JPH0662669B2 - Method for purifying glutathione - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to glutathione-containing amino acids such as glutamic acid, for example, yeast cell extract, plant cell extract, glutathione-containing solution obtained by a synthetic method, and the like. , A method for separating glutathione.

[Prior Art] Glutathione is a bioactive tripeptide that is widely distributed in yeast and animal livers, is involved in the redox system in vivo, and plays an important role in activating and detoxifying various enzymes. , A very important substance in medicine.

When glutathione is isolated from a glutathione-containing solution containing impurities such as yeast extract, it is generally recovered by a crystallization operation or the like, but amino acids such as glutamic acid are factors that significantly reduce the crystal purity and yield of glutathione. Has become. Therefore, it is indispensable to remove amino acids such as glutamic acid before the final crystallization operation in order to obtain a high-purity product of glutathione, but glutathione contains amino groups and amino groups in the molecule like amino acids such as glutamate. Mutual separation between glutathione and amino acids is extremely difficult because they have a carboxyl group and their physicochemical properties are similar.

Conventionally, the copper salt method is widely known as a method for isolating and purifying glutathione from a glutathione-containing liquid, but the general process of the copper salt method is a copper salt forming reaction, a copper salt washing, and a copper salt decomposing reaction. It consists of stages, complicated operation, and
It has a drawback that the recovery rate of glutathione is also low.

Further, a method for obtaining a high-concentration glutathione-containing solution from a yeast cell extract using an ion-exchange resin is disclosed in JP-B-44-2.
39, 46-4755, 46-2838 and the like, in these methods, in order to eliminate glutathione and amino acids such as glutamic acid at the same time using high-concentration salts, acid, and aqueous alkali hydroxide solution, Separation of glutathione and amino acids is incomplete, and it becomes impossible to obtain a highly pure glutathione aqueous solution, and further steps are required. Moreover, in these methods, the salts, acids, and alkali hydroxides used for desorption are mixed in the eluate, and it is necessary to remove these before the crystallization operation. Further, if a low-concentration acid, salt, or alkaline aqueous solution is used as a desorbing solution, the purity of the purified solution is improved, but the concentration of the purified solution is extremely lowered, and it is said that it is not suitable for practical use. Therefore, development of a process by which a highly purified glutathione purified solution with high concentration can be easily obtained has been strongly desired in industrialization.

[Structure of the Invention] The present inventors have conducted various studies on a method of isolating high-purity, high-concentration glutathione in high yield from a glutathione-containing solution in which impurities such as glutamic acid coexist. It was found that by using a porous strongly acidic ion exchange resin as an adsorbent and water as a desorbent, it is possible to separate glutathione from impurities and a high-concentration glutathione aqueous solution can be obtained. Was completed.

That is, in the present invention, the porous strongly acidic ion exchange resin is housed inside, and the front end and the rear end are joined by the liquid passage to form an endless form, and the liquid is circulated in one direction. Into the bed, water is introduced as a glutathione-containing solution in which amino acids coexist and a desorption solution, and at the same time, a glutathione aqueous solution and an amino acid solution are extracted from the packed bed,
In the packed bed, a glutathione-containing solution inlet port containing amino acid, a glutathione aqueous solution outlet port, a water inlet port and an amino acid aqueous solution outlet port are arranged in this order along the direction of fluid flow, and It is intended to provide a method for purifying glutathione, which is characterized by using a simulated moving bed which comprises sequentially moving those positions in the flow direction intermittently.

That is, the present invention is a yeast cell extract, plant cell extract, from the glutathione-containing solution obtained by the synthetic method, a porous strongly acidic ion exchange resin as an adsorbent, by a simulated moving bed using water as a desorbent, The present invention relates to a method for obtaining a highly pure and high-concentration glutathione-containing liquid.

