JPH045436B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a novel method for producing optically active alcohol, which is characterized by the action of 3α-hydroxysteroid dehydrogenase.

[Conventional technology]

Optically active alcohols are used in medicines, agricultural chemicals, synthetic intermediates for physiologically active substances, and ferroelectric materials. For example, D-carnitine, which is a derivative of ethyl 4-chloro-3(S)-hydroxybutanoate, has physiological activity that competitively inhibits carnitine acetyltransferase. Also, optically active R-(-)-2-octanol and 2-methyl-
4(S)-hydroxypentane can be used as a raw material for ferroelectric materials.

Conventionally, methods for converting ketone compounds into optically active alcohols include methods using chemical catalysts,
Alternatively, a method using a biocatalyst is known.
In methods using chemical catalysts, when reduced using NaBH ₄ or LiAlH ₄ , the optical yield is very low and a racemate is produced. In other words, this racemate is mixed with tartaric acid or D-
After optical resolution using an optical resolving agent such as mandelic acid, a complicated step must be taken to obtain an optically active alcohol. Recently, attempts have been made to obtain optically active alcohols using chiral catalysts with optically active ligands, but these efforts require reactions at extremely low temperatures, and chiral catalysts are expensive and difficult to regenerate. is becoming a problem.

On the other hand, methods using biocatalysts such as microorganisms, plants, and animals generally have the advantage of high optical yield. For example, ethyl 4-chloro-3(S)-hydroxybutanoate is
(1984), it has become clear that the enzymes are fermented and produced by various microorganisms.

[Problem to be solved by the invention]

At present, there are technical difficulties in synthesizing optically active alcohols using chemical catalysts. On the other hand, the method of fermentation production using microorganisms has the problem that it is not possible to use a large amount of raw materials because the growth of bacterial cells is inhibited by the raw materials or products. Another problem is that it takes time and effort to purify the product, as by-products must be removed when the product is collected from the fermentation liquid.

The purpose of the present invention is to solve these problems,
An object of the present invention is to provide a method for efficiently producing an optically active alcohol with few by-products.

[Means to solve the problem]

The present inventors have intensively investigated a method for producing optically active alcohol using enzymes derived from microorganisms, and have found that ketone compounds can be efficiently converted to optically active alcohol using 3α-hydroxysteroid dehydrogenase. They discovered this and completed the present invention.

That is, the present invention uses 3α-hydroxysteroid dehydrogenase to synthesize the general formula [1] (In the formula, R ₁ represents a methyl group or a halogen-substituted methyl group, and R ₂ represents an alkyl group having 1 to 5 carbon atoms),
Or general formula [2] (wherein n represents an integer of 1 to 10) is a method for producing an optically active alcohol, which is characterized by converting a ketone compound represented by the formula into an optically active alcohol.

The 3α-hydroxysteroid dehydrogenase (hereinafter abbreviated as 3α-HSDH) used in the present invention is Cellulomonas tourbata strain KE31
No.] is preferred, but is not limited thereto. This enzyme can be obtained in pure form by the purification method described in Japanese Patent Application No. 62-69598. Of course, this pure enzyme can be used, but semi-purified products obtained by ammonium sulfate fractionation or DEAE-Sepharose chromatography can also be used. When producing optically active alcohol using such an enzyme, for example, 0.01
~200mg PH5.5~8.0, preferably PH6.5~7.5
Dissolve in 1 ml of 0.1M phosphate buffer and add the starting ketone compound (e.g. ethyl 4-chloroacetoacetate, 2
-octanone, methyl isobutyl ketone, etc.) and reduced nicotinamide adenine dinucleotide (NADH) in an equimolar amount to the raw material. In this reaction, the added NADH is oxidized to nicotinamide adenine dinucleotide (NAD), so it is necessary to add an equimolar amount of NADH to the product. However, in order to effectively utilize expensive NADH, enzymes that convert NAD to NADH,
For example, glucose dehydrogenase (hereinafter abbreviated as GDH)
Amano Pharmaceutical Co., Ltd.), heat-stable glucose-6-phosphate dehydrogenase (Unitika Co., Ltd.), formate dehydrogenase (Amano Pharmaceutical Co., Ltd., Boehringer Co., Ltd., Merck Co., Ltd., etc.), or 3α-HSDH (derived from KE3 strain). If purified enzymes (manufactured by Sigma), etc. coexist, the amount will be 1/10 of the raw material.
The reaction can also be carried out simply by adding 1/10000 mole of NAD(H). The amount of ketone compound added is
It varies depending on the type, but about 10 to 10% of the enzyme weight
It is 1000 times more. These are added and the reaction is carried out at 15-40°C, preferably 25-35°C, with stirring for 1 hour to 1 week. Aerosol OT or Tween in the reaction solution
When a surfactant such as 80 is added, the reaction can be made more efficient. Furthermore, by adding an ion exchange resin such as DEAE-Sepharose or Duolite A561 to the reaction system, the reaction rate can be increased or the reaction can be continued while keeping the enzyme stable. Gas chromatography (PEG2 capillary column, 25 m,
50 → 150℃, temperature increase analysis at 10℃/min)
The reaction is stopped when almost all of the raw material is consumed, and the aqueous layer is separated by centrifugation and the product is collected. To increase the recovery rate, the product dissolved in water is extracted using a solvent such as ethyl acetate, hexane, diethyl ether, 1,2-dichloroethane, or chloroform. If a purified product is required, these products can be purified by distillation or the like to obtain the desired optically active alcohol.

