JPS62236500A - Production of cephalosporin-type antibiotic substance - Google Patents

Abstract

PURPOSE:To obtain a cephalosporin-type antibiotic substance in high yield, without using expensive methionine, by adding a thiosulfate and a specific compound to a medium for the culture of microbial strain capable of producing the cephalosporin-type antibiotic substance. CONSTITUTION:A medium for the cultivation of a microbial strain belonging to Cephalosporium genus and capable of producing a cephalosporin-type antibiotic substance is added with (A) a thiosulfate and (B) one or more compounds selected from alanine, serine, pyruvic acid, alpha-ketoglutaric acid, 2-ketobutyric acid, citric acid, oxaloacetic acid and their salts. The thiosulfate is preferably an inorganic salt of thiosulfuric acid such as ammonium thiosulfate, sodium thiosulfate, etc. The amount of the thiosulfate added to the medium is preferably 0.5-2wt% and that of alanine, serine, pyruvic acid, alpha-ketoglutaric acid, etc., is preferably 0.3-1.5W/V%.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing cephalosporin antibiotics by fermentation.

Prior Art Cephalosporin antibiotics, especially cephalothin (Cep
halothin), Cephal-0 lysine (Cephal
oridinc).

Cephalexin is one of the few antibiotics that is highly effective against Durham-positive and -negative bacterial infections, especially against various antibiotic-resistant bacterial infections.
Cefmenoxime), Cephalothin (Cefsu
It is well known that cephalosporins (lodin) and the like are highly evaluated clinically as second and third generation cephalosporins.

Cephalosporin antibiotics produced by microorganisms are used as starting materials for these semisynthetic cephalosporin antibiotics.

Many studies have been conducted to date to find ways to accumulate large amounts of these cephalosporin antibiotics in fermentation broth. For example, how to improve the strain used [
For example, Anti-microbial agents and chemotherapies
ents and Chemotherapy)13.
7-13 (1971!l)] and methods by improving culture medium components [JP-A-55-144896. Special Publication No. 52-48196].

Regarding the improvement of culture medium components, thiosulfate, sulfate, dithionite (also called sulfite or dithionite),
Metasulfite, S-sulfocysteine, and the like are added to the culture medium as sulfur sources, but with the improvement of fermentation yield, it has become essential to add methionine in addition to these sulfur-containing compounds.

Problems to be Solved by the Invention Cephalosporin antibiotics produced by microorganisms are industrially important substances as starting materials for semi-synthetic cephalosporin antibiotics, and it is difficult to produce them cheaply and in large quantities. This is extremely significant. However, when the above-mentioned methionine is used as a raw material for fermentation, it has drawbacks such as being expensive, producing a bad odor during fermentation, and requiring a long fermentation time. Methods for producing phosphorus antibiotics are not always satisfactory.

Means for Solving the Problems The present inventors have conducted various efforts in the production of cephalosporin antibiotics in order to establish a method for producing cephalosporin antibiotics with high yield without using methionine as a fermentation raw material. As a result of repeated research, the present invention was completed.

That is, the present invention provides a method for producing cephalosporin antibiotics by culturing microorganisms belonging to the genus Cephalosporium and having the ability to produce cephalosporin antibiotics in a medium, in which thiosulfate is added to the medium, and alanine is added to the medium. , Serin,
The present invention provides a production method characterized by adding one or more compounds selected from pyruvic acid, &-ketoglutaric acid, 2-ketobutyric acid, citric acid, and oxaloacetic acid.

The cephalosporin antibiotic may be either a cephalosporin or cephamycin antibiotic, but for example, cephalosporin C (in the following formula, R is OC
Compound of OCH3. (hereinafter abbreviated as "rCPC"), and deacetylcephalosporin C (a compound in the following formula in which R is OH; hereinafter abbreviated as rDcPcJ).

As a specific example of thiosulfate, inorganic salts of thiosulfate are preferred, such as ammonium thiosulfate.

