KR960004036B1 - Microorganism that produces cephalosporin c and the processing method of cephalosporin c with using it - Google Patents

Abstract

Cephalosporium acremonium (I) KFCC 10783 (II) for cephalosporin C (III) fermentation is screened with NTG treatment from (I) KFCC 10679 media containing 50 microgram per milliliter of nistatin. (I) KFCC 10784 (IV) is screened with 0.1 microgram per milliliter of amphotericin B. For (III) fermentation, (II) or (IV) is cultivating in the medium containing glucose-fructose mixture 4.5%, mixed oil 7.0%, peanut powder 2.5%, corn steep liquor 3.5%, corn meal 1.5%, gluten 3.0%. The final concentration of (III) is 40.2 gram per liter in (II), and 39.7 gram per liter in (IV).

Description

Microorganisms That Produce Cephalosporin C and Methods of Making Cephalosporin C Using the Same

The present invention relates to a microorganism producing cephalosporin C and a method for producing cephalosporin C using the same, and more specifically, as a variant of cephalosporium acremonium KFCC 10679, a polyene antibiotic nystatin or The present invention relates to a microorganism that is resistant to amphotericin B and capable of producing high concentrations of cephalosporin C when cultured in a medium whose main components are isomerized liquid sugar, mixed oil, peanut powder and corn steep liquor.

In the case of the method for producing cephalosporin C using a conventional microorganism, if cephalosporin C produced in the cells is not easily secreted out of the cells, the cephalosporin C is excessively accumulated in the cells, thereby causing the biosynthesis of cephalosporin C. Enzymes involved in the inhibition has been a problem that the productivity of cephalosporin C can be reduced.

In general, in the case of bacteria, strains that obtain resistance to antibiotics such as polymyxin B, penicillin G, and bacitracin are obtained as a method for improving membrane permeability by changing the structure of cell membranes and obtaining a mutant strain having excellent double fermentation ability. The development method is used.

In view of the fact that the microorganism used in the present invention is not a bacterium but a fungus, an antifungal agent that inhibits the growth of fungi has been found, and thus polyene antibiotics have been used. Polyene antibiotics are antibiotics with 26-28 lactones and 3-7 conjugated double bonds, such as nystatin, amphotericin B, and trichomycin. Polyene antibiotics are effective against molds and yeasts, and against bacteria. That is, the present invention was completed by discovering the effect of eukaryotic membrane sterols to change the membrane permeability to improve the productivity of cephalosporin C.

Microorganisms of the present invention, namely, nystatin-resistant and amphotericin B-resistant strains were treated with cephalosporium acremonium KFCC 10679 as ultraviolet light, N-methyl-N'-nitro-N-nitrosoguanidine (NTG). Liquid or agar medium containing 10-300 µg / ml and 0.01-0.5 µg / ml of statistatin or amphotericin B, respectively, after treatment according to conventional methods with mutagenesis agents such as , 1 g / L asparagine, 0.5 g / L magnesium sulfate, 0.5 g / L potassium phosphate 1g / L potassium phosphate 1 g / L, pH 7.0). At this time, some strains cannot grow due to high concentration of polyene antibiotics, and strains with high antifungal resistance can be grown so that the minimum inhibitory concentration (MIC) of polyene antibiotics is more than twice that of parent strain. Of these resistant strains, the cephalosporium acremonium KFCC10783 and the amphotericin B-resistant cephalosporium acremonium KFCC 10784, which improved the productivity of cephalosporin C among these resistant strains, It was deposited with the Korean spawn association on March 3rd. The bacteriological properties of the strains are identical to those of the parent strain, except for polyene antibiotic resistance and the productivity of cephalosporin C.

The characteristics of the microorganism of the present invention are as described in the following table.

[Table 1] Comparison of resistance to nystatin concentration

[Table 2] Comparison of resistance to amphotericin B concentration

(Note) use medium; Agar Badge

+; Growth,-; Lack of growth

Hereinafter, the present invention will be described in more detail in the following examples.

Example 1

Use strain: Microorganism of the present invention (KFCC10783, KFCC10784)

Primary seed medium: soy flour 3.0%, corn steep liquor 0.75%, sugar 2.5%, glucose 1.0%, calcium carbonate 0.5%, antifoam 0.15%, pH 7.0

Secondary seed medium: soy flour 3.0%, peanut powder 3.0%, corn steep liquor 1.5%, sugar 2.5%, glucose 1.0%, calcium carbonate 0.5%, antifoam 0.15%, pH 6.9

Fermentation medium: 4.5% per isomerized solution, mixed oil 7.0%, peanut powder 2.5%, corn steep liquor 3.5%, corn wheat 1.5%, gluten wheat 3.0%, D, L-methionine 0075%, calcium sulfate 1.0%, antifoam 0.1 %, pH 6.9

Primary seed culture: The primary seed medium was placed in a 500 ml shake Erlenmeyer flask with 50 ml each, and the bacteria were sterilized and shaken at 30 ° C. at 220 rpm for 90-96 hours.

Secondary seed culture: Dispense the secondary seed medium into each 7 liter fermenter for 5 liters each, inoculate 15 ml of the culture complete solution obtained from the primary seed culture after sterilization and cooling for 30 minutes at 121 ° C, and It was incubated for 80-90 hours at 30 ℃ at 600-900rpm feeding 0.5-1.0L per minute.

Fermentation method: Dispense 19.4ℓ of fermentation broth into 30L test fermenter, sterilize and cool at 121 ℃ for 30 minutes, inoculate 1.6L of secondary culture medium, and supply air 0.5-1.0ℓ per minute. Incubated at 300-600 rpm 25-30 ° C. When the residual mixed oil concentration in the culture was 4-5%, sterilized mixed oil was frequently supplied.

Culture was continued until the sum of the mixed oil added and the mixed oil initially added to the fermentation broth was 12-17% of the fermentation broth. The pH of the culture was adjusted to 5.0-6.0 using ammonia water. After incubation, cephalosporin C concentrations were 35.7 g / L KFCC 10679RK and 40.2 g / L KFCC 1078301.

Example 2

As a result of the same composition as in Example 1, the strains used in the culture, the primary and secondary seed media, the fermentation broth, and the cultivation method were the same as those of Example 1. g / L.

As described above, it was confirmed that the microorganisms of the present invention are given resistance to polyene antibiotics, and thus the production concentration of cephalosporin C is improved as membrane permeability is changed.

Claims (4)

Microbial cephalosporium acremonium that produces cephalosporin C that can grow even under the concentration of nitstatin, a polyene antibiotic, at a concentration of 50 µg / ml or more KFCC 10783. Microbial cephalosporium acremonium that produces cephalosporin C that can grow under 0.1 µg / ml or more of the polyene antibiotic amphotericin B. KFCC 10784. The method for producing cephalosporin C, wherein the microorganism of claim 1 is cultured in a medium containing isomerized sugar, mixed oil, peanut powder, corn steep liquor and the like to obtain cephalosporin C. The method for producing cephalosporin C, wherein the microorganism of claim 2 is cultured in a medium containing isomerized sugar, mixed oil, peanut powder, corn steep liquor and the like to obtain cephalosporin C.