U.S. patent application number 09/725287 was filed with the patent office on 2001-06-07 for process for preparing (s)-3-halogeno-1,2-propanediol by microorganism.
Invention is credited to Idogaki, Hideaki, Kasai, Naoya, Nakagawa, Atsushi, Suzuki, Toshio.
Application Number | 20010003046 09/725287 |
Document ID | / |
Family ID | 18312211 |
Filed Date | 2001-06-07 |
United States Patent
Application |
20010003046 |
Kind Code |
A1 |
Suzuki, Toshio ; et
al. |
June 7, 2001 |
Process for preparing (S)-3-halogeno-1,2-propanediol by
microorganism
Abstract
An industrial and economical method for obtaining a
(S)-3-halogeno-1,2-propanediol, which comprises cultivating a
microorganism belonging to the genus Pseudomonas which has an
ability to assimilate (R)-3-halogeno-1,2-propanediol and can grow
by assimilating (R)-3-halogeno-1,2-propanediol as a single carbon
source, in a culture medium containing a racemic
3-halogeno-1,2-propanediol as a substrate, and isolating the
(S)-3-halogeno-1,2-propanediol from the culture medium.
Inventors: |
Suzuki, Toshio; (Osaka-shi,
JP) ; Idogaki, Hideaki; (Osaka-shi, JP) ;
Nakagawa, Atsushi; (Osaka-shi, JP) ; Kasai,
Naoya; (Sennan-gun, JP) |
Correspondence
Address: |
WENDEROTH, LIND & PONACK, L.L.P.
Suite 800
2033 K Street, N.W.
Washington
DC
20006
US
|
Family ID: |
18312211 |
Appl. No.: |
09/725287 |
Filed: |
November 29, 2000 |
Current U.S.
Class: |
435/158 |
Current CPC
Class: |
C12P 41/001 20130101;
C12P 7/18 20130101 |
Class at
Publication: |
435/158 |
International
Class: |
C12P 007/18 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 29, 1999 |
JP |
337812/1999 |
Claims
What is claimed is:
1. A method for obtaining a (S)-3-halogeno-1,2-propanediol, which
comprises cultivating a microorganism belonging to the genus
Pseudomonas which has an ability to assimilate
(R)-3-halogeno-1,2-propanediol and can grow by assimilating
(R)-3-halogeno-1,2-propanediol as a single carbon source, in a
culture medium containing a racemic 3-halogeno-1,2-propanedi- ol as
a substrate, and isolating the (S)-3-halogeno-1,2-propanediol from
the culture medium.
2. The method of claim 1, which comprises cultivating the
microorganism in the culture medium containing a racemic
3-halogeno-1,2-propanediol as a single carbon source.
3. The method of claim 1 or 2 for obtaining a (S)-3-chloro or
bromo-1,2-propanediol, which comprises using a racemic 3-chloro or
bromo-1,2-propanediol as a substrate.
4. The method of claim 1, 2 or 3, wherein the microorganism is
Pseudomonas sp. DS-SI-5 (FERM BP-7080).
Description
TECHNICAL FIELD
[0001] The present invention relates to a process for preparing a
(S)-3-halogeno-1,2-propanediol (abbreviated as (S)-[1]) by reacting
a racemic 3-halogeno-1,2-propanediol (abbreviated as racemate [1])
with a microorganism which has an ability to grow by assimilating a
(R)-3-halogeno-1,2-propanediol (abbreviated as (R)-[1]) as a single
carbon source and obtaining (S)-[1].
[0002] (S)-3-halogeno-1,2-propanediols are very important as
intermediates in making optically active compounds, such as
pharmaceuticals, agrochemicals or physiologically active
compounds.
PRIOR ART
[0003] As to biological or enzymatic methods for preparing a
(S)-3-halogeno-1,2-propanediol the following methods are known.
[0004] There are two known methods by Takahasi et al. (Japanese
Patent Publication A 62-122596, Japanese Patent Publication A
63-36798) and Nikaido et al. (Japanese Patent Application A
6-209781), which comprise reacting racemate [1] with a
microorganism to degrade (R)-[1] and recovering remained (S)-[l].
