US3929577A - Production of cephalosporin C - Google Patents

Production of cephalosporin C Download PDF

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Publication number
US3929577A
US3929577A US510861A US51086174A US3929577A US 3929577 A US3929577 A US 3929577A US 510861 A US510861 A US 510861A US 51086174 A US51086174 A US 51086174A US 3929577 A US3929577 A US 3929577A
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US
United States
Prior art keywords
cpc
cephalosporin
cephalosporium
atcc
polyploid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
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US510861A
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English (en)
Inventor
Toshihiko Kanzaki
Tadazumi Fukita
Yoichiro Kono
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Takeda Pharmaceutical Co Ltd
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Takeda Chemical Industries Ltd
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
    • C12P35/06—Cephalosporin C; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/44—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D317/46—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D317/48—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring
    • C07D317/62—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to atoms of the carbocyclic ring
    • C07D317/64—Oxygen atoms
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/911—Microorganisms using fungi
    • Y10S435/925—Cephalosporium
    • Y10S435/926—Cephalosporium acremonium

Definitions

  • This invention relates to the production of cephalosporin-C (hereinafter abbreviated as CPC) by fermentation processes, and is particularly concerned with processes for the production of CPC using a CPC- producing polyploid mold belonging to the genus Cephalosporium.
  • CPC cephalosporin-C
  • the fermentation yields are 760 Lg/ml at best in the case of the Japanese patent application laid open No.1828/l971, about 500 ug/ml in the case of US. Pat. No. 3,082,155 and, even in the most satisfactory of all the cases heretofore reported, Japanese patent application laid open No. 26986/1973, the attainable potency is not more than 5,000 ug/ml.
  • the present inventors have attempted to find a way to produce CPC in an increased yield through intensive research directed to a genetic improvement of micoorganisms, and have ultimately found that molds possessing excellent CPC-producing abilities can be obtained at a higher frequency from among polyploid molds induced by polyploidizing treatment of CPC-producing microorganisms belonging to the genus-Cephalosporium, than from among other mutants induced by other conventional mutation treatment, and that some of the polyploid molds are able to accumulate CPC extracellularly in unprecedentedly high concentrations.
  • This invention is directed to a method for the production of CPC comprising cultivating the polyploid microorganisms induced from parent microorganisms of the genus Cephalosporium having CPC producing ability in a culture medium to let the microorganism accumulate CPC extracellularly.
  • any suitable CPC producer of the genus Cephalosporium for example, Cephalosporium acremonium or Cephalo- 5 ATCC-14553, etc., and various mutants induced from these strains.
  • the FERM-P and ATCC numbers indicate the accession number of the organisms at the Research lnstitute of Fermentation of the Agency of Industrial Science and Technology, Chiba, Japan and at American Type Culture Collection, Rockville, Md., U.S.A., respectively.
  • One of them is the procedure comprising polyploidizing microbial cells with a chemical agent.
  • a chemical agent there may be mentioned camphor, acenaphthene, colchicine, etc. While the conditions of treatment vary with the particular mold, type of chemical agent and other factors, the following procedure generally can be followed with success.
  • camphor or acenaphthene for instance, 1 microbial cells are exposed to a vapor'of the chemical for a few to tens of hours, after which they are removed from the ambient vapor and further cultivated.
  • the cells are cultivated from the beginning on an agar plate containing the chemical. The grown cells taken from the resulting colonies are examined, e.g., under a microscope. The colonies made up of giant cells are selectively taken.
  • a polyploid mold can be obtained by the method described in Journal of General and Applied Microbiology 2, 345(1956). These procedures cannot only be followed in repetition but can be followed in combination with other methods of breeding and selecting some specific microbial strains and, in such instances, im-
  • polyploid strains thus obtained can be easily isolated, if desired, by a conventional pure-isolation method, for example by micromanipulation.
  • Cephalosporium acremonium ATCC- 14553 as a parent strain was treated with camphor and the CPC-producing abilities of the resulting polyploid strains were investigated.
  • the investigation resulted in the finding of the number of strains which can accumulate CPC in the medium in the increased yields, for
  • the [F0 number indicates the accession numbers of the organisms at the lnstitute for Fermentation, Osaka,
