acid; Hydrolases; Plant Leaves

Theanine, a unique amino acid in

Topics: Alanine; Camellia sinensis; Carboxy-Lyases; Cloning, Molecular; Escherichia coli; Ethylamines; Gene Expression; Gene Expression Regulation, Plant; Genetic Vectors; Glutamates; Nitrogen; Organ Specificity; Phylogeny; Plant Leaves; Plant Proteins; Plant Roots; Recombinant Proteins; Seedlings; Serine; Tea

This study investigated the association between serum ethylamine levels as an indicator of l-theanine consumption and the development of type 2 diabetes in a Japanese community.. A total of 2,253 community-dwelling Japanese individuals aged 40-79 years without diabetes were monitored for 7 years. Serum ethylamine levels were divided into quartiles: ≤0.86, 0.87-2.10, 2.11-5.28, and ≥5.29 ng/mL. Kinetic analysis of serum ethylamine concentrations was performed after ingestion of l-theanine-rich green tea products containing 8 mg of l-theanine by 12 healthy volunteers.. Higher serum ethylamine was significantly associated with lower risk of the development of type 2 diabetes in a general Japanese population. The measurement of serum ethylamine concentration would be a useful biomarker for the objective estimation of l-theanine consumption.

Topics: Adult; Aged; Biomarkers; Diabetes Mellitus, Type 2; Drinking Behavior; Eating; Ethylamines; Female; Glutamates; Humans; Incidence; Insulin Resistance; Japan; Male; Middle Aged; Prediabetic State; Risk Factors; Tea

Recombinant γ-glutamyl transpeptidase (rBLGGT) from Bacillus licheniformis ER-15 was purified to homogeneity by ion-exchange chromatography. Molecular masses of large and small subunits were 42 and 22 kDa, respectively. The enzyme was optimally active at pH 9.0 and 60 °C and was alkali stable. K(m) and V(max) for γ-glutamyl-p-nitroanilide hydrochloride were 45 μM and 0.34 mM/min, respectively. L-Theanine synthesis was standardized using a one variable at a time approach followed by response surface methodology, which resulted in approximately 85-87% conversion of L-glutamine to L-theanine within 4 h. The standardized reaction contained 80 mM L-glutamine, 600 mM ethylamine, and 1.0 U/mL rBLGGTin 50 mM Tris-Cl (pH 9.0) at 37 °C. Similar conversions were also obtained with the enzyme immobilized in calcium alginate. Using immobilized enzyme, 35.2 g of L-theanine was obtained in three cycles of 1 L each. The product was purified by Dowex 50W X 8 hydrogen form resin and was confirmed by HPLC and proton NMR spectroscopy.

Topics: Bacillus; Enzymes, Immobilized; Ethylamines; gamma-Glutamyltransferase; Glutamates; Glutamine; Recombinant Proteins

Glutamic acid gamma-methyl ester (GAME) was used as substrate for theanine synthesis catalyzed by Escherichia coli cells possessing gamma-glutamyltranspeptidase activity. The yield was about 1.2-fold higher than with glutamine as substrate. The reaction was optimal at pH 10 and 45 degrees C, and the optimal substrate ratio of GAME to ethylamine was 1:10 (mol/mol). With GAME at 100 mmol, 95 mmol theanine was obtained after 8 h.

Topics: Catalysis; Escherichia coli; Ethylamines; gamma-Glutamyltransferase; Glutamates; Glutamic Acid; Hydrogen-Ion Concentration; Methyl Ethers; Substrate Specificity; Temperature

Theanine, a unique amino acid found almost exclusively in tea plants, has various favourable physiological and pharmacological functions in humans. Gamma-glutamyltranspeptidase (GGT, EC 2.3.2.2) is considered to be the most effective enzyme for the production of theanine. In fact, GGT can catalyse the transfer of γ-glutamyl moieties from γ-glutamyl compounds to water (hydrolysis) or to amino acids and peptides (transpeptidation).. A novel strain, SK11.004, which produces GGT with high theanine-forming ability was isolated from fermented shrimp paste and identified as Bacillus subtilis through its physiological and biochemical properties as well as its 16S rDNA sequence analysis. Theanine (18.9 mmol L(-1)) was synthesised by GGT (0.06 U mL(-1)) through transfer reaction in the presence of glutamine (20 mmol L(-1)) as a donor and ethylamine HCl (50 mmol L(-1)) as an acceptor at pH 10 and 37 °C for 4 h, the conversion rate being up to 94%.. The enzymatic synthesis of theanine using GGT from a newly isolated strain Bacillus subtilis SK11.004 was found to be an efficient method. Moreover, compared with others, the GGT from B. subtilis SK11.004 exhibited the highest ratio of transferring activity to hydrolytic activity using glutamine, suggesting a high potential application in the production of theanine and other functional γ-glutamyl compounds.

