JPH0947294A - Separation of component - Google Patents
Separation of componentInfo
- Publication number
- JPH0947294A JPH0947294A JP20015695A JP20015695A JPH0947294A JP H0947294 A JPH0947294 A JP H0947294A JP 20015695 A JP20015695 A JP 20015695A JP 20015695 A JP20015695 A JP 20015695A JP H0947294 A JPH0947294 A JP H0947294A
- Authority
- JP
- Japan
- Prior art keywords
- substrate
- group
- organic phase
- target component
- 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
Links
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は疎水性膜を用いて効率よ
く目的成分をエマルション抽出する方法に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for efficiently extracting an objective component by emulsion using a hydrophobic membrane.
【０００２】[0002]
【従来の技術】多成分水溶液から目的物質を分離する方
法の１つとして、液−液抽出があり、この液−液抽出を
利用したものとして、具体的にはタンパク質分解酵素の
逆反応によるペプチドの合成がある。2. Description of the Related Art Liquid-liquid extraction is one of the methods for separating a target substance from a multi-component aqueous solution. As a method utilizing this liquid-liquid extraction, specifically, a peptide produced by the reverse reaction of proteolytic enzyme There is a synthesis of.
【０００３】上記タンパク質分解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
とで見掛け上の平衡を合成側にシフトさせる方法とし
て、従来から沈殿法（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）、微水系反応
法（特開平２−３９８９５号公報）、二相系反応法が知
られている。Since the synthetic reaction equilibrium by the above-mentioned proteolytic enzymes is low, a precipitation method has been conventionally used as a method for shifting the apparent equilibrium to the synthesis side by efficiently taking out the product from the reaction system (Chemical Engineering Society No. 20). Abstracts of research presentations at the Autumn Meeting have been known in 1987, p. 343), a fine water-based reaction method (Japanese Patent Laid-Open No. 2-39895), and a two-phase reaction method.
【０００４】沈殿法は、水溶性基質から水不溶性生成物
を合成する方法であり、高純度の生成物を得ることがで
きるが、酵素が沈殿物とともに系外に持出されるため酵
素を補給しなければならない。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
を抑制する方法であり、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。The precipitation method is a method for synthesizing a water-insoluble product from a water-soluble substrate, and a high-purity product can be obtained. However, since the enzyme is taken out of the system together with the precipitate, the enzyme is supplemented. There must be. The slightly water-based reaction method is a method of suppressing the hydrolysis reaction, which is a reverse reaction, by limiting the amount of water in the system, and it is difficult to increase the purity because the reaction equilibrium of the product is low. There is also a problem with the stability of the enzyme present in the solvent.
【０００５】更に、二相系反応法は水相で酵素反応を行
い、有機相に目的生成物を抽出する方法であり、本発明
者等は先に二相系反応法に属する方法を特開平６−２１
７７８５号公報及び特願平６−１９１５５５号として提
案している。前者は、反応槽を水相と有機相で構成し、
水相に基質Ａ、基質Ｂ及び酵素を供給して反応物Ｐ（ジ
ペプチド）を、水相−有機相間の抽出平衡を利用して基
質Ｂと反応物Ｐを有機相側に分離し、更に抽出槽で基質
Ｂを含む有機相から反応物Ｐを分離するようにした方法
である。後者は、反応槽内に荷電膜を設けることで、水
相−有機相間の抽出平衡を利用して基質Ａの有機相側へ
の移行を阻止し、荷電膜によって基質Ｂの有機相側への
移行を阻止するようにした方法である。Further, the two-phase reaction method is a method in which an enzymatic reaction is carried out in an aqueous phase to extract a target product into an organic phase. The present inventors have previously described a method belonging to the two-phase reaction method. 6-21
7785 and Japanese Patent Application No. 6-191555. In the former, the reaction tank is composed of an aqueous phase and an organic phase,
The substrate A, the substrate B and the enzyme are supplied to the aqueous phase to separate the reaction product P (dipeptide), and the substrate B and the reaction product P are separated to the organic phase side by utilizing the extraction equilibrium between the aqueous phase and the organic phase, and further extracted. In this method, the reaction product P is separated from the organic phase containing the substrate B in a tank. In the latter, by providing a charged film in the reaction tank, the transfer equilibrium of the substrate A to the organic phase side is prevented by utilizing the extraction equilibrium between the aqueous phase and the organic phase, and the charged film transfers the substrate B to the organic phase side. It is a method that prevents migration.
