JPH08183768A - Method for isolating l-proline - Google Patents

Abstract

PURPOSE: To easily isolate L-proline used in the areas of foods, medicines and cosmetics, in a high purity and yield by adsorbing and eluting the same at a specific temperature by an ion exchange chromatography using a specific ion exchanging resin from the mixture solution of amino acids. CONSTITUTION: This method for isolating L-proline comprises adsorbing and eluting L-proline from the mixture solution of amino acids containing L-proline as a major component by an ion exchange chromatography using H<+> -type strongly acidic cation exchanging resin (e.g. a gel-type) at an operating temperature of 40-80 deg.C, and further if necessary treating the eluted solution containing mainly L-proline by an OH-type strongly basic anion exchanging resin to remove acidic amino acids from this eluted solution to isolate the objective L-proline.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for isolating L-proline.

[0002]

2. Description of the Related Art L-proline, which is used in a wide range of fields such as food, feed, medicine, cosmetics, and industry, is
Usually, it is produced by a fermentation method using a sugar such as glucose as a carbon source. The fermentation broth containing L-proline obtained by this method contains not only L-proline but also several kinds of by-produced amino acids. In addition, it also contains impurities such as organic acids, inorganic cations, anions, and dyes. Therefore, in order to obtain L-proline produced by the fermentation method in a pure form from the fermentation solution, it is a major problem to efficiently remove many impurities and by-produced amino acids. Such a fermentation broth is a typical example of the L-proline aqueous solution to be treated in the present invention, as described later.

As a method of isolating L-proline from an L-proline fermentation broth, after adsorbing it on a strongly acidic cation exchange resin at room temperature or a lower temperature to allow contaminants to flow through,
A very general method is known in which L-proline is eluted with aqueous ammonia and then separated by crystallization (for example, JP-A-58-60995 and JP-A-60-1).
41284, JP-A-3-195493).

[0004]

However, in practice, a by-product amino acid having an isoelectric point close to that of L-proline is used as L-proline.
Separation from proline by an ion exchange resin is difficult, and inevitably a contaminated state is obtained, and therefore recrystallization must be repeated in order to produce higher purity L-proline. However, there is a problem in that the yield is reduced.

[0005]

[Means for Solving the Problems] The present inventors have completed the present invention as a result of intensive research to find a method for isolating high-purity L-proline in a high yield.

That is, the present invention uses an H ⁺ -type strongly acidic cation exchange resin from an amino acid mixture containing L-proline as a main component, and adsorbs and elutes at an operating temperature of ion exchange chromatography in the range of 40 to 80 ° C. The method for isolating L-proline is characterized in that

Further, a mixed solution containing L-proline as a main component (including an L-proline fermented solution containing a by-product amino acid) is adsorbed on an H ⁺ type strongly acidic cation exchange resin to give an H ⁺ type strongly acidic cation exchange resin. the eluate from the resin L- proline are obtained in concentrated form OH ^- in contact with the mold strongly basic anion exchange resin OH ^- removing the mold strongly basic anion exchange resin adsorbate, high purity L- This is a method for efficiently isolating proline.

Generally, in ion exchange chromatography in high performance liquid chromatography, the diffusion rate of sample components in the stationary phase is increased by raising the temperature of the column, and the number of theoretical plates is increased to promote the ion exchange reaction. Therefore, the separation efficiency can be improved by operating at high temperature, and it becomes possible to separate even overlapping peaks at room temperature. However, in high performance liquid chromatography, the amount of sample that can be used for separation is extremely small in view of the exchange capacity of the ion exchange resin, the processing efficiency per resin used is low, and a large amount of sample is processed. It is not possible.

Further, when an ion exchange resin is used to remove impurities having the same electric charge by adsorption to the resin,
Part of the target substance is also adsorbed on the resin, and there is a problem that the yield decreases.

INDUSTRIAL APPLICABILITY The present invention opens a new way of application on an industrial scale for injecting a sample to the limit of the exchange capacity of an ion exchange resin to produce a high-purity amino acid in a large amount and effectively. .

The present invention will be described in more detail below. L-
Proline and other neutral amino acids such as L-alanine, L
-Valine and the like show no significant difference in the adsorptive power to the H ⁺ type strongly acidic cation exchange resin. When the operating temperature is room temperature,
Upon elution with aqueous ammonia, the by-produced amino acids are L
-It elutes almost simultaneously with proline. Therefore, the yield of L-proline was low. In addition, L, which is an acidic amino acid
-Glutamic acid and the like tend to elute earlier than L-proline by such a separation operation, but most of them elute at the same time as L-proline.

