JP3055711B2 - Method for producing optically active (S) -3-phenyl-1,3-propanediol - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an optically active (S) -3-.
The present invention relates to a method for producing phenyl-1,3-propanediol. More specifically, a microorganism or a processed product thereof is allowed to act on an enantiomeric mixture of 3-phenyl-1,3-propanediol to obtain an optically active 3-phenyl-1,3-propanediol.
The present invention relates to a method for producing optically active (S) -3-phenyl-1,3-propanediol, which comprises collecting 3-propanediol.

Optically active (S) -3-phenyl-1,3
-Propanediol is an important synthetic intermediate for various pharmaceuticals.

[0003]

2. Description of the Related Art
As a method for producing optically active (S) -3-phenyl-1,3-propanediol, a regioselective chemical reduction method of 2.3-epoxycinnamyl alcohol (J. Org. Chem., 53) (17), 4081
(1988)) and a method by chemical reduction of optically active 3-phenyl-3-hydroxypropionic acid (Tetr
ahedron Lett. , 26 (3), 351 (1
985). However, the former method has poor regioselectivity and poor chemical purity,
The latter method has disadvantages such as the necessity of resolving the optically active organic acid with an optical resolving agent, and the low optical purity of the obtained product. Under these circumstances, there is a demand for a method of obtaining optically active (S) -3-phenyl-1,3-propanediol having high optical purity by economical and simple means.

[0004]

Means for Solving the Problems The present inventors have developed an optically active (S)-having a high optical purity by an economically excellent and simple method.
As a method of obtaining 3-phenyl-1,3-propanediol, a method of causing microorganisms to act was searched for microorganisms suitable for this purpose with respect to soil-isolated bacteria. As a result, Serratia genus or Pseudomonas (Pseu) was obtained.
A microorganism selected from the group of microorganisms belonging to the genus (domonas) acts on an enantiomeric mixture of 3-phenyl-1,3-propanediol to produce (S) -3-phenyl-
It has been found that 1,3-propanediol can be obtained, and the present invention has been completed. The microorganism used in the present invention is a microorganism belonging to the genus Serratia or the genus Pseudomonas.
Any microorganism can be used as long as it acts on an enantiomeric mixture of -phenyl-1,3-propanediol and has the ability to obtain (S) -3-phenyl-1,3-propanediol.

Specifically, a microorganism strain having the following properties, which is isolated from soil according to the present invention, namely Serratia sp. 2664 strains (Serratia
sp. No. 2664) Or Serratia SP N
o. 2666 strain (Serratia sp. No. 26)
66) and Pseudomonas putida no. 2145B
(Pseudomonas Putida No. 21
45B) is preferred.

[0006] These microbial strains are Serratia sp. No. 2664 strain is a micro-organism bacterium No. 12268 (F
ERM P-12268) as Serratia SP No. The 2666 strain is a micro-organisms bacterium No. 12269 (FE
RM P-12269) as Pseudomonas putida No. The strain 2145B has been deposited with the Research Institute of Microbial Industry as No. 12270 (FERM P-12270). The bacteriological properties and identification results of these strains are as follows. Serratia SP No. 2664 strain (a) Morphology (1) Cell shape and size Bacillus 0.5-0.7μ × 1.2-3.0μ (2) Motility Available (3) Presence or absence of spores No (4) Gram stainability Negative (b) Physiological properties (1) Oxidase negative (2) Catalase positive (3) Aminopeptidase positive (4) Indole formation negative (5) VP test positive (6) Denitrification reaction positive (7) Nitrate reduction positive (8) Utilization of citric acid (Simons') positive (9) Urease positive (10) Phenylalanine deaminase negative (11) Malonic acid utilization negative (12) Levan production from sucrose negative (13) Lecithinase positive (14) Starch (15) Gelatin hydrolysis positive (16) Casein hydrolysis positive (17) DNA hydrolysis positive (18) Tween80 hydrolysis positive (19) Extrin hydrolysis positive (20) 3% KOH lysis positive (21) Attitude to oxygen Facultative anaerobic (22) Growing at 37 ° C (23) Growing at 41 ° C (24) pH5. 6) (25) Growing on Mac-Conkey-Agar medium (26) Growing on SS-Agar medium (27) Generation of pigment None (28) OF test F (29) Generation of gas from glucose- (30) Generation of acid from sugar Glucose + fructose + xylose + rhamnose-sucrose + L-arabinose + melibiose + trehalose + lactose-raffinose-mannose + maltose + cellobiose-melezitol-adnitol + inositol + mannitol + mannitol + mannitol + mannitol -Salicin + g Serine + (31) ONPG (β-galactosidase) positive (32) arginine dihydrolase negative (33) lysine decarboxylase positive (34) ornithine decarboxylase positive (35) availability of carbon compounds adipic acid-caproic acid + citric acid + Hippuric acid-maleic acid + phenylacetic acid + glucose + mannose + maltose + raffinose-cellobiose-adonitol + mannitol + nicotinic acid.
´s Manualof Systematic B
acteriology (1986)], the strain was determined to belong to the genus Serratia. Therefore, this strain was designated Serratia sp. 2664 (S
erratia sp. No. 2664).

