JPS6262158B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a method for producing an epoxide having a corresponding aromatic ring from an olefin having an aromatic ring using microorganisms. Incidentally, 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. Prior Art Methods for producing epoxide using microorganisms include Nocardia genus, Mycobacterium genus,
Genus Methylococcus, Genus Methylosinus,
Pseudomonas spp., Corynebacterium spp.
It is known that epoxides are produced from linear olefins by microorganisms belonging to the genus Methylobacterium or Candida, but the production of epoxides with aromatic rings by microorganisms belonging to the genus Pseudomonas and
The only known method is to utilize microorganisms belonging to the genus Brevibacterium. Purpose of the Invention The present invention was made based on the knowledge that among the above-mentioned microorganisms, epoxide-producing bacteria belonging to the genus Nocardia can also produce epoxides having an aromatic ring from olefins having an aromatic ring. An object of the present invention is to provide a method for producing an epoxide having a corresponding aromatic ring from an olefin having an aromatic ring using a microorganism having an epoxide-producing ability belonging to the genus Nocardia. The present invention will be explained in detail below. Composition of the Invention The compositional feature of the present invention is that a microorganism having an epoxide-producing ability belonging to the genus Nocardia is allowed to act on an olefin having an aromatic ring under aerobic conditions in the presence of a water-insoluble organic solvent to produce an aromatic ring. to produce an epoxide with and separate the obtained epoxide,
It is about collecting. The microorganism used in the present invention belongs to the genus Nocardia, and can be exemplified by Nocardia corallina. This bacterium was submitted to the Institute of Microbial Technology of the Agency of Industrial Science and Technology with the receipt number FERM-P-4094 in 1972.
It has been entrusted to storage as of June 15, 2013, and its mycological properties are detailed in the Japanese Patent Publication No. 1983-40. In the present invention, the olefin having an aromatic ring (hereinafter referred to as raw material olefin) used as a reaction substrate for producing epoxide using the above-mentioned microorganisms is a linear or branched olefin having an aromatic ring such as a phenyl group or a naphthyl group. olefins (wherein the double bond is located at the α, β or γ position from any terminal carbon atom); such olefins include styrene, α-methylstyrene, β-methylstyrene, Examples include methylstyrene, allylbenzene, 4-phenyl-1-butene, 1-phenyl-2-butene, and 2-vinylnaphthalene, and further olefins having substituents on the aromatic ring, such as p-methylstyrene,
o-chlorostyrene, m-chlorostyrene, p-
Includes chlorostyrene, p-methoxystyrene, etc. These raw material olefins are used alone or as a mixture of two or more as a reaction substrate. In the present invention, the water-insoluble organic solvent (hereinafter simply referred to as organic solvent) that is present when epoxidizing the raw material olefin by the action of the microorganism is paraffin having 9 to 17 carbon atoms, paraffin having 10 to 18 carbon atoms, and the like. an organic solvent selected from the group consisting of olefins having a carbon number of 9 to 16, halogenated paraffins having a carbon number of 9 to 16, and alkylbenzenes having a side chain having a chain length of 6 to 15, which may be used alone or as a mixture of two or more. can also be used. A detailed explanation of these organic solvents is as follows:
Among the paraffins with carbon numbers 9 to 17, normal paraffins are present in approximately 20% of the kerosene and gas oil fractions of petroleum.
It contains ~25%. In other words, it can be separated and recovered using zeolite (molecular sieve) after hydrodesulfurizing the fraction with a boiling point of about 160°C to 350°C, and is generally used as a raw material for soft detergents. . Among the above paraffins, those with a large number of carbon atoms have a higher effect of promoting epoxidation, and paraffins with 12 to 16 carbon atoms are particularly preferred. Incidentally, if the number of carbon atoms is less than 9, no promoting effect on epoxidation will be observed, while if the number of carbon atoms is greater than 17, the promoting effect will decrease and, in addition, it will solidify at room temperature, which is not practical. Among the above paraffins, isoparaffin coexists with normal paraffin in the above-mentioned fraction, and can be separated from normal paraffin by precision distillation, but in reality, isoparaffin is used as a mixture with normal paraffin. is convenient. Although those with short side chains such as methyl and ethyl are generally used, isoparaffins having 12 to 16 carbon atoms are preferred in terms of promoting epoxidation. Next, carbon number 10-18 used as an organic solvent
The olefin having the above may be a low polymer or oligomer of propylene or butylene, and those commercially available as reagents may also be used. Generally, it is a linear or less branched monoolefin. It should be noted that olefins having less than 8 carbon atoms do not have the effect of promoting epoxidation, while those having more than 18 carbon atoms have a low effect and are not practical because of their high viscosity. Halogenated paraffins having 9 to 16 carbon atoms as organic solvents include chlorinated and brominated paraffins, such as decyl chloride, undecyl chloride, dodecyl chloride, tridecyl chloride, tetradecyl chloride,
Includes decyl bromide, undecyl bromide, dodecyl bromide, tetradecyl bromide, hexadecyl bromide, and the like. Incidentally, even if the number of carbon atoms is less than 9 or more than 16, the effect of promoting epoxidation is not observed. Next, alkylbenzene having a side chain with a chain length of 6 to 16 is usually used as an intermediate for hard or soft detergents, and has a straight chain or branched alkyl group having 6 to 16 carbon atoms in the side chain. It is something that you have. In addition, for those whose chain length is outside the range of 6 to 16,
