JP2000106891A - Production of alfa-halo-alfa, beta-saturated carbonyl compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject α-halo-α,β-saturated carbonyl compound that is useful as a starting substance for medicines and agrochemicals by reducing an α-halocarbonyl compound bearing an α,β-carbon, carbon double bond with a micro-organism in Acetobacter (or its treated product). SOLUTION: An α-halocarbonyl compound bearing an α,β-carbon, carbon double bond represented by formula I [R1 is a halogen; R2 and R3 are each H, a halogen, a 1-6C aliphatic hydrocarbon, a 1-6C alkoxy, OH, COOH, (substituted) aromatic group or the like; R4 is OH, alkoxy, amino or the like] is reduced by using a microorganism (or its treated product) in Acetobacter, Actinomyces, Acinetobacter, Agrobacterium, Aeromonas, Alcaligenes, Arthrobacter, Bacillus, Brevibacterium, Cellulomonas, and the like to give the objective compound of α-halo-α,β-saturated carbonyl compound represented by formula II, as a variety of conditions, for example, its operability, economy, safety, reaction properties and the like are satisfied.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an Acetobacter (A)
cetobacter), especially microorganisms belonging to the genus Pseudomonas or Burkholderia, more particularly Pseudomonas sp. SD81
0 strain (Pseudomonas sp.SD810), Pseudomonas sp.SD811 strain (Pseudomonas sp.SD811), Pseudomonas sp.SD812 strain (Pseudomonas sp.SD81)
2), Burk-Holidia sp. SD816 strain (Burkh
olderia sp. SD816) or a specific strain thereof, or an α-β-carbon-carbon double bond-containing α-β-carbon double bond.
The present invention relates to a method for producing a corresponding α-halo-α, β-saturated carbonyl compound by hydrogenating an α, β-carbon-carbon double bond of a halocarbonyl compound. Furthermore, microorganisms belonging to the genus Pseudomonas or Burkholderia, in particular, Pseudomonas sp. SD810 strain (P.
seudomonas sp.SD810), Pseudomonas sp. S
Strain D811 (Pseudomonas sp. SD811), Pseudomonas sp.
Sp. SD812 strain (Pseudomonas sp. SD812) and Burkholderia sp. SD816 strain (Burkholde
ria sp.SD816).

Further, the present invention relates to an α-halocarbonyl compound having an α, β-carbon-carbon double bond, which is a prochiral molecule at the α-position. For the corresponding α-position, the S-form α-halo-
The present invention relates to a method for producing an α, β-saturated carbonyl compound. This novel method can be used in the field of producing optically active carbonyl compounds such as saturated carboxylic acids and amides having various optical activities (absolute configuration S at α-position). Optically active carbonyl compounds are valuable chiral building blocks that are difficult to prepare with classical chemical processes,
In particular, it is a group of substances useful as raw materials for medicines and agricultural chemicals.

[0003]

2. Description of the Related Art In recent years, methods for producing various compounds, particularly optically active substances, by reducing carbon-carbon double bonds of microorganisms have attracted attention, and have various α, β-carbon-carbon double bonds. A method has been reported for producing a corresponding α, β-saturated carbonyl compound having a substituent at the α-position by microbially reducing the carbon-carbon double bond of a carbonyl compound having a substituent at the α-position (H .Simon.et.al., Hoppe-Seyler's Z.Phys
iol.Chem.362,33 (1981), H.Giesel.et.al., Arch.Microb
iol. 135, 51 (1983), HGWLeuenberger.et.al., Helv. Ch
im, Acta., 62, 455 (1979), R. Matuno., et.al., J. Ferm. Bio
eng., 84, 195 (1997)). However, for example, in a method using bacteria as a microorganism, Clostridium kluyveri (DSM-555), Clostridium sp. La-1 (Clostridium sp.
Since an anaerobic bacterium such as M-1460)) is used, the growth rate of the microorganism is slow, and it is difficult to increase the cell concentration, so that the reaction rate is not sufficient and there is a problem in economy and operability.

A Clostridium thermosaccharilytic cam (C) disclosed in JP-A-63-003794 is also disclosed.
method using lostridium theremosaccharolyticum)
The problem was solved by using thermophilic bacteria, but the growth rate and reaction rate were not sufficient because anaerobic bacteria were still used. Has not been resolved. In addition, α, β- having a substituent at the α-position, which is produced by the reduction of a carbonyl compound having a α, β-carbon-carbon double bond and having a substituent at the α-position by this microorganism.
The saturated carbonyl compound has an absolute configuration of R-form, and cannot produce an S-form.

The method of reducing α, β-carbon-carbon double bonds using baker's yeast as a microorganism has a wide range of compounds that can be reduced, and thus has general versatility. Both S-form and R-form are known and the most reported examples are produced, but yeast grows slower than bacteria, and a reduction reaction aimed at producing an optically active product. In many cases, optical selectivity is not sufficient, and α, β-carbon-carbon double bond having α
-The reduction reaction of the halocarbonyl compound is not known.

As described above, the carbon-carbon double bond of an α-halocarbonyl compound having an α, β-carbon double bond is reduced using a microorganism to form the corresponding α-halo-α, β
-In the method for producing a saturated carbonyl compound, a method that satisfies all conditions such as operability, economy, safety, and reaction characteristics has not been known.

[0007]

SUMMARY OF THE INVENTION Accordingly, the present invention provides an α-halocarbonyl compound having an α, β-carbon-carbon double bond by reducing the carbon-carbon double bond of the α-halocarbonyl compound using a microorganism. It is an object of the present invention to provide a method for producing a halo-α, β-saturated carbonyl compound which satisfies all conditions such as operability, economy, safety and reaction characteristics, and is excellent in optical selectivity. And

[0008]

Means for Solving the Problems The present inventors have reduced the carbon-carbon double bond of an α-halocarbonyl compound having an α, β-carbon-carbon double bond to reduce the corresponding α-halo-carbon double bond.
Extensive screening from soil has found that microorganisms producing α, β-saturated carbonyl compounds are surprisingly distributed over a relatively large number of aerobic or facultative anaerobic bacteria. Was. In particular, Pseudomonas or Burkholderia
There are many strains having this activity among microorganisms belonging to ia). Among these strains, α-halocarbonyl compounds having α, β-carbon-carbon double bonds are reduced to have extremely high purity. The present inventors have found that there is a compound capable of producing an α-halo-α, β-saturated carbonyl compound in the S-configuration in the absolute configuration at the α-position, and completed the present invention.

That is, the present invention relates to the following matters. [1] An α-halocarbonyl compound having an α, β-carbon-carbon double bond is a compound of the genus Acetobacter,
Actinomyces, Acinetobacter
(Acinetobacter) genus, Agrobacterium (Agrobacteriu)
m) genus, Aeromonas genus, alkaline genes
(Alcaligenes) genus, Arthrobacter
Genus, Azotobacter, Bacill
us), Brevibacterium, Burkholderia, Cellulomonas
monas), Corynebacterium,
Enterobacter, Enterococcus
(Enterococcus) genus, Escherichia
Genus, genus Flavobacterium, genus Gluconobacter, halobacterium (Haloba
cterium), Halococcus, Klebsiella
(Klebsiella) genus, Lactobacillus (Lactobacillus) genus,
Microbacterium, Micrococcus, Micropolyspor
a) Genus, Mycobacterium (Mycobacterium) genus, Nocardia (Nocardia) genus, Pseudomonas (Pseudomonas)
Genus, Pseudonocardia, Rhodococcus, Rhodobacter
Genus, Serratia, Staphylococcus (Staph
ylococcus), Streptococcus
Genus, Streptomyces (Streptomyces) genus, using a microorganism belonging to any of the genus Xanthomonas (Xanthomonas) or a processed product thereof, α- halo characterized by reducing the α, β- carbon-carbon double bond A method for producing an α, β-saturated carbonyl compound.

[2] An α-halocarbonyl compound having an α, β-carbon-carbon double bond is prepared by using Pseud
omonas), wherein the α, β-carbon-carbon double bond is reduced using a microorganism belonging to the genus omonas) or a treated product thereof, wherein the α-halo-α, β-saturated carbonyl compound according to [1] is reduced. Production method. [3] An α-halocarbonyl compound having an α, β-carbon-carbon double bond is prepared by using a microorganism belonging to the genus Burkholderia or a processed product thereof, and converting the α, β-carbon-carbon double bond to the compound. The method for producing an α-halo-α, β-saturated carbonyl compound according to [1], which comprises reducing.

[4] The microorganism belonging to the genus Pseudomonas is Pseudomonas sp. SD810 strain (Pseudom
onas sp. SD810). [5] The microorganism belonging to the genus Pseudomonas is Pseudomonas sp. SD811 (Pseudomonas sp.
The production method according to [2], which is 1). [6] The microorganism belonging to the genus Pseudomonas is Pseudomonas sp. SD812 strain (Pseudomonas sp. SD81).
The production method according to [2], which is 2). [7] The microorganism belonging to the genus Burkholderia is Burkholderia sp. SD816 (Burkholderia ssp.
p.SD816). [8] The production method according to any one of [1] to [7], wherein an S-form compound in which the α-position is chiral is produced by reduction of a carbon-carbon double bond.