〔Example〕

Example 1 Method for producing optically active ethyl 4-chloro-3(S)-hydroxybutanoate 0.1M phosphate buffer containing 0.2M Nacl (PH7.0)
1.5 g (8.33 mmol) glucose in 18 ml, 7
mg of 3α-HSDH (purified enzyme derived from strain KE31,
20U/mg protein), 2.5mg as NADH regenerating enzyme
GDH (40 U/mg protein) and 191 mg (0.25 mmol) of NADH were dissolved, and 1.2 g (7.29 mmol) of ethyl 4-chloroacetoacetate was added thereto, followed by reaction at 20° C. with thorough stirring. As the reaction progresses, the PH decreases, so adjust the PH with 1M _{Na2CO3 .}
I adjusted it to 7.0 and continued the reaction. After reacting for 3.5 hours, ethyl acetate (20 ml/twice) was added to the reaction solution to extract the product. After adding Na ₂ SO ₄ to the extract to remove moisture, a molecular sieve was further added for drying. Ethyl acetate present in the sample was removed under reduced pressure to obtain 750 mg (4.5 mmol) of ethyl 4-chloro-3-hydroxybutanoate. This is 3,5-
After derivatization with dinitrophenyl isocyanate (hereinafter abbreviated as DNPI), liquid chromatography (column: OA-2100, manufactured by Sumitomo Chemical, solvent: hexane/chloroform/ethanol = 50/15/
1, flow rate 1 ml/min). The 3-hydroxyne produced by the above enzymatic reaction contains 99.1% ethyl 4-chloro-3(S)-hydroxybutanoate and 0.9% ethyl 4-chloro-3(R)-hydroxybutanoate. Includes 3(S)
-Hydroxy form was preferentially produced. The yield of ethyl 4-chloro-3(S)-hydroxybutanoate is 62
It was %.

Example 2 Method for producing optically active ethyl 4-chloro-3(S)-hydroxybutanoate 3α-HSDH (purified enzyme similar to Example 1) was used as the enzyme for regenerating NADH, and methylisobutylcarbinol was used as the substrate for regenerating NADH. there was. 25μ
2.33 mg 3α− in 0.1 M phosphate buffer (PH7.0)
Add and dissolve HSDH and 25μ of 5mM NADH (0.125μmol), add 260μ methylisobutylcarbinol and 40μ (295μmol) of ethyl 4-chloroacetoacetate and incubate at 30°C with stirring.
I reacted. 66 hours after the start of the reaction, 40μ of 4-
Ethyl chloroacetoacetate, 40μ of ethyl 4-chloroacetoacetate, 260μ of methylisobutyl carbinol, and 10μ of phosphate buffer were added at 21 hours, and the reaction was continued for an additional 60 hours (total reaction time 81 hours). After the reaction was completed, analysis by gas chromatography revealed that 810 μmol of 3-hydroxy compound had been produced. After removing methyl isobutyl carbinol and methyl isobutyl ketone mixed in this solution at 40° C. under reduced pressure, the isomer purity was measured by measuring the degree of light exposure and liquid chromatography. The specific optical rotation when dissolved in chloroform is [α] ²⁵ ₅₈₉ = -20.63deg, which is 4-chloro-3
Ethyl (S)-hydroxybutanoate was preferentially produced. In addition, as a result of liquid chromatography analysis of the sample derivatized with DNPI, 99% of ethyl 4-chloro-33(S)-hydroxybutanoate and 1% of ethyl 4-chloro-3(R)-hydroxybutanoate were found. was being generated. The yield of ethyl 4-chloro-3(S)-hydroxybutanoate was 91%.

