JPS608116B2 - New antibiotic tridecaptin C and its production method - Google Patents

Description

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

Tridecaptin C (Tridecaptj) according to the invention
nC) is a peptide antibiotic and has activity primarily against Gram-negative bacteria. Next, the physicochemical properties of tridecaptin C will be shown.

However, the physical constants shown below are for tridecaptin C hydrochloride. (a) Pale yellow powder (b) Melting point 190-197o0 (decomposition) (c) Elemental analysis value C, 49.92, 50.52: 7.08, 7.15
;N, 13.63, 13.46: ○, 20.11, 20
．． 77; CI, 5.89, 5.84: optical rotation [Q] customer. 〇-3.0±1.4 (c0.299, acetic acid/
Water (1:・)) E ultraviolet absorption spectrum shame 9m (E supervisor) 217 (S) (210), 275 (
25), 281.5 (27), 289 (23). (1st
(see figure) to infrared absorbing svectorre general support skin - 13285, 3060, 1725, 1637
, 1528, 1230, 1167, 1075, 740 (
(See Figure 2).

Amino acid analysis value (added 4% thioglycolic acid, hydrochloric acid,
20-hour hydrolysis; 40-hour hydrolysis #mole/female) serine (
1.41; 1.29), glutamic acid (0.58:0.
59), glycine (0.60; 0.62), valine (1
．． 25; 1.35), alloisoleucine (0.09; 0
．． 12), phenylalanine (0.50; 0.47),
Tryptophan (0.60; 0.61), 2,4-diaminobutyric acid (1.61:1.61), ammonia (0.3
4; 0.44).

Constituent fatty acid 8-hydroxyantisoundecanoic acid,
8-hydroxyisodecanoic acid, 8-hydroxydecanoic acid.

1' molecular weight 1650 (calculated from amino acid analysis values).

This compound was previously developed by the inventors from Bacillus polymyxa A.
R-110 (Bacill ship polym〆aAR-11
Antibiotic AR-1 found to be produced by
10 (Patent Publication No. 51-144796) and Bacillus Polymyxa B-2 (Bacm Peng Polymy Net B-2).
) is a peptide antibiotic related to the antibiotic tridecaptin B (Patent Publication No. 144502/1989), which was discovered to be produced by They differ in some of their components and in their constituent fatty acids.

Table 1 Comparison of constituent amino acids *valine, alloisoleusone,
of isoleucine residues.

Table 2 Comparison of constituent fatty acids Comparison of three antibiotics Thin layer chromatography (abbreviated as TLC in Table 3, using silica gel GF)
and paper chromatography**- (in Table 3, PC
Abbreviated as Toyoro Paper No. 51) are shown below.

Table 3 Comparison of Rf values CEN = Chloroform/ethanol/14 polyammonium water (4:7:2) CEA = Chloroform/ethanol/10 polyacetic acid (4:7:2) AHAN = Acetone water/
Acetic acid/2N anthenia water (15:5:1:2) BAW=
Butanol/acetic acid/water (4:1:2) BPAW=butanol/pyridine/acetic acid/water (10:6:1:4) Tridecaptin C obtained according to the present invention has activity against Gram-negative bacteria.

The antibacterial spectrum of tridecaptin C (hydrochloride) is shown below. *The results of a therapeutic trial of tridecaptin C supplemented with 10% horse serum are as follows.

1 Test method Test bacteria were administered subcutaneously to JCL-ICR female mice, and tri*decaptin C was administered twice 1 hour and 5 hours later.
Administer hydrochloride subcutaneously.

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

As shown above, tridecaptin C has activity against Gram-negative bacteria and exhibits a therapeutic effect against Klebsiella pneumoniae and Escherichia pneumoniae.

Its toxicity was determined in mice by a single intraperitoneal administration.
Its value is 150 fox/k9, and it can be used as a medicine, veterinary drug, disinfectant, etc. When administering tridecaptin C 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 tridecaptin C is administered to humans, it is usually administered orally or by injection in an amount of about 18 days to about 4 days to adults. Various acid addition salts of tridecaptin C, such as hydrochloride, sulfate, oxalate, and succinate, can be used similarly to tridecaptin C as a medicine, veterinary drug, and disinfectant. As the tridecaptin C-producing bacteria, a strain belonging to the genus Bacillus is used.

For example, Bacillus polymyxa E-23 (Bacillus polymyxa E-23)
spol mpine aE-23) has good tridecaptin C
produce. The strain has the following mycological properties. 1. Morphological properties (GIy-IM toji*1, 3000, cultured for 1-2 days) [11. Shape and arrangement: Antroccus is round, has bacterial ends, and exists singly or in clusters.

