JPS62148459A - Separation and purification of glutamine - Google Patents

Abstract

PURPOSE:To carry out the separation and purification of glutamine from an aqueous solution of glutamine containing impurities such as glutamic acid, pyrrolidonecarboxylic acid, sulfate radical, etc., by treating the solution with ion exclusion chromatography using a strongly acidic cation exchange resin. CONSTITUTION:Glutamine can be separated and purified from an aqueous solution of glutamine containing impurities composed mainly of glutamic acid, pyrrolidonecarboxylic acid, sulfate radical and pigment (e.g. glutamine fermentation liquid, dissolved liquid of crude glutamine crystal separated from the fermentation liquid, mother liquor of glutamine crystallization, etc.) by subjecting the solution to ion exclusion chromatography. The strongly acidic cation exchange resin used in the ion exclusion chromatography is preferably a styrene resin having a crosslinking degree of 4-8% to attain the highest separation performance. The operation temperature is selected below the heat-resistant temperature of the strongly acidic cation exchange resin.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for separating and purifying glutamine, and more specifically, the present invention relates to a method for separating and purifying glutamine.
A glutamine aqueous solution containing impurities mainly consisting of one or more sulfate groups (hereinafter referred to as PCA) is subjected to ion exclusion chromatography using a strongly acidic cation exchange resin to remove such glutamine from the glutamine aqueous solution. The present invention relates to a method for fractionally purifying high-purity glutamine with high yield by removing impurities.

Glutamine is usually produced by a fermentation method, one of which is a fermentation method using glucose as the main raw material. The glutamine fermentation liquid obtained by this method contains glutamic acid, PC
A. Contains impurities mainly consisting of sulfate groups and pigments. Such a fermentation liquid is a typical example of an aqueous glutamine solution to be treated in the present invention, as described below. Incidentally, the same can be said of glutamine obtained by other methods.

In general, for glutamine, the − of its solution is lower than its isoelectric point (pl
-5,65) It easily decomposes into glutamic acid and PCA at low or high temperatures or at high temperatures. Therefore, glutamic acid and PCA are inevitably produced in the fermentation liquid, and sulfate radicals are present as medium components.

Therefore, methods for separating and purifying glutamine in the glutamine fermentation solution include a method in which impurities are ion-exchanged at the isoelectric point of glutamine using an OH-type anion exchange resin and flowed through the glutamine, or conversely, a method in which glutamine is extracted in the low-range region. A method in which glutamine is made to exist as a cation, adsorbed on a strongly acidic cation exchange resin, and eluted after passing through the impurities (in these cases, bacterial cells and pigments are removed before or after).

JP-A-49-81587, JP-A-50-89590 and JP-A-56-3040) and a method of purification by repeated crystallization (JP-A-50-95481''), but in the case of the resin method, - fluctuations The point is that siglutamine causes a yield loss when ion-exchanged with W fat, and the point that siglutamine decomposes due to fluctuations in - and changes to glutamic acid and PCA ("Ch@m1stry of th@ Am1
n.

Ac1ds JP, 1933 (1961)
John Willoy & 5ons in
a), the use of an alkali such as /aOH for resin regeneration and the complexity of the operation, and in the case of crystallization, the yield may decrease due to repeated crystallization. There is a problem with nine points.

As a result of extensive research, the present inventor has developed a method for separating and refining extremely pure glutamine from a glutamine fermentation liquid contaminated with impurities mainly consisting of one or more of glutamic acid, PCA, and sulfate groups. The present inventors have found that glutamine can be obtained in good yield and with high purity through extremely simple procedures by treatment with ion exclusion chromatography using a strongly acidic cation exchange resin, and the present invention has been completed. However, the application of the present invention is not limited to such treatment of glutamine fermentation liquid, as will be described later.

Generally, non-electrolyte or weak electrolyte compounds can be separated from strong electrolyte compounds by ion exclusion chromatography. This is because strong electrolyte compounds are excluded by the Donnan potential due to their charged ion exchange groups and cannot penetrate into the ion exchange resin, but non-electrolyte or weak electrolyte compounds can freely penetrate. . The present invention is based on this law.

