JPS63202388A - Production of alkene derivative - Google Patents

Abstract

PURPOSE:To industrially and advantageously obtain an alkene derivative useful for synthesizing pheromones, perfumes, etc., by cultivating a microorganism, belonging to the genus Rhodococcus and capable of producing unsaturated hydrocarbon chain compounds in a culture medium containing an alkane (derivative). CONSTITUTION:A strain of the genus Rhodococcus separated from soil is irradiated with ultraviolet rays to provide Rhodococcus sp. KSM-B-3M exhibiting the following mycological properties. Shape; Bacilli having 0.5-0.8X1-5mum size. Growth range: 15-37 deg.C. pH5-9.5. Assimilating D-xylose, etc. The resultant strain is then aerobically cultivated in a culture medium, such as glucose or peptone,containing 5-10wt.% alkane (derivative) expressed by formula I [R is >=2C, preferably 12-22C saturated straight-chain or branched hydrocarbon group; A is H, halogen, -OH, -CN or formula II (R1 and R2 are H or 1-20C alkyl group)] (e.g. n-tetradecane) at pH6.5-8 and 25-35 deg.C for 1-3 days. The resultant alkene derivative (e.g. n-tetradecene) produced in the culture is subsequently extracted and recovered.

Description

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

[Conventional technology and its problems]

Many alkenes and their derivatives are useful as synthetic intermediates for pheromones, fragrances, or organic compounds.

Conventionally, methods for producing alkenes and their derivatives include combining halogenated monoalkenes and monoalkenes.
A method using a metathesis reaction using a catalyst such as a halogen-containing metal is known.

However, in this method, the activity of the catalyst decreases in the presence of water, so the raw materials, catalyst components, solvent, reaction equipment, etc. used must be thoroughly dehydrated and dried, and the reaction must be carried out in an inert gas stream. In addition to requiring complicated operations such as the following, there were disadvantages in that the reaction product was a mixture of cis and trans isomers.

On the other hand, a method using microorganisms is also known (US Pat. No. 362QO72), but this method also takes a long reaction time and
The yield was low and the process was not satisfactory as an industrial method.

[Means for solving problems]

Under these circumstances, the present inventor conducted intensive research and found that the strain isolated by the present inventor from the soil of the main island of Okinawa and irradiated with ultraviolet rays has the ability to efficiently convert alkanes or derivatives thereof into alkenes or derivatives thereof. The present invention was completed based on the discovery that the present invention has the following properties.

That is, the present invention is characterized in that an unsaturated hydrocarbon chain compound producing bacterium belonging to the genus Rhodococcus is cultured in a medium containing an alkane or a derivative thereof, or the resting bacterium is allowed to act on an alkane or a derivative thereof. The present invention provides a method for producing alkenes or derivatives thereof.

The unsaturated hydrocarbon chain compound producing bacterium belonging to the genus Rhodococcus used in the present invention has the following mycological properties.

(4) Morphology It is a bacillus, and the cells are pleomorphic, becoming rod-shaped in young cultures and spherical in old cultures. The size is 0.5~0, 8 x 1.
O ~ 5.0 pm.

(B) Growth status on each medium■ Sucrose/nitrate agar medium Growth was poor, and the colonies were pale flesh-colored, smooth, and glossy.

■ Glucose-asparagine agar medium Growth is poor, and colonies are pale flesh-colored, smooth and shiny.

■ Growth on glycerin-asparaquine agar medium is moderate, and the colony color is milky white, smooth and glossy. Smooth and rough colonies can be seen.

■ Growth on starch agar medium is moderate, and the colony color is milky white with a clear and shiny luster.

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

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

■ Yeast/malt agar medium Growth is abundant, and colonies are orange in color, wrinkled, and glossy. Some colonies become slime-like.

■ Oatmeal agar medium Growth is moderate, colonies are pale orange in color and have a dull luster.

