JPH01277494A - Production of optical active alcohol - Google Patents

Abstract

PURPOSE:To produce an optically active alcohol in high efficiency, with little production of by-product, by reacting a ketone compound with a 3alpha- hydroxysteroid dehydrogenase (HSDH). CONSTITUTION:HSDH is prepared by purifying an enzyme originated from Cellulomonas turbata KE31 strain. An enzyme liquid is produced by dissolving 0.01-200mg of the HSDH in 1ml of a 0.1M phosphate buffer solution having pH of 5.5-8. The enzyme liquid is added with a ketone compound (e.g. 4- chloroaceto-acetic acid ethyl ester) of an amount corresponding to 10-1,000 times the weight of the enzyme and with a reduced nicotinamide adenine dinucleotide of an amount equimolar to the ketone compound used as a raw material and optionally Tween 80, etc., and the components are made to react at 15-40 deg.C for 1hr-7 days under agitation. The progress of the reaction is traced by gas chromatography and the reaction is terminated when the raw material is disappeared. The obtained reaction liquid is subjected to centrifugal separation, extraction and purification to recover an optically active alcohol.

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.

[Prior Art] Optically active alcohols are used in pharmaceuticals, agricultural chemicals, synthetic intermediates for physiologically active substances, and ferroelectric +A materials. For example, D-carnitine, which is a derivative of ethyl 4-chloro-3(S)-hydroxybutanoate, has physiological activity that competitively inhibits carnitine acetyltransferase. Furthermore, optically active R-(-)-2-octatool and 2-methyl-4(S)-hydroxypenkune can be used as raw materials for ferroelectric materials.

Conventionally, methods using chemical catalysts or methods using biocatalysts are known as methods for converting ketone compounds into optically active alcohols.

In the method using a chemical catalyst, when reduction is performed using NaBH4 or LiAllI4, the optical yield is very low and a racemate is produced. That is, it is necessary to take the complicated steps of optically resolving this racemate using an optical resolving agent such as tartaric acid or D-mandelic acid, and then obtaining an optically active alcohol. Recently, attempts have been made to obtain optically active alcohols using chiral catalysts with optically active ligands, but the reaction must be carried out at extremely low temperatures, and chiral catalysts are expensive and difficult to regenerate. The problem is that

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 clearly produced by fermentation by various microorganisms, as described in "Annual New York, Academic Science Vol. 434, 186493 (1984) 1. It has become.

(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, fermentation production using microorganisms There is a problem in 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.Also, when collecting the products from the fermentation liquid, by-products must be removed, making purification time-consuming. There is a problem.

An object of the present invention is to solve these problems and provide a method for efficiently producing optically active alcohol with fewer by-products.

[Means for Solving the Problems] 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 produced using 3α-hydroxysteroid dehydrogenase. The present invention was completed based on the discovery that the compound can be converted into an optically active alcohol.

That is, the present invention is a method for producing an optically active alcohol, which is characterized by converting a ηton compound into an optically active alcohol using 3α-hydroxysteroid dehydrogenase.

The 3α-hydroxysteroid dehydrogenase (hereinafter abbreviated as 3α-HS D I+) used in the present invention is preferably derived from Cellulomonas turbata strain KE31 [Feikoken Bibori No. 9059], 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-finished products obtained by ammonium sulfate fractionation or DEAE-Sepharose chromatography can also be used. When producing an optically active alcohol using such an enzyme, for example, 0.01 to 200 mg of the purified enzyme is mixed at pH 5.5 to a, 6,
Preferably pH 6.5-7.5 (7) Dissolve in 0.1N phosphate buffer 1-, and add the starting ketone compound (e.g. 4-
(chloroacetic acid 1 to ethyl acetate, 2-octanone, methyl isobutyl ketone, etc.) and reduced nicotinamide adenine dinucleotide (NADH) in an equimolar amount to the raw material are added. 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, N
Enzymes that convert AD to NADH, such as glucose dehydrogenase (hereinafter abbreviated as GDH, manufactured by Amano Pharmaceutical Co., Ltd.), heat-stable glucose-6-phosphate dehydrogenase (manufactured by Uniaca Co., Ltd.), and formate dehydrogenase [Amano Pharmaceutical Co., Ltd.] Pharmaceutical, Heringer, Merck, etc.), or 3α-H3DII (KE3
If a purified enzyme derived from a single strain (manufactured by Sigma) is coexisting, NAD (1(
) can also be reacted by simply adding. The amount of ketone compound added varies depending on the type, but is about 10 to 1000 times the weight of the enzyme. Add these, 15~
The reaction is carried out at 40° C., preferably from 25 to 35° C., with stirring for 1 hour to 1 week. When a surfactant such as Aerosol OT or Tween 80 is added to the reaction solution, the reaction can be carried out more efficiently. Furthermore, by adding an ion exchange resin such as DEAE-Sepharose or Duolite 8561 to the reaction system, the reaction rate can be increased or the reaction can be continued while keeping the enzyme stable.

