JPH0361434B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing ω-halocarboxylic acids from alkyl (or alkenyl) halides using yeast. Conventionally, it has been considered industrially difficult to selectively oxidize only the ω-terminus of fatty acids. for example,
ω-Hydroxypalmitic acid is used in the production of hexadecanolide, which is the main component of lactone musk (synthetic fragrance), but the reason why musk is so expensive is because of its precursor, ω-hydroxypalmitic acid. This is due to the difficulty in producing mitic acid. That is, it is difficult in terms of synthetic chemistry to selectively oxidize the ω-terminus of palmitic acid to form ω-hydroxypalmitic acid. On the other hand, it has been reported that ω-hydroxy higher fatty acids are also obtained as a by-product when normal paraffin is assimilated by microorganisms to produce dicarboxylic acids (e.g., Japanese Patent Publication No. 26238-1973).
-Hydroxy higher fatty acids are metabolic intermediates of normal paraffin into dicarboxylic acids by alkane-assimilating bacteria, but it is difficult to produce them in significant quantities. The reason for this is presumed to be that the rate of conversion to dicarboxylic acids is much higher than the rate of production of ω-hydroxy higher fatty acids. Further, similar to ω-hydroxy fatty acids, ω-halocarboxylic acids are useful intermediates for macrocyclic lactones, which are the main components of lactone musks. ω-
Halocarboxylic acids can also be derived into various derivatives by introducing a functional group into the halogen.
Regarding this ω-halocarboxylic acid, there is a method of producing ω-halocarboxylic acid by culturing bacteria that belong to the genus Arthrobacter, Corynebacterium, and Nocardia and have the ability to produce ω-halocarboxylic acid from alkyl halides. It has been reported (Japanese Patent Application Laid-Open No. 57-50893). However, no yeast that has such an ability is known yet. Therefore, in view of the current situation, the present inventor proposed that the alkyl (or alkenyl) halide correspond to ω-
As a result of a wide search for yeast that has the ability to convert into halocarboxylic acids, it was discovered that some microorganisms belonging to the genus Candeida have such an ability, and the present invention was completed. That is, the present invention involves culturing ω-halocarboxylic acid-producing bacteria belonging to the genus Candeida in a medium supplemented with an alkyl (or alkenyl) halide having 6 to 22 carbon atoms, and producing ω-halocarboxylic acid having 6 to 22 carbon atoms in the medium. A method for producing ω-halocarboxylic acid using a microorganism, which is characterized by producing and accumulating ω-halocarboxylic acid and collecting the same. The yeast used in the present invention belongs to the genus Candeida and has an alkyl (or alkenyl) halide ω-
It is capable of selectively oxidizing the terminal to produce ω-halocarboxylic acids, such as Candeida.
Guilli Monday KSM-B-24 (Candida)
guilliermondii・KSM-B-24).
This strain was isolated from soil by the present inventors and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiology Research Institute No. 7309, and has the following mycological properties. ing. Table 1 shows the results of a comparison between Candeida Guillimondei KSM-B-24 and the standard strain regarding the assimilation of various carbon sources. (a) Cell size: 2.2-2.5μ in diameter (b) Growth status in each medium (1) MY agar medium: produces circular, dull colonies. The color has white. (2) MY liquid medium: No skin is formed, it is moderately cloudy and a precipitate is formed. (3) Slide culture on cornmeal agar medium: Forms pseudohyphae. Conidia have a budding form. (4) Formation of ascospores Godorochova medium: No formation Malt extract agar medium: No formation (5) Formation of extruded spores MY agar flat medium: No formation (c) Physiological properties (1) Growth conditions: Temperature 14~ 47℃ (optimum 20-40℃) PH 2.2-9.6 (optimum 4.3-9.0) (2) Nitrate assimilation: Not assimilated (3) Lipid decomposition (fats and oils): Not decomposed (no lipase activity) (4) Urea Decomposition: Decomposes (5) Liquefaction of gelatin: No liquefaction (6) Osmotic pressure resistance (NaCl resistance): 12-13% (7) Production of carotenoids: No production (8) Production of significant organic acids: Organic acids (9) Requirements for vitamins: Not required (10) Assimilation of each carbon source:

【table】 (11) Degradation of albumin: Does not degrade. (12) Utilization of nitrogen sources Bactopeptone: Use Ammonium sulfate: use DL-Asparagine: Use Urea: Use (13) Productivity of starch-like substances: None (14) Collection site: Separated from soil

