DE1227192B - Process for the preparation of cycloserine - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

GERMAN

PATENT OFFICE

EDITORIAL

Int. CL:

Number:
File number:
Registration date:
Display day:

C12d

German class: 30h-6

1227 192
C 25343IV a / 30 h
October 24, 1961
October 20, 1966

The invention relates to a process for the production of cycloserine by culturing Streptomyces orchidaceus. ·

Cycloserine is an antibiotic with a broad spectrum of activity against both gram-negative and gram-negative drugs also against gram positive bacteria and has been found to be particularly effective for the treatment of tuberculosis and male urinary tract infections. - ν,

According to German patent specification 958 242, it is known to produce cycloserine by cultivating the microorganism Streptomyces orchidaceus under submerged conditions in an aqueous, calcium carbonate containing nutrient medium at 20 to 30 ° C. As nitrogen sources for the nutrient medium So far, proteins of plant or animal origin of undefined composition were used used as alfalfa flour, amino acid cake, milk solids, whey nutrients, dried Wheat Sludge and Casein / Soybean products such as soybean meal have been most successful. The use of these vegetable or animal substances of indefinite composition as nitrogen sources, however, has the consequence that the nutrient medium numerous impurities contains, which have to be removed in cumbersome procedures for the purification of the cycloserine. For example, when using soybean products, the fermentation mash has a dark color and contains a large amount of residues from non-assimilable soybean materials. These impurities have to be removed through time-consuming cleaning processes with the help of ion exchangers, Animal charcoal and falling over of the cycloserine salts removed. In this way, the process for the preparation and work-up of the cycloserine not only cumbersome and time-consuming, but larger amounts of the antibiotic are also possible, normally more than 25% lost, so that the final yield of cycloserine is greatly reduced will.

It is also known in the cultivation of other microorganisms for the production of antibiotics in addition to proteins of vegetable or animal origin, urea or ammonium salts Add nutrient medium. Finally, one has to produce in the cultivation of Streptomyces arsitiensis of arsimycin already uses an inorganic substance, namely sodium nitrate, as a nitrogen source. However, all these known methods have nothing in common with the method according to the invention, since neither the same microorganisms and nitrogen processes for making cycloserine

Applicant:

Commercial Solvents Corporation,

TerreHaute, Ind. (V.St. A.)

Representative: "] '...

Dr. HH Willrath and Dipl.-Ing. H. Roever,
Patent attorneys, Wiesbaden, Hildastr. 18th

Named as inventor:

Roger L. Harned,:.

Terre Haute, Ind. (V. St. A.), '

Claimed priority:

V. St. v. America October 25, 1960 (64732)

sources are used while the same product is obtained.

The object of the invention, however, was to determine the yields and the degree of purity of the cycloserine during production using Streptomyces prchidaceus to increase. The method of manufacture of cycloserine by growing Streptomyces orchidaceus in a carbohydrate, a nitrogen source and nutrient medium containing mineral salts according to the invention, wherein the ratio of carbohydrate to assimilable nitrogen maintained at about 10: 1 to 20: 1 under aerobic conditions is characterized in that urea, ammonia or exclusively as a nitrogen source Ammonium carbonate is used. Urea is preferably used here.

As mineral salts, the nutrient medium is known to contain magnesium, potassium, iron, zinc and manganese salts and phosphorus compounds are added. Here are for example manganese sulfate, Magnesium sulfate, potassium hydrogen phosphate, iron sulfate and zinc sulfate should be mentioned. Suitable carbohydrates are for example starch and glucose, xylose, maltose, fructose and dextrans, peanut oil, Olive oil, tree wort oil and others It is preferred to use starch.

In general, about 3 to 8% carbohydrate, about 0.05% magnetic

609 707/378

Claims (1)

