JPS59198993A - Preparation of l-aspartyl-l-phenylalanine - Google Patents

Abstract

PURPOSE:To obtain the titled compound useful as a raw material of sweetener, in high efficiency, by utilizing a microbial strain capable of producing L-aspartyl-L-phenylalanine by the condensation of L-aspartic acid and L-phenylalanine. CONSTITUTION:The objective L-aspartyl-L-phenylalanine (abbreviated as AP) useful as a raw material of a sweetener, L-aspartyl-L-phenylalanine methyl ester, can be prepared by condensing L-aspartic acid with L-phenylalanine in an aqueous medium by the action of a microbial strain belonging to Alcaligenes genus, Achromobacter genus, Corynebaterium genus, etc. and capable of condensing L-aspartic acid and L-phenylalanine to produce AP.

Description

[Detailed Description of the Invention] The invention comprises 1. -Regarding a method for producing asbardi-L-phenylalanine (hereinafter abbreviated as APL).

L-aspartyl-L-phenylalanine methyl ester (hereinafter abbreviated as APM) is a peptide that has recently attracted attention as a sweetener. AP is a substance useful as a raw material for APM and can be easily converted into APM.

The present inventors have been researching to find an efficient production method for AP, and by using microorganisms, AP, the raw material of 8PM, can be directly and efficiently produced from L-aspartic acid and L-phenylalanine. I found something to do.

That is, the present invention provides alkaline earth metals, Achromobacterium, Corynebacterium, Candida, Cryptococcus, Escherichia, Flavobacterium, Geotrichum, Micrococcus, Paxoren, Zarcina, and Subo. 1 Bo 1 Mrs. bile, Zaccharomyces curvature, Trichosbo
In an aqueous medium, I7- This is a method for producing AP, which is characterized in that 1f is produced by reacting aspartic acid 7I7-f with nylalanine to produce AI). ■、−
A by condensing aspartic acid and ■7-fuynylalanine
A method of condensing 17-aspartic acid and L-phenylalanine in an aqueous medium to convert it into AI' by the action of microorganisms capable of producing P is as follows: The acid and L-phenylalanine may be brought into contact with the cells, culture solution, or treated product of the microorganism described in 1.

Examples of microorganisms that have the ability to condense I7-aspartic acid and I7-phenylalanine to convert them into AP used in the present invention include Alcaligenes faecalis ATCC8750 Aku'1 Mobacter bupuli 8J 2438 FERM-
r' 705 + Corynebacterium sp.
8TCC2125];-1 Lineobacterium xerosis ATCC3734-Yandida intermedia ΔJ 4fl19 FERM P 10
G2 Cryptococcus neoformans IFo 4
289 Niji Area Cori 8J
2000 PERM-P 7055 Flavobacterium sewanens ^J 247 (i PERM-
1' 7052 Geotrichum candium
IFO4599 Micrococcus luteus
8TCC4698 Paxolene Tannophilus
iPo 1007 Zarutina Alvida
8J 1210 PERM-P 7048 Sborobo ``1 Mrs. Odorus IPo 159G Trichosporon cavitatum IFO1197 Torj, j Psis Inconspiqua IPO0 (i
21 “1F Torla Lactoza IFO1
424 Xanthomonas campestris ^J 2
787 PERM-r' 7059 Pseudomonas
Erginosa ATCC14210 Kruihe I+
Mrs. Thermotoleranos IPO0flG2 Endomyces obedensis 11101201 Zakka I-1 Mrs. Cereviche Il'02003
There is a hat.

To obtain cells of these microorganisms, they may be cultured using a normal medium. Alternatively, 7-aspartic acid, L-], and nylalanine may be added to the culture from the beginning of the culture or during the J-1 feeding.

The medium used for culturing the microorganisms is a conventional medium containing conventional carbon sources, nitrogen sources, and inorganic ions.

Furthermore, desirable results can often be obtained by adding trace amounts of nutrients 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 may be used as appropriate. As the nitrogen source, ammonia gas, aqueous ammonia, ammonium salt, and others are used. Inorganic ions include magnesium ions, phosphate ions, potassium ions, and iron ions.

Others may be used as appropriate.

-5- Cultivation was carried out under aerobic conditions, rJII 4 to 8, temperature 25
Desired results can be obtained by culturing for 1 to 10 days while controlling the temperature within an appropriate range of 40°C to 40°C.

As the bacterial cells, any of the culture solution after completion of cultivation, the bacterial cells isolated from the culture solution, and the washed bacterial cells can be used. 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, and mechanically ground bacterial cells. In addition to bacterial cells, enzyme protein fractions having enzymatic activity for converting L-aspartic acid and L-phenylalanine into AP obtained from these processed bacterial cells, as well as immobilized products of which bacterial cells and bacteria. Insolubilized products of body-treated products and others can also be used.

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

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

6-1゛4 Bigyumono f'1. The f-form was cultured in an aqueous medium, and the 1'II- and 1. -Aspartic acid and 17-
When contacting phenylalanine to make it work, use an aqueous medium that contains ■7-aspartic acid and ■7-phenylalanine, and also contains nutrients such as a carbon source, nitrogen source, and inorganic ions necessary for the growth of microorganisms. It will be done.

