JPH01144982A - Production of unsaturated fatty acid and derivative thereof - Google Patents

Abstract

PURPOSE:To obtain the title compound useful as a synthetic raw material for perfumery, etc., on an industrial scale at a low cost, by using cells of microorganism belonging to genus Rhodococcus and capable of producing an unsaturated fatty acid and reacting the cells with a fatty acid in the presence of thiamine, inorganic metal salt, etc., and in the presence of a carbohydrate or an amino acid. CONSTITUTION:An unsaturated fatty acid and its derivative (e.g., cis-9- hexadecenoic acid methyl ester) is produced by using cells of microorganism belonging to genus Rhodococcus and capable of producing an unsaturated fatty acid [e.g., Rhodococcus sp. KSM-B-3M (FERM BP-1531)] and reacting the cells with a fatty acid or its derivative (e.g., methyl palminate) 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 [Industrial Field of Application] The present invention relates to a method for producing unsaturated fatty acids and derivatives thereof using microorganisms.

[Conventional technology and its problems]

Unsaturated fatty acids and their derivatives are used in fragrances, drugs, paints,
It is widely used as a surfactant, cosmetics, etc., and as a raw material for their synthesis.

Conventionally, such unsaturated fatty acids and their derivatives have been produced by hydrolyzing animal or vegetable oils or by chemically synthesizing them.

However, the method of hydrolyzing fats and oils produces a wide variety of products with different carbon chain lengths, and chemical synthesis requires multiple steps, and a mixture of cis and trans stereoisomers is obtained. This method also had the disadvantage that separation was difficult.

On the other hand, methods of utilizing microorganisms in the production of unsaturated fatty acids include T-lilunic acid fermentation using Tamnibuium spp. described in Japanese Patent Publication No. 61-37096, and Japanese Patent Publication No. 58-2219.
The problem was that it contained Mortierella bacteria No. 9 and was difficult to recover.

Acyl-CoA desaturase (^) is an enzyme derived from microorganisms, animals, and plants that desaturates fatty acid derivatives.
cyl-Co eight desat, E, C, 1,
14,99°5) are known, but they have low enzymatic activity and are extremely unstable, making them unsuitable for industrial production.

In addition, the present inventors reported a method for producing unsaturated fatty acids etc. using an unsaturated fatty acid producing bacterium belonging to the genus Rhodococcus in Japanese Patent Application No. 62-33363; In this case, the production volume was still not sufficient, and it could not be said that industrial productivity was fully satisfied.

[Means for solving problems]

Under these circumstances, the present inventors conducted intensive research and found that microorganisms isolated from soil and further mutated as shown in Reference Example 1 were able to transform fatty acids and their derivatives into unsaturated fatty acids and their derivatives. It has been found that the unsaturated fatty acids and their derivatives produced are excreted outside the bacterial cells and can be easily recovered.

Alternatively, the present invention was completed by discovering that the productivity of unsaturated fatty acids and their derivatives can be greatly improved by adding inorganic metal salts such as magnesium sulfate and manganese sulfate.

That is, the present invention uses bacterial cells of unsaturated fatty acid-producing bacteria belonging to the genus Rhodococcus to produce unsaturated fatty acids or derivatives thereof by applying fatty acids or derivatives thereof to cough bacterial cells in the presence of carbohydrates or amino acids. The present invention provides a method for producing unsaturated fatty acids and derivatives thereof, characterized in that thiamine or an inorganic metal salt is present therein.

The unsaturated fatty acid-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 soil in Okinawa Prefecture by the method of Reference Example 1, and the following bacterium: It has scientific 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 μm x 1
．． It is 0 to 5.0 μm.

■Glucose-asparagine agar medium Growth is poor, and the 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. Colony mosquitoes 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 (optimum 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-fructose ■D-Mannitol + (I chemical taxonomic properties ■Glycolyl test Glycolyl type.

■Menaquinone system MK-8 (Hz).

