JP3210080B2 - Method for producing theanine - Google Patents
Method for producing theanineInfo
- Publication number
- JP3210080B2 JP3210080B2 JP18431892A JP18431892A JP3210080B2 JP 3210080 B2 JP3210080 B2 JP 3210080B2 JP 18431892 A JP18431892 A JP 18431892A JP 18431892 A JP18431892 A JP 18431892A JP 3210080 B2 JP3210080 B2 JP 3210080B2
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- Japan
- Prior art keywords
- theanine
- cells
- reaction
- immobilized
- ethylamine
- Prior art date
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Description
【０００１】[0001]
【産業上の利用分野】本発明はテアニンの製造方法に関
する。The present invention relates to a method for producing theanine.
【０００２】[0002]
【従来の技術・発明が解決しようとする課題】テアニン
は玉露の旨味成分として知られ、茶をはじめとする食品
の香味および調味成分として重要でありその需要が高ま
りつつある。また一方では、テアニンを含めたγーグル
タミル誘導体は、動・植物体における生理活性物質とし
て作用することが知られている。BACKGROUND OF THE INVENTION Theanine is known as an umami component of gyokuro, and is important as a flavor and seasoning component of tea and other foods, and its demand is increasing. On the other hand, it is known that γ-glutamyl derivatives including theanine act as physiologically active substances in animals and plants.
【０００３】例えば、Chem.Pharm.Bull., 19(7) 1301-1
307(1971), 同19(6)1257-1261(1971), 同 34(7) 3053-
3057(1986), 薬学雑誌 95(7) 892-895(1975), Agric.Bi
ol.Chem., 51,3281-3286(1987), 同52, 3173-3174(198
8) には、テアニンがカフェインの中枢興奮作用を抑制
する物質であると考えられ、その生理活性物質としての
有用性が期待されている。For example, Chem. Pharm. Bull., 19 (7) 1301-1
307 (1971), 19 (6) 1257-1261 (1971), 34 (7) 3053-
3057 (1986), Pharmaceutical Journal 95 (7) 892-895 (1975), Agric.Bi
ol.Chem., 51, 3281-3286 (1987), 52, 3173-3174 (198
In 8), theanine is considered to be a substance that suppresses the central excitatory action of caffeine, and its usefulness as a bioactive substance is expected.
【０００４】従来より、テアニンの製造方法としては玉
露などの茶葉から抽出する方法が一般的であるが、この
場合、テアニンは茶葉乾燥物あたりわずか1.5 ％しか蓄
積されず、また一般の茶園では光合成が活発であるため
ほとんど蓄積されないのが実状である。従って、茶葉か
らの抽出法では工業的に実用的でない。Conventionally, as a method for producing theanine, extraction from tea leaves such as gyokuro is generally performed. In this case, only 1.5% of theanine accumulates in dried tea leaves, and photosynthesis is not performed in general tea gardens. The fact is that it is hardly accumulated because it is active. Therefore, the extraction method from tea leaves is not industrially practical.
【０００５】また他の製造方法としてテアニンを化学的
に有機合成する方法が報告されている（Chem.Pharm.Bul
l., 19(7), 1301-1307(1971), Biosci.Biotech.Bioche
m., 56(4), 689(1992) ）。しかし、このような有機合
成反応では収率が低く、生成物の分離精製等において煩
雑な操作を必要とするという問題点が指摘されている。As another production method, a method of chemically synthesizing theanine organically has been reported (Chem. Pharm.
l., 19 (7), 1301-1307 (1971), Biosci.Biotech.Bioche
m., 56 (4), 689 (1992)). However, it has been pointed out that such an organic synthesis reaction has a low yield and requires a complicated operation in the separation and purification of a product.
