DK147602B - PROCEDURE FOR PREPARING COENZYM Q - Google Patents

Description

147602

The present invention relates to a process for producing Coenzyme Q, in which a microorganism belonging to the genus Pseudomonas, which is capable of producing Coenzyme Q, is grown in a culture substrate and isolates the Coenzyme Q produced.

The term "Coenzyme Q", used in the present specification with claims, is generally understood to mean 2,3-dimethoxy-5-methyl-1,4-benzoquinones containing an isoprene side chain at the 6-position of the quinone nucleus represented by the general formula.

ISLAND

H3COV / ^ V \ / CH3 H, CO''A''Vw ^ NcH -CH = C -CH ~ ·) - H 3 2 | 2 n O CH 3 wherein n = 8, 9 and 10, i.e. Coenzyme Qg, Coenzyme Qg and Coenzyme Q

Coenzyme Q is widely used in animals, plants and microorganisms, etc. and plays an important role as an essential element of the end-of-life electron transfer system.

Recently, it has been established that Coenzyme Q exerts excellent medical and physiological effects on various diseases. In particular, Coenzyme Q1Q is considered highly valuable as a drug, as Coenzyme Q in humans is Coenzyme.

Coenzyme Q can be obtained by extracting it from animal or plant cells or microorganisms or by chemically synthesizing it. However, it is difficult to produce Coenzyme Q by extracting large-scale animal or plant cells. It is also difficult to prepare Coenzyme Q by organic synthesis, since one has a disadvantage in the form of poor yields. Thus, these processes are not satisfactory for industrial purposes. Extracting Coenzyme Q from microorganisms is thus most attractive from an economic point of view.

It is well known that microorganisms belonging to the genus Pseudomonas produce Coenzyme Qg, Qg and Q ^ q, and that p-hydroxybenzoic acid and acetic acid and its salts are capable of increasing the content of Coenzyme Q cell unit if added to the culture substrate (Japanese Patent Specification No. 20396/1972). It is well known that the isoprene side chain in Coenzyme Q is prepared through geranyl and farnesyl pyrophosphate by biosynthesis in which the condensation of isopentenyl and dimethylallyl pyrophosphate is repeated.

However, since these precursors are difficult to penetrate through the cell membrane, no attempt has been made to increase the content of Coenzyme Q by adding such precursors to the culture substrate.

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It has now been found that isopentenyl alcohol, dimethylallyl alcohol, geraniol, isopentenyl acetate, dimethylallyl acetate, geranyl acetate and β-methylcrotonic acid can be readily used by microorganisms belonging to the genus Pseudomonas if they are added to the culture substrate and are substantially capable of to increase the content of Coenzyme Q per cell unit.

The process of the invention for the preparation of Coenzyme Q is accordingly characterized in that at least one compound selected from the group consisting of isopentenyl alcohol, dimethylallyl alcohol, geraniol, isopentenyl acetate, dimethylallyl acetate, geranyl acetate and o-methylcrotonic acid is added to the culture substrate.

Any of the microorganisms belonging to the genus Pseudomonas and capable of producing Coenzyme Q can be used in the method of the present invention. Examples of microorganisms capable of producing Coenzyme Q Q q are Pseudomonas diminuta ATCC 11568 (IAM-1513), of those capable of producing Coenzyme Qg, Pseudomonas schuylkilliensis ATCC 31419 (IAM-1126), Pseudomonas denitrificans ATCC 13867 (IAM-12023), Pseudomonas olevorans ATCC 8062 (IAM-1508) and Pseudomonas putre-faciens ATCC 8071 (IAM-1509), and on those capable of producing Coenzym Qg, Pseudomonas rubescens ATCC 12099 (IAM-1510), Pseudomonas fulva ATCC 31418 (IAM-1529) and Pseudomonas putida ATCC 4359 (IAM-1506).

In the culture substrate used in carrying out the process of the invention, sugars such as glucose, molasses, etc., and any other carbon sources which these microorganisms are capable of utilizing, can be used as carbon sources. Inorganic nitrogen compounds such as, for example, ammonium sulfate, ammonium chloride and the like, organic nitrogen compounds such as corn molding liquid, fish meat extracts, peptone, yeast extract and the like can be used as nitrogen sources. In addition, inorganic salts such as potassium salts, sodium salts, magnesium salts, phosphoric and sulfuric acid salts and the like may be added.

Cultivation is usually carried out with stirring with air at pH 4-8, at a temperature of 25-35 ° C and within 10-50 hours.

