JP2818461B2 - Method for producing optically active 1,3-butanediol - Google Patents

Description

The present invention relates to a method for producing optically active 1,3-butanediol.

Optically active 1,3-butanediol is an important raw material for various pharmaceuticals such as antibiotics and optically active physiologically active substances.

[Problems to be solved by conventional technology and invention]

Conventionally, methods for producing optically active 1,3-butanediol include (1) a method of producing a chemically synthesized racemic 1,3-butanediol.
A method of optically resolving butanediol using an optical resolving agent (JP-A-61-191631), (2) 2-hydroxy-butanediol using a Raney nickel catalyst treated with an optically active compound.
Method for obtaining from 4-butanone by an asymmetric synthesis reaction
58-204187 and Bull.Chem. Soc. Jpn., 53, 1356-1360.
(1980)) and the like, but both (1) and (2) require the use of expensive optical resolving agents and catalysts, and (2)
Has a drawback such as low optical purity, and is economically excellent, and has a high optical purity by a simple means.
It has been desired to establish a method for obtaining butanediol.

[Means for solving the problem]

The present invention provides a simple method and high optical purity optical activity
Focusing on the asymmetric reduction method using microorganisms as a method for obtaining 1,3-butanediol, and searching for microorganisms suitable for this purpose, we found that the optical activity by asymmetric reduction method using certain microorganisms
A method for producing 1,3-butanediol has been found, and a patent application has already been filed (Japanese Patent Application No. 1-107017).

The present inventors have further studied the production method of optically active 1,3-butanediol by an asymmetric reduction method, and as a result, Agrobacterium genus, Azotobacter genus, Bordetella genus,
Brettanomyces genus, Deckera (Dek
genus kera, genus Endomyces, genus Eremascus, genus Erwinia, genus Fusarium, Geotrichum
Genus, Gibberella, Glomerel
la), Gonatobotryum, Klebsiella, Micrococ
cus), Mycobacterium, Neosartorya, Oospora
Genus, Pachysolen, Paecilomyces (Paec
ilomyrces, Paracoccus, Preussia, Saccharomy
ces), Saccharomycopsis
Genus, Serratia, Syringospora
ospora, Spetoria, Sporopachydermia, Talaromise (Tala)
romyces), Westerdykella
Genus, Zygoascus, Zygozym
a) a microorganism belonging to the genus asymmetrically reducing 4-hydroxy-2-butanone to produce (R) -1,3-butanediol; and acycloconidium (Aciculoconidiu).
m) genera, genus Brettanomyces, genus Clavispora, Cochliobol
genus, Corynespora genus, Dactylium genus, Echinopodospo
ra), Enterobacter, Hamigera, Helminthosporium
porium, genus Kloeckera, genus Nectria, genus Pseudomonas, genus Philalocephala, rhodotorra
dotorula), Saccharomyces
Microorganisms belonging to the genera, genus Sterigmatomyces and genus Schizoblastosporion asymmetrically reduce 4-hydroxy-2-butanone to produce (S) -1,3-butanediol Microorganisms that can be used in the present invention include genus Agrobacterium and Azotobact
er), Bordetella, Brettanomyces, Dekkera, Endomyces, Eremascus
s) Genus, Erwinia, Fusarium
genus, Geotrichum, Gibberella, Glomerella, Gonatobotryum, Klebzilla
bsiella), Micrococcus, Mycobacterium, Neosartoluya (N
eosartorya), Oospora, Pachysolen, Paecilomyrces
Genus, Paracoccus, Preucia
ssia), Saccharomyces, Saccharomycopsis, Serratia, Syringospora,
Genus Spetoria, Sporopathic dermia (Sp
oropachydermia), Talaromyces
Asymmetric reduction of 4-hydroxy-2-butanone by a microorganism belonging to the genera, genus Westerdykella, genus Zygoascus, genus Zygozyma to produce (R) -1,3-butanediol A microorganism capable of producing, or Aciculocium (Aciculoc)
onidium), Brettanomyces,
Clavispora spp., Cochliobolus (Coch)
liobolus, Corynespora, Dactylium, Echinopodospora
odospora), Enterobacter,
Genus Hamigera, Helminsosporium (Helm
genus inthosporium, genus Kloeckera, genus Nectria, Pseudomonas
Genus, genus Phialocephala, genus Rhodotorula, Saccharomyc
es) genus, Sterigmatomyces
Genus, Schizoblastosporion
Any microorganism can be used as long as it is a microorganism belonging to the genus and capable of producing (S) -1,3-butanediol by asymmetric reduction of 4-hydroxy-2-butanone.