The epoxidation promoting effect is not observed or is so low that it is not practical. In the present invention, by allowing each of the above-mentioned organic solvents to exist in the system, the yield of epoxide can be significantly improved, as shown in Examples below. In the present invention, in order to cause an epoxide-producing bacterium belonging to the genus Nocardia to act on the raw material olefin in the presence of the above-mentioned organic solvent, for example, (a) the bacterium is cultured in advance and grown, and then the raw material olefin is added to the resulting bacterial body. A method of reacting by aerobic contact in the presence of a solvent, (b) the above bacteria is mixed with a raw material olefin and an organic solvent,
and possibly other carbon sources as well as nitrogen sources,
It is preferable to apply a method of culturing under aerobic conditions in a nutrient medium containing inorganic salts and, if necessary, growth-promoting substances. In the method of (a) above, in which the grown bacterial cells are brought into contact with the raw material olefin in an aerobic manner in the presence of an organic solvent, carbohydrates are first used as the carbon source, such as glucose, sucrose, molasses, and starch hydrolyzate. , cellulose hydrolyzate, hydrocarbons such as propane, butane, dodecane, tetradecane, and other substances with high bacterial growth effects such as acetic acid, and ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, aqueous ammonia, and amino acids. and other assimilated organic nitrogen compounds, inorganic salts such as potassium phosphate, sodium phosphate, magnesium sulfate, manganese sulfate, ferrous sulfate, ferric chloride, calcium chloride, manganese chloride, and as necessary. Inoculum of epoxide-producing bacteria belonging to the genus Nocardia is brought into contact with a medium supplemented with growth-promoting substances such as vitamins, yeast extract, and corn staple liquor, and cultured under aerobic conditions to multiply the bacterial cells. . Then, the bacterial cell culture obtained in this way,
Alternatively, the raw material olefin and an organic solvent are added to a suspension of bacterial cells isolated from the culture or to an immobilized bacterial cell, and an oxygen-containing gas such as air, oxygen, or oxygen-enriched gas is supplied. Make it react. The ratio of the raw material olefin and the organic solvent to the bacterial cell-containing aqueous solution such as the bacterial cell culture solution or bacterial cell suspension may vary depending on the type of organic solvent, but the raw material olefin is usually 0.1 to 25 vol/
vol%, preferably 0.5 to 5 vol/vol%, and 1 to 200 vol/vol%, preferably 10 to 5 vol% for organic solvents.
It is 100vol/vol%. The reaction is carried out at a pH range of 5 to 9, preferably 6 to 8, at a temperature of 20 to 50°C, preferably 25 to 45°C.
Do this for ~6 days. The reaction is usually carried out under normal pressure,
Epoxide productivity can also be improved by carrying out the process under pressure. In addition, the epoxide production activity of the bacterial cells can be maintained or increased by appropriately adding the carbon source, nitrogen source, and other components used for bacterial cell proliferation 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 can be supplied continuously or intermittently during the reaction. Since the epoxide produced by the above reaction mainly exists in the organic solvent phase, it can be separated and collected by applying known techniques such as phase separation, distillation, and extraction. Next, in the culture method (b) above, the raw material olefin and an organic solvent are added during bacterial growth in the method (b) above, and epoxide is produced in one step. Regarding the culture conditions (PH, temperature, pressure, etc.), culture method, and separation and collection of the produced epoxide, the reaction conditions, reaction method, separation, and collection method described in (a) above can be similarly used. The epoxide obtained according to the present invention can be used in a wide variety of conventionally known applications as mentioned above. The present invention will be explained in more detail with reference to Examples below. Example 1 Preparation of bacterial cell suspension Three platinum loops of Nocardia corallina B-276 (FERM-P-4094, Institute of Microbial Technology, Agency of Industrial Science and Technology) were mixed with NBG medium (“Lavrenko” manufactured by Oxoid).
Powder 10g, Bacteriological Peptone 10
g, glucose 10g, and sodium chloride 5g, add tap water to make 1000ml, adjust the pH to 7.5 with 1N caustic soda aqueous solution, and then in an autoclave at 120℃ for 15 minutes.
500ml containing 100ml of liquid culture medium (heat sterilized for minutes)
ml Sakaguchi flask was inoculated and cultured with shaking (150 times/min) at 30°C for 48 hours. The bacterial cells produced by this culture were dissolved in 0.01 molar phosphate buffer (PH7.5).
The cells were washed twice, and then washed with the reaction medium shown below, and then resuspended in the reaction medium shown below at a dry cell concentration of 3.8 mg/ml to obtain a cell suspension. Reaction medium K ₂ HPO ₄ 1.74g MgSO ₄・7H ₂ O 1.50g FeSO ₄・7H ₂ O 50mg Deionized water 1 (PH was adjusted to 8.0 with 2-N sulfuric acid aqueous solution) Reaction and product analysis As described above 20ml of the bacterial suspension prepared in the above manner, and 0.1ml of each raw material olefin and n-
2 ml of hexadecane was placed in each 500 ml Sakaguchi flask and the flasks were sealed tightly. Then, after 24 hours of reciprocating shaking at 30°C and 150 times/min, the reaction solution in the flask was
It was extracted with 50 ml of ether and analyzed. For analysis
DEGS (diethylene glycol succinate) is used as the liquid phase, Uniport B (manufactured by Gas Cross Kogyo Co., Ltd.)
Shimadzu equipped with a glass column with an inner diameter of 3 mm and a length of 2 m filled with a packing material containing 80 to 100 mesh.
A GC-7A ionization flame gas chromatograph was used. The product produced by the reaction was measured using a gas chromatograph, and the retention time and mass spectrum measured using a mass spectrometer connected to the gas chromatograph were compared with the retention time and mass spectrum of a standard sample. It was confirmed that it was a corresponding epoxide. The results of the above analysis are shown in Table 1.
Note that the amount of epoxide produced was determined by gas chromatography. In addition, as a comparative example, the results of a reaction carried out in the same manner as above except that 1 ml of raw material olefin was used instead of 0.1 ml without adding n-hexadecane are also shown in Table 1. Indicated.

