JPS6388196A - Purifying method for glutathione - Google Patents

Abstract

PURPOSE:To industrially and advantageously obtain the titled compound having detoxifying action, by intermittently and successively moving inlet ports and taking outlets for each solution in passing an amino acid-containing glutathione solution through a bed filled with a porous strong acidic ion exchange resin and eliminating the glutathione. CONSTITUTION:A pseudo-moving bed, having an inlet port 19 for a glutathione- containing solution containing an amino acid, taking outlet 23 for an aqueous solution of glutathione, water inlet port 11 for an eliminating solution and taking outlet 15 for the aqueous solution of the amino acid arranged in the order mentioned in a packed bed of a structure for circulating a liquid in one direction in which a porous strong acidic ion exchange resin is contained therein and the front and back ends are connected with a fluid passage to form an endless shape in the flow direction of a fluid and capable intermittently and successively moving the positions of the inlets and taking outlets is used to separate and purify the aimed glutathione from a glutathione-containing solution containing the coexisting amino acid.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is directed to extracts containing amino acids such as glutamic acid together with glutathione, such as yeast cell extracts, plant cell extracts, glutathione-containing liquids obtained by synthetic methods, etc. ,
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 important substance in medicine.

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 gelcumic acid before hot cooking to obtain a highly purified glutathione product, but glutathione, like amino acids such as glutamic acid, is a molecule. It is extremely difficult to separate glutathione and amino acids from each other because they have amino groups and carboxyl groups and their physicochemical properties are similar.

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 a copper salt production reaction, copper salt washing, and a copper salt decomposition reaction. It has the disadvantage that it consists of three steps, the operation is complicated, and the recovery rate of glutathione is low.

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 use highly concentrated salts, acids, and aqueous alkali hydroxide solutions to simultaneously eliminate glutathione and amino acids such as glutamic acid. Separation of glutathione and amino acids is incomplete, making it impossible to obtain a highly pure aqueous glutathione solution and requiring additional steps. In addition, in these methods, salts, acids, and alkali hydroxides used for desorption are mixed into the eluate, so it is necessary to remove these before the crystallization operation. Furthermore, if a low concentration acid, salt, or alkaline aqueous solution is used as the desorption liquid, the purity of the purified liquid can be improved, but the concentration of the purified liquid is extremely reduced, so it was considered not to be suitable for practical use. Therefore, the development of a process by which a purified glutathione solution with high purity and high concentration can be easily obtained is strongly desired for industrialization.

[Structure of the Invention] As a result of various studies conducted by the present inventors on a method for isolating high-purity, high-concentration glutathione in high yield from geltadeone-containing liquids in which impurities such as glutamic acid coexist, the present inventors have developed a pseudo-moving bed. The inventors have discovered that, by using a porous strongly acidic ion exchange resin as an adsorbent and water as a desorption liquid, it is possible to separate glutathione from impurities, and a highly concentrated aqueous glutathione solution can be obtained. has been completed.

That is, in the present invention, 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 structure, and the liquid circulates in one direction. The method consists of introducing a glutathione-containing liquid in which amino acids coexist and water as a desorption liquid into the bed, and simultaneously extracting a glutathione aqueous solution and an amino acid aqueous solution from the packed bed. A glutathione aqueous solution outlet, a water inlet, and an amino acid aqueous solution outlet are arranged in this order along the direction of fluid flow, and their positions are intermittently moved one after another in the direction of fluid flow within the bed. The subject matter is a method for purifying glutathione, which is characterized by using a simulated moving bed.

That is, the present invention uses a yeast cell extract, a plant cell extract, and a glutathione-containing liquid obtained by a synthetic method.
Keiji Pass Isun Graduated Fisherman Enomoto Nishi Wakasho 1 and 1-.

The present invention relates to a method for obtaining a highly pure and highly concentrated glutathione-containing liquid using a simulated moving bed using water as a desorption liquid.

A packed bed containing a solid adsorbent and connected between the leading and trailing ends by a fluid passageway to allow fluid to circulate through the bed, along the flow of fluid within the bed. Desorption liquid fluid inlet, adsorbate fluid outlet, raw material fluid inlet,
A non-adsorbent fluid outlet is provided, each fluid is continuously introduced or extracted from each inlet and outlet, and the raw material fluid is fixed by sequentially switching each inlet and outlet to the downstream one for a certain period of time. A so-called pseudo moving bed that separates components into components that are relatively easily adsorbed by an adsorbent (adsorbate components) and components that are relatively difficult to adsorb (non-adsorbate components) is known (Japanese Patent Publication No. 15681/1973).
Examples of the use of such techniques include a method for producing fructose (Japanese Unexamined Patent Publication No. 53-88335) and a method for separating maltose (Japanese Unexamined Patent Publication No. 60-67000). However, no application example has yet been reported regarding a method for separating high-purity glutathione from a system containing glutathione and amino acids using a simulated moving bed, and it has been considered difficult to apply this method.

The present invention will be explained in more detail below.

The porous ion exchange resin used in the present invention includes:
Various products including SO1 group as an exchange group and a copolymer of styrene and vinylbenzene as a backbone, such as Amberlite IR2.00C manufactured by Rohm & Haas Co., Ltd. and Diaion pk228 manufactured by Mitsubishi Kasei Co., Ltd. Yes, but
Not limited to these. Among these, H+ type porous ion exchange resins are preferred. The particle size of the ion exchange resin is not particularly limited, but in order to prevent uneven flow within the bed, the particle size is 300~
600 μm order is desirable. 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 to this.

