JPH0755154B2 - Theanine manufacturing method - Google Patents

Description

Detailed Description of the Invention

[Industrial applications]

The present invention relates to a novel method for producing γ-glutamylethylamide (hereinafter referred to as theanine).

[0002]

2. Description of the Related Art Theanine is known as a main component of gyokuro's umami, and is an important substance as a flavoring substance for foods such as 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, Chem. Pharm. Bull., 19 (7) 1301-1307
(1971), 19 (6), 1257-1261 (1971), 34 (7), 3053
-3057 (1986), Pharmaceutical Journal, 95 (7), 892-895 (1975), reported that theanine and glutamine antagonize the spasms induced by caffeine, which suggests that these compounds Is considered to act on the central nervous system and is expected to be useful as a physiologically active substance.

[0003] Conventionally, as a method for producing theanine, a method of extracting it from a dried tea leaf obtained in a tea garden for producing gyokuro containing theanine has been generally used. However, in this case, theanine is accumulated in only about 1.5% per dry matter of the tea leaf, and the photosynthesis is active in a general tea garden for sencha, so that theanine is hardly accumulated. Therefore, it has been pointed out that the extraction method from dried tea leaves is not industrially practical.

From the above, development of an industrial production method is expected, and as one of them, a method of chemically organically synthesizing theanine has been reported (Chem. Pharm.
Bull., 19 (7) 1301-1307 (1971)). However, it has been pointed out that such an organic synthesis reaction has a low yield and requires a complicated operation for separating and purifying the product. Further, Japanese Patent Publication No. 63-28596 discloses a method for synthesizing theanine from glutamic acid in the presence of glutamine synthetase, using ATP produced by yeast during sugar fermentation. However, in the method disclosed in this publication, the optimum pH of yeast is neutral (6 to 8), whereas the optimum pH of the theanine synthesis reaction by glutamine synthetase is 10 to 11. It has been pointed out that combining both reactions is not easy and difficult to carry out.

By the way, glutaminase hydrolyzes glutamine to produce glutamic acid and ammonia.
It is known that γ-glutamylhydroxamic acid is produced by adding hydroxylamine and selecting reaction conditions (SC Hartman, Journal of Biological Ch
emistry, 243, 853-863, 1968). However, a method for producing theanine using such a glutaminase is not known. Therefore, an object of the present invention is to provide a method for producing theanine simply and industrially advantageously.

[0006]

In order to solve the above problems, the present inventors have conducted various experiments by replacing the above hydroxylamine with ethylamine. As a result, they have unexpectedly found that theanine can be obtained by reacting glutaminase under specific reaction conditions, and have completed the present invention. That is, the gist of the present invention is to use a mixture of glutamine and ethylamine at pH 9 to 1
The present invention relates to a method for producing theanine, which comprises allowing glutaminase to act under the conditions of 2.

In the present invention, it is necessary to carry out under the condition of pH 9-12. This is because when the pH is lower than 9, the hydrolysis reaction of glutamine becomes large as shown in Fig. 1 and thus the synthesis of theanine is suppressed, and when the pH is higher than 12, the stability of glutaminase becomes poor as shown in Fig. 2. Theanine synthesis stops.

The glutaminase in the present invention is not particularly limited in its origin. For example, as a microorganism-derived one, bacteria such as Pseudomonas genus, Saccharomyce
Enzymes derived from yeasts such as s genus and fungi such as Aspergillus genus. Here, the microorganisms of the genus Pseudomonas include Pseudomonas nitroreducens, Pseudomonas aptata,
Alternatively, Pseudomonas denitrificans and the like can be mentioned as suitable examples. Other sources of glutaminase may be plants, animals and the like.

The glutaminase in the present invention has the following physicochemical properties. (1) Action Under the condition of pH 9 to 12, catalyzes the transfer reaction at the γ-position of glutamine, and theanine is synthesized by the reaction between glutamine and ethylamine. Under conditions of pH less than 9, glutamine is mainly hydrolyzed to produce glutamic acid. (2) Substrate specificity Glutamine is used as a substrate. (3) Optimum pH and stable pH The optimum pH for theanine synthesis is 9 to 12 (see FIG. 1), and the stable pH is stable up to pH 11.5 by heat treatment at 45 ° C. for 10 minutes (see FIG. 2). (4) Optimum temperature and stable temperature The optimum temperature is 35 to 40 ° C, and the stable temperature is stable up to 40 ° C in a heat treatment for 10 minutes under the condition of pH 11 (Fig. 2).
However, it is preferable to set the reaction temperature to 30 ° C. in the reaction for several hours to several tens hours. The optimum temperatures are the amounts of theanine produced when the temperature conditions are set to 10, 20, 30, 35, 40, and 50 ° C (the amount produced at 30 ° C is 100) are 22, 52, 100, 147, and 15, respectively.
It is based on 0 and 12.

