U.S. patent application number 09/369144 was filed with the patent office on 2002-03-07 for method of producing alpha-halo-alpha, beta-saturated carbonyl compounds from the corresponding alpha, beta unsurated compounds.
Invention is credited to ESAKI, NOBUYOSHI, KAMACHI, HARUMI, KAMACHI, MOTOAKI, YONEDA, TADASHI.
Application Number | 20020028491 09/369144 |
Document ID | / |
Family ID | 27330956 |
Filed Date | 2002-03-07 |
United States Patent
Application |
20020028491 |
Kind Code |
A1 |
KAMACHI, HARUMI ; et
al. |
March 7, 2002 |
METHOD OF PRODUCING ALPHA-HALO-ALPHA, BETA-SATURATED CARBONYL
COMPOUNDS FROM THE CORRESPONDING ALPHA, BETA UNSURATED
COMPOUNDS
Abstract
A method of producing an .alpha.-halo-.alpha.,.beta.-saturated
carbonyl compound from an .alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond by reducing said
.alpha.,.beta.-carbon-carbon double bond using a microorganism
belonging to any one of the genera Acetobacter, Actinomyces,
Acinetobacter, Agrobacterium, Aeromonas, Alcaligenes, Arthrobacter,
Azotobacter, Bacillus, Brevibacterium, Burkholderia, Cellulomonas,
Corynebacterium, Enterobacter, Enterococcus, Escherichia,
Flavobacterium, Gluconobacter, Halobacteium, Halococccus,
Klebsiella, Lactobacillus, Microbacterium, Micrococcus,
Micropolyspora, Mycobacterium, Nocardia, Pseudomonas,
Pseudonocardia, Rhodococcus, Rhodobacter, Serratia, Staphylococcus,
Streptococcus and Streptomyces, Xanthomonas, or a microbial product
thereof. Pseudomonas sp. SD810, SD811 and SD812, Burkholderia sp.
SD 816, and mutants thereof having an activity of reducing the
.alpha.,.beta.-carbon-carbon double bond of an .alpha.-halocarbonyl
compound having an .alpha.,.beta.-carbon-carbon double bond.
Inventors: |
KAMACHI, HARUMI; (CHIBA,
JP) ; YONEDA, TADASHI; (CHIBA, JP) ; KAMACHI,
MOTOAKI; (CHIBA, JP) ; ESAKI, NOBUYOSHI;
(SHIGA, JP) |
Correspondence
Address: |
SUGHRUE MION ZINN MACPEAK & SEAS PLLC
2100 PENNSYLVAINIA AVENUE N W
WASHINGTON
DC
200373202
|
Family ID: |
27330956 |
Appl. No.: |
09/369144 |
Filed: |
August 5, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60138085 |
Jun 8, 1999 |
|
|
|
Current U.S.
Class: |
435/121 |
Current CPC
Class: |
C12P 7/52 20130101; C12P
7/24 20130101; C12P 7/40 20130101 |
Class at
Publication: |
435/121 |
International
Class: |
C12P 017/10; C07C
001/00; C07F 001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 7, 1998 |
JP |
HEI. 10-224821 |
Claims
What is claimed is:
1. A method of producing an .alpha.-halo-.alpha.,.beta.-saturated
carbonyl compound from an .alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond by reducing said
.alpha.,.beta.-carbon-carbon double bond comprising culturing a
microorganism belonging to any one of the genera Acetobacter,
Actinomyces, Acinetobacter, Agrobacterium, Aeromonas, Alcaligenes,
Arthrobacter, Azotobacter, Bacillus, Brevibacterium, Burkholderia,
Cellulomonas, Corynebacterium, Enterobacter, Enterococcus,
Escherichia, Flavobacterium, Gluconobacter, Halobacteium,
Halococccus, Klebsiella, Lactobacillus, Microbacterium,
Micrococcus, Micropolyspora, Mycobacterium, Nocardia, Pseudomonas,
Pseudonocardia, Rhodococcus, Rhodobacter, Serratia, Staphylococcus,
Streptococcus, Streptomyces and Xanthomonas, or a microbial product
thereof in the presence of said .alpha.-halocarbonyl compound
having an .alpha.,.beta.-carbon-carbon double bond and recovering
said .alpha.-halo-.alpha.,.beta.-saturated carbonyl compound.
2. The method of producing an .alpha.-halo-.alpha.,.beta.-saturated
carbonyl compound as claimed in claim 1, wherein said method
comprises reducing said .alpha.,.beta.-carbon-carbon double bond of
the .alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond using a microorganism
belonging to the genus Pseudomonas or a microbial product
thereof.
3. The method of producing an .alpha.-halo-.alpha.,.beta.-saturated
carbonyl compound as claimed in claim 1, wherein said method
comprises reducing said .alpha.,.beta.-carbon-carbon double bond of
the .alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond using a microorganism
belonging to the genus Burkholderia or a microbial product
thereof.
4. The method of producing an .alpha.-halo-.alpha.,.beta.-saturated
carbonyl compound as claimed in claim 2, wherein the microorganism
belonging to the genus Pseudomonas is Pseudomonas sp. SD810.
5. The method of producing an .alpha.-halo-.alpha.,.beta.-saturated
carbonyl compound as claimed in claim 2, wherein the microorganism
belonging to the genus Pseudomonas is Pseudomonas sp. SD811.
6. The method of producing an .alpha.-halo-.alpha.,.beta.-saturated
carbonyl compound as claimed in claim 2, wherein the microorganism
belonging to the genus Pseudomonas is Pseudomonas sp. SD812.
7. The method of producing an .alpha.-halo-.alpha.,.beta.-saturated
carbonyl compound as claimed in claim 3, wherein the microorganism
belonging to the genus Burkholderia is Burkholderia sp. SD816.
8. The method of producing an .alpha.-halo-.alpha.,.beta.-saturated
carbonyl compound as claimed in claim 1, wherein said method
comprises producing an S-form compound chiral at the
.alpha.-position by the reduction of the carbon-carbon double
bond.
9. The method of producing an .alpha.-halo-.alpha.,.beta.-saturated
carbonyl compound as claimed in claim 1, wherein the
.alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond is a compound represented
by the following formula (1): 4wherein R.sub.1 represents a halogen
atom, R.sub.2 and R.sub.3 each independently represents a hydrogen
atom, a halogen atom, a linear or branched aliphatic hydrocarbon
group having from 1 to 6 carbon atoms, a linear or branched alkoxy
group having from 1 to 6 carbon atoms, a hydroxyl group, a carboxyl
group, an aromatic group which may be substituted, or a nitrogen-,
oxygen- or sulfur-containing heterocyclic group, and R.sub.4
represents a hydroxyl group, a linear or branched alkoxy group
having from 1 to 3 carbon atoms or a primary, secondary or tertiary
amino group) and the .alpha.-halo-.alpha.,.beta.-saturated carbonyl
compound is a compound represented by the following formula (2):
5wherein R.sub.1 to R.sub.4 have the same meanings as defined
above.
10. The method of producing an
.alpha.-halo-.alpha.,.beta.-saturated carbonyl compound as claimed
in claim 9, wherein the compound represented by formula (1) is an
.alpha.-haloacrylic acid and the compound represented by formula
(2) is an .alpha.-halopropionic acid having an absolute S form
configuration.
11. The method of producing an
.alpha.-halo-.alpha.,.beta.-saturated carbonyl compound as claimed
in claim 10, wherein the halogen atom is a bromine atom.
12. The method of producing an
.alpha.-halo-.alpha.,.beta.-saturated carbonyl compound as claimed
in claim 10, wherein the halogen atom is a chlorine atom.
13. The method of producing an
.alpha.-halo-.alpha.,.beta.-saturated carbonyl compound as claimed
in claim 1, wherein the microbial product of a microorganism is a
microbial culture, a microbial extract, a microbial cell suspension
or a microbial cell fixed to a support.
14. The method of producing an
.alpha.-halo-.alpha.,.beta.-saturated carbonyl compound as claimed
in claim 1, wherein the microorganism used varies so as to not
decompose the .alpha.-halo-.alpha.,.beta.-saturated carbonyl
compound produced, thereby increasing the amount of the product
accumulated.
15. The method of producing an
.alpha.-halo-.alpha.,.beta.-saturated carbonyl compound as claimed
in claim 1, wherein the .alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond and a compound capable of
being oxidized by the microorganism used are present together in
the reaction system and thereby the reaction continuing time is
prolonged.
16. The method of producing an
.alpha.-halo-.alpha.,.beta.-saturated carbonyl compound as claimed
in claim 15, wherein the compound capable of being oxidized by the
microorganism used is a sugar having from 3 to 6 carbon atoms.
17. The method of producing an
.alpha.-halo-.alpha.,.beta.-saturated carbonyl compound as claimed
in claim 15, wherein the compound capable of being oxidized by the
microorganism used is an organic acid having from 2 to 4 carbon
atoms.
18. Pseudomonas sp. SD810 and mutants thereof having an activity of
reducing the .alpha.,.beta.-carbon-carbon double bond of an
.alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond.
19. Pseudomonas sp. SD811 and mutants thereof having an activity of
reducing the .alpha.,.beta.-carbon-carbon double bond of an
.alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond.
20. Pseudomonas sp. SD812 and mutants thereof having an activity of
reducing the .alpha.,.beta.-carbon-carbon double bond of an
.alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond.
21. Burkholderia sp. SD816 and mutants thereof having an activity
of reducing the .alpha.,.beta.-carbon-carbon double bond of an
.alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond.
22. A microbial product containing the microorganism described in
any one of claims 18 to 21.
23. The microbial product as claimed in claim 22, which is a
microbial culture, a microbial extract, a microbial cell suspension
or a microbial cell fixed to a support.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is an application filed under 35 U.S.C.
.sctn.111(a) claiming benefit pursuant to 35 U.S.C. .sctn.119(e)(i)
of the filing date of the Provisional Application No. 60/138,085
filed Jun. 8, 1999 pursuant to 35 U.S.C. .sctn.111(b).
