DE3416854A1 - Process for the preparation of ubiquinone 10 - Google Patents

Abstract

A process for the preparation of ubiquinone 10 from microorganisms is described and entails bacterial cells of the species Rhodospirillum rubrum being cultivated in aqueous culture medium under aerobic conditions and with simultaneous illumination, and the ubiquinone 10 subsequently being obtained from the cells.

Description

description

The invention relates to a process for the production of ubiquinone-10 from microorganisms, in particular from bacteria of the species Rhodospirillum rubrum.

Ubiquinone-10, a benzoquinone of the following formula occurs in animals, plants and microorganisms (cf.

R.L. Lester and F.L. Crane, The Journal of Biological Chemistry 234, 2169 (1959)). As an electron carrier, it is part of the respiratory chain as well in some organisms part of the photosynthetic system. Ubiquinone-10 will too found in humans and has more recently been used as an active pharmaceutical ingredient, in particular for the therapy of certain heart diseases, gained importance.

The production of ubiquinone-10 from microorganisms is from one Series of publications known per se. For example, DE-OS 27 08 149 describes the extraction from Basidiomycetes of the genus Sporidiobulus or from Oosporidium and EP application 0 040 536 from Aureobasidium or Trichosporon. DE-OS 27 47 510 and DE-OS 29 38 377 describe special cultivation methods for yeast, which should lead to improved ubiquinone yields and in DE-OS 28 34 952 discloses a production process from bacteria of the species Xanthomonas.

Ubiquinon-10 is currently either commercially produced semi-synthetic or through the production of biologically formed ubiquinone-10 from beef hearts or from fermentation of yeasts of the genus Rhodotorula. the currently used methods are time consuming and expensive and lead to proportionate low yields. Even with the fermentation of yeast only one is insufficient Productivity, i.e. yield / unit of time achieved.

It is the object of the invention to provide a process for the production of ubiquinone-10 to create with high productivity, in which inexpensive starting products, for example organic agricultural waste such as liquid manure, can be used and which leads to high yields of ubiquinone-10 with relatively short cultivation times leads.

To solve the problem, a method is proposed that thereby is characterized in that one bacterial cells of the species Rhodospirillum rubrum in aqueous Culture medium at pH values of about 6 to 9 under aerobic conditions and in the light cultivated and then the ubiquinone-10 formed from the cells wins.

Rhodospirillum rubrum is a well-known member of the Rhodospirillaceen family belonging, facultatively phototrophic bacterium. It can easily be made up of mud or standing Isolated from water and grows either chemotrophically, i.e. aerobically in the dark or phototrophic, i.e.

anaerobic in light (see Hans G. Schlegel, Allgemeine Mikrobiologie, 3rd edition, page 298).

The formation of ubiquinone-10 in Rhodospirillum rubrum was reported by J. Ölze, W. Palke and S. Bohm, Arch. Microbiol. 102, 65-69 (1975). the The authors conclude that when the cells grow chemotrophically, i.e. aerobically in the dark, have a relatively high ubiquinone content, while under phototrophic conditions lead to the formation of bacterial chlorophyll at the expense of ubiquinone is favored.

It has now been found, surprisingly, that bacterial cells of the species Rhodospirillum rubrum, in particular the strain DSM 50914 with a combination of chemotrophs and phototrophic growth conditions and that the cells develop excellently at the same time have a high ubiquinone content. So it is with the invention Process achieved high productivity.

Rhodospirillum rubrum can be grown in the aqueous medium, its pH is about 6 to 9, under the conditions selected according to the invention organic media give better results. In a preferred embodiment According to the invention, liquid manure or liquid manure is used as the culture medium.

During the cultivation, the medium is mixed with 0.1 to 1 VVM (parts by volume Air / fermenter volume / minute). The ventilation can be used for smaller batches by stirring or shaking, while in large-scale fermentations sterile air is blown into the fermenter by conventional methods with stirring. The speed of the stirrer should be chosen so that there is sufficient ventilation is taken care of, but the medium is not moved so fast that the development of the Cells is affected. Usually a stirrer rotation of 50 to 200 rpm, preferably 150 to 160 rpm, with the stirring speed as a rule decreases with increasing fermenter size.

The exposure can be with any light source during the cultivation period be carried out that has a sufficient proportion of red light, in particular Fluorescent tubes of the type Osram Fluora are suitable.

The light intensity on the surface of the culture medium can range from 500 to 10,000, preferably 1,000 to 2,000 and particularly preferably 1,500 lx.

The cultivation takes place at temperatures of 25 to 40 ° C., preferably at 30 to 350C. The cultivation time required to achieve optimal productivity is 12 to 72 hours.

The fermentation can be carried out semi-continuously or in a batch process will. The cultivation is when a cell count of about 5 x 10 is reached 1 x 1010 cells / ml completed. The cells are after the cultivation period separated from the medium, for example by centrifugation, and then suitably dried, preferably freeze-dried.

The ubiquinone-10 is then prepared from the dry according to known methods Cell mass obtained; compared to fermentation processes with other microorganisms such as yeast, it saves time and money that the invention saponification can be dispensed with.