The addition of isopentenyl alcohol, dimethyl allyl alcohol, geraniol, isopentenyl acetate, dimethyl allyl acetate, geranyl acetate and 4-147602 (3-methylcrotonic acid in the process of the present invention can be carried out by any desired process and at any desired time. For example, the entire amount of additive can be added the additive can be used alone or with other additives The amount of additive, -5 - 3 added, is usually 1x10 to 5x10 mol / s. per liter (as final -5 -4 concentration), preferably 5x10 to 5x10 mol / liter.

After culture, the Coenzyme Q formed is extracted from the cells and separated from the other material. For example, the wet cells obtained by centrifugation are extracted with a hydrophilic solvent such as, for example, acetone and the like; then the Coenzyme Q-containing fraction is transferred to a solvent such as, for example, petro, leum ether and the like; the Coenzyme Q-containing fraction is subjected to fractional purification by alumina column, etc., whereby Coenzym Q can be isolated.

In the present invention, the identification of Coenzyme Q is performed by comparing the subject product to a standard sample by UV spectrum, melting point measurement and reverse phase thin layer chromatography, in which a mixture of acetone: water (95: 5) is used as solvent. ·

The following examples are given to further explain the method of the invention.

Example 1 in a 30-liter fermentor was placed 15 liters of a culture substrate (pH 7.0) containing 0.05% KH2PO4, 0.15% NA2HPO4, 0.05% MgSO4.7H20, 1% glucose, 1% peptone and 0 , 2% yeast extract. After steam sterilization, 645 mg of isopentenyl alcohol dissolved in 10 ml of ethanol was added. Pseudomonas schuylkilliensis ATCC 31419 (IAM-1126) first grown in 500 ml of the culture substrate having the same composition as described above for 24 hours, seeded in the above culture substrate and grown for 24 hours with aeration of 15 liter / minute at 27 ° C.

After culture, the culture broth was centrifuged to obtain 418 g of wet cells (87 g in the form of dry cells).

5 147602

The wet cells were added to 2 liters of acetone and extracted with stirring. The cells were separated by centrifugation. This procedure was repeated twice more. The acetone extracts were combined, then evaporated under reduced pressure to remove the acetone. Thereafter, the residual solution was extracted three times with 1 liter of petroleum ether per ml. time, and the resulting petroleum ether layers were combined. The combined petroleum ether layers were washed with water, dried and condensed under reduced pressure.

The residual oil was dissolved in a small amount of petroleum ether and then chromatographed on an alumina column by elution with a mixture of petroleum ether and ethyl ether. The solvent was distilled off from the above obtained eluate containing Coenzyme Q. The residual oil was dissolved in a small amount of ethanol and then left to refrigerate to crystallize crystals of Coenzyme Qg. These crystals were recrystallized with ethanol three times to give 142.6 mg of Coenzyme Qg crystals.

On the other hand, the same culture as above was performed using 15 liters of a culture substrate to which no isopentenyl alcohol was added. As above, 359 g of wet cells were obtained (69 g of dry cells). Further, the same procedure was used as above to obtain 81.42 mg of Coenzyme Qg crystals.

From the above data, the effect of isopentenyl alcohol on the preparation of Coenzyme Qg was calculated. Addition of isopentenyl alcohol to the culture substrate increased the yield of Coenzyme Qg per ml. liter of broth with 75% and 39% per liter. g dry cells.

Thus, it was clearly confirmed that the addition of isopentenyl alcohol was effective in increasing the yield of Coenzyme Qg.

Example 2

The procedure of Example 1 was followed except that 1.16 g of geraniol was used instead of isopentenyl alcohol to give 390 mg of wet cells (68 g in the form of dry cells).

The wet cells were subjected to the same procedure as described in Example 1 and 104.7 mg of Coenzjty Qg crystals were obtained.

On the other hand, the same microorganism was grown using a culture substrate to which no geraniol was added. 327 g of wet cells (63 g in the form of dry cells) were obtained. From the cells, 74.3 mg of crystals of Coenzyme were obtained.

From the data given above, the effect of geraniol on the preparation of Coenzyme Qg was investigated. Addition of geraniol to the culture substrate increased the yield of Coenzyme Q lil cultivation broth and with 30% per. g. dry cells.

Example 3

Pseudomonas rubescens ATCC 12099 (IAM-1510) was grown in the same culture medium and in the same manner as described in Example 2 to give 410 g of wet cells (73 g in the form of dry cells). These were treated in the same manner as described in Example 1 and 58.4 mg of Coenzyme Qg were obtained.