【table】

[Table] As seen in Table 1, n as an organic solvent
- The presence of hexadecane in the system significantly improves the amount of epoxide produced from the raw material olefin. Example 2 A bacterial suspension was prepared in the same manner as described in Example 1. Analysis of reaction and products: 20 ml of bacterial suspension, 0.5 ml of styrene as raw material olefin, and various organic solvents shown in Table 2.
10 ml of each was placed in a 500 ml Sakaguchi flask and reacted in the same manner as described in Example 1. The organic solvent layer obtained after the reaction was analyzed and quantified by gas chromatography. The results are shown in Table 2. As a comparative example, the results of the same analysis conducted in the case where no organic solvent was added and the case where other types of organic solvents were added are also shown in Table 2.

【table】

[Table] Compound.
Example 3 Preparation of bacterial suspension A bacterial suspension was prepared in the same manner as described in Example 1. Reaction and Product Analysis 20 ml of the bacterial suspension and each amount of styrene as the raw material olefin and n-hexadecane as the organic solvent shown in Table 3 were placed in a 500 ml Sakaguchi flask, and the same amount as in Example 1 was added. The reaction was carried out in the same manner as described above, and styrene oxide in the product obtained after the reaction was extracted with 50 ml of ether and quantified by gas chromatography. The results are shown in Table 3. For comparison, Table 3 also shows the results of the same analysis in the case where no organic solvent (n-hexadecane) was added.

【table】

Claims (1)

[Claims] 1. Microorganisms belonging to the genus Nocardia that have epoxide-producing ability are treated in the presence of a water-insoluble organic solvent,
A method for producing an epoxide, which comprises reacting an olefin having an aromatic ring under aerobic conditions to produce an epoxide having an aromatic ring, and separating and collecting the obtained epoxide. 2. The water-insoluble organic solvent consists of paraffin having 9 to 17 carbon atoms, olefin having 10 to 18 carbon atoms, halogenated paraffin having 9 to 16 carbon atoms, and alkylbenzene having a side chain having a chain length of 6 to 16. The manufacturing method according to claim 1, which is one type or a mixture of two or more types selected from the group. 3. Claim 1 in which the microorganism having epoxide-producing ability belonging to the genus Nocardia is Nocardia corallina.
Manufacturing method described in section.