JPH06217785A - Production of dipeptide - Google Patents

Production of dipeptide

Info

Publication number
JPH06217785A
JPH06217785A JP4358471A JP35847192A JPH06217785A JP H06217785 A JPH06217785 A JP H06217785A JP 4358471 A JP4358471 A JP 4358471A JP 35847192 A JP35847192 A JP 35847192A JP H06217785 A JPH06217785 A JP H06217785A
Authority
JP
Japan
Prior art keywords
substrate
phase
group
organic phase
dipeptide
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.)
Granted
Application number
JP4358471A
Other languages
Japanese (ja)
Other versions
JP2972903B2 (en
Inventor
Mitsutoshi Nakajima
光敏 中嶋
Hiroshi Nabeya
浩志 鍋谷
Nobuo Hoshino
信夫 星野
Akira Hoshino
明 星野
Yasuyuki Isono
康幸 礒野
Genichirou Araya
弦一郎 荒谷
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dainichiseika Color and Chemicals Mfg Co Ltd
National Food Research Institute
Original Assignee
Dainichiseika Color and Chemicals Mfg Co Ltd
National Food Research Institute
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Dainichiseika Color and Chemicals Mfg Co Ltd, National Food Research Institute filed Critical Dainichiseika Color and Chemicals Mfg Co Ltd
Priority to JP4358471A priority Critical patent/JP2972903B2/en
Publication of JPH06217785A publication Critical patent/JPH06217785A/en
Application granted granted Critical
Publication of JP2972903B2 publication Critical patent/JP2972903B2/en
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

PURPOSE:To produce a peptide capable of preventing an outflow of a substrate and providing a high-purity product. CONSTITUTION:A membrane type device is installed as a reaction tank 1 and aspartic acid whose amino group is protected with benzyloxycarbonyl group, phenylalanine methyl ester and thermolysin are added to a buffer solution for a water phase 2 to prepare an aqueous solution for reaction. Ethyl acetate is used as an organic solvent for forming an organic phase and fed to an organic phase 3 of the reaction tank 1. The aqueous solution for reaction is supplied and circulated at 1L/minute to give the water phase 2 and ethyl acetate of the organic phase 3 is circulated at 2L/minute rate. Further, an extracting tank 5 is laid separately from the reaction tank 1 and an extracted solution is sent to an extraction phase 6. The organic solvent flowed out from the organic phase 3 of the reaction tank 1 is introduced into an organic phase 7 of the extracting tank 5 and the outflow solution is returned from the extraction phase to the organic phase 3.

Description

【発明の詳細な説明】Detailed Description of the Invention

【0001】[0001]

【産業上の利用分野】本発明は、タンパク質分解酵素の
逆反応を利用してアミノ酸からジペプチドを製造する方
法に関し、特に基質の流失を防いで効率的な反応を行う
ことの可能なジペプチドの製造法に関するものである。
BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a dipeptide from an amino acid by utilizing the reverse reaction of a proteolytic enzyme, and particularly to the production of a dipeptide capable of preventing the substrate from being washed away and carrying out an efficient reaction. It is about law.

【0002】[0002]

【従来の技術】タンパク質分解酵素の逆反応を利用した
ペプチド合成において、酵素による合成反応平衡は低い
ため、生成物を効率よく反応系外に取り出すことで見掛
上の平衡を合成側にシフトさせる方法がとられている。
2. Description of the Related Art In peptide synthesis utilizing the reverse reaction of a proteolytic enzyme, the enzymatic reaction equilibrium is low, so the apparent equilibrium is shifted to the synthesis side by efficiently taking out the product from the reaction system. The method is taken.

【0003】上述の酵素によるペプチド合成法としては
沈殿法、微水系反応法、二相系反応法等が知られてい
る。このうち沈殿法は、水溶性基質から水不溶性生成物
を合成する場合に用いられる。このような沈殿法の例と
しては、平田らが二相系で行った沈殿ろ過による連続合
成に関する、化学工学会第20回秋期大会研究発表講演
要旨集(1987年、343頁)の報告がある。
As the peptide synthesizing method using the above-mentioned enzymes, a precipitation method, a fine water reaction method, a two-phase reaction method and the like are known. Of these, the precipitation method is used when a water-insoluble product is synthesized from a water-soluble substrate. As an example of such a precipitation method, there is a report by the Hirata et al., Proceedings of the 20th Autumn Meeting of the Chemical Engineering Society of Japan, on the continuous synthesis by precipitation filtration in a two-phase system (1987, p.343). .

【0004】また微水系反応法は、系内の水量を制限す
ることにより、逆反応である加水分解反応を抑制しよう
とするものである。特開平2−39895号公報には、
この微水系反応法を用いたジペプチドの連続製造法が記
載されている。また小山らはメソッズ・イン・エンザイ
モロジー(Methods in Enzymology;1987 Vol.136 p503-
516)に酵素固定化カラムを用いたジペプチドの製造法
を報告している。
Further, the slightly water-based reaction method is intended to suppress the hydrolysis reaction, which is a reverse reaction, by limiting the amount of water in the system. Japanese Patent Laid-Open No. 2-39895 discloses that
A continuous method for producing a dipeptide using this slightly water-based reaction method is described. Also, Koyama et al. (Methods in Enzymology; 1987 Vol.136 p503-
516) reported a method for producing a dipeptide using an enzyme-immobilized column.

