JPS603475B2 - New antibiotic tridecaptin B and its production method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antibiotic tridecaptin B and a method for producing the same.

Tridecaptin B according to the present invention is a peptide antibiotic and has activity primarily against Gram-negative bacteria.

Next, the physicochemical properties of tridecaptin B are shown. However, the physical constants shown below are for tridecaptin B hydrochloride. A Colorless powder B Gradually decomposes from melting point 235℃.

C Elemental analysis value C, 49-70; Sun, 7-19; N, 14-65; C So, 4.87.

D Optical rotation [Q] color 3.5-21.2 ± 2.70 (co. female 60
．． Hydrochloric acid) E Ultraviolet absorption spectrum ^ band 29mr (B king porcelain) 220 (s), 2 bulbs (s), 2
65(s), 275(260), 281(260), 2
89(19) (see Figure 1).

Infrared absorption spectrum -13 total of three branches 0, 3280, 3060, 2960
, 2 chicks 0, 1 mouse 5, 1 mouse 0, 1460, 1400, 12
35, 1165, 107ul040 (see Figure 2).

Amino acid analysis value (addition of 4% thioglycolic acid, constant point hydrochloric acid, 110°C, 2 feeding hours hydrolysis, 4q hour hydrolysis mountain mo
le/female) serine (1.4 f1. spots), glutamic acid (
0.53, 0.55), glycine (0.891.02)
, valine (0.63 0.73), alloisoleucine (
0.30, 0.36), isoleucine (0.39, 0.
42), tryptophan (0.42, 0.39), 2,
4-diaminobutyric acid (1.59, 1.59), ammonia (0.27, 0.40).

H Constituent fatty acid anteisononanoic acid molecular weight It is estimated to be about 1800 based on amino acid analysis values.

This compound is: Bacillus polymixer AR-110 (mother cmuspolymy Pine AR), which the present inventors discovered earlier.
-110), an antibiotic produced by Bacillus polymyxa E
-23 (mother cillus spore m. a. E-23) produces antibiotic tridecaptin C
47002), but differs from these antibiotics in some of the constituent amino acids and fatty acids as shown in Tables 1 and 2. Table 1 Comparison of constituent amino acids* The sum of the number of palin, alloisoleucine, and isoleucine residues is 3.

- None Table 2 Comparison of constituent fatty acids In order to compare the three substances, thin layer chromatography (abbreviated as TLC in Table 3, using silica gel GF) and vapor chromatography (abbreviated as PC in Table 3, Toyoro Paper M.
51) are shown below.

Table 3 Comparison of Rf values* OEN dichloroform/ethanol/14-polyammonium water (4:7:2) OBA = chloroform/cotanol/10% acetic acid (4:7:2) AHAN = acetone/water/acetic acid/2N ammonia Water (15:5:1:2) BAW = Butanol/acetic acid/water (4:1:2) BPAW
=butanol/pyridine/acetic acid/water (10:6:1:4
) Tridecaptin 8 obtained according to the present invention has activity against Gram-negative bacteria.

The antibacterial spectrum of tridecaptin B (hydrochloride) is shown below. The results of the tridecaptin B therapeutic trial are as follows. 1. Test method The bacteria to be saponified are subcutaneously administered to JCL-ICR female mice, and tridecaptin B hydrochloride is subcutaneously administered 1 and 5 hours later.

2 Results The results are as shown in the table below, and the numbers indicate the number of surviving mice/number of test mice after 7 days.

As mentioned above, tridecaptin B exhibits activity against Gram-negative bacteria and has a therapeutic effect against Klebsiella pneumoniae.

The plexus pattern of L was determined by a single intraperitoneal administration in mice. The value is 7
5 to 10 o/k9, and can be used as medicine, veterinary medicine, sterilizing agent, etc. When administering tridecabutin B as a medicine or veterinary drug, it can be administered orally as a tablet, capsule, powder, etc., or parenterally as an injection, liniment, suppository, etc.

When administering tridecaptin B to an adult, it is usually administered orally or by injection for about 4 days from one female per day. Various acid addition salts produced from tridecabutin B, such as hydrochloride, sulfate, oxalate, and succinate, can be used similarly to tridecabutin B as medicine, veterinary medicine, and disinfectant. Tridecaptin B
A strain belonging to the genus Bacillus is used as the producing bacterium.

For example, Bacillus polymyxa B-2
ly-m pine aB-2). The strain has the following mycological properties. 1 Morphological properties (GIy-IM tower *13, cultured for 1 to 2 days) '11 Shape and arrangement Amphoteric is round, has bacterial ends, and exists singly or in clusters.

'21 Mobility Yes '3' Size 0.7-0.9 x 2.0-4.0 mounds (main body is 0.7 x 3-4 m).

■ Irregular bacterial shape Not observed.

'51 Sporangium Has a distinct bulge.

'6' spore size 1.0-1.2 x 1.5-2.0
oval shape. [71 Gram staining positive.

'8' No Nada vertical land.