[9] The α-halocarbonyl compound having an α, β-carbon-carbon double bond is represented by the following general formula (1)

Embedded image (Wherein, R ₁ represents a halogen atom; R ₂ and R ₃ independently represent a hydrogen atom, a halogen atom, a linear or branched aliphatic hydrocarbon group having 1 to 6 carbon atoms, and a carbon atom having 1 to 6 carbon atoms) A linear or branched alkoxy group, a hydroxyl group,
R ₄ represents a carboxyl group, an aromatic group which may be substituted or a nitrogen-containing, oxygen-containing or sulfur-containing heterocyclic group;
Represents a hydroxyl group, a linear or branched alkoxy group having 1 to 3 carbon atoms or a primary to tertiary amino group. ) Wherein the α-halo-α, β-saturated carbonyl compound is represented by the following general formula (2)

Embedded image (Wherein, R _{1 to} R ₄ represent the same meaning as described above.)
The method for producing an α-halo-α, β-saturated carbonyl compound according to [1] to [8], which is a compound represented by the formula:

[10] The compound represented by the general formula (1) is α-haloacrylic acid, and the compound represented by the general formula (2) is α-halopropionic acid having an absolute configuration S. The method for producing the α-halo-α, β-saturated carbonyl compound described in the above. [11] The production method according to [10], wherein the halogen atom is a bromine atom. [12] The production method according to [10], wherein the halogen atom is a chlorine atom. [13] The production method according to [1] to [12], wherein the treated microorganism is a microorganism culture, a microorganism extract, a microorganism cell suspension, or a microorganism cell fixed substance. [14] A method of producing a α-halo-α, β-saturated carbonyl compound that does not decompose the generated α-halo-α, β-saturated carbonyl compound in a microorganism to be used to increase the amount of accumulated product [1] to [13]. The production method described in 1. [15] The reaction duration is extended by coexisting an α-halocarbonyl compound having an α, β-carbon double bond and a compound that can be oxidized by a microorganism to be used in the reaction system [1] to [14]. ] The production method according to [1]. [16] The production method according to [15], wherein the compound that can be oxidized by the microorganism used is a saccharide having 3 to 6 carbon atoms. [17] The production method according to [15], wherein the compound that can be oxidized by the microorganism used is an organic acid having 2 to 4 carbon atoms.

[18] Pseudomonas sp. Strain SD810 (Pseudomonas sp.) Having an activity of reducing an α, β-carbon-carbon double bond of an α-halocarbonyl compound having an α, β-carbon-carbon double bond SD810) or a mutant thereof. [19] Pseudomonas sp. SD8 having an activity of reducing an α, β-carbon-carbon double bond of an α-halocarbonyl compound having an α, β-carbon-carbon double bond
11 strains (Pseudomonas sp. SD811) or mutants thereof. [20] Pseudomonas sp. SD8 having an activity of reducing an α, β-carbon-carbon double bond of an α-halocarbonyl compound having an α, β-carbon-carbon double bond
12 strains (Pseudomonas sp. SD812) or mutants thereof.

[21] Burkholderia sp. SD816 strain (Burkholderia) having an activity of reducing an α, β-carbon-carbon double bond of an α-halocarbonyl compound having an α, β-carbon-carbon double bond sp. SD816) or a mutant thereof. [22] A processed product of the microorganism according to [18] to [21]. [23] The processed product of the microorganism according to [22], which comprises a microorganism culture, a microorganism extract, a microorganism cell suspension or a microorganism cell fixed substance.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The microorganisms that can be used in the present invention include the genus Acetobacter, the genus Actinomyces, and the genus Acinetobacter.
Genus, Agrobacterium, Aeromonas, Alcaligenes
Genus, Arthrobacter, Azotobacter, Bacillus, Brevibacterium, Burkholderia
kholderia), Cellulomonas, Corynebacterium, Enterobacter
(Enterobacter), Enterococcus
Genus, Escherichia, Flavobacterium, Gluconobacter
bacter), Halobacterium, Halococcus, Klebsiella,
Lactobacillus genus, Microbacterium genus, Micrococcus
Genus, Micropolyspora, Mycobacterium, Nocardia
Genus, Pseudomonas, Pseudonocardia, Rhodococcu
s), Rhodobacter, Serratia
ia), a microorganism belonging to the genus Staphylococcus, the genus Streptococcus, the genus Streptomyces, or the genus Xanthomonas.

Preferably, it is a microorganism belonging to the genus Pseudomonas or the genus Burkholderia. α, β of α-halocarbonyl compound having α, β-carbon-carbon double bond
There is no particular limitation as long as the strain has an activity of reducing a carbon-carbon double bond. Examples thereof include Pseudomonas sp. SD810 strain and Pseudomonas sp.
One strain, Pseudomonas sp. SD812 strain or Burkholderia sp. SD816 strain is preferably used. Above all, Pseudomonas SP SD8
11 strains or Burkholderia sp. SD816
Strains are particularly preferably used. Pseudomonas SP SD810, Pseudomonas SP SD81
One strain, Pseudomonas sp. SD812 strain or Burkholderia sp. SD816 strain, has been isolated from soil and has the ability to decompose and assimilate various carbonyl compounds.

[0018] Further, the microorganism may be an α, β-carbon compound having an α, β-carbon-carbon double bond.
As long as it has the activity of reducing carbon-carbon double bonds,
Any strain such as a wild strain, a mutant strain, or a recombinant derived by cell fusion or genetic engineering can be used. For example, mutants with reduced or deficient product degradation activity, mutants and recombinants with improved reducing activity, mutants with improved resistance to high concentrations of substrates and products, and the like are particularly preferably used. Can be.

Next, an example of the separation and culture will be described. That is, an inorganic salt medium (for example, (N
H ₄ ) ₂ SO ₄ 2 g / l, NaH ₂ PO ₄ 1 g / l, K ₂ HPO ₄
1 g / l, MgSO ₄ 0.1 g / l, yeast extract 0.5 g / l)
For example, a test tube containing 5 ml of a minimal medium containing 2 g / l of an α, β-unsaturated carbonyl compound having a halogen atom at the α-position as a substituent, such as α-chloroacrylic acid, is added as a substantially sole carbon source. After sterilization, about 0.1 g of soil is added, shaking culture is performed at 28 ° C., and subculture is performed every day. After repeating this enrichment culture for 6 days, 20 g / l of agar was added to the above minimal medium, and the plate was plated and applied.
After culturing at 28 ° C. for 3 days and the resulting colonies are purely isolated, Pseudomonas sp. SD810 strain, Pseudomonas sp. SD811 strain or Pseudomonas sp. SD812 strain can be obtained. The Pseudomonas sp. SD810 strain, Pseudomonas sp. SD811 strain and Pseudomonas sp.
No. BP-6767 (FERM BP-6767)
-16746)], BF-6768 (FERM BP-
6768) [Transferred from Life Science Bacteria No. 16747 (FERM 16747)]
And deposited in the Life Science and Technology Laboratory as BP-6769 (FERM BP-6769) [transferred from FERM 16748].

An example of isolation and culture of a strain having a relatively low population in a relatively isolated source will be described.

That is, microorganisms having a low population, microorganisms having a low assimilation ability, and α, β having a halogen atom at the α-position as a substituent, such as α-chloroacrylic acid, obtained by accumulating and accumulating culture using soil or the like as a separation source. -Microorganisms with low resistance to unsaturated carbonyl compounds (hereinafter also referred to as "halogen-containing compounds") can be isolated. Yeast extract (0.5 g / l), ammonium sulfate (2 g / l), sodium dihydrogen phosphate (1 g / l), dipotassium hydrogen phosphate (1 g / l), magnesium sulfate (0.1 g / l) Various basic halogen-containing compounds may be used in a basic medium containing a basic medium (for separating some microorganisms that grow with a high salt concentration, sodium chloride is added to the basic medium at a concentration of 10 to 20%). 0.01% (where "%" represents "weight (g) / volume (100 ml) x 100" unless otherwise specified) as a carbon source together with substantially only or the same concentration of glucose. 1 g of the collected soil is suspended in 10 ml of the obtained culture medium and cultured with shaking at 30 ° C. Twenty-four hours later, 5 ml of the culture supernatant is taken, 5 ml of a new medium in which only the concentration of the halogen-containing compound is 0.02% of the above composition is added, and the cells are shake-cultured at 30 ° C. (first passage). Twenty-four hours later, 5 ml of the supernatant of the culture was taken, and the concentration of the halogen-containing compound in the medium was adjusted to 0.
Add 5 ml of a medium raised from 02% to 0.1%, and culture with shaking at 30 ° C (second passage). This operation is repeated three more times every 24 hours (the third to fifth subcultures). At the time of the sixth subculture, the concentration of the halogen-containing compound in the medium added to the culture solution is increased to 0.2%, 5 ml of the supernatant is taken, 5 ml of a new medium is added, and the cells are cultured at 30 ° C with shaking. This work 2
Repeat 6 times every 4 hours (subculture 6-12 times), 6
At each passage, 2% agar was added to the medium containing 0.2% of the halogen-containing compound to form a plate medium, a part of the culture supernatant was applied, and the mixture was cultured at 30 ° C. When pure colonies are isolated, for example, Burkholderia
SP SD816 strain can be obtained. Burkholderia sp. SD816 strain was purchased from Life Science
No. -6770 (FERM BP-6770).

