DD248144A1 - PROCESS FOR THE PREPARATION OF 1,4-ANDROSTADIENE-3,17-DION BY FERMENTATIVE STEROL DEGRADATION - Google Patents

Abstract

The invention relates to a process for the fermentative side chain degradation of sitosterol to 1,4-androstadiene-3,17-dione (ADD) as starting substance for the production of steroid drugs. It is based on the task of adjusting the physiological state of the biomass necessary for the education of high ADD formation rates by means of an easily reproducible process regime. This is achieved by the fact that, in agreement with the growth kinetics of the biomass, in the course of the fermentation, temporarily or continuously low values of the relative residual oxygen saturation are realized. In a preferred embodiment, the process is carried out with respect to the introduction of the oxygen into the culture broth as a pulse regime, the relative residual oxygen content being kept within the range of this target value after falling to the 10% level.

Description

Field of application of the invention

The invention relates to a process for the fermentative side chain degradation of sterols to 1,4-androstadiene-3,17-dione, which is of importance as starting material for the production of steroidal drugs. The field of application of the invention thus lies in the pharmaceutical industry and in technical microbiology.

Characteristic of the known technical solutions

It is known that sterols, such as. As sitosterol, cholesterol, stigmasterol, campesterol and ergosterol, by microorganisms from numerous genera, such. B. Arthrobacter, Brevibacterium, Nocardia, Microbacterium, Protaminobacter, Bacillus, Streptomyces, Corynebacterium and especially Mycobacterium et al., About the intermediate product 1,4-androstadiene-3,17-dione (hereinafter referred to as ADD) can be degraded. This product is enriched in the fermentation medium only if no 9a hydroxylation occurs. This in turn will u. a. achieved by using microorganism mutants with defects in said enzymatic power for fermentation.

The first method of producing ADD from sterols using enzyme defective mutants (GDSearle & Co., US 3684657, 1970, Mycobacterium spec. NFSFtL B-3683) describes using 1 g of sitosterol per liter of culture solution and a fermentation time of 186 hours to 216 hours theoretical yields of 40% ADD, based on the proportion of converted in the fermentation sitosterol.

The von I.Sauerbaum et al. (I.European Congress for Biotechnology, Interlaken, 1978, 139-143) indicated fermentation of sitosterol with Mycobacterium spec. NRRL B-3683 * in the presence of the nonpolar adsorbent XAD-2 and the nonionic surfactant Tween 20 in which 10g of sitosterol per liter of culture solution is converted to a mixture of ADD and AD in 96 hours in 96 hours is considered to be the best method solution so far , According to these authors, product formation is stimulated by high dissolved oxygen concentrations and the formation of ADD is favored over the formation of 4-androstene-3,17-dione (AD). The optimal condition, and thus the lower limit, is a relative dissolved oxygen saturation of the culture broth of 50%. This results in the process management in the fermentor requirements for the ventilation and circulation of the fermentation broth, which are unfavorable in terms of foaming, creaming of substrates and biomass, Scherkraft- and mixing ratios, especially in the presence of adorbers in the fermentation broth.

Object of the invention

The invention aims to produce 1,4-androstadiene-3,17-dione by microbial route in high yields from sterols.

* According to JP 105289 (Kokai), this Mycobacterium spec. Strain was classified as Mycobacterium vaccae.

Explanation of the essence of the invention

The invention has for its object to describe an improved process for the preparation of 1,4-androstadiene-3,17-dione by microbial transformation of sterols, in which with regard to the fermentation, in particular with regard to the guidance of the relative dissolved oxygen saturation of the culture broth, a solution is realized, which avoids the disadvantages of the known technical solutions.

It has been found that the oxidative process of formation of 1,4-androstadiene-3,17-dione can be achieved by microbial transformation of sterols or sterol mixtures, preferably sterol mixtures with sitosterol as the major component, with surprisingly high yield, although the relative dissolved oxygen saturation the culture broth in the course of the fermentation after waste of saturation value 100% temporarily or constantly less than 10%.

The method is carried out in its basic form as follows:

Sterols such as sitosterol, cholesterol, campesterol, stigmasterol or ergosterol or mixtures of these sterols are used in the form of sterol surfactant preparations, the sterol concentration based on the liter of fermentation medium

1 gram to 50 grams. For fermentation, microorganisms are used which are capable of transforming to form the product ADD or the product mixture ADD / AD. Such microorganisms can be selected from the genera Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Norcardia, Pseudomonas, Protaminobacter, Serratia, Streptomyces, etc. In particular, for the present method of producing ADD, the strain Mycobacterium vaccae NRRL B-3683 or a selectin of this strain is used. A selectant in the culture collection of ZIMET has been deposited under the registration number I MET SK113. The sterol surfactant preparations are obtained by the known methods of lyophilization, grinding or spray drying.

