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

Definitions

• Culture broths containing antibiotics are usually worked up after the maximum content has been reached.
• the first step is to separate cells and solids by filtration or centrifugation. Afterwards, a valuable substance enrichment can take place by extraction or adsorption.
• the purified product is then usually crystallized and can be further processed into semi-synthetic antibiotics.
• CPC Cephalosporin C
• the CPC is already degraded during the fermentation. This can be both purely chemical, attack of water on the ⁇ -lactam ring, and enzymatic, e.g. by esterases (Konecny et al., 1973, J. of Antib., 26, 3, 135-141). Furthermore, a number of by-products, such as deacetoxycephalosporin C (DOCPC) and deacetylcephalosporin C (DCPC), occur during fermentation, which have to be separated off from CPC during the purification. This is associated with considerable losses in yield.
• DOCPC deacetoxycephalosporin C
• DCPC deacetylcephalosporin C
• the object of the present invention is to find a process with which the breakdown of cephalosporin C and the formation of by-products are reduced, and the yield based on the amount of substrate used can be increased.
• the invention therefore relates to a process for the production of CPC, which is characterized in that the fermentation solution is filtered during the fermentation via a cross-flow filtration system and the removed amount of filtrate in the fermenter can be replaced.
• Acremonium chrysogenum (Cephalosporium acremonium), as well as mutants and selectants, as long as they produce CPC derivatives, are suitable for use in the process according to the invention.
• the nutrient solution contains carbon sources such as sucrose, corn starch, dextrose or molasses and nitrogen sources such as soybean meal, peanut meal, malt extract or ammonium acetate.
• the nutrient medium also contains inorganic salts such as sodium hydrogen phosphate, sodium chloride, calcium chloride, calcium sulfate, calcium carbonate, magnesium sulfate or potassium hydrogen phosphate.
• Fat such as methyl oleic acid or soybean oil can also be added to the nutrient medium.
• trace elements such as iron, manganese, copper, zinc, cobalt or other metal salts are also added.
• the cultivation of Acremonium chrysogenum takes place at temperatures between 20 ° C and 30 ° C, preferably at 25 ° C and at pH values between 5 and 8, preferably at pH 7.
• the cultivation is carried out aerobically first in the shake flask and then in the fermenter with stirring and aeration with air or pure oxygen.
• the microorganisms are cultivated in the fermenters over a period of 120 to 240 hours, preferably between 130 and 170 hours. Plate, tube, capillary tube, winding or hollow fiber membrane modules made of polymers, carbon or ceramic with separation limits from the ultrafiltration to the sterile filtration range can be used as the cross-flow filtration system.
• Filtration modules with a pore size of 0.2 / m or 4 nm are preferably used.
• Polysulfones, polyamides, cellulose acetate, aluminum oxide or zirconium oxide can be used as membrane materials.
• the filtration can be carried out continuously or batchwise. It begins about 2 - 3 days after inoculation of the fermenter and can be continued until the end of the fermentation.
• the overflow speed of the fermenter solution over the filtration surface is between 0.5 and 20 m / s, preferably 1 to 10 m / s.
• the filtrate which is withdrawn from the fermenter during the fermentation can be replaced by an appropriate amount of liquid or, after separation of the product of value, e.g. by absorption, the filtrate can be returned to the fermenter.
• water, enriched with appropriate salts or other nutrient components can be pumped in.
• the liquid volume not permeated through the membrane is returned to the fermenter.
• the fermenter and the cross-flow filtration system are connected with appropriate pipes or hoses and sterilized before fermentation.
• appropriate pipes or hoses for a 1001 fermenter, approximately 0.2 m 2 of filtration area is required. Larger and smaller filtration areas can also be used.
• Slant agar cultures are used to inoculate 100 ml of pre-culture medium (500 ml shake flask with 4 baffles). The pistons are at 150 for 48 hours Revolutions per minute (rpm) incubated at 25 to 28 ° C. These cultures are used to inoculate a further preculture (1000 ml medium, 5000 ml flask, 25 to 28 ° C, 120 rpm, 58 to 60 hours). The second preculture is used to inoculate 60 l of fermentation medium in a stirred fermenter. The fermentation takes place at 25 ° C. The gassing is controlled so that the p0 2 is over 20% in the fermentation nutrient solution.
• the filtration begins with a cross-flow ceramic module (Membraflow, ⁇ -AI 2 0 3 ) with a filter area of 0.2 m 2 and a pore size of 0.2 ⁇ m.
• a cross-flow ceramic module Meltaflow, ⁇ -AI 2 0 3
• the following process parameters were observed: Overflow speed: 2 m / s
• Example 2 shows the results after 167 hours of fermentation with cross-flow filtration (B) compared to a parallel fermentation without filtration which was stopped after 142 hours: Table 2:

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• Organic Chemistry (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Life Sciences & Earth Sciences (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Health & Medical Sciences (AREA)
• General Engineering & Computer Science (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Biotechnology (AREA)
• Biochemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Microbiology (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Cephalosporin Compounds (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
• Soy Sauces And Products Related Thereto (AREA)
• Data Exchanges In Wide-Area Networks (AREA)

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• 1991
  • 1991-08-21 DE DE4127648A patent/DE4127648C1/de not_active Expired - Fee Related
• 1992
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  • 1992-08-08 WO PCT/EP1992/001811 patent/WO1993004188A1/de not_active Ceased
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• 1994
  • 1994-02-18 FI FI940778A patent/FI103988B1/fi active
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