JPS63216485A - Production of mevalonic acid - Google Patents

Abstract

PURPOSE:To produce mevalonic acid in high yield, by culturing a specific microbial strain in a medium added with a nonionic surfactant having solubilizing action to cell membrane. CONSTITUTION:A microbial strain capable of producing mevalonic acid (e.g. Saccharomycopsis fibuligera IFO 0107, etc.) is inoculated in a medium composed of 5-15% carbon source, 0.5-3% organic nitrogen source, 0.002-0.2%, preferably 0.02-0.1% nonionic surfactant and polyoxyethylene glycol octylphenyl ether-type surfactant and polyoxyethylene glycol sorbitan ester-type surfactant (especially Triton(R) X-100) and 0.01-5% each of phosphoric acid salt, potassium salt, magnesium salt and calcium salt and cultured at 20-40 deg.C, preferably 25-35 deg.C under shaking or aeration and agitation. The objective mevalonic acid is separated from the cultured product.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing mevalonic acid, and particularly to a method for obtaining mevalonic acid in high yield.

Note that mevalonic acid has two structures, an acid type and a lactone type, but it is known that they can be converted into each other, and hereinafter in this specification, unless otherwise specified, both types will be collectively referred to as mevalonic acid.

(Prior art and problems to be solved by the invention) Mevalonic acid is a substance that was first isolated by Wright et al. (JAC 5, 5273.1956) and is an important intermediate of various isoprenoid compounds including cholesterol. known as the body.

In addition, mevalonic acid plays an important role in the metabolism of living organisms, having a growth-promoting effect on various microorganisms and plants, and is therefore used as a growth promoter for microorganisms, plants, etc., and is also used as a pyrethroid pesticide. It is used as a precursor of , ubiquinone (respiratory coenzyme), dolichol (essential factor for polysaccharide biosynthesis), and fat-soluble vitamins.

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

The production of natural mevalonic acid by fermentation is carried out by Applied Microbiology.
.

Although examples using Satucharomycobsis fibrigera NRRL Y-9069 are described in 1 Mutual, 965 (1968) and U.S. Patent No. 3,617,447, the yield is low and 700 ~
At present, the amount is only 1000 μg/-, and industrial production has not yet been achieved.

Therefore, an object of the present invention is to provide a method for producing mevalonic acid with a high yield that is industrially practicable.

[Means for solving problems]

The present invention aims to achieve the above object by producing mevalonic acid by culturing microorganisms in a medium, adding a nonionic surfactant having a cell membrane solubilizing effect to the medium, and then culturing the microorganisms. This has been achieved by providing a method for producing mevalonic acid, which is characterized by obtaining mevalonic acid from.

