JPH02234682A - Production of protocatechuic acid - Google Patents

Abstract

PURPOSE:To obtain protocatechuic acid in high yield by culturing a bacterium capable of producing protocatechuic acid in a medium containing isophthalic acid as a carbon source. CONSTITUTION:A bacterium belonging to the genus Rhodococcus, capable of producing protocatechuic acid, preferably Rhodococcus sp. L-1 strain (FERM P-10614) separated from soil in Tokyo Metropolis by the inventor of this patent is prepared. Then the bacterium is cultured in a medium containing especially isophthalic acid as a main carbon source, a nitrogen source, an inorganic salt, a vitamin and other nutrient factor properly under the following condition. Namely, the culture is carried out under an aerobic condition by shaking culture at 20-35 deg.C at pH 15-10 of the medium. Then, protocatechuic acid formed and accumulated during the culture is separated by using conventional method useful for separation natural organic compound and collected.

Description

[Detailed Description of the Invention] [Object of the Invention] (Industrial Application Field) The present invention is applicable to the production of rust preventives, food antioxidants, vanillin used as fragrances, and the production of various pharmaceutical products. This invention relates to a method for producing protocatechuic acid, which is widely used as a raw material for the production of alcohol, using microorganisms.

(Prior Art) There are two methods for producing protocatechuic acid: one is an organic synthesis reaction, and the other is an enzymatic reaction using a microorganism with high selective specificity. Among these, fermentation methods using microbial enzymatic reactions are more effective than methods using organic synthesis reactions. Because the reaction proceeds even under mild conditions at room temperature and pressure, no harmful catalysts are used, and there are few by-products, it saves energy and resources, and is highly safe with little pollution. It is an efficient manufacturing method with low cost and high yield.

Conventionally, protocatechuic acid has been produced by fermentation using mutant strains of Previbacterium or Corynebacterium. A known method is to culture it in a medium containing glucose as the main carbon source (
Unexamined Japanese Patent Publication No. 189592 and Unexamined Japanese Patent Publication No. 6 0-9
(See Publication No. 8989).

However, in these methods using 5-glucose as the main carbon source, glucose must be used in high concentrations, expensive medium components must be used, and... As a result, even when glucose is used at high concentrations, the yield is low. There was a drawback that the production cost of protocatechuic acid was high.

(Problems to be Solved by the Invention) As a result of intensive studies, the present inventors have discovered a microorganism recognized to belong to the genus Rhodocosoccus that was isolated from soil in Tokyo.
We found that protocatechuic acid was produced in high yield in the culture when cultured in a medium containing isophthalic acid as the main carbon source. This completes the present invention.

(Structure of the Invention) (Means for Solving the Problems) The present invention consists of culturing a microorganism belonging to the genus Rhodococcus and having the ability to produce protocatechuic acid in a medium containing isophthalic acid as the main carbon source. This is a method for producing protocatechuic acid, which is characterized by collecting protocatechuic acid from a substance.

The microorganisms used in the present invention include those belonging to the genus Rhodochosoccus and having the ability to produce protocatechuic acid.A representative example of the microorganism is Rhodocosoccus sp., which the present inventors isolated from soil in Tokyo. Ll strain (Rhod
ococcus sp. An example of this is strain L-1, Microtechnical Research Institute No. 10614). The mycological properties of this strain (L-1 strain) are as shown below.

■． Morphological properties (11 bacteria Shape: Bacillus (2) Size: 0.8x
1.5 to 2.0 pm (3) Polymorphism: When cultured at 30°C, hyphae will form in 1 to 2 days, and then fragmentation will occur to form rods. Aerial mycelium does not form.

(4) Acid-fast: None (5) Durham staining: Positive (6) Whip
Hair: None (7) Hair
Child: None■. Cultural properties (1) Broth agar plate culture: Good growth.

The colony shape is circular, the circumference is It is smooth and milky white in color.

(2) Meat juice agar slant culture: filamentous.

Colony color is milky white.

(3) Meat juice liquid culture: Uniformly cloudy.

(4) Isophthalic acid agar plate culture (medium composition, Table 1):
Good growth.

The colony shape is circular, with a is smooth and white in color.

■． physiological properties (11. Optimum growth temperature (2) Rent temperature (3) Optimal pH (4) Growth p　H 《5) Oxygen requirement (6) Production of pigments 《7》Gelatin liquefaction properties (8) Catalase activity (9) Oxidase activity αω urease activity αυ Production of indole (6) Starch hydrolysis activity (b) Reduction of nitrates 00 Growth in citric acid medium Q51 Methyl red test Q*VP reaction (Hereafter, margin) 25-33℃ 20~35℃ 6.0-8.0 5.0-10.0 aerobic none positive positive negative positive negative negative negative grow negative negative : Not produced.

0 cell wall diaminobimelic acid isomer; meso-
DAP. 001 Cell wall acyl type (glycolyl test): Glycolyl group is present.

(21.) Presence of mycolic acid: Exist.

(22) Source of isolation: Based on the mycological properties of soil in Tokyo, it was found in the Hergiei Manual of Systemic Hacteriology Volume 2 (Bergei Manual of Systemic Hacteriology, Vol.
y's Manual of Systematic
Bacteriology, Volume 2) revealed that one strain was of the genus Rhodochosoccus (Rh).
It is recognized that it belongs to the genus Oclococcus. This strain was Rhodococcus sp. L-1 strain (Rhodococcus
us sp. The strain was named L-1 strain). This strain is modeled after the National Institute of Microbiology, Agency of Industrial Science and Technology.
It has been deposited as No. 4.