【０００６】[0006]
【発明が解決しようとする課題】本発明者等が提案した
上述の方法において、膜を用いた方法では水相と有機相
とを混合せず、膜を介して抽出するため、膜の物質移動
抵抗のため抽出時間が長くかかってしまう。In the above-mentioned method proposed by the present inventors, in the method using a membrane, the aqueous phase and the organic phase are not mixed and extraction is performed through the membrane. It takes a long time to extract because of resistance.
【０００７】[0007]
【課題を解決するための手段】上記課題を解決するた
め、本願に係る成分分離方法は、目的成分群、水相／有
機相の分配係数が前記目的成分群よりも大きい第１の非
目的成分群及び水相／有機相の分配係数が前記目的成分
群の分配係数と近似した第２の非目的成分群を含む水相
と有機相とを混合攪拌することで、水相とこの水相より
も目的成分群の濃度が濃い有機相とからなるエマルショ
ンを調製し、このエマルションを疎水性膜を用いて濾過
することで目的成分群と第２の非目的成分群を含む有機
相を分離し、次いでこの有機相から目的成分群を回収す
るようにした。In order to solve the above-mentioned problems, the component separation method according to the present invention is a first non-target component having a target component group and an aqueous / organic phase distribution coefficient larger than that of the target component group. Group and an aqueous phase / organic phase having a partition coefficient close to the partition coefficient of the target component group and a second non-target component group-containing water phase and an organic phase are mixed and stirred, whereby the water phase and the water phase Also prepare an emulsion consisting of an organic phase having a high concentration of the target component group, and separating the organic phase containing the target component group and the second non-target component group by filtering this emulsion using a hydrophobic membrane, Then, the target component group was recovered from this organic phase.
【０００８】また、ジペプチドと、水相／有機相の分配
係数がジペプチドよりも大きい基質Ａとを含む２成分系
のエマルションを作成した場合には、当該エマルション
を構成する有機相中に水相のジペプチドを移行せしめ、
次いでエマルションを疎水性膜を用いて濾過すること
で、水相よりも多量にジペプチドを含む有機相を分離
し、この有機相からジペプチドを回収する。Further, when a two-component system emulsion containing dipeptide and a substrate A having a partition coefficient of aqueous phase / organic phase larger than that of dipeptide is prepared, the organic phase forming the emulsion contains the aqueous phase. Transfer the dipeptide,
Then, the emulsion is filtered using a hydrophobic membrane to separate an organic phase containing a larger amount of dipeptide than the aqueous phase, and the dipeptide is recovered from this organic phase.
【０００９】更に、ジペプチドと、水相／有機相の分配
係数がジペプチドの分配係数と近似した基質Ｂとを含む
２成分系のエマルションを作成した場合には、当該エマ
ルションを構成する有機相中に水相のジペプチドを移行
せしめ、次いで前記エマルションを疎水性膜を用いて濾
過することで、水相よりも多量にジペプチドを含む有機
相を分離し、この有機相からジペプチドまたは基質Ｂの
一方を抽出する。Further, when a two-component emulsion containing a dipeptide and a substrate B in which the partition coefficient of the aqueous phase / organic phase is close to the partition coefficient of the dipeptide is prepared, in the organic phase constituting the emulsion, The dipeptide in the aqueous phase is transferred, and then the emulsion is filtered using a hydrophobic membrane to separate an organic phase containing a larger amount of the dipeptide than the aqueous phase, and one of the dipeptide and the substrate B is extracted from this organic phase. To do.