*I GIy-1M Toji Glycerin 0.5%, Beptone 0.25%, Beef Extract 0.25%, Yeast Extract 0.
25%, salt 0.3%, and agar 1.0-1.2% (pH 6.8).

2 Culture properties'1) Agar colony (GIy-IM Tochi, 3bi○, 1~
3-day culture) Shape: Circular surface, smooth edges, ridges on all twills, convex circular transparency Slightly translucent at the beginning of culture, becoming opaque as culture time passes.

Composition Slightly gummy and adheres to agar medium.

'21 Agar slant (GIy-skin medium, 1-3 days culture)
Growth Moderately growing.

Growth shape: zuzu-like or wart-like.

Production of intracellular pigments None Production of extracellular pigments None It has a glossy surface.

When cultured for 7 days, it becomes wrinkled.

Composition Slightly gummy and adheres to agar medium.

Transparency Slightly translucent at the initial stage of culture, becoming opaque and waxy as time passes.

'3l liquid medium (NAM Nagisaji*2, 30℃, 1-3
(daily culture) Growth Grows uniformly and moderately, producing a powdery precipitate.

■ Gelatin high-rise culture (gelatin tower containing yeast extract, 30 pm, culture for 1 to 20 days) Liquefaction Liquefy.

'5' Litmus milk (370, cultured for 1-7 days) Litmus reaction Produces acid.

Coagulation Yes *2 NAM Toji Beptone 0.5%, beef extract 0.
3%, yeast extract 0.2%, and potassium nitrate 0.2% (pH 6.8).

3 Physiological properties '1' Oxygen requirement (S medium *3, 3000, 1-5 day culture) Suitability anaerobic (agar puncture method).

■ Optimum growth temperature (MV Tower *4) Optimum is around 32 to 370, 25oo, 28℃, 320
Growth is good for 0 and 3.

1100, no growth at 45°C.

'3} ○-F test (S tower area) Facultatively anaerobic, producing acids and gases.

'41 Reducing properties of nitrate Enteric (produces nitrogen dioxide).

'51 Fouges-Proskawell reaction dominant '61
Hydrogen sulfide production (Beptone, iron, agar tower, manufactured by Difco Laboratories) Negative {7' Starch hydrolysis Positive '81 Carbon source availability (medium 1 * 5, 3 B○, 1-2
day culture) Acid is produced from L-arabinose, D-xylose, D-mannitol, and D-glucose.

*3S Tochi Glucose 1%, Beptone 0.5%, Beef extract 0.3%, Yeast extract 0.2%, Salt 0.3%,
From cresol purple 0.008%, agar 0.4%
(pH 6.8).

*4MV medium 2.0% soluble starch, 0 glycerin
．． 5%, soytone 1.5%, corn steep liquor 0.5%, sodium chloride 0.3%, and agar 1.5% (pH 6.8). *5 Medium 1 Ammonium hydrogen phosphate 0.1%, potassium chloride 0.02%, magnesium sulfate 0.02%, yeast extract 0.02%, glucose 0
．． 5%, bromcresol purple 0.008%, and agar 1.5%. The strain with the above mycological properties is Bacillus polymyxa (Female Bacillus poMmatsua).
[R. E. BMha Asahi n & N
．． E. Gibbo ship; Iber crab's Ma
nnal of DetennativeIBac
teriology, breast hed. (1974; TheW
miams & Wilkin Shark Company), B.
M. Gibhiro & D. ”A. Shapton; l
dentification Methods
ormierobiologists (1968, Ac
academicpress), AI. Mountain askin &
Day. ALeChevalier; HDndb.

〇k. fMicrobio! ogy, vol. 1 (1
972. CRCpress) and other publications].
Therefore, this strain was used as Bacillus polymyxa B-2 (Bacillus polymyxa B-2).
The vessel was named ill ship Polymatsua B-2) and has been deposited with the National Institute of Microbial Technology, Agency of Industrial Science and Technology as National Institute of Industrial Science and Technology Deposit No. 3973. In the present invention, the above-mentioned Bacillus polymyxa B-2 and its natural or artificial mutant strains can of course be used, and all tridecaptin B-producing bacteria belonging to the genus Bacillus can be used. Next, the method for producing tridecaptin B according to the present invention will be described.

Tridecaptin B-producing bacteria are cultured under aerobic conditions in a medium containing various nutrients.

The culture conditions and medium composition may be similar to those used in the production of general antibiotics. That is, the medium basically contains a carbon source, a nitrogen source, and an inorganic salt, and vitamins and precursors may be added as necessary. As a carbon source, for example, glucose, arabinose, xylose, starch, dextrin, glycerin, mannitol, organic acid, molasses, potato, etc. are used alone or in a mixture, and as a nitrogen source, for example, beptone, soybean meal, corn steep. - Liquor, malt extract, amino sugar, rice sugar, barley skin, urea, ammonium salt, etc. or a mixture thereof are used. The medium is preferably a liquid medium, and in the case of mass production, aerated deep culture is preferable.