Examples include sodium thiosulfate and potassium thiosulfate.

Regarding the above alanine, serine, pyruvate, α-ketoglutaric acid, 2-ketobutyric acid, citric acid, and oxaloacetic acid, if these compounds can exist as optically active forms, they may be optically active forms, and racemic forms are also advantageous. It can be used for Furthermore, when these compounds can form salts, they can be used as salts.

These compounds may be used alone, or one or more kinds, for example, two or three kinds, may be used in combination.

Furthermore, if desired, glutamic acid or glycine can be further added in addition to the above compounds.

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 those that belong to the genus Cephalosporium.
ycologia) Vol. 55, p. 563 (1963),
Volume 58, page 351 (1966), Rabenhorst, Cryptogamenflora (Raben-horst)
Kryptogamenflora der Mark
brandenburg ■.

1907), etc., which belong to the genus Cephalosporium.

Specific examples of microorganisms used in the method of the present invention include, for example, Cephalosporium spp.
Polyallerium (Cephalospori-ua+
polyaleriun+)199 (F E RM
-PNo, lI59; IFo9394. A
TCC-20359), Cephalosporium polyallerium Y505 (FERM-PNo, 1160; IFO
9535; ATCC-20360), Cephalosporium acremonium
acremonium) 2 M-16 (FERM-
PNo. 2283; IFO9999; ATCC-204
25), Cephalosporium acremonium LA-1
01 (FERM-PNo, 2284; IFo 300
1; ATCC-20426), Cephalosborium acremonium -121 (FERM PNo, 228
5; IFO9998; ATCC-20427), Cephalosporium acremonium N-75 (PERM-
PNo. 2286; I Fo 9997, ATCC
-20428), etc.

In addition, in the production of DCPC, cephalosporium
Acremonium C-28 (C-28 (FER, I430)
; IFO9537; ATCC-20370, and this strain is also called Cephalosporium sp, c-28).

In the above, the number represented by FERM-P is the microbial accession number of the National Institute of Microbial Technology, Agency of Industrial Science and Technology.
The number represented by O is the microorganism accession number of the Fermentation Research Institute, and the number represented by ATCC is the number of the American Fermentation Research Institute.
Type Culture Collection (The Amer)
ican Type CultureCol 1ect
ion) (USA) (hereinafter abbreviated as rATccJ), respectively.

Among the above bacteria, those with ATCC deposit numbers are listed in ATCC's 1978 Catalog of Strains, 13th edition, and are bacteria that can be handled manually by ATCC. In addition, Cephalosporium polyallerium IP0 9394 strain is included in the 1978 List of Cultures of the Fermentation Research Institute (Inst.
Itute for Fermentation 0s
aka Li5t of Cultures
1978 Sixth Edition) and is available from the Fermentation Research Institute.

When culturing the bacteria used in the present invention, a medium containing an assimilable carbon source, an assimilable nitrogen source, and the like is used. As a carbon source, any assimilable material can be used, such as glucose, sucrose, starch,
Soluble starch, glycerin.

Organic acids such as n-paraffin, acetic acid, fumaric acid, and benzoic acid; alcohols such as ethanol and butanol;
Fats and oils (lard oil) are used alone or in combination. Examples of nitrogen sources include peptone, soybean flour, meat extract, cottonseed flour, dried yeast, yeast extract, corn steep liquor, and proflo (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). Used alone or in combination. Other inorganic salts of medium components include various phosphates (e.g., potassium phosphate), sulfates (e.g.,
(sodium sulfate), hydrochloride (eg, magnesium chloride), etc. iron, magnesium, calcium.

The addition of ions such as 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.

Any amount of thiosulfate can be added to the medium as long as it does not inhibit the growth of the bacterial strain, and it can be added to both the basic growth medium and the feed medium, for example, about 0.3 to 5% ( W/V), preferably about 0.5-2%
(ml/V). In particular, it is preferred to add thiosulfate to the base medium in a ratio of 1.5 to the feed medium in a ratio of about 1.0.

