JPH0528117B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a method for producing optically active 3-halogeno-1,2-propanediol. Optically active 3-halogeno-1,2-propanediol is a substance useful as a raw material for the synthesis of various pharmaceuticals and optically active physiologically active substances. For example (R)−
3-Chloro-1,2-propanediol is used in the synthesis of L-carnitine (Japanese Patent Application Laid-Open No. 1987-1999).
165352). (Prior art and problems) Regarding the production method of (R)-3-halogeno-1,2-propanediol, Hayden F. Jones describes methyl-5-chloro-5-deoxy-α-L-arabinofuranoside. How to get more (Chemistry and Industry)
P.538, 15July, 1978], and the method for obtaining it from 1,2,5,6-diacetonyl-D-mannitol by H. Jackson et al. [Chemistry and Biological Interactions (Chem.-
Biol. Interactions), 13 , 193 (1976)], as well as Y.
The method of Kawakami et al. [Journal of Organic Chemistry]
Chemistry, 47 , 3581 (1982)]. Regarding the method for producing (S)-3-halogeno-1,2-propanediol, HFJones' method for obtaining it from methyl-6-chloro-6-deoxy-α-D-glucopyranoside (West German Patent No. 2743858);
Method for obtaining 1,2,5,6-diacetonyl-D-mannitol by Porter KE et al. [Chem.-
Biol. Interactions. 41 , 95 (1982)]. However, these methods cannot be called industrial production methods because the raw materials are difficult to obtain and the steps are complicated.
-Production of halogen-1,2-propanediol was desired. The present inventors discovered that optically active 3-halogeno-1,2, which had been obtained through complicated reactions using expensive raw materials,
- As a result of research aimed at industrial production of propanediol, we found that cheap (R,S)-3-halogeno-1,
By allowing microorganisms to act on 2-propanediol, (S)-3-halogeno-1,2-propanediol is selectively metabolized, and (R)-3-halogeno-
1,2-propanediol can remain (Patent application 1986-264568, 1981-000409), or (R)
-3-halogeno-1,2-propanediol can be selectively metabolized and (S)-3-halogeno-1,2-propanediol can remain (patent application filed in 1983-
264567) have already been found. (Means for Solving the Problems) As a result of intensive research in order to increase the productivity of optically active 3-halogeno-1,2-propanediol by utilizing microbial reactions, the present inventors discovered that the SH group-containing compound was added to the reaction solution. The present inventors have discovered that the addition of these substances to the substrate can increase the substrate concentration and lead to improved productivity, and have completed the present invention. Examples of SH group-containing compounds that can be used in the present invention include dithiothreitol, dithioerythritol, glutathione, cysteine, mercaptoethanol, thioglycerin, 2,3-dimercaptopropanol, thioacetic acid, dimercaptosuccinic acid,
-Mercaptopropionic acid, 3-mercaptopropionic acid, thioglycolic acid, tert-butylmercaptan, sodium hydrosulfide, potassium hydrosulfide, and the like. When selectively metabolizing only the optically active (S)-3-halogeno-1,2-propanediol and accumulating the (R) form, microorganisms of the genus Brettanomyces and Candida can be used.
Genus, Endomycea, Geotrichum, Nadsonia, Pachysolen,
Rhodosporidium, Saccharomycopsis, Sporidiobolus, Torulopsis, Bacillus
Genus, Enterobacter, Escherichia, Klebsiella, Arthrobacter, Aspergillus, Beauveria
Genus, Calonectoria, Conichhaetidium, Backusella, Cladobotryum, Chloridium, Corticium
Genus, Cunninghamella, Ciricinella, Gliocladium, Gliocephalotrichum, Echinopodospora, Gelasinospora, Dichotomomyces ), Pycnoporus, Neosartorya, Penicillium, Thamnostylum, Zygorhynchus, monascus
Microorganisms belonging to the genus can be mentioned. For more information, see Brettanomyces custerianus IFO 1585, Candida utilis IFO
0626, Endomyces magnusui
magnusii) CBS 164.32, Geotrichum candidum CBS
187.67, Nadsonia elongata
Pachysolen tannophilus IFO 1007, Rhodosporidium toruloides IFO 0871, Saccharomycopsis lipolytica IFO 0717, Sporidiobol us johnsonii ) IFO 6903, Torulopsis grotspenguieseli (Torulopsis
gropengiesseri) IFO 0659, Bacillus cereus (Bacillus cereus) IFO 3001, Enterobacter aerogenes (Enterobacter aerogenes)
IFO 13534, Escherichia coli
coli IFO 12734, Klebsiella pneumoniae IFO 12009, Arthrobacter simplex
simplex) IFO 12069, Aspergillus ficuum IFO 4034, Beaveria bassiana IFO 8554,
Calonectria hedera (Calonectria
hederae), Conichhaetidium savoryi IFO 30424, Backusella circina IFO
9231, Cladobotyryum apiculatum IFO 7795, Choridium chlamydospolis
chlamydosporis) IFO 7070, corteicium
Corticium rolfsii IFO 30071, Cunninghamella
echinulata) IFO 4441, Circinella simplex (Circinella simplex) IFO 6412, Gliocladium delicecens (Gliocladium
deliguescens) IFO 6790, Gliocephalotrichum cylindrosporum (Gliocephalotrichum cylindrosporum) IFO
9326, Echinopodospora jamaicensis IFO 30406,
Gelasinospora cerealis (Gelasinospora
cerealis) IFO 6759, Dichotomomyces cejpii IFO 8396, Pynoporus coccineus
IFO 9768, Neosartorya aurata, Penicillium janthinellum IFO
4651, Thamnostylum piriforme IFO 6117, Zygorhynchus moelleri
