JPS6350995B2 - - 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. Epoxide compounds are widely used as raw materials or intermediates for the production of various organic chemical products, including synthetic resins, surfactants, pharmaceuticals, and concentrates. Prior Art Conventionally, methods for producing epoxides from corresponding olefins include chemical methods using peroxides such as hydrogen peroxide and organic peracids as oxidizing agents, and biochemical methods using oxygen oxidation using microorganisms. Are known. In addition, in the method of producing epoxide using microorganisms, the genus Nocardia, Pseudomonas, Brevibacterium, Corynebacterium, Mycobacterium, Arthrobacter, Acinetobacter, Alcaligenes, and Methylobacterium are used. It is known that epoxides are produced from linear olefins by microorganisms belonging to the genus Methylococcus, Methylocinus, and the like. However, as a method for producing the corresponding epoxide from an olefin having an aromatic ring using microorganisms, only methods using microorganisms belonging to the genus Pseudomonas and Brevibacterium are known. That is, the only microorganisms that belong to the above-mentioned genus are known as microorganisms that have the ability to produce epoxides from olefins having aromatic rings. As a result of searching for microorganisms belonging to various genera that have the ability to produce epoxides from olefins having aromatic rings, the present inventor found that epoxide-producing bacteria belonging to the genus Micrococcus can also produce epoxides from olefins having aromatic rings. The present inventors have discovered that it is possible to produce the following, and have come up with the present invention. OBJECT OF THE INVENTION Therefore, 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 an epoxide-producing bacterium belonging to the genus Micrococcus. The present invention will be explained in detail below. Structure of the Invention The structural feature of the present invention is to produce an epoxide having a corresponding aromatic ring by causing a microorganism having an epoxide-producing ability belonging to the genus Micrococcus to act on an olefin having an aromatic ring under aerobic conditions.
The purpose is to separate and collect the obtained epoxide. The present invention is also characterized in that the epoxide is produced more advantageously by allowing the microorganism to act in the presence of a water-insoluble organic solvent. The microorganism used in the present invention belongs to the genus Micrococcus, and is Micrococcus paraphynolyteicus.
paraffinolyticus) ATCC15589. This strain is from the American Type Culture Collection.
Collection) and is easily available. 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 olefin having an aromatic ring such as a phenyl group or a naphthyl group. or branched olefins (olefins in which the double bond is in the α, β or γ position from any terminal carbon atom), such as styrene, α-methylstyrene, β-methylstyrene, allylbenzene, 4-phenyl- 1-butene, 1-phenyl-2-butene, 2-vinylnaphthalene, etc., and olefins having substituents on the aromatic ring, such as p-methylstyrene, o-chlorostyrene, m-chlorostyrene, p-chlorostyrene. etc. are included. In the present invention, these raw material olefins are used alone or as a mixture of two or more as a reaction substrate. In the present invention, in order to perform epoxidation by causing the above-mentioned microorganism to act on the above-mentioned raw material olefin,
For example, (a) a method in which the microorganisms are cultured and grown in advance and the raw material olefin is brought into contact with the bacterial cells obtained under aerobic conditions to react; (b) the above microorganism is grown in an aerobic culture medium containing the raw material olefin. A method of culturing under suitable conditions can be applied. In the above method (a) in which the raw material olefin is brought into contact with the growing bacterial cells and reacted, carbohydrates such as glucose, sucrose, molasses, starch hydrolyzate, hydrocarbons such as propane, butane, dodecane, tetradecane, etc. are used as carbon sources. Use a substance with a high bacterial growth effect such as acetic acid or ethanol, and add a nitrogen source such as ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, aqueous ammonia, amino acids and other assimilable organic nitrogen compounds, or phosphorus. Inorganic salts such as potassium chloride, sodium phosphate, magnesium sulfate, manganese sulfate, ferrous sulfate, ferric chloride, calcium chloride, manganese chloride, and optionally vitamins, yeast extract, and cornstarch liquor. The above-mentioned microorganisms are inoculated with the inoculum into a medium supplemented with a growth-promoting substance, and the microorganisms are cultured under aerobic conditions to multiply the bacterial cells. The bacterial cell culture obtained in this way, a suspension of bacterial cells isolated from the culture, or an immobilized bacterial cell is added to the raw material olefin and oxygen such as air, oxygen, or an oxygen-enriched gas. Containing gas is supplied and reacted. The reaction is carried out for half a day to six days at a pH of 5 to 9 and a temperature of 20 to 50°C, which is determined as appropriate depending on the microorganism used and the type of raw material olefin. 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. In addition, by appropriately adding the carbon source, nitrogen source, and other components used for cell proliferation during the reaction, the cell concentration and the epoxide production ability of the cell can be maintained or increased. The ratio of the raw material olefin used in the reaction to the bacterial cell-containing aqueous solution is usually 0.1 to 50 vol/vol%, preferably 0.5 to 20 vol/vol%. The reaction can be carried out either batchwise or continuously, or semi-batchwise in which the raw material olefin or other components are continuously or intermittently replenished 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 intended to produce epoxide in one step by adding the raw material olefin during cell proliferation in the method (a). Regarding the culture conditions (PH, temperature, pressure, amount of raw material olefin added, 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. As mentioned above, the present invention includes an embodiment in which the epoxidation reaction of the raw material olefin by the microorganism is carried out in the presence of a water-insoluble organic solvent, and this embodiment will be explained below. 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 paraffins having carbon numbers of 9 to 17, normal paraffins are present in about 20 to 17 carbon atoms in kerosene and gas oil fractions.
It contains 25%. That is, after hydrodesulfurizing the fraction with a boiling point of approximately 160°C to 350°C, it is separated using zeolite (molecular sieve), etc.
It can be recovered 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 on promoting epoxidation, especially those with a carbon number of 12
~16 are preferred. Incidentally, when the number of carbon atoms is less than 9, no promoting effect on epoxidation is observed;
Even if the amount is larger, the accelerating effect will be reduced 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, the olefin having 10 to 18 carbon atoms 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 9 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 will not be observed. Next, alkylbenzene having a side chain with a chain length of 6 to 15 is usually used as an intermediate for hard or soft detergents, and has a straight or branched alkyl group with a carbon number of 6 to 15 in the side chain. It is something that you have. If the chain length is outside the above range of 6 to 15, the effect of promoting epoxidation is not observed or is so low that it is not practical. The ratio of the above-mentioned organic solvent to a bacterial cell-containing aqueous liquid such as a bacterial cell culture solution or a bacterial cell suspension may vary depending on the type of organic solvent, but it is usually 1 to 1.
200vol/vol%, preferably 10-100vol/vol%. In addition, the reaction conditions when an organic solvent is present,
The reaction method and method for separating and collecting the produced epoxide can be applied in the same manner as described above, and the productivity of the desired epoxide can be further significantly increased by the reaction in the presence of this organic solvent. 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 Micrococcus paraphynolyteicus
Three platinum loops of ATCC1589 were prepared by adding tap water to NBG medium (10 g of Labrenco powder manufactured by Oxoid, 10 g of bacteriological peptone, 10 g of glycose, and 5 g of sodium chloride), and adjusting the pH to 7.5 with a 1N aqueous sodium chloride solution. 100ml of liquid medium (100ml) heated and sterilized in an autoclave at 120°C for 15 minutes.
Inoculated into a 500 ml Sakaguchi flask containing 30
The cells were cultured with shaking at ℃ for 48 hours. The cells produced by this culture 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. A bacterial suspension was prepared. In addition, the bacterial concentration of the bacterial suspension is 3.5 to 4.0 as a dry bacterial cell concentration.
g/. 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 . To 20 ml of this bacterial suspension, 0.1 ml of the raw material olefin and 2 ml of n-tetradecane listed in Table 1 were added, cultured with shaking at 30°C for 24 hours in a tightly stoppered 500 ml Sakaguchi flask, and then extracted with 50 ml of ether. The epoxide was analyzed. The results are shown in Table 1.

