JPS6047677A - 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-21 (FERM-P No.7006) 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 Tucartia.

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. Producing dicarboxylic acids was a national problem.

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
Only the genus erium (Japanese Patent Publication No. 17075, 1983, etc.) 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. They discovered that some of them have such ability, and completed the present invention.

That is, the present invention relates to a novel Nocardia sp. KSM-B-21 (Feikoken Bacteria Serial No. 7006) which belongs to the genus Nocardia and has the ability to assimilate normal paraffin, fatty acids or fatty acid derivatives to produce dicarboxylic acids. (No.).

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 1.5 l. (b) Growth status on each medium 1) Sucrose, nitrate agar medium: Growth is moderate, and the color of the colonies is white to cream. Niffy and shiny.

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

It has a dull luster.

3) Glycerol-based asparagine 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) Growth on tyrosine agar medium is moderate, and the colony color is flesh-colored to pale orange. It has a dull luster.

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

7] Yeast/malt agar medium: Growth is most abundant, with colonies of pale pink to pale orange.

Shiny and shiny.

8) Oatmeal agar medium: Medium growth, white colonies,
is shiny.

(C) Physiological properties 1) Growth range: temperature IEI ~ 386C (optimal 27 ~ 346C) pH 5,
0-9.2 (optimal 6.0-8.3) 2) Liquefaction of gelatin (glucose peptone gelatin medium): Negative 3) Hydrolysis of starch (starch agar medium negative 4) Coagulation of skim milk, peptone 5) Production of melanin-like pigment: Negative (d) Assimilation of carbon source L-arabinose di- D-xylose: - D-glucose: + D-fructose: + Sucrose: + Inositol: + L- Rhamnose di-raffinose: - D-mannitol: + (e) Production of acids and gases from s Fructose di- (no gas produced) Sorbitol di- (no gas produced) (f) Cell wall composition Diaminopimelic acid: meso -DAP sugar:
Arabinose, galactose (g) 5-flu to uracil resistance (40mg/18)
: Resistant (h) Isolation source: Bergey's Manual of Bacteria with better mycological properties than soil
Dete-rminative Bacteriol
As a result of a search based on the 8th edition (1975) of 8th edition (1975), this strain was recognized as a new strain belonging to the genus Nocardia, and was classified as Nocardia axsub* KSM-
8-21 (Nocardiasp, KSM-B-20
) was named. In addition, this strain
It has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as the No.

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 amount of carbon, a nitrogen source, or an organic Consists of nutrients, inorganic salts, etc. Carbon sources include carbohydrates (e.g.
Assimilated substances such as glucose, fructose, sucrose, mannitol, etc.), organic acids (e.g., citric acid, succinic acid, fatty acids and their esters, etc.), and hydrocarbons (e.g., n-dodecane, n-hexadecane, etc.) If so, you can use either one. Examples of nitrogen sources or organic nutritional sources include nitrates such as sodium nitrate, potassium nitrate, and ammonium nitrate, yeast extract, meat extract,
Examples include peptone.

Moreover, various phosphates, magnesium sulfate, etc. can be used as the reki salt. In addition, trace amounts of heavy metal salts are used, but media containing natural products do not necessarily require additions. 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,
It may be shaken or aerated at 35°C for 3 to 5 days. pH
Good results can be obtained by adjusting the value 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 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 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 target substance, dicarboxylic acid, 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 Approximately 0.5 g of the collected soil sample was suspended in 10 m of sterilized water and thoroughly stirred. 0.2 m of this soil suspension was poured into a 10 m volume (50 m 9. volume test tube) of liquid medium (I) with the following composition. hand)
and cultured with shaking at 30°C for 4 days.

This liquid medium (1) Composition n-hexadecane 100 g (NH4)I SO4', , 20 g KHQPO42
g Yeast extract 2g MgSO4・? H10500mg Fe504・7H2010mg Mr3S04・4~B) 120 8+ng ion exchange water 1 sentence* 1.5hiio and R, Uchiio.

Agr, Biol, Cheffl, 35(13
), 2033-2042 (19771) The culture solution that showed growth by the above culture was appropriately diluted with sterilized water, then transferred to a meat juice agar medium (manufactured by Eiken Chemical; ordinary agar medium), and incubated at 30'C for 2 days. The cells are cultured and transplanted onto a broth agar medium repeatedly until it can be confirmed macroscopically and microscopically that the resulting multiple colonies are not different from each other.

10 of the above colonies were inoculated onto slanted agar medium (II) with the following composition and cultured at 30°C for 3 days, and the strains on the 10 wooden slanted medium were macroscopically and microscopically the same strain. It was confirmed that these 10 strains had the same properties on each medium and physiological properties.

Slant agar 4 medium (II) Composition Mg50 4 Self 7 H20500mgFeSO4m
? ) 120 10 mg MnSO4*4 ~ flHlo 8 mg polyoxyethylene-sorbitan monolaurate (average EO number 20 moles) 50 mg agar 20 g ion-exchanged water ii The properties and physiological properties of the above bacterial strain on the medium are as described above. As a result of the above test, each lO aqueous solution (2ml
) in a cryopreservation vial and freeze-preserved 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 on each medium were examined under the same conditions as above. No change was observed from before.

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 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 citrate (7 hydrate) 0.0113g, manganese sulfate (
0.018 g of 4-6 hydrate salt and 2 g of yeast extract were dissolved in tap water 12 to adjust the pH to 7.0. This liquid medium 5
1 in a 1-volume shaking tester and heated at 120°C for 15 minutes.
After steam sterilization for a minute, Nocardia eSPS# KS
M-8-21 (Nocardiaesp, KSM-8
-21) was inoculated with a platinum loop and cultured with shaking at 30°C for 86 hours.

After the culture was completed, 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 (22 L) mixture. This extract was concentrated under reduced pressure, then methylated using a methanol-BF3 catalyst, and the product was quantified by gas chromatography. The result is the first
Shown in the table.

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).

Table 1 M Display of 1 incident 1981 Patent Application No. 53850 2. Name of the invention new microorganisms 3. Person who makes corrections Specification and drawings 6 Contents of amendment As per attached sheet /ノ；上-ai-１、 The following is inserted after Table 1 on page 17 of the specification.

[4. Brief explanation of the drawings

Claims (1)

[Claims] Spi@KSM-B-21 strain. (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 l. (b) Growth status on each medium 1) Sucrose, nitrate agar medium: Growth is moderate, and the color of the colonies is white to cream. It has a dull shine. 2) Glucose-asparagine agar medium: Growth is moderate, 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) Starch agar medium: Growth is poor and colonies are white in color. The colony is dull and dry. 5) Tyrosine agar medium: ■or medium, colony color is flesh-colored to pale orange. It has a bright shine. 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 l) Raw mushroom range: Temperature 16-38°C (optimum 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 nostarch (Starch agar medium) Negative 4) Coagulation and peptonization of skim milk: Both negative 5) Production of melanin-like pigment: Negative (d) Assimilation of carbon source L- Acibinose: - D-xylose - D-glucose: + D-fructose: + Sucrose: + Inositol: + L-rhamnose - Raffinose: - D-mannite: + (e) Generation of acid and gas from sugar flag)・-su2- (gas is not produced) sorbitol di- (gas is not produced) (f) Cell wall composition Diaminopimeline #: meso-D A P sugar
=7 Rabinose, galactose (g) 5-fluorouracil resistance (40mg/18)
: resistant