【００１０】ここで、前記第１の非目的成分群と第２の
非目的成分群は、例えば水相中で酵素触媒により反応し
て目的成分群であるジペプチドを生産する基質Ａ及び基
質Ｂであり、基質Ａ及び基質Ｂとしては例えばは以下の
組み合わせのいずれかとすることができる。 組み合わせ 基質Ａ：Ｌｙｓ（リジン）、Ｈｉｓ（ヒスチジン）、Ａ
ｒｇ（アルギニン）、Ｏｒｎ（オルニチン）、Ａｓｐ
（アスパラギン酸）及びＧｌｕ（グルタミン酸）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Ｂ：Ａｌａ（アラニン）、Ｖａｌ（バリン）、Ｌｅ
ｕ（ロイシン）、Ｉｌｅ（イソロイシン）、Ｍｅｔ（メ
チオニン）、Ｔｒｐ（トリプトファン）、Ｐｈｅ（フェ
ニルアラニン）、Ｐｒｏ（プロリン）、Ｇｌｙ（グリシ
ン）、Ｓｅｒ（セリン）、Ｔｈｒ（トレオニン）、Ｃｙ
ｓ（システイン）、Ｔｙｒ（チロシン）、Ａｓｎ（アス
パラギン）、Ｇｌｎ（グルタミン）、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 組み合わせ 基質Ａ：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Ｂ：前記Ｉ群から選ばれるアミノ酸であって、その
カルボキシル基がアルキルエステル化されたアミノ酸Here, the first non-target component group and the second non-target component group are, for example, a substrate A and a substrate B that react with an enzyme catalyst in an aqueous phase to produce a target component dipeptide. The substrate A and the substrate B can be, for example, any of the following combinations. Combination Substrate A: Lys (lysine), His (histidine), A
rg (arginine), Orn (ornithine), Asp
An amino acid selected from the group (group I) of (aspartic acid) and Glu (glutamic acid), the amino group of which is protected by a protecting group. Substrate B: Ala (alanine), Val (valine), Le
u (leucine), Ile (isoleucine), Met (methionine), Trp (tryptophan), Phe (phenylalanine), Pro (proline), Gly (glycine), Ser (serine), Thr (threonine), Cy
s (cysteine), Tyr (tyrosine), Asn (asparagine), Gln (glutamine), 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).
It is. ) Amino acid combination Substrate A: an amino acid selected from Group II above, whose amino group is protected by a protecting group Substrate B: an amino acid selected from Group I above, whose carboxyl group is an alkyl ester Amino acid
【００１１】基質Ａのアミノ基の保護基としては、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。Examples of the amino group-protecting group for the substrate A include diphenylmethyl group, triphenylmethyl group, aromatic group and aliphatic oxycarbonyl group, which are halogen, nitro group, lower alkyl group and lower alkoxy group. Etc. may be substituted. 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.
【００１２】また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 number of moles of the substrate B added to 1 mole of the substrate A is 1 to 5, preferably 1 to 2. When the number of moles is less than 1, substrate B accumulates, and when it exceeds 5, the number of substrate B is increased.
In some cases, the object of the present invention cannot be achieved because the outflow from the system becomes significant.
【００１３】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).
【００１４】前記水相及び抽出相には、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ｐＨを
調整することができる。また有機相に使用する有機溶媒
としては、水に殆んど溶解しないものであればどのよう
なものでもよいが、酵素への影響、食品製造ということ
を考慮した場合には、酢酸ブチルがよいが、膜へのダメ
ージ、水／溶媒の分離の良さを考慮するとブタノール、
ヘキサノール、ペンタノール等のアルコール類がよい。The water phase and the extraction phase have, for example, 0.05
The pH of each phase can be adjusted using a known buffer solution such as a mol acetate buffer solution. As the organic solvent used in the organic phase, any solvent may be used as long as it is hardly soluble in water, but butyl acetate is preferable in consideration of the influence on enzymes and food production. However, considering the damage to the membrane and the good separation of water / solvent, butanol,
Alcohols such as hexanol and pentanol are preferable.
【００１５】[0015]
【作用】水相中には基質Ａ、基質Ｂ、これら基質Ａと基
質Ｂの反応によって生成された目的成分群Ｐ及び酵素触
媒Ｃが存在するが、この水相が有機相と接触すると、水
相／有機相の分配係数の相違によって、目的成分群Ｐが
有機相に移行し分離される。そして、本発明にあって
は、攪拌によって水相と有機相とを混合接触させた後、
直ちに有機相を疎水性膜にて分離する。The substrate A, the substrate B, the target component group P and the enzyme catalyst C produced by the reaction of the substrate A and the substrate B are present in the aqueous phase. When the aqueous phase comes into contact with the organic phase, The target component group P is transferred to the organic phase and separated due to the difference in the distribution coefficient between the organic phase and the organic phase. And in the present invention, after mixing and contacting the aqueous phase and the organic phase by stirring,
Immediately separate the organic phase with a hydrophobic membrane.