FIELD OF THE INVENTION
[0002] The present invention relates to a method of producing a
corresponding .alpha.-halo-.alpha.,.beta.-saturated carbonyl
compound from an .alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-car- bon double bond by hydrogenating the
.alpha.,.beta.-carbon-carbon double bond using a microorganism
belonging to the genus Acetobacter, Actinomyces, Acinetobacter,
Agrobacterium, Aeromonas, Alcaligenes, Arthrobacter, Azotobacter,
Bacillus, Brevibacterium, Burkholderia, Cellulomonas,
Corynebacterium, Enterobacter, Enterococcus, Escherichia,
Flavobacterium, Gluconobacter, Halobacteium, Halococccus,
Klebsiella, Lactobacillus, Microbacterium, Micrococcus,
Micropolyspora, Mycobacterium, Nocardia, Pseudomonas,
Pseudonocardia, Rhodococcus, Rhodobacter, Serratia, Staphylococcus,
Streptococcus, Streptomyces or Xanthomonas, preferably a
microorganism belonging to the genus Pseudomonas or Burkholderia,
more preferably Pseudomonas sp. SD810, Pseudomonas sp. SD811,
Pseudomonas sp. SD812 or Burkholderia sp. SD816, or a microbial
product thereof. The present invention also relates to novel
microorganisms belonging to the genera Pseudomonas and
Burkholderia, particularly Pseudomonas sp. SD810, Pseudomonas sp.
SD811, Pseudomonas sp. SD812 and Burkholderia sp. SD816.
[0003] Furthermore, the present invention relates to a method of
producing a corresponding .alpha.-halo-.alpha.,.beta.-saturated
carbonyl compound as an S form compound with respect to the
.alpha.-position from an .alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond by hydrogenating the
carbon-carbon double bond. This method can be used in the
production of optically active carbonyl compounds such as various
optically active (having an absolute S form configuration at the
.alpha.-position) saturated carboxylic acids or amides. The
optically active carbonyl compounds are a highly valuable chiral
building block which is difficult to prepare by classical chemical
processes, and are materials useful particularly as a raw material
of medical or agricultural chemicals.
BACKGROUND OF THE INVENTION
[0004] In recent years, a method of producing various compounds,
particularly optically active substances, by the reduction of a
carbon-carbon double bond using a microorganism is drawing
attention. To this effect, various methods of producing a
corresponding .alpha.,.beta.-saturated carbonyl compound having a
substituent at the .alpha.-position from a carbonyl compound having
an .alpha.,.beta.-carbon-carbon double bond and having a
substituent at the .alpha.-position by microbially reducing the
carbon-carbon double bond have been reported (see, H. Simon, et
al., Hoppe-Seyler's Z. Physiol. Chem., 362, 33 (1981), H. Giesel,
et al., Arch. Microbiol., 135, 51 (1983), H. G. W. Leuenberger, et
al., Helv. Chim. Acta., 62, 455 (1979), R. Matsuno, et al., J.
Ferm. Bioeng., 84, 195 (1997)). However, for example, according to
the method of using bacteria as the microorganism, an anaerobe such
as Clostridium kluyveri (DSM-555) or Clostridium sp. La-1
(DSM-1460) is used. Therefore, the growing rate of the
microorganism is slow, it is difficult to increase the cell
concentration and accordingly, the reaction rate is not
satisfactorily high. Thus, these methods have a problem in
profitability and operability.
[0005] The method using Clostridium theremosaccharolyticum
disclosed in JP-A-63-003794 (the term "JP-A" as used herein means
an "unexamined published Japanese patent application") has an
object of solving the above-descried problem by using a
thermophilic bacterium. However, the bacterium used is still
anaerobic, therefore, the growing rate and the reaction rate both
are not satisfactorily high and the process involves use of
hydrogen. Thus, the method fails in solving the problems in
profitability and safety. Furthermore, the .alpha.,.beta.-saturated
carbonyl compound having a substituent at the .alpha.-position
produced by reducing a prochiral carbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond and having a substituent
at the .alpha.-position using a microorganism is a compound having
an absolute R form configuration and an S form configuration
compound cannot be produced.
[0006] The method of reducing the .alpha.,.beta.-carbon-carbon
double bond using a bread yeast as the microorganism has
general-purpose applicability because compounds over a wide range
can be reduced. In addition, since the microorganism used is
aerobic, good operability can be attained. Furthermore, the
optically active substances produced include both S form and R
form, therefore, this method is most abundant in the cases
reported. However, the yeast grows slowly as compared with
bacteria, the optical selectivity is not sufficiently high in many
cases in the reduction reaction for obtaining a more optically
active product, and reduction of an .alpha.-halocarbonyl compound
having an .alpha.,.beta.-carbon-carbon double bond is not
known.
[0007] As described above, in the technique of producing a
corresponding .alpha.-halo-.alpha.,.beta.-saturated carbonyl
compound from an .alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond by reducing the
carbon-carbon double bond using a microorganism, a method
satisfying all of the requirements regarding operability,
profitability, safety and reaction properties is not yet known.
SUMMARY OF THE INVENTION
[0008] An object of the present invention is to provide a method of
producing a corresponding .alpha.-halo-.alpha.,.beta.-saturated
carbonyl compound from an .alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond by reducing the
carbon-carbon double bond using a microorganism, which method can
satisfy all of the requirements for operability, profitability,
safety and reaction properties and ensure excellent optical
selectivity.
[0009] As a result of thorough screening from soil, the present
inventors have found that surprisingly, microorganisms capable of
producing a corresponding .alpha.-halo-.alpha.,.beta.-saturated
carbonyl compound from an .alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-car- bon double bond by reducing the
carbon-carbon double bond are distributed over a relatively wide
genus range of the aerobes and facultative anaerobes. In
particular, it has been found that strains having this activity are
present in a large number in microorganisms belonging to the genera
Pseudomonas and Burkholderia, and some of these strains can reduce
an .alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond and thereby produce an
extremely high-purity .alpha.-halo-.alpha.,.beta.-saturated
carbonyl compound having an absolute configuration of S form at the
.alpha.-position. The present invention has been accomplished based
on these findings.
[0010] More specifically, the present invention relates to the
following embodiments:
[0011] [1] a method of producing an
.alpha.-halo-.alpha.,.beta.-saturated carbonyl compound from an
.alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond by reducing the
.alpha.,.beta.-carbon-carbon double bond using a microorganism
belonging to any one of the genera Acetobacter, Actinomyces,
Acinetobacter, Agrobacterium, Aeromonas, Alcaligenes, Arthrobacter,
Azotobacter, Bacillus, Brevibacterium, Burkholderia, Cellulomonas,
Corynebacterium, Enterobacter, Enterococcus, Escherichia,
Flavobacterium, Gluconobacter, Halobacteium, Halococccus,
Klebsiella, Lactobacillus, Microbacterium, Micrococcus,
Micropolyspora, Mycobacterium, Nocardia, Pseudomonas,
Pseudonocardia, Rhodococcus, Rhodobacter, Serratia, Staphylococcus,
Streptococcus, Streptomyces and Xanthomonas, or a microbial product
thereof;
[0012] [2] the method of producing an
.alpha.-halo-.alpha.,.beta.-saturate- d carbonyl compound as
described in [1], wherein the .alpha.,.beta.-carbon-carbon double
bond of the .alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond is reduced using a
microorganism belonging to the genus Pseudomonas or a microbial
product thereof;
[0013] [3] the method of producing an
.alpha.-halo-.alpha.,.beta.-saturate- d carbonyl compound as
described in [1], wherein the .alpha.,.beta.-carbon-carbon double
bond of the .alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond is reduced using a
microorganism belonging to the genus Burkholderia or a microbial
product thereof;
[0014] [4] the method of producing an
.alpha.-halo-.alpha.,.beta.-saturate- d carbonyl compound as
described in [2], wherein the microorganism belonging to the genus
Pseudomonas is Pseudomonas sp. SD810;
[0015] [5] the method of producing an
.alpha.-halo-.alpha.,.beta.-saturate- d carbonyl compound as
described in [2], wherein the microorganism belonging to the genus
Pseudomonas is Pseudomonas sp. SD811;
[0016] [6] the method of producing an
.alpha.-halo-.alpha.,.beta.-saturate- d carbonyl compound as
described in [2], wherein the microorganism belonging to the genus
Pseudomonas is Pseudomonas sp. SD812;
[0017] [7] the method of producing an
.alpha.-halo-.alpha.,.beta.-saturate- d carbonyl compound as
described in [3], wherein the microorganism belonging to the genus
Burkholderia is Burkholderia sp. SD816;
[0018] [8] the method of producing an
.alpha.-halo-.alpha.,.beta.-saturate- d carbonyl compound as
described in [1] to [7], wherein an S-form compound chiral at the
.alpha.-position is produced by the reduction of the carbon-carbon
double bond;
[0019] [9] the method of producing an
.alpha.-halo-.alpha.,.beta.-saturate- d carbonyl compound as
described in [1] to [8], wherein the .alpha.-halocarbonyl compound
having an .alpha.,.beta.-carbon-carbon double bond is a compound
represented by the following formula (1): 1
[0020] wherein R.sub.1 represents a halogen atom, R.sub.2 and
R.sub.3 each independently represents a hydrogen atom, a halogen
atom, a linear or branched aliphatic hydrocarbon group having from
1 to 6 carbon atoms, a linear or branched alkoxy group having from
1 to 6 carbon atoms, a hydroxyl group, a carboxyl group, an
aromatic group which may be substituted, or a nitrogen-, oxygen- or
sulfur-containing heterocyclic group, and R.sub.4 represents a
hydroxyl group, a linear or branched alkoxy group having from 1 to
3 carbon atoms or a primary, secondary or tertiary amino group) and
the .alpha.-halo-.alpha.,.beta.-saturated carbonyl compound is a
compound represented by the following formula (2): 2
[0021] wherein R.sub.1 to R.sub.4 have the same meanings as defined
above;
[0022] [10] the method of producing an
.alpha.-halo-.alpha.,.beta.-saturat- ed carbonyl compound as
described in [9], wherein the compound represented by formula (1)
is an .alpha.-haloacrylic acid and the compound represented by
formula (2) is an .alpha.-halopropionic acid having an absolute S
form configuration;
[0023] [11] the method of producing an
.alpha.-halo-.alpha.,.beta.-saturat- ed carbonyl compound as
described in [10], wherein the halogen atom is a bromine atom;
[0024] [12] the method of producing an
.alpha.-halo-.alpha.,.beta.-saturat- ed carbonyl compound as
described in [10], wherein the halogen atom is a chlorine atom;
[0025] [13] the method of producing an
.alpha.-halo-.alpha.,.beta.-saturat- ed carbonyl compound as
described in [1] to [12], wherein the microbial product of a
microorganism is a microorganism culture, a microbial extract, a
microbial cell suspension or a microbial cell fixed to a
support;
[0026] [14] the method of producing an
.alpha.-halo-.alpha.,.beta.-saturat- ed carbonyl compound as
described in [1] to [13], wherein the microorganism used is varied
not to decompose the .alpha.-halo-.alpha.,.b- eta.-saturated
carbonyl compound produced, thereby increasing the amount of the
product accumulated;
[0027] [15] the method of producing an
.alpha.-halo-.alpha.,.beta.-saturat- ed carbonyl compound as
described in [1] to [14], wherein the .alpha.-halocarbonyl compound
having an .alpha.,.beta.-carbon-carbon double bond and a compound
capable of being oxidized by the microorganism used are present
together in the reaction system and thereby the reaction continuing
time is prolonged;
[0028] [16] the method of producing an
.alpha.-halo-.alpha.,.beta.-saturat- ed carbonyl compound as
described in [15], wherein the compound capable of being oxidized
by the microorganism used is a sugar having from 3 to 6 carbon
atoms;
[0029] [17] the method of producing an
.alpha.-halo-.alpha.,.beta.-saturat- ed carbonyl compound as
described in [15], wherein the compound capable of being oxidized
by the microorganism used is an organic acid having from 2 to 4
carbon atoms;
[0030] [18] Pseudomonas sp. SD810 and mutants thereof having an
activity of reducing the .alpha.,.beta.-carbon-carbon double bond
of an .alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond;
[0031] [19] Pseudomonas sp. SD811 and mutants thereof having an
activity of reducing the .alpha.,.beta.-carbon-carbon double bond
of an .alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond;
[0032] [20] Pseudomonas sp. SD812 and mutants thereof having an
activity of reducing the .alpha.,.beta.-carbon-carbon double bond
of an .alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond;
[0033] [21] Burkholderia sp. SD816 and mutants thereof having an
activity of reducing the .alpha.,.beta.-carbon-carbon double bond
of an .alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond;
[0034] [22] a microbial product containing the microorganism
described in [12] to [21]; and
[0035] [23] the microbial product as described in [22], which is a
microbial culture, a microbial extract, a microbial cell suspension
or a microbial cell fixed to a support.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 shows an example of the accumulative production of
.alpha.-chloropropionic acid in the cases where a substance to be
oxidized is not present and where such is present.