As also described in our two copending applications, surfactants are compounds from the group of the polyethylene oxide-polypropylene oxide adducts of the N, N'-tetra (oxypropylpropyol) -N '- (2-oxypropyl) -diethylenetriamine with molecular weights of the polypropylene glycol around 1400 to 2000 for use.

The fermentation media contain carbon sources such as glycerol, glucose, sucrose, starch and / or molasses, etc., nitrogen sources such as yeast extract, urea, ammonium salts, corn steep liquor, plant meals and / or other complex media ingredients (such as meat extract, hydrolysates etc.) as well as various mineral salts.

To the fermentation medium is added an adsorbent which is of the organic polymer type and contains polar groups (copolymer of styrene and divinylbenzene with acrylic acid derivative portion). The quantity ratio is between

2 grams per liter and 200 grams per liter. For further characterization of the adsorber reference is made to one of our parallel applications.

The duration of the fermentation is 24 hours to 192 hours. It is carried out under aerobic conditions at temperatures of 2O ⁰ C to 45 ⁰ C and pH values in the range of pH 6 to pH 8. The fermentation is carried out in Rührfermentor or in fermentors with other Umwälzprinzipien, z. B. in air-lift fermentors.

For the fermenter used in each case an apparatus characterization is carried out; This is done for the purpose of the invention using the oxygen transport rate as a function of ventilation and circulation. The measurement of the oxygen transport rate is carried out in a conventional manner.

For the microorganism used for the sterol transformation are used in preliminary experiments in a conventional manner (see, eg.

F. Bergter, Growth of Microorganisms, G. Fischer Verlag Jena, 1983)

The yield coefficients for the biomass from the determining substrates,

- the specific growth rate and

- the specific oxygen uptake rate

certainly. Furthermore, during the fermentation process, the relative dissolved oxygen saturation is continuously measured as the pO ₂ value of the fermentation broth in a manner known per se.

For the phase of biomass growth proceeding at the beginning of the fermentation process, the operating point for the regime of aeration and circulation of the fermentation broth is selected on the basis of the dependence of the oxygen transport rate determined for the fermentor used above so that at the earliest 0.5 hours to no later than 45 Hours fermentation time of the pO ₂ value of the fermentation broth differentiates the 10% level. With this oxygen consumption enters a favorable for the achievement of high ADD formation rates physiological state of the biomass.

In the subsequent stationary biomass phase, there is an increase in the relative dissolved oxygen saturation of the fermentation broth, provided further control measures, e.g. Reduction of circulation and ventilation, is dispensed with. In this case, the pO2 value can again be 100% at the end of the fermentation.

In a preferred embodiment, the process is carried out in the stationary biomass phase with regard to the oxygen transport, ie with respect to aeration and circulation of the fermentation broth, in a pulse regime based on a pO ₂ setpoint control. The p02 setpoint is selected from the range of 5% to 30%; preferably it is set to 10%.

The pO ₂ setpoint control at a setpoint of, for example, 10% here becomes effective in order to suppress the increase in the relative dissolved oxygen saturation of the fermentation broth in the stationary biomass phase.

The pulse regime for air entry and recirculation parameters (eg speed at the stirring fermentor) has the two states

- Reduced or stopped Lufteirvirag and reduced circulation for pO ₂ -stwert> p0 ₂ setpoint,

- Air intake and activated circulation for pO ₂ -value <pO ₂ setpoint.

The change ranges of circulation parameters and air intake are to be determined from the oxygen transport characteristics of the respective fermenter used.

In extreme cases, this embodiment is modified in such a way that the pulse regime already becomes effective in the phase of biomass growth. This process variant is characterized in that the pO ₂ value of the fermentation broth immediately after the start of fermentation as a result of Zuluftstop below the setpoint level, for example, below the 10% level drops, so the pO ₂ setpoint control by means of coincident intensification of ventilation and circulation at setpoint below begins early. With this process control are in a particularly advantageous manner

and foaming problems avoided and favorable contact and mixing conditions for the solid phases (adsorber, sitosterol, biomass) guaranteed. At the same time high ADD yields are realized.

