JPS6257317B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing aspartyl phenylalanine alkyl ester, and more specifically, the present invention relates to a method for producing aspartyl phenylalanine alkyl ester, and more specifically, it relates to a method for producing aspartyl phenylalanine alkyl ester, and more specifically, α-L is produced from L-aspartic acid and L-phenylalanine lower alkyl ester using a microorganism. This invention relates to a method for producing aspartyl-L-phenylalanine lower alkyl ester. α-L-aspartyl-L-phenylalanine lower alkyl ester (hereinafter referred to as α-APE),
In particular, methyl ester is a useful substance that is attracting attention as a new sweetener. The method for producing α-APE is to react N-protected L-aspartic acid anhydride and L-phenylalanine lower alkyl ester to form N-protected α-APE, and remove the protecting group to form α-APE. Method: N-protected L-aspartic acid and phenylalanine lower alkyl ester are reacted in the presence of a proteolytic enzyme to produce N-protected α-APE, or N-protected α-APE and phenylalanine lower alkyl ester. A known method is to create an addition compound of α-APE and remove the protecting group to obtain α-APE. The former method uses N-protection α-APE as well as N
-Protection β-There is a problem that APE is produced as a by-product.
The latter method is an excellent method in that it does not have such problems and can use a racemate as a raw material. However, in either method, it was necessary to protect the amino group of the raw material aspartic acid or its anhydride with a protecting group such as a benzyloxycarbonyl group before use. If we could develop a method that eliminates the steps of introducing and removing protecting groups from amino groups, which are naturally required in these known techniques, it would be possible to simplify the process and avoid the associated loss of raw materials, products, etc. is possible,
Significant industrial advantages result. From this perspective, the present inventors have conducted intensive studies on a method for directly synthesizing α-APE from aspartic acid and phenylalanine lower alkyl ester, and have found that L-asparagine can be synthesized directly from aspartic acid and phenylalanine lower alkyl ester by using bacteria belonging to the genus Pseudomonas. α- from acid and L-phenylalanine lower alkyl ester
We found that APE is generated. The present invention is directed to asparagus, which is characterized by culturing α-APE-producing bacteria belonging to the genus Pseudomonas in a medium containing L-aspartic acid and L-phenylalanine lower alkyl ester to produce α-APE, and collecting the α-APE. A method for producing rutile phenylalanine alkyl ester is provided. The microorganism used in the present invention is an α-APE producing bacterium belonging to the genus Pseudomonas. The mycological properties of such a microorganism isolated from the soil of Shinnanyo City, Yamaguchi Prefecture by the present inventors are as follows. (a) Morphology When grown on broth agar medium at 37℃ for 6 to 24 hours Cell shape and size Bacillus (0.5 to 0.7) x (1.0 to 1.5)μ Presence or absence of cell pleomorphism Monobacterial or dipobacterial Presence or absence of motility Polar Flagella Presence or absence of spores Absent Gram staining Negative Anti-acidic Absence (b) Growth status in each medium Broth agar plate culture (cultured at 37°C for 2 days) A Slow rate of colony formation Normal Approximately 6 mm in diameter B Colony shape Circular C Colony surface shape Smooth D Colony terrestrial condition Semi-lenticular E Colony periphery Full edge F Colony contents Homogeneous G Colony color milky white H Colony transparency Translucent L Colony luster Dull light N Production of soluble pigment Production of soluble pale colored pigment Broth agar slant culture (cultured at 37°C for 2 days) B Quality of growth Good growth B Colony shape Smooth C Terrestrial condition of colony cross section Flattened D Colony luster Dull Light E Colony surface shape Smooth F Colony transparency Translucent G Colony color Milky white H Colony quality Buttery Meat liquid culture (37℃, 2 days culture) A Surface growth None B Turbidity Slightly cloudy C Sedimentation Powdered gas generation None E Medium coloring None Meat juice agar puncture culture (37℃, 2 days culture) A Place of growth Uniform top and bottom B Colony shape Papillary Meat juice gelatin puncture culture (37℃ and 20℃, 14
(day culture) i Gelatin liquefaction None Litmus milk (cultured at 37℃ for 7 days) A Reaction Turns BCP blue and litmus bluish-purple B Coagulation, liquefaction None (c) Physiological properties Reduction of nitrate - denitrification Reaction - MR test - VP test - Production of indole - Production of hydrogen sulfide + (W) Hydrolysis of starch - Use of citric acid + Use of inorganic nitrogen source Only use of ammonia form Production of pigment Production of green-yellow water-soluble fluorescent pigment Urease - Oxidase + Catalase + Growth range PH 5-9.5 Temperature 10-43℃ Attitude towards oxygen Aerobic O-F test 0 Presence or absence of acid and gas production from sugars [Table] [Table] Arginine Dihydrolase + Utilization of carbon sources (cultivation at 37°C for 1 to 7 days) What is used: D-glucose, L-valine, β-L-alanine, L-arginine What is not used: trehalose, meso-inositol, geraniol Virgies manual Of Determinative Bacteriology (Bergey's Manual
of Determinative Bacteriology), 8th edition (1974
According to the identification method described in 2010, this bacterium has characteristics of the genus Pseudomonas. Furthermore, poly-β-hydroxybutyric acid (poly-
There should be no accumulation of β-hydroxybutrate. To produce fluorescent pigments. Due to the presence of arginine dihydrolase, Pseudomonas aeroginosa species ( P.
