JPH0789938B2 - Method for producing mevalonic acid - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing mevalonic acid, and more particularly to a method for obtaining mevalonic acid in high yield.

Mevalonic acid has two types of structures, an acid type and a lactone type, but it is known that they are mutually converted, and hereinafter, unless otherwise specified, both types are generically referred to as mevalonic acid.

[Problems to be Solved by Prior Art and Invention]

Mevalonic acid is a substance first isolated by Wright et al. (Journal of the American Chemical Society).
78), 5273-5275, 1956) Known as an important intermediate of various isoprenoid compounds such as cholesterol.

Further, mevalonic acid is used as a growth promoter for microorganisms, plants, etc. because it has an important role in the metabolism of organisms, such as having a growth promoting action on various microorganisms and plants, and it is also a pyrethroid pesticide. , Ubiquinone (respiratory coenzyme), dolichol (polysaccharide biosynthesis essential factor), and precursors of fat-soluble vitamins and the like.

Conventionally, chemically synthesized racemates have been used in these studies, and natural mevalonic acid was difficult to obtain.

Fermentation of natural mevalonic acid is carried out by Applied Microbiology 16 ,
965 (1968) and U.S. Pat. No. 3,617,447 describe examples using Saccharomycopsis fibriguera NRRL Y-7069, but the yield is low, 700-1000 μg.
The current situation is that the production is still limited to / ml and has not reached industrial production.

Therefore, an object of the present invention is to provide a method for producing mevalonic acid which is industrially practicable and has a good yield.

[Means for solving problems]

The present invention has the above-mentioned object of producing a mevalonic acid by culturing a microorganism belonging to the genus Saccharomycopsis in a medium, and adding a substrate containing at least a carbon source to the culture during the culturing. Then, it was achieved by providing a method for producing mevalonic acid, which comprises obtaining mevalonic acid from the culture.

More preferably, in the method for producing mevalonic acid of the present invention, the carbon source concentration in the culture solution (culture filtrate) in the culture during culture is maintained at 2 to 15% by weight, particularly preferably 5 to 10% by weight. Thus, the rapid growth of microorganisms and the rapid production of mevalonic acid are measured to obtain a large amount of mevalonic acid.

Saccharomyces used in the present invention
Microorganisms belonging to the genus copsis include Saccharomucopsis fibuliger.
a), Saccharomycopsis capsularis (Saccharom
ucopsis capsularis) and the like, and of these, the most preferred of these is Saccharomycopsis fibrigella (Sa
ccharomycopsis fibuligera), and strains belonging to these include, for example, IFO 0107, IFO 0103, IFO 010.
4, IFO 0105, IFO 0106, IFO 0109, IFO 0111, IFO
1665, IFO 1711, IFO 1744, IFO 1745, AHU 411
3, IAM 4247, OUT 6071, HUT 7234, ATCC 2080, AT
CC 2082, ATCC 2088, ATCC 9947, ATCC 20145, ATC
C 24945, ATCC 44872, ATCC 46252, ATCC 46253, A
TCC 46949, ATCC 52921, NRRL Y-1060, NRRL Y
-1064, NRRL Y-2385, NRRL Y-7061, NRRL Y-
7221, NRRL Y-7324, NRRL Y-7464, NRRL Y-90
69, DSM 70554, IAM 4025, IFO 1342, IFO 0800, I
FO 1880, IFO 0980, IFO 1477, IFO 1254, IFO 09
75, IFO 0807, IFO 0984, IAM 4945, IFO 1850, IA
M 4307, IFO 0672, IAM 4771, IAM 12236, IAM 12
241, IFO 1002, IFO 1003, IFO 1626, IFO 1572, I
FO 1574, IFO 1146, IFO 1213, IFO 1287, IFO 09
54, IFO 0941, IFO 8812 can be mentioned.

IFO, ATCC, NRRL, DSM, AHU, IAM, OUT, and HUT are the fermentation strains preserved by the Fermentation Institute, the American Type Culture Collection preserved strains, the ARS Northern Regional Research Center preserved strains, and Deutsche Zammulung, respectively. The von Microorganismen conserved strain, the Hokkaido University Faculty of Agriculture conserved strain, the University of Tokyo Applied Microbiology Institute conserved strain, the Osaka University Faculty of Engineering fermentation engineering department preserved strain, Hiroshima University Faculty of Engineering fermentation engineering department preserved strain are shown.

