JPH0617344B2 - Valine separation and purification method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for separating and purifying valine, and more specifically,
An aqueous solution of valine containing at least an impurity mainly containing one or more of an acidic amino acid, a sulfate group, a chloride ion and a dye is subjected to ion exclusion chromatography using a strongly acidic cation exchange resin to remove the valine solution from the aqueous solution of valine. The present invention relates to a method for separating and purifying high-purity valine in high yield by removing such impurities.

Valine is usually produced by a fermentation method, and one method is a fermentation method using glucose as a main raw material. The valine fermentation broth obtained by this method contains several kinds of by-produced amino acids. In addition, it also contains impurities such as sulfate radicals, chloride ions, and pigments. Such a fermented liquor is a typical example of an aqueous valine solution to be treated in the present invention, as described below. The same applies to valine obtained by other methods.

As a method for separating and purifying valine in a valine fermentation solution, a method of purifying by repeating crystallization, particularly a method of separately crystallizing and separating a solution containing leucine and isoleucine having a similar structure by a specific method (Patent Document 1 56-1645
0), and there is a method of adsorbing valine after adsorbing it on a strongly acidic cation exchange resin and then passing through contaminants, but valine flows through without ion exchange with the resin due to pH fluctuation,
There are problems in that yield loss is caused, that chemicals such as acid and alkali are used to regenerate the resin, and that the operation is complicated. Further, in the case of the crystallization method, there is a problem in that the yield is lowered because the crystallization is repeated.

As a result of diligent research, the present inventors have found that acidic amino acids, sulfate radicals,
In a method for separating and purifying extremely high purity valine from a valine fermentation liquor which is contaminated with impurities mainly containing one or more of chlorine ions and pigments, as a step, ion exclusion chromatography using a strongly acidic cation exchange resin is used. The present invention has been completed by finding that valine of high purity can be obtained in high yield by an extremely simple operation by treating. However, the application of the present invention is not limited to the treatment of such a valine fermentation broth as described later.

In general, non-electrolyte or weakly electrolyte compounds can be separated from strong electrolyte compounds by ion exclusion chromatography. This is because the strong electrolyte compound is excluded by the Donnan potential due to the charged ion-exchange group, so that it cannot penetrate into the ion-exchange resin, but the non-electrolyte or weak-electrolyte compound can freely penetrate. . The present invention is based on this law.

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

The valine aqueous solution containing at least an acidic amino acid, a sulfate group, a chloride ion, and an impurity mainly composed of one or more of pigments in the present invention means a valine fermentation solution, a valine sterilization fermentation solution obtained from the fermentation solution, Examples thereof include a valine crude crystal solution and a valine crystallization mother liquor. In addition to this, acidic amino acids, sulfates, chloride ions, and pigments 1
Alternatively, the present invention can be applied to any aqueous solution containing valine mainly containing two or more impurities. There is no particular limitation on the concentration of valine in such an aqueous solution,
It is sufficient if valine is dissolved.

When subjecting an aqueous solution of valine containing impurities to ion-exclusion chromatography, first, the aqueous solution of valine is adjusted to an isoelectric point (pH = 5.96) of valine or a pH in the vicinity thereof so that most of valine is brought into an uncharged state. To do. Acidic amino acids, sulfates and chlorides exist as anions at that pH.

On the other hand, the strongly acidic cation exchange resin is in the form of a cation that is a counterion of such anion. For example,
In the case of valine fermented liquor, since the acidic amino acid, sulfate and chloride ion are usually in the form of ammonium salt, the strongly acidic cation exchange resin is used in the ammonium salt form.

By the way, if there are multiple cations in the aqueous solution to be subjected to ion exclusion chromatography, it is advisable to treat the cation exchange resin with an aqueous solution containing the multiple cations in advance. Sex decreases. Therefore, in order not to lower the separability, it is preferable to remove the contaminating cations by performing a pretreatment such as ion exchange in the cation exchange resin in advance. Although ion-exclusion chromatography can be performed using an anion exchange resin, in the case of valine which is the object of the present invention, at the isoelectric point of valine, acidic amino acids, sulfate radicals and chloride ions are present in the form of anion, that is, Since there are many anionic species, the separability is reduced and it is not practical.

The strongly acidic cation exchange resin used in the present invention is Diaion SK-102, SK-104, SK-106, SK1B, SK-104S, SK1BS and U.
BK-101L (Made by Mitsubishi Kasei), XFS-43279, XFS-43280, XFS
-43281, HCR-W2 and TG8500A (Dow Chemical Co.), C-2
0, C-25D, ES-26 and C-3 (made by Duolite), S-10
0, S-109, SP-112 and SP-120 (manufactured by Levatit) and
IR-116, IR-118, IR-120B, IR-122, IR-124, IR-252, I
Mainly styrene resins such as R-200C and IR-200CT (manufactured by Amberlite) can be used. Among these, the separation performance of the resin having a cross-linking degree of 4-8% is the best.