【０００６】このようなことから、植物細胞もしくは微
生物を利用した生合成法が報告されている。茶の細胞培
養による方法（特開平３−１８７３８８号）では、培養
細胞の増殖率が極めて低いことが問題となっており実用
的には難しい。また、特公昭６３−２８５９６号公報で
は酵母が糖の発酵の際に生成するＡＴＰを利用して、グ
ルタミン合成酵素の存在下でグルタミン酸からテアニン
を合成する方法が開示されている。しかしながら、この
方法では酵母の至適ｐＨが中性（８−６）であるのに対
して、グルタミン合成酵素の反応至適ｐＨが10−11であ
るために両反応を組み合わせることは容易ではなく、工
業的規模での実施が困難であることが指摘されている。[0006] Under such circumstances, a biosynthesis method using a plant cell or a microorganism has been reported. In the method based on tea cell culture (Japanese Patent Application Laid-Open No. 3-187388), it is problematic that the proliferation rate of the cultured cells is extremely low, which is practically difficult. JP-B-63-28596 discloses a method for synthesizing theanine from glutamic acid in the presence of glutamine synthetase using ATP produced by yeast during fermentation of sugar. However, in this method, the optimum pH of yeast is neutral (8-6), whereas the optimum pH of glutamine synthetase is 10-11, so that it is not easy to combine both reactions. It is pointed out that implementation on an industrial scale is difficult.
【０００７】そこで、Pseudomonas 属細菌から得られる
グルタミナーゼにグルタミンとエチルアミンをｐＨ９−
12の条件下で作用させることを特徴とするテアニンの製
造方法を提案している（平成３年特許願第２６３１２０
号）。しかし、この方法ではいくつかの問題点がある。
すなわち、酵素の精製が煩雑であること、ｐＨおよび温
度に対する酵素の安定性に問題があること、さらに反応
後の酵素と生成物の分離操作が煩雑であること、および
連続的な反応によるテアニンの生産が困難である等の多
くの問題点を有する。Therefore, glutamine and ethylamine were added to glutaminase obtained from bacteria of the genus Pseudomonas at pH 9-.
A method for producing theanine characterized by acting under 12 conditions has been proposed (Japanese Patent Application No. 263120, 1991).
issue). However, this method has several problems.
That is, the purification of the enzyme is complicated, there is a problem with the stability of the enzyme with respect to pH and temperature, the separation operation of the enzyme and the product after the reaction is complicated, and the production of theanine by a continuous reaction. There are many problems such as difficulty in production.
【０００８】[0008]
【課題を解決するための手段】本発明者らは前記の課題
を解決すべく鋭意研究を重ねた結果、Pseudomonas 属細
菌の固定化菌体を用いることにより従来の方法に比べ操
作が簡単で、かつテアニンが高収率・高安定的・連続的
に得られることを見いだし、本発明を完成するに至っ
た。Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems, and as a result, the use of immobilized cells of the genus Pseudomonas simplifies the operation as compared with the conventional method. In addition, they have found that theanine can be obtained continuously with high yield, high stability, and have completed the present invention.
【０００９】以下、本発明を詳細に説明する。本発明の
固定化菌体に用いられる菌体はPseudomonas 属細菌、Sa
ccharomyces属等の酵母、Asperigillus属等のかび等の
菌体が用いられる。好ましくは、Pseudomonas 属細菌の
菌体が好適であり、Pseudomonas 属細菌としては、Pseu
domonas nitroreducens, Pseudomonas aptata, Pseudom
onas denitrificans等が挙げられる。これらの菌体を得
るためには通常の培養方法でよい。好ましくは、高いテ
アニン生成能を有する菌体を得るために培地中にグルタ
ミン酸ナトリウムおよび塩酸エチルアミンを添加するこ
とが効果的である。Hereinafter, the present invention will be described in detail. The cells used for the immobilized cells of the present invention are bacteria of the genus Pseudomonas, Sa
Bacteria such as yeasts of the genus ccharomyces and molds of the genus Asperigillus are used. Pseudomonas bacteria are preferably used, and Pseudomonas bacteria include Pseu.
domonas nitroreducens, Pseudomonas aptata, Pseudom
onas denitrificans and the like. A usual culture method may be used to obtain these cells. Preferably, it is effective to add sodium glutamate and ethylamine hydrochloride to the medium in order to obtain cells having a high theanine-producing ability.
【００１０】本発明の固定化菌体とは、ある一定の空間
に閉じ込められた状態にある微生物菌体であり、さらに
連続的に酵素反応を行うことができ、反応後、微生物菌
体を回収し、再利用できる状態にある微生物菌体のこと
であり、公知の定義と同様である。[0010] The immobilized cells of the present invention are microbial cells confined in a certain space, and can be continuously subjected to an enzymatic reaction. After the reaction, the microbial cells are recovered. It is a microbial cell in a reusable state, which is the same as a known definition.