The medium composition used in the present invention may be a commonly used medium composition, but may be a nonionic surfactant having a cell membrane solubilizing effect in the medium, for example, polyoxyethylene glycol-pt-octylphenyl ether type. (Known as tratoin type, nonidet type), polyoxyethylene glycol alkyl ether type (known as brij type, emulgen type), polyoxyethylene glycol sorbitan ester type (known as tween type, span type) One or more surfactants selected from the group consisting of Triton X-100 (registered trademark), Triton X-114 (registered trademark), Triton X-102 (registered trademark). ), Nonidet P-40 (registered trademark), Brij 35 (registered trademark), Brij 76 (registered trademark), Brij 96 ( Recorded trademark), Brij
56 (registered trademark), Emulgen 120 (registered trademark),
It is preferable to culture by adding Emulgen 109P (registered trademark), Tween 20 (registered trademark), Span 20 (registered trademark) or the like, and particularly preferable is Triton X-10.
0 (registered trademark), and the yield of mevalonic acid can be further improved by adding these at a proportion of 0.002 to 0.2% by weight, preferably 0.02 to 0.1% by weight of the medium, and culturing. When the cell membrane is solubilized, mevalonic acid is more likely to come out of the cell, so that the yield is increased.

As the nitrogen source, peptone, which is an organic nitrogen source,
It is preferable to use yeast extract, meat extract, casein, and soybean powder without using an inorganic nitrogen source because the yield can be further improved.

As an example of the specific medium composition used in the present invention, for example, a medium composed of 5 to 15% by weight of carbon source, 0.5 to 3% by weight of organic nitrogen source, 0.02 to 0.1% by weight of surfactant and water (the balance) is used. Inorganic salts such as phosphates, potassium salts, magnesium salts, calcium salts, etc. can be added each in an amount of 0.01 to 5
The thing containing a weight% can also be mentioned.

In the present invention, the substrate is added so that the carbon source concentration in the culture solution (culture filtrate) in the culture during culture is 2 to 15% by weight,
It is preferable to carry out so as to be maintained at 5 to 10% by weight. However, since it generally takes a long time to measure the carbon source concentration, it is preferable to use the pH of the culture or the amount of dissolved oxygen as an index to judge the time of adding the substrate.

When pH is used as an index, the point that pH is 7 or more
When the dissolved oxygen amount is used as an index, the dissolved oxygen amount once decreases and starts to rise again, that is, immediately after the minimum value of the curve of the dissolved oxygen amount (saturation is the initial value) has passed.

The amount of the substrate to be added at this time may be obtained by previously obtaining the carbon source concentration at the time point of the index and adding the amount according to the concentration.

The substrate to be added may be at least a carbon source, and may be the same as or different from the medium composition before culturing, but preferable carbon sources include, for example, glucose, fructose, maltose, malt extract, glycerin,
An acetic acid salt etc. are mentioned. Further, the form of the substrate at the time of addition is not limited as long as it is aseptic, but it is preferably used as an aqueous solution from the standpoint of sterilization and the like, and the concentration in that case is also not limited, but high concentration (for example, 40% or more) ) Is preferred.

Preferred specific embodiments of the production method of the present invention are as follows.

That is, a medium containing a predetermined amount of a carbon source, an organic nitrogen source, a nonionic surfactant having a cell membrane solubilizing action and inorganic salts is inoculated with a microorganism, and shaken at 20 to 40 ° C, preferably 25 to 35 ° C. Culture or 100-500 rpm, preferably 200-400rp
m, 0.2-1.5VVM, preferably 0.5-1.0VVM aeration stirring culture, after culturing for a predetermined time, by measuring the carbon source concentration in the culture medium in the culture, pH measurement, or by measuring the dissolved oxygen content, The substrate is added (added) to the culture. Such feeding of the substrate is performed a required number of times, and the culture is terminated when it is determined that the mevalonic acid production amount is not further improved. After culturing, the culture is centrifuged or filtered to remove the cells by a known method and concentrated by reverse osmosis or vacuum distillation, or a countercurrent distribution method using butanol or ethyl acetate or silica gel or a porous polymer resin. , Mevalonic acid can be obtained by treatment with a combination of known purification techniques such as column chromatography using an ion exchange resin, molecular distillation, crystallization and the like.

If desired, an antifoaming agent or the like can be added to the medium in the method of the present invention within the scope of the object of the present invention.
In addition, the measurement of the carbon source concentration in the culture solution is carried out by an enzymatic method using glucose oxidase, and the "culture solution" in this case means a solution part obtained by centrifuging the culture, filtering the cells, etc. Say.

〔Example〕

Examples of the present invention will be shown below, but the present invention is not limited thereto.

The quantitative determination of mevalonic acid in Examples and Comparative Examples was performed as follows).

