JP2001316342A - Method for purifying isoleucine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for purifying isoleucine easily, in high yield and purity using an inexpensive precipitant. SOLUTION: This method for purifying isoleucine comprises the following process: p-vinylbenzenesulfonic acid or a water-soluble salt thereof is made to act on an isoleucine-containing aqueous solution to form isoleucine-p- vinylbenzenesulfonic acid salt crystal followed by separating the salt and then decomposing it to purify and obtain the objective isoleucine; wherein the p- vinylbenzenesulfonic acid may be either of free acid form or in the form of alkali metal salt or ammonium salt.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel isoleucine / p-vinylbenzenesulfonate crystal suitable for isoleucine purification and a method for purifying isoleucine using said salt. Isoleucine is very useful as a raw material for pharmaceutical amino acid preparations and nutrients, and as a synthetic intermediate for various pharmaceuticals.

[0002]

2. Description of the Related Art Isoleucine is produced by a method of hydrolyzing proteins such as soybean protein or a method of culturing a microorganism having an ability to produce isoleucine. As a method for separating and purifying isoleucine from an aqueous solution containing isoleucine such as a protein hydrolyzate or a fermentation solution obtained by these methods, for example, (1) separation of acidic and basic amino acids by treating with an ion exchange resin After removal, neutral amino acid sections are fractionated and recrystallized repeatedly to remove neutral amino acids other than isoleucine (Biochem. J., 48 , 313 (195)
1)) (2) Hydrochloric acid is added to an aqueous solution containing isoleucine,
Conventionally, a method of repeating generation and precipitation of isoleucine hydrochloride crystals (J. Biologc. Chem., 118 , 78 (1937)) has been carried out. However, the former method is very complicated, and the method using valine and leucine is difficult. Separation is difficult, and the latter has a problem that the solubility of isoleucine hydrochloride crystals in water is high, resulting in a low yield.

On the other hand, as another purification method, a method using a precipitant which selectively produces an adduct (poorly soluble salt) with isoleucine includes, for example, (1) 4-nitro-4'-methyldiphenylamine-2
-Method of reacting sulfonic acid (J. Biologc. Chem., 143 , 121 (1942)) (2) 2-naphthol-6-sulfonic acid which is a naphthalenesulfonic acid derivative (JP-A-48-13515), Method of reacting 5-naphthalenedisulfonic acid (Japanese Patent Application Laid-Open No. 54-109953) and 2-naphthalenesulfonic acid (Japanese Patent Application Laid-Open No. 54-163519) (3) p-ethylbenzenesulfonic acid which is a benzenesulfonic acid derivative (Japanese Patent Application Laid-Open No. 237030), a method of reacting p-toluenesulfonic acid is known. However, (1) has problems such as that the precipitant used is expensive and is difficult to obtain industrially, and that the method for isolating isoleucine from an adduct is complicated, and (2) has problems such as leucine. There are problems such as difficulty in separating isoleucine from amino acids having similar properties, and high toxicity of the precipitant itself. (3) has high solubility of isoleucine in the water of the salt crystals of the precipitant. Therefore, there was a problem that the yield was lowered.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a simple, high-yield and high-purity isoleucine purification method using a relatively inexpensive precipitant.

[0005]

The present inventors have conducted intensive studies in view of the above-mentioned circumstances. As a result, p-vinylbenzenesulfonic acid was added to an aqueous solution containing isoleucine and allowed to react. -It has been found that p-ethylbenzenesulfonate crystals are selectively precipitated, and the present invention has been completed.

That is, the present invention relates to a novel isoleucine / p-vinylbenzenesulfonic acid crystal comprising (1) 1 mol of isoleucine and 1 mol of p-vinylbenzenesulfonic acid, and (2) an aqueous solution containing isoleucine. p-Vinylbenzenesulfonic acid or a water-soluble salt thereof is added to generate and precipitate isoleucine / p-vinylbenzenesulfonic acid salt crystals, the salt is separated and obtained, and then the salt is decomposed to form isoleucine. The present invention relates to a method for purifying isoleucine, characterized by being obtained.

The isoleucine which can be applied in the method of the present invention may be either an L- or D-form optically active form, a racemic form, or a mixture of both. As an aqueous solution containing isoleucine, a basic amino acid is separated from a hydrolyzed solution obtained by hydrolyzing a protein such as soybean protein, an amino acid mixture obtained by removing the amino acid, a fermented solution obtained by culturing a microorganism having an ability to produce and accumulate isoleucine, or It is possible to use various aqueous solutions such as a solution obtained by removing cells from the fermented solution, or an aqueous solution of crude DL-isoleucine obtained by a chemical synthesis method such as a method using a hydantoin derivative.

