DD248144B1 - PROCESS FOR THE PREPARATION OF 1,4-ANDROSTADIENE-3,17-DION BY FERMENTATIVE STEROL DEGRADATION - Google Patents

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 state of the art

It is known that sterols, such as e.g. Sitosterol, cholesterol, stigmasterol, campesterol and ergosterol, by microorganisms from many 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 is 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 (GD Searle & Co., US 3684657, 1970, Mycobacterium spec. NRRLB-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 I. Sauerbaum et. al. (1st European Congress for Biotechnology, Interlaken, 1978, 139-143) indicated fermentation of sitosterol with Mycobacterium spec. NRRL B-3683 * ¹ 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 are converted to 80% of theory in a mixture of ADD and AD in 96 hours, is the best so far View process solution. According to these authors, the 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), with the relative best condition and thus the lower limit being a relative dissolved oxygen saturation of the culture broth of 50 % is specified.

This results in requirements for the process control in the fermenter, which are unfavorable in terms of foaming, creaming of substrates and biomass, Scherkraft- and mixing ratios, especially in the presence of adsorbers in the fermentation broth.

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

Object of the invention

The aim of the invention is to produce 1,4-androstadiene-3,17-dione in high yields from sterols.

Explanation of the essence of the invention

The invention has for its object to describe an improved process for the preparation of 1,4-Androstatien-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.

According to the invention, this object is achieved in its basic feature 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 sterol transformation to form the product ADD or the product mixture ADD / AD, for example those of the genus Mycobacterium, especially those of the species Mycobacterium vaccae.

The sterol surfactant preparations are obtained by the known methods of lyophilization, grinding or spray drying.

As described in the patents DD-WP 248143 A3 and DD-WP 248145 A3, come as surfactants compounds from the group of polyethylene oxide-polypropylene oxide adducts of N, N "-Tra - (- oxypropylpropyol) -N '- (2-oxypropyl ) -diethylenetriamine having molecular weights of polypropylene glycol around 1400 to 2000 for use.

The aqueous fermentation media contain at least one carbon source from the collection glycerol, glucose, sucrose, starch and / or molasses, etc., furthermore at least one nitrogen source from the collection yeast extract, urea, ammonium salts and / or complex media components (such as corn steep liquor, plant flours , Meat extract, hydrolysates) and various mineral salts.

To the fermentation medium, an adsorbent is added, 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 (see again the writings DD-WP 248143A3 and DD-WP 248145A3).

The duration of the fermentation is 24 hours to 192 hours. It is cultured under aerobic conditions at temperatures of 20 ⁰ C to 45 ⁰ C and pH values in the range of pH6 to pH8.

The fermentation is carried out in Rührfermentor or in fermentors with other Umwälzungsprinzipien, 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 pGy 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 to be selected on the basis of the dependence of the oxygen transport rate determined above for the fermentor used. In this context, it is essential to the invention, by this regime, the relative dissolved oxygen saturation of the fermentation solution, measured as pO ₂ value, after 0.5 hours to 45 hours, preferably after 1.0 hours to 6 hours, below the 5% level to lower to 30% level, preferably below the 15% level, and to keep at least 10 hours at this level. This protocol is based on the finding 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 main component, with surprisingly high yield, although the relative dissolved oxygen saturation of the culture broth in the course of the fermentation after falling from the saturation value is 100% temporarily or constantly less than 10%. With this oxygen consumption enters a favorable for the achievement of high ADD formation rates physiological state of the biomass.

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 pO _{2 set} point is chosen 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.

For the pulsed regime of aeration and recirculation (eg, agitator rotor speed), the two states coincide in both quantities

- Reduced or stopped air entry and reduced circulation for pO ₂ -value> p0 ₂ setpoint,

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

The change ranges of recirculation parameters and air intake are to be determined from the oxygen transport characteristics of the fermenter used in each case.

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, whereby the рОг setpoint control by means of coincident intensification of ventilation and circulation at setpoint below begins early. With this process control creaming and foaming problems are avoided in a particularly advantageous manner and ensures favorable contact and Durchmischungsbedingungeri for the solid phases (adsorber, sitosterol, biomass). At the same time high ADD yields are realized. When carrying out the process on an industrial scale, for example in the 10m ³ -Rührfermentor, the above pulse regime is modified according to the advantageous technical realization. Preferred embodiments are then

- Continuous, for example, linear increase of the speed up to a specified upper limit when falling 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 the air entry when exceeding the pO 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 the production of ADD via microbial sterol degradation, which is characterized by low levels of relative dissolved oxygen saturation of the fermentation broth during long stretches 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 NRRI B-3683, the seed culture for the ADD fermentation is cultivated. In the first step, a Sägägagarkultur by means of four-day incubation at 35 ⁰ C is created. 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 10g, glycerol 10g, KH ₂ PO ₄ 0.5g, K ₂ HPO ₄ 0.5g, (NH ₄ J ₂ HPO ₄ 1.5g, MgSO ₄ .7H ₂ O 0.2g, FeSO ₄ .7H ₂ O 0.01 g, ZnSO ₄ 7H ₂ O 0.002 g with pH 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 5ml of the first preculture are used to inoculate round bottom flask of the second preculture stage with the filling conditions as in the first precultivation stage, which are also shaken for 36 hours at 28 ° C.

