JPS62246594A - Purification of glutathione - Google Patents

Abstract

PURPOSE:To obtain high-purity glutathione useful as a raw material for pharmaceuticals in high yield, by purifying a glutathione-containing liquid with a pseudo-moving bed using a porous strong acid-type ion-exchange resin as an adsorbent and using a low concentration acid aqueous solution s a desorbing liquid. CONSTITUTION:A front end of a packed bed receiving a porous strong acid-type ion-exchange resin therein is first connected to a rear end thereof with a liquid passage to constitute endless pass so that the liquid may recycle in one direction. Then an inlet of an amino acid-coexisting, glutathione-containing liquid as a raw material, an outlet of the glutathione aqueous solution, an inlet of a desorbing liquid and an outlet of the amino acid aqueous solution are equipped successively along the direction of the stream of liquid, and these positions are gradually moved intermittently along the direction of the stream. Into the pseudo-moving bed constituted as mentioned above, said glutathione-containing liquid as the raw material and 0.01-0.5N acid or alkali aqueous solution as the desorbing liquid are introduced and the purified glutathione aqueous solution and the impurity amino acid aqueous solution are taken out.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is directed to extracts from yeast cell extracts, plant cell extracts, glutathione-containing liquids obtained by synthetic methods, etc. that contain amino acids such as glutamic acid together with glutathione. ,
The present invention relates to a method for separating glutathione.

[Prior art] Glutathione is a physiologically active tripeptide that is widely distributed in yeast and animal livers, and is involved in the redox system in living organisms and plays important roles such as activation of various enzymes and detoxification. , is an extremely useful substance medicinally.

When glutathione is isolated from a glutathione-containing liquid containing impurities, such as a yeast extract, it is generally recovered by crystallization, but amino acids such as glutamic acid are a factor that greatly reduces the crystal purity and yield of glutathione. It becomes. Therefore, it is essential to remove amino acids such as glutamic acid before the final crystallization operation in order to obtain a highly pure product, but glutathione, like amino acids such as glutamic acid, has an amino group and a carboxyl group in its molecule. It is extremely difficult to separate glutathione and amino acids from each other because they have similar physicochemical properties.

Conventionally, the copper salt method is widely known as a method for isolating and purifying glutathione from glutathione-containing solutions, but the general process of the copper salt method involves three steps: copper salt production reaction, copper salt washing, and copper salt decomposition reaction. It consists of stages, the operation is complicated, and
It also has the disadvantage of a low recovery rate of glutathione.

In addition, a method for obtaining a liquid containing high concentration of glutathione from a yeast cell extract using an ion exchange resin was published in Japanese Patent Publication No. 44-2.
39, 46-4755, 46-2838, etc., these methods simultaneously elute glutathione and amino acids such as glutamic acid using highly concentrated salts, acids, and aqueous alkali hydroxide solutions. Separation of glutathione and amino acids is incomplete, making it impossible to obtain a highly pure aqueous glutathione solution and requiring additional steps. Therefore, the development of a process that can easily purify glutathione to a high degree of purity has been strongly desired for industrialization.

[Structure of the Invention] As a result of various studies conducted by the present inventors on a method for isolating glutathione in high yield from a glutathione-containing liquid in which impurities such as glutamic acid coexist, the present inventors found that a porous strongly acidic ion exchange resin was used as an adsorbent. The present inventors have discovered that it is possible to separate glutathione from impurities using a simulated moving bed by using a low-concentration acid aqueous solution or alkali aqueous solution as a desorption liquid, leading to the completion of the present invention.

That is, the present invention provides a packing system in which a porous strongly acidic ion exchange resin is housed inside, and the front end and the rear end are connected by a liquid passage to form an endless shape, and the liquid circulates in one direction. The method consists of introducing a glutathione-containing solution in which amino acids coexist and a 0.01 to 0.5 N acid or alkali aqueous solution as a desorption solution into the bed, and simultaneously extracting the glutathione aqueous solution and the amino acid aqueous solution from the packed bed. A glutathione-containing liquid inlet containing an amino acid, a glutathione aqueous solution outlet, an acid or alkali aqueous solution inlet, and an amino acid aqueous solution outlet are arranged in this order along the direction of fluid flow, and these are connected to the flow of fluid in the bed. The present invention is directed to a method for purifying glutathione, which is characterized by using a simulated moving bed in which the positions thereof are moved intermittently and sequentially in the direction of flow.