The p-vinylbenzenesulfonic acid used in the present invention is commercially available at relatively low cost. Also,
p-Vinylbenzenesulfonic acid may be used as a free acid, and water-soluble salts thereof, for example, alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt, ammonium salt and the like It may be used in the form of The amount of p-vinylbenzenesulfonic acid or its water-soluble salt used is 0.5 to 2.0 times mol, preferably 1.0 to 1.1 times mol, based on isoleucine contained in the aqueous solution, and particularly large excess. You do not need to use an amount.

The isoleucine / p-vinylbenzenesulfonic acid salt is prepared by adding p-vinylbenzenesulfonic acid or an aqueous solution thereof to an aqueous solution having an isoleucine concentration of 40 g / L or more and adjusting the pH to about 1.5.
The target compound isoleucine / p-vinylbenzenesulfonate can be easily formed and precipitated. The pH of the solution suitable for the formation and precipitation of isoleucine / p-vinylbenzenesulfonate crystals is 0.1 to 2.3, preferably 1.0 to 1.7. As the acid used for the pH adjustment, an inorganic acid such as hydrochloric acid or sulfuric acid is used. If necessary, a seed crystal of isoleucine / p-vinylbenzenesulfonic acid salt is added to a mixed solution of isoleucine and p-vinylbenzenesulfonic acid, whereby crystals can be efficiently precipitated. In this case, precipitation can be easily performed with an aqueous solution having an isoleucine concentration of 25 g / L or more. When the aqueous solution of isoleucine is diluted, the salt crystals can be precipitated by concentration. In this case, p-vinylbenzenesulfonic acid may be added at any stage before and after concentration.
When the aqueous solution of isoleucine is concentrated to neutrality, crystals of free isoleucine precipitate, but an appropriate amount of p-
By adding vinylbenzenesulfonic acid and adjusting the pH to about 1.5, isoleucine / p-vinylbenzenesulfonic acid salt crystals can be easily generated. It is also possible to precipitate an isoleucine / p-vinylbenzenesulfonic acid salt crystal by adjusting a diluted solution of isoleucine coexisting with an appropriate amount of p-vinylbenzenesulfonic acid to about pH 2 in advance and then performing concentration. .

In order to separate and obtain the precipitated isoleucine / p-vinylbenzenesulfonate crystals, a usual solid-liquid separation method such as filtration and centrifugation may be employed. The separated crystals have high purity, but can be further purified by a commonly used purification method such as a treatment such as washing and dehydration, and a recrystallization operation.

The thus obtained isoleucine / p-vinylbenzenesulfonate crystal comprising 1 mol of isoleucine and 1 mol of p-ethylbenzenesulfonic acid is a novel substance and exhibits the following physical properties. Colorless and transparent plate-like crystals. Soluble in water and ethanol. Solubility in water: 5.1 wt% (pH 1.5, 10
C) Crystal structure: monoclinic Crystal density: 1.29 g / cm ³ Elemental analysis: C: 50.4%, H: 7.0%, N: 4.0
%, S; 9.7% (calc. C; 50.4%, H;
7.0%, N; 4.0%, S; 9.7%)

By the same operation, leucine / p-vinylbenzenesulfonate crystals can be obtained. However, the solubility of these crystals in water is 10.2 wt% (pH 1.5,
10 ° C.), which is higher than the solubility of isoleucine / p-vinylbenzenesulfonate crystals. By the same operation, an attempt was made to obtain a valine / p-vinylbenzenesulfonate crystal, but it could not be obtained.

To isolate free isoleucine from isoleucine / p-vinylbenzene sulfonate crystals, a solution dissolved in a large amount of hot water is contacted with a weakly basic ion exchange resin (OH type) or An alkali such as sodium oxide may be added. When an ion exchange resin is used, p-vinylbenzenesulfonic acid is adsorbed and free isoleucine is obtained in the permeate. The isoleucine crystal is obtained by a conventional method, for example, concentration crystallization. The precipitant (p-vinylbenzenesulfonic acid) adsorbed on the resin elutes as an alkali salt when the resin is regenerated with an alkali solution such as sodium hydroxide.
In the method of adding an alkali, isoleucine-p
-An alkali such as sodium hydroxide or the like is added to the aqueous suspension of the vinylbenzene sulfonate crystals as it is or in the form of an aqueous solution, and the pH is adjusted to about 5 to 8, preferably 6 to 7 to obtain p-vinylbenzene. Sulfonic acid is dissolved in a solution as an alkali salt to precipitate free isoleucine, and the separated isoleucine is separated and obtained. The precipitant (p-vinylbenzenesulfonic acid) separated and recovered as an alkali salt can be reused as it is as the next precipitant.

