JPH0728748B2 - Method for producing menaquinone-4 - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing menaquinone-4 by a microorganism.

Menaquinone-4 is known as vitamin K ₂ for pharmaceuticals, is a fat-soluble vitamin that promotes blood coagulation, and is an important physiologically active substance having an anti-hemorrhagic action.

[Problems to be Solved by Prior Art and Invention]

As a method for producing menaquinone-4, a synthetic method and an extraction method from animal and plant tissues are known. However, the former is difficult to obtain the raw material, and the cost is high, while the latter cannot avoid the high cost due to the limited supply amount and complicated extraction operation, and both methods are suitable for practical use. It's hard to say.

On the other hand, if it is possible to find a microorganism or condition that efficiently accumulates menaquinone-4 outside the microbial cell, it is very advantageous because it can be industrially produced in large quantities. No microorganisms or conditions that efficiently accumulate outside have been found.

The present invention has been made in view of the above circumstances, and is directed to the production of menaquinone-4 by microorganisms.

[Means for solving problems]

In order to achieve the above-mentioned object, as a result of the present inventors' earnest studies, a mutation process was performed using a strain that does not produce menaquinone-4 as a parent strain, and succeeded in obtaining a strain that produces menaquinone-4 in the cells. Applied for a patent (Japanese Patent Application No. 60-5545)
No. 8). After that, as a result of further research on the menaquinone-4 producing strain, the menaquinone-4 producing strain accumulates the produced menaquinone-4 in the bacterial cells and discharges it to the outside of the bacterial cells. The present invention was completed by finding that a large amount of menaquinone-4 was excreted out of the cells by adding a surfactant to the medium or the culture solution in the above culture.

That is, the present invention is a method for producing menaquinone-4, which comprises collecting menaquinone-4 discharged from the microbial cells of a microorganism having a capacity to produce menaquinone-4.

The strain used in the present invention may be any strain as long as it is a strain that produces menaquinone-4 and excretes it outside the cells.
However, at the present time, no wild strain is known that excretes menaquinone-4 to the outside of the cell, and due to mutation treatment, etc.,
A menaquinone-4 producer must be created. The method for preparing this menaquinone-4 producing strain is the same as in Japanese Patent Application No. 60-55458.

That is, as the parent strain to be subjected to the mutation treatment, a microorganism that produces menaquinone having 5 or more side chain isoprene is used. Generally, the menaquinone-producing microorganisms are actinomycetes and bacteria, and the menaquinone types are menaquinone-5 to menaquinone-14. Therefore, all of these menaquinone-producing microorganisms can be used as parent strains. It is possible to obtain a mutant strain that produces menaquinone-4 by performing mutation treatment using all of these menaquinone-producing microorganisms as parent strains.
A preferred microorganism having a high probability of obtaining a 4-producing strain is a menaquinone-6-producing microorganism.

Menaquinone-6-producing microorganisms include, for example, Staphy
lococcus sciuri, Flavobacterium aquatile, Flavobacte
rium breve, Flavobacterium odoratum, Flavobacterium
sulfureum var miyamizu, Flavobacterium menigosaptic
um, Cytophaga aquatilis and Cytophaga johnsonal
Etc., all of which are known bacteria.

The mutation treatment method may be a commonly used method, but among them, UV treatment and NTG (N-methyl-N'-nitro-
N-nitrosoguanidine) treatment and EMS (ethyl methane sulphonate) treatment are preferred.

Selection of mutant strains that produce menaquinone-4 after mutation treatment (hereinafter referred to as menaquinone-4 producing strain) is performed by a commonly used method-for example, random screening method, or drug resistant strain, screening from drug sensitive strain. And the like. As the drug, a menaquinone biosynthesis inhibitor is usually used, but as the menaquinone biosynthesis inhibitor, for example, 1-hydroxy-
2-naphthoic acid, usnic acid and menadione.

Menaquinone-4 producing strains are, for example, Flavobacterium aquatile having the ability to produce menaquinone-6.
Flavobacterium aquatile HNA 250-15 (minor) was used as a strain having the ability to produce menaquinone-4 by repeating mutation and selection using No. 238-7 (Microtechnical Research Institute No. 8113) as a parent strain. Institute of Microbiology 8114), Flavobacterium aquatile USN ^γ -2 (Minister of Microbiology 811)
No. 5) and Flavobacterium aquatile K ₃ -1
5 (Microtechnical Lab. No. 8116) were sequentially obtained. Thus the content of menaquinone-4 intracellular increases in this order, the highest content of menaquinone-4 Flavobacterium Aqua tile K ₃ -15 of these. The Flavobacterium Aqua tile K ₃ -15 together with accumulating menaquinone -4 generated intracellularly, also discharged to extracellular.

In addition, when culturing this strain, in the medium or culture solution,
The addition of the surfactant increases the amount of menaquinone-4 released outside the cells.

As the surfactant used in the present invention, a nonionic surfactant is preferable, and as typical examples thereof (displayed as trade names), union (higher alcohol), emulgen (polyoxyethylene oleyl ether), and brownon ( Nonylphenyl, Castor oil), Leodol (Polyoxyethylene sorbitan, Polyoxyethylene tetraoleate), Licanone (Polyoxyethylene oleyl ether), Sylvan (Sorbitan monopalcitate), Amizetto (Palm fatty acid monoethanolamide) and Newcol (Special) Nonion) etc.
These surfactants are added to the medium or the culture medium, but it is preferable to add them to the medium or the culture medium at the initial stage of the culture.

