KR950005424B1 - Process for producing l-aspartyl-l-phenylalanine and its diketo piperazine - Google Patents

Abstract

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Description

Method for preparing L-aspartyl-L-phenylalanine and diketopiperazine thereof

The present invention provides a method for producing L-aspartyl-L-phenylalanine (hereinafter abbreviated as "AP") which is a precursor of L-aspartyl-L-phenylalanine methyl ester (hereinafter abbreviated as "AMP"). It is about. AMP is a dipeptide sweetener that has attracted attention in recent years.

Known dipeptide synthesis methods using microorganisms or enzymes include introducing a suitable protecting group to a functional group that is not involved in the condensation reaction between two amino acids to limit the location of the condensation reaction. Next, the microantigen condensation product is obtained using a microorganism or an enzyme capable of forming a poptide bond between these amino acid derivatives, and then the unnecessary protecting group is removed from the condensation product to obtain a dipeptide product. Using this method, a satisfactory yield can be obtained but a step of introducing and removing the protecting group is required. Therefore, expensive protection groups are needed and complicated and complex reaction steps are required.

For the biochemical synthesis method of AMP, for example, benzyloxy group (-Z) is used as a protecting group for the amino group of aspartic acid, and N-benzyloxy-L-aspartic acid and phenylalanine methyl by the action of an endotype protease. Methods of condensing esters are known. These methods have the drawbacks described above.

In addition, no protecting group is necessary and a biochemical synthesis method of AMP is also known (Japanese Patent Laid-Open No. 129796/83). This type of method does not require protection groups, but yields poorly and poses a problem in the synthesis of APM.

On the other hand, in the chemical synthesis of APM, it is necessary to use L-aspartic acid and L-phenylalanine to prepare a sweetener APM. This is because of the four optical isomers of aspartyl-phenylalanine, only L-aspartyl-L-phenylalanine methyl ester (APM) consisting solely of L-amino acids is sweet.

Since no superior method for separating these four optical isomers is known until now, DL-amino acids which are cheaper than L-amino acids cannot be used as raw materials.

In addition, in order for the reaction to proceed, the reaction parallel must be directed towards the adverse condensation reaction accompanying the AP synthesis. In the enzymatic condensation reaction of L-aspartic acid and L-phenylalanine, the AP should be removed from the reaction system so that the synthetic equilibrium reaction proceeds to the AP synthesis. For this reason, in the process where these condensation reactions are used, The AP is removed by adsorbing onto the ion exchange resin or by incorporating a precipitant in such a way that the AP is selectively insoluble.

The methods in these prior arts have some serious drawbacks: conventional physiological synthetic methods that do not require protecting groups are poor in yield, and expensive protective group reagents and unnecessary methods for methods involving the protection of functional groups The reaction step is used.

It is therefore an object of the present invention to provide a method for preparing AP that does not require the use of expensive protective group drugs or reaction steps following the protection of functional groups.

It is a second object of the present invention to provide a method for producing an AP in high yield without using a protecting group.

It is also an object of the present invention to provide a novel method of producing expensive APM and AP on a large scale with good efficiency.

These and other objects of the present invention will become apparent from the following detailed description, which can hydrolyze L-aspartyl-L-phenylalanine diketopiperazine to produce L-aspartyl-L-phenylalanine. A method for producing an AP of the present invention is characterized by contacting a culture, cell or cell-treatment material of a microorganism with L-aspartyl-L-phenylalanine diketopiperazine in an aqueous medium.

The method of the present invention also provides a culture, cell or cell-treatment material of a microorganism capable of intermolecular condensation of L-aspartyl-L-phenylalanine to produce L-aspartyl-L-phenylalanine diketopiperazine. Also included are methods for preparing L-aspartyl-L-phenylalanine diketopiperazine characterized by contacting with L-aspartyl-L-phenylalanine in an aqueous medium.

AP can be converted to APM by a simple methyl esterification reaction. The present invention relates to a process for producing AP, more particularly to a process for producing AP from L-aspartyl-L-phenylalanine diketopiperazine (hereinafter abbreviated as "DKP").

Since the above-mentioned reaction is an equilibrium reaction, it is also possible to synthesize DKP from AP by reverse reaction. DKP is also an important material from APM precursors. After chemically synthesizing the AP, the AP can be converted to DKP. In this method, optically active AP can be separated from the reaction mixture, for example D-amino acid-mixed aspartylphenylalanine in the reaction mixture, so that optically active AP is easily obtained.

The yield can be increased by using microorganisms that can induce intermolecular condensation of the AP and convert the AP into DKP. Because this method uses an equilibrium reaction system that is very disadvantageous for AP synthesis, for example, due to the direct binding of aspartic acid with phenylalanine by enzymes, the traces of AP produced are converted to DKP and the equilibrium is changed towards synthesis.

It has been found that the hydrolysis of DKP takes place at specific locations and can be used to synthesize AP using reverse reactions as shown below.

