JPH0361437B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing L-threo-β-hydroxyaspartic acid. L-threo-β-hydroxyaspartic acid itself has antibacterial activity. That is, Bacillus subtilis
subtilis), Xanthomonas oryzae, Mycobacterium
It is known to be effective against bacteria such as Mycobacterium phlei and Botrytis cinerea (Reference 1).
Ishiyama et al., Journal of Antibiotics, vol. 23,
821 pages, 1975). On the other hand, this substance can be regarded as an analog of L-aspartic acid, which is a type of amino acid that plays an important role in living organisms, and is known to be involved in biological enzyme systems and have important physiological activities. There is. For example, L
-Aspartate β-decaboxylase (L-
Aspartate β-decarboxylase) is known to be an inhibitor of (Reference 2. EWMiles and
A. Meister, Biochemistry vol. 6, p. 1734, 1967
Year). In addition, N-
It has been reported that L-threo-β-hydroxyaspartate suppresses the absorption of methyl-D-aspartate in the cerebral cortex (Reference 3.JH
Skerritt and G. AR Johnston, Journal of
Neurochemistry, vol. 36, p. 881, 1981). As mentioned above, L-threo-β-hydroxyaspartic acid is not only useful in itself as an antibacterial agent and a physiologically active substance, but also in the synthesis of pharmaceuticals and other industrially useful compounds if it can be supplied at low cost as a type of amino acid. It is likely to be used as a raw material or raw material. Although it is possible to produce this substance by extracting and purifying it from animals and plants, it is industrially disadvantageous because of the limitations on raw materials and the low content. Although synthetic methods are possible and various methods are known, β-hydroxyaspartic acid has four stereoisomers: the threo type, the erythro type, and their D and L forms. It is not necessarily an industrially advantageous method because it is necessary to generate L-threo isomers and isolate only the L-threo isomers. In contrast to these methods, the fermentation method using microorganisms can be said to be an industrially extremely advantageous production method because it can efficiently produce only compounds limited to specific stereoisomers. Arthrinium phaeospermum, a type of fungus, is a microorganism that ferments and produces L-threo-β-hydroxyaspartic acid.
phaeospermum) and one strain of Streptomyces, an actinomycete, is known (Reference 1). The present inventors discovered that a strain of the actinomycete Dactylosporangium was found to be L-threo-
The present invention was completed based on the discovery that it has the ability to ferment and produce β-hydroxyaspartic acid, and that it can be efficiently produced and accumulated in the culture solution at a high concentration. The method will be explained below. An example of a microbial strain used in the present invention is strain SF-2253, which belongs to the genus Dactylosporangium and was isolated by the present inventors from soil in Kyoto City. science fiction
The mycological properties of strain -2253 are as described below. Observations were mainly made using the methods of EBShirling and D. Gottlieb (International Journal of Systematic
Bacteriology, Vol. 16, pp. 313-340, 1966). Morphology The basal hyphae are well branched and elongate in a wavy manner, with a diameter of
It is 0.5-0.6 micron. No fragmentation of the basal hyphae was observed in either the agar medium or the liquid medium. Aerial hyphae are rarely seen and appear to virtually never form. SF-2253 strain forms a single sporangium or a tuft-like sporangium on the surface of the agar medium. Sporangia are found in relatively large numbers on starch agar media. The sporangia are finger-shaped and approximately 0.7 in size.
~1.0 x 2.0~4.0 microns. Each sporangium usually has 3 to 4 spores in a row within it. Spores are mostly cylindrical or oval in shape, with smooth surfaces and approximately 0.6-0.9 x 0.9-1.8 microns in size. The spores were suspended in, for example, a soil extract, left to stand for 15 to 30 minutes, and then examined under a microscope and found to be motile. Growth status on various media The growth status of SF-2253 strain on various media is as shown in the following table. In the description of colors, the standards shown in [ ] are those of Container Corporation of
The one described in the "Color Harmony Manual" manufactured by America Inc. was used. Observations were made at 28℃ 14
This was done after ~28 days of culture.

