KR820000031B1 - Process for the preparation of rifamycin b by micro-organism - Google Patents

Abstract

High conc.-rifamycin B is produced by the starch assimilable mutant Streptomyces mediterrane; CKD 1129 under aerobic culture conditions. Thus, 100 ml of an inoculum of S. mediterrane; is cultured in 1L of a medium contg. glucose 2 %, yeast ext. 0.5 %. peptone 0.5 %, beef ext. 0.5 %, Natl 0.1 %, pH 6.7 for 48 hr at 28≰C. This medium is moved to main-culture medium in fermentor, cultured to give rifamycin B for 200 hr at 28≰C with RPM 1000, ventilation amount 1.2 VVm.

Description

Preparation of rifamycin B by microorganism

The present invention relates to a method of accumulating rifamycin B at a high concentration by using a Streptomyces genus mutant having high starch magnetizing power in a high concentration substrate containing starch.

Since lipamycin has been reported by Sensi et al. Of the Italian Institute of Repetite in 1959, many lipamycin derivatives have been developed by fermentation or chemical treatment, and these derivatives have great antimicrobial activity against Gram-positive groups including tuberculosis bacteria. It is one of the antibiotics that attracted.

In addition, lipamycin B is known as an industrial production method by Streptomyces mediterranei (US Pat. No. 3,150,046) because lipamycin B is very important as a starting material for the preparation of rifamycin derivatives. And since rifamycin B is produced as a complex with other homologs of the other four species (rifamycin A, C, D, E), the method of adding barbital to the medium (US Pat. No. 2,988,490) or using mutant strains (US Pat. 3,871,965) has also been developed to selectively produce rifamycin B.

More specifically, the conventional lipamycin B-producing Streptomyces mediterranei is relatively good in the use of disaccharides such as glucose and fructose and sucrose and maltose, while polysaccharides such as starch Since it is not available, there is a drawback that the high concentration fermentation is inhibited by the substrate concentration to inhibit the growth of bacteria or to significantly reduce the yield of the unit substrate.

The present inventors have isolated a new Streptomyces spp. Actinomycete strain having lipamycin B production ability in soil, and examined the sugar availability. Therefore, the present inventors have the same sugar availability as the conventional bacteria, and thus, there is a high concentration of rifamycin B in the medium. There was a problem. Therefore, in order to solve this drawback, strain Streptomyces mediterranean SM-153, which has the highest productivity of rifamycin B, is isolated from N-methyl-N'-nitro-guanidine (N-methyl-N'-nitro-). guanidine) found that the mutant strain Streptomyces mediterranean CKD 1129 (KFCC 10028) has strong starch availability.

Hereinafter, the separation method of the new mutant strain in the present invention will be described in more detail.The separation of the rifamycin B producing strain is inoculated with soil collected from all over the country by adding 50 micrograms per ml of tetracycline to the medium containing glucose. The actinomycetes grown by the method of cultivation were collected, and the morphomycetes B were transplanted into a juicy liquid medium containing glucose, shaken and cultured under aerobic conditions at 28 ° C. for 7 days, and the culture solution was filtered to obtain rifamycin B. Quantitatively determined by colorimetry, Streptomyces SM-153 was isolated.

The isolated strain of Streptomyces genus SM-153 was treated with N-methyl-N'-nitro-guanidine on starch-inorganic agar medium to select strains with high growth rate and high starch-resolving power. Cultured in the medium was isolated Streptomyces mediterranean CKD 1129 (KFCC 10028) excellent in the production capacity of rifamycin B. Streptomyces SP.SM-153, the ancestor of such mutant strains, was described as "The Burgess Menu" 8th edition (Bergey's manual of Determinative Bacteriology. 8th edition, Williams & Wilkins, 1974) and G. Sykes. Numerical Taxonomy of Streptomycetes, polish medical publishers (1975), Margaris et al. (Actinomysetales, Academic Press, 1973) and W. Kurylowies. Margalith, PG.Beretta, Rifomycin.XI.Taxonomic Study on Streptomyces Mediterranei nov.SP.Mycopathol. Et Mycol.Appl. 13: 321-1961. The taxonomic properties were identified as Streptomyces mediterranei. The differences between mutant and parent strains are shown in Table 1 and their properties are the same as their parent strains.

