JPS6047690A - Production of dicarboxylic acid by microorganism - Google Patents

Abstract

PURPOSE:To produce a 6-22C dicarboxylic acid, in high yield, by culturing a dicarboxylic acid-producing strain belonging to Nocardia genus in a medium added with a 6-22C n-paraffin, fatty acid and fatty acid derivative. CONSTITUTION:Nocardia sp. KSM-B-20 (FERM-P No.7005) or Nocardia sp. KSM-B-21 (FERM-P No.7006) separated newly from the soil is cultured in a medium obtained by adding a component selected from 6-22C n-paraffin, fatty acid and fatty acid derivative to conventional medium. The n-paraffin, fatty acid and fatty acid derivative is converted to the corresponding 6-22C dicarboxylic acid, and accumulated in the medium.

Description

[Detailed description of the invention] The present invention relates to a method for producing luponic acid.

Dicarboxylic acid is a useful substance as a raw material for producing synthetic resins, high-grade lubricants, plasticizers, fragrances, etc. However, dicarboxylic acids produced by synthetic methods have a limited number of carbon atoms. 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, Candida K (Special Publication No. 50-1
No. 9630, etc.), Pichia (Special Public Service No. 4)
5-24392, etc.), and bacteria such as Corynebacterium
cterium ) genus (Special Publication No. 56-17075, etc.)
only had been 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 provides paraffins selected from normal paraffins, fatty acids, and fatty acid derivatives having 6 to 22 carbon atoms. A dicarboxylic acid produced by a microorganism characterized by culturing a dicarboxylic acid-producing bacterium belonging to Nocardia in a medium to which a substrate has been added, producing and accumulating dicarboxylic acid having 6 to 22 carbon atoms in the medium, and collecting the dicarboxylic acid. It is in the manufacturing method.

The microorganism used in the present invention is Nocardia (Nocardia).
dia) H, which can selectively oxidize the ω-terminus of normal paraffins, fatty acids or fatty acid derivatives to produce dicarboxylic acids; examples include Nocardia sp.
sp, KSM-B-20) and Nocardia sp. a KSM-B-21 (Nocardia@sp, K
SM-B-21). These two strains were isolated from soil by the present inventors, and have been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiology Research Institute No. 7005 and No. 7006, respectively, and are shown in the table below. It has the mycological properties shown in 1.

About bacteria with the above mycological properties/Bergay's Manual of D
e-terminal Bacteriology
y) As a result of searching based on the 8th edition (1975), it was found that both of the above bacterial strains belong to the genus Nocardia.

The normal, < rough-in, fatty acid or fatty acid derivative used as a raw material in the present invention is one having 6 to 22 carbon atoms, and the normal, < rough-in, fatty acid or fatty acid derivative has 6 to 22 carbon atoms. I of a mixture of two or more different normal paraffins
/z−f may be used. For example, normal hexane, normal heptane, normal nonane, normal decane,
Normal undecane, normal dodecane, normal tridecane, normal undecane, normal hexadecane,
Examples include normal octadecane, normal nonadecane, normal icosane, normal icosane, and normal tocosane. Examples of fatty acids include capronic acid, heptanebutyric acid, caprylic nonanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, icosanoic acid, and tocosanoic acid. Further, the fatty acid derivative in the present invention is preferably a lower alkyl ester of a fatty acid, such as methyl ester, ethyl ester, normal (or iso) propyl ester, normal (or 5ec- or tert-) butyl ester of the above fatty acid. .

The composition of the medium used in the present invention is such that it contains a carbon source, nitrogen source, or organic nutrient source suitable for the growth of the strain used and for the smooth production of dicarboxylic acids from normal paraffin, fatty acids, or fatty acid derivatives. , 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-dodecane,
- Hexadecane, etc.) Any substance that can be assimilated can be used. In addition, as a nitrogen source or an organic nutrient source, for example, 7. Examples include 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. Furthermore, trace amounts of heavy metal salts are used, but supplements (2) are 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, and
What is necessary is just to shake or aerate and stir at 37 degreeC for 3 to 5 days. Good results can be obtained by adjusting the pH to about 6.0 to 8.3.

