JP2831833B2 - Process for producing optically active 3-hydroxybutyrate - Google Patents

Description

The present invention relates to an optically active 3-hydroxybutyrate, ie, 3-acetoacetate, obtained by microbially reducing acetoacetate.
The present invention relates to a method for producing (R) -hydroxybutyrate or 3 (S) -hydroxybutyrate.
3 (R) -hydroxybutyrate and 3 (S) -hydroxybutyrate are important synthetic raw materials for various pharmaceuticals such as antibiotics.

[Conventional technology]

As a method for producing 3 (R) -hydroxybutyrate by asymmetric reduction of acetoacetate, a method based on organic synthesis using (R, R) -tartaric acid-modified Raney nickel as a catalyst (The Chemical Society of Japan, 10 , 1270 (1986) ), JP-A-60
−36442), Geotrical Candidam (Geotric
um Candidum), Rhodotorula palida
microbial method using a lida) (Helv. Chim. Acta., 66, 4
85 (1983), JP-A-1-117792).

As a method for producing 3 (S) -hydroxybutyrate by asymmetric reduction of acetoacetate, an organic synthesis method using an asymmetrically modified nickel catalyst (Oil Chemistry, 29, 44).
(1980)), baker's yeast (Saccharomyces cerevisiae),
Thermoanaerobium Blocky
brockii) and the like (Angew. Chem. Int. E
d.Engl., 23,570 (1984); Angew.Chem.Int.Ed.Engl., 23,
151 (1984)).

[Problems to be solved by the invention]

3 (R) -hydroxybutyrate and 3
There are still many problems to be solved in order to industrially produce optically active 3-hydroxybutyrate such as (S) -hydroxybutyrate. Above all, it is important to obtain a simple production method with high optical activity and yield.

[Means for solving the problem]

The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, the acetoacetate ester was changed to the genus Clostridium or Methanobacterium.
bacterium), a microorganism selected from the genus Methanosarcina and the genus Acetogenium or a processed product thereof, the optical activity
They have found that hydroxybutyrate can be obtained, and have completed the present invention.

The starting material acetoacetate used in the present invention is:
For example, it is a compound represented by the general formula CH ₃ COCH ₂ COOR obtained by reacting alcohol and diketene in the presence of a catalyst. R is an alkyl group, preferably a lower alkyl group such as methyl acetoacetate and ethyl acetoacetate are useful.

Examples of useful strains for use in the present invention are as follows. Clostridium thermoaceticum DSM1754, Methanobacterium thermooutroficum (Methanobacteri)
um thermoauto-trophicum) DSM1503, Methanosarcina barkeri DSM804, Methanosarcina sp. DSM2906,
Methanosarcina sp. DSM2
980, Clostridium thermoautotrohicum (Clo
stridium thermoautotrophicum) DSM1974; Acetogeniump kivui DSM2030. These mutants can also be used.

The cultivation of the strain used in the present invention is basically the same as that of general microorganisms, but it is necessary to prevent contamination of oxygen, and in the laboratory, in a culture vessel sealed with a rubber stopper or the like. Then, a method of standing or shaking is used. Industrially, fermenters commonly used can be used as they are, and an anaerobic atmosphere can be created by replacing the oxygen in the device with an inert gas such as nitrogen or a gas such as carbon dioxide or hydrogen used in culture. It is possible. The type of fermentation tank is not particularly limited, but a one-stage or multi-stage bubble column type or draft tube type fermentation tank can be used in addition to a commonly used stirring and mixing tank.

Various types of media can be used for the culture. Examples of the carbon source include various sugars, acetic acid, methanol, and carbon dioxide, and examples of the nitrogen source include inorganic salts, yeast extract, meat extract, peptone, and corn steep liquor. In addition, if necessary, inorganic compounds such as potassium dihydrogen phosphate, magnesium sulfate, manganese sulfate, sodium chloride, zinc sulfate, copper sulfate, alum, sodium molybdate, and vitamins such as bition and pantothenic acid may not be added. As usual.