Next, the taxonomic test results of these strains are shown. Pseudomonas sp. SD810 strain Form; (1) Cell shape and size Bacillus 0.6-1.0 μm × 1.2-3.0 μm (2) Motile (3) Gram stain negative (4) Spore Presence or absence None (5) 3% KOH lysis positive physiological activity; (1) aminopeptidase positive (2) oxidase positive (3) catalase positive (4) indole formation negative (5) VP test negative (6) nitrate Reduction negative (7) Denitrification reaction negative (8) Utilization of citric acid (Simons) positive (9) Urease negative (10) Phenylalanine deaminase negative (11) Use of malonic acid positive (12) Levan production from sucrose positive (13) Lecithinase negative (14) Starch hydrolysis negative (15) Gelatin hydrolysis negative (16) Casein hydrolysis Sex (17) Hydrolysis negative DNA

(18) Tween 80 hydrolysis negative (19) Extrin hydrolysis negative (20) Growth Oxygen behavior Absolute aerobic Growth at 37 ° C.-Growth at 41 ° C.-Growth at pH 5.6 + Mac -Growth on Conkey-Agar medium-Growth on SS-Agar medium-Growth on Centrimid-Agar-(21) Pigment formation Non-diffusible negative Diffusible negative Fluorescent negative Pyrocyanine negative (22) OF test No (23) Acid generation Glucose negative Fructose positive Xylose negative (24) ONPG (β-galactosidase) negative (25) Arginine dihydrolase negative (26) Gas production from glucose negative (27) Tyrosine degradation negative (28) Growth factor Requirement None (29) Availability of various carbon compounds Acetic acid + adipic acid −

Capric acid + citric acid + citraconic acid + glycolic acid + levulinic acid + maleic acid + malonic acid + mesaconic acid + muconic acid + phenylacetic acid + sugar acid + sebacic acid + D-tartaric acid + m-tartaric acid + L-arabinose -Cellobiose-fructose + D-fucose-glucose-mannose-maltose-ribose-rhamnose-xylose-mannitol-gluconic acid-2-ketogluconic acid + N-acetylglucosamine-

Tryptamine-ethanolamine-D-alanine + L-ornithine-L-serine-L-threonine-glutamic acid + benzoic acid + m-hydroxybenzoic acid + sodium salicinate-2,3-butylene glycol Bergey's Manua
According to the classification based on l of Systematic Bacteriology (1986), this strain belongs to the genus Pseudomonas, but none of these strains was found to be consistent with the standard strain. Therefore, this strain is Pseudomonas sp.
The strain (Pseudomonas sp. SD810) was called.

Pseudomonas sp. SD811 strain Form; (1) Cell shape and size Bacillus 0.7-0.9 μm × 1.5-3.0 μm (2) Motile (3) Gram stain negative ( 4) Presence or absence of spores None (5) Positive lysis by 3% KOH Physiological activity; (1) Positive for aminopeptidase (2) Positive for oxidase

(3) Catalase positive (4) Indole formation negative (5) VP test negative (6) Nitrate reduction negative (7) Denitrification reaction negative (8) Use of citric acid (Simons) positive (9) Urease Negative (10) Phenylalanine deaminase Negative (11) Use of malonic acid Positive (12) Levan formation from sucrose Negative (13) Lecitinase negative (14) Starch hydrolysis negative (15) Gelatin hydrolysis negative (16) Casein hydrolysis negative (17) DNA hydrolysis negative (18) Tween 80 hydrolysis positive (19) Extrin hydrolysis negative (20) Growth Oxygen behavior Absolute aerobic Growth at 37 ° C-41 ° C Growth-Growth at pH 5.6 + Growth on Mac-Conkey-Agar medium-Growth on SS-Agar medium-Growth on Cetrimid-Agar- (21) Dye formation Non-diffusible negative Diffusible negative

(22) OF test Does not decompose sugar (23) Generates acid Glucose positive Fructose positive Xylose positive (24) ONPG (β-galactosidase) negative (25) Arginine dihydrolase negative (26) Glucose (27) Tyrosine degradation negative (28) Growth factor requirement None (29) Utilization of various carbon compounds Acetic acid + adipic acid-capric acid-citric acid-citraconic acid + glycolic acid + levulinic acid-maleic acid + Malonic acid + mesaconic acid-muconic acid + phenylacetic acid + sugar acid + sebacic acid-D-tartaric acid +

M-tartaric acid-L-arabinose + cellobiose-fructose + D-fucose + glucose + mannose-maltose-ribose + rhamnose + xylose-mannitol + gluconic acid-2-ketogluconic acid + N-acetylglucosamine + tryptamine-ethanolamine -D-alanine-L-ornithine-L-serine-L-threonine-glutamic acid + benzoic acid + m-hydroxybenzoic acid + sodium salicinate-2,3-butylene glycol According to this classification, this strain belongs to the genus Pseudomonas, but none of these strains coincided with the standard strain.
Therefore, this strain is Pseudomonas sp.
One strain (Pseudomonas sp. SD811) was selected.

Pseudomonas sp. SD812 strain Form; (1) Cell shape and size Bacillus 0.5-0.8 μm × 1.5-3.0 μm (2) Motile (3) Gram stain negative ( 4) Presence or absence of spores None (5) Positive lysis by 3% KOH Physiological activity; (1) Aminopeptidase positive (2) Oxidase positive (3) Catalase positive (4) Indole production negative (5) VP test negative (6) ) Nitrate reduction negative (7) Denitrification reaction negative (8) Use of citric acid (Simons) positive (9) Urease positive (10) Phenylalanine deaminase negative (11) Use of malonic acid positive (12) Levan from sucrose (13) Lecithinase negative (14) Starch hydrolysis negative (15) Gelatin hydrolysis negative (16) Casei Hydrolysis negative hydrolysis negative (17) DNA

(18) Tween 80 hydrolysis negative (19) Extrin hydrolysis negative (20) Growth Oxygen behavior Absolute aerobic Growth at 37 ° C.-Growth at 41 ° C.-Growth at pH 5.6 + Mac -Growth on Conkey-Agar medium-Growth on SS-Agar medium-Growth on Cetrimid-Agar-(21) Pigment formation Non-diffusible Positive Diffusive negative Fluorescent negative Pyrocyanine negative (22) OF test Decomposes sugar (23) Acid production Glucose negative Fructose negative Xylose negative (24) ONPG (β-galactosidase) negative (25) Arginine dihydrolase negative (26) Gaseous production from glucose negative (27) Tyrosine degradation positive (28) Growth factor Requirement None (29) Utilization of various carbon compounds Acetic acid + adipic acid +

Capric acid-citric acid + citraconic acid-glycolic acid + levulinic acid-maleic acid + malonic acid + mesaconic acid + muconic acid + phenylacetic acid + sugar acid + sebacic acid + D-tartaric acid-m-tartaric acid + L-arabinose -Cellobiose-fructose-D-fucose-glucose-mannose-maltose-ribose-rhamnose-xylose-mannitol-gluconic acid + 2-ketogluconic acid + N-acetylglucosamine-

Tryptamine-ethanolamine-D-alanine + L-ornithine-L-serine-L-threonine + glutamic acid + benzoic acid + m-hydroxybenzoic acid + sodium salicinate-2,3-butylene glycol Similarly, according to the classification based on Berjay's bacterial taxonomy, this strain belongs to the genus Pseudomonas, but none of those properties was found to be consistent with the standard strain.
Therefore, this strain is Pseudomonas sp.
2 strains (Pseudomonas sp. SD812).

Burkholderia sp. SD816 strain Form; (1) Cell form Bacillus (2) Motility present (3) Gram stain negative (4) Presence or absence of spores None (5) Flagella formation unknown physiological Activity; (1) oxidase positive (2) catalase positive (3) cleavage of protocatechinic acid ortho-type (4) reduction of nitrate negative

(5) Denitrification reaction negative (6) PHB accumulation positive (7) Starch hydrolysis negative (8) Gelatin hydrolysis negative (9) Growth Oxygen behavior Absolute aerobic Growth at 40 ° C. + ( 10) Pigment production Color tone of colonies Characteristic colony pigments are not produced Water soluble pigments are produced negative (11) OF test Does not decompose sugar (12) Arginine dihydrolase negative (13) Availability of various carbon compounds Levulinic acid- Mesaconic acid-D-tartaric acid + ribose + rhamnose + xylose + tryptamine-2,3-butyleneglycol- (14) quinone-type Q-8 (15) GC content (mol%) of intracellular DNA 62 Bacterial taxonomy (Bergey's Manua
According to the classification based on l of Systematic Bacteriology (1986, 1994)), this strain belongs to the genus Burkholderia, but none of those properties was found to be consistent with the standard strain. Therefore, this strain is Burkholderia sp.
SD816 strain (Burkholderia sp. SD816).

In the present invention, α, β which can be suitably used
The α-halocarbonyl compound having a carbon-carbon double bond is represented by the following general formula (1).

