JPH01144983A - Production of unsaturated hydrocarbon and derivative thereof - Google Patents

Abstract

PURPOSE:To obtain the title compound useful as a synthetic raw material for pheromone, etc., on an industrial scale at a low cost, by using cells of microorganism belonging to genus Rhodococcus and capable of producing an unsaturated hydrocarbon and reacting the cells with a carbohydrate in the presence of thiamine, inorganic metal salt, etc., and in the presence of a carbohydrate or an amino acid. CONSTITUTION:An unsaturated hydricarbon and its derivative (e.g., cis-3- dodecane) is produced by using cells of microorganism belonging to genus Rhodococcus and capable of producing an unsaturated hydrocarbon [e.g., Rhodococcus sp. KSM-B-3M (FERM BP-1531)] and reacting the cells with a hydrocarbon or its derivative (e.g., n-dodecane) in the presence of thiamine or an inorganic metal salt (e.g., magnesium sulfate) and in the presence of a carbohydrate (e.g., glucose) or an amino acid (e.g., L-glutamic acid).

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for producing unsaturated hydrocarbons and derivatives thereof using microorganisms.

[Prior Art and its Problems] Unsaturated hydrocarbons and their derivatives are widely used as pheromones, fragrances, and drugs, as raw materials for their synthesis, or as intermediates for the synthesis of organic compounds.

Conventionally, such unsaturated hydrocarbons and their derivatives have been known to be chemically synthesized using metathesis reactions.

However, chemical synthesis requires multiple steps and has the disadvantage that cis and trans stereoisomers are obtained in a mixed state.

On the other hand, as a method of using microorganisms in the production of alkenes, there is a method of using bacteria of the genus Nocardia (US Pat. No. 3,629,072), but this method has the disadvantages of low yield and long reaction time. However, when this method was used, the production volume was still not sufficient and it could not be said that industrial productivity was fully satisfied.

Furthermore, although there is a desire for a simple method for producing dialkenes from industrially easily available monoalkenes, no method has yet been established that satisfies this requirement.

Means for Solving Problem C] Under the current situation, the present inventors conducted intensive research and found that microorganisms isolated from Okinawan soil and further obtained through mutational manipulation as shown in Reference Example 1. It was discovered that the microorganism has the ability to convert hydrocarbons into unsaturated hydrocarbons and their derivatives, and that the produced unsaturated hydrocarbons and their derivatives are excreted outside the microbial cell and can be easily recovered. Furthermore, we have developed a method for producing unsaturated hydrocarbons and their derivatives in high yields through cell reactions using this strain, in which L-glutamic acid, L-glutamic acid, L- He discovered the Aspergillus japonica and completed the present invention.

That is, the present invention uses cells of an unsaturated hydrocarbon-producing bacterium belonging to the genus Rhodococcus, and allows hydrocarbons or derivatives thereof to act on the cells in the presence of carbohydrates or amino acids to produce unsaturated hydrocarbons or derivatives thereof. The present invention provides a method for producing unsaturated hydrocarbons and derivatives thereof, characterized in that thiamine or an inorganic metal salt is present in the system.

The unsaturated hydrocarbon-producing bacterium belonging to the genus Rhodococcus discovered by the present inventors and used in the present invention is a strain bred by mutation of a strain obtained from Okinawan soil by the method of Reference Example 1. It has mycological properties.

(A) Morphology: Bacilli; the cells are pleomorphic, becoming rod-shaped in young cultures and spherical in older cultures. Size is 0.5~0.8 um
Xl, O~5. Ott m.

(B) Growth status in each medium Growth is poor, colonies are pale flesh-colored, smooth and shiny.

■Glycerin-asparagine agar medium Growth is moderate, and the colonies are milky-white, smooth and glossy. Smooth and rough colonies can be seen.

■Growth on starch agar medium is moderate, and the colonies are milky-white, smooth and glossy.

■Tyrosine agar medium Growth is vigorous, and the colonies are flesh-colored, smooth and shiny. Colonies that become slime-like can be seen.

■Nutritional agar medium Growth is vigorous, and the colonies are pale orange in color, smooth and glossy. Smooth and rough colonies can be seen.

■Yeast/malt agar medium Growth is vigorous, and colonies are orange to dark brown with a dull luster.