[0037] FIG. 2 shows an example of the accumulative production of
.alpha.-chloropropionic acid in the cases where glucose is not
present and where such is present.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0038] The microorganisms which can be used in the present
invention are microorganisms belonging to any one of the genera
Acetobacter, Actinomyces, Acinetobacter, Agrobacterium, Aeromonas,
Alcaligenes, Arthrobacter, Azotobacter, Bacillus, Brevibacterium,
Burkholderia, Cellulomonas, Corynebacterium, Enterobacter,
Enterococcus, Escherichia, Flavobacterium, Gluconobacter,
Halobacteium, Halococccus, Klebsiella, Lactobacillus,
Microbacterium, Micrococcus, Micropolyspora, Mycobacterium,
Nocardia, Pseudomonas, Pseudonocardia, Rhodococcus, Rhodobacter,
Serratia, Staphylococcus, Streptococcus, Streptomyces and
Xanthomonas.
[0039] Among these, microorganisms belonging to the genera
Pseudomonas and Burkholderia are preferred. The strain is not
particularly limited as long as it has an activity of reducing the
.alpha.,.beta.-carbon-carbon double bond of an .alpha.-halocarbonyl
compound having an .alpha.,.beta.-carbon-carbon double bond.
However, for example, Pseudomonas sp. SD810, Pseudomonas sp. SD811,
Pseudomonas sp. SD812 and Burkholderia sp. SD816 are preferably
used. Among these, Pseudomonas sp. SD811 and Burkholderia sp. SD816
are more preferred. The strains Pseudomonas sp. SD810, Pseudomonas
sp. SD811, Pseudomonas sp. SD812 and Burkholderia sp. SD816 are
strains isolated from soil and have an activity of decomposing and
assimilating various carbonyl compounds.
[0040] The above-described microorganisms may be any of a wild
type, variant and recombinant induced by cell fusion or genetic
engineering as long as the strain has an activity of reducing the
carbon-carbon double bond of an .alpha.-halocarbonyl compound
having an .alpha.,.beta.-carbon-carbon double bond. For example, a
variant reduced or defective in activity of decomposing the
product, a variant or recombinant improved in reducing activity or
a variant improved in resistance against a high-concentration
substrate or product may be preferably used.
[0041] Examples of the isolation and cultivation of these strains
are described below.
[0042] 5 ml of a minimum medium obtained by adding, as a
substantially sole carbon source, 2 g/l of an
.alpha.,.beta.-unsaturated carbonyl compound having a halogen atom
as a substituent at the .alpha.-position, such as
.alpha.-chloroacrylic acid, to an inorganic salt culture medium
(for example, (NH.sub.4).sub.2SO.sub.4: 2 g/l, NaH.sub.2PO.sub.4: 1
g/l, K.sub.2HPO.sub.4: 1 g/l, MgSO.sub.4: 0.1 g/l, yeast extract:
0.5 g/l) used for normal bacteria is poured in a test tube and
sterilized. Thereafter, about 0.1 g of soil is added thereto and
cultivation by shaking is performed at 28.degree. C. Subculture is
performed every single day. After repeating this accumulative
cultivation for 6 days, the culture is spread on the agar plate of
minimum medium described above containing 20 g/l of agar as a
solidifying agent, and cultured at 28.degree. C. for 3 days. By the
isolation of colonies produced, the strain Pseudomonas sp. SD810,
Pseudomonas sp. SD811 or Pseudomonas sp. SD812 may be obtained.
These strains Pseudomonas sp. SD810, Pseudomonas sp. SD811 and
Pseudomonas sp. SD812 are deposited with National Institute of
Bioscience and Human-Technology under the accession numbers BP-6767
(FERM BP-6767) (transferred from accession number 16746
(FERM-16746)), BP-6768 (FERM BP-6768) (transferred from accession
number 16747 (FERM-16747)) and BP-6769 (FERM BP-6769) (transferred
from accession number 16748 (FERM-16748)), respectively.
[0043] Examples of the isolation and cultivation of the strains
having a relatively small population in isolation source are
described below.
[0044] By accumulative acclimatization using soil as the separation
source, microorganisms having a small population, microorganisms
having a low assimilating activity or microorganisms having a low
resistance against .alpha.,.beta.-unsaturated carbonyl compounds
having a halogen atom as the substituent at the .alpha.-position
(hereinafter sometimes referred to as a "halogen-containing
compound"), such as .alpha.-chloroacrylic acid, may be separated.
More specifically, 0.01% (in the present specification, unless
otherwise indicated, "%" is "weight (g)/volume (100 ml).times.100")
of various halogen-containing compounds are added as a
substantially sole carbon source or added together with glucose in
the same concentration to a basal medium containing yeast extract
(0.5 g/l), ammonium sulfate (2 g/l), sodium dihydrogenphosphate (1
g/l), dipotassium hyrogenphosphate (1 g/l) and magnesium sulfate
(0.1 g/l) (for the isolation of some microorganisms which require a
high salt concentration to grow, a medium obtained by adding sodium
chloride in a concentration of from 10 to 20% to the above-descried
basal medium is used as the basal medium). In 10 ml of the
thus-prepared medium, 1 g of soil collected is suspended and
cultured by shaking at 30.degree. C. After 24 hours, 5 ml of the
supernatant of the culture is sampled and after adding thereto 5 ml
of a new medium having the same composition as above except that
the concentration of halogen-containing compounds is 0.02%,
cultured by shaking at 30.degree. C. (first subculture). After 24
hours, 5 ml of the supernatant of the culture was sampled and after
adding thereto 5 ml of the medium where the concentration of
halogen-containing compounds in the medium is increased from 0.02%
to 0.1%, cultured by shaking at 30.degree. C. (second subculture).
This operation is further repeated 3 times every 24 hours (third to
fifth subculture). At the sixth subculture, the concentration of
halogen-containing compounds in the medium added to the culture is
increased to 0.2% and 5 ml of the supernatant is sampled and after
adding thereto 5 ml of a new medium, cultured by shaking at
30.degree. C. This operation is repeated 6 times (sixth to twelfth
subculture) every 24 hours. At each time of these 6 subculture
operations, a part of the supernatant of the culture is spread on a
agar plate obtained by adding 2% of agar to the above-described
medium containing 0.2% of halogen containing compounds, and
cultured at 30.degree. C. The colonies produced are pure separated
and then, for example, Burkholderia sp. SD816 may be obtained.
Burkholderia sp. SD816 is deposited with National Institute of
Bioscience and Human-Technology under the accession number BP-6770
(FERM BP-6770).
[0045] The taxonomic test results of these strains are shown
below.
