JPH0379990B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for obtaining L-alanine crystals,
More specifically, the present invention relates to a method for obtaining high-purity L-alanine crystals by a simple means from a crude L-alanine solution containing ammonium salts of fumaric acid, L-malic acid, and L-aspartic acid as contaminants.

An industrial method for producing L-alanine is to produce L-aspartic acid using fumaric acid and ammonia as starting materials using a microorganism having aspartase activity, or simultaneously to produce L-aspartic acid.
A method for producing L-alanine using microorganisms having β-decarboxylase activity is known, and this method is said to be efficient because it can omit the crystallization and separation operation of L-aspartic acid. There is. Furthermore, recently, when the above enzymatic reaction is carried out using immobilized microorganisms, impurities such as the culture materials of microbial cells and various proteins are not contained in the enzyme reaction finished solution, and therefore, the enzyme reaction finished solution is efficiently extracted. It is known that L-alanine can be obtained. L using immobilized microorganisms in this way
-Although the method for producing alanine is an excellent method, since the above-mentioned microorganisms that have aspartase activity also have fumarase activity, the enzymatic reaction using this microorganism also involves the conversion reaction of fumaric acid to L-malic acid. occur at the same time. Also, from fumaric acid to L-
There is a reaction equilibrium in the reaction to produce aspartic acid, and the starting material, fumaric acid, remains.
Furthermore, in the reaction to produce L-alanine from L-aspartic acid, 100% L-aspartic acid is used.
The reaction cannot be carried out, and L-aspartic acid remains, and in addition, carbon dioxide is produced as a by-product in the reaction, and this reacts with the raw material ammonia to produce ammonium carbonate. That is, even in the method for producing L-alanine using the above-mentioned immobilized microorganism, the enzyme reaction finished solution contains fumaric acid, L-malic acid, L-aspartic acid (these Since the acid is a strong acid, it contains impurities such as its ammonium salt (in the form of its ammonium salt) and ammonium carbonate. For this reason, the conventional method for obtaining high-purity L-alanine crystals from an L-alanine solution containing such impurities was to treat the solution with an ion exchange resin and fractionate the fraction corresponding to L-alanine. This method requires complicated purification and separation operations, such as subsequent concentration and crystallization, or once separated as crude L-alanine crystals and then recrystallization, which is not necessarily satisfactory from an industrial perspective. .

As a result of intensive research into the above problem, the present inventors found that L-alanine crystals were obtained from a crude L-alanine solution containing ammonium salts of fumaric acid, L-malic acid, and L-aspartic acid as impurities. When obtaining L-alanine, the present invention was completed based on the discovery that by adding an alkali metal to the crude L-alanine solution, high-purity crystals of L-alanine can be obtained in high yield with a simple operation. That is, the present invention provides a method for obtaining L-alanine crystals from a crude L-alanine solution containing ammonium salts of fumaric acid, L-malic acid, and L-aspartic acid as impurities. Fumaric acid, L-malic acid, and L-malic acid coexisting in
- A method for obtaining L-alanine crystals, which comprises performing crystallization after concentration in the presence of about 0.5 to 1.5 equivalents of alkali metal based on the total amount of aspartic acid.

The crude L-alanine solution used in the present invention can be obtained, for example, as described above, by a known method of reacting with an immobilized enzyme using fumaric acid and ammonia as substrates. For example, microbial cells having aspartase activity or a processed product thereof and microbial cells having L-aspartate-β-decarboxylase activity or a processed product thereof are collected using known methods (e.g., carrageenan gel entrapment method, polyacrylamide gel entrapment method, etc.). It can be easily produced by carrying out an enzymatic reaction using an immobilized product obtained by immobilization using a method such as a method. As the microorganism having aspartase activity, any microorganism having this activity can be used, such as Escherichia coli (ATCC-11303), and as the microorganism having L-aspartate-β-decarboxylase activity, Any active substance can be used, and for example, Pseudomonas dacne (IAM-1152) can be suitably used.