■Mobility (3) Size 0.7-0.9 x 2.0-5.5 Buddha.

■Irregular bacterial shapes were not observed. {5) Sporangia have a distinct bulge.

{6) Spore size: 1.0 to 1.2 x 1.5 to 20 oval, located in the center or near the end).

'71 Gram stain positive {81 liquid cannon not observed *IGIy-IM Toji glycerin 0.5%, beptone 0
．． 25%, beef extract 0.25%, yeast extract 0.25
%, salt 0.3%, agar 1.0-1.2% (pH 6.8
).

2 Culture properties {1' Agar colony (GIy-IM tower, 30°C, cultured for 1 to 3 days) Shape: Circular surface, smooth throughout, but becomes wavy with time.

Raised, convex, circular (transparent) Semi-transparent at the initial stage of culture, becoming opaque as culture time progresses.

The composition is slightly gummy and adheres to the agar medium.

'2) Agar slope (GIy-IM Toji, 30qo, 1-3
(day culture) Growth Grows moderately.

The shape of growth is citron-like or wart-like.

No intracellular pigment production No extracellular pigment production It has a glossy surface.

It becomes wrinkled after 7 days of culture.

Composition Slightly gummy and does not adhere to agar medium. Ru.

Transparency: At the beginning of culture, it is translucent, but as the culture time passes, it becomes opaque and becomes waxy.

'Milk liquid medium (NAM medium *2, 30q0, cultured for 1 to 3 days) Growth uniformly and moderately, producing powdery precipitate.

'41 gelatin high-rise culture (gelatin tower containing yeast extract, 30 qo, cultured for 1-20 days) Liquefaction Liquefaction.

'51 litmus milk (370, cultured for 1-7 days) produces litmus reaction acid.

With coagulation *2 NAM medium bepton 0.5%, beef extract 0.3%
, yeast extract 0.2%, potassium nitrate 0.2% (pH 6
．． 8).

3 Physiological properties '1 - Oxygen requirement (S beach area *3, 30℃, 1-5 days culture) Suitability anaerobic (agar puncture method) ■ Optimum growth temperature (MV land *4) 32-3 Optimal around C, 2yo, 2800, 3〆○
and 370 and grows well.

Does not grow at 11°C and 45qo.

{3}○-F test (S tower area) Produces acid and gas in suitable anaerobic form.

t4 - Reducing enteric nature of nitrates (producing nitrogen dioxide).

■Forges-Proscawell reaction enteric [6] Hydrogen sulfide production (Beptone/iron/agar medium, manufactured by Difuco Laboratories) Negative '7] Starch hydrolysis enteric [8] Utilization of carbon source (medium 1* 5, 30qo, 197
day culture) Acid is produced from L-arabinose, D-xylose, D-mannitol, and D-glucose.

*3S medium glucose 1%, beptone 0.5%, beef extract 0.3%, yeast extract 0.2%, salt 0.3%, fromcresol purple 0.008%, agar 0.4% (
pH6.8).

*4MV medium soluble starch 2.0%, glycerin 0.
5%, soytone 1.5%, corn steep liquor 0.5%, sodium chloride 0.3%, agar 1.5% (p
H6.8). *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%, agar 1.5%.

Bacterial strains with the above boxlike characteristics are recognized to belong to Bacillus polymyxa (Bacill female poMmWa) [R. E. Buchanan & N. E. Gibbons
: Bergey's Manual of Detennin
active European plow, hed. (197
4, meWilliams & Wilkin Shark Compa
ny), B. M. Gib & D. AShapton;l
denti8cationMehioshipforMicro
biologists (1968, Aeademicp.
ress), A. 1. LaSkin&Japan・ALeChe
valier; day is ndbo.

k. fMicrobiology Vol. 1 (1972
, CRCpress) and other publications]. Therefore, this strain was used as Bacillus vorimixa R-2 (Bacillus
It was named female polymy Shigeru 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. 3974. In the present invention, the above-mentioned bacterial strains and their natural or artificial mutant strains can of course be used, and all tridecaptin C-producing bacteria belonging to the genus Bacillus can be used. The method for producing tridecaptin C will be described below.

Tridecaptin C is produced by culturing ToIJ decaptin C producing bacteria in a medium containing various nutrients under aerobic conditions.