Scheme 1

L-aspartyl-L-phenylalanine diketopiperazine (DKP) Aspartyl-L-phenylalanine (AP)

Examples of microorganisms that can be used in the present invention include, but are not limited to: Streptomyces flavovirens IFO 3197 [Korea Accession No .: KFCC-10287, Deposit period: Korea Communist Association, Deposition date: October 17, 1986], Streptomyces parvulus AJ 9037 FERM-P 8920 FERM BP 1168 17], Achromobacter lacticum AJ 2394 FERM-P 7401, Agrobacterium tumefaciens ATCC 4452, Alcaligenes faecails AJ 2565 FERM-P 8460, Bacillus Bacillus circulas ATCC 9966, Enterobacter agglomerans ATCC 12287, Ervinia amylovora FERM-P 7056, Flavobacterium rhenanum AJ 2468 -P 8459, microphone Micrococcus varians ATCC 339, Serratia marcescence AJ 2763 FERM-P 8461, Xanthomonas citri AJ 2797 FERM-P 8462.

In order for these microorganisms to act on the DKP or AP, the microorganisms are cultured and the culture medium or cell or cell treatment material of the microorganism is contacted with the DKP or AP in an aqueous medium.

The culture medium of the above-mentioned microorganisms is a conventional medium containing a crude carbon source, a nitrogen source and inorganic ions. Sometimes desirable results are obtained by further incorporation of organic micronutrients such as vitamins, amino acids and the like.

As the carbon source, carbohydrates such as glucose, sucrose and the like, organic acids such as acetic acid and alcohol, and the like can be appropriately used. As the nitrogen source, ammonia gas, ammonia water, ammonium salt and the like can be used. As the inorganic ions, magnesium ions, phosphate ions, potassium ions, iron ions and the like can be appropriately used as necessary.

Cultivation is carried out for 1 to 30 days under aerobic conditions, adjusting the pH and temperature to appropriate ranges of 2 to 8 and 15 to 30 ° C, respectively.

In order for the culture, cells or cell-treatment of the microorganism to act on the DKP or AP in an aqueous medium, the cell or cell-treatment may be suspended or lysed in an aqueous medium containing DKP or AP, and the aqueous medium Can be stirred or allowed to settle for a short time while adjusting the temperature to a preferred range of 10 to 70 ° C.

In the reaction to generate AP from DKP, the amount of DKP used is not particularly limited, but in the case of a Batch system, the amount of use is generally about 0.01 to 2.0M, preferably 0.01 to 1.0M.

In the case of the reverse reaction producing DKP from the AP, the amount of AP used is not particularly limited but in the case of the Batch system, the amount is generally approximately 0.00001 to 2.0M, preferably 0.01 to 1.0M.

DKP or AP can also be added in portions as the reaction proceeds.

The reaction is generally carried out at 10 to 60 ° C., preferably 20 to 40 ° C. in aqueous vegetation.

In the case of using bacterial cells, the culture solution containing the cells can also be used as it is. The cells can also be used after washing off from the culture or with or without washing.

Suitable cell-treatments include, but are not limited to, mechanically ground cells, ultrasonically treated cells, lyophilized cells, cells acetone dried, cells treated with enzymes such as lysozyme, cells treated with surfactants, toluene, and the like. Protein fractions, immobilized materials thereof, and the like.

In order to obtain such cell strains, the above-mentioned media and culture methods can be applied without modification.

After culturing the microorganisms, a small amount of DKP, AP, or both DKP and AP can be mixed in the medium to obtain cells with high ability to produce AP from DKP or DKP from AP.

In the equilibrium reaction of biochemical condensation of L-aspartic acid with L-phenylalanine, generally, a method for removing the generated AP from the reaction system (e.g., treatment with an ion exchange resin, extraction with a precipitant, a solvent, specific for AP Reaction, etc.) to drive the reaction towards the condensation reaction. These methods can also be used in the present invention using the reaction of converting DKP to AP. That is, by incorporating a microorganism capable of converting AP into DKP or a treatment material thereof (as described above), the reaction can be propagated toward the condensation reaction. These microorganisms can be added to the above equilibrium reaction system at the initial stage of the reaction or during the reaction. In this case, various conditions for the reaction for converting AP to DKPl may follow the conditions as described above.

Other features of the present invention will become apparent in the course of describing the following examples, which are intended to illustrate the invention, but not to limit it.

EXAMPLE

Example 1

Polypeptone 1.0 g / dl, yeast juice 1.0 g / dl, K ₂ HPO ₄ 0.3 g / dl, KH ₂ PO ₄ 0.1 g / dl, MgSO ₄ 7H ₂ 0 0.05 g / dl and DKP 0.5 50 ml of medium (pH 7.0) containing g / dl was added and sterilized at 115 ° C for 15 minutes.

Cultures obtained from the microorganisms of Table 1, which were incubated at 30 ° C. for 2 days in the broth agar medium, were inoculated into the above-mentioned medium with I platinum each and then incubated at 30 ° C. for 2 days. Cells are harvested from these cultures by filtration. After washing once with saline saline, cells are collected.

