JPH03228686A - Production of optically active 1,3-butane diol - Google Patents

Abstract

PURPOSE:To readily obtain high optical purity by affecting a specific microorganism to 4-hydroxy-2-butanone (HB). CONSTITUTION:A microorganism converting HB to (R)-1,3 butane diol with asymmetrically reducing is searched to obtain a group of microorganism (A) belonging to Agrobacterium, Azotobacter, Bordetella, Brettanomyces, Dekkera, Endmyces, Eremascus, Erwinia, Fusarium, Geotrichum, Gibberella, Glomerella, Gonatobotryum, Klebsiella, Micrococcus, Mycobacterium and Neosartorya, etc. Next, a microorganism selected from the group A is cultured in a medium containing glucose, etc., at pH3.0-9.5 and at 20-45 deg.C for 5-120hr and centrifuged to obtain a fungus body (B). Then, the fungus body B is added to an aqueous solution containing 0.1-10wt.% HB, etc., and subjected to reducing reaction at pH3-9 and at 10-60 deg.C for 1-120hr, then a resultant reacted solution is extracted, thus purified to produce the aimed optically active 1,3-butane diol.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] 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 techniques and inventions] Conventionally,
As a method for producing optically active 1,3-butanediol, (1) a method of optically resolving chemically synthesized racemic 1,3-butanediol using an optical resolving agent (Japanese Patent Application Laid-open No. 1983-1999-1); 191631), (2) A method of obtaining it by asymmetric synthesis reaction from 2-hydroxy-4-butanone using a Raney nickel catalyst treated with an optically active compound (
JP-A-58-204187 and Bull, Ch.
ewr, Soc, Jpn.

53, 1356-1360 (1980)), but both (1) and (2) have drawbacks such as the need to use expensive optical resolution agents and catalysts, and (2) low optical purity. Therefore, it has been desired to establish an economically superior and simple method for obtaining optically active 1,3-butanediol with high optical purity.

[Means to solve the problem]

The present inventor focused on the asymmetric reduction method using microorganisms as a simple method to obtain optically active 1,3-butanediol with high optical purity, and as a result of searching for microorganisms suitable for this purpose, certain types of We have discovered a method for producing optically active 1,3-butanediol using an asymmetric reduction method using microorganisms, and have already filed a patent application (Japanese Patent Application No. 1-107017).

The present inventors further intensively researched the method for producing optically active 1,3-butanediol by an asymmetric reduction method, and as a result, newly discovered that the genus Agrobacterium, Azotobacter, Bordetella ( Bordatella spp., Brettanomyces spp., Dekkella spp.
genus Kera, Endomyces
Genus, Eremascus, Erwinia, Fusari
um), Geot-richum, Gibberella, Glomerella (
Glomerel la), Gonatobothrium (Go
genus natobotryum, Klebzilla
siella) genus, Micrococcus
cus), Mycobacterium
ium), Neosartorya
) genus, Oospora genus, Baxolene (
Pachysolen genus, Paeci
Lo-myces) genus, Paracoccus
ccus), Preussia genus, Saccharomyces genus, Saccharomycopsis
s) genus, Serratia genus, Syringospora genus, Spectoria (S
petoria), Sporopathy Dermia (Spor)
opachyder+1ia), Talaromyces (Ta
Wes
terdykella), Zygoa
microorganisms belonging to the genus Zygozyscus and Zygozy++a asymmetrically reduce 4-hydroxy-2-butanone to produce (R)-1,3-butanediol;
and Acycloconidium (Aciculoc.

ni d i u + *) genus, Brettanomyces (Br
ettanomyces), Tarabis borra (Clav)
ispora), Coryneporus (Cochl 1ob)
olus), Corynespora
a) Genus Dactylium, Genus Echinopodospora,
Enterobacter genus, Hasigera genus, Helminthosporium genus, Kloeckera genus, Nectria genus
ria), Pseudomonas
) genus, Phialocephala
), Rhodotorula genus, Sa-ccharomyces genus, Sterigma tomyce
s), genus Schizoplastosporion (Sch i -zo
Microorganisms belonging to the genus Blastosporion are 4-
Asymmetric reduction of hydroxy-2-butanone produces (S)-1,
The present invention was completed by discovering that 3-butanediol can be produced.