Furthermore, desirable results can often be obtained by adding essential micronutrients such as vitamins and amino acids.

As carbon sources, carbohydrates such as glucose and 1-su, acetic acids such as acetic acid, alcohols, and others are used as appropriate.As nitrogen sources, ammonia gas, aqueous ammonia, ammonium salts, and others are used. Examples of the inorganic ions include magnesylanoyl ions, phosphate ions, potassium ions, iron ions, and others, as appropriate.

The culture was carried out under aerobic conditions (under 'I, pH/l to 8, temperature 2
If you control it within the appropriate range of 5 to 40'C, you can get the desired results.

Thus, if the culture is continued for 1 to 1011 hours, ■.

- Asparagus - 1 gonic acid and 1. -Phenylalanine is AP
It is efficiently converted into

On the other hand, when the culture solution of the microorganism mentioned in 1. is used as is, the cultured bacterial cells or the treated bacterial cells are brought into contact with L-aspartic acid and 17-phenylalanine,
Adjust the aqueous medium in which L-aspartic acid and (1) 7-fuylinalani/ and the culture solution, cultured cells, or treated bacterial cells to an appropriate temperature of 10°C to 70°C, and It is sufficient to let it stand for a while or stir it while keeping the temperature between 8 and 8.

Thus, a large amount of AP is produced and accumulated in the aqueous medium after 5 to 100 hours have passed.

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

Example 1 Glucose 2.0 g/di, (NII4)>5040.
5g/dl, KllUPO40, Ig/dl, KA1I
P040.1g/dl, Mg5O4・711□00.0
5g/dl, Pe5O4411,01mg/dl, M
n5O-411zO++++g/dl, yeast extract]
, Og/dl, Malt extract 0.5g/di, calcium carbonate 4,0g/dl (separately killed 1°rf) containing 50ml of culture medium (pH 7,0) was added to 50ml (11ml volume) and heated at 120°C. Sterilized for 15 minutes.

This was fed 24 hours at 30'C in a buoy agar medium.
One platinum loop of the microorganisms shown in Table 2, which had been cultured in 100 g, was inoculated and cultured at 30°C for 20 hours. The bacterial cells are centrifuged from this culture solution.
One sample was taken, washed once with physiological saline in the same amount as the culture solution, and the bacterial cells were collected.

Add 5 g/d+Ninal-like bacteria of these bacteria to the reaction solution shown in Table 1 (final pH 5, 4, 5 ml), 37°C.
The t1j reaction was maintained for 16 hours. AP generated at this time
was measured using an amino acid analyzer, and the results are shown in Table 2.
It was shown to.

Table 1 -1) - Table 2 -10- Example 2 Flavobacterium sewanens AJ 247 (E, FF', R11l) cultured and washed in the same manner as in Example 1.
- P 70,525g was added to 100ml of reaction solution A, and 3
The reaction was carried out at 7°C for 24 hours.

This reaction i (k is for adjustment T I, C is 5 pots
Developed with a developing solvent of L,, n-butanol: acetic acid: water = 2:1:1, scraped off the formed AP portion, and crystallized the reaction product after Senzan with distilled water to obtain 250 mg of crystals. Ta. As a result of measuring the optical rotation, melting point, and specific rotation of this crystal, the product was completely identical to the standard product.

Example 3 Eschlichia fluri AJ 2606PER cultured at 30°C for 12 hours using the same medium as in Example 1
M-1' 7055 was added to the culture medium with 5 g of l,-aspartic acid and l. - Aqueous solution 101 containing 7 g of phenylalanine
(prepared to pl+ 5.4) was aseptically introduced, and the culture solution was aseptically adjusted to p II of 5.4, followed by further culturing for 10 hours. During culture, p I-1 was aseptically adjusted 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 180 mg/dl of AP was produced.

Patent applicant Ajinomoto Co., Ltd. Continuation of page 1 9 Int, C1, 3 identification symbol Internal organization - (C1
2P 21102 C12R1/20 ) (C12P 21102 C12R1/88 ) (C12P 21102 CI2 R1/38 ) (C12P 21102 C12R1785) (C12P 21102 C12R1101) (, CI2 P 21102 C I2 R1/645) 0 Inventor Koji Kubota Tama, Kawasaki City 6-4-6-5 Nagao, Ward

Claims (1)

[Claims] Alkaline earth metals, Achromobacter genus, Corynebacterium vulgaris, Candida genus, Cryptococcus genus, Niji, Lichia spp., Flavobacterium 14eta, Geotrichum spp., Micrococcus spp., Baxoles spp., Zardiri spp.
Sporobolomyces, Zacca '1 Mrs. curvature, Triph 1 Suboron spp., Torulopsis spp., 1 Dotorula spp.; - The action of microorganisms capable of condensing phenylalanine to produce I,-aspartyl-cy-fugnilalanine by the use of I,-aspartic acid and L-7 enylalanine in an aqueous medium. A method for producing I7-asbardyl-L-phenyl 7''172, characterized by producing I7-asbardyl-L-phenylalanine.