JP3135367B2 - Method for producing L-imino acid - Google Patents
Method for producing L-imino acidInfo
- Publication number
- JP3135367B2 JP3135367B2 JP04183698A JP18369892A JP3135367B2 JP 3135367 B2 JP3135367 B2 JP 3135367B2 JP 04183698 A JP04183698 A JP 04183698A JP 18369892 A JP18369892 A JP 18369892A JP 3135367 B2 JP3135367 B2 JP 3135367B2
- Authority
- JP
- Japan
- Prior art keywords
- acid
- imino
- producing
- pipecolic
- reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
【０００１】[0001]
【産業上の利用分野】本発明は、Ｄ−またはＤＬ−イミ
ノ酸からＬ−イミノ酸を製造する方法に関する。Ｌ−プ
ロリン、Ｌ−ピペコリン酸などのＬ−イミノ酸は、医薬
品などの原料として工業的に有用な物質である。The present invention relates to a process for producing L-imino acid from D- or DL-imino acid. L-imino acids such as L-proline and L-pipecolic acid are industrially useful substances as raw materials for pharmaceuticals and the like.
【０００２】[0002]
【従来の技術】プロリン、ヒドロキシプロリン、ピペコ
リン酸、サルコシン等の第２級アミンを持つアミノ酸は
慣習的にイミノ酸と呼ばれる。そのうち、ラセミ体のイ
ミノ酸は化学合成法により大量・安価に生産可能され
る。Ｌ−イミノ酸はこのラセミ体のイミノ酸を原料と
し、各種の光学分割法により得ることが出来る。例え
ば、Ｌ−イミノ酸の一種であるＬ−ピペコリン酸を得る
方法としては、ＤＬ−ピペコリン酸にＤ−酒石酸を添加
し、Ｄ−ピペコリン酸ーＤ−酒石酸塩を生成させ、該塩
を沈澱として除去した上澄液からＬ−ピペコリン酸を回
収する方法がある（Methods in Enzymology, Vol.17B,
p.174-178 (1971)）。しかし、この方法では、残ったＤ
−ピペコリン酸を利用するためには一度Ｄ−ピペコリン
酸ーＤ−酒石酸塩からＤ−ピペコリン酸を単離し、これ
を再びラセミ化する必要があり、効率的な方法とは言い
難い。また、酵素を用いてＬ−ピペコリン酸を得る方法
も知られている（Methods in Enzymology, Vol.17B, p.
174-178 (1971)）。即ち、ＤＬ−ピペコリン酸にＤ−ア
ミノ酸オキシダーゼを作用させ、Ｄ−ピペコリン酸を分
解除去し、Ｌ−ピペコリン酸を得るというものである。
しかし、この方法ではＬ−ピペコリン酸の収率は５０％
を越えることはなく、生産性において甚だ不経済であ
り、加えて操作も非常に複雑である。2. Description of the Related Art Amino acids having secondary amines such as proline, hydroxyproline, pipecolic acid and sarcosine are conventionally called imino acids. Among them, racemic imino acids can be produced in large quantities and at low cost by chemical synthesis. L-imino acid can be obtained by various optical resolution methods using this racemic imino acid as a raw material. For example, as a method for obtaining L-pipecolic acid which is a kind of L-imino acid, D-tartaric acid is added to DL-pipecolic acid to generate D-pipecolic acid-D-tartrate, and the salt is precipitated. There is a method of recovering L-pipecolic acid from the removed supernatant (Methods in Enzymology, Vol. 17B,
174-178 (1971)). However, in this method, the remaining D
In order to utilize -pipecolic acid, it is necessary to isolate D-pipecolic acid from D-pipecolic acid-D-tartrate once and to re-racemize it, which is not an efficient method. A method for obtaining L-pipecolic acid using an enzyme is also known (Methods in Enzymology, Vol. 17B, p.
174-178 (1971)). That is, D-amino acid oxidase is allowed to act on DL-pipecolic acid to decompose and remove D-pipecolic acid to obtain L-pipecolic acid.
However, in this method, the yield of L-pipecolic acid is 50%.
, Which is extremely uneconomical in productivity and, in addition, the operation is very complicated.
【０００３】[0003]
【発明が解決しょうとする課題】本発明はＤ−またはＤ
Ｌ−イミノ酸からＬ−イミノ酸を効率よく製造すること
を目的とする。SUMMARY OF THE INVENTION The present invention relates to D- or D-
An object is to efficiently produce L-imino acid from L-imino acid.