【００１１】本発明に用いられる固定化菌体は、各種固
定化担体に公知の方法に従って調製できる。菌体の固定
化方法は無機担体、有機担体、陰イオンおよび陽イオン
交換体に菌体を結合させる方法、グルタルアルデヒドの
ような試薬によって菌体と菌体を架橋する方法および高
分子素材を用いて菌体を包括する方法のいずれでも良
い。好ましくは、テアニンの高収率および固定化菌体の
安定性等の観点からカラギーナン、ポリアクリルアミ
ド、寒天およびアルギン酸を用いた包括法が好適に使用
できる。The immobilized cells used in the present invention can be prepared according to a known method for various immobilized carriers. The method of immobilizing the cells uses a method of binding the cells to an inorganic carrier, an organic carrier, an anion and a cation exchanger, a method of crosslinking the cells with a reagent such as glutaraldehyde, and a polymer material. Any method may be used to cover the cells. Preferably, an inclusive method using carrageenan, polyacrylamide, agar, and alginic acid can be suitably used from the viewpoints of high theanine yield and stability of the immobilized cells.
【００１２】以下、固定化菌体によるテアニン製造時の
反応条件について説明する。用いた固定化菌体はκ−カ
ラギーナンを固定化担体とし、公知の方法により調製し
たものである。 （１）反応ｐＨ テアニン生成に対する本固定化菌体による反応のｐＨは
30℃, 30分間の条件ではｐＨ７−10の範囲で安定であ
り、9.5 が最適である。本反応に用いられる緩衝液は通
常の緩衝液でよく、好ましくはホウ砂−水酸化ナトリウ
ム緩衝液、炭酸ナトリウムー炭酸水素ナトリウム緩衝液
が好適に使用できる。その濃度は適宜選択できる。Hereinafter, the reaction conditions when theanine is produced by the immobilized cells will be described. The immobilized bacterial cells used were prepared by a known method using κ-carrageenan as an immobilizing carrier. (1) Reaction pH The reaction pH of the immobilized cells for theanine production is
Under conditions of 30 ° C. for 30 minutes, the pH is stable in the range of 7-10, and 9.5 is optimal. The buffer used in this reaction may be a conventional buffer, and preferably a borax-sodium hydroxide buffer or a sodium carbonate-sodium bicarbonate buffer can be suitably used. The concentration can be appropriately selected.
【００１３】（２）反応温度 テアニン生成に対する本反応の温度はｐＨ9.5, 30 分間
の条件では35℃まで安定であり、30℃が最適である。(2) Reaction temperature The reaction temperature for theanine production is stable up to 35 ° C. under conditions of pH 9.5 and 30 minutes, and optimally 30 ° C.
【００１４】（３）基質濃度 基質として添加するグルタミンおよびエチルアミンの濃
度には特に制限はなく、基質溶液の流速および反応温度
等により適宜決定できる。すなわち反応後に未反応のグ
ルタミンおよびエチルアミンが残存しないように決定す
ればよく、このことにより反応液からのテアニンの分離
・精製が容易となる。(3) Substrate Concentration The concentration of glutamine and ethylamine to be added as a substrate is not particularly limited, and can be appropriately determined depending on the flow rate of the substrate solution, the reaction temperature, and the like. That is, it is only necessary to determine such that unreacted glutamine and ethylamine do not remain after the reaction. This facilitates separation and purification of theanine from the reaction solution.
【００１５】テアニン生成に対する至適グルタミン濃度
は、エチルアミンを0.7Mに固定し、グルタミンの濃度を
0.05-0.7M の範囲まで変化させ、ｐＨ9.5, 30 ℃, 60分
間反応させた場合、グルタミン濃度が0.3M付近が最適で
あった。なお、テアニン生成におけるグルタミンに対す
るＫｍ値は約0.025Mである。The optimal glutamine concentration for theanine production is determined by fixing ethylamine at 0.7 M and adjusting the glutamine concentration.
When the reaction was carried out for 60 minutes at pH 9.5, 30 ° C., with a change in the range of 0.05-0.7 M, the optimum glutamine concentration was around 0.3 M. In addition, the Km value for glutamine in theanine production is about 0.025M.
