JPH0378100B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel microorganism belonging to the genus Micrococcus. 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-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 Norman 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, bacteria belonging to the genus Arthrobacter, Corynebacterium, and Norcaldeia and having the ability to produce ω-halocarboxylic acid from alkyl halides were cultured.
A method for producing halocarboxylic acids has been reported (Japanese Patent Application Laid-Open No. 57-50893). Therefore, in view of the current situation, the present inventors converted an alkyl (or alkenyl) halide into a corresponding ω-
As a result of a wide search in nature for bacteria that have the ability to convert into halocarboxylic acids, it was discovered that some microorganisms belonging to the genus Micrococcus have such an ability, and the present invention was completed. That is, the present invention belongs to the genus Micrococcus,
The present invention relates to a novel Micrococcus sp. KSM-B-1 (Feikoken Bibori No. 7002) that has the ability to assimilate alkyl (or alkenyl) halides to produce ω-halocarboxylic acids. Next, the mycological properties of this strain isolated and collected by the present inventors will be described in detail. (a) Morphology (1) Cell shape and size: Coccus, diameter 0.5
~1μ (2) Population: Irregular linkage and reversion are not observed (3) Motility: None (4) Spores: Not formed (5) Gram staining: Positive (6) Acid-fastness: Hardly observed ( b) Growth status in each culture medium (1) Broth agar plate culture: Produces perfectly circular colonies with smooth surfaces. Light green, glossy, convex circular shape (2) Juicy agar slant culture: Shows medium growth, producing colonies with smooth surface. It has a light green luster. (3) Meat juice liquid culture: No film is formed on the surface, it is moderately turbid, and a slight precipitate is formed. (4) Meat juice gelatin puncture culture: Bacteria grows after 3 days of culture, and only the upper part liquefies (5) Litmus milk: Slightly alkaline and does not peptonize. There is no litmus reduction. (c) Physiological properties (1) Nitrate reduction: No reduction (2) Denitrification reaction: Negative (3) MR test: Negative (4) VP test: Negative (5) Indole formation: Negative (6) Hydrogen sulfide (TSI agar): Weakly positive (7) Starch hydrolysis: Negative (8) Utilization of citric acid Koser's medium: Not used Christensen's medium: Used (9) Utilization of inorganic nitrogen sources Nitrate: Positive Ammonium salt : Negative (10) Pigment production: Water-soluble green pigment (11) Urease Christensen medium: Negative SSR medium: Negative (12) Oxidase: Negative (13) Catalase: Positive (14) Growth range: 14-42℃ (Optimal 28-35℃) PH5.0-9.5 (Optimal 6.5-8.0) (15) Attitude toward oxygen: Aerobic (16) OF test: Negative (17) Generation of acids and gases from sugars: L-arabinose: - D-xylose: - D-glucose: - D-mannose: - D-fructose: - D-galactose: - Maltose: - Sucrose: - Lactose: - Trehalose: - D-sorbitol: - D-mannitol: - Wild boar: - Glycerin: - Starch: - (18) 5% sodium chloride tolerance: Grows (19) Collection location: Isolated from the soil of Yuzawa Onsen, Niigata Prefecture Bergey's manual for bacteria with the above mycological properties. s Manual of
Determinative Bacteriology) 8th edition (1975)
As a result of the search based on
(Micrococcus sp.KSM-B-1).
This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiological Research Institute No. 7002. The bacterial strain was isolated from the source soil using a normal paraffin-containing medium using a conventional method. The composition of the medium used for culturing this strain is such that it contains an appropriate carbon source, nitrogen source, or Consists of organic nutrients, inorganic salts, etc. Carbon sources include carbohydrates (e.g., glucose, fructose, sucrose, sorbitol, etc.);
Any organic acid (for example, citric acid, succinic acid, etc.), hydrocarbon (for example, n-dodecane, n-hexadecane, etc.), alkyl (or alkenyl) halide, etc. that can be assimilated can be used. 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. The culture obtained as described above can be used as it is as an enzyme source, but if the bacterial cells are to be separated from the culture solution, ordinary solid-liquid separation means can be used. The live bacterial cells isolated in this way and their processed products (freeze-dried bacterial cells, etc.) can also be used as enzyme sources. When this strain is cultured as described above using an alkyl (or alkenyl) halide as a reaction substrate, ω-halocarboxylic acid is produced. Collection and purification of the target substance, ω-halocarboxylic acid, from these culture solutions can be carried out in accordance with the methods for collection and purification of 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. Hereinafter, the present invention will be explained in more detail with reference to Examples. Example 1 One small spatula (approximately 0.5 g) of collected soil and fallen leaves attached to the soil was suspended in 10 ml of sterilized water.
Stir thoroughly and leave to stand. 0.1 ml of the 100-fold dilution of the soil suspension supernatant obtained in this way was mixed with n-
Transfer directly to a hexadecane-containing agar medium () and culture at 30°C for 5 days.