[0007] Serratia sp. 2666 strain (a) Morphology (1) Cell shape and size Bacillus 0.5-0.7μ × 1.2-3.0μ (2) Motility Yes (3) Presence or absence of spores No (4) Gram stainability Negative (b) Physiological properties (1) Oxidase negative (2) Catalase positive (3) Aminopeptidase positive (4) Indole formation negative (5) VP test positive (6) Denitrification reaction positive (7) Nitrate reduction positive (8) Utilization of citric acid (Simons') positive (9) Urease negative (10) Phenylalanine deaminase negative (11) Malonic acid utilization negative (12) Levan production from sucrose negative (13) Lecithinase positive (14) Starch (15) Gelatin hydrolysis positive (16) Casein hydrolysis positive (17) DNA hydrolysis positive (18) Tween80 hydrolysis positive (19) Positive hydrolysis of exulin (20) Positive hydrolysis of 3% KOH (21) Attitude to oxygen Facultative anaerobic (22) Growing at 37 ° C (23) Growing at 41 ° C (24) pH5 (25) Growing on Mac-Conkey-Agar medium (26) Growing on SS-Agar medium (27) Pigment formation None (28) OF test F (29) Gas from glucose Production-(30) Production of acid from sugar Glucose + fructose + xylose + rhamnose-sucrose + L-arabinose + melibiose + trehalose + lactose-raffinose-mannose + maltose + melezitol-adnitol + inositol-lysitol + solitol + itolitol − Salicin + glycerin + (3 1) ONPG (β-galactosidase) positive (32) Arginine dihydrolase negative (33) Lysine decarboxylase positive (34) Ornithine decarboxylase positive (35) Availability of carbon compounds adipic acid-caproic acid + citric acid + hippuric acid- Maleic acid + phenylacetic acid + glucose + mannose + maltose + raffinose-cellobiose-adonitol + mannitol + nicotinic acid +
'S Mannualof Sistematic B
acteriology (1986)], the strain was determined to belong to the genus Serratia. Therefore, this strain was designated Serratia sp. 2666 (S
erratia sp. No. 2666).

