We developed a scheme of consecutive replacement of complex components of a known Brucella medium containing peptones and blood with simple analogs and created a synthetic medium for Helicobacter pylori culturing. H. pylori cells require hemic iron f
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K. N. Gabinskaya, V. M. Ryzhkova, and N. N. Suvorov
UDC 615,357,453,012.6 : 576,083.3
Epicortisol is an intermediate product in the synthesis of cortisone and prednisolone - steroid compounds, which have wide use in medicine. Epicortisol (II) is produced microbiologically from cortexolone acetate (I) with the aid of the fungus Tieghemella hyalospora:
c~oooc~ GO -
CO --- OH
o~ k - - s ~
In the existing method of microbiological ll~-hydroxylation of eortexolone acetate, a corn-glucose medium is used . Corn extract is a substance whose composition is nonstandard and varies from lot to lot. Its presence in the medium hinders the isolation of the reaction products. It is advisable to grow the fungus and conduct the transformation on a medium with standard sources of nutrition without inert substances. It was established that Tieghemella hyalospora can be cultured on a synthetic medium, and the carbon sources (glucose, sucrose, glycerin, and starch) proved equivalent. The nitrogen sources (ammonium salts of sulfuric, phosphoric, nitric, and succinic acids) provide for growth of the fungus and a 73% yield of the transformation only when calcium carbonate is added to the medium or less acid nitrogen sources are used, such as amino acids, since the fungus is sensitive to high acidity. The purpose of this work was to conduct the transformation on synthetic medium on a semiindustrial scale and to characterize the preparatively isolated product. The data obtained when the fermentation was conducted on medium No. 1 (glucose, ammonium succinate, and asparagine) and on medium No. 2 (sugar, ammonium nitrate, and calcium carbonate) indicated that the yield of epicortisol on these media is approximately the same and equal to the average yield obtained in the laboratory on corn-glucose medium: the yield of epicortisol on media Nos. i and 2 and corn-glucose medium is 73, 66.18, and 66%, respectively. EXPERIMENTAL
The fungus Tieghemella hyalospora [i] was cultured in flasks on wort-agar for seven days. The experiments were conducted in a 20-1iter glass fermenter, equipped with an electrical spiral with automatic temperature regulation, a propeller mixer (260 rpm), and a bubbler (delivery of air 1 liter/liter of medium per min). The mycelium was grown and the step of steroid transformation was conducted at the temperature 27-28 ~ using medium No. 1 (5% glucose, 1% ammonium suceinate, 0.25% asparagine, 0.1% monosub-
Ordzhonikidze All-Union Scientific-Research Chemico-Pharmaceutical Institute, Moscow. Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 5, No. 11, pp. 35-36, November, 1971. Original article submitted June 29, 1970.
9 1972 Consultants Bureau, a division of Plenum Publishing Corporation, 227 ~/est 17th Street, New York, N. Y. 10011. All rights reserved. This article cannot be reproduced for any purpose whatsoever without permission of the publisher. A copy of this article i.s available from the publisher for $15.00.
stituted p o t a s s i u m phosphate, 0.3% m a g n e s i u m sulfate, pH 5) and m e d i u m No. 2 (5% s u c r o s e , 1% a m m o n i u m nitrate, 0.1% monosubstituted p o t a s s i u m phosphate, 0.3% m a g n e s i u m sulfate, 0.3% calcium carbonate, pH 5). A 1 0 - l i t e r portion of the m e d i u m was inoculated with 200 ml of s p o r e suspension (3 9107.+ 4.107 s p o r e s per ml). A f t e r 48 h of growth, 5 l i t e r s of s t e r i l e w a t e r was added to the m y c e l i u m and 5 g cortexolone, dissolved in 280 ml of acetone, was introduced. The c o u r s e of the t r a n s f o r m a t i o n was monitored by a c o l o r i m e t r i c method, based on the c o l o r a t i o n of eortexolone and its acetate in the p r e s e n c e of sulfuric acid. F o r the analysis, 25 ml of the culture fluid with m y c e l i u m was extracted with 25 ml of a mixture of c a r b o n t e t r a e h l o r i d e and c h l o r o f o r m , and 10 ml of the e x t r a c t was evaporated to d r y n e s s . The residue was dissolved in 10 ml concentrated sulfuric acid, and a f t e r 15 min the intensity of the c o l o r obtained was m e a s u r e d on a p h o t o e l e c t r o c o l o r i m e t e r - n e p h e l o m e t e r (Ft~K-56) with a No. 5 filter (X 490 mm). After 24 h of fermentation, the m y c e l i u m was filtered off f r o m the culture fluid and washed w i t h w a t e r . The culture fluid, together with the wash w a t e r s , w e r e extracted three t i m e s with methylene chloride in a 1 : 1 r a t i o . The solvent was distilled off under v a c u u m to a residual volume of 125 ml and left in a r e f r i g e r a t o r at 0 ~ f o r complete c r y s t a l l i z a t i o n of the product for 24 h. The precipitate of epicortisol f o r m e d was filtered off, washed three times with chilled methylene chloride, and dried to constant weight at a t e m p e r a t u r e up to 50 ~. E p i c o r t i s o l with vitrification point 204-205 ~ was isolated f r o m the culture fluid in the case of f e r m e n tation on m e d i u m No. 1. The content of the adduct was 9.63%, admixture of h y d r o c o r t i s o n e m o r e than 1%, admixture of cortexolone and its acetate l e s s than 1%. After concentration of the m o t h e r liquor, another 0.7 g of e p i c o r t i s o l was isolated, which was additionally r e c r y s t a l l i z e d . The total amount of epicortisol, c o n s i d e r i n g the adduct, was 3.41 g, a yield of 73%. E p i c o r t i s o l with vitrification point 202-203 ~ was isolated f r o m the culture fluid in the case of f e r mentation on m e d i u m No. 2. The content of the adduct was 2.05%, h y d r o c o r t i s o n e impurity about 1%, no admixture of cortexolone and its acetate. The total amount of epicortisol, considering the adduct, was 3.085 g, a yield of 66.18%. LITERATURE 1.
L . V . Sokolova, T. A. Grinyuk, Z. A. Y a r o s l a v t s e v a , et al., Med. P r o m . SSSR, No. 6, 36 (1966).