JPS6225358B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing a cephalosporin antibiotic. Semi-synthetic cephalosporin antibiotics such as Cephalothin, Cephaloridine, and Cephalexin, which are known as antibiotics that are highly effective against Gram-positive and -negative bacterial infections, especially against various antibiotic-resistant bacterial infections. It is well known that cephalosporin antibiotics obtained from nature are used as starting materials. Much research has been carried out to date to find ways to accumulate large amounts of these starting materials in the fermentation broth. However, most of these methods involve improving the bacterial strains used [for example,
58289, JP 50-58290, JP 52-105290, Antimicrobial Agents and Chemotherapy
Chemotherapy) 13 , 7-13 (1978), etc.], and not much research has been conducted on methods by improving culture medium components. Based on the above-mentioned background, the present inventors have continued research on a method for industrially advantageous production of cephalosporin antibiotics, mainly with the aim of improving culture medium components. By adding S-sulfocysteine, which was not used as a medium component when producing cephalosporin antibiotics by fermentation, to the culture medium, the production of cephalosporin antibiotics was significantly promoted. As a result of discovering the facts and conducting further research based on them, the present invention has been completed. The present invention is a method for producing a cephalosporin antibiotic by culturing a microorganism belonging to the genus Cephalosporium and having the ability to produce a cephalosporin antibiotic in a medium, which is characterized in that S-sulfocysteine is added to the medium. This is a method for producing cephalosporin antibiotics. Examples of cephalosporin antibiotics include cephalosporin C (in the following formula, R is
Compound of OCOCH ₃ . Hereinafter, it will be abbreviated as "CPC". ), deacetylcephalosporin C (compound in the following formula, where R is OH; hereinafter abbreviated as "DCPC"), deacetoxycephalosporin C (compound in the following formula, where R is H; hereinafter abbreviated as "DCPC"). , abbreviated as "DACPC"). Any microorganism that belongs to the genus Cephalosporium and has the ability to produce one or more cephalosporin antibiotics can be used in the method of the present invention. Microorganisms belonging to the genus Cephalosporium are defined as microorganisms such as Mycologia, Vol. 55, p. 563 (1963), Mycologia, Vol. 58, p. 351 (1966), Rabenhorst, Cryptogamenflora. ,
Kryptogenflora der Markbrandenburg,
1907), Cephalosporium Essays by Durrell (Durrell, Colorado State Univ. No. 248, 1963)
This refers to species belonging to the genus Cephalosporium, which is classified by As a specific example of the microorganism used in the method of the present invention, for example, in the production of CPC, Cephalosporium polyallerium 199 (FERM-
P No. 1159; IFO 9394; ATCC-20359), Cephalosporium polyallerium Y505 (FERM
-P No. 1160; IFO 9535; ATCC-20360), Cephalosporium acremonium 2M-16
(FERM-P No.2283; IFO 9999; ATCC-
20425), Cephalosporium acremonium
LA-101 (FERM-P No.2284; IFO 30001;
ATCC-20426), Cephalosporium acremonium K-121 (FERM-P No.2285; IFO
9998; ATCC-20427), Cephalosporium acremonium N-75 (FERM-P No.2286;
IFO 9997; ATCC-20428), etc., but in the production of DCPC, Cephalosporium sp.C-28
(FERM-P No.1430; IFO 9537; ATCC-
20370), but in the production of DACPC, Cephalosporium acremonium M105
(FERM-P No.2626; IFA 9919), Cephalosporium sp.M-20 (FERM-P No.2625; IFO
9921) and others. In the above, the number represented by FERM-P is the microorganism deposit number of the Institute of Microbial Technology, Agency of Industrial Science and Technology, the number represented by IFO is the microorganism deposit number of the Fermentation Research Institute, and the number represented by ATCC is the microorganism deposit number.・The American Type Culture Collection
(hereinafter abbreviated as "ATCC"), respectively. All of the above-mentioned bacteria with ATCC deposit numbers are listed in ATCC's 1978 Catalog of Strains.
