JPH04360697A - Production of r-secondary alcohol - Google Patents

Abstract

PURPOSE:To obtain in an industrially advantageous way the title compound as a raw material for synthesizing optically active substances by oxidation, using Aureobasidium microorganisms, of only the D-modification of an optically inactive secondary alcohol into the corresponding ketone followed by collecting the remaining R-modification. CONSTITUTION:Microorganisms belonging to Aureobasidium capable of selectively oxidizing only the S-modification in the optically inactive racemic modification of a secondary alcohol of formula R1-CH(OH)-R2 (R1 is 1-4C aliphatic hydrocarbon which may have C=C bond; R2 is 3-12C divalent aliphatic hydrocarbon which may have C=C bond; the number of carbon atoms for R1 and R2 totaling 4-13)(e.g. n-pentan-2-ol) into the corresponding ketone [e.g. Aureobasidium sp. SM-25 (FERM P-11277)] are cultured in a medium containing said racemic modification followed by collecting or fractionating the R- modification of the secondary alcohol left in the reaction system, thus obtaining the objective R-secondary alcohol.

Description

[Detailed description of the invention]

0001

[Industrial Field of Application] The present invention is directed to the production of optically active R-isomer secondary alcohols by microorganisms.
The present invention relates to a method for producing grade alcohol.

0002

[Prior Art and Problems to be Solved by the Invention] A method for obtaining a ketone by oxidizing a secondary alcohol using microorganisms is well known, as exemplified in Japanese Patent Publication No. 48518/1983. There have been no known attempts to obtain optically active secondary alcohols using this method. The present invention provides a method for producing an optically active R-form secondary alcohol by reacting a racemic secondary alcohol with a microorganism capable of oxidizing only the S-form secondary alcohol into ketone. It is something to do.

0003

[Means for Solving the Problems] While carrying out detailed research on the properties of microorganisms that have the ability to assimilate fatty acids or their esters and convert them into the corresponding methyl ketones, the present inventors discovered that Aureobasidium (Aure
When certain microorganisms belonging to the genus Obasidium are allowed to act on racemic secondary alcohols, only the S-form secondary alcohols are specifically oxidized to ketones, and by removing the ketones from the system. , and completed the present invention by discovering that R-configured secondary alcohols can be obtained in high yield. That is, Aureobasidium (Aureobasidium)
belongs to the genus idium) and has the general formula R1-CH(OH)-
R2 (wherein R1 is an aliphatic hydrocarbon group having 1 to 4 carbon atoms which may have a carbon-carbon double bond, R2 is an aliphatic hydrocarbon group having 3 to 12 carbon atoms which may have a carbon-carbon double bond) is a divalent aliphatic hydrocarbon group, and the total number of carbon atoms of R1 and R2 is 4 or more and 13 or less). A microorganism having the ability to oxidize to This is a method for producing an R-form secondary alcohol, which comprises converting the S-form secondary alcohol into the ketone, and then collecting or fractionating the remaining R-form secondary alcohol.

One of the microorganisms having such properties is Aureobasidium sp.
ureobasidium sp. SM-25).
P-11277) and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.

[0005] The production of R-isomer secondary alcohol according to the present invention may be carried out by culturing the above-mentioned microorganism in a medium containing racemic secondary alcohol (hereinafter referred to as the first method). After culturing the microorganisms in a normal medium, the racemic form 2 is grown in an aqueous medium, either subsequently or by harvesting.
This may also be done by contacting with alcohol (
(hereinafter referred to as Law 2).

[0006] First, regarding the first method, these microorganisms have a general formula of R1-CH(OH)-R2 (wherein R1 is a carbon number 1 which may have a carbon-carbon double bond).
-4 aliphatic hydrocarbon group, R2 is a divalent aliphatic hydrocarbon group having 3 to 12 carbon atoms which may have a carbon-carbon double bond, and the total number of carbon atoms of R1 and R2 is 4 or more, 13
Culture is carried out under aerobic conditions while controlling temperature, pH, etc., in a medium containing a suitable amount of racemic secondary alcohol (as shown below), a nitrogen source, inorganic substances, micronutrients, etc. Other carbon sources that are commonly used for culturing filamentous fungi, such as glucose, sucrose, glycerin, dextrin, carboxymethyl cellulose, and kerosene, may also be used in combination. Nitrogen sources include ammonium sulfate, ammonium nitrate, ammonium tartrate, sodium nitrate, asparagine, peptone, corn steep liquor, etc. Inorganic substances and micronutrients include magnesium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, and sodium chloride. , manganese sulfate, zinc sulfate, copper sulfate, ferric chloride, ammonium molybdate, potassium iodide, boric acid, various vitamins (calcium pantothenate, inositol, pyridoxine, etc.), and the like are used. The appropriate concentration of the precursor in the medium is usually 1 to 100 grams/liter.

