JPH0412718B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a method for producing unsaturated fatty acids and derivatives thereof using microorganisms. [Prior art and its problems] Unsaturated fatty acids and their derivatives are widely used as fragrances, drugs, paints, surfactants, cosmetics, etc., and as raw materials 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 using microorganisms in the production of unsaturated fatty acids include γ-linolenic acid fermentation using Tamnidium bacteria as described in Japanese Patent Publication No. 61-37096;
58-22199 using γ-linolenic acid fermentation using Morteiera bacteria is known, but the yield is low, the reaction time is long, and the unsaturated fatty acids produced are accumulated in the bacterial cells. Therefore, there was a drawback that its recovery was difficult. Acyl-CoA desaturase (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 JP-A-63-202392; 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 the Problems] Under the current situation, the present inventors have conducted intensive research and found that microorganisms isolated from soil and further mutated as shown in Reference Example 1 were obtained. It has been found that this strain has the ability to convert fatty acids and their derivatives into unsaturated fatty acids and their derivatives, and that the produced unsaturated fatty acids and their derivatives are excreted outside the bacterial cells and can be easily recovered. Furthermore, a method for producing unsaturated fatty acids and their derivatives in high yield by a reaction using the cells of this strain, that is, adding L-glutamic acid, L-glutamic acid,
Amino acids such as L-aspartic acid, citric acid,
The present inventors have discovered that the productivity of unsaturated fatty acids and their derivatives can be greatly improved by adding carbohydrates such as glucose acid and further adding thiamine or magnesium salts or manganese salts such as magnesium sulfate or manganese sulfate. . That is, the present invention uses the cells of an unsaturated fatty acid-producing bacterium belonging to the genus Rhodococcus, and in the production of unsaturated fatty acids or derivatives thereof, a fatty acid or a derivative thereof is applied to the cells in the presence of a carbohydrate or an amino acid. 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 Rhodocoscus 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 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~
The size is 0.8 μm×1.0 to 5.0 μm. (B) Growth status on each medium Seuucrose/nitrate agar medium Growth is poor, and the colonies are pale flesh-colored, smooth, and glossy. Glucose-Asparagine Agar Medium Growth is poor, colonies are pale flesh-colored, smooth and shiny. Glycerin-asparagine agar medium Growth is moderate, colonies are milky white, smooth and glossy. Smooth and rough colonies can be seen. Starch agar medium Growth is moderate, colonies are milky white, smooth and glossy. Tyrosine agar medium Growth is vigorous, colonies are flesh-colored, smooth and shiny. Colonies that become slime-like can be seen. Nutrient agar medium Growth is vigorous, colonies are light orange in color, smooth and shiny. Smooth and rough colonies can be seen. Yeast/malt agar medium Growth is vigorous, colonies are orange in color, wrinkled and shiny. Colonies that become slime-like can be seen. 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℃ (optimum 25-35℃) PH: 5-9.5 (optimum 6-8) Liquefaction of gelatin (glucose, peptone,
gelatin medium) negative. Hydrolysis of starch (starch agar medium)
negative. Both skimmed milk coagulation and peptization tests were negative. Formation of melanin-like pigment (tyrosine medium, peptone/yeast/iron medium) was negative. (D) Assimilation of carbon source L-arabinose + D-xylose + D-glucose + D-fructose + sucrose + inositol + L-rhamnose + D-mannitol + (E) Chemical taxonomic properties Glycolyl test Glycolyl type . Menaquinone system MK-8 (H ₂ ). Microorganisms with the above mycological properties are described in Virgie's Manual of Systematics.
Bacteriology (Bergey's Manual of
Systematic Bacteriology), Volume 2 (1986)
As a result of the search, it was recognized as a strain belonging to the genus Rhodococcus;
KSM-B-3M (Rhodococcus sp.
KSM-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. Examples of fatty acids and derivatives thereof that can be used as raw materials for carrying out the method of the present invention include those represented by the following general formula (). R-COCR ₁ () [In the formula, R represents a straight chain or branched, saturated or unsaturated hydrocarbon group having 1 to 22 carbon atoms, and R ₁ is a hydrogen atom or a straight chain or branched hydrocarbon group having 1 to 10 carbon atoms. Hydrogen group or aromatic hydrocarbon group, alkali metal or (R ₂ and R ₃ represent a hydrogen atom or a straight chain or branched hydrocarbon group having 1 to 5 carbon atoms)] In the present invention, R is a straight chain carbonized group having 14 to 20 carbon atoms. The hydrogen group R ₁ is preferably a straight chain or branched hydrocarbon group having 2 to 4 carbon atoms. According to the invention, the fatty acids and their derivatives are converted into corresponding unsaturated fatty acid derivatives. Specific examples include methyl, ethyl, n-propyl, isopropyl, n- of fatty acids such as n-hexadecanoic acid and n-tetradecanoic acid.
