JPH0751546B2 - Method for separating leucine - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel method for separating leucine, and more particularly to a method for selectively separating leucine from an amino acid mixture containing at least leucine and isoleucine.

2. Description of the Related Art Among neutral amino acids, leucine and isoleucine have a wide range of uses as foods, pharmaceuticals, and raw materials thereof. These amino acids are produced by a method such as a fermentation method or an extraction method, and in each case, leucine or isoleucine is contained as an impurity.

Since leucine and isoleucine have a branched side chain as a common structural feature, they have very similar physical and scientific properties, and it was difficult to efficiently separate them industrially.

Conventionally, as a method for separating leucine, a method using an ion exchange resin, a hydrochloride crystallization method, a method using a precipitating agent, and the like are known.

The method using an ion exchange resin has already been established as an excellent amino acid analysis method, but it can be separated only with a very small amount of amino acid. That is, the amount of amino acid that can be separated with one resin is about 1 mg. Furthermore, as the eluate, a buffer solution whose pH is adjusted accurately must be used.
Therefore, it is necessary to separate the amino acid and the buffer solution component, which is industrially unsuitable.

Next, the hydrochloride crystallization method is the most efficient method of separating leucine and isoleucine up to now, and is industrially available. However, this method reduces the difference in solubility between leucine and isoleucine hydrochlorides. It is used, but its selectivity is insufficient. Therefore, in order to obtain high-purity leucine and isoleucine, it is necessary to repeat recrystallization several times, which is problematic in that the cost becomes high (JP-A-56-16450).

Also, as has been known for a long time, there is a method using a precipitant. As a precipitating agent, for example, there is a method of selectively precipitating and separating leucine using benzenesulfonic acid (JP-A-51-149222).

However, when a precipitating agent is used, the precipitation yield is often low, and in particular, the precipitating agent is expensive and has problems such as toxicity. To solve these problems,
It is important to separate, recover and recycle the precipitant and amino acid. The precipitant can be recycled by separating the amino acid and then adjusting the pH again with a small amount of the amino acid, and then removing the precipitant from the separated amino acid.
It's extremely difficult. This is because the precipitating agent is a compound having a strong interaction with the amino acid, and therefore, when the amino acid is crystallized, the precipitating agent is contained as an impurity in the amino acid, and separation is not easy.

Problems to be Solved by the Invention There are few troublesome operations such as recrystallization and pH adjustment, simple and safe, industrial separation of leucine and isoleucine at low cost, and development of a method for obtaining high-purity leucine has been developed. Is desired.

Means for Solving the Problems The present inventors have conducted extensive studies for the purpose of developing a method for separating leucine from an amino acid mixture containing leucine and isoleucine, and as a result, a chelate resin known for optical resolution of amino acids. , That is, chelating resin with L-proline immobilized (VA Dabvankov, SV Rogozhin, JC
hromatogr., 60 (1971) 280-283) It was found that leucine can be selectively separated and the above-mentioned object can be achieved by using, and the present invention has been completed based on this finding.

That is, in the method for separating leucine of the present invention, when separating leucine from an amino acid mixture containing at least leucine and isoleucine, the leucine is selectively contacted with the L-proline-metal complex salt-immobilized resin. It is characterized by being adsorbed and separating leucine from isoleucine and other amino acids.

In the present invention, the amino acid mixed solution used as a starting material may be one containing at least leucine and isoleucine, and may of course contain other amino acids and impurities.

In the separation of leucine and isoleucine using a chelate resin, first, the chelate resin is chelated with a metal ion, and this is passed through an aqueous solution containing leucine and isoleucine. As a result, leucine is selectively adsorbed and isoleucine is eluted. Then, leucine is eluted by elution with an alkaline or acidic aqueous solution. At this time, since the metal ions are eluted together with the amino acid, they are passed through a chelating resin after the separation column to remove the metal ions and produce amino acids. The chelate resin can almost completely remove the eluted metal ions.

Any chelate resin may be used as long as it can selectively chelate leucine. For example, one having the above structure can be used. The amino acid load of this resin is 20
It is about 30 g / l-resin, and amino acids are almost completely recovered. To chelate this chelating resin with metal ions, use a 0.1N aqueous solution of a metal complex salt and add 1 g of chelating resin.
You need 10 to 30 ml per. At this time, it is desirable to keep the pH of this aqueous solution alkaline. The pH at this time and the pH at which the metal complex salt is dissolved and the metal ion is chelate-immobilized on the chelate resin may be used, but it is usually preferable that the pH is 10 or more.