【table】

[Table] Physiological properties (1) Growth temperature range: 15 to 40 on starch agar
It grows in a temperature range of 25°C to 37°C. (2) Liquefaction of gelatin: Negative (3) Hydrolysis of starch: Positive (4) Reduction of nitrate: Negative (5) Coagulation of skim milk: Negative Peptonization of skim milk: Negative (6) Production of melanin-like pigments: Negative (7) Salt tolerance: Grows at 1.5%, but does not grow at 3.0% or higher. Carbon source availability

[Table] Cell wall composition Method of Betzker et al. [Appl.Microbiol.13:236
(1965)] showed that diaminopimelic acid in the cell wall composition was mainly in the hydroxy type. Based on the above properties, strain SF-2253 is a strain that belongs to the genus Dactylosporangium among actinomycetes.
It is not known that strains belonging to the genus Dactylosporangium produce L-threo-β-hydroxyaspartic acid. Therefore, the present inventors named the SF-2253 strain Dactyrosporangium escipi SF-2253. In addition, Dactyrosporangium sp.
Strain SF-2253 has been deposited with the Institute of Microbiology, Agency of Industrial Science and Technology under accession number 6858. The outline of the production of L-threo-β-hydroxyaspartic acid using the SF-2253 strain whose properties have been clarified above will be explained below. Dactylosporangium sp. SF-2253
Strains, as seen in the case of other actinomycetes, are susceptible to changes in their properties and can be mutated by artificial mutagenic means using, for example, ultraviolet rays, X-rays, radiation, chemicals, etc.; All strains of Dactyrosporangium that have the ability to produce L-threo-β-hydroxyaspartic acid can be used in the method of the present invention. In the method of the present invention, strain SF-2253 is cultured in a medium containing nutrients that can be used by microorganisms.
For example, glucose, sucrose,
Dextrin, starch, starch syrup, molasses, soybean oil, etc. can be used. Further, soybean flour, wheat germ, peptone, meat extract, yeast extract, cornstarch liquor, sodium nitrate, ammonium sulfate, etc. can be used as the nitrogen source. In addition to adding inorganic salts such as calcium carbonate, potassium chloride, and phosphates as necessary, L-threo-β
- Organic and inorganic substances that promote the production of hydroxyaspartic acid can be appropriately added. The most suitable culture method is the liquid culture method, especially the deep agitation culture method, as is the case with general microbial culture methods. Cultivation is performed under aerobic conditions, and the temperature suitable for cultivation is 25°C to 40°C, but it is usually cultured at around 30°C, and the pH is preferably neutral to slightly alkaline.
When cultured for 3 to 10 days using the liquid culture method, L
-Threo-β-hydroxyaspartic acid is produced and accumulated in the culture solution. As a method for assaying L-threo-β-hydroxyaspartic acid, a bioassay method generally used for assaying antibiotics can be applied since this substance has antibacterial activity. That is, using Bacillus subtilis PCI-219 (Bacillus subtilis) as the test bacterium, a test dish consisting of 0.5% peptone and 1.5% agar (PH7 before sterilization) was prepared, and a paper disk (diameter 8 mm) soaked with the test solution was placed. Place it on the certification sheet 37
After culturing at ℃ for 17 hours, a growth inhibition circle corresponding to the concentration of the test substance can be seen. Inhibition circle diameter and L-threo-β
- The logarithm of the concentration of hydroxyaspartic acid has a linear correlation within a certain range, in this case 100μ
It can be quantified in the concentration range of g/ml to 2000 μg/ml. In order to isolate and purify the SF-2253 substance produced and accumulated in the culture solution, any means commonly used for purifying water-soluble acidic substances can be used as appropriate. Namely, Diaion HP-20 (manufactured by Mitsubishi Kasei), Amberlite XAD-2 (manufactured by Rohm and Haas),