That is, the present invention provides yeast cell extract, plant cell extract,
From a glutathione-containing liquid etc. obtained by a synthetic method, a high-quality glutathione-containing liquid is obtained using a rotary bed using a porous strongly acidic ion exchange resin as an adsorbent and an acid or alkaline aqueous solution as a desorption liquid. It is about the method.

A packed bed containing a solid adsorbent and having a leading end and a trailing end connected by a fluid passageway to allow fluid to circulate through the bed. A desorbed liquid fluid inlet, an adsorbate fluid outlet, a raw material fluid inlet, and a non-adsorbent fluid outlet are provided along the direction, and the respective fluids are continuously introduced or extracted from each inlet and outlet to maintain a constant flow rate. By sequentially switching each inlet and outlet with those downstream, the feed fluid can be divided into components that are relatively easily adsorbed by the solid adsorbent (adsorbate components) and components that are relatively difficult to adsorb (non-adsorbate components). ) is known (Japanese Patent Publication No. 42-15681), and examples using such technology include a method for producing fructose (Japanese Patent Application Publication No. 88335-1988) and a method for separating maltose ( Tokukai Showa 6
O-67000), etc. However, no application example has been reported yet regarding the method of separating high-purity glutathione from a system containing glutathione and amino acids using a simulated moving bed, and it is considered difficult to apply it.
The present invention will be explained in more detail.

Porous strongly acidic ion exchange resins used in the present invention include those having an SOs group as an exchange group and a copolymer of styrene and divinylbenzene as a backbone, such as Amberlite IR200C manufactured by Rohm & Haas Co., Ltd., Mitsubishi Kasei Co., Ltd. There are various products such as Diaion Pk22g manufactured by Co., Ltd., but the product is not limited to these. Further, the particle size of the ion exchange resin is not particularly limited, but it is preferably 300 to 600 μm in order to prevent uneven flow within the bed. Further, the pore diameter of the ion exchange resin is preferably 50 to 150 pores from the viewpoint of selectivity, but is not particularly limited thereto.

If the desorption liquid used is an acid, the type of ion exchange resin is
[H'''''' In the case of a U-type aqueous alkali solution, it is necessary to use the same type of metal as the alkali (for example, [Na''] type when the desorbing liquid is a NaOH aqueous solution), but the former is better than the latter. high resolution can be obtained.

In addition, the glutathione-containing liquid may be used as is, but
The separation ability can be improved by adjusting the concentration of the same type of acid or alkali as that of the desorbing liquid to be equal to that of the desorbing liquid.

On the other hand, the higher the temperature during liquid passage, the higher the resolution can be obtained.
At temperatures above 0°C, the decomposition of glutathione becomes significant, so 10
~40°C is desirable.

Further, examples of acids used as the desorption liquid include hydrochloric acid, sulfuric acid, etc., and examples of alkalis include sodium hydroxide, barium hydroxide, etc., but the invention is not particularly limited to these. However, the migration speed of amino acids such as glutathione and glutamic acid in the adsorbent packed bed differs depending on the concentration of the desorbing solution used, so in order to obtain high separation power, the difference in migration speed between the two must be 0.01. It is preferable to use a desorption solution with a concentration of ~0.5N.

Hereinafter, the method of the present invention will be explained in more detail based on the drawings.

FIG. 1 is a schematic diagram of an example of a simulated moving bed used in the method of the present invention. In Figure 1, the inside of the packed bed, which is the main part of the simulated moving bed, is divided into 16 unit packed beds, but the number varies depending on the composition and concentration of the glutathione-containing liquid, the size of the equipment, etc. Can be appropriately determined according to the factors. In FIG. 1, each unit packed bed is filled with a porous strongly acidic ion exchange resin, and a space is provided between each unit packed bed. Each space has an inlet for introducing a glutathione-containing liquid containing amino acids such as glutamic acid into the packed bed, an inlet for an acid or aqueous alkali solution, an outlet for extracting a purified glutathione liquid from the packed bed, and an outlet for extracting an amino acid-containing liquid such as glutamic acid from the packed bed. The type is open (however, the first
Most of them are omitted in the figure. ). Although the installation of this space is not essential, when the glutathione-containing liquid containing amino acids such as glutamic acid and the desorbed liquid are introduced into this space, they are quickly absorbed into the fluid flowing down and circulating in the bed. This is preferable because it allows for diffusion mixing.

