Process for producing coenzyme Q10

Abstract

A process for producing on an industrial scale the oxidized coenzyme Q.sub.10, includes culturing a reduced coenzyme Q.sub.10-producing microorganism selected from the group consisting of the genus Rhodobacter, the genus Saitoella, the genus Schizosaccharomyces and the genus Trichosporon, to obtain microbial cells containing reduced coenzyme Q.sub.10 at a ratio of not less than 70 mole % among the entire coenzymes Q.sub.10; and one of: (a) oxidizing thus-obtained reduced coenzyme Q.sub.10 to oxidized coenzyme Q.sub.10 and then extracting the oxidized coenzyme Q.sub.10 by an organic solvent; or (b) extracting reduced coenzyme Q.sub.10 by an organic solvent and oxidizing the extracted reduced coenzyme Q.sub.10 to oxidized coenzyme Q.sub.10.

Claims

1. A process for producing on an industrial scale the oxidized coenzyme Q.sub.10, comprising: aerobically culturing a reduced coenzyme Q.sub.10-producing microorganism selected from the group consisting of the genus Rhodobacter, the genus Saitoella, the genus Schizosaccharomyces and the genus Trichosporon, to obtain microbial cells containing reduced coenzyme Q.sub.10 at a ratio of not less than 70 mole % among the entire coenzymes Q.sub.10; and one of: (a) oxidizing thus-obtained reduced coenzyme Q.sub.10 to oxidized coenzyme Q.sub.10 and then extracting the oxidized coenzyme Q.sub.10 by an organic solvent; or (b) extracting reduced coenzyme Q.sub.10 by an organic solvent and oxidizing the extracted reduced coenzyme Q.sub.10 to oxidized coenzyme Q.sub.10.

2. The process according to claim 1, wherein the extraction of the oxidized coenzyme Q.sub.10 is carried out by using a hydrophilic organic solvent.

3. The process according to claim 1, wherein the extraction of reduced coenzyme Q.sub.10 is carried out using a hydrophilic organic solvent.

4. The process according to claim 1, wherein the extraction of the oxidized coenzyme Q.sub.10 is carried out by using a hydrophobic organic solvent.

5. The process according to claim 1, wherein the extraction of reduced coenzyme Q.sub.10 is carried out using a hydrophobic organic solvent.

6. The process according to claim 1, wherein the reduced coenzyme Q.sub.10 is oxidized with an oxidizing agent.

7. The process according to claim 6, wherein the oxidizing agent is manganese dioxide.

8. The process according to claim 1, wherein the oxidized coenzyme Q.sub.10 is extracted by a continuous extraction.

9. The process according to claim 1, wherein the reduced coenzyme Q.sub.10 is extracted by a continuous extraction.

10. The process according to claim 8, wherein the continuous extraction is a countercurrent multistage extraction.

11. The process according to claim 9, wherein the continuous extraction is a countercurrent multistage extraction.

12. The process according to claim 1, wherein the reduced coenzyme Q.sub.10 has a ratio of not less than 70 mole % among the entire coenzymes Q.sub.10 when measured under the condition that the reduced coenzyme Q.sub.10 is protected from an oxidation reaction.

13. The process according to claim 1, wherein step (a) or step (b) is conducted under an inert gas atmosphere.

14. The process according to claim 13, wherein the inert gas atmosphere comprises nitrogen gas.

15. The process according to claim 1, wherein the culturing is carried out with at least 750 L of culture medium.

16. The process according to claim 1, further comprising the step of disrupting the microbial cells.

17. The process according to claim 1, wherein the culturing is conducted in a culture medium containing a carbon source, a nitrogen source, a phosphorus source and a micronutrient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a schematic diagram of a countercurrent 3-step continuous extraction apparatus used in Example 8.

BEST MODE FOR CARRYING OUT THE INVENTION

(2) The following examples illustrate the present invention in further detail. These examples are, however, by no means limitative of the scope of the present invention.

