JPS60214893A - Method of epoxide production from olefin utilizing microorganism - Google Patents

Abstract

PURPOSE:A microorganism in Nocardia is allowed to act on an olefin bearing aromatic rings in the presence of a water-insoluble organic solvent under aerobic conditions to enable high-yeild production of an epoxide which is used as a starting material of synthetic resin, surfactants, medicines or agricultural chemicals or their intermediates. CONSTITUTION:A microorganism in Nocardia such as Nocardia corallina (FERM P-4094) is allowed to act on an olefin bearing aromatic rins such as styrene in the presence of a water-insouble organic solvent, preferably paraffin of 9-17C, olefin of 10-18C, halogenated paraffin of 9-16C or alkylbenzene with side chains of 6-15C, under aerobic conditions to produce an epoxide with aromatic rings, which is isolated and collected.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for producing epoxy 1 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.

■The method for producing epoxide using microorganisms is as follows:
Genus Nocardia, Mycobacterium I
I, MeLlly-1ococcus sp., Methyl
osinus spp., Pseudomonas spp., Cory
Nebacterium spp., Metl+ylobact
It is known that epoxides are produced from linear olefins by microorganisms belonging to the genus Erium or Candid. s
Microorganisms belonging to the genus Brevibacterium and the genus Brevibacterium
．． There are only ζ known ways to use things.

The present invention is based on the knowledge that the producing bacteria can produce epoxides having aromatic rings from olefins having aromatic rings. The object of the present invention is to provide a method for producing an epoxide having an aromatic ring fi from an olefin having an aromatic ring a using a microorganism belonging to the genus Nocardia that has an ability to produce epoxide. purpose.

The present invention will be explained below.

Published 0. )" IH formation The structural feature of the present invention is that a microorganism belonging to the genus Nocardia that has an epoxy l-producing ability is subjected to aerobic conditions to the A-refin, which whitens the aromatic ring, in the presence of a water-insoluble organic solvent. It acts below to produce an epoxide with an aromatic ring,
The purpose is to separate and collect the obtained epoxy 1.

The microorganism used in the present invention belongs to the genus Nocardia, and is Nocardia corallina.
-I can give an example. This bacterium was sent to the Institute of Microbial Technology of the Agency of Industrial Science and Technology with the receipt number FERM-P-4094 in 1932.
June 15, 1983 (=J), and its mycological properties are described in detail in 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 straight or straight type having an aromatic ring such as a phenyl group or a naphthyl group. Branched olefins (olefins in which the double bond is located α, β, or γ from any terminal carbon atom) are included. Examples include styrene, α-mer styrene, β-methylstyrene, rilhensen, 4-fuco-nyl-1-butene, 1-), :1-nyl-2-phthene, and 2-hinylnaphthalene. , history is 1-,;♂ open ring is ii'
Olefins having x substituents include, for example, i) methylstyrene, 0-chlorostyrene, m-1j11-styrene, p-chlorostyrene, p-m-1-xostyrene, and the like.

These l++44J reflexes can be used as GDR or a mixture of two or more of them as a C reaction substrate.

In the present invention, it is present when the above-mentioned microorganism is applied to the raw material olefin to carry out the °C1 boxylation.
1. Water insolubility - organic solvent (hereinafter referred to as organic solvent)
is a paraffin having a carbon number of 9 to 17, a carbon number of 10
-C1 is an autosolvent selected from the group consisting of olefins having from 18 to 18 carbon atoms, halogenated paraffins having from 9 to 16 carbon atoms, and alkylhenzene having side chains having a chain length of 6 to 15; It can also be used as a small dish or as a mixture of two or more kinds.

For a detailed explanation of these organic solvents, normal paraffins, which are paraffins having 9 to 17 carbon atoms, are contained in approximately 20 to 25% of the kerosene and gas oil fractions of petroleum. Straw, boiling point approximately 160℃~35
After hydrodesulfurizing the 0°C fraction, it can be separated and recovered using zeolite (Molecular Bu), etc., and is generally used as a raw material for rough detergents.

Among the above paraffins, paraffins with a large number of carbon atoms have a strong 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 promotion effect on epoxidation will be observed, while if the number is more than 17, the promotion effect will decrease and, in addition, it will solidify at room temperature, which is not practical. Furthermore, among the above paraffins, isoparaffins coexist with normal paraffins in the above-mentioned fraction (and can be separated from normal paraffins by precision distillation, or are actually mixed with normal paraffins). It is convenient to use C.Although those with a short side chain such as methyl or ethyl are generally used, isobaffin having 12 to 16 carbon atoms is preferred in terms of promoting epoxidation.

Next, the olefin having a carbon number of 10 to 18 used as the organic solvent may be a low polymer or oligomer of propylene or butylene, and those commercially available as reagents may also be used. It is generally a straight chain or less branched monomolefin.