【００１６】次に、至適エチルアミン濃度は、グルタミ
ンを0.3Mに固定し、エチルアミン濃度を0.3-1.0Mの範囲
まで変化させ、ｐＨ9.5, 30 ℃, 60分間反応させた場
合、0.75M 以上の濃度でテアニンの生成量が一定となっ
た。この結果から、エチルアミン濃度は0.7M付近が最適
である。Next, the optimum ethylamine concentration is 0.75M or more when glutamine is fixed at 0.3M, the ethylamine concentration is changed to a range of 0.3-1.0M, and the reaction is carried out at pH 9.5, 30 ° C. and 60 minutes. The production amount of theanine became constant at the concentration of. From these results, it is optimal that the concentration of ethylamine is around 0.7M.
【００１７】（４）基質溶液の流速 κ−カラギーナンで固定化した菌体200 mlをジャケット
付きガラスカラム（1.7 ×40cm, 30℃）に充填し、0.3M
グルタミンおよび0.7Mエチルアミンの基質溶液を流速
（SV）を0.1-0.5 の範囲まで変化させ、最適流速を求め
た。その結果、SV=0.3からテアニン生成は一定となり、
最適流速SVは約0.3 付近である。(4) Flow Rate of Substrate Solution 200 ml of the cells immobilized with κ-carrageenan were packed in a jacketed glass column (1.7 × 40 cm, 30 ° C.), and 0.3 M
The optimal flow rate was determined by changing the flow rate (SV) of the substrate solution of glutamine and 0.7 M ethylamine to a range of 0.1-0.5. As a result, theanine production becomes constant from SV = 0.3,
The optimum flow velocity SV is around 0.3.
【００１８】このようにして得られる本反応液中には反
応産物のテアニンとわずかな未反応の基質のみが含まれ
ている。従って本反応液液からのテアニンの分離・精製
は、極めて簡単である。すなわち濃縮、膜による分離・
濃縮、溶媒分配、透析および各種クロマトグラフィー、
ＨＰＬＣを組み合わせることにより容易に行うことがで
きる。The reaction solution thus obtained contains only the unreacted substrate with theanine as a reaction product. Therefore, separation and purification of theanine from the reaction solution is extremely simple. That is, concentration, separation by membrane,
Concentration, solvent distribution, dialysis and various chromatography,
It can be easily performed by combining HPLC.
【００１９】[0019]
【実施例】以下、実施例により本発明をさらに詳しく説
明するが、本発明はこれらにより何ら限定されるもので
はない。 実施例１．Pseudomonas nitroreducens IFO 12694 の培
養 1.0 ％グルコース、0.05％ 酵母エキス、0.75％ グル
タミン酸ナトリウム、0.1 ％塩酸エチルアミン、0.15％
リン酸第二カリウム、0.15％ リン酸第一カリウム、
0.01％ ＥＤＴＡ- Ｆｅおよび0.07％ 硫酸マグネシウ
ム７水和物を含む培養液（ｐＨ5.5 ）を用いて30℃, 20
時間、好気培養を行い、遠心分離により菌体を得た。The present invention will be described in more detail with reference to the following examples, which should not be construed as limiting the invention thereto. Embodiment 1 FIG. Culture of Pseudomonas nitroreducens IFO 12694 1.0% glucose, 0.05% yeast extract, 0.75% sodium glutamate, 0.1% ethylamine hydrochloride, 0.15%
Potassium phosphate, 0.15% Potassium phosphate,
Using a culture solution (pH 5.5) containing 0.01% EDTA-Fe and 0.07% magnesium sulfate heptahydrate at 30.degree.
Aerobic culture was performed for a time, and cells were obtained by centrifugation.
