JPS6047676A - Novel microorganism - Google Patents

Abstract

PURPOSE:To provide a novel microbial strain belonging to Nocardia genus and capable of converting a n-paraffin and a fatty acid (derivative) to the corresponding dicarboxylic acid in high efficiency. CONSTITUTION:Nocardia sp. KSM-B-20 (FERM-P No.7005) separated from a source soil using a medium containing n-paraffin by a conventional process. The microbial strain is cultured in a medium containing a n-paraffin or a fatty acid (derivative) as a reaction substrate aerobically at about 28-35 deg.C and about 6.5- 8pH for about 3-5 days. Preferably after separating the microbial cells, the dicarboxylic acid is extracted from the culture liquid with an organic solvent (e.g. mixture of chloroform and methanol) under acidic condition.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel microorganism belonging to the genus Nocardia.

Dicarboxylic acids are useful substances as raw materials for producing 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 genus Candida (Special Publication No. 50-196
30, etc.), Pichia genus (Special Publication No. 1973-
24392, etc.), and bacteria such as Corynebacterium (C).
Only the genus Terium (Japanese Patent Publication No. 17075, 1983) was found.

Therefore, in view of the current situation, the present inventors conducted a wide search in nature for bacteria that have the ability to convert normal paraffins, fatty acids, or fatty acid derivatives into the corresponding dicarboxylic acids, and found that microorganisms belonging to the genus Nocardia. The present invention has been completed based on the discovery that some species have such an ability, and thus the present invention relates to plants belonging to the genus Nocardia that have the ability to produce dicarboxylic acids by assimilating normal paraffins, fatty acids or fatty acid derivatives. New Nocardia SP KSM-B-20 (Feikoken Bacterial F57005)
It is related to.

Next, the mycological properties of this strain isolated and collected by the inventors will be described in detail.

(a) Morphology Young cells grow like hyphae and branching is observed. As the culture progresses, the hyphae divide into coccoid or rod shapes.

The diameter of the hyphae is 1.5k (b) Growth status on each medium l) Sucrose, nitrate agar medium: Growth is moderate, and the color of the colony is white to cream. It has a dull luster.

2) Glucose φ asparagine agar medium: Growth is moderate, and colonies are white to cream colored.

It has a dull luster.

3) Glycerin-asparaquine agar medium: Growth is moderate, and colonies are white to cream colored.

However, it is not shiny.

4) Growth on starch agar medium is poor, and colonies are white in color. The colony is dull and dry.

5) Tyrosine agar medium: Growth is moderate, and colonies are flesh-colored to pale orange in color. It has a dull luster.

6) Nutrient agar medium: Growth is abundant and colonies are flesh-colored to light pink in color. Shiny.

? ) Yeast/malt agar: Growth is most abundant, colonies pink to pale orange in color.

Shiny and shiny.

8) Oatmeal agar medium; growth is moderate and colonies are white in color. colony
is shiny.

(c) Physiological properties 1) Growth range: Temperature 16-38°G (optimal 27-34°C) pH 5,
0-8.2 (optimal 6.0-8,3) 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: VA property (d) Assimilation of carbon source L-7 rabinose: - D-xylose - D-glucose: + D-fructose: + Sucrose: + Inositol: + L - Rhamnose: - Raffinose: - D-Mannireduction: + (e) Production of gas from sugar Fructose: + (No gas is produced) Sorbitol: + (No gas is produced) (f) Cell wall composition Diaminopimelic acid: meso-DAP sugar:
Arabinose, galactose (g) 5-fluorouracil resistance (40mg718)
: Resistant (h) Isolation source: Regarding bacteria with mycological properties superior to soil/<-Bergey's Manuat of D
ate-rminative Bacteriology
y) As a result of the search based on the 8th edition (1975), this strain was recognized as a new strain belonging to the genus Nocardia, and was classified as Nocardia sp. *KSM-B-
20 (Nocardiasp, KSM-8-20). This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiological Research Institute No. 7005.

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.), organic acids (e.g., citric acid, succinic acid, fatty acids and their esters, etc.), hydrocarbons (e.g., n-dodecane, n-hexadecane, etc.), etc. Either 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 culture, after sterilizing the medium by heating etc., inoculate the bacteria,
What is necessary is just to shake or aerate and stir at 35 degreeC for 3 to 5 days. Good results can be obtained by adjusting the pH to about 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 sorbicun to the medium.

The culture obtained as above can be used as it is as an enzyme source, but when the bacterial cells are separated from the culture solution, a conventional solid-liquid separation means is 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. The substrate preferably has 6 to 22 carbon atoms, and the fatty acid derivative is preferably a lower alkyl ester of fatty acid.

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 made acidic,
Extract 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 Perform intermittent shaking culture.

*Liquid medium (I) composition -11111111----------------------pH7 *1. 5hiio and R, υchiio.

Agr. Biol. Chem., 35(13).
2033-2042 (1977) The culture solution that showed growth in the above culture was appropriately diluted with sterilized water, then transferred to a broth agar medium (manufactured by Eiken Chemical; ordinary agar medium), and cultured at 30°C for 2 days. It was confirmed macroscopically and microscopically that the resulting multiple colonies were not different from each other. After culturing at 30°C for 3 days, the strains on the 10 slanted media were macroscopically and microscopically the same bacterial strain. It was also confirmed that the properties and physiological properties of these IO strains on each medium were the same.