[0007] In the first method, the microorganisms are cultured in liquid culture under aerobic conditions, such as a shaking culture method or an aerated agitation culture method. Culture is usually carried out at a temperature of 20-35°C and a pH of 4.
．． It can be carried out at 0 to 7.5. The culture is preferably carried out until the stationary phase, and the culture time is usually 3 to 14 days. As a result, only the S-form in the racemic secondary alcohol is oxidized to a ketone, and the R-form secondary alcohol remains in the system.

Next, to discuss the second method, first, the microorganisms used in the present invention are treated with carbon sources, nitrogen sources, inorganic substances, etc.
Culture is carried out under aerobic conditions in a medium containing moderate amounts of micronutrients, etc. while controlling temperature, pH, etc. As the carbon source, nitrogen source, and inorganic substances/micronutrients, those normally used for culturing the above-mentioned filamentous fungi can be used as they are. The culture conditions are also the same as those in the first method, but the culture period is preferably from the latter half of the logarithmic growth phase to the stationary phase, and is usually about 3 to 7 days. The microorganism culture obtained in this way can be used as it is or by treating the culture in various ways to obtain a racemic 2nd form represented by chemical formula 1.
contact with grade alcohol. Examples of the processed product include concentrates of the culture, bacterial cells obtained by subjecting the culture to centrifugation, immobilized bacterial cells, crushed bacterial cells, enzyme preparations extracted from the bacterial cells, etc. can. The contact reaction can be carried out in any aqueous medium, but it is preferable to use a culture solution of the bacteria to be used or its concentrate, water after harvesting the bacteria, or a medium without a carbon source (other than the one in the previous stage except for the lack of a carbon source). The same medium used for culture may be used as a suspension.
A racemic secondary alcohol whose general formula is represented by Chemical Formula 1 is present, and the pH is adjusted to 4.0 to 7.5, more preferably 6.0 to 7.5, and at 20 to 32°C. By reacting with shaking or aeration for 7 days,
The R-form secondary alcohol can remain in the system. The concentration of the racemic secondary alcohol in the reaction solution is suitably 1 to 500 g/liter. Further, the second method can also be carried out by a bioreactor method using immobilized bacterial cells obtained by immobilizing the bacteria used in the present invention by a conventional method.

The racemic secondary alcohol suitably used in the present invention has a general formula of R1-CH(OH)-R2 (wherein R1 has a carbon number of 1 and may have a carbon-carbon double bond).
-4 aliphatic hydrocarbon group, R2 is a divalent aliphatic hydrocarbon group having 3 to 12 carbon atoms which may have a carbon-carbon double bond, and the total number of carbon atoms of R1 and R2 is 4 or more, 13
It is a racemic secondary alcohol represented by the following). In addition, as mentioned above, R1 in the formula is an aliphatic hydrocarbon group having 1 to 4 carbon atoms which may have a carbon-carbon double bond,
R2 has 3 to 1 carbon atoms and may have a carbon-carbon double bond
2 divalent aliphatic hydrocarbon group, and the total number of carbon atoms of R1 and R2 is 4 or more and 13 or less, both of which are linear or branched alkyl (alkydene) or alkenyl (alkenydene). , alkadienyl (alkadienidene), alkatrienyl (alkatrienidene), and the like.

[0010] In both the first method and the second method, the target product can be isolated and purified from the reaction-completed solution by a conventional method. That is, the target product is extracted from the conversion reaction completion liquid or its bacterial cell removal liquid with a solvent such as chloroform, hexane, petroleum ether, etc., and the extract is further subjected to liquid chromatography using silica gel, molecular distillation, etc. , the desired object can be obtained.

[0011]

Effects of the Invention According to the present invention, optically active R-isomer secondary alcohol is produced by using a microorganism whose productivity has not been previously known and using racemic secondary alcohol whose general formula is represented by chemical formula 1 as a raw material. Secondary alcohol can be produced. The secondary alcohol is expected to be used as a raw material for the synthesis of optically active substances.