【００２０】実施例２．固定化菌体の調製（１） 得られたPseudomonas nitroreducens IFO 12694 の菌体
をκ- カラギーナンに固定化した。可溶化した3.4 ％κ
−カラギーナン450ml に、実施例１で得た菌体80g を加
えすばやく攪拌後、４℃で30分間静置した。同量の0.3M
KClを加え、４℃、１時間静置したゲルを適当な大きさ
（約¢2mm ×10mm）に細断した。このゲルに0.3M KClで
可溶化した1 ％ヘキサメチレンジアミン500 mlを加え、
４℃、10分間静置後、水洗した。さらに0.5 ％グルタル
アルデヒド250 mlを加え4 ℃、10分間静置後、水洗し本
発明の固定化菌体を得た。固定化菌体800 mlをジャッケ
ト付きガラスカラム（1.7 ×40cm、１本当りの容量200
ml）４本に充填し30℃に保温した。Embodiment 2 FIG. Preparation of immobilized cells (1) The obtained cells of Pseudomonas nitroreducens IFO 12694 were immobilized on κ-carrageenan. 3.4% κ solubilized
-To 450 ml of carrageenan, 80 g of the bacterial cells obtained in Example 1 were added, and the mixture was stirred rapidly and allowed to stand at 4 ° C for 30 minutes. 0.3M of the same amount
KCl was added, and the gel left at 4 ° C. for 1 hour was shredded to an appropriate size (about 2 mm × 10 mm). To this gel was added 500 ml of 1% hexamethylenediamine solubilized with 0.3 M KCl,
After leaving still at 4 ° C. for 10 minutes, it was washed with water. Further, 250 ml of 0.5% glutaraldehyde was added, the mixture was allowed to stand at 4 ° C. for 10 minutes, and then washed with water to obtain the immobilized cells of the present invention. Place 800 ml of immobilized cells on a glass column with a jacket (1.7 x 40 cm, capacity 200 per tube).
ml), and the mixture was kept at 30 ° C.
【００２１】実施例３．固定化菌体の調製（２） 実施例１で得られた菌体をポリアクリルアミドに固定化
した。ポリアクリルアミド60g 、Ｎ，Ｎ’−メチレン−
ビス（アクリルアミド）3.2gを溶かした水溶液275ml に
菌体60g 、Ｎ，Ｎ，Ｎ’，Ｎ’−テトラメチルエチレン
ジアミン1.0g、亜硫酸アンモニウム1.0gを順次添加後、
10℃、30分間冷却しゲルを得た。実施例２と同様に、こ
のゲルを細断、ヘキサメチレンジアミン、グルタルアル
デヒドにて処理し、本発明の固定化菌体を得た。Embodiment 3 FIG. Preparation of immobilized cells (2) The cells obtained in Example 1 were immobilized on polyacrylamide. 60 g of polyacrylamide, N, N'-methylene-
60 g of cells, 1.0 g of N, N, N ', N'-tetramethylethylenediamine and 1.0 g of ammonium sulfite were sequentially added to 275 ml of an aqueous solution in which 3.2 g of bis (acrylamide) was dissolved.
The gel was obtained by cooling at 10 ° C. for 30 minutes. As in Example 2, this gel was shredded and treated with hexamethylenediamine and glutaraldehyde to obtain the immobilized cells of the present invention.
【００２２】実施例４．テアニンの連続製造 0.3Mグルタミンおよび0.7Mエチルアミンの0.05M ホウ酸
緩衝液（ｐＨ9.5 ）を調製し、実施例２で調製した固定
化菌体を用いてテアニンの連続製造を行った。４本のガ
ラスカラムを第１のカラム、第２のカラム、第３のカラ
ムおよび第４のカラムとつなぎ、SV＝0.3 の条件で２２
日間反応を行った。それぞれのカラムから出た反応液を
サンプリングしテアニンの生成量を測定した。結果を図
１に示した。図１の相対活性とは、最も高いテアニン生
成量を示した１２日目の値（反応液１リットル当り285
mmol）を１００とした活性である。また表中の記号は、
第１カラム（▲）、第２カラム（■）、第３カラム
（△）および第４カラム（○）の反応液である。Embodiment 4 FIG. Continuous Production of Theanine A 0.05 M borate buffer (pH 9.5) of 0.3 M glutamine and 0.7 M ethylamine was prepared, and the theanine was continuously produced using the immobilized cells prepared in Example 2. Four glass columns were connected to the first column, the second column, the third column, and the fourth column.
The reaction was performed for a day. The reaction solution discharged from each column was sampled and the amount of theanine produced was measured. The results are shown in FIG. The relative activity in FIG. 1 is the value on the 12th day showing the highest amount of theanine produced (285 per liter of reaction solution).
(mmol) as 100. The symbols in the table are
Reaction liquids of the first column (カ ラ ム), the second column (■), the third column (△), and the fourth column (○).
【００２３】図１から明かなように、各カラムとも反応
１２日目以降テアニン生成量が一定となり、最後の第４
カラムの反応液の場合、１リットル当り280-285 mmolと
高い値を示し、グルタミンからテアニンへの変換率は約
９５％であった。また本固定化菌体によるテアニン生成
活性は、反応開始から２２日間全く安定であり、長期間
にわたるテアニンの連続製造が可能となった。As is clear from FIG. 1, the amount of theanine produced in each column became constant after the twelfth day of the reaction.
