JP2009225705A - Method for producing theanine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method for efficiently producing L-theanine and to simply and industrially advantageously produce L-theanine in a high yield by solving the problem that although a method for extracting dried tea leaves obtained in a tea field for producing refined green tea containing theanine is generally known as a method for producing theanine from conventionally, however, in the method, only approximately about 1.5% of theanine is accumulated based on dried tea leaves, since photosynthesis is active in a general tea field for green tea, theanine is hardly accumulated in the real situation. <P>SOLUTION: The method for producing theanine comprises using Methylovorus mays TGMS No.9 (accession number: NITE P-298, indication identification: MMTGMS-9). <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a novel process for producing theanine (γ-glutamylethylamide).

  Theanine is known as the main ingredient of umami contained in green tea and is an important substance as a flavor substance for foods including tea. On the other hand, it has been pointed out that γ-glutamyl derivatives including theanine act as physiologically active substances in animals and plants. For example, theanine and glutamine have been reported to antagonize convulsions induced by caffeine, and these compounds may act on the central nervous system, and are expected to be useful as bioactive substances. Has been.

  Conventionally, as a method for producing theanine, a method of extracting from a dried tea leaf obtained in a tea garden for producing gyokuro containing theanine is generally used. However, in this case, theanine accumulates only about 1.5% per tea leaf dry matter, and photosynthesis is active in a general sencha tea garden. Therefore, it is pointed out that the extraction method from dried tea leaves is not industrially practical.

  For these reasons, development of industrial production methods is expected, and as one of them, a method for chemically organic synthesis of theanine has been reported (see Non-Patent Document 1). However, it has been pointed out that such an organic synthesis reaction has a low yield and requires a complicated operation in separating and purifying the target product from a mixed solution with unreacted raw materials and by-products. Patent Document 1 discloses a method of synthesizing theanine from glutamic acid in the presence of glutamine synthase using ATP produced by yeast during the fermentation of sugar. However, in the method disclosed in the publication, the optimum pH of yeast is neutral (6.0 to 8.0), whereas the optimum pH of the theanine synthesis reaction by glutamine synthase is 10.0. Since it is ˜11.0, it is pointed out that it is not easy to combine both reactions and it is difficult to carry out. Furthermore, a method for producing theanine characterized in that glutaminase obtained from Pseudomonas bacteria is allowed to act on glutamine and ethylamine under conditions of pH 9.0 to 12.0 (see Patent Document 2), A method for producing theanine (see Patent Document 3) characterized in that it is used is disclosed. However, since ethylamine has a very low boiling point of 16.6 ° C, the ethylamine vapor that volatilizes during production has an adverse effect on workers and the environment, and attempts to react at temperatures above the boiling point for the purpose of improving reaction efficiency. Then, there is a problem that special equipment is required. Further, these production methods have practical problems because the substrate concentration is low and the product yield is low. In addition, the enzyme disclosed in Patent Document 2 is complicated to purify, has a problem with the stability of the enzyme with respect to pH and temperature, and has a complicated operation for separating the enzyme and product after the reaction, In addition, there are many problems such as difficulty in producing theanine by continuous reaction.

Chem. Pharm. Bull. , 19 (7) 1301-1307 (1971) JP 58-04-0094 Japanese Patent Laid-Open No. 05-055154 JP 05-328986 A

  An object of the present invention is to provide an efficient method for producing theanine, and to enable theanine production that is simple and industrially advantageous.

  In order to solve the above-mentioned problems, the present inventors have studied γ-glutamylmethylamide synthase (GMAS) possessed by methylotrophic bacteria and a part of methylotrophic bacteria. GMAS is an enzyme that synthesizes γ-glutamylmethylamide from glutamic acid, methylamine, and ATP, but it is said that ethylamine, which is a homolog of methylamine, is also highly reactive.

  According to the present invention, a novel efficient method for producing theanine can be provided, and simple and industrially advantageous production can be realized.

  As the GMAS in the present invention, those derived from methanol-assimilating microorganisms can be used. Examples include methylotrophic (C1 compound-assimilating bacteria) bacteria and yeasts. Most preferably, the Mylovorous mays TGMS No. 9 etc. are mentioned. As an enzyme source for this reaction, viable cells or various treated samples of viable cells, for example, crushed cells, sonicated cells, solvent-treated cells, low-temperature dried cells, and ammonium sulfate salted-out products can be used. GMAS can use a crude enzyme or a purified enzyme, and is not particularly limited as a purification method, but gel filtration, ion exchange, membrane separation, acetone treatment, ammonium sulfate fractionation, and the like are conceivable. In addition, purified enzyme preparations, bacterial cells and the like can be used as they are or immobilized on a carrier. The carrier is for immobilizing GMAS, for example, celite, diatomaceous earth, kaolinite, silica gel, molecular sieves, porous glass, activated carbon, calcium carbonate, ceramics and other inorganic carriers, ceramic powder, polyvinyl alcohol, polypropylene, Examples include, but are not limited to, organic polymers such as chitosan, ion exchange resin, hydrophobic adsorption resin, chelate resin, and synthetic adsorption resin.