The present invention will be explained in more detail below.

In the present invention, the glutamine aqueous solution containing impurities mainly consisting of at least one or more of glutamic acid, PCA, and sulfate radicals refers to the glutamine fermentation liquid itself, the solution of crude glutamine crystals obtained from the fermentation liquid, and the glutamine crystals. The present invention can be applied to any aqueous solution containing glutamine contaminated with impurities mainly consisting of one or more of glutamic acid, PCA, and sulfate groups.

There is no particular limit to the glutamine concentration of such an aqueous solution;
It is fine as long as glutamine is dissolved.

When subjecting an aqueous glutamine solution containing impurities to ion exclusion chromatography, first the PH of the aqueous glutamine solution is adjusted to a PL (before or near the isoelectric point of glutamine) to render siglutamine in an uncharged state. Glutamic acid, PCA and sulfate radicals exist as anions in the -.

On the other hand, a strongly acidic cation exchange resin is made of a cation that serves as a counter ion to its more anion. for example,
In the case of glutamine fermentation liquid, usually glutamic acid, PCA
Since the and sulfate groups are in the form of ammonium salts, a strongly acidic cation exchange resin is used in the form of ammonium salts.

Incidentally, if the aqueous solution to be subjected to ion exclusion chromatography contains multiple types of cations, it is best to treat the cation-exchanged stool fat with an aqueous solution containing the multiple types of cations in advance. Separability decreases. Ion exclusion chromatography can also be achieved using an anion exchange resin, but in the case of glutamine, which is the subject of the present invention, the isoelectric point of glutamine is
Since CA and sulfuric acid exist in the form of anions, that is, there are many anion species, the separation property is reduced and it is not practical.

The strongly acidic cation exchange resin used in the present invention is Diaion 5K-102, 5K-104, 5K-106, 8KI.
B.

5K-1048 and 5KIBS (manufactured by Mitsubishi Kasei), XF
S-XL.

HCR-W2 and TG 8500A, (manufactured by Dow Chemical Company), C-20, 0-250, E8-26 and C-
3 (manufactured by Duolite), S-100, S-109
, 5P-112 and 5p-t20 (manufactured by Revachit) and [R-116.

IR-118, IR-120B, IR-122, 1
1-124.

rR-252, IR-200C and [R-2000T (
Mainly styrene-based materials such as Amberlite Co., Ltd.) can be used. Among these, resins with a degree of crosslinking of 4 to 8 have the best separation performance.

The amount of strongly acidic cation exchange resin to be used is, in the case of an aqueous solution with a glutamine concentration of about 6% and an impurity concentration of about 1%,
About 4 to 5 times the amount of the aqueous solution is sufficient. If the overall concentration of glutamine and impurities in the aqueous solution is reduced, the amount of resin may be further reduced. Appropriate amounts of resin can be readily determined by those skilled in the art through preliminary experimentation.

There is no particular restriction on the operating temperature, as long as it is within the heat resistance temperature of the strongly acidic cation-exchanged fat. Raising the temperature will increase the rate of separation of impurities and glutamine, but this will accelerate the decomposition of glutamine, so it is best to carry out the process at the optimum temperature depending on the solution.

A column is filled with a strongly acidic cation exchange resin shaped according to the cations 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 guideline. For example, in the case of a glutamine fermentation solution, fill a column with a strongly acidic cation exchange resin from an ammonium shop, adjust the temperature to the isoelectric point of glutamine or its vicinity, and inject the glutamine fermentation solution into the top of the column as appropriate.

Next, when water is passed through the solution, the aforementioned contaminant impurities are first eluted, and then glutamine is eluted. Incidentally, even if the glutamine fermentation liquid to be subjected to the ion exclusion chromatography of the present invention contains bacterial cells and/or pigments, these behave in the same way as ammonium salts of glutamic acid, PCA, or sulfuric acid, so they are usually not a problem. However, if necessary, remove bacterial cells from the glutamine fermentation solution in advance to prevent clogging of the resin layer.

