JPS5820597B2 - Manufacturing method of rifamycin S - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the production of rifamycin S, which comprises culturing Nocardia bacteria capable of producing rifamycin S, and collecting rifamycin S from the culture. Regarding the law.

Conventionally, rifamycin S, the raw material for the anti-tuberculosis drug rifampicin, is produced by culturing Streptomyces mediterranei, or by chemically oxidizing famycin B to rifamycin O, and then hydrolyzing it. [Il Farmaco Nido Si (I 1
, Farmaco, Ed.

5ci) 16.165 (1961)], or by culturing a mutant strain of Streptomyces mediterranei, or by chemical oxidation of famycin S.

In addition, regarding the fermentation production of rifamycin S, Micromonospora thiarcia (Micyoikonospra
chalcea) (U.S. Patent No. 3,884,763) and Micromonospora roughstris new species Routhien (U.S. Patent No. 3,884,763)
ris sp, nov, Routien) (Unexamined Japanese Patent Publication No. 5
It has been reported that it exists as a trace component in the ansamycin mixture component due to the following.

The present inventors found a newly isolated strain of bacteria belonging to the genus Nocardia that produces a significant amount of the antibiotic rifamycin S in the culture medium, and completed this research.

The strain most advantageously used in the present invention is designated Nocardia-468, and was deposited with the Microbial Technology Research Institute on February 7, 1971 (Feikoken Bacterial Deposit No. 4391).
.

The mycological properties and findings on the culture medium of Nocardia ni-468 are as follows.

Cultivation properties are 1 at 28°C unless otherwise specified.
The cells were cultured for 4 days and observed.

1 Morphological properties As shown in Table 1, Nocardia -468 shows growth that is pale yellow to yellowish brown or dark brown to reddish brown, forms aerial mycelia in most media, and is white to yellowish orange. exhibits.

The branches of the vegetative hyphae are branched, the shape of the sporophyte is bent, and the shape of the spore is oval to oblong, and its size is 0.4
~0.6X0.7~1.0μ, the surface is smooth.

It is not stained by the Zweel-Nielsen acid-fast staining method.

2. Findings on Medium The properties of Nocardia -468 were observed using various media commonly used for bacterial strain identification, and the results are shown in Table 1 below.

3 Availability of carbon source Adding 1 carbon source to Bridham and Gottlieb basal medium
Table 2 shows the results of examining the usability of the microorganisms, which were added at a concentration of 1.5% and cultured at 28° C. for 2 weeks after inoculation.

4 Physiological Properties The physiological properties of Nocardia-468 are shown in Table 3 below.

5 Cell wall composition Dry cells of Nocardia -468 were hydrolyzed with 6N hydrochloric acid and prepared using the method of Becker et al. [Appl-Microbial]
12.421 (1964)) and examined the constituent amino acids.
also gave monodiaminopimelic acid.

In addition, when the same bacterial cells were hydrolyzed with 2N sulfuric acid and paper chromatography was performed using a developing solvent of n-butanol-acetic acid-water (4:1:5), galactose and arabinose were observed as constituent sugars. .

Regarding the above test results, please refer to the Bersey's Manual of Determinative Bacteriology (8th edition,
The Williams & Wilkins Company, 1974) [Bergy's manual
f Determinative Micro-obi
ology (8th Ed, The William
Sand Wilkins Co., 1974), it is clear that this strain belongs to Nocardia, which has cell wall type II.

-468 forms well-developed hyphae on Nocardia,
It does not fragment within 5 days, belongs to the morphological type ■, and is included in the 1-AA group because it is non-acid-fast, but this bacterium does not substantially grow on inorganic media that do not contain carbon sources. As a heterotrophic microorganism, among the known Nocardia, Nocardia croissii (
It seems to be a strain closely related to Noca-rdia kuroishii).

However, Nocardia croissii is partially acid-fast (
Partially acid fast),
-468 forms aerial mycelia on potato sections, whereas -468 on Nocardia is non-acid fast.
Ast) and does not form aerial mycelia in potato section culture medium, and Nocardia kuroishii does not liquefy in gelatin culture medium, grows yellowish brown, produces a yellowish soluble pigment, and also produces yellowish soluble pigment. In contrast, Nocardia-468 clearly liquefied the gelatin medium, albeit incompletely, showing pale yellow growth and producing a brownish soluble pigment. They differ in many respects, such as the fact that they do not form, are positive for clotting ability even in lidmus milk, and do not produce soluble pigments.

Furthermore, Micromonospora spp., such as Micromonospora cialsia and Micromonospora roughstris new species Routhien, which have been reported as Norifamycin S-producing bacteria, are different from Micromonospora spp., which do not form aerial hyphae and have specific spore attachment. It is clearly different from Nocardia-468 in that it exhibits a chemical composition and that it has xylose in its cell wall composition.