[0046] Pseudomonas sp. SD810
1 Morphology: (1) Shape and size of cell rod 0.6-1.0 .mu.m .times.
1.2-3.0 .mu.m (2) Motility motile (3) Gram staining negative (4)
Spore none (5) Bacteriolysis by 3% KOH positive Physiological
activity: (1) Aminopeptidase positive (2) Oxidase positive (3)
Catalase positive (4) Production of indole negative (5) VP test
negative (6) Reduction of nitrate negative (7) Denitrification
negative (8) Use of citric acid (Simons) positive (9) Urease
negative (10) Phenylalanine deaminase negative (11) Use of maronic
acid positive (12) Production of levan from sucrose positive (13)
Lecithinase negative (14) Hydrolysis of starch negative (15)
Hydrolysis of gelatin negative (16) Hydrolysis of casein negative
(17) Hydrolysis of DNA negative (18) Hydrolysis of Tween 80
negative (19) Hydrolysis of exrin negative (20) Growth Behavior to
oxygen obligately aerobic Growth at 37.degree. C. - Growth at
41.degree. C. - Growth at pH 5.6 + Growth in Mac-Conkey-Agar medium
- Growth in SS-Agar medium - Growth in Cetrimid-Agar - (21)
Production of dye Nondiffusive negative Diffusive negative
Fluorescent negative Pyrocyanine negative (22) OF Test no
decomposition of sugar (23) Formation of acids Glucose negative
Fructose positive Xylose negative (24) ONPG (.beta.-Galactosidase)
negative (25) Arginine dihydrolase negative (26) Production of gas
from glucose negative (27) Tyrosine decomposition negative (28)
Growth factor request none (29) Use 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 + Saccharic
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 -
[0047] When these results were taxonomically examined based on
Bergey's Manual of Systematic Bacteriology (1986), it was found
that this strain belongs to the genus Pseudomonas but the
properties thereof did not coincide with those of standard strains.
Therefore, this strain was named Pseudomonas sp. SD810.
[0048] Pseudomonas sp. SD811
2 Morphology: (1) Shape and size of cell rod 0.7-0.9 .mu.m .times.
1.5-3.0 .mu.m (2) Motility motile (3) Gram staining negative (4)
Spore none (5) Bacteriolysis by 3% KOH positive Physiological
activity: (1) Aminopeptidase positive (2) Oxidase positive (3)
Catalase positive (4) Production of indole negative (5) VP test
negative (6) Reduction of nitrate negative (7) Denitrification
negative (8) Use of citric acid (Simons) positive (9) Urease
negative (10) Phenylalanine deaminase negative (11) Use of malonic
acid positive (12) Production of levan from sucrose negative (13)
Lecithinase negative (14) Hydrolysis of starch negative (15)
Hydrolysis of gelatin negative (16) Hydrolysis of casein negative
(17) Hydrolysis of DNA negative (18) Hydrolysis of Tween 80
positive (19) Hydrolysis of exrin negative (20) Growth Behavior to
oxygen obligately aerobic Growth at 37.degree. C. - Growth at
41.degree. C. - Growth at pH 5.6 + Growth in Mac-Conkey-Agar medium
- Growth in SS-Agar medium - Growth in Cetrimid-Agar - (21)
Production of dye Nondiffusive negative Diffusive negative
Fluorescent negative Pyrocyanine negative (22) OF Test no
decomposition of sugar (23) Formation of acids Glucose positive
Fructose positive Xylose positive (24) ONPG (.beta.-galactosidase)
negative (25) Arginine dihydrolase negative (26) Production of gas
from glucose negative (27) Tyrosine decomposition negative (28)
Growth factor request none (29) Use 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 + Saccharic
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 -
[0049] When these results were taxonomically examined in the same
manner based on Bergey's Manual of Systematic Bacteriology, it was
found that this strain belongs to the genus Pseudomonas but the
properties thereof did not coincide with those of standard strains.
Therefore, this strain was named Pseudomonas sp. SD811.
[0050] Pseudomonas sp. SD812
3 Morphology: (1) Shape and size of cell rod 0.5-0.8 .mu.m .times.
1.5-3.0 .mu.m (2) Motility motile (3) Gram staining negative (4)
Spore none (5) Bacteriolysis by 3% KOH positive Physiological
activity: (1) Aminopeptidase positive (2) Oxidase positive (3)
Catalase positive (4) Production of indole negative (5) VP Test
negative (6) Reduction of nitrate negative (7) Denitrification
negative (8) Use of citric acid (Simons) positive (9) Urease
positive (10) Phenylalanine deaminase negative (11) Use of malonic
acid positive (12) Production of levan from sucrose negative (13)
Lecithinase negative (14) Hydrolysis of starch negative (15)
Hydrolysis of gelatin negative (16) Hydrolysis of casein negative
(17) Hydrolysis of DNA negative (18) Hydrolysis of Tween 80
negative (19) Hydrolysis of exrin negative (20) Growth Behavior to
oxygen obligately aerobic Growth at 37.degree. C. - Growth at
41.degree. C. - Growth at pH 5.6 + Growth in Mac-Conkey-Agar medium
- Growth in SS-Agar medium - Growth in Cetrimid-Agar - (21)
Production of dye Nondiffusive positive Diffusive negative
Fluorescent negative Pyrocyanine negative (22) OF Test no
decomposition of sugar (23) Formation of acids Glucose negative
Fructose negative Xylose negative (24) ONPG (.beta.-galactosidase)
negative (25) Arginine dihydrolase negative (26) Production of gas
from glucose negative (27) Tyrosine decomposition positive (28)
Growth factor request none (29) Use 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 + Saccharic
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 -
[0051] When these results were taxonomically examined in the same
manner based on Bergey's Manual of Systematic Bacteriology, it was
found that this strain belongs to the genus Pseudomonas but the
properties thereof did not coincide with those of standard strains.
Therefore, this strain was named Pseudomonas sp. SD812.
[0052] Burkholderia sp. SD816
4 Morphology: (1) Shape and size of cell rod (2) Motility motile
(3) Gram staining negative (4) Spore none (5) Flagella Flagallation
state is unknown. Physiological activity: (1) Oxidase positive (2)
Catalase positive (3) Cleavage of protocatechinic acid ortho type
(4) Reduction of nitrate negative (5) Denitrification negative (6)
Accumulation of PHB positive (7) Hydrolysis of starch negative (8)
Hydrolysis of gelatin negative (9) Growth Behavior to oxygen
obligately aerobic Growth at 40.degree. C. + (10) Production of dye
Hue of colony no production of characteristic colonial dye
Production of water-soluble dye negative (11) OF Test no
decomposition of sugar (12) Arginine dihydrolase negative (13) Use
of various carbon compounds Levulinic acid - Mesaconic acid -
D-Tartaric acid + Ribose + Rhamnose + Xylose + Tryptamine -
2,3-Butylene glycol - (14) Quinone type Q-8 (15) GC content of DNA
in cell (mol %) 62
[0053] When these results were taxonomically examined in the same
manner based on Bergey's Manual of Systematic Bacteriology (1986,
1994), it was found that this strain belongs to the genus
Burkholderia but the properties thereof did not coincide with those
of standard strains. Therefore, this strain was named Burkholderia
sp. SD816.
[0054] .alpha.-Halocarbonyl compounds having an
.alpha.,.beta.-carbon-carb- on double bond, which can be suitably
used in the present invention, is represented by the following
formula (1): 3
[0055] wherein R.sub.1 represents a halogen atom, preferably a
chlorine atom or a bromine atom; R.sub.2 and R.sub.3 each
independently represents a hydrogen atom, a halogen atom, a linear
or branched aliphatic hydrocarbon group having from 1 to 6 carbon
atoms, a linear or branched alkoxy group having from 1 to 6 carbon
atoms, a hydroxyl group, a carboxyl group, an aromatic group which
may be substituted, or a saturated or unsaturated nitrogen-,
oxygen- or sulfur-containing heterocyclic group, preferably a
hydrogen atom; R.sub.4 represents a hydroxyl group, a linear or
branched alkoxy group having from 1 to 4 carbon atoms, or a
primary, secondary or tertiary amino group, preferably a hydroxyl
group.
[0056] Specific examples of the compounds include
.alpha.-chloroacrylic acid, .alpha.-bromoacrylic acid,
2-chloro-2-butenoic acid, 2-bromo-2-butenoic acid,
2-chloro-2-pentenoic acid, 2-bromo-2-pentenoic acid, and the methyl
ester and the ethyl ester thereof. Among these,
.alpha.-chloroacrylic acid and .alpha.-bromoacrylic acid are
preferred.
[0057] In the present invention, the reaction is performed by
contacting a microorganism belonging to the genus Pseudomonas or
Burkholderia, specifically a microorganism such as Pseudomonas sp.
SD811 or Burkholderia sp. SD816 strain, with an
.alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond to reduce the
carbon-carbon double bond, thereby producing a corresponding
.alpha.-halo-.alpha.,.beta.-saturated carbonyl compound.
[0058] For the reduction of the carbon-carbon double bond of an
.alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond performed in the present
invention, a microbial cell obtained by the cultivation according
to the above-described method or a microbial product of the
microorganism, such as a cell-free extract obtained by disrupting a
cell culture according to the above-described method, may be used
under conditions such that the reducing activity of the
microorganism can be stably achieved.
[0059] More specifically, in the case of using a cell obtained by
cultivation, an .alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond is continuously or
batchwise added to a culture suspension as a substrate to a
concentration of from 0.1 to 10%, preferably from 0.2 to 2%, and
cultivation is performed at a growth temperature of from 15 to
35.degree. C., preferably from 25 to 30.degree. C., thereby
producing a corresponding .alpha.,.beta.-saturated carbonyl
compound in the culture suspension.
[0060] Alternatively, the culture obtained by the above-described
method is subjected to centrifugation or the like to recover cells
and the cells are suspended in an appropriate solution, for
example, an aqueous solution such as a diluted pH buffer solution.
To this suspension, an .alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond is continuously or
batchwise added as a substrate to a concentration, for example, of
from 0.1 to 10% and reacted at a temperature of from 15 to
50.degree. C., preferably from 25 to 30.degree. C., while adjusting
the reaction pH to from 6.0 to 9.0, preferably from 6.5 to 7.3,
thereby producing a corresponding .alpha.-halo-.alpha.,.beta.-
-saturated carbonyl compound in the cell suspension.