【0005】二相系反応法は水相で酵素反応を行い、有
機相に生成物を抽出する方法であり、生成物側へ平衡を
移動させて純度を向上させることが可能である。例えば
平田らは化学工学論文集17巻3号(1991年、58
6−588頁)においてソルベントリフト型リアクター
を用いた半連続の製造法を報告している。
The two-phase reaction method is a method in which an enzyme reaction is carried out in an aqueous phase to extract a product in an organic phase, and it is possible to shift the equilibrium to the product side to improve the purity. For example, Hirata et al. Chemical Engineering Papers Vol. 17 No. 3 (1991, 58
6-588), a semi-continuous production method using a solvent lift type reactor is reported.

【0006】[0006]

【発明が解決しようとする課題】上述の沈殿法は、生成
物が沈殿するため高純度の生成物を得ることができるが
連続化は困難である。また、沈殿物と酵素とが複合体を
形成することにより、酵素が系外へ漏出するため酵素の
再利用も困難である。
The precipitation method described above makes it possible to obtain a high-purity product because the product precipitates, but it is difficult to make it continuous. In addition, since the precipitate and the enzyme form a complex, the enzyme leaks out of the system, so that it is difficult to reuse the enzyme.

【0007】また微水系反応法については、一般に生成
物の反応平衡度が低いため純度を高めることが困難であ
り、更に有機溶媒中に存在する酵素の安定性にも問題が
ある。上記特開平2−39895号公報においては、カ
ルボキシル基保護の基質Bの濃度をアミノ基保護の基質
Aの濃度よりも高くすることによって基質Aに関しては
高い転換率を得ているが、反応液中の過剰の基質Bにつ
いては再利用ができていない。また前記小山の方法にお
いては、有機溶媒が酵素を失活させる問題が未解決であ
る。
[0007] In addition, in the fine water reaction method, it is generally difficult to increase the purity because the reaction equilibrium of the product is low, and there is a problem in the stability of the enzyme existing in the organic solvent. In the above-mentioned JP-A-2-39895, a high conversion rate is obtained for the substrate A by increasing the concentration of the substrate B for protecting the carboxyl group higher than the concentration of the substrate A for protecting the amino group. Excess Substrate B was not reused. Further, in the method of Koyama, the problem that the organic solvent deactivates the enzyme remains unsolved.

【0008】更に二相系反応法は、前記のように酵素を
触媒とした基質Aと基質Bとの反応を水相で行い、生成
したジペプチドを有機相へ抽出させる方法であるため、
反応生成物が除去される水相においては、平衡を保つた
めに更に反応が進行する理想的なものである。しかしこ
の方法には、ジペプチドと共に基質Bの一部も有機相へ
抽出されるという問題があり、このため有機相から高純
度のジペプチドを得ることは困難である。前記平田らの
報告においてもこの問題は解決されていない。また基質
Bは流失による現象を考慮して、基質Aに比較して高濃
度で反応系に供給されるため、前記有機相への流出によ
る損失量は膨大となる。更に、浮遊する酵素についても
エマルションの形で有機相に巻き込まれるために、この
有機相の取り出しに伴って酵素の一部が漏出してしまう
という難点もあった。
Further, the two-phase reaction method is a method in which the reaction between the substrate A and the substrate B using an enzyme as a catalyst is carried out in the aqueous phase as described above, and the produced dipeptide is extracted into the organic phase.
In the aqueous phase where the reaction products are removed, it is ideal that the reaction proceeds further to maintain equilibrium. However, this method has a problem that a part of the substrate B is extracted into the organic phase together with the dipeptide, which makes it difficult to obtain a high-purity dipeptide from the organic phase. This problem has not been solved even in the report by Hirata et al. Further, since the substrate B is supplied to the reaction system at a higher concentration than that of the substrate A in consideration of the phenomenon due to the flow-out, the amount of loss due to the outflow into the organic phase becomes enormous. Furthermore, since the floating enzyme is also caught in the organic phase in the form of an emulsion, there is a problem that a part of the enzyme leaks when the organic phase is taken out.

【0009】本発明は、従来の技術が有するこのような
問題点に鑑みてなされたものであり、その目的とすると
ころは、有機相に抽出される前記基質Bを回収して利用
効率を高めることにより、生成物への転換率を高め、ま
た高純度の生成物を得、更に酵素の漏出も防止すること
の可能なジペプチドの製造法を提供するものである。
The present invention has been made in view of the above problems of the prior art, and its object is to enhance the utilization efficiency by recovering the substrate B extracted in the organic phase. This provides a method for producing a dipeptide capable of increasing the conversion rate to a product, obtaining a highly pure product, and preventing the leakage of an enzyme.

【0010】[0010]