[0008] Pseudomonas putida no. 2145
B strain (a) Morphology (1) Cell shape and size 0.5-0.8μ × 1.5-3.0μ (2) Motility available (3) Presence or absence of spores None (4) Gram stain negative (5) flagella polar flagella (1 or more) (b) physiological properties (1) oxidase positive (2) catalase positive (3) aminopeptidase positive (4) indole formation negative (5) VP test negative (6) denitrification Reaction Negative (7) Nitrate reduction Negative (8) Urease negative (9) Phenylalanine deaminase negative (10) Levan formation from sucrose negative (11) Lecithinase negative (12) Starch hydrolysis negative (13) Gelatin hydrolysis Negative (14) Casein hydrolysis Negative (15) DNA hydrolysis Negative (16) Tween 80 hydrolysis Negative (17) Extrin hydrolysis negative (18) Tyrosine Negative (19) Hydrolysis of 3% KOH Positive (20) Attitude to oxygen Aerobic (21) Growing at 37 ° C (22) No growing at 41 ° C (23) Growing at pH 5.6 (24) Growing on Mac-Conkey-Agar medium (25) Growing on SS-Agar medium (26) Growing on Cetrimid-Agar medium (27) Pigment generation Yes (28) OF test O (29) From glucose Gas generation-(30) Generation of acid from sugar Glucose + fructose + xylose + (31) ONPG (β-galactosidase) negative (32) Arginine dihydrolase positive (33) Growth factor requirement negative (34) Use of carbon compounds Acetic acid + glucose + adipic acid-mannose + caproic acid + maltose + citric acid + xylose-citraconic acid-mannitol + Glycolic acid + sorbitol-levulinic acid-gluconic acid + maleic acid + 2-ketogluconic acid + malonic acid + N-acetylglucosamine + D / L mandelic acid-glycine-phenylacetic acid + D-tryptophan-sebacic acid-L-tryptophan- L-tartaric acid + hippocampus-L-arabinose + benzoylformic acid-trehalose-benzylamine + fructose +
'S Mannualof Sistematic B
acteriology (1986)], the strain is fixed as belonging to Pseudomonas putida, and the strain is Pseudomonas putida No. 21
45B (Pseudomonas Putida N)
o. 2145B).

Any of these microorganisms can be suitably used, such as a wild-type strain, a heterozygous strain, or a recombinant strain derived by a genetic technique such as cell fusion or genetic engineering.

[0010] The medium for culturing the microorganism used in the present invention is not particularly limited as long as the microorganism can grow. For example, as the carbon source, any of the above microorganisms can be used, and specifically, glucose, fructose, sucrose, saccharides such as dextrin,
Alcohols such as sorbitol, ethanol and glycerol, organic acids and salts thereof such as fumaric acid, citric acid, acetic acid and propionic acid, hydrocarbons such as paraffin and mixtures thereof can be used. Examples of the nitrogen source include ammonium salts of inorganic acids such as ammonium chloride, ammonium sulfate and ammonium phosphate, ammonium salts of organic acids such as ammonium fumarate and ammonium citrate, meat extract, yeast extract, corn steep liquor, casein hydrolysate. A decomposition product, an inorganic organic nitrogen-containing compound such as urea, or a mixture thereof can be used. In addition, nutrients used in ordinary culture, such as inorganic salts, trace metal salts, and vitamins, can be appropriately mixed and used. If necessary, a factor that promotes the growth of the microorganism, a factor that enhances the ability to produce the target compound of the present invention, or a substance that is effective in maintaining the pH of the medium can be added.

[0011] As a culture method, the pH of the medium is from 3.0 to 9.0.
5, preferably 4 to 8 and a culture temperature of 20 to 45 ° C, preferably 25 to 37 ° C, under anaerobic or aerobic conditions suitable for growth of the microorganism for 5 to 120 hours, preferably 12 to Incubate for about 72 hours. 3-phenyl-1,3
As a method for producing optically active (S) -3-phenyl-1,3-propanediol from a mixture of enantiomers of propanediol, 3-phenyl-1,3-propanediol is used in a culture medium as it is. The cells were separated by a centrifugal separator or the like, and the resulting cells were directly or washed, and then resuspended in a buffer solution, water, or the like, to give 3-phenyl-1,3-propanediol. There is a method in which an enantiomeric mixture is added and reacted. The cells may be live cells, or may be crushed cells, treated with acetone, freeze-dried, or the like. In addition, these cells or treated cells are immobilized by a known method such as a polyacrylamide gel method, a sulfur-containing polysaccharide gel method (such as a carrageenan gel method), an alginic acid gel method, and an agar gel method. You can also. Further, an enzyme purified and obtained from a treated product of the cells by a combination of known methods can also be used. 3-phenyl-1,3
-The enantiomeric mixture of propanediol can be used as it is, or dissolved in water, or dissolved in an organic solvent that does not affect the reaction, or dispersed in a surfactant, etc. May be added.