Although, each microorganism used in both methods has an ability to
stereoselectively degrade and metabolize (R)-[1] in racemate [1],
but the microorganism has not an ability to assimilate (R)-[l] as a
single carbon source and therefore, the said microorganism can not
grow and propagate in a completely synthetic medium containing
racemate [1] as a single carbon source, and an inorganic nitrogen
compound such as ammonium sulfate or ammonium nitrate as a nitrogen
source. In these methods, in order to obtain (S)-[1] from racemate
[1], after the microorganism was separately in a large amount
cultivated in a culture medium in which the microorganism can grow,
the cells are made to react with racemate [1], or otherwise
racemate [1] must be added to a nutrient culture medium in which
the microorganism can grow.
[0005] Especially, the method of Takahasi et al. belongs to the
reaction utilizing the degradatively metabolizing reaction by
oxidation, and in order to efficiently progress the reaction, it is
necessary to add glutathione or a compound having a SH group such
as sodium hydrosulfide or potassium hydrosulfide.
[0006] On the other hand, the method of Nikaido et al. is one
utilizing a strain belonging to the same genus Pseudomonas as in
the present invention, but the strain has not have an ability to
assimilate (R)-[1] and therefore, the degradatively assimilating
reaction of (R)-[1] with growth of the strain in a synthetic medium
containing racemate[1] as a single carbon source does not occur and
it is impossible to get (S)-[1].
[0007] These two known methods are not convenient and practical
from the viewpoints of optical resolution of racemate [1], recovery
and purification of (S)-[l] obtained, and are not economical from
the viewpoint of the industrial production scale.
DETAILED DESCRIPTION OF INVENTION
[0008] The problem to be solved is to provide a method for
preparing (S)-[1] from racemate [1] more economically, cheaper and
more simply in technique.
[0009] The present inventors extensively engaged in seeking a
microorganism which has an ability to preferentially assimilate
(R)-[l] in racemate [1] and furthermore, can grow by assimilating
(R)-[l] as a single carbon source, and have found such a
microorganism to complete the present invention.
[0010] The present invention relates to a method for obtaining
(S)-[1] which comprises cultivating a microorganism belonging to
the genus Pseudomonas (abbreviated as the microorganism of the
present invention) which has an ability to assimilate (R)-[1] and
can grow by assimilating (R)-[1] as a single carbon source, in a
culture medium containing racemate [1] as a substrate, and
isolating (S)-[1] from the culture medium.
[0011] Halogen atoms of racemate [1] used as a substrate in the
present invention are preferably chlorine atom and bromine
atom.
[0012] The present invention is in more detail explained as
follows.
[0013] The microorganism of the present invention is inoculated
into a completely synthetic medium containing racemate [1] as a
single carbon source and inorganic compounds such as many kinds of
ammonium salts or nitrates as nitrogen sources, and a small amount
of metalic salts or inorganic salts such as phosphoric acid salts,
and cultivated or reacted to assimilate (R)-[1], and then (S)-[1]
remaining in the culture broth is recovered, or the microorganism
of the present invention may be cultivated in a nutrient culture
medium usually used, such as a bouillon culture medium or a peptone
culture medium containing organic carbon sources and nitrogen
sources, if necessary inorganic salts, a small amount of metalic
salts, or vitamines to assimilate (R)-[1], and then (S)-[1]
remaining in the culture broth is recovered.
[0014] The present invention, namely relates to the method for
recovering (S)-[1] remained in the culture broth or the reaction
solution by preferentially degradative assimilating racemate [1]
with the microorganism of the present invention.
[0015] The assimilating reaction is preferably carried out within
optimum pH of the strain used herein and optimum temperature. When
the microorganism of the present invention grows by assimilating
(R)-[1] as a carbon source, hydrochloric acid in the same amount as
(R)-[1] which is degraded with dehalogenation is generated.