【課題を解決するための手段】上記の課題を解決するた
めに本発明は、リジン(Lys)、ヒスチジン(Hi
s)、アルギニン(Arg)、オルニチン(Orn)、
アスパラギン酸(Asp)及びグルタミン酸(Glu)
の群(I群)から選ばれるアミノ酸であって、そのアミ
ノ基が保護基によって保護されたアミノ酸(基質A)
と、アラニン(Ala)、バリン(Val)、ロイシン
(Leu)、イソロイシン(Ile)、メチオニン(M
et)、トリプトファン(Trp)、フェニルアラニン
(Phe)、プロリン(Pro)、グリシン(Gl
y)、セリン(Ser)、トレオニン(Thr)、シス
テイン(Cys)、チロシン(Tyr)、アスパラギン
(Asn)、グルタミン(Gln)、Lys、His、
Arg、Orn、Asp及びGluの群(II群)から
選ばれるアミノ酸であって、そのカルボキシル基がアル
キルエステル化(但し、このアルキルは炭素数1乃至4
である。)されたアミノ酸(基質B)との組合わせ、又
は前記II群から選ばれるアミノ酸であって、そのアミ
ノ基が保護基によって保護されたアミノ酸(基質A)
と、前記I群から選ばれるアミノ酸であって、そのカル
ボキシル基が前記条件のアルキルエステル化されたアミ
ノ酸(基質B)との組合わせを、二相系の反応槽中で酵
素触媒により反応させるジペプチドの製造法において、
前記反応槽の反応液を水相、及び水と混和しない有機溶
媒からなる有機相の二相から構成し、これら水相又は有
機相のいずれかに前記基質A、基質B及び酵素を供給し
てジペプチドを合成し、この合成されたジペプチドが主
として含有される前記有機相を構成する溶液を、水性の
抽出相を有する抽出槽へ導入し、この導入した溶液から
抽出相へジペプチドを抽出し、抽出後の溶液は前記反応
槽へ戻すようにする。
To solve the above problems, the present invention provides lysine (Lys), histidine (Hi).
s), arginine (Arg), ornithine (Orn),
Aspartic acid (Asp) and glutamic acid (Glu)
Amino acid selected from the group (group I), the amino group of which is protected by a protecting group (substrate A)
And alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (M
et), tryptophan (Trp), phenylalanine (Phe), proline (Pro), glycine (Gl)
y), serine (Ser), threonine (Thr), cysteine (Cys), tyrosine (Tyr), asparagine (Asn), glutamine (Gln), Lys, His,
An amino acid selected from the group consisting of Arg, Orn, Asp and Glu (group II), the carboxyl group of which is alkyl esterified (provided that the alkyl has 1 to 4 carbon atoms).
Is. A combined amino acid (substrate B), or an amino acid selected from the above group II, the amino group of which is protected by a protecting group (substrate A)
And an amino acid selected from Group I, the carboxyl group of which is an alkylesterified amino acid under the above conditions (substrate B), which is reacted with an enzyme catalyst in a two-phase reaction tank. In the manufacturing method of
The reaction solution in the reaction tank is composed of two phases, an aqueous phase and an organic phase composed of an organic solvent immiscible with water, and the substrate A, the substrate B and the enzyme are supplied to either of the aqueous phase and the organic phase. The dipeptide is synthesized, and the solution constituting the organic phase mainly containing the synthesized dipeptide is introduced into an extraction tank having an aqueous extraction phase, and the dipeptide is extracted from the introduced solution into the extraction phase and extracted. The latter solution is returned to the reaction tank.

【0011】本発明に係る前記基質Aは例えばアミノ基
を保護されたアスパラギン酸であり、また前記基質Bは
例えばフェニルアラニンのアルキルエステル(但し、こ
のアルキルは炭素数1乃至4である。)である。
The substrate A according to the present invention is, for example, an amino group-protected aspartic acid, and the substrate B is, for example, an alkyl ester of phenylalanine (provided that the alkyl has 1 to 4 carbon atoms). .

【0012】本発明の製造法においては、前記反応槽及
び/又は抽出槽中の水相及び有機相が限外ろ過膜又は低
阻止率逆浸透膜を介して接触していることが好ましい。
In the production method of the present invention, it is preferable that the aqueous phase and the organic phase in the reaction tank and / or the extraction tank are in contact with each other through an ultrafiltration membrane or a low rejection reverse osmosis membrane.

【0013】本発明は上述の二相系反応法に属する連続
式、回分式のどちらにも適用可能な製造法であり、生成
物の純度を上げ、且つ前記基質Bの有機相への流失を防
ぐために反応槽の他に抽出槽を設けたことを特徴として
いる。即ち、反応槽の有機相から直接ジペプチドを取り
出さず、抽出槽において再度水性の抽出相へ移動させ、
有機相中に溶解している基質Bと分離した後に取り出
す。従って、この抽出相から得られるジペプチドは高純
度である。またジペプチドを除去した基質Bを含有する
有機相の溶液は前記反応槽に戻すため、基質Bを無駄に
することもなくその使用量を減らすことができる。な
お、本発明に係る上記反応層及び抽出槽中の水系の相の
pHは、生成するジペプチドの有する側鎖解離基の種類
によって適宜調整する。
The present invention is a production method applicable to both the continuous system and the batch system belonging to the above-mentioned two-phase system reaction method, which raises the purity of the product and prevents the substrate B from flowing out to the organic phase. In order to prevent this, an extraction tank is provided in addition to the reaction tank. That is, the dipeptide was not directly taken out from the organic phase of the reaction tank, but moved to the aqueous extraction phase again in the extraction tank,
It is taken out after separating from the substrate B dissolved in the organic phase. Therefore, the dipeptide obtained from this extraction phase is highly pure. In addition, since the solution of the organic phase containing the substrate B from which the dipeptide has been removed is returned to the reaction tank, the amount of the substrate B used can be reduced without wasting it. The pH of the aqueous phase in the reaction layer and the extraction tank according to the present invention is appropriately adjusted depending on the kind of side chain dissociating group contained in the dipeptide to be produced.

【0014】また、反応槽及び/又は抽出槽中の水相及
び有機相を限外ろ過膜又は低阻止率逆浸透膜を介して接
触させることによって、浮遊酵素がエマルションの形で
有機相に巻き込まれることを防止することができる。
Further, by bringing the aqueous phase and the organic phase in the reaction tank and / or the extraction tank into contact with each other through an ultrafiltration membrane or a low rejection reverse osmosis membrane, the floating enzyme is caught in the organic phase in the form of an emulsion. Can be prevented.