[0014]

The present invention will be described below in detail with reference to examples. In addition, quantification of isoleucine and other amino acids in the examples was performed using a Hitachi L-8500 amino acid analyzer.

Example 1 10 g of L-isoleucine and p
-After adding 100 mL of water to 14 g of vinylbenzenesulfonic acid, the liquid temperature was set to 40 ° C, and the solid content was dissolved. Next, the solution was cooled to 10 ° C and L-isoleucine-p-
Vinyl benzene sulfonate crystals were precipitated, and the precipitated crystals were separated by filtration and dried under reduced pressure. The obtained L-isoleucine / p-vinylbenzenesulfonate is a colorless and transparent plate-like crystal, the crystal system is monoclinic, and the crystal specific gravity is 1.29 g.
/ Cm ³ . FIG. 1 shows a powder X-ray diffraction pattern of the crystal. X-ray diffraction was measured using Cu-Kα radiation as a radiation source. Elemental analysis: C; 50.4%; H; 7.0.
%, N; 4.0%, S; 9.7%. The solubility of the crystals in water was 5.1 wt% (pH 1.5,
10 ° C.).

Comparative Example 1 10 g of L-isoleucine and p
-After adding 100 mL of water to 13 g of toluenesulfonic acid, the liquid temperature was set to 40 ° C, and the solid content was dissolved. Next, the solution was cooled to 10 ° C. to precipitate L-isoleucine / p-toluenesulfonate crystals. The precipitated crystals were separated by filtration and dried under reduced pressure. The solubility of this crystal in water is 1
1.3 wt% (pH 1.5, 10 ° C.).

Comparative Example 2 25 g of L-isoleucine and p
After adding 100 mL of water to 36 g of -ethylbenzenesulfonic acid, the liquid temperature was adjusted to 40 ° C, and the solid content was dissolved. Next, the solution was cooled to 10 ° C and L-isoleucine-p-
Ethylbenzenesulfonate crystals were precipitated, and the precipitated crystals were separated by filtration and dried under reduced pressure. The solubility of the crystals in water was 17.9 wt% (pH 1.5, 10 ° C.).

Comparative Example 3 25 g of L-isoleucine and p
-38 g of normal propylbenzene sulfonic acid and 10 parts of water
After adding 0 mL, the liquid temperature was adjusted to 40 ° C., and the solid content was dissolved. Next, the solution was cooled to 10 ° C., but no crystals were obtained.

The solubility of various benzenesulfonic acid derivatives and isoleucine salts is shown in Table 1 below.

[0020]

[Table 1]

L-isoleucine / p-vinylbenzenesulfonate has the lowest solubility among the salts of benzenesulfonic acid derivatives and isoleucine reported so far. The solubility of isoleucine salts differs greatly even between precipitants having a very similar structure, and the solubility of isoleucine / p-vinylbenzenesulfonic acid is unexpectedly low.

Example 2 10 g of L-isoleucine and 0.2 g each of L-leucine and L-valine were added to water 1
Then, 16 g (equimolar to isoleucine) of sodium salt of p-vinylbenzenesulfonic acid was added thereto, and dissolved by heating.
Adjusted to 5. Next, the solution was cooled to precipitate L-isoleucine / p-vinylbenzenesulfonic acid salt crystals. After the precipitated crystals were collected by centrifugation, they were dissolved in a large amount of hot water, and this solution was passed through an OH-type weakly basic ion exchange resin to remove p-vinylbenzenesulfonic acid. The filtrate was concentrated and crystallized to give free L-
7 g of isoleucine crystals were obtained. Analysis of the mother liquor revealed that the precipitation of isoleucine was 73%, the purity of free L-isoleucine was 98%, the content of other amino acids was 2% or less, and about 68% of impurities before crystallization were eliminated.