C) Physiological properties ■ Growth range Temperature 15-37℃ (optimal 25-35℃) pH 5.0-
9.5 (optimal 6.0 to 8.0) ■ Liquefaction of gelatin (glucose peptone/gelatin medium) negative ■ Hydrolysis of starch (starch agar medium) negative ■ Both coagulation and peptonization of skim milk negative ■ Melanin-like Pigment production (Tyrosine medium, Befton's yeast iron ferrous medium) Assimilation of carbon sources ■L-arabinose + ■D-xylose + ■D-glucose + ■D-7 lactose ■Sucrose + ■Inositol + ■L-Rhamnose + ■Raffinose + ■D-Mannitol + (E) Chemical taxonomic properties ■Glycolyl test Glycolyl type ■Menaquinone system MK-8 (for microorganisms with mycological properties higher than H) Bergey's Manual of
As a result of a search based on Systematic Bacteriology, Volume 2 (1986)), it was recognized as a new bacterial strain belonging to the genus Rhodococcus, and was named Rhodococcus sp. It has been deposited at the Institute of Fine Arts and Technology as No. 9060.

Alkanes and their derivatives as raw materials of the present invention have the following general formula (1) % formula % () [wherein R is a saturated straight-chain or branched hydrocarbon having 2 or more carbon atoms, preferably 12 to 22 carbon atoms] Indicates a group, A is a hydrogen atom,
a halogen atom, -OH, -CN, or an alkyl group of ~20]. Among these, those in which R is a saturated linear hydrocarbon group having 12 to 22 carbon atoms and A is a hydrogen atom or a halogen atom (% chlorine atom) are preferred. Specific examples of such preferred alkanes and derivatives thereof include n-tetradecane, n-pentadecane, n-hexadecane, n-octadecane, n-eicosane, 1-
Chloro-n-tetradecane, 1-chloro-n-hexadeca7.1-lya-n-oftatecane, 1-chloro-n
-Eicosane and the like.

According to the present invention, the alkanes and their derivatives include n-tetradecene, n-pentadecene, n-hexadecene, n-octadecene, n-eicosene, 1-chloro-n-tetradecene, 1-chloro-n- It is converted into alkenes such as xadecene, 1-chloro-n-octadecene, 1-chloro-〇-eicosene, or derivatives thereof.

In the present invention, a medium containing a carbon source, a nitrogen source, and an inorganic salt for good growth of the bacteria is used. Carbon sources include hydrocarbons such as glucose, fructose, sucrose, and nrubitol, organic acids such as acetic acid, citric acid, and succinic acid, fatty acids, alkanes, and halogenated alkanes; nitrogen sources include sodium nitrate, potassium nitrate, Yeast extract, peptone, etc.; examples of inorganic salts include various phosphates, magnesium sulfate, etc.

It is also possible to add trace amounts of heavy metals.

The raw material alkane or its derivative is about 5-10% in the medium.
It is preferable to add If the raw material is #I soluble in water,
Preferably, a surfactant such as polyoxyethylene sorbitan is added. The pH of the culture solution is preferably 6.5 to 8.

The culture is preferably carried out at a temperature of 25 to 35° C. for 1 to 3 days with shaking or aeration.

In addition, a method using resting microorganisms involves suspending the resting cells of the microorganism grown in the above-mentioned medium and isolated from this in a buffer solution such as a phosphate buffer solution, and adding an alkane or its derivative to the suspension. This is done by shaking and reacting.

In this way, alkenes or derivatives thereof are produced in the culture solution or the reaction solution of resting bacterial cells. In order to isolate the target alkene or its derivative from the culture solution or the reaction solution of resting bacterial cells, general organic compound separation and purification methods are used.

For example, the liquid is filtered to remove bacterial cells and the like, and the F liquid is extracted with an organic solvent such as ethyl ether, n-hexane, ethyl acetate, or chloroform. If this extract is purified by column chromatography or the like, a highly pure target product can be obtained.