On the other hand, by raising the operating temperature to 40 to 80 ° C., the ion exchange reaction of the resin is promoted, and the adsorption power of L-proline and the by-product amino acid to the resin becomes different during adsorption. Further, during elution with aqueous ammonia, the resin is heated to improve the separation efficiency, so that L-proline and the by-product amino acid are efficiently separated.
-It is believed that the yield of proline is significantly improved.

In both the adsorption and elution operations, when the operating temperature exceeds 80 ° C., bubbles are generated in the column and the resin is accelerated to deteriorate, which is not practical, but less than 40 ° C. Then, the impurity amino acid and L-proline are not sufficiently separated, and further purification is required to obtain high-purity L-proline in good yield.

When the L-proline aqueous solution containing impurities is applied to the strongly acidic cation exchange resin of the type, first, L-proline is added.
The aqueous solution of proline is adjusted to be acidic to bring L-proline into a charged state. In the vicinity of the isoelectric point of L-proline, the adsorptivity for resin decreases.

On the other hand, the strongly acidic cation exchange resin is used in the H ⁺ type. Other cation types, such as ammonium salt types, are not practical because the ability to adsorb L-proline to the resin is reduced.

The concentration of aqueous ammonia used for elution is 0.
About 25 to 1% is suitable. When the concentration of aqueous ammonia increases, the elution rate of L-proline increases.

As the strongly acidic cation exchange resin used in the present invention, any known one can be used. As a commercially available product, for example, Diaion UBK-530K, S
K-102, SK-104, SK-106, SK-1B
And SK-104S (all manufactured by Mitsubishi Kasei Co., Ltd.), Levatit S-100, S-109, SP-112, SP-1.
20 and MDS-1368 (all manufactured by Bayer),
IR-116, IR-118, IR-120B, IR-
122 and IR-124 (all manufactured by Amberlite Co., Ltd.). Of these, the gel type has better separation ability than the macroporous type.
The degree of crosslinking is generally 4-8%.

The amount of the strongly acidic cation exchange resin used is appropriately determined depending on the concentrations of L-proline in the aqueous solution and the total amount of impurities. However, when L-proline is adsorbed on the resin, L-proline does not leak. The L-proline concentration is about 5% and the inorganic salt concentration is 5%.
In the case of an aqueous solution having a certain amount, about 1 to 2 times the amount of the aqueous solution is sufficient.

In the method of the present invention, first, a column of H ⁺ type strongly acidic cation exchange resin is packed and the column temperature is kept at 40 to 40 ° C.
After constant temperature in the range of 80 ° C., the above standard amount of the L-proline aqueous solution is injected into the upper part of the column.

For example, in the case of an L-proline aqueous solution containing impurities, the column is filled with H ⁺ type strongly acidic cation exchange resin, the column temperature is thermostatted at 40 to 80 ° C., and the pH above the column is preferably 1 to 5; An appropriate amount of L-proline aqueous solution adjusted to 2 to 4 is injected. Then, water is passed through to elute anions, uncharged substances, and in the fermentation liquid, contaminants such as various pigments. Then 0.25
By passing a ˜1% ammonia solution, first, the acidic amino acid is eluted first, followed by L-proline, and then the neutral and basic amino acids in this order, and the fraction containing L-proline as the main component is collected.

The liquid passing speed (SV) is usually 0.5
It may be about 10 to 10. The target fractionation position is p of the eluent
It is possible to trace and determine changes with time such as H, coloring degree, and electrical conductivity.

In the present invention, if L-proline is the main component, L-proline can be efficiently isolated as described above. Particularly, when an acidic amino acid such as L-glutamic acid is contained, the above-mentioned results are obtained. It can be isolated but is often not sufficiently separated from L-proline.

Preferably, the L-proline fraction eluted from the H ⁺ type strongly acidic cation exchange resin is passed through an OH ⁻ type strongly basic anion exchange resin to adsorb and remove the acidic amino acid L-glutamic acid and the like. . L-proline is also adsorbed on the OH ^- type strongly basic anion exchange resin at the same time.
It is replaced with L-glutamic acid or the like, which has a stronger adsorptive power to the resin, and is eluted sequentially. Therefore, by properly setting the amount of resin, 95% or more of L-proline can be recovered and acidic amino acids such as L-glutamic acid can be removed.