[0016] When pH gradually becomes lower by hydrochloric acid
released from (R)-[1] with progress of the assimilating reaction,
it is necessary to adjust pH of the reaction solution to optimum pH
by addition of a suitable alkali. For example, the solution is
preferably controlled in the range of optimum pH by using a known
acid-neutralizing agent, such as an aqueous alkali carbonate
solution, e.g. an aqueous calcium carbonate solution, an aqueous
sodium carbonate solution, an aqueous potassium carbonate solution
or an aqueous ammonium carbonate solution, an aqueous alkali
hydroxide solution, e.g. sodium hydroxide solution, an aqueous
potassium hydroxide solution or an aqueous calcium hydroxide
solution, or an aqueous ammonium solution.
[0017] The culture medium for cultivation of the microorganism of
the present invention and for making assimilating reaction of
(R)-[1] is preferably a completely synthetic medium containing
racemate [1] as a single carbon source, inorganic compounds such as
many kinds of ammonium salts or nitric acid salts as nitrogen
sources, and a small amount of metalic salts, or inorganic salts
such as a phosphoric acid salt in the economical viewpoint, but is
not limited as long as the conventional culture medium containing
racemate [1] as a substrate in which the microorganism of the
present invention can grow. For example, carbohydrates such as
glucose or fructose, alcohols such as glycerol, sorbitol, or
mannitol, organic acids, such as acetic acid, citric acid, malic
acid, maleic acid, fumaric acid or gluconic acid, or a salt
thereof, or a mixture thereof, can be used as carbon source.
[0018] Inorganic nitrogen compounds such as ammonium sulfate,
ammonium nitrate or ammonium phosphate, organic nitrogen compounds
such as urea, peptone, casein, yeast extract, meat extract, corn
steep liquor or a mixture thereof, can be used as nitrogen source.
Furthermore, inorganic salts such as a phosphoric acid salt,
metalic salts such as a magnesium salt, a potassium salt, a
manganese salt, an iron salt, a zinc salt, or a copper salt, or if
suitable, vitamins may be used.
[0019] The cultivation mentioned above is aerobically carried out
in an usual manner, at pH 4-10, preferably 5-9, at 15-50.degree.
C., preferably 20-37.degree. C. under stirring or agitating for
20-96 hours.
[0020] The microorganism of the present invention may be previously
cultivated in a nutrient culture medium usually used, such as a
bouillon culture medium or a peptone culture medium containing
organic carbon sources and nitrogen sources, and if necessary
inorganic salts, a small amount of metalic salts, or vitamines.
[0021] As enzyme-inducing additives to obtain the microorganism
having high enzyme activity, to the above mentioned culture medium,
or a nutrient culture medium such as a peptone culture medium or a
bouillon culture medium may be added a 3-halogeno-1,2-propanediol
such as racemic 3-chloro-1,2-propanediol or racemic
3-bromo-1,2-propanediol.
[0022] The concentration of the substrate in the reaction mixture
is preferably 0.1-15% (v/v) and the substrate may be added at once
in the initial stage or in several times.
[0023] The reaction is aerobically carried out under shaking or
agitation according to the usual method, and the reaction is
preferably completed in 24-120 hours, depending on the
concentration of the substrate or other reaction conditions. When
the residual amount of the substrate (racemate [1]) becomes 50%
comparing with the initial concentration of the substrate by gas
chromatography, the reaction is preferably finished, or with
measurement of optical purity on the optically active
object-compound ((S)-[1]), the end point may be preferably
determined. That is, it is preferable to quench the reaction at the
time when (R)-[1] in racemate [1] is completely assimilated.
[0024] Thus obtained (S)-[1] remaining in the reaction solution is
recovered, separated and purified by the conventional method. For
example, after removal of the cells from the reaction medium by
centrifugation, the supernatant is condensed with an evaporator,
extracted with a solvent such as ethyl acetate, ethanol, etc. The
extract is dried over anhydrous magnesium sulfate, and then the
solvent is evaporated in vacuo to obtain (S)-[1] in syrup.
Additionally, the purification by distillation may be carried
out.
