JPH0789939B2 - 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, which has a growth promoting action on various microorganisms and plants, plays an important role in the metabolism of organisms, and therefore, microorganisms belonging to the genus Saccharomycopsis,
It is used as a growth promoter for plants and the like, and is also used as a precursor for pyrethroid pesticides, ubiquinone (respiratory coenzyme), dolichol (an essential factor for polysaccharide biosynthesis), 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 of producing a mevalonic acid by culturing a microorganism belonging to the genus Saccharomycopsis in a medium, by adding a nonionic surfactant having a solubilizing action of a cell membrane to the medium. This has been achieved by providing a method for producing mevalonic acid, which comprises culturing and then 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, 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 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.

The medium used in the present invention includes a nonionic surfactant having a cell membrane solubilizing action, such as polyoxyethylene glycol-pt-octylphenyl ether type (known as Triton type or nonidet type), poly 1 or 2 selected from the group consisting of oxyethylene glycol alkyl ether type (known as bridging type and emulgen type) and polyoxyethylene glycol sorbitan alkyl ester type (known as tween type, span type) One or more surfactants, specifically,
Triton X-100 (registered trademark), Triton X-1
14 (registered trademark), Triton X-102 (registered trademark),
Nonidet P-40 (registered trademark), Bridget 35 (registered trademark), Bridget 76 (registered trademark), Bridget 96 (registered trademark), Bridget 56 (registered trademark), Emulgen 120
(Registered trademark), Emulgen 109P (registered trademark), Toein 20 (registered trademark), Span 20 (registered trademark), preferably Tratoin X-100 (registered trademark), preferably 0.002 to 0.2% by weight of the medium, more preferably Is 0.02 to 0.
The inclusion of 1% by weight makes it possible to achieve the object of the present invention to obtain mevalonic acid in a high yield.

The nitrogen source added to the medium of the present invention is an organic nitrogen source, peptone, yeast extract, meat extract, casein, using soybean powder, it is more mevalonic acid without using an inorganic nitrogen source. It is preferable because the yield can be 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. Examples thereof include those containing 0.01 to 5% by weight of each of phosphate, potassium salt, magnesium salt and calcium salt.

If desired, an antifoaming agent or the like can be added to the medium used in the present invention within the scope of the object of the present invention.

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 and agitation culture, and after culturing for a predetermined time, measurement of carbon source concentration in culture solution (culture filtrate), measurement of culture pH Alternatively, the substrate is fed (added) to the culture by measuring the amount of dissolved oxygen. Fed-batch of such a substrate is performed as many times as necessary,
The culture is terminated when it is determined that the mevalonic acid production cannot be further improved. After culturing, centrifuge the culture,
The cells are removed by a known method such as filtration, concentrated by reverse osmosis, vacuum distillation, etc., or countercurrent distribution using butanol or ethyl acetate, or column chromatography using silica gel, porous polymer resin, ion exchange resin, etc. Law,
Mevalonic acid can be obtained by treatment with a combination of known purification techniques such as molecular distillation and crystallization.

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. Take the lower layer and match with the previous layer. This operation is repeated again to obtain 6.0 ml of ethyl acetate layer, which is evaporated to dryness. This dry matter
It was quantified by gas chromatography using 3,4-dimethoxybenzaldehyde as an internal standard. The conditions for gas chromatography are as follows.

Cam 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
% And Tratoin X-100 (registered trademark) 0.05% by weight, and 20 and 200 ml of a medium consisting of the balance water were prepared.
Saccharomycopsis fibrigella IFO 174 in culture medium
4 platinum loops were inoculated and cultured at 28 ° C for 3 days with shaking, and then inoculated into the above 20 medium at 28 ° C, rotation speed 300 rpm,
The culture was performed with aeration and stirring at an aeration rate of 20 / min.

The amount of mevalonic acid obtained after continuing the culture for 6 days was 14
It was 10 μg / ml.

In addition, when the same culture was performed except for Tratoin X-100 (registered trademark) from the above medium (Comparative Example 1), 6
The amount of mevaloic acid obtained after culturing for 1 day was 1160μ.
It was g / ml.

[Example 2 and Comparative Example 2] The culture was carried out for 6 days in the same manner as in Example 1 except that 0.3% by weight of ammonium chloride was further added to the medium having the composition of Example 1. The amount of mevalonic acid obtained as a result was 1320 μg / ml.

Also, from the above medium, Triton X-100 (registered trademark)
When culturing was carried out except for (Comparative Example 2), the amount of mevalonic acid obtained after culturing for 6 days was 910 μg / ml.

[Example 3] Culture was carried out for 6 days in the same manner as in Example 1 except that Toritoin X-100 (registered trademark) was replaced with Brij 35 (registered trademark). The amount of mevalonic acid obtained as a result was 1380 μg / ml.

[Example 4] Culture was carried out for 6 days in the same manner as in Example 1 except that Saccharomycopsis fibrigella IFO 1744 was replaced with Saccharomycopsis fibriguera IFO 0107. The amount of mevalonic acid obtained as a result was 5560 μg / ml.

[Example 5] The culture 24 obtained in Example 1 was centrifuged at a rotation speed of 5000 rpm to obtain a filtrate 16. This was concentrated to 5 using a reverse osmosis membrane, adjusted to pH 2 with 50% by weight aqueous phosphoric acid, and extracted 3 times with ethyl acetate of 5 to obtain an ethyl acetate layer 15. 0.02 for this
The solution was dissolved in N-sodium hydroxide water 5 and adjusted to pH 2 with 50% by weight phosphoric acid water, and then 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 11.2 g of a colorless oily substance.

The specific optical rotation [α] ²⁵ _D = -21.4 ° (C = 2.54, ethanol).

[Effects of the Invention] According to the method for producing mevalonic acid of the present invention, mevalonic acid can be obtained in a light yield that is industrially feasible.

Claims (6)

[Claims] 1. When producing mevalonic acid by culturing a microorganism belonging to the genus Saccharomycopsis in a medium, a nonionic surfactant having a solubilizing effect on a cell membrane is added to the medium for culturing. Then, a method for producing mevalonic acid, which comprises obtaining mevalonic acid from the culture. 2. A nonionic surfactant selected from the group consisting of polyoxyethylene glycol octylphenyl ether type, polyoxyethylene glycol alkyl ether type and polyoxyethylene glycol sorbitan ester type, or two or more types thereof. The method for producing mevalonic acid according to claim (1), characterized in that a nonionic surfactant is used. 3. Triton as a nonionic surfactant
X-100 (registered trademark) is used, The manufacturing method of the mevalonic acid of Claim (1) characterized by the above-mentioned. 4. The addition amount of nonionic surfactant is 0.00 in the medium.
The method for producing mevalonic acid according to claim (1), characterized in that the content is 2 to 0.2% by weight. 5. The method for producing mevalonic acid according to any one of claims (1) to (4), wherein the nitrogen source added to the medium is composed of only an organic nitrogen source. . 6. 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 item (5).