Various kinds of metal ions can be used as the metal ions to be chelated by the chelating resin. That is, copper ion, zinc ion, nickel ion, cobalt ion, iron ion,
Any metal ion having a coordination number of four or more, such as lead ion, rhodium ion, and palladium ion, can be used. In addition, as the salt, many salts such as sulfate, hydrochloride, carbonate, or organic acid salt such as amino acid salt can be used.

When actually separating leucine and isoleucine, the concentrations of these amino acid supply solutions differ depending on the pH. pH is 3
Approximately 9 is desirable, and within this range, almost the same separability is exhibited up to a concentration of about 10 g / dl, and the concentration dependence is small.

However, it is desirable that the feed rate and the elution rate of the feed solution be SV5 or less, and if it exceeds this, it may be difficult to separate leucine and isoleucine.

Usually, when a separation operation is carried out using such a chelate resin, metal ions are eluted, but by utilizing this phenomenon, the L-proline resin was made to be an L-proline-metal complex salt-immobilized resin.

This method uses about one-fifth of the resin in the separation column for metal ion chelation and removal when it is used for optical resolution, but in the present invention, it is used under the column for separating leucine. A similar column is set up to chelate metal ions and remove metal ions, and after this column is saturated with metal ions, it is used for separation. By alternating operation of these two columns, continuous leucine and isoleucine separation steps became possible.

Also, there was a concern that the purified amino acids would be mixed by the elution of metal ions, but this was solved by placing an L-proline resin column next to the leucine separation column. However, it goes without saying that such a recycling method does not limit the method for separating leucine and isoleucine.

As described above, according to the present invention, a chelate resin that selectively adsorbs leucine is used to separate from isoleucine via a metal ion, and a chelate resin similar to this is used to remove a metal ion from an amino acid. By further recycling the metal ion and the chelate resin, leucine and isoleucine can be continuously separated.

Examples Hereinafter, the present invention will be described in detail with reference to Examples.

Example 1 (Separation of leucine and isoleucine) 20 cc chelating resin manufactured by Sumitomo Chemical Co., Ltd. was added to 300 ml of 0.1N copper sulfate-0.5N ammonia aqueous solution and shaken for 1 hour. Next, the resin was filtered and washed with water until the pH became neutral. This resin was packed in a column of 15 mm x 300 mm, and the resin which had not been exposed to copper chelate was packed in the column and linked, and 1.25 g / dl of isoleucine and leucine were respectively added.
20 ml was loaded and eluted with SV4 with water. As a result, isoleucine is first eluted as shown in FIG. In RV13
Leucine was eluted by elution with a 1N aqueous ammonia solution and could be separated almost completely. Isoleucine,
The recovery rate of leucine was about 95%. In addition, the separated amino acid contains about 3 ppm of copper.
It was found to be below the amino acid standard, and copper leakage was also extremely small.

Example 2 (Creation of breakthrough curve) 20cc chelating resin Sumitomo Chemical Co., Ltd. 1N copper sulfate-
It was made into a copper chelate with 300 ml of 0.5N aqueous ammonia solution and washed with water until the pH became neutral.

This resin was packed in a column (15 mm x 300 mm), and
Two columns (15 mm × 300 mm) packed with 20 cc of chelating resin that did not chelate copper ions were connected.

The isoleucine and leucine aqueous solutions adjusted to the respective concentrations of 1.25 g / dl were fed to this column by SV1.5, and the fractions were analyzed.

The breakthrough curve was as shown in FIG.

Almost only isoleucine is eluted up to 4 RV, and a resin loading of 20 g / l-resin or more can be expected.

EFFECTS OF THE INVENTION As is clear from the above, according to the present invention, leucine and isoleucine and other amino acids can be easily and safely separated, so that the present invention is industrially very useful.

[Brief description of drawings]

FIG. 1 shows SVs of leucine and isoleucine in Example 1.
6 is a graph showing a separation concentration curve by RV when it is set to 4. FIG. 2 shows SVs of leucine and isoleucine in Example 2.
It is a graph which shows a breakthrough curve when it is set to 1.5. A: Leucine, B: Isoleucine

Claims (5)

[Claims] 1. When separating leucine from an amino acid mixture containing at least leucine and isoleucine, the amino acid mixture is brought into contact with an L-proline-metal complex salt-immobilized resin to selectively adsorb leucine. Method for separating leucine. 2. The method according to claim 1, wherein the amino acid mixture has a pH of 3 to 9 and a concentration of 10 g / dl or less. 3. The method according to claim 1, wherein the metal complex salt-immobilized resin is packed in a column. 4. The method according to claim 3, wherein the supply rate of the mixed solution of amino acids to the column packed with the immobilized resin is SV5 or less. 5. The method according to claim 3, wherein a column packed with the L-proline-metal complex salt-immobilized resin is followed by a column packed with the L-proline chelate resin to pass the amino acid mixture.