【0015】図1に本発明の製造法の一例を示す。同図
において、反応槽1は水相2及び水と混和しない有機溶
媒からなる有機相3を擁している。そしてこの水相2は
反応性を良好にするために循環されており、この循環系
4には基質A及び基質Bを供給する手段が設けてある。
また触媒となる酵素は水相2中に予め分散させるか又は
基質A、Bと共に供給される。一方、抽出槽5は、水性
の抽出相6及び前記有機相3から流入する有機溶媒から
なる有機相7を擁している。そして有機相7の溶液流出
口8は有機相3の流入口9へ連結されている。
FIG. 1 shows an example of the manufacturing method of the present invention. In the figure, a reaction tank 1 has an aqueous phase 2 and an organic phase 3 made of an organic solvent immiscible with water. The aqueous phase 2 is circulated to improve the reactivity, and the circulation system 4 is provided with a means for supplying the substrate A and the substrate B.
Further, the enzyme serving as a catalyst is previously dispersed in the aqueous phase 2 or supplied together with the substrates A and B. On the other hand, the extraction tank 5 has an aqueous extraction phase 6 and an organic phase 7 consisting of an organic solvent flowing from the organic phase 3. The solution outlet 8 of the organic phase 7 is connected to the inlet 9 of the organic phase 3.

【0016】図1において、循環系4を通じて反応槽1
の水相2へ供給された基質A及び基質Bは酵素触媒によ
り水相2内で反応して下記の平衡状態となる。 基質A+基質B←→ジペプチド そしてここで形成されたジペプチドは有機相3へ移行す
るが、同時に基質Bの一部も移行する。なお、触媒も一
部有機相3に巻き込まれるが、水相2と有機相3との間
に前記限外ろ過膜又は低阻止率逆浸透膜を設けることに
よってこれを阻止することが可能である。
In FIG. 1, a reaction tank 1 is provided through a circulation system 4.
Substrate A and substrate B supplied to the aqueous phase 2 of the above react with each other in the aqueous phase 2 by the enzyme catalyst to be in the following equilibrium state. Substrate A + Substrate B ← → dipeptide Then, the dipeptide formed here moves to the organic phase 3, but at the same time, part of the substrate B also moves. Although the catalyst is also partly caught in the organic phase 3, it is possible to prevent this by providing the ultrafiltration membrane or the low rejection reverse osmosis membrane between the aqueous phase 2 and the organic phase 3. .

【0017】上記のジペプチド、基質B(及び上記膜を
設けない場合は酵素)の一部を含有する有機相3の溶液
は抽出槽5の有機相7へ送られる。この抽出槽5におい
ては、抽出相6のpHを前記水相2のpHと異なる値に
設定してあるため、有機相7に含まれるジペプチドのみ
が抽出相6へ移行し、基質B(及び酵素)は残留する。
そしてこの基質B(及び酵素)を含有する有機相7の溶
液は有機相3へ戻されて再利用される。なお、前記膜に
ついては、反応槽1に設けなかった場合にはこの抽出槽
5に設けてもよく、反応槽1及び抽出槽5の両方に設け
てもよい。また、反応槽1及び抽出槽5における反応或
いは抽出の温度は、20〜80℃とすることが好まし
い。
The solution of the organic phase 3 containing a part of the above-mentioned dipeptide and substrate B (and the enzyme when the above-mentioned membrane is not provided) is sent to the organic phase 7 of the extraction tank 5. In this extraction tank 5, since the pH of the extraction phase 6 is set to a value different from the pH of the aqueous phase 2, only the dipeptide contained in the organic phase 7 moves to the extraction phase 6 and the substrate B (and enzyme ) Remains.
Then, the solution of the organic phase 7 containing the substrate B (and the enzyme) is returned to the organic phase 3 for reuse. The membrane may be provided in the extraction tank 5 when it is not provided in the reaction tank 1, or may be provided in both the reaction tank 1 and the extraction tank 5. The reaction or extraction temperature in the reaction tank 1 and the extraction tank 5 is preferably 20 to 80 ° C.

【0018】生成物であるジペプチドは、抽出相6のジ
ペプチドを含有する水性液を取り出し、必要に応じて酸
分解等によって保護基をはずし、再結晶等で精製して高
純度の結晶として析出させる。
For the dipeptide product, an aqueous liquid containing the dipeptide of the extraction phase 6 is taken out, if necessary, the protecting group is removed by acid decomposition or the like, and purified by recrystallization or the like to precipitate as highly pure crystals. .

【0019】本発明の製造法に使用できる基質Aは、前
記のアミノ基を保護されたアミノ酸であり、この保護基
としては、ペプチド合成反応に慣用される例えばジフェ
ニルメチル基、トリフェニルメチル基、芳香族基及び脂
肪族オキシカルボニル基が挙げられ、これらはハロゲ
ン、ニトロ基、低級アルキル基、低級アルコキシ基等に
よって置換されていてもよい。その他、ファルマシアレ
ビューNo,3(日本薬学会、1980年)の第31
頁、表1に記載される保護基も好適に使用できるが、最
も好ましい保護基はベンジルオキシカルボニル基であ
る。
Substrate A that can be used in the production method of the present invention is the amino acid whose amino group is protected. Examples of the protecting group include diphenylmethyl group and triphenylmethyl group, which are commonly used in peptide synthesis reactions. Examples thereof include aromatic groups and aliphatic oxycarbonyl groups, which may be substituted with halogen, nitro group, lower alkyl group, lower alkoxy group and the like. Others, 31st of Pharmacia Review No, 3 (Japan Pharmaceutical Association, 1980)
The protecting groups described in page 1 and Table 1 can be preferably used, but the most preferred protecting group is a benzyloxycarbonyl group.

