JPS6314951B2 - - Google Patents

Description

[Detailed description of the invention]

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, medicines, and agricultural chemicals. 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 others. 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 discovered that epoxide-producing bacteria belonging to the genus Arthrobacter, Corynebacterium, or Rhodococcus have aromatic rings. It was discovered that an epoxide having an aromatic ring can also be produced from an olefin having the following, and the present invention was completed. 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 epoxide-producing bacteria belonging to each of the above genera. The present invention will be explained in detail below. Structure of the Invention The structural feature of the present invention is that a microorganism having an epoxide-producing ability belonging to the genus Arthrobacter, Corynebacterium, or Rhodocotcus is allowed to act on an olefin having an aromatic ring under aerobic conditions. The purpose is to produce an epoxide having a corresponding aromatic ring, and to separate and collect the resulting 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 microorganisms used in the present invention belong to the genus Arthrobacter, Corynebacterium, or Rhodocotcus, and include the following strains. These strains are from the American Type Culture Collection.
It has been deposited with the following number and is easily available. Arthrobacter rubellus ATCC 21495 Arthrobacter simplex ATCC 21032 Arthrobacter sp. ATCC 27778 Corynebacterium fujiokense ATCC 21496 Corynebacterium hydrocarboclastus ATCC
15108 Rhodococcus rhodochrous ATCC 29670 Rhodococcus rhodochrous ATCC 29672 Rhodococcus rhodochrous ATCC 29675 Rhodococcus sp. ATCC 21499 Rhodococcus sp. ATCC 29673 In the present invention, an olefin having an aromatic ring ( The raw material olefin (hereinafter referred to as raw material olefin) is a linear or branched olefin having an aromatic ring such as a phenyl group or a naphthyl group (however, the double bond is located at the α, β or γ position from any terminal carbon atom). certain olefins), such as styrene, α-methylstyrene, β-methylstyrene, allylbenzene, 4-phenyl-1-butene, 1-phenyl-2-butene, 2-vinylnaphthalene, etc., and further substituents on the aromatic ring. Examples include p-methylstyrene, o-chlorostyrene, m-chlorostyrene, p-chlorostyrene, and the like. 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 obtained by culturing and propagating the microorganism in advance are brought into contact with the raw material olefin under aerobic conditions to react; (b) the above microorganism is grown aerobically in a culture medium containing the raw material olefin. A method of culturing under certain 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 and ethanol, and add nitrogen sources such as ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, aqueous ammonia, amino acids and other assimilable organic nitrogen compounds, and potassium phosphate. , sodium phosphate, magnesium sulfate, manganese sulfate, primary sulfate
The inoculum of the above-mentioned microorganisms is inoculated into a medium to which inorganic salts such as iron, ferric chloride, calcium chloride, and manganese chloride are added, and if necessary, growth-promoting substances such as vitamins, yeast extract, and corn staple liquor. Then, the cells are grown under aerobic conditions. The bacterial cell culture obtained in this way,
Alternatively, the raw material olefin and an oxygen-containing gas such as air, oxygen, or oxygen-enriched gas are supplied to a suspension of bacterial cells or immobilized bacterial cells separated from the culture to cause a reaction. 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 appropriately determined depending on the type of microorganism and raw material olefin. Although the reaction is usually carried out under normal pressure, it is also possible to improve the productivity of epoxide by carrying out the reaction under increased pressure. In addition, by appropriately adding the carbon source, nitrogen source, and other components used for bacterial cell proliferation during the reaction, the bacterial cell concentration and the epoxide production activity of the bacterial cells 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 by the above reaction undergoes phase separation.
Separate and collect by applying known methods such as extraction and distillation. Next, in the method (b) using culture, a raw material olefin is added during bacterial growth in the method (a) above to produce epoxide in one step.
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 applied. 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. may 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%. That is, 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 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 accelerating 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 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 by 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 This example illustrates a method for producing styrene oxide from styrene using various bacterial strains. Preparation of bacterial suspension Three platinum loops of the ten bacterial strains listed in Table 1 were
NBG medium (Lavrenko powder manufactured by Oxoid)
10g of bacteriological peptone, 10g of glucose, and 5g of sodium chloride were added with tap water to make 1, adjusted to pH 7.5 with 1N caustic soda aqueous solution, and then heated and sterilized in an autoclave at 120°C for 15 minutes.Contains 100ml of liquid medium. The mixture was inoculated into a 500 ml Sakaguchi flask, and cultured with shaking at 30°C for 48 hours. The bacterial 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. Bacterial suspensions were prepared for each of the following bacterial strains. The bacterial concentration in 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 adjusted to 8.0 with 2N−H ₂ SO ₄ Reaction and analysis of products Analysis of the above bacteria suspension 20 ml of the solution, 0.1 ml of styrene, and 2 ml of n-hexadecane were placed in a 500 ml Sakaguchi flask, the flask was tightly stoppered, and the flask was cultured with shaking at 30°C for 24 hours, followed by extraction with 50 ml of ether to quantify the amount of styrene oxide produced. Quantification was carried out using a gas chromatograph equipped with a column carrying diethylene glycol succinate on a Uniport B (manufactured by Gascro Kogyo Co., Ltd.) 80-100 mesh and an ionization flame detector. Results Table 1 shows the types of bacterial strains used and the amount of styrene oxide produced in each case.

【table】

[Table] Example 2 This example is an example in which various raw material olefins were subjected to epoxidation by allowing the same microorganism to act on them. Rhodococcus rhodochrous ATCC 29670 was cultured by the method described in Example 1 to prepare a bacterial suspension. To 20 ml of this bacterial suspension, 0.1 ml of the raw material olefin and 2 ml of n-tetradecane listed in Table 2 were added, and the reaction was carried out in a tightly stoppered 500 ml Sakaguchi flask in the same manner as described in Example 1. analyzed. The results are shown in Table 2.

[Table] Example 3 This example shows an example in which the reaction was carried out in the presence of various organic solvents. Preparation of bacterial suspension A bacterial suspension was prepared in the same manner as in Example 1 using the bacterial strain Arthrobacter sp. ATCC 27778. Reaction and product analysis 20 ml of the above bacterial suspension, p-
0.5 ml of chlorostyrene and 10 ml of the organic solvent shown in Table 3 were placed in a 500 ml Sakaguchi flask and reacted in the manner described in Example 1. After the reaction, the organic solvent layer was analyzed by gas chromatography to determine the produced p- Chlorostyrene oxide was quantified. Results Table 3 shows the type of organic solvent used and the amount of p-chlorostyrene oxide produced. Note that Table 3 also shows the case where no organic solvent was present as a control.

[Table] Compound

Claims (1)

[Claims] 1. A microorganism having epoxide-producing ability belonging to the genus Arthrobacter, Corynebacterium, or Rhodocotcus is allowed to act on an olefin having an aromatic ring under aerobic conditions to produce the corresponding aromatic ring. 1. A method for producing an epoxide, which comprises producing an epoxide containing the epoxide, and separating and collecting the obtained epoxide. 2 A microorganism having epoxide-producing ability belonging to the genus Arthrobacter, Corynebacterium, or Rhodocotcus is allowed to act on an olefin having an aromatic ring under aerobic conditions in the presence of a water-insoluble organic solvent. A method for producing an epoxide, which comprises producing an epoxide having an aromatic ring, and separating and collecting the obtained epoxide. 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.