JPS63159355A - Recovery of l-phenylalanine and l-aspartic acid - Google Patents

Abstract

PURPOSE:To recover a compound for the production of alpha-APM useful as a dipeptide sweetener, in the form of a high-quality amino acid, by concentrating a mother liquor of alpha-APM production process under a specific condition and hydrolyzing the concentrate with a mineral acid. CONSTITUTION:One or more kinds of mother liquors produced in each process in the production of alpha-aspartyl-L-phenylalanine methyl ester (alpha-APM) are concentrated at <=70 deg.C preferably in the presence of a mineral acid. The concentrated liquid is hydrolyzed with a mineral acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, at a temperature between 90 deg.C and the refluxing temperature to recover L-phenylalanine and L-aspartic acid. The side reactions such as racemization, etc., in the recovering process can be suppressed. Two kinds of amino acids having high quality can be provided as raw materials for the production of alpha-APM without using repurification process.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to the production of α-L-aspartyl-L-phenylalanine methyl ester (hereinafter abbreviated as α-APM). α-APM
and a method for hydrolyzing related dipeptide compounds and recovering them as L-phenylalanine and L-aspartic acid.

More specifically, heat one or more of the mother liquors to 70°C.
After concentrating to a predetermined concentration under the following temperature conditions and optionally in the presence of a mineral acid, this concentrated solution is treated in the presence of a mineral acid and concentrated to a predetermined concentration. The present invention relates to a method for recovering L-phenylalanine and aspartic acid, which comprises hydrolyzing α-APM and related dipeptide compounds contained in the mother liquor in the presence of a mineral acid, and relates to an industrial method for producing α-APM.

α-APM according to the present invention is a substance useful as a new dipeptide sweetener. It is a highly sweet substance that is nearly 200 times more sweet than sucrose and is in growing demand as a dietary sweetener.

(Prior art) Regarding the production method of α-APM, various methods have already been proposed, but when considering the stability of the intermediate, ease of production, and the overall process, the N-protected A method using -L-aspartic anhydride, particularly N-formyl-aspartic anhydride, is a relatively simple process and is industrially suitable. Japanese Patent Publication No. 61-1433
According to the description in No. 97, N-formyl-α-L-aspartic acid anhydride can be directly condensed with L-phenylalanine in water, and N-formyl-α-L-aspartic acid anhydride is an important intermediate for the production of α-APM.
Formyl-α-L-aspartyl-L-phenylalanine can be produced. Furthermore, this N-formyl-α-L-
Aspartyl-L-phenylalanine is converted into α-APM hydrochloride by removing the formyl group and performing an esterification reaction in a hydrochloric acid-methanol medium (Japanese Patent Publication No. 50200/1983), which was also discovered by the present inventors. It is converted into α-APM hydrochloride by removing the formyl group and carrying out an esterification reaction in a medium consisting of sulfuric acid, water and methanol in the presence of a metal chloride. In these methods, α-APM products are produced through further neutralization and purification steps.

Also, N-formyl-aspartic anhydride and L-
A method for producing α-APM hydrochloride from the condensation of phenylalanine in one pot without isolating intermediate intermediates has also been disclosed (Japanese Patent Application Laid-open No. 218597/1983).

These production methods can be carried out in an aqueous medium from the condensation step to the purification step.

By the way, the α-APM production method using N-formyl-L-aspartic acid anhydride produces a non-negligible amount of β-isomer in addition to the target α-isomer during the condensation reaction with L-phenylalanine, i.e. N-formyl-β-mono-aspartyl-L-phenylalanine is produced as a by-product. Also α-APM
During the hydrochloride production process, neutralization process, and even purification process, a non-negligible amount of α-APM and related compounds are dissolved and lost in the mother liquor, so the total yield of α-APM is usually reduced to L-phenylalanine. The yield is less than 50%. Therefore, it is important to efficiently recover active ingredients from these mother liquor systems and improve the raw material consumption rate.
As a method for zero-recovery, which is essential for reducing the production cost of PM, various methods can be considered to recover the active ingredient in different forms (compounds) depending on the type of mother liquor, but each step of the reaction Hydrolyze the resulting mother liquor all at once,
It is preferable to recover the raw materials as l,-phenylalanine and L-aspartic acid because the recovery process is simplified.