Preferred microorganisms used in the present invention include, for example, Candida genus, Guillemondella (G.
huilliermondella), Ransentaapora, Hansenula (H
Ansenula genus, Pichia genus, Saccharomycopai
s), genus Saccharomyces
) genus, Citeros+yces genus, Endomycops is
Examples include microorganisms selected from the genus Endomyces, the genus Sordaria, and especially Candida.
), genus Bansenula - Pichia (
Genus Pichia, Saccharomycobsis
arosycopsis), Saccharomyces (Sac
charo+myces) genus, Endomycobsis (
Endos + ycops is) genus, Eidomyces (
Examples include microorganisms of the genus Endo+myces, specifically Candida citrea.
a cttrea), Candida fragicola (C
andida flagj cola), candida
Lambica (Candida 1aa+bica), Candida salmon (Candida 5ake), Candida scotchie (CandidascotH),
Hansanula dime
nnae), Hansenula californica (Hans
enuIa californica), Hansenula holstii
, A Hansenula h.
enrtcii), Hansenula fabianii (I(a)
nsenula fabianii), Hansenula minuta (Hansenula +5tnuta), Hansenula silvicola (llansenula 5ilv)
icola), Hansenula capsulata (Hanse
nula capsulata), Hanseniaspora pagerinkii (Hanseniaspora p.
eijerinckii), Pichia binus (Pich
ia pinus), Pichta membranaefaciens
)% Pichia saitoi (Pichia 5aitoi
), Sacc.
baromycopsis fibuligera)
, Saccharomycobsis cabusularis (Saccha
romycopsis capsularis), Saccharomyces 6 famentatei (Saccharomyces
yces fermentati), Satucharomyces
Delpur Equi-(3a((haro+*yces de
lbrueckti), Saccharomyces cytoanus (Saccharomyces 5aitoanus)
)-, Saccha 0 Mrs 0 Celebisiae (Saccha
romyces cerevisiae), Saccharon+yces
vafer), Endomycobsis fibrigera (E
ndowycopsis fibuligera),
Endoe+yces
fibuligera), Guillier+mondella selenospora (Guillier+mondella 5ele)
nospora), Cytelomyces matritelasis (
Cttero+myces matriteusis)
, Sordaria fimicola
micola), etc., and the preferable meat amount is Sacc.
haromycopsis fibultgera) - Strains belonging to these include, for example, IFO01
07, IFO0103, IFO0104, IFO010
5, IFO0106, IFO0109, IFO01) 1
, IPO1665, IFO171) 1FO1744, I
FO1745, AHU 41)3, IAM
4247.0UT6071, HUT7234,A
TCC2080, ATCC2082, ATCC2088
, ATCC9947, ATCC20145, ATCC2
4945, ATCC44B72, ATCC46252,
ATCC46253, ATCC46949, ATCC5
2921, NRRL Y-1060, NRRL
Y-1064, NRRL Y-2385, NRR
LY-7061, NRRL Y-7221,
NRRL Y-7324, NRRL Y-74
64, NRRL Y-9069, DSM 70
554, IAM 4025, IFO1342,
IFo 0800, IFo 1880, I
FO0980, IFO1477, IFO1254, IF
O0975, IFO0807, IFO0984, IAM
4945, IFO1850, IAM 4307,
IFO0672, IAM4771. IAM122
36, IAM 12241, IFOL0O2,
IFO1003, IFO1626, IFO1572, I
FO1574, IFO1)46, IFO1213, IF
O1287, IFO0954, IFO0941, IFO
8812 can be mentioned.

In addition, I FOSATCC, NRRL, DSM, AHU,
IAM, OUT, and HUT are the preserved strains of Fermentation Research Institute, American Type Culture Corexilan, ARS Northern Regional Research Center, Teutje Zamlung Phono Microorganismen, and Hokkaido University Faculty of Agriculture. The following are preserved strains, preserved strains of the Institute of Applied Microbiology, University of Tokyo, preserved strains of the Department of Fermentation Engineering, Faculty of Engineering, Osaka University, and preserved strains of the Department of Fermentation Engineering, Faculty of Engineering, Hiroshima University.

The medium used in the present invention contains nonionic surfactants that have a solubilizing effect on cell membranes, such as polyoxyethylene glycol-pt-octylphenyl ether type (known as Triton type and nonideal type), One or two selected from the group consisting of oxyethylene glycol alkyl ether type (known as bridi type and emuldiene type) and polyoxyethylene glycol sorbitan alkyl ester type (known as triton type and span type) More than one type of surfactant, specifically:
Triton X-100 (registered trademark), Triton X
-1) 4 (registered trademark), Triton X-102 (registered trademark), Nonideft P-40 (registered trademark), Bridge 3
5 (registered trademark), Bridge 76 (registered trademark), Bridge
96 (registered trademark), Prigi 56 (registered trademark), Emuldiene 120 (registered trademark), Emuldiene 109P
(registered trademark), Touin 20 (registered trademark), Span 20 (registered trademark 1), preferably Triton X-1
00 (registered trademark), preferably 0.002 to 0 of the medium.
．． By containing it in a proportion of 2% by weight, more preferably 0.02 to 0.1% by weight, the objective of the present invention of obtaining mevalonic acid in high yield can be achieved.

As nitrogen sources to be added to the culture medium of the present invention, peptone, yeast extract, meat extract, casein, and soybean flour, which are organic nitrogen sources, are used; it is better to use mevalonate without using an inorganic nitrogen source. This is preferable because it can improve the yield.

Examples of specific culture medium compositions used in the present invention include, for example, 5 to 15% by weight of carbon source and 0.5 to 3% by weight of organic nitrogen source.
, a medium consisting of 0.02 to 0.1% by weight of a surfactant and water (the balance), phosphates, potassium salts,
Examples include those containing 0.01 to 5% by weight of each of magnesium salt and calcium salt.