When the L-proline fraction of the eluate of the H ⁺ type strongly acidic cation exchange resin column is applied to the strongly basic anion exchange resin, it is not necessary to adjust the pH of the eluate, but it is usually 3 to 7 Often ranges.

The strongly basic anion exchange resin is used in the form of OH ⁻ . Other anion types, for example, Cl ⁻ type, are not practical because Cl ⁻ is mixed in the eluate and the adsorbability of L-glutamic acid and the like to the resin decreases.

As the strong basic anion exchange resin that can be used here, any known strong basic anion exchange resin can be used, and as a commercially available product, for example, Diaion PA.
306, PA312 and PA318 (above, manufactured by Mitsubishi Kasei Co., Ltd.), Levatit M500, MP500 and MP5
00A (above, Bayer), Amberlite IRA
-430, IRA-900 and IRA-904 (above, manufactured by Amberlite Co.) are sold. Of these, the macroporous type has a better adsorptivity than the gel type and can be preferably used.

The amount of strongly basic anion exchange resin used is
The amount that can completely capture the acidic components such as L-glutamic acid contained in the L-proline-containing liquid is sufficient. For example, when the acidic amino acid component is only L-glutamic acid, when the concentration is about 0.3%. Is 1/3 of the amount of the aqueous solution
0 is enough. The amount of resin is appropriately set depending on the concentration of L-glutamic acid in the aqueous solution.

In the present invention, an OH ^- type strongly basic anion exchange resin is packed in a column, and the above standard amount of the L-proline aqueous solution is injected into the upper part of the column.

For example, in the case of an L-proline aqueous solution containing L-glutamic acid, a column is filled with an OH ^- type strongly basic anion exchange resin, and an appropriate amount of the L-proline aqueous solution is injected onto the column. As the solution is passed, L-glutamic acid is preferentially adsorbed on the resin, and L-proline is eluted with almost no adsorption, and is recovered as a plain effluent. Then, by washing with an appropriate amount of water, L-proline remaining in the column is recovered.

The liquid passing speed (SV) is usually 0.5
It may be about 20. The operating temperature of the column may be room temperature. When the temperature is raised, the L-glutamic acid adsorption capacity is increased, but at 50 ° C. or higher, the performance of the resin is significantly deteriorated, which is not practical.

L-proline can be isolated from the obtained L-proline eluate by a conventionally known method. For example, after removing the remaining dye with activated carbon, L-proline is obtained by drying by spray drying. You can

[0032]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 L-proline 50 g / l and L-alanine 0.5 g / l
1, L-valine 3.0 g / l, NaCl 10 g / l, MgSO ₄
The pH of the L-proline aqueous solution containing 0.5 g / l of 7H ₂ O and 20 g / l of (NH ₄ ) ₂ SO ₄ was adjusted to 3.0 with hydrochloric acid and then adjusted to 70 ° C., 110 ml of which was strongly acidic ion exchange resin. Diaion UBK-530K (6% cross-linking degree, gel type, manufactured by Mitsubishi Kasei) was injected into the upper part of a column (φ1.6 × 55 cm) filled with 110 ml of H ⁺ type and kept at 70 ° C.
Then, after washing with 440 ml of water at 70 ° C., it was washed with water at 70 ° C.
Elution was performed with 1 l of 5% aqueous ammonia. The operations of adsorption, washing with water and elution were performed under the condition of SV = 2.0. The ammonia water eluate was fractionated every 12 ml with a fraction collector, and amino acids were measured by amino acid HPLC. The pH was also measured. The results are shown in Fig. 1. Fractions from the start of L-proline elution to the start of L-alanine and L-valine elution were collected as an L-proline fraction and analyzed by amino acid HPLC to determine the removal rate of L-alanine and L-valine. , L-proline recovery rate was calculated by the following formula. The results are shown in Table 1. Removal rate (%) of L-alanine (L-valine) = 100-
(A / B) × 100 A: L-alanine (L-valine) absolute amount in the L-proline fraction liquid (g) B: L-alanine (L-valine) in the L-proline aqueous solution injected into the column Absolute amount (g) L-proline recovery rate (%) = (a / b) × 100 a: L-proline absolute amount in the L-proline fraction liquid (g) b: L-proline aqueous solution injected into the column Absolute amount of L-proline (g)

Examples 2 to 5 Example 1 was repeated except that the operating temperature for adsorption, washing with water and elution was 40, 50, 60 or 80 ° C. The results are shown in Figure 2
5 and Table 1.

Comparative Example 1 Example 1 was repeated except that the operating temperature for adsorption, washing with water and elution was 30 ° C. The results are shown in FIG. 6 and Table 1.

