JPH0361436B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing protocatechuic acid by fermentation. Protocatechuic acid is useful as a raw material for food antioxidants, as a raw material for vanillin, which has uses as a flavoring agent, and as a raw material for various pharmaceutical products. Conventional bacteria that accumulate protocate acid in the culture medium include a mutant strain of the genus Neurospora (Biochemical Journal 68, 168, 1958), a mutant strain of the genus Corynebacterium (Japanese Patent Publication No. 45-39036), and a mutant strain of the genus Brevibacterium. Mutant strains of the bacterium (Japanese Unexamined Patent Publication No. 1989-8952) have been reported. As a result of repeated research aimed at developing a more efficient fermentation production method for protocatechuic acid, the present inventors found that the genus Brevibacterium or Corynebacterium cannot grow on protocatechuic acid as the sole carbon source. We found that among the mutant strains, there are strains that produce and accumulate large amounts of protocatechuic acid. The present invention was completed based on this knowledge. In the present invention, the microorganism capable of producing protocatechuic acid belongs to coryneform bacteria such as the genus Brevibacterium or the genus Corynebacterium, and has properties necessary for producing protocatechuic acid, such as parafluorophenylalanine, metafluorophenylalanine,
Resistance to aromatic amino acid analogs such as tyrosine hydroxamate or 5-methyltryptophan, individual or combined requirements for aromatic amino acids such as tyrosine, phenylalanine, and tryptophan, and intermediates in the aromatic amino acid biosynthesis system It is a microorganism that requires shikimic acid and chorismic acid. The mutant strain used in the present invention belongs to coryneform bacteria such as the genus Brevibacterium or Corynebacterium and exhibits the above-mentioned ability to produce protocatechuic acid, and also has the property of not being able to grow on protocatechuic acid as the sole carbon source. It is a mutant strain. The microorganism used in the present invention is specifically, for example, Brevibacterium Brevibacterium lactofermentum AJ 12106 FERM P-73
33 (mFP ^r , PCA degrading activity deleted) Brevibacterium Brevibacterium lactofermentum AJ 12107 FERM P-73
34 (PFP ^r , Try ^- , PCA degrading activity deleted) Corynebacterium Corynebacterium acetoacidophyllum AJ 12108 FERM P-73
35 (PFP ^r , PCA decomposition activity deficiency) mFP ^r : Metafluorophenylalanine resistance PCA decomposition activity deficiency : Protocatechuic acid decomposition ability deficiency Try ^- : L-tyrosine requirement pFP ^r : Parafluorophenylalanine resistance These books The mutant strain of the invention is produced by using a microorganism of the genus Brevibacterium or Corynebacterium as a parent strain, and subjecting it to conventional mutagenesis operations such as ultraviolet rays, X-rays, etc.
irradiation or N-methyl-N'-nitro-N-
The mutagenized bacterial cells were treated with chemical agents such as nitrosoguanidine (abbreviated as NTG) and nitrous acid, and cultured on agar plates containing complete medium, and minimal agar plates containing glucose or protocatechuic acid, respectively, as the sole carbon source. Replica onto the medium. In this way, it is obtained by isolating colonies that can grow on glucose as the sole carbon source, but not on protocatechuic acid. In addition to protocatechuic acid, the only carbon source contained in the minimal medium is
There is no problem even if it is quinic acid or shikimic acid. Next, a method for inducing mutations in mutant strains used in the present invention will be shown in the following experimental examples. Experimental example 1 Brevibacterium lactofermentum
AJ12105 (FERMP-7332) was cultured on yeast broth agar slant medium, and the grown cells were collected and 1/
Suspend in 50M phosphate buffer (PH7.0) (10 ⁸ - 10 ⁹
NTG was added to this (NTG concentration was 1000 μg/ml), and the mixture was kept at room temperature for 30 minutes. After thoroughly washing the NTG-treated bacterial cells with the same phosphate buffer, the bacterial solution was appropriately diluted and applied on a complete agar plate so that the number of colonies was approximately 200, and the bacterial solution was spread on a complete agar plate for 3 to 30 minutes. The cells were cultured at 31.5°C for 4 days. Next, it is replicated on a minimal agar plate medium each containing glucose or protocatechuic acid as the sole carbon source as shown in Table 1, and grown on a minimal agar medium containing glucose as the sole carbon source without growing on the protocatechuic acid medium. Growing colonies were isolated. Among the mutant strains thus obtained, there were many strains exhibiting excellent protocatechuic acid productivity. Among these, strain AJ12106 with the highest production capacity was selected. Brevibacterium bacterium
Lactofermentum AJ3436 (FERM−P1913)
AJ12107 was selected as the parent stock. Table 1 Composition of minimal medium Glucose 10g/or protocatechuic acid 5 Ammonium sulfate 5 Urea 2 KH ₂ PO ₄ 1 MgSO ₄・7H ₂ O 1 Fe ⁺⁺ , Mn ⁺⁺ ions 2 ppm each Biotin 50μg/ VB ₁・HCl 2000 〃 DL-Methionine 400mg/g L-Tyr 100 〃 PH7.0 (KOH) Corynebacterium spp.
AJ12108 was induced in the same manner using acetoacidophyllum ATCC13870 as the parent strain. Next, Table 2 shows the results of growth of the mutant strains thus obtained in a minimal medium containing glucose or plutocatechuic acid as the sole carbon source.