Furthermore, an antifoaming agent or the like may be added to the culture medium used in the present invention, if desired, within the scope of the purpose of the present invention.

A preferred specific implementation B of the manufacturing method of the present invention is as follows.

That is, microorganisms are inoculated into a medium containing a carbon source, an organic nitrogen source, a nonionic surfactant that has a solubilizing effect on cell membranes, and inorganic salts in predetermined amounts, and the mixture is heated at 20-40°C, preferably at 25-40°C.
Culture with shaking at 35°C or 100-500 rpm, preferably 200-400 rpm, 0.2-1.5 VVM
, preferably 0.5 to 1, aerated agitation culture with OVVM,
After culturing for a predetermined time, the substrate is fed (added) to the culture by measuring the carbon source concentration in the culture solution (culture filtrate), measuring the pH of the culture, or measuring the amount of dissolved oxygen. Such feeding of the substrate is carried out a necessary number of times, and the culture is terminated when it is judged that the production amount of mevalonic acid does not increase any further. After culturing, remove the bacterial cells from the culture using known methods such as centrifugation and filtration, and concentrate using reverse osmosis, vacuum distillation, etc., countercurrent distribution using butanol or ethyl acetate, silica gel, Diaion, etc. Column chromatography using ion exchange resin, molecular distillation,
Mevalonic acid can be obtained by processing by a combination of known purification techniques such as crystallization methods.

It should be noted that an antifoaming agent or the like may be added to the culture medium in the method of the present invention, if desired, within the scope of the purpose of the present invention.

In addition, the carbon source concentration in the culture solution is measured by an enzymatic method using glucose oxidase, etc. In this case, the "culture solution" refers to the solution part from which the facepieces, etc. are removed by centrifugation, filtration, etc. say.

〔Example〕

Examples of the present invention are 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.

[Mevalonic acid determination method]

Take 0.8 ml of the sample solution into a subic tube and incubate it with I N-HC.
Adjust the pH to 2 using Z solution. Nal 5 for this
After adding 041 g of ethyl acetate and 2.0 ml of ethyl acetate and stirring, remove the upper layer. Take the bottom layer and combine it with the previous top layer.

This operation is repeated again to obtain an ethyl acetate layer of 6.0+*1, which is evaporated to dryness. This dried product was quantified by gas chromatography using 3,4-dimethoxybenzaldehyde as an internal standard.

The conditions for gas chromatography are as follows.

Column size: diameter 3m length 10100O made of stainless steel) Column liquid phase: 10% Thermon-3000 Column support: chromosorb WAW-DMC38
0-100 mesh column temperature: 180°C mesh temperature: 230°C carrier gas: NtC40d/min) [Example 1,
Comparative Example 1] Glucose 10% by weight, malt extract 1% by weight
, peptone 0.5% by weight, yeast extract 0.
1% by weight, KHz PO40, 3% by weight, Mg 304
・7 Hz 00.05% by weight, CaCO51% by weight
, Triton X-100 (registered trademark) 0.05% by weight
, media 201 and 200- consisting of the remainder water are prepared,
Satucharomycobsis fibrigera I in 00m medium
One platinum loopful of FO1744 was inoculated and cultured with shaking at 28°C for 3 days, then inoculated into the medium of 201 above and incubated at 28°C.
The aerated culture was carried out at a rotation speed of 30 rpm and aeration rate of 120 It/min.

The amount of mevalonic acid obtained after continuing the culture for 6 days was 1
It was 410 μg/−.

In addition, when cultured in the same manner except for Triton X-100 (registered trademark) from the above medium (Comparative Example 1), 6
The amount of mevalonic acid obtained after continuing the culture for days is 1)
It was 60 μg/d.

[Example 2, Comparative Example 2] In addition to the medium having the composition of Example 1, 0.3 ammonium chloride was added.
Culture was carried out for 6 days in the same manner as in Example 1 except that % by weight was added. The amount of mevalonic acid obtained as a result was 1320μ
g/-.

In addition, when culturing was performed without Triton
It was /-.

[Example 3] Triton X-100 (registered trademark) was used as Bridge 35 (
The cells were cultured for 6 days in the same manner as in Example 1 except that the cells were replaced with (registered trademark). The amount of mevalonic acid obtained as a result was 1380
It was μg/ml.

