JPH0629227B2 - D-beta-lysylmethanediamine and method for producing the same - Google Patents

Description

The present invention relates to a novel antibiotic D-beta-lysylmethanediamine and a method for producing this compound by culturing a microorganism belonging to the genus Streptomyces.

The inventors of the present invention actinomycete MD743-GF4 isolated from a soil sample in the Institute for Microbial Chemistry, Kamiosaki, Shinagawa-ku, Tokyo.
It was discovered that the strain can be cultured to produce and accumulate D-beta-lysylmethanediamine, and the novel compound, D-beta-lysylmethanediamine, can be collected from the culture. D-beta-lysylmethanediamine is an effective antibiotic against Gram-positive bacteria and has an immunopotentiating action. Further, D-beta-lysine obtained by hydrolysis of D-beta-lysylmethanediamine is useful as a starting material for synthesizing the deoxynegamycin, an antibiotic effective against Gram-negative bacteria invented by the present inventors. is there. (JP-A-52-59112, May 16, 1977)
Published on the day).

The gist of the first aspect of the present invention is to provide a compound of the following formula (I) D-beta-lysylmethanediamine and its acid addition salt represented by

The physicochemical and physiological properties of this novel compound are as follows.

D-beta-lysyl methanediamine is a strongly basic substance, and D-beta-lysyl methanediamine isolated as its 1/2 carbonate is a hygroscopic colorless powder whose clear melting point cannot be measured. The specific rotation of this 1/2 carbonate is [α] _D ²⁶ =-
7.4 ° (c0.5, water) and [α] _D ²⁶ = -21.3 ° (c0.46, 0.08N
Hydrochloric acid), elemental analysis values are C44.75%, H8.63%, N2
6.50%, O20.62%, C ₇ H ₁₈ N ₄ O ・ 1 / 2H ₂ CO ₃ molecular formula (theoretical value: C43.89%, H9.33%, N27.30%, O19.49%)
Was estimated, and m / z 175 was obtained by mass spectrometry (SIMS).
(MH ⁺ ) was shown. The ultraviolet absorption spectrum curve of D-beta-lysylmethanediamine in aqueous solution shows no specific absorption. The infrared absorption spectrum curve measured with a potassium bromide tablet is as shown in FIG. 1 of the accompanying drawings. The chemical shifts (δ, ppm) of the nuclear magnetic resonance spectrum measured in heavy water (pD6) are shown in Table 1.

D-beta-lysylmethanediamine is well soluble in water, but is generally insoluble or insoluble in organic solvents. Ninhydrin reaction and Lydon-Smith reaction are positive. Thin-layer chromatography of cellulose (Avicel SF, Funakoshi Chemical) (Development system: propanol / pyridine / acetic acid / water,
15: 10: 3: 12), a single spot (ninhydrin reaction) was exhibited at Rf 0.38.

The mobility of alanine was 1. by high-pressure paper electrophoresis (3,300 V, 15 minutes) using formic acid, acetic acid, and water (1: 3: 36 volume).
When the value was 0, the mobility of D-beta-lysylmethanediamine was 2.50.

Picrates of D- beta lysylmethanediamine is a decomposition point 181-183 ° C. Yellow crystals, elemental analytical values C34.74%, H3.21%, N21.40% , at O40.95%, C ₇ H ₁₈
Theoretical value of N ₄ O ・ 3C ₆ H ₃ N ₃ O ₇ (C34.85%, H3.16%, N21.1
3%, O40.85%).

The D-beta-lysylmethanediamine (1/2 carbonate) obtained in the present invention was used for measuring Staphylococcus aureus (Smith strain) and Bacillus subtilis (B-558) measured on a 0.5% peptone agar plate.
Strain and PCI219 strain) were 50 mcg / ml, respectively. Furthermore, when sheep red blood cells (SRBC) were administered as an antigen to a mouse that was intravenously injected with D-beta-lysylmethanediamine every day, the spleen was excised several days later, and the number of antibody-producing cells in the spleen cells was measured. It was found that the number of antibody-producing cells was increased as compared with the control test, and it was confirmed that D-beta-lysylmethanediamine has a humoral immunity enhancing action. In addition, D-beta-lysyl methanediamine daily,
SRBC as an antigen was administered to the mouse footpads administered intraperitoneally, and the immunity to SRBC was established again.
Delayed type hypersensitivity (Delayed Type Hypersensitivity)
Sensitivity) response was examined, and a cell-mediated immunity enhancing effect was observed.