Example 1

(3) Various coenzyme Q.sub.10-producing microorganisms shown in the following Tables 1 to 3 were cultured with shaking (amplitude: 2 cm, 310 reciprocation/min) at 25 C. for 72 hours in 10 mL of culture media [(glucose: 20 g, peptone: 5 g, yeast extract: 3 g, malt extract: 3 g)/L, pH: 6.0] using test tubes (inner diameter: 21 mm, entire length: 200 mm), and the obtained broth were optionally concentrated. Under a nitrogen atmosphere, in the concomitant presence of 3 parts by volume of isopropanol and 18.5 parts by volume of n-hexane relative to 10 parts by volume of the broth, the obtained solutions were vigorously shaken for 3 minutes using 10 parts by volume of glass beads (425 to 600 m) to carry out cell disruption and extraction. The obtained hexane phases were evaporated (at 40 C.) under reduced pressure and analyzed by high performance liquid chromatography (HPLC) to determine the ratio and the production amount of reduced coenzyme Q.sub.10.

(4) HPLC Conditions Column: YMC-Pack 4.6250 mm (manufactured by YMC. Co., Ltd.) Mobile phase: methanol/n-hexane=85/15 Flow rate: 1 mL/min Detection: UV 275 nm

(5) The results are shown in Tables 1 to 3. The ratio of reduced coenzyme Q.sub.10 means a mole percentage value of the ratio of reduced coenzyme Q.sub.10 relative to the total of oxidized coenzyme Q.sub.10 and reduced coenzyme Q.sub.10 on the basis of the areas of the peaks of reduced coenzyme Q.sub.10 and oxidized coenzyme Q.sub.10 and the ratio of the mole absorption coefficients thereof (1:7.5).

(6) TABLE-US-00001 TABLE 1 Upper stand: Ratio of reduced coenzyme Q10 (%) Lower stand: Production amount of reduced coenzyme Strain name Q10 (g/ml) Agrobacterium tumefacience IFO 13263 82 7 Agrobacterium radiobacter ATCC 4718 78 7 Aspergillus clavatus JCM 1718 83 2 Acetobacter xylinum IFO 15237 77 2 Aminobacter aganouensis JCM 7854 70 3 Agromonas oligotrophica JCM 1494 75 2 Acidiphilium multivorum JCM 8867 73 3 Bulleromyces albus IFO 1192 72 2 Bullera armeniaca IFO 10112 85 7 Brevundimonas diminuta JCM 2788 82 5 Cryptococcus laurentii IFO 0609 79 6 Chionosphaera apobasidialis CBS 7430 71 2 Candida curvata ATCC 10567 74 3 Cerinosterus luteoalbus JCM 2923 79 5 Exisophiala alcalophila JCM 12519 77 3 Exobasidium gracile IFO 7788 79 2 Fellomyces fuzhouensis IFO 10374 70 2 Filobasidiella neoformans CBS 132 88 2 Filobasidium capsuloigenum CBS 1906 82 3 Geotrichum capitatum JCM 6258 77 3 Graphiola cylindrica IFO 6426 75 4 Gluconobacter suboxydans IFO 3257 86 6 Kockovaella imperatae JCM 7826 78 2