HUT 1305, Monascus
anka) There is IFO 5965. On the other hand, when selectively metabolizing only optically active (R)-3-halogeno-1,2-propanediol and accumulating the (S) form, microorganisms of the genus Pichia and Trichosporon
Mention may be made of microorganisms belonging to the genus Corynebacterium. For more information, see Pichia farinosa
IFO 1003, Trichosporon fermentans CBS 2264, Corynebacterium acetoacidophilum ATCC
There are 21476. As the medium composition for culturing the above-mentioned microorganisms, any medium in which these microorganisms can grow can be used. For example, sugars such as glucose, sucrose, and maltose as carbon sources;
Alcohols such as ethanol, glycerol, 1,2-propanediol; organic acids such as acetic acid, lactic acid, or mixtures thereof; nitrogen sources such as ammonium sulfate, ammonium phosphate, urea, yeast extract, meat extract, peptone, etc.; Nutrient sources used in normal culture, such as inorganic salts, trace metal salts, and vitamins, can be appropriately mixed and used. The above microorganisms may be cultured by conventional methods, for example, the culture medium pH should be in the range of 4.0 to 9.5, and the culture temperature should be in the range of 20 to 45.
It is preferable to culture aerobically at a temperature in the range of 10 to 96 hours. As a method for obtaining optically active 3-halogeno-1,2-propanediol by allowing microorganisms to act on (R,S)-3-halogeno-1,2-propanediol, a culture solution cultured as described above or this culture solution is used. A method in which a substrate is added to a suspension of microbial cells obtained by centrifugation, a method in which a substrate is added to a medium and culture and reaction are carried out simultaneously, or a method in which microorganisms immobilized by a conventional method are placed in an appropriate buffer. There is a method of adding a substrate to a suspension in a liquid. The reaction temperature is preferably 15 to 50°C and the reaction pH is preferably 4.0 to 10.0, and a buffer or the like can be used as appropriate to maintain the pH. The substrate concentration in the reaction solution is preferably 0.1 to 10 (W/V)%, and the substrate may be added all at once at the initial stage or may be added in portions. At that time, an SH group-containing compound is added to the reaction solution. The concentration of the SH group-containing compound added is 0.05
The amount is preferably ~10 (w/v)%, and the addition method may be to add it all at once together with the substrate at the initial stage, or to add it in portions. The reaction is usually carried out with shaking or stirring,
Although the reaction time varies depending on the substrate concentration, enzyme amount, and other reaction conditions, it is preferable to select conditions so that the reaction can be completed within 72 hours. To stop the reaction, analyze the remaining substrate using gas chromatography, etc.
In terms of yield, it is preferable to stop the process when about 50% of the substrate has been consumed. In order to collect the optically active 3-halogeno-1,2-propanediol thus obtained from the reaction solution, a general optically inactive 3-halogeno-1,2-propanediol is used.
A method for collecting 2-propanediol can be used. For example, after removing bacterial cells from the reaction solution by centrifugation or the like, the supernatant is appropriately concentrated and extracted with a solvent such as ethyl acetate. After dehydrating the extract over anhydrous sodium sulfate or the like, the solvent is removed under reduced pressure to obtain a syrup of optically active 3-halogeno-1,2-propanediol. Further, it may be further purified by distillation. (Example) The present invention will be specifically described below with reference to Examples. Example 1 Glycerol 4%, (NH ₄ ) ₂ HPO ₄ 1.3%,
_{KH2PO4 0.7} %, _MgSO4・_7H2O 800ppm,
_ZnSO4・_7H2O 60ppm, _FeSO4・_7H2O 90ppm,
_CuSO4・_5H2O 5ppm, _MnSO4・_4H2O 10ppm,
A medium consisting of 100 ppm NaCl and 0.3% yeast extract was prepared with deionized water (PH 7.0), dispensed into 500 ml Sakaguchi flasks in 50 ml portions, and sterilized at 120°C for 20 minutes. Satucharomycopsis lipolyteica in the above medium.
IFO 0717 was inoculated and cultured with shaking at 30°C for 48 hours. Centrifuge 150ml of this culture solution to advance the bacterial cells. After washing with water, transfer the bacterial cells to 0.3M phosphate buffer (PH7.0).
(R,S)-3-chloro-1,
0.5g of 2-propanediol and as shown in Table 1
0.15g of SH group-containing compound was added and reacted with shaking at 30°C for 24 hours. The decomposition rate of the substrate was determined by extracting 1 ml of the reaction solution with 2 ml of ethyl acetate and analyzing it using gas chromatography.
It was measured. (Conditions) Column length: 50cm Packing material: FAL-M6% Carrier: TENAX GC (manufactured by Shimazu) Carrier gas: N ₂ (22.5ml/min) Column temperature: 175℃ Detection: FID Substrate retention time: 1.8min (3 -chloro-1,2-
(propanediol) Substrate retention time: 3.0 min (3-bromo-1,2
-propanediol)