Already, the mother liquor produced through the α-APM manufacturing process has been treated to extract the raw material compounds L-phenylalanine and L-
A method for hydrolyzing and recovering aspartic acid is already known (JP-A-48-97812 and JP-A-57-130958).

The method described in JP-A No. 48-97812 discloses that N-benzyloxycarbonyl-cycloaspartic anhydride and L-
A mixture of α and β of APM protected with a hensyloxycarbonyl group is obtained by condensation with phenylalanine methyl ester, which is then catalytically reduced to remove the protecting group, and treated with an aqueous mineral acid solution to form α-APM into a single mixture. The method involves separating β-L-aspartyl-L-phenylalanine methyl ester contained in the remaining mother liquor to recover L-phenylalanine and L-aspartic acid, and relates to a separation technique after hydrolysis.

In this method, the separation after hydrolysis is performed by first adjusting the aqueous solution obtained by hydrolysis treatment in mineral acid to pH 114-8 to separate L-phenylalanine, and then separating the mother liquor into PL.
) 1 to 3, L-aspartic acid is precipitated and separated.

Furthermore, the method described in JP-A-57-130958 condenses N-formyl-L-aspartic acid anhydride and L-phenylalanine in acetic acid to obtain N-formyl-β-L-aspartyl-L-phenylalanine. Then deformylated to α-aspartyl-phenylalanine,
Then, α-
This technology relates to the technology of isolating APM and recovering the active ingredient from the mother liquor system. Specifically, the mother liquor after separating the α-isomer is subjected to i4 condensation, and then the α-APM hydrochloride production mother liquor and hydrochloric acid and water are After that, L-phenylalanine is first precipitated as a hydrochloride and separated, and then the mother liquor is adjusted to the isoelectric point of L-aspartic acid to crystallize L-aspartic acid. This is the way to do it.

Incidentally, both L-phenylalanine and L-aspartic acid used in the production of α-APM are required to be of high quality with high optical purity. If L-phenylalanine or L-aspartic acid with low optical purity is used as a raw material, not only will the yield of α-APM decrease, but in some cases, the quality of the α-APM product may be deteriorated. Therefore, during recovery, it is required to establish a recovery process that suppresses side reactions such as racemization reactions and recovers high quality optical purity.

The problem of racemization in the recovery process has not yet been sufficiently investigated, and high-quality L-
A method for recovering phenylalanine and L-aspartic acid has also not been established.

(Problems to be Solved by the Invention) An object of the present invention is to provide a method for recovering monophenylalanine and monoaspartic acid from the reaction mother liquor produced in the production process of α-APM with high quality. be.

The present inventors are currently considering recovering two types of amino acids that can be hydrolyzed and reused from the mother liquor generated in each step in the production of α-APM. For example, N-formyl-thi-aspartic acid anhydride and L-phenylalanine are condensed in water, and the mixture is diluted with hydrochloric acid to pH 3. 0 to precipitate N-formyl-α-L-aspartyl-L-phenylalanine, and the mother liquor containing the β-isomer obtained by separating this was fAkm to a predetermined concentration under normal pressure, and then hydrochloric acid was added. Teka.

Water splitting was performed. As a result of analyzing the degree of racemization of the two types of amino acids in the obtained hydrolyzed product solution using an amino acid optical resolution column, the content ratio of L-phenylalanine as a unit/D unit was 9773, as that of L-aspartic acid/ The D content ratio was 88/12, and the degree of racemization of aspartic acid was particularly large.

Furthermore, as a result of recovering each amino acid from this hydrolyzed solution according to the method of JP-A-48-97812, a non-negligible amount of racemate was mixed into the recovered amino acids. In particular, the quality of recovered aspartic acid is 94.615.4.
As a result, the product was of poor quality and contained about 10% racemate.

(Means for solving the problem) Based on the above-mentioned results, the present inventors conducted a detailed study of the factors that induce the racemization reaction in the recovery process through various model experiments, and found that heat and pH promote racemization. In particular, if the temperature is raised to about 80°C or higher at around pH 3, the aspartic acid side tends to racemize significantly regardless of the substrate, and hydrolysis under these conditions tends to cause racemization. It was found that the degree of racemization of the recovered aspartic acid obtained was increased. On the other hand, it was also found that the degree of racemization was smaller under acidic mineral acid conditions even under the same temperature conditions.