[Method of quantifying mevalonic acid]

0.8 ml of the sample solution is put into a pitch tube, and the pH is adjusted to 2 with 1N-HCl solution. Na ₂ SO ₄ lg was added to this, 2.0 ml of ethyl acetate was further added, and after stirring, the upper layer was taken. 2.0 ml of ethyl acetate was added to the lower layer, and after stirring, the upper layer was taken and combined with the previous upper layer. Repeat this operation again, total 6.
0 mlb is obtained, which is evaporated to dryness. The dried solid was quantified by gas chromatography using 3,4-dimethoxybenzaldehyde as an internal standard. The conditions for gas chromatography are as follows.

Column size: Diameter 3mm Length 1000mm (stainless steel) Column liquid phase: 10% Thermon-3000 Column support: chromosorb W AW-DMCS 80-100 mesh Column temperature: 180 ℃ Injection temperature: 230 ℃ Carrier gas: N ₂ (40ml / Min) [Example 1, Comparative Example 1] Glucose 10% by weight, malto extract 1% by weight,
Peptone 0.5%, yeast extract 0.1 wt%, KH ₂ PO ₄ 0.3 wt%, MgSO ₄ · 7H ₂ O0.05 wt%, CaCO ₃ 1
Prepare 20 and 200 ml of a medium consisting of weight% and the balance water,
Saccharomycopsis fibriguera IFO in 00 ml medium
1 platinum loop inoculated with 1744 loops and shake-cultured at 28 ° C for 3 days was inoculated into the above 20 medium, which was inoculated at 28 ° C, rotation speed 300 rpm, aeration rate 20 / min, glucose concentration, pH, dissolved Aeration-agitation culture was performed while measuring the oxygen content.

On the third day of culture, the amount of glucose in the culture solution was measured to confirm that the glucose concentration was 5% or less. At this point, 2.0 kg of a 50% by weight aqueous glucose solution was added to continue the culture. Furthermore, 2.0 kg of a 50 wt% aqueous solution of glucose was fed to each of the 6th day of culture and the 9th day of culture to give a total of 12
The culture was continued for a day and the culture was completed. After completion of the culture, mevalonic acid obtained from the culture was measured by the above-mentioned quantitative method, and the amount of mevalonic acid was 2010 μg / ml. When glucose was not fed (Comparative Example 1), the amount of mevalonic acid did not increase after 6 days, and the amount of mevalonic acid was 1160 μg /
It was ml.

FIG. 1 shows the culture time, dissolved oxygen amount (saturation = 100), pH, glucose amount (% by weight), and
And the amount of mevalonic acid (% by weight). From this graph, the pH exceeds 7 and the amount of dissolved oxygen immediately after the glucose concentration in the culture solution becomes 5% or less on the 3rd day of culture. It can be seen that is passing the minimum value.

[Example 2, Comparative Example 2] Tratoin X-100 (registered trademark) was added to the medium of Example 1.
0.05% by weight was added, and the culture was continued for 12 days as in Example 1. The amount of mevalonic acid obtained as a result was 2430 μg / ml
Met. When fed-batch was not used (Comparative Example 2), the amount of mevalonic acid accumulated did not increase after 6 days, and the amount of mevalonic acid obtained was 1410 μg / ml.

[Example 3 and Comparative Example 3] 0.3% by weight of ammonium chloride was further added to the medium of Example 2, and the culture was continued for 12 days in the same manner as in Example 2. The amount of mevalonic acid obtained as a result was 2270 μg / ml. Without fed-batch (Comparative Example 3), the amount of mevalonic acid accumulated did not increase after 6 days, and the amount of mevalonic acid obtained was 1320.
It was μg / ml.

[Example 4, Comparative Example 4] 0.3% by weight of ammonium chloride was further added to the medium of Example 1, and the culture was continued for 12 days as in Example 1. The amount of mevalonic acid obtained as a result was 1960 μg / ml. Without fed-batch (Comparative Example 4), the amount of mevalonic acid accumulated did not increase after 6 days, and the amount of mevalonic acid obtained was 910 μg.
It was / ml.

[Example 5] Maltose was used in place of glucose in the medium of Example 2, and the cultivation was continued for 12 days in the same manner as in Example 2 with the time point when the pH exceeded 7 as the feeding period. The amount of mevalonic acid obtained as a result was 2410 μg / ml.

[Example 6] Sucrose was used instead of glucose in the medium of Example 2, fed immediately after passing the minimum value of the dissolved oxygen amount, and the culture was continued for 12 days as in Example 2. The amount of mevalonic acid obtained as a result was 2400 μg / ml.