The alkali metals present in the crude L-alanine solution obtained by the above enzymatic reaction are as follows:
For example, alkali metals such as sodium and potassium are preferably mentioned, and these include, for example, alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates such as sodium carbonate and potassium carbonate, and sodium hydrogen carbonate and potassium hydrogen carbonate. Preferably, it is supplied as an alkali metal bicarbonate such as. The amount of the alkali metal added is 0.5 equivalent or more, preferably 1 to 1.5 equivalent, based on the total amount of impurities coexisting in the crude L-alanine solution, that is, fumaric acid, L-malic acid, and L-aspartic acid. is preferable. These alkali metal compounds are preferably added to the crude L-alanine solution after the enzymatic reaction, but they may also be added in advance according to the amount when preparing the substrate or at the end of the converting enzyme reaction to L-aspartic acid. good. When added at the time of substrate preparation, it is in the form of an alkali metal salt of fumarate such as sodium fumarate and potassium fumarate, and when added at the end of the conversion enzyme reaction to L-aspartic acid, it is added in the form of sodium L-aspartate, L-aspartate, etc. - It can also be supplied in the form of an alkali metal salt of L-aspartate, such as potassium aspartate.

In obtaining L-alanine crystals from a crude L-alanine solution in the presence of an alkali metal, it can be carried out, for example, by treating the solution with activated carbon, concentrating it, and then crystallizing L-alanine crystals. .

Activated carbon treatment removes pyrogenic substances (pyrogienes) derived from solid particles and microbial cells contained in the solution.
In order to perform this operation efficiently, it is preferable to carry out the operation under heating (about 80°C to about 60°C). A treatment time of approximately 10 to 20 minutes is sufficient. This activated carbon treatment may be carried out directly on the crude L-alanine solution, or may be carried out after first concentrating the L-alanine solution to some extent.

The purpose of the concentration operation is to remove ammonia contained in the solution, and the concentration operation is performed at a relatively low temperature (approximately 80°C or lower, preferably approximately 60°C or lower) to avoid racemization or decomposition of L-alanine. It is preferable to carry out the process under reduced pressure. In addition, according to the present invention method of concentration in the presence of an alkali metal, since the solution is alkaline, ammonium ions (NH ₄ ⁺ ) contained in the solution are converted into dissolved ammonia (NH ₃ ). Ammonia can be efficiently removed in this concentration operation.

In order to crystallize L-alanine crystals from the L-alanine solution subjected to the concentration operation as described above, it is easy to further concentrate the solution, cool it, or add an aqueous solvent such as methanol or ethanol. It can be implemented. These operations can be performed by methods commonly used for crystallizing amino acids. In addition, since the liquid property during the crystallization operation affects the yield of the target product, if necessary, adjust the liquid property of the crystallization system to PH5.0 to 9.0, preferably 6.0 to 8.0 for crystallization. is preferable. To adjust the pH, use hydrochloric acid,
Acetic acid, sulfuric acid, etc. can be used.

According to the method of the present invention as described above, fumaric acid, L
- Contains no impurities such as malic acid or L-aspartic acid, and no pyrodiene contamination.Furthermore, extremely high purity L- with low ammonium content.
Alanine crystals can be obtained in high yield with a very simple operation without complicated operations such as recrystallization or ion exchange resin treatment. As mentioned above, the L-alanine crystals obtained by the method of the present invention do not contain impurities or pyrodiene, so they can be used as they are for medical purposes (for example, as bulk powder for infusions).

Therefore, the method of the present invention is an excellent method for industrially producing high-purity crystals of L-alanine.

The present invention will be further explained in detail with reference to Examples below.