By the way, the concentration of α-APM and related compounds contained in various mother liquors produced in the α-APM production process is usually low, so these compounds in the mother liquor are hydrolyzed to produce two types of raw materials. Two concentration operations are essential to recover the amino acids. After hydrolysis? Concentration may be considered, but when considering the volumetric efficiency during hydrolysis and the rate of hydrolysis, it is more efficient to am the mother liquor to a predetermined concentration before hydrolysis treatment.

Based on these findings, the present invention suppresses racemization in the recovery process of recovering L-phenylalanine and L-aspartic acid from the mother liquor produced in each process in the production of α-APM, and produces amino acids with optically good quality. It has now been completed as a method for collecting

That is, the present invention involves concentrating one or more mother liquors generated in the α-APM production process to a predetermined concentration under a temperature condition of 70° C. or lower, and then treating the concentrated solution in the presence of a mineral acid. This method is characterized by recovering L-phenylalanine and L-aspartic acid obtained by hydrolysis.

The mother liquor recovered in the method of the present invention includes (1) N
- N-formyl-α produced by condensing L-phenylalanine with formyl-L-aspartic anhydride in water
- Mother liquor produced by separating L-aspartyl-L-phenylalanine, (2) N-formyl-α-L-
The mother liquor produced when aspartyl-L-phenylalanine is converted to α-APM hydrochloride, (3) the mother liquor produced when α-APM hydrochloride is neutralized in an aqueous medium, and (
4) This is the mother liquor produced when α-APM is purified with water or a mixed solvent of lower alcohol and water, and α-APM is produced using N-formyl-cyaspartic acid anhydride and L-phenylalanine as raw materials. A series of mother liquors in the process are applied. Although the mother liquors may be treated individually, it is more efficient to treat a series of mother liquors in combination.

The method of the present invention comprises the steps of concentrating these mother liquors to a predetermined concentration and then hydrolyzing them in the presence of mineral acids.

The first concentration step is performed at a temperature of 70°C or lower.

This suppresses racemization in the concentration step as much as possible. 7
When heated at a temperature of over 0°C, especially at a temperature of 80°C or higher, and at a pH of around 3, the α-
Aspartic acid produced by hydrolysis of APM and related dipeptide compounds is particularly susceptible to racemization. The degree of concentration cannot be absolutely limited depending on the type and amount of inorganic salts contained, but it must be to the extent that the two types of amino acids produced in the subsequent hydrolysis treatment can be separated and recovered without the need for further concentration operations. ? Better to concentrate.

Usually, the concentration in terms of L-phenylalanine is 3% by weight or more, preferably 5% by weight or more.

The upper limit of concentration is preferably about 15% by weight. If it is too concentrated, inorganic salts may precipitate depending on the type of mother liquor.
This is not preferable because it reduces the quality of the recovered amino acids, requires further separation from inorganic salts, and impairs workability.

This concentration step is more preferably carried out in the presence of a mineral acid in order to further suppress racemization of the substrate. The mineral acid to be present in the system is hydrochloric acid, sulfuric acid or hydrobromic acid,
The amount is not particularly limited, but the p of the mother liquor subjected to f:4 condensation is
Concentration operation is performed by adding these mineral acids so that l+ is approximately smaller than l.

Further, concentration is not limited to concentration under reduced pressure as long as the temperature during concentration is 70° C. or lower. However, usually, a vacuum concentration method of concentrating under reduced pressure is often used. Concentration using a membrane such as a reverse osmosis membrane is also possible.

In the present invention, after the concentration treatment under the above-mentioned conditions, various dipeptide compounds in the concentrated mother liquor are hydrolyzed in the presence of mineral acids to produce two amino acids as raw materials, namely L-phenylalanine and L-phenylalanine. -converted to aspartic acid.

Although this hydrolysis depends on the concentration and amount of mineral acid used, it is preferable to carry out it at a somewhat elevated temperature in terms of the rate of hydrolysis. It is usually carried out in the range of 90°C to reflux temperature.

The mineral acid used in this hydrolysis step is often hydrochloric acid, sulfuric acid, or hydrobromic acid, and the amount used is 3 to 30% in the system concentration.
% by weight.

L-phenylalanine and L- produced after hydrolysis
Aspartic acid can be easily separated and recovered by the method disclosed in JP-A-48-97812 or JP-A-57-130958.