In the case of the reaction solution of the column, the value was as high as 280-285 mmol per liter, and the conversion from glutamine to theanine was about 95%. The theanine producing activity of the immobilized cells was completely stable for 22 days from the start of the reaction, and continuous production of theanine over a long period of time became possible.
【００２４】実施例５．テアニンの分離・精製（１） 実施例４で得られた反応液１リットルを200 mlまで減圧
濃縮後、10℃, 30分間冷却した。それに400 mlのイソプ
ロピルアルコールを加え攪拌後、濾過を行い、風乾しテ
アニンの結晶48ｇを得た。Embodiment 5 FIG. Separation and Purification of Theanine (1) One liter of the reaction solution obtained in Example 4 was concentrated under reduced pressure to 200 ml, and then cooled at 10 ° C. for 30 minutes. 400 ml of isopropyl alcohol was added thereto, and the mixture was stirred, filtered, and air-dried to obtain 48 g of theanine crystals.
【００２５】実施例６．テアニンの分離・精製（２） 実施例４のようにして得られた反応液５リットルを市販
の脱塩装置にて脱塩し、１リットルまで減圧濃縮後、噴
霧乾燥してテアニンの粉末220 ｇを得た。Embodiment 6 FIG. Separation / Purification of Theanine (2) 5 L of the reaction solution obtained as in Example 4 was desalted with a commercially available desalting apparatus, concentrated under reduced pressure to 1 L, and then spray-dried to obtain 220 g of theanine powder. I got
【００２６】この結晶をアミノ酸アナライザーによる分
析を行うと、標準物質と同じ挙動を示した。また、この
結晶を塩酸あるいはグルタミナーゼで加水分解を行う
と、１：１の割合でグルタミン酸とエチルアミンを生じ
たことから、エチルアミンがグルタミン酸のγ位に結合
していたことが示される。また、加水分解で生じたグル
タミン酸がＬ型であることも、グルタミン酸デヒドロゲ
ナーゼ（GluDH ）により確認された。図２はテアニンの
結晶の1H-NMRスペクトルであり、標準物質および本結晶
とも同一のスペクトルが得られた。When the crystals were analyzed by an amino acid analyzer, they showed the same behavior as a standard substance. Further, when the crystals were hydrolyzed with hydrochloric acid or glutaminase, glutamic acid and ethylamine were produced at a ratio of 1: 1. This indicates that ethylamine was bonded to the γ-position of glutamic acid. It was also confirmed by glutamate dehydrogenase (GluDH) that the glutamic acid produced by the hydrolysis was L-type. FIG. 2 shows the 1 H-NMR spectrum of the theanine crystal, and the same spectrum was obtained for both the standard substance and the present crystal.
【００２７】1H-NMRの分析条件および結果は次の通りで
ある。溶媒（D2O ）、内部標準（Sodium 4,4-dimethyl-
4-silapentanesulfonate）、1H-NMR(D2): 1.109 (1H,t,
J=7.15 Hz, b-CH3), 2.129 (2H, dd,J=6.05, 13.75 Hz,
4-CH2), 2.394 (2H, dd, J=6.05, 13.75 Hz, 3 -CH2),
3.200 (2H, dd, J=7.15, 14.3 Hz, a-CH2), 3.763 (1
H, t, J=6.05 Hz, 2 -CH)。これらの分析結果から得ら
れた結晶がテアニンであることが示された。The analysis conditions and results of 1 H-NMR are as follows. Solvent (D 2 O), internal standard (Sodium 4,4-dimethyl-
4-silapentanesulfonate), 1 H-NMR (D 2 ): 1.109 (1H, t,
J = 7.15 Hz, b-CH 3 ), 2.129 (2H, dd, J = 6.05, 13.75 Hz,
4-CH 2 ), 2.394 (2H, dd, J = 6.05, 13.75 Hz, 3 -CH 2 ),
3.200 (2H, dd, J = 7.15, 14.3 Hz, a-CH 2 ), 3.763 (1
H, t, J = 6.05 Hz, 2-CH). The results of these analyzes indicated that the crystals obtained were theanine.