Specifically, from 4-hydroxy-2-butanone to (R)
Microorganisms that can produce -1,3-butanediol include:
Agrobacterium radiobacter
ratdiobacter) IFO 12664, Azotobacter chroococcum IFO 12994, Bordetella bronchiseptic
a) IFO 13691, Brettanomyces abstines (Bret
tanomyces abstines) DSM 70726, Dekkera bruxellensis IFO 1590, Endomyces decipiens IFO 010
2. Eremascus fertili
s) IFO 0691, Erwinia carotovora subsp. carrotbora (Erwinia carotovora subsp.carot)
ovora) IFO 3830, Fusarium oxysporum
ium oxysporum IFO 7152, Fusarium soianii (F
usarium soiani) IFO 5232, Geotrichum fragrans JCM 1749, Gibberella fujikuroi IFO 5268, Glomerella cingulata IAM 8050, gonatobotorium apicratum
ulatum) IFO 9098, Klebzilla pneumonia
siella pneumoniae) IFO 12059, Micrococcus luteus (Micrococcus Iuteus) IFO 3333, Micrococcus roseus (Micrococcus roseus) IFO 3764, Mycobacterium smegmati
s) IFO 3153, Neosartorya fischeri var.spinosa
IFO 5955, Oospora
astringenes) IFO 7001, Paxolene tanophilus (P
achysolen tannophilus) IFO 1007, Paecilomyces variotii IFO 4855, Paracoccus denitrifica
ns) IFO 12442, Preussia terricola
rricola) IFO 7893, Saccharomyces cerevisiae IAM 0216, Saccharomycopsis fibuliger
a) IFO 0103, Serratia marsessens
rcescens) IAM 1105; Syringospora claussenii IFO 0759;
Glycines (Spetoria glycines) IFO 5294, Sporopachydermia lac
tativora) IFO 1867, Talaromyces flavas var.flavas IFO
7231, Westerdyk
ella multispora) IFO 5813, Zygoascus hellenicus (IFO 1575), and Zygozyma oligophaga (IFO 10360).

Also, from 4-hydroxy-2-butanone to (S) -1,
Microorganisms that can produce 3-butanediol include Aciculoconidium aculetum
eatum) IFO 10124, Bretanomyces anomaras (Br
ettanomyces anomalus IFO 0796, Clavispora lusitaniae IFO 1019, Cochliobolus miyabeanus I
FO 6331, Corynespora c
assiicolla) IFO 6724, Dactylium dentroides ATCC 46032, Echinopodospora jamaicensi
s) IFO 9819, Enterobacter cloase (Enterobac)
ter cloacae) ATCC 7256, Hamiguella Avellanea
gera avellanea) IFO 7721, Helminthosporium, sigmoidium, baryiti illegrale (Helminth)
osporium sigmoideumvar.irregulare) IFO 5273, Kloeckera africana IFO 086
9. Nectria cinnabarina IF
O 6821, Pseudomonas di
minuta) IFO 12697, Fialocephala bactrospora IFO 8770, Rhodotorula glutinis IFO 0395,
Saccharomyces cere
visiae) AHU 3402, Sterigmatomyces elviae DSM 70852, Schizoblastosporion kobayasi
i) IFO 1644.

