SU582772A3 - Method of preparing deacetoxycephalosporinc - Google Patents

Description

I

This invention relates to microbiological severity, namely the production of antibiotics.

A known method of producing deacetoxycephalosporin C by chemical means is 1.

However, this method is complicated and time consuming.

In order to simplify the method of producing deacetoxycephalosporin C of the formula

b

Oh and

Sy- (P1,) z-s4t-A

Tsho

Shz

soy.

strain - producer of penicillin N, belongs to the genus Ceph, atosporlum Emertcettopsis, Scopula gSor, Paecitomy ses or B1Ke1e "Hogs1"

cultivated under aerobic conditions on a nutrient medium containing sources of nitrogen, carbon and mineral salts, and then the desired product is isolated from the culture fluid.

From the genus dephaEosporLum. choose strains

dephatospoKuTTi Korda NRR-t.

Cepkatosporcum chrysogftnum YASS

Cepttatosporium up, NRRL

CephaEosporium sp. NR.KL

CepKatosporiom 6p.NR.Tlb

daphatosporCum sp, NRB.L

CepKci ospDritjm lp. NR.R.L.

Ce.ph.a ObporCuni sp. fyfRRL

CupKaCosporCum sp. NR.R.L

CeptiatobporLUTn. bp. NRRt

CepKatosf orium sp, NR.KL,

CcpKatospoKum bp.Kft.Rb

From the genus EmerCcettopstsraut strain fcJ:

bmerLce op "is sp. NRE.U

5446, Emericef otisis sp. 5713, Emari-ct opsrs sp. NRfib 5714 or Emerice opSS sp. NRRt. 5717.

From the genus ScoputaT-LOps.LS, choose the Strain ScoputariopsiS er. NRRb 5715, from the genus PacCLfomifces, select the strain: Carriftus strain

NRRL. 5711, and from the genus DLHetegoEroch and choose the strain DLfteterospora cKEaiTiLdospara NRRb 5728.

The proposed strains are known, described in the literature and they are used

for the preparation of penicillin N (cephalosporin L).

The product strains were cultured with stirring in 1 L flasks and a small amount of deacetoxycephalosporin C was obtained. To obtain a large amount of strain, they were cultivated in large-size fermenters under conditions of deep aerobic fermentation.

To obtain deacetoxycephalosporin C, the body spores are kept on oblique agar. Spores from oblique nagar are used for (grafting medium-volume solvent solvents. The vegetation medium is kept in a thermostat until the culture of the microorganism is replenished. This vegetation medium is then used as a grafting material for the fermentation medium used in large quantities.

In some cases, the vegetative medium is additionally introduced as a graft material for the fermentation medium. Such a secondary vegetation medium is usually used if the volume of the fermentation medium is significantly greater than the volume of the first vegetation medium. A large-volume vegetation medium is then introduced with a suitable concentration of the microorganism to initiate the beginning fermentation in large-sized fermenters. The vegetation environment may have the same composition as the fermentation medium or it may contain additional components for the growth and development of microorganisms.

The formation of deacetoxycephalosporin C occurs at a pH of from 6.2 to 8.0, preferably a pH of 5-7.5.

Cultivation is carried out at a temperature of 30-35 s. The optimal yield of deacetoxycephalosporin C occurs at a temperature of 26 ° C.

The maximum yield of deacetoxycephalosporin C during the cultivation of the strain in large containers occurs within 4-7 days, but if the cultivation is given in 1 l flasks, the strain grows much faster and the production of deacetoxycephalosporin C occurs in 2-3 days.

If the pH of the enzyme medium reaches 8.0 or higher, this adversely affects the yield of deacetoxycephasosporin C, therefore it is necessary to control the time and pH value of the fermentation medium during the fermentation process. The pH of the medium is lowered by adding a suitable acid or a suitable buffer to the fermentation medium.