These bacteria are added to 100 ml of 0.1 M Tris-HCl buffer (pH 8.0) containing DKP (2.0 g / dl) to a concentration of 1.0 g / dl, respectively, and reacted at 30 ° C. for 2 hours. The amount of AP generated from each bacterium is listed in Table 1.

TABLE 1

Example 2

Streptomyces flavovirens IFO 3197 (Korean Deposit No .: KFCC-10287, Deposit Organization: Korean spawn association, Deposit Date: October 17, 1986) was cultured and washed in a similar manner as in Example 1. Reaction was carried out at 30 ° C. for 48 hours using the same reaction solution as in Example 1. The amount of AP obtained in this case is 420 mg / dl. L-Asp and L-Phe obtained by further degradation of the produced AP are produced in amounts of 43 mg / dl and 57 mg / dl, respectively.

Example 3

The microorganisms described in Table 2 were cultured in a similar manner as in Example 1 to obtain bacterial cells. Each of these cells is added to 100 ml of 0.1 M phosphate buffer (pH 6.0) containing AP (1.0 g / dl) to a concentration of 1.0 g / dl, followed by reaction at 30 ° C. for 2 hours. The amount of DKP obtained is listed in Table 2:

TABLE 2

Example 4

Streptomyces flavovirens IFO 3197 (Korea Deposit No .: KFCC-10287, Deposit Organization: Korean spawn association, Deposit Date: October 17, 1986) was produced in a similar manner to Example 1, and 1 g of deionized water 4 Suspension in ml. After ice cooling, 750 mg of acrylamide and 45 mg of methylenebisacrylamide were added. After introducing nitrogen gas to purge the oxygen, 3.5 mg of ammonium persulfate and 8 µl of N, N'-dimethylaminopropionitrile are added and the mixture is allowed to settle with ice cooling. After 1 hour, the cell-containing gel is passed through a 50 mesh metal mesh and washed with physiological saline to prepare a gel-fixed product. 2 g of immobilized product are added to the reaction solution prepared in a similar manner to Examples 1 and 3, respectively. The amount of AP and DKP obtained in these cases was 370 mg / dl and 700 mg / dl, respectively.

Example 5

Streptomyces parbulus AJ 9037 FERM BP 1168 (Korean Deposit No .: KFCC-10288, Deposit Duration: Korean Communistant Association, Deposit Date: October 17, 1986) 0.1M carbon containing DKP (2.0 g / dl), each of Streptomyces flavovirens IFO 3197 (Korean Deposit No .: KFCC-10287, Deposit Organization: Korean spawn association, Deposit Date: October 17, 1986) Add 100 ml of each of the acid buffer (pH 9.8) and 0.1 M Tris-HCl buffer (pH 8.0) to a concentration of 5 g / dl, and then react for 10 hours at 30 ° C. The results obtained are shown in Table 3.

TABLE 3

It is clear that the present invention can be variously modified and changed in view of the above fact. It is, therefore, to be understood that the invention may be practiced otherwise than as described in the detailed description herein within the scope of the appended claims.

Claims (12)

Culture of L-aspartyl-L-phenylalanine diketopiperazine to produce L-aspartyl-L-phenylalanine, cells or cell-treatments of microorganisms in L-aspartic acid in aqueous medium A method for producing L-aspartyl-L-phenylalanine, characterized by contacting with yl-L-phenylalanine diketopiperazine. The microorganism of claim 1, wherein the microorganism is Streptomyces genus, Bacillus genus, Flavoacterium genus, Agrobacterium genus, Xanthomonas genus, Erbinia. (Ervinia), Serratia, Alcaligenes, Micrococcus, Achromobacter and Enterobacter. . The method of claim 1, wherein the microorganism belongs to the genus Streptomyces. Cultures, cells, or cell-treatments of microorganisms capable of intermolecular condensation of L-aspartyl-L-phenylalanine to produce L-aspartyl-L-phenylalanine diketopiperazine, L-As in an aqueous medium A process for producing L-aspartyl-L-phenylalanine diketopiperazine characterized by contacting with partyl-L-phenylalanine. The genus of claim 4, wherein the microorganism is Genus Streptomyces, Genus Bacillus, Genus Flavobacterium, Genus Agrobacterium, Genus Xanthomonas, Erbinia, Certiatia, Alkogen genes, Micrococcus , A genus selected from the group consisting of the genus Accomobacter and enterobacter. The method of claim 4, which belongs to the genus Microbial Streptomyces. The process of claim 1 wherein the contacting reaction is carried out under aerobic conditions at a temperature of about 2 to 8 and about 10 to 70 ° C. The method of claim 7 wherein the temperature is about 20-40 ° C. 9. The method of claim 1, wherein the contact reaction is carried out in a batch system. The process of claim 4, wherein the contact reaction is carried out under aerobic conditions at a pH of about 2 to 8 and a temperature of about 10 to 70 ° C. 6. The method of claim 10, wherein the temperature is about 20-40 ° C. 12. The method of claim 4, wherein the contact reaction is carried out in a batch system.