0012

[Example] Example 1 Aureobasidium sp.sm-25 (A
ureobasidium sp. SM-25) 5
The cells were inoculated into 100 ml of basal medium (composition below) in a 00 ml Sakaguchi flask, and cultured with reciprocating shaking (120 rpm, 7 cm amplitude) at 28°C for 3 days. Composition of medium used (the following is made up to 100 ml with deionized water): Glucose
0.5 grams
dipotassium hydrogen phosphate
0.7 Sodium sulfate
0.2 Sodium nitrate
0.2 Potassium dihydrogen phosphate
0.2 Magnesium sulfate (
7 hydrate salt) 0.06 Sodium chloride
0.05 Calcium chloride (dihydrate)
0.01 Vitamin preservation solution
0.1
ml mineral preservation solution
0.1 1% chloramphenicol 0.2 Composition of vitamin preservation solution (milligrams/liter) Biotin
2 Calcium pantothenate
400 folic acid
2 inositol
2000 niacin
400 p-aminobenzoic acid 200 Pyridoxine hydrochloride 400
riboflavin
200 Thiamine Hydrochloride
Composition of 400 mineral preservation solution (milligrams/liter) Manganese sulfate (tetra-pentahydrate)
60 Zinc sulfate (heptahydrate)
300 Copper sulfate (pentahydrate)
40 Ferric chloride (hexahydrate salt)
250 boric acid
60 Ammonium molybdate (tetrahydrate) 25 Potassium iodide
100 milligrams of various racemic secondary alcohols were added to the medium after 100 min of pre-culture, and the culture was continued for an additional 24 hours under the same conditions as above. 2 of the cultured solution
The liquid extracted twice with 0 ml of chloroform was subjected to gas chromatography (column: 20% PPGS on Chromosorb WAW, 2 m glass column, column temperature: 7
0 to 170°C, heating rate 5°C/min Inlet and detector temperature: 230°C Detector: FID carrier gas (nitrogen) to measure the residual amount of 2-ol and the amount of 2-one produced. 2-ol was purified by thin layer chromatography, and then its specific rotation was measured. As a result, as shown in Table 1, using each racemic secondary alcohol as a substrate, R-form secondary alcohol was obtained in a high yield of 60 to 100% of the theoretical yield.

[Table 1] ──────────────────────────
──────────── 2-Types of all
Amount of remaining 2-ol (specific optical rotation)
Amount of 2-on produced──────────────────
──────────────────── (RS)
-n-pentan-2-ol 31 mg (-
13.2 degrees) 1 mg (RS
)-n-heptan-2-ol 47 (
-10.5) 10 (RS)-
n-nonan-2-ol 53 (-
8.0) 4──────
──────────────────────────
──────

Example 2 In the basic medium of Example 1, instead of glucose (RS)
- 100 ml of a medium with the same composition except that 0.5% of n-heptan-2-ol was added was placed in a 500 ml Sakaguchi flask, and Aureobasidium sp.
ium sp. SM-25) was inoculated and cultured at 28°C for 4 days with reciprocal shaking (120 rpm, amplitude 7 cm). The amounts of 2-ol and 2-one in the culture solution and the specific optical rotation of the former were measured in the same manner as in Example 1. As shown in Table 2, the R-form secondary alcohol was approximately 8% of the theoretical yield.
A high yield of 0% was obtained.

[Table 2]

Claims (1)

[Claims] Claim 1: Aureobasidium (Aureob)
belongs to the genus Acidium, and its general formula is R1-CH(OH
)-R2 (wherein, R1 is an aliphatic hydrocarbon group having 1 to 4 carbon atoms that may have a carbon-carbon double bond, and R2 is a 3-carbon group that may have a carbon-carbon double bond) -12 divalent aliphatic hydrocarbon groups, and the total number of carbon atoms of R1 and R2 is 4 or more and 13 or less A microorganism having the ability to oxidize into ketone is cultured in a medium containing the racemic secondary alcohol, or a culture of the microorganism or a treated product thereof is brought into contact with the racemic secondary alcohol in an aqueous medium. A method for producing an R-form secondary alcohol, which comprises converting the S-form secondary alcohol into the ketone, and then collecting or fractionating the remaining R-form secondary alcohol.