Esters such as butyl, isobutyl, and tert-butyl are derived from methyl, ethyl, n-
Esters such as propyl, isopropyl, n-butyl, isobutyl, and tert-butyl are produced. In the present invention, the cells of the bacteria can be obtained by a normal microorganism culturing method, and when culturing, the culture medium, pH, temperature, culture time, etc., can be adjusted as long as the strain grows. Any condition may be used. 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. 0.1-90% of the above culture solution or collected bacteria,
Preferably, 0.2 to 0.5% of the dry weight of the bacterial cells is suspended in water or a buffer solution (PH6 to 8), and 1 to 70% of the fatty acids and their derivatives to be reacted, preferably 10%.
A basic reaction solution containing 0.05 to 10%, preferably 0.5 to 2%, of amino acids such as L-glutamic acid and L-aspartic acid or carbohydrates such as glucose and succinate is used as the basic reaction solution. Add 0.001 to 2% of hydrochloride, preferably 0.01 to 0.3% as thiamin content, or 0.01 to 2% of magnesium salt or manganese salt such as magnesium sulfate or manganese sulfate, preferably 0.01 to 0.3% of magnesium sulfate. As the reaction solution of the invention,
The reaction is carried out under aerobic conditions at 20-37°C, preferably 25-30°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. , in combination with other carbohydrates,
Each may be reacted alone or 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. It can be in either state.
Examples of the magnesium salt or manganese salt include magnesium sulfate and manganese sulfate. 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. 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. Furthermore, it is also possible to recover the once-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, a certain amount of the reaction solution is extracted with an organic solvent, and then extracted using a gas chromatography (GC) or gas chromatograph/mass spectrometer (GC-
It can be identified and quantified by analysis using MS). In addition, the methylthioetherification method (Shibahara et al., Oil Chemistry 34, p. 619 (1985), etc.), which involves GC-MS analysis after treatment with dimethyl disulfide, is used to determine the position of the double bond in an unsaturated compound. The unsaturated fatty acids and their derivatives produced in the reaction solution can be used for the extraction and extraction of organic compounds from ordinary natural products.
It can be recovered and separated depending on the isolation method. For example, by centrifuging the reaction solution to separate the bacterial cells, oil layer, and water layer and separating the oil layer, or by using the reaction solution as it is, or after removing the bacterial cells by filtration, etc., and extracting with an organic solvent. You can easily collect the target object. In addition, if further purification is required, purification and isolation can be carried out by conventional column chromatography, partition extraction, etc. [Effects of the Invention] According to the present invention, unsaturated fatty acids and derivatives thereof can be industrially advantageously produced using cells of unsaturated fatty acid-producing bacteria belonging to the Rhodocotcus family. [Example] Next, an example will be given and explained. Example 1 Glucose 2.5g, polypeptone [Daigo Nutrition Co., Ltd.]
[manufactured by Daigo Nutrition Co., Ltd.] 17 g, Polypeptone S [manufactured by Daigo Nutrition Co., Ltd.] 3 g,
Rhodococcus sp. KSM-B-3M strain was transplanted into a 500 ml Sakaguchi flask containing 50 ml of a medium consisting of 2.5 g of potassium dihydrogen phosphate, 5 g of sodium chloride, and 1 part of ion-exchanged water.
A shaking culture was performed for 1 day. Thereafter, 1 ml of this culture solution was transferred to a 500 ml Sakaguchi 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 5000× g to obtain 2 g of bacterial cells. 1 g of bacterial cells was suspended in 20 ml of 0.25 M phosphate buffer (PH7.0) containing 1% monosodium glutamate, 0.1% thiamine hydrochloride, and 0.1% magnesium sulfate, and propyl palmitate (Palmitic acid n)
-propyl ester, propyl hexadecanoate) 4ml
was added, and the reaction was carried out by shaking in a 500 ml Sakaguchi Fusco at 26°C for 2 days. After the reaction, the main products contained in the reaction solution are extracted with ethyl acetate and analyzed using gas chromatography (GC), gas chromatograph/mass spectrometer (GC-MS), or gas chromatograph/infrared spectrometer (GC-MS).
(FT-IR) analysis (Fig. 1), the main product was propyl cis-6-hexadecenoate (propyl
cis-6-hexadecenoate). The position of the double bond was determined by mass spectrum analysis after dimethyl disulfide derivatization (FIG. 2). The mass spectrum of the main product is shown in FIG. After the reaction, 1 ml was extracted with 3 ml of ethyl acetate, and cis-
When the amount of propyl 6-hexadecenoate was measured by gas chromatography, it was found to be 15 g/2
It was productivity of the day. Example 2 Instead of propyl palmitate in Example 1,
Methyl palmitate (methyl hexadecanoate),
Ethyl palmitate (ethyl hexadecanoate),
Isopropyl palmitate
hexadeca-noate) butyl palmitate (iso
-butyl hexadecanoate) and butyl palmitate (n-butyl hexadecanoate) were used, but the same operation as in Example 1 was performed to cause a reaction. 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 the table below were obtained.