The higher the temperature during the wake, the higher the separation power can be obtained, but at 50
At temperatures above 10°C, the decomposition of glutathione becomes significant.
40°C is desirable. In addition, the water used as the desorption liquid in the present invention can be general tap water, industrial water, ion exchange water, distilled water, etc. However, if ions such as calcium are contained, the ability of the ion exchange resin may be reduced. It is desirable to use ion-exchanged water or distilled water.

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 pseudo moving bed used in the present invention. In Figure 1, the inside of the packed bed, which is the main part of the pseudo moving bed, is divided into 16 unit packed beds.
The number can be appropriately determined according to factors such as the composition of the glutathione-containing liquid, the concentration and the size of the device. 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 four types of openings: an inlet for introducing a glutathione-containing liquid containing amino acids such as glutamic acid into the packed bed, an inlet for water, an outlet for extracting purified glutathione liquid from the packed bed, and an outlet for extracting a liquid containing amino acids such as glutamic acid. (This is mostly omitted in Figure 1.)

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 added to 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 water is introduced into the space 11 as a desorption liquid. Further, a liquid containing amino acids such as glutamic 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 II2, a primary purification zone consisting of four unit packed beds 113 to 116, and a desorption zone consisting of four unit packed beds 101 to 104. It consists of four zones: a zone and a secondary purification zone consisting of four unit packed beds of 105 to 108 units. The action of each zone is based on amino acids such as glutamic acid as adsorbent components.
A concentration distribution of amino acids such as glutathione and glutamic acid is formed in the liquid in the packed bed that does not adsorb glutathione, and this concentration distribution moves downstream while maintaining its shape. Following this movement, the inlet for the 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 amino acid-containing liquid such as glutamic acid from the packed bed are sequentially switched to the lower one. . The switching may be performed simultaneously for the four types of openings, or may be performed at different times for each opening. The time for which liquid is continued to be introduced or withdrawn from the same opening varies depending on the size of the unit packed bed, the flow rate within the bed, etc., but is usually several minutes to several tens of minutes. 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, maintaining 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 pseudo-polarized bed using a porous strongly acidic ion exchange resin as an adsorbent is influenced by various factors, but the most important factors are the flow rate of the liquid in the bed, This is the time during which liquid is continued to be introduced or withdrawn from the same opening. This means that switching to the downstream openings of the liquid inlet and outlet is equivalent to moving the porous strong acidic resin upstream while keeping the positions of the inlet and outlet constant. It is also inferred from the fact that the concentration distribution of each component in the bed is formed by interaction with the liquid moving in the upstream direction. Further, this moving speed corresponds to the length of each unit packed bed divided by the time (T) for which liquid is continuously introduced or withdrawn from the same opening (CQ/T). As is well known, in order to separate two or more components using a simulated moving bed, the moving velocity of the non-adsorbate component in the packed bed, V, in the adsorption zone is v + > Q / T - in the primary purification zone. v + <Q/'rs In the desorption band, v + > Q/T.

In the secondary purification zone, v+>c/'r, and the moving speed of the adsorbate component in the packed bed, V, is v in the adsorption zone.
t < Q/T, in the primary purification zone Vz <
Q/T, Vv>Q/T in the desorption zone, and v t <Q/T in the secondary purification zone. Therefore, the time for continuous flow of liquid and introduction or withdrawal of liquid from the same opening is necessarily determined from these relationships. On the other hand, the movement speed 1 of the non-adsorbate component and the movement speed V of the adsorbate component in the packed bed are determined by the liquid flow rate in the packed bed, but these can be easily determined using a batch-type packed bed. Needless to say, it can be measured.

Next, the present invention will be specifically explained using Examples, but the present invention is not limited thereto.

[Example] Glutathione was isolated from a glutathione-containing liquid containing glutamine using a simulated moving bed consisting of 16 columns with 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 109/Q and 59/Q, respectively. H as an adsorbent
A porous strongly acidic ion exchange resin of ``'' type (lR200C, manufactured by Rohm and Haas Co., Ltd.) was used, and water was used as the desorption liquid.

The raw material liquid supply rate is 2.8:Lo2/min, and the desorption liquid supply rate is 12.25mN! Furthermore, the glutathione purified liquid is extracted at a flow rate of 3.30if2/min, and the glutamic acid-containing liquid is extracted at a flow rate of 11.78+C/min. Movements were made every 60 minutes.

FIG. 2 shows temporal changes in the concentrations of glutathione and glutamic acid contained in the purified glutathione solution. Second
As shown in the figure, the glutathione purified solution does not contain any glutamic acid and reaches a steady state in about 300 minutes.
At steady state, 99% of the glutathione contained in the raw material solution
was recovered.

[Effects of the Invention] According to the present invention, a highly purified glutathione purified liquid can be obtained without reducing the concentration of glutathione, and since the desorbed liquid is water, it is possible to directly perform the crystallization operation to obtain glucthione crystals. can.

In addition, since the method of the present invention does not require continuous operation, it is possible to easily perform Ippaku glaze (P).Furthermore, an advantage of the simulated moving bed is that the amount of water, which is the adsorbent and the desorbing liquid, can be saved. are cited as effects of the present invention.

[Brief explanation of the drawing]

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 glutamic acid contained in the purified glutathione solution in Examples. be. Patent applicant Kanebuchi Chemical Industry Co., Ltd. Agent Patent attorney Maeda Ao and two others Figure 1 Figure 2 Saimon (Min)

Claims (2)

[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. Water is introduced as a glutathione-containing solution in which amino acids coexist and water is removed as a desorption solution, and at the same time, the glutathione aqueous solution and the amino acid aqueous solution are extracted from the packed bed. The port, water inlet, and amino acid aqueous solution outlet are arranged in this order along the direction of fluid flow,
A method for purifying glutathione, which comprises using a pseudo-moving bed in which the positions of the glutathione are successively moved intermittently in the direction of fluid flow within the bed. (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.