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

Microorganisms with IFO numbers are described in Fermentation Research Institute (IFO) List of Cultures of Fermentation, 8th edition, vol.
8) and can be obtained from the IFO. Microorganisms with AHU numbers can be found in the Catalog of Cultures, 4th edition (198
It is described in 7) and can be obtained from the Faculty of Agriculture, Hokkaido University. Microorganisms with JCM numbers are listed in the third edition of the Microorganism Catalog (19
1986) and can be obtained from the facility. Microorganisms with ATCC numbers are American Type Cu
Catalog of Bacteri issued by the lture Collection (ATCC)
a Phages rDNA Vectors, 16th edition (1985) and Catalogue
of Fungi / Yeast, 17th edition (1987)
Available from C. Microorganisms with DSM numbers are published by Deutsch Sammlungvon Mikroorganismen (DSM)
Catalog of strains (1983)
Can be obtained from Microorganisms with IAM numbers can be obtained from the Institute of Applied Microbiology, The University of Tokyo.

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,
Sugars such as sucrose and dextrin, sorbitol,
Alcohols such as 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. As a nitrogen source, for example,
Ammonium chloride, ammonium sulfate, ammonium salts of inorganic acids such as ammonium phosphate, ammonium fumarate, ammonium salts of organic acids such as ammonium citrate, meat extract, yeast extract, corn steep liquor,
Casein hydrolysates, inorganic and organic nitrogen-containing compounds such as urea,
Alternatively, mixtures 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.

As a culture method, the medium pH is 3.0 to 9.5, preferably 4 to
8. The cultivation temperature is 20-45 ° C, preferably 25-37 ° C, under anaerobic or aerobic conditions suitable for the growth of the microorganism.
The culture is performed for about 120 hours, preferably about 12 to 72 hours.

In the present invention, as the method of the reduction reaction, a method using the culture solution as it is, centrifugation, etc., to separate the cells
There is a method in which 4-hydroxy-2-butanone is added to a suspension resuspended in a buffer solution, water or the like as it is or after washing, and reacted. At the time of this reaction, it may be better to add a carbon source such as glucose or sucrose as an energy source. The cells may be live cells, or may be cells that have been crushed, treated with acetone, freeze-dried, or the like. In addition, these cells or treated cells are immobilized by a known method such as polyacrylamide gel method, sulfur-containing polysaccharide gel method (carrageenan gel method, etc.), alginic acid gel method, agar gel method and the like. 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.

4-hydroxy-2-butanone as it is, or
It may be dissolved in water, dissolved in an organic solvent that does not affect the reaction, or dispersed in a surfactant or the like, and added all at once from the beginning of the reaction or dividedly.

In the present invention, the reduction reaction is carried out at pH 3 to 9, preferably at pH 5.
The reaction is carried out at a temperature of 10 to 60 ° C., preferably 20 to 40 ° C. for about 1 to 120 hours under stirring or standing.
The concentration of the substrate used is not particularly limited, but is preferably about 0.1 to 10%.

The optically active 1,3-butanediol produced by the reaction can be easily collected by using a normal purification method such as extraction with an organic solvent, distillation, column chromatography, etc., directly from the reaction solution or after isolation of cells. it can.

〔Example〕

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

In the examples, the amount of 1,3-butanediol in the reaction solution was determined by gas chromatography (column: Salmon 30).
00 (2 m), detection: FID, carrier: He, column temperature: 130
° C), and the optical purity can be easily determined by acetylating the obtained optically active 1,3-butanediol with acetyl chloride by a conventional method, and then performing high performance liquid chromatography using an optical resolution column (column: Daicel Chemical Industry Chiral Cell OB, solvent: n-hexane / 2-propanol = 19:
1, at a wavelength of 220 nm and a flow rate of 0.5 ml / min (retention time (S): 15 minutes, (R): 19.3 minutes).