The presence of deacetoxycephalosporin C in the fermentation broth is determined by chromatography, as well as by means of bioautographs. As

defining organism for bioautographs use pseudomonos boylsmgeij

Sterile air is passed through the culture medium to increase the release of deacetoxycephalosporin C. The volume of air passing through the culture medium is 0.2 ob.h. air and 1 min per 1) culture medium.

In order to extract deacetobbitfaco spbrin C from the fermentation medium, the entire fermentation broth is treated during short time in an acidic medium to decompose some of the accompanying impurities.

6

Deacetoxycephalosporin; C is separated from the other components of the fermentation medium by chromatography on an ion exchange resin, and then purified by chromatography on cellulose or silica gel, followed by precipitation and recrystallization.

First, the filtered fermentation broth is pre-purified by extraction with an organic solvent that is not miscible with water, for example n-butanol or amyl acetate, in order to remove impurities. Extracted Broth Purified by Chromatography

0 on activated carbon, and the column is washed with water to remove water. soluble colored impurities and water-soluble inorganic substances. The fermentation products are then eluted from the coal with a 50% acetone / water mixture.

The eluate is concentrated to an aqueous phase, which is then chromatographed over a basic anion exchange resin. Resins use hydroxyl, acetate or formate type, polystyrene resin type of quaternary ammonium or other suitable types. Concentrated liquate coming from an activated carbon column is poured onto an anion exchange resin, after which the resin is washed with water. Deacetoxycephalosporin C is peeled from the exchange resin as a salt with a suitable weak base, for example,

0 When using an acetate type resin column, an aqueous solution of sodium acetate at a concentration of Hi or less is used as the eluting solvent. To prevent the formation of an excess of the inorganic salt (acetate) in the zlute from the exchange column, the concentration of sodium acetate should be 0.10, 5 n.

ABOUT

When using a formate or other suitable type of exchange resin, use the corresponding 1N salt as a solvent for elution, for example,

5 formate ammonium. Numerous fractions of sobirkkot, and fractions containing deacetoxyFalospoinS, combine

The combined eluates are chromatographed over activated carbon to remove excess inorganic salts, such as sodium acetate, used as an elution solution. The activated carbon column is rescued with water to remove inorganic water-soluble salts, and deacetoxycephalosporin C is eluted from the column with a 50% mixture of acetate and water. The eluate is evaporated to dryness or evaporated to remove acetone, and the concentrated aqueous solution is lyophilized and deacetoxycephalosporin C is obtained as a crude solid.

The crude product of deacetoxycephalosporin C is further purified by chromatography over cellulose or silica gel or over another suitable non-ionic adsorbent. Deacetoxycephalosporin C is eluted from the column with a solvent containing acetonitrile and water (80:20), and numerous fractions are collected. Fractions containing deacetoxycephalosporin C are determined by paper chromatography, combined and concentrated to a small volume or to dryness. The highly concentrated aqueous residue or dry residue is then dissolved in a minimum amount of isopropyl alcohol, and the alcohol solution is poured into a large amount of diethyl ether.

Purified deacetoxycephalosporin is obtained in the form of a salt corresponding to an aqueous eluting agent used to elute the anion exchange resin. For example, using sodium acetate as an eluent, deacetoxycephalosporin C is obtained as a monosodium salt.

The deacetoxydephalosporin C in the form of a salt is filtered and dried. The salt of deacetoxycephalosporin C can be converted into a free acid, for example, by passing through a cation exchange resin.

Chromatography to identify deadetoxycephalosporin C in untreated fermentation broths or in the fractions of eluates of the resins is carried out on Whatman paper on a chromatographic paper using an upstream method. The solvent system used for the development consists of acetonitrile and water (80:20 v.p.); the chamber is saturated with solvent vapors of the following composition: p-propanol - pyridine acetic acid.-acetonitrile-water in the ratio 45: 30: 9: 40: 36 (parts by volume). To simplify chromatography, the test sample is pre-treated with penicillinase.

