KR930008970B1 - Vancomycin producing microorganism - Google Patents

Abstract

A osmotolerant, vancomycin resistant mutant strain producing vancomycin claimed, has been selected by UV treatment of Nocardia orientalis (ATCC 19795). The vancomycin was produced by aerobic culture of the mutant strain (KFCC 10744) on a nutrient medium containing starch, potato protein, soy bean powder, sodium chloride at 30-35 deg.C, pH 7.8-8.0.

Description

Microorganisms Producing Vancomycin

The present invention relates to a novel microorganism producing vancomycin, a method for obtaining the same, and a method for producing vancomycin by fermentation using the novel microorganism.

A feature of the present invention relates to a microorganism and a method for producing vancomycin in a high yield by culturing aerobically in a nutrient medium containing a high concentration of sugar, soy flour, potato protein, and the like as a main ingredient. 1991.11.2. It was deposited with the Korean spawn association (KFCC 10744).

Vancomycin is a glycopeptide-based antibiotic that inhibits bacterial cell wall synthesis. Therefore, its usefulness is increasing for the treatment of all Gram-positive bacterial diseases or for the treatment of allergic patients.

Conventional fermentation technology (US Pat. No. 3,067,099) has a disadvantage in that the production cost is high due to the low titer (0.2 mg / ml) of the strain. In view of the above problems, an object of the present invention is to isolate the mutant strains greatly improving the production of vancomycin to produce a large amount of vancomycin industrially as a low-cost medium source. In the present invention, Nocardia Orientalis received from the American Type Culure Collection (ATCC), an international microbial deposit institution (ATCC) ATCC 19795) strain was used as parent strain. In more detail, the present invention is treated with ultraviolet light by the method of Example 1 as a mutagen, and complete medium adjusted to pH 7.0 with starch 1.0%, Bacto soytone 1.0%, and agar 20.% added with sodium chloride by concentration. Bacillus subtilis, which is grown on vancomycin after obtaining a mutant strain capable of growing in 2.0 mol of sodium chloride by incubating in a thermostat at 25 ° C. to 30 ° C. The culture solution of ATCC 6677) was aliquoted and incubated for another day at 30 ° C. At this time, the agar slant consisting of 1.0% glucose, 0.3% yeast juice, 0.3% wort, 0.5% peptone, and 2.0% agar and adjusted to pH 7.2 by selecting mutants having a larger inhibitory ring size than the parent strain : Incubated for 5 to 10 days in Note 2). The productivity of vancomycin for the selected strains was carried out in the same manner as in Example 2.

Investigation of vancomycin productivity in the Erlenmeyer flask showed that the osmotic resistance strain of vancomycin was superior to the parental strain of nocardia orientalis ATCC 19795 (0.2 mg / ml of vancomycin), and nocardia orientalis CVA-1 (KFCC 10744: vancomycin production 4.4). Mg / ml) was selected. When the selected mutants were subjected to aggregation of production titers during 20 passages, the mutants were found to be excellent mutants with no decrease in production titers.

As a result of the experiment by the triangular flask of the present invention, the genetic variation occurs due to the treatment of the mutagen, and the availability of the carbon source in the high concentration sugar increases, and thus the optimum concentration of the carbon source added in the production medium tends to increase gradually. A comparison is shown in Table 1. As shown in Table 1, the parent strain showed the maximum production amount of vancomycin in 2% of the hydrolyzed sugar, the carbon source, and the production of vancomycin was suppressed when added more than 5%. On the contrary, the mutant strain CVA-1 shows an increase in the concentration of the increased carbon source to 8% for maximum vancomycin production.

TABLE 1 Carbon Source Availability of Parent and Mutant.

Nocardia orientalis CVA-1 1100 (KFCC 10744) strain obtained in the present invention has the following characteristics microbiologically.

1. Growth on medium.

1) Microscopic findings

Morphology of feeder cells (Y.M medium): Eggplant mycelia.

Proliferation of feeder cells (Y.M medium): Mycelia.

2) Growth characteristics on agar slope.

* +: Growth,-: no growth.

2. Physiological features.

Oxygen composition: Aerobic

Optimal growth temperature: 25 ℃ ~ 35 ℃

Optimal Growth pH: 6.5 ~ 8.0

Gram Dye: +

AFB Staining:-

Nitrate Test: +

Catalase Test: +

Urease Test: +

β-hemolysis test: +

3. Availability of sugars.

Glucose: +

Fructose: +

Mannose: +

Lactose: +

Maltose: +

Trihalose: +

Mannitol: +

Raffinose: +

As the medium composition bred for the production of vancomycin, as shown in Example 2, a synthetic medium containing a carbon source, an organic nutrient source and a small amount of inorganic matter may be used. As a carbon source, glucose, fructose, starch, hydrolyzed starch, etc. are good. Yeast juice, wort, tuna juice, soybean meal, soy flour, potato protein, etc. are good as an organic nutrient source, and sodium chloride is used as an inorganic substance.