  • Table 1 Size of Number of DNA content Amount of DNA arthrospore nuclei per per per nucleus Major Minor arthrospore arthrospore Strain diameter diameter (A) (B) (BIA) (n) (u) (us) (#2) Cephalosporium Tablel-continued Size of Number of DNA content Amount of DNA arthrospore nuclei per per per nucleus Major Minor arthrospore arthrospore Strain diameter diameter (A) (B) (B/A) (F) (n) (us) (as) acremonium 7.2 3.6 1.0 0.30 X 0.30 X 10" ATCC-14553 I Cephalosporium acremonium 11.2 4.7 3.2 1.98 X 10 0.62 X 10 2M-l6 (Note: All the values given in the above table are the 1 results on analysis of the arthrospores obtained after 2 many superior CFC-producing strains which can accumulate CPC extracellularly in the concentration of, for
  • throspore and the number of nuclei are the mean of The taxonomic characters of this strain were found to results for approximately 100 arthrospores'The mean be essentially identical with the characters of Cephalodimensions of conidia are 3.5 p X 7.1 p. in the case of sporium sp. ATCC-14553 except the differences noted ATCC-l4553 and 4.3 a X 8.6 u in the case of 2M-l6). in Table 2.
  • the two strains to be analysis. of the conidia harvested from a slant culture crossed are differently marked beforehand, e.g., with grown by the procedure set forth in the footnote to different amino acid or vitamin requirements. They are Table 1.
  • the dimensions of conidia and the number of crossed and grown on a minimal medium e.g., a menuclei are the mean values for about 100 spores.
  • dium made up of 3.0 sucrose, 0.3 NH N0 ,0.l
  • the 00- Cephalosporium or those of the genus Emericellopsis nidia of grown colonies are collected and plated onto a can be employed to advantage.
  • minimal medium such as the one indicated above and As the carbon sources that can be. employed, there the colonies formed are investigated. Most of the may be mentioned, for example, glucose, sucrose, growth consists of polyploid strains. starch, soluble starch, waste molasses, n-paraffins, If necessary, these methods cannot only be practiced acetic acid, methanol, ethanol, etc.
  • metal methionine-leucine-requiring mutant, and Cephalosposalts as the 80,, Cl, N0 CO P0 and other salts of rium acremonium AP-78, an arginine-phenylalanine- Na, K, Ca, Mg, Mn, Zn, Fe, Cu, etc. requiring mutant, both induced from Cephalosporium
  • acremonium ATCC-14553 as the parent strain, a numamino acids (e.g., methionine, cysteine, serine), thiosulfates, fatty acid esters, oils, e.g., lard oil, olive oil,
  • aerated submerged culture is generally advantageous.
  • the temperature is desirably within the range of 18 to 35C. Satisfactory results are obtained when the pH is maintained within the range of about 2 to and, preferably, between 4 and 9. A cultivation time of 3 to days is sufficient.
  • Most of the CPC elaborated occurs in filtered broth and, therefore, to recover the antibiotic, it is advantageous to remove the cells from the broth by centrifugation or filtration and, then, purify CPC from the supernatant or filtrate. To isolate the CPC, the procedures generally used for the fractional recovery of CPC can be followed with success.
  • the desired result can be obtained with advantage by utilizing an ion exchange resin, activated carbon, nonionic copolymer resin, gel filtration medium, etc. in a suitable combination.
  • an ion exchange resin, activated carbon, nonionic copolymer resin, gel filtration medium, etc. in a suitable combination.
  • a Sakaguchi flask of 2-liter capacity is filled with 500 ml of an inoculum medium made up of 3.0 of sucrose, 1.5 of meat extract, 0.5 of corn steep liquor and 0.15 of CaCO and, after sterilization, inoculated with Cephalosporium acremonium 2M-16.
  • the flask is incubated on a reciprocating shaker at 28C for 3 days.
  • a 50-1iter fermentation tank of stainless steel is charged with 30 liters of a medium made up of 6 of sucrose, 5 of glucose, 3 of peanut cake, 3 of soybean meal, 1.0 of DL-methionine and 0.15 of CaCO and the medium is sterilized and cooled in the routine manner.
  • the medium is then aseptically inoculated with the above seed culture and incubated at 28C under sparging and agitation (30 l. per min. aeration, 250 rpm.)
  • the fractional isolation of CPC is carried out in the following manner.
  • the filtrate (25 l.) is run down a column of activated carbon (20 1.), whereby the CPC is adsorbed on the carbon.
  • the carbon is first washed well with water and, then, eluted with 5 aqueous butanol containing 0.01 N NaOH, whereupon 31 l. of CPC fractions are obtained.
  • These fractions are pooled, concentrated and neutralized to pH 7.0 with NaOH. Finally, ethanol is added to the concentrate to a concentration of 50 (V/V).
  • the potency of CPC is measured by an enzymatic assay method using the cephalosporinase of Aerobacter cloaceae [Biochemical Journal, 116, 385 (1970)].
  • EXAMPLE 2 The same cultivation as in Example 1 is carried out, except that the strain used is Cephalosporium aoremonium LA-101.
  • the potency of CPC in the filtered broth is 7,900 pg/ml.
  • a process for producing cephalosporin C which comprises cultivating Cephalosporium acremom'um ATCC 20425 or Cephalosporium acremonium ATCC 20426 in a culture medium containing an assimilable carbon source and a digestible nitrogen source, to accumulate cephalosporin C in the culture medium, and recovering the accumulated cephalosporin C from the culture medium.
  • microorganism is Cephalosporium acremonium ATCC-20425 3.
  • microorganism is Cephalosporium acremonium ATCC- 20426.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
US510861A 1973-09-28 1974-09-30 Production of cephalosporin C Expired - Lifetime US3929577A (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP10968073A JPS577719B2 (enrdf_load_stackoverflow) 1973-09-28 1973-09-28

