GB1591048A

GB1591048A - Microbiological production of ubiquinones

Info

Publication number: GB1591048A
Application number: GB13021/80A
Authority: GB
Original assignee: Laboratoire Roger Bellon SA
Current assignee: Laboratoire Roger Bellon SA
Priority date: 1978-05-31
Filing date: 1978-05-31
Publication date: 1981-06-10

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 8-, 9- and/or 10-ubiquinones 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 denitrificans, Agrobacterium tumefaciens, Ochromonas malhamensis and Rhodopseudomonas spheroides; from yeasts such as Cryptococcus, Rhodotorula, Sporobolomyces and Rhodo spondium; and from fungi such as Polyporus schfrarzeti, Neurospora crassa and Aspergillus fumigatus.

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 8-, 9- or 10-ubiquinone can be obtained by a method which comprises culturing a Gram-negative bacteria capable of producing 8-, 9- or 10-ubiquinone in a culture medium therefor, separating bacterial solids from the culture medium, and recovering the ubiquinone from the bacterial solids by drying the latter, extracting the dry residue with an organic solvent, and concentrating and purifying the organic extract.

Suitable strains of Gram-negative bacteria for use in the method according to the invention include, for example, Escherichia coli, Enterobacter aerogenes, Enterobacter liquefaciens, Serratia marcescens, Vibrio, Achromobacter alcaligenes and Acinetobacter anitratum (all for 8-ubiquinone), Pseudomonas aeruginosa (serotypes P1 to P12), Pseudomonas fluorescens, Pseudomonas tomato, Phytobacterium saliciperda, Acineto bacter antiratrum and Acinetobacter Iwoffi (all for 9-ubiquinone) and Xanthomonas stewartii (for 10-ubiquinone).

A method of producing 10-ubiquinone, which comprises culturing bacteria of the genus Xanthomonas (for example, Xanthomonas stewartii) in a culture medium therefor and recovering 10-ubiquinone from the culture medium is described and claimed in our Application 34584/77 (Serial No. 1,591,047), from which the present application has been divided. Particularly preferred strains of Xanthomonas stewartii have been deposited with the Institut Pasteur under accession numbers 1035 and 1036.

The Gram-negative bacteria may be conducted 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 sterilized 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 400C, more especially from 280C to 35"C, 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.

Bacterial solids may be separated from the culture medium by, for example, centrifuging or filtrations; in this way, between 10 and 40 kg of culture, expressed as dry weight, per m3 of fermented medium, may be harvested.

Water is then removed from the centrifuged or filtered medium, for example, by washing with a hydrophilic solvent or, preferably, by spray-drying or by some other drying process.

The washing may be carried out with methanol at a temperature of from 10 to 30"C for 15 to 60 minutes; the addition of from 1 to 15% by volume of butanol to the methanol enables pigments to be removed from the ubiquinone.

The spray-drying should be carried out under conditions such that the ubiquinone present in the cells is not degraded. It has been found, unexpectedly, that by carrying out the spray-drying at a temperature of from 140 to 250"C (preferably 160 to 1800C) at the injection point and from 40 to 110 C (preferably 60 to 85"C) at the recovery cyclone, the ubiquinone is not significantly degraded.

Under these conditions, the powder obtained still contains from 3 to 10% by weight of residual water, for example from 5 to 7%. This proportion of residual water is important since more extensive dehydration runs the risk of degrading the ubiquinone contained in the bacterial cells.

The ubiquinone present in the dehydrated bacterial cells is then extracted with a solvent such as acetone or an aliphatic alcohol having 1 to 4 carbon atoms (for example, methanol, ethanol, propanol or isopropanol), at a temperature of from 20 to 65"C, for 20 to 60 minutes.

Preferably, methanol to which from 5 to 20% by volume, more preferably 8 to 15% by volume, of butanol, has been added is used; the use of butanol improves the solubilisation of the ubiquinone. 2 to 3 successive extractions may be carried out with 1.5 to 4 volumes of solvent relative to the weight of dehydrated bacterial culture. The extracts may then be concentrated to half their volume in vacuo at a temperature of from 30 to 60"C and preferably from 30 to 45"C and the ubiquinone in these concentrated solvents extrated 2 to 3 times with hexane, heptane, petroleum ether or cyclohexane. The solvent phases containing the ubiquinone may then be concentrated in vacuo at a temperature between 30 and 45"C.

The resulting concentrate may then be purified conventionally, for example, by chromatography on a column of alumina, silicic acid, aluminium silicate or magnesium silicate. Residual impurities, in the main consisting of phospholipids, may be removed by precipitation with acetone followed by crystallisation of the ubiquinone. For this purpose, 4 to 6 volumes of acetone per volume of organic extract concentrate at a tem perature of 20 to 40"C may be added to the concentrate, whereby a precipitate forms.