In an acute toxicity test by intravenous administration of D-beta-lysylmethanediamine to mice, no death occurred at a dose of 250 mg / kg.

The first D-beta-lysylmethanediamine according to the invention can be obtained as the free base or its hydrates or carbonates, but in addition to the pharmaceutically acceptable acids by the usual methods any other non-toxic It can be an addition salt produced. Acids to be added include hydrochloric acid, hydrobromic acid, sulfuric acid,
Inorganic salts such as phosphoric acid and nitric acid, acetic acid, malic acid, citric acid,
Organic acids such as ascorbic acid, methanesulfonic acid and picric acid are used.

The gist of the second invention is to cultivate a D-beta-lysylmethanediamine-producing bacterium belonging to the genus Streptomyces and collect D-beta-lysylmethanediamine from the culture. It is in the manufacturing method.

According to the method of the present invention, the D-beta-lysylmethanediamine is in the pure state or in the crude state, respectively.
Further, it is collected in a solution state or in a solid state.

An example of a D-beta-lysylmethanediamine-producing bacterium is an actinomycete isolated by soil in the laboratory premises at the Institute for Microbial Chemistry in February 1973, which is a strain with the strain number MD743-GF4. The mycological properties of the MD743-GF4 strain are as follows.

1. Morphology The MD743-GF4 strain forms spiral aerial hyphae from the basal hyphae branched under the microscope, and the limbus is not observed. The mature spore chain has a chain of 20 or more spores, and the size of the spore is 0.6 to 0.7 x 1.4 to 1.7.
At the micron level, the surface of spores is smooth.

2. Regarding the description of colors in various media, the standard shown in [] is the Color Harmony Manual of Container Corporation of America (Container Corporation of America).
Color harmony manual).

(1) Sucrose / nitrate agar medium (27 ° C. culture) On the growth of colorless, aerial hyphae of light brown ash [4ge, Rose Beige] are attached to the mycelium, and no soluble pigment is observed.

(2) Glycerin / nitrate agar medium (27 ° C culture) On the development of light yellow [2gc, Bamboo] to Nifu yellow [3pc, Amber], white aerial hyphae grow slightly and the soluble pigment is ,
It becomes yellowish.

(3) Glucose-asparagine agar medium (27 ° C culture) Light brown ash [4ge, on growth of light yellow [2gc, Bamboo]
Rose Beige] grows on the aerial mycelium, and no soluble pigment is found.

(4) Yeast-malt agar medium (ISP-medium 2,27 ° C culture) On the growth of light yellow tea [3ng, Yellow Maple], aerial hyphae of bright brown ash [3dc, Natural-5fe, Ashes] were settled. However, no soluble dye was found.

(5) Oatmeal agar medium (ISP-medium at 3,27 ° C) Bright brown ash [5fe, A] on the growth of light yellow [3ie, Camel]
shes] aerial hyphae are settled, and soluble pigments are not found.

(6) Starch / inorganic salt agar medium (ISP-medium 4, 27 ° C culture) On the development of colorless to light tea [3 lg, Lt Brown], aerial mycelia of bright brown ash [5 fe, Ashes] are grown and dissolved. No sex pigments are found.

(7) Glycerin / asparagine agar medium (ISP-medium 5,
(27 ° C culture) On the growth of light tea [3 pg, Golden Brown], aerial hyphae of white to bright brown ash [5 fe, Ashes] were grown, and the soluble pigment was
It is only yellowish.

(7) Shiroshi agar medium (ISP-medium 7, 27 ° C culture) On the growth of light tea [3 ng, Yellow Maple], aerial mycelia of light ash [3 dc, Natural] to light brown ash [5 fe, Ashes] were settled. However, almost no soluble dye is found.

(8) Lime malate agar medium (27 ° C. culture) Growth was pale yellow [2ea, Lt Wheat], aerial hyphae were pale and white, and no soluble pigment was observed.

(9) Nutrient agar medium (cultivated at 27 ° C) Growth is light tea [2pi, Mustard Brown], and aerial hyphae do not grow and soluble pigments are not found.

