JPH0751597B2 - Daitocidins and production method thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) The present invention relates to physiological activity, in particular, H ⁺ , K ⁺ -adenosine triphosphatase (hereinafter referred to as HK-ATPase) inhibitory activity and / or phospholipase A ₂ (hereinafter referred to as PLA). ₂ ) a novel daitocidins having inhibitory activity and a method for producing the same by fermentation.

(Solution) The present inventors searched for a microorganism that produces a physiologically active substance,
When we proceeded with screening for that product,
The present inventors have completed the present invention by finding that bacteria isolated from soil of Minamidaito Island produce daitocydins having both HK-ATPase inhibitory activity and PLA ₂ inhibitory activity.

The daitocydins of the present invention are cyclic acylheptapeptides specified by the following general formula (I).

(In the formula, R is an alkyl group having 12 to 14 carbon atoms, and X is Val.
The present invention also includes a method for producing a compound represented by the above general formula (I) by a fermentation method. The microorganism used in the production method, the production method and the compound (I) of the present invention will be described in detail below.

(Microorganism) The microorganism used in the production method of the present invention is Bacillus (Baci).
llus) belonging to the genus llus) and having the ability to produce the daitosidines represented by the general formula (I). Specific examples of the microorganism include Bacillus sp. Q-
There are 55 shares. This Q-55 strain was isolated from the field soil of Minamidaito Island, Okinawa Prefecture. The mycological properties of this strain are as follows.

(1) Morphological characteristics The cells cultured on broth agar medium at 37 ° C for 5 days showed 0.5 to 0.6.
× 1.5-3.0 μm bacillus with motility. Gram stain is positive and does not show acid resistance. Also, one spore is formed from the center to the end of the cell. The spores are resistant to heating at 100 ° C for 1 hour.

(2) Growth state on various media Meat agar medium Yellowish white to light yellow tea, forming shiny thin circular colonies. The area around the colony is smooth, but sometimes it has a rough appearance and the outer edges are hairy.

 Meat juice medium The whole medium becomes cloudy and no film is formed.

 Meat broth gelatin stab culture Good growth.

Liquefy slowly.

 Litmus milk Peptone remains neutral. No coagulation is observed.

(3) Physiological properties Nitrate reduction: Negative denitrification reaction: Negative MR test: Positive VP test: Positive Indole formation: Negative Hydrogen sulfide formation: Negative Starch hydrolysis: Negative Use of citric acid: Positive Inorganic nitrogen source (A) Sodium nitrate: Negative (b) Ammonium sulfate: Positive Dye formation: Negative urease: Positive oxidase: Negative catalase: Positive Growth temperature: 15 to 50 ° C Optimal growth temperature: 33 to 40 ° C Growth pH: 4.5 to 8.5 Optimum pH: 5.5 to 7.0 Growth under anaerobic conditions: Grows.

 OF test: Non-degrading auxotrophy: None Arginine decomposition reaction: Negative Poly β-hydroxybutyrate intracellular accumulation: Negative NaCl Growth in broth medium: Growing up to 9%.

Summarizing the above mycological properties, this strain is a Gram-positive, facultatively anaerobic spore-forming bacillus and has motility. The growth temperature range is 15 to 50 ° C. VP reaction, MR test, and catalase test are positive, but starch degradation, indole formation, hydrogen sulfide formation, and oxidase test are negative. Bacteria having such properties are referred to as Vergi's Manual of Determinating Bacteriology,
8th Edition (Bergey's Manual of Determinative Bacteriol
ogy, 8th ed., 1975), Vergy's Manual of Systematic Bacteriology, Volume 1 (Berg
ery's Manual of Systematic Bacteriology vol 1,198
Based on 4) and other references, this strain is judged to belong to the genus Bacillus. The genus Bacillus is classified into two groups according to
Two species group II, 26 species total 48 species are described, but as a strain similar to this strain, Bacillus licheniformis (B.li
cheniformis). As a result of comparative examination, the outline of the difference between this strain and B. licheniformis is described below.