Differences other than circumference (Streptomyces SP.SM-153) and mutant strain (Streptomyces mediterraneia) CKD 1129 (KFCC 10028)

TABLE 1

end. Morphological properties

)로서 균락은 담등황색을 띤다.(1) Hyphae: Multi-trophic hyphae (0.8-1.0μ) without septum

) Is pale yellow in color.

(2) Kiyaksa: simple branches, curved in shape, phantom spore disease.

(3) Spores: 0.8-1.0μ ellipsoidal chain form, spore surface is smooth and no spore bag.

(4) Gram dyeing: positive

I. Culture properties

(1) sucrose nitrate agar medium

It has good growth and the surface is milky white or yellowish, yellow to pink backside, and the aerial mycelium is milky white to produce pale yellow soluble pigment.

(2) glucose asparagine agar medium

It has good growth and the surface is milky or yellow with yellow to pink back side, and the mycelia are milky white to produce pale yellow soluble pigment.

(3) Glycerin Asparagine Agar Medium

Good growth, dark orange or pink surface, yellow backside, milky white mycelia, yellow soluble pigment.

(4) starch agar medium

It grows vigorously, the surface is white or pink, has an orange yellow back side, and produces yellow soluble pigment.

(5) Chirosine Agar Badge

It grows well and the surface is orange yellow, pink back side, pink mycelia, and yellow soluble pigment.

(6) East Malt Agar Badges

It grows vigorously, its surface is yellow or pink, and the mycelia are poor and do not produce soluble pigment.

(7) Oatmeal Agar Badge

It is well grown and its surface is yellow or orange with pink back side, and the mycelia are pale yellow with white or yellow color.

(8) Nutritional Agar Medium

Growth Normal, surface is pale yellowish yellow with pink surface, and mycelia are yellow-pink and do not form soluble pigment.

All. Physiological properties

(1) Optimum growth temperature 25-37 ℃

(2) Optimal Growth PH 5.5 ~ 7.0

(3) oxygen composition aerobic

(4) acetate-reducing negative

(5) litmus milk blue

(6) starch gas resolution

(7) hydrogen sulfide production negative

(8) No gelatin liquefaction

(9) melanin pigmentation negative

la. Assimilation of carbon sources

Arabinose, rhamnose, chisirose, glucose, garactose, fructose, sucrose, ribose, sorbose, melibiose, lactose, trehalose, cerubiose, maltose, raffinose, glycerol, mannitol, inositol, salicycin, acetate, citrate, Dextrin, inulin, starch.

The above properties were compared to the classification identification method described in Burgy's manual of Determinative Bacteriology, 8th edition, 1974, and found to be in the Streptomyces family of Streptomycetaceae. The results of this study were based on the taxonomic study of Streptomyces mediterranean by Margaris et al., And it was identified as Streptomyces mediterranean, and the previously released lipamycin B-producing Streptomyces mediterranean ATCC 13685 As a result, it was confirmed that the strain was a new strain because there was a significant difference in the availability of starch degrading raffinose, citrate and acetate.

In addition, the results of comparing the morphological and physiological characteristics between the present strain and Streptomyces mediterranei ATCC 13685 are shown in Table 2.

TABLE 2

As shown in Table 2, the difference from Streptomyces mediterranean ATCC 13685 is that the present strain has strong magnetization of starch, raffinose, sodium citrate, and sodium acetate and generates soluble pigments on juicy agar media, while the existing strain Streptomyces Mediterani ATCC-13685 has no magnetization to the above carbon sources and does not produce soluble pigments on juicy cloth media.

As a medium used for the production of rifamycin B using the new mutant strain of the present invention, the carbon source is liquefied using alpha amirase together with starch in order to prevent an increase in viscosity resulting from gelatinization when the soluble starch is heated or when the starch is heated. Combined with glucose, glycerin, sucrose and maltose at a certain ratio.For nitrogen sources, mineral nitrogen sources such as yuan, salt eye, nitrate ammonia, and dropping meal, soybean meal, cottonseed meal, meat meal, fishmeal slicka, and yeast extract Using the same organic nitrogen source alone or in combination, the inorganic salts and other media components are the same as in the known methods.

Cultivation is carried out at a temperature of 25-35 ° C., PH 5.5-7.0, and aerobic conditions for 160-240 hours.