(10) Starch agar medium (27 ° C culture) Light brown ash [3dc, on the growth of light yellow [2gc, Bamboo]
Natural ~ 5fe, Ashes] aerial hyphae are settled, and soluble pigments are not found.

(11) Skim milk (37 ° C culture) On the development of light yellow [2ic, Honey Gold], aerial hyphae of tea ash are settled, and the soluble pigment is slightly brownish.

(12) Gelatin puncture culture In simple gelatin medium (20 ° C culture), light yellow tea [3n
e, Topaz] develops, and white to gray aerial hyphae grow on the surface and the soluble pigment becomes brownish. In the glucose-peptone-gelatin medium (cultured at 27 ° C), the growth was light yellow tea [2ic, Honey Gold], the aerial hyphae did not settle, and the soluble pigment exhibited a dark brown color.

(13) Cellulose (cultivated at 27 ° C.) An aerial mycelium of tea ash grows on the colorless growth, and no soluble pigment is found.

3. Physiological properties (1) Growth temperature range Yeast / starch agar (yeast extract (manufactured by Daigo Nutrition Chemicals) 0.2%, soluble starch (manufactured by Komune Chemical) 1.0)
%, String agar 3.0%, pH 7.0), 20 ° C, 2
As a result of the test at each temperature of 4 ° C., 27 ° C., 30 ° C., 37 ° C., and 50 ° C., except for 50 ° C., growth occurred at any temperature, but the optimum growth temperature is around 24 ° C. to 27 ° C. I think that the.

(2) Liquefaction of gelatin (15% simple gelatin, 20 ° C culture; glucose / peptone / gelatin, 27 ° C culture) About 14 days after culturing in simple gelatin medium, in the case of glucose / peptone / gelatin medium, after culturing Liquefaction starts from about the 10th day. Both actions are moderate.

(3) Hydrolysis of starch (starch / inorganic salt agar medium and starch agar medium, both are cultured at 27 ° C.) Starch inorganic salt agar medium is about 3 days after culturing, and starch agar medium is 7 days after culturing. From the time of the beginning, the water-disintegratability was found, and the action was strong.

(4) Coagulation and peptone conversion of defatted milk (10% skim milk (Difco), 37 ° C culture) Weak peptone conversion started about 9 days after the culture and was completed about the 13th day. The action is stronger. Coagulation cannot be discerned.

(5) Production of melanin-like pigment (tryptone yeast broth, ISP-medium 1; peptone yeast iron agar,
(ISP-medium 6; tyrosine agar, ISP-medium 7, both were cultured at 27 ° C.) The production of melanin-like pigment was observed in any medium.
The action is moderate in tryptone yeast broth, strong in peptone yeast iron agar, and weak in tyrosine agar.

(6) Utilization of carbon source (Pridham-Gotrieve agar medium, ISP-medium 9, 27 ° C. culture) Glucose and raffinose are used for growth, and L-arabinose, D-xylose, D-fructose, sucrose, inositol, L-rhamnose and D-mannitol are not used.

(7) Dissolution of lime malate (calcium malate agar, cultured at 27 ° C) Dissolution of lime malate was observed, and its action was moderate to
Strong one.

(8) Reduction reaction of nitrate (peptone water containing 0.1% potassium nitrate, ISP-medium 8, 27 ° C. culture) Positive.

To summarize the above properties, the MD743-GF4 strain belongs to the genus Streptomyces, and 2,6-diaminopimelic acid contained in the cell wall is of the LL-type. Also, spore carcasses are not observed, aerial hyphae have helix formation, and ring branches are not observed. The surface of spores is smooth. In various mediums, colorless to light yellow tea, or white to light brown ash aerial hyphae grow on the development of light tea, and almost no soluble pigment is observed, or slightly yellowish. Melanin pigment is positive, proteolytic activity is moderate, and starch is highly hydrolyzable.

From these properties, a search for known bacterial strains closely related to the MD743-GF4 strain gives the following species.