[0061] In the case of using a microbial product of a microorganism,
for example, the culture obtained by the above-described culture
method is subjected to centrifugation to recover cells, the cells
are disrupted by French pressing or a like method to obtain a
cell-free extract, and the cell-free extract is added to a reaction
mixture containing an .alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond as a substrate, in a
concentration of from 0.1 to 10%, preferably from 0.2 to 2%, and
also containing an ingredient effective in maintaining the pH of
the reaction mixture, in a concentration of from 10 mM to 1 M, and
reacted with continuous or batch addition of substrate at a
temperature of from 15 to 50.degree. C., preferably from 28 to
35.degree. C., thereby producing a corresponding
.alpha.,.beta.-saturated carbonyl compound.
[0062] In the present invention, the reaction may be performed
while continuously or batchwise adding a substance effective in
maintaining the activity of reducing the .alpha.-halocarbonyl
compound having an .alpha.,.beta.-carbon-carbon double bond, for
example, a compound capable of being oxidized by the microorganism
used, such as sugar or organic acid, preferably glucose or L-lactic
acid, by itself or as a mixed solution with an .alpha.-halocarbonyl
compound having an .alpha.,.beta.-carbon-carbon double bond to have
a concentration of from 0.1 to 10%, preferably from 0.2 to 1%
during the reaction. The ratio of the .alpha.-halocarbonyl compound
having an .alpha.,.beta.-carbon-carbon double bond to the
additional substance to be oxidized may be freely selected between
1:1 and 20:1 on a molar basis. By this addition of sugar or organic
acid, the reaction time may be prolonged and in turn, the
concentration of the objective product
.alpha.-halo-.alpha.,.beta.-satura- ted carbonyl compound in the
reaction suspension may be increased. This is advantageous for
collecting the product by isolation. Except for the cultivation
time, the reaction may be performed either in an aerobic or
anaerobic environment. The ratio of the cell or cell-free extract
to the .alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond as the substrate, and the
timing and rate or frequency of addition may be freely selected in
the range capable of attaining the completion of reaction within
the objective time.
[0063] In the present invention, the
.alpha.-halo-.alpha.,.beta.-saturated carbonyl compound obtained by
the reduction of an .alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond is a metabolic
intermediate for the microorganism used and may be further
decomposed. For example, some microorganisms relatively swiftly
decompose the .alpha.-chloropropionic acid produced from
.alpha.-chloroacrylic acid. If the case is so, the decomposition
reaction may be stopped by using a mutant defective in decomposing
activity, decreasing the pH value, heat-treating the cells or
cell-free extract, or adding an appropriate inhibitor of the
decomposing enzyme. To speak more specifically, under the
conditions where an .alpha.-chloropropionic acid is produced from
an .alpha.-chloroacrylic acid at an optimal rate, the Pseudomonas
sp. SD811 strain usually swiftly decomposes the
.alpha.-chloropropionic acid produced in the culture suspension or
reaction suspension, whereby the conversion ratio into
.alpha.-chloropropionic acid based on the .alpha.-chloroacrylic
acid decreases. However, since the optimal reaction pH at the stage
of producing .alpha.-chloropropionic acid from
.alpha.-chloroacrylic acid is from 5 to 7 and the optimal reaction
pH at the decomposition stage of a-chloropropionic acid is from 7.0
to 7.3, thus, the optimal reaction pH at the decomposition stage of
.alpha.-chloropropionic acid is higher than the optimal reaction pH
at the stage of producing .alpha.-chloropropionic acid from
.alpha.-chloroacrylic acid. Accordingly, the amount of
.alpha.-chloropropionic acid produced can be increased by
performing the reaction at a low pH of from 5 to 7 which is the
optimal reaction pH range for the stage of producing
.alpha.-chloropropionic acid. Furthermore, certain enzymes which
decompose the .alpha.-chloropropionic acid are known to be
effectively inhibited by hydroxylamine (see, Soda K. et al., J.
Biol. Chem., 272, 3363-3368 (1997)) and also in the present
invention, the decomposition reaction can be inhibited by adding an
appropriate amount of hydroxylamine to the culture suspension or
reaction suspension.
[0064] The cell or cell-free extract of the microorganism for use
in the present invention may be used by fixing it to an
immobilizing support of various types by a commonly known method
such as adsorption, inclusion or crosslinking. The kind of the
support is not particularly limited and for example, a
polysaccharide-type material such as cellulose, a polymer-type
material, or a protein-type material such as collagen, may be
used.
[0065] The .alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-c- arbon double bond for use in the present
invention is a molecule prochiral at the .alpha.-position, however,
by reducing the carbon-carbon double bond, a corresponding
.alpha.-halo-.alpha.,.beta.-saturated carbonyl compound having an
absolute S form configuration at the .alpha.-position may be
produced.
[0066] The culture method of the microorganism used in the present
invention is not particularly limited as long as aerobic
microorganisms in general can grow. The carbon source of the medium
may be any source as long as the above-described microorganisms can
be used and examples thereof include saccharides, acetic acid,
lactic acid and a mixture thereof. Examples of nitrogen sources
which can be used include ammonium salts such as ammonium sulfate
and ammonium phosphate, nitrogen-containing compounds such as meat
extract and yeast extract, and mixtures thereof.
[0067] In addition to these ingredients, nutrients commonly used in
cultivation, such as inorganic salts, trace metal salts and
vitamins, may be used in the medium by appropriately mixing them.
Furthermore, if desired, a factor of accelerating the growth of the
microorganism and an ingredient effective in maintaining the pH of
the medium may be added. Also, a compound effective in increasing
the reductive activity of the microorganism, for example, an
.alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond, such as
.alpha.-chloroacrylic acid, may be used as a sole carbon source or
may be used by mixing it with a plurality of carbon sources.
[0068] The microorganism for use in the present invention may be
cultured under aerobic conditions used for the cultivation of
almost all aerobes or facultative anaerobes, for example, under
conditions such that the pH of the medium is from 5.5 to 8.0,
preferably from 6.5 to 7.0, and the growth temperature is from 15
to 35.degree. C., preferably from 25 to 30.degree. C. The
cultivation time is, for example, from 1 to 144 hours, preferably
from 12 to 72 hours.
[0069] The .alpha.-halo-.alpha.,.beta.-saturated carbonyl compound
produced according to the present invention may be obtained using
an ordinary purification method such as organic solvent extraction
or distillation. For example, .alpha.-chloropropionic acid produced
from .alpha.-chloroacrylic acid may be obtained by subjecting the
culture suspension or reaction suspension to organic solvent
extraction, distillation or the like. Furthermore, although the
.alpha.-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond is a molecule prochiral at
the .alpha.-position, the .alpha.-halo-.alpha.,.beta.-satura- ted
carbonyl compound produced by the reducing method of the present
invention is a chiral compound and it is advantageous to determine
the purity of the enantiomer thereof by GC and/or HPLC with a
chiral column or by a polarimeter.
[0070] As described in the foregoing, in the method of the present
invention for producing a corresponding
.alpha.-halo-.alpha.,.beta.-satur- ated carbonyl compound having an
absolute S form configuration from an .alpha.-halocarbonyl compound
having an .alpha.,.beta.-carbon-carbon double bond by reducing the
carbon-carbon double bond, an aerobe or facultative anaerobe is
used, therefore, the method is favored with high profitability,
good operability and excellent processing safety.
[0071] The present invention is described in greater detail below
by referring to the Examples, however, the present invention should
not be construed as being limited to these Examples.
EXAMPLE 1
[0072] Detection of Activity of Reducing .alpha.-halocarbonyl
Compound Having an .alpha.,.beta.-Carbon-Carbon Double Bond
[0073] Microorganisms cultured by accumulative cultivation or
accumulative acclimatization and isolated were cultured at
30.degree. C. using a medium obtained by adding 0.2% of
.alpha.-chloroacrylic aid or .alpha.-chloro-.alpha.,.beta.-butenoic
acid as a carbon source to a basal medium containing yeast extract
(0.5 g/l), ammonium sulfate (2 g/l), sodium dihydrogenphosphate (1
g/l), dipotassium hyrogenphosphate (1 g/l) and magnesium sulfate
(0.1 g/l) (for the isolation of some microorganisms which grow only
in a high salt concentration medium, a medium obtained by adding
sodium chloride in a concentration of from 10 to 20% to the
above-descried basal medium was used as the basal medium). Whether
or not .alpha.-chloropropionic acid or .alpha.-chlorobutyric acid
as the corresponding reduction product appeared in the culture
suspension was examined using gas chromatography. From each
solution, 0.5 ml was sampled at a specific time and centrifuged to
remove cells. Thereafter, 0.4 ml of the supernatant was mixed with
0.4 ml of 2N HCl and analyzed under the following conditions:
[0074] Apparatus: GC-7A (manufactured by Shimadzu Seisakusho)
[0075] Column: Thermon-3000/SHINCARBON A, 2.6 mm.times.2.1 m
[0076] Carrier gas: nitrogen, 50 ml/min.
[0077] Detection: FID
[0078] Column temperature: 200.degree. C. (constant)
[0079] Injection: 2 to 10 .mu.l, 260.degree. C.
[0080] Recording: CHROMATOCODER 12 (SIC)
[0081] By this detection, peaks appeared swiftly after the
initiation of cultivation at the position of
.alpha.-chloropropionic acid or .alpha.-chlorobutyric acid
accompanying the consumption of .alpha.-chloroacrylic in the
culture suspension of a plurality of microorganisms. The peaks were
analyzed by GC-MS and found to coincide with the mass spectrum of
.alpha.-chloropropionic acid or .alpha.-chlorobutyric acid as the
standard substance, therefore, the products were confirmed to be
.alpha.-chloropropionic acid or .alpha.-chlorobutyric acid. The
genera of microorganisms of which cultivation brought about
generation of the reduction product were identified, as a result,
it was found that microorganisms belonging to various genera have
reducing activity. Microorganisms recognized to have the activity
were aerobes or facultative anaerobes belonging to the genera
Acetobacter, Actinomyces, Acinetobacter, Agrobacterium, Aeromonas,
Alcaligenes, Arthrobacter, Azotobacter, Bacillus, Brevibacterium,
Burkholderia, Cellulomonas, Corynebacterium, Enterobacter,
Enterococcus, Escherichia, Flavobacterium, Gluconobacter,
Halobacteium, Halococccus, Klebsiella, Lactobacillus,
Microbacterium, Micrococcus, Micropolyspora, Mycobacterium,
Nocardia, Pseudomonas, Pseudonocardia, Rhodococcus, Rhodobacter,
Serratia, Staphylococcus, Streptococcus, Streptomyces and
Xanthomonas. Strains of the genus Pseudomonas prevailed in the
strains recognized. Microorganisms relatively surpassed in the
amount of the reduction product were the microorganisms belonging
to the genera Pseudomonas and Burkholderia. With any strain, the
reduction product recognized in the culture suspension was in a
small amount (0.01% or less), therefore, the following elite
strains were examined for optimization of product accumulating
conditions.