In addition, an olefin having less than 8 carbon atoms does not have an effect of promoting epoxidation, and an olefin having more than 18 carbon atoms has a low effect, and in addition, the viscosity becomes high, which is not practical.

Halogenated paraffins having 9 to 16 carbon atoms as organic solvents are chlorinated and nitrogenated paraffins,
Includes decyl chloride, undecyl chloride, dodecyl chloride, tridecyl chloride, tetramonodecyl chloride, decyl bromide, undecyl bromide, 1-decyl bromide, tetradecyl bromide, hexadecyl bromide, and the like.

Note that even if the number of carbon atoms is less than 9 and ζ is also more than 1G, the effect of promoting epoxidation will not be observed.

Next, alkyl hanzene having a side chain with a chain length of 6 to 15 is used as an intermediate for energizing hearts or soft detergents, and straight chain having 6 to 15 carbon atoms is also used, and < is branched. The alkyl group is white on the side chain.

If the chain length is outside the above range of 6 to 15, the effect of promoting epoxidation (9) will not be seen or will be too low to be 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 the examples below.

In the present invention, in the presence of the above-mentioned organic solvent, in order to use a cohoquine-producing bacterium belonging to the genus Nocardia as a raw material olefin, for example, (a) culturing and propagating a Streptococcus bacterium. (The raw material olefin is organically dissolved in the obtained bacterial cells)
(1) A method of aerobically contacting ll+ with 7i to react. Furthermore, it is preferable to apply a method of culturing under aerobic conditions in a nutrient medium consisting of C with the addition of growth-promoting substances as necessary.

” Add raw material olefin to the grown bacterial cells in Section 2 (A).
In a method in which the reaction is carried out by aerobic contact with the presence of a solvent F, carbohydrates such as glutinose, sucrose, molasses, starch hydrolyzate, cellutrace hydrolyzate, hydrocarbons are first used as carbon sources. For example, a substance with a high bacterial growth effect such as 1-copane, butane, dodecane, 1-radecane, and other l"i+ acids is used, and ammonium chloride,
Ammonium sulfate, ammonium phosphate, lit'! 1
Nitrogen sources such as m-j' ammonium, urea, aqueous ammonia, amino acids and other assimilated organic nitrogen compounds, potassium phosphate, #! 1-lium i acid, magnesium sulfate,
Inorganic salts such as manganese sulfate, ferrous sulfate, ferric chloride, calcium chloride, and manganese chloride, and if necessary, growth promoting substances such as vitamins, yeast extract, and corn staple liquor are added to the medium. An inoculum of an epoxide-producing bacterium belonging to the genus Nocardia is inoculated and cultured under aerobic conditions to multiply the bacterium.

Next, the raw material olefin and an organic solvent are added to the bacterial cell culture thus obtained, a suspension of bacterial cells isolated from the culture, or an immobilized product of the bacterial cells, and air, m The reaction is carried out by supplying an oxygen-containing gas such as an oxygen-enriched gas.

The above bacterial cells of raw material olefin and organic solvent) 8'jl#
The ratio to be used in aqueous liquid containing bacteria such as liquid or bacteria suspension may vary depending on the type of organic solvent, but it is 0.1 to 25 VC11/VOI for olefin as a raw material for energization.
%, 61' or 0.5 to 5 vol/vol %, and tJM'1g agent is l -200 vol/v.
ol %, preferably I O-1(1(] vol/v
ol%.

The reaction is carried out in the pH range of 5 to 9, preferably 6 to 8.
ζ1~ at a temperature of 0~50℃, preferably 25~45℃
Performed for 611 minutes. Although the reaction is 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, during the reaction, the number of bacterial cells increased by 911! By appropriately adding the carbon source, nitrogen source, and other components used in the above, the epoxyl production activity of the bacterial cells can be maintained or increased.

The reaction can be carried out either batchwise or continuously.
'、4ru. 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 reaction described in -1- mainly exists in the autosolvent phase, it is separated and collected by applying known techniques such as phase separation, distillation, and extraction17.

Next, in the culture method (IJ), the raw material "refin" and an organic solvent are added during bacterial growth in the method (A) above, and Evo4-So1 is produced in one step. Culture conditions (plL temperature, pressure, etc.), culture method, and amount of epoxide produced411.

Collection is performed using the reaction conditions, reaction force formula, and minute 811 of (a) above.
Harvesting methods can be used as well.

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 Three platinum loops of Materials Technology Research Institute PERM-P-4094)
NRG medium (“Lavrenko” powder manufactured by Oxoid Co., Ltd.)
ug, hakuuichisu” 1-I nononorbezotone +-OIN
, Crunice 10 +
500m7 containing +2 (1'(+, 15 times); J-sterilized liquid culture) 1i IO [1mp!
volume] and 1-1 Solas, and cultured at 30°C for 48 hours with shaking (150 times/min). The bacterial cells produced by this culture were
, (pH 7.5) once, and then as shown below.
” in reaction medium? After purifying 5L, add 1 liter to the following reaction medium so that the concentration of dried bacterial cells is 3.8 mg/mρ.
[1] The ζ bacterial cells were suspended to make a 1'f3 suspension.