Example 1 In the case of a strain belonging to a yeast filamentous fungus, a YM medium (yeast extract
0.3%, malt extract 0.3%, peptone 0.5%, glucose 2%, pH 6.0) 100ml, and for strains belonging to bacteria, YP
M medium (glucose 2%, yeast extract 0.5%, peptone 0.
3%, meat extract 0.3%, (NH ₄ ) ₂ HPO ₄ 0.2%, KH ₂ PO ₄ 0.1
%, PH 7) was placed in a 500-ml Sakaguchi flask, sterilized, and inoculated with the microorganisms described in Table 1, followed by shaking culture at 27 ° C. for 48 hours. Subsequently, the cells were separated by centrifugation and washed once with physiological saline to obtain viable cells.

Next, 50 ml of distilled water was placed in a 500 ml Sakaguchi flask, the viable cells were suspended therein, and 5 g of glucose was added. 27 ℃
For 10 minutes, 0.5 g of 4-hydroxy-2-butanone was added, and a reciprocal shaking reaction was performed at 27 ° C. for 20 hours.

After the completion of the reaction, the bacteria were removed by centrifugation, and the obtained supernatant was saturated with sodium chloride, extracted with 50 ml of ethyl acetate, and the ethyl acetate layer was analyzed by gas chromatography to obtain a reaction yield. Was examined.

Next, the ethyl acetate layer was dehydrated with anhydrous sodium sulfate, and the solvent was removed to obtain a syrup. This was acetylated with acetyl chloride by a conventional method, dissolved in a solvent, and the absolute configuration and optical purity of the product were measured by high performance liquid chromatography. Table 1 shows the results.

[Effect of the Invention] The method for producing an optically active 1,3-butanediol using the microorganism of the present invention can be easily performed using an optically active 1,3-butanediol having high optical purity.
This makes it possible to produce butanediol, which is extremely advantageous industrially.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12P 7/18 C12R 1:18) (C12P 7/18 C12R 1:22) (C12P 7/18 C12R 1: 265) (C12P 7/18 C12R 1:34) (C12P 7/18 C12R 1:38) (C12P 7/18 C12R 1: 425) (C12P 7/18 C12R 1:77) (C12P 7/18 C12R 1:79) (C12P 7/18 C12R 1: 865)

Claims (2)

(57) [Claims] (1) 4-hydroxy-2-butanone is selected from the group consisting of Agrobacterium and Azotobacter (A)
genus zotobacter, genus Bordetella, genus Brettanomyces, Dekkera
Genus, Endomyces genus, Elemascus (E
genus remascus, genus Erwinia, genus Fusarium, genus Geotrichum, genus Gibberella, Glomerella
Genus, Gonatobotryum, Klebsiella, Micrococcu
s) genus, Mycobacterium genus, Neosartorya genus, Oospora
Genus, Pachysolen, Paecilomyces (Paec
ilomyrces, Paracoccus, Preussia, Saccharomy
ces), Saccharomycopsis
Genus, Serratia, Syringospora
ospora, Spetoria, Sporopachydermia, Talaromise (Tala)
romyces), Westerdykella
Genus, Zygoascus, Zygozym
a) selected from the group of microorganisms belonging to the genus, 4-hydroxy-
A method of treating a microorganism having the ability to asymmetrically reduce 2-butanone to (R) -1,3-butanediol or a processed product thereof, and collecting (R) -1,3-butanediol produced. A method for producing an optically active 1,3-butanediol, which is characterized by: 2. A method according to claim 2, wherein the 4-hydroxy-2-butanone is selected from the group consisting of genus Aiculoconidium, genus Brettanomyces, and Clavispor.
a) genera, genus Cochliobolus, genus Corynespora, Dactylium
Genus, Echinopodospora, Enterobacter, Hamigera
Genus, Helminthosporium, Kloeckera, Nectria,
Genus Pseudomonas, genus Phialocephala, genus Rhodotorula,
4-hydroxy-2-butanone is selected from the group of microorganisms belonging to the genera Saccharomyces, Sterigmatomyces, and Schizoblastosporion, and 4-hydroxy-2-butanone is (S) -1,3.
(S) -1,3 which is formed by reacting butanediol with a microorganism capable of asymmetric reduction or a processed product thereof.
-Optical activity characterized by collecting butanediol
A method for producing 1,3-butanediol.