GB2034353A - A process for increasing the substrate concentration of fermentation broths in the microbiological conversion of cardenolide derivatives - Google Patents

Abstract

A process for increasing the substrate concentration of fermentation broths in microbiological conversions of cardenolides having the general formula (I> <IMAGE> [wherein X represents a hydrogen atom or a hydroxy group, and R represents a hydrogen atom or a group of formula (II), (III), (IV) or (V)], <IMAGE> the said starting substance of the general formula (I) being introduced into the fermentation broth in a solution formed with a water-miscible solvent of electron donor character or with a mixture of such solvents.

Description

SPECIFICATION A process for increasing the substrate concentration of fermentation broths in the microbiological conversion of cardenolide derivatives The invention relates to a process for increasing the substrate concentration of fermentation broths in the microbiological conversion of cardenolides having the general formula (I),

wherein X represents a hydrogen atom or a hydroxy group, and R represents a hydrogen atom or a group of formula (11), (III), (IV) or (V).

As known, certain representatives of the cardenolides obtained from plant extracts can be converted into pharmaceutically applicable cardiac glycosides by fermentation processes (Nazaki et al.: Agric. and Biol. Chem./Japan/29, 783/1965/).

It is also known that, beside fermentation time and degree of conversion, the substrate concentration of the fermentation broth, also has a marked influence on the efficiency of microbiological conversions since, owing to the large energy requirement of the sterilization of culture media and the aeration and stirring of fermentation broths, the amount of substrate convertible in unit volume is not irrelevant at all.

There are several factors which limit the efforts to increase the concentration of the substrate.

The methods used to overcome these difficulties differ from each other in accordance with the nature of the limiting factors. Thus e.g. when the substrate suppresses the microbiological process, the undisturbed action of the enzyme is ensured by introducing the substrate continuously or batchwise (Hungarian patent specifications Nos. 146,307 and 150,245). In some of the microbiological conversions the high substrate concentration suppresses only the formation of the appropriate enzyme, not affecting, however, the function of the enzyme already formed. This effect was utilized by the authors of the Hungarian patent specification No.

149,062. The process described in the Hungarian patent specification No. 153,499 enables one to apply high substrate concentrations in the conversion of certain steroids where dehydrogenation is suppressed by the formed product itself.

The invention aims at increasing the cardenolide concentration of fermentation liquids in microbiological conversions of cardenolides.

As microbiological conversions proceed almost exclusively in aqueous media, it is a common requirement in the majority of conversions that the steroids or cardiac glycosides, of low water solubility, are supplied to the fermentation liquid in an appropriate distribution. This can generally be ensured by adding the substrate to the fermentation liquid in the form of a solution formed with an organic solvent well miscible with water (such as acetone or methanol).

In addition to dissolving the substrate, the organic solvent also acts on the cell membranes of lipoprotein character, being able to influence not only the diffusion of the substrate but also the enzymatic reactions proceeding in the cell. Therefore we started to examine the effects of the various solvents, applied to dissolve the glycoside, on the microbiological conversion.

We could not find any correlation between the conversion and the dielectric constant of the solvent applied. According to our experiences the solubility of glycosides in the most frequently applied protic solvents is generally unsatisfactory, and the use of these solvents in amounts required to attain higher substrate concentrations suppresses the conversion. No unambiguous correlation could be found between the apolar aprotic and dipolar aprotic solvents tested and the concentration of the substrate applicable in the conversion.

However, it was found that when solvents of strong electron donor character, e.g. Lewis bases, are used regardless of their protic or aprotic, apolar or dipolar nature, larger amounts (i.e.

700 to 1000 jug/ml instead of the usual 50 yg/ml) of glycoside can be converted in an aqueous system of given volume in comparison to the methods applying conventional solvents for introducing the substrate.

Consequently, according to the present invention there is provided a process for increasing the substrate concentration of fermentation broths in the microbiological conversion of cardeno lides having the general formula (I) [wherein X represents a hydrogen atom or a hydroxy group and R represents a hydrogen atom or a group of formula (if), (III), (IV) or (V)], the said starting substance of the general formula (I) being introduced into the fermentation broth in a solution formed with a water-miscible solvent of electron donor character, capable of dissolving 5 to 20% of the substrate concerned, or with a mixture of such solvents, fermentation then being performed in a manner known per se.

According to our experiences tetrahydrofuran, dioxans, tetrahydrofurfuryl alcohol, dimethyl formamide, dimethyl sulfoxide, diethyl formamide, diethyl acetamide, pyridine, pyrrolidine, N methyl-morpholine, hexamethylphosphoric triamide or mixtures thereof can be applied to advantage as solvents.

The main advantage of the process according to the invention is to highly increase the amount of substrate it is possible to convert in a given volume of the fermentation broth during the microbiological conversion of cardiac glycosides with only a minimum change in the fermentation time.

Further advantages result from the fact that some of the electron donor solvents specified above can be sterilized by heating. Utilizing such solvents in aseptic microbiological conversions, the sterile administration of otherwise heat stable substrates can be made much simpler and safer.

The process according to the invention is illustrated in detail by the aid of the following nonlimiting Examples.

Example 1 A five to six week old slant culture of Streptomyces praecox MNG 127, cultivated on potatoagar, is suspended in 10 ml of sterile water, and 1 ml each of the resulting suspension is applied to inoculate 100 ml each of "PS" culture medium filled into conic flasks 500 ml in volume. The "PS" culture medium is prepared by sterilizing an 0.8% aqueous solution of pulverulent soybean extract (pH = 7.0) at 120"C for 45 minutes, and adding a 50% aqueous solution of glucose, sterilized for 30 minutes at 120"C, in an amount sufficient to adjust the glucose content of the culture medium to 3.0%.