[0025] The microorganism of the present invention is one belonging
to the genus Pseudomonas, illustratively Pseudomonas sp. DS-SI-5.
This strain was identified to a strain belonging to species of the
genus Pseudomonas from its physiological and bacteriological
properties and already deposited with the National Institute of
Bioscience and Human-Technology Agency of Industrial Science and
Technology, Japan under Budapest Treaty with an accession number of
FERM BP-7080.
[0026] The present invention is illustratively explained by
following examples, but should not be limited by these examples.
Percentage (%) in examples means % (w/v), if not defined
otherwise.
EXAMPLE 1
[0027] A culture medium (100 ml, pH 6.9) consisting of
1 Ammonium sulfate 0.5% Sodium monohydrogen phosphate 0.02%
Potassium monohydrogen phosphate 0.02% Sodium dihydrogen phosphate
0.04% Magnesium sulfate 0.05% Copper sulfate 0.0001% Manganese
nitrate 0.0001% and Calcium carbonate 0.45%
[0028] were poured into a 500 ml Erlenmeyer flask with baffles and
the flask was sterilized at 121.degree. C. for 15 minutes by
autoclaving. Thereto racemic 3-chloro-1,2-propanediol (1 ml, 1.3 g)
was added to prepare a completely synthetic medium containing
racemic 3-chloro-1,2-propanediol as a single carbon source. And
then, Pseudomonas sp. DS-SI-5 was previously incubated in a
gradient agar culture medium containing polypeptone (1.0%), yeast
extract (1.0%) and D-glucose (1.0%) and a loopful of the strain was
aseptically inoculated into the above synthetic medium. The culture
medium was cultivated with rotary shaking (130 rpm) at 30.degree.
C. for 2 days. At that time the remaining amount of racemic
3-chloro-1,2-propanediol was measured with gas chromatography (GL
Science Co., Ltd. column support: PEG20M, 60-80 mesh, 0.31-0.42 mm)
to be 45% in the remaining ratio. After the cultivation being over,
the culture broth was taken out, and the cells were removed by
centrifugation to give a supernatant. The supernatant was
concentrated to about 2 ml by evaporator and extracted with ethyl
acetate. The extract was dried over magnesium sulfate and the
solvent was removed in vacuo to give 0.51 g of
3-chloro-1,2-propanediol as a syrup.
[0029] The measurement of optical purity of the product thus
obtained was carried out by subjecting the product to gas
chromatography with Capillary column: astec CHIRALDEX G-TA (inner
diameter; 0.25 mm.times.30 m) after the conversion of the product
(an optical isomer of 3-chloro-1,2-propanediol) into the
corresponding optical isomer of glycidol by alkali-treating with an
aqueous sodium hydroxide solution [Suzuki et al., Appln. Microbiol.
Biotechnol., Vol.40,273-278 (1993)].
[0030] As a result, 3-chloro-1,2-propanediol obtained was 99% ee in
the optical purity and was (S)-form.
[0031] Conditions on the above gas chromatography analysis were as
follows:
[0032] Retention time of glycidol: (R)-form, 80.6 min.; (S)-form,
82.1 min.
[0033] Analysis temperature: Column temp. (45.degree. C.), Inject
temp.: 200.degree. C.
[0034] Carrier gas: nitrogen (flow 0.5 ml/min.), Split ratio:
200/1, Detection: FID 200.degree. C.
EXAMPLE 2
[0035] A nutrient culture medium (100 ml, pH 7.2) consisting of
polypeptone (1.0%), yeast extract (1.0%) and D-glucose (1.0%) were
poured into a 500ml Erlenmeyer flask with baffles and the flask was
sterilized under vapor pressure at 121.degree. C. for 15 minuets to
prepare a liquid nutrient culture medium. A loopful of Pseudomonas
sp. DS-SI-5 which was previously cultivated in a gradient agar
nutrient culture medium of the above ingredients was inoculated
into the above liquid medium. The culture medium was cultivated
with rotary shaking (130 rpm) at 30.degree. C. for 24 hours. The
cells were harvested by centrifugation and washed twice by
phosphate buffer (50 m M, pH 7.2) to prepare washed cells. The
cells were suspended in the culture medium (101 ml) containing
racemic 3-chloro-1,2-propanediol as a single carbon source shown in
Example 1 and were reacted by agitation (130 rpm) at 30.degree. C.
