JPH0320228B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel microorganism belonging to the genus Candeida guillimondei. 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 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). Therefore, in view of the current situation, the present inventors converted alkyl (or arekenyl) halide into a corresponding ω-
As a result of a wide search in nature for bacteria that have the ability to convert 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 belongs to the genus Candeida, and utilizes an alkyl (or arekenyl) halide to produce ω
-Relates to the new Candeida Sp. KSM-B-24 (Feikoken No. 7309) which has the ability to produce halocarboxylic acids. Next, the mycological properties of this strain isolated and collected by the present inventors will be described in detail. (a) Cell size: 2.2 to 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 film is formed, moderately turbid and precipitation occurs. (3) Slide culture on cornmeal agar medium: Forms pseudohyphae. Conidia have a budding form. (4) Formation of ascospores Godorochova medium: Not formed Malt extract agar medium: Not formed (5) Formation of extruded spores MY agar flat medium: Not formed (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】

[Table] (11) Decomposition of albumin: Not decomposed (12) Use of nitrogen sources Bactopeptone: Used Ammonium sulfate: Used DL-Asparagine: Used Urea: Used (13) Productivity of starch-like substances: None ( 14) Collection site: Isolated from soil 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, Cyandida Guiramondi KSM
-B-24 (Candida・guilliermondii・KSM-B
−24). This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiological Research Institute No. 7309. The present bacterial strain was isolated from the source soil using a conventional method using a medium containing an alkyl (or alkenyl) halide. 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 (eg, citric acid, succinic acid, etc.), carbohydrate (eg, 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 or the like that is poorly soluble in water, it is also possible to add various surfactants such as polyethylene 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. The ω-halocarboxylic acid is produced by culturing this strain as described above using an alkyl (or alkenyl) halide as a reaction substrate. Particularly suitable substrates have 2 to 18 carbon atoms. 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. 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 thus obtained is directly transferred to a cetyl chloride-containing separation agar medium (2) having the composition shown below, and cultured at 30°C for 5 days.

【table】

[Table] The preparation method for the above separation agar medium (I) is _{as follows} :
Only the 7H ₂ O component was sterilized by filtration, and added to the medium component, which had been autoclaved beforehand and cooled to around 60°C. After thoroughly stirring and emulsifying in a Dynamitsukushiker, it was divided into sterilized petals. Please note this. 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 visually and microscopically that the multiple colonies that have arisen are not different from each other. In addition,
Isolation agar medium () is Isolation agar medium ()
Based on the composition, cetyl chloride and polyoxyethylene sorbitan monolaurate were removed, and a filter paper impregnated with cetyl chloride was placed on the bottom of the agar medium to supply cetyl chloride. 10 of the above colonies were inoculated onto slanted agar medium with the same composition as the isolation agar medium () and cultured for 3 days at 30°C, and the bacterial strains on the 10 slanted medium were visually and microscopically identical. It was confirmed that these 10 strains were the same, 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 (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 this liquid medium into a 5 ml shaking test tube and heat it at 120℃ for 15 minutes.
After steam sterilization for a minute, Candida gulliermondii KSM-B-24 (Candida gulliermondii
KSM-B-24) was inoculated with 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-BF ₃ catalyst, and the amount of ω-chloropalmitic acid in the product was determined by gas chromatography. The results are shown in Table 1. 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】

Claims (1)

[Scope of Claims] 1. A novel Candeida guillimondii KSM-B-24 strain deposited as FIKEN Bibliography No. 7309 having the following properties. (a) Cell size: 2.2 to 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 film is formed, moderately turbid and precipitation occurs. (3) Slide culture on cornmeal agar medium: Forms pseudohyphae. Conidia have a budding form. (4) Formation of ascospores Godorochova medium: Not formed Malt extract agar medium: Not formed (5) Formation of extruded spores MY agar flat medium: Not formed (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] [Table] (11) Decomposition of albumin: Not decomposed (12) Use of nitrogen sources Bactopeptone: Used Ammonium sulfate: Used DL-Asparagine: Used Urea: Used (13) Productivity of starch-like substances: None