The influence of glutaminase activity by metal ions in the present invention is such that, when an enzymatic reaction is carried out in the presence of nickel, cobalt, cadmium or zinc, the reaction in which glutamine is hydrolyzed by glutaminase to produce glutamic acid is suppressed. However, it does not directly affect the synthesis of theanine. That is, L is used to measure the hydrolysis activity.
In a test conducted using -γ-glutamyl p-nitroanilide (pH 9) and L-glutamine + ethylamine (pH 11) for measuring the transfer reaction activity, as shown in Table 1, nickel, cobalt, cadmium, Alternatively, when the enzymatic reaction is carried out in the presence of zinc, the hydrolysis of glutamine to produce glutamic acid is suppressed, and it does not directly affect the synthesis of theanine.

Therefore, when the reaction is carried out in the presence of nickel, cobalt, cadmium, or zinc, the production of glutamic acid is suppressed, so that theanine can be more effectively synthesized. For example, when this nickel is added to the actual reaction solution, the production of theanine is increased and the production of glutamic acid is suppressed by the addition of nickel as shown in FIG.

[0012]

[Table 1]

The optimum glutamine concentration in the present invention is as follows.
The reaction is carried out in the range of 1 to 0.7 M and borate buffer (Na ₂
B ₄ O ₇ -NaOH, in pH 11), the amount of 30 obtained by reacting 1 to 7 hours at ° C. (glutaminase 1.0
U / ml). As shown in FIG. 4, 0.7 M glutamine produced about 280 mM (about 50 g / L) theanine in 5 hours, and after 5 hours, it tends to undergo hydrolysis and decrease. From these results, the glutamine concentration is preferably 0.3 to 0.7 M, and considering the conversion rate from glutamine and the maximum production amount, the glutamine concentration of around 0.3 M is optimal. The Km value for glutamine in theanine production is about 20 mM.

The optimum ethylamine concentration in the present invention is
Glutamine was fixed at 0.3M and ethylamine concentration was adjusted to 2
When it is changed to the range of M, the amount of theanine produced becomes constant at a concentration of 1 M or more. That is, glutamine 0.3M
The concentration was determined by changing the concentration of ethylamine to a range of 2 M and reacting in borate buffer (pH 11) at 30 ° C. for 5 hours. As shown in FIG. 5, ethylamine is 1
The amount of theanine produced becomes constant at a concentration of M or higher. In addition,
The Km value for ethylamine is about 130 mM.

The relationship between the amount of glutaminase and the incubation time is as follows: 0.3M glutamine and 1.5M ethylamine in a borate buffer (pH 11) at 30 ° C. in the range of 0.05 to 0.5 U / ml of glutaminase for 23 hours. When reacted up to 0.1 to 0.
At 3 U / ml it increases linearly and reaches a certain amount of production, which is a prolongation of the time.

From these points, in order to carry out an efficient reaction in the present invention, the glutamine concentration is usually 0.3 to 0.7 M and the ethylamine concentration is 0.3 M to glutamine under the condition of pH 9 to 12. In general, 1.0 M or more is preferable. The amount of glutaminase is 1 reaction time.
If it is less than 0 hours, it is usually about 0.8 to 1.0 U / ml,
In the case of 10 hours to 30 hours, 0.3 to 0.4 U / ml is preferable. When the reaction time is several hours or longer, the reaction temperature is usually preferably 30 to 35 ° C. The isolation and purification of the thus obtained theanine from the reaction solution can be easily carried out by a commonly known method, for example, by combining solvent distribution and various chromatographies and HPLC.