It is preferable to culture the medium at a pH of 3.0 to 9.5 and a culture temperature of 20 to 75 ° C.

The reaction method is to use the culture solution as it is, to separate the cells by centrifugation, etc., to wash the cells as they are or to wash them. Alternatively, a method in which crushed cells, treated with acetone, freeze-dried or the like are used instead of living cells, or a method in which these are immobilized on a carrier or the like can be applied. In this reaction, the yield is often improved by adding glucose, fructose, acetic acid, methanol, hydrogen or the like as an energy source.

As a medium for such a reaction, not only water but also an organic solvent compatible with water, for example, a water / organic solvent mixed system such as alcohol and acetone is used. It is, of course, necessary to select an organic solvent that does not harm microorganisms.

The acetoacetate is added to the system as it is, or dissolved or dispersed in an organic solvent. The concentration range of the ester in the system is usually 0.01 to 50% by weight, and 0.01% by weight.
If it is less than 10%, there is no disadvantage in terms of reactivity, but it is not economically practical. On the other hand, if it is more than 50% by weight, the load on the cells is too high, and problems such as a decrease in yield are likely to occur.

The reaction is preferably carried out at a pH of 3 to 9.5 at a temperature of 4 to 75 ° C., and is carried out for 1 to 200 hours with stirring or standing.

After completion of the reaction, the reaction solution is extracted with an organic solvent such as ethyl acetate or hexane, and then the solvent is removed. 3-hydroxyacetic acid ester is obtained.

In the present invention, the desired optically active 3-hydroxybutyric acid ester can be obtained by appropriately combining the starting materials and the cells.

〔Example〕

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.

The optical purity and yield in the examples were determined by high performance liquid chromatography (mobile phase; mobile phase; using a normal phase column (Finepack SILC ₁ ; manufactured by JASCO Corporation) and an optical resolution column (Chiral Cell OD; manufactured by Daicel Chemical). n-hexane / 2
-Propanol = 19/1, detection 210 nm).

Example _{1 K 2 HPO 4 0.3g: KH} 2 PO 4 0.2g: NH 4 Cl 0.5g: MgSO 4 · 7H 2 O
_{0.5g: CaCl 2 · 2H 2 O} 0.3g: NaCl 2.0g: NaHCO 3 1.0g: FeSO 4
・ 7H ₂ O 0.002g: Yeast extract 2.0g: Peptone 2.0g: Vitamin solution (biotin 2.0mg: folic acid 2.0mg: pyridoxine hydrochloride 1
0.0 mg: Thiamine hydrochloride 5.0 mg: Riboflavin 5.0 mg: Nicotinic acid 5.0 mg: Calcium pantothenate 5.0 mg: Vitamin B ₁₂ 0.1
mg: D-aminobenzoic acid 5.0mg: Lipoic acid 5.0mg: water 1) 10m
l: trace elements solution _{(ZnSO 4 · 7H 2 O 0.1g} : MnCl 2 · 4H 2 O 0.03
g: H ₃ BO ₃ 0.3g: CoCl ₂・ 6H ₂ O 0.2g: CuCl ₂・ 2H ₂ O 0.01g: Ni
_{Cl 2 · 6H 2 O 0.02g:} NaMoO 4 · 2H 2 O 0.03g: water 1) 3ml: Na ₂
0.3 g of S · 9H ₂ O: 0.4 g of L-cysteine · HCl: 0.4 g of titanium citrate: 8 g of methanol: 8 g of methanol: inoculated with Methanosarcina sp. Culture was performed at 55 ° C. for 10 days using the mixed gas of (1). After completion of the culture, isolate the cells by centrifugation,
The cells were washed twice with mM phosphate buffer (pH 7.0) to obtain viable cells.