Example 3 Method for producing optically active ethyl 4-chloro-3(S)-hydroxybutanoate In order to increase the reaction rate and stability of the enzyme, 70
A reaction was carried out in the same manner as in Example 2 by adding mg of swollen DEAE-Sepharose. Gas chromatography analysis of the reaction completed solution revealed 860 μmol of 3
-Hydroxy forms were produced. After removing methyl isobutyl carbinol and methyl isobutyl ketone, the isomer purity was measured by optical rotation and liquid chromatography. The specific rotation in chloroform is [α] ²⁵ ₅₈₉ =-
20.71 degrees, and ethyl 4-chloro-3(S)-hydroxybutanoate was preferentially produced. Also
Analysis of the DNPI derivative by liquid chromatography revealed that 99.2% of ethyl 4-chloro-3(S)-hydroxybutanoate and 0.8% of ethyl 4-chloro-3(R)-hydroxybutanoate were produced. The yield of ethyl 4-chloro-3(S)-hydroxybutanoate was 97%.

Example 4 Production method of optically active R-(-)-2-octal 0.1M phosphate buffer containing 0.2M NaCl (PH7.0)
0.51 g (2.83 mmol) glucose per 1 ml, 1
mg of 3α-HSDH (purified enzyme from KE31 strain, 2.6 mg
of GDH and 10 mg of NADH (13 μmol) were dissolved, 0.1 ml (82 mg, 640 μmol) of 2-octanone was added thereto, and the reaction was carried out at 2°C. As the pH decreases with the reaction, constantly stir with a stirrer.
The reaction was carried out while adjusting the pH to 7.0 with 1M Na ₂ CO ₃ .
After 15 hours of reaction (1 mg of GDH and 6 mg of NADH were further added at 6 hours), 1,2-dichloroethane (1.5 mm x 2) was added to the reaction solution to extract the product. After centrifugation, the 1,2-dichloroethane layer (oil layer) was collected and analyzed using a gas chromatograph. 64mg (490μmol) in oil layer
2-octanol was produced. When the optical rotation of this sample was measured, the specific rotation [α] ²⁵ ₅₈₉ = -
It was 6.7 degrees, and the R form was preferentially produced. On the other hand, the specific optical rotation of pure R-(-)-2-octanol was [α] ²⁵ ₅₈₉ =-9.745 degrees. Next, 1.6 g of Na ₂ SO ₄ was added to the above-mentioned oil layer, and the mixture was stirred overnight, and then 0.3 ml was collected, and molecuura sieve was added thereto, and the mixture was left to stand for another night. To this solution, 33 mg of DNPI was added and stirred thoroughly, and 30 μm of dry pyridine was further added and stirred, and then left for 4 hours. When this derivative was analyzed by liquid chromatography, 84.5% of the 2-octanol produced was R-(-)-2.
-octanol and 15.5% is S-(+)-2-
It was hot with octanol. The yield of R-(-)-2-octanol was 67%.

Example 5 Method for producing optically active 2-methyl-4(S)-hydroxypentane 0.1M phosphate buffer containing 0.1M NaCl (PH7.0)
1.99 g (11.1 mmol) glucose in 2.8 ml,
2.6 mg of 3α-HSDH (purified enzyme derived from strain K31E), 2.7
mg of GDH and 20 mg of NADH (26 μmol) were dissolved, and 0.6 ml (480 mg, 4.97 mmol) of methyl isobutyl ketone was added thereto, and the reaction was carried out at 25° C. while adjusting the pH to 7.0. After 15 hours of reaction (in the middle of 6
2 mg of GDH and 12 mg of NADH were further added to the reaction mixture), 1,2-dichloroethane (2.5 ml x 2) was added to the reaction solution for extraction, and the mixture was centrifuged to form a 1,2-dichloroethane layer (oil layer). was recovered.
When this was analyzed using a gas chromatograph, 215
mg of methylisobutylcarbinol was produced. When the optical rotation of this sample was measured, it was found to be specific optical rotation [α] ²⁵ ₅₈₉ = +0.127 deg. In addition, after derivatization with DNPI, by liquid chromatography,
As a result of measuring the purity of the isomer, 2-methyl-4
It contained 62.4% (S)-hydroxypentane and 37.6% 2-methyl-4(R)-hydroxypentane, and the 4(S)-hydroxy form was preferentially produced. The yield of 2-methyl-4(S)-hydroxypentane was 27.8%.

〔Effect of the invention〕

According to the method of the present invention, there are few by-products and the method is effective.
Optically active alcohols can be produced.

Claims (1)

[Claims] Using 13α-hydroxysteroid dehydrogenase, the general formula [1] (In the formula, R ₁ represents a methyl group or a halogen-substituted methyl group, and R ₂ represents an alkyl group having 1 to 5 carbon atoms), or general formula [2] (In the formula, n represents an integer of 1 to 10.) A method for producing an optically active alcohol, which comprises converting a ketone compound represented by the following into an optically active alcohol. 2. The production method according to claim 1, wherein the 23α-hydroxysteroid dehydrogenase is an enzyme derived from the genus Cellulomonas.