Comparative Example 2 The procedure of Example 1 was repeated except that the operating temperature for adsorption, washing with water and elution was 90 ° C. However, the inside of the column could not be adsorbed and the experiment was stopped.

[0037]

[Table 1]

Example 6, Comparative Example 3 As a strongly acidic ion exchange resin, Levatit MDS1368
Example 1 or Comparative Example 1 was repeated except that the H ⁺ type (crosslinking degree 6%, gel type, manufactured by Bayer) was used. The elution curves were as shown in FIGS. 7 and 8, respectively. Also, L-
The alanine, L-valine removal rate and L-proline recovery rate were as shown in Table 2.

[0039]

[Table 2]

Example 7 L-proline 55 obtained by sterilizing the L-proline fermentation broth
g / l and L-glutamic acid 6.2 g / l, L-alanine 0.3 g / l, L-valine 3.5 g / l, Na ⁺ 5
pH of L-proline aqueous solution containing g / l, Mg ²⁺ 0.05 g / l, NH ₄ ⁺ 3 g / l was adjusted to 3.0 with hydrochloric acid, and 110 ml thereof was strongly acidic ion exchange resin DIAION UBK
-530K (6% cross-linking, Mitsubishi Kasei) H ⁺ type 1
It was injected into the upper part of a column (φ1.6 × 55 cm) packed with 10 ml. The amount of cation contained was determined by ion chromatography. Then, after washing with 440 ml of water, elution was carried out with 1.0 l of 0.5% aqueous ammonia. The operations of adsorption, washing with water and elution were carried out at a temperature of 70 ° C. under the condition of SV = 2.0. The elution curve of the above example was as shown in FIG. The removal rates of L-alanine and L-valine were both 99.3%, and the L-proline recovery rate was 87%. Liquid 2 of the obtained L-proline fraction
The 64 ml contained 2.5 g / l of L-glutamic acid and had a pH of 4.2. Next, OH of strong basic anion exchange resin Diaion PA306 (manufactured by Mitsubishi Kasei)
^- mold 9ml packed column flow rates at the top SV = 2.0 of (φ1.6 × 4.5cm), were injected under the conditions of operating temperature 25 ° C., followed by washing with water with water 36 ml, and L- glutamic acid After removal, 300 ml of an aqueous L-proline solution was obtained. This aqueous solution was freeze-dried to obtain 5.0 g of L-proline crystals.
The final purity of L-proline was 99.9%, and the recovery rate was 83% based on the L-proline fermentation broth.

[0041]

As described above, it is clear that the method of the present invention has a better separability of L-proline than the conventional method of separating the operating temperature of the H ⁺ type strongly acidic cation exchange resin at room temperature. And the acidic amino acid that cannot be completely removed by the H ⁺ -type strongly acidic cation exchange resin is O
The H ^- type strongly basic anion exchange resin can be removed by appropriate use, and L-proline can be easily obtained in high purity and high yield, so that its industrial value is extremely high.

[Brief description of drawings]

[1] Elution curve of Example 1 (Operation Temperature 70 ⁰ C).

[Figure 2] in Example 2 eluting curve (operating temperature 40 ⁰ C).

Elution curves in Figure 3 Example 3 (operating temperature 50 ⁰ C).

[4] Elution curve of Example 4 (operating temperature 60 ⁰ C).

[5] Elution curve of Example 5 (operating temperature 80 ⁰ C).

FIG. 6 is an elution curve of Comparative Example 1 (operating temperature: 30 ⁰ C).

[7] Elution curve of Example 6 (operating temperature 70 ⁰ C).

FIG. 8: Elution curve of Comparative Example 3 (operating temperature: 30 ⁰ C).

FIG. 9: Elution curve of Example 7 (operating temperature 70 ⁰ C).

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Takeshi Nakamura 1 Toyonuma-cho, Sunagawa-shi, Hokkaido Mitsui Toatsu Chemical Co., Ltd.

Claims (2)

[Claims] 1. Adsorption and elution from an amino acid mixture containing L-proline as a main component using an H ⁺ type strongly acidic cation exchange resin at an operating temperature of ion exchange chromatography in the range of 40 to 80 ° C. A method for isolating L-proline characterized. 2. An H ⁺ -type strongly acidic cation exchange resin is used for L
-The method for isolating L-proline according to claim 1, wherein after the isolation of proline, the eluent mainly containing L-proline is further subjected to removal of acidic amino acids with an OH ^- type strongly basic anion exchange resin.