SK19794A3 - Method for the fermentative production of cephalosporin "c" using acremonium chrysogenum - Google Patents

Abstract

Described is a method for the fermentative production of cephalosporin C, the fermentation solution being filtered during fermentation through a cross-flow filtration system. The filtrate obtained from the fermenter can be used again.

Description

Technical field

The invention relates to a process for the fermentative production of cephalosporin C from Acremonium chrysogenum.

BACKGROUND OF THE INVENTION

Treatment of the culture medium containing the antibiotics is generally carried out only after the maximum antibiotic content in the culture medium has been reached. Usually, as a first processing step, the cells and solids are separated by filtration or centrifugation. Subsequently, the liquid medium of the culture medium is enriched by the substance or substances which, if appropriate, are the target of the cultivation process, this enrichment being carried out by extraction or adsorption. The product thus purified is then generally allowed to crystallize and then further processed to semi-synthetic antibiotics.

In this processing method, which involves several subsequent processing steps, the instability of the melts of many antibiotics, such as cephalosporin C, is disadvantageous. enzymatically, for example by the action of esterases (Konečný et al., 1973,

J. of Antib., 26, 3, 135-141). Furthermore, a number of by-products such as deacethoxycephorosporin n C and deacetylcephorosporin n C are formed during fermentation, which must be separated during purification of cephalosporin, resulting in a significant reduction in the yield of cephalosporin C.

Cross-flow filtration systems with polymeric or ceramic membranes have already been used to treat antibiotic-containing culture media (Harris et al., J. Chem. Techn. Biotechnol., 1988, 42, 19). 30). However, it has not yet been found that the decomposition of cepha 1osporin C can be limited in this process.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a process in which the decomposition of cephalosporin C and the formation of flawed products is reduced and an increase in the yield of cephalosporin C relative to the amount of substrate used can be achieved.

Surprisingly, it has now been found that by using a cross-flow filter module during fermentation of Acremonium chrysogenum, the yield of cepha 1osporin C is increased, the formation of deacetylcephalosporin C is reduced and production time can be extended. This filtration and low formation of deacetylcephalosporin C also greatly simplifies the processing of cephalosporin C.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a process for the production of cephalosporin C, wherein the fermentation solution is filtered through a cross-flow filtration system during fermentation, and the amount of filtrate collected can be replaced in the fermenter.

With regard to the production of cephalosporin C derivatives, Acremonium chrysogenum (Cephalosporium acremonium), mutants and selectants thereof are suitable for use in the method of the invention.

The nutrient solution comprises 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 medium contains such inorganic salts as sodium hydrogen phosphate, sodium chloride, calcium chloride, calcium sulfate, calcium carbonate, magnesium sulfate or potassium hydrogen phosphate. In addition, a fat such as methyl oleic acid or soybean oil may also be added to the culture medium. In addition, trace elements such as iron, manganese, copper, zinc, cobalt in the form of salts or salts of other metals may also be added.

The cultivation of Acremonium chrysogenum (Cephaosporum um acremonium), preferably DSM 6473, is carried out at a temperature of 20 to 30 ° C, preferably at a temperature of 25 ° C and at a pH between 5 and 8, preferably at a pH of 7. This cultivation is in a shake flask and then in a fermenter with stirring and aeration with air or pure oxygen. The cultivation of the microorganisms in the fermenter is carried out for 120 to 240 hours, preferably for 130 to 170 hours.

As cross-flow filtration systems, polymeric, hydrocarbon or ceramic modules consisting of membranes of platelets, tubes, capillaries, coils or hollow fibers and having a separation threshold corresponding to a separation threshold of 1 to 8 and to sterile may be used. . Preferably, filter modules with a pore size of 0.2 µm or 4 nm are used. Polysulfones, polyamides, cellulose acetate, alumina or zirconia can be used as materials for making these membranes. The filtration can be carried out continuously or discontinuously. Filtration is initiated about 2 to 3 days after inoculation of the fermenter and may be continued until the end of the fermentation process. The flow rate of the fermentation solution through the filter surface is 0.5 to 20 m / s, preferably 1 to 10 m / s.