That is, Streptomyces nashvillensis (Reference International Jou
rnal of Systematic Bacteriology, 19: 455, 196
9; Antibiotics and Chomotherapy, 11: 312, 196
1), Streptomyce
s misakiensis, Literature International Journal of Systema
tic Bacteriology, 18: 348, 1968), and Streptomyces xanth
ocidicus, Literature International Journal of Systematic B
Acteriology, Vol. 22, p. 372, 1972). These 3
Species ISP strains were obtained and compared with MD743-GF4 strain. As a result, Streptomyces misakiensis has an extremely weak proteolytic activity, and M is used in terms of utilizing D-xylose, D-fructose and sucrose in utilizing sugar.
It is distinguished from the D743-GF4 strain. On the other hand, Streptomyces xanthosidicus means that this strain does not show proteolytic activity, and that it develops by utilizing L-arabinose, D-xylose, D-fructose and sucrose in its sugar utilization ability. -It showed a big difference with the GF4 strain. Table 2 below shows the results of the comparative experiments with the remaining Streptomyces nutsyuvilleensis.

As shown in Table 2, the MD743-GF4 strain and Streptomyces natsuyuvirensis are in good agreement except for the availability of L-arabinose, D-xylose.

Regarding the use of L-arabinose in the MD743-GF4 strain, variations were observed in repeated experiments, and in some cases, it was only slightly used. Considering this point, it can be considered that the reliable difference between the two is only the use of D-xylose.
From these results, the MD743-GF4 strain was identified as Streptomyces nashvil
lensis), and was identified as Streptomyces nautilus MD743-GF4. The MD743-GF4 strain was applied for storage consignment to the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology on September 7, 1985, and the consignment number is Micromachinery Research Institute No. 8442. In addition, this MD
The 743-GF4 strain was re-deposited on Dec. 25, 1991, and was assigned the deposit number of Micro Engineering Institute No. 12676.

Although actinomycetes are prone to mutations artificially and in nature, Streptomyces nutsyuvilleensis according to the present invention includes all of the mutants. D referred to in the present invention
The -beta-lysylmethanediamine-producing bacterium produces D-beta-lysylmethanediamine to produce MD743-
It includes all strains belonging to the genus Streptomyces that produce D-beta-lysylmethanediamine, including strains that are not clearly distinguished from the GF4 strain and its mutant strain.

In the method of the present invention, a D-beta-lysylmethanediamine-producing bacterium, particularly the MD743-GF4 strain described above, is inoculated into a nutrient source-containing medium that can be used by ordinary microorganisms and cultured, preferably under aerobic conditions. By growing, D-beta-lysylmethanediamine is mainly produced and accumulated in the culture medium, and the target product is separated from the culture medium, particularly the culture medium.

As a nutrient source in the medium to be used, a known one used as a nutrient source for actinomycetes can be used. For example, commercially available soybean flour, peanut flour, cotton seed flour, dried yeast, peptone,
Nitrogen sources such as meat extract, casein, corn steep liquor, sodium nitrate, and ammonium sulfate, and commercially available glucose, galactol, starch, glycerin, maltose, dextrin, sucrose, lactose and other carbohydrates,
Alternatively, carbon sources such as soybean oil and fat and, if necessary, inorganic salts such as sodium chloride, calcium carbonate, magnesium sulfate, manganese chloride, ammonium sulfate and phosphate, and various amino acids can be used. These may be used by D-beta-lysylmethanediamine-producing bacteria and can be useful for the production of D-beta-lysylmethanediamine, and all known actinomycete culture materials can be used. Liquid aeration and agitation culture is preferable for the large-scale production, and the culture temperature can be applied within a range in which the D-beta-lysylmethanediamine-producing bacterium grows to produce D-beta-lysylmethanediamine, but particularly preferably 25 ~ 30 ° C. The culture is usually continued until the D-beta-lysylmethanediamine is sufficiently accumulated, and usually the culture is performed for 2 to 7 days.

The quantification of D-beta-lysylmethanediamine was carried out using Bacillus subtilis PCI219 strain as a test strain,
The purified D-beta-lysyl methanediamine obtained by the present invention was used as a standard substance at 1000 mcg (unit) / mg by the usual cylindrical plate method used for the determination of antibiotics.

D-beta-lysyl methanediamine and its salts are well soluble in water and are mainly present in the liquid part in cultures of D-beta-lysyl methanediamine-producing bacteria. Since D-beta-lysylmethanediamine in the liquid is substantially not extracted with an organic solvent such as butanol, butyl acetate or chloroform, treatment with these solvents can be used if necessary for removing contaminants. D-beta-lysylmethanediamine in the culture solution or aqueous solution can be collected by adsorbing it with various adsorbents. When activated carbon is used as the adsorbent, the adsorbed antibiotics are weakly acidic water and weakly acidic methanol water, propanol water,
It is eluted with acetone water.