On the other hand, microorganisms were grown in the same culture substrate as above, except that no geraniol was added. 390 g of wet cells were obtained (74 g in the form of dry cells). From the cells 44.4 mg of Coenzyme Qg were obtained.

From the above mentioned data, the same comparison was made as in Example 1. Addition of geraniol to a culture medium increased the yield of Coenzyme Qg by 32% per ml. liters of culture broth and 33% per liter. g dry cells.

Example 4

Pseudomonas diminuta ATCC 11568 (IAM-1513) was grown by the same procedure as in Example 1 except that sodium acetate was used instead of glucose in the composition of the substrate, and three 300 mg fractions of isopentenyl alcohol (900 mg total) were added separately. In this way, 380 g of wet cells (69 g in the form of dry cells) were obtained, which were then subjected to the same treatment as in Example 1 to give 30.5 mg of crystals of Coenzyme.

On the other hand, the same microorganism was grown in the culture substrate to which no isopentenyl alcohol was added.

320 g of wet cells (61 g in the form of dry cells) were obtained, from which 19.8 mg crystals of Coenzyme Q ^ q were obtained.

From the above data, the same comparison was made as in Example 1. The addition of isopentenyl alcohol increased the yield of Coenzyme by 54% per liter. per liter of culture broth and at 38% per liter. g dry cells.

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Example 5

The procedure as described in Example 1 was followed except that pseudomonas denitrificans ATCC 13867 (IAM-12023) was used as a microorganism and two 1 g portions of geraniol were added separately to the culture substrate (2 g total) at different times during culture. In this way, 395 g of wet cells (76 g in the form of dry cells) were obtained, which were then subjected to the same treatment as described in Example 1 to obtain 63.1 mg crystals of Coenzyme Qg.

On the other hand, the same microorganism was grown in the culture medium to which no geraniol was added and 328 g of wet cells (63 g of dry cells) were obtained, from which 37.2 mg of crystals of Coenzyme Qg were obtained.

From the data given above, the same comparison was made as in Example 1. The addition of geraniol increased the yield of Coenzyra Q. by 69% per liter. liters of culture broth and 41% per liter. g dry y cells.

Example 6

The same procedure was used as in Example 1 except that Pseudomonas fulva ATCC 31418 (IAM-1529) was used as a microorganism and three portions of isopentenyl alcohol (total 3 g) were added separately during culture at different times. In this way, 390 g of wet cells (73 g in the form of dry cells) were obtained, which were then subjected to the same treatment as in Example 1 to obtain 86.4 mg crystals of Coenzyme Qg.

On the other hand, the same microorganism was grown in the culture substrate to which no isopentenyl alcohol was added and 350 g of wet cells (67 g of dry cells) were obtained, from which 60.5 crystals of Coenzyme Qg were obtained.

From the data given above, the same comparison was made as in Example 1. The addition of isopentenyl alcohol increased the yield of Coenzyme Qg by 43% per liter of culture broth and by 31% per liter. g dry cells.

147602 8.

Example 7

The same procedure as in Example 1 was used except that Pseudomonas rubescens ATCC 12099 (IAM-1510) was used as a microorganism and 645 mg of dimethylallyl alcohol was added. In this way, 430 g of wet cells (80 g in the form of dry cells) were obtained, which were subjected to the same treatment as in Example 1 to give 86 mg crystals of Coenzyme Qg.

On the other hand, the same microorganism was grown in the culture substrate to which no dimethyl allyl alcohol had been added. There were thus obtained 390 g of wet cells (77 g in the form of dry cells) from which 68 mg crystals of Coenzyme Qg were obtained.

From the data given above, the same comparison was made as in Example 1. By adding dimethyl allyl alcohol, the yield of Coenzyme Qg was increased by 26% per day. per liter of culture broth and at 20% per liter. g dry cells.

Example 8

The same procedure was used as in Example 1 except that Pseudomonas fulva ATCC 31418 (IAM-1529) was used as a microorganism and 750 mg of o-methylcrotonic acid was added. In this way, 540 g of wet cells (105 g in the form of dry cells) were obtained, which were subjected to the same treatment as in Example 1 to obtain 102 mg crystals of Coenzyme Qg.