Strains) and is available from ATCC. The mycological properties of Cephalosporium sp.C-28 are as follows. 1 Properties on agar medium (1) Malt extract agar medium Growth is poor, with many irregular wrinkles on the surface, and colorless to light brown growth. The underside is colorless or light brown. Almost no formation of aerial mycelium is observed. No soluble dyes are produced. (2) Potato glucose agar medium Poor growth and warty growth. Colorless or light brown. Underside colorless or light brown. Almost no formation of aerial mycelium is observed. No soluble dyes are produced. (3) Oatmeal agar medium Good growth and dispersibility. Growth is colorless. Back side colorless. Almost no formation of aerial mycelium is observed. No soluble dyes are produced. (4) Sweetened bouillon medium Good growth and dispersibility. Growth is colorless or light brown. Underside colorless or light brown. Aerial mycelium formation is poor. No soluble dyes are produced. 2. Microscopic properties The microscopic properties of the bacteria grown on the various media mentioned above are as follows. Aerial hyphae are branched, 1.5-3.0μ wide, and colorless. Conidiophores are erect from aerial or basal hyphae and are 30 mm long.
Forms a conidial mass of ~70μ, width 1.5-2.0μ, colorless 8-14μ at the tip. Conidia are elliptic, unicellular, and colorless with somewhat pointed ends. The size is 2
~3μ x 8~13μ. Conidia are poorly formed on malt extract agar, potato-glucose agar, and oatmeal agar, but are abundantly formed on sweetened bouillon agar. Cephalosporium sp. C-28 is considered to belong to Cephalosporium acremonium based on the descriptions of the above-mentioned literature regarding its mycological properties. Cephalosporium Acremonium M105
The mycological properties of Cephalosporium and Cephalosporium sp.M-20 are almost the same except for the ability to produce DACPC, and are as follows. (1) Properties on agar medium (1) Malt extract agar medium Growth is not very good, the surface has many irregular wrinkles, and is colorless to light brown and moist. The underside is colorless or light brown. Almost no formation of aerial mycelium is observed. No soluble dyes are produced. (2) Potato Glucose Agar Medium Growth is not very good, showing irregular, mounded growth. It is colorless or light brown and moist. The underside is colorless or light brown. Almost no formation of aerial mycelium is observed. No soluble dyes are produced. (3) Oatmeal agar medium: Slightly good growth and dispersibility. Growth is colorless. Back side colorless. Aerial mycelium is rarely formed. No bathable dyes are produced. (4) Sweetened bouillon medium: Slightly good growth and dispersibility. Growth is colorless or light brown. The underside is colorless or light brown. Aerial mycelium formation is poor. No soluble dyes are produced. (2) Microscopic properties The microscopic properties of the bacteria grown on the various media mentioned above are as follows. Aerial hyphae are branched, 1-3.5μ wide, and colorless. Conidiophores are erect from aerial or basal hyphae;
Forms a colorless conidial mass of 8-14μ at the tip, 30-60μ long and 2-3.5μ wide (base). Conidia are cylindrical to somewhat curved oval, unicellular, and colorless. Size is 2~3μ x 6~12
μ. In general, the properties of Cephalosporium bacteria are easily changeable, and they can be easily mutated by, for example, X-ray irradiation, ultraviolet irradiation, radiation irradiation, or artificial mutagenesis methods using artificial mutagenic agents (e.g., nitrosoguanidine, ethyleneimine, etc.). I can do it. Even among such mutant strains, any strain capable of producing a cephalosporin antibiotic can be used in the method of the present invention. When culturing the bacteria used in the present invention, a medium containing a carbon source that can be assimilated by the bacteria, a nitrogen source that can be assimilated, etc. is used. The carbon source may be anything as long as it can be assimilated, such as glucose, sucrose, starch, soluble starch, glycerin, n-paraffin, organic acids such as acetic acid, fumaric acid, and benzoic acid, alcohols such as ethanol and butanol, Fats and oils (eg, lard oil) can be used alone or in combination. Examples of nitrogen sources include peptone, soy flour, meat extract, cotton seed flour, dried yeast, yeast extract, corn steep liquor, Proflo (manufactured by Traders Protein Division), corn gluten meal, urea, ammonium salts (e.g. , ammonium chloride), nitrates (e.g., potassium nitrate), and other organic or inorganic nitrogen-containing substances (e.g., NZ amine (A), ammonium sulfate) are used alone or in combination. Other inorganic salts of medium components include various phosphates (e.g.