Side agar medium (II) Composition n-hexadecane 20 g (NH4)2S0420 g KH, PO42 g Yeast extract 2 g MgSO4a 7) 110 500 m'gFeSO4.
7J0 10mg Ion-exchanged water 1 sentence The properties and physiological properties of the above bacterial strain on the culture medium are as described above. As a result of the above test, one platinum loopful from each of the 10 bacterial strains was placed in a cryopreservation vial containing a sterilized 10% glycerin aqueous solution (2 mfL) and stored frozen at -80°C. After 3 months of frozen storage, a loopful of the resulting suspension was quickly thawed and resuscitated on a broth agar medium.
As a result of examining the properties and physiological properties on each medium under the same conditions as above, no changes were observed from before freezing.

In addition, when the properties and physiological properties of the strains on each culture medium were similarly examined after the above-mentioned freezing and thawing was 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 valmitate 50g, ammonium phosphate 1
0 g, potassium phosphate 2 g, magnesium sulfate (7 hydrate) 0.2 g, ferrous sulfate (7 hydrate) 0.02 g, zinc sulfate (7 hydrate) o, oteg, carb sulfate 47 *
x Subi* KSM-B-20 (Nocardia
*sp, KSM-8-20) was inoculated with a platinum loop, 30
Shaking culture was carried out at ℃ for 8θ hours.

After culturing, 1 ml of 9N sulfuric acid was added to this culture solution to make the pH strongly acidic, and chloroform-methanol (2:1
) Extracted with 20ml of 1m drop. This extract was concentrated under reduced pressure, then methylated using a methanol-BF3 catalyst, and the product was quantified by gas chromatography. As a result, it was found that 29 mg of α,ω-tetradecanedicarboxylic acid was obtained per culture solution.

The product is determined by gas-mass (GC-MS) as α, ω-
It was confirmed to be tetradecanedicarboxylic acid. FIG. 1 shows a mass pattern of dimethyl ester of α,ω-tetradecanedicarboxylic acid (esterified for measurement).

Prepared as in Reference Example 2. 50+nl of this liquid medium was shaken to a volume of 500ml, and Nocardia*sp, KSM-B-20) was inoculated into a loopful, followed by shaking culture at 30°C for 89 hours.

After culturing, add 10 ml of 9N sulfuric acid to this culture solution.
The mixture was extracted with 100 ml of ethyl ether using H as a strong acid. After drying this extract with anhydrous thorium sulfate,
The mixture was concentrated under reduced pressure, methylated with methanol-BF3, and the product was quantified by gas chromatography. As a result, it was found that 12 mg of α,ω-tetradecanedicarboxylic acid was obtained per culture solution.

The product is determined by gas-mass (GC-MS) as α, ω-
It was confirmed that it was tetradecanedicarboxylic acid.

Display of items 1981 Patent Application No. 53E149 2. Name of the invention new microorganisms 3. Person who makes corrections Specification and drawings 6ηli positive content As per attached sheet The following is inserted after line 15 on page 18 of the specification.

[4. Brief explanation of the drawings

Claims (1)

[Scope of Claims] 1. A new Mecardia SP-KSM-B deposited as FIKEN Bibliography No. 7005 having the following properties:
-20 stocks. (a) Morphology Young cells grow like hyphae and branching is observed. As the culture progresses, the hyphae divide into coccoid or rod shapes. The diameter of the hyphae is 1.5 sho (b) Growth status on each medium 1) Sucrose, nitrate agar medium = Growth is moderate, and the colony color is white to cream colored. It has a dull luster. 2) Glucose asparagine agar medium; freshness is medium, and colonies are white to cream colored. It has a dull luster. 3) Glycerin-asparagine agar medium: Growth is moderate, and colonies are white to cream colored. However, it is not shiny. 4) Scooch agar medium 22-year-old Yr is poor and the colony color is white. The colony lacked luster and had a 1:t appearance. 5) Tyrosine agar medium: Growth is moderate, and colonies are flesh-colored to pale orange in color. It has a dull shine. 6) Nutrient agar medium: Growth is yyM, and colony color is flesh-colored to swirly pink. Shiny. 7 Yeast φ malt agar medium: Growth is most abundant, and colonies are pink to light orange in color. Shiny and shiny. 8) Oatmeal agar medium: Growth is moderate and colonies are white in color. colony
is shiny. (C) Physiological properties l) Growth range: Temperature 16-38°C (optimum 27-346°C) pH 5.0
~8.2 (optimal 6.0~8.3) 2) Liquefaction of gelatin (
Glucose ψ peptone gelatin medium): Negative 3) Hydrolysis of starch (Starch agar medium) Negative 4) Coagulation and peptonization of skim milk: Both negative Formation of melanin-like pigment: Negative (d) *Assimilation of the source L - arabinose: - D-xylose: - D-glucose: + D-fructose: + sucrose: + inositol: + L-rhamnose two - raffinose: - D-mannitol: ten (e) a! Production of acid and gas from i Fructose: + (No gas produced) Sorbitol; + (No gas produced) (f) Cell wall composition Diaminopimelic acid: meso -D A P sugar:
Arabinose, galactose (g) 5-fluorouracil resistance (40mg/18)
: resistant