For identification and quantitative determination of alkenes or derivatives thereof produced in a culture solution or a reaction solution of resting bacterial cells, make the solution alkaline with potassium hydroxide, stir thoroughly, and then hold for a certain amount of time.]
, n-hexane, and the extract is analyzed and quantified by gas chromatography or GC-MS.

〔Effect of the invention〕

According to the present invention, alkenes or derivatives thereof can be industrially advantageously produced using specific bacteria belonging to the genus Rhodococcus.

〔Example〕

Next, an example will be given and explained.

Example 1 Glucose 2.5f, Polypeptone 172, Polypeptone 783 P, KH2PO42,,5? , NaC15? and 50ml of a medium solution consisting of 1t of ion-exchanged water.
The mixture was placed in a 0-Yosaka flask and steam sterilized at 120°C for 15 minutes to prepare a culture medium. Rhodococcus sp. KSM
- After pre-culturing the B-3M strain in the same medium, 0.5
ml was inoculated into a medium with the same composition, and cultured at 30°C for 24 hours. Next, the bacterial cells obtained by centrifugation were
．． Wash with 5 M IJ acid buffer (pH 7,0) and
．． 0.5M phosphate buffer containing 5% glucose 4.75
After suspending in -, 0.25 g of n-pentadecane or n-hexadecane was added and shaking was performed at 30°C for 72 hours.

When the monoalkene in the reaction solution was analyzed, identified (GC-MS analysis of monoalkene, GC-FT-IR analysis, and GC-MS analysis of dimethyl disulfide derivative) and quantified, the results shown in Table 1 below were obtained. Ta.

Table 1 GC-MS spectrum of 6-cis-pentadecene is the first
Figure 2 shows the GC-FT-IR spectrum of the dimethyl disulfide derivative of 6-cis-pentadecene.
The MS spectrum is shown in FIG.

Example 2 Example 1 except that halogenated alkanes such as 1-chlorohexadecane and 1-chlorooctadecane were used instead of n-heptadecane and n-hexadecane used in Example 1.
The same operation was performed.

When the halogenated monoalkene in the reaction solution was analyzed, identified, and quantified in the same manner as in Example 1, the following table-
The results shown in 2 were obtained.

Below is the margin Example 3 Glucose 5? , NH4NOs 10? , K.H.
zPO41,2f, Na2HPO48f, MgSO4@
7 H2O0,5? , 5 m of a medium solution (pH 7.0) consisting of 2F of yeast extract and 1 t of ion-exchanged water was placed in a test tube (φ25 x 200 ms), and after adding 0.25 m of n-hexadecane or 1-chlorohexadecane, the mixture was heated to 120°C. The cells were steam sterilized for 15 minutes and used as a culture medium.

Rhodococcus sp. KSM-B-3M strain was used as a raw material (
)・halogenated) Alkane-free medium was precultured, and then 0.14 of the culture was added to the medium containing the raw material (halogenated) alkane, and cultured at 30° C. for 72 hours.

The monoalkenes or halogenated monoalkenes in the culture solution were analyzed, identified, and quantified in the same manner as in Example 1, and the results shown in Table 3 below were obtained.

Table-3

[Brief explanation of the drawing]

Figure 1 Tri, GC of 6-cis-pentadecene
The MS spectrum, Figure 2 is the QC-FT-IR spectrum of the same substance, and Figure 3 is the GC-MS spectrum of the dimethyl disulfide derivative of 6-7 subventadecene. that's all

Claims (1)

[Scope of Claims] 1. A method characterized by culturing an unsaturated hydrocarbon chain compound-producing bacterium belonging to the genus Rhodococcus in a medium containing an alkane or a derivative thereof, or allowing the resting bacterium to act on an alkane or a derivative thereof. A method for producing alkenes or derivatives thereof. 2. The method for producing an alkene according to claim 1, characterized in that Rhodococcus sp. KSM-B-3M is cultured in a medium containing an alkane, or the resting bacterial cells are allowed to act on an alkane.