The precipitate may be allowed to ripen at +4 C for 5 to 15 hours since phospholipids are quantitatively and selectively precipitable by means of acetone at +4 C.

The precipitate thus formed may be removed by centrifuging or filtration, while the acetone solution, freed from phospholipids and containing ubiquinone, may be con centrated to dryness in vacuo at a temperature from 30 to 50"C.

The oily residue thus obtained may be taken up in an alcohol such as methanol or ethanol and crystallised at a temperature of from 0 to 20"C. From 2 to 5 crystallisations may then be carried out; and the resulting purity may be determined by spectrophotometric measurement. Under these conditions, ubiquinone crystals having a purity of 90 to 95 % can be obtained.

When using the strain of Xanthornonas stewartii having the Pasteur accession number 1035 or 1036 in accordance with the invention, 2 to 25 g of 10-ubiquinone may be obtained per mS of fermented broth, depending on the strain and the medium used.

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 30"C 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 65"C, 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 50"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 phospholipids, which was left to ripen at +4 C for 10 hours or more. The precipi tated phospholipids were removed by filtration and the ketone filtrate containing the ubi quinone was concentrated to dryness in vacuo at a temperature of 30 to 40"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, 0.1% of dipotassium phosphate and 0.5% of sodium chloride.

The medium was sterilised by passing it through a heat exchanger at 140"C for 2 minutes and its pH was adjusted to 7.

The medium was inoculated with a preculture of Xanthomonas stewartii (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.

Example 3.

100 litres of an aqueous medium of the following composition were prepared: 1% of glucose, 0.5% of peptone, 0.7% of yeast extract, 0.5% O/o of meat extract and 0.3% of sodium chloride. The medium was sterilised at 1200C for 30 minutes and its pH was adjusted to 7.

The medium was inoculated with 10 litres of a preculture of Pseudomonas fluor- escens. Fermentation was carried out with stirring and aeration at 35"C for 7 to 12 hours, up to the end of the exponential phase of growth, the pH being maintained between 6.5 and 7.

Bacterial bodies were centrifuged and spray-dried as in Example 1 and 12 kg of powder were thus obtained.

The ubiquinone contained in the spray-dried powder was extracted with 4 volumes (relative to the weight of material) of methanol containing 8% of butanol, for 30 minutes at 6S"C, that is under reflux.

Thereafter the procedure described in Example 1 was followed and after two crystallisations from ethanol, 1.5 to 2.5 g of 9-ubiquinone were obtained.

This example shows that the process of extraction and purification according to the invention is applicable to all fermentation media of bacteria producing 8-, 9- or 1 0-ubiquinone.

WHAT WE CLAIM IS: 1. A method of producing 8-, 9- or 10-ubiquinone which comprises culturing a Gram-negative bacteria capable of producing 8-, 9- or 10-ubiquinone in a culture medium therefor, separating bacterial solids from the culture medium, and recovering the ubiquinone from the bacterial solids by drying the latter, extracting the dry residue with an organic solvent, and concentrating and purifying the organic extract.

2. A method according to claim 1, in which the drying of the bacterial solids is effected by washing them with a hydrophilic solvent.

3. A method according to claim 2, in which the hydrophilic solvent is methanol containing 1 to 15% by volume of butanol and washing is effected at a temperature of 10 to 30"C for 15 to 60 minutes.

4. A method according to claim 1, in which drying of the bacterial solids is effected by spray drying.

5. A method according to claim 4, in which spray drying is effected with an injection temperature of 140 to 250"C and a temperature in the recovery cyclone of 40 to llO"C.

6. A method according to claim 5, in which the injection temperature is from 1600 to 1800C and the temperature in the recovery cyclone is from 60 to 85"C.

7. A method according to any of claims 1 to 6, in which the extraction of the dry residue is effected with acetone or an aliphatic alcohol having 1 to 4 carbon atoms.

8. A method according to claim 7, in which extraction is effected with methanol containing 5 to 20% by volume of butanol at 20 to 65"C.

9. A method according to claim 8, in which the organic extract concentrate is purified by extraction two or three times with hexane, heptane, petroleum ether or cyclohexane, concentration of the resulting extract, adding acetone to the resulting concentrate to precipitate phospholipids and crystallisation of the ubiquinone.

10. A method according to claim 9, in which from 4 to 6 volumes of acetone are added per volume of organic extract concentrate at a temperature of 20 to 40"C, the precipitate formed is allowed to stand at +4 C for 5 to 15 hours and is then separated by centrifugation or filtration.