【００２８】[0028]
【発明の効果】本発明の製造方法により、テアニンが高
収率、高安定的かつ連続的に得られる。また、本発明の
固定化菌体を得る方法、ならびにテアニンの分離・精製
などの操作が極めて簡単であり、工業的規模での大量製
造に貢献すること大である。According to the production method of the present invention, theanine can be obtained with high yield, high stability and continuously. In addition, the method for obtaining the immobilized bacterial cells of the present invention, and the operations such as separation / purification of theanine are extremely simple, and greatly contribute to mass production on an industrial scale.
【図１】テアニンの生成量を示したものである。FIG. 1 shows the amount of theanine produced.
【図２】テアニンの結晶の1H-NMRスペクトルである。FIG. 2 is a 1 H-NMR spectrum of a crystal of theanine.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 ＦＩ (Ｃ１２Ｐ 13/04 (Ｃ１２Ｐ 13/04 Ｃ１２Ｒ 1:66） Ｃ１２Ｒ 1:66） (72)発明者 山本 武彦 三重県四日市市赤堀新町９番５号 太陽 化学株式会社内 (72)発明者 金 武祚 三重県四日市市赤堀新町９番５号 太陽 化学株式会社内 審査官 本間 夏子 (56)参考文献 特開 昭58−40094（ＪＰ，Ａ) 特開 昭57−39792（ＪＰ，Ａ) 特開 昭54−143593（ＪＰ，Ａ) (58)調査した分野(Int.Cl.7，ＤＢ名) C12P 13/04 - 13/24 ＢＩＯＳＩＳ（ＤＩＡＬＯＧ) ＷＰＩ（ＤＩＡＬＯＧ) ＣＡ（ＳＴＮ) ＲＥＧＩＳＴＲＹ（ＳＴＮ)──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. 7 Identification symbol FI (C12P 13/04 (C12P 13/04 C12R 1:66) C12R 1:66) (72) Inventor Takehiko Yamamoto Akahori, Yokkaichi-shi, Mie Prefecture 9-5 Shinmachi, Taiyo Chemical Co., Ltd. (72) Inventor Kin Takezo 9-5, Shinmachi, Akahori Shinmachi, Yokkaichi, Mie Prefecture Examiner, Taiyo Chemical Co., Ltd. Natsuko Homma (56) References JP-A-57-39792 (JP, A) JP-A-54-143593 (JP, A) (58) Fields investigated (Int. Cl. 7 , DB name) C12P 13/04-13/24 BIOSIS (DIALOG) WPI (DIALOG) CA (STN) REGISTRY (STN)
Claims (1)
ａｒｏｍｙｃｅｓ属菌及びＡｓｐｅｒｉｇｉｌｌｕｓ属
菌の群より選ばれる菌を固定化した固定化菌体を用いる
ことを特徴とするテアニンの製造方法。1. Pseudomonas sp., Sacch
A method for producing theanine, comprising using immobilized cells immobilized with bacteria selected from the group consisting of aromyces and Asperigillus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP18431892A JP3210080B2 (en) | 1992-05-30 | 1992-05-30 | Method for producing theanine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP18431892A JP3210080B2 (en) | 1992-05-30 | 1992-05-30 | Method for producing theanine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH05328986A JPH05328986A (en) | 1993-12-14 |
| JP3210080B2 true JP3210080B2 (en) | 2001-09-17 |
Family
ID=16151240
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP18431892A Expired - Fee Related JP3210080B2 (en) | 1992-05-30 | 1992-05-30 | Method for producing theanine |
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| Country | Link |
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|---|---|---|---|---|
| JP4210325B2 (en) | 2002-08-06 | 2009-01-14 | 太陽化学株式会社 | Theanine production method |
| KR100606553B1 (en) * | 2004-06-22 | 2006-08-01 | 씨제이 주식회사 | Functional Beverage Composition for Improving Ability of Learning, Concentration and Memory |
| CA2570828C (en) * | 2004-06-28 | 2014-11-04 | Taiyokagaku Co., Ltd. | Method of making theanine |
| AU2014209028C1 (en) | 2013-01-28 | 2019-08-29 | Hector L. Lopez | Methods of improving tolerability, pharmacodynamics, and efficacy of b-alanine and use therefor |
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1992
- 1992-05-30 JP JP18431892A patent/JP3210080B2/en not_active Expired - Fee Related
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|---|---|
| JPH05328986A (en) | 1993-12-14 |
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