JPH0361433B2 - - 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 microorganisms. 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 Kraton-based musk (Jiya synthetic fragrance), but it is this precursor, ω-hydroxypalmitic acid, that makes musk expensive. This is due to the difficulty in producing 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, when microorganisms assimilate normal paraffin to produce dicarboxylic acids, ω-
It has been reported that hydroxyl higher fatty acids can also be obtained (for example, Japanese Patent Publication No. 48-26238). Thus, ω-hydroxyl higher fatty acids are metabolic intermediates of normal paraffin to dicarboxylic acids by alkane-assimilating bacteria; Its mass production is said to be difficult. 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). In view of the current situation, the present inventors conducted a wide search for bacteria that have the ability to convert alkyl (or alkenyl) halides into the corresponding ω-halocarboxylic acids, and as a result, found microorganisms belonging to the genus Micrococcus that have such an ability. They discovered that there is, and completed the present invention. That is, the present invention involves culturing ω-halocarboxylic acid-producing bacteria belonging to the genus Micrococcus 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 microorganisms used in the present invention belong to the genus Micrococcus and are capable of selectively oxidizing the ω-terminus of alkyl (or alkenyl) halides to produce ω-halocarboxylic acids. KSM-B-1 (Micrococcus
sp. KSM-B-1) and Micrococcus sp. KSM-B-8 (Micrococcus sp. KSM-B
-8) and Micrococcus sp. KSM -
B-16 (Micrococcus sp. SM-B-16) is mentioned. These three strains were isolated from soil by the present inventors, and are
It has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Nos. 7003 and 7004, and has the mycological properties shown in Table 1 below.

【table】

[Table] ** +: Produces -: Does not produce Bacteria with the above mycological properties are listed in Bergey's Manual of
Determinative Bacteriology) 8th edition (1975)
As a result of a search based on the above, it was found that the three strains mentioned above belong to the genus Micrococcus. 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. For example, n-hexyl chloride, n-heptyl chloride, n-octyl chloride, n-decyl chloride, n-undecyl chloride, n-dodecyl chloride (lauryl chloride), 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. For culturing, the culture medium may be sterilized by heating or the like, inoculated with bacteria, and shaken or stirred with aeration at 28 to 35°C for 3 to 5 days. 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
- 0.016g of hexahydrate salt, 2g of yeast extract, 1 part of tap water
The pH was adjusted to 7.0. This liquid medium 5
ml into a 50 ml shaking test tube, steam sterilize it at 120℃ for 15 minutes, and then add Micrococcus sp.
KSM-B-1 (Microco-ccus sp.KSM-B-
A loopful of 1) was inoculated and cultured at 30% shaking for 96 hours. At the end of the culture, 1 ml of 9N sulfuric acid was added to the culture solution to make the pH strongly acidic, and the mixture was extracted with 20 ml of a chloroform-methanol (2:1) mixture. After concentrating this extract under reduced pressure, it was methylated using a methanol- _BF3 catalyst, and the product was analyzed using gas chromatography.
- Chlorpalmitic acid was determined. The results are shown in Table 2. The product was confirmed to be ω-chlorpalmitic acid by gas mass analysis (GC-MS). Figure 1 shows the mass pattern of methyl ester of ω-chlorpalmitic acid (esterified for measurement). Indicated.

[Table] Examples 2-3 Micrococcus sp. KSM as bacterial strain
Micrococcus sp. KSM-B instead of -B-1 (Microco-ccus sp. KSM-B-1)
-8 (Micrococcus sp. KSM-B-8) and Micrococcus sp. KSM-B-16 (Micrococcus sp. KSM-B-16)
-ccus sp.KSM-B-16) and cultured under the same conditions as in Example 1. The results are shown in Table 3.

【table】 [Brief explanation of drawings]

FIG. 1 is a schematic diagram of the mass pattern of the product and standard material of Example 1 of the present invention.

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 Micrococcus 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.