Example 1 L-alanine solution obtained in Reference Example 2 (L-alanine 1.4 mol, fumaric acid 28 mmol, L
- containing 52 mmol of malic acid, 15 mmol of L-aspartic acid and ammonium carbonate) 1,
170 mmol of potassium hydroxide and activated carbon "Snow A"
After adding 2 g of activated carbon (manufactured by Fujisawa Pharmaceutical Co., Ltd.) and heating at 60°C for 20 minutes, the activated carbon is separated. After concentrating the filtrate under reduced pressure to a liquid volume of approximately 180 ml,
Cool to 15°C (PH of the coolant: 8.0). The precipitated crystals were collected by filtration, washed with a small amount of water-containing methanol, and then dried to obtain 114.9 g of L-alanine crystals.
Yield 92.1% [α] ²⁰ _D +14.8° (C = 10, 6N-HCl) Fumaric acid (Note 1); Not detected, L-malic acid (Note 1); Not detected, L-Aspartic acid (Note 2); Not detected and pyrogen (Note 3); Not recognized (0.05°, 0.10°,
0.15°) Ammonium content (Note 4); 9ppm Note 1: Capillary isotachophoresis analysis method (Shimadzu 1P-
Type 2A). Detection limit 0.01% Note 2: Analyzed by high performance liquid chromatography. Detection limit 0.005% Note 3: 10% L-alanine 5% solution by rabbit method
Conducted under mg/Kg conditions. Other conditions were based on the 10th edition of the Japanese Pharmacopoeia.

Note 4: Analyzed by indophenol method.

Example 2 L-alanine solution obtained in Reference Example 2 (L-alanine 1.41 mol, fumaric acid 29 mmol,
L-malic acid 53 mmol, L-aspartic acid 2
0.2 mol of potassium hydroxide was added to 1 (containing mmol and ammonium carbonate), treated with activated carbon and concentrated under reduced pressure under the same conditions as in Example 1 to a total volume of about 200 ml, and then cooled to 15°C. (of the cooling liquid)
PH9.6). Next, adjust the pH of the solution to 7.6 with concentrated hydrochloric acid, and let it stand for about 1 hour. The precipitated crystals were collected by filtration, washed with a small amount of water-containing methanol, and then dried to obtain 113.7 g of L-alanine crystals. Yield 90.5% [α] ²⁰ _D +14.8° (C = 10, 6N-HCl) Fumaric acid: not detected L-malic acid: not detected L-aspartic acid: not detected Pyrogen; not recognized (0.20°, 0.15°, 0.10°) Ammonium content: 11 ppm Example 3 Reference Example 2 described later using a 1.25 M ammonium fumarate aqueous solution containing 10 ^-3 M magnesium chloride and 0.08 M dipotassium fumarate as a substrate solution.
An enzymatic reaction was carried out in the same manner as in (3), and L-alanine solution (1.26 mol/L-alanine, 13.5 mol/fumaric acid) was prepared.
mmol/, L-malic acid 49.4 mmol/,
1.5 mmol of L-aspartic acid/containing ammonium carbonate) is obtained. Add 4 g of activated carbon "Yuki A" to 2.2 of the L-alanine solution and heat at 80°C.
Separate the activated carbon after heating for 10 minutes. The filtrate is concentrated under reduced pressure to a total volume of about 280 ml, and then cooled to 15°C (PH of the cooling liquid: 9.0). By filtering the precipitated crystals, washing them with a small amount of water-containing methanol, and drying them,
228.7 g of L-alanine crystals are obtained. Yield 92.4% [α] ²⁰ _D +14.8° (C = 10, 6N-HCl) Fumaric acid: not detected L-malic acid: not detected L-aspartic acid: not detected Pyrogen; not recognized (0.10°, 0.20°, 0.00°) Ammonium content: 10ppm Reference example 1 An example in which no alkali metal is used, showing that the purity of the obtained target product is greatly affected.

2 g of activated carbon "Yuki A" was added to L-alanine solution 1 similar to that used in Example 1, and the mixture was heated at 60°C.
Separate the activated carbon after heating for 20 minutes. The filtrate is concentrated under reduced pressure to a total volume of about 200 ml, and then cooled to 15°C (PH of the cooling liquid: 5.6). By filtering the precipitated crystals, washing them with a small amount of water-containing methanol, and drying them, L
- Obtain 110.6 g of alanine crystals. Yield 88.7% [α] ²⁰ _D +14.7° (C = 10, 6N-HCl) Fumaric acid; 0.04% L-malic acid; 0.02% L-aspartic acid; 0.013% Ammonium content: 164 ppm This product is Compared to the crystals obtained by the invention method, there is contamination and the ammonium content is about 10 times higher.