The amounts of alanine, serine, pyruvate, α-ketoglutaric acid, 2-ketobutyric acid, citric acid, and oxaloacetic acid added to the medium are, for example, about 0.2 to 3.0% U/V.
), preferably 4!0.3 to 1.5% (W/V) is added. Even when two or more kinds of compounds are added, the total amount of each compound is preferably about 0.2 to 3.0% (W/V).

These compounds are preferably added to the feed medium, and when further adding glutamic acid, glycine, etc.
It can be added in the same manner as above.

Culture temperature, culture period, and medium p during actual culture
Culture conditions such as H1 aeration and stirring 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 most cases, the culture temperature is 20 to 37°C, the culture period is 96 to 336 hours, and the pH of the medium is 5.0 to 9.0, and it is preferable to perform the culture aerobically.

Effects and Examples The present invention will be specifically explained below using Examples, but the content of the present invention is not limited thereby.

In addition, for the bacterial strains Cephalosporium acremonium-121 and Cephalosporium acremonium C-18 disclosed in the Examples, F.
FERM-P No. for each RI.

2285 and FERM-PNo. 1430.

Example! cpc when the additive of the present invention is added to the medium.

The amount of produced DCPC is shown.

The microorganisms used were Cephalosporium acremonium -121 for CPC-producing bacteria, and Cephalosporium acremonium C-28 for DCPC-producing bacteria.
The basal medium and feed medium shown in Table 3 below were used, and ammonium thiosulfate as thiosulfate and each compound were added in the amounts shown in Table 1. Culture at 28℃
for 60 hours, followed by shaking culture at 25° C. for 240 hours.

Table 1 shows the amounts of CPC and DCPC produced in the medium.

Table 1 (Similar results were obtained using sodium salt and potassium salt as thiosulfate) As described above, by adding the additive of the present invention to the culture medium and culturing, CPC, which is equivalent to methionine-added culture, DCP
You can get a C main PC.

Example 2 After growing Cephalosporium acremonium -121 on a bouillon slant at 28°C for 120 hours, a liquid medium containing the nutrients shown in Table 2 (pH 7,6) 30
One platinum loopful was inoculated into 0ml and incubated at 28°C for 168 hours! iL
It was cultured. Next, this culture solution was inoculated into the basal medium 1.612 shown in Table 3 in 4Q Jahuamenta.

Furthermore, feed medium 2Q shown in the table was added continuously at a constant rate, and the temperature was 24-32°C.
! After aeration and stirring for 3 days, the CPC content of the culture filtrate was measured. The results are shown in Table 4. Additionally, additives to the basic medium and feed medium are shown in Table 4.

Table 2 Table 3 Table 4 Example 3 After growing Cephalosporium acremonium C-28 on a bouillon slant at 28°C for 120 hours, it was grown in a liquid medium (pH 7,6) containing the nutrients shown in Table 2.
0IIll was inoculated with one platinum loop, and cultured with shaking at 28°C for 168 hours.

Next, the basic medium 1 shown in Table 3 in 412 Jarhuamenta
．． 6 (inoculated with this culture solution.Furthermore, feed medium 2e shown in Table 3 was added continuously at a constant rate,
°(:, pH 6.0 to 7.6. After aeration and stirring for 10 to 13 days, the DCPC content of the culture filtrate was measured.

The results are shown in Table 5. Additionally, additives to the basic medium and feed medium are shown in Table 5.

Effects of the Invention According to the production method of the present invention, cephalosporin antibiotics can be produced in high yield without adding methionine to the culture medium, and the culture time can also be shortened.

Claims (1)

[Claims] In a method for producing cephalosporin antibiotics by culturing microorganisms belonging to the genus Cephalosporium and having the ability to produce cephalosporin antibiotics in a medium, in addition to thiosulfate, alanine, serine, and pyruvate are added to the medium. , α-ketoglutaric acid, 2-ketobutyric acid, citric acid, and oxaloacetic acid.