There is no particular restriction on the water passing rate (SV), and it may be within the usual range of about 0.5 to 4. A fraction of the target product is obtained by tracking temporal changes in the components of the eluent.

Example I 61.5 g/l L-glutamine and 7 I/l ammonium salt of L-glutamate! 4 QtJ of an aqueous glutamine solution containing 200 d of NH4 type SK-1048 was injected into the top of a 9 column (φ3.23×H25cIn). Water was passed through the solution at pH-5°, 65.45° C., and SV-1o conditions to carry out dissolution for 111 hours.

L-glutamic acid ammonium salt was eluted first, followed by L-glutamine. Eluate volume 70-310
Fractions of m were collected, of which 70-160d were defined as a sub-fraction and 170-3101jt-main fraction.

The main fraction contained only glutamine, 100% of L-glutamate ammonium salt was removed, and the recovery rate of L-glutamine was 1001.

Example 2 5611/l of glutamine was obtained by dissolving L-glutamine crude crystals obtained from L-glutamine fermentation liquid.

Ammonicum L-glutamate salt 0.61/1% PCA
Ammonicum salt 0.91/l, ffl acid 7 ammonium 9
Glutamine aqueous solution 401d containing 0.41//71
The NH4+ form of '1XFS-XL was injected into the upper part of a column (φ3.2 cm x H25 cIn) filled with 2001 nl. Elution was performed by passing water under the conditions of pl (-5.59, 45°C, and 5V-1.4).

L-glutamic acid anthonium salt, PCA heptammonium salt, and ammonium sulfate salt were eluted first, and then L-glutamine was eluted. Eluate volume 70~3'yo
ag't- were collected, of which 70 to 150 d were taken as a sub-fraction and 160 to 37 od were made into a main fraction.

The main fraction was a fraction containing only L-glutamine.

The sub-fraction contains L-glutamic acid ammonium salt.

PCA ammonium salt and ammonium sulfate salt are approximately 1
004 was included. The removal rate of impurities in the main fraction is L-
Glutamate ammonium salt, PCA7 ammonium salt 1) was 1100%, and aN ammonium was 99.4%. Moreover, the recovery rate of L-glutamine was 98.2%. Note that the degree of coloring of the initial solution of crude L-glutamine crystals is 0.
．． 039 (spectrophotometer 400 nm), but that of the main fraction was 0.003 on average, and the color removal rate was 78.
．． It was 0%.

Example 3 L-glutamine 1 obtained by sterilizing L-glutamine fermentation liquid
9.311/l, L-glutamic acid ammonium salt 1.
09/l, 40 ml of L-glutamine aqueous solution containing 1.51/l of PCA ammonium salt and 991/l of ammonium sulfate! r XFS-XL NH4m k 2
Column packed with 00rnl (φ3.2cInX H2
5 crn).

pH=5.70.45°C, 5V=1. Elution was carried out by passing water under OO conditions.

L-glutamic acid ammonium FA, PCA ammonium salt, and ammonium sulfate salt were eluted first, and then L-glutamic acid was eluted. Eluate volume 90-35
0m/i sampling, of which 90~150d was divided into regular fractions,
The main fraction was 160-350di.

The removal rate of impurities in the main fraction was 84.8% for L-glutamic acid ammonium salt and 86.6% for PCA ammonium salt.
%, ammonium sulfate was 93.0%. Also, L-
The recovery rate of glutamine was 97.5%. The degree of coloration of the initial glutamine solution was 0.641 (spectrophotometer 400 nm), but that of the main fraction was 0.641 on average.
．． 046, and the color removal rate was 51.5%.

Claims (1)

[Claims] A process for purifying glutamine by subjecting an aqueous glutamine solution containing impurities mainly consisting of at least one or more of glutamic acid, pyrrolidone carboxylic acid, and sulfate groups to ion exclusion chromatography using a strongly acidic cation exchange resin. Separation and purification method.