【００１６】即ち、従来の水相と有機相とを混合せず、
膜を介して水相中の目的成分群Ｐを有機相に抽出する方
法であると、膜の物質移動抵抗のため抽出時間が長くか
かっているが、本発明のように水相と有機相とを混合し
てエマルション抽出を行い、２相の分離は疎水性膜を用
いて行うことで、大幅な時間短縮が可能になる。That is, without mixing the conventional aqueous phase and organic phase,
According to the method of extracting the target component group P in the aqueous phase into the organic phase through the membrane, the extraction time is long due to the mass transfer resistance of the membrane. It is possible to drastically shorten the time by mixing the two to perform emulsion extraction and separating the two phases using a hydrophobic membrane.
【００１７】[0017]
【実施例】以下に本発明の実施例を添付図面に基づいて
説明する。図１は本発明方法を実施する連続式装置の一
例を示すものである。この装置は、タンク１、攪拌槽３
及び回収槽４から構成され、タンク１には基質Ａ及び基
質Ｂを溶解した酢酸エチル（有機溶媒）５が満たされ、
攪拌槽３には酵素Ｃを含む水溶液６が満たされている。Embodiments of the present invention will be described below with reference to the accompanying drawings. FIG. 1 shows an example of a continuous apparatus for carrying out the method of the present invention. This equipment consists of tank 1, agitation tank 3
And a recovery tank 4, the tank 1 is filled with ethyl acetate (organic solvent) 5 in which the substrate A and the substrate B are dissolved,
The stirring tank 3 is filled with an aqueous solution 6 containing the enzyme C.
【００１８】基質Ａとしてベンジルオキシカルボニルア
スパラギン酸、基質Ｂとしてフェニルアラニンメチルエ
ステル、酵素Ｃとしてサーモライシンを本実施例では用
いている。更に水溶液６はｐＨ６に調整されている。Benzyloxycarbonyl aspartic acid is used as the substrate A, phenylalanine methyl ester is used as the substrate B, and thermolysin is used as the enzyme C in this example. Furthermore, the pH of the aqueous solution 6 is adjusted to 6.
【００１９】また攪拌槽３には、攪拌羽根７が設けら
れ、この攪拌槽３にはポンプを備えた配管８を介して酢
酸エチル５が供給され、更に攪拌槽３の上面は密閉さ
れ、タンク１内には圧気源１０から空気や窒素等の加圧
ガスを供給するようにしている。Further, the stirring tank 3 is provided with a stirring blade 7, ethyl acetate 5 is supplied to the stirring tank 3 through a pipe 8 equipped with a pump, and the upper surface of the stirring tank 3 is hermetically sealed. A pressurized gas such as air or nitrogen is supplied from the compressed air source 10 into the inside 1.
【００２０】ここで、攪拌槽３内の圧力（ゲージ圧）と
しては0.5kg／cm2〜4.0kg／cm2程度が好適である。圧力
が低いと濾過に時間がかかり、逆に圧力が高過ぎると攪
拌槽３の中間部に設けた疎水性膜１１に負担がかかり過
ぎることになる。また、疎水性膜１１は有機溶媒の透過
を許容し、水及び酵素の透過を阻止するものを選定す
し、例えば、テフロン、ポリエチレン等の材料から構成
される。[0020] Here, the pressure inside the agitation tank 3 (gauge pressure) is preferably about 2 0.5kg / cm 2 ~4.0kg / cm. If the pressure is low, it takes time to filter, and if the pressure is too high, the hydrophobic membrane 11 provided in the middle part of the stirring tank 3 is overloaded. Further, the hydrophobic film 11 is selected from those which allow the permeation of the organic solvent and prevent the permeation of water and the enzyme, and is made of a material such as Teflon or polyethylene.
【００２１】以上において、タンク１から基質Ａ，Ｂが
溶解した酢酸エチル５を攪拌槽３内の水溶液６に供給し
攪拌する。すると、図１に示すように酢酸エチル５及び
水溶液６とが混合せしめられ攪拌槽３内はエマルション
状態となる。In the above, ethyl acetate 5 in which substrates A and B are dissolved is supplied from tank 1 to aqueous solution 6 in stirring tank 3 and stirred. Then, as shown in FIG. 1, the ethyl acetate 5 and the aqueous solution 6 are mixed and the inside of the stirring tank 3 becomes an emulsion state.