Examples of microorganisms that can be used in the present invention include the genus Agrobacterium, the genus Azotobacter, and the genus Bordetella.
genus Plechlamyces (Bretta)
genus noa+yces), genus Dekkera, genus Endomyces, genus Eren+ascus-Erwinia (E
Genus rwinia, Genus Fusarium, Genus Geotrichum, Genus Gibbe-rella, Genus Glomerella.
erella), Gonatobtrium (Gonatob)
otryum genus, Talebsiell
a) Genus Micrococcus, Mycobacterium iu
m) genus Neosartorya, genus 00-spora, genus Baxoren (P
achysolen), Paecilomyces (Paeci)
1osyces), Para-coc
cus), Preussia genus, Saccharomyces genus, Saccharomycopsis
), Serra-tia, Syringospora, Spetria
petoria), Sporopathy Dermia (Spor)
opachydermia), Talaromyces (Tal)
aro-myces), Westerdichella (Wes
terdyke 11a), Zygoascus (Zy
goascus) genus, Zy-gozyma
A microorganism belonging to the genus Acycloconidium (
Aciculoconidium genus, Plechlamyces genus (Brettanomyces), Clavisbora (
Genus Clavispora, Cochliobolus
Corynespora (Corynespora)
genus spora, Dactylium+
) genus, Echinopodosvora
ra) genus, Enterobacter
ter), Hamigera, Helminthosporium
Genus Kloeckera, Genus Nect-ria, Genus Pseudomonas
domonas), Phialocephala (Phialocephala)
cephala), Rhodotoru
genus Saccha-romyces (la), Saccha-romyces
ces), the genus Sterigmadomyces (Sterigms)
-tosyces) genus, Schizophrestosporion (S
Any microorganism belonging to the genus Chizo-blastosporion that has the ability to asymmetrically reduce 4-hydroxy-2-butanone to produce (S)-1,3-butanediol can be used.

Specifically, from 4-hydroxy-2-butanone (R)-
Microorganisms that can produce 1,3-butanediol include:
Agrobacterium radiobacter (Agrobac
teriua + radiobacter) IFO12
664, Azotobacter kurukotsucum (Azoto
bacterium chroococcum) IFO129
94, Bordetella pronchepti force
lla bronchiseptica) IFO13
691, Bre-t
DSM 7
0726, Dekkera purxerensis (Dekker
a bruxellensis) IFO1590, Endomyces decibiens (Endomyces
decipiens) IFO0102, Eremascus fertilis
is) IFO0691, Erwinia carotovora
Subspecies carotovora (t!rvinia c
arotovora 5ubsp, ca-rotov
ora) IPo 3830, Fusarium oxysporus IF
O7152, Fusarium soianii
s 5oiani) IPO5232, Geotrichum
Fragrance (Geotrichum frag-ra)
ns) JCM 1749, Gibberella flagroy (G
ibbe-rella fujikuroi) IPo
526B, Glomerella singulata (Glose
IAM 8050
, Gonatobothrium aviculatum (Gonatob)
o-tryus apiculatuo+) IFo
9098, Talebzilla pneumonia
ella pneumoniae) IFO12059,
Micrococcus luteus
1uteus) IFO3333, Micrococcus roseus I
FO3764, Mycobacterium smegmatis (M
ycobacteria+ smegmatis) I
FO3153, Neosartoria fischeri varitei 8 Spinosa
eri var.

5pinosa) IFO5955, Oospora astrinimigenes (Oospora astringen)
es) IFO7001, Paxolene Taraphyllus (
Pachysolen tannophilus)IF
O1007, Paecilomyces variotii
omyces variotii) IFO4855,
Paracoccus denitrificans
us denitrificans) IFO12442
, Preussia tericola
ricola) IPo 7893, Saccharomyces 0 cerevisiae s (Saccharon+yces
cerevisiae) IAM 0216, Saccharomycopsis fibrigera (Saccharomycop
sis fibuligera) IFO0103, Serratia marcescens (Serratia a+ar
cescens) IAM 1105, Syringosbora
Syringospora claus
senii) IFO0759, Spetria glycines IFO5
294, Sporopathy Dermia lactativora (Sp
or.

pachydermia 1 act, ativora)
IPo 1867, Claromyces flavus variitii flavus
as var, flavas) IFO7231, Westerdychella multispora (Westerdyk
ellamultispora) IPo 5813,
Zygoascus helenicus (Zygoascus he
llenicus) IFO1575, Zygozys+a oligophaga
) IFO10360.

Also, from 4-hydroxy-2-butanone (S)=1.
A microorganism that can produce 3-butanediol is Acycloconidium aculatum (Aciculoconidium aculatum).
idium aculeatum) IFO10124
, Brettanomyces anomalous (Brettano)
mycesanomalus) IFO0796, Tarabispora lusitaniae (Clavispora 1u)
sitaniae) IFO1019, Cochliobolus m1ya
beanus) IFO6631, Corynespora cassiico
la) IFO6724, Dactylium dentr.