Microorganisms with the above mycological properties are described in 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 fatty acids was significantly different from that of other Rhodococcus bacteria, so it was classified as Rhodococcus sp. KS.
M-B-3M (Rhodococcus sp,
KSM-B-3M), and was sent to the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, with the name No. 1531 (FERM).
BP-1531).

Examples of fatty acids and derivatives thereof that can be used as raw materials for carrying out the method of the present invention include the following general formula [wherein R represents a straight chain or branched, saturated or unsaturated hydrocarbon group having 1 to 22 carbon atoms, R1 is a hydrogen atom, a straight chain or branched hydrocarbon group having 1 to 10 carbon atoms, or an aromatic hydrocarbon group,
represents an alkali metal or a group represented by (Rz, Rz represents a hydrogen atom or a straight chain or branched hydrocarbon group having 1 to 5 carbon atoms),
] In the present invention, R is preferably a straight chain hydrocarbon group having 14 to 20 carbon atoms, and R1 is preferably a straight chain or branched hydrocarbon group having 2 to 4 carbon atoms.

According to the present invention, the fatty acids and their derivatives are converted into corresponding unsaturated fatty acids and their derivatives. Specific examples include methyl of fatty acids such as n-hexadecanoic acid, n-tetradecanoic acid, ethyl, n-propyl, isopropyl, n-methyl and ethyl of fatty acids such as tetradecenoic acid, To obtain the microorganisms, it is sufficient to use the usual culture method of microorganisms.
Any conditions may be used as long as the strain concerned grows. 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 fatty acids and 1 to 70% of the derivative, preferably 10 to 30%,
Amino acids such as L-glutamic acid and L-aspartic acid, or carbohydrates such as glucose and succinic acid, from 0.05 to 1
The basic reaction solution is one containing 0%, preferably 0.5 to 2%, and further 2% of thiamine hydrochloride, preferably 0.01 to 0.3% of magnesium sulfate is added to carry out the reaction of the present invention. liquid, 20-37°C1 preferably 25-30°C
The reaction is carried out under aerobic conditions at °C. This reaction produces unsaturated fatty acids and derivatives thereof corresponding to the fatty acids and derivatives thereof added to the reaction solution.

The addition of thiamine or magnesium sulfate to the reaction solution is to significantly improve the productivity of unsaturated fatty acids and their derivatives, and combinations with amino acids such as L-glutamic acid and L-aspartic acid are preferred. , may 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.

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

It can also produce amino acids, carbohydrates, thiamin, and inorganic gold.

When performing a batch reaction, the reaction time may be about 1 to 3 days, but it can be extended further by supplementing raw materials such as fatty acids and derivatives thereof, amino acids, vitamins, metal salts, etc.

It is also possible to collect the used bacterial cells by centrifugation, filtration, etc. and re-react them.

The unsaturated fatty acids 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 (QC-MS). can. Also,
To determine the position of the double bond in an unsaturated compound, the methylthioetherification method, which involves GC-MS analysis after treatment with dimethyl disulfide [2 Hara et al., Oil Chemistry Vol. 34, p. 619 (198
5 years) and others] are available.

After removing the unsaturated fatty acid produced in the reaction solution and, preferably, the bacterial cells by filtration or the like, the desired product can be easily recovered by extraction with an organic solvent. In addition, if further purification is required, purification and isolation can be carried out by ordinary column chromatography, partition extraction, etc.