The amount of strongly acidic cation exchange resin used is 7 for valine concentration.
%, And in the case of an aqueous solution having an impurity concentration of about 3%, about 4-5 times the amount of the aqueous solution is sufficient. If the total concentration of valine and impurities in the aqueous solution is reduced, the amount of resin may be further reduced. The appropriate amount of resin can be easily determined by those skilled in the art by preliminary experiments.

The operating concentration is not particularly limited as long as it is within the heat resistant temperature of the strongly acidic cation exchange resin. Increasing the temperature increases the rate of separation of contaminants and valine.

A column is filled with a strongly acidic cation exchange resin in a form corresponding to the cation contained in the liquid to be treated, and the liquid to be treated is injected into the upper part of the column according to the above-mentioned standard. For example, in the case of a valine fermentation broth, a column is filled with an ammonium-type strongly acidic cation exchange resin, and an appropriate amount of a valine fermentation broth whose pH is adjusted to or near the isoelectric point of valine is injected into the column.

Then, when water is passed, valine is eluted first after the above-mentioned contaminant impurities are eluted.

Incidentally, even if the valine fermentation liquor to be subjected to the ion exclusion chromatography of the present invention contains bacterial cells and / or pigments, they usually behave as acidic amino acids, sulfate radicals and ammonium salts of chloride ions, and therefore usually cause problems. However, in order to prevent clogging of the resin layer, bacterial cells are removed from the valine fermentation broth in advance, if necessary.

There is no particular limitation on the water passage speed (SV), which is normally 0.
It may be about 5-4. The pH of the eluent, the refractive index, and other changes over time are followed to obtain the target fraction. A desired method may be used to isolate the desired product from the desired product fraction.

Thus, in the purification of valine containing impurities, in the ion exclusion chromatography, the pH of the aqueous solution of valine containing impurities is adjusted to the vicinity of the isoelectric point of the amino acid, and the type of the cation that is the counterion and the anion of the impurity is adjusted. If it is fed to the strongly acidic cation exchange resin, the target product can be obtained by simply elution with water (ion exchanged water), and subsequent washing of the resin is not required. Very easy operability and regeneration of the resin There is also an economic merit that washing, etc. are unnecessary and acids and alkalis for this are also unnecessary.

Example 1 40 ml of an L-valine aqueous solution containing 60 g of L-valine and 2 g of glutamic acid ammonium salt / 15 g / of ammonium sulfate and 15 g / ammonium chloride obtained by removing the L-valine fermentation broth was treated with XFS-43279 (degree of crosslinking 4 %) NH ₄ type (200 ml) was injected into the upper part of a column (φ3.2 cm × H25 cm). Elution was carried out by passing water under the conditions of pH = 5.96, 45 ° C. and SV = 1.0.

First, glutamic acid ammonium salt, ammonium sulfate and ammonium chloride were eluted, and then L-valine was eluted. Collect a fractional fraction with an eluate volume of 80-350 ml,
80-160 ml of which is the sub-fractionation portion, 170-350 ml
It was the main fractionation section. The main fraction was mostly L-valine, and the removal rates of ammonium glutamic acid salt, ammonium sulfate and ammonium chloride were 91% and 93%, respectively.
%, 89%, and the recovery rate of L-valine was 99%. The initial coloring degree of the valine aqueous solution was 2.38 (spectrophotometer 400 nm), but that of the main fractionation part was 0.1 on average.
The color removal rate was 78%.

Example 2 20 ml of an aqueous L-valine solution containing 50 g / l of L-valine and 10 g / ammonium sulfate was treated with NH ₄ of SK104S (degree of crosslinking 4%).
The mold was injected into the upper part of a column (L / D = 12) packed with 100 ml. Elution was carried out by passing water under the conditions of pH = 9.0, 60 ° C. and SV = 1.5.

Ammonium sulphate was eluted first, followed by L-valine. Fractions with an eluate volume of 60-150 ml were collected, of which 60-100 ml was a subfraction, 110-15
0 ml was used as the main fractionation part. Most of the main fraction was L-valine, and the ammonium sulfate removal rate was 96.0%.
-The recovery of valine was 99.0%.

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Claims (1)

[Claims] 1. A pH of an aqueous valine solution containing at least one or more of an acidic amino acid, a sulfate group, a chloride ion, and a pigment as a main component is adjusted to a pH near the isoelectric point of valine, and the anion pair of the impurity is previously prepared. A method for separating and purifying valine by ion exclusion chromatography, which comprises feeding to a strongly acidic cation exchange resin adjusted to the form of cations, and then eluting with water or ion exchange water.