For culturing in the present invention, ordinary bacterial culturing methods can be used. The following can be used as a nutrient source for culture.

That is, isophthalic acid as the main carbon source, nitrogen source,
As long as the medium contains appropriate amounts of inorganic salts, vitamins, and other nutritional factors. Both natural and synthetic media can be used.

Ammonium nitrate and sodium nitrate are sources of nitrogen. Inorganic nitrogen sources such as ammonium sulfate, ammonium chloride, ammonium phosphate, peptone. Meat extract, cornsteep liquor, yeast extract, casamino acids, soybean flour, etc. can be used alone or in combination. Inorganic salts include potassium phosphate and sodium phosphate. Magnesium sulfate. Manganese sulfate, iron sulfate, zinc sulfate, copper sulfate, sodium molybdate, calcium chloride potassium dichloride, etc. may be added. In addition to these, nutritional factors necessary for growth include vitamins, amino acids, nucleic acids, and their salts. Also peptone, meat extract, and consteep liquor. Natural organic nutrients containing the above-mentioned nutritional factors, such as yeast extract and casamino acids, can also be added as appropriate. Appropriate amounts of each of the above-mentioned medium components are mixed, and conditions suitable for producing protocatechuic acid are determined and used.

For culturing, aerobic conditions such as shaking culture method and aerated agitation culture method are suitable. A liquid static culture method can also be combined as appropriate. The culture temperature is 20-35℃, especially 25-3
3℃ is good. The pH of the medium may be between 5 and 10.

The concentration of isophthalic acid in the medium is usually 0.1 to 10%, preferably 0.5 to 3.0%. Using isofucric acid as the main carbon source. Medium pH 7. Near 0, 30℃
When cultured with shaking or with aeration. Prol-catechuic acid is produced and accumulated in the culture from the third day of culture. Cultivation is usually completed in 3 to 8 days, and ends when the production amount of pro-1 catechuic acid reaches its maximum.

Protocatechuic acid can be separated from the culture by a method commonly used for separating natural organic compounds. for example. After removing the bacterial cells, the culture solution is acidified with hydrochloric acid, extracted with a chlorine-containing, oxygen-containing, or hydrocarbon-based solvent, and concentrated under reduced pressure. In the present invention, this concentrate is subjected to TLC
(thin layer chromatography), the target sposoto was separated, and analyzed by GC-MS (gas chromatography mass spectrometer) and FDMS (electrolytic desorption ion mass spectrometer), and identified as protocatechuic acid. . In addition, the determination of brol-catechuic acid in the culture solution is as follows. HPLC (
This can be done by high performance liquid chromatography).

(Example) The following is an example. The present invention will be explained in further detail.

Example 1 A mixture having the composition shown in Table 2 was prepared. The mixture was dispensed into a 100 m β Erlenmeyer flask and autoclaved at 121° C. for 15 minutes to obtain a protocatechuic acid production medium.

In the obtained protocatechuic acid production medium, Rhodococcus sp. L-1 strain (Rhodococcus sp.
One platinum loop of L-1 strain (Feikoken Bacterial Serial No. 10614) was inoculated and cultured with shaking at 30°C for 4 days. After sterilizing the obtained culture by centrifugation. The amount of protocatechuic acid produced was measured by HPLC. It was 6.0g/j2.

Example 2 A mixture having the composition shown in Table 2 was prepared in the same manner as in Example 1.
A medium for protocatechuic acid production was obtained, and Rhodococcus sp.
L-1 strain (Rhodococcus sp.
- Inoculated with 1 strain, Sokoken Bacteria No. 10614) 2 3
Culture was carried out with shaking at 0°C, and 0.4 g of isophthalic acid was added 72 hours after the start of culture, and 0.4 g of isophthalic acid was added 120 hours after the start of culture.
2 g of each was added, and the culture was terminated 140 hours after the start of culture. The obtained culture was treated in the same manner as in Example 1. When the amount of protocatechuic acid produced was measured, it was found that 1
It was 6.5g/Il.

Example 3 3 liters of the mixture having the composition ratio shown in Table 1 was dispensed into a 51-volume jar fermenter and autoclaved at 121°C for 15 minutes to obtain a protocatechuic acid production medium.

Rhodococcus sp. was added to the obtained protocatechuic acid production medium. L-1 strain (Rhodococcus sp.
- 1 share. Inoculated with ``Feikoken Bacteria No. 10614'', and the aeration rate was 0. 3 1/min. Culture was carried out for 120 hours at a stirring rotation speed of 32 rpm and a culture temperature of 30°C. The obtained culture was treated in the same manner as in Example 1. When the amount of protocatechuic acid produced was measured, it was 10.6 g/N [
Effects of the invention] According to the present invention. Lower cost than conventional fermentation methods that use glucose as the main carbon source. With high yield. Protocatechuic acid can now be produced.

Claims (1)

[Claims] 1. Production of protocatechuic acid, which is characterized by culturing a microorganism belonging to the genus Rhodococcus and having the ability to produce protocatechuic acid in a medium containing isophthalic acid as the main carbon source, and collecting protocatechuic acid from the resulting culture. Method.