(7) TABLE-US-00002 TABLE 2 Upper stand: Ratio of reduced coenzyme Q10 (%) Lower stand: Production amount of reduced coenzyme Strain name Q10 (g/ml) Kurtzmanomyces nectairei IFO 10118 79 2 Lalaria cerasi CBS 275.28 75 2 Leucosporidium scottii IFO 1212 88 6 Legionella anisa JCM 7573 73 3 Methylobacterium extorguens JCM 2802 72 2 Mycoplana ramosa JCM 7822 80 2 Oosporidium margaritiferum CBS 2531 76 2 Pseudomonas denitrificans IAM 12023 85 8 Pseudomonas shuylkilliensis IAM 1092 84 6 Psedozyma aphidis CBS 517.23 79 5 Paracoccus denitrificans JCM 6892 83 5 Petromyces alliaceus IFO 7538 72 2 Rhodotorula glutinis IFO 1125 79 7 Rhodotorula minuta IFO 0387 74 8 Rhodosporidium diobovatum ATCC 1830 86 4 Rhizomonas suberifaciens IFO 15212 82 2 Rhodobium orients JCM 9337 80 2 Rhodoplanes elegans JCM 9224 74 2 Rhodopseudomonas palustris JCM 2524 90 6 Rhodobacter capsulatus SB 1003 95 6 Sporobolomyces holsaticus IFO 1034 72 9 Sporobolomyces pararoseus IFO 0471 93 8 Sporidiobolus johnsonii IFO 1840 73 7 Saitoella complicata IFO 10748 97 9

(8) TABLE-US-00003 TABLE 3 Upper stand: Ratio of reduced coenzyme Q10 (%) Lower stand: Production amount of reduced coenzyme Strain name Q10 (g/ml) Schizosaccharomyces pombe IFO 0347 90 8 Sphingomonas parapaucimobilis IFO 15100 78 7 Sporotrichum cellulophilium ATCC 20493 73 6 Sympodiomycopsis paphiopedili JCM 8318 80 6 Sterigmatosporidium polymorphum IFO 10121 72 2 Sphingomonas adhesiva JCM 7370 80 3 Tapharina caerulescens CBS 351.35 81 2 Tremella mesenterica ATCC 24438 89 3 Trichosporon cutaneum IFO 1198 95 8 Tilletiaria anomala CBS 436.72 75 4 Tilletia caries JCM 1761 80 3 Tolyposporium bullatum JCM 2006 73 4 Tilletiopsis washintonesis CBS 544 76 2 Ustilago esculenta IFO 9887 78 2 Udeniomyces megalosporus JCM 5269 87 2 Xanthophilomyces dendrorhous IFO 10129 84 2 Xanthobacter flavus JCM 1204 80 2 Paecilomyces lilacinus ATCC 10114 80 5 Acremonium chrysogenum ATCC 11550 75 5 Hyphomonas hirschiana ATCC 33886 72 3 Rhizobium meliloti ATCC 9930 85 10

Example 2

(9) Rhodotorula glutinis IFO1125 was aerobically cultured at 25 C. for 48 hours in a culture medium (peptone: 5 g, yeast extract: 3 g, malt extract: 3 g, glucose: 20 g/L, pH: 6.0). The cells after the culture were collected by centrifugation and suspended in a phosphoric acid buffer solution at pH 7 to which N-methyl-N-nitro-N-nitrosoguanidine have been added so as to have its concentration of 200 g/mL. After maintaining the solution at 25 C. for 1 hour, the cells were washed for 5 times with a 0.9% NaCl solution and further suspended in a 0.9% NaCl solution. The obtained cell suspension was properly diluted and a colony was to be formed on an agar plate of the above-mentioned culture medium. The production amount and the ratio of reduced coenzyme Q.sub.10 in the isolated mutant strain were determined in the same manner as Example 1. The strains having higher production amount and the ratio of reduced coenzyme Q.sub.10 as compared with those of wild strains was further mutated repeatedly. As the result, by repeating the mutagenesis for 10 times, mutant strains with productivity of not less than 15 g/mL were obtained. In this case, the ratio of reduced coenzyme Q.sub.10 was not less than 80 mole %.

Example 3

(10) Saitoella complicata IFO 10748 was aerobically cultured at 25 C. for 72 hours in 10 L of a culture medium (peptone: 5 g, yeast extract: 3 g, malt extract: 3 g, glucose: 20 g/L, pH: 6.0). The obtained cells were disrupted for 2 times at 80 MPa of disruption pressure by a pressure homogenizer (manufactured by Lanni Co.) sealed with nitrogen gas to obtain a cell-disrupted solution. The cell-disrupted solution was subjected to extraction with 30 parts by volume of isopropanol and 40 parts by volume of hexane for 3 times to obtain an extract. The extraction ratio was 99%. The ratio of reduced coenzyme Q.sub.10 was 97 mole %.