〔Effect of the invention〕

According to the present invention, unsaturated fatty acids and derivatives thereof can be advantageously produced industrially using cells of unsaturated fatty acid 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.)
, 1 potassium dihydrogen phosphate 2.5 g, 5 sodium chloride
500 mj containing 50 m2 of culture medium consisting of g and 12 ml of ion-exchanged water! A culture solution of Rhodococcus sp. KSM-B was centrifuged at 5000x in a Yosaka Rof flask to obtain 2 g of bacterial cells. 1 g of bacterial cells was added to 0.25M phosphate buffer (pH 7.0) containing 1% monosodium glutamate, 0.1% thiamine hydrochloride, and 0.1% magnesium sulfate.
Q, 33 to 20 mffi, propyl balmitate (
Palmitic acid n-propyl es
ter.

propyl hexadecanoate) 4
ml and heated at 26°C in a 500 m2 Slope flask.
The reaction was carried out by shaking 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/RM analyzer (GC-MS), or gas chromatograph/infrared analyzer (GC-FT-). IR) analysis (Figure 1) was performed, and the main product was propyl cis-6-hexadenoate.
cenoate). The position of the double bond was determined by mass spectrum analysis after dimethyl disulfide derivatization (FIG. 2). The mass spectrum β of the main product was the productivity for 2 days.

Example 2 Methyl hexadecanoat was used instead of propyl balmitate in Example 1.
e), ethyl hexade
canoate), isopropyl valmitate (iso
-propyl hexadeca-noate),
Isobutyl valmitate (1so-butylhexa
decanoate), butyl valmitate (n-b
The reaction was carried out in the same manner as in Example 1 except that util hexadecanoate) was used.

When the unsaturated fatty acid ester in the reaction solution was identified and quantified in the same manner as in Example 1, the results shown in Table 1 below were obtained.

Example 3 The same procedure as in Example 1 was performed except that sodium valmitate, isopropyl myristate, and ethyl myristate were used in place of propyl palmitate in Example 1 to cause a reaction.

Comparative Example 1: 50 m of culture medium consisting of 2.5 g of glucose, 17 g of polypeptone, 3 g of polypeptone S, 2.5 g of monopotassium dihydrogen phosphate, 5 g of sodium chloride, and 1N of ion-exchanged water
500mj including 2! Rhodococcus sp. KSM-B-3M strain was transplanted into a Yosaka Rof flask and cultured with shaking at 30°C for 1 day. After that, this culture solution 1mff1
were transplanted into a 500 mf Yosaka flask containing 100 ml of a medium with the same composition, and cultured with shaking at 30°C for 1 day. This culture solution was centrifuged at 5000X for 2
The reaction was carried out by shaking for several days.

1 m2 of the reaction solution was extracted with 3 mj2 of ethyl acetate, and isopropyl hexadecenate was identified and quantified in the same manner as in Example 1, and the productivity was 4.0 g/2.2 days.

Comparative Example 2 Comparative Example 1 except that 0.25 MU phosphate buffer containing 1.0% monosodium L-glutamate was used instead of the 0.25 M phosphate buffer containing 1.0% glucose in Comparative Example 1. The reaction was carried out in the same manner.

Example 1
Identification and quantification were performed using the same procedure as in 5.
The productivity was 2 g/l for 2 days.

Example 4 Instead of the 0.25M phosphate buffer containing 1.0% glucose in Comparative Example 1, 01.0% glucose, 0.1% thiamine hydrochloride acid buffer ■ 1.0% monosodium L-glutamate, 0.1%
0.25M phosphate buffer containing thiamine hydrochloride, ■1.0% monosodium L-glutamate, 0.1%
Thiamine hydrochloride, 0.1% magnesium sulfate.
25 MIJ acid buffer, ■ 0.25 MIJ acid buffer containing 1.0% monosodium L-aspartate, 0.1% thiamine hydrochloride, 0.1% magnesium sulfate, 01
．． 0.2 containing 0% glycine, 0.1% thiamine hydrochloride
5M phosphate buffer, ■1.0% L-tyrosine, 0.1% thiamine hydrochloride, 0
．． 0.25M phosphate buffer containing 1% magnesium sulfate, ■1.0% L-threonine, 0.1% thiamine hydrochloride,
0.25M phosphate buffer containing 0.1% magnesium sulfate, ■1.0% L-proline, 0.1% thiamine hydrochloride, 0
．． The reaction was carried out in the same manner as in Comparative Example 1, except that a 0.25M solution containing 1% magnesium sulfate was used.