Also, Streptomyces mediterranei, which is well known as a rifamycin-producing bacterium (this strain was later renamed to the genus Nocardia).

[Ar-ch, Mik
Robiol) 67, 147 (1969) Streptomyces mediterranee does not use raffinose or starch as a carbon source, does not coagulate or peptonize lidmus milk, and does not use peptone-
It differs from Nocardia-468 in that it does not produce hydrogen sulfide on yeast extract iron agar, and it also has growth characteristics on typical media such as Nutriendo agar, Bennett agar, and yeast extract. - Glucose agar, Tresner-Coleniaco agar, Emmerthun glucose agar, Siabeck-sucrose agar, potato agar, skim milk agar, etc. are completely different.

Based on the above facts, no strain matching this strain was found, and the strain was designated Nokanogeia -468.

It is well known that the properties of microorganisms are not fixed and easily mutate naturally or artificially, and Nocardia-468 is no exception.

Therefore, it goes without saying that even the -468 variant or mutant strain of Nocardia can be used in the method of the present invention as long as it does not lose the ability to produce the antibiotic rifamycin S.

In order to culture 1468 bacteria in Nocardia and produce rifamycin S, a known method for culturing actinomycetes or a modified method thereof is used, but as an industrial culture method,
It is preferable to employ a shaking culture method or a deep aeration agitation culture method in a liquid medium.

In addition, the culture medium components include carbon sources, nitrogen sources, and inorganic salts that can be assimilated by the cultured bacteria, as well as fine amounts as necessary.

Those containing appropriate amounts of nutritional substances, etc. are used.

For example, carbon sources include sugars such as glucose, sucrose, starch, and glycerin, as well as hydrocarbons, ethanol, and other nitrogen sources. ammonium, organic acid ammonium salts such as ammonium tartrate;
Ammonium sulfate, ammonium chloride, urea and other inorganic or organic nitrogen compounds can be used.

Further, as inorganic salts, metal salts such as phosphates, common salt, calcium carbonate, zinc sulfate, and manganese chloride, and as micronutrients, vitamins such as bioxy, vitamin B1□, and others may be added as necessary.

Alternatively, glyoxylate or the like may be added to zero.

Culture conditions such as culture temperature, culture time, and pH during culture are as follows:
It should be selected or adjusted appropriately to obtain the best yield of rifamycin S, but in the case of liquid shaking or aeration agitation culture, the culture temperature is 520 to 40°C, and the culture period is usually 2 to 10 days. .

The appropriate state at the start of culture and during culture is 5.0 to 9.0.

Rifamycin S is produced and accumulated in the culture thus obtained.

Since this antibiotic is also contained in the supercellular part of the culture, the desired product can be extracted and recovered directly from the culture or from the sap and cells.

Extraction from the culture or its r-liquid is preferably carried out after adjusting the pH to 6 to 8, followed by extraction with an organic solvent that is not completely miscible with water, such as ethyl acetate, benzene, toluene, or chloroform.

In addition, the antibacterial substances in the bacterial cells are extracted with ethanol, acetone, etc., concentrated under reduced pressure, extracted with an organic solvent, and combined with the culture liquid extract.

The extract is washed with pH 7 OIJ phosphate buffer and concentrated to yield orange-yellow crystals.

By recrystallizing and purifying this crystal with ethanol, pure crystals of famycin S can be obtained.

Additionally, a column can be used as necessary during the purification operation.

The chemical properties and antibacterial spectrum of rifamycin S obtained according to the present invention were consistent with known compounds obtained from rifamycin B by chemical manipulation.

Melting point: 180° C. Specific light absorption [α]20=+480 (C=1 methanol; ultraviolet and visible absorption spectra are as shown in FIG. 1).

The infrared absorption spectrum is as shown in FIG.

The minimum inhibitory concentrations against various microorganisms are shown in Table 4.

Next, examples will be described.

Example 1 Nocardia -468 was grown on Bennett agar at 28°C for 6
Cultivate for ~8 days and add 100 m to a 500 ml Erlenmeyer flask.
Inoculate one platinum loopful into the seed medium injected with A.

The culture solution obtained by culturing at 28°C for 48 hours is used as the seed mother liquor.

The seed medium used had the following composition.

Glycerin 2% Defatted soybean flour 0.5% Peptone 0.5% Salt 0.2 coefficient Adjust the pH to 7.0 with caustic soda, pour 100 ml into a 500 ml Erlenmeyer flask, and use after pressure sterilization.

