JPS6350996B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a method for producing a corresponding halogenated epoxide from a halogenated olefin using microorganisms. Prior Art Epoxide compounds are widely used as raw materials or intermediates for the production of various organic chemical products including synthetic resins, surfactants, medicines, and agricultural chemicals. Examples of methods for producing epoxide using microorganisms include Nocardia, Mycobacterium, Methylococcus, Methylosinus, pseudomonas, and Corynebacterium. ), Methylobacterium, and Candida are known to produce epoxides from linear olefins. However, the only known example of production of halogenated epoxides is by microorganisms belonging to the genus Nocardia. Purpose of the Invention The present invention provides that epoxide-producing bacteria belonging to the genus Micrococcus, Arthrobacter, Corynebacterium, Mycobacterium, Rhodococcus and Brevibacterium can produce corresponding halogenated epoxides from halogenated olefins. The purpose of this invention is to provide a method for producing a corresponding halogenated epoxide from a halogenated olefin by using microorganisms having epoxide-producing ability belonging to each of the above-mentioned genera. shall be. The present invention will be explained in detail below. Structure of the Invention The structural feature of the present invention is that Micrococcus spp.
A microorganism capable of producing epoxide selected from the group consisting of Arthrobacter, Corynebacterium, Mycobacterium, Rhodococcus and Brevibacterium is allowed to act on halogenated olefin under aerobic conditions. The goal is to separate and collect the halogenated epoxide that is produced. The microorganisms used in the present invention belong to each of the above-mentioned genera, and include the strains shown in Table 1 below. Please note that these strains are American type.
Culture Your Collection (American Type
It has been deposited in the Culture Collection under the following number and can be easily obtained.

[Table] Halogenated olefin used as a reaction substrate for producing epoxide using the above microorganisms in the present invention (hereinafter referred to as raw material olefin)
Examples include halogenated linear or branched olefins (olefins in which the double bond is in the α, β or γ position from any terminal carbon atom), such as allyl chloride, allyl bromide, allyl iodide. , 3-chloro-1-butene, 3-chloro-2-methyl-propene, 1-chloro-2-butene, 2-fluoropropene, 3,
Examples include 3,3-trifluoro-propene. In the present invention, these raw material olefins are used alone or as a mixture of two or more, or as a mixture with a hydrocarbon other than olefin, such as a saturated hydrocarbon or an aromatic hydrocarbon, as a reaction substrate. In the present invention, in order to cause the microorganism to act on the raw material olefin, for example, (a) a method in which the raw material olefin is brought into contact with bacterial cells obtained by culturing and propagating the microorganism in advance under aerobic conditions to react; (b) A method of culturing the above-mentioned microorganisms under aerobic conditions in a nutrient medium consisting of raw material olefin or raw material olefin and other carbon sources, a nitrogen source, inorganic salts, and, if necessary, a growth promoting substance. can be applied. In the method of (a) above, in which the raw material olefin is brought into contact with the growing bacterial cells and reacted, first, as a carbon source, carbohydrates such as glucose, sucrose, molasses, starch hydrolysates, cellulose hydrolysates, hydrocarbons such as propane, butane, etc. Dodecane, tetradecane, and other substances with high bacterial growth effects such as acetic acid are used, and nitrogen such as ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, aqueous ammonia, amino acids, and other assimilable organic nitrogen compounds are used. sources, inorganic salts such as potassium phosphate, sodium phosphate, magnesium sulfate, manganese sulfate, ferrous sulfate, ferric chloride, calcium chloride, manganese chloride, and optionally vitamins, yeast extract, and cornstarch. Inoculum of each of the above microorganisms is inoculated into a medium to which a growth promoting substance such as Epliquor is added, and the microorganisms are cultured under aerobic conditions to multiply the microbial cells. The raw material olefin and air, oxygen, or oxygen-enriched gas are added directly to the bacterial cell culture obtained in this way, or to a suspension of bacterial cells isolated from the culture, or to a suspension of bacterial cells isolated from the culture. A reaction is caused by supplying an oxygen-containing gas such as The reaction takes place in the pH range of 5 to 9, preferably 6 to 8.
1-6 at a temperature of 20-50℃, preferably 25-45℃
Do it for days. The reaction is usually carried out under normal pressure, but the productivity of epoxide can also be improved by carrying out the reaction under increased pressure. The activity of the reaction between the bacterial cells and the raw material olefin can be maintained or increased by appropriately adding the carbon source, nitrogen source, and other components used for bacterial growth during the reaction. The reaction can be carried out either batchwise or continuously. In the case of a batch reaction method, the raw material olefin can be supplied in its entirety at the start of the reaction, or it can also be supplied continuously or intermittently during the reaction. The epoxide produced in the reaction solution by the above reaction is separated and collected using known techniques such as phase separation, extraction, and distillation. Next, the culture method (b) is a method in which raw material olefin is added during cell growth in the method (a) to produce epoxide in one step. For the culture conditions (PH, temperature, pressure, etc.), culture method, and separation and collection of the produced epoxide, the reaction conditions, reaction method, and separation and collection method described in (a) above can be similarly used. The epoxide obtained by the present invention can be used for various conventionally known uses as mentioned above. The present invention will be explained in more detail with reference to Examples below. Example 1 Three platinum loops of each of the 19 types of bacterial cells listed in Table 2 below were cultured in NBG medium (10 g of Labrenco powder manufactured by Oxoid, 10 g of bacteriological peptone, 10 g of glucose, and 5 g of sodium chloride with tap water added to it. After adjusting the pH to 7.5 with a 1N aqueous solution of caustic soda, the culture medium was inoculated into a 500 ml Sakaguchi flask containing 100 ml of a liquid medium that had been sterilized by heating at 120°C for 15 minutes in an autoclave, and cultured with shaking at 30°C for 48 hours. The cells produced by these cultures were washed once with 0.01M phosphate buffer (PH7.5), then washed once with the reaction medium shown below, and then resuspended in the same reaction medium. Bacterial suspensions were prepared for each of the 19 strains. In addition, the bacterial concentration of the bacterial suspension was adjusted to be in the range of 3.5 to 4.0 g/dry bacterial cell concentration. Reaction medium K ₂ HPO ₄ 1.74g MgSO ₄・7H ₂ O 1.50g FeSO ₄・7H ₂ O 0.05g Deionized water 1 PH was adjusted to 8.0 with 2N−H ₂ SO ₄ . 20ml of the above bacterial suspension and 200μ of allyl chloride
was placed in a 500 ml Sakaguchi flask and cultured with shaking at 30°C for 24 hours, after which 2μ of the culture solution was sampled and analyzed. For analysis, porapak Q (Waters
A gas chromatograph with a 2 m column packed with 80-100 mesh (manufactured by Associates, Inc.) and an ionization flame detector was used. Table 2 shows the types of bacterial cells used and the amount of epichlorohydrin produced.