Potassium phosphate), sulfate (eg, sodium sulfate), hydrochloride (eg, magnesium chloride), etc. are used. The addition of ions such as iron, magnesium, calcium, manganese, and cobalt is closely related to the growth of bacteria and the production and stability of cephalosporin antibiotics. These medium materials to be used can be appropriately combined or selected depending on the bacterial strain used, the conditions used for culture, etc. S-sulfocysteine used in the present invention can be obtained by the method of Segel et al., Analytical Biochemistry 5 , 330.
(1963). The concentration of S-sulfocysteine added to the medium depends on the strain used,
Although it varies depending on the medium composition etc., it is generally about
It is preferably 0.01 to 3.0 W/V%, more preferably about 0.5 to 2.0 W/V%. Moreover, the addition method to the medium may be either from the beginning of the culture or from the middle of the culture. In short, the amount of the target cephalosporin antibiotic is adjusted appropriately so that the amount accumulated is maximized. Culture temperature, culture period, and
Culture conditions such as pH of the medium and aeration/agitation vary depending on the strain used, medium composition, etc., but may be selectively adjusted so as to maximize the accumulation of the desired cephalosporin antibiotic. In many cases,
When culturing is carried out aerobically at a culture temperature of 20 to 37°C, a culture period of 4 to 14 days, and a medium pH of 5.0 to 9.0, the accumulation of cephalosporin antibiotics in the culture solution reaches its maximum. Cephalosporin antibiotics are produced and accumulated in the culture solution obtained as a result of culturing. Since most of the cephalosporin antibiotic is present in the culture solution, it is preferably obtained from the liquid portion of the culture from which bacterial cells have been removed by centrifugation or filtration. To collect cephalosporin antibiotics separately,
Known methods, such as general fractional collection methods for weakly acidic organic substances, can be applied accordingly. i.e. ion exchange resin (e.g. Amberlite IRA-900), activated carbon, cellulose, silica gel, Cephadex, XAD
(manufactured by Rohm and Haas) or the like, the target substance can be advantageously collected by combining chromatography or gel filtration methods. To quantify cephalosporin antibiotics, separate each component using thin layer chromatography,
A method for measuring the antibacterial activity against the test bacteria or a method using cephalosporinase described in Nature, Vol. 246, p. 154 (1973) is used. In addition, elemental analysis, nuclear magnetic resonance spectroscopy, paper electrophoresis, thin layer chromatography, etc. are used to identify cephalosporin antibiotics. The content of the present invention will be explained in more detail with reference to Examples below. The present invention is not limited thereby. Note that the percentages used below represent weight/volume percentages (W/V%) unless otherwise specified. Example 1 50 ml of a seed medium consisting of 3.0% sucrose, 1.5% meat extract, 0.5% corn steep liquor, and 0.15% CaCO ₃ was dispensed into a 200 ml Erlenmeyer flask, and after sterilization,
In addition to this, the cephalosporin C producing strain Cephalosporium polyallerium 199 (FERM-P
No. 1159; IFO 9394; ATCC-20359), or Cephalosporium polyallerium Y505
(FERM-P No.1160; IFO 9535; ATCC-
20360) in each of the above-mentioned media, and cultured for 2 days at 28°C on a rotary shaking incubator. On the other hand, seuucrose 3.0%, raw soybean flour 3.0
%, CaCO ₃ 0.15% to which 0.5% of DL-methinion was added and 0.5% of S-sulfocysteine were prepared, and 30 ml of each was dispensed into a 200 ml Erlenmeyer flask.
Keep it sterile. These main fermentation media were inoculated with 1 ml of one of the seed cultures, and cultured on the shaker at 24°C for 6 days. Table 1 shows the measurement results of CPC in the culture solution after completion of the culture.

[Table] Stain

Claims (1)

[Claims] 1. A method for producing a cephalosporin antibiotic by culturing a microorganism belonging to the genus Cephalosporium and having the ability to produce a cephalosporin antibiotic in a medium, which comprises adding S-sulfocysteine to the medium. Method of manufacturing antibiotics.