(C) Physiological properties ■Growth range Temperature: 15-37°C (optimal 25-35°C) pH:
5-9.5 (optimal 6-8) ■ Liquefaction of gelatin (glucose/peptone/gelatin medium) Negative.

■Starch hydrolysis (starch agar medium) negative.

■Coagulation and peptonization of skimmed milk Both tested negative.

■Melanin-like pigment production (tyrosine medium, peptone/yeast/iron medium) Negative.

(D) Assimilation of carbon sources ■L-arabinose + ■D-xylose + ■D-glucose + ■D-Mannitol + (E) Chemotaxonomic properties ■Glycolyl test Glycolyl type.

■ Menaquinone System MK8 (Hz) - Regarding microorganisms with the above mycological properties, please refer to Bergey's Manual of Syst
ematic Bacteriology) + Volume 2 (
As a result of the search based on Rhodococcus (1986), Rhodococcus (R
Although it was recognized as a strain belonging to the genus Rhodococcus, the productivity of unsaturated hydrocarbons is significantly different from that of other Rhodococcus bacteria, so Rhodococcus sp.
SM-B-3M (Rhodococcus sp,
K S M -B -3M) and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, as FERM BP-1531.

Hydrocarbons and derivatives thereof as raw materials of the present invention [in the formula R
represents a straight chain or branched, saturated or unsaturated hydrocarbon group having 6 to 22 carbon atoms, and A is a hydrogen atom, an aromatic hydrocarbon group, a halogen atom, -0H1-CN, -CO-R, (
R represents a straight chain or branched hydrocarbon group or an aromatic hydrocarbon group having 1 to 10 carbon atoms), o R2 (Rz
represents a linear or branched hydrocarbon group or an aromatic hydrocarbon group having 1 to 10 carbon atoms), a metal or (Rff
, R4 represents a hydrogen atom or a straight chain or branched hydrocarbon group having 1 to 5 carbon atoms. ] In the present invention, those in which R is a straight chain hydrocarbon having 14 to 20 carbon atoms are preferably used.

According to the invention, the hydrocarbons and their derivatives are the corresponding unsaturated hydrocarbons and their decane
tadecane), eicosane
), chloro-hexadecane (chloro-hexad
ecane), chloro-octadecane (chloro-
octadeane), hexadecanol (hexa
decanol), hexadecylbenzene (hexa
decylbenzene), heptadecanonitrile (
heptadecanonitrile), hexadecene (hexa-decene), eicosene (ei
cosene), tetradecene (te tradece)
From hydrocarbons such as ne) and their derivatives, respectively,
hexadecene, octadecene, tetradecene
ade-cene), pentadecene
cene), eicosene, chloro-hexadecene
e) , chloro-octadecene (chloro-octadecene)
adecene), hexadecadiene (hexade)
cenol), hexadecenylbenzene (hexade
cenylbenzene), hebutadecenonitrile (
Heptadecenoni torile, hexadeca-di-ene, eicosa-di-ene, tetradecadiene, etc. are produced.

In the present invention, the microbial cells to be used are obtained through regular use. Preferably, the culture is carried out aerobically at 30°C for 1 to 2 days.

The culture fluid thus obtained is used as it is, or the bacterial cells are obtained by centrifugation, filtration, etc. and subjected to the following reaction.

Preparation of a reaction solution using bacterial cells and reaction are performed as follows.

The above culture solution or collected bacterial cells are suspended in 0.1 to 90%, preferably 0.2 to 0.5% as dry weight of the bacterial cells, in water or a buffer solution (pH 6 to 8) and reacted with hydrocarbons. and its derivatives from 1 to 70%, preferably from 10 to 30%
, L-glutamic acid, L-aspartic acid, or other amino acids, or glucose, citric acid, or other carbohydrates from 0.05 to
The basic reaction solution is one containing 10%, preferably 0.5 to 2%, and 0.001% of thiamin hydrochloride, etc.
~2%, preferably 0.01~0.0 as thiamin content.
The addition of 3% or 0.001 to 0.01% of inorganic metal salts such as magnesium sulfate and manganese sulfate is to significantly improve the productivity of unsaturated hydrocarbons and their derivatives.
Although combinations with amino acids such as glutamic acid and L-aspartic acid are preferred, they may also be reacted in combination with other carbohydrates or individually in combination with amino acids or carbohydrates. Further, these may be added at the time of obtaining the bacterial cells.