  The presence of a metal salt is important for the production of theanine in the present invention, and a divalent metal salt is desirable as the metal salt. Examples of divalent metal salts include manganese ions, magnesium ions, copper ions, etc. Among them, manganese ions such as manganese chloride and magnesium ions such as magnesium chloride are preferably present, and water-soluble manganese salts and magnesium salts are preferred. Can be used. The presence of a combination of two or more of these divalent metal ions shifts the optimum pH for theanine production from the alkali side to the neutral side, thereby obtaining the target condition of the present invention. The concentration in the reaction solution is not particularly limited, but is preferably 0.01 to 500 mM, more preferably 0.1 to 100 mM, and most preferably 1 to 50 mM.

  To activate GMAS in the present invention, adenosine triphosphate (ATP), which is fermentation chemical energy, is essential, and a microorganism capable of producing fermentation chemical energy can be cited as an ATP source. As this fermentative chemical energy producing microorganism, yeasts, particularly microorganisms belonging to the genus Saccharomyces, which are microorganisms capable of producing ATP by sugar fermentation can be generally used. A normal culture method is used to obtain cells of these microorganisms. These microorganisms can be used in various forms such as solvent-treated cells, dried cells, disrupted cells, and cell-free preparations. In addition, a compound such as polyphosphoric acid having a high phosphate bond is also conceivable as another ATP source.

  The glutamic acid in the present invention is produced by a chemical synthesis method, a fermentation method or the like, and may be either L-form or D-form, but L-form is preferred. In addition to the free acid, the form includes a metal salt and the like, and a metal salt having high solubility in water is preferable, and a sodium salt is most preferable. The optimal glutamic acid concentration for the theanine production reaction is not particularly limited because enzyme inhibition by glutamic acid does not occur, but it is desirable to adjust so that no unreacted substrate remains in consideration of subsequent purification.

  The form of ethylamine according to the present invention includes hydrochloride and the like in addition to the anhydride. Any form may be used, but hydrochloride is preferred from the viewpoint of safety. The optimal ethylamine concentration for theanine production reaction is desirably 3.0M or less, preferably 2.0M or less, more preferably, because high concentration of ethylamine reduces the ATP production rate in the glycolysis system of yeast. 1.0M or less. However, according to the method in which ethylamine is added in several portions, ethylamine can be added in a large amount as long as this concentration is not exceeded at each addition, and the production amount of theanine can be improved. The reaction of 1: 0.1-5 is also desired in the ratio of glutamic acid and ethylamine serving as a substrate, but 1: 0.5-3 is preferred. In order to perform an efficient reaction in the present invention, pH 6.0 to 9.0 is preferable, and 6.5 to 7.5 is more preferable. Moreover, 0-55 degreeC of reaction temperature is preferable, More preferably, it is 10-40 degreeC, Most preferably, it is 20-35 degreeC. Isolation and purification of the theanine thus obtained from the reaction solution can be carried out by a common known method, for example, by combining solvent partitioning, various chromatography and HPLC.

  EXAMPLES The present invention will be further described below with reference to examples, but the present invention is not limited to these examples.

Example 1
Separation of bacteria using C1 compound assimilation performance as an index. Medium using methanol as carbon source and methylamine as nitrogen source (methylamine 0.25%, methanol 0.25%, NaCl 0.2%, KCl 0.1%, MgSO ₄ 0.03%, KH ₂ PO ₄ 0.005%, K ₂ HPO ₄ 0.005%, pH 7.0) was subcultured 3 times to isolate about 100 strains of methylotrophic bacteria.

Example 2
Separation of bacteria by theanine-producing activity Reaction solution containing a substrate for theanine synthesis, divalent metal ions and surfactant, viable cells of the isolated bacteria (glutamate 50 mM, ethylamine 150 mM, MgCl ₂ 30 mM or MnCl ₂ 3 mM, ATP 7.5 mM) , Imidazole Buffer 100 mM, CTAB 0.1 Mg / ml, bacterial cell OD _{610 nm} = 1.0, pH 7.7), the activity was measured by theanine quantification, and 6 types of candidate bacteria were obtained from about 100 strains of methylotrophic bacteria. Among them, TGMS No. 99 having a homology of 99% with Methylovorus mays recently reported as an obligate methanol-assimilating bacterium. We paid attention to 9 shares. The results are shown in Table 1.