When the unsaturated fatty acid ester in the reaction solution was identified and quantified in the same manner as in Example 1, the results shown in Table 3 below were obtained.

Reference example 1: Approximately 0.5 g of the collected soil sample from Okinawa Prefecture was placed in a sterile liquid medium (I): n-hexadecane 100 g (N114)2SO4 20 g Kll□Pop 20 g Yeast extract 2 g Gso, 711□OO, 5 g FeSO4・7LOo,ot g MnSO-・4～6LOO,008g Ion exchange water II! .

pH 7 After appropriately diluting the culture solution that showed growth in 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. The transplantation to the ordinary agar medium was repeated until it was confirmed macroscopically and microscopically that there was no difference between the two.

This isolated bacterial strain was suspended in 0.1 MIJ acid buffer (pH 7.0) and irradiated with ultraviolet light for 90 seconds.

After that, the obtained colonies were treated with 2.5 g of glucose and 30 g of glucose.
Shaking culture was performed at °C for 1 day. Bacterial cells obtained from this culture solution by centrifugation were added to 0.2 μg containing 0.5% glucose
The suspension was suspended in 19 m2 of 5M phosphate buffer (pH 7,0), 1 mf of hexadecane was added, and the mixture was shaken at 30°C for 3 days in a 500 mj 2-volume Slope flask. After shaking, the main product contained in the reaction solution was extracted and subjected to gas chromatography (CC
), etc., and strains that produce hexadecene were obtained.

After appropriately diluting this hexadecene-producing strain with sterilized water, it was transferred to an ordinary agar medium and cultured at 30°C for 2 days, and it was confirmed visually and microscopically that the resulting colonies were not different from each other. The transplantation to ordinary agar culture medium was repeated until confirmation was made.

After that, among the colonies that grew on ordinary agar medium,
Each of the 10 colonies was inoculated onto an agar slant (II) with the following composition and cultured at 30°C for 3 days, and the strains on the 10 agar slants were macroscopically and microscopically the same strain. and n-hexadecane
20 g(NHa)*SOn 20
gKH2POn 2g yeast extract 2g Mg5Oa・7HzOO,5g FeSOa・7thOO,01g MnSO4・4~6tlzOO,008g polyoxyethylene-sorbitan monolauric acid 0.05g ester (
Average added EO number: 20 moles) Agar: 20 g Ion-exchanged water: IP Next, for the bacterial strain obtained by the above procedure, a loopful of it was taken from the slanted agar medium and placed in a cryopreservation solution containing a sterilized 10% glycerin aqueous solution (2rr+42). The suspension was suspended in a vial 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 above-mentioned shape and physiological properties were examined, but no changes were observed.

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

[Brief explanation of the drawing]

FIG. 1 shows a GC-FT-IR spectrum of propyl hexadecenoate produced according to the present invention. FIG. 2 shows the mass spectrum of propyl hexadecenate bismethylthioether 1H4C produced according to the present invention. FIG. 3 shows the mass spectrum of propyl hexadecenoate produced according to the present invention. that's all

Claims (1)

[Scope of Claims] 1. Production of unsaturated fatty acids or derivatives thereof by using bacterial cells of unsaturated fatty acid-producing bacteria belonging to the genus Rhodococcus and allowing fatty acids or derivatives thereof to act on the bacterial cells in the presence of carbohydrates or amino acids. 1. A method for producing unsaturated fatty acids and derivatives thereof, characterized in that thiamine or an inorganic metal salt is present in the system. 2. The unsaturated fatty acid producing bacterium is Rhodococcus sp.
The production method according to claim 1, which is the SM-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 fatty acid or its derivative is n-heptadecanoic acid, n
- from the group consisting of hexadecanoic acid, n-pentadecanoic acid, n-tetradecanoic acid, or their methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-heptyl, n-hexyl esters; The manufacturing method according to claim 1, which is selected.