In FIG. 1, a glutathione-containing liquid containing an amino acid such as glutamic acid is introduced into the space 19, and an acid or alkaline aqueous solution is introduced into the space 11 as a desorption liquid. Further, an amino acid-containing liquid exclusively for glutathionic acid is extracted from the space 15, and a purified glutathione liquid is extracted from the space 23. Therefore, the packed beds include an adsorption zone consisting of four unit packed beds 109 to 112, a primary purification zone consisting of four unit packed beds 113 to 116, and a four unit packed bed 101 to 104.
a desorption zone consisting of unit packed beds of 105 to 108
The secondary purification zone consists of four unit packed beds. The action of each zone is equivalent to that of a known simulated moving bed in which the adsorbate component is an amino acid such as glutamic acid and the non-adsorbate component is glutathione.

A concentration distribution of amino acids such as glutathione and glutamic acid is formed in the liquid in the packed bed, and this concentration distribution moves downstream while maintaining its shape. Following this movement, the inlet for the glutathione-containing liquid containing amino acids such as glutamic acid and the acid or alkaline aqueous solution to the packed bed, and the outlet for the extraction of the purified glutathione liquid and the amino acid-containing liquid such as glutamic acid from the packed bed are sequentially opened downward. It can be switched to that. The switching may be performed simultaneously for the four types of openings, or may be performed at different times for each opening. The time to continue introducing or withdrawing liquid from the same opening varies depending on the size of the unit packed bed, the concentration of the acid or alkaline aqueous solution used, the flow rate within the bed, etc., but it is usually several minutes to several tens of minutes. It is. By this switching, the four zones described above sequentially move their positions in the packed bed. However, the length of each zone is always approximately constant and maintains its size and relative position as it circulates through the packed bed.

The degree of separation of amino acids such as glutathione and glutamic acid in a simulated moving bed using a porous strongly acidic ion exchange resin as an adsorbent is affected by various factors, but the most important factor is the flow rate of the liquid in the bed, the same The time period during which liquid is continued to be introduced or withdrawn from the opening, and the concentration of the acid or alkaline aqueous solution used as the desorption liquid. This means that switching to the openings downstream of the liquid inlet and outlet is equivalent to moving the porous strong acidic ion exchange resin upstream while keeping the inlet and outlet at a constant position. It is also inferred from the fact that the concentration distribution of each component in the bed is formed by this interaction with the liquid moving in the upstream direction. Further, this moving speed corresponds to the length (12) of each unit packed bed divided by the time (T) for which liquid is continued to be introduced or extracted from the same opening (Q/T).

As is well known, in order to separate two or more components using a simulated moving bed, the moving velocity V of the non-adsorbate component in the packed bed is set to v in the adsorption zone, >(1/T in the primary purification zone), is v I < 121 T s In the desorption band, Vl
>12/T% In the secondary purification zone, Vt > Q/T, and the moving velocity V of the adsorbate component in the packed bed is Vt < Q/T in the adsorption zone, and v2 < in the 1-blowing purification zone. Q/T.

In the desorption zone, v>Q/T, and in the secondary purification zone, v*<Q/T. Therefore, among the above factors, the flow rate of the liquid and the time for which the liquid is continuously introduced or extracted from the same opening are necessarily determined from these relationships. On the other hand, the moving speed V of non-adsorbable components within the packed bed and the moving speed V of adsorptive components within the packed bed depend on the concentration of the acid or alkaline aqueous solution used as the desorption liquid and the liquid flow rate within the packed bed. However, it goes without saying that these can be easily measured using a batch-type packed bed.

Next, the present invention will be specifically explained using examples.
The present invention is not limited thereto.

[Example 1] Glutathione was isolated from a glutathione-containing liquid containing glutamic acid using a simulated moving bed consisting of 16 columns each having an inner diameter of 1 am and a length of 20 cm.