11. A method according to claim 10, in which the solution from which the precipitate has been removed is concentrated to dryness under reduced pressure at a temperature of 30 to 50or, the oily residue obtained is taken up in an alcohol and subjected to from 2 to 5 successive recrystallisations at a temperature of from 0 to 20"C in order to obtain ubiquinone crystals of 90 to 95% purity.

12. A method according to any of claims 1 to 11, in which the bacteria used in the fermentation are of the genus Xanthomonas and the ubiquinone obtained is 10ubiquinone.

13. A method according to claim 12, in which the bacteria used is Xanthomonas stewartii.

14. A method according to claim 12, in which the bacteria used is the strain of Xanthomonas stewartii deposited with the Institut Pasteur under accession number 1035 or 1036.

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

Claims

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

Example 3.

100 litres of an aqueous medium of the following composition were prepared: 1% of glucose, 0.5% of peptone, 0.7% of yeast extract, 0.5% O/o of meat extract and 0.3% of sodium chloride. The medium was sterilised at 1200C for 30 minutes and its pH was adjusted to 7.

The medium was inoculated with 10 litres of a preculture of Pseudomonas fluor- escens. Fermentation was carried out with stirring and aeration at 35"C for 7 to 12 hours, up to the end of the exponential phase of growth, the pH being maintained between 6.5 and 7.

Bacterial bodies were centrifuged and spray-dried as in Example 1 and 12 kg of powder were thus obtained.

The ubiquinone contained in the spray-dried powder was extracted with 4 volumes (relative to the weight of material) of methanol containing 8% of butanol, for 30 minutes at 6S"C, that is under reflux.

Thereafter the procedure described in Example 1 was followed and after two crystallisations from ethanol, 1.5 to 2.5 g of 9-ubiquinone were obtained.

This example shows that the process of extraction and purification according to the invention is applicable to all fermentation media of bacteria producing 8-, 9- or 1 0-ubiquinone.

WHAT WE CLAIM IS: 1. A method of producing 8-, 9- or 10-ubiquinone which comprises culturing a Gram-negative bacteria capable of producing 8-, 9- or 10-ubiquinone in a culture medium therefor, separating bacterial solids from the culture medium, and recovering the ubiquinone from the bacterial solids by drying the latter, extracting the dry residue with an organic solvent, and concentrating and purifying the organic extract.
2. A method according to claim 1, in which the drying of the bacterial solids is effected by washing them with a hydrophilic solvent.
3. A method according to claim 2, in which the hydrophilic solvent is methanol containing 1 to 15% by volume of butanol and washing is effected at a temperature of 10 to 30"C for 15 to 60 minutes.
4. A method according to claim 1, in which drying of the bacterial solids is effected by spray drying.
5. A method according to claim 4, in which spray drying is effected with an injection temperature of 140 to 250"C and a temperature in the recovery cyclone of 40 to llO"C.
6. A method according to claim 5, in which the injection temperature is from 1600 to 1800C and the temperature in the recovery cyclone is from 60 to 85"C.
7. A method according to any of claims 1 to 6, in which the extraction of the dry residue is effected with acetone or an aliphatic alcohol having 1 to 4 carbon atoms.
8. A method according to claim 7, in which extraction is effected with methanol containing 5 to 20% by volume of butanol at 20 to 65"C.
9. A method according to claim 8, in which the organic extract concentrate is purified by extraction two or three times with hexane, heptane, petroleum ether or cyclohexane, concentration of the resulting extract, adding acetone to the resulting concentrate to precipitate phospholipids and crystallisation of the ubiquinone.
10. A method according to claim 9, in which from 4 to 6 volumes of acetone are added per volume of organic extract concentrate at a temperature of 20 to 40"C, the precipitate formed is allowed to stand at +4 C for 5 to 15 hours and is then separated by centrifugation or filtration.
11. A method according to claim 10, in which the solution from which the precipitate has been removed is concentrated to dryness under reduced pressure at a temperature of 30 to 50or, the oily residue obtained is taken up in an alcohol and subjected to from 2 to 5 successive recrystallisations at a temperature of from 0 to 20"C in order to obtain ubiquinone crystals of 90 to 95% purity.
12. A method according to any of claims 1 to 11, in which the bacteria used in the fermentation are of the genus Xanthomonas and the ubiquinone obtained is 10ubiquinone.
13. A method according to claim 12, in which the bacteria used is Xanthomonas stewartii.
14. A method according to claim 12, in which the bacteria used is the strain of Xanthomonas stewartii deposited with the Institut Pasteur under accession number 1035 or 1036.
15. A method according to claim 1 of producing 8-, 9- or 10-ubiquinone substan

tially as herein described in any of the Examples.
16. 8-, 9- or 10-Ubiquinone when produced by the method claimed in any of the preceding claims.