Thiamine may be in any state such as salt-free, hydrochloride, sulfate, etc. The inorganic metal salt is preferably magnesium sulfate, but sulfuric acid, such as manganese sulfate, or inorganic metal salts containing magnesium or manganese can also be used.

Furthermore, as a mixture containing amino acids, carbohydrates, thiamine, inorganic metal salts, etc., extracts derived from natural products and amino acid mixtures, such as meat extracts, yeast extracts, casamino acids, etc., can also be added and used.

The reaction time may be about 1 to 3 days when performing a batch reaction, but the reaction time may be approximately 1 to 3 days, but the reaction time may be approximately 1 to 3 days, but the reaction time may be approximately 1 to 3 days. Derivatives thereof are produced.

The unsaturated hydrocarbons and their derivatives produced in the reaction solution can be identified and quantified using conventional methods for identifying and quantifying organic compounds contained in natural products.

For example, identification and quantification can be performed by extracting a certain amount of the reaction solution with an organic solvent and then analyzing it using gas chromatography (GC) or gas chromatograph/mass spectrometer (GC-MS). can. To determine the position of the double bond of an unsaturated compound, for example, a methylthioetherification method in which QC-MS analysis is performed after treatment with dimethyl disulfide [2 Hara et al., Oil Chemistry Vol. 34, p. 619 (19
1985) and others] are available.

The unsaturated hydrocarbons and their derivatives produced in the reaction solution are
It can be recovered and separated using ordinary methods for extracting and isolating organic compounds from natural products. For example, the reaction solution can be centrifuged to separate bacterial cells, an oil layer, and an aqueous layer, which can be purified and isolated by oil column chromatography, partition extraction, or the like.

〔Effect of the invention〕

According to the present invention, unsaturated hydrocarbons and derivatives thereof can be industrially advantageously produced using cells of unsaturated hydrocarbon-producing bacteria belonging to the genus Rhodococcus.

〔Example〕

Next, an example will be given and explained.

Example 1 2.5 g of glucose, 17 g of polypeptone (manufactured by Otama Nutrition Co., Ltd.), 3 g of polypeptone S (manufactured by Otama Nutrition Co., Ltd.),
Dihydrogen phosphate l Potassium 2.5g, Sodium chloride 5g
500 mj containing 50 ml of culture medium consisting of ion exchange water and 11 ml of ion-exchanged water! Rhodococcus sp. KSM-B-3M strain was transplanted into a Yosaka Rof flask and incubated with shaking at 30°C for 1 day. After that, 1 ml of this culture solution was added to a 0.25 M phosphate buffer (pH 7) containing the same composition of medium sodium, 0.1% thiamine hydrochloride, and 0.1% magnesium sulfate.
,O)20mj! Suspend in 500 mj! and add 5 ml of n-hexadecane. The reaction was carried out by shaking in a Yosaka Lough flask at 30°C for 2 days.

After the reaction, the main product contained in the reaction solution is extracted with ethyl acetate and subjected to gas chromatography (GC), gas chromatograph/mass spectrometer (GC-MS), or gas chromatograph/infrared analyzer (GC-FT-TR). ) (Figure 1), and the main product was cis-7-hexadecene (c
is-7-hexadecene). The position of the double bond was determined by mass spectrum analysis after dimethyl disulfide t tetramerization (FIG. 2), and FIG. 3 shows the mass spectrum of the main product.

1 mff1 of the reaction solution was extracted with 3 ml of ethyl acetate, and cis-
When the amount of 7-hexadecene was measured by gas chromatography, the productivity was 18 g/l in 2 days.

decane), n-octadecane (n-octad
ecane), n-eicosane
), 1-chloro-tetradecane (1-chloro-t
ettradecane), ■-chlorohexadecane (
1-chloro-hexadecane), l-chloro-octadecane (1-chloro-octade)
cane), or l-chloro-eicosane (1-
The reaction was carried out in the same manner as in Example 1 except that chloro-eicosane was used.

