JPS6269993A - Production of optically active alpha-monochlorohydrin by bacterium treatment - Google Patents

Abstract

PURPOSE:To selectively collect R-(-)-3-chloro-1,2-propanediol simply without forming another derivative, by treating racemic modification of 2,3- dichloro-1-propanol with a bacterium belonging to the genus Pseudomonas, etc. CONSTITUTION:Racemic modification of 2,3-dichloro-1-propanol is treated with a bacterium such as Pseudomonas OS-K-29(FERM P-7846), etc., belonging to the genus Pseudomonas, capable of assimilating R-(+)-2,3-dichloro-1-propanol or its culture mold. To be concrete, the bacterium is cultivated in a medium containing the racemic modification of the propanol as a carbon source, a nitrogen source, an inorganic salt, etc., or the culture mold is treated in a medium containing the racemic modification of the propanol and alpha-monochlorohydrin is selectively collected. The selective collection of the aimed substance is carried out by active carbon column treatment, ether extraction, etc., of supernatant liquid of culture.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for separating optically active α-monochlorohydrin from caries using microorganisms.

(Prior art) Optically active 3-chloro-1,2-propanediol (α
-monochlorohydrin) is a substance useful as a synthetic raw material for pharmaceuticals and the like. Optically active α-monochlorohydrin can be produced synthetically from D-mannitol, but since lead tetraacetate is used, there are problems in terms of toxicity.

There is no known method for producing α-monochlorohydrin, which is more optically active than racemic 2,3-dichloro-1-propanol.

(Objective of the invention) The present invention aims to directly assimilate racemic 2.3-propanol into microorganisms without passing through other derivatives, and to separate optically active α-monochlorohydrin. purpose.

(Structure of the Invention) The present invention refers to R-(+)-2,3-dichloro-1
- Bacteria belonging to the genus Pseudomonas having the ability to assimilate propanol, or cultured cells thereof, are allowed to react with racemic 2,3-dichloro-1-propanol to produce R-(-)-3.
This is a method for producing optically active α-monochlorohydrino through microbial treatment, which is characterized by separating -chloro-1,2-propanediol.

The mycological properties of the microorganisms isolated and collected from soil by the present inventors and used in the present invention are shown in Table 1.

Table 1 a. Morphology ■Cell shape and size Bacillus, 0.4-0.6
X 1,2-1.8 μm■Cell pleomorphism
Absence ■ Presence of motility Yes, polar flagella ■ Presence of spores Absent ■ Durham staining Negative ■ Anti-acidity None, Growth status in each medium ■ Broth agar plate culture (30℃, 3 days culture) a) Slow colony shape Normal Approximately 3 to 4 squares in diameter 10) Shape of the colony: circular C) Shape of the colony surface: smooth 2) Protruding state of the colony: convex circular e) Periphery of the colony To the golden edge) Contents of the colony: homogeneous G) Color of the colony: milky white C) Colony Transparency of translucent January colony gloss
Blunt light (nu) Production of soluble pigments None ■ Broth agar slant culture (cultured at 30°C for 3 days) (a) Quality of growth Good growth, filamentous mouth (colony shape) Smooth (c) Protruding state of colony cross section (flat) (ii) Colony Gloss Brightness E) Colony surface shape Smooth) Colony transparency Translucent G) Colony color Milky white■ Meat juice liquid culture! ! (Cultivated at 30℃ for 3 days) a) Growth characteristics Membrane-like mouth) Turbidity Slightly cloudy.

C) Gas generation None e) Coloring of the chopsticks material None ■Meat juice gelatin puncture culture Does not liquefy gelatin.

■Lidmus Milk It does not solidify and turns lidmus pale blue or colorless.

C0 Physiological Test 1 Nitrate Reduction 12 MR Test - 3 VP Test - 4 Indole Production - 5 Hydrogen Generation - 6 Starch Hydrolysis - 7 Denitrification Reaction - 8 Citric Acid Utilization 19 Inorganic Nitrogen Source Utilization 110 Production of dye Fluorescent dye production in Sellers' medium 11 Urease +
12 Oxidase +13 Catalase +14 Growth range
pH5.5-9.0. Temperature 20-37℃15
Attitude towards oxygen Aerobic 16 0-F test (1-1ugh 1-eifson method) 017
Presence or absence of sugars in the production of acids and gases from sugars Acid Gas (1) D-glucose 10 - (2) D-galactose 10 - (3) Sucrose 10
− (4) Trehalose + −(5) Starch −− Based on the above results, Purge Aids Manual of
Determinative bacteriology (3ergey)
' s Manuat of l) etermin
tiveB acteriology) @ Based on the description in the B version, the origin is identified as having characteristics of the genus Pseudomonas.

Hereinafter, the present inventors will refer to the home country as Pseudomonas P seud.
omonas OS-K-29
No. 846: FERM P-7846).