1des) ATCC46032, Ecinobodosbora
jamaicensis (Echinopodospora)
jamaicensis) IFO9819, Enterobacter cl.
oacae) ATCC7256, Hamigera avellanea (l(a+sigera avellanea) I
FO7721, Helsinthosborium sigmoidium baritei 9 irregular
poriuo+ sigmoideuavar, ir
regular) IFO5273, Kloeckera africana
IFo 0869, Nectria cinabarina (Nec
tria cinnabarina) IFO6821,
Pseudomonas diminuta
s diainuta) IFO12697, Phialocephala bactrospora (Phialocephala
a bactro-spora) IFO8770, Rhodotorula glutinis (Rhodotorula g
Saccharomyces lutinis) IFO0395, Saccharomyces ce
re-visiae)^HU 3402, Sterigmadomyces elvis, (Sterigsatosyc
es elviae) DSM 70852, Schizoblastosporion kobayashi
sporion kobayasii) IFO164
4 can be mentioned.

Any of these microbial strains, wild strains, mutant strains, or recombinant strains induced by genetic techniques such as cell fusion or genetic manipulation methods can be suitably used.

The microorganisms with IFO numbers are Li5t of Cultures of R Fermentation, Institute of Fermentation (IFO).
, 8th edition, Volume 1 (198B),
It can be obtained from FO. Microorganisms with AHU numbers are C.
It is described in the atalogue of Cultures, 4th edition (1987) and is available from the Faculty of Agriculture, Hokkaido University. Microorganisms with JCM numbers are listed in the Microbial Strain Catalog, 3rd edition (1986) published by the RIKEN Microbial System Preservation Facility, and can be obtained from the facility. Microorganisms with ATCC numbers are Aa+erican Type Cultur.
Catalog of BacteriaPha published by eCo 11 eCti on (ATCC)
ges rDNA Vectors+ 16th edition (1
985) and Catalog of Fungi/
Yeast, 17th edition (1987), available from the ATCC.

Microorganisms with DSM numbers are Deutsch Sam
Mlungvon Mikroorganismen
Catalog of strains published by (DSM)
(1983) and can be obtained from the DSM. Microorganisms with IAM numbers can be obtained from the Institute of Applied Microbiology, University of Tokyo.

The medium for culturing the microorganisms used in the present invention is not particularly limited as long as the microorganisms can grow therein. For example, as a carbon source, any of the above-mentioned microorganisms can be used as long as it can be used. Specifically, sugars such as glucose, fructose, sucrose, and dextrin, alcohols such as sorbitol, ethanol, and glycerol, fumaric acid, Organic acids such as citric acid, acetic acid and propionic acid and their salts, hydrocarbons such as paraffin, etc., or mixtures thereof can be used. Examples of nitrogen sources 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;
Inorganic organic nitrogen-containing compounds such as Uchiko Kiss, yeast extract, cornstarch liquor, casein hydrolyzate, urea, or mixtures thereof can be used. In addition, nutrient sources used in normal culture, such as inorganic salts, trace metal salts, and vitamins, can be appropriately mixed and used. Further, if necessary, factors that promote the growth of microorganisms, factors that enhance the ability to produce the target compound of the present invention, or substances that are effective in maintaining the pH of the medium can also be added.

As for the culture method, the culture medium pH is 3.0 to 9.5, preferably 4 to 8, and the culture temperature is 20 to 45°C, preferably 25 to 8.
The microorganism is cultured at 37° C. anaerobically or aerobically for 5 to 120 hours, preferably 12 to 72 hours, under conditions suitable for the growth of the microorganism.

In the present invention, the method for the reduction reaction is to use the culture solution as it is, to separate the bacterial cells by centrifugation, etc., and to use them directly or after washing and resuspending them in a buffer solution, water, etc. There are methods such as adding 4-hydroxy-2-butanone and causing the reaction. During this reaction, it may be better to add a carbon source such as glucose or sucrose as an energy source. Furthermore, the bacterial cells may be kept as viable cells, or may be crushed bacterial cells, treated with acetone, freeze-dried, or the like.

Furthermore, these bacterial cells or treated bacterial cells may be immobilized and used by a known method such as a polyacrylamide gel method, a synthetic polysaccharide gel method (carrageenan gel method, etc.), an alginate gel method, an agar gel method, etc. You can also do it. Furthermore, it is also possible to use enzymes that have been purified from processed bacterial cells using a combination of known methods.