【００２２】このエマルション状態では水相と有機相と
が十分に接触する。そして、図２に示すように、水相／
有機相の分配係数の相違によって、有機相中の基質Ａが
水相に移行し、基質Ｂ及び目的成分群Ｐが、有機相に移
行する。そして、基質Ｂ及び目的成分群Ｐであるベンジ
ルオキシカルボニルアスパラギルフェニルアラニンメチ
ルエステルが移行した有機相を疎水性膜１１を通して回
収槽４に移し、目的成分群Ｐを沈殿せしめて回収する。In this emulsion state, the water phase and the organic phase are in sufficient contact with each other. Then, as shown in FIG.
Due to the difference in the partition coefficient of the organic phase, the substrate A in the organic phase transfers to the aqueous phase, and the substrate B and the target component group P transfer to the organic phase. Then, the substrate B and the organic phase to which benzyloxycarbonylasparagylphenylalanine methyl ester which is the target component group P has transferred is transferred to the recovery tank 4 through the hydrophobic membrane 11 and the target component group P is precipitated and recovered.
【００２３】上記の操作は連続して行われ、攪拌槽３か
ら疎水性膜１１を透過した有機相の減少分をタンク１か
ら攪拌槽３に補給する。尚、酵素Ｃは水溶液中に残るた
め、原則として補給は不要である。The above operation is continuously performed, and the depletion amount of the organic phase which has permeated the hydrophobic membrane 11 from the stirring tank 3 is replenished from the tank 1 to the stirring tank 3. In addition, since the enzyme C remains in the aqueous solution, in principle, supplementation is unnecessary.
【００２４】図３は回分式装置の全体概略図であって、
この装置は、タンク１、タンク２、攪拌槽３及び回収槽
４から構成され、タンク１には酢酸エチル（有機溶媒）
５が満たされ、タンク２には水溶液６が満たされ、この
水溶液６には、前記基質Ａが１０ミリモル、基質Ｂが１
０ミリモル、及びこれらの反応によって生成される目的
成分群Ｐが５ミリモル、及び酵素Ｃが含まれ、更にｐＨ
６に調整されている。FIG. 3 is an overall schematic view of the batch type apparatus,
This apparatus is composed of a tank 1, a tank 2, a stirring tank 3 and a recovery tank 4, and the tank 1 contains ethyl acetate (organic solvent).
5 and the tank 2 is filled with an aqueous solution 6, and the aqueous solution 6 contains 10 millimoles of the substrate A and 1 milliliter of the substrate B.
0 mmol, 5 mmol of the target component group P produced by these reactions, and enzyme C are contained, and
It has been adjusted to 6.
【００２５】この回分式装置と前記連続式装置との相違
は、回分式装置にあっては水溶液用タンク２を別に設
け、このタンク２に基質Ａ及び基質Ｂを溶解している点
であり、他の構成については略同一であるので、同一の
符号を付して説明を省略する。The difference between this batch type apparatus and the continuous type apparatus is that the batch type apparatus is provided with an aqueous solution tank 2 separately and the substrate A and the substrate B are dissolved in this tank 2. Since other configurations are substantially the same, the same reference numerals are given and the description is omitted.
【００２６】そして、回分式装置にあっては、タンク
１，２から酢酸エチル５及び水溶液６を供給し攪拌す
る。すると、図４に示すように酢酸エチル５及び水溶液
６とが混合せしめられ攪拌槽３内はエマルション状態と
なる。In the batch type apparatus, ethyl acetate 5 and aqueous solution 6 are supplied from tanks 1 and 2 and stirred. Then, as shown in FIG. 4, the ethyl acetate 5 and the aqueous solution 6 are mixed and the inside of the stirring tank 3 becomes an emulsion state.
【００２７】このエマルション状態では前記したように
水相と有機相とが十分に接触するので、水相中の基質Ｂ
及び目的成分群Ｐが、水相／有機相の分配係数の相違に
よって、有機相に移行する。そして、基質Ｂ及び目的成
分群Ｐが移行した有機相を疎水性膜１１を通して回収槽
４に移し、目的成分群Ｐを沈殿せしめて回収する。In this emulsion state, since the aqueous phase and the organic phase are in sufficient contact with each other as described above, the substrate B in the aqueous phase is
And the target component group P is transferred to the organic phase due to the difference in the partition coefficient between the aqueous phase and the organic phase. Then, the organic phase to which the substrate B and the target component group P have transferred is transferred to the recovery tank 4 through the hydrophobic membrane 11, and the target component group P is precipitated and recovered.