[Example 7] The culture was continued for 12 days in the same manner as in Example 2 except that Prizi 35 (registered trademark) was used instead of Triton X-100 (registered trademark) in the medium of Example 2. The amount of mevalonic acid obtained as a result was 2290 μg / ml.

[Example 8] 10 ml of the medium of Example 2 was placed in a test tube and sterilized at 121 ° C for 15 minutes to give 1 strain of Saccharomycopsis fibrigella IFO0107.
A platinum loop was inoculated and cultured at 28 ° C. and a rotation speed of 250 rpm. Day four,
On the 8th day, 1.0 g of a 50% by weight glucose aqueous solution was fed to each, and the mixture was cultured for 12 days and then centrifuged at 2500 rpm for 10 minutes to quantify mevalonic acid in the filtrate. The amount of mevalonic acid obtained as a result was 12010 μg / ml.

[Example 9] The culture 25 obtained in Example 2 was centrifuged at a rotation speed of 5000 rpm to obtain a filtrate 17. This was concentrated to 5 using a reverse osmosis membrane, adjusted to pH 2 with 50% by volume aqueous phosphoric acid, and extracted 3 times with 5 ethyl acetate to obtain an ethyl acetate layer 15. 0.02N
After being redissolved in aqueous sodium hydroxide 5 and adjusted to pH 2 with 50 wt% phosphoric acid water, it was passed through Diaion HP-20 (registered trademark) column (1). The effluent was extracted 3 times with ethyl acetate of 5 to obtain an ethyl acetate layer 15. This was dried over anhydrous sodium sulfate and evaporated to dryness. This dried solid was dissolved in a small amount of acetone / benzene (1/7) and subjected to silica gel column chromatography (Wakogel C-200).
(Registered trademark), 1500 g). Then, the fraction containing mevalonic acid was dried to obtain 19.0 g of a colorless oily substance.

This substance had a specific optical rotation [α] ²⁵ _D ▼ -21.9 ° (C = 3.89, ethanol).

〔The invention's effect〕

According to the method for producing mevalonic acid of the present invention, mevalonic acid can be obtained in a high yield as industrially feasible.

[Brief description of drawings]

FIG. 1 shows the relationship between the culture time and the dissolved oxygen content (saturation = 100), pH, glucose content (wt%), and mevalonic acid content (μg / ml) in Example 1 of the present invention. In the figure, 1 is the amount of mevalonic acid, 2 is the amount of glucose, 3 is pH, and 4 is the amount of dissolved oxygen.

Claims (11)

[Claims] 1. When producing a mevalonic acid by culturing a microorganism belonging to the genus Saccharomycopsis in a medium, the culture medium during culturing contains a substrate containing at least a carbon source.
A method for producing mevalonic acid, which comprises adding once or a plurality of times to continue culturing, and then obtaining mevalonic acid from the culture. 2. The substrate is added so that the carbon source concentration in the culture broth (filtrate of the culture) is maintained at 2 to 15% by weight. Method for producing mevalonic acid. 3. The method for producing mevalonic acid according to claim 1, wherein the substrate is added when the pH of the culture reaches 7 or more. 4. The method according to claim 1, wherein the addition of the substrate is carried out when the curve showing the dissolved oxygen amount (the initial value is saturation) of the culture has passed the minimum value. A method for producing mevalonic acid according to the description. 5. The method for producing mevalonic acid according to claim 1, wherein the culture is performed by adding a nonionic surfactant having a cell membrane solubilizing effect to the medium. . 6. One or more nonionic surfactants selected from the group consisting of polyoxyethylene glycol octyl phenyl ether type, polyoxyethylene glycol alkyl ether type and polyoxyethylene glycol sorbitan ester type. The method for producing mevalonic acid according to claim (5), characterized in that a nonionic surfactant is used. 7. A nonionic surfactant, Triton.
X-100 (Triton X-100, registered trademark) is used, The manufacturing method of the mevalonic acid of Claim (5) characterized by the above-mentioned. 8. The method for producing mevalonic acid according to claim 5, wherein the added amount of the surfactant is 0.002 to 0.2% by weight of the medium. 9. The nitrogen source in the medium is constituted only by an organic nitrogen source, according to claim (1).
The method for producing mevalonic acid according to the item. 10. An organic nitrogen source comprising one or more substances selected from the group consisting of peptone, yeast extract, meat extract and casein soybean powder. A method for producing mevalonic acid according to the range (9). 11. The method for producing mevalonic acid according to any one of claims (1) to (4), wherein the substrate is added as an aqueous solution.