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 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 ₃ 3g, MgSO ₄ .7H ₂ O 0.2 g, FeSO 4 ₄ · 7H ₂ O 0.01 g, ZnSO ₄ · 7H ₂ O 0.002 g Before sterilization, the pH is adjusted to 7.0 The sterilization is carried out by autoclaving the fermenter vessel containing the medium including the necessary amount of adsorber for 60 minutes at 121 ° C. the Sitosterolpräparation is sterilized twice 60 minutes steam pot and introduced in one step with the inoculation in the rear, cooled to 31 ⁰ C fermentor. per liter of fermentation broth 15g sitosterol are used, the preparation obtained by wet grinding with 10% of a surfactant from Group of the polyethylene oxide-polypropylene oxide adducts of N, N "-Tra - (- oxypropylpropyol) -N '- (2-oxypropyl) -diethylenetriamine with molecular weights of the polypropylene glycol around 1400 to 2000 in a colloid mill 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 is measured and recorded as the pO ₂ value (100% calibration in the uninoculated stirred and aerated fermentation broth); 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 values 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 proceed as in Example 1.

The fermentation medium of the main culture has per liter of distilled water. the composition: urea 0.25 g, glucose 5 g, (NH ₄ ) ₂ SO ₄ 3 g, KH ₂ PO ₄ 1 g, K ₂ HPO _{4 4} g, MgSO ₄ .7H ₂ O 0.2 g, 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 рОг sensor.

Within 0.5 hours, the drop in the pO ₂ value to 10% occurs as a result of oxygen depletion as a result of the uptake of oxygen 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 is 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 450min " ¹ and Zuluftstop.

This pulse regime for compliance with the theoretical setpoint value of 10% has the process-technically extremely important effect that no creaming of biomass and substrates and no foaming is achieved. 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 the fermentation) there are 3.01 g / l ADD. The plateau value of the dry biomass of about 2.6 g / l is around the

30th fermentation hour reached. From the 130th fermentation hour, the pO ₂ value rises to about 20%, although it works without air and at 450 min ^-1 .

Table 1: Kinetics for ADD, dry biomass and pO ₂ 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

10

10

20

50

83

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 setpoint control (target value 10%, other process conditions according to 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

ie the pO ₂ setpoint control started from 0.5h.

Claims (6)

1. A process for preparing 1,4-androstadiene-3,17-dione by fermentative sterol degradation at aerobic culture conditions for microorganisms capable of sterol side-chain degradation, for example those of the genus Mycobacterium, especially of the species Mycobacterium vaccae, in aqueous fermentation media containing at least one Carbon source, at least one nitrogen source and in addition to mineral salts, a styrene-surfactant preparation and a particulate present polar adsorber type of organic polymer whose acidity during the 24 hours to 192 hours at temperatures between 20 ° C and 45 ⁰ C carried out fermentation is in the range of pH 6 to pH 8, characterized in that the relative dissolved oxygen saturation of the fermentation solution, measured as pO ₂ value, via the regime of aeration and circulation of this solution after 0.5 hours to 45 hours below the 5% to 30 % Level lowers and at least 10 hours at this m level holds. 2. The method according to claim 1, characterized in that one lowers the relative dissolved oxygen saturation after 1 hour to 6 hours below the 15% level. 3. The method according to claim 1 and 2, characterized in that, after lowering the relative dissolved oxygen saturation to a level value of the above labeling retains this as the desired value for the remaining fermentation period. 4. The method according to claim 2 and 3, characterized in that one lowers the relative dissolved oxygen saturation to the 10% level and this maintains as the set value for the remaining fermentation period. 5. The method according to claim 1 to 3, characterized in that one applies after lowering the relative dissolved oxygen saturation to a level value above labeling a ventilation and circulation of the fermentation solution in coincidence detected setpoint control to maintain this level value. 6. The method according to claim 2 and 5, characterized in that one applies after lowering the relative dissolved oxygen saturation to the 10% level ventilation and circulation of the fermentation solution in coincidence detected setpoint control for maintaining this level value.