【００２８】そして、回分式にあっては、目的成分群Ｐ
を沈殿せしめて回収した後、攪拌槽３内に残っている水
溶液を排出して図３に示す状態に戻し、その後は前記同
様の操作を繰り返す。In the batch system, the target component group P
Is recovered by settling, the aqueous solution remaining in the stirring tank 3 is discharged to return to the state shown in FIG. 3, and thereafter the same operation as described above is repeated.
【００２９】尚、目的成分群Ｐを回収する方法として
は、回収槽４にて基質Ｂ及び目的成分群Ｐが移行した有
機相と前記水相とは異なるｐＨに調製された水相（逆抽
出用水相）とを接触せしめ、目的成分群Ｐのみを異なる
ｐＨに調製された水相側に移行せしめることも可能であ
る。また目的成分群Ｐのみを有機相側に残し、基質Ｂを
水相側に移行せしめるようにしてもよい。As a method for recovering the target component group P, the organic phase to which the substrate B and the target component group P have transferred in the recovery tank 4 and the aqueous phase prepared at a different pH (back extraction) It is also possible to bring the target component group P into contact with the aqueous phase) and transfer only the target component group P to the aqueous phase side adjusted to a different pH. Alternatively, only the target component group P may be left on the organic phase side and the substrate B may be transferred to the aqueous phase side.
【００３０】[0030]
【発明の効果】以上に説明したように、本発明によれ
ば、二相系反応法を利用して水相から有機相に目的生成
物を抽出するにあたり、水相と有機相とを混合した後、
直ちにエマルションの状態から疎水性膜によって有機相
を濾過するようにしたので、一旦混合した後に二相に分
れるまで静置する必要がなくなり、分離に要する時間を
大巾に短縮することができる。As described above, according to the present invention, the aqueous phase and the organic phase are mixed in extracting the target product from the aqueous phase to the organic phase by using the two-phase reaction method. rear,
Since the organic phase is immediately filtered from the emulsion state by the hydrophobic membrane, it is not necessary to stand still until the two phases are separated after mixing once, and the time required for separation can be greatly shortened.
【００３１】特に、疎水性膜を用いない従来のエマルシ
ョン法でもって二相を分離するには、遠心分離による方
法や電気的な解乳も考えられるが、前者にあっては高回
転を与えなければならず、連続化が困難であり、後者に
あっては数十ｋＶの高電圧を印加しなければならない。
これに対し、本発明によれば、エマルションを約0.5kg
／cm2（ゲージ圧）〜数kg／cm2の圧力で濾過するだけで
二相を分離することができ、連続化も容易である。In particular, in order to separate the two phases by the conventional emulsion method which does not use a hydrophobic membrane, a method by centrifugation or electric demulsification may be considered, but in the former case, high rotation should be applied. However, it is difficult to make it continuous, and in the latter case, a high voltage of several tens of kV must be applied.
On the other hand, according to the present invention, about 0.5 kg of emulsion is used.
The two phases can be separated only by filtration at a pressure of / cm 2 (gauge pressure) to several kg / cm 2 , and continuous formation is easy.
【００３２】また、本発明によれば、疎水性膜によって
酵素の流失を阻止できるので、酵素の使用量を低減する
ことができる。Further, according to the present invention, since the hydrophobic membrane can prevent the enzyme from being washed away, the amount of the enzyme used can be reduced.
【図１】本発明方法を実施する連続式装置の全体概略図FIG. 1 is an overall schematic diagram of a continuous apparatus for carrying out the method of the present invention.
【図２】有機相と水相との分配を示す概念図FIG. 2 is a conceptual diagram showing partitioning between an organic phase and an aqueous phase.
【図３】本発明方法を実施する回分式装置の全体概略図
であって、エマルションを作る前の状態を示す図FIG. 3 is an overall schematic view of a batch type apparatus for carrying out the method of the present invention, showing a state before making an emulsion.