The reaction is carried out at pH 3 to 10, preferably at pH 5
9, the temperature is 10-60 ° C, preferably 20-4 ° C.
The reaction is carried out at 0 ° C. for about 1 to 120 hours under stirring or standing. When the reaction time is extended, 3-phenyl-
Although the residual amount of 1,3-propanediol is often reduced, optically active 3-phenyl-1,3-phenyl with high optical purity is obtained.
It is possible to obtain propanediol. The concentration of the substrate used is not particularly limited, but is preferably about 0.1 to 20%.

The optically active 3-phenyl-1,3-propanediol remaining by the reaction can be collected by a usual production method such as extraction with an organic solvent, distillation, column chromatography, etc., directly from the reaction solution or after separation of cells. It is easily obtained if used.

[0014]

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

In the examples, the quantification and optical purity of 3-phenyl-1,3-propanediol in the reaction solution were determined based on the optical activity (S) -3-phenyl- obtained by the reaction.
High-performance liquid chromatography using 1,3-propanediol directly using an optical resolution column (column: Chiral Cell OB manufactured by Daicel Chemical Industries, solvent: n-hexane / 2-propanol = 19/1, wavelength 254 nm, flow rate 1.0 m)
1 / min, column temperature 40 ° C., injection volume 10 ul). (Retention time; (S) body 13.1 minutes, (R) body 1
(6.4 minutes)

[0016]

Example 1 500 ml of a medium containing 1% glucose, 0.5% yeast extract, 0.3% polypeptone, 0.2% ammonium sulfate, and 0.05% magnesium sulfate heptahydrate (pH 7) was placed in a 2 L Sakaguchi flask. And heat sterilized. Serratia sp. 2664 and inoculated at 30 ° C for 2 hours.
The cells were cultured with shaking for 4 hours. Thereafter, the cells were separated by centrifugation and washed with physiological saline. The cells were suspended in water to make up to 95 ml, placed in a 500 ml Sakaguchi flask, and then 5 ml of a 20% (W / V) aqueous solution of racemic 3-phenyl-1,3-propanediol was added.
A reciprocal shaking reaction was performed at 0 ° C. for 72 hours. After completion of the reaction, the bacteria were removed by a centrifugal separator, and the obtained supernatant was added to ethyl acetate 10%.
0 ml was added and extraction was performed twice. The organic layers were combined, dehydrated with anhydrous sodium sulfate, and desolvated to obtain a syrup.

This syrup was dissolved in 10 ml of a hexane / isopropyl alcohol (50:50) solution, and the yield, optical purity and absolute configuration were analyzed by high performance liquid chromatography. As a result, the yield was 37%, the absolute configuration was S-form, and the optical purity was 96% e. e. Met.

[0018]

Example 2 The strain used was Serratia sp. 2
Culture, reaction and purification were carried out in exactly the same manner as in Example 1 except that the product was changed to 666, and the product was analyzed.

As a result, the yield was 40% and the absolute configuration was S
Body, optical purity 98% e. e. Met.

[0020]

Example 3 The strain used was changed to Pseudomonas putida N
o. Culture, reaction, and purification were performed in exactly the same manner as in Example except that the product was changed to 2145B, and the product was analyzed.

As a result, the yield was 38% and the absolute configuration was S
Body, optical purity 90% e. e. Met.

[0022]

The process for producing optically active (S) -3-phenyl-1,3-propanediol using the microorganism according to the present invention is simple and convenient. This makes it possible to produce propanediol, which is extremely advantageous industrially.

────────────────────────────────────────────────── ─── Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) C12P 41/00 BIOSIS (DIALOG) CA (STN)

Claims (1)

(57) [Claims] 1. An enantiomeric mixture of 3-phenyl-1,3-propanediol is added to Serratia (Serrat).
ia) genus or Pseudomona
s) An optically active (S) -3-phenyl, which is obtained by allowing a microorganism belonging to the genus or a processed product thereof to act thereon and collecting the obtained optically active (S) -3-phenyl-1,3-propanediol. A method for producing -1,3-propanediol.