Example 4

(11) When mutant strains of Rhodotorula glutinis IFO1125 were aerobically cultured at 25 C. in 10 L of a culture medium (peptone: 10 g, yeast extract: 5 g, malt extract: 3 g, glucose: 20 g/L, pH: 6.0), glucose was fed at the rate of 4 g/h after the lapse of 48 hours to 96 hours (fed glucose amount: 190 g). The production amount of reduced coenzyme Q.sub.10 per culture medium was not less than 20 g/mL and the ratio of reduced coenzyme Q.sub.10 was not less than 80 mole %.

Example 5

(12) The extract obtained in Example 3 was subjected to solvent substitution with a hexane solution, the resultant was adsorbed in a column filled with silica gel and subjected to development and elution by a solution of n-hexane/diethyl ether (9/1) to obtain a fraction containing reduced coenzyme Q.sub.10. Furthermore, the fraction was cooled to 2 C. with stirring to obtain a white slurry. All the above-mentioned operations were carried out in a nitrogen atmosphere. The obtained slurry was filtered under reduced pressure, the resulting wet crystals were washed with the development solution same as used above (the temperature of the solvent used for washing was 2 C.), and the wet crystals were dried under reduced pressure (20 to 40 C., 1 to 30 mmHg) to obtain 81 mg of white dry crystals. The purity of the obtained crystals was 99.9% and the ratio of reduced coenzyme Q.sub.10 was 90 mole %.

Example 6

(13) The extract obtained in Example 3 was subjected to solvent substitution with n-hexane, the resultant was added with 50 mg of manganese dioxide, and the mixture was stirred at 30 C. for 30 minutes. Thus-obtained reaction solution was fractionated and purified in the same manner as Example 5 to obtain 74 mg of high-purity oxidized coenzyme Q.sub.10.

Example 7

(14) Saitoella complicata IFO 10748 was aerobically cultured at 25 C. for 72 hours in 500 mL of a culture medium (peptone: 5 g, yeast extract: 3 g, malt extract: 3 g, glucose: 20 g/L, pH: 6.0). The obtained cells were disrupted for 2 times at 80 MPa of disruption pressure by a pressure homogenizer (manufactured by Lanni Co.) sealed with nitrogen gas to obtain a cell-disrupted solution. The ratio of reduced coenzyme Q.sub.10 in the cell-disrupted solution was 97% relative to the entire coenzymes Q.sub.10 including oxidized coenzyme Q.sub.10. 200 mL of the cell-disrupted solution was mixed with isopropanol and n-hexane at the ratios shown in the first extraction section in the following Table 4 so as to adjust the total solvent amount to be 500 mL and the mixtures were stirred at 40 C. for 30 minutes to carry out the first extraction. After completion of the extraction, the resultants were kept standing for 10 minutes and the separated upper layers were collected. The volume ratios of the lower layers (residues) relative to the total solution amounts were defined as indexes of separability and shown as the interface positions in Table 4.

(15) Furthermore, in order to carry out the second extraction, the solvent concentrations of the residual layers were measured and isopropanol and hexane were further added so as to keep the solvent ratios in the entire solutions be the ratios shown in the second extraction section in Table 4. The resulting solutions were stirred at 40 C. for 30 minutes. Then, the solutions were kept standing for 10 minutes and the upper layers were collected in the same manner as described above to determine the solvent concentrations of the residual layers. Isopropanol and hexane were added thereto so as to keep the solvent ratios in the entire solutions be the ratios shown in the third extraction section in Table 4, and the solutions were stirred at 25 C. for 30 minutes to carry out the third extraction.

