JPH06253870A - Production of (r)-3-hydroxybutyric acid and its ester - Google Patents

Abstract

PURPOSE:To obtain the subject compound useful as a raw material for synthesizing antibiotics, etc., in high purity and in high yield by allowing a strain belonging to the genus Paracoccus to act on an acetoacetate ester. CONSTITUTION:A strain (preferably Pracoccus denitrificans IFO-13301, etc.) belonging to the genus Paracoccus is allowed to act on an acetoacetate ester (preferably methyl acetoacetate, ethyl acetoacetate) to obtain the objective compound. The reaction is preferably performed at a pH of 4-9 at a temperature of 25-40 deg.C for 3-120hrs.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a process for producing (R) -3-hydroxybutyric acid and its ester,
More particularly, it relates to a method for producing (R) -3-hydroxybutyric acid and its ester by allowing a specific microorganism to act on acetoacetic acid ester. (R) -3-Hydroxybutyric acid and its ester are important synthetic raw materials for various pharmaceuticals such as antibiotics.

[0002]

2. Description of the Related Art As a method for producing (R) -3-hydroxybutyric acid and its ester by asymmetric reduction of acetoacetic acid ester, there are (1) a method by organic synthesis, (2) a method by a microorganism and the like. As a method of (1), a report using (R, R) -tartaric acid modified Raney nickel as a catalyst is reported (Journal of the Chemical Society of Japan, 10, 1270 (1986), JP-A-60-36442).
As a method of (2), there is a report using Geotricum as a bacterial cell (Helv.Chim.Acta., 66, 485 (1983)),
There are reports using Rhodotorula and the like (JP-A-1-117792) and reports using anaerobic bacteria (JP-A-4-148689).

[0003]

Although the above-mentioned method is known as a method for producing (R) -3-hydroxybutyric acid and its ester, from (A) acetoacetic acid ester to (R)
There are still many problems to be solved in order to carry out industrial production of -3-hydroxybutyric acid and its ester. Above all, it is important to provide a production method with high optical activity and high yield.

[0004]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that when acetoacetate is reacted with a strain belonging to the genus Paracoccus (R)- The inventors have found that 3-hydroxybutyric acid and its ester can be obtained, and completed the present invention. That is, the present invention is characterized in that a strain belonging to the genus Paracoccus is allowed to act on acetoacetic acid ester to collect (R) -3-hydroxybutyric acid and its ester, and (R) -3-hydroxybutyric acid and its A method for producing an ester is provided.

The starting material acetoacetic acid ester used in the present invention is, for example, a compound of the general formula CH ₃ COCH ₂ CO obtained by reacting an alcohol with diketene in the presence of a catalyst.
A compound represented by OR (in the formula, R represents an alkyl group). Particularly, methyl acetoacetate and ethyl acetoacetate are useful.

The strain used in the present invention is Paracoccus (Para).
coccus) strains belonging to the genus, such as Paracoccus
Denitrificans IFO 1
3301 and the like, and any strain such as a wild strain, a mutant strain, or a recombinant strain derived by cell fusion or a genetic engineering method can be preferably used. This microorganism is described in List of Cultures, 9th edition (1992) published by Fermentation Research Institute, and can be obtained from the IFO.

The culture of the strain used in the present invention is basically the same as that of a general microorganism, but it can also be cultured while preventing the contamination of oxygen. In the laboratory, a method of standing or shaking in an incubator tightly sealed with a rubber stopper or the like is used. A fermenter that is usually used industrially can be used as it is, and it is possible to create an anaerobic atmosphere by substituting oxygen in the apparatus with an inert gas such as nitrogen or a gas used in culture. The form of the fermenter is not particularly limited, and in addition to the commonly used stirring and mixing tank, a single-stage or multi-stage bubble column type or draft tube type fermenter can also be used. The culture temperature is 20 to 50 ° C, preferably 25 to
Incubate at 40 ° C. for 5 to 120 hours, preferably 12 to 72 hours.

Various media can be used for the culture, but various sugars such as glucose, organic acids such as acetic acid and succinic acid and salts thereof as carbon sources, alcohols such as methanol and glycerol, and hydrocarbons such as paraffin. , Potato extract, etc., or a mixture of these,
As a nitrogen source, potassium nitrate, ammonium chloride, ammonium sulfate, inorganic salts such as ammonium nitrate, ammonium acetate, organic acid salts such as ammonium succinate, yeast extract, meat extract, peptone, corn steep liquor,
Examples include casein hydrolyzate, urea, liver extract and the like, or a mixture thereof. 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 biotin and pantothenic acid should not be added. As usual. The medium pH is 3 to 9.5, preferably 4 to 8.5.

As the reaction method, the culture solution is used as it is, or the cells are separated by centrifugation or the like, and the cells are directly or washed and then resuspended in a buffer solution, water or the like, and acetoacetic acid ester is added thereto. A method of adding and reacting, a method of using a cell lysate, a method of treating with acetone, freeze-drying or the like instead of viable cells, a method of immobilizing these on a carrier or the like, and the like can be applied. In this reaction, the yield is often improved by adding glucose, acetic acid, methanol, nitric acid, 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 of alcohol, acetone or the like is used. It is of course necessary to choose an organic solvent that is not harmful to microorganisms.