aeroginosa ), putida species ( P. putida ), florescene species ( P. flurescene ), Chlorafuis species ( P.
chloraphis ), Aureophaciens species ( P.
aureofaciens ). The growth temperature range is higher than that of Putida species and close to that of Aeroginosa species, but it does not reduce nitrate. Pseudomonas spp.
It was identified as a variant of (Putida ). Pseudomonas putida TS-15001, a representative strain of this fungus, has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (National Institute of Industrial Science and Technology, Microbiological Research Institute No. 6035). Cultivation of Pseudomonas putida TS-15001 is no different from known methods. Carbon sources include carbohydrates such as glucose, organic acids such as tartaric acid, fumaric acid, maleic acid, malic acid, and their salts, and nitrogen sources include ammonium sulfate, ammonium chloride, ammonia, ammonium phosphate, and ammonium nitrate, which are used in normal fermentation. Inorganic nitrogen compounds such as urea, corn stave liquor, casein, peptone, yeast extract, meat extract and the like can be used. Other inorganic salts that can be used include, for example, calcium salts, magnesium salts, potassium salts, phosphates, iron salts, manganese salts, zinc salts, copper salts, and the like. From the viewpoint of promoting bacterial growth, it is effective to add organic substances such as soybean protein hydrolyzate, yeast extract, vitamins, and amino acids to the medium. In addition to the above-mentioned nutritional sources, the culture of the present invention uses α-
This is carried out by adding L-aspartic acid and L-phenylalanine lower alkyl ester, which are substrates for APE synthesis, to the medium. There is no particular limit to the amount of L-aspartic acid and L-phenylalanine lower alkyl ester added, but both are usually about 1% by weight to a range permitted by solubility, preferably about 5% to about 40% by weight. That's about it. Cultivation is usually carried out at a temperature of about 20 to about 40°C, preferably
The reaction is carried out aerobically at a temperature of about 25 to about 38° C. and a pH of about 5 to about 9.5, preferably about 5.5 to about 7.5, by means such as shaking and aeration. L-aspartic acid and L-phenylalanine lower alkyl ester, which are substrates for the synthesis of α-APE, may be added at any time during the cultivation of microorganisms, but L-phenylalanine lower alkyl ester and Inhibition of hydrolysis of α-APE,
Also, it is preferably added in the latter stage of the culture for the reason that the synthesis reaction proceeds more rapidly under high bacterial cell concentration than under low bacterial cell concentration. The lower alkyl group of L-phenylalanine lower alkyl ester is a group such as a methyl group, an ethyl group, or an isopropyl group. Since the D-forms of aspartic acid and phenylalanine lower alkyl ester are not utilized and do not participate in the reaction, their racemic forms may be used in place of their L-forms. The produced α-APE can be separated and purified by known separation and purification means. Separation from solid content such as bacterial cells in the culture solution can be carried out by a conventional method such as centrifugation or filtration. α-APE can be purified and isolated from the culture solution by conventional separation and purification means such as column chromatography, thin layer chromatography, crystallization, and drying under reduced pressure. According to the present invention, there is no need to protect the amino group of the raw material L-aspartic acid, and α-
It can be used in the production of APE. Moreover, since it utilizes a biochemical reaction, only the LL- form of α-APE can be selectively produced even if racemic form is used as a raw material. Furthermore, in the present invention, there is no by-product of β-APE. The present invention will be explained in more detail with reference to Examples below. In the examples, all percentages indicate weight percentages. Example 1 Ammonium fumarate 2%, dihydrogen phosphate 1