【図４】回分式装置の全体概略図であって、エマルショ
ン抽出を行っている状態を示す図FIG. 4 is an overall schematic view of a batch-type apparatus showing a state in which emulsion extraction is being performed.
１，２…タンク、３…攪拌槽、４…回収槽、５…有機溶
媒、６…水溶液、７…攪拌羽根、１１…疎水性膜、Ａ，
Ｂ…基質、Ｃ…酵素、Ｐ…目的成分群。1, 2 ... Tank, 3 ... Stirring tank, 4 ... Recovery tank, 5 ... Organic solvent, 6 ... Aqueous solution, 7 ... Stirring blade, 11 ... Hydrophobic membrane, A,
B ... Substrate, C ... Enzyme, P ... Target component group.
フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｃ１２Ｍ 1/40 Ｃ１２Ｍ 1/40 Ａ (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 星野 信夫 東京都練馬区西大泉５−５−４ (72)発明者 星野 明 埼玉県越谷市越谷本町３−４ (72)発明者 福島 健司 埼玉県川口市安行領根岸2087−８ コトー 根岸台ＣContinuation of the front page (51) Int.Cl. 6 Identification number Internal reference number for FI FI technical display location C12M 1/40 C12M 1/40 A (72) Inventor Yasuyuki Isono 1-8 Wakaba, Kukizaki-cho, Inashiki-gun, Ibaraki Condole BA (72) Inventor Nobuo Hoshino 5-5-4 Nishioizumi, Nerima-ku, Tokyo (72) Inventor Akira Hoshino 3-4 Koshigayahoncho, Koshigaya City, Saitama Prefecture (72) Kenji Fukushima Yasukuryo, Kawaguchi City, Saitama Prefecture Negishi 2087-8 Cotto Negishidai C
Claims (4)
が前記目的成分群よりも大きい第１の非目的成分群と、
水相／有機相の分配係数が前記目的成分群の分配係数と
近似した第２の非目的成分群とを含む水相に、有機相を
混合攪拌することで、水相とこの水相よりも目的成分群
の濃度が濃い有機相とからなるエマルションとし、この
エマルションを疎水性膜を用いて濾過することで目的成
分群と第２の非目的成分群を含む有機相を分離し、次い
でこの有機相から目的成分群を回収するようにしたこと
を特徴とする成分分離方法。1. A target component group, and a first non-target component group having a partition coefficient of an aqueous phase / organic phase larger than that of the target component group.
By mixing and stirring the organic phase with the aqueous phase containing the second non-target component group in which the partition coefficient of the aqueous phase / organic phase is similar to the partition coefficient of the target component group, the aqueous phase and this aqueous phase An emulsion consisting of an organic phase having a high concentration of the target component group is prepared, and the emulsion is filtered through a hydrophobic membrane to separate the organic phase containing the target component group and the second non-target component group. A method for separating components, characterized in that a target component group is recovered from the phase.
るジペプチドを分離する方法であって、この方法は、ジ
ペプチドと、水相／有機相の分配係数がジペプチドより
も大きい基質Ａとを含むエマルションを作成すること
で、エマルションを構成する有機相中に水相のジペプチ
ドを移行せしめ、次いでエマルションを疎水性膜を用い
て濾過することで、水相よりも多量にジペプチドを含む
有機相を分離し、この有機相からジペプチドを回収する
ようにしたことを特徴とする成分分離方法。2. A method for separating a dipeptide produced by the reaction of a substrate A and a substrate B, which comprises dipeptide and substrate A having a partition coefficient of an aqueous phase / organic phase larger than that of the dipeptide. By creating an emulsion, the dipeptide in the aqueous phase is transferred to the organic phase that constitutes the emulsion, and then the emulsion is filtered using a hydrophobic membrane to separate the organic phase containing the dipeptide in a larger amount than the aqueous phase. Then, the dipeptide is recovered from this organic phase.