For the system, the acetoacetic acid ester is as it is,
Alternatively, it is added after being dissolved or dispersed in an organic solvent.
The concentration range of the ester in the system is usually 0.01 to 50% by weight. When it is less than 0.01% by weight, there is no disadvantage in terms of reaction but it is economically poor in practical use. Problems such as a decrease in yield tend to occur because the body is overloaded.

The reaction is pH 3 to 10, preferably pH 4 to 9.
At a temperature of 15 to 60 ° C., preferably 25 to 40 ° C.
~ 200 hours, preferably 3 to 120 hours, with stirring or standing. After completion of the reaction, (R) -3-hydroxybutyric acid and its ester can be obtained directly or by centrifugation from the reaction solution, and then by using a usual separation method such as extraction with an organic solvent such as ethyl acetate or hexane, distillation, or column chromatography. Can be easily obtained.

[0012]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. In the case of ethyl 3-hydroxybutyrate, the yields and optical purities in the examples are high performance liquid chromatography using a normal phase column (Fine Pack SILC; manufactured by JASCO) and an optical resolution column (Chiralcel OD; manufactured by Daicel Chemical Industries). It was measured by chromatography (mobile phase; n-hexane / 2-propanol = 29/1, detection 210 nm). In the case of 3-hydroxybutyric acid, the yield is high performance liquid chromatography using an anion exchange column (Ion Pack S-801; Showa Denko) (mobile phase: 1% phosphoric acid solution, detection 210 nm).
The optical purity was measured by high performance liquid chromatography (mobile phase; 2 mM copper sulfate / 10% acetonitrile aqueous solution, detection 254 nm) using an optical resolution column (ChiralPak MA; manufactured by Daicel Chemical Industries, Ltd.). The yield is expressed by the following formula.

Yield (%) = product concentration (M) / initial substrate concentration (M) × 100 product; 3-hydroxybutyric acid and ethyl 3-hydroxybutyrate substrate; ethyl acetoacetate Example 1 <Preparation of bacterial cells Culture medium 1> KNO ₃ 10.8 g / liter Na ₂ MoO ₄ · 2H ₂ O 4 mg / liter polypeptone 2 g / liter yeast extract 1 g / liter MgSO ₄ / 7H ₂ O 1 g / liter pH 7.0 gas phase; N ₂ Put 400 ml of the above-mentioned cell culture medium 1 in a 700 ml vial bottle, sterilize, and then paracoccus denitrificans IFO
13301 was inoculated and cultured at 28 ° C for 72 hours. After culturing,
The cells were separated by centrifugation and washed twice with a phosphate buffer to obtain viable cells. Ethyl acetoacetate in a 30 ml vial
15 ml of a reaction solution containing 100 mM and 10 g / liter of KNO ₃ was added, and the above-mentioned viable cells were suspended in this, and the reaction was carried out at 28 ° C. for 66 hours. After completion of the reaction, the mixture was extracted with 15 ml of ethyl acetate. This extract was subjected to high performance liquid chromatography to quantify (R) -3-hydroxybutyric acid and ethyl (R) -3-hydroxybutyrate produced and to measure optical purity. As a result, the yield was 33% and the optical purity was 92% ee.

[0014] Example 2 <Culture medium for preparation of microbial cells 2> KNO ₃ 10.8 g / l _{Na 2 MoO 4 · 2H 2 O} 4 mg / l polypeptone 2 g / l yeast extract 1 g / liter MgSO ₄ · 7H ₂ O 1 g / liter Glucose 4 g / liter pH 7.0 Gas phase; 40 ml of N ₂ cell preparation medium 2 was put into a 700 ml vial bottle and sterilized, and then Paracoccus denitrificans IFO 13301
Was inoculated and cultured at 28 ° C. for 72 hours. After the completion of the culture, cells were separated by centrifugation and washed twice with a phosphate buffer to obtain viable cells. 100 mM ethyl acetoacetate in a 30 ml vial
, 15 g of a reaction solution containing 10 g / liter of KNO ₃ was added, and the above-mentioned viable cells were suspended in the reaction solution and reacted at 28 ° C. for 24 hours.
After completion of the reaction, the mixture was extracted with 15 ml of ethyl acetate. This extract is
(R) -generated using high performance liquid chromatography
3-Hydroxybutyric acid and ethyl (R) -3-hydroxybutyrate were quantified and the optical purity was measured. As a result, the yield was 83% and the optical purity was 92% ee.

Example 3 The reaction was carried out in the same manner as in Example 2. The reaction time was 8 hours and 24 hours. The product was (R) -3-
Ethyl hydroxybutyrate 31 mM, (R) -3-hydroxybutyric acid 18 mM, and reaction 24 hours were ethyl (R) -3-hydroxybutyrate 25 mM and (R) -3-hydroxybutyric acid 48 mM.

[0016]

INDUSTRIAL APPLICABILITY According to the method of the present invention, Paracoccus (Paracocus)
The use of a strain belonging to the genus ccus makes it possible to produce (R) -3-hydroxybutyric acid and its ester with high optical purity in a high yield, which is extremely advantageous as an industrial production method. .

Front page continuation (72) Inventor Naoki Kawada 1-14-14 Sengen, Tsukuba

Claims (1)

[Claims] 1. Paracoccus (Pa
A process for producing (R) -3-hydroxybutyric acid and its ester, which comprises reacting a strain belonging to the genus racoccus) to collect (R) -3-hydroxybutyric acid and its ester.