Embedded image In the formula, R ₁ represents a halogen atom, and preferred R ₁ includes a chlorine atom and a bromine atom. R ₂ , R
₃ is independently a hydrogen atom, a halogen atom, a linear or branched aliphatic hydrocarbon group having 1 to 6 carbon atoms, a linear or branched alkoxy group having 1 to 6 carbon atoms, a hydroxyl group, a carboxyl group, Represents an optionally substituted aromatic group or a saturated or unsaturated nitrogen-containing, oxygen-containing or sulfur-containing heterocyclic group, and preferable R ₂ and R ₃ include a hydrogen atom. R ₄ represents a hydroxyl group, a linear or branched alkoxy group having 1 to 4 carbon atoms, or a primary to tertiary amino group. Preferred examples of R ₄ include a hydroxyl group.

Specifically, α-chloroacrylic acid, α-bromoacrylic acid, 2-chloro-2-butenoic acid, 2-bromo-2-butenoic acid, 2-chloro-2-pentenoic acid, 2-chloro-2-pentenoic acid,
Bromo-2-pentenoic acid, methyl ester, ethyl ester thereof and the like are mentioned, and α-chloroacrylic acid and α-bromoacrylic acid are preferable.

The reaction method of the present invention is a method for producing microorganisms belonging to the genus Pseudomonas or Burkholderia, such as microorganisms such as Pseudomonas sp. SD811, and Burkholderia sp. The method is carried out by contacting an α-halocarbonyl compound having a carbon double bond to reduce the carbon-carbon double bond to obtain a corresponding α-halo-α, β-saturated carbonyl compound.

The reduction reaction of the carbon-carbon double bond of the α-halocarbonyl compound having an α, β-carbon-carbon double bond carried out in the present invention is carried out under conditions where the reducing power of the microorganism is stably exhibited. If present, in the culture solution of the microorganism, any of the cells obtained by culturing by the above method or the processed product of the microorganism such as a cell-free extract obtained by crushing the cells obtained by culturing by the above method Can also be used.

That is, when cells obtained by culturing are used, α, β-carbon-carbon double bond
-0.1% to 10% of the halocarbonyl compound in the culture solution
, Preferably at a concentration of 0.2% to 2% at a culture temperature of 15 ° C to 35 ° C.
C., preferably at 25.degree. C. to 30.degree. C. to produce the corresponding .alpha.-halo-.alpha.,. Beta.-saturated carbonyl compound in the culture.

Alternatively, the cells recovered from the culture obtained by culturing according to the above-described method by centrifugation or the like may be added to a suitable solution, for example, a solution suspended in an aqueous solution such as a dilute pH buffer solution. The α-halocarbonyl compound having an α, β-carbon-carbon double bond is, for example, 0.1%
It is added continuously or batchwise to a concentration of 10% to 10%, and the reaction temperature is 15 ° C to 35 ° C, preferably 25 ° C to 3 ° C.
0 ° C., reaction pH 6.0-9.0, preferably pH 6.5
The reaction is adjusted to ７7.3 to produce the corresponding α-halo α, β-saturated carbonyl compound in the cell suspension. The culture by the culture method is subjected to centrifugation to collect the cells, and an α-halocarbonyl compound having an α, β-carbon-carbon double bond as a substrate, for example,
A component containing 0.1% to 10%, preferably 0.2% to 2%, effective for maintaining the pH of the reaction solution, preferably an aqueous buffer such as potassium phosphate or Tris / HCl at 10 mM. The reaction solution is suspended in a reaction solution containing the solution at a reaction temperature of 15 ° C. to 50 ° C., preferably 28 ° C.
C. to 35.degree. C., and the corresponding .alpha.-halo-.alpha.
β-saturated carbonyl compounds can be produced.
The timing, rate or number of additions of the α-halocarbonyl compound having an α, β-carbon-carbon double bond can be freely selected within a range such that the reaction is completed within a target time.

In the case of using a processed microorganism, for example,
A cell-free extract obtained by subjecting a cell obtained by subjecting the culture obtained by the culture method to centrifugal separation and destroying by a method such as a French press has an α, β-carbon-carbon double bond serving as a substrate. The concentration of the α-halocarbonyl compound is 0.1% to
10%, preferably 0.2% to 2%,
H is added to a reaction solution containing a component effective for maintaining H in the range of 10 mM to 1 M, and reacted at a reaction temperature of 15 ° C to 50 ° C, preferably 28 ° C to 35 ° C, and the corresponding α-halo- α, β-saturated carbonyl compounds can be produced.

Further, in the present invention, α, β-carbon
A substance effective for maintaining the reducing activity of the α-halocarbonyl compound having a carbon double bond, for example, a compound such as a sugar or an organic acid that can be oxidized by a microorganism used, preferably glucose or L-lactic acid, During the reaction, alone or as a mixed solution with α-chloroacrylic acid, the reaction was carried out at 0.
The reaction can be carried out while adding continuously or batchwise to a concentration of 1% to 10%, preferably 0.2% to 1%. The addition ratio of α-chloroacrylic acid and these oxidizable substances can be arbitrarily selected between 1: 1 and 20: 1. The reaction time can be extended by the addition of the sugar or organic acid. This makes it possible to increase the concentration of the α-halo-α, β-saturated carbonyl compound, which is the target product, in the reaction solution, which is preferable for isolating and obtaining the product. When the reaction is not being performed during the culture, the reaction can be performed in either an aerobic or anaerobic environment. The reaction is terminated within the target time, depending on the ratio of the bacterial cell or cell-free extract to the α-halocarbonyl compound having an α, β-carbon-carbon double bond as a substrate, or the timing and rate or number of additions of the substrate. You can choose freely within such a range.

In the present invention, the α-halo-α, β-saturated carbonyl compound produced by the reduction of the α-halocarbonyl compound having an α, β-carbon-carbon double bond is a metabolic intermediate for the microorganism used. And may undergo further decomposition. For example, α-chloropropionic acid produced from α-chloroacrylic acid is relatively quickly degraded in some microorganisms. In such cases, use a mutant strain that has lost the degrading activity, or stop the degradation reaction by lowering the pH value, heating the cells or cell-free extract, or adding an appropriate inhibitor of the degrading enzyme. Can be. For example, Pseudomonas SP SD
Strain 811 usually gives the optimal rate of production of α-chloropropionic acid from α-chloroacrylic acid,
The conversion rate of α-chloroacrylic acid to α-chloropropionic acid decreases due to the rapid decomposition of α-chloropropionic acid generated in the culture solution or reaction solution. The pH range of the optimal reaction in the α-chloropropionic acid production step is pH 5.0 to pH 5.0.
7.0, the optimum reaction pH range for the decomposition stage of α-chloropropionic acid is pH 7.0 to 7.3,
Since the range of the optimum reaction pH in the decomposition step of chloropropionic acid is high, the reaction is carried out in the range of the optimum reaction pH in the low α-chloropropionic acid production step of pH 5.0 to 7.0, so that α-chloroform can be obtained. The amount of propionic acid produced can be increased. It is known that one kind of the enzyme that decomposes α-chloropropionic acid is effectively inhibited by hydroxylamine (Soda K. et. Al., J. Bi.
ol. Chem. 272, 3363-3368, 1997).
The decomposition reaction can be inhibited by adding an appropriate amount of hydroxylamine to the culture solution or the reaction solution.

Further, the microbial cells or the cell-free extract used in the present invention can be immobilized on various immobilized carriers by a conventional method, for example, a method such as adsorption, entrapment, or crosslinking. . The type of the carrier is not particularly limited, and for example, a polysaccharide-based material such as cellulose, a polymer-based material, and a protein-based material such as collagen can be used.

The α-halocarbonyl compound having an α, β-carbon-carbon double bond used in the present invention is a prochiral molecule at the α-position. The corresponding α-halo-α, β-saturated carbonyl compound in the absolute configuration S form can be prepared.

The method of culturing the microorganism used in the present invention is not particularly limited as long as it is a method capable of proliferating general aerobic microorganisms, and any carbon source can be used as a carbon source of the medium as long as the microorganism can be used. For example, sugars, acetic acid, lactic acid, and mixtures thereof can be used. As the nitrogen source, for example, ammonium salts such as ammonium sulfate and ammonium phosphate, nitrogen-containing compounds such as meat extract and yeast extract, and mixtures thereof can be used.

In addition, nutrients used in normal culture, such as inorganic salts, trace metal salts, vitamins, etc., in addition to the above components, can be appropriately mixed and used in the medium. Furthermore, if necessary, factors that promote the growth of microorganisms, components effective for maintaining the pH of the medium, and the like can be added. Compounds effective for enhancing the reducing activity of microorganisms, for example, α, β-carbons such as α-chloroacrylic acid.
The α-halocarbonyl compound having a carbon double bond can be used alone or as a mixture with a plurality of carbon sources.

Cultivation of the microorganism used in the present invention can be performed under aerobic conditions used for culturing most aerobic or facultative anaerobic bacteria. For example, medium pH
It can be carried out under the conditions of 5.5 to 8.0, preferably pH 6.5 to 7.0, and the culture temperature of 15 to 35 ° C, preferably 25 to 30 ° C. The culturing time is, for example, 1-144.
Hours, preferably 12 hours to 72 hours.