【table】

[Table] Example 2 Two platinum loops of each of the 15 strains listed in Table 3 below were added to a synthetic medium [(NH ₄ ) ₂ HP0 ₄ 4g, Na ₂ HPO ₄ .
12H ₂ O2.5g, KH ₂ PO ₄ 2g, MgSO ₄・7H ₂ O0.5
g, FeSO ₄・7H ₂ O 30 mg, CaC1 ₂・2H ₂ O 60 mg,
After adding ion-exchanged water to 200 mg of Difco yeast extract and making it 1, it was heated to 150℃ in an autoclave.
500ml containing 20ml of liquid medium sterilized by heating for minutes
The flask was inoculated into a Sakaguchi flask, and after the flask was tightly stoppered, 120 ml of propane was pressurized and cultured with shaking at 30°C for one week. The bacterial bodies produced by the culture were washed by the method described in Example 1, and bacterial suspensions were prepared for each of the 15 types of bacterial strains. Reaction and analysis were performed using the above bacterial suspension according to the method described in Example 1. Table 3 shows the bacterial concentration (expressed as OD) in the bacterial suspension used in the reaction and the amount of epichlorohydrin produced.

[Table] Example 3 Rhodococcus rhodochrous ATCC29675 was cultured by the method described in Example 2 to prepare a bacterial suspension. After putting 20 ml of the bacterial suspension into a 500 ml Sakaguchi flask and sealing the flask, each raw material olefin shown in Table 4 was added through an injection port fitted with a rubber gasket. Note that 100 µ of the raw material olefin, which is liquid at room temperature, and 40 ml (at room temperature and under normal pressure) when it is gas at room temperature, were respectively pressurized into the flask. then 30
After 24 hours of culture with reciprocating shaking at 150 times/min at ℃, 1 ml of the gas phase above the reaction solution in the flask and 1μ
The remaining reaction solution was extracted with 50 ml of diethyl ether, and 1 μm of the ether layer was sampled and analyzed. The product produced by the reaction was measured with the gas chromatograph described in Example 1, and the retention time and mass spectrum measured with a mass spectrometer connected to the gas chromatograph were compared with the retention time and mass spectrum of a standard sample. The substance was hydrolyzed under hydrochloric acid acidity and was confirmed to be the corresponding epoxide. Table 4 shows the amount of epoxide produced corresponding to the type of raw material olefin. The amount of epoxide produced is
The amount produced by 20 ml of bacterial suspension is displayed.

【table】

Claims (1)

[Claims] 1 A microorganism capable of producing epoxide selected from the group consisting of the genus Micrococcus, Arthrobacter, Corynebacterium, Mycobacterium, Rhodococcus and Brevibacterium is added to a halogenated olefin under aerobic conditions. The halogenated epoxide produced is separated.
A method for producing epoxide, which comprises collecting epoxide.