Topics: Bacillus subtilis; Ethylamines; gamma-Glutamyltransferase; Glutamates; Glutamine; Hydrochloric Acid; Hydrolysis

Gamma-glutamylcysteine synthetase (gammaGCS, EC 6.3.2.2) catalyzes the formation of gamma-glutamylcysteine from L-glutamic acid (Glu) and L-cysteine (Cys) in an ATP-dependent manner. While gammaGCS 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 gammaGCS from Escherichia coli has the ability to combine Glu and amines to form gamma-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 gammaGCS was used in the production of theanine (gamma-glutamylethylamine). The resting cells of E. coli expressing gammaGCS, in which ATP was regenerated through glycolysis, synthesized 12.1 mM theanine (18 h) from 429 mM ethylamine.

Topics: Biocatalysis; Escherichia coli; Ethylamines; Glutamate-Cysteine Ligase; Glutamates; Hydrogen-Ion Concentration; Substrate Specificity

Gamma-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 Mn(2+) to the reaction mixture, whereas Mg(2+) 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 MnCl(2), 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).

Topics: Carbon-Nitrogen Ligases; Cations, Divalent; Energy Transfer; Ethylamines; Fermentation; Glucose; Glutamates; Methylophilaceae; Recombinant Proteins; Sodium Glutamate

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 gamma-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.

Topics: Adenosine Triphosphate; Ammonium Sulfate; Cations, Divalent; Cell Membrane Permeability; Chromatography, DEAE-Cellulose; Culture Media; DNA, Bacterial; Electrophoresis, Polyacrylamide Gel; Energy Transfer; Ethylamines; Fermentation; Glutamates; Glutamic Acid; Hydrogen-Ion Concentration; Indicators and Reagents; Methanobacteriaceae; Reverse Transcriptase Polymerase Chain Reaction

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.

Topics: Adenosine Monophosphate; Cations, Divalent; Desiccation; Energy Transfer; Ethylamines; Fermentation; Glutamate-Ammonia Ligase; Glutamates; Magnesium; Pseudomonas; Saccharomyces cerevisiae

The distribution of theanine-degrading activity in Wistar rats was examined and this activity was detected only in the kidney. Judging from polyacrylamide gel electrophoresis, theanine-degrading enzyme from rat kidney was purified almost to homogeneity. Theanine-degrading activity was co-purified with glutaminase activity, and the relative activity for theanine was about 85% of that for L-glutamine throughout purification. Substrate specificity of purified enzyme preparation coincided well with the data of phosphate-independent glutaminase [EC 3.5.1.2], which had been previously reported. It was very curious that gamma-glutamyl methyl and ethyl esters were more effectively hydrolyzed than theanine and L-glutamine, in view of relative activity and K(m) value. It was suggested that gamma-glutamyl moiety in theanine molecule was transferred to form gamma-glutamylglycylglycine with relative ease in the presence of glycylglycine. On the other hand, purified phosphate-dependent glutaminase did not show theanine-degrading activity at all. Thus, it was concluded that theanine was hydrolyzed by phosphate-independent glutaminase in kidney and suggested that, as for the metabolic fate of theanine, its glutamyl moiety might be transferred by means of gamma-glutamyl transpeptidase reaction to other peptides in vivo.

Topics: Animals; Chromatography, Thin Layer; Electrophoresis, Polyacrylamide Gel; Ethylamines; gamma-Glutamyltransferase; Glutamates; Glutamic Acid; Glutaminase; Glycylglycine; Isoenzymes; Kidney; Phosphates; Rats; Rats, Wistar; Substrate Specificity