On the other hand, the same microorganism was grown in the culture substrate to which no β-methylcrotonic acid had been added. 525 g of wet cells (103 g in the form of dry cells) were obtained, from which 79 mg crystals of Coenzyme Qg were obtained.

From the data given above, the same comparison was made as in Example 1. The addition of β-methylcrotonic acid increased the yield of Coenzyme Qg by 28% per liter of culture broth and by 26% per liter. g dry cells.

Example 9

The procedure of Example 1 was used except that Pseudomonas olevorans ATCC 8062 (IAM-1508) was used as a microorganism and 960 mg of dimethyl allyl acetate was added. In this way, 490 g of wet cells (94 g in the form of dry cells) were obtained, which were then subjected to the same treatment as in Example 1 to obtain 96 mg crystals of Coenzyme Qg.

On the other hand, the same microorganism was grown in the culture substrate to which no dimethyl allylate tate was added, thereby obtaining 470 g of wet cells (90 g in the form of dry cells) from which 68 mg crystals of Coenzyme Qg were obtained.

From the above mentioned data, the same comparison was made as in Example 1. The addition of dimethyl allyl acetate increased the yield of Coenzyme Qg by 42% per ml. per liter of culture broth and at 34% per liter. g. dry cells.

Example 10

The procedure of Example 1 was followed except that Pseudomonas schuylkilliensis ATCC 31419 (IAM-1126) was used as a microorganism and 1.47 g of geranyl acetate was added. In this way, 530 g of wet cells (93 g in the form of dry cells) were obtained, which were subjected to the same treatment as in Example 1 to obtain 120 mg crystals of Coenzyme Qg.

On the other hand, the same microorganism was grown in the culture substrate to which no geranyl acetate had been added. This yielded 525 g of wet cells (90 g in the form of dry cells) from which 98 mg of crystals of Coenzyme Qg were obtained.

From the above mentioned data, the same comparison was made as in Example 1. The addition of geranyl acetate increased the yield of Coenzyme Qg by 23% per ml. per liter of culture broth and at 19% per liter. g dry cells.

Example 11

The procedure was followed as in Example 1 except that Pseudomonas denitrificans ATCC 13867 (IAM-12023) was used as a microorganism and the mixture of 960 mg of isopentenyl acetate and 1.47 g of geranyl acetate was added. In this way, 480 g of wet cells (96 g of dry cells) were subjected to the same treatment as in Example 1 to obtain 115 mg of Coenzyme crystals

V

On the other hand, the same microorganism was grown in the culture medium to which the mixture was not added. There were thus obtained 490 g of wet cells (98 g of dry cells) from which

Claims (4)

147 mg of 76 mg crystals of Coenzyme were obtained Q g · From the above data the same comparison was made as in Example 1. The addition of isopentenyl acetate and geranyl acetate increased the yield of Coenzyme and by 51% per ml. per liter of culture broth and at 54% per liter. g. dry cells. Example 12 Pseudomonas diminuta ATCC 11568 (IAM-1513) was grown by the same procedure as in Example 4 except that a mixture of 960 mg of isopentenyl acetate and 960 mg of dimethylallyl acetate was added. In this way, 495 g of wet cells (98 g in the form of dry cells) were obtained, which were subjected to the same treatment as in Example 1 to obtain 53 mg crystals of Coenzyme Q1Q. On the other hand, the same microorganism was grown in the culture substrate to which the mixture was not added. In this way 480 g of wet cells (94 g in the form of dry cells) were obtained, from which 39 mg crystals of Coenzyme Q ^ q were obtained. dimethyl allyl acetate increased the yield of Coenzyme Q1Q by 36% per liter of culture broth and at 32% per liter. g dry cells. A process for producing Coenzyme Q, in which a microorganism belonging to the genus Pseudomonas, capable of producing Coenzyme Q, is grown in a culture substrate and isolates the produced Coenzyme Q, characterized in that at least one compound selected from the group consisting of isopentenyl alcohol, dimethylallyl alcohol, geraniol, isopentenyl acetate, dimethylallyl acetate, geranyl acetate and β-methylcrotonic acid for the culture substrate. Process according to claim 1 for the preparation of Coenzyme Q1Q, characterized in that the microorganism is Pseudomonas diminuta ATCC 11568. Method according to claim 1 for the preparation of Coenzyme Qg, characterized in that the microorganism is Pseudomonas schuylkilliensis ATCC 31419, Pseudomonas denitrificans ATCC 13867 or Pseudomonas olevorans ATCC 8062. A process according to claim 1 for the preparation of Coenzyme