The filtrate which is removed from the fermenter during fermentation can be replaced by the corresponding amount of liquid or the filtrate can be returned to the fermenter after separation of the desired product, for example by absorption. It can be pumped with water enriched with corresponding salts or other constituents of the medium.

The volume of liquid that did not permeate across the membrane is also returned to the fermontor.

The fermenter and the cross-flow filtration system are connected by corresponding tubes or hoses which have been sterilized prior to fermentation. For a 100 L fermenter, about 0.2 m ^{and a} filtration area are needed. However, larger and smaller filtration areas may also be used.

In the following, the invention will be further elucidated by means of examples of specific embodiments thereof, which are intended to be illustrative only, and are not intended to limit the scope of the invention as defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Fermentation of Acremonium chrysogenum DSM 6473

This fermentation is carried out in the following nutrient solution:

Pre-heating medium / 1

Maize leach11,75

Ammonium acetate4,5

Sacharóza.20,0

Calcium sulfate dihydrate 0.5

Magnesium sulphate heptahydrate 0.5 pH 7.0 (adjusted 15% (w / w) NaOH)

Fermentation environment 9/1 Fat-free groundnut flour Ammonium acetate Glucose monohydrate Mety 101eát D, L-Methionine 100.0 6, 0 5.0 5.0 3.0 Calcium sulphate dihydrate Magnesium sulphate heptahydrate Calcium carbonate Odpeňovad10 5.0 5.0 5.0 0, 5

Solution to supplement the fermentation broth

Glucose monohydrate D, L-Methionine 500.0 24.75

To inoculate 100 ml of pre-culture medium, use:

live slant agar cultures (shaking and agar) bank 0 content 500

ml with 4 bullying). These flasks are incubated for 48 hours at room temperature

150 revolutions per minute and temperature.25 to Deň: 28 ° C. These cultures

are then used to inoculate another preculture medium (1000 ml of medium in a 5000 ml flask is incubated at 120 rpm at 25-28 ° C for 58-60 hours). 60 liters of fermentation broth are inoculated in the mixed fermentation broth. This fermentation is carried out at 25 ° C. The aeration is regulated such that pO? in the fermentation broth is greater than 20%.

After 74 hours, filtration begins through a cross-flow ceramic module (commercially available from Membraflow, alphaA12O3) having a filtration area of 0.2 m ² and a pore size of 0.2 µm. This filtration is carried out under the following operating conditions:

flow rate: 2 m / s pumping speed: 1500 1 / h filtration power: 2 1 / h a f i 1 traction time: 68 h.

The following table shows the results of the experiment after 142 hours of fermentation without cross-flow filtration (A) and using cross-flow filtration (B).

Table 1

Cefalo- Deacetyl- Productivity cefaloProduct of cephalosporin cephalosporin deacetylcephine / losporin cefa 1osporin Max.

After 6 days

After 6 days

U) (%) (%)

A 100

B 140

100 100

200

100

400

100

Example 2

The fermentation was carried out as in Example 1. Table 2 shows the results after 167 hours of fermentation using cross-flow filtration (B) as compared to para-

fermentation without filtration, hours. Table 2 which ended after 142 cephalosporin (WITH) Deacetylcephalosporin / cephalosporin (x) A (142 h) 100 100 B (167 h) 129 69

Claims (8)

Process for the fermentative production of cephalosporin (1), characterized in that the fermentation solution is filtered through a cross-flow filtration system during fermentation, wherein the amount of filtrate collected can be replaced in the fermenter. Method according to claim 1, characterized in that Acremonium chrysogenum is used. Method according to claim 1 or 2, characterized in that a polymeric or ceramic filter is used as the cross-flow filtration system. Method according to one or more of Claims 1 to 3, characterized in that the filter has a pore size of between 4 and 200 nm. Method according to one or more of Claims 1 to 4, characterized in that the flow rate through the filtering surface is 1 to 10 m / s. Method according to one or more of Claims 1 to 5, characterized in that the filtrate is replaced with water in the ferment. Method according to claim 6, characterized in that the filtrate is replaced by a nutrient solution. A process according to claim 6, characterized in that the filtrate is returned to the fermenter after removal of the desired product.