Further, D-beta-lysylmethanediamine is adsorbed on the cation exchange resin in good yield and eluted with a suitable eluent based on its basic property. This adsorption elution method is the most suitable method for collecting from a large amount of culture solution. As the cation exchanger, Amberlite IRC-50 and CG-50 (Rohm and.
Haas Co., Rewacht CNP (Bayer Co.), C
H-type, such as M-Sefadex (manufactured by Pharmacia)
Na type, NH ₄ type and the like and mixed types thereof are used, and the adsorbed antibiotics are eluted with good yield by acidic water, diluted ammonia water and an aqueous solution of an inorganic salt. 0.2N-1N ammonia water is used. Since D-beta-lysylmethanediamine does not substantially adsorb to the anion exchange resin, it is a preferable material for neutralizing the acidic solution and removing acidic contaminants.

D-beta-lysylmethanediamine can be collected purely by appropriately combining or repeating the above-mentioned extraction method and separation method.

Hereinafter, the present invention will be described with reference to Examples. Since the properties of D-beta-lysylmethanediamine have been clarified by the present invention, the production of D-beta-lysylmethanediamine is based on those properties. Various methods can be devised. Therefore, the present invention is not limited to the examples, and the known means based on the properties of D-beta-lysylmethanediamine revealed by the present invention, as well as the modifying means of the examples, can be used. It includes all methods for producing, concentrating, extracting and purifying D-beta-lysylmethanediamine.

Example 1 Streptomyces natsuyuvirensis MD743-GF4 strain cultured in an agar slant medium (Microtechnological Research Institute, 844th)
No. 2) containing 2.0% galactose, 2.0% dextrin, 1.0% peptone (Bacto Soyton, Difco), 0.5% corn staple liquor (Ajinomoto), 0.2% ammonium sulfate, 0.2% calcium carbonate (5)
Inoculate 100 ml in a 00 ml Erlenmeyer flask and incubate at 28 ° C for 2
Seed culture was obtained by rotary shaking culture (180 rpm) for one day. 2 ml each of this seed culture is the same medium as above (110 ml)
And 90 Erlenmeyer flasks were cultured for 3 days under the same conditions.

The culture solution was collected and collected to obtain 9,000 ml of liquid (pH 6.0,
(8.0 mcg / ml) was obtained. Add this solution to Amberlite I
It was passed through a column packed with 550 ml of RC-50 (NH ₄ type: H type, 7: 3) to adsorb D-beta-lysylmethanediamine, washed with water (1.100 ml), and eluted with 1.2N aqueous ammonia. The active eluent (1370 ml) was collected and concentrated under reduced pressure to dryness to obtain 636 mg (644 mcg / mg) of crude powder.

Dissolve this crude powder in 10 mg of water and use Amberlite CG.
-50 (NH ₄ type) 60 ml was adsorbed on a packed column, washed with 120 ml of water (minus 1-18) and 300 ml of 0.6N ammonia water (fraction 19-60), and then 300 ml of 1.2N ammonia water ( Fraction 61-101) was eluted. Fractions 77-95 were combined, concentrated under reduced pressure to dryness, and further dried to obtain 1/2 carbonate of D-beta-lysylmethanediamine as a colorless powdery substance (hygroscopic). Yield is 3
70mg (1,000mcg / mg) yield of 50.8 from culture
It was in%. Specific rotation of this material [α] _D ²⁶ -7.4 °
(C0.5, water). It should be noted that this substance became colorless and candy-like when it absorbed moisture.

[Brief description of drawings]

FIG. 1 shows an infrared absorption spectrum curve of D-beta-lysylmethanediamine carbonate according to the present invention measured in a potassium bromide tablet.

Claims (2)

[Claims] 1. The following formula (I) D-beta-lysyl methanediamine and its acid addition salt represented by. 2. A method for producing D-beta-lysylmethanediamine, which comprises culturing a D-beta-lysylmethanediamine-producing bacterium belonging to the genus Streptomyces and collecting D-beta-lysylmethanediamine from the culture. .