JPS62286A - Novel substance cv-1 and production thereof - Google Patents

Abstract

PURPOSE:To produce novel substance CV-1 having enhancing action on antimicrobial activity and a specific chemical structure, by cultivating a bacterium belonging to the genus Streptomyces. CONSTITUTION:A bacterium such as Streptomyces sp. CO-1 (FERM P-8311) belonging to the genus Streptomyces, capable of producing novel substance CV-1 having a chemical structure shown by the formula, is subjected to liquid culture in a nutritive medium at 25-37 deg.C at 4-10pH for 1-7 days and CV-1 is collected from the culture solution.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a new substance having an antibacterial activity-enhancing effect and a method for producing the same.

More particularly, the present invention relates to the novel substance CV-1 and the fermentative production of CV-1 by a microorganism belonging to the genus Streptomyces.

BACKGROUND OF THE INVENTION Various fermentation products that exhibit antibacterial activity-enhancing effects have been known as substances that have antibacterial activity-enhancing effects.

For example, enhancing the antibacterial activity of macrolide antibiotics,
Cultured products of the genus Bacillus or Pseudomonas are known (The Journal of Anti
biotics, vol, XXX No, 3.2O
9 (1977)).

It was discovered that a bacterium isolated from soil in Machida City, Tokyo (hereinafter referred to as CO-1 strain) produces CV-1, a new substance that has an antibacterial activity-enhancing effect in culture.

DISCLOSURE OF THE INVENTION According to the present invention, the new substance CV-1 is a microorganism belonging to the genus Streptomyces and having the ability to produce CV-1.
CV-1 is produced by culturing in a nutrient medium until at least substantially CV-1 is detected in the culture and isolating CV-1 from the culture.

The CV-1 thus obtained has an antibacterial activity enhancing effect,
When used together with existing antibacterial compounds, it is useful as a disinfectant or detergent for instruments in laboratories, hospitals, etc.

The mycological characteristics and physiological properties of the CO-1 strain are as follows.

(A) Mycological properties of strain Co-1 Strain C0-1 shows good or normal growth on various natural and synthetic media generally used for the growth of actinomycetes. Adhesion of aerial mycelia was also moderate or better.

In addition, growth on peptone/yeast extract/iron agar medium and synicrose/nitrate agar medium is poor. Long-growing hyphae exhibit ridge-like growth on a well-grown medium. The color tone of aerial hyphae is gray ranging from light gray to black, and long-growing hyphae is gray with a light brown tone. There is no production of soluble pigments and melanin-like pigments.

The spores form a chain of 20 or more spores at the tip of a simple branch from the aerial hyphae, and exhibit a spiral shape.

The shape of the spore is oval and the size is 1.1-1.2 x 1
．． According to electron microscopy, the surface is smooth and no flagella are observed. Also, no sporangia were found.

(8) Growth status on various media The characteristics of growth and color when cultured on various media at 28°C for 2 weeks are shown below. Color display is Co1 or H
armony Manual (ContainerC
It follows the color classification by the organization of America).

Note that no soluble pigment was produced in any of the media.

(1) Growth on Shiny Claus/Nitrate agar medium: Poor Aerial hyphae and its color: Poor, bistu (3ec) Color of long hyphae: Natural (2dC) (2) Growth on Glucose/Asparagine agar medium: Good, raised aerial Mycelium and its color: Abundant, natural (2dc) to beige gray (3ih) Color of long-growing mycelium: Natural (2dC) (3) Growth on glycerin/asparagine agar medium: Good,
Raised aerial hyphae and their color: Abundant, bistu (3eC) to silver gray (3fe) Long hyphae color: Natural (2dC) (4) Growth on starch agar medium: Good, ridged aerial hyphae and their color: Abundant , black plum (10po
) ~ Assi 5 (5f e) Color of long hyphae bi-beige (3ge) (5) Growth on tyrosine agar medium: Moderate Aerial hyphae and its color: Medium, cobalt gray (2f
e) Color of long-growing hyphae: Bistu (3ac) (6) Growth on nutrient agar medium: Good, raised aerial hyphae and its color: medium, silver/gray (3f)
e> Color of long-growing hyphae: Natural (2dC) (7) Growth on yeast/malt agar medium: Good, raised aerial hyphae and their color: Abundant, black plum (10po
) ~ Ash (5'e) Color of long-growing hyphae Nirite mustard tan (2i e) (8) Growth on oatmeal agar medium: Good, raised aerial hyphae and their color: Abundant, Pearl (3ba) ~ Cobalt gray (2fe) Color of long mycelia: Ash (5fe) (C) Various physiological properties The physiological properties of strain C0-1 are shown below. Regarding the growth temperature range, the results after 2 days, skim milk, gelatin and fiber The effects on the elements were observed after 1 month at 28°C, and the other results were observed after 3 weeks at 28°C.

(1) Assimilation of carbon sources: D-glucose, L-arabinose, D-fructose, D-xylose, and sicucrose are assimilated, but D-mannitol, l-inositol, L-rhamnose, and raffinose are assimilated. do not.