Adsorbents such as charcoal powder; Gel filtration agents such as Cephadex G-10 (manufactured by Pharmacia) and Toyopearl HW-40 (manufactured by Toyo Soda Co.); (manufactured by Chemicals), DEAE-Sephadex (manufactured by Pharmacia)
Although chromatography using an anion exchange resin such as the following is used, the following purification method is efficient. The culture solution of SF-2253 strain is filtered using a filter aid such as Hyflo Super Cell to remove bacterial cells and other solid substances, and then the active ingredients during filtration are removed using Dowex 1x.
2 (Cl ^- ) and elute with 0.5M NaCl water. This eluent is subjected to column chromatography such as a cellulose column or Sephadex G-10; precipitation with an organic solvent such as methanol or ethanol, etc., to obtain high purity SF-2253.
substance can be obtained. Below, L-threo-β obtained by the present invention
- Shows the physical and chemical properties of hydroxyaspartic acid. (1) Appearance: White needle-like crystals (2) Melting point: Gradually turns brown from around 200℃. (3) Elemental analysis values: C 33.36%, H 4.69%, N
9.13%, O 52.82% (4) Molecular weight: 149 (according to FD mass spectrum) (5) Ultraviolet absorption spectrum: No characteristic absorption maximum between 220 nm and 370 nm. (6) Infrared absorption spectrum (potassium bromide method):
Shown in Figure 1. 1725, 1660, 1500, 1160, 1100, 1040 and
A characteristic absorption is seen at 970 cm ^-1 . (7) Solubility Well soluble in alkaline or acidic water and hot water, soluble in water, and virtually insoluble in organic solvents such as methanol, chloroform, ethyl acetate, hexane, benzene, and ether. (8) Stability Stable in acidic, neutral, and alkaline conditions. (9) R value of silica gel thin layer chromatography n-propanol-pyridine-acetic acid-water (15:
10:3:12) 0.5 Ethanol-Water (7:3) 0.3 (10) Paper chromatography R value (ascending method) n-Propanol-Pyridine-acetic acid-Water (15:10:3:12) 0.22 n -Butanol-acetic acid-water (2:1:1) 0.14 (11) Positive color reaction: Ninhydrin, Remieux, iodine reaction Negative: sulfuric acid, Sakaguchi reaction (12) High-pressure filter paper electrophoresis (3500V, 20 minutes) Formic acid-acetic acid -Water (PH1.9) (20:80:900, V/
V) Rm (Lys) 0.19 Pyridine-acetic acid-water (PH6.4) (200:8:2890,
V/V) Rm (Glu) 1.14 (13) Specific rotation [α] ₃₆₅ +38.0° (C 0.5, 1N HCl) (14) Hydrogen nuclear magnetic resonance spectrum measured in heavy water (100MPH, TMS external standard) So 4.07
(1Hd, J=2Hz) and 4.55ppm (1Hd, J=2Hz)
Hz). (15) Carbon nuclear magnetic resonance spectrum (25MHz, dioxane external standard) measured in heavy water shows 57.8(d);
Signals can be seen at 71.5(d), 173.0(s) and 177.2(s). The method of the present invention will be explained below based on examples. Example 1 Starch 2.0%, glucose 1.0 as seed medium
%, wheat germ 0.6%, peptone 0.5%, yeast extract 0.3%, soybean flour 0.2% and calcium carbonate 0.1%. In addition, as production medium, starch syrup 2.0%, soybean oil 0.15%, soybean flour 1.0%,
"Sungrain" (manufactured by Suntory) 0.25%, cottonseed meal 0.5%, calcium carbonate 0.1%, ferrous sulfate
A medium containing nickel chloride 0.00005% and cobalt chloride 0.00005% was used. The pH of the culture medium before sterilization was adjusted to 7 in all cases. Dactyrosporangium sp. SF-2253 strain (Fiber Science and Technology Research Institute), which grew well on yeast extract/malt extract agar slant.
6858) into two 100 ml Erlenmeyer flasks containing 20 ml of the above sterilized seed medium, and inoculated with 6 to 7 platinum loops at 28℃.