When carrying out the process on an industrial scale, for example in the 10-m ³ Rührfermentor, the above pulse regime is modified according to the advantageous technical realization. Preferred embodiments are

- Continuous, for example, linear increase in speed up to a specified upper limit falls below the pO ₂ setpoint and intensification of air intake starting with the achievement of the upper speed plateau; Speed reduction and reduction or stop of air entry when exceeding the p0 ₂ setpoint or

- Maintaining a consistently constant speed and execution of the pulse regime only for the fermentor supply air. The procedural solution has the following advantages:

The development of the present process for producing ADD via microbial sterol degradation, characterized by low levels of relative dissolved oxygen saturation of the fermentation broth over long periods of the process, ensures high product yields with simultaneously improved fermentation regime without foaming and creaming of biomass, adsorbent and sitosterol , An additional advantage is seen in the savings of fermenter supply air and electrical energy to be provided.

embodiments

The invention will be explained in more detail by way of examples:

example 1

From a preserve of the strain Mycobacterium vaccae NRRL B-3683, the seed culture for the ADD fermentation is cultivated. In the first step, a slant culture is applied by means of four-day incubation at 35 ° C. The nutrient agar has per liter of distilled water. the composition: glycerol 20g, bacto-peptone 5g, meat extract 3g, agar agar 15g. Its pH is adjusted to 7.0 in the liquid state before sterilization; the sterilization takes 20 minutes at 121 ⁰ C. Subsequently, two submerse Vorkulturstufen done. For the first submerged preculture, the slant culture is washed off with 5 ml of the culture medium for the two submerged preculture stages. This medium has per liter of distilled water. the composition: yeast extract 10 g, glycerol 10 g, KH ₂ PO ₄ , 0.5 g, K ₂ HPO ₄ 0.5 g, (NH ₄ I ₂ HPO ₄ 1.5 g, MgSO ₄ .7H ₂ O 0.2 g, FeSO ₄ . 7H ₂ O 0.01 g, ZnSO ₄ · 7H ₂ O0,002g mitpH value adjustment to 7.0 before sterilization for 20 minutes at 121 ⁰ C.

From the flooding 500 ml round-bottomed flasks are inoculated, each containing 50 ml of the latter medium and shaken for 36 hours at 28 ° C on the rotary table. Each 5 ml of the first preculture are used for inoculation of resting piston of the second preculture stage with the filling conditions as in the first precultivation stage, which are also shaken at 28 ° C for 36 hours.

The subsequent Fermentor main culture is carried out over 94 hours with a start volume (including pre-culture and sitosterol without adsorber) of 2.51 in a stainless steel glass-Rührfermentor 4.51 gross volume at 31 ⁰ C. The amount of inoculum from the second Vorkulturstufe forms 10 % of startup volume. To the liter of fermentation broth, 140 g adsorber of the type of a copolymer of styrene and divinylbenzene with acrylic acid derivative content are added. The adsorber is previously washed out with methanol and rinsed with water. The lower limit for the grain diameter is 0.4mm. The fermentation medium of the main culture has the following composition per liter of distilled water: urea 0.125 g, glucose 5 g, KH ₂ PO ₄ , ₂ g, K ₂ HPO ₄ 8 g, (NH ₄ J ₂ SO ₄ 3 g, MgSO ₄ .7H ₂ O 0 takes place ₄ 7 H ₂ O 0,002g. Before sterilization, the pH is adjusted to 7.0. sterilization of the containing 2g, FeSO ₄ · 7H ₂ O 0.01 g, ZnSO by autoclaving the medium, including the necessary of adsorber Fermenter vessel for 60 minutes at 121 ° C. The sitosterol preparation is sterilized twice in the steam pot for 60 minutes and introduced in one step with the inoculation into the fermenter cooled to 31 ° C. 15 g of sitosterol are used per liter of fermentation broth, the preparation of which is wet-ground by 10%. a surfactant from the group of polyethylene oxide-polypropylene oxide adducts of N, N "-Tra - (- oxypropylpropyol) -N '- (2-Oxypropy!) - Diethylentriamins with molecular weights of the polypropylene glycol around 1400 to 2000 in a colloid mill.

The main culture fermentation is carried out under the initially set constant conditions of 0.331 of air per liter of fermentation broth and minute at 450 revolutions per minute (6-blade stirrer) for aeration and agitation, during fermentation the relative dissolved oxygen saturation as pO ₂ value (100% calibration in uninoculated stirred and aerated fermentation broth) measured and recorded; Sampling determines the kinetics of biomass and ADD formation. Upon completion of the fermentation, the yield of ADD is 5.1 g / l. The kinetics for ADD, biomass and pO ₂ value are summarized in Table 1.