Preferably, the dry cell mass is extracted with n-hexane, the The extract is filtered and concentrated, and the residue is extracted with acetone. That after narrowing the oil obtained from the acetone extract is extracted again with hexane and the extract purified by chromatography.

The productivity achieved with the process according to the invention is about 4.6 mg ubiquinone / l / day and is thus significantly above that of the more well-known Procedure.

It is also advantageous that agricultural waste products such as liquid manure or manure using the method according to the invention for obtaining a high-quality active ingredient can be used and thus eliminated.

The process is explained below with the aid of examples.

Example 1 Composition of the culture medium: Ingredients% by weight Glucose 2 yeast extract 1 CaCO3 0.2 H2O double dist. ad 100 pH value: 6.8 A preculture of Rhodospirillum rubrum was in the culture medium given above at 25 0C below Air to enter is attracted for about 1 week. The number of cells in the preculture was about 5 x 109 cells / ml.

250 ml of the above culture medium were placed in a 1 l Erlenmeyer flask Sterilized for 20 minutes at 121 ° C. After cooling to about 250C, the medium became with such an amount of the above-mentioned preculture of Rhodospirillum rubrum DSM 50914 inoculates that a baseline cell count of approximately 10 cells / ml has been achieved. The culture mixture was shaken for 72 hours at 250C Incubated with access to air. The shaking frequency was 160 rpm. During the cultivation period was exposed with a 2000 lx lamp.

After the incubation time had elapsed, the cells were centrifuged off and freeze-dried; the cell yield was 10 g dry matter / l culture medium.

100 g of the dried cell mass with an ubiquinone-10 content of 130 mg were used with 1 liter of n-hexane for 30 minutes long at reflux boiled, the cell suspension obtained was vacuum filtered and the filtrate at Concentrated to dryness at 400C. The residue was taken up in a little acetone, thoroughly suspended and the suspension centrifuged. The supernatant obtained was concentrated again. There was thus obtained 2 g of a reddish oil. The oil was taken up in a little hexane and using n-hexane / ethyl acetate (95: 5) as the mobile phase Purified by thin-layer chromatography on a silica gel plate. The ubiquinone fraction was eluted with ethyl acetate and the eluate was concentrated to dryness.

100 mg of a yellow oil were obtained. After recrystallization 60 mg of yellow crystals were obtained from ethanol, which were determined by means of HPLC and mass spectroscopy identified as pure ubiquinone-10. The productivity was 3.3 9 cell dry matter / l / day.

Example 2 The fermentation was carried out as described in Example 1, except that a culture medium of the following composition is used was: Ingredients% by weight yeast extract 1 KH2P04 0.1 MgSO4.7H2O 0.05 H20 dist. ad 100 pH value: 6.8 The dry cell yield was 5 g / l of culture medium with an ubiquinone-10 content of 85 mg / 100 cell dry matter. The productivity was 1.7 g cell dry matter / l / day.

It was found that with the lower organic content in the culture medium according to Example 2, both in terms of total cell mass also with regard to the ubiquinone-10 content considerably lower yields than with the culture medium according to Example 1 can be obtained.

Example 3 Composition of the culture medium: Ingredients wt.

Glucose 2 yeast extract 1 K2HPO4 0.1 MgS04.7H2O 0.05 H20 dist. ad 100 The pH of the culture medium was adjusted with dipotassium hydrogen phosphate during the cultivation period held between 6 and 7.

16 1 of the culture medium given above were in a 20 1 laboratory stirred fermenter Sterilized for 20 minutes at 121 ° C. After cooling to 300C, the medium became inoculated with 2.0 l of the preculture according to Example 1, so that a starting cell number from approximately 5 x 10 8 cells / ml of culture medium. The fermentation approach was incubated for 24 hours at 30 ° C. with stirring.

During the cultivation period, sterile air was released at a rate of 3 to 4 1 / min (0.2 VVM) passed into the fermenter. The stirring intensity during the The cultivation time was 150 rpm and the light intensity on the fermenter surface 1000 lx. The cells were collected and processed according to the method according to Example 1, whereby a cell yield of 6.5 g dry matter / l culture medium was obtained. The ubiquinone-10 content the dry cell mass was 90 mg / 100 g and the productivity 6.5 g dry cell mass / l / day.

Claims (5)

Process for the preparation of ubiquinone-10 Claims 1. Process for the production of ubiquinone-10 from microorganisms, characterized in that one bacterial cells of the species Rhodospirillum rubrum in an aqueous culture medium in a Cultivated under aerobic conditions in the light and then pH of about 6 to 9 the ubiquinone-? O wins from the cells. 2. The method according to claim 1, characterized in that the Strain Rhodospirillum rubrum DSM 50914 used. 3. The method according to claim 1 and 2, characterized in that one the culture medium during the cultivation period with 0.1 to 1 VVM (parts by volume Air / fermenter volume / minute). 4. The method according to claims 1 to 3, characterized in that that the culture medium is exposed to 500 to 1000 lx during the cultivation period. 5. Process according to claims 1 to 4, characterized in that that manure or liquid manure is used as a culture medium.