for 2 days. The remaining amount of racemic
3-chloro-1,2-propanediol was measured in a same manner as in
Example 1 to be 46% in the remaining ratio. After the reaction, the
cells were removed by centrifugation to give a supernatant. The
recovery of 3-chloro-1,2-propanediol from the supernatant was
conducted in the same method as in Example 1 to obtain 0.52 g of
the product.
[0036] As a result of measurement on optical purity of the product
in the same method as in Example 1, 3-chloro-1,2-propanediol
obtained was 99% ee in the optical purity and was (S)-form.
EXAMPLE 3
[0037] A culture medium (2.5 L, pH 6.9) consisting of
2 Ammonium sulfate 0.5% Sodium monohydrogen phosphate 0.02%
Potassium monohydrogen phosphate 0.02% Sodium dihydrogen phosphate
0.04% Magnesium sulfate 0.05% Ferric sulfate 0.001% Copper sulfate
0.0001% and Manganese nitrate 0.0001%
[0038] were put into a 5 L fermenter (jar fermenter, Mitsuwa
Rikagaku Co., Ltd. Model KMJ5B) and sterilized under vapor pressure
at 121.degree. C. for 15 minutes. Thereto racemic
3-chloro-1,2-propanediol (25 ml, 32.5 g) was added to prepare a
completely synthetic medium containing racemic
3-chloro-1,2-propanediol as a single carbon source. And then,
Pseudomonas sp. DS-SI-5 was previously incubated under shaking in a
nutrient culture medium containing polypeptone (1.0%), yeast
extract (1.0%) and D-glucose (1.0%) at 30.degree. C. for 24 hours,
and 50 ml [2% (v/v)] of the culture broth were aseptically
inoculated into the above synthetic medium. The culture medium was
aerobically (airation: 0.5 L/min) cultivated with agitation (500
rpm) at 30.degree. C. for 3 days.
[0039] Measurement of pH and its control was conducted with a pH
controller and pH was adjusted to 6.9 with an aqueous sodium
hydroxide solution (3 mmol/L).
[0040] The measurement and identification of the product was
conducted in the same method as in Example 1.
[0041] After the cultivation being over, the culture broth was
taken out, and the cells were removed by centrifugation to give a
supernatant. The recovery of 3-chloro-1, 2-propanediol was
conducted in the same method as in Example 1 to give 13.7 g of the
product. As a result of measurement on optical purity of the
product in the same method as in Example 1,
3-chloro-1,2-propanediol obtained was 99% ee in the optical purity
and was (S)-form.
EXAMPLE 4-6
[0042] Experiments (three kinds) were conducted according to the
procedures of the above Examples 1-3, except for using racemic
3-bromo-1,2-propanediol as a substrate instead of racemic
3-chloro-1,2-propanediol.
[0043] As results, optically purity and optical isomer of
3-bromo-1,2-propanediol obtained were 96% ee and (S)-form,
respectively. The remaining amount of 3-bromo-1,2-propanediol was
0.24 g, 0.28 g and 6.2 g, respectively.
EFFECT OF INVENTION
[0044] According to the present invention, by cultivating a
microorganism belonging to the genus Pseudomonas, especially
Pseudomonas sp. DS-SI-5 having an ability to assimilate (R)-[1] in
the culture medium containing racemate [1] as a substrate,
especially in the culture medium containing the racemate as a
single carbon source, and by preferentially degradative
assimilating (R)-[1] to obtain (S)-[1] economically and in
industrial simple.
[0045] On the other hand, according to the present invention, even
if (S)-[1] is prepared in industrial scale, it is not necessary to
prepare a large amount of cells by cultivation the strain
separately and it is enough to cultivate the amount of the strain
useful as starter and inoculate it, namely it is enough if only one
microorganism is there.
* * * * *