3 4 siumsulfat, about 0.05 ⁰ zo potassium hydrogen phosphate, slurried in water. This porridge would about 0.02% iron sulfate, about 0.2% zinc sulfate, then mixed with hydrochloric acid to form solid silver chloride 0.1% manganese sulphate and about 0.1 to 0.5% of the stick precipitates and the cycloserine in aqueous solution source of material added. Often it is necessary to release. The solid silver chloride was now the nutrient medium an antifoam agent, such as 5 "removed by filtration, through the / aqueous cyclo- Add lard oil, mineral oil, etc. In order to make serine solution, hydrogen sulfide was blown around to precipitate residual amounts of silver for good growth of the air microorganism, and that Distributed throughout the nutrient medium, this purpose-precipitated silver sulfide was filtered off. The accruing moderately stirred. '"The filtrate was then poured into cold isopropyl alcohol According to the process of the invention, an io is obtained to add 369 g of white crystalline cycloserine Nutrient medium that will receive about 2000 to 3000 micrograms. This means a 62% yield Contains cycloserine per milliliter, even if you have cycloserine. The product showed a purity of works on an industrial scale, easily 95.0%. are worked up according to known methods Example 2 can to get a very pure product. 15th The following examples serve to further illustrate the procedure of Example 1 was followed in the manner Purification ^{1 of} the invention. modified that 7% starch and 0.62% urea . · _{Τ1 were} used, being, 3000 micrograms Example! Cycloserine per milliliter at the end of a 72 hour period Streptomyces orchidaceus was obtained in an aqueous fermentation period .: gen nutrient medium of the following composition Example 3 breeds: ------ ■ .... ..... Cerelose 1% The procedure of Example 1 was followed in the manner . * "η ς 0 / modified that 0.48% ammonia was used Cjrelatine U, 5/0 _{35 d} J _11n results similar to those in Example 1 Keep NaNO ₃ 0.3%. MgSO ₄ -TH ₂ O.-.-. 0.05% Example 4 K ₂ HPO ₄ 0.05% The procedure of Example L was followed with the Ab> The cultivation was for 24 hours at 30 ⁰ C 30 conversion throughput repeats that: 0.48% Ammoniumcarbo- guided. nat have been used to get results similar to 34,0001 of an aqueous solution of the following additives in Example 1 to be obtained. . . . ; ....., composition were then placed in a fermentation vessel:. . ., - ■, and sterilized at 121 ° C for 15 minutes. Comparative example ■ Strength: ..... 5% ■ _; z _U j- Explanation of a procedure according to the status - MgSO ₄ -TH ₂ O .'......-..-. Tr.t. 0.05% - the technique was a nutrient medium with a K HPO 0.05% contains the following substances manufactured: ...: FeSO ₄ 7 H ₂ O 0.002% ^ soybean meal 3 percent by weight '' ZnSO ₄ .; ......, ........... .... ........ .0.002% , glucose., ... '2 percent by weight _: MnSO ₄ 0.001% calcium carbonate 0.5% by weight Lard oil .v .......... 0.4% Lard oil .............. 0.04 percent by weight Then the sterilized nutrient medium 45 would be The nutrient medium was 15 minutes at 121? C. ■ 0.48% sterile urea added. Then it was sterilized. Then the nutrient medium was in.einem Fermentation vessel with 30281 inoculation culture ^ added, which like fermentation vessel with Streptomyces. oichidaceus inoculates, was made above. After an incubation time of 8.6 hours at 30? C. 30 ° C: for 86 hours: incubated while it was aerated with the medium. After completion of the 22'65 <D1 air per minute was ventilated. At the end of the 50-86 hour fermentation period, the nutrient 86-hour fermentation period, the medium showed a medium filtered off with a filter aid to remove the mycelium Remove cyclose content of 2600 micrograms per muli-. The filtered nutrient medium was liter. then treated with 1% activated charcoal, filtered and with 261,21-a sewing medium obtained as; above '; nitric acid adjusted to a pH value of 3.0, with 2256 micrograms of cycloserine per milliliter A total of 592 g) were treated with a filter aid film silver salt in order to remove the silver cycloserine. The filtrate was then precipitated at 1 percent by weight. This was in the activated carbon described in Example 1 added, -the mixture · worked up with nitric acid in a manner .- = .... to pH 3.0. set .imd with 1131g AgNO ₃ The white crystalline cycloserine thus obtained puts. The mixture then became intimate for 30 minutes and turned brown very quickly. Analysis showed that stirred: and - ■ then filtered. The filtrate was only 86.5% pure. the Then aqueous sodium hydroxide solution diluted to pH was only 36.2%, which is the same 6ß adjusted to the silver salt, from: cycloserine as the degree of purity significantly below the yield fall .. The ¹ precipitated, moist .silver cycloserin of example 1: .. .- ... ...:. .-...-. ; was dried and gave 1011 g of silver cycloserin ', 65. - - Claim · "■ which contained 442g cycloserine. This means a -.-. . ... ··. ..'..... ·: Recovery of 74.5 i / o of the cycloserine in the nutrient process for making cycloserine medium. Now it became: silver cycloserin in deioni-breeding of Streptomyces orchidaceus in one 5 6 Carbohydrates, a nitrogen source and minerals - finally urea, ammonia or ammo nutrient medium containing salts, the sodium carbonate being used. ratio of carbohydrate to assimilable Nitrogen to about 10: 1 to 20: 1 under aerobic Pamphlets Considered: Conditions is maintained, thereby becoming 5 German Patent No. 958242; indicates that the nitrogen source from German interpretation no. 1022230. 609 707/378 10.66 ® Bundesdruckerei Berlin