【０００４】[0004]
【課題を解決するための手段】このような状況のもと
で、本発明者らは、Ｄ−またはＤＬ−イミノ酸からＬ−
イミノ酸を製造する新しい製造法について研究を重ねた
結果、以下の事実を見い出した。即ち、Ｄ−プロリン及
びＤ−ピペコリン酸等のＤ−イミノ酸は、Ｄ−アミノ酸
オキシダーゼを作用させると、Δ1−ピロリン−２−カ
ルボン酸及びΔ1−ピペリデイン−２−カルボン酸にそ
れぞれ酸化される。そして、該両カルボン酸は水素化ホ
ウ素等の還元剤の存在下で還元され、再びもとのイミノ
酸のラセミ体に転換される。これら２つの反応を組み合
わせると、系内のＤ−イミノ酸はＤ−アミノ酸オキシダ
ーゼにより選択的に酸化されて次第に減少し、代わって
Ｌ−イミノ酸が増加すようになる。このようにＤ−体の
みに対する選択的な酸化反応と、還元剤によるラセミ体
の生成反応の共役反応を用いることによりＤ−またはＤ
Ｌ−イミノ酸からＬ−イミノ酸が高収率で生成すること
を見出し、本発明を完成するに至った。Under such circumstances, the present inventors have proposed to convert L- or DL-imino acid from L- or L-imino acid.
As a result of repeated research on a new production method for producing imino acids, the following facts were found. That, D- imino acids such as D- proline and D- pipecolic acid, when allowed to act D- amino acid oxidase, delta 1 - pyrroline-2-carboxylic acid and delta 1 - is oxidized respectively Piperidein 2-carboxylic acid You. Then, both carboxylic acids are reduced in the presence of a reducing agent such as borohydride, and converted back to the original racemic imino acid. When these two reactions are combined, D-imino acid in the system is selectively oxidized by D-amino acid oxidase, and gradually decreases, and L-imino acid increases instead. As described above, by using a selective oxidation reaction for only the D-form and a conjugate reaction of a racemic formation reaction with a reducing agent, D- or D
The present inventors have found that L-imino acid is produced in a high yield from L-imino acid, and have completed the present invention.
【０００５】即ち、本発明はＤ−アミノ酸オキシダーゼ
および還元剤の存在下、Ｄ−またはＤＬ−イミノ酸から
Ｌ−イミノ酸を生成させることを特徴とするＬ−イミノ
酸の製造方法を提供するものである。That is, the present invention provides a method for producing L-imino acid, which comprises producing L-imino acid from D- or DL-imino acid in the presence of D-amino acid oxidase and a reducing agent. It is.
【０００６】本発明において使用するＤ−アミノ酸オキ
シダーゼは、ブタ腎などの動物起源のもの、ノイロスポ
ラ属、シュードモナス属、大腸菌及びメタノール資化性
菌などの微生物起源のものなどを用いることが出来る。
一方、還元剤としては水素化ホウ素ナトリウムが好適に
用いることが出来る。Ｄ−またはＤＬ−イミノ酸からＬ
−イミノ酸を生成する反応は、通常 10〜50℃、pH 7〜1
0の範囲で、ゆるやかな攪拌下にＤ−アミノ酸オキシダ
ーゼ、水素化ホウ素ナトリウムおよびＤ−またはＤＬ−
イミノ酸を緩衝液等の水性媒体中で接触させることによ
り行われる。基質であるＤ−またはＤＬ−イミノ酸の濃
度には特に制限はないが、通常は0.1 〜10重量％程度で
ある。Ｄ−アミノ酸オキシダーゼ、水素化ホウ素ナトリ
ウムおよびＤ−またはＤＬ−イミノ酸は反応液に連続ま
たは間欠的に添加しても良い。また、反応には、Ｄ−ア
ミノ酸オキシダーゼの補酵素であるフラビンアデニンジ
ヌクレオチド（以後、ＦＡＤと略記する）を添加するこ
とが望ましい。反応時間は、Ｄ−アミノ酸オキシダーゼ
の酵素力価や基質濃度等の反応条件により異なるが、通
常は10分〜30時間程度である。このようにして反応を行
うと反応液中にはＬ−イミノ酸が生成する。反応液から
Ｌ−イミノ酸を採取するには、イオン交換樹脂を用いる
方法や濃縮晶析法等公知の方法を用いることができる。As the D-amino acid oxidase used in the present invention, those derived from animals such as pig kidney and those derived from microorganisms such as Neurospora, Pseudomonas, Escherichia coli and methanol assimilating bacteria can be used.