The pulverulent soybean extract is prepared as follows: A 10% tap water suspension of ground, extracted soybean, sieved through a sieve of 0.4 mm gap diameter, is subjected to heat treatment for one hour at an overpressure of 1 atm. Thereafter the suspension is cooled to 37"C, 0.4% of pancreatine are added, and hydrolysis is continued for 2 hours under continuous stirring. During this period the pH of the mixture is maintained at 7.5 to 8.0 by occasionally adding sodium hydroxide to the mixture. The suspension is centrifuged and the supernatant fluid is spray-dried. The nitrogen content of the resulting pulverulent soybean extract is 9%.

The inoculated flasks are shaken at 32"C for 2 days, and then 70 mg each of digitoxin are introduced into the flasks dissolved in the following solvents: Flask 1: 1 ml of tetrahydrofuran, Flask 2:1 ml of dioxane, Flask 3:1 ml of tetrahydrofurfuryl alcohol, Flask 4:1 ml of dimethyl formamide, Flask 5:1 ml of dimethyl acetamide, Flask 6:1 ml of dimethyl sulfoxide, Flask 7:1 ml of pyridine, Flask 8:1 ml of hexamethylphosphoric triamide.

The flasks are shaken at 37"C for 3 days, and then the cultures are extracted twice with 50 ml of chloroform each. The glycoside contents of the extracts are determined by photometry, utilizing the colour reaction with dixanthyl urea, after separating the desired component by layer chromatography.

Accordingly, the first step of this analytical procedure is layer chromatography. Kieselgel He264 layers (Merck, Darmstadt, German Federal Republic), 0.25 mm in thickness, are applied as adsorbent, and the operation is performed in a vat with saturated vapour phase. With primary glycosides a 90:10 mixture of chloroform and methanol, whereas with secondary glycosides a 55:35:10 mixture of ethyl acetate, cyclohexane and absolute ethanol is applied as solvent, and the system is run thrice.

The sample to be examined is applied to the layer in an amount containing 10 to 50 jL9 of the individual glycosides.

After running, the layer is dried thoroughly, and the chromatogram is developed in iodine vapour. The spots are marked according to the standards, and then iodine is removed from the adsorbent by sublimation in air flow.

Thereafter the marked spots are collected separately into test tubes equipped with cut glass stoppers. 3 ml of a dixanthyl urea reagent, prepared as described below, are filled into the test tubes. The test tubes are closed, thoroughly shaken, and then immersed for 3 minutes into a 100"C water bath. Thereafter the test tubes are cooled in a cold water bath, the suspension is shaken, and the solid is removed by centrifuging. The colour intensity of the clear supernatant fluid is measured at 535 nm using a glycoside-free solution as reference.

The reference solution is prepared by scraping a glycoside-free (blank) spot of the same area from the adsorbent at the same height as the glycoside-containing spot, and treating it as described above.

The concentrations of the individual glycosides are calculated from the extinctions by considering the volume applied and the degree of optional dilution, using the multiplication factor obtained from the standard concentration curve.

The dixanthyl urea reagent is prepared by dissolving 10 mg of dixanthyl urea in a mixture of 50 ml of acetic acid and 1 ml of concentrated hydrochloric acid, and adjusting the volume of the solution to 100 ml with acetic acid. The reagent is prepared freshly before measurement.

The concentrations of the individual glycosides are given in Table 1.

Table 1 Glycoside content, mg No. of the flask Digitoxin Digoxin and 7P-hydroxydigoxin 1 0 60 2 below 5 41 3 5 54 4 8 38 5 5.6 50 6 6.3 35 7 7 35 8 9 37 Example 2 One proceeds as described in Example 1 with the difference that the microorganism suspensions are prepared from cultures of Streptomyces purpurascens KA-26 (MNG 179), Streptomyces purpurascens KA-43 (MNG 178), Streptomyces purpurascens KC-157 (MNG 176) or Streptomyces alboniger AB-318-ST (MNG 180), respectively. According to the colourimetric data digitoxin is converted with the formation of 20 to 30% of digoxin and 7ss- hydroxydigoxin.

Example 3 One proceeds as described in Example 2 with the difference that acetyldigitoxin or lanatoside A is applied as substrate. According to the colourimetric data 80 to 90% of the substrate is converted. The product consists of digoxin and 7ss-hydroxydigoxin.

Claims (4)

1. A process for increasing the substrate concentration of fermentation broths in microbiological conversions of cardenolides having the general formula (I)

[wherein X represents a hydrogen atom or a hydroxy group, and R represents a hydrogen atom or a group of formula (II), (III), (IV) or (V)],

the said starting substance of the general formula (I) being introduced into the fermentation broth in a solution formed with a water-miscible solvent of electron donor character or with a mixture of such solvents.

2. A process, as claimed in claim 1, wherein the said solvent comprises tetrahyfuran, tetrahydrofurfuryl, alcohol, dioxane, dimethyl formamide, dimethyl acetamide, dimethyl sulfoxide, pyridine or hexamethylphosphoric triamide or a mixture of two or more thereof.

3. A process, as claimed in claim 1, substantially as herein described.

4. A process, as claimed in claim 1, substantially as herein described in any of the Examples.