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

In the present invention, the reduction reaction is performed at pH 3 to 9, preferably at p
H5 to 8, temperature 10 to 60°C, preferably 2
The concentration of the substrate to be 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 directly from the reaction solution or after bacterial cell isolation using conventional purification methods such as extraction with organic solvents, distillation, and column chromatography. can.

〔Example〕

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

In addition, in the examples, the determination of 1,3-butanediol in the reaction solution was performed using gas chromatography (column: Salmon 3).
000 (2■), Detection: FID, Carrier: He,
Column temperature: 130°C).
Optical purity was determined by acetylating the obtained optically active 1,3-butanediol with acetyl chloride in a conventional manner, followed by high performance liquid chromatography using an optical resolution column (column:
Measured using Chiralcel OB manufactured by Daicel Chemical Industries, solvent: n-hexane/2-propanol = 111, wavelength 220n, flow rate 0.5 d/min) (retention time (S) body 1
5 minutes, (R) body 19.3 minutes).

Example 1 In the case of strains belonging to yeast filamentous fungi, sin medium (yeast extract 0
．． 3%, malt extract 0.3%, peptone 0.5%, glucose 2%, pH 6.0) 100-, or YPM medium (glucose 2%, yeast extract 0.5%, Peptone 0.3%, meat extract 0.3%, (
NH4) JPO40.2%, KH! PO40.1%, p
H7 > 100- was placed in a 5001 volume slope flask, sterilized, inoculated with the microorganisms listed in Table 1, and incubated at 27°C for 48 hours.
Reciprocating shaking culture was performed for hours. Subsequently, the bacterial cells were separated by centrifugation and washed once with physiological saline to obtain viable bacterial cells.

Next, put 50wJ of distilled water into the 500-Yosaka flask,
The above-mentioned live bacterial cells were suspended in this, and 5 g of glucose was added. After reciprocating shaking at 27°C for 10 minutes, 0.5 g of 4-hydroxy-2-butanone was added, and the mixture was subjected to reciprocating shaking reaction at 27°C for 20 hours.

After the reaction was completed, bacteria were removed by centrifugation, the resulting supernatant was saturated with sodium chloride, and extraction was performed using ethyl acetate, and the ethyl acetate layer was analyzed by gas chromatography to determine the reaction yield. I checked the rate.

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

The results are shown in Table 1.

Table 1 continued Table 1 continued Table 1 continued Table 1 continued [Effects of the invention] The method for producing optically active 1,3-butanediol using microorganisms of the present invention can easily produce optically active 1,3-butanediol with high optical purity. 1.3
-It makes it possible to produce butanediol and is extremely advantageous industrially.

Claims (1)

[Scope of Claims] 1,4-hydroxy-2-butanone, Agrobacterium genus, Azotobacter genus, Bordetella genus
genus Brettanomyces, Brettanomyces
genus Dekkera
Genus, Endomyces, Eremas-cus, Erwinia (E
Genus rwinia, Genus Fusarium, Genus Geotrichum, Genus Gibberella, Genus Glomerella.
gonato-b
otryum genus, Klebsiell
a) Genus Micrococcus, Genus Mycobacterium, Genus Neosartorya, Genus Oospora, Pachy
genus solen, Paecilomyces
es), Paracoccus, Preussia, Saccharomyces, Saccharomycopsis, Serratia, Syringospora
ngospora), Spectoria
) genus, Sporopachyd
ermia), Talaromyces
s), genus Westerdykel
4-hydroxy-2-butanone (R)-1,3
-(R)-1 produced by reacting with a microorganism or its treated product having the ability to asymmetrically reduce to butanediol,
A method for producing optically active 1,3-butanediol, which comprises collecting 3-butanediol. 2,4-Hydroxy-2-butanone is added to the genus Aciculoconidium, the genus Brettanomyces, the genus Clavispora, and the genus Cociliobolus (
Coch-liobolus genus, Corynespora (Co
rynespora), Dactylium
ium), Echinopodospora
spora), Enterobacter (Enterobacter)
cter), Hamigera genus, Helminthosporium (Helminthosporium)
), Kloeckera, Nectria, Pseudomonas
omonas), Phialocephala (Phialoc)
genus ephala, Rhodotorul
a) Genus, Saccharomyces
s) genus, Sterigmatomyces
myces), Schizoblastosporion (Schiz
4-hydroxy-2-butanone (S)-1
, 3-butanediol by acting on a microorganism or its treated product that has the ability to asymmetrically reduce it to (S)-
A method for producing optically active 1,3-butanediol, which comprises collecting 1,3-butanediol.