On the other hand, a 500m7 Erlenmeyer flask containing 50mt of a medium (adjusted to pH 7.0 before sterilization) consisting of 3% glycerin, 1.5% defatted soybean flour, and 0.2% salt was planted in a 50m7 Erlenmeyer flask with a 5% volume ratio of seed mother liquor. The cells were cultured for 150 to 190 hours at 200 rpm in a rotary shaker with a stroke of 5 CWl.

Rifamycin S titer was 60 μg/mt.

Example 2 A microorganism called Nocardia-468 is grown under the same conditions as shown in Example 1 to obtain a seed mother liquor.

The fermentation medium was a medium consisting of glycerin, 6-row defatted soybean flour, 3 steamed rice, salt 0.2%, sodium diethyl barbylate 0.1%, sodium glyoxylate 0.1% (adjusted to pH 7.0 with caustic soda before sterilization). Pour 50 ml into a 500 ml Erlenmeyer flask, sterilize under pressure, and add 50 ml of seed mother liquor.
% and cultured in the same manner as in Example 1 for 160 to 200 hours.

The highest titer of rifamycin S was 220 μg/mt.

Example 3 Nocardia -468 was grown under the same conditions as in Example 1 to obtain a seed mother liquor.

The fermentation medium consisted of 6% glycerin, 3% defatted soybean flour, 0.2% salt, 0.1% sodium glyoxylate, 0.2% n-amyl alcohol, 0.1% sodium propionate, and 0.5 ppm of vitamin B1□. After injecting 50 mt of (pre-sterilization pH 7.0) into a 500 ml Erlenmeyer flask, the seeds were sterilized under pressure, and 5% seed mother liquor was planted, followed by culturing for 160 to 200 hours in the same manner as in Example 1.

Rifamycin S titer was 240 μg/mt.

Example 4 Nocardia -468 was grown under the same conditions as in Example 1 to obtain a seed mother liquor.

Eight bottles of this seed mother liquor were combined and inoculated into 30-ton stainless steel jar fermenters each containing 15 tons of the fermentation medium shown in Example 2.

Cultivation is carried out at 28° C. for 6 days using an aeration agitation method (rotation speed: 300 rpm, aeration volume: 1 v, v, m).

After the culture is completed, the culture solution is filtered once, and the r solution is adjusted to pH 7.0 and extracted with an equal amount of toluene.

In addition, the bacterial cells were suspended in 3 times the volume of ethanol, stirred for 30 minutes, and the ethanol layer was concentrated under reduced pressure to pH 7.01/15M.
After adding phosphate buffer and dissolving, extract with toluene.

After combining the exudates from both sleeves and washing with an equal volume of pH 7.01/15 M'J acid buffer, the solvent layer is concentrated to obtain 1.8 g of an orange-yellow substance.

This orange-yellow substance is dissolved in 20 mt of ethanol and crystallized to obtain 0.48 g of Norifamycin S crystals.

Example 5 Nocardia -468 was grown under the same conditions as in Example 1 to obtain a seed mother liquor.

Obtain this seed mother liquor. Combine 8 bottles of this seed mother liquor, content 3
The fermentation medium of Example 3 (15A) was placed in a stainless steel Jarfunmentor (No. 01), and cultured according to the method of Example 4.

After the culture is completed, the culture solution is filtered, the pH of the solution is adjusted to 7.0, and the solution is extracted with an equal amount of butyl acetate.

In addition, the bacterial cells were suspended in 3 times the volume of ethanol, stirred for 30 minutes, and the ethanol layer was concentrated under reduced pressure to a pH of 7.01/15.
After adding MIJ acid buffer and dissolving, extract with butyl acetate.

Combine the exudate from both sleeves, wash with an equal volume of phosphate buffer, and then concentrate and dry.

The obtained yellow substance is dissolved in 100 mA toluene and purified by silica gel column chromatography.

Chloroform:methanol is used as the elution solvent.

After concentrating the eluate, 1.25 g of crude crystals of rifamycin S were obtained.
is obtained.

If this orange-yellow substance is dissolved in 20 m7 of ethanol and crystallized, 0.52 g of pure crystals will be obtained.

[Brief explanation of the drawing]

FIG. 1 shows the absorption spectrum of rifamycin S in the ultraviolet and visible regions, and FIG. 2 shows its infrared absorption vector.

Claims (1)

[Scope of Claims] 1 Rifamycin S, characterized in that Nocardia bacteria capable of producing Rifamycin S are cultured aerobically in a nutrient medium, and Rifamycin S is collected from the culture. manufacturing method. 2. The production method according to claim 1, wherein the Nocardia bacterium capable of producing rifamycin S is Nocardia -468 and a corresponding mutant strain.