JPS6135797A - Production of l-aspartyl-l-phenylalanine or its methyl ester - Google Patents

Abstract

PURPOSE:To produce the titled compound useful as a sweetener and its raw material, in high yield, by condensing L-aspartic acid with L-phenylalanine (or its methyl ester) using a specific microorganism. CONSTITUTION:L-aspartyl-L-phenylalanine (abbreviated as AP) or L-aspartyl-L- phenylalanine methyl ester (abbreviated as APM) can be produced in high efficiency, by treating L-aspartic acid and L-phenylalanine or its methyl ester with a microbial strain belonging to Arthrobacter genus, Cellulomonas genus, or Brevibacterium genus and capable of condensing L-aspartic acid with L-phenylalanine or L-phenylalanine methyl ester to produce AP or APM.

Description

DETAILED DESCRIPTION OF THE INVENTION This invention relates to L-aspartyl-phenylalanine (
Hereinafter, it will be abbreviated as AP. ) and aspartyl-phenylalanine methyl ester (hereinafter abbreviated as APM)
Concerning the manufacturing method.

APM is a peptide that has recently attracted attention as a sweetening agent.

Chemical synthesis methods and enzymatic synthesis methods are known as methods for producing APM.

As a chemical synthesis method, there is a method in which N-protected L-aspartic acid anhydride and L-phenylalanine methyl ester are condensed to form N-protected APM, and then the protecting group is removed, and as an enzymatic synthesis method, there is a method in which the protecting group is removed. N-protected L-aspartic acid and L-phenylalanine methyl ester are treated with a protease to form N-protected APM or a phenylalanine methyl ester adduct of N-protected APM, and then the M-protecting group is removed. A method is known in which APM is obtained by converting a protective group into APM, but both methods require introduction and removal of a protecting group, making the process complicated.

In addition, a method for producing 8PM without using a protecting group (Japanese Unexamined Patent Publication No. 58
-43793, JP-A-58-63394, JP-A-58-
126796, 1981 Japanese Agricultural Chemistry Conference Abstracts P4
2) is also known, and is a microbial synthesis method using any of the genus Pseudomonas, alkaline earth metal, Torulopsis, Rhodotorula, or Sporobolomyces, but the yield is very low and it is difficult to produce industrial APM. Not necessarily suitable for production.

The present inventors conducted research to find a more efficient method for producing APM or its raw material AP, and found that by using microorganisms, L-aspartic acid and L-phenylalanine or It has been found that AP or APM can be directly and efficiently produced from L-phenylalanine methyl ester.

That is, the present invention provides L-aspartyl-phenylalanine or L-aspartyl-L by condensing L-aspartic acid and L-phenylalanine or L-phenylalanine methyl ester belonging to the genus Arthrobacter, Cellulomonas, and Brevibacterium. -A microorganism capable of producing phenylalanine methyl ester is allowed to act on [-aspartic acid and L-phenylalanine or -phenylalanine methyl ester to produce L-aspartyl-phenylalanine or L-aspartyl-phenylalanine methyl ester. This is a method for producing AP or APM.

AP or AP is obtained by condensing L-aspartic acid with L-phenylalanine or L-phenylalanine methyl ester.
By the action of microorganisms capable of producing M, L-aspartic acid and L-)]-nylalanine or L-phenylalanine methyl ester are condensed in an aqueous medium to produce A.
The conversion to P or APM can be carried out by bringing L-aspartic acid and L-phenylalanine or L-phenylalanine methyl ester into contact with the cells, culture solution, or treated product of the above-mentioned microorganisms in an aqueous medium. .

Examples of microorganisms that have the ability to condense L-aspartic acid and L-phenylalanine or L-phenylalanine methyl ester and convert them into AP or APM used in the present invention include Arthrobacter citreus ATCC 11624 Cellulomonas flavigena ATCo 8183 Brevibacterium - Linen ATCC8377 is an example.

In order to obtain cells of these microorganisms, it is sufficient to culture them using a normal medium and adding L-aspartic acid and L-phenylalanine or L-phenylalanine methyl ester from the beginning of the culture or during the culture. .