EXAMPLE 2
[0082] (1) Cultivation of Pseudomonas sp. SD811 Strain
[0083] Pseudomonas sp. SD811 strain was cultured in a medium
containing the following ingredients (amount: g/L):
.alpha.-chloroacrylic acid (2), yeast extract (0.5), ammonium
sulfate (2), sodium dihydrogenphosphate (1), dipotassium
hydrogenphosphate (1) and magnesium sulfate (0.1). The medium was
prepared as follows. All ingredients except for
.alpha.-chloroacrylic acid and magnesium sulfate were dissolved in
950 ml of water and after the pH was adjusted to 7.0, the solution
was poured into a 5 l-volume flask and sterilized at 121.degree. C.
for 20 minutes. After the temperature of this medium decreased to
about 70.degree. C., a solution obtained by dissolving
.alpha.-chloroacrylic acid and magnesium sulfate in 50 ml of water,
adjusted to a pH of 7.0 and then sterilized through a sterilization
filter was mixed with the medium prepared above. Without supplying
oxygen or adjusting the pH any more, a 5% seed culture (OD 660 nm:
1.10) was inoculated in this medium and the strain was cultured at
30.degree. C.
[0084] (2) Detection of .alpha.-chloropropionic Acid in
.alpha.-chloroacrylic Acid Culture Medium
[0085] During cultivation of the Pseudomonas sp. SD811 strain with
.alpha.-chloroacrylic acid, 0.5 ml was sampled at a specific time.
The sample was centrifuged to remove cells and 0.4 ml of the
supernatant was mixed with 0.4 ml of 2N HCl. The solution obtained
was analyzed under the conditions described in Example 1.
[0086] By the detection, a peak appeared swiftly after the
initiation of culturing at the position of .alpha.-chloropropionic
acid accompanying the consumption of .alpha.-chloroacrylic acid in
the culture suspension of Pseudomonas sp. SD811 strain. The
production was about 0.02% of the culture suspension at the maximum
time and the conversion ratio based on the substrate
.alpha.-chloroacrylic acid was about 10%.
EXAMPLE 3
[0087] (1) Cell Suspension Reaction 1 Using .alpha.-chloroacrylic
Acid as Substrate
[0088] A culture obtained by culturing the Pseudomonas sp. SD811
strain in a {fraction (1/10)} scale of the method in Example 2 was
subjected to centrifugation to recover the cells. The cells were
suspended in 20 ml of a solution (adjusted to a pH of 7.3)
containing 0.2% of .alpha.-chloroacrylic acid and 100 mM of
phosphate buffer (pH: 7.3) and reacted by shaking at 28.degree.
C.
[0089] From the reaction mixture, 0.5 ml was sampled at a specific
time and centrifuged to remove cells. Thereafter, 0.4 ml of the
supernatant and 0.1 ml of 6N HCl were mixed and then the product
was extracted with 0.4 ml of ethyl acetate. The sample extracted
was analyzed by the method of Example 1. As a result, a peak
appeared at the position of .alpha.,.beta.-chloropropionic acid
accompanying the consumption of .alpha.-chloroacrylic acid in the
reaction mixture. The production was about 0.05% of the reaction
suspension at the maximum time and the conversion ratio was about
25% based on the substrate .alpha.-chloroacrylic acid.
[0090] (2) Isolation of .alpha.-chloropropionic Acid from Cell
Suspension Reaction Using .alpha.-chloroacrylic Acid as
Substrate
[0091] A culture obtained by culturing the Pseudomonas sp. SD811
strain in a 1/5 scale of the method in Example 1 was subjected to
centrifugation to recover the cells and the cells were suspended in
100 ml of a solution (adjusted to a pH of 7.3) containing 0.2% of
.alpha.-chloroacrylic acid and 100 mM of potassium phosphate-sodium
hydroxide buffer and reacted by stirring at 28.degree. C. At the
time when the .alpha.-chloroacrylic acid was exhausted during the
reaction, .alpha.-chloroacrylic acid was added in a concentration
of 0.2% of the reaction mixture and the reaction was continued.
From the reaction suspension, 0.5 ml was sampled at a specific
time, the product was separated by extraction according to the
method of Example 3, and the production of .alpha.-chloropropionic
acid was monitored by the method of Example 1. After about 9 hours
when .alpha.-chloroacrylic acid was not detected in the reaction
suspension, the reaction was completed and the entire amount of the
reaction suspension was subjected to centrifugation to remove
cells. To 95 ml of the supernatant obtained, 20 ml of 6N HCl was
added, and the product was extracted with 95 ml of ethyl acetate.
The ethyl acetate layer was washed with 100 ml of saturated saline
and then concentrated by removing ethyl acetate by evaporation. The
concentrated sample was analyzed by the method of Example 1, as a
result, a slight amount of .alpha.-chloroacrylic acid and
.alpha.-chloropropionic acid were detected. The
.alpha.-chloropropionic acid present in the concentrated sample was
about 100 mg in total and the conversion ratio was about 25% based
on the substrate .alpha.-chloroacrylic acid.
EXAMPLE 4
[0092] (1) Methylation of .alpha.-chloropropionic Acid
[0093] When optical resolution is performed by an optical
resolution GC column, unmodified carboxylic acid exhibits poor
separability due to the effect of the carboxyl group in many cases.
Therefore, the product was methyl esterified by a boron
trifluoride-methanol complex salt method. More specifically, 4 ml
of methanol was added to 3 mg of .alpha.-chloropropionic acid and
mixed. Thereafter, 1 ml of a 14% methanol solution of boron
trifluoride-methanol complex was further added and the resulting
solution was refluxed on an oil bath at 80.degree. C. for 1 hour
while stirring. After 1 hour, 30 ml of water was added to the
reaction mixture and the reactant was extracted with 10 ml of ethyl
acetate. The ethyl acetate was then distilled off by a centrifugal
evaporator to concentrate the sample to the entire amount of about
0.1 ml. The concentrated sample was analyzed by the method of
Example 1 except that the column temperature only was changed to
120.degree. C. As a result, one main peak was observed at the
position of methyl .alpha.-chloropropionate. By the GC-MS analysis
of the peak, the product was identified to be methyl
.alpha.-chloropropionate.
[0094] (2) Optical Resolution GC Analysis of
.alpha.-chloropropionic Acid
[0095] After the methylation by the above-described method, the
S-form .alpha.-chloropropionic acid and the R-form
.alpha.-chloropropionic acid could be successfully separated under
the following analysis conditions.
5 Apparatus: GC-14A (manufactured by Shimadzu Seisakusho) Column:
CP-Chirasil-DEX CB, 0.32 mm I.D. .times. 25 m, df = 0.25 mm
(manufactured by GL Science K.K.) Carrier gas: He, 0.38 kg/cm.sup.2
Detection: FID, 275.degree. C. Column temperature: 70.degree. C.
(constant) Injection: 0.1 to 0.2 .mu.l, split, about 1:100,
250.degree. C. Recording: CHROMATOPACK C-6A (manufactured by
Shimadzu Seisakusho)
[0096] The methyl .alpha.-chloropropionate obtained from the
reaction mixture of Example 3 under the above-described conditions
was analyzed, then a peak was observed only at the position of
S-form methyl .alpha.-chloropropionate. The optical purity thereof
was 99% or more.
EXAMPLE 5
[0097] Cell Suspension Reaction 2 Using .alpha.-chloroacrylic Acid
as Substrate
[0098] A culture obtained by culturing Pseudomonas sp. SD811 strain
in a {fraction (1/10)} scale of the method as described in Example
2 was subjected to centrifugation to recover the cells. The cells
were suspended in 20 ml of a solution (adjusted to a pH of 5.7)
containing 0.2% of .alpha.-chloroacrylic acid and 100 mM of
phosphate buffer (pH: 5.7) and reacted by shaking at 28.degree. C.
From the reaction mixture, 0.5 ml was sampled at a specific time
and centrifuged to remove cells. Thereafter, 0.4 ml of the
supernatant and 0.1 ml of 6N HCl were mixed and then the product
was extracted with 0.4 ml of ethyl acetate. The sample extracted
was analyzed by the method of Example 1, as a result, a peak
appeared at the position of .alpha.-chloropropionic acid
accompanying the consumption of .alpha.-chloroacrylic acid in the
reaction mixture. The production was about 0.08% of the reaction
suspension at the maximum time and the conversion ratio was about
41% based on the substrate .alpha.-chloroacrylic acid.
EXAMPLE 6
[0099] Preparation of .alpha.-chloropropionic Acid-undecomposable
Mutant
[0100] A culture obtained by culturing Pseudomonas sp. SD811 strain
in a {fraction (1/200)} scale of the method in Example 2 was
subjected to centrifugation to recover the cells and the cells were
washed with physiological saline. The washed cells were
re-suspended in 1 ml of 10 to 100 mM phosphate buffer (pH: 7). To
the resulting cell suspension, N-methyl-N'-nitro-N-nitrosoguanidine
(NTG) was added to a final concentration of from 50 to 100 ppm and
the cells were treated at room temperature for from 2 to 20
minutes. After the treatment, the cell suspension was subjected to
centrifugation to recover the cells, the cells were washed with
sterilized physiological saline, and the entire amount thereof was
inoculated in 5 ml of an L medium (polypeptone:
[0101] 10 g/l, yeast extract: 5 g/l, sodium chloride: 5 g/l, pH: 7)
and cultured by shaking at 28.degree. C. After the completion of
cultivation, the culture was subjected to centrifugation to recover
the cells and the cells were suspended in a 20% glycerin solution.