Reference Example 2 (Production of L-alanine using immobilized bacterial cells) (1) Preparation of immobilized bacterial cells having aspartase activity Corn steep liquor 2%, meat 2%,
Fumaric acid 1.14%, diammonium fumarate 0.5
%, 100 ml of medium (PH7.0) containing monopotassium phosphate 0.2% and magnesium sulfate 0.05%.
Pour into a 500 ml Sakaguchi flask and inoculate it with Escherichia coli (ATCC-11303). 30
After culturing with shaking at ℃ for 16 hours, 23 g (wet weight) of E. coli cells were obtained by centrifugation.

Separately, add 6 g of Genyugel WG (carrageenan manufactured by Copenhagen Pectin Factory).
A carrageenan aqueous solution is prepared by dissolving it in 129 ml of warm water at 45°C, and 23 g of E. coli cells obtained above suspended in 23 ml of physiological saline are added to this solution and mixed at 40°C. Mixed liquid at 4℃
Cool it to 30 minutes and leave it for 30 minutes.
Form into a mm cube. The obtained molded gel is
- immersed in 400 ml of an aqueous solution containing 10 mmol of aspartic acid and 2% potassium chloride at 37°C for 24 hours.
Leave it for a while. The resulting gel was separated and washed with a 2% potassium chloride aqueous solution to obtain 180 g (immersion weight) of immobilized E. coli cells.

(2) Preparation of immobilized bacterial cells having L-aspartate-β-decarboxylase activity Sodium glutamate 3.2%, Meast 0.5
%, monobasic potassium phosphate 0.05%, and magnesium sulfate 0.01% (PH7.3) into 10 500 ml Sakaguchi flasks, 120 ml each.
This is combined with Syedomonas dacne (IAM-
1152). After culturing with shaking at 30°C for 24 hours, 20 g (wet weight) of Pseudomonas dacne cells were obtained by centrifugation.

Separately, add Genyugel WG 4.03g to 50℃ warm water 85
Prepare a carrageenan aqueous solution by dissolving 20 g of the Pseudomonas dacne cells obtained above in 20 ml of physiological saline.
Add and mix at ℃. Cool the mixture to 4°C
Immobilized Pseudomonas by molding the resulting gel into a cube with a side of approximately 3 mm.
Obtain 130 g (soaked weight) of Daknei bacterial cells.

(3) Enzyme reaction Pack 40 g of the immobilized bacterial cells obtained in (1) into a column with an outer shell (inner diameter 1.6 cm, length 19 cm). to this,
A 1.5M ammonium fumarate aqueous solution containing 10 ^-3 M magnesium chloride (pH 8.5 solution prepared with 1.5M fumaric acid and ammonia) was heated at 37℃ for 20 minutes.
Conduction was conducted at a flow rate of ml/hr. By adding L-aspartic acid crystals to this effluent, pH6.0 was achieved.
6.5 and add 10 ⁻⁴ M pyridoxal phosphate to the solution. This solution was poured upward at 37°C from the bottom of a column with an outer jacket tube (inner diameter 2.1 cm, length 34.8 cm) filled with 120 g of the immobilized bacterial cells obtained in (2) at a flow rate of 20 ml/hr. Conduct. By collecting the effluent, an L-alanine-containing enzyme reaction completed solution is obtained.

Claims (1)

[Claims] 1. When obtaining L-alanine crystals from a crude L-alanine solution containing ammonium salts of fumaric acid, L-malic acid, and L-aspartic acid as impurities, fumaric acid coexisting in the crude L-alanine solution , L-malic acid and L-aspartic acid are concentrated in the presence of about 0.5 to 1.5 equivalents of an alkali metal relative to the total amount, and then crystallized.