(2) Liquefaction of gelatin: No liquefaction (3) Coagulation and peptonization of skim milk: Peptonization and coagulation were shown.

(4)　JJ! Fiber decomposition effect: None.

(5) Starch hydrolysis effect: Yes.

(6) Growth temperature range: 24-37°C. The optimum temperature range is 27-29°C.

(7) No production of melanin-like pigments.

The CO-1 strain is a mesophilic bacterium and forms abundant aerial hyphae on an agar medium. 2 on sporophytes that are simply branched from aerial hyphae.
Creates O or more spore chains.

In addition, no division of hyphae was observed. On the other hand, cell wall analysis revealed that the bacterium contained LL-type diaminopimelic acid, and thus the bacterium was classified as a member of the genus Streptomyces in the order Actinobacteria.

Therefore, this strain is Streptomyces Sp.

(Streptomyces sp, ) Co −
1 and was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiology Research Institute No. 8311 (Deposit date: 1985).
June 13th).

Next, the physicochemical properties and biological properties of CV-1 will be described.

(^)Physicochemical properties of CV-1 Molecular formula: C7HI4N2O& Ultraviolet absorption spectrum: Shows no characteristic absorption.

Infrared absorption spectrum: Shown in Figure 1. Measured by KBr method.

Solubility: Well soluble in water and dimethyl sulfoxide,
Barely soluble in methanol, ethanolopacetone, ethyl acetate, chloroform, benzene, and hexane.

PMR spectrum (measured in DO, internal standard HDO)
δ (ppm): 3.6-3.9 (6H, many peaks are seen).

5.2 (LH, doublet) CMR spectrum (measured in D 2 O, external standard DS
S) δ (ppm): 63.6, 64.4, 70.4, 71.2, 71.6° 80.0.163.9 (8) Antibacterial enhancing effect of CV-1 Next, antibacterial enhancing substances I will talk about the biological properties of CV-1. It is clear from Table 1 that spiramycin and novobiocin at concentrations that do not affect growth exhibit antibacterial activity against E. coli in the coexistence of CV-1.

Table 1: Antibacterial enhancement effect of spiramycin and novobiocin by cv-1 obtained in Example 1 (Test bacteria: Escherichia coli ATCC Table 1) A broth agar medium containing the drugs listed in Table 1. A paper disk with an 8-diameter diameter was soaked with 25% of the sample aqueous solution and placed on agar. It shows the effect of enhancing the antibacterial effects of mycin and novobiocin.

The novel substance CV-1 obtained by the method of the present invention exhibits the effect of enhancing the antibacterial activity of macrolide antibiotics such as spiramycin, erythromycin, leucomine, tylosin, and carbomycin, and antibiotics such as novobiocin and coumamycin.

Therefore, CV-1 can be used in combination with these antibiotics to treat the above-mentioned infectious diseases in mammals (e.g., mice, rats, dogs, humans) and poultry (e.g., chickens, ducks). can.

Next, the manufacturing method of CV-1 will be explained in detail.

According to the present invention, any microorganism belonging to the genus Streptomyces and having the ability to produce CV-1 can be used. One example is Streptomyces Sρ.

One example is the C0-1 strain.

Furthermore, as long as it has the ability to produce CV-1, it can be mutated by known methods for increasing the productivity of metabolic products of the above-mentioned producing bacteria, such as ultraviolet irradiation, X-ray irradiation, or mutagenic treatment using mutagenic substances. Bacterial strains can also be used.

In the cultivation of the present invention, ordinary methods for culturing actinomycetes are applied. Any culture medium can be used as long as it contains appropriate amounts of carbon sources, inorganic substances, etc. As the carbon source, glucose, starch, glyceronopemannose, fructose, shakerose, molasses, etc. are used alone or in combination. Furthermore, depending on the assimilation ability of the bacteria, hydrocarbons, alcohols, organic acids, etc. may also be used. Nitrogen sources include nitrogen-containing compounds such as ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, urea, and nitrogen-containing natural products such as peptone, meat extract, yeast extract, dried yeast, corn stew liquor, soy flour, and casamino acids. are used alone or in combination. In addition, inorganic salts such as common salt, potassium chloride, ferrous sulfate, zinc sulfate, manganese sulfate, copper sulfate, calcium carbonate, and phosphates may be added as necessary, and organic and inorganic substances that promote CV-1 production, such as Biotin, vitamins, etc. can be appropriately added.

The most suitable culture method is a liquid culture method, especially a deep agitation culture method. The optimum culture temperature is 25 to 37°C, especially 28 to 30°C, and the pH of the culture medium is 4 to 10, preferably 6 to 8, by adding aqueous ammonia or aqueous ammonia carbonate solution. When culture is carried out in liquid culture for usually 1 to 7 days, the target substance is produced and accumulated in the culture solution.

Stop the culture when the production amount in the culture medium reaches the maximum,
The target product is purified and isolated from the culture solution or supernatant obtained by centrifuging the bacterial cells.

Isolation and purification of CV-1 from culture p fluid or supernatant fluid utilizes separation and purification methods commonly used to isolate metabolic products of microorganisms from their culture fluids. For example, culture p solution etc. can be mixed with ion exchange resin such as Dowex.
1X2 (trade name, manufactured by Dow Chemical Company) to adsorb impurities.