【0020】また基質Bとしては、前記のカルボキシル
基がアルキルエステル化(但し、このアルキルは炭素数
1〜4である。)されたアミノ酸である。1モルの基質
Aに対する基質Bの添加モル数は1〜5、好ましくは1
〜2である。このモル数が1未満では基質Bが蓄積し、
また5を超えると基質Bの系外流出が著しくなるため本
発明の目的を達成できないことがある。
The substrate B is an amino acid in which the above-mentioned carboxyl group is alkyl esterified (wherein the alkyl has 1 to 4 carbon atoms). The number of moles of substrate B added to 1 mole of substrate A is 1 to 5, preferably 1.
~ 2. If this number of moles is less than 1, substrate B will accumulate,
On the other hand, when it exceeds 5, the outflow of the substrate B from the system becomes remarkable and the object of the present invention may not be achieved.

【0021】触媒として使用する酵素としては、メタロ
プロティナーゼの一種であるサーモライシンが好適に使
用できるが、その他の酵素の例を挙げれば、Staphyloco
ccalプロティナーゼ(EC 3.4.21)、パパイン(EC 3.4.
22.2)がある。
As an enzyme used as a catalyst, thermolysin, which is a kind of metalloproteinase, can be preferably used, but other examples of the enzyme include Staphyloco
ccal proteinase (EC 3.4.21), papain (EC 3.4.21)
22.2).

【0022】前記水相2及び抽出相6には、例えば0.
05モル酢酸緩衝液等、公知の緩衝液を用いて各相のp
Hを調整することができる。また有機相3に使用する有
機溶媒としては、水に殆ど溶解しないものであればどの
ようなものでもよいが、例えばアスパルテーム等の食用
ジペプチドを製造する場合には、食品添加物として認可
されている酢酸エチルを使用することが好ましい。
The water phase 2 and the extraction phase 6 contain, for example, 0.
A well-known buffer such as a 05 mol acetate buffer was used to adjust the p
H can be adjusted. The organic solvent used in the organic phase 3 may be any one as long as it is almost insoluble in water. For example, when producing edible dipeptides such as aspartame, it is approved as a food additive. Preference is given to using ethyl acetate.

【0023】本発明に係る反応槽1及び抽出槽5として
は、共に図2に示すような形式の装置を使用することが
できる。同図(a)は攪拌槽型装置の模式図を示したも
のであり、水相及び有機相は攪拌槽10内では攪拌羽根
11によってミキシングされる。そしてこの混合液が分
離槽12に移送されて分離され、水相13の水溶液は攪
拌槽10に戻され、有機相14の有機溶液は先の工程に
送られる。なお、分離槽12における水相13及び有機
相14は各相を構成する水(若しくは緩衝液)及び有機
溶媒の比重次第で上下が逆転する場合があるが、いずれ
にしても水相を攪拌槽10に戻す。
As the reaction tank 1 and the extraction tank 5 according to the present invention, an apparatus of the type as shown in FIG. 2 can be used. FIG. 1A shows a schematic diagram of a stirring tank type device, in which the water phase and the organic phase are mixed by stirring blades 11 in the stirring tank 10. Then, this mixed liquid is transferred to the separation tank 12 and separated, the aqueous solution of the aqueous phase 13 is returned to the stirring tank 10, and the organic solution of the organic phase 14 is sent to the previous step. The water phase 13 and the organic phase 14 in the separation tank 12 may be turned upside down depending on the specific gravity of the water (or buffer solution) and the organic solvent forming each phase. Return to 10.

【0024】また同図(b)はソルベントリフト型装置
の模式図示したものであり、例えば水より比重の小さい
有機溶媒を用いる時は、抽出塔15内に水(若しくは緩
衝液)を仕込み、塔底16の仕込口17から有機溶媒を
供給する。この際、邪魔板或いはパルス付与等によって
有機溶媒を小滴状としておく。この有機溶媒は比重差に
よって塔内の水相18中を上昇し塔頂19に達する。分
配はこの上昇中に両液の接触により生ずるため、分配後
の有機溶媒を塔頂19から取り出すことができる。
Further, FIG. 2B is a schematic diagram of a solvent lift type apparatus. For example, when an organic solvent having a specific gravity smaller than that of water is used, water (or a buffer solution) is charged in the extraction column 15 to The organic solvent is supplied from the charging port 17 of the bottom 16. At this time, the organic solvent is made into a droplet shape by baffle plate or pulse application. This organic solvent rises in the aqueous phase 18 in the tower due to the difference in specific gravity and reaches the top 19 of the tower. Since the distribution occurs due to the contact of both liquids during this rising, the organic solvent after the distribution can be taken out from the top 19.

【0025】更に同図(c)は膜型装置の模式図を示し
たものであり、接触槽20内は限外ろ過膜或いは低阻止
率逆浸透膜等の膜21を介して有機相22及び水相23
が接触している。この装置を反応槽1として用いる場合
には、水相23側に各原料基質及び酵素を導入すること
が好ましい。なお、膜型装置は前記攪拌槽型に比較して
二相を分離する時間が不要であるという特徴がある。
Further, FIG. 3C is a schematic diagram of a membrane type device, in which the organic phase 22 and the organic phase 22 are provided in the contact tank 20 through a membrane 21 such as an ultrafiltration membrane or a low rejection reverse osmosis membrane. Water phase 23
Are in contact. When this apparatus is used as the reaction tank 1, it is preferable to introduce each raw material substrate and enzyme on the side of the aqueous phase 23. The membrane type device is characterized in that it does not require time for separating two phases as compared with the stirring tank type.

