JPH0738792B2 - Method for producing protocatechuic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Object of the Invention] (Industrial field of application) The present invention is directed to a raw material for producing rust preventives, antioxidants for foods, a raw material for producing vanillin used as flavors, and various pharmaceuticals. The present invention relates to a method for producing protocatechuic acid, which is widely used as a raw material for the production of the above, by using a microorganism.

(Prior Art) As a method for producing protocatechuic acid, there are a method by an organic synthetic reaction and a method by an enzymatic reaction of a microorganism having a high selective specificity. Of these, the fermentation method using the enzyme reaction of microorganisms has the advantage that the reaction proceeds even under mild conditions near room temperature and atmospheric pressure, does not use harmful catalysts, and has few by-products, as compared with the method using the organic synthesis reaction. Therefore, it is an efficient manufacturing method that is energy-saving and resource-saving, and is highly safe with low pollution and low cost, high yield.

And, as a conventional method for producing by protocatechuic acid fermentation, there is known a method in which a mutant strain of Brevibacterium or a mutant strain of Corynebacterium is cultured in a medium containing glucose as a main carbon source. (See JP-A-50-89592 and JP-A-60-98989).

However, in these methods using glucose as a main carbon source, glucose must be used at a high concentration, expensive medium components must be used, and even if glucose is used at a high concentration, the yield is high. The rate was low, and as a result, the production cost of protocatechuic acid was high, which was a drawback.

(Problems to be Solved by the Invention) As a result of diligent studies, the present inventors have found that the microorganisms identified as belonging to the genus Rhodococcus isolated from the soil in Tokyo are
The present invention has been completed by finding that protocatechuic acid is produced in a high yield in the culture when it is cultured in a medium containing isophthalic acid as a main carbon source.

[Structure of Invention]

(Means for Solving the Problem) The present invention cultivates a microorganism belonging to the genus Rhodococcus and capable of producing protocatechuic acid in a medium containing isophthalic acid as a main carbon source, and producing protocatechuic acid from the obtained culture. A method for producing protocatechuic acid, which comprises collecting the protocatechuic acid.

Examples of the microorganism used in the present invention include a bacterium belonging to the genus Rhodococcus and having protocatechuic acid-producing ability. As a typical bacterium, the present inventors isolated Rhodococcus sp. L- from soil in Tokyo. 1 strain (Rhodococcus sp.L
-1 strain, Micro Engineering Research Institute, No. 10614). The mycological properties of this strain (L-1 strain) are as follows.

I. Morphological properties (1) Bacterial form: bacillus (2) Size: 0.8 × 1.5 to 2.0 μm (3) Polymorphism: When cultured at 30 ° C, hyphae are formed in 1 to 2 days, and then It causes fragmentation and becomes a bacillus. It does not form aerial mycelium.

(4) Anti-acidity: None (5) Gram stainability: Positive (6) Flagella: None (7) Spores: None II. Culture properties (1) Meat agar plate culture: Good growth.

The colony shape is circular, the periphery is smooth, and the color is milky white.

(2) Meat broth agar culture: filamentous.

The color of the colony is milky white.

(3) Broth liquid culture: uniformly cloudy.

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

The colony shape is circular, the periphery is smooth, and the color is white.

III. Physiological properties (1) Optimal growth temperature: 25-33 ℃ (2) Growth temperature: 20-35 ℃ (3) Optimum pH: 6.0-8.0 (4) Growth pH: 5.0-1.0 (5) Oxygen requirement : Aerobic (6) Dye production: None (7) Gelatin liquefaction: Positive (8) Catalase activity: Positive (9) Oxidase activity: Negative (10) Urease activity: Positive (11) Indole production: Negative ( 12) Starch hydrolysis activity: negative (13) Nitrate reduction: negative (14) Growth in citric acid medium: grows (15) Methyl red test: negative (16) VP reaction: negative (19) Cell wall diaminopimelic acid isomer: meso-DA
P.

(20) Cell wall acyl type (glycolyl test): There is a glycolyl group.

(21) Presence of mycolic acid: present.

(22) Separation source: Soil in Tokyo From the above mycological properties, Berjay Manual of Cysticatic Bacteriology, Volume 2 (Be
rgey's Manual of Systematic Bacteri-ology, Volume
The strain was identified by Rhodococcus genus (R
This strain was identified as Rhodococcus sp. L-1 strain (Rhodococcus sp. L-1 strain). This strain has been deposited in the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as Microbiology Research Institute No. 10614.