As shown in the above table, this strain was found to have the ability to hydrolyze starch and the productivity of pigments, such as B. lichenei
formis) was different. Therefore, this strain was named Bacillus sp. Q-55 for the time being as a new strain belonging to the genus Balance (Bacillus).
In addition, this strain is
It has been deposited with I) under the accession number Microindustrial Research Institute No. 9299 (FERM P-9299).

The microorganism used in the present invention is characterized in that it has the ability to produce a compound having an inhibitory activity on HK-ATPase and / or PLA ₂ , in addition to the above-mentioned characteristics of mycological properties.

The microorganisms used in the present invention are naturally or ultraviolet rays, X-rays,
It is easy to artificially mutate by chemical agents. Therefore,
Examples of the microorganism used in the present invention include the above-mentioned Q-55 strain isolated from nature, a strain belonging to the genus Bacillus and having a compound (I) -producing ability, as well as natural mutants and artificial mutants thereof.

The microorganism used in the present invention was obtained by separating it from natural soil, but it can be easily obtained by reconstitution of the freeze-dried product of the strain deposited at the Institute of Microbial Technology.

(Production Method) The production method of the present invention is characterized by culturing a microorganism belonging to the genus Bacillus and capable of producing the compound (I) of the present invention, and obtaining the compound (I) of the present invention from the culture.

The culturing is advantageously performed using a medium containing a nutrient source utilized by the microorganism. The medium may be synthetic, semi-synthetic or natural, solid or liquid medium, but a liquid medium containing a natural nutrient source is generally preferable. Among the nutrient sources added to the medium, the carbon source may be any assimilable carbon compound, such as L-arabinose, D-xylose, D-glucose, D-fructose, maltose, sucrose, lactose, trehalose, D. Mannitol, glycerin, starch, salicin, starch, glucose, dextrin, coconut oil, soybean oil, α-melibiose, etc., which may be used alone or in combination. Furthermore, alcohols, organic acids, etc. can also be used. Inorganic and organic nitrogen sources include ammonium chloride, ammonium sulfate, ammonium nitrate, and urea, and organic nitrogen sources include peptone, yeast extract, and
Dried yeast, meat extract, gluten meal, corn steep liquor, soybean flour, fish meal, peanut flour, cottonseed meal, casamino acids and various amino acids (eg glutamic acid, alanine, lysine, etc.) are used alone or in combination.

If necessary, the medium may be supplemented with metal salts such as sodium, potassium, magnesium, calcium, zinc, iron and cobalt such as sulfates, nitrates, chlorides, carbonates, phosphates and organic acid salts such as sodium citrate. It can be added.

The culture is preferably carried out under aerobic conditions, and any of static culture, shaking, and aeration-agitation culture is possible, but shaking or aeration-agitation culture is advantageous. The culture temperature is preferably in the range of about 15 to 50 ° C, particularly about 33 to 40 ° C. Also,
Suitably the pH of the medium is maintained at about 4.5-8.5, preferably 5.5-7.0. The culture period varies depending on the culture conditions such as medium composition and temperature, but it is usually about 2 to 14 days, preferably about 3 to 5 days. Finish the culture.

In order to isolate and purify the physiologically active substance accumulated in the culture thus cultivated, a commonly used isolation and purification means may be applied.

When Bacillus sp. Q-55 strain is cultured under the culture conditions described in Examples, at least 5 kinds of physiologically active substances are accumulated in the culture medium.

Isolation and purification of physiologically active substances are performed by centrifuging or passing the culture to remove the bacterial cells, and then the solubility and solubility in a suitable solvent, the difference in the rate of precipitation from the solution, and various adsorbents. It is preferable to apply a method utilizing a difference in adsorption affinity, a difference in partition between two kinds of liquid phases, and the like. These methods may be used alone, or combined in any order, and vice versa.