【0026】上記膜型装置に使用する膜21は、疎水性
膜及び親水性膜のいずれも使用可能であるが、pH調整
の点で疎水性膜がより好ましい。即ち、疎水性の膜21
を用いると、膜の組織内には水性液が侵入し難いため、
例えば水相のpHを7に設定した場合に、膜組織内のp
Hが6.5となるようなpHの不一致あるいはpH値の
安定に時間が掛かる等の問題を軽減することができる。
The membrane 21 used in the membrane type device may be either a hydrophobic membrane or a hydrophilic membrane, but a hydrophobic membrane is more preferable from the viewpoint of pH adjustment. That is, the hydrophobic film 21
When using, because it is difficult for an aqueous liquid to enter the tissue of the membrane,
For example, when the pH of the aqueous phase is set to 7, p in the membrane tissue
It is possible to reduce problems such as a pH mismatch where H becomes 6.5 or a long time required for stabilizing the pH value.

【0027】上記3種の装置の特性を下記(表1)に示
す。反応槽1又は抽出槽5の一方或いは双方を膜型装置
とすると、浮遊酵素の漏出を抑えることができるため特
に好ましい。
The characteristics of the above three types of devices are shown in the following (Table 1). It is particularly preferable to use one or both of the reaction tank 1 and the extraction tank 5 as a membrane type device because leakage of floating enzyme can be suppressed.

【0028】[0028]

【表1】 [Table 1]

【0029】[0029]

【作用】本発明の製造法は、有機相から生成物であるジ
ペプチドを直接取り出さず、この有機相の溶液を抽出槽
へ移し、ジペプチドを再度水性の抽出相へ移動してから
取り出すため、有機相中に溶解している基質Bと製品で
あるジペプチドとを容易に分離することができる。また
ジペプチドを除去した基質Bを含有する溶液は前記反応
槽に戻すため、基質Bを無駄にすることもなくなる。
In the production method of the present invention, the product dipeptide is not directly taken out from the organic phase, but the solution of this organic phase is transferred to the extraction tank, and the dipeptide is again transferred to the aqueous extraction phase and then taken out. The substrate B dissolved in the phase and the product dipeptide can be easily separated. Further, since the solution containing the substrate B from which the dipeptide has been removed is returned to the reaction tank, the substrate B is not wasted.

【0030】[0030]

【実施例】以下に本発明に基づく実施例を説明する。実施例 本実施例は生成物としてアスパルテーム(L-アスパルチ
ル-L-フェニルアラニンメチルエステル)を合成した例
である。反応槽1として膜型の装置を設置し、水相2用
の緩衝液(0.05モル酢酸緩衝液、pH5)中に、基
質Aとしてベンジルオキシカルボニル基でアミノ基を保
護したアスパラギン酸(試薬特級;和光純薬(株)製)
を5ミリモル、また基質Bとしてフェニルアラニンのメ
チルエステル(試薬特級;アルドリッヒ(Aldrich Chemi
cal Company Inc.)製)を同じく5ミリモル添加し、更
に触媒用の酵素としてサーモライシン(「サモアー
ゼ」;大和化成(株)製)を8Kg/m3用いて反応用
水溶液を調製した。また有機相形成用の有機溶媒として
酢酸エチルを用い、反応槽1の有機相3へ仕込んだ。そ
して上記反応用水溶液を1L/分の速度で供給、循環し
て水相2とし、また上記有機相3の酢酸エチルも1L/
分の速度で循環させた。
EXAMPLES Examples according to the present invention will be described below. Example This example is an example in which aspartame (L-aspartyl-L-phenylalanine methyl ester) was synthesized as a product. A membrane type device was installed as the reaction tank 1, and aspartic acid (reagent for protecting the amino group with a benzyloxycarbonyl group as a substrate A was used as a substrate A in a buffer solution (0.05 mol acetate buffer solution, pH 5) for the aqueous phase 2. Special grade; manufactured by Wako Pure Chemical Industries, Ltd.)
And 5 ml of methyl ester of phenylalanine as a substrate B (reagent grade; Aldrich Chemi
Cal Company Inc.) was also added in an amount of 5 mmol and thermolysin (“Samoaase”; manufactured by Daiwa Kasei Co., Ltd.) was used as an enzyme for the catalyst at 8 kg / m 3 to prepare an aqueous solution for reaction. Further, ethyl acetate was used as the organic solvent for forming the organic phase, and the organic phase 3 in the reaction tank 1 was charged. Then, the aqueous solution for reaction is supplied at a rate of 1 L / min and circulated to make an aqueous phase 2, and the ethyl acetate in the organic phase 3 is also 1 L / min.
Circulated at the rate of minutes.

【0031】更に、上記反応槽1とは別に抽出槽5を設
置し、1規定の水酸化ナトリウムによってpH7に調製
した抽出液をこの抽出槽5の抽出相6に送液した。また
反応槽1の有機相3から流出した有機溶媒は抽出槽5の
有機相7に導入し、ここからの流出液は有機相3に戻す
ようにした。
Further, an extraction tank 5 was installed separately from the reaction tank 1, and an extract liquid adjusted to pH 7 with 1N sodium hydroxide was sent to the extraction phase 6 of the extraction tank 5. Further, the organic solvent flowing out from the organic phase 3 of the reaction tank 1 was introduced into the organic phase 7 of the extraction tank 5, and the effluent from this was returned to the organic phase 3.