The α-halo-α, β-saturated carbonyl compound produced in the present invention can be obtained by a conventional purification method such as solvent extraction and distillation. For example, α-chloropropionic acid formed from α-chloroacrylic acid is
It can be obtained by subjecting the culture solution or reaction solution to extraction with an organic solvent, distillation, or the like. An α-halocarbonyl compound having an α, β-carbon-carbon double bond is a prochiral molecule at the α-position, but a chiral α-halo-α, β-saturated carbonyl formed by the reduction method of the present invention. The enantiomeric purity of the compound is determined by the G
Advantageously, it is determined by C, HPLC or polarimeter.

As described above, the present invention uses an aerobic or facultative anaerobic bacterium, and therefore has an α-, β-carbon-carbon double bond and an α-β-carbon double bond excellent in economy, operability and process safety. An object of the present invention is to provide a method for producing a corresponding α-halo-α, β-saturated carbonyl compound in the absolute configuration S by reducing a carbon-carbon double bond of a halocarbonyl compound.

[0052]

EXAMPLES The present invention will be described in more detail in the following examples, but the present invention is not limited to these examples.

Example 1 Detection of reducing activity of α-halocarbonyl compound having α, β-carbon-carbon double bond Yeast extract (0.5 g / l), ammonium sulfate (2 g / l)
l), a basic medium containing sodium dihydrogen phosphate (1 g / l), dipotassium hydrogen phosphate (1 g / l), and magnesium sulfate (0.1 g / l). The microorganisms used were those obtained by adding sodium chloride to the basic medium at a concentration of 10 to 20% as the basic medium), and α-chloroacrylic acid or α-chloro-α, β-
Using a medium supplemented with 0.2% butenoic acid, microorganisms isolated by enrichment culture or conditioned enrichment culture are cultured at 30 ° C., and α-chloropropionic acid, α-chloropropionic acid, The appearance of chlorobutyric acid was examined using gas chromatography. At a specific point in time, 0.5 ml of a sample was collected, and 0.4 ml of 2N HCl was mixed with 0.4 ml of supernatant obtained by subjecting to centrifugation to remove cells,
The analysis was performed under the following conditions.

Main unit: GC-7A (Shimadzu Corporation) Column: Thermon-3000 / SHINCARBON A 2.6 mm × 2.1 m Carrier gas; Nitrogen 50 ml / min. Detection; FID column temperature; 200 ° C (constant) Injection; 2 to 10 microliters, 260 ° C Record; Chromatographic coder 12 (SIC) By the above detection, α- Chloroacrylic acid or α-chloro-
With the consumption of α, β-butenoic acid, the reduction product α
-A peak appeared at the position of chloropropionic acid or α-chlorobutyric acid. When this peak was analyzed by GC-MS, α-chloropropionic acid or α
Is consistent with the mass spectrum of -chlorobutyric acid.
-Chloropropionic acid, α-chlorobutyric acid. When the genus of microorganisms that produced the reduction product in the culture was identified, it was found that microorganisms of various genera exhibited this reducing activity.

The microorganisms whose activity was recognized include those of the genus Acetobacter and Actinomyces.
Genus, Acinetobacter, Agrobacterium, Aeromona
s), Alcaligenes, Arthrobacter, Azotobacte
r), Bacillus, Brevibacterium (Bre
vibacterium), Burkholderia (Burkholderia),
Cellulomonas, Corynebacterium (C
orynebacterium), Enterobacter
Genus, Enterococcus, Escherichia, Flavobacte
rium), Gluconobacter, Halobacterium, Halococc
us), Klebsiella, Lactobacillus (La
ctobacillus), Microbacterium
Genus, micrococcus, micropolyspora, Mycobacterium
rium), Nocardia, Pseudomonas
(Pseudomonas) genus, Pseudonocardia
ia), Rhodococcus, Rhodococcus (R)
hodobacter), Serratia, Staphylococcus, Streptococcus
tococcus), Streptomyces (Streptomyces),
It was an aerobic or facultative anaerobic bacterium belonging to the genus Xanthomonas. In particular, a large number of strains were found in the genus Pseudomonas, and the amount of reduction products was relatively large in Pseudomonas (Pseudomonas).
as) and a microorganism belonging to the genus Burkholderia. Since the amount of reduction products observed in the culture solution was very small (0.01% or less) in all strains, the product accumulation conditions were optimized for the excellent strains below.

Example 2 (1) Culture of Pseudomonas sp. SD811 strain Pseudomonas sp. SD811 strain was cultured in the following medium (amount g / l): α-chloroacrylic acid (2), yeast extract ( 0.5), ammonium sulfate (2), sodium dihydrogen phosphate (1), dipotassium hydrogen phosphate (1), magnesium sulfate (0.1). Preparation of the medium was performed as follows. Of the above components, all components except α-chloroacrylic acid and magnesium sulfate were 9
The pH was adjusted to 7.0 by dissolving in 50 ml of water, placed in a 5 l flask and sterilized at 121 ° C. for 20 minutes. Subsequently, after the temperature of the medium dropped to about 70 ° C, α-
A solution in which chloroacrylic acid and magnesium sulfate were dissolved in 50 ml of water to adjust the pH value to 7.0 was sterilized with a sterilizing filter, and then mixed. Supply more oxygen,
Without adjusting the pH, the medium was inoculated with a 5% preculture (OD660nm = 1.10) and the strain was cultured at 30 ° C.

(2) Detection of α-Chloropropionic Acid in α-Chloroacrylic Acid Culture Solution The α-chloropropionic acid strain of the above-mentioned Pseudomonas sp.
In the culture using chloroacrylic acid, at a specific time point, 0.5 ml of a sample was collected, and 0.4 ml of 2N HCl was mixed with 0.4 ml of the supernatant from which the cells were removed by centrifugation. The analysis was carried out under the conditions shown in FIG.

As a result of the above detection, a peak appears at the position of α-chloropropionic acid in the culture of Pseudomonas sp. The maximum production was about 0.02% of the culture solution, and the conversion to the substrate α-chloroacrylic acid was about 10%.

Example 3 (1) Cell suspension reaction using α-chloroacrylic acid as a substrate-1 The above Pseudomonas sp. SD811 strain was cultured on a 1/10 scale of the method of Example 2. The obtained culture was subjected to centrifugation to collect the cells. This cell is called α
-0.2% chloroacrylic acid, 100 mM phosphate buffer
20 ml of a solution containing (pH 7.3) (adjusted to pH 7.3)
And reacted by shaking at 28 ° C.

At a specific point in time, a 0.5 ml sample was collected from the reaction solution and subjected to centrifugation to remove the bacterial cells.
After mixing 0.1 ml of 6N HCl with 0.4 ml,
The product was extracted with ethyl acetate and the extracted sample was analyzed by the method of Example 1.
-With the consumption of chloroacrylic acid, a peak appears at the position of α-chloropropionic acid, the production of which is at a maximum about 0.05% of the reaction solution, relative to the substrate α-chloroacrylic acid. The conversion was about 25%.

(2) Isolation of α-chloropropionic acid from a cell suspension reaction using α-chloroacrylic acid as a substrate The above Pseudomonas sp. SD811 strain (Pseudom
onas sp. SD811) was cultured on a 1/5 scale of the method of Example 1 and subjected to centrifugation to collect the collected cells, and α-chloroacrylic acid 0.2%, potassium phosphate-
Solution containing 100 mM sodium hydroxide buffer (pH 7.
The mixture was suspended in 100 ml and reacted by stirring at 28 ° C. When α-chloroacrylic acid disappeared during the reaction, 0.2% α-chloroacrylic acid of the reaction solution was further added, and the reaction was continued. Samples at specific time points.
5 ml was collected and the product was extracted and separated by the method of Example 3,
The production amount of α-chloropropionic acid was monitored by the method of Example 1. After about 9 hours, when α-chloroacrylic acid in the reaction solution could not be detected any more, the reaction was terminated, and the entire amount of the reaction solution was subjected to centrifugation to remove cells. 20 ml of 6N HCl was added to 95 ml of the obtained supernatant,
The product was extracted with ml of ethyl acetate. After the ethyl acetate layer was washed with 100 ml of saturated saline, the ethyl acetate was removed with an evaporator and concentrated. When the concentrated sample was analyzed by the method of Example 1, trace amounts of α-chloroacrylic acid and α-chloropropionic acid were detected, the total amount of α-chloropropionic acid contained in the concentrated sample was about 100 mg, and the substrate α-chloropropionic acid was contained. The conversion with respect to acrylic acid was about 25%.