Example 1. Separated spores of Cr | ha ospOT-ium & p. NRRL 5445, Cepka QsporCum ch.rySQgenum ATCC 14 615, pT4ericeetopsi.s sp. NR-RL 5446 and Emarice-EEopSLS sp. NRRL

5447 no separately grafted on oblique agar with medium of the following composition,%:

Lactose1,000

Glycerin1,000

Soy Peptone0,250

Soluble dried corn and malt0,250

Magnesium sulphate heptahydrate 0.050 Ferrous sulphate ferrous sulfate 0.001 Calcium carbonate 0.200 Trace element solution O, 625 Agar 2,000 Water Remaining Oblique agar is incubated for 7 days at a temperature. Mature cultures are poured with sterile distilled water and gently scraped with a sterile stick to obtain a spore suspension.

The resulting spore suspensions are used for grafting two separate sterile vegetation media of the following composition,%:

Soya flour2,000

Malt Extract2,000

Corn extract0,500

Magnesium sulfate heptahydrate 0.025 monopotassium phosphate0,100 dipotassium phosphate0.050 calcium chloride dihydrate 0.010 trace element solution0.625 water Remaining

Adjust the pH to 6.5 before autoclaving. The grafted vegetation medium is thermostated for 48 hours at 26 ° C using a rotating agitator. These media are then used as a graft material for the fermentation medium, at a ratio of 1% of the growing season to 1 part by volume. fermentation. The media used for this purpose have the following composition,%:

Sucrose4.0

Glycerin1,0

Sodium glutamate 0.5

Peanut Flour2.0

Hexahydrate of double ammonium sulfate and ferrous iron0.3 Potassium nitrate 2.0

Calcium Carbonate 0.3

Osaal water

During the fermentation of a large amount of product, surface active substances or antifoaming agents are used. The pH of the medium is adjusted to 6.5 before sterilization. Then the unvaccinated fermentation medium is incubated for 5 days at 26 ° C and with stirring. During the fermentation, air is passed through the fermentation medium at a rate of 1/2 volume per 1 volume of culture medium per minute. The final pH of fermentation media is 7.5. 15 liters of the resulting broth are acidified to pH 2 with sulfuric acid. The acidified whole broths are stirred for 1 hour at room temperature, then alkalinized to pH 6.0 with sodium hydroxide solution.

The broths are filtered off and the aqueous filtrate is extracted with p-butanol to remove insoluble substances in suspension. The aqueous phases are then passed through columns filled with activated carbon, and the columns are distilled water. The eluate prokivnyh waters discarded. Next, the columns are eluted with 50% aqueous acetone. The eluate is evaporated under vacuum to an aqueous phase, which is chromatographed in columns filled with polystyrene resin containing quaternary ammonium and acetate type groups. The columns are first washed with water and the washings are removed.

Active fermentation products elute from the column with 0.15 n. sodium acetate solution. Numerous fractions are collected and fractions containing deacetoxycephalosporin C are combined.

The presence of deacetoxycephalosporin C in eluate fractions was determined by chromatography using an upstream method using Whatman paper.

A mixture of acetonitrile and water (80:20) is used as the eluting solvent. A mixture of solvents containing 300 ml of the composition solution,%: p-propanol 37.5, pyridine 25, acetic acid 7.5, water 30, which is diluted to 100 ml with acetonitrile, is layered on the bottom of the chromatographic chamber.

Deacetoxycephalosporin C is also determined by the microorganism Pseudomo-nou bofancearcum. The obtained fractions of deacetoxycephalosporin C after chromatography are combined. The combined fractions are absorbed on a column filled with activated carbon. The column is washed with distilled water to remove soluble inorganic substances, for example sodium acetate, and then eluted with 50% aqueous acetone. vacuum under vacuum. A crude solid preparation of the deacetoxycetafaposporin C sodium salt is obtained, which is purified by chromatography on a column filled with cellulose.