Gas chromatography (PEG2
0M capillary column, 25m, 50→150'
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 NaC (pH
7,0) 1.5 in 18m1. (8,33 mmol) of glucose, 7 mg of 3a-H3DI (KE3
Purified enzyme derived from one strain, 200/mg protein), 2.5 mg of GDH (40 U/mg protein) as an enzyme for NADH regeneration, and 191+++ g (0.25 mmol) of NA
Dissolve DH and add 1.2 g (7.29 mmol) to it.
Ethyl 4-chloroacetoacetate was added thereto, and the mixture was reacted at 20°C with good stirring. As the reaction progressed, the pH decreased, so the pH was adjusted to 7.0 with IM NazCO, and the reaction was continued. After reacting for 3.5 hours, ethyl acetate (20ml x 2) was added to the reaction solution to extract the product. After adding Na25O to the extract to remove moisture, molecular sieves were further added for drying.

Ethyl acetate mixed in the sample was removed under reduced pressure, and 750 m
g (4.5 mmol) of ethyl 4-chloro-3-hydroxybutanoate was obtained. This was derivatized with 3,5-dani-1-lophenylisocyanate-1 (hereinafter abbreviated as DNPI) and then subjected to liquid chromatography (column: 0A-2).
100, manufactured by Sumitomo Chemical, solvent: hexane/chloroform/
Ethanol-50/15/1, flow rate lIn1/m1n
) was analyzed. 99.1% of 4-chloro-3 is present in the 3-hydroxyne body produced by the above-mentioned enzymatic reaction.
It contained ethyl (S)-hydroxybutanoate and 0.9% of ethyl 4-chloro-3(R)-hydroxybutanoate, and the 3(S)-hydroxy form was preferentially produced. 4
The yield of ethyl -chloro-3(S)-hydroxybutanoate was 62%.

Example 2. Method for producing optically active ethyl 4-chloro-3(S)-hydroxybutanoate 3α-113DII (purified enzyme similar to Example 1) was used as the enzyme for regenerating NADH, and methylisobutylcarbinol was used as the substrate for regenerating NADH. 25μ! 0.
2.33 mg of 3 in 1M phosphate buffer (pH 7,0)
α-IIsDII and 25μ! of 5mM N.A.D.
Add H (0.125 umol)? Understand, 260
μl of methyl isophyl carbinol and 40 μf
fi (295 μmol) of ethyl 4-chloroacetoacetate was added and reacted at 30° C. with stirring. 6 hours after the start of the reaction, 40 μl of ethyl 4-chloroacetoacetate, 2
40ul for the 11th hour! , of ethyl 4-chloroacetoacetate and 260 μl of methylisobutylcarbinol,
10 μl of phosphate buffer was added and the reaction 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 optical rotation measurement and liquid chromatography. The specific rotation when dissolved in chloroform is [α] 5N-2
0.63 deH, and ethyl 4-chloro-3(S)-hydroxybutanoate was preferentially produced. Also DNP
Analysis of the sample derivatized with I by liquid chromatography revealed that 99% of ethyl 4-chloro-3(S)-hydroxybutanoate and 1% of ethyl 4-chloro-3(R)-hydroxybutanoate were present. It was generating. 4-chloro-3(s
)-Ethyl hydroxybutanoate yield was 91%.