A solid adsorbent is housed, and a fluid passage is connected between the front end and the rear end of the bed so that the fluid can circulate in the bed. Desorbed fluid inlet, adsorbate fluid outlet, raw material fluid inlet,
Non-adsorbate fluid outlet is provided, and each fluid is continuously introduced or withdrawn from each inlet and outlet, and the source fluid is fixed by switching each inlet and outlet with the downstream one at regular intervals. A so-called simulated moving bed, which separates into a component that is relatively easily adsorbed by an adsorbent (adsorbate component) and a component that is relatively difficult to be adsorbed (non-adsorbate component), is known (Japanese Patent Publication No. 42-15681).
Examples of utilizing such a technique include a method for producing fructose (JP-A-53-88335) and a method for separating maltose (JP-A-60-67000). However, an application example of a method for separating glutathione with high purity from a system containing glutathione or amino acids using a simulated moving bed has not been reported at all, and it has been considered difficult to apply.

Hereinafter, the present invention will be described in more detail.

The porous ion exchange resin used in the present invention includes
Having SO ₃ group as an exchange group and a skeleton of a copolymer of styrene and divinylbenzene, for example, Rohm & Haas Co., Ltd.
There are various products such as Amberlite IR200C manufactured by Mitsubishi Kasei Co., Ltd., Diaion pk228 manufactured by Mitsubishi Kasei Co., Ltd., but not limited to these. Among them, H ⁺ type porous ion exchange resin is preferable. The particle size of the ion exchange resin is not particularly limited, but in order to prevent uneven flow in the bed, it is 300 to 600.
μm is preferable. Further, the ion exchange resin having a pore size of 50 to 150 Å is desirable in terms of selectivity, but is not particularly limited thereto.

The higher the temperature at which the solution is passed, the higher the resolution is.
Degradation of glutathione becomes significant above ℃, so 10-
40 ° C is desirable. Further, as the water used as the desorbing liquid in the present invention, general tap water, industrial water, ion-exchanged water, distilled water and the like can be applied, but if ions such as calcium are contained, the capacity of the ion-exchange resin is lowered. It is desirable to use ion-exchanged water or distilled water because it causes the cause.

Hereinafter, the method of the present invention will be described in more detail with reference to the drawings.

FIG. 1 is a schematic view of an example of a simulated moving bed used in the present invention. In FIG. 1, the inside of the packed bed, which is the main part of the simulated moving bed, is divided into 16 unit packed beds.
The number can be appropriately determined according to factors such as the composition and concentration of the glutathione-containing liquid, the size of the device, and the like. 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. There are four types of openings in each space: the inlet for the glutathione-containing solution containing amino acids such as glutamic acid to the packed bed and the inlet for water, and the outlet for the purified glutathione solution from the packed bed and the outlet for the liquid containing amino acids such as glutamic acid. (However, most of them are omitted in FIG. 1). It is not essential to install this space, but if the glutathione-containing liquid containing amino acids such as glutamic acid and the desorbed liquid, which are introduced into the packed bed, are introduced into this space, they are quickly introduced into the fluid flowing down and circulating in the bed. It is preferable because it can be mixed by diffusion.

In FIG. 1, a glutathione-containing liquid containing an amino acid such as glutamic acid is introduced into the space portion 19, and water is introduced into the space portion 11 as a release liquid. In addition, an amino acid-containing liquid such as glutamic acid is extracted from the space portion 15, and a glutathione purified liquid is extracted from the space portion 23. Therefore, the packed bed is an adsorption zone consisting of 4 unit packed beds of 109 to 112, a primary purification zone consisting of 4 unit packed beds of 113 to 116, and a desorption consisting of 4 unit packed beds of 101 to 104. The zone consists of four zones, a secondary purification zone consisting of four unit packed beds of 105 to 108. The action of each zone is to use amino acids such as glutamic acid as an adsorbate component,
This is equivalent to that of the known simulated moving bed when glutathione is used as the non-adsorbate component.

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. In order to follow this movement, the inlet for glutathione-containing liquid or water containing amino acids such as glutamic acid to the packed bed and the outlet for the purified glutathione liquid and the liquid containing amino acids such as glutamic acid from the packed bed are sequentially switched to that downward. . The switching may be performed for four types of openings at the same time, or may be shifted for each opening in time. The time for continuing the introduction or withdrawal of the liquid from the same opening varies depending on the size of the unit packed bed, the flow rate of the liquid flowing through the bed, etc., but is usually several minutes to several tens of minutes. By this switching, the above-mentioned four zones sequentially move to the positions occupied in the packed bed. However, the length of each zone is always almost constant and circulates through the packed bed while maintaining its size and relative position.