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

That is, isophthalic acid as a main carbon source, a nitrogen source,
Either a natural medium or a synthetic medium can be used as long as the medium appropriately contains inorganic salts, vitamins and other nutritional factors.
As the nitrogen source, inorganic nitrate sources such as ammonium nitrate, sodium nitrate, ammonium sulfate, ammonium chloride, ammonium phosphate, peptone, meat extract, constip liquor, yeast extract, casamino acid, soybean powder, etc., alone or in combination. Can be used.
As inorganic salts, potassium phosphate, sodium phosphate,
Magnesium sulfate, manganese sulfate, iron sulfate, zinc sulfate,
Copper sulfate, sodium molybdate, calcium chloride, potassium chloride, etc. may be added. In addition to these, nutritional factors necessary for growth include vitamins, amino acids, nucleic acids and salts thereof. Also, peptone, meat extract,
Natural organic nutrients containing the above nutritional factors such as constip liquor, yeast extract, and casamino acid can also be added as appropriate. Appropriate conditions for the production of protocatechuic acid are determined and used by mixing the above-mentioned medium components in appropriate amounts.

The culture is preferably carried out under aerobic conditions, for example, shaking culture method and aeration-agitation culture method, but liquid static culture method can be appropriately combined. The culture temperature is preferably 20 to 35 ° C, especially 25 to 33 ° C, and the pH of the medium may be 5 to 10. The concentration of isophthalic acid in the medium is usually 0.1-10%, preferably 0.5-3.0%
Is preferred. Isophthalic acid as the main carbon source, medium pH
When shaking culture or aeration stirring culture at around 7.0 at 30 ℃,
Protocatechuic acid is produced and accumulated in the culture from the 3rd day of culture. The culturing is usually completed in 3 to 8 days, but is terminated when the production amount of protocatechuic acid reaches the maximum.

Separation of protocatechuic acid from the culture may be performed by a method usually used for separating natural organic compounds. For example, after acidifying the culture solution after removing the bacterial cells with hydrochloric acid,
Extract with an oxygen- or hydrocarbon-based organic solvent and concentrate under source pressure. In the present invention, this concentrate is used as TLC.
Analyze by (thin layer chromatography), collect the target spots, analyze by GC-MS (gas chromatography mass spectrometer) and FD-MS (electrolytic desorption ion mass spectrometer), and extract protocatechuic acid. Identified. Further, the quantification of protocatechuic acid in the culture medium can be carried out by HPLC (high performance liquid chromatography).

(Examples) Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1 40 ml of a mixture having the composition shown in Table 2 was placed in a 100 ml Erlenmeyer flask.
The mixture was dispensed and autoclaved at 121 ° C for 15 minutes to obtain a medium for protocatechuic acid production.

The obtained protocatechuic acid production medium was mixed with Rhodococcus.
sp. L-1 strain (Rhodococcus sp. L-1 strain, Micro Inst.
No. 614) was inoculated with 1 platinum loop and cultured at 30 ° C. for 4 days with shaking.
The resulting culture is sterilized by centrifugation and then HPLC
The amount of protocatechuic acid produced was measured and found to be 6.0 g /.

Example 2 In the same manner as in Example 1 from the mixture having the composition shown in Table 2,
Protocatechuic acid production medium was obtained and Rhodococcus sp. L-
1 strain (Rhodococcus sp. L-1 strain, Microcosm Research Institute No. 10614
No.) was inoculated and cultured at 30 ° C. with shaking, 0.4 g of isophthalic acid was added 72 hours after the start of the culture, 0.2 g of isophthalic acid was added 120 hours after the start of the culture, and the culture was completed 140 hours after the start of the culture. The production amount of protocatechuic acid in the obtained culture was measured in the same manner as in Example 1.
It was 5 g /.

Example 3 The mixture having the composition ratio shown in Table 1 was poured into a 5-volume jar fermenter for 3 minutes and autoclaved at 121 ° C for 15 minutes to obtain a medium for producing protocatechuic acid.

The obtained protocatechuic acid production medium was mixed with Rhodococcus.
sp. L-1 strain (Rhodococcus sp. L-1 strain, Micro Inst.
No. 614) inoculated, aeration rate 0.3 / min, stirring speed 320rp
The cells were cultured for 120 hours under the conditions of m and culture temperature of 30 ℃. The amount of protocatechuic acid produced in the obtained culture was measured in the same manner as in Example 1, and it was 10.6 g /.

[Advantages of the Invention] According to the present invention, protocatechuic acid can be produced at low cost and in high yield as compared with the conventional fermentation method using glucose as a main carbon source.

Claims (1)

[Claims] 1. A protocatechuic acid characterized by culturing a microorganism belonging to the genus Rhodococcus and capable of producing protocatechuic acid in a medium containing isophthalic acid as a main carbon source, and collecting protocatechuic acid from the obtained culture. Method for producing acid.