On the other hand, sodium borohydride can be suitably used as the reducing agent. D- or DL-imino acid to L
-The reaction for producing imino acid is usually performed at 10 to 50 ° C. and pH 7-1.
In the range of 0, D-amino acid oxidase, sodium borohydride and D- or DL-
This is performed by bringing the imino acid into contact with an aqueous medium such as a buffer solution. The concentration of D- or DL-imino acid as a substrate is not particularly limited, but is usually about 0.1 to 10% by weight. D-amino acid oxidase, sodium borohydride and D- or DL-imino acid may be added to the reaction solution continuously or intermittently. In addition, it is desirable to add flavin adenine dinucleotide (hereinafter abbreviated as FAD), which is a coenzyme of D-amino acid oxidase, to the reaction. The reaction time varies depending on the reaction conditions such as the enzyme titer of D-amino acid oxidase and the substrate concentration, but is usually about 10 minutes to 30 hours. When the reaction is performed in this manner, L-imino acid is generated in the reaction solution. In order to collect L-imino acid from the reaction solution, a known method such as a method using an ion exchange resin or a concentration crystallization method can be used.
【０００７】[0007]
【実施例】以下、実施例により本発明を具体的に説明す
るがＤ−およびＬ−イミノ酸の定量は、キラルパックＷ
Ｈカラム（ダイセル化学製）を用いた液体クロマトグラ
フィーにより行った。EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the determination of D- and L-imino acids was determined by using Chiralpak W
This was performed by liquid chromatography using an H column (manufactured by Daicel Chemical Industries, Ltd.).
【０００８】実施例１ Ｄ−アミノ酸オキシダーゼ（ブタ腎起源、0.09単位／mg
蛋白、シグマ社製）0.3単位／ml、ＤＬ−ピペコリン酸
2.5mM、ＦＡＤ 0.03mM および牛血清アルブミン 1mg／m
lを含む 500mM 燐酸緩衝液（pH 8.0） 20mlに 10分毎に
Ｄ−アミノ酸オキシダーゼ 2単位および水素化ホウ素ナ
トリウム 8mg を添加しながら 37℃で 80分間振盪しな
がら反応した。反応終了後、液中のＤ−およびＬ−ピペ
コリン酸を分析したところ、Ｄ−ピペコリン酸は完全に
消失し、Ｌ−ピペコリン酸は反応前の２倍に増加してい
た。そこで、反応の終わった液に塩酸を 0.7M になるよ
うに添加した。蛋白を遠心分離により除去した上澄液の
pHを 3.4 に調整し、イオン交換樹脂カラム（Dowex 50W
X-8 (H+) 2.5 X 33cm）に通液しピペコリン酸を吸着さ
せた。このカラムを蒸留水で洗浄した後、1M のアンモ
ニア水で溶離し 6.4mg のＬ−ピペコリン酸を得た。Ｄ
Ｌ−ピペコリン酸に対するＬ−ピペコリン酸の収率は 9
9% であった。Example 1 D-amino acid oxidase (porcine kidney origin, 0.09 units / mg)
Protein, Sigma) 0.3 unit / ml, DL-pipecolic acid
2.5 mM, FAD 0.03 mM and bovine serum albumin 1 mg / m
The reaction was carried out with shaking at 37 ° C. for 80 minutes while adding 2 units of D-amino acid oxidase and 8 mg of sodium borohydride to 20 ml of 500 mM phosphate buffer (pH 8.0) containing l every 10 minutes. After the reaction was completed, D- and L-pipecolic acid in the solution was analyzed. As a result, D-pipecolic acid completely disappeared, and L-pipecolic acid increased twice as much as before the reaction. Therefore, hydrochloric acid was added to the solution after the reaction so as to have a concentration of 0.7M. The supernatant liquid from which proteins were removed by centrifugation
Adjust the pH to 3.4 and use an ion exchange resin column (Dowex 50W).
X-8 (H + ) 2.5 X 33 cm) to adsorb pipecolic acid. The column was washed with distilled water and eluted with 1M aqueous ammonia to obtain 6.4 mg of L-pipecolic acid. D
The yield of L-pipecolic acid relative to L-pipecolic acid is 9
9%.