Potassium 0.1%, dipotassium monohydrogen phosphate 0.1%,
Magnesium sulfate / heptahydrate 0.05%, ferric sulfate /
Medium consisting of 0.01% heptahydrate, 0.01% manganese chloride, 0.01% sodium chloride, and balance water (PH5.5) 1.0
Place in a 2-volume mini-jar type fermenter and heat to 120℃15
Sterilization was performed for minutes. A medium with the same composition (PH5.5) was inoculated with 50 ml of the preculture solution obtained by culturing Pseudomonas putida TS-15001 at 37℃ for 16 hours, and during the culture period the pH
2N-HC aqueous solution, 2N to maintain 5.5-6.0
- While adding an aqueous NaOH solution, aerated agitation culture was carried out under the conditions of a culture temperature of 37°C, a stirrer rotation speed of 500 rpm, and an aeration rate of 1 air/min. 70g of L-aspartic acid and L-phenylalanine methyl ester adjusted to pH 6.0 after 16 hours
200 ml of an aqueous solution containing 90 g was added aseptically and cultured for a further 8 hours. After the cultivation was completed, the culture solution was centrifuged at 15° C. and 10,000 rpm for 30 minutes to remove bacterial cells and solid matter. The obtained supernatant liquid was mixed with water:ethanol (volume ratio 80:
Column chromatography using 20) as eluent (filling material, Toyo Pearl 55F, trademark, Toyo Soda Kogyo Co., Ltd.)
The fractionation was carried out using the following method (manufactured by Co., Ltd.). The fraction corresponding to α-L-aspartyl-L-phenylalanine methyl ester is concentrated under reduced pressure to produce a white powder.
1.2g was obtained. The elemental analysis values and physicochemical properties of this product were as follows. [Table] The molecular weight of the product in which the amino group was trifluoroacetylated and the carboxyl group was methylated was 404. In addition, this can be used as
L-phenylalanine, L-phenylalanine methyl ester, L-aspartic acid, L-aspartyl-L-phenylalanine, L-aspartyl-L-aspartic acid, α-L-aspartyl-L-phenylalanine methyl ester, β-L
-Aspartyl-L-phenylalanine was used as a standard substance for analysis using thin layer chromatography, high performance liquid chromatography, and an amino acid analyzer, and the product was identified as α-L-aspartyl-L-phenylalanine lower alkyl ester. Furthermore, gas chromatography and gas chromatography/mass spectrography analyzes of samples that had been methylated with hydrochloric acid and methanol and trifluoroacetylated with trifluoroacetate methyl ester revealed that this was α-L-aspartyl-L-phenylalanine. It was confirmed that it was a methyl ester. Example 2 Example 1 was repeated using 200 ml of an aqueous solution containing 50 g of each of L-aspartic acid and 90 g of L-phenylalanine methyl ester instead of 200 ml of the aqueous solution containing 70 g of L-aspartic acid and 90 g of L-phenylalanine methyl ester. α-L-aspartyl-
White powder of L-phenylalanine methyl ester
0.7g was obtained.

Claims (1)

[Claims] 1. α-L-aspartyl-L-phenylalanine lower alkyl ester-producing bacteria belonging to the genus Pseudomonas are cultured in a medium containing L-aspartic acid and L-phenylalanine lower alkyl ester. - A method for producing aspartyl phenylalanine alkyl ester, which comprises producing and collecting L-aspartyl-L-phenylalanine lower alkyl ester. 2 α-L belonging to the genus Pseudomonas in the nutrient medium
-Production according to claim 1, wherein aspartyl-L-phenylalanine lower alkyl ester-producing bacteria are cultured, L-aspartic acid and L-phenylalanine lower alkyl ester are added thereto, and further culture is carried out. Law. 3. The manufacturing method according to claim 1, wherein the lower alkyl group of L-phenylalanine lower alkyl ester and α-L-aspartyl-L-phenylalanine lower alkyl ester is a methyl group.