A

SIMPLE

THE USING

SYNTHETIC

Ai-DEHYDROGENAT Mycobaeterium N. N. Klimontovieh, and V. M. Ryzhkova

MEDIUM ION

FOR OF

globiforme E.

V.

PERFORMING

HYDROCORTISONE 193 UDC

Markova,

615.357.453.012.6

Prednisolone is a valuable broad-aetion-profilemedicinal preparation. The preparation of prednisolone is carried out in a corn-glucose medium [i] with the aid of Mycobaeterium globiforme 193 [2]; in it a corn extraet is used which contains various amino acids (leueine, isoleueine, valine, and glutamic acid) [3] and vitamins (riboflavin, pantothenie acid, pyridoxine, and biotin) [4]. The wide use of corn extractis caused by the fact that its use offers an opportunity for growing a broad group of microorganisms without a detailed study of the requirements of the latter in carbon and nitrogen sources. However, the composition of corn extract is variable and changes from lot to lot. This impedes the preparation of a finished product ina standardyield. Moreover, the presence of corn extract in the medium hinders isolation of prednisolone. The purpose of the present work was to select a simple synthetic medium of constant composition which does not contain inert matter, to carry out the Ai-dehydrogenation of hydroeortisone using Mye. globiforme 193. The work was performed with the use of the mathematicalexperiment-planning method of [5]. As a result of the work performed, a synthetic medium (No. i) of the following composition was selected (figures are given in percent): glucose, 2; ammomum sulfate, 0.I; monobasic potassium phosphate, 0.5; calcium carbonate, 0.4; magnesium sulfate, 0.03; sodium chloride, 0.01; calcium chloride, 0.05; ferric chloride, 0.001; water, from tap (pH after sterilization, 5.2-5.4). It was shown that Myc. globiforme 193 does notneed vitamins or amino acids for normal growth and transforming activity. Ammonium or nitrate forms of nitrogen can be used as nitrogen sources. The yield of prednisolone on medium No. 1 was equivalent to the yield of finished product when a corn-glucose medium is used. EXPE

RIME

NTA

The work was done with a culture of Mye. globiforme corn-glucose agar for three days.

L

193 which had been grown in flasks containing

Into 250-rnl flasks containing i00 ml of synthetic medium (the composition of the medium was varied as a function of matrix conditions) was introduced 1 ml of an aqueous suspension containing 4 mg of cells. Growth of the culture and transformation of the steroid were performed on a rocker at 28 ~ A 48-h growth culture (7.570) was transferred into 250-mi flasks with 80 ml of sterile distilled water. Then 80 nag each of hydrocortisone dissolved in 1.6 ml of ethanol was introduced into these same flasks. The transformation was carried out for 24 h. Monitoring was performed by a colorimetrie method. The amount of dehydrogenated product was expressed in percent relative to the amount of steroid introduced. As a control, a medium was used which contained 170 glucose and 170 corn extract (No. 2). The conditions of growth and conversion were the same as in the preceding experiments. In the use of the mathematical experiment-planning method, the yield of prednisolone (in percent relative to starting steroid) served as the optimization parameter. The experiment planning was carried out in the first stage of the research, when it was unknown what sources of nitrogen and phosphorus it was desirable to use, and what combination of other components to make the basis of the synthetic medium. Eight feeS. Ordzhonikidze All-Union Scientific-Research Institute of Pharmaceutical Chemistry, Moscow. Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 7, No. i, pp. 20-21, January, 1973. Original article submitted June 3, 1971. 9 1973 Consultants Bureau, a division of Plenum Publishing Corporation, 227 Test 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 is available from the publisher for $15.00.

23

TABLE 1. Experiment Planning Matrix. o

~o Base Level

,teral C/Q Upp~ r level, level,

LO+W~~) ~)

X,

"

X,

2

0,003 0,03 0,03 '0,0125 0 , 2

r162

1

0,002 0,02 0,02 ),0075 0,1

8--K,HPO4--0,4

3

0,005 0,05 0,05

B--(NH4)=SO,-0,16 ,0200 0,3 6--NaH=PO4--0,4 C--NaNOs--0,20

0,01

,0050 0,1 ~--Na=HPO4--0,4 D--KNO3--0,25

0,001 i0,01

A--NH4NO=--0,10

-

Experiment NO,

1 2 3 4 5 0 7 8 9 10 11 12 13 14 15 10

,p ~2

6 8 ,p

+

A D D D D A A A B C C C C B B B

29,0 44,0 26,5

4~,o 44,0 54,5

29,0

45,0 13,0 06,5 44,0

Note: Experiments 4, 5, 10, and 11werenot realized, sincethe combination of cations proved unsuitable f o r the present purposes. The optimization p a r a m e t e r is Y, the mean arithmetic value f r o m four duplicate experiments; mean figures are shown here. tors were studied (see Table 1). The concentration of the fi rst six components was varied at two levels. The seventh factor was four sources of phosphorus; and the eighth factor was four sources of nitrogen. These two factors were varied at four levels. F o r the planning we used a complex plan - a combination of a 26-2 m atr ix with a 4 • Greco--Latin square. This plan permitted us to study factors at different numbers of levels (the f i r s t six factors at two levels, and the last two factors at four levels). It was decided to pick those experiments in which the yield of dehydrogenated product exceeded 60%. Experiment No. 15 is the best in the table. It gives a predrdsolone yield of 66.5%. In this experiment monobasic potassium phosphate (0.4%) was used in combination with ammonium sulfate (0.16%), and the concentration of the other components was as follows: glucose, 170; f e r r i c chloride, 0.001%; magnesium sulfate, 0.0570; sodium chloride, 0.01%; ,calcium chloride, 0.005%; and calcium carbonate, 0.37o. F o r each of the four fo rms of phosphorus salts and also f or each of the four form s of nitrogen salts we made a calculation of the prednisolone yield. With respect to the mean values, the phosphorus sources can be put in the following ranging series: o~>fl > ~ > 7. The ranging s e ri es for the nitrogen sources has the appearance A > B > D > C. Consequently, the best results were shown by monobasic potassium phosphate, ammonium nitrate, and ammonium sulfate. In the second stage of the research, we selected the best concentrations of the eight components of the medium, taking into account the chosen combination of nitrogen and phosphorus sources which gave good r e suits in the preceding experiments. All factors were varied at two levels. A 28-4 fractional replica was used. When the experiment was per f or m ed in conformity with the matrix, high yields of prednisolone were obtained in four runs (85.5, 81, 85.5, and 84%, respectively). When one compares the yield of prednisolone obtained on the optimum synthetic medium (No. 1) and that obtained on corn-glucose medium (No. 2), the yield of dehydrogenated products ar e 85.5 and 75.5%, respectively. LITERATURE 1. 2. 3. 4. 5. 6.

24

CITED

G.K. Skryabin, I. S. Zvyagintseva, and L. V. Sokolova, Izv. Akad. Nauk SSSP~ Ser. Biol., No. 5, 715 (1964). N.A. Krasil'nikov, G. K. Skryabin, I. V. Aseeva, et al., Dokl. Akad. Nauk SSSI~ 128, No. 4, 836 (1959). E . V . Cardinal and L. R. Hedrica, J. Biol. Chem., 174, 609 (1948). E . K . Tanner, C. R. Pleifler, and A. van Lamen, Arch. Biochem., 8~ 24 (1945). E . V . Markova, Zavod. Labor., No. 1, 60 (1968). A. Mizsei and A. Szabo, J. Biochem. Microbiol. Technol., 8, 119 (1961).