Example 1
Identification and quantification were performed in the same manner as above, and the results shown in Table 1 below were obtained.

Below is a blank space Example 3: One heptadecanonitrile (n-heptadecanonitrile)
The reaction was carried out in the same manner as in Example 1 except that nitorile) was used.

When the unsaturated hydrocarbons in the reaction solution were identified and quantified in the same manner as in Example 1, the results shown in Table 2 below were obtained.

Below is a blank space Example 4 In place of n-hexadecane in Example 1, 1-hexadecene (1-hexadecene), 1-octadecene (1-octadecene), 1-eicosene (1-
eicosene), 1-tetradecene (1-te t
Example 1 except that radecene) was used. ! : The same operation was performed to cause a reaction.

The unsaturated hydrocarbons in the reaction solution were identified and determined in the same manner as in Example 1.
-3M strain was transplanted and cultured with shaking at 30°C for 1 day. After that, this culture solution ImA was mixed with 100 mj of a medium of the same composition.
2 into a 500mff1 Yosaka flask,
Shaking culture was performed at 30°C for 1 day. Add this culture solution to 5
Centrifugation was performed at 000x to obtain 2 g of bacterial cells. 1g of bacterial cells
0.25M phosphate buffer FL containing 1.0% glucose
The suspension was suspended in 20mj2 (pH 7,0), 4mβ of 1-chloro-hexadecane was added, and the reaction was carried out by shaking in a 500ml Sakaro flask at 30°C for 2 days.

1 mf of the reaction solution was extracted with 3 ml of ethyl acetate, and 1-chloro-hexadecene was identified and quantified in the same manner as in Example 1, and the productivity was 5.5 g/l for 2 days. .

Comparative Example 2 A 0.25MIJ acid buffer roller containing 1.0% L-glutamate monosodium instead of the 0.25M U acid buffer containing 1.0% glucose of Comparative Example 1, 6.2 g/
F!・It was productive for 2 days.

Example 5 0.25M phosphate buffer containing 01.0% glucose and 0.1% thiamine hydrochloride instead of the 0.25M phosphate buffer containing 1.0% glucose of Comparative Example 1, 1. 0.25M phosphate buffer containing 0% L-monosodium glutamate and magnesium sulfate, ■1.0%L
- Monosodium glutamate, 0.25M phosphate buffer containing 0.1% thiamine hydrochloride, ■ 1.0% L-monosodium glutamate, 0.1%
Thiamine hydrochloride, 0.1% magnesium sulfate.
25MIJ phosphate buffer, ■ 0.25M phosphate buffer containing 1.0% monosodium L-aspartate, 0.1% thiamine hydrochloride, 0.1% magnesium sulfate, ■ 1.0% glycine, 0 .1% thiamine hydrochloride, 0.
0.25 MIJ containing 1% magnesium sulfate 1.0
% L-Threonine, 0.1% Thiamine Hydrochloride, 0-11
0.25M IJ acid buffer containing % magnesium sulfate, ■ 1.0% L-proline, 0.1% thiamine hydrochloride, 0
．． 0.25M phosphate buffer containing 1% magnesium sulfate @ 1.0% monosodium L-glutamate, 0.1%
The reaction was carried out in the same manner as in Comparative Example 1, except that 0.25M+J acid buffer containing manganese sulfate was used.

When 1-chloro-hexadecene in the reaction solution was identified and quantified in the same manner as in Example 1, the results shown in Table 4 below were obtained.

Reference example 1: Approximately 0.5g of the soil sample collected from Okinawa was added to 100ml of sterilized water.
After stirring thoroughly, 0.2 mff of this suspension was added.
1 into a liquid medium (1) with the following composition: 10 mjl! (Test tube φ25 x 200 mm) was inoculated and cultured with shaking at 30°C for 4 days.

Kll□p0420 g Yeast extract 2 g Mg5Oa -TIIZOQ, 5 g Peso, = 711.0 0.01
gMnSO4・4～6tlz0 0.008
g Ion-exchanged water 12 pH 7 After diluting the culture solution that showed growth from the above culture with sterilized water, it was transferred to an ordinary agar medium (manufactured by Eiken Chemical Co., Ltd.) and cultured at 30°C for 2 days. Transfers to plain agar plates were repeated until it was confirmed macroscopically and microscopically that the colonies were not different from each other.