(16) The ratios of the amounts of reduced coenzyme Q.sub.10 contained in the collected upper layers of each of the first, second and third steps relative to the amount of reduced coenzyme Q.sub.10 contained in the cell-disrupted solution or the extraction residue before the extraction were defined as the extraction ratios of reduced coenzyme Q.sub.10 in the respective steps. The calculation results are shown in Table 4. The integrated extraction ratios of reduced coenzyme Q.sub.10 in the second and third extraction steps are also shown. In any steps, the static separability was excellent and the integrated extraction ratio in the case where extraction was repeated for 3 times was as high as not less than 90% to show high recovery ratio. Particularly, in the case where the isopropanol concentration was adjusted to be not less than 30%, the recovery ratio was as high as not less than 99%.

(17) TABLE-US-00004 TABLE 4 Extraction ratio (%) Solvent ratio (vol %) Interface Respective Integrated Isopropanol Hexane position extraction ratio extraction ratio Case1 First 18.8 52.7 0.492 73.6 73.6 Second 19.0 52.4 0.624 47.6 86.2 Third 29.7 41.7 0.645 55.5 93.8 Case2 First 31.3 40.2 0.499 90.7 90.7 Second 37.7 33.7 0.549 83.7 98.5 Third 40.6 30.9 0.565 40.1 99.1 Case3 First 31.3 40.2 0.526 89.0 89.0 Second 34.1 37.3 0.553 85.8 98.3 Third 36.8 34.6 0.555 46.6 99.1 Case4 First 31.3 40.2 0.526 89.0 89.0 Second 34.1 37.3 0.553 85.8 98.3 Third 42.4 29.0 0.644 50.0 99.0 Case5 First 31.3 40.2 0.526 89.0 89.0 Second 40.1 31.4 0.595 88.1 98.6 Third 40.7 30.7 0.593 45.3 99.1 Case6 First 31.3 40.2 0.526 89.0 89.0 Second 40.1 31.4 0.595 88.1 98.6 Third 45.8 25.7 0.663 40.7 99.0

Example 8

(18) Saitoella complicata IFO 10748 was aerobically cultured at 25 C. for 72 hours in 750 L of a culture medium (peptone: 5 g, yeast extract: 3 g, malt extract: 3 g, glucose: 20 g/L, pH: 6.0). The obtained cells were disrupted for 2 times at 140 MPa of disruption pressure by a pressure homogenizer (manufactured by Lanni Co.) sealed with nitrogen gas to obtain a cell-disrupted solution. The cell-disrupted solution was subjected to continuous extraction by a countercurrent 3-step continuous extraction apparatus shown in FIG. 1. The capacity of the stirring tank was 630 L and the capacity of the static separation tank was 200 L. The cell-disrupted solution was supplied to the first stirring tank and isopropanol and n-hexane were supplied to respective steps. The supply amount of the cell-disrupted solution was 2 L/min and the supply amounts of isopropanol and n-hexane were adjusted to be 1.3 L/min for isopropanol and 3.7 L/min for n-hexane as the total of the supply amounts in respective steps. In this case, the solvent concentration in respective steps was properly adjusted so that the isopropanol concentration of 5 to 50 v/v % and the n-hexane concentration of 25 to 65 v/v % were kept. The extraction temperature was 40 C. and the treatment duration was 6 hours. At the point after the lapse of 6 hours, the recovery ratio of reduced coenzyme Q.sub.10 extracted from the cell-disrupted solution was calculated on the basis of reduced coenzyme Q.sub.10 remaining in the extraction residue in the static separation tank in the third step to find the recovery ratio of 98.9%. The static separation was well carried out during the entire operation period and stable continuous extraction was possible.

INDUSTRIAL APPLICABILITY

(19) According to the processes of the present invention, reduced coenzyme Q.sub.10 can be produced cheaply on the industrial scale by considerably simple steps comprising culturing microorganisms and recovering reduced coenzyme Q.sub.10. In addition, oxidized coenzyme Q.sub.10 can also be produced by simple processes.