The cultivation conditions under aerobic conditions, the cultivation temperature of 30 ℃ ~ 35 ℃, pH was 7.8 ~ 8.0 at the beginning of the cultivation, the pH was automatically controlled to 6.6 ~ 7.0 using ammonia water when the pH drops during the cultivation. In addition, RPM, internal pressure, and aeration amount were adjusted to supply sufficient oxygen as the strain grew.

When described in more detail by the embodiment of the present invention as follows.

Example 1

Method for separating vancomycin resistant strain by mutation.

Mutant strain separation was carried out by culturing the parent strain Nocardia orientalis ATCC 19795, sterile physiological saline and filtration and separation of mycelia using glass wool to prepare a spore suspension. This spore suspension was treated with ultraviolet rays for 40 to 60 seconds with a killing rate of 99.9%, and then plated into a complete medium (Note 1) containing 1.0 to 2.5 moles (Table 2) of sodium chloride by concentration and 3 to 6 at 30 ° C. After culturing for one day, colonies of mutant strains capable of growing even at 2.0 mol of sodium chloride were obtained, and a culture solution of Bacillus subtilis ATCC 6677 whose growth was inhibited by vancomycin was aliquoted and incubated for another day at 30 ° C. Mutant strains larger than the parent strain appearing at this time were selected and incubated for 5 to 10 days in agar oblique medium (Note 2).

The isolated Nocardia orientalis ATCC 19795 mutants were produced and cultured by a triangular flask, and the titers were measured to obtain a Nocardia orientalis CVA-1 having excellent vancomycin titers.

The isolated excellent strains were stored for a long time in a liquid nitrogen tank using 10% skim milk, and when reused, they were used by inoculating the whole culture medium after melting.

[Table 2] Comparison of resistance to osmotic pressure

* +: Growth,-: no growth.

Example 2

Comparison of Vancomycin Productivity of Parental Variants and Mutants of the Invention by Erlenmeyer Flask

Preculture Badge

Glucose 1.7%

Wort 0.3%

Yeast Juice 0.3%

Peptone 1.1%

pH 7.2

Seed culture badge

Starch oxide 5.0%

Potato Protein 0.5%

Soybean oil 0.5%

Calcium Carbonate 0.3%

Antifoam 0.2%

pH 7.2

Production medium

Hydrolyzed Starch 3.0 ~ 10.0%

Potato Protein 1.8%

Soy flour 1.8%

Sodium Chloride 0.12%

Antifoam 0.2%

pH 7.8

[Cultivation method]

25 ml of the preculture medium of the composition was put into a 250 ml Erlenmeyer flask and autoclaved at 121 ° C. for 15 minutes. At this time, glucose was added after sterilization separately. Each strain was inoculated and incubated at 30 ° C. for 20 to 30 hours under a rotating agitator.

25 ml of the culture medium of the above composition was put into a 500 ml Erlenmeyer flask and pressurized and sterilized at 121 ° C. for 30 minutes, followed by 0.1% inoculation of the complete culture medium, which was incubated at 30 ° C. for 30 to 40 hours. After 25 ml of the production medium was put into a 500 ml Erlenmeyer flask, pressure sterilization was carried out at 121 ° C. for 30 minutes, and the pH was adjusted to 7.8 using 1 N NaOH, which was inoculated, and then the incubator was incubated at 34 ° C. under RPM 200 for 100 hours. The results are shown in Table 3, wherein USP vancomycin was used as a standard and quantified by HPLC.

[Table 3] Titer Comparison of Parent and Mutantism.

Example 3

Comparison of vancomycin production capacity of parent strain and fermented strain of the present invention.

Medium composition of the present Example, quantification method of vancomycin and the like are the same as in Example 2, 30L fermentation tank was filled with 20L seed culture medium and sterilized for 25 minutes. After sterilization, the pH was adjusted to 7.2 using sterilized 1N NaOH, followed by seed culture of 0.1% of the preculture culture cultured in the same manner as in Example 2. After filling 20L of the production medium in a 30L fermenter, it was autoclaved for 30 minutes. At this time, the hydrolyzed starch was added after sterilization separately. The pH of the medium immediately before phagocytosis was adjusted to 7.8 using sterile 5N NaOH, and the culture medium was cultured for 5% in the prepared production culture tank. It was incubated for 110 hours at the conditions of 34 ℃, 250 ~ 350 RPM, aeration amount 0.5 ~ 1.0VVM, internal pressure 0.5 ~ 1.0atm. At this time, the pH was automatically adjusted to 6.8 using ammonia water when the pH was less than 6.8 during the culture. The results are shown in Table 4.

[Table 4] Comparison of Productivity of Parent and Mutant in the Presentation Group

Example 4

Optimization experiment of sugar concentration by fermentation tank.

The composition, culture method and quantification method of vancomycin in this example were the same as in Example 3, and the hydrolyzed starch concentration in the production medium was changed to 8%, 10%, 12%, and 14%.

The results are shown in Table 5.

[Table 5] Availability of Hydrolyzed Starch

Claims (1)

The mutant strain KFCC 10744, which belongs to the nocardia orientalis mutant strain, is resistant to osmotic pressure, and has an increased vancomycin production at a carbon source concentration of 5.0-10.0%.