Example 4 (1) Methylation of α-chloropropionic acid When optical resolution is carried out using an optical resolution GC column, unmodified carboxylic acids often have poor separation properties on the GC column due to the effect of carboxyl groups. Therefore, the product was subjected to methyl esterification by a boron trifluoride-methanol complex salt method.
That is, 3 mg of α-chloropropionic acid and methanol 4
After adding and mixing, 1 ml of a 14% methanol solution of boron trifluoride-methanol complex was further added, and the mixture was refluxed for 1 hour on an 80 ° C. oil bath with stirring. One hour later, 30 ml of water was added to the reaction solution, and the reaction product was extracted with 10 ml of ethyl acetate. Ethyl acetate was distilled off with an evaporator to concentrate the entire sample to about 0.1 ml. When the concentrated sample was analyzed under the condition of only the column temperature of 120 ° C. in the method of Example 1, one main peak was observed at the position of α-chloropropionic acid methyl ester. This peak was analyzed by GC-MS to confirm that it was α-chloropropionic acid methyl ester. (2) Optical Resolution GC Analysis of α-Chloropropionic Acid S-form and R-form α-chloropropionic acid methylated by the above method were well separated under the following analysis conditions.

Main unit: GC-14A (Shimadzu Corporation) Column: CP-Chirasil-DEX-DEX CB 0.32 mm ID × 25 m df = 0.25 mm (GL Science Co., Ltd.) Carrier gas; He, 0.38 kg / cm ² detection; FID 275 ° C. column temperature: 70 ° C. (constant) injection; 0.1-0.2 micro litter split about 1: 100, 250 ° C. recorded; Chromatopack C-R6A (Shimadzu) performed in this condition When the α-chloropropionic acid methyl ester obtained from the reaction solution of Example 3 was analyzed, a peak was observed only at the position of the S-form α-chloropropionic acid methyl ester, and the optical purity was 99% or more.

Example 5 Cell suspension reaction using α-chloroacrylic acid as a substrate-2 Culture obtained by culturing the Pseudomonas sp. SD811 strain on a 1/10 scale of the method of Example 2 Is subjected to centrifugation to collect the cells, and the cells are subjected to α-chloroacrylic acid 0.2%, phosphate buffer 100 mM (pH
The solution was suspended in 20 ml of a solution containing 5.7) (adjusted to pH 5.7) and reacted by shaking at 28 ° C. At a specific time, a 0.5 ml sample was collected from the reaction solution, subjected to centrifugation to remove the cells, and 0.4 ml of the supernatant was mixed with 0.1 ml of 6N HCl, followed by 0.4 ml of ethyl acetate. When the product was extracted and the extracted sample was analyzed by the method of Example 1, a peak appeared at the position of α-chloropropionic acid in the reaction solution with the consumption of α-chloroacrylic acid,
The maximum production is about 0.08% of the reaction solution and the substrate α
The conversion to chloroacrylic acid was about 41%.

Example 6 Preparation of α-chloropropionic acid non-degrading mutant strain Pseudomonas sp. SD811 strain and Burkholderia sp. SD816 strain were cultured on a 1/200 scale of the method of Example 2 and obtained. The culture thus obtained was subjected to centrifugation to collect the cells, and each was washed with physiological saline. The washed cells were added to a 10 to 100 mM phosphate buffer (pH
7) Add a final concentration of 50-
N-methyl-N'-nitro-
Add N-nitrosoguanidine (NTG) and add
Treated for ~ 20 minutes. The cell suspension after the treatment is subjected to centrifugation to collect the cells, and the cells are washed with a sterilized physiological saline, and then the whole amount is subjected to L medium (polypeptone 10 g / l, yeast extract 5 g / l,
5 g / l sodium chloride, pH 7)
The cells were cultured with shaking at ℃ overnight. After completion of the culture, the culture was subjected to centrifugation to collect the cells, suspended in a 20% glycerin solution, aliquoted in appropriate amounts, and frozen to obtain glycerin stocks for selection of mutant strains.

Example 7 Selection of α-chloropropionic acid non-degrading mutant strain The glycerin stock for selection of the mutant strain was inoculated into 5 ml of the medium used in the method of Example 2, and cultured at 28 ° C. for 5 hours. . When the turbidity rises several times, the final concentration of 10
0-1000 ppm of penicillin G is added,
Culture was continued at 8 ° C. The culture broth after culturing for 5 to 16 hours is subjected to centrifugation to collect the cells, washed twice with sterile physiological saline, and inoculated in 5 ml of L-broth at 28 ° C.
And cultured overnight with shaking.

In the medium used in Example 2, a medium in which α-chloroacrylic acid as a carbon source was replaced with an equal amount of lactic acid, and a plate obtained by adding 2% agar to the medium and plating the same, The solution was diluted, applied, and cultured at 28 ° C. After 1-2 days, the colony formed on the plate was replicated on a plate obtained by adding 2% agar to the medium used in Example 2, and cultured at 28 ° C for 1 to 2 days. Strains that grew on the lactic acid plate but did not grow on the α-chloroacrylic acid plate were isolated as candidates for α-chloropropionic acid non-degradable mutants.

The isolated candidate strain was inoculated with a platinum loop into 5 ml of a medium containing 2 g / l of lactic acid as a viable carbon source in the medium used in Example 2, and cultured at 28 ° C. with shaking. When the culture was analyzed by the method of Example 1, the maximum amount of α-chloropropionic acid produced in the culture varied depending on the candidate strain, but the maximum amount of α-chloropropionic acid varied depending on the candidate strain. In the strain, a maximum of about 0.17% of the culture solution, a conversion rate to the substrate α-chloroacrylic acid of about 85%, and in a mutant strain of Burkholderia sp.
%, About 75% conversion to the substrate α-chloroacrylic acid
%Met. These strains were selected as non-α-chloropropionic acid-degrading mutants.

Example 8 Bacterial Cell Suspension Reaction Using α-Chloroacrylic Acid as a Substrate-3 The α-chloropropionic acid non-degradable mutant of Pseudomonas sp. SD811 and the Burkholderia sp. SD816 strain Cultures obtained by culturing the α-chloropropionic acid non-degrading mutants in the same medium as used in Example 7 were subjected to centrifugation to collect cells, and the cells were subjected to α-chloropropionic acid-degrading mutants. Chloroacrylic acid 0.2%,
Solution containing 100 mM phosphate buffer (pH 7.3) (pH
Each was suspended in 20 ml, and reacted by shaking at 28 ° C.

From the reaction solution, a 0.5 ml sample was collected at a specific point in time, and subjected to centrifugation to remove the bacterial cells.
After mixing 0.1 ml of 6N HCl with 0.4 ml of ethyl acetate, the product was extracted with 0.4 ml of ethyl acetate, and the extracted sample was analyzed by the method of Example 2.
With the consumption of chloroacrylic acid, a peak appeared at the position of α-chloropropionic acid. When the amount of α-chloroacrylic acid disappeared from the reaction solution, about 0.19% of the reaction solution in the mutant strain of Pseudomonas sp. About 95%, Burke Holderia SP S
About 0.2% and about 100% for the D816 mutant strain, respectively.
%Met. In all strains, the produced α-chloropropionic acid did not decrease over time. As a result of analyzing the optical activity of this α-chloropropionic acid according to the method of Example 4, both were S-forms and the optical purity was 99% or more.

Example 9 Cell Suspension Reaction Using α-Chloroacrylic Acid as Substrate-4 α-Chloropropionic Acid Non-Degrading Mutant of Pseudomonas sp. SD811 and Burkholderia sp. SD816 The culture obtained by culturing the α-chloropropionic acid non-degradable mutant in 100 ml of a medium supplemented with 0.2% glucose as a carbon source in the basic medium used in Example 2 was subjected to centrifugation. The cells were collected, and the cells were treated with α-chloroacrylic acid 0.2%.
%, And 20 ml of a solution (adjusted to pH 7.3) containing 100 mM phosphate buffer (pH 7.3) at 28 ° C.
And reacted.

At a specific time, a 0.5 ml sample was collected from the reaction solution and subjected to centrifugation to remove the supernatant.
After mixing 0.1 ml of 6N HCl with 0.4 ml of ethyl acetate, the product was extracted with 0.4 ml of ethyl acetate, and the extracted sample was analyzed by the method of Example 1. After a lag time of time, a peak appeared at the position of α-chloropropionic acid with the consumption of α-chloroacrylic acid. When the amount of α-chloroacrylic acid disappeared from the reaction solution, about 0.19 of the reaction solution of the mutant strain of Pseudomonas sp.
%, Conversion rate about 95% for the substrate α-chloroacrylic acid
% For the mutant strain of Burkholderia sp. SD816, which was about 0.2% and about 100%, respectively.

Example 10 Culture by Jar Fermenter A non-degrading α-chloropropionic acid-degrading mutant of Burkholderia sp. SD816 was used as a medium (5 times the concentration of the medium used in Example 9). A culture solution cultured in 100 ml of 5 × medium (pH 7.0) for 16 hours was inoculated into 2 liters of 5 × medium stuck in a 5 liter jar fermenter, and cultured at 28 ° C., 800 rpm and aeration rate of 1 ml / min. About 15 to 20 hours after the start of cultivation, glucose consumed in the initial input is completely consumed.
-20% / ammonium sulfate 5-15% / yeast extract 1
A ５5% solution was separately or individually added as a mixture, and continuously added by a Peristack pump. During this time, the rate of addition was adjusted so that the glucose concentration was maintained between 0.02 and 2%. The pH of the culture was adjusted to be maintained between 6.3 and 7.3 with a 20% aqueous ammonia solution. The OD 660 nm after culturing for about 30 to 48 hours by this method was about 30 to 50.

