GB1591047A - Microbiological production of ubiquinones - Google Patents

Description

(54) MICROBIOLOGICAL PRODUCTION OF UBIQUINONES (71) We, LABORATOIRE ROGER BELLON, S.A., a French body corporate, of 159, avenue du Roule, 92201 Neuilly sur Seine, France, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- The present invention relates to the production of 10-ubiquinone from bacterial fermentation broths.

Ubiquinones are of the general formula:

in which n is a number of from 1 to 10. These compounds are known to be present in certain animals, plants and micro-organisms, including bacteria, yeasts and fungi. The ubiquinone homologue present in micro-organisms varies according to the species.

10-Ubiquinone has been isolated from bacteria such as Pseudomonas denitrlficans, Agrobacterium tumefaciens, Ochromonas malhamensis and Rhodopseudomonas spheroides; from yeasts such as Cryptococcus, Rhodotorula, Sporobolomyces and Rhodosporidium; and from fungi such as Folyporus schwarzeti, Neurospora crassa and Aspergillus fumigatus. The isolation of 10-ubiquinone from Xanthomonas has not, however, previously been described.

It is known to extract ubiquinones from the cells of micro-organisms by extraction with hexane, heptane or petroleum ether after saponification in an alcoholic potassium hydroxide medium in the presence of pyrogallol, or by direct extraction with a solvent such as ethanol or acetone or mixtures of solvents such as chloroform-methanol and ether-ethanol, followed by extraction with hexane, heptane or petroleum ether. The ubiquinone is subsequently isolated by chromatography on a column of alumina, silicic acid, aluminium silicate or magnesium silicate, using mixtures of solvents or different solvents in succession, always in large volumes. The ubiquinone obtained can then be crystallized from ethanol.

In general, yields of ubiquinone are mediocre either because the previously used microbial strains are poor producers of ubiquinone or because the methods of extraction used, especially saponification, are complex and difficult to use on an industrial scale.

We have now found that good yields of 10-ubiquinone can be obtained from Xanthomonas fermentation cultures and, particularly, from Xanthomonas stewartii cultures. We have also developed strains of Xanthomonas stewartEi which, when cultured in suitable media, give very good yields of 10-ubiquinone. These strains of Xanthomonas stewartii have been deposited at the Institut Pasteur, Paris, France, under the accession numbers 1035 and 1036, respectively; and have the following morphological characteristics: 1. Characteristics of the Cells: Small rods of 0.2 to 0.8 ssm by 0.6 to 2,um, mobile by means of polar flagellae, non-sporulated.

2. Staining characteristics: Gram-negative.

3. Culture Characteristics: on nutrient agar: opaque, of a golden yellow to orange colour, non-diffusible, circular, smooth and convex colonies.

on broth: slight cloudiness, deposit and surface film.

4. Physiological Characteristics pH: culture at pH 6.8.0, optimum at about pH 7.

Temperature: growth between 20 and 40"C, optimum from 30 to 400 C.

Oxygen: aero-anaerobic behaviour.

Gelatine: no liquefaction.

Oxidase: positive.

Cytochrome oxidase: positive.

Catalase: positive.

Assimilation: urea negative.

Production of indole: (1036)'positive, (1035) negative.

Production of hydrogen sulphide: negative.

Reduction of nitrates: negative.

Production of acetyl-methyl-carbinol: positive.

Methyl red: negative Acidification: Arabinose (1035) positive (1036) positive Sucrose (1035) positive (1036) positive Rhamnose (1035) positive (1036) negative Glucose (1035) positive (1036) positive Mannose (1035) negative (1036) negative Lactose (1035) negative (1036) negative Sorbitol (1035) negative (1036) negative Inositol (1035) negative (1036) negative Lysine decarboxylase: negative.

Ornithine decarboxylase: negative.

Tryptophane desaminase: (1036) positive, (1035) negative.

Arginine deshydrolase: negative.

Production of gas: negative.

According to the invention, there is provided a method of producing 10ubiquinone, which comprises culturing bacteria of the genus Xanthomonas preferably of the species Xanthomonas stewartii, and more preferably the strain of Xanthomonas stewartii deposited at the Institut Pasteur under accession number 1035 or 1036, in a culture medium therefor, and recovering 10-ubiquinone from the culture medium.

The bacteria used in the method according to the invention (which are Gramnegative bacteria) may be cultured in conventional manner in the method according to the invention, using media containing a source of carbon, a source of nitrogen, and inorganic salts.

The source of carbon used may be, for example, sucrose, starches or paraffin hydrocarbons of chain length between 9 and 26 carbon atoms.

The source of nitrogen used may be, for example, ammonium salts, corn steep liquor (C.S.L.), meat peptones, casein peptones, yeast extracts, casein hydrolysis products, or various flours such as soya flour, corn flour, potato flour and wheat flour.

The culture medium should be sterilised before inoculation with the bacteria, for example, at 1200 to 1500C for 60 to 2 minutes, depending on the method of sterilisation used.

The culture conditions, such as the temperature, pH of the medium and aeration, which depend on the strain(s) used, should be controlled so that the growth of the micro-organism is rapid and so that the maximum amount of ubiquinone is produced. A preferred range of temperatures is from 25 to 400 C, more especially from 280C to 350C, for Xanthomonas stewartii. During culture, the pH is preferably maintained between 6 and 8 and more preferably between 6.5 and 7.5. The aeration is preferably regulated so as to be between 0.2 and 1.5 volumes per volume per minute, depending on the pressure and the rate of stirring. Under these conditions the fermentation takes from 7 to 24 hours, depending on the strain of bacteria used. The time at which the bacteria are harvested is determined by the end of the exponential phase of growth.