【0032】上述の状態を24時間継続して平衡状態と
し、この時の各成分の濃度を下記(表2)に示した。本
実施例においては、基質Bの流失がないため、基質Aに
対して基質Bを過剰に添加する必要はなかった。また得
られたアスパルテームの純度は89.3%、基質を基準
としたアスパルテームへの転換率は94.4%、装置の
単位容積当たりの生産性は36モル/(m3・日)であっ
た。
The above state was maintained for 24 hours to reach an equilibrium state, and the concentration of each component at this time is shown in the following (Table 2). In this example, since the substrate B was not washed away, it was not necessary to add the substrate B in excess to the substrate A. The purity of the obtained aspartame was 89.3%, the conversion rate to substrate-based aspartame was 94.4%, and the productivity per unit volume of the apparatus was 36 mol / (m 3 · day). .

【0033】[0033]

【表2】 [Table 2]

【0034】比較例 ソルベントリフト型反応器を用いて、pHが約5.2の
水中に、基質Aを25ミリモル、基質Bを100ミリモ
ル、更にサーモライシンを20Kg/m3加えて調製し
た反応液を0.001L/分の速度で有機相に供給し
た。なお、上記基質A、基質B、サーモライシン及び有
機溶剤については実施例1と同様のものを使用した。
Comparative Example A reaction liquid prepared by adding 25 mmol of substrate A, 100 mmol of substrate B and 20 kg / m 3 of thermolysin in water having a pH of about 5.2 using a solvent lift type reactor. The organic phase was fed at a rate of 0.001 L / min. The same substances as in Example 1 were used for the above-mentioned substrate A, substrate B, thermolysin and organic solvent.

【0035】上述の状態を24時間継続して平衡状態と
なった時の各成分の濃度を下記(表3)に示した。得ら
れたアスパルテームの純度は17.6%、基質Aを基準
としたアスパルテームへの転換率は60.0%、装置の
単位容積当たりの生産性は80モル/(m3・日)であっ
た。
The concentrations of the respective components when the above state is maintained for 24 hours to reach an equilibrium state are shown in the following (Table 3). The purity of the obtained aspartame was 17.6%, the conversion rate to aspartame based on the substrate A was 60.0%, and the productivity per unit volume of the apparatus was 80 mol / (m 3 · day). .

【0036】[0036]

【表3】 [Table 3]

【0037】[0037]

【発明の効果】以上説明したように本発明の製造法によ
れば、有機相から生成物であるジペプチドを直接取り出
さず、この有機相の溶液を抽出槽へ移し、ジペプチドを
再度水性の抽出相へ移動してから取り出すため、有機相
中に溶解している基質Bとジペプチドとを容易に分離す
ることができる。また、上記のような手段をとるため抽
出相から得られるジペプチドの純度は高くなる。更に、
上記ジペプチドを除去した基質Bを含有する有機相は、
前記反応槽に戻すため基質Bの無駄を防げ、その使用量
を減らすことができる。
As described above, according to the production method of the present invention, the product dipeptide is not directly taken out from the organic phase, the solution of this organic phase is transferred to the extraction tank, and the dipeptide is again extracted with an aqueous extraction phase. The substrate B and the dipeptide which are dissolved in the organic phase can be easily separated since they are taken out after moving to. In addition, the purity of the dipeptide obtained from the extraction phase is increased due to the above measures. Furthermore,
The organic phase containing the substrate B from which the above dipeptide has been removed is
Since the substrate B is returned to the reaction tank, it is possible to prevent the substrate B from being wasted and reduce the amount used.

【0038】また、反応槽及び/又は抽出槽中の水相及
び有機相を限外ろ過膜又は低阻止率逆浸透膜を介して接
触させることによって、浮遊酵素がエマルションの形態
で有機相に巻き込まれることを防ぎ、これにより酵素再
利用効率の低下を防止することができる。
In addition, by bringing the aqueous phase and the organic phase in the reaction tank and / or the extraction tank into contact with each other through an ultrafiltration membrane or a low rejection reverse osmosis membrane, the floating enzyme is entrained in the organic phase in the form of an emulsion. It is possible to prevent the degradation of the enzyme reuse efficiency.

【図面の簡単な説明】[Brief description of drawings]

【図1】本発明の製造法の一例を示す模式図FIG. 1 is a schematic diagram showing an example of a production method of the present invention.

【図2】本発明に係る(a)攪拌槽型装置、(b)ソル
ベントリフト型装置及び(c)膜型装置の各模式図
FIG. 2 is a schematic view of (a) a stirring tank type device, (b) a solvent lift type device, and (c) a membrane type device according to the present invention.

【符号の説明】[Explanation of symbols]

1…反応槽、2、13、23…水相、3、7、14、2
2…有機相、4…循環系、5…抽出槽、6…抽出相、1
0…攪拌槽、12…分離槽、15…抽出塔、18、20
…接触槽、21…膜。
1 ... Reactor, 2, 13, 23 ... Aqueous phase, 3, 7, 14, 2
2 ... Organic phase, 4 ... Circulation system, 5 ... Extraction tank, 6 ... Extraction phase, 1
0 ... Stirring tank, 12 ... Separation tank, 15 ... Extraction tower, 18, 20
... Contact tank, 21 ... Membrane.