Example 3 10 g of L-isoleucine and 0.8 g each of L-leucine and L-valine were added to water 1
Then, 16 g (equimolar to isoleucine) of sodium salt of p-vinylbenzenesulfonic acid was added thereto, and dissolved by heating.
Adjusted to 5. Next, the solution was cooled to precipitate L-isoleucine / p-vinylbenzenesulfonic acid salt crystals. After the precipitated crystals were collected by centrifugation, they were dissolved in a large amount of hot water, and this solution was passed through an OH-type weakly basic ion exchange resin to remove p-vinylbenzenesulfonic acid. The filtrate was concentrated and crystallized to give free L-
6 g of isoleucine crystals were obtained. Analysis of the mother liquor showed that the precipitation of isoleucine was 62%, the purity of free L-isoleucine was 91%, the content of other amino acids was 9% or less, and about 65% of impurities before crystallization were eliminated.

Example 4 5 g of D-isoleucine and 0.1 g each of D-leucine and D-valine were added to water 5
0 g, and 8 g of sodium salt of p-vinylbenzenesulfonic acid (equimolar to isoleucine)
Was added and dissolved by heating, and the pH was adjusted to 1.5 with concentrated sulfuric acid. Next, the solution was cooled to precipitate crystals of D-isoleucine / p-vinylbenzenesulfonate.
After the precipitated crystals were collected by centrifugation, they were dissolved in a large amount of hot water, and this solution was passed through an OH-type weakly basic ion exchange resin to remove p-vinylbenzenesulfonic acid. The permeate was concentrated and crystallized to obtain 3 g of free D-isoleucine crystals. According to the analysis of the mother liquor, the precipitation rate of isoleucine was 74% and the purity of free D-isoleucine was 9%.
The content of other amino acids was 8% or less, and impurities before crystallization were removed by about 68%.

Example 5 10 g of DL-isoleucine and 0.2 g each of L-leucine and L-valine were dispersed in 100 ml of water, and 16 g of sodium salt of p-vinylbenzenesulfonic acid was added thereto (for isoleucine and the like). Mol) was added and dissolved by heating, and the pH was adjusted to 1.5 with concentrated sulfuric acid. Next, the solution was cooled to precipitate crystals of DL-isoleucine / p-vinylbenzenesulfonate. After the precipitated crystals were collected by centrifugation, they were dissolved in a large amount of hot water, and this solution was passed through an OH-type weakly basic ion exchange resin to remove p-vinylbenzenesulfonic acid. The filtrate was concentrated and crystallized to obtain 7 g of free DL-isoleucine crystals. Analysis of the mother liquor showed that the precipitation of isoleucine was 74%, the purity of free DL-isoleucine was 98%, the content of other amino acids was 2% or less, and about 68% of impurities before crystallization were eliminated.

[0026]

As described above, the isoleucine / p-vinylbenzenesulfonate crystals comprising isoleucine and p-vinylbenzenesulfonic acid obtained according to the present invention can be produced inexpensively and easily when used for isoleucine purification. High purity isoleucine can be produced in high yield,
Very useful. That is, the operation of producing isoleucine / p-vinylbenzene sulfonate crystals and purifying isoleucine is highly effective in separating isoleucine from other amino acids that are not easily separated from isoleucine due to the specificity of the salt. Since the solubility of the salt is low, there is an effect that the two features of high yield can be simultaneously used. Also, p
-Vinyl benzene sulfonic acid is industrially manufactured as a polymer raw material, is easily available and inexpensive, and can easily separate and obtain isoleucine from the salt and recover and reuse p-vinyl benzene sulfonic acid. For this reason, the present invention is easy to adapt industrially.

[Brief description of the drawings]

FIG. 1 is a powder X-ray diffraction chart of L-isoleucine.p-vinylbenzenesulfonate crystals obtained in Example 1.

Claims (3)

[Claims] An isoleucine / p-vinylbenzenesulfonic acid crystal comprising 1 mol of isoleucine and 1 mol of p-vinylbenzenesulfonic acid (p-styrenesulfonic acid). 2. An isoleucine-containing aqueous solution is treated with p-vinylbenzenesulfonic acid or a water-soluble salt thereof to produce isoleucine / p-vinylbenzenesulfonic acid crystals, and then the salt is separated. A method for purifying isoleucine, characterized in that isoleucine is obtained by decomposing. 3. The method according to claim 2, wherein the water-soluble salt of p-vinylbenzenesulfonic acid is an alkali metal salt.