[Example 4] Cells were cultured for 6 days in the same manner as in Example 1, except that S. fibrigera IFO1744 was replaced with S. fibrigera IFO0107.
The amount of mevalonic acid obtained as a result was 5560 μg/d.

[Example 5] 24 J of the culture obtained in Example 1 was centrifuged at a rotation speed of 500° rpm to obtain 16 J of filtrate. This was concentrated to 51 using a reverse osmosis membrane, and then adjusted to pH 2 using 50% by weight phosphoric acid water.
51 was extracted three times with ethyl acetate to obtain an ethyl acetate layer 151. This was transferred to 0.02N sodium hydroxide solution 51, adjusted to pH 2 with 50% by weight phosphoric acid solution, and then passed through a Diaion HP-20 column (1). This effluent was extracted three times with 51 ethyl acetate, and the ethyl acetate layer 151
I got it. This was dehydrated with anhydrous sodium sulfate and then evaporated to dryness. This dry matter was mixed with a small amount of acetone/benzene (1/
7) and separated by silica gel column chromatography (150 g). Thereafter, the fraction containing mevalonic acid was dried to form a colorless oily substance 1). 2g was obtained.

Specific optical rotation [α]'--21.4@ (C-2,54, ethanol).

〔Effect of the invention〕

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

Procedural amendment April 6, 1988 1, Indication of the case, Patent Application No. 1982-49621 2, Title of the invention: Process for producing mevalonic acid 3, Person making the amendment Relationship with the case Patent applicant (03B) Asahi Denka Kogyo Co., Ltd. 4, Agent 601 Pacific Nogizaka, 6-29 Akasaka, Minato-ku, Tokyo 5, Voluntary amendment of date of amendment order (amendment within 1 year and 3 months from filing date) 6, Target of amendment 7, Amendment Contents (1) r on page 3, line 15 (JAC5, 7B, 527
3.1956) J to ``(Journal of the American Chemical Society)
the A+merican Chemical 5o
cfety) Vol. 78, pp. 5273-5275, 195
6 years)” was corrected.

(2) "Diaion" on page 12, line 13 was corrected to "porous polymer resin."

(3) “Diaion HP-” on page 16, lines 14-15
Corrected 20J to "Diaion HP-20 (registered trademark)".

(4) Corrected "Silica gel column chromatography (150g) J" on page 16, lines 19-20 to "Silica gel column chromatography (Wakogel C-200 (registered trademark), 1500g) J.

that's all

Claims (7)

[Claims] (1) When producing mevalonic acid by culturing microorganisms in a medium, a nonionic surfactant with a cell membrane solubilizing effect is added to the medium, and then mevalonic acid is extracted from the culture. A method for producing mevalonic acid, characterized in that it obtains. (2) As the nonionic surfactant, one or more nonionic surfactants selected from the group consisting of polyoxyethylene glycol octylphenyl ether type, polyoxyethylene glycol alkyl ether type, and polyoxyethylene glycol sorbitan ester type. The method for producing mevalonic acid according to claim (1), characterized in that a surfactant is used. (3) Triton X-1 as a nonionic surfactant
00 (registered trademark) is used. The method for producing mevalonic acid according to claim (1). (4) The amount of nonionic surfactant added is 0.002 of the culture medium.
The method for producing mevalonic acid according to claim (1), wherein the content is 0.2% by weight. (5) Claim No. 1, characterized in that the nitrogen source added to the culture medium is composed only of organic nitrogen sources.
) to (4). (6) As an organic nitrogen source, one or more substances selected from the group consisting of peptone, yeast extract, meat extract, and casein soy flour are used. The method for producing mevalonic acid as described in (5). (7) Candida as a microorganism
Genus, Guilliermondella
a) Genus Hanseniaspora
a) Genus Hansenula, Genus Pichia, Genus Saccharomycopsis (Sacc)
genus haromycopsis, genus Saccharomyces (sa
genus ccharomyces, genus Citellomyces
eromyces), Endomycopsis (Endo mycopsis)
mycopsis), Eidomyces (Endomyces)
The method for producing mevalonic acid according to claim (1), characterized in that a microorganism selected from the group consisting of the genus Ces and the genus Sordaria is used.