The medium used for culturing this microorganism is a normal medium containing L-aspartic acid and L-7 enylalanine or L-phenylalanine methyl ester, as well as normal carbon sources, nitrogen sources, and inorganic ions. . Additionally, desirable results can often be obtained by adding organic micronutrients such as vitamins and amino acids.

As the carbon source, carbohydrates such as glucose and sucrose, B-containers such as acetic acid, alcohols, and others are used as appropriate. As the nitrogen source, ammonia gas, aqueous ammonia, ammonium salt, and others are used. As the inorganic ions, magnesium ions, phosphate ions, potassium ions, iron ions, and others are used as appropriate.

Desired results can be obtained by culturing for 1 to 10 days under aerobic conditions with I) H4 to 8 and temperature controlled at an appropriate range of 25 to 40°C.

As the bacterial cells, any of the following can be used: the culture solution as it is after completion of culturing, the bacterial cells isolated from the culture solution, and the washed bacterial cells. The bacterial cell treatments include freeze-dried bacterial cells, acetone-dried bacterial cells, bacterial cells that have been brought into contact with toluene, surfactants, etc., bacterial cells that have been treated with lysozyme, bacterial cells that have been exposed to ultrasound,
In addition to mechanically ground bacterial cells, L-aspartic acid and L-phenylalanine or L-phenylalanine methyl ester obtained from these treated bacterial cells are used as AP or AP.
Enzyme protein fractions having enzymatic activity for converting into M, furthermore, immobilized products of these bacterial cells, insolubilized products of treated bacterial cells,
Any of the others can be used.

As the aqueous medium, those containing water, buffers, and organic solvents such as ethanol can be used.

Furthermore, nutrients, antioxidants, surfactants, coenzymes, hydroxylamine, metal ions, etc. necessary for the growth of microorganisms can be added to the aqueous medium, if necessary.

While culturing the cells of the above microorganism in an aqueous medium, the cells, L-aspartic acid, L-phenylalanine or L-
When phenylalanine methyl ester is brought into contact with the phenylalanine methyl ester, an aqueous solution containing L-aspartic acid, monophenylalanine or L-phenylalanine methyl ester, and nutrients such as a carbon source, nitrogen source, and inorganic ions necessary for the growth of microorganisms is used. A medium is used. Additionally, desirable results can often be obtained by adding organic micronutrients such as vitamins and amino acids.

As the carbon source, carbohydrates such as glucose and sucrose, organic acids such as acetic acid, alcohols, and others are used as appropriate. As the nitrogen source, ammonia gas, aqueous ammonia, ammonium salt, and others are used. As the inorganic ions, magnesium ions, phosphate ions, potassium ions, iron ions, and others are used as appropriate.

Desired results can be obtained if the culture is carried out under aerobic conditions, with pH 4 to 8 and temperature controlled within an appropriate range of 25 to 40°C.

Thus, by culturing for 1 to 10 days, L-aspartic acid and monophenylalanine or L-phenylalanine methyl ester are efficiently converted to only AP or APM.

On the other hand, the above-mentioned microorganism culture solution was used as it was, cultured bacterial cells or treated bacterial cells were mixed with L-aspartic acid and L-
In the case of contacting with phenylalanine or L-phenylalanine methyl ester, an aqueous solution containing L-aspartic acid, L-phenylalanine or L-phenylalanine methyl ester, and a culture solution, cultured bacterial cells, or treated bacterial cells dissolved or suspended. Temperature the medium from 10℃ to 70℃
While adjusting the temperature to an appropriate temperature of .degree. C. and keeping pt-1 at 4 to 8, the mixture may be allowed to stand for a while or be stirred. Thus, after 5 to 100 hours, a large amount of AP or A is present in the aqueous medium.
PM is generated and accumulated.

The produced AP or APM can be separated and purified by a known separation method. The produced AP or APM was measured using an amino acid analyzer.