The cells were cultured for days and used as a type 1 culture solution. Next, 4 ml of the above-mentioned type 1 culture solution was inoculated into two 500 ml Erlenmeyer flasks containing 100 ml of sterilized seed medium, and incubated at 28°C for 2 hours.
The culture was cultured with shaking for several days, and this was used as a second type culture solution. Next, 50 ml each of the second type culture solution was inoculated into two 5-volume Erlenmeyer flasks containing sterilized seed medium 1,
The culture was cultured with shaking at 28° C. for 2 days, and this was used as a third type culture solution. Next, this third type culture solution was inoculated into two 50-volume jar fermenters containing 35 sterilized production medium, and cultured with aeration and stirring at 28°C for 5 days (rotation speed 270 rpm, aeration rate 35/min). . After culturing,
Filter using diatomaceous earth as an auxiliary agent, and culture filtrate 40
I got it. L-threo-β- by bioassay
The production amount of hydroxyaspartic acid is 1100μg/
It was hot in ml. Example 2 After adjusting the culture filtrate 40 obtained in Example 1 to pH 2,
It was passed through a column filled with activated carbon 4. The passing liquid was treated with Dowex 1×2 (Cl ^- ) (manufactured by Dow Chemical Company).
The active ingredients were adsorbed through a column packed with 4.
After adsorption, the column was washed with 12 portions of water and eluted with 0.2M sodium chloride solution, and the active ingredient was eluted in fractions No. 1 and 2 in 5 fractions. This fraction was concentrated under reduced pressure to about 30 ml.
The sodium chloride crystals precipitated at this time were removed by filtration, and the filtrate was passed through a tower filled with 1.2 Cefdex G-10 (manufactured by Pharmacia) and developed with water, resulting in fractions No. 27 to 49 (18 ml fractions). The active substance was eluted over time. When 360 ml of the active fraction was collected, concentrated under reduced pressure and lyophilized, a pale yellow L-threo-β-
Coarse powder of sodium hydroxyaspartate
31.5g was obtained. Next, after dissolving this coarse powder in 50% aqueous ethanol, cellulose for column chromatography (manufactured by Watmann) 1, which had been previously washed with 90% ethanol water, was dissolved.
After washing with 1 part of 90% ethanol water and developing with 3.5 parts of 70% ethanol water and 2 parts of 50% ethanol water, fraction No.
The active substance was eluted between 137 and 278 (18 ml fraction). This active fraction 2.5 was concentrated under reduced pressure and freeze-dried to produce a pale yellow L-threo-β.
-14.8g of purified powder of sodium hydroxyaspartate was obtained. Next, dissolve 3.5g of this in 5ml of water,
After adjusting the pH to 2, add 50ml of ethanol to form a precipitate.
2.72g was produced. This precipitate was added to 20 ml of warm water, adjusted to pH 2 with hydrochloric acid, and dissolved. Add ethanol until the solution becomes slightly cloudy and leave it at 5°C overnight to precipitate white crystals of L-threo-β-hydroxyaspartic acid, which are filtered and dried under reduced pressure to produce white needle-like crystals. 2.41g was obtained.

[Brief explanation of drawings]

FIG. 1 is an infrared absorption spectrum of L-threo-β-hydroxyaspartic acid in potassium bromide tablets.

Claims (1)

[Scope of Claims] 1. A strain belonging to the genus Dactylosporangium and having the ability to produce L-threo-β-hydroxyaspartic acid is cultured in a nutrient medium, and L-threo-β-hydroxyaspartic acid is extracted from the culture. 1. A method for producing L-threo-β-hydroxyaspartic acid, which comprises collecting L-threo-β-hydroxyaspartic acid. 2. A strain that belongs to the genus Dactylosporangium and has the ability to produce L-threo-β-hydroxyaspartic acid is Dactylosporangium sp.
-2253 strain (FERM P-6858).