Example 11 Cell suspension reaction using α-chloroacrylic acid as a substrate-5 An α-chloropropionic acid non-degradable mutant of Burkholderia sp. SD816 was cultured according to the method of Example 10. did. For 48 to 72 hours after the start of the culture, the addition of α-chloroacrylic acid was added to the culture solution in which the growth was stopped by stopping the addition of glucose and the like so that the final concentration was about 0.2%, and the reaction was performed under the same conditions as during the culture. I let it. At a specific point in time, a 0.5 ml sample was collected and subjected to centrifugation to remove the supernatant.
After mixing 4 ml of 0.4 ml of 2N HCl, the concentration of α-chloroacrylic acid in the solution was determined under the following conditions.

LC-9A (Shimadzu Corporation) Column; ODSpak F-511 / 4.6 mm × 250 mm (Shodex) Eluent; Acetonitrile / water = 2/8 + 0.1% trifluoroacetic acid, 1 ml / min Detection; SPD -6AV UV-VIS Spectrophotometer (Shimadzu Corporation), 256nm Column temperature; 25 ° C Injection; Autosampler Model123 (SIC) with 20 microliter / sample loop Record; Chromatocoder 12 (SIC) After an hour of lag time, α-chloroacrylic acid began to be consumed. α-
When the concentration of chloroacrylic acid became about 0.02%, α-chloroacrylic acid was additionally added so that the concentration of α-chloroacrylic acid became about 0.2%. This operation was repeated until the consumption of α-chloroacrylic acid substantially stopped. The accumulated production amount of α-chloropropionic acid 32 hours after the reaction was substantially stopped was about 1.0 to 1.0% of the reaction solution.
The conversion was about 97% with respect to the substrate α-chloroacrylic acid. The optical activity of the accumulated α-chloropropionic acid was analyzed according to the method of Example 4, and as a result, it was found to be the S-isomer and the optical purity was 99% or more.

Example 12 Cell suspension reaction using α-chloroacrylic acid as a substrate-6 An α-chloropropionic acid non-degradable mutant of Burkholderia sp. A culture obtained by culturing for ~ 72 hours is subjected to centrifugation to collect cells, and the cells are dissolved in a solution containing 0.2% α-chloroacrylic acid and 60 mM phosphate buffer (pH 7.1). (Adjust to pH 7.1) Suspend in 2 liters, 28 ° C, 800
The reaction was performed at a rpm of 1 ml / min. At a specific point in time, 0.5 ml of the sample was collected, and 0.4 ml of 2N HCl was mixed with 0.4 ml of the supernatant from which the cells were removed by centrifugation. -The chloroacrylic acid concentration was determined.

In this reaction, after a lag time of 5 to 10 hours at the beginning of the reaction, α-chloroacrylic acid began to be consumed. The concentration of α-chloroacrylic acid is about 0.02%
At which point the concentration of α-chloroacrylic acid is 0.2
%, Α-chloroacrylic acid was additionally added.
This operation was repeated until the consumption of α-chloroacrylic acid substantially stopped. 28 hours after the reaction was substantially stopped, the accumulated production of α-chloropropionic acid was about 0.8 to 1.0% of the reaction solution, and the conversion to the substrate α-chloroacrylic acid was about 98%. Met.

Example 13 Microbial cell suspension reaction using α-chloroacrylic acid as a substrate-7 An α-chloropropionic acid non-degradable mutant of Burkholderia sp. The culture obtained by culturing for ~ 72 hours was subjected to centrifugation to collect the cells, and the cells were transferred to a 60 mM phosphate buffer.
(PH 7.1) (suspended to pH 7.1). The microbial cell suspension was reacted at 28 ° C., 800 rpm and aeration rate of 1 ml / min while adding a small amount of a 5-10% α-chloroacrylic acid solution using a Peristack pump. At a specific point in time, a 0.5 ml sample is taken,
0.4 ml of the supernatant, which was subjected to centrifugation to remove the cells,
Was mixed, and the concentration of α-chloroacrylic acid in the liquid was determined by the method described in Example 11.

In this reaction, after a lag time of 5 to 10 hours at the beginning of the reaction, α-chloroacrylic acid began to be consumed. The concentration of α-chloroacrylic acid is about 0.02-
Between 0.2%, preferably around 0.1%,
The addition speed of the α-chloroacrylic acid solution was adjusted according to the consumption speed. This operation was continued until the consumption of α-chloroacrylic acid was substantially stopped. The accumulated production of α-chloropropionic acid 24 hours after the reaction was substantially stopped was about 0.8 to 1.2% of the reaction solution, and the conversion rate to the substrate α-chloroacrylic acid was about 95%. Met.

Example 14 Cell suspension reaction using α-chloroacrylic acid as a substrate-8 An α-chloropropionic acid non-degradable mutant of Burkholderia sp. The culture obtained by culturing for ~ 72 hours was subjected to centrifugation to collect the cells, and the cells were transferred to a 60 mM phosphate buffer.
(PH 7.1) (suspended to pH 7.1). The microbial cell suspension was reacted at 28 ° C., 800 rpm and aeration rate of 1 ml / min while adding a small amount of a 5-10% α-chloroacrylic acid solution using a Peristack pump. At the same time, a 5 to 10% glucose solution or a 5 to 10% sodium lactate solution was added alone or as a mixed solution with α-chloroacrylic acid in small amounts using a Peristack pump. The ratio of α-chloroacrylic acid to these oxidizable substances is from 1: 1 to 20:
It was adjusted to be in the range of 1. At a specific point in time, 0.5 ml of the sample was collected, and 0.4 ml of 2N HCl was mixed with 0.4 ml of the supernatant from which the cells were removed by centrifugation. -The chloroacrylic acid concentration was determined. When glucose was added, the concentration was quantified using a glucose analyzer, and when lactic acid was added, the concentration was quantified using lactate dehydrogenase. The pH of the reaction system was adjusted to 6.5 to 7.3 using 20% aqueous ammonia and 2N HCl.

In this reaction, α-chloroacrylic acid began to be consumed after a lag time of 5 to 10 hours at the beginning of the reaction. The substance to be oxidized was quickly consumed immediately after the start of the reaction. The concentration of α-chloroacrylic acid is about 0.02-
Between 0.2%, preferably around 0.1%,
The addition speed of the α-chloroacrylic acid solution is adjusted according to the consumption speed, and at the same time the concentration of the oxidized substance is 0.4%.
The addition amount was adjusted within the range not exceeding. About 60
Continued until time. This reduction reaction was remarkably sustained by the coexistence of the substance to be oxidized, and proceeded at a constant rate even after about 60 hours. After 60 hours, the accumulated production amount of α-chloropropionic acid was about 2.8 to 3.2% of the reaction solution, and the conversion to the substrate α-chloroacrylic acid was about 99% or more.

Example 15 Cell suspension reaction using α-chloroacrylic acid as a substrate-9 An α-chloropropionic acid non-degrading mutant of Burkholderia sp. The culture obtained by culturing for ~ 72 hours was subjected to centrifugation to collect the cells, and the cells were transferred to a 60 mM phosphate buffer.
(PH 7.1) (suspended to pH 7.1). The mixed solution of 10% α-chloroacrylic acid and 10% glucose was added to the cell suspension at a temperature of 28 ° C. and 80
The reaction was carried out at 0 rpm at a flow rate of 1 ml / min. At a specific point in time, 0.5 ml of a sample was collected, and 0.4 ml of 2N HCl was mixed with 0.4 ml of supernatant obtained by subjecting to centrifugation to remove cells,
The concentration of α-chloroacrylic acid in the liquid was determined by the method described in Example 11. The glucose concentration was determined by subjecting the reaction supernatant obtained by centrifugation to an undiluted solution and using a glucose analyzer. The pH of the reaction system was adjusted to 6.5 to 7.3 using 20% aqueous ammonia and 2N HCl. In this reaction, α-chloroacrylic acid began to be consumed after a lag time of 6 to 12 hours at the beginning of the reaction. Glucose was consumed immediately immediately after the start of the reaction, but was consumed at a constant rate after the reduction activity was stabilized. The mixed solution, taking care that the concentration of α-chloroacrylic acid is between about 0.02 and 0.2%, preferably around 0.1%, and at the same time that the glucose concentration does not exceed 0.4%. The addition speed was adjusted. This operation was continued for about 60 hours. This reduction reaction was remarkably sustained by the coexistence of glucose, and proceeded at a constant rate even after about 60 hours. The conversion to α-chloropropionic acid after 60 hours was about 99% or more.