[Table] The preparation method for the above isolation agar medium () is as follows:
Only the FeSO ₄ 7H ₂ O component was sterilized by filtration, and this was added to the medium components, which had been previously autoclaved and cooled to around 60°C. After thorough stirring and emulsification in a Dynamikshaker, it was placed in a sterilized shear dish. It is to be dispensed. A loopful of colonies generated by the above culture is diluted 100 times with sterilized water, 0.1 ml of this diluted solution is transferred to the following separation agar medium (), and cultured at 30°C for 3 days. Confirm macroscopically and microscopically that the multiple colonies produced are not different from each other. The separation agar medium () is prepared by placing a filter paper impregnated with n-hexadecane on the bottom of an agar medium with n-hexadecane and polyoxyethylene sorbitan monolaurate removed from the composition of the separation agar medium (). - fed with hexadecane. 10 of the above colonies were inoculated onto slanted agar medium with the same composition as the isolation agar medium (), and cultured at 30℃ for 3 days. It was confirmed that they were the same strain, and that the properties and physiological properties on each medium of these 10 strains were the same. The properties and physiological properties of the above strains on each medium are as described above. As a result of the above test, it was found that each of the 10 cultured bacteria was a single strain isolated from nature. Next, a loopful of the pure cultured bacterial strain on the slant medium was suspended in a cryopreservation vial containing a sterilized 10% glycerin aqueous solution (2 ml), and -
Store frozen at 80℃. After 3 months of frozen storage, a loopful of the suspension obtained by rapid thawing was resuscitated on an ordinary agar medium, and the properties and physiological properties on each medium were investigated under the same conditions as above. There was no discernible change from before. Furthermore, the properties and physiological properties of the strains on each culture medium were similarly investigated after the above-mentioned freezing and thawing was repeated five times every month, and no changes were observed. Next, an example in which ω-halocarboxylic acid was produced using this strain will be listed as a reference example. Reference 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 (heptahydrate), 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 (Micrococcus sp.KSM-B-1)
was inoculated with one platinum loop and cultured with shaking at 30°C for 96 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-BF ₃ catalyst, and the product ω-chloropalmitic acid was determined by gas chromatography. The results are shown in Table 1. The product was confirmed to be ω-chlorpalmitic acid by gas-mass (GC-MS). FIG. 1 shows a mass pattern of methyl ester of ω-chlorpalmitic acid (esterified for measurement).

【table】 [Brief explanation of drawings]

FIG. 1 is a schematic diagram of mass patterns of the product of Reference Example 1 of the present invention and a standard substance.

Claims (1)

[Scope of Claims] 1. A novel Micrococcoccus sp.
KSM-B-1 strain. (a) Morphology (1) Cell shape and size: Coccus, diameter 0.5
~1μ (2) Population: Irregular linkage and reversion are not observed (3) Motility: None (4) Spores: Not formed (5) Gram staining: Positive (6) Acid-fastness: Hardly observed ( b) Growth status in each culture medium (1) Broth agar plate culture: Produces perfectly circular colonies with smooth surfaces. Light green, glossy, convex circular shape (2) Juicy agar slant culture: Shows medium growth, producing colonies with smooth surface. It has a light green luster. (3) Meat juice liquid culture: No film is formed on the surface, it is moderately turbid, and a slight precipitate is formed. (4) Meat juice gelatin puncture culture: Bacteria grows after 3 days of culture, and only the upper part liquefies (5) Litmus milk: Slightly alkaline and does not peptonize. There is no litmus reduction. (c) Physiological properties (1) Nitrate reduction: No reduction (2) Denitrification reaction: Negative (3) MR test: Negative (4) VP test: Negative (5) Indole formation: Negative (6) Hydrogen sulfide (TSI agar): Weakly positive (7) Starch hydrolysis: Negative (8) Use of citric acid Koser's medium: Not used Christensen's medium: Used (9) Use of inorganic nitrogen sources Nitrate: Positive Ammonium salt : Negative (10) Pigment production: Water-soluble green pigment (11) Urease Christensen medium: Negative SSR medium: Negative (12) Oxidase: Negative (13) Catalase: Positive (14) Growth range: 14-42℃ (Optimal 28-35℃) PH5.0-9.5 (Optimal 6.5-8.0) (15) Attitude toward oxygen: Aerobic (16) OF test: Negative (17) Generation of acids and gases from sugars: L-arabinose: - D-xylose: - D-glucose: - D-mannose: - D-fructose: - D-galactose: - Maltose: - Sucrose: - Lactose: - Trehalose: - D-sorbitol: - D-mannitol: - Inosite: - Glycerin: - Starch: - (18) 5% sodium chloride tolerance: Grows.