Reaction -■ KtllPO, +1.74g in NgSO 71120 1.50g Fe5% 71120 5[]mg Deionized water In (p If was adjusted to 8.0 with 2-N sulfuric acid aqueous solution) 2μL reaction - 1 pair of bacterial suspensions prepared in duplicate, 2 (1 mff) and 5 (Hl) of each raw material melefin [1,1 mf! m 1
．． Pour into a Yonosaka flask and seal it tightly. Next: l(1
After 24 hours of reciprocating shaking at 15 () times/min, the reaction solution in Solas was mixed at 50 mj! Extract with ether of ζ min 1ji' L, friend. For analysis, DIEGS (diconithylene glycol tacone 2.1.) was used as the liquid phase, and a Uniball 11L (cask +, 31 liters or more) 80 to 100 units was filled with a filler containing 80 to 100 methane as a carrier, with an inner diameter of 3 mm and length. 2m
A Shimabara GC-7A type ionizing flame gask 1''7 glass column with a fj:ii glass column was used. The products produced by the reaction were measured using a gas filter l:I-/l-graph, and the retention time and mass spectrum measured using a mass spectrometer connected to the Casta 1-1 Futoclub were compared with the retention time and mass spectrum of the standard sample. Comparison was made, and furthermore, it was confirmed that the product was hydrolyzed under hydrochloric acid acidity, and it was confirmed that it was the corresponding epoxide.

The results of the above analysis are shown in Table 1.

Note that) the amount of niboxito produced is determined by Gas Chroma 1~Graphy! ';1 In addition, as a comparative example, 0.1 m7 of raw material olefin was added without adding n-hexadecane! The reaction was carried out in the same manner as described above, except that 1mβ was used instead of .
Shown in the table.

As shown in Table 1, the presence of n-hexadecane in the system as a self-organic solvent causes the raw olefin to burn out. The production amount has been significantly improved.

Example 2 A bacterial suspension V& was prepared in the same manner as described in Example 1 11+1.

1k2 (l m j!) of bacterial suspension, 0.5m7 of styrene as a raw material olefin, and 9 various organic solvents Hymn shown in Table 2 in 500m7 !Put in Yosaka 1-1 flask and react in the same manner as described in Example 1.
After the reaction, the six-layer solvent layer was analyzed by gas chromatography for quantitative determination.

The results are shown in Table 2. In addition, as a comparative example, the case where ζfi organic solvent is not used at all and the case where other types of Y-it
When adding a solvent, do the same thing.
The results of 1 are shown in Table 2, divided by IN.

Example 3 J4':l'Al Night' Qa Zhou Army 1 A bacterial suspension was prepared in the same manner as described in Example 1.

r;-HノL+ -L-? 4', Castle JIIJ □□o2
□5 minutes JE1 - 20 mρ of the bacterial suspension liquid and each intoxicant shown in Table 3 of styrene as the raw material olefin and tohekidi-decane as the organic solvent were added to 500 nl of each in a Yosaka 1-1 flask I. The reaction was carried out in the same manner as described in Example 1, and after the reaction, 5f1m7! The mixture was extracted with ether and the M was determined by gas chromatography.

The results are shown in Table 3. For comparison, in the case where the organic solvent (formerly Kikadecane) is not added,
Shown in the table]:.

Table 3 Procedural Amendment 1. Indication of the case: 1982 Patent Application No. 71949 2,
Title of the invention: Method for producing epoxides from olefins having an aromatic ring using microorganisms 3; Relationship to the amended case Name of patent applicant: Nippon Mining Co., Ltd. 4; Agent address: 2 Higashi-Shinbashi, Minato-ku, Tokyo Shinbashi Kokusai Building 8, 7-7-chome, amend the statement of contents of the amendment as follows.

(1) In the table on page 16, [l-methyl-2-)゛tene[
Corrected to [1-phenyl-2-buchikari.

Claims (3)

[Claims] (1) The production of epoxyl belonging to the genus Nocalteia was carried out under aerobic conditions in the presence of a water-insoluble autogenous solvent, and the fi-1 ring was exposed to a white olefin. 1. A method for producing epoxy 1, which is characterized in that the epoxy produced by the reaction is separated and collected. (2) Water-insoluble rJ1 solvents include paraffins having 9 to 17 carbon atoms, olefins having 10 to 18 carbon atoms, halogenated vasosoin having 9 to 16 carbon atoms, and side chains with chain lengths of 6 to 15. The manufacturing method according to claim 8'1 (Ili, In. (3) A microorganism that has the ability to produce epoxy 1 and belongs to the genus Nocardia is Nocardia cosolina (N4B).
cora l l 1na)
The manufacturing method described in section 1).