Example 2

The procedure is as in Example 1 to obtain the preculture for the ADD fermentation. The main culture is carried out in a 4.51 gross volume stainless steel glass stirred fermentor at 28 ° C. With regard to the amount of the starting volume of 2.51, 250 ml of seed culture of the second preemergence stage and 7 g / l of sitosterol preparation are included, the preparation and addition of which proceeds as in Example 1.

The fermentation medium of the main culture has per liter of distilled water. the composition: urea 0.25g, glucose 5g, (NH ₄ ) ₂ SO ₄ 3g, KH ₂ PO ₄ 1g, K ₂ HPO ₄ 4g, MgSO ₄ .7H ₂ O 0.2g, FeSO ₄ .7H ₂ O 0 , 01 g, ZnSO ₄ .7H ₂ O 0.002 g. Furthermore, 60 g of the adsorber of Example 1 are added to the liter of fermentation broth. The sterilization conditions are identical to those in Example 1 with the exception of the longer sterilization time of 90 minutes.

The main culture fermentation begins in the state of dissolved oxygen saturation of the fermentation broth, which is also used to calibrate the pO ₂ probe. The control threshold for the pO ₂ value is 10%. The starting speed is 450 ~ min ~ ¹ , the air supply is interrupted after the calibration of the pO ₂ probe.

It takes place within 0.5 hours. The drop of the pO ₂ value to 10% by oxygen depletion as a result of the oxygen uptake by the growing biomass. If the pO ₂ control threshold is undershot, the speed is increased to 700 min ^-1 and the air supply path coincidentally released, which then flows through the air quantity of 0.331 per liter of fermentation broth and minute After increasing the pO ₂ value above 10%, the process conditions are reset to 450min " ¹ and Zuluftstop.

This pulse regime for compliance with the p0 ₂ nominal value of 10% has the process technology extremely important effect that no creaming of biomass and substrates and no foaming comes about. In addition, there is a limited need for air. After 92 hours fermentation time, the yield of ADD is 1.87 g / l; after 164 hours (end of fermentation) there are 3.01 g / i ADD. The plateau value of the dry biomass of about 2.6g / l is around the

30th fermentation hour reached. From the 130th fermentation hour, the pO2 value rises to about 20%, although it is operated without air intake and at 450 min ^-1 .

Table 1: Kinetics for ADD, dry biomass and pO2 value for non-application of a pO ₂ setpoint control (process conditions according to embodiment 1)

ADD (g / l)

Dry biomass (g / l)

pO ₂ (%)

0 (n.b.) 11 22 24 30 35 43 46 50 52 55 60 70 94

pO ₂ -value <10% for 10 hours pO ₂ -value <20% for 20 hours

0 0.91

2.42

4,41 5,10 ^;

0.965 2.24

100

100

100

Table 2: Kinetics for ADD, dry biomass and pO ₂ value at feedstop after start of fermentation and subsequent application of a pO ₂ nominal value control (target value 10%, other process conditions according to exemplary embodiment 2)

ADD (g / l)

Dry biomass (g / l)

pO ₂ (%)

0 (n.b.) 0.25 0.5 21 44 68 92 128 164

0.221

0.824

1,447

1,867

2.79

3.01

1.68 2.44 2.55 2.59 2.54 2.03

100 80 10

10 20

i.e. the pCV setpoint control started at 0.5h.

Claims (3)

1. A process for the preparation of 1,4-androstadiene-3,17-dione by fermentative sterol degradation, preferably by fermentative degradation of sito- and / or cholesterol, characterized in that in Rührfermentoren or in fermentors with other Umwälzprinzipien, preferably in the air Lift fermentor, during the fermentation period from 24 hours to 192 hours, via the aeration and recirculation regime, the relative dissolved oxygen saturation, measured as the pCV of the fermentation broth, after 0.5 hours to 45 hours below the 5% to 30% level, preferably below the 10% level, lowered and held at this level for at least 10 hours. 2. The method according to item 1, characterized in that after lowering the relative dissolved oxygen saturation to the 5% to 30% level, preferably to the 10% level, a setpoint selected from this range, preferably the 10% setpoint, during the remaining duration of the fermentative process is maintained. 3. Method according to item 2, characterized in that, to maintain the desired value selected from the range of 5% to 30%, preferably the 10% set point, the relative dissolved oxygen saturation during the remaining duration of the fermentative process, the circulation coinciding with the aeration Setpoint control is used.