Starch is late in culture because when used in high concentration alone, high viscosity of the medium hinders oxygen migration in the medium. However, when starch is mixed with monosaccharides such as glucose, sucrose and maltose, or disaccharides and glycerin, it is possible to avoid the inhibition of substrate concentration that is common when monosaccharides, disaccharides and glycerin are used alone. It can also improve caring. In addition, the same effect can be obtained even if starch is added during the culture.

The accumulation concentration of rifamycin B in the medium according to the mixing ratio of starch and various sugars varies depending on the type of sugar as shown in Table 3, but the starch concentration is high within the range of 20-60% of the total sugar concentration. For each type of sugar, the method using a mixture of glucose may give the best results compared to other sugars.

TABLE 3

When starch is mixed with monosaccharides, disaccharides, or glycerin, as shown in Table 4, it is possible to alleviate the inhibition of substrate concentration when the substrate concentration in the medium is high, thus increasing the yield without lowering the yield even when the substrate is initially used. You can.

TABLE 4

Note: starch is soluble starch

As described above, the present invention removes the substrate inhibitory phenomenon that occurs when monosaccharides or glycerin are used at a high concentration of 10% or more by using a mixture of monosaccharides, disaccharides or glycerin using starch CKD 1129 of Streptomyces genus having strong starch magnetization power. In addition, it is possible to increase the concentration of substrate up to 50% without lowering the yield, thereby increasing the productivity of rifamycin B and reducing the raw material cost because the starch price is lower than that of glucose. It is a manufacturing method of B.

The following examples will illustrate the invention in more detail, but the scope of the invention is not limited thereto.

Example 1

Number of bacteria used: Streptomyces SM-153, the mutant, and Streptotomyces mediterranean CKD 1129

Germination medium: Glucose 2%, Yeast extract 0.5%, Peptone 0.5%, Juicy 0.5%, Sodium chloride 0.1%, PH 6.7

Seed cultures: glucose 3%, soy flour 1%, ferrous phosphate 0.1%, magnesium sulfate 0.1%, calcium carbonate 0.5%, ferrous sulfate 0.001%, zinc sulfate 0.005%, manganese sulfate 0.005%, antifoam 0.05%, PH 6.7

Main culture medium: glucose 5%, soluble starch 5%, peanut starch 3%, soy flour 1%, monobasic phosphate 0.2%, magnesium sulfate 0.2%, calcium carbonate 1%, zinc sulfate 0.005%, ferrous sulfate 0.001 %, Ammonia sulfate 0.5%, Sulfuric acid lamp 0.0004%, Molybdate ammonium 0.0001%, Defoamer 0.05%, PH 6.7

Cultivation method: Inoculate the seedlings which were shaken in a 500 ml sagagucci shake flask containing 100 ml of germination medium for 72 hours at 28 ° C. in a 5 l shake flask containing 1 liter of culture medium, and incubate at 28 ° C. for 48 hours. The batch was inoculated into a 20 L test fermenter in which 9 L was added and incubated at 28 ° C. for 200 hours at RPM: 1000 and an aeration of 1.2 VVm.

Incubation results: Table 5.

TABLE 5

Example 2

Strains used: Streptomyces mutant strain CKD 1129

Fermentation medium: This culture medium is the same as in Example 1 except that 7.5% of soluble starch and 7.5 of glucose are used instead of 10% of glucose in the culture medium.

Cultivation method: same as Example 1

Cultivation result: Rifamycin B, 3510 µg / ml was obtained.

Example 3

Strains used: same as Example 1

Fermentation broth: in Example 2, excluding glucose and using 7.5% glycerin

Cultivation method: same as Example 1

Culture results: Rifamycin B, 2980 ㎍ / ㎖ was obtained.

Example 4

Strains used: same as Example 1

Fermentation Medium: Reduced glucose to 4% and used glycerin 3.5% in Example 2

Cultivation method: same as Example 1

Culture results: Rifamycin B, 2750g / ㎖ was obtained.

Claims (1)

A microorganism characterized by producing a high concentration of rifamycin B by culturing starch magnetizing strain Streptomicros mediterranean CKD 1129 (KFCC 10028) in batches where starch is used at 20-60% of the total sugar concentration. Method for preparing rifamycin B.