The degree of separation of glutathione and amino acids such as glutamic acid in a simulated moving bed using a porous strongly acidic ion-exchange resin as an adsorbent is affected by various factors. It is the time to continue introducing or withdrawing the liquid from the opening. This means that switching the inlet and outlet of the liquid to the downstream opening is equivalent to moving the porous strong acid resin in the upstream direction while keeping the inlet and outlet positions constant. It is also inferred from the fact that the concentration distribution of each component in the bed is formed by the interaction with the liquid moving in the upstream direction. The moving speed corresponds to the length (l) of each unit packed bed divided by the time (T) during which the liquid is continuously introduced or withdrawn from the same opening (l / T). As is well known, in order to separate two or more components by a simulated moving bed, the moving speed v ₁ of the non-adsorbate component in the packed bed is v ₁ > l / T in the adsorption zone and in the primary purification zone. v ₁ <l /
T, v ₁ in the desorption zone> l / T, in the secondary refining zone and v _1> l / T, in the adsorption zone the moving speed v ₂ in the packed bed adsorptive component v ₂ <l / T in the primary purification zone, v ₂ <1 / T, in the desorption zone v ₂ > 1 / T,
In the secondary purification zone, v ₂ <1 / T may be set. Therefore, the time during which the liquid flows down and the liquid is continuously introduced or withdrawn from the same opening is inevitably obtained from these relationships. On the other hand, the moving velocity v ₂ of the non-adsorbates in the packed bed moving speed v ₁ and adsorbate component ingredients, is determined by the liquid flow rate in the packed bed, easily using a packed bed of these batch It goes without saying that you can actually measure.

Next, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

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

The concentrations of glutathione and glutamic acid in the raw material liquid were 10 g / l and 5 g / l, respectively. An H ⁺ type strongly acidic ion exchange resin (IR200C manufactured by Rohm and Haas Co., Ltd.) was used as the adsorbent, and water was used as the desorption liquid.

Raw material liquid supply rate is 2.83 ml / min, desorption liquid supply rate is 12.25 m
l / min, withdrawal of glutathione purified liquid 3.30 ml /
Min., The glutamic acid-containing liquid is withdrawn at a flow rate of 11.78 ml / min, and the movement of the raw material liquid and the desorbed liquid supply port and the glutathione purified liquid and the glutamic acid-containing liquid withdrawal port is 6
It was done every 0 minutes.

FIG. 2 shows the time-dependent changes in the concentrations of glutathione and glutamate contained in the purified glutathione solution. Second
As shown in the figure, the glutathione purified solution does not contain glutamate at all, and the steady state is reached in about 300 minutes.
99% of glutathione contained in the raw material liquid in the steady state
Was recovered.

[Effects of the Invention] According to the present invention, a highly purified glutathione purified liquid can be obtained without lowering the concentration of glutathione, and since the desorbed liquid is water, the crystallization operation can be performed as it is to obtain glutathione crystals. it can. Further, since the method of the present invention is a continuous operation, it can be easily automated. Further, as an advantage of the simulated moving bed, it is possible to save the amount of adsorbent and desorbed water as an effect of the present invention.

[Brief description 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 FIG. 2 is a graph showing temporal changes in the concentrations of glutathione and glutamate contained in a purified glutathione solution in Examples. is there.

Claims (2)

[Claims] 1. A packed bed in which a porous strongly acidic ion-exchange resin is housed, the front end and the rear end are connected by a liquid passage to form an endless form, and the liquid circulates in one direction. In, water is introduced as a glutathione-containing solution and a desorption solution in which amino acids coexist, and at the same time, the glutathione aqueous solution and the amino acid aqueous solution are extracted from the packed bed, and the packed bed contains a glutathione-containing solution inlet containing amino acids, glutathione. Arranging the aqueous solution outlet, the water inlet, and the amino acid aqueous solution outlet in this order along the fluid flow direction, and intermittently moving their positions in the fluid flow direction in the bed. A method for purifying glutathione, which comprises using a simulated moving bed comprising 2. The method according to claim 1, wherein a H ⁺ type porous strongly acidic ion exchange resin is used as the ion exchange resin.