Example 3. Preparation of optically active ethyl 4-chloro-3(S)-hydroxybutanoate The reaction was carried out in the same manner as in Example 2, with the addition of 70 mg of swollen DEAE-Sepharose to increase the reaction rate and stability of the enzyme. I did it. Gas chromatography analysis of the reaction-completed solution revealed that 860 μmol of 3-hydroxy compound had been 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 chloroporum is [α] sH=
It was 20.71 deg, and ethyl 4-chloro-3(S)-hydroxybutanoate was preferentially produced. Furthermore, as a result of liquid chromatography analysis of DNPI derivatives,
99.2% of ethyl 4-chloro-3(S)-hydroxybutanoate and 0.8% of ethyl 4-chloro-3(R)-hydroxybutanoate were produced. 4-chloro-3(S
)-Ethyl hydroxybutanoate yield was 97%.

Example 4. Production method of optically active R-(-)-2-octatool 0.1M phosphate buffer containing 0.2M NaCl (pH
7,0) 0.51 g (2,83 mmol) of glucose in 1 ml, 1 g of 3 α-) ISDH (KE31
Purified enzyme from strain, 2.6 mg G D H and 1
Dissolve 0 ■ NADH (13 μmol) and add 0.1
ml (82 mg, 640 μmol) of 2-octanone was added and the reaction was carried out at 25°C. pH along with reaction
Since the Na
The reaction was carried out while adjusting the pH to 7.0 with zCO:+. 1
After 5 hours of reaction (1 mg of CI H and 6 μm of NADH were further added at 6 hours), 1.
The product was extracted by adding 2-dichloroethane (1.5 mm x 2). 1,2-dichloroethane layer after centrifugation,
(oil layer) was collected and analyzed using a gas chromatograph. 64 mg (490 μmol) of 2-octatool was produced in the oil layer. When the optical rotation of this sample was measured, the specific optical rotation [α]5H = -6.7 deg, 9
The body was producing it preferentially.

On the other hand, the specific optical rotation of pure R-(-)-2-octatool was [α]sH=9.745 deg. Next, 1.6 g of Na2SO4 was added to the above oil layer, stirred overnight, and after one more time, 0.3 m! The sample was collected, added with molecular sieve, and left for one night. To this solution, 3 mg of 1) NPI was added and stirred well, and then 30 μl of dry pyridine was added and stirred, and then left for 4 hours. When this derivative was analyzed by liquid chromatography, 84.5% of the 2-octatool produced was R-(-)-2-octatool, and 15.5% was S-(+)-2- It was an octatool. The yield of R-(-)-2-octatool is 67
%Met.

Example 5. Preparation method of optically active 2-methyl-4(S)-hydroxypenkune 0, IM 0.1M phosphate buffer containing NaCl (pH
7,0) 1.99 g (11,1 mmol) of glucose in 2,8 ml, 2.6 mg of 3α-H3Dtl (
K31E strain (purified enzyme), 2.7 mg of GDH and 20+ng of NADH (26 μmol) were dissolved, and 0.6 ml (480 mg, 4.97 mmol) of methyl isobutyl ketone was added to adjust the pH to 7.0. The reaction was then carried out at 25°C. After 15 hours of reaction (2 mg of G D H and 12n + g of N at 6 hours)
1,2-dichloroethane layer (oil layer) was collected by centrifugation. When this was analyzed with a gas chromatograph, it was 215 m.
g of methylisobutylcarbinol was produced. When the optical rotation of this sample was measured, the specific rotation [α] s
H=+0.127deg. After derivatization with DNPI, the purity of the isomer was measured by liquid chromatography. As a result, 2-methyl-4(S)-hydroxypenkune was 62.4%, 2-methyl-4(R)-hydroxy Penkun contains 37.6%, 4 (S)
-Hydroxy form was preferentially produced. 2-methyl-
The yield of 4(S)-hydroxypenkune was 27.8%.

〔Effect of the invention〕

According to the method of the present invention, optically active alcohol can be efficiently produced with few by-products.

Claims (2)

[Claims] (1) A method for producing an optically active alcohol, which comprises converting a ketone compound into an optically active alcohol using 3α-hydroxysteroid dehydrogenase. (2) The production method according to claim 1, wherein the 3α-hydroxysteroid dehydrogenase is an enzyme derived from the genus Cellulomonas.