[Table] Example 2 Bacterial strain Micrococcus paraphynolyteicus
A bacterial suspension was prepared in the same manner as in Example 1 using ATCC15589. 20 ml of the bacterial suspension, 0.5 ml of p-chlorostyrene as the raw material olefin, and 10 ml of the organic solvent shown in Table 2 were placed in a 500 ml Sakaguchi flask and reacted in the manner described in Example 1. After the reaction, an organic solvent layer was formed. was analyzed by gas chromatography to quantify the p-chlorostyrene oxide produced. Table 2 shows the type of organic solvent used and the amount of p-chlorostyrene oxide produced. Note that Table 2 also shows the case where no organic solvent was present as a control.

【table】

[Table] Mixture with %.

Claims (1)

[Claims] 1. A microorganism having an epoxide-producing ability belonging to the genus Micrococcus is allowed to act on an olefin having an aromatic ring under aerobic conditions to produce an epoxide having the corresponding aromatic ring, and the resulting epoxide is A method for producing epoxide, characterized by separating and collecting it. 2 A microorganism that has epoxide-producing ability belonging to the genus Micrococcus 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 the corresponding epoxide, and the resulting epoxide is separated. , a method for producing epoxide, which comprises collecting . 3. 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 15. The manufacturing method according to claim 2, which is one type or a mixture of two or more types selected from the group.