In particular, five kinds of physiologically active substances, which are products produced by culturing the Q-55 strain, utilize the adsorption affinity for various carriers and are separated from the products having no physiological activity using the physiological activity as an index. In addition, each of these physiologically active substances is ODS (trade name)
Yamamura Scientific Co., Ltd.) was used as a carrier for high performance liquid chromatography, and acetonitrile: tetrahydrofuran: water:
Each can be isolated by an operation of eluting with acetic acid (900: 100: 99: 9).

(Production substances) The five kinds of physiologically active substances produced by the Q-55 strain were named Daitocidin A ₁ , A ₂ , B ₁ , B ₂ , B ₃ . The physicochemical properties of these daitocydins are as follows.

The ultraviolet absorption spectrum (methanol), infrared absorption spectrum (KBr) and hydrogen nuclear magnetic resonance spectrum (deuteriomethanol) of each of the daitocidins are shown in FIGS. 1, 2 and 3, respectively.

From the above physicochemical properties, the fermentation product of the present invention was identified as having the following structure.

(Effects of the Invention) The ditocidins (I) of the present invention have HK-ATPase inhibitory activity and are useful as a gastric acid secretion inhibitor and a gastric ulcer preventive / therapeutic agent. The compound (I) of the present invention also has PLA ₂ inhibitory activity, and is a preventive / therapeutic agent for various diseases associated with the overproduction of eicosanoids by PLA ₂ , such as an anti-inflammatory agent, a platelet aggregation inhibitor, and an anti-inflammatory agent. It is also useful as an allergic agent.

Below, the HK-ATPase inhibitory activity of ditocydins (I) and P
The pharmacological test method for LA ₂ inhibitory activity and the results thereof are shown.

(I) NK-ATPase inhibitory activity Biochimica e.
t Biophisica acta), 7 2, 8, 31-38 (1983), and the inhibitory activity was determined as IC ₅₀ (50% inhibitory concentration).

The results are shown in the table.

IC ₅₀ Daitocidin A ₁ 2.03 × 10 ^-6 M 〃 A ₂ 1.98 × 10 ^-6 〃 B ₁ 2.10 × 10 ^-6 〃 〃 B ₂ 1.18 × 10 ^-6 〃 〃 B ₃ 1.58 × 10 ^-6 〃 (ii) PLA ₂ inhibitory activity The inhibitory effect of the compound of the present invention on phospholipase A ₂ is B.
It was measured based on the method of Rothhut et al. [Biochemical a
nd Biophysical Research Communications, 1 1 7 No.3,87
8-884 (1983)] The measurement method is as follows. Bovine serum albumin, the compound in 150mM Tris buffer containing calcium chloride (pH 8), adding E. coli cells labeled with phospholipase A _2, ³ H- oleic acid obtained from pig pancreas was reacted 60 ° C. 10 minutes .

The phospholipase A ₂ inhibitory activity of this compound is shown in the table.

IC ₅₀ Daitocidin A ₁ 7.3 × 10 ^-5 M 〃 A ₂ 8.5 × 10 ^-5 〃 〃 B ₁ 5.7 × 10 ^-5 〃 〃 B ₂ 4.9 × 10 ^-5 〃 〃 B ₃ 5.9 × 10 ^-5 〃 (Example ) Examples will be given below to explain the present invention in more detail.
However, the present invention is not limited to the embodiment.

Example 1. (1) 60 ml of seed culture medium (sterilant pH 7.0) containing glucose 1.0%, potato starch 2.0%, yeast extract 0.5%, polypeptone 0.5%, calcium carbonate 0.4%
Was dispensed into a 500 ml Erlenmeyer flask and sterilized by a conventional method, and the bacterial cells of Bacillus sp. Q-55 strain grown on Bennett's agar medium were scraped off and inoculated, and the rotary shaker was rotated at 200 rpm. Shake culture at 28 ℃ for 48 hours.