Next, the active ingredient is adsorbed onto activated carbon, washed with water, and then eluted with a 20% acetone aqueous solution. After distilling off the acetone, the active fraction is freeze-dried to obtain a crude powder. Further, this can be subjected to column chromatography using gel filtration resin, high performance liquid chromatography, etc. to separate cv-i.

Aspects of the present invention are illustrated below in Examples.

Example Streptomyces sp, C○-1 (Feikoken Bacterial Serial No. 2311) is used as the seed fungus. 21 strains!
Bacto-tryptone (Difc) in a volume Erlenmeyer flask
(manufactured by company o) 5g/j! , yeast extract 5g/j! , meat extract 3g/l, soluble starch 10g/j2, glucose 10g
/j! , inoculated into 300 ml of seed medium (pH 7.2 before sterilization) having a composition of 5 g/42 calcium carbonate, and incubated at 30°C.
Culture with shaking for 48 hours. The resulting seed culture was placed in a fermentation medium of the following composition in a 301 volume jar fermenter.
Inoculate the bacteria into a tube and heat it at 30℃ with aeration stirring method (rotation speed 250 rpm).
Culture is performed by aeration (maximum 15β/min).

Fermentation medium composition: dextrin 40g/l.

KH2PO40.5g/j! 5Mg5O<・7aq0,
5 g/β, dry yeast powder 20 g/β, calcium carbonate 5 g/1 (pH 7.0, prepared with NaOH before sterilization).

Adjust the pH of the medium during culture using aqueous ammonia.
Culture for 72 hours while adjusting the temperature from 6.5 to 7.5.

Bacterial cells are separated from the culture solution to obtain p-liquid 13j2.

After adjusting the P solution 13β to pH 8.0, it is passed through a column of IIl ion exchange resin Dowex 1X2 (carbonate type) (trade name, manufactured by Dow Chemical Company) to adsorb impurities, and the active substance that flows out is adsorbed on activated carbon. After washing with water, elute with 20% acetone solution. After distilling off the acetone in the eluate fraction, it is freeze-dried. The powder obtained here was dissolved in water and gel filtration resin (TSK-Gel Toyopearl) IW-40F
, Toyo Soda) and develop with water to obtain the active fraction.

This fraction is lyophilized to a powder.

Both fractions were dissolved in a small amount of water and subjected to high performance liquid chromatography (Wakoge I LCNH2-10゜Wako Tsumugi Pharmaceutical Co., Ltd.), and the active fractions eluted using a concentration gradient method of 100% acetonitrile and 70% acetonitrile were collected. Pure CV-1 was obtained by lyophilization. The CV-1 obtained here is
It exhibited the physicochemical properties and biological properties shown above.

Effects of the Invention According to the present invention, a novel substance CV-1 exhibiting an antibacterial activity enhancing effect is produced by culturing a microorganism belonging to the genus Streptomyces.

[Brief explanation of drawings]

FIG. 1 shows the infrared absorption spectrum of CV-1. Patent applicant (102) Kyowa Hakko Kogyo Co., Ltd. Procedures
Amendment: January 2, 1985 1, Indication of the case: Patent Application No. 13'8135, 1985 2, Name of the invention: New substance CV-1 and its manufacturing method 3, Person making the amendment: Relationship with the case Patent Applicant postal code: 100 Address: 6-1 Otemachi, Chiyoda-ku, Tokyo Name
(102) Claims column and Detailed Description of the Invention column 5 of Kyowa Hakko Kogyo Co., Ltd. specification, content of amendment: “From the above physicochemical properties, CV-1 has the following chemical structure. Determined; H2OH” Claims (1) A substance represented by the formula CV-1゜(2) A microorganism belonging to the genus Streptomyces and having the ability to produce C'l-1 represented by the formula A method for producing CV-1, which comprises culturing in a nutrient medium until CV-1 is at least substantially detected in the culture solution, and collecting CV-1 from the culture.

Claims (1)

[Claims] (1) Substance CV specified by the following physicochemical properties - Molecular formula: C_7H_1_4N_2O_6 Ultraviolet absorption spectrum: Shows no characteristic absorption. Infrared absorption spectrum: Shown in Figure 1. Measured by KBr method. Solubility: Well soluble in water and dimethyl sulfoxide,
Barely soluble in methanol, ethanol, acetone, ethyl acetate, chloroform, benzene, and hexane. PMR spectrum (measured in D_2O, internal standard HDO
) δ (ppm): 3.6-3.9 (6H, many peaks seen), 5
．． 2 (1H, doublet) CMR spectrum (measured in D_2O, external standard DSS
) δ (ppm): 63.6, 64.4, 70.4, 71.2, 71.6,
80.0, 163.9 (2) Cultivate a microorganism belonging to the genus Streptomyces and having the ability to produce CV-1 in a nutrient medium until CV-1 is at least substantially detected in the culture solution. A method for producing CV-1, which comprises collecting CV-1 from a substance.