Example 16 Cell suspension reaction using α-chloroacrylic acid as a substrate-10 An α-chloropropionic acid non-degradable mutant of Burkholderia sp. A culture obtained by culturing for 48 to 72 hours at a scale of / 2 was subjected to centrifugation to collect the cells, and the cells were separated from α-chloroacrylic acid 0.2%, glucose 0.2%, phosphorus Suspended in 1 L of a solution (adjusted to pH 7.1) containing 60 mM (pH 7.1) of an acid buffer, 28 ° C., 800 rpm, aeration rate 1 mL
/ Min. At the end of the lag time of 5 to 10 hours after the start of the reaction, the ventilation was changed from air to nitrogen gas, and then the reaction was performed in an anaerobic environment. At a specific point in time, a 0.5 ml sample was collected and subjected to centrifugation to remove the supernatant.
After mixing 4 ml with 0.4 ml of 2N HCl, Example 1
The α-chloroacrylic acid concentration in the liquid was determined by the method shown in FIG. At the same time, the glucose concentration
It was quantified with an analyzer. The pH of the reaction system was adjusted to 6.5 to 7.3 using 20% ammonia and 2N HCl.

In this reaction, after a lag time of 5 to 10 hours at the beginning of the reaction, α-chloroacrylic acid began to be consumed. The concentration of α-chloroacrylic acid is about 0.02%
At which point the concentration of α-chloroacrylic acid is 0.2
% Or the concentration of glucose was about 0.1%, α-chloroacrylic acid or glucose was additionally added. This operation was continued for about 60 hours. This reduction reaction is remarkably sustained by the coexistence of the substance to be oxidized.
Even after 0 hour, it progressed at a constant speed. Glucose consumption is drastically reduced immediately after the reaction system is moved from the aerobic environment to the anaerobic environment, and the total consumption is about 1/1 of the reaction in the aerobic environment.
0 or less. After 60 hours, the accumulated production amount of α-chloropropionic acid is about 2.5 to 3.2% of the reaction solution,
-The conversion to chloroacrylic acid was about 99% or more.

[0085]

According to the present invention, since an aerobic bacterium or a facultative anaerobic bacterium is used, an α-halo having an α, β-carbon-carbon double bond excellent in economy, operability, and process safety is obtained. Reducing the carbon-carbon double bond of the carbonyl compound,
A method for producing the corresponding α-halo-α, β-saturated carbonyl compound is provided. Furthermore, the carbon-carbon double bond of a prochiral α-halocarbonyl compound having an α, β-carbon-carbon double bond is reduced to obtain a high-purity absolute configuration S useful as a chiral building block for medical and agricultural chemicals. Body α-
Provided is a method for producing a halo-α, β-saturated carbonyl compound.

[Brief description of the drawings]

FIG. 1 shows an example of the accumulated production amount of α-chloropropionic acid when no oxidized substance is added and when it is added.

FIG. 2 shows an example of a comparison of the amount of accumulated and produced α-chloropropionic acid when glucose was not added and when glucose was added.

────────────────────────────────────────────────── ───

[Procedure amendment]

[Document name] Notification of change of accession number

[Submission date] July 28, 1999

[Name of former depositary institution] Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry

[Old Accession Number] P.K.

[Name of the new deposited organization] Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry

[New accession number] FERM BP-6767

[Name of former depositary institution] Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry

[Old Accession Number] P.K.

[Name of the new deposited organization] Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry

[New accession number] FERM BP-6768

[Name of former depositary institution] Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry

[Old Accession Number] P.K.

[Name of the new deposited organization] Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry

[New accession number] FERM BP-6767

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court ゛ (Reference) (C12N 1/20 C12R 1:38) (72) Inventor Motoaki Kamachi 1-chome Onodai, Midori-ku, Chiba-shi, Chiba No. 1-1 Showa Denko KK Research Institute (72) Inventor Nobuyoshi Ezaki 2--21 Kurosu, Otsu City, Shiga Prefecture

Claims (23)

[Claims] (1) α having an α, β-carbon-carbon double bond
-The halocarbonyl compound in Acetobacte (Acetobacte
r) genus, Actinomyces genus, Acinetobacter genus, Agrobacterium (Agroba
cterium), Aeromonas, Alcaligenes, Arthrobact
er), Azotobacter, Bacillus (Bac
illus), Brevibacterium, Burkholderia, Cellulomonas
lomonas), Corynebacterium
Genus, Enterobacter, Enterococcus, Escherichia
a) genus, Flavobacterium genus, Gluconobacter genus, halobacterium (Halo
bacterium), Halococcus (Halococcus), Klebsiella, Lactobacillus
Genus, Microbacterium, Micrococcus, Micropolyspora
pora), Mycobacterium, Nocardia, Pseudomonas
Genus, Pseudonocardia, Rhodococcus, Rhodobacter
Genus, Serratia, Staphylococcus (Staph
ylococcus), Streptococcus
Genus, Streptomyces genus, Xanthomonas (Xanthomonas) using a microorganism belonging to any of the genus or a processed product thereof, α-β-carbon-carbon double bond is characterized by reducing the α-halo A method for producing an α, β-saturated carbonyl compound. 2. An α having an α, β-carbon-carbon double bond.
-Halocarbonyl compound is used in Pseudomonas (Pseudomona
s) using a microorganism belonging to the genus or a processed product thereof,
The method for producing an α-halo-α, β-saturated carbonyl compound according to claim 1, wherein the carbon-carbon double bond is reduced. 3. An α-β having an α, β-carbon-carbon double bond.
Burkholderia
a) using a microorganism belonging to the genus or a processed product thereof,
The method for producing an α-halo-α, β-saturated carbonyl compound according to claim 1, wherein the carbon-carbon double bond is reduced. 4. The microorganism belonging to the genus Pseudomonas sp. Strain SD810 (Pseudomonas sp.
p.SD810). 5. The microorganism belonging to the genus Pseudomonas sp. Strain SD811 (Pseudomonas sp.
The production method according to claim 2, which is p.SD811). 6. The microorganism belonging to the genus Pseudomonas is Pseudomonas sp. Strain SD812 (Pseudomonas sp.
p.SD812). 7. The microorganism belonging to the genus Burkholderia,
Burkholderia sp. SD816 strain (Burkholde
ria sp. SD816). 8. The process according to claim 1, wherein the S-form compound having a chiral α-position is produced by reducing a carbon-carbon double bond. 9. α having an α, β-carbon-carbon double bond
The halocarbonyl compound is represented by the following general formula (1): (Wherein, R ₁ represents a halogen atom; R ₂ and R ₃ independently represent a hydrogen atom, a halogen atom, a linear or branched aliphatic hydrocarbon group having 1 to 6 carbon atoms, and a carbon atom having 1 to 6 carbon atoms) A linear or branched alkoxy group, a hydroxyl group,
R ₄ represents a carboxyl group, an aromatic group which may be substituted or a nitrogen-containing, oxygen-containing or sulfur-containing heterocyclic group;
Represents a hydroxyl group, a linear or branched alkoxy group having 1 to 3 carbon atoms or a primary to tertiary amino group. ), Wherein the α-halo-α, β-saturated carbonyl compound is represented by the following general formula (2): (Wherein, R _{1 to} R ₄ represent the same meaning as described above.)
The method for producing an α-halo-α, β-saturated carbonyl compound according to any one of claims 1 to 8, which is a compound represented by the formula: 10. A compound represented by the general formula (1):
And the compound represented by the general formula (2) is α-halopropionic acid having an absolute configuration S.
The method for producing an α-halo-α, β-saturated carbonyl compound according to the above. 11. The method according to claim 10, wherein the halogen atom is a bromine atom. 12. The method according to claim 10, wherein the halogen atom is a chlorine atom. 13. The production method according to claim 1, wherein the treated microorganism is a microorganism culture, a microorganism extract, a microorganism cell suspension or a microorganism cell fixed substance. 14. The method according to claim 1, wherein a mutation is used in the microorganism to be used so as not to decompose the α-halo-α, β-saturated carbonyl compound to be produced, thereby increasing the accumulation of the product. The production method according to any one of the above. 15. The reaction duration is extended by coexisting an α-halocarbonyl compound having an α, β-carbon-carbon double bond and a compound capable of oxidizing a microorganism to be used in a reaction system. 15. The production method according to any one of 14. 16. The production method according to claim 15, wherein the compound which can be oxidized by the microorganism used is a sugar having 3 to 6 carbon atoms. 17. The production method according to claim 15, wherein the compound which can be oxidized by the microorganism used is an organic acid having 2 to 4 carbon atoms. 18. A Pseudomonas sp. Having an activity of reducing an α, β-carbon-carbon double bond of an α-halocarbonyl compound having an α, β-carbon-carbon double bond.
SD810 strain (Pseudomonas sp. SD810) or a mutant thereof. 19. A Pseudomonas sp. Having an activity of reducing an α, β-carbon-carbon double bond of an α-halocarbonyl compound having an α, β-carbon-carbon double bond.
SD811 strain (Pseudomonas sp. SD811) or a mutant thereof. 20. A Pseudomonas sp. Having an activity of reducing an α, β-carbon-carbon double bond of an α-halocarbonyl compound having an α, β-carbon-carbon double bond.
SD812 strain (Pseudomonas sp. SD812) or a mutant thereof. 21. A Burkholderia sp. SD816 strain having an activity of reducing an α, β-carbon-carbon double bond of an α-halocarbonyl compound having an α, β-carbon-carbon double bond. .SD816) or a mutant thereof. A processed product of the microorganism according to any one of claims 19 to 21. 23. A microorganism culture, a microorganism extract, a microorganism cell suspension or a microorganism cell fixed substance.
A treated product of the microorganism according to the above.