[Table] Example 3 Instead of propyl palmitate in Example 1,
The reaction was carried out in the same manner as in Example 1 except that sodium palmitate, isopropyl myristate, and ethyl myristate were used. When the unsaturated fatty acid or 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 2 below were obtained.

[Table] Comparative Example 1 Rhodocoscus sp. KSM was placed in a 500 ml Sakaguchi flask containing 50 ml of a 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 1 portion of ion-exchanged water. -B-3M strain was transplanted and cultured with shaking at 30°C for 1 day. After that, add 1 ml of this culture solution to 500 ml containing 100 ml of medium with the same composition.
The cells were transplanted into a Sakaguchi flask and cultured with shaking at 30°C for 1 day. This culture solution was centrifuged at 5000× g to obtain 2 g of bacterial cells. Suspend 1 g of bacterial cells in 20 ml of 0.25 M phosphate buffer (PH7.0) containing 1.0% glucose, add 4 ml of isopropyl palmitate,
The reaction was carried out by shaking in a 500 ml Sakaguchi Fusco at 26°C for 2 days. 1 ml of the reaction solution was extracted with 3 ml of ethyl acetate, and the isopropyl hexadecenate was identified and quantified in the same manner as in Example 1. As a result, 4.0 g/.
It was two days of productivity. Comparative Example 2 Reaction was carried out in the same manner as in Comparative Example 1 except that 0.25 M 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. I let it happen. The amount of isopropyl hexadecenate in the reaction solution was identified and quantified by the same procedure as in Example 1, and the productivity was 5.2 g/2 days. Example 4 Instead of the 0.25M phosphate buffer containing 1.0% glucose in Comparative Example 1, 0.25M phosphate buffer containing 1.0% glucose, 0.1% thiamine hydrochloride, 1.0% monosodium L-glutamate, 0.1
0.25M phosphate buffer containing % magnesium sulfate 1.0% monosodium L-glutamate, 0.1
0.25M phosphate buffer containing % thiamine hydrochloride, 1.0% monosodium L-glutamate, 0.1
% thiamine hydrochloride, 0.25M phosphate buffer containing 0.1 magnesium sulfate, 1.0% monosodium L-aspartate,
0.25M phosphate buffer containing 0.1% Thiamine Hydrochloride, 0.1% Magnesium Sulfate, 1.0% Glycine, 0.1% Thiamine Hydrochloride
0.25M phosphate buffer, 1.0% L-tyrosine, 0.1% thiamine hydrochloride,
0.25M phosphate buffer containing 0.1% magnesium sulfate, 1.0% L-threonine, 0.1% thiamine hydrochloride, 0.25M phosphate buffer containing 0.1% magnesium sulfate, 1.0L-proline, 0.1% thiamine hydrochloride, 0.1
0.25M phosphate buffer containing % 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 phosphate buffer containing % manganese 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.