るジペプチドを分離する方法であって、この方法は、ジ
ペプチドと、水相／有機相の分配係数がジペプチドの分
配係数と近似した基質Ｂとを含むエマルションを作成す
ることで、エマルションを構成する有機相中に水相のジ
ペプチドを移行せしめ、次いで前記エマルションを疎水
性膜を用いて濾過することで、水相よりも多量にジペプ
チドを含む有機相を分離し、この有機相からジペプチド
または基質Ｂの一方を抽出するようにしたことを特徴と
する成分分離方法。3. A method for separating a dipeptide produced by the reaction of a substrate A and a substrate B, which comprises a dipeptide and a substrate B in which the partition coefficient of the aqueous phase / organic phase is similar to that of the dipeptide. By creating an emulsion containing and, by transferring the dipeptide of the aqueous phase into the organic phase that constitutes the emulsion, and then filtering the emulsion using a hydrophobic membrane, the dipeptide is contained in a larger amount than the aqueous phase. A method for separating components, comprising separating an organic phase and extracting one of the dipeptide and the substrate B from the organic phase.
方法において、前記第１の非目的成分群と第２の非目的
成分群は水相中で酵素触媒により反応して目的成分群で
あるジペプチドを生産する基質Ａ及び基質Ｂであり、基
質Ａ及び基質Ｂは以下の組み合わせのいずれかであるこ
とを特徴とする成分分離方法。 組み合わせ 基質Ａ：Ｌｙｓ、Ｈｉｓ、Ａｒｇ、Ｏｒｎ、Ａｓｐ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Ｂ：Ａｌａ、Ｖａｌ、Ｌｅｕ、Ｉｌｅ、Ｍｅｔ、Ｔ
ｒｐ、Ｐｈｅ、Ｐｒｏ、Ｇｌｙ、Ｓｅｒ、Ｔｈｒ、Ｃｙ
ｓ、Ｔｙｒ、Ａｓｎ、Ｇｌｎ、Ｌｙｓ、Ｈｉｓ、Ａｒ
ｇ、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 組み合わせ 基質Ａ：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Ｂ：前記Ｉ群から選ばれるアミノ酸であって、その
カルボキシル基がアルキルエステル化されたアミノ酸4. The component separation method according to claim 1, wherein the first non-target component group and the second non-target component group react with each other in an aqueous phase by an enzyme catalyst to obtain a target component group. Which is a substrate A and a substrate B which produce the dipeptide, wherein the substrate A and the substrate B are any of the following combinations. Combination Substrate A: an amino acid selected from the group (group I) of Lys, His, Arg, Orn, Asp and Glu, the amino group of which is protected by a protecting group. Substrate B: Ala, Val, Leu, Ile. , Met, T
rp, Phe, Pro, Gly, Ser, Thr, Cy
s, Tyr, Asn, Gln, Lys, His, Ar
An amino acid selected from the group (group II) of g, Orn, Asp and Glu, wherein the carboxyl group is alkyl esterified (wherein the alkyl has 1 to 4 carbon atoms) A combination A substrate A: An amino acid selected from Group II, the amino group of which is protected by a protecting group. Substrate B: an amino acid selected from Group I, whose carboxyl group is alkyl esterified.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7200156A JP3012493B2 (en) | 1995-08-07 | 1995-08-07 | Component separation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7200156A JP3012493B2 (en) | 1995-08-07 | 1995-08-07 | Component separation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0947294A true JPH0947294A (en) | 1997-02-18 |
| JP3012493B2 JP3012493B2 (en) | 2000-02-21 |
Family
ID=16419728
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP7200156A Expired - Fee Related JP3012493B2 (en) | 1995-08-07 | 1995-08-07 | Component separation method |
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| Country | Link |
|---|---|
| JP (1) | JP3012493B2 (en) |
Cited By (2)
| 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 |
| WO2020230492A1 (en) | 2019-05-16 | 2020-11-19 | 日曹エンジニアリング株式会社 | Membrane-based continuous phase separation system and device suitable for low flow rates |
-
1995
- 1995-08-07 JP JP7200156A patent/JP3012493B2/en not_active Expired - Fee Related
Cited By (4)
| 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 |
| WO2020230492A1 (en) | 2019-05-16 | 2020-11-19 | 日曹エンジニアリング株式会社 | Membrane-based continuous phase separation system and device suitable for low flow rates |
| KR20220009398A (en) | 2019-05-16 | 2022-01-24 | 닛소 엔지니아링 가부시키가이샤 | Continuous Phase Separation System and Apparatus by Membrane Suitable for Low Flow Flow |
Also Published As
| Publication number | Publication date |
|---|---|
| JP3012493B2 (en) | 2000-02-21 |
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