[Table] (Note) ±: Weak As a result of searching for bacteria with the above mycological properties based on Lodder's The Yeasts, 2nd edition (1971), the above yeast was found to be Candeida.・Recognized as a new strain belonging to Guillimondei, Kyandida Guillimondei, KSM
-B-24 (Candida・guilliermondii・KSM-B
−24). The alkyl (or alkenyl) halide used as a raw material in the present invention is suitably an alkyl (or alkenyl) chloride or an alkyl (or alkenyl) bromide having 6 to 22 carbon atoms. Among these, those having 12 to 18 carbon atoms are particularly preferred. Examples of alkyl (or alkenyl) halides include n-hexyl chloride, n-heptyl chloride, n-octyl chloride, n-decyl chloride, n-undecyl chloride, n-dodecyl chloride (lauryl chloride), and n-tetradecyl chloride. (myristyl chloride),
n-pentadecyl chloride, n-hexadecyl chloride (cetyl chloride), n-octadecyl chloride, n-eicosyl chloride, n
-docosyl chloride, undecenyl chloride, octadecenyl chloride (oleyl chloride), mixtures thereof, and their corresponding bromides. The composition of the medium used in the present invention is such that it contains appropriate carbon sources, nitrogen sources, or organic nutrients in order to allow the strain used to grow well and to smoothly produce ω-halocarboxylic acids from alkyl (or alkenyl) halides. It consists of sources, inorganic salts, etc. Carbon sources include carbohydrates (e.g., glucose, fructose, sucrose, sorbitol, etc.), organic acids (e.g., citric acid, succinic acid, etc.), and hydrocarbons (e.g., n-dodecane, n-hexadecane, etc.). You can use any of the following. Examples of the nitrogen source or organic nutrient source include nitrates such as sodium nitrate, potassium nitrate, and ammonium nitrate, yeast extract, meat extract, and peptone. Moreover, various phosphates, magnesium sulfate, etc. can be used as inorganic salts.
In addition, trace amounts of heavy metal salts are used, but their addition is not necessarily required in media containing natural products. Furthermore, when using a mutant strain that requires nutritional requirements, a substance that satisfies the nutritional requirements must be added to the medium. The culture medium may be sterilized by heating or the like, inoculated with bacteria, and shaken or aerated for 3 to 5 days at 28 to 35°C. Good results can be obtained by adjusting the pH to around 6.5 to 8. When using a carbon source that is poorly soluble in water, it is also possible to add various surfactants such as polyoxyethylene sorbitan to the medium. Collection and purification of the target substance, ω-halocarboxylic acid, from these culture solutions can be carried out in accordance with the methods used to collect and purify general organic compounds. For example, the filtrate from which bacterial cells have been removed from the culture solution or the culture solution itself is acidified and extracted with an organic solvent such as ethyl ether, ethyl acetate, or a chloroform-methanol mixture. The ω-halocarboxylic acid can be isolated from this extract using a method such as column chromatography or recrystallization. EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto. Example 1 Cetyl chloride 50g, diammonium phosphate
10 g, monopotassium phosphate 2 g, magnesium sulfate (7 hydrate) 0.2 g, ferrous sulfate (7 hydrate) 0.02 g,
Zinc sulfate (7 hydrate) 0.016g, manganese sulfate (4
- 0.016 g of hexahydrate) and 2 g of yeast extract were dissolved in tap water to make the pH 1 and adjust the pH to 6.5. Pour 5 ml of this liquid medium into a 50 ml shaking test tube and heat it to 200°C.
After steam sterilizing for 15 minutes in
guilliermondii KSM-B-24) was inoculated in one platinum loop and cultured with shaking at 30°C for 168 hours. After culturing, add 1 ml of 9N sulfuric acid to this culture solution.
The pH was made strongly acidic and extraction was performed with 20 ml of a chloroform-methanol (2:1) mixture. This extract was concentrated under reduced pressure, then methylated using a methanol- _BF3 catalyst, and the amount of ω-chloropalmitic acid in the product was determined by gas chromatography. The results are shown in Table 2. The gas mass (GC-MS) data of the product matched that of the standard product, and it was confirmed that it was ω-chloropalmitic acid.

[Table] Example 2 Cetyl chloride 50g, diammonium phosphate
10g, monopotassium phosphate 2g, magnesium sulfate (heptahydrate) 0.2g, polypeptone 1g, yeast extract
0.5g was dissolved in 1 part of tap water and the pH was adjusted to 6.5. Pour 50 ml of this liquid medium into a 500 ml shaking flask, steam sterilize it at 120°C for 15 minutes, and then
(Candida guilliermondii KSM-B-24) was inoculated with one loopful and cultured with shaking at 30°C for 168 hours. After culturing, add 10ml of 9N sulfuric acid to this culture solution.
The pH was made strongly acidic and extraction was performed with 200 ml of a chloroform-methanol (2:1) mixture. This extract was concentrated under reduced pressure, then methylated using a methanol- _BF3 catalyst, and the amount of ω-chloropalmitic acid in the product was determined by gas chromatography. the result,
It was found that 16 mg of ω-chloropalmitic acid was obtained per 1 culture solution. The gas mass (GC-MS) data of the product is consistent with that of the standard product.
It was confirmed that it was ω-chloropalmitic acid. Example 3 Various carbon sources shown in Table 3 were used instead of cetyl chloride as the reaction substrate, and Candeida Guillimondei KSM-B-24 was used as the bacterial strain.
(Candida guilliermondii KSM-B-24) was cultured under the same conditions as in Example 1. The results are shown in Table 3.

【table】

Claims (1)

[Claims] 1 Alkyl (or alkenyl) having 6 to 22 carbon atoms
A microorganism characterized by culturing ω-halocarboxylic acid-producing bacteria belonging to the genus Candeida in a medium to which a halide has been added, producing and accumulating ω-halocarboxylic acids having 6 to 22 carbon atoms in the medium, and collecting the ω-halocarboxylic acids. Method for producing ω-halocarboxylic acid.