In the present invention, the microorganism is brought into contact with cymic 2,3-dichloro-1-propanol to produce R-(-)-3-chloro-1,2-propanediol (hereinafter referred to as R-(-)-α-monochloro-1-propanol). (referred to as ruhydrin).Specifically, the creeping semiform 2,3-dichloro-1-propanol is used as the carbon source, inorganic nitrogen (various ammonia salts and nitrates) is used as the nitrogen source, and other inorganic salts are separated. or culture the bacteria in a commonly used nutrient medium containing a carbon source, a nitrogen source, an organic nutrient source, and an inorganic nutrient source, such as a bouillon medium or a sweetened bouillon medium. After allowing the cells to grow well and allowing the cells obtained to react in a medium containing racemic 2,3-dichloro-1-propanol, α-monochlorohydrin may be fractionated.

Glucose and sucrose are carbon sources.

Carbohydrates such as glycerin, or citric acid.

Organic acids such as maleic acid and malic acid and their salts are used as nitrogen sources, such as ammonium sulfate, ammonium chloride, ammonium nitrate, 1. Inorganic nitrogen such as ammonium phosphate,
and urea, peptone.

Organic nitrogen such as casein, yeast extract, meat extract, etc. can be used. Other inorganic salts include phosphates,
Magnesium salt, potash salt, manganese salt, iron salt, zinc salt,
Copper salts etc. are used.

Cultivation in the home country can be performed by conventional methods. usually,
The reaction is carried out aerobically at a temperature of about 20 to 40°C, preferably 25 to 37°C, and a pH of about 6 to 9, preferably l) 86.5 to 7.5, by means such as shaking or aerated stirring.

Microorganisms used in the present invention and racemic 2,3-dichloro-1
- The concentration of racemic 2,3-dichloro-1-propanol when contacting with propanol is approximately 0.6 to 1% of the width of the medium.
．． It may be about 0% by volume, and the contact time is usually 2 to 10 days.

After the culture is completed, the culture solution is taken out and centrifuged to separate the microbial cells and supernatant, and α-monochlorohydrin in the supernatant is separated by activated carbon column treatment, ether extraction, vacuum distillation, etc. take.

This will be explained below using examples. In the examples, % represents weight % unless otherwise specified.

Example 1 Medium with racemic 2.3-dichloro-1-propanol as the sole carbon source, i.e. racemic 2.3-dichloro-1-propanol 0.6% by volume Ammonium sulfate O, OS% Ammonium nitrate
0.05% Potassium hydrogen phosphate 0.1% Sodium phosphate 0.1
% Sodium phosphate 0.2% Magnesium sulfate 0.05% Iron sulfate, copper sulfate, manganese sulfate Trace amounts p) 4 6.5 Medium 10
Home country O8- to Sakaro flask (500-volume) with 0-
One platinum loopful of each strain of 129 strains is inoculated onto a slanted agar medium, and cultured with shaking at 30° C. for 3 to 5 days. Next, the above-mentioned columnar nutrients were added to 40 500-Sakaro flasks containing 100-ml of the above-mentioned medium at a concentration of 2%, and cultured with shaking under the following conditions for 3 to 5 days.

Temperature 30℃ 1) H initial 6.5 (-0.5% CaCO3 added)
-1 is maintained. ) (Rice count: 125rDIIll) After the culture is completed, the culture solution is taken out and separated into microbial cells and their supernatant using a centrifuge, and the remaining α
- 3.29 g of monochlorohydrin was collected as an oily substance by activated carbon column treatment, ether extraction, and vacuum distillation. The substance was identified using the following method.

1) Identification column carrier P by gas chromatography
As a result of comparison with commercially available α-monochlorohydrin using EG-20M P, 5%, 60-80 meshes, the retention times were exactly the same.

Purity 90.5% or higher.

2) Identification by IR (infrared absorption spectrum) As shown in the chart shown in FIG. 1, its absorption pattern was exactly the same as that of commercially available α-monochlorohydrin.

From the above, this substance was clearly found to be α-monochlorohydrin. The following method was used to confirm that this substance was R-(-)-α-monochlorohydrin.

1) Measurement of optical rotation The optical rotations of commercially available α-monochlorohydrin and this substance are as follows.

Commercially available α-monochlorohydrin [α]. =0.0° (
C=1. H20) This substance [α]25--4.3°
(c=1°D H2O) From the above results, it was found that this substance was R-(-)-α-monochlorohydrin, and its optical purity was 63% or more.

(Effects of the Invention) According to the present invention, optically active 3-chloro-1,2-propanediol can be obtained using O3-ni-29 strain belonging to the genus Pseudomonas isolated from soil.

[Brief explanation of drawings]

FIG. 1 shows infrared absorption spectra of R-(-)-3-chloro-1,2-propanediol obtained in Example 1 and a commercially available product of the same substance.

Claims (1)

[Claims] A bacterium belonging to the genus Pseudomonas or a culture thereof having the ability to assimilate R-(+)-2,3-dichloro-1-propanol is reacted with racemic 2,3-dichloro-1-propanol to produce R- A method for producing optically active α-monochlorohydrin by microbial treatment, which comprises separating (-)-3-chloro-1,2-propanediol.