The pH of the medium is preferably about 55 to about 8.5, and the culture temperature is preferably adjusted to about 20 to about 40°C, particularly 32 to 370°C. If necessary, an antifoaming agent may be added as appropriate before or during culturing. After completion of the culture, tridecaptin B can be collected from the culture according to a conventional method for separating and collecting fermentation products from the culture.

For example, tridecaptin B is collected by appropriately combining extraction methods using various organic solvents, chromatography using various active adsorbents, and the like. Furthermore, if necessary, tridecaptin B can be converted into a salt as described above. Salt production follows conventional methods. Next, an example of the production of tridecaptin B, the object compound of the present invention, will be shown, but this example is not intended to limit the present invention in any way. Example T medium Glucose 1.0%, Beptone 0.5%, Meat extract 0.5%, Sodium chloride 0.1%, Potassium dihydrogen phosphate 0.05%, Magnesium sulfate 0.05%, Manganese sulfate 0. 001% and 0.001% ferric sulfate (PH7.0).

C tower base glucose 1.0%, soybean flour 4.0%, ammonium sulfate 2.0%, potassium dihydrogen sulfate 0.2%, magnesium sulfate 0.05%, calcium carbonate 1.0%,
Consists of 0.01% sodium chloride (pH 7.0).

Bacillus polymixa B-12 (portion ci
ll PoIMmyxaB-2, Chokoken Bacterial Serial Number 3973
No.) and cultured in a shaking plate for 1 day at 28oo.

Using this culture solution 3 as a seed, incubate it in a tank of 120 liters of liquid C having the above composition in 500 individual Sakaguchi flasks,
Culture in honey at 28°C for 3 days. The resulting culture solution was adjusted to pH 5 with hydrochloric acid and mixed with butanolnomethanol (1:
Add 5 portions of the mixture of 1) and filter.

After concentrating the furnace liquid, it is extracted three times with putanol. 2% extract
After sequentially washing with sodium hydrogen carbonate solution, water, dilute hydrochloric acid (pH 2), and water, concentrate and dry. Approximately 20 minutes of the residue was extracted three times with water-saturated butanol (40 points), and after adding the same amount of ethyl acetate, it was extracted three times with dilute hydrochloric acid (pH 3) (30 points). The extract is freeze-dried to obtain crude product 110c. The crude product is extracted with a small amount of a mixture of chloroform/ethanol/14% ammonium water (4:7:2).

The extract was concentrated, and the resulting residue 300:9 was subjected to paper chromatography [Toyoro Paper Yuzu. 525 butanopyridine/acetic acid/water (10:6:1:4)] and extracted with 80% acetone adjusted to pH 2 with hydrochloric acid.
Neutralize the extract with Amberlite (OH type) to pH 5.
0 and lyophilize. Dissolve the product in methanol,
Tridecaptin B hydrochloride 123 when precipitated with acetone
o is obtained as a colorless powder.

[Brief explanation of drawings]

Figure 1 shows an aqueous solution of tridecaptin B hydrochloride and part 3.
Figure 2 shows the infrared absorption spectrum of tridecaptin B hydrochloride in potassium bromide tablets. Figure 1 Figure 2

Claims (1)

[Scope of Claims] 1. Antibiotic tridecaptin B, the hydrochloride of which has the following properties. A Colorless powder B Gradually decomposes from melting point 235°C C Elemental analysis value C, 49.70; H, 7.19; N, 14.65; Cl
, 4.87. D Optical rotation [α] ^2^3^5_D_. -21.2±2.7°(c
0.226 0.5N hydrochloric acid) E Ultraviolet absorption spectrum λ^H^O_m_a_xmμ(E^1^%_1_c_m
) 220(s), 258(s), 265(s), 273
(26.0), 281 (26.0), 289 (19) (
(See Figure 1). F Infrared absorption spectrum ν^K^B^r_m_a_xcm^-^13380, 3
280, 3060, 2960, 2920, 1645, 1
540, 1460, 1400, 1235, 1165, 1
070, 1040 (see Figure 2). Amino acid analysis value (addition of 4% thioglycolic acid, constant boiling point hydrochloric acid, 110°C, 20 hours hydrolysis, 40 hours hydrolysis μmo
le/mg) serine (1.45, 1.38), glutamic acid (0.53, 0.55), glycine (0.89, 1
．． 02), valine (0.63, 0.73), alloisoleucine (0.30, 0.36), isoleucine (0.3
9, 0.42), tryptophan (0.42, (0.3
9), 2,4-diaminobutyric acid (1.59, 1.59),
Ammonia (0.27, 0.70). H Constituent fatty acid anteisononanoic acid Molecular weight approximately 1800 (estimated from amino acid analysis values). 2. A method for producing the novel antibiotic tridecaptin B, which comprises culturing tridecaptin B-producing bacteria belonging to the genus Bacillus in a medium and collecting tridecaptin B from the resulting culture.