JPH0378106B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel microorganism belonging to the genus Mycobacterium. Dicarboxylic acids are useful substances as raw materials for the production of synthetic resins, high-grade lubricants, plasticizers, fragrances, etc. However, dicarboxylic acids produced by synthetic methods have a limited number of carbon atoms. It has been difficult to produce dicarboxylic acids. Therefore, in recent years, a method for producing dicarboxylic acids by fermentation using microorganisms has attracted attention. Conventionally, as a method for producing dicarboxylic acids using microorganisms, the method of producing dicarboxylic acids using microorganisms has been
19630, etc.), Pichia genus (Special Publication No. 1963-
24392, etc.), and bacteria of the genus Corynebacterium (Special Publication No. 17075, 1975, etc.)
Only one thing had been discovered. In particular, there were no bacteria that could be cultured at around 45°C. Therefore, in view of the current situation, the present inventors conducted a wide search in the natural world for bacteria that have the ability to convert normal paraffins, fatty acids, or fatty acid derivatives into the corresponding dicarboxylic acids, and found that they belong to the genus Mycobacterium. The present invention was completed based on the discovery that some microorganisms have such an ability. That is, the present invention relates to a novel Mycobacterium sp. KSM- which belongs to the genus Mycobacterium and has the ability to assimilate normal paraffin, fatty acids or fatty acid derivatives to produce dicarboxylic acids.
This is related to B-33 (Feikoken Bibori No. 7310). Next, the mycological properties of this strain isolated and collected by the present inventors will be described in detail. (a) Morphology Slightly curved short rod, non-motile and does not form spores. Positive Gram staining. Anti-acidity is not very strong, but it is recognized. It does not grow in mycelial form. (b) Growth status on each medium (1) Sucrose/nitrate agar medium: Growth is abundant, producing pale orange convex colonies. It has a dull luster. (2) Glucose-asparagine agar medium: Growth is abundant, producing orange wrinkled colonies. There is no shine. (3) Glycerin-asparagine agar medium: Growth is moderate, producing pink convex colonies. The edges of the colony are not sharp. (4) Starch agar medium: Growth is moderate, producing orange convex colonies. It has a dull luster. (5) Tyrosine agar medium: Growth is moderate and produces pale orange colonies. The edges of the colony are not sharp. There is no shine. (6) Nutrient agar medium: Growth is moderate, producing pale orange convex colonies. It has a dull luster. (7) Yeast/malt agar medium: Growth is most abundant, with large convex colonies of dark orange color and gloss. (8) Oatmeal agar medium: Growth is abundant, producing orange wrinkled colonies. It has a dull luster. (c) Physiological properties (1) Growth range: Temperature 23-51℃ (optimum 35-50℃) PH 2.8-9.3 (optimum 5.3-8.5) (2) Liquefaction of gelatin (glucose-peptone gelatin medium): Negative (3) Hydrolysis of starch (starch agar medium): negative (4) Coagulation and peptonization of skim milk: both negative (5) Production of melanin-like pigment: negative (d) Assimilable carbon source L-arabinose: - D-xylose: - D-glucose: + D-fructose: + Sucrose: ± Inositol: - L-rhamnose: - Raffinose: - D-mannitol: ± (e) Production of acid and gas from sugar Fructose: + (gas Sorbitol: + (No gas is produced) (f) Cell wall composition Diaminopimelic acid: Meso-type sugars: amabinose, galactose (g) Mycolic acid (TLC analysis): Yes (h) Antiacidity: Yes (i) Separation Source: Soil Bargey's Manual of fungi with the following mycological properties:
Determinative Bacteriology) 8th edition (1975)
As a result of the search based on
33 (Mycobacterium sp.KSM-B-33). This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiological Research Institute No. 7310. 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 determined by adding an appropriate carbon source, nitrogen source, or organic Consists of nutrients, inorganic salts, etc. Carbon sources include carbohydrates (e.g. glucose,
fructose, sucrose, mannitol, etc.)
Any organic acid (e.g., citric acid, succinic acid, fatty acid and its ester, etc.), hydrocarbon (e.g., n-dodecane, n-hexadecane, etc.) can be used as long as it can be assimilated. Nutrient sources include, for example, nitrates such as sodium nitrate, potassium nitrate, and ammonium nitrate, yeast extract, meat extract, and peptone. In addition, as inorganic salts, various phosphates, magnesium sulfate, etc. can be used. Heavy metal salts are used, but they do not necessarily need to be added in media containing natural products.Also, when using mutant strains that require nutritional requirements, substances that meet the nutritional requirements must be added to the media. For culture, after sterilizing the medium by heating etc., inoculate the bacteria and shake or aerate at 40-50℃ for 3-5 days.Good results can be obtained by adjusting the pH to about 6.5-8. When using a poorly soluble carbon source, 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. However, when separating the bacterial cells from the culture medium, ordinary solid-liquid separation means are 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 normal paraffin, fatty acids, or fatty acid derivatives as reaction substrates, dicarboxylic acids are produced.
18 are particularly suitable, and lower alkyl esters of fatty acids are preferred as fatty acid derivatives. The dicarboxylic acid, which is the target substance, can be collected and purified from these culture solutions 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 dicarboxylic 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 Approximately 0.5 g of the collected soil sample was suspended in 10 ml of sterilized water and thoroughly stirred, then 0.2 ml of this soil suspension was added to 10 ml of a liquid medium () with the following composition (in a 50 ml test tube).
and culture with shaking at 30°C for 4 days.

