JPS6399088A - Kbs3-p1002 substance and production thereof - Google Patents

Abstract

NEW MATERIAL:KBS3-P1002 substance having the following physical and chemical properties. Elemental analysis (%), C 48.27, H 7.47, N 11.98, ash 1.69; ash point, 183-185 deg.C; appearance, white powder; solubility, soluble in water, methanol, ethanol and acetonitrile and insoluble in n-propyl alcohol, acetone, ethyl acetate, ether, chloroform, diethyl ether and petroleum ether; color reaction, negative to ninhydrin test; amino acid composition (ratio), Asp 3, Glu 1, Ser 1, Pro 1, Tyr 1; containing 1mol of fatty acid having unknown structure per 1mol of the substance. USE:Antimycotic agent. PREPARATION:A microbial strain belonging to Bacillus genus and capable of producing KBS3-P1002 substance [e.g. Bacillus KB-S3 (FERM P-8986)] is cultured preferably under aerobic condition at 25-30 deg.C for 4 days.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a substance having antifungal activity, KBS3-P1002.
and its manufacturing method.

While searching for antibacterial low-molecular-weight substances produced by bacteria of the genus Bacillus, the present inventors discovered that a strain of the genus Bacillus produces an active antifungal substance, and further confirmed that the substance was a new substance. This was named substance KBS3-P1002, and the present invention was completed.

The bacteria used in the present invention may be any species as long as it produces the substance KBS3-P1002, and an example thereof is Bacillus KB-33.

Bacillus KB-S3 is FERM P-8986 to Microtech Institute
The mycological properties are summarized as follows.

Durham staining Decaspore staining Decascatalase test + Oxidase test + The culture medium for the substance KBS3-P1002 producing bacteria contains appropriate nutrients such as assimilable carbon sources, nitrogen sources, vitamins, etc. Any medium may be used as long as it produces and accumulates.

Examples of suitable media for culturing Bacillus KB-S3 include the following.

Polypeptone 30g Yeast extract 5g NaC 15g Deionized water 1111 (pH 7,0) Cultivation is carried out aerobically for about 4 days at 25-30°C with aeration, stirring, shaking, etc.

The obtained culture solution was filtered to obtain a filtrate, and 6N lI was added to this.
Add cI2 and adjust the pH to 3.0 to obtain a precipitate.

The precipitate was dissolved in water and NaHCO was added to pH=7.
0 and then add ethanol to 80%. The obtained synovial fluid was vacuum dried, and the dried product was dissolved in distilled water.

Add 6N H (Jl) again to adjust the pH to 3.0 to obtain a precipitate. Dissolve the obtained precipitate in distilled water and add NaH
Add COi to pH=7.0 and dialyze against distilled water.

The substance KBS3-P was obtained by freeze-drying the dialysate fluid.
A crude product containing 1002 is obtained as a yellow powder.

The obtained yellow powder is dissolved in water, subjected to high performance liquid chromatography using an acetonitrile system, isolated, and freeze-dried to obtain an almost pure substance KBS3-P1002 as a white amorphous powder.

The physical and chemical properties of the obtained substance B53-P4O10 are as follows.

1. Elemental analysis value C: 48.27% H: 7.47% N: 11.98% Ash: 1.69% 2. Ashing point 183-185℃ 3. Color and properties of substance: white powder 4. Ultraviolet absorption spectrum: As shown in Figure 1.

5. Infrared absorption spectrum: As shown in Figure 2.

6. Solubility in solvents: Soluble in water, methanol, ethanol, acetonitrile.

Insoluble in n-propyl alcohol, acetone, ethyl acetate, ether, chloroform, diethyl ether, petroleum ether.

7. Color reaction ninhydrin test - 8. Amino acid composition The amino acid composition determined by an amino acid analyzer is as follows.

9. There is one molecule of undetermined fatty acid molecule per molecule of substance.

10. This substance exhibits strong antibacterial properties against fungi.

11. Purification method: An almost pure product can be obtained by repeating the steps of precipitating at about pH 3.0, dissolving the precipitate at pH 7.0, and finally applying to high performance liquid chromatography. The absorbance curve of human 220 in high performance liquid chromatography is as shown in FIG. The arrow is the material KBS
3-P1002 is shown.

In addition, the column size for high performance liquid chromatography is φ1.0×20
as, the gel is Hitachi Gel 3053 (wrinkle strength type), the elution conditions are initial acetonitrile 40%, trifluoroacetic acid 0.
1% aqueous solvent, final (after 40 min) acetonitrile 6
A linear concentration gradient of 0% trifluoroacetic acid to 0.1% aqueous solvent was performed.

12. Antibacterial spectrum: The antibacterial activity of substance KBS3-P1002 is shown in Table 1 below.

Table 1 Next, examples of the present invention will be shown.

Example 1 Bacillus KB-53, FERN P-8986 was inoculated into the following medium and cultured with shaking at 27°C for 4 days.

Polypeptone 30g Yeast extract 5g NaCΩ 5g Deionized water 1Ω (PH7,0) The obtained culture solution was filtered to obtain a culture filtrate, which was diluted with 6N
Adjust ptt to 3.0 with HCQ and leave for a while to obtain a precipitate. Dissolve the obtained precipitate in distilled water, add NaHCO, and adjust the pH to 7.0.

Further add ethanol to 80% and filter. The obtained supernatant is vacuum dried to obtain a dried product. This dried product is dissolved in distilled water, adjusted to pH=3.0 with 6N HCfi, and left to stand for a while to obtain a precipitate. This precipitate is dissolved in distilled water, brought to ptt = 7.0 with NaHCO3, and the solution is dialyzed against distilled water.

The obtained dialysate was lyophilized and the substance KBS3-P100
A crude product containing 2 was obtained as a yellow powder.

The obtained yellow powder was subjected to high-performance liquid chromatography, and the corresponding part of the substance KBS3-PIO02 indicated by the arrow in Fig. 3 was taken.
By freeze-drying this, Habo pure substance KBS
3-PIO02 was obtained as a white amorphous powder.

[Brief explanation of the drawing]

Figure 1 is a diagram showing the ultraviolet absorption spectrum of the substance 853-P4O10, Figure 2 is a diagram showing the infrared absorption spectrum, and Figure 3 is a diagram showing the infrared absorption spectrum of the substance KBS3-P1002 as measured by high performance liquid chromatography. It is a figure showing the absorbance curve by. The arrow indicates the substance KBS3-P1002
shows.

Claims (2)

[Claims] (1) Substance KBS3-P1 with the following physical and chemical properties
002. 1. Elemental analysis value C: 48.27% H: 7.47% N: 11.98% Ash: 1.69% 2. Ashing point 183-185°C 3. Color and properties of substance: White powder 4 , UV absorption spectrum: as shown in FIG. 5. Infrared absorption spectrum: As shown in Figure 2. 6. Solubility in solvents: Soluble in water, methanol, ethanol, acetonitrile. Insoluble in n-propyl alcohol, acetone, ethyl acetate, ether, chloroform, diethyl ether, petroleum ether. 7. Color reaction ninhydrin test 8. Amino acid composition: The amino acid composition determined by an amino acid analyzer is as follows. 9. There is one molecule of undetermined fatty acid molecule per molecule of substance. (2) A method for producing the substance KBS3-P1002, which comprises culturing a substance KBS3-P1002-producing bacteria belonging to the genus Bacillus and collecting the substance KBS3-P1002.