For example, an aqueous solution obtained by hydrolysis treatment in a mineral acid is first adjusted to pH 4 to 8 to separate L-phenylalanine, and then the mother liquor is adjusted to pH 111 to 3 to precipitate and separate L-aspartic acid. ing.

(Actions and Effects) The method of the present invention involves hydrolyzing the mother liquor produced in each step in the production of α-APM to recover the raw materials L-phenylalanine and L-aspartic acid, which undergo side reactions such as racemization. This is an extremely effective method for suppressing . Therefore, the quality of the two types of amino acids recovered is also extremely good, and they can be used as raw materials for α-APM production without the need for repurification.

(Example) The present invention will be explained below with reference to Examples.

In the examples, the degree of racemization of L-phenylalanine and L-aspartic acid was analyzed by high performance liquid chromatography under the following conditions.

Separation condition column TSK gel Enantio Ll 4.
6ms+I DX25cm (manufactured by Toyo Soda H) Mobile phase 0.5mM CuSO4aq Flow rate 1.0ml/sin Detector Ultraviolet spectrophotometer wavelength 254nm Production example 1 (a) Production of N-formyl-α-L-aspartyl-L-phenylalanine 165.2 g (1.0 mol) of L-phenylalanine was suspended in 1650 sl of water, and 88.9 g of a 45% aqueous sodium hydroxide solution was added dropwise to dissolve it. This solution was cooled to below 5°C, and 150.2 g (1.05 mol) of N-formyl-di-aspartic anhydride was added at approximately 1 to 5°C.
It took some time and was gradually added. During this time, 97 g of a 45% aqueous sodium hydroxide solution was added dropwise to maintain the pH of the reaction solution at around 10. Thereafter, the reaction was continued for an additional hour at the same temperature, and then concentrated hydrochloric acid was added dropwise to the reaction solution until the pH reached 3.1. After stirring at 5°C for 1 hour, the precipitated crystals were filtered, washed with cold water, and dried under vacuum to obtain N-formyl-α-L-aspartyl containing 1.0% β-isomer.
221 g of L-phenylalanine was obtained. On the other hand, the filtrate and washing liquid were combined to obtain 2130 g of mother liquor.

(b) Production of α-APM-11cI A solution prepared by adding 68.2 g of methanol to 585 g of a 25% aqueous sulfuric acid solution was heated to 50"C, and the N-formyl-α- produced in (a) was added to the solution. 221 g of L-aspartyl-L-phenylalanine was gradually charged, and then 50 g of
After reacting at ~55°C for 2 hours, the solution was cooled to 25°C, and 119°5 g of anhydrous magnesium chloride was charged and dissolved in the solution, followed by reaction at room temperature for 4 days. The precipitated crystals were filtered and washed with cold water to obtain α-APM hydrochloride. α-APM coverage in the wet cake was 168.2 g. On the other hand, the filtrate and washing liquid were combined to obtain 780 g of mother liquor.

(c) Production of α-APM (neutralization and purification of α-APM hydrochloride!!) The α-APM hydrochloride obtained in (b) was suspended in 1700 ml of water, and the concentrated ammonia water was heated at 20 to 25°C. and pH
= 5.2. Thereafter, it was filtered while cooling to 5°C and washed with cold water to obtain a wet cake of α-APM. At this time, 1850 g of mother liquor was obtained.

The α-APM wet cake obtained here was 50% (ν/
V) 144 g of purified α-APM was obtained by recrystallization purification from 1600 ml of methanol aqueous solution and drying under reduced pressure. This purification step yielded 1820 g of mother liquor.

Example 1 1820 g of the α-APM purified mother liquor obtained in the above production example was concentrated to about 173 ml under reduced pressure over a period of 4 hours, taking care not to allow the internal temperature to exceed 70°C. Next, 2130 g of the mother liquor from the production of N-formyl-α-L-aspartyl-L-phenylalanine was charged, and 50 g of the mother liquor 1B from the neutralization of α-APM hydrochloride and 780 g of the mother liquor from the production of α-APM hydrochloride were charged. While gradually charging the liquid, reduce the pressure to 1 so that the liquid temperature does not exceed 70°C! The mixture was concentrated until the concentrate weighed 1320 g. The time required during this period was approximately 12 hours.