Example 3
Preparation of Glutamylmethylamide Synthetic Oxygen (GMAS) (a) Methylovorus mays TGMS No. 9 cultures Methanol 0.25%
Methylamine hydrochloride 0.25%
Yeast extract 0.03%
NaCl 1.0%
KCl 0.02%
MgSO ₄ · 7H ₂ O 0.03%
KH ₂ PO ₄ 0.005%
K ₂ HPO ₄ 0.005%
Cyanocobalamine 1 × 10 ⁻⁷ %
In a 5 liter jar fermenter (30 ° C., rotation speed: 100 rpm) containing Methylovorus mays TGMS No. 9 was cultured for 5 days.
(B) Cell-free extract After washing 1 liter of cells, the suspension is suspended in 50 ml of 30 mM potassium phosphate buffer (pH 7.0) and subjected to ultrasonic disruption at 5 to 20 ° C. Obtained.
(C) Ammonium sulfate fraction Ammonium sulfate fractionation was performed while adjusting the pH to 7.0 with 7% aqueous ammonia to obtain a 45 to 90% saturated fraction. This was dissolved in 0.01 M potassium phosphate buffer and dialyzed against the same buffer.
(D) DEAE-cellulose column chromatography The dialyzed enzyme solution was buffered with 0.01 M potassium phosphate buffer. Next, it was made to adsorb | suck to a DEAE-cellulose column (15 * 60 cm), and the adsorbed enzyme was eluted with the buffer solution which contains a 0.1-M salt.

  By the above operation, glutamylmethylamide synthetic oxygen (GMAS) having practically sufficient purity could be obtained. A single sample was then obtained electrophoretically. The purification rate was 31 times, and the recovery rate in terms of total activity was 21%.

Example 4
Production of theanine by energy conjugation The activity of GMAS was simply measured by a reaction of synthesizing γ-glutamyl hydroxalic acid from glutamic acid, hydroxylamine, and ATP. One unit of enzyme was defined as the amount of enzyme that synthesizes 1 μmol of γ-glutamyl hydroxalic acid per minute. As a result of carrying out the reaction by adding 1 unit of GMAS to the conjugation reaction solution, the glycolysis intermediate fructose 1,6-bisphosphate (FBP) temporarily accumulates with the consumption of glucose by yeast cells. Then, theanine was produced with a decrease in FBP. When GMAS or yeast cells were removed from the complete reaction system, theanine was not generated, indicating that theanine production observed in the complete reaction system proceeded by conjugation of GMAS and yeast cells. The results are shown in FIG.

Example 5
Effect of divalent metal ions on theanine production When metal ions are not added to the reaction solution containing yeast cells as a source of common energy (Figure 2 left), sugar fermentation of yeast cells proceeds normally. Regardless, theanine was hardly produced. As metal ions, divalent metal ions such as Mg ²⁺ and Mn ²⁺ were used. When compared with the amount of theanine produced when Mg ²⁺ was added to the reaction solution, it was found that Mn ²⁺ was more effective for theanine synthesis. In addition, it was possible that more theanine was produced when Mg ²⁺ and Mn ²⁺ were added simultaneously than when Mn ²⁺ and Mg ²⁺ were added alone. The results are shown in FIG.

Example 6
The influence of pH on the theanine synthesis reaction by GMAS in the presence of Mg ²⁺ or Mn ²⁺ , a metal ion useful for theanine production, was investigated. As a result, the activity of the Mn ²⁺ -dependent reaction at the optimum pH for Mg ²⁺ -dependent reaction (7.75, 100%) was about 28%. At the optimum pH of Mn ²⁺ -dependent reaction (7.0), the activity of Mg ²⁺ -dependent reaction was comparable to that of Mn ²⁺ . The results are shown in FIG.

Example 7
Changes in theanine-producing ability depending on substrate concentration Next, when the concentrations of glutamic acid and ethylamine, which are substrates, were increased together with the glucose concentration in producing a high concentration of theanine, the amount of theanine produced decreased conversely. Since the consumption rate of glucose by yeast cells is reduced, it can be considered that a high concentration of the substrate inhibits the glycolysis reaction of yeast, thereby reducing the production amount of theanine. The results are shown in FIG.

Example 8
Concentration of components in the reaction solution and the effect on the yeast glycolysis system Thus, the effects of each component in the reaction solution on the yeast glycolysis system were investigated. The concentrations of potassium phosphate buffer (KPB) and ethylamine were determined in the yeast glycolysis system. It was confirmed that it has a significant effect on As shown in the figure, as the concentration of KPB increases, the consumption rate of glucose increases and the yeast glycolysis proceeds smoothly. Moreover, even if the ethylamine concentration was lowered, the inhibition of the yeast glycolysis reaction was alleviated. The results are shown in FIG.