Recovery of the 10-ubiquinone from the culture medium may be conventional or may be as described and claimed in our Patent Application 8013021 (Serial No.

1591048), which has been divided out of the present application. In the method described in the above-mentioned divisional application, bacterial solids are separated from the culture medium, and the ubiquinone is recovered from the bacterial solids by drying the latter, the dry residue being extracted with an organic solvent, and the organic extract being concentrated and purified.

When using the strain of Xanthomonas stewartii deposited with the Institut Pasteur under accession number 1035 or 1036 in accordance with the invention, 2 to 25g of 10-ubiquinone may be obtained per m3 of fermented broth, depending on the strain and the medium used, on purification by the method of the abovementioned divisional application.

In- order that the present invention may be more fully understood, the following Examples, in which all percentages are by weight unless otherwise indicated, are given by way of illustration only.

Example 1 1,000 litres of an aqueous medium of the following composition were prepared: 4% of glucose, 3% of soya flour, 0.02% of magnesium sulphate, 0.1% of monopotassium phosphate, 0.1% of dipotassium phosphate and 0.5% of sodium chloride. The medium was sterilised at 1300C for 30 minutes and its pH was adjusted to 7.

The medium was inoculated with a preculture of Xanthomonas stewartii (Pasteur accession number 1036) in an amount of 10% by volume of the culture.

Fermentation was carried out with stirring and aeration at 300C for 18 to 22 hours, that is up to the end of the exponential phase of growth, the pH being maintained between 6.5 and 7.5. Bacterial solids were recovered by centrifuging and a concentrate was obtained, which was spray-dried using an inlet temperature of 1800C and an outlet temperature of 85"C.

Under these conditions, 25 to 30 kg of powder were obtained.

The ubiquinone contained in the spray-dried powder was extracted with 4 volumes (relative to the weight of the material) of methanol containing 8% of butanol at 650C, that is, at the reflux temperature, for 30 minutes; this operation was carried out twice in succession. After filtration, the extracts were combined and concentrated to half their volume at a temperature of 40 to 500 C.

The ubiquinone contained in the concentrates was extracted twice in succession with 20%, based on the volume of the concentrates, of hexane, and 5% of water was added to the first extract to obtain a two-phase system.

The hexane phases were concentrated in vacuo at 40"C until an oil was obtained, into which 5 volumes of acetone was poured. A precipitate formed, consisting essentially of phosholipids, which was left to ripen at +40C for 10 hours or more. The precipitated phospholipids were removed by filtration and the ketone filtrate containing the ubiquinone was concentrated to dryness in vacuo at a temperature of 30 to 400 C.

The concentrate obtained was then taken up in 40 to 50 litres of methanol and was crystallised at +4"C. Three successive crystallisations were carried out and 12 g of 10-ubiquinone were thus obtained.

Example 2 1,000 litres of an aqueous medium having the following composition were prepared 4% of glucose, 4% of corn steep liquor (C.S.L.), 0.12% of yeast extract, 0.02% of magnesium sulphate, 0.1% of monopotassium phosphate, 0.10/, of dipotassium phosphate and 0.5% of sodium chloride.

The medium was sterilised by passing it through a heat exchanger at 1400C for 2 minutes and its pH was adjusted to 7.

The medium was inoculated with a preculture of Xanthomonas stewartil (Pasteur accession number 1035) and fermentation was carried out as described in Example 1.

The bacterial solids were recovered by centrifuging and 100 kg of a concentrate were obtained; this was dehydrated by washing with 2 volumes of methanol containing 4% of butanol (volume/weight) for 30 minutes at ambient temperature.

After centrifuging, the ubiquinone contained in the dehydrated bacterial cake was extracted with 2 volumes of methanol containing 10% of butanol. The remainder of the extraction was carried out as in Example 1.

After three successive crystallisations from ethanol, 2.5 g of 10-ubiquinone were obtained.

WHAT WE CLAIM IS: 1. A method of producing l0-ubiquinone, which comprises culturing bacteria of the genus Xanthomonas in a culture medium therefor and recovering 10ubiquinone from the culture medium.

2. A method according to claim 1, in which the bacteria used is Xanthomcnas stewartii.

3. A method according to claim 1, in which the bacteria used is the strain of Xanthomonas stewartii deposited with the Institut Pasteur under accession number 1035 or 1036.

4. 10-ubiquinone when produced by the method claimedin any of claims 1 to 3.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (4)

**WARNING** start of CLMS field may overlap end of DESC **. (Pasteur accession number 1035) and fermentation was carried out as described in Example 1. The bacterial solids were recovered by centrifuging and 100 kg of a concentrate were obtained; this was dehydrated by washing with 2 volumes of methanol containing 4% of butanol (volume/weight) for 30 minutes at ambient temperature. After centrifuging, the ubiquinone contained in the dehydrated bacterial cake was extracted with 2 volumes of methanol containing 10% of butanol. The remainder of the extraction was carried out as in Example 1. After three successive crystallisations from ethanol, 2.5 g of 10-ubiquinone were obtained. WHAT WE CLAIM IS:

1. A method of producing l0-ubiquinone, which comprises culturing bacteria of the genus Xanthomonas in a culture medium therefor and recovering 10ubiquinone from the culture medium.

2. A method according to claim 1, in which the bacteria used is Xanthomcnas stewartii.

3. A method according to claim 1, in which the bacteria used is the strain of Xanthomonas stewartii deposited with the Institut Pasteur under accession number 1035 or 1036.

4. 10-ubiquinone when produced by the method claimedin any of claims 1 to 3.