The concentrations of glutathione and glutamic acid in the raw material solution were 10 p/(1.5 v/I), and 0.05 N hydrochloric acid was contained. 1R20 manufactured by Haas Co., Ltd.
0C), and 0.05N hydrochloric acid was used as the desorption liquid. The raw material liquid supply rate is 2.83x (2/min, the desorption liquid supply rate LtlO,83z&/min, the extraction of the glutathione purified liquid is 5゜65m<2/min, and the withdrawal of the glutamic acid-containing liquid is 8゜01x (1 The flow rate of the raw material liquid and the desorbed liquid was changed every 20 minutes.

FIG. 2 shows temporal changes in the concentrations of glutathione and glutamic acid contained in the purified glutathione solution. As shown in FIG. 2, the purified glutathione solution contained no glutamic acid and reached a steady state in about 250 minutes, and in the steady state, 99% of the glutathione contained in the raw material solution was recovered.

In addition, glutathione was separated from the glutathione-containing solution using 1N hydrochloric acid as a desorption solution, but in this case, the glutathione purity in the glutathione purified solution in a steady state was 75%, and the yield was low. The rate was 60%.

[Example 2] Example! Glutathione was isolated from the same glutathione-containing solution as that used in the above using Na'-type porous ion exchange resin Amberlite 1R200C as an adsorbent.
The reaction was carried out using a simulated moving bed using 01N sodium hydroxide as a desorption liquid. However, there is no hydrochloric acid in the raw material liquid.
It contained 0.01 normal sodium hydroxide. The supply of the raw material liquid and the desorbed liquid and the withdrawal of the purified glutathione liquid and the glutamic acid-containing liquid were performed at flow rates of 2.83πQ/min, 9.49xQ/min, 4.25zlJ/min, and 8.07ml/min, respectively. The raw material liquid and desorbed liquid supply ports and the glutathione purified liquid and glutamic acid-containing liquid outlet were moved every minute. FIG. 3 shows the time changes in the glutathione and glutamic acid concentrations in the glutathione purified solution.

As shown in FIG. 3, the glutathione concentration in the purified geltadeone solution reached a steady state in about 30 minutes, and in the steady state, a purified geltadeone solution with a purity of 78% was obtained with a glutathione recovery rate of 70%.

In addition, as a desorption liquid! Glutathione was isolated from the glutathione-containing solution using the specified sodium hydroxide, but in this case, the glutathione purity in the glutathione purified solution in steady state was 66%, and the yield was 58%. Met.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram of an example of a simulated moving bed used in the method of the present invention, and FIGS. 2 and 3 are graphs showing temporal changes in the concentrations of glutathione and glutamic acid contained in the purified glutathione solution. . Patent applicant Kanebuchi Kagaku Kogyo Co., Ltd. Agent Patent attorney Aohaku Ao and 2 others 4 degrees (9/R)
) Concentration [a/JP]

Claims (3)

[Claims] (1) A packed bed in which a porous strong acidic ion exchange resin is housed, the front end and the rear end are connected by a liquid passage to form an endless structure, and the liquid circulates in one direction. The method consists of introducing a glutathione-containing solution in which amino acids coexist and a 0.01 to 0.5 N acid or alkaline aqueous solution as a desorption solution, and simultaneously extracting a glutathione aqueous solution and an amino acid aqueous solution from a packed bed. A glutathione-containing liquid inlet, a glutathione aqueous solution outlet, an acid or alkali aqueous solution inlet, and an amino acid aqueous solution outlet are arranged in this order along the direction of fluid flow, and these are arranged in the direction of fluid flow in the bed. 1. A method for purifying glutathione, which comprises using a pseudo moving bed in which the positions of the glutathione are sequentially moved intermittently. (2) The method according to claim 1, in which an H^+ type porous strongly acidic ion exchange resin is used as the ion exchange resin, and a 0.01 to 0.5 N acid aqueous solution is used as the desorption liquid. (3) The method according to claim 1, in which a salt-type porous strongly acidic ion-exchange resin is used as the ion-exchange resin, and a 0.01-0.5N aqueous alkaline solution is used as the desorption liquid.