[Table] Reference example 1 Approximately 0.5 g of the collected soil sample from Okinawa Prefecture was suspended in 10 ml of sterile water, and after thorough stirring, 0.2 ml of this suspension was added.
10 ml of liquid medium () with the following composition (test tube φ25 x
200mm) and cultured with shaking at 30°C for 4 days. Liquid medium (): n-hexadecane 100g (NH ₄ ) ₂ SO ₄ 20g KH ₂ PO ₄ 20g Yeast extract 2g MgSO ₄・7H ₂ O 0.5g FeSO _{4 ・}7H 2 O 0.01g MnSO ₄・4～6H ₂ O 0.008 g Ion-exchanged water 1 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. The transplantation onto the regular agar medium was repeated until it was confirmed macroscopically and microscopically that the colonies were not different from each other. This isolated bacterial strain was suspended in 0.1M phosphate buffer (PH7.0) and irradiated with ultraviolet light for 90 seconds. After that, the obtained colonies were added to glucose 2.5g, polypeptone 17
The cells were transplanted into a 500 ml Sakaguchi flask containing 50 ml of a medium consisting of 3 g of polypeptone S, 2.5 g of monopotassium dihydrogen phosphate, 5 g of sodium chloride, and 1 portion of ion-exchanged water, and cultured with shaking at 30° C. for 1 day. The bacterial cells obtained from this culture solution by centrifugation are
0.25M phosphate buffer containing 0.5% glucose (PH
7.0) Suspend in 19ml, add 1ml of hexadecane,
The mixture was shaken at 30°C for 3 days in a 500ml Sakaguchi flask. After shaking, the main product contained in the reaction solution was extracted and analyzed by gas chromatography (GC) or the like to obtain a strain that produces hexadecene. After appropriately diluting this hexadecene-producing strain with sterile water, it was transferred to an ordinary agar medium for 30 minutes.
The colonies were cultured at ℃ for 2 days, and the transplantation onto ordinary agar medium was repeated until it was confirmed macroscopically and microscopically that the resulting colonies were not different from each other. After that, 10 colonies among the colonies that had grown on the regular agar medium were inoculated onto the slanted agar medium () with the following composition, and cultured at 30℃ for 3 days. are macroscopically and microscopically the same strain;
It was confirmed that the properties and physiological properties of the 10 strains on each medium were the same. Slanted agar medium (): n-hexadecane 20g (NH ₄ ) ₂ SO ₄ 20g KH ₂ PO ₄ 2g Yeast extract 2g MgSO ₄・7H ₂ O 0.5g FeSO ₄・7H ₂ O 0.01g MnSO ₄・4～6H ₂ O 0.008g polyoxyethylene-sorbitan monolauric acid 0.05g ester (average number of added EO 20 moles) agar 20g ion-exchanged water 1 PH 7 Next, for the strain obtained by the above procedure, one platinum loop was sterilized from the slanted agar medium. Ten%
The suspension was suspended in a cryopreservation vial containing an aqueous glycerin solution (2 ml) and stored frozen at -80°C. After storage for 3 months, 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. As a result, no changes were found. I confirmed that there was no. Also. Even when the strain was subjected to the above freezing and thawing process repeated five times every month, its properties and physiological properties on each medium were examined in the same manner, but no changes were observed. The properties and physiological properties of this strain on each medium are as described above.

[Brief explanation of drawings]

FIG. 1 shows a GC-FT-IR spectrum of propyl hexadecenoate produced according to the present invention. FIG. 2 shows the mass spectrum of the bismethylthioether derivative of propyl hexadecenoate produced according to the present invention. FIG. 3 shows the mass spectrum of propyl hexadecenoate produced according to the present invention.

Claims (1)

[Claims] 1. In the production of unsaturated fatty acids or derivatives thereof by using the cells of an unsaturated fatty acid-producing bacterium belonging to the genus Rhodococcus and allowing fatty acids or derivatives thereof to act on the cells in the presence of carbohydrates or amino acids. , a method for producing unsaturated fatty acids and derivatives thereof, characterized by the presence of thiamine or a magnesium salt or a manganese salt in the system. 2 Claim 1 in which the unsaturated fatty acid-producing bacterium is Rhodococcus sp. KSM-B-3M strain
Manufacturing method described in section. 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 fatty acid or its derivative is n-heptadecanoic acid, n-hexadecanoic acid, n-pentadecanoic acid,
n-tetradecanoic acid, or its methyl, ethyl, n-propyl, isopropyl, n-butyl,
The method according to claim 1, wherein the ester is selected from the group consisting of isobutyl, tert-butyl, n-heptyl, and n-hexyl esters.