[Table] The thioglycerin and potassium hydrosulfide addition groups, which were particularly effective, were purified using the following flow. Sterilize the reaction solution by centrifugation, and save approximately 10ml of the supernatant.
Concentrate to
Extracted. The extract was dehydrated with anhydrous sodium sulfate and the solvent was removed under reduced pressure to obtain a syrup. After distillation of this product, the specific rotation was measured and the following values were obtained. [a] ²⁰ _D = -6.23° (c = 2.0 H ₂ O) ... Thioglycerin addition group [a] ²⁰ _D = -5.35° (c = 2.0 H ₂ O) ... Potassium hydrosulfide addition group (R) Literature value for -3-chloro-1,2-propanediol [a] ²² _D = -6.9° (c = 2.0, H ₂ O) From this, even the addition of SH group-containing compounds does not affect the identification of optical activity. Same as additive-free system.
It can be seen that (R) form of 3-halogeno-1,2-propanediol can be obtained. Examples 2 to 40 (number 6 is omitted) The same method as Example 1 was carried out except that the bacterial strain was changed to those shown in Table 2. However, in 17 to 37, glucose was used as the carbon source. The results shown in Table 2 were obtained (however, thioglycerin was used as the SH group-containing compound).

【table】

【table】

【table】

[Table] In addition, Konicaeteidium sapori IFO
Using the reaction solution of the thioglycerin-added group of 30424 (Example 20), after the same extraction and purification operations as in Example 1,
When the specific optical rotation was determined, the following values were obtained. [a] ²⁰ _D = -7.28° (c = 2.0, H ₂ O) Examples 41 to 80 (however, 46 is a missing number) Using the same bacterial strain as in Examples 1 to 40, only the substrate (R, S) -3-Bromo-1,2-propanediol was used, and the same procedure as in Examples 2 to 40 was carried out to obtain the results shown in Table 3. ).