The antibiotic is eluted from the cellulose column with a mixture of solvents containing acetonitrile and water (80:20). Numerous fractions are collected, and using the chromatographic system described above, the fractions in which cephalosporin C is detected are combined. The combined fractions are concentrated to dryness, and the dry residue containing deacetoxycephalosporin C is dissolved in a minimum amount of isopropanol. The alcohol solution is poured into ether, the amount of which is 20 times the volume of isopropanol. When stirred, a precipitate of the deacetoxycephalosporin C sodium salt is formed. It is filtered and dried in air. 126 mg of antibiotic are obtained in the form of sodium salt.

Example2. According to the method described in example 1, the controversy EmcriceftopSLS lp. NRRL 5714 cultivated in a growing environment. The culture medium used for grafting the production medium has the following composition,%:

Soluble starch 1.0 Dextrose4.0

Soyeva krupa1,0

Calcium carbonate 0.5 Antifoam 0.1 Water Else

The pH of the production medium was adjusted to 6.5 before autoclaving and grafting. Fermentation is carried out for 5 days at 26 ° C. Deacetoxycephalosporin C is recovered and recovered according to Example 1.

Example 3. Analogously to Example 1, ScopyCarCopsis LR-NRRU 5715 spores are cultivated on the vegetative medium described in Example 1. The vegetation medium is used for grafting fermentation medium, the composition of which is also described in Example 1.

Fermentation is carried out for 5 days at, and deacetoxycephalosporin C is removed from the fermentation medium according to example 1.

P RIM e p. Analogously to Example 1, POWiEom y es was cultivated and fermented to obtain deacetoxycephalosporin C.

EXAMPLE 5. DeKeterospOY-a ckCawy dnapo Ca was obtained using the same method of cultivation and isolation, as well as using the medium described in Example J, to produce deaietoxycephalosporin C.

Claims (2)

1. A method for producing deacetoxycephalosporin C of the formula HE HOOC-CH- (CH2) 3-C-i YaND soon characterized in that, in order to simplify the method, the strain producing penicillin K, belonging to the genus depkatosporCuTn, ScopyfartopsCa, EmftrictpeopsilS, Paeci fonriMces .DLheterospora is cultivated under aerobic conditions on a nutrient medium containing sources of nitrogen, carbon and mineral salts, and then the desired product is separated from the culture fluid. 2. Fsoba POP.1, about aphids and rota and with the fact that a strain is selected from the genus Ceph.atoSporLum; Korda NRRL 5445, c.Krysog-enum ATCC 14615, sp. fJRRL 5712, sp klRRL 5716, 6p NRRL 718, CeptiaEosporiutn sp, KRRL5719, CepfiaEosportum sp. NR.RL5720, Cephatosporiom sp. HRRL5721, CepfiaEasporLum sp.HRRu5722, Ccphaeosportum sp WRRL5723, Cephatosporium 6p NRRL5724 or C ephaEobpotium sp. NRRU5725, from the genus Erne rice sis knock out the strain: EmerCcegfopSLS sp. NRRb5446, EmerLcetEopSLs sp. NRRL5447, EmericettopSLS sp. NRRL, 5713, EmerCceftopSLS sp. NRRL5714 or EmericeEtopsLS sp. NRRL5717, from the genus ScDpuEartopsis choose the strain ScopufariopSLS sp. NRRU 5715, from the genus PaecilEoTnyces, choose the strain Paecuiomyces carTi "us NRRL 5711, from the genus DLKettrospora choose the strain DihetCi ospora tWamydospora NRRL5728. Priority no to items and features. 04/26/72 according to claim 1 and on the grounds of paragraph 2 regarding the use of strains 5445, 14615, 5446, 5447; 09. 04., 73 for the rest of signs p. 2. Sources of information taken into account in the examination: 1. US patent 3124576, CL. 260-243,1964.