Culture conditions and medium composition may be selected according 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, precursors, etc. may be added as necessary. As the carbon source, for example, glucose, arapinose, xylose, starch, dextrin, glycerin, mannitol, organic acid, bran syrup, potato, etc. are used alone or in a mixture, and as the nitrogen source, for example, beptone, soybean flour, Corn steep liquor, malt extract, amino sugar, rice sugar, wheat hulls, urea, ammonium salts, etc. or mixtures thereof are used. The medium is preferably a liquid medium;
For mass production, aerated deep culture is preferable.

The solution of the medium is preferably about 5.5 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 C can be separated and collected from the culture in accordance with a conventional method for separating and collecting fermentation products from the culture.

That is, tridecaptin C is collected by appropriately combining extraction methods using various organic solvents, chromatography using various active adsorbents, and the like. The collected tridecaptiso C can be further converted into a desired acid addition salt, if necessary. Conversion to salt is carried out according to conventional methods. Next, a production example of tridecaptin C, the object compound of the present invention, will be shown, but this example is not intended to limit the scope of the present invention in any way. Example T medium glucose 1.0%, veptone 0.5%, meat extract 0.5%, sodium chloride 0.1%, potassium dihydrogen phosphate 0.05%, magnesium sulfate 0.05%, manganese sulfate 0. 001%, ferric sulfate 0.001% (pH 7
．． 0).

Bacillus polymixa E-23 was added to the 'T medium 120 in the customer Sakaguchi flask' in page 15, lines 9 to 12 of the S column.
(Bacillus spoIMm Matsu aE-2 Iriko Koken Bacterial Serial No. 3974) was plated and cultured at 2800 for 1 day.

This culture solution 3 skin was used as a seed fungus to be planted in 500 pieces of S medium 120 pieces in a Sakaguchi flask, and cultured in a shaking moat at 28°C for 2 days. Obtained culture solution 5.5 butanol/methanol
After adding 1:1 solution for 5 days, the pH was adjusted to 2, and the mixture was filtered.

After the furnace solution was brought to pH 4.0, it was concentrated, adjusted to pH 2.0, and extracted with butanol. The extract was washed with dilute sodium bicarbonate solution and water, and then concentrated to a concentration of about 500 ml. Add 250 ml of ethyl acetate to this concentrated solution, then adjust the pH to 2.0.
Extract with water. When the extract is freeze-dried, crude substance 1
．． Get 41 evenings. This product was analyzed by paper chromatography [Toyoro Paper M. 527, developed with butanol/pyridinenoacetic acid/water (10:6:1:4)], the antibiotic-containing part (detected by ninhydrin reaction and bioautography) was extracted with 50% methanol water (pH 2.0). do. The extract is freeze-dried to obtain 318 mg of crude powder.
This was applied to a Sephadex LH-20 column (2.5 x 9
0 skin) and developed with 20% methanol. When the fraction with antibacterial activity was concentrated to dryness, tridecaptin C hydrochloride 14
9 of 0 is obtained.

[Brief explanation of the drawing]

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

Claims (1)

[Scope of Claims] 1. Antibiotic tridecaptin C, the hydrochloride of which has the following properties. A Pale yellow powder B Melting point 190-197°C (decomposition) C Elemental analysis value C, 49.92, 50.52; H, 7.08, 7.15
;N,13.63,13.46;O,20.11,20
．． 77; Cl, 5.89, 5.84; D Optical rotation [α] ^2^3^0_D_. -3.0±1.4 (c0.
299, Acetic acid/water (1:1)) E Ultraviolet absorption spectrum λ^H^O_m_a_xmμ(E^1^%_1_c_m
) 217 (S) (210), 275 (25), 281.
5(27), 289(23). (See Figure 1) Infrared absorption spectrum ν^K^B^r_m_a_xcm^-^13285,3
060,1725,1637,1528,1230,1
167,1075,740 (see Figure 2). Amino acid analysis values (4% thioglycolic acid added, hydrochloric acid, 20-hour hydrolysis; 40-hour hydrolysis μmole/mg) serine (1.41; 1.29), glutamic acid (0.58;
0.59), glycine (0.60; 0.62), valine (1.25; 1.35), alloisoleucine (0.09)
;0.12), phenylalanine (0.50;0.47
), tryptophan (0.60; 0.61), 2,4-
Diaminobutyric acid (1.61; 1.61), ammonia (0
．． 34; 0.44). H Constituent fatty acids β-hydroxyanteisoundecanoic acid, β-hydroxyisodecanoic acid, β-hydroxydecanoic acid. Molecular weight: 1650 (calculated from amino acid analysis values). 2. A method for producing the novel antibiotic tridecaptin C, which comprises culturing tridecaptin C-producing bacteria belonging to the genus Bacillus in a medium and collecting tridecaptin C from the resulting culture.