───────────────────────────────────────────────────── フロントページの続き (72)発明者 星野 明 埼玉県越谷市越谷本町3−4 (72)発明者 礒野 康幸 茨城県稲敷郡茎崎町若葉1−8 コンドレ アB−A (72)発明者 荒谷 弦一郎 東京都北区志茂5−27−7 山上荘 ─────────────────────────────────────────────────── --- Continuation of the front page (72) Inventor Akira Hoshino 3-4 Koshigaya Honcho, Koshigaya City, Saitama Prefecture (72) Inventor Yasuyuki Isono 1-8 Wakaba, Kukizaki-cho, Inashiki-gun, Ibaraki Condrea B-A (72) Inventor Zenichiro Araya 5-27-7 Shimo, Kita-ku, Tokyo Yamajoso

Claims (3)

【特許請求の範囲】[Claims] 【請求項1】 Lys、His、Arg、Orn、As
p及びGluの群(I群)から選ばれるアミノ酸であっ
て、そのアミノ基が保護基によって保護されたアミノ酸
(基質A)と、Ala、Val、Leu、Ile、Me
t、Trp、Phe、Pro、Gly、Ser、Th
r、Cys、Tyr、Asn、Gln、Lys、Hi
s、Arg、Orn、Asp及びGluの群(II群)
から選ばれるアミノ酸であって、そのカルボキシル基が
アルキルエステル化(但し、このアルキルは炭素数1乃
至4である。)されたアミノ酸(基質B)との組合わ
せ、又は前記II群から選ばれるアミノ酸であって、そ
のアミノ基が保護基によって保護されたアミノ酸(基質
A)と、前記I群から選ばれるアミノ酸であって、その
カルボキシル基が前記条件のアルキルエステル化された
アミノ酸(基質B)との組合わせを、二相系の反応槽中
で酵素触媒により反応させるジペプチドの製造法におい
て、前記反応槽の反応液を水相、及び水と混和しない有
機溶媒からなる有機相の二相から構成し、これら水相又
は有機相のいずれかに前記基質A、基質B及び酵素を供
給してジペプチドを合成し、この合成されたジペプチド
が主として含有される前記有機相を構成する溶液を、水
性の抽出相を有する抽出槽へ導入し、この導入した溶液
から抽出相へジペプチドを抽出し、抽出後の溶液は前記
反応槽へ戻すことを特徴とするジペプチドの製造法。
1. Lys, His, Arg, Orn, As
an amino acid selected from the group of p and Glu (group I), the amino group of which is protected by a protecting group (substrate A); and Ala, Val, Leu, Ile, Me.
t, Trp, Phe, Pro, Gly, Ser, Th
r, Cys, Tyr, Asn, Gln, Lys, Hi
Group of s, Arg, Orn, Asp and Glu (Group II)
An amino acid selected from the group II or a combination with an amino acid (substrate B) whose carboxyl group is alkyl esterified (wherein the alkyl has 1 to 4 carbon atoms). And an amino acid whose amino group is protected by a protecting group (substrate A), and an amino acid which is an amino acid selected from Group I and whose carboxyl group is alkyl esterified under the above conditions (substrate B) In the method for producing a dipeptide in which a combination of the above is reacted with an enzyme catalyst in a two-phase reaction tank, the reaction solution in the reaction tank is composed of two phases: an aqueous phase and an organic phase composed of an organic solvent immiscible with water Then, the substrate A, the substrate B and the enzyme are supplied to either the aqueous phase or the organic phase to synthesize a dipeptide, and the synthesized dipeptide is mainly contained. The solution constituting the organic phase is introduced into an extraction tank having an aqueous extraction phase, the dipeptide is extracted from the introduced solution into the extraction phase, and the solution after extraction is returned to the reaction tank. Manufacturing method.
【請求項2】 前記基質Aはアミノ基を保護されたアス
パラギン酸であり、前記基質Bはフェニルアラニンのア
ルキルエステル(但し、このアルキルは炭素数1乃至4
である。)であることを特徴とする請求項1に記載のジ
ペプチドの製造法。
2. The substrate A is an amino group-protected aspartic acid, and the substrate B is an alkyl ester of phenylalanine (wherein the alkyl has 1 to 4 carbon atoms).
Is. The manufacturing method of the dipeptide of Claim 1 characterized by these.
【請求項3】 前記反応槽及び/又は抽出槽中の水相及
び有機相が、限外ろ過膜又は低阻止率逆浸透膜を介して
接触していることを特徴とする請求項1又は2に記載の
ジペプチドの製造法。
3. The aqueous phase and the organic phase in the reaction tank and / or the extraction tank are in contact with each other through an ultrafiltration membrane or a low rejection reverse osmosis membrane. The method for producing a dipeptide according to 1.
JP4358471A 1992-12-25 1992-12-25 Dipeptide production method Expired - Fee Related JP2972903B2 (en)

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Country Link
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003010189A1 (en) * 2001-07-26 2003-02-06 Ajinomoto Co., Inc. Process for producing dipeptide, peptide synthase to be used therein and process for producing peptide synthase
JP2018121648A (en) * 2012-09-12 2018-08-09 ビュータマックス・アドバンスド・バイオフューエルズ・エルエルシー Processes and systems for the production of fermentation products

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003010189A1 (en) * 2001-07-26 2003-02-06 Ajinomoto Co., Inc. Process for producing dipeptide, peptide synthase to be used therein and process for producing peptide synthase
US7754466B2 (en) 2001-07-26 2010-07-13 Ajinomoto Co., Inc. Method for producing dipeptides
JP2018121648A (en) * 2012-09-12 2018-08-09 ビュータマックス・アドバンスド・バイオフューエルズ・エルエルシー Processes and systems for the production of fermentation products

Also Published As

Publication number Publication date
JP2972903B2 (en) 1999-11-08

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