Example 1 Glucose 2.09/djj, (NH4) 28040
．． 5g/dJl, KH2PO40,1'j/dJ1
11VH+ 804 ・7H200,05g/df,
Fe 804 ・'7H201rIt! J/ df, M
nSO4・4H2011Rg/dJl, yeast extract 1.
0g/d4. Medium containing malt extract 0.5g/dffi, calcium carbonate 4.0g/df (separately sterilized)
Pour 50d of pH 1.0) into a 5'OOd volume flask 12
Sterilized at 0°C for 15 minutes.

One platinum loop of the microorganisms shown in Table 3, which had been cultured on a bouillon agar medium at 30°C for 24 hours, was inoculated thereto and cultured at 30°C for +20 hours. Bacterial cells were collected from this culture solution by centrifugation, washed once with physiological saline in the same amount as the culture solution, and collected.

These bacterial cells were added to reaction solution A shown in Table 1 to a concentration of 5 g/dJl, and the final pH was 5, 4, 5 #11, and the mixture was heated at 37°C.
A holding reaction was carried out for 16 hours. The APM produced at this time was measured using an amino acid analyzer, and the results are shown in Table 2.

Table 1 *0. Containing the above substrate in IM phosphate buffer (final pH 5.4) Table 2 Example 2 Arthrobacter cultured and washed in the same manner as in Example 1.
Citreus ATCC1162459 was added to the reaction solution A100#.
Ii! and reacted at 37°C for 24 hours.

This reaction solution was developed in 3 pots on a TLC for preparation with a developing solvent of n-butanol:acetic acid:water-2:1:1.
A portion of the produced APM was scraped off, and the reaction product after extraction with distilled water was crystallized to obtain crystals of 552. As a result of measuring the optical rotation, melting point, and specific rotation of this crystal, it was found that the product from reaction solution A was APM Example 3 Brevibacterium linens cultured at 30°C for 12 hours using the same medium as in Example 1. 10 d of an aqueous solution (pH 5,
4) was added aseptically, and the pH of the culture solution was adjusted aseptically to 5.4, followed by further culturing for 10 hours. During the culture, IIH was aseptically prepared to a concentration of 5.4 every 2 hours.

As a result of measuring the products in this culture solution using an amino acid analyzer, it was found that 315 mg/l of APM was produced.

Example 4 Cells of the microorganisms shown in Table 4, which were cultured and cleaned in the same manner as in Example 1, were added to the reaction solution B shown in Table 3 at a concentration of 5 y/dJl. A holding reaction was carried out for 16 hours. The AP produced at this time was measured using an amino acid analyzer, and the results are shown in Table 4.

Table 3 Containing the above substrate in 0.1M phosphate buffer (final pH 5.4) Table 4 Example 5 Arthrobacter chloride cultured and washed in the same manner as in Example 1.
Citruus A'TO'C'11624! M was added to reaction solution B10C1 and reacted at 37°C for 24 hours.

This reaction solution was developed in 5 pots on a TLC for adjustment using a developing solvent of n-butanol:acetic acid:water=2:1:1.
The produced AP portion was scraped off, and the reaction product after extraction with distilled water was crystallized to obtain 243 mg of crystals. As a result of measuring the optical rotation, melting point, and specific rotation of this crystal, it was found that the product was A.
Completely matched with the PI product.

Example 6 Using the same medium as in Example 1, 10 d of an aqueous solution (pH 5, 4) was added aseptically, and the culture solution was aseptically adjusted to l)H of 5.4, followed by further culturing for 10 hours. During culturing, the pH was adjusted aseptically to 5.4 every 2 hours.

As a result of measuring the products in this culture solution using an amino acid analyzer, it was found that 176 #1 g/df of AP was produced.

Claims (1)

[Claims] L-aspartic acid belonging to the genus Arthrobacter, Cellulomonas, and Brevibacterium is condensed with L-phenylalanine or L-phenylalanine methyl ester to produce L-aspartyl-L-phenylalanine or L-aspartyl-L-phenylalanine methyl ester. Microorganisms capable of producing L-aspartic acid and L-
- L-aspartyl-L-phenylalanine or its methyl ester, characterized in that it acts on phenylalanine or L-phenylalanine methyl ester to produce L-aspartyl-L-phenylalanine or L-aspartyl-L-phenylalanine methyl ester. Production method.