Example 9
Concentration of each reaction solution component and the amount of theanine produced Based on the examples so far, the concentration of components that can affect theanine production was adjusted to examine the most efficient reaction conditions. By changing the concentration of ethylamine from 900 mM to 600 mM and the concentration of KPB from 50 mM to 200 mM, the inhibition of yeast glycolysis was eliminated, and the theanine production increased accordingly. The results are shown in FIG.

Example 10
Preparation of Immobilized GMAS Using Chitopearl 4010 A commercially available chitopearl 4010 (Fujibo Co., Ltd.), which is chitosan beads, was immersed in a 50 mM sodium phosphate buffer (pH 7.4) for 24 hours. After this equilibration, 10 mL of Chitopearl 4010 was immersed in 25 mL of GMAS prepared in Example 3 and shaken for about 2 hours. Thereafter, Chitopearl 4010 from which the adhering liquid had been removed was added to a 2.5% glutaraldehyde solution, and the mixture was further shaken for 2 hours. After the glutaraldehyde treatment, an immobilized GMAS using Chitopearl 4010 is prepared by washing with 30 volumes of 50 mM sodium phosphate buffer (pH 7.4) until the absorbance (280 nm) is 0.01 or less. be able to.

Example 11
Preparation of Immobilized GMAS Using Anion Exchange Resin 10 mL of Diaion HPA25 (Mitsubishi Chemical Corporation), an anion exchange resin, was added to 25 mL of purified GMAS obtained in Example 3 and then shaken for about 2 hours. did. Thereafter, HPA25 from which the adhering liquid had been removed was added to a 2.5% glutaraldehyde solution, and the mixture was further shaken for 2 hours. After treatment with glutaraldehyde, an immobilized GMAS using Diaion HPA25 is prepared by washing with 30 volumes of 50 mM sodium phosphate buffer (pH 7.4) until the absorbance (280 nm) is 0.01 or less. can do.

Production of theanine As a final condition, 40 mg / ml of dried yeast in 200 mM potassium phosphate buffer (pH 7.0) containing 600 mM glutamic acid, 600 mM ethylamine, 300 mM glucose, 30 mM MgCl ₂ , 3 mM MnCl ₂ and 5 mM AMP The mixture was added and reacted at 30 ° C. for 27 hours in 30.0 unit / ml GMAS. As a result, about 600 mM of theanine was isolated from 1 L of the reaction solution. Isolation and purification from the theanine reaction solution was performed by subjecting the reaction solution to Dowex 50 × 8 and Dowex 1 × 2 column chromatography and treating it with ethanol. When this isolated material was subjected to amino acid analyzer and paper chromatography, it showed the same peak as the standard material. When hydrolyzed with hydrochloric acid or glutaminase, glutamic acid and ethylamine were produced at a ratio of 1: 1. Thus, the isolated substance was hydrolyzed by glutaminase, indicating that ethylamine was bound to the γ-position of glutamic acid. It was also confirmed by glutamate dehydrogenase (GluDH) that glutamic acid generated by hydrolysis was in the L form.

  Methylovous mays TGMS no. By using γ-glutamylmethylamide synthase (GMAS) derived from No. 9 (Accession Number: NITE P-298 Identification: MMTGMS-9), an efficient method for producing theanine is provided in comparison with the prior art. Simple and industrially advantageous production can be made possible.

It is the figure which showed that the reaction system of GMAS requires conjugate energy. It is the figure which looked at the influence on theanine production in presence of a bivalent metal ion. It is a figure regarding the influence of theanine productivity by a bivalent metal ion and pH. It is a figure regarding the change of the productivity of theanine by the density | concentration of a substrate. It is the figure which showed the influence which the component density | concentration in a reaction liquid has on a glycolysis system. It is a figure regarding the change of the theanine production rate by the density | concentration change of a substrate component.

Claims (4)

Methylovous mays TGMS No. 9 (Accession number: NITE P-298 Identification display: MMTGMS-9). Methylovous mays TGMS No. A method for producing theanine, comprising using γ-glutamylmethylamide synthase (GMAS) derived from No. 9 (accession number: NITE P-298, identification: MMTGMS-9). The method for producing theanine according to claim 1 or 2, wherein γ-glutamylmethylamide synthase (GMAS) is allowed to act on a mixture of glutamic acid and ethylamine under conditions of pH 6.0 to 7.5. The method for producing theanine according to any one of claims 1 to 3, wherein γ-glutamylmethylamide synthase (GMAS) is allowed to act in the presence of a divalent metal ion.

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