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 target substance, dicarboxylic acid, can be collected and purified from these culture solutions in accordance with the methods used for collecting and purifying 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 ++F crystallization.

Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is limited thereto.Rum 10g, Lymbutyric potassium 2g, Magnesium sulfate (7 hydrate) 0.
Dissolve 2g, ferric ester (heptahydrate) 0.02g, zinc sulfate (heptahydrate) O,018g, manganese sulfate (tetra-hexahydrate) 0.018g, yeast extract 2g in tap water IJit. After adjusting the pH to 7.0, 5 ml of this liquid medium was placed in a 50 ml shaking tester and steam sterilized at 120°C for 15 minutes.

Nocardia ax speed e KSM-B-20 (Noc
ardia-sp, KSM-B-20) was inoculated using a platinum loop, and cultured with shaking at 30°C for 86 hours.

After the culture was completed, 11 parts 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 (2:1) mixture. 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 28 rag of α,ω-tetradecanedicarboxylic acid was obtained per culture solution 19.

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).

Example 2 Nocardia No. SP KSM-8-20 as a bacterial strain
(Nocardia * sp, KSM-B-20) instead of Nocardia AX Subie KSM-8-21
(Nocardiaesp, KSM-B-21) and culture was performed under the same conditions as in Example 1. The results are shown in Table 2.

Table 2 Example 3 Using isopropyl balmitate instead of methyl valmitate as a reaction substrate, the bacterial strain te/Cardia sp. KSM-B-20 (Nocardia sp.
, KSM-B-20) and Nocardia sp, KSM-8-20 (Nocardia*sp, KSM-8-20) and Nocardia sp.
SM-B-20) and Tukartia Sp. KSM
-B-21 (Nocardia sp, KSM-B-
21) were cultured under the same conditions as in Example 1. The results are shown in Table 4.

Table 4 Example 5 50 g of n-hexadecane, 10 ammonium phosphate
g, 2 g of potassium phosphate, 0.2 g of magnesium 4&acid (heptahydrate), polypeptone Ig, and 2 g of Naumo extract were dissolved in tap water 11 and the pH was adjusted to 7.0. Pour 50ml of this liquid medium into a 500ml shaking flask,
After incubating with FA for 15 minutes at 20°C,
SP〇KSM-B-20 (Nocardia #
sp, KSM-B-20) was inoculated with a platinum loop and 30°
The cells were cultured with shaking at C for 89 hours.

After the completion of culturing, add 10 ml of 9N sulfuric acid to this culture solution.
It was found that when H was made to be a strong acid, 100% of ethyl ether was used, and 12 m8 of α,ω-tetradecanedicarboxylic acid could be obtained per culture solution. The product was confirmed by GC-MS to be α,ω-tetradecanedicarboxylic acid.

Example 6 Using n-dodecane instead of n-hexadecane as the reaction substrate, Nocardia Φ sp. aKSM was used as the bacterial strain.
-B-20 (NocardiaIIsp, KSM-B
-20) and Nocardia sp.-KSM-B-2
1 (Nocardia * sp, KSM-8-21
) were cultured under the same conditions as in Example 5.

The results are shown in Table 5.

Table 5 l Incident display Patent Application No. 53E of 1988! No. 52 2. Name of the invention Method for producing dicarboxylic acid using microorganisms 3. Person who makes corrections Relationship to the incident: Patent applicant 3-1 Kasumikuseki-chome, Chiyoda-ku, Tokyo Specification and drawings 6 Contents of amendment As per attached sheet The following is inserted after Table 5 on page 15 of the specification.

4. Brief explanation of the drawing

Claims (1)

[Claims] 1. A dicarboxylic acid-producing bacterium belonging to the genus Nocardia is cultured in a medium supplemented with a substrate selected from normal paraffins having 6 to 22 carbon atoms, fatty acids, or fatty acid derivatives, and dicarboxylic acids having 6 to 22 carbon atoms are added to the medium. Generate and accumulate,
A method for producing dicarboxylic acid using microorganisms, which comprises collecting the dicarboxylic acid.