2 ml each of this culture is inoculated into 20 medium prepared and sterilized in the same manner, and shake culture is performed under the same conditions to obtain a seed culture.
Next, dextrin 8.0%, gluten meal 2.5%, soybean powder 2.5%, corn steep liquor 1.5%, sodium citrate 0.5%, ferric sulfate 0.29%, magnesium sulfate.
50 ml each of 24 l of yeast medium (sterilizer pH 7.2) containing 0.02%
Dispense into a 0 ml Erlenmeyer flask and sterilize by a conventional method.
Inoculate 2 ml each of the above seed culture medium and perform shaking culture at 28 ° C for 90 hours on a rotary shaker at 230 rpm. The resulting culture fluid has a pH of 9.2.

(2) Add 20 l of culture to pH 2.8 by adding hydrochloric acid and extract with 40 l of ethyl acetate. The extract is concentrated under reduced pressure to 5 liters. The concentrate is extracted with 2 l of 5% sodium bicarbonate. Adjust the pH of the extract to 2.8 with hydrochloric acid and extract with 5 l of ethyl acetate. The ethyl acetate layer is washed with water and then concentrated to dryness under reduced pressure. Silica gel column chromatography [Wakogel C-2
00 (product made by Wako Pure Chemical Industries)] 2.2 l. (Elution solvent, chloroform: methanol: acetic acid 20: 1: 0.4) Collect the active fractions and concentrate. The concentrate is applied to 500 cc of silica gel chromatography [Kieselgel 60 (trade name, Merck)] and eluted with chloroform: methanol: acetic acid (40: 1: 0.4). The eluate is concentrated and then subjected to LH-20 (trade name, manufactured by Pharmacia) 450 cc column chromatography. Elute with methanol. After collecting and concentrating the active fractions, LH-20
(Pharmacia) Subject to column chromatography.
After elution with methanol and concentration, 400 mg of the active substance is obtained. High-performance liquid chromatography (column: φ20
* 250mm ODS (trade name, manufactured by Yamamura Scientific Co., Ltd.), acetonitrile: tetrahydrofuran: water: acetic acid (900: 100:
Elute with 99: 1). The active substances A ₁ , A ₂ , B ₁ , B ₂ , B ₃ were separated. Further, each active substance is subjected to high performance liquid chromatography (column: ODS, Yamamura Kagaku) and eluted with acetonitrile: tetrahydrofuran: water: acetic acid (900: 100: 99: 1). Active substance Daitocidin A ₁ , 49mg A ₂ 8mg, B ₁ 146mg, B ₂
4.6 mg and B ₃ 26 mg were obtained.

[Brief description of drawings]

Figure 1 shows the ultraviolet absorption spectra of daitocydins.
(A) is Daitosaidin A ₁ , (ii) is Daitosaidin
A ₂ , (c) shows Daitosaidin B ₁ , (d) shows Daitoshidin B ₂ , and (e) shows Daitosaidin B ₃ . Figure 2 shows the infrared absorption spectra of daitocydins.
(A) is Daitosaidin A ₁ , (ii) is Daitosaidin
A ₂ , (c) shows Daitosaidin B ₁ , (d) shows Daitoshidin B ₂ , and (e) shows Daitosaidin B ₃ . Fig. 3 shows the ¹ H-nuclear magnetic resonance spectra of daitocydins. (A) is daitosaidin A ¹ , (b) is daitosaidin A ₂ , (ha) is daitosaidin B ₁ , (d) is daitosaidin B ₂ , ( E) shows that of Daitosaidin B ₃ .

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location A61K 38/08 ACL C12N 9/99 9152-4B (C12P 21/02 C12R 1:07) (72) Inventor Hisaka Takashi 2-3-1-220 Kaga, Itabashi-ku, Tokyo (72) Akiko Ota 1-8-20 Minami, Wako-shi, Saitama

Claims (2)

[Claims] 1. A general formula (Wherein R represents an alkyl group having 12 to 14 carbon atoms, and X represents Val or ILeu). 2. A method for producing daitocidins, which comprises culturing bacteria belonging to the genus Bacillus capable of producing daitocydins and collecting the daitocydins from the culture.