[Table] The culture solution that showed growth in the above culture was appropriately diluted with sterilized water, transferred to a broth agar medium (manufactured by Eiken Chemical; ordinary agar medium), and cultured at 30°C for 2 days. until it can be confirmed macroscopically and microscopically that the colonies are not different from each other.
Repeat transfer to broth agar medium. Inoculate 10 of the above colonies onto slanted agar medium () with the following composition and culture at 30°C for 3 days, and confirm that the bacterial strains on the 10 slanted medium are macroscopically and microscopically the same strain. It was also confirmed that the properties and physiological properties of these 10 strains on each medium were the same.

[Table] The properties and physiological properties of the above bacterial strain on the 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 is suspended in a cryopreservation vial containing a sterilized 10% glycerin aqueous solution (2 ml) and stored frozen at -80°C. After three months of frozen storage, a loopful of the suspension obtained by rapid thawing was resuscitated on a broth agar medium, and the properties and physiological properties were examined on each medium under the same conditions as in the previous period.
No change was observed from before freezing. Furthermore, as a result of similarly examining the properties and physiological properties on each culture medium of the strains subjected to the above-mentioned freezing and thawing process repeated five times every month, no changes were observed. Next, an example in which dicarboxylic acid was produced using this strain will be listed as a reference example. Reference example 1 Methyl palmitate 50g, diammonium phosphate 10g, monopotassium phosphate 2g, magnesium sulfate (heptahydrate) 0.2g, ferrous sulfate (heptahydrate) 0.02
g, 0.016 g of zinc sulfate (heptahydrate), 0.016 g of manganese sulfate (tetra-hexahydrate), and 2 g of yeast extract were dissolved in tap water to adjust the pH to 1 and adjust the pH to 7.0. Pour 5 ml of this liquid medium into a 50 ml shaking tester, and
After steam sterilization for 15 min at °C, Mycobacterium spp.
KSM-B-33 (Mycobacterium sp.
One loopful of KSM-B-33) was inoculated and cultured with shaking at 45°C for 120 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 and then methylated using a methanol BF ₃ catalyst, and the product was quantified by gas chromatography. As a result, 13 mg of α per culture solution,
It was found that ω-tetradecanedicarboxylic acid was obtained. The gas-mass (GC-MS) data of the product matched that of the standard product, and it was confirmed that it was α,ω-tetradecanedicarboxylic acid. Reference example 2 Dissolve 50 g of methyl palmitate, 10 g of diammonium phosphate, 2 g of monopotassium phosphate, 0.2 g of magnesium sulfate (heptahydrate), 1 g of polypeptone, and 2 g of yeast extract in tap water to bring the pH to 1 and adjust the pH to 7.0.
It was prepared as follows. 100 ml of this liquid medium was placed in a 500 ml shaking flask, and after steam sterilization at 120°C for 15 minutes,
Mycobacterium sp. KSM-B-33
(Mycobacterium sp. KSM-B-33) was inoculated with one platinum loop and cultured with shaking at 45°C for 132 hours. After culturing, add 10ml of 9N sulfuric acid to this culture solution.
The pH was made strongly acidic and the mixture was extracted with 100 ml of ethyl ether. This extract was dried over anhydrous sodium sulfate, concentrated under reduced pressure, methylated with a methanol- _BF3 catalyst, and the product was quantified by gas chromatography. As a result, per 1 culture solution
It was found that 13 mg of α,ω-tetradecanedicarboxylic acid was obtained. The gas-mass (GO-MS) data of the product is consistent with that of the standard product, and α,
It was confirmed that it was ω-tetradecanedicarboxylic acid.

Claims (1)

[Scope of Claims] 1. A novel Mycobacterium sp. KSM-B-33 strain deposited as Fiber Science and Technology Research Institute No. 7310 having the following properties. (a) Morphology Slightly curved short rod, non-motile and does not form spores. Positive Gram staining. Anti-acidity is not very strong, but it is recognized. It does not grow in mycelial form. (b) Growth status on each medium (1) Sucrose/nitrate agar medium: Growth is abundant, producing pale orange convex colonies. It has a dull luster. (2) Glucose-asparagine agar medium: Growth is abundant, producing orange wrinkled colonies. There is no shine. (3) Glycerin-asparagine agar medium: Growth is moderate, producing pink convex colonies. The edges of the colony are not sharp. (4) Starch agar medium: Growth is moderate, producing orange convex colonies. It has a dull luster. (5) Tyrosine agar medium: Growth is moderate and produces pale orange colonies. The edges of the colony are not sharp. There is no shine. (6) Nutrient agar medium: Growth is moderate, producing pale orange convex colonies. It has a dull luster. (7) Yeast/malt agar medium: Growth is most abundant, with large convex colonies of dark orange color and gloss. (8) Oatmeal agar medium: Growth is abundant, producing orange wrinkled colonies. It has a dull luster. (c) Physiological properties (1) Growth range: Temperature 23-51℃ (optimum 35-50℃) PH 2.8-9.3 (optimum 5.3-8.5) (2) Liquefaction of gelatin (glucose-peptone gelatin medium): Negative (3) Hydrolysis of starch (starch agar medium): negative (4) Coagulation and peptonization of skim milk: both negative (5) Production of melanin-like pigment: negative (d) Assimilable carbon source L-arabinose: - D-xylose: - D-glucose: + D-fructose: + Sucrose: ± Inositol: - L-rhamnose: - Raffinose: - D-mannitol: ± (e) Production of acid and gas from sugar Fructose: + (gas Sorbitol: + (No gas is produced) (f) Cell wall composition Diaminopimelic acid: Meso-type sugars: amabinose, galactose (g) Mycolic acid (TLC analysis): Yes (h) Anti-acidity: Yes.