[Table] The experimental method was to thoroughly wash the bacterial cells of each mutant strain with the minimal medium shown in Table 1, and then place them in a minimal medium (4 ml) containing the specified amount of carbon source shown in Table 2, which was placed in a small tester. Inoculate a certain amount and culture with shaking at 31.5℃ for 24 hours.
The degree of growth was determined by measuring the absorbance of the culture solution at 560 nm. Table 2 shows the relative growth values. The medium used in the present invention contains carbon sources, nitrogen sources, inorganic salts, amino acids, vitamins, etc. as necessary.
Conventional nutrient media containing organic micronutrients such as nucleic acids are used. The carbon source may be any carbon source that can be used by the mutant strain used; for example, glucose, fructose, sucrose, maltose, saccharides such as starch decomposition product molasses can be used, and other carbon sources include alcohols such as ethanol and propanol, acetic acid, Organic acids such as citric acid, and depending on the strain, normal paraffin and the like are also used alone or in combination with other carbon sources. As the nitrogen source, inorganic or organic nitrogen sources such as ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, nitrates, urea, ammonia, and meat extracts are used. As organic micronutrients, amino acids, vitamins, fatty acids, nucleic acids, peptones containing these things, casamino acids, yeast extracts, protein decomposition products, etc. are used.
When using an auxotrophic mutant strain that requires amino acids and the like for growth, it is necessary to supplement the required nutrients. It is desirable to culture under aerobic conditions, with the pH of the medium being between 5 and 9 and the temperature between 20°C and 20°C.
A large amount of protocatechuic acid is accumulated in the culture solution by culturing with shaking or stirring with aeration for 1 to 4 days while controlling the temperature at 40°C. Protocatechuic acid may be collected from the culture solution by any known method, and after separating and removing the bacterial cells from the culture solution, protocatechuic acid is collected by a method using an ion exchange resin or the like. Examples will be described below. Example 1 A protocatechuic acid production medium shown in Table 3 below was prepared and dispensed to 20 ml into a 500 ml shaking flask.
Heat sterilization was performed at 120°C for 10 minutes. To this was added 1.0 g of calcium carbonate powder which had been separately heat sterilized.

[Table] One platinum loop of the protocatechuic acid-producing bacteria shown in Table 4 was inoculated into this medium, and cultured with shaking at 30°C for 72 hours.
The amount of protocatechuic acid produced in the culture solution was measured, and the results are shown in Table 4.

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Claims (1)

[Claims] 1 Cultivating a mutant strain belonging to the genus Brevibacterium or Corynebacterium that cannot grow on protocatechuic acid as a sole carbon source and has the ability to produce protocatechuic acid, produces and accumulates protocatechuic acid in the medium, and collects it. A method for producing protocatechuic acid by a fermentation method, characterized by: