JPH0751068B2 - 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-9069, 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 essential component of the method for producing mevalonic acid of the present invention is Saccharomycopsis f.
ibuligera) IFO 0107, IFO 0103, IFO 0104, IFO 0
105, IFO 0106, IFO 0109, IFO 0111, IFO 1665, I
FO 1711, IFO 1745, AHU 4113, IAM 4247, OUT 60
71, HUT 7234, ATCC 2080, ATCC 2082, ATCC 208
8, ATCC 20145, ATCC 24945, ATCC 44872, ATCC 46
252, ATCC 46253, ATCC 46949, ATCC 52921, NRRL
Y-1060, NRRL Y-1064, NRRL Y-2385, NRRL Y
-7061, NRRL Y-7221, NRRL Y-7324, NRRL Y-
7464, DSM 70554 and one or more microorganisms selected from the group consisting of these mutants are cultivated, preferably IFO 010
Culturing 7 and then obtaining mevalonic acid from that culture.

In addition, 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 Zamrumung von, respectively. -Preserved strains of microorganismen, preserved strains of Hokkaido University Faculty of Agriculture, preserved strains of Institute of Applied Microbiology, University of Tokyo, preserved strains of fermentation engineering department of Osaka University, and preserved strains of fermentation engineering department of Hiroshima University.

According to the present invention, that is, in order to produce mevalonic acid using the above-mentioned microorganism, for example, as shown in U.S. Pat.No. 3,617,447, a known method can be used, but preferably the following method is used. good.

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 to 1.5 VVM, preferably 0.5 to 1.0 VVM, aeration and agitation culture, and after culturing for a predetermined time, measurement of carbon source concentration in the culture solution (culture filtrate of the culture), pH of the culture The substrate is fed (added) to the culture by measurement or measurement of the amount of dissolved oxygen. 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 performed by an enzyme method using glucose oxidase, and the above-mentioned "culture solution"
Is a solution part in which the bacterial cells and the like have been removed from the culture by centrifugation, filtration and the like.

〔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. To this, 1 g of Na ₂ SO ₄ was added, 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 3mm Length 1000mm
(Stain column liquid phase: 10% Thermon-3000 column support: chromosorb W AW-DMCS 80-100 mesh Column temperature: 180 ° C Injection temperature: 230 ° C Carrier gas: N ₂ (40 ml / min) [Example 1] Glucose 10 % By weight, malt 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
A medium consisting of wt%, Triton X-100 (nonionic surfactant having a solubilizing effect on the membrane) 0.05 wt%, and the balance water.
Prepare 20 and 200 ml, and inoculate 1 200 ml of Saccharomycopsis fibrillella IFO 0107 into the medium and incubate at 28 ° C.
The medium which had been shake-cultured for 3 days was inoculated into the above-mentioned 20 medium, and the culture was carried out with aeration and stirring while measuring the glucose concentration, pH and dissolved oxygen content at 28 ° C., a rotation speed of 300 rpm, and an aeration rate of 20 / min.

It was confirmed that the glucose concentration in the culture solution became 5% or less on the third day of the culture, and immediately after that, the pH exceeded 7, and the dissolved oxygen amount passed the minimum value.
2.0 kg of the aqueous solution was fed and the culture was continued. Furthermore, culture 6
On the day and the 9th day of the culture, 2.0 kg of a 50% by weight aqueous solution of glucose was fed, respectively, and the culture was continued for a total of 12 days to complete the culture. 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 1210.
It was 0 μg / ml.

Example 2 and 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 20% by weight, 0.3% by weight of ammonium chloride, and the balance of water, and inoculate 1 ml of Saccharomycopsis fibrigella IFO 0107 into the 200 ml of the medium.
The medium which had been shake-cultured at 8 ° C. for 3 days was inoculated into the above-mentioned 20 medium, and culture was carried out with aeration and stirring at 28 ° C., a rotation number of 300 rpm, and an aeration rate of 20 / min.

The amount of mevalonic acid obtained by continuing the culture for 6 days was 3010μ.
It was g / ml.

In addition, Saccharomycopsis fibriguera IFO 010
When 7 was replaced with NRRL Y-9069 and cultured in the same manner (Comparative Example 1), the amount of mevalonic acid obtained by continuing the culture for 6 days was 780 μg / ml.

[Example 3] 10 ml of the medium of Example 1 was placed in a test tube, sterilized at 121 ° C for 15 minutes, and 1 platinum loop of each strain of Saccharomycopsis fibrigella shown in Table 1 was inoculated. Culturing was carried out by shaking at 250C and 250 rpm. On the 4th and 8th days, 1.0 g of 50 wt% glucose aqueous solution was fed to each well and cultured for 12 days.
After centrifugation at rpm for 10 minutes, mevalonic acid in the filtrate was quantified. The amount of mevalonic acid thus obtained is shown in Table 1.

Example 4 The culture 22 obtained in Example 1 was centrifuged at a rotation speed of 5000 rpm to obtain a filtrate 15. 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 (Wagel (-200
(Registered trademark), 1500 g). Then, the fraction containing mevalonic acid was dried to obtain 91.4 g of a colorless oily substance.

The specific optical rotation [α ▲] ²⁵ _D ▼ = -22.0 ° (C = 3.20, 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.

Claims (1)

[Claims] 1. Saccharomycopsis fibrigella (Sa
ccharomycopsis fibuligera) IFO 0107, IFO 0103, I
FO 0104, IFO 0105, IFO 0106, IFO 0109, IFO 01
11, IFO 1665, IFO 1711, IFO 1745, AHU 4113, IA
M 4247, OUT 6071, HUT 7234, ATCC 2080, ATCC 2
082, ATCC 2088, ATCC 20145, ATCC 24945, ATCC 4
4872, ATCC 46252, ATCC 46253, ATCC 46949, ATCC
52921, NRRL Y-1060, NRRL Y-1064, NRRL Y
-2385, NRRL Y-7061, NRRL Y-7221, NRRL Y-
A method for producing mevalonic acid, which comprises culturing at least one microorganism selected from the group consisting of 7324, NRRL Y-7464, DSM 70554 and mutants thereof, and then obtaining mevalonic acid from the culture.