The suspension was equally divided by an appropriate amount and
frozen to prepare glycerin stocks for selection of a mutant.
EXAMPLE 7
[0102] Selection of .alpha.-chloropropionic Acid-undecomposanble
Mutant
[0103] A glycerin stock for selection of a mutant prepared above
was inoculated in 5 ml of a medium used in the method of Example 2
and cultured at 28.degree. C. for 5 hours. At the time when the
turbidity was increased as high as several times, penicillin G was
added to the culture suspension in an amount of giving a final
concentration of from 100 to 1,000 ppm and the cultivation was
continued at 28.degree. C. After the cultivation for from 5 to 16
hours, the culture suspension was subjected to centrifugation to
recover the cells. The cells were washed twice with sterilized
physiological saline and the entire amount thereof was inoculated
in 5 ml of L-broth and cultured by shaking at 28.degree. C. an
entire day and night.
[0104] The culture suspension obtained above was diluted, spread on
a medium the same as used in Example 2 except that the
.alpha.-chloroacrylic acid as a carbon source was replaced by an
equivalent amount of lactic acid and the medium was solidified by
adding 2% agar, and cultured at 28.degree. C. After 1 or 2 days,
colonies formed on the plate were replicated on the medium used as
described in Example 2 which was solidified by adding 2% agar, and
cultured at 28.degree. C. for 1 or 2 days. Strains which grew on
the lactic acid plate but did not grow on the .alpha.-chloroacrylic
acid plate were isolated as candidates for the
.alpha.-chloropropionic acid-undecomposable mutant.
[0105] The candidate strains each isolated were inoculated by an
inoculating loop in 5 ml of a medium the same as used in Example 2
except that 2 g/l of lactic acid was added as a carbon source
capable of growing, and cultured by shaking at 28.degree. C. The
culture suspensions obtained were analyzed by the method of Example
1, as a result, the production maximum of .alpha.-chloropropionic
acid produced in the culture was distributed depending on the
candidate strains, however, the production maximum with the mutant
of Pseudomonas sp. SD811 was about 0.17% of the culture suspension
and the conversion ratio was about 85% based on the substrate
.alpha.-chloroacrylic acid. With the variation strain of
Burkholderia sp. SD816, the production maximum was about 0.15% of
the culture suspension and the conversion ratio was about 75% based
on the substrate .alpha.-chloroacrylic acid. These strains were
selected as the .alpha.-chloropropionic acid-undecomposable
mutant.
EXAMPLE 8
[0106] Cell Suspension Reaction 3 Using .alpha.-chloroacrylic Acid
as Substrate
[0107] The .alpha.-chloropropionic acid-undecomposable mutant of
Pseudomonas sp. SD811 and the .alpha.-chloropropionic
acid-undecomposable mutant of Burkholderia sp. SD816 each was
cultured in the same medium of lactic acid as used in Example 7,
and each of the cultures obtained was subjected to centrifugation
to recover the cells. The cells of each strain were suspended in 20
ml of a solution (adjusted to a pH of 7.3) containing 0.2% of
.alpha.-chloroacrylic acid and 100 mM of phosphate buffer (pH: 7.3)
and reacted by shaking at 28.degree. C.
[0108] From each reaction suspension, 0.5 ml was sampled at a
specific time and centrifuged to remove cells. To 0.4 ml of the
supernatant, 0.1 ml of 6N HCl was mixed and then, the product was
extracted with 0.4 ml of ethyl acetate. The sample extracted was
analyzed by the method of Example 1. As a result, a peak appeared
at the position of .alpha.-chloropropionic acid accompanying the
consumption of .alpha.-chloroacrylic acid in the reaction
suspension. With the mutant of Pseudomonas sp. SD811, the
production was about 0.19% of the reaction suspension at the time
when the .alpha.-chloroacrylic acid disappeared in the reaction
mixture and the conversion ratio was about 95% based on the
substrate .alpha.-chloroacrylic acid. With the mutant of
Burkholderia sp. SD816, those were about 0.2% and about 100%,
respectively. In either case, the .alpha.-chloropropionic acid
produced did not decrease with the passing of time. The optical
activity of the .alpha.-chloropropionic acid was analyzed according
to the method of Example 4. As a result, in both cases, the product
was an S-form compound and the optical purity thereof was 99% or
more.
EXAMPLE 9
[0109] Cell Suspension Reaction 4 Using .alpha.-chloroacrylic Acid
as Substrate
[0110] The .alpha.-chloropropionic acid-undecomposable mutant of
Pseudomonas sp. SD811 and the .alpha.-chloropropionic
acid-undecomposable mutant of Burkholderia sp. SD816 each was
cultured in 100 ml of a medium obtained by adding 0.2% of glucose
as a carbon source to the basal medium used in Example 2, and the
cultures obtained each was subjected to centrifugation to recover
the cells. The cells of each strain were suspended in 20 ml of a
solution (adjusted to a pH of 7.3) containing 0.2% of
.alpha.-chloroacrylic acid and 100 mM of phosphate buffer (pH: 7.3)
and reacted by shaking at 28.degree. C.
[0111] From each reaction suspension, 0.5 ml was sampled at a
specific time and centrifuged to remove cells. To 0.4 ml of the
supernatant, 0.1 ml of 6N HCl was mixed and then, the product was
extracted with 0.4 ml of ethyl acetate. The sample extracted was
analyzed by the method of Example 1. As a result, after the lag
time of a few hours at the initial stage when the reaction was
started, a peak appeared at the position of .alpha.-chloropropionic
acid accompanying the consumption of .alpha.-chloroacrylic acid in
the reaction suspension. With the mutant of Pseudomonas sp. SD811,
the production was about 0.19% of the reaction suspension at the
time when the .alpha.-chloroacrylic acid disappeared in the
reaction mixture and the conversion ratio was about 95% based on
the substrate .alpha.-chloroacrylic acid. With the mutant of
Burkholderia sp. SD816, those were about 0.2% and about 100%,
respectively.
EXAMPLE 10
[0112] Cultivation in Jar Fermenter:
[0113] The .alpha.-chloropropionic acid-undecomposable mutant of
Burkholderia sp. SD816 was cultured for 16 hours in 100 ml of a
medium (5.times.medium, pH: 7.0) the same as used in Example 9
except that the medium ingredients each had a five-fold
concentration. The culture obtained was inoculated in 2 L of
5.times.medium filled in a 5 L-volume jar fermenter and cultured at
28.degree. C., 800 rpm and an aeration rate of 1 ml/min. When from
about 15 to 20 hours passed after the initiation of cultivation,
the glucose initially charged were completely consumed,
accordingly, a 5 to 20% glucose solution, a 5 to 15% ammonium
sulfate solution and a 1 to 5% yeast extract solution as individual
solutions or a mixed solution were further continuously added by
means of PERISTACK pump. The addition rate was controlled so that
the glucose concentration of from 0.02 to 2% could be maintained
during the addition. The pH of the culture suspension was adjusted
by a 20% aqueous ammonia to lie in the range of from 6.3 to 7.3.
After the cultivation for from about 30 to 48 hours by this method,
OD 660 nm was about 30 to 50.
EXAMPLE 11
[0114] Cell Suspension Reaction 5 Using .alpha.-chloroacrylic Acid
as Substrate
[0115] The .alpha.-chloropropionic acid-undecomposable mutant of
Burkholderia sp. SD816 was cultured according to the method of
Example 10. When from 48 to 72 hours passed after the initiation of
cultivation, the addition of glucose and the like was stopped to
interrupt growth. To this culture suspension, .alpha.-chloroacrylic
acid was added to a final concentration of about 0.2% and reacted
under the same conditions as in the cultivation. From the reaction
suspension, 0.5 ml was sampled at a specific time and centrifuged
to remove cells. Thereafter, 0.4 ml of the supernatant and 0.4 ml
of 2N HCl were mixed and the .alpha.-chloroacrylic acid
concentration in the solution was determined under the following
conditions.
6 Apparatus: LC-9A (manufactured by Shimadzu Seisakusho) Column:
ODSpak F-511/4.6 mm .times. 250 mm (Shodex) Eluent
acetonitrile/water = 2/8 + 0.1% trifluoroacetic acid, 1 ml/min.
Detection: SPD-6AV UV-VIS Spectrophotometer (manufactured by
Shimadzu Seisakusho) Column temperature: 25.degree. C. Injection:
Autosampler Model 23 (SIC) with 20 .mu.l sample loop Recording:
CHROMATOCODER 12 (SIC)
[0116] In this reaction, after the lag time of from 3 to 7 hours at
the initial stage when the reaction was started, consumption of
.alpha.-chloroacrylic acid started. When the .alpha.-chloroacrylic
acid concentration became about 0.02%, .alpha.-chloroacrylic acid
was further added so that the .alpha.-chloroacrylic acid
concentration could be increased to about 0.2%. This operation was
repeated until the consumption of .alpha.-chloroacrylic acid
substantially terminated. The accumulative production of
.alpha.-chloropropionic acid after 32 hours where the reaction was
substantially stopped was from about 1.0 to 1.2% of the reaction
suspension and the conversion ratio was about 97% based on the
substrate .alpha.-chloroacrylic acid. The optical activity of the
.alpha.-chloropropionic acid accumulated was analyzed according to
the method of Example 4. As a result, the product was an S-form
compound and the optical purity thereof was 99% or more.
EXAMPLE 12
[0117] Cell Suspension Reaction 6 Using .alpha.-chloroacrylic Acid
as Substrate
[0118] The .alpha.-chloropropionic acid-undecomposable mutant of
Burkholderia sp. SD816 was cultured for from 48 to 72 hours
according to the method of Example 10 and the culture obtained was
subjected to centrifugation to recover the cells. The cells were
suspended in 2 l of a solution (adjusted to a pH of 7.1) containing
0.2% .alpha.-chloroacrylic acid and 60 mM of phosphate buffer (pH:
7.1) and reacted at 28.degree. C., 800 rpm and an aeration rate of
1 ml/min. From the reaction suspension, 0.5 ml was sampled at a
specific time and centrifuged to remove cells. Thereafter, 0.4 ml
of the supernatant and 0.4 ml of 2N HCl were mixed and the
.alpha.-chloroacrylic acid concentration in the solution was
determined by the method described in Example 11.