Next, 120 g of concentrated hydrochloric acid was added to this concentrated solution, and the
The hydrolysis reaction was carried out at −150° C., yielding 98% of theoretical phenylalanine. The time required for this hydrolysis was 15 hours. The degree of racemization of the two produced amino acids was analyzed by high performance liquid chromatography, and the results were as follows.

L-phenylalanine: D-phenylalanine = 99.
2: 0.8 L-aspartic acid: D-aspartic acid = 97.4:
2. The solution obtained by treating the hydrolyzed solution with activated carbon and filtering it hot was cooled to 30 °C, neutralized to po = 5.6 with 45% aqueous sodium hydroxide solution, cooled to 5 °C, and filtered. By washing with cold water and drying, L-
Phenylalanine was recovered. The L-phenylalanine recovered here did not contain any D. Further, the results of measurement of specific optical rotation were as follows.

[α] 1°=-34,3 (C=2.1110) On the other hand, the filtrate and washing liquid after recovering L-phenylalanine was adjusted to pi+=2.3 with concentrated hydrochloric acid. After cooling, the precipitated mono-aspartic acid was treated with 2 It, washed with water and dried, resulting in a theoretical recovery rate of 76%. As a result of analysis of the mono-aspartic acid recovered here, the content of D-aspartic acid was 0.3% or less, and L-phenylalanine was not detected. In addition, the specific optical rotation was as follows.

[α]C' = +25.5 (C = 8.6NHCI) Example 2 When concentrating in Example 1, 4 ml of concentrated hydrochloric acid was added in advance.
Concentration and hydrolysis were performed in the same manner as in Example 1 except that 2 g was added (the scale, concentration conditions, and hydrolysis conditions were almost the same).

As a result of analysis of the hydrolyzed solution, the degree of racemization was as follows.

L-phenylalanine: D-phenylalanine = 99.
5F 0.4 L-aspartic acid: D-aspartic acid = 98.2:
1.8 Comparative Example 1 In Example 1, the concentration before hydrolysis was carried out under normal pressure for 14 hours. The final internal temperature was 109 degrees. After that, 120g of concentrated hydrochloric acid was charged and the temperature was 100~105° under normal pressure.
A hydrolysis reaction was carried out with C to produce 99% of the theoretical phenylalanine. The time required for this hydrolysis reaction was 13
It was 11r. The results of analysis of the two produced amino acids by high performance liquid chromatography were as follows.

L-phenylalanine: D-phenylalanine = 98.
l: 1.9 L-aspartic acid: D-aspartic acid-80,5:
19.5 L-phenylalanine and L-aspartic acid were recovered from the hydrolysis solution in the same manner as in Example 1.

L-phenylalanine Recovery rate: 81.3% L-phenylalanine: D-phenylalanine = 99.77 0.3 L-Aspartic acid Recovery rate: 63.5% L-aspartic acid: D-aspartic acid = 83.5: 16.5 Comparative Example 2 In Example 1, the concentration before hydrolysis was carried out at 80 to 85% under normal pressure.
The same procedure as in Example 1 was carried out except that the temperature was 0.degree.

The degree of racemization of the two types of amino acids after hydrolysis and the quality of the two types of amino acids recovered were as follows.

Analysis results of hydrolyzed solution L-phenylalanine: D-phenylalanine = 98.
5: 1.5 L-aspartic acid: D-aspartic acid = 9o, a:
7.8 Recovered amino acids L-phenylalanine: D-phenylalanine = 9
9.8: 0.2 L-aspartic acid: D-aspartic acid-96,4:
3.6

Claims (1)

[Scope of Claims] 1) After concentrating one or more mother liquors produced in the process of producing α-aspartyl-L-phenylalanine methyl ester under a temperature condition of 70°C or lower, this concentrated liquid is concentrated in the presence of a mineral acid. 1. A method for recovering L-phenylalanine and L-aspartic acid, the method comprising: 2) The method according to claim 1, wherein the concentration is carried out in the presence of a mineral acid. 3) The method according to claim 2, wherein the mineral acid is hydrochloric acid, hydrobromic acid or sulfuric acid. 4) The mother liquor is α-aspartyl using N-formyl-L-aspartic anhydride and L-phenylalanine as raw materials
The method according to claim 1 or 2, which is produced in a method for producing L-phenylalanine methyl ester.