【０００９】実施例２ Ｄ−アミノ酸オキシダーゼ（ブタ腎起源、0.09単位／mg
蛋白、シグマ社製）0.3単位／ml、ＤＬ−プロリン 2.5m
M、ＦＡＤ 0.03mM および牛血清アルブミン1mg／mlを含
む 500mM 燐酸緩衝液（pH 8.0） 20mlに 10分毎にＤ−
アミノ酸オキシダーゼ 2単位および水素化ホウ素ナトリ
ウム 8mg を添加しながら 37℃で 60分間振盪しながら
反応した。反応終了後、液中のＤ−およびＬ−プロリン
を分析したところ、Ｄ−プロリンは完全に消失し、Ｌ−
プロリンは反応前の２倍に増加していた。そこで、反応
の終わった液に塩酸を 0.7M になるように添加した。蛋
白を遠心分離により除去した上澄液のpHを 3.4 に調整
し、イオン交換樹脂カラム（Dowex 50W X-8 (H+) 2.5 X
33cm）に通液しプロリンを吸着させた。このカラムを
蒸留水で洗浄した後、1M のアンモニア水で溶離し 5.74
mg のＬ−プロリンを得た。ＤＬ−プロリンに対するＬ
−プロリンの収率は 97% であった。Example 2 D-amino acid oxidase (porcine kidney origin, 0.09 units / mg)
Protein, Sigma) 0.3 units / ml, DL-proline 2.5m
M, FAD 0.03 mM and bovine serum albumin 1 mg / ml 500 mM phosphate buffer (pH 8.0)
The reaction was carried out with shaking at 37 ° C. for 60 minutes while adding 2 units of amino acid oxidase and 8 mg of sodium borohydride. After the reaction was completed, D- and L-proline in the solution were analyzed.
Proline was increased twice as much as before the reaction. Therefore, hydrochloric acid was added to the solution after the reaction so as to have a concentration of 0.7M. The pH of the supernatant, from which the protein was removed by centrifugation, was adjusted to 3.4, and the ion exchange resin column (Dowex 50W X-8 (H + ) 2.5 X
33 cm) to adsorb proline. The column was washed with distilled water and eluted with 1M aqueous ammonia.
mg of L-proline were obtained. L for DL-proline
-Proline yield was 97%.
【００１０】[0010]
【発明の効果】本発明によれば、Ｄ−アミノ酸オキシダ
ーゼおよび還元剤の存在下、Ｄ−またはＤＬ−イミノ酸
からＬ−イミノ酸が高収率で生産することが出来る。According to the present invention, L-imino acid can be produced from D- or DL-imino acid in high yield in the presence of D-amino acid oxidase and a reducing agent.
Claims (4)
の存在下、Ｄ−またはＤＬ−イミノ酸からＬ−イミノ酸
を生成させることを特徴とするＬ−イミノ酸の製造方
法。1. A method for producing L-imino acid, comprising producing L-imino acid from D- or DL-imino acid in the presence of D-amino acid oxidase and a reducing agent.
の存在下、Ｄ−またはＤＬ−プロリンからＬ−プロリン
を生成させることを特徴とするＬ−プロリンの製造方
法。2. A method for producing L-proline, comprising producing L-proline from D- or DL-proline in the presence of D-amino acid oxidase and a reducing agent.
の存在下、Ｄ−またはＤＬ−ピペコリン酸からＬ−ピペ
コリン酸を生成させることを特徴とするＬ−ピペコリン
酸の製造方法。3. A method for producing L-pipecolic acid, comprising producing L-pipecolic acid from D- or DL-pipecolic acid in the presence of D-amino acid oxidase and a reducing agent.
ことを特徴とする請求項１〜３の何れか一項に記載の製
造方法。4. The method according to claim 1, wherein the reducing agent is sodium borohydride.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP04183698A JP3135367B2 (en) | 1992-07-10 | 1992-07-10 | Method for producing L-imino acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP04183698A JP3135367B2 (en) | 1992-07-10 | 1992-07-10 | Method for producing L-imino acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0630789A JPH0630789A (en) | 1994-02-08 |
| JP3135367B2 true JP3135367B2 (en) | 2001-02-13 |
Family
ID=16140383
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP04183698A Expired - Fee Related JP3135367B2 (en) | 1992-07-10 | 1992-07-10 | Method for producing L-imino acid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3135367B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101946051B1 (en) * | 2017-04-24 | 2019-02-08 | 주식회사 에프에스티 | Cutting apparatus |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7683175B2 (en) | 2005-06-06 | 2010-03-23 | Navinta, Llc | Process of making optically pure L-pipecolic acid and process of making anesthetics and intermediates therefrom |
-
1992
- 1992-07-10 JP JP04183698A patent/JP3135367B2/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101946051B1 (en) * | 2017-04-24 | 2019-02-08 | 주식회사 에프에스티 | Cutting apparatus |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0630789A (en) | 1994-02-08 |
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|---|---|---|---|
| LAPS | Cancellation because of no payment of annual fees |