[0017]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. Example of preparation of glutaminase (a) Cultivation of Pseudomonas nitroreducens IFO 12694 Sodium glutamate 0.6%, yeast extract 0.1
%, Glucose 1.0%, KH ₂ PO ₄ 0.05%,
_{K 2 HPO 4 0.05%, MgSO} 4 · 7H 2 O
Using a culture solution (pH 7) containing 0.07% and EDTA-Fe 0.01%, a 30 liter jar fermenter (30 ° C., aeration 1 vvm = 25 liter / min,
2,000 rpm), Pseudomonas nitroreduc
The ens IFO 12694 strain was cultured for about 20 hours. (B) Cell-free extract After washing 175 liters of cells, the cells were suspended in 7.5 liters of 30 mM potassium phosphate buffer (pH 7.0), and 5
Ultrasonication was performed at -20 ° C to obtain a cell-free extract. (C) Ammonium sulfate fraction Ammonium sulfate fractionation was performed while adjusting the pH to 7 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, DEAE-cellulose column (15x60c
m), and glutaminase was eluted with a buffer containing 0.1 M sodium chloride.

By the above operation, glutaminase of practically sufficient purity can be obtained, but CM-cellulose column chromatography and Sephadex G150 are further used.
By performing column chromatography, hydroxyapatite column chromatography, and butyltoyopearl column chromatography, a single sample can be obtained by disc electrophoresis. The purification rate is 250 times and the recovery rate is 10%.

Example 1 0.3M glutamine and 1.5M ethylamine in a borate buffer (Na ₂ B ₄ O ₇ -NaOH, pH 11)
The reaction was carried out with 0.3 U / ml glutaminase at 30 ° C. for 22 hours. 225 mmole of theanine was isolated from 1 liter of the reaction solution. The by-product glutamic acid is 2
It was 0 mmole. For the isolation and purification of theanine from the reaction solution, the reaction solution was Dowex 50 × 8, Dowex 1 × 2
It was carried out by subjecting it to column chromatography and treating it with ethanol.

When this isolated substance was subjected to an amino acid analyzer and paper chromatography, it showed the same behavior as the standard substance. When hydrolyzed with hydrochloric acid or glutaminase, glutamic acid and ethylamine were produced in a ratio of 1: 1. . Thus, the isolated substance was hydrolyzed by glutaminase, indicating that ethylamine was bound to the gamma position of glutamic acid. Also,
It was also confirmed by glutamate dehydrogenase (GluDH) that the glutamate produced by hydrolysis was L-type. FIG. 7 is an IR spectrum of theanine, and the same spectrum was obtained for both the standard and the isolated substance. From these, it was confirmed that the isolated substance was theanine.

Example 2 When 1 mM of NiCl ₂ was added to the same reaction solution as in Example 1 and the reaction was carried out under the same conditions, 240 mmole of theanine was isolated from 1 liter of the reaction solution. The by-product glutamic acid was 10 mmole.

[0022]

INDUSTRIAL APPLICABILITY The present invention provides a novel method for efficiently producing theanine, which enables simple and industrially advantageous production.

[Brief description of drawings]

FIG. 1 is a diagram showing the optimum pH for theanine synthesis by glutaminase.

FIG. 2 is a diagram showing stable pH and stable temperature of glutaminase.

FIG. 3 is a diagram showing the influence of nickel addition on theanine synthesis.

FIG. 4 is a diagram showing the influence of glutamine concentration on theanine synthesis.

FIG. 5 is a diagram showing the influence of ethylamine concentration on theanine synthesis.

FIG. 6 is a diagram showing the relationship between the amount of glutaminase and the incubation time.

FIG. 7 is a diagram showing IR spectra of the theanine preparation and an isolated substance.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kim Takehisa 9-5 Akahori Shinmachi, Yokkaichi-shi, Mie Taiyo Kagaku Co., Ltd. (72) Inventor Nobuyuki Hagiwara 9-5 Akahori-shinmachi, Yokkaichi-shi, Mie Solarization Gaku Co., Ltd. (72) Inventor Masaya Tateishi 9-5 Akahori-shinmachi, Yokkaichi-shi, Mie Taiyo Kagaku Co., Ltd.

Claims (4)

[Claims] 1. A mixture of glutamine and ethylamine is added with p.
A method for producing theanine, which comprises causing glutaminase to act under the conditions of H9 to 12. 2. The production method according to claim 1, wherein the glutaminase is allowed to act in the presence of nickel, cobalt, cadmium or zinc. 3. The method according to claim 1, wherein the glutaminase is an enzyme obtained from a microorganism belonging to the genus Pseudomonas. 4. The microorganism of the genus Pseudomonas according to claim 3, wherein Pseudomonas nitroreducens, Pseudomonas aptata,
The method according to claim 3, which is Pseudomonas denitrificans.