JPH0789940B2 - Manufacturing method of 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) It is 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 object, when producing mevalonic acid by culturing a microorganism belonging to the genus Saccharomycopsis in a medium, culturing using a medium to which only an organic nitrogen source is added as a nitrogen source, Then, it was achieved by providing a method for producing mevalonic acid, which comprises obtaining mevalonic acid from the culture.

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, NRR Y-906
9, DSM 70554, IAM 4025, IFO 1342, IFO 0800, IF
O 1880, IFO 0980, IFO 1477, IFO 1254, IFO 097
5, IFO 0807, IFO 0984, IAM 4945, IFO 1850, IAM
4307, IFO 0672, IAM 4771, IAM 12236, IAM 122
41, IFO 1002, IFO 1003, IFO 1626, IFO 1572, IF
O 1574, IFO 1146, IFO 1213, IFO 1287, IFO 095
4, IFO 0941, IFO 8812 can be mentioned.

IFO, ATCC, NRRL, DSM, AHU, IAM, OUT, and HUT are the strains preserved by the Fermentation Research Institute, American type, respectively.
Culture Collection Preserved Strain, ARS Northern Regional Research Center Preserved Strain, Deutsche Zammulung von Microorganizmen Preserved Strain, Hokkaido University Faculty of Agriculture Preserved Strain, University of Tokyo Applied Microbial Research Institute Preserved Strain, Osaka University Faculty of Engineering Preservation Strain , Shows the preserved strains of the Department of Fermentation Engineering, Faculty of Engineering, Hiroshima University.

As the medium composition used in the present invention, only an organic nitrogen source is used as the nitrogen source, for example, peptone, yeast extract, meat extract, casein, soybean flour and the like are used, and are usually used unless an inorganic nitrogen source is added. The medium can be used, and specific examples of the medium composition include, for example, 5 to 15% by weight of carbon source and 0.5 to 3 of organic nitrogen source.
A medium consisting of wt% and water (the balance) can be mentioned, and further one containing 0.01 to 5 wt% each of inorganic salts such as phosphate, potassium salt, magnesium salt, calcium salt and the like.

In addition, it is preferable that the amount of mevalonic acid obtained is increased when the medium contains 0.02 to 0.1% by weight of a nonionic surfactant.

In the method for producing mevalonic acid of the present invention, when the carbon source concentration of the culture solution (culture filtrate) in the culture during culture is maintained at 2 to 15% by weight, preferably 5 to 10% by weight, mevalon It is preferable because the amount of acid obtained can be increased.

Examples of the method of maintaining the carbon source concentration within the above range include a method of adding a substrate containing at least a carbon source to the culture during culture.

When the above-mentioned substrate is added, it is preferable to add it to the culture so that the carbon source concentration in the culture solution (culture filtrate) in the culture is maintained in the above range. However, since it generally takes a long time to measure the carbon source concentration, it is better to use the pH of the culture or the amount of dissolved oxygen as an index to judge the timing 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 added at this time may be obtained by measuring the carbon source concentration at the time point of the index in advance 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 fed (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 completion of the culture, the culture is centrifuged or filtered to remove the cells by a known method, and then concentrated by a reverse osmosis method, vacuum distillation or the like, or a countercurrent distribution method using butanol or ethyl acetate, 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.

In addition, 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 ml 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 3 mm Length 1000 mm (stainless steel) Column liquid phase: 10% Thermon-3000 Column support: chromsorb 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
One loop of 1744 was inoculated and shake-cultured at 28 ° C for 3 days, then inoculated into the above 20 medium, 28 ° C, rotation speed 300r
The culture was performed with aeration and stirring at pm and an aeration rate of 20 / min.

The amount of mevalonic acid obtained by continuing the culture for 6 days was 1160μ.
It was g / ml.

Further, when 0.3% by weight of ammonium chloride was further added to the above medium for culturing (Comparative Example 1), the amount of mevalonic acid was 910 μg / ml after culturing for 6 days.

[Example 2] The same medium as in Example 1 was used at 28 ° C, a rotation speed of 300 rpm, and an aeration rate.
The culture was performed with aeration and stirring while measuring the glucose concentration, pH, and dissolved oxygen amount at 20 / min.

After confirming that the glucose concentration was 5% or less by measuring the glucose amount in the culture solution on the 3rd day of culture, 2.0 kg of a 50% by weight aqueous solution of glucose was fed at this point 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.

FIG. 1 shows the culture time, dissolved oxygen amount (saturation = 100), pH, glucose amount (wt%), 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 3] Culture was performed in the same manner as in Example 2 except that the yeast extract was replaced with the meat extract. The amount of mevalonic acid obtained as a result was 2070 μg / ml.

[Example 4] Culture was performed in the same manner as in Example 2 except that casein was used instead of peptone. The resulting amount of mevalonic acid was 1970μ
It was g / ml.

[Example 5] Culture was carried out in the same manner as in Example 2 except that Saccharomycopsis fibriguera IFO 1744 was replaced with Saccharomycopsis fibriguera IFO 0107. The amount of mevalonic acid obtained as a result was 8590 μg / ml.

[Example 6] The culture obtained in Example 2 was centrifuged at 22 rpm to obtain a filtrate 14. This was concentrated to 4 using a reverse osmosis membrane, adjusted to pH 2 with 50% by weight aqueous phosphoric acid, and extracted 3 times with 4 ethyl acetate to obtain an ethyl acetate layer 12. This is 0.
After being redissolved in 02N sodium hydroxide water 5 and adjusted to pH 2 with 50% by weight phosphoric acid water, Diaion HP-20 (registered trademark)
Passed through column (1). The effluent was extracted 3 times with ethyl acetate of 4 to obtain an ethyl acetate layer 12. 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-20
0 (registered trademark), 1500 g). The fraction containing mevalonic acid was dried to obtain 14.4 g of a colorless oily substance.

The specific optical rotation [α] ²⁵ _D = -21.6 ° (C = 2.31, 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 (6)

[Claims] 1. When producing mevalonic acid by culturing a microorganism belonging to the genus Saccharomycopsis in a medium, culturing is performed using a medium to which only an organic nitrogen source is added as a nitrogen source, and then the culture. A method for producing mevalonic acid, which comprises obtaining mevalonic acid from 2. 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 process for producing mevalonic acid according to item (1). 3. The method for producing mevalonic acid according to claim 1, wherein a culture containing at least a carbon source is added to the culture during the culture. 4. The substrate is added so that the carbon concentration in the culture broth (filtrate of the culture) is maintained at 2 to 15% by weight. Of mevalonic acid. 5. The method for producing mevalonic acid according to claim (3), wherein the addition of the substrate is carried out when the pH of the culture becomes 7 or more. 6. The method according to claim 3, wherein the addition of the substrate is carried out at a time point when it passes through an inflection point of a curve showing the dissolved oxygen content of the culture (vs. the number of culture days). Manufacturing method of mevalonic acid.