JPH0617343B2 - Method for separating and purifying isoleucine - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for separating and purifying isoleucine, and more specifically, a strong acid isoleucine aqueous solution containing at least an impurity containing at least one of an acidic amino acid, a sulfate group, a chloride ion and a dye. The present invention relates to a method for separating and purifying high-purity isoleucine in high yield by removing such impurities from such an aqueous solution of isoleucine by subjecting it to ion exclusion chromatography using a cationic cation exchange resin.

Isoleucine is usually produced by a fermentation method, and one method is a fermentation method using glucose as a main raw material. The isoleucine 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 fermentation broth is a typical example of an isoleucine aqueous solution to be treated in the present invention, as described below. The same applies to isoleucine obtained by other methods.

As a method for separating and purifying isoleucine in an isoleucine fermentation solution, a method of purifying by repeating crystallization (a method for purifying hydrochloride, JP-A-59-62554), particularly for a solution containing isoleucine and valine having similar structures, A method of separating crystals by fractional crystallization by a specific method (Japanese Patent Laid-Open No. 56-1645).
No. 0, JP-A-50-123622), and a method of adsorbing isoleucine after adsorbing it on a strongly acidic cation-exchange resin to allow impurities to flow through (JP-A-50-126878, JP-A-56-131550). The problem is that isoleucine flows through the resin without ion exchange due to pH fluctuations, resulting in yield loss, the use of chemicals such as acids and alkalis for resin regeneration, and the complicated operation. There is. 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 of separating and purifying extremely high-purity isoleucine from an isoleucine 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 it is possible to obtain high-purity isoleucine in a high yield with an extremely simple operation by treatment. However, the application of the present invention is not limited to the treatment of such an isoleucine 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 isoleucine 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 according to the present invention is an isoleucine fermentation solution, an isoleucine-sterilized fermentation solution obtained from the fermentation solution,
Examples thereof include a solution of crude crystals of isoleucine and a mother liquor of crystallization of isoleucine. In addition to the above, the present invention can be applied to any aqueous solution containing isoleucine contaminated with an acidic amino acid, a sulfate group, a chloride ion, and impurities mainly containing one or more of dyes. There is no particular limitation on the concentration of isoleucine in such an aqueous solution, as long as isoleucine is dissolved.

When subjecting an isoleucine aqueous solution containing impurities to ion-exclusion chromatography, first, the isoleucine aqueous solution should be at or near the isoelectric point (pH = 5.94) of isoleucine.
Most of the isoleucine becomes uncharged by adjusting the pH. 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 isoleucine fermented liquor, the acidic amino acid, sulfate and chloride ion are usually in the form of ammonium salt, so 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 established using an anion exchange resin, in the case of isoleucine which is the object of the present invention, at the isoleucine isoelectric point, acidic amino acids, sulfate radicals and chloride ions exist in the form of anions, that is, anions. Since there are many species, the separability is reduced and it is not practical.

The strongly acidic cation exchange resin used in the present invention includes Diaion SK-102, SK-104, SK-106, SK1B, SK-104S, SK1BS and
UBK-101L (Made by Mitsubishi Kasei), XFS-43279, XFS-43280, XF
S-43281, HCR-W2 and TG8500A (manufactured by Dow Chemical Co.), C-
20, 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.

In the case of an aqueous solution having an isoleucine concentration of about 3% and an impurity concentration of about 2%, the amount of the strongly acidic cation exchange resin used is about 4 to 5 times the amount of the aqueous solution. If the concentration of isoleucine and impurities in the aqueous solution becomes smaller,
The amount of resin may be smaller. The appropriate amount of resin can be easily determined by those skilled in the art by preliminary experiments.

The operating temperature 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 degree of separation between contaminants and isoleucine.

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 isoleucine fermentation broth, a column of ammonium-type strongly acidic cation exchange resin is packed, and an appropriate amount of isoleucine fermentation broth whose pH is adjusted to or near the isoelectric point of isoleucine is injected into the column.

Next, when water is passed, first, the above-mentioned contaminant impurities are eluted and then isoleucine is eluted.

Incidentally, even if the isoleucine fermentation liquor to be subjected to the ion-exclusion chromatography of the present invention contains bacterial cells and / or pigments, since they behave together with acidic amino acids, sulfate radicals and ammonium salts of chloride ions, they are usually problematic. However, in order to prevent clogging of the resin layer, bacterial cells are removed from the isoleucine fermentation solution 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 separation and purification of isoleucine containing impurities, by adjusting the aqueous solution of isoleucine containing impurities in the vicinity of its isoelectric point by ion exclusion chromatography, most of the amino acids are brought into the non-charged state, thereby removing impurities from anions. It is present in a state of being present, and by feeding it to a strongly acidic positive on-exchange resin in the form of a cation that is a counter ion with this anion, subsequent elution and re-feeding can be performed only with water (ion exchanged water), which is extremely simple Operability and elution,
There is an economic merit that no acid or alkali is required for cleaning or regeneration.

Example 1 40 g of an aqueous L-isoleucine solution containing 29 g / l of L-isoleucine and 2 g / l of glutamic acid ammonium salt, 10 g / l of ammonium sulfate and 9 g / l of ammonium chloride obtained by removing the L-isoleucine fermentation broth was treated with XFS-43279 ( Column filled with 200 ml of NH ₄ type (crosslinking degree 4%) (φ3.2 cm x
H25 cm). pH = 5.96, 60 ℃, SV
Elution was carried out by passing water under the condition of 1.0.

First, glutamic acid ammonium salt, ammonium sulfate and ammonium chloride were eluted, and then L-isoleucine was eluted. Fractions with 80-350 ml eluate were collected, 80-160 ml of which was the sub-fraction, 170-350 ml
Was the main fractionation section. The main fraction is mostly L-isoleucine, and the removal rates of ammonium glutamic acid salt, ammonium sulfate and ammonium chloride are 100.0 each.
%, 98.3% and 97.2%, and the recovery rate of L-isoleucine was 99.9%. The initial isoleucine aqueous solution had a coloring degree of 1.79 (400 nm spectrophotometer), but that of the main fractionated portion was 0.113 on average, and the color removal rate was 75.6%.

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Claims (1)

[Claims] 1. A pH of an aqueous solution of isoleucine containing at least an acidic amino acid, a sulfate group, a chloride ion and an impurity mainly composed of one or more of dyes is adjusted to be near the isoelectric point of isoleucine, and most of isoleucine is uncharged. Ion-exclusion chromatography, characterized in that it is fed to a strongly acidic cation exchange resin that has been adjusted to the type of cation that is the counterion of the impurity anion in advance, and then eluted with water or ion-exchanged water. Isolation method of isoleucine.