25 ml of the reaction solution (ethyl acetoacetate 100 mM, methanol 250 mM, 50 mM phosphate buffer pH7.
0) was added thereto, and the viable cells were suspended therein, and the gas phase was reacted at 55 ° C. for 12 hours as a mixed gas of nitrogen / carbon dioxide = 4/1. After the completion of the reaction, the mixture was extracted with 100 ml of ethyl acetate. After the solvent was removed from the extract, quantitative determination of the generated ethyl 3 (R) -hydroxybutyrate and measurement of optical purity were performed using high performance liquid chromatography. Table 1 shows the obtained results.

Examples 2 to 8 When the cells shown in Table 1 were cultured using the carbon sources shown in Table 1 according to Example 1, and reacted using the energy sources shown in Table 1, the corresponding 3 (R)- A hydroxybutyrate was obtained. Table 1 also shows the results.

Example _{9 K 2 HPO 4 0.3g: KH} 2 PO 4 0.2g: NH 4 Cl 0.5g: MgSO 4 · 7H 2 O
_{0.5g: CaCl 2 · 2H 2 O} 0.3g: NaCl 2.0g: NaHCO 3 1.0g: FeSO 4
・ 7H ₂ O 0.002g: Yeast extract 2.0g: Peptone 2.0g: Vitamin solution (biotin 2.0mg: folic acid 2.0mg: pyridoxine hydrochloride 1
0.0 mg: Thiamine hydrochloride 5.0 mg: Riboflavin 5.0 mg: Nicotinic acid 5.0 mg: Calcium pantothenate 5.0 mg: Vitamin B ₁₂ 0.1
mg: D-aminobenzoic acid 5.0mg: Lipoic acid 5.0mg: water 1) 10m
l: trace elements solution _{(ZnSO 4 · 7H 2 O 0.1g} : MnCl 2 · 4H 2 O 0.03
g: H ₃ BO ₃ 0.3g: CoCl ₂・ 6H ₂ O 0.2g: CuCl ₂・ 2H ₂ O 0.01g: Ni
_{Cl 2 · 6H 2 O 0.02g:} NaMoO 4 · 2H 2 O 0.03g: water 1) 3ml: Na ₂
0.3 g of S · 9H ₂ O: L-cysteine · HCl 0.4 g: titanium citrate 0.075 mM: 8 g of methanol: 8 g of water: A medium having a composition of 1 was inoculated with Methanosarcina sp. Culture was performed at 55 ° C. for 10 days using the mixed gas of (1). After completion of the culture, isolate the cells by centrifugation,
The cells were washed twice with mM phosphate buffer (pH 7.0) to obtain viable cells.

25 ml of a reaction solution (methyl acetoacetate 100 mM, methanol 250 mM, 50 mM phosphate buffer pH7.
0) was added thereto, and the viable cells were suspended therein, and the gas phase was reacted at 55 ° C. for 12 hours as a mixed gas of nitrogen / carbon dioxide = 4/1. After the completion of the reaction, the mixture was extracted with 100 ml of ethyl acetate. After the solvent was removed from the extract, quantitative determination of the generated methyl 3 (S) -hydroxybutyrate and measurement of optical purity were performed using high performance liquid chromatography. Table 2 shows the obtained results.

Examples 10 to 13 When the cells shown in Table 2 were cultured with the carbon sources shown in Table 2 according to Example 9, and reacted using the energy sources shown in Table 2, the corresponding 3 (S)- A hydroxybutyrate was obtained. Table 2 also shows the results.

[Effect of the Invention] The present invention provides a method for producing 3 (R) -hydroxybutyrate or 3 (R) -hydroxybutyrate having high optical purity by using the aforementioned strain.
(S) -Hydroxybutyrate can be easily produced in a high yield and is extremely advantageous as an industrial production method.

Claims (1)

(57) [Claims] 1. The method of claim 1 wherein the acetoacetate is a genus Clostridium or Methanobact.
erium), a microorganism selected from the group consisting of Methanosarcina and Acetogenium, or a processed product thereof, and collecting the resulting optically active 3-hydroxybutyrate ester. -A method for producing hydroxybutyric acid esters.