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US (1) US3929577A (enrdf_load_stackoverflow)
JP (1) JPS577719B2 (enrdf_load_stackoverflow)
BE (1) BE820456A (enrdf_load_stackoverflow)
CH (1) CH606014A5 (enrdf_load_stackoverflow)
DE (1) DE2445616A1 (enrdf_load_stackoverflow)
FR (1) FR2245763B1 (enrdf_load_stackoverflow)
GB (1) GB1488822A (enrdf_load_stackoverflow)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4368263A (en) * 1978-04-26 1983-01-11 Glaxo Group Limited Production of cephalosporins
US5215620A (en) * 1989-09-19 1993-06-01 Shin-Etsu Handotai Co. Ltd. Method for pulling a silicon single crystal by imposing a periodic rotation rate on a constant rotation rate
CN102808012A (zh) * 2012-08-28 2012-12-05 伊犁川宁生物技术有限公司 一种头孢菌素c的发酵方法以及顶头孢霉发酵培养基
CN102808013A (zh) * 2012-08-28 2012-12-05 伊犁川宁生物技术有限公司 一种头孢菌素c的发酵方法
CN102808011A (zh) * 2012-08-28 2012-12-05 伊犁川宁生物技术有限公司 一种头孢菌素c的发酵方法

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5842515A (ja) * 1981-09-08 1983-03-12 Sumitomo Heavy Ind Ltd コンベア制御装置
US4520101A (en) * 1983-03-07 1985-05-28 Bristol-Myers Company Production of cephalosporin C

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3776815A (en) * 1970-01-21 1973-12-04 Ciba Geigy Corp Process for the manufacture of cephalosporin c
US3816257A (en) * 1971-11-15 1974-06-11 Takeda Chemical Industries Ltd Method for producing cephalosporin c

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3776815A (en) * 1970-01-21 1973-12-04 Ciba Geigy Corp Process for the manufacture of cephalosporin c
US3816257A (en) * 1971-11-15 1974-06-11 Takeda Chemical Industries Ltd Method for producing cephalosporin c

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4368263A (en) * 1978-04-26 1983-01-11 Glaxo Group Limited Production of cephalosporins
US5215620A (en) * 1989-09-19 1993-06-01 Shin-Etsu Handotai Co. Ltd. Method for pulling a silicon single crystal by imposing a periodic rotation rate on a constant rotation rate
CN102808012A (zh) * 2012-08-28 2012-12-05 伊犁川宁生物技术有限公司 一种头孢菌素c的发酵方法以及顶头孢霉发酵培养基
CN102808013A (zh) * 2012-08-28 2012-12-05 伊犁川宁生物技术有限公司 一种头孢菌素c的发酵方法
CN102808011A (zh) * 2012-08-28 2012-12-05 伊犁川宁生物技术有限公司 一种头孢菌素c的发酵方法
CN102808013B (zh) * 2012-08-28 2013-10-16 伊犁川宁生物技术有限公司 一种头孢菌素c的发酵方法

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FR2245763A1 (enrdf_load_stackoverflow) 1975-04-25
DE2445616A1 (de) 1975-04-03
JPS577719B2 (enrdf_load_stackoverflow) 1982-02-12
BE820456A (fr) 1975-01-16
GB1488822A (en) 1977-10-12
JPS5058289A (enrdf_load_stackoverflow) 1975-05-21
FR2245763B1 (enrdf_load_stackoverflow) 1976-12-31
CH606014A5 (enrdf_load_stackoverflow) 1978-10-13

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