This isolated strain was transferred to O, IM phosphate buffer i! (pH 7.0) and irradiated with ultraviolet light for 90 seconds.

After that, the obtained colonies were combined with 2.5 g of glucose, 17 g of polypeptone, 33 g of polypeptone, and 1 g of dihydrogen phosphate.
500 mj2 containing 50 ml of a medium consisting of 2.5 g of potassium, 5 g of sodium chloride, and 12 g of ion-exchanged water.
Transplanted to Yosaka Lough flask, suspended in 19 m2, added 1 mj2 of hexadecane, and 500 mj! Shake at 30°C for 3 days in a Yosaka Lough flask. After shaking, the main product contained in the reaction solution was extracted and subjected to gas chromatography (GC).
We obtained strains that produce hexadecene.

After appropriately diluting this hexadecene-producing strain with sterilized water, it was transplanted onto an ordinary agar medium and cultured at 30°C for 2 days, resulting in multiple colonies that had grown on the ordinary agar medium. Ten colonies were each inoculated onto a slanted agar medium (U) with the following composition, and 30
After culturing at °C for 3 days, it was confirmed that the strains on the 10 slanted agar plates were macroscopically and microscopically the same strain, and that the properties and physiological properties of these 10 strains were the same on each medium. I confirmed that there is.

Slanted agar medium (■): n-hexadecane 20 g (Ni14)zsO420g KlhPO42g Yeast extract 2g Mg5Oa・7i120 0.5 gF
eSOa -7HzOO,01g MnSOn 4-6HzO0,008g polyoxyethylene- Sorbitan monolauric acid 0.05g ester (
Average number of added EOs: 20 moles) Agar
20 g ion-exchanged water 11 The suspension was suspended in a cryopreservation vial containing an aqueous lyserin solution (2 mff) and stored frozen at -80°C.

After 3 months of storage, a loopful of the suspension obtained by rapid thawing was resuscitated on an agar medium, and the properties and physiological properties were examined on each medium under the same conditions as above. I confirmed that there was no change.

Furthermore, the properties and physiological properties of the bacterial strains that had been subjected to the above freezing and thawing process repeated five times every month were examined in the same manner on each medium, but no changes were observed.

The properties and physiological properties of this strain on each medium are as described above.

[Brief explanation of the drawing]

Figure 1 shows GC of cis-hexadecene produced according to the present invention.
-FT-I H spectra are shown. FIG. 2 shows a mass spectrum of the bismethylthioether derivative of cis-hexadecene produced according to the present invention. FIG. 3 shows the mass spectrum of cis-hexadecene produced according to the present invention. that's all

Claims (1)

[Claims] 1. Using the cells of an unsaturated hydrocarbon-producing bacterium belonging to the genus Rhodococcus, hydrocarbons or derivatives thereof are applied to the cells in the presence of carbohydrates or amino acids to produce unsaturated hydrocarbons or derivatives thereof. 1. A method for producing unsaturated hydrocarbons and derivatives thereof, characterized in that thiamine or an inorganic metal salt is present in the system. 2. The unsaturated hydrocarbon-producing bacterium is Rhodococcus sp.
The production method according to claim 1, which is the KSM-B-3M strain. 3. The production method according to claim 1, wherein the carbohydrate is glucose, sorbitol, arabinose, xylose, fructose, inositol, rhamnose, mannitol, acetic acid, succinic acid, or citric acid. 4. The production method according to claim 1, wherein the amino acids are L-glutamic acid, L-aspartic acid, glycine, L-threonine, L-tyrosine, L-isoleucine, and L-proline. 5. The production method according to claim 1, wherein the inorganic metal salt is an inorganic metal salt containing sulfuric acid, magnesium, or manganese. 6. The hydrocarbon or its derivative is n-dodecane, n-heptadecane, n-hexadecane, n-octadecane, n-
-tetradecane, n-pentadecane, n-eicosane,
2. The method according to claim 1, wherein the compound is selected from the group consisting of chloro-heptadecane, chloro-hexadecane, chloro-octadecane, hexadecyl alcohol, hexadecylbenzene, heptadecanonitrile, hexadecene, eicosene, octadecene, and tetradecene.