[0119] In this reaction, after a lag time of from 3 to 7 hours at
the initial stage when the reaction was started, consumption of
.alpha.-chloroacrylic acid started. When the .alpha.-chloroacrylic
acid concentration became about 0.02%, .alpha.-chloroacrylic acid
was further added so that the .alpha.-chloroacrylic acid
concentration could be increased to about 0.2%. This operation was
repeated until the consumption of a-chloroacrylic acid
substantially terminated. The accumulative production of
.alpha.-chloropropionic acid after 28 hours where the reaction was
substantially stopped was from about 0.8 to 1.0% of the reaction
suspension and the conversion ratio was about 98% based on the
substrate .alpha.-chloroacrylic acid.
EXAMPLE 13
[0120] Cell Suspension Reaction 7 Using .alpha.-chloroacrylic Acid
as Substrate
[0121] The .alpha.-chloropropionic acid-undecomposable mutant of
Burkholderia sp. SD816 was cultured for from 48 to 72 hours
according to the method of Example 10 and the culture obtained was
subjected to centrifugation to recover the cells. The cells were
suspended in 2 l of 60 mM phosphate buffer (pH: 7.1) and reacted at
28.degree. C., 800 rpm and an aeration rate of 1 ml/min while
adding from 5 to 10% of an .alpha.-chloroacrylic acid solution to
the cell suspension little by little by means of PERISTACK pump.
From the reaction suspension, 0.5 ml was sampled at a specific time
and centrifuged to remove cells. Thereafter, 0.4 ml of the
supernatant and 0.4 ml of 2N HCl were mixed and the
.alpha.-chloroacrylic acid concentration in the solution was
determined by the method described in Example 11.
[0122] In this reaction, after a lag time of from 5 to 10 hours at
the initial stage when the reaction was started, consumption of
.alpha.-chloroacrylic acid started. The addition rate of the
.alpha.-chloroacrylic acid solution was controlled in accordance
with the consuming rate so that the .alpha.-chloroacrylic acid
concentration could be in the range of from about 0.02 to 0.2%,
preferably around 0.1%. This operation was continued until the
consumption of .alpha.-chloroacrylic acid substantially terminated.
The accumulative production of .alpha.-chloropropionic acid after
24 hours where the reaction was substantially stopped was from
about 0.8 to 1.2% of the reaction suspension and the conversion
ratio was about 95% based on the substrate .alpha.-chloroacrylic
acid.
EXAMPLE 14
[0123] Cell Suspension Reaction 8 Using .alpha.-chloroacrylic Acid
as Substrate
[0124] The .alpha.-chloropropionic acid-undecomposable mutant of
Burkholderia sp. SD816 was cultured for from 48 to 72 hours
according to the method of Example 10 and the culture obtained was
subjected to centrifugation to recover the cells. The cells were
suspended in 2 l of 60 mM phosphate buffer (pH: 7.1) and reacted at
28.degree. C., 800 rpm and an aeration rate of 1 ml/min while
adding from 5 to 10% of an .alpha.-chloroacrylic acid solution to
the cell suspension little by little by means of PERISTACK pump. At
the same time, from 5 to 10% of a sodium lactate solution was added
alone or as a mixed solution with .alpha.-chloroacrylic acid little
by little by means of PERISTACK pump. From the reaction suspension,
0.5 ml was sampled at a specific time and centrifuged to remove
cells. Thereafter, 0.4 ml of the supernatant and 0.4 ml of 2N HCl
were mixed and the .alpha.-chloroacrylic acid concentration in the
solution was determined by the method described in Example 11. The
concentration of lactic acid was determined using lactate
dehydrogenase. The pH of the reaction system was adjusted to from
6.5 to 7.3 using 20% aqueous ammonia and 2N HCl.
[0125] In this reaction, after the lag time of from 5 to 10 hours
at the initial stage when the reaction was started, consumption of
.alpha.-chloroacrylic acid started. Lactic acid was swiftly
consumed immediately after the initiation of reaction. The addition
rate of the .alpha.-chloroacrylic acid solution was controlled in
accordance with the consuming rate so that the
.alpha.-chloroacrylic acid concentration could lie in the range of
from about 0.02 to 0.2%, preferably around 0.1%. At the same time,
the added amount of lactic acid was controlled to prevent the
concentration from exceeding 0.4%. This operation was continued for
about 60 hours. In this Example, the reduction clearly continued
and proceeded at a constant rate even after about 60 hours because
lactic acid was present together. The accumulative production of
.alpha.-chloropropionic acid after the 60-hour reaction was from
about 2.8 to 3.2% of the reaction suspension and the conversion
ratio was about 99% or more based on the substrate
.alpha.-chloroacrylic acid.
EXAMPLE 15
[0126] Cell Suspension Reaction 9 Using .alpha.-chloroacrylic Acid
as Substrate
[0127] The .alpha.-chloropropionic acid-undecomposable mutant of
Burkholderia sp. SD816 was cultured for from 48 to 72 hours
according to the method of Example 10 and the culture obtained was
subjected to centrifugation to recover the cells. The cells were
suspended in 2 l of 60 mM phosphate buffer (pH: 7.1) and reacted at
28.degree. C., 800 rpm and an aeration rate of 1 ml/min while
adding a mixed aqueous solution containing 10% of an
.alpha.-chloroacrylic acid and 10% of glucose to the cell
suspension little by little by means of PERISTACK pump. From the
reaction suspension, 0.5 ml was sampled at a specific time and
centrifuged to remove cells. Thereafter, 0.4 ml of the supernatant
and 0.4 ml of 2N HCl were mixed and the .alpha.-chloroacrylic acid
concentration in the solution was determined by the method
described in Example 11. In the determination of the glucose
concentration, the reaction supernatant as the original solution
obtained after the centrifugation was used intact and subjected to
the measurement by a glucose analyzer. The pH of the reaction
system was adjusted to from 6.5 to 7.3 using 20% aqueous ammonia
and 2N HCl. In this reaction, after a lag time of from 6 to 12
hours at the initial stage when the reaction was started,
consumption of .alpha.-chloroacrylic acid started. The glucose was
swiftly consumed particularly right after the initiation of
reaction but after the reducing activity was induced, the glucose
was consumed at a constant rate. The addition rate of the mixed
solution was controlled so that the .alpha.-chloroacrylic acid
concentration could lie in the range of from about 0.02 to 0.2%,
preferably around 1%, and at the same time, the glucose
concentration could be prevented from exceeding 0.4%. This
operation was continued for about 60 hours. In this Example, the
reduction reaction clearly continued and proceeded at a constant
rate even after about 60 hours because glucose was present
together. The conversion ratio was about 99% or more based on the
.alpha.-chloroacrylic acid after the 60-hour reaction.
EXAMPLE 16
[0128] Cell Suspension Reaction 10 Using .alpha.-chloroacrylic Acid
as Substrate
[0129] The .alpha.-chloropropionic acid-undecomposable mutant of
Burkholderia sp. SD816 was cultured for from 48 to 72 hours in the
1/2 scale of the method of Example 10 and the culture obtained was
subjected to centrifugation to recover the cells. The cells were
suspended in 1 l of a solution (adjusted to a pH of 7.1) containing
0.2% of .alpha.-chloroacrylic acid, 0.2% of glucose and 60 mM of
phosphate buffer (pH: 7.1), and reacted at 28.degree. C., 800 rpm
and an aeration rate of 1 ml/min. At the end point in a lag time of
from 5 to 10 hours after the initiation of reaction, air for the
aeration was changed to nitrogen gas and thereafter, the reaction
was performed in an anaerobic environment. From the reaction
suspension, 0.5 ml was sampled at a specific time and centrifuged
to remove cells. Then, 0.4 ml of the supernatant and 0.4 ml of 2N
HCl were mixed and the .alpha.-chloroacrylic acid concentration in
the solution was determined by the method described in Example 11.
At the same time, the glucose concentration was determined by a
glucose analyzer. The pH of the reaction system was adjusted to
from 6.5 to 7.3 using 20% aqueous ammonia and 2N HCl.
[0130] In this reaction, after a lag time of from 5 to 10 hours at
the initial stage when the reaction was started, consumption of
.alpha.-chloroacrylic acid started. When the .alpha.-chloroacrylic
acid concentration became about 0.02%, .alpha.-chloroacrylic acid
or glucose was further added so that the .alpha.-chloroacrylic acid
concentration could be increased to 0.2% or the glucose
concentration could be around 0.1%. This operation was continued
for about 60 hours. In this Example, the reduction reaction clearly
continued and proceeded at a constant rate even after about 60
hours because a substance to be oxidized was present together. The
consumption of glucose showed a marked decrease immediately after
the reaction system was changed from an aerobic environment to an
anaerobic environment and the total consumption was reduced to
about {fraction (1/10)} or less that in the reaction in an aerobic
environment. The accumulative production of .alpha.-chloropropionic
acid after the 60-hour reaction was from about 2.5 to 3.2% of the
reaction suspension and the conversion ratio was about 99% or more
based on the substrate .alpha.-chloroacrylic acid.
[0131] In the method of the present invention for producing a
corresponding .alpha.-halo-.alpha.,.beta.-saturated carbonyl
compound from an a-halocarbonyl compound having an
.alpha.,.beta.-carbon-carbon double bond by reducing the
carbon-carbon double bond, an aerobe or facultative anaerobe is
used, therefore, the method is favored with high profitability,
good operability and excellent processing safety. Furthermore,
according to the method of the present invention, a high-purity
.alpha.-halo-.alpha.,.beta.-saturated carbonyl compound useful as a
chiral building block of medical and agricultural chemicals and the
like is produced.
[0132] While the invention has been described in detail and with
reference to specific embodiments thereof, it will be apparent to
one skilled in the art that various changes and modifications can
be made therein without departing from the spirit and scope
thereof.
* * * * *