【table】

【table】

【table】

[Table] (Effects of the invention) According to the present invention, optically active 3-halogeno-1,
In the production of 2-propanediol, the concentration of the substrate to be charged increases, leading to improved productivity.

Claims (1)

[Claims] 1. Optically active (R) or (S)-3-halogeno-1,
Brettanomyces genus, which has the ability to selectively metabolize 2-propanediol;
Genus Candida, Genus Endomyces, Geotrichum
Genus, Nadsonia, Pachysolen, Rhodosporidium, Saccharomycopsis, Sporidiobolus, Torulopsis, Bacillus ), Enterobacter, Escherichia, Klebsiella, Arthrobacter, Aspergillus, Beauveria
Genus, Calonectria, Conichaeidium, Backusella, Cladobotryum, Chloridium, Corticium
Genus, Cunninghamella, Ciricinella, Gliocladium, Gliocephalotrichum, Echinopodospora, Gelasinospora, Dichotomomyces ), Pycnoporus, Neosartorya, Penicillium, Thamnostylum, Zygorhynchus, Monascus
Microorganisms belonging to the genus Pichia,
Microorganisms belonging to the genus Trichosporon and Corynebacterium were treated with racemic (R,S)-3-halogeno-1,
Optically active 3-halogeno-1 characterized in that an SH group-containing compound is added to the reaction solution when reacting on 2-propanediol and collecting the remaining optically active 3-halogeno-1,2-propanediol. , 2-propanediol manufacturing method. 2 The SH group-containing compound is dithiothreitol,
dithioerythritol, glutathione, cysteine, mercaptoethanol, thioglycerin,
2,3-dimercaptopropanol, thioacetic acid,
The manufacturing method according to claim 1, which is dimercaptosuccinic acid, 2-mercaptopropionic acid, 3-mercaptopropionic acid, thioglycolic acid, tert-butylmercaptan, sodium hydrosulfide, and potassium hydrosulfide. 3. The manufacturing method according to claim 1 or 2, wherein the remaining optically active 3-halogeno-1,2-propanediol is (R)-3-halogeno-1,2-propanediol. 4. The manufacturing method according to claim 3, wherein the (R)-3-halogeno-1,2-propanediol is (R)-3-chloro-1,2-propanediol. 5. The manufacturing method according to claim 3, wherein the (R)-3-halogeno-1,2-propanediol is (R)-3-bromo-1,2-propanediol. 6 Microorganisms such as Brettanomyces custerianus, Candeida
Candida utilis, Endomyces magnusii, Geotrichum
candidum), Nadsonia elongata, Pachysolen tannophilus, Rhodosporidium toruroides
toruloides), Saccharomycopsis lipolytica, Sporidiobolus zijonsonii
johnsonii), Torulopsis gropengiesseri, Bacillus cereus, Enterobacter aerogenes, Escherichia coli, Klebsiella pneumoniae,
Arthrabacter simplex, Aspergillus ficuum, Beauveria bassiana, Calonectria hederae, Conichhaetidium
savoryi), Backusella sarcina (backusella
circina), Cladobotyryum apiculatum, Chloridium chlamydospolis
chlamydosporis), Corticium rolfsii, Cunninghamella echinulata, Ciricinella simplex, Gliocladium
deliquescens), Gliocephalotrichum (Gliocephalotrichum)
cylindrosporum), Echnopodospora jamaicensis, Gelasinospora cerealis
cerealis), Dichotomomyces cejpii, Pycnoporus coccineus, Neosartorya aurata, Penicillium
janthinellum), Thamnostylum piriforme, Zygorhynchus moelleri, Monascus anka. 7. The manufacturing method according to claim 1 or 2, wherein the remaining optically active 3-halogeno-1,2-propanediol is (S)-3-halogeno-1,2-propanediol. 8. The manufacturing method according to claim 7, wherein the (S)-3-halogeno-1,2-propanediol is (S)-3-chloro-1,2-propanediol. 9. The manufacturing method according to claim 7, wherein the (S)-3-halogeno-1,2-propanediol is (S)-3-bromo-1,2-propanediol. 10 The microorganism is Pichia falinosa
farinosa), Trichosporon fermentans, Corynebacterium
acetoacidophilum)
or the manufacturing method described in paragraph 7.