The concentration of the surfactant in the medium or the culture medium may be within the range of 0.01 wt / v% (hereinafter referred to as%) to the growth inhibitory concentration, and the strain to be used, the bacterial cell concentration in the culture medium and the kind of the surfactant. It is appropriately selected depending on factors such as 0.01 to 1%
The degree is preferably about 0.01 to 0.3%.

As the nutrient medium used for culturing the menaquinone-producing microorganism, an ordinary medium used for culturing a microorganism containing a carbon source, a nitrogen source, an inorganic salt and the like is used.

The carbon source may be any as long as these microorganisms can assimilate, glucose, fructose, sucrose, maltose, molasses, carbohydrates such as starch and starch hydrolysates, sugar alcohols such as glycerol and sorbitol. , Aspartic acid,
Amino acids such as glutamic acid, lysine, alanine, glycine, proline and methionine, lactic acid, pyruvic acid,
Organic acids such as acetic acid, malic acid, formic acid, succinic acid, fumaric acid, citric acid, propionic acid and fatty acids and alcohols such as ethanol, propanol and butanol can be used alone or in combination. Of these, glycerol is most preferred.

As the nitrogen source, phosphates, magnesium salts, iron salts and, if necessary, trace metal salts are used, and further, growth promoting substances such as amino acids, nucleic acids, vitamins, yeast extracts and malt extracts are also used. When the strain used shows auxotrophy, the auxotrophic substance is added to the medium.

Cultivation of the menaquinone-4 producing microorganism is carried out by aerobically shaking or aerating and stirring culture at pH 5 to 8.5 and a culture temperature of 20 to 40 ° C for 1 to 10 days.

Menaquinone-4 is extracted and isolated from the culture supernatant after the cells have been separated from the cells by solid-liquid separation means such as centrifugation and filtration.

In order to isolate menaquinone-4 from the culture supernatant, a conventional method can be applied as it is. That is, for example, the culture supernatant as it is, or after concentration, an organic solvent insoluble in water, such as hexane, benzene, and ethyl ether, is added, and menaquinone-4 is transferred to the organic solvent layer to give menaquinone- After the 4 extracts are concentrated, they are dissolved in ether and subjected to silica gel column chromatography to separate and concentrate the menaquinone-4 fraction.

The identification of menaquinone-4 was performed by high performance liquid chromatography, thin layer chromatography, UV and IR spectra,
This is done by confirming that it is menaquinone-4 from the results of mass spectrum and the like. A high performance liquid chromatography method is used as a quantitative method.

〔Example〕

 Specific examples will be described below.

Example 1 A Acquisition of A Menaquinone-4 Producing Strain As a parent strain, a Menaquinone-6 producing strain, Flavobacterium aquatile No. 238-7 (Ministry of Industrial Science and Technology, Contribution No. 8113)
No.) was used. Although this strain was newly isolated from the natural world, it was identified as Flavobacterium aquatile according to Virgin AIDS Manual of Systematic Bacteriology Vol. 1 (1984) based on its taxonomical characteristics. The mycological properties of this bacterium are described, for example, in "Vitamin Volume 58, p.409-419 (1984)".

Beptone-glycerol liquid medium (M31 medium-glycerol 10 g, polypeptone 15 g, yeast extract 1 g, K ₂ HPO _{4
3g, NaCl 2g, MgSO 4 ·} 7H 2 O 0.2g and pure water 1000m
Flavobacterium Aquatile No. 238-7 cells grown at 30 ° C for 24 hours at pH 7.0) were treated with N-methyl-N'-nitro-N-nitrosoguanidine 100 µg / ml.
The suspension was suspended in 0.1 M phosphate buffer (pH 7.0) containing 10 ⁸ cells / ml and shaken at 30 ° C. for 30 minutes. Then, the bacterial cells collected by the centrifuge were washed with 0.1M phosphate buffer,
The cells were resuspended in the same buffer at 10 ⁶ / ml. 1
-Hydroxy-2-naphthoic acid Peptone-glycerol agar plate supplemented with 350 μg / mg (20 g / agar supplemented with the M-31 medium) was coated with 0.1 ml of the suspension, and the emerged colonies were collected. It was collected as a 1-hydroxy-2-naphthoic acid resistant strain. From these resistant strains, Flavobacterium aquatile HNA 250-15 (Microtechnical Research Institute No. 8114) was obtained as a menaquinone-4 producing strain. In addition, Flavobacterium aquatile HNA
By using 250-15 as a parent strain, a bacterium having higher productivity of menaquinone-4 in the bacterium was obtained as follows. That is, Flavobacterium aquatyl HNA 2 grown in a peptone-glycerol liquid medium at 30 ° C for 24 hours.
Suspended 50-15 in 0.1M phosphate buffer (pH 7.0) at 10 ⁸ cells / ml and treated with UV (2 10W UV lamps, 4
0 cm) for 30 seconds. 0.1 ml of the above-mentioned treatment solution was applied on a peptone-glycerol agar plate medium to which 4 μg / ml of usnic acid was added, and a colony that appeared was collected as a usnic acid resistant strain. The parent strain (HNA
250-15), with a clearly higher menaquinone-4 production capacity, Flavobacterium aquatile USN ^γ -2
(Ministry of Microbiology, No. 8115).

In addition, Flavobacterium aquatile USN ^γ −
2 was used as a parent strain, peptone-containing 20 μg / ml of menadione was subjected to mutation treatment by UV treatment in the same manner as above.
0.1 ml of the treatment solution was applied onto a glycerol agar plate medium, and the colonies that appeared here were collected as menadione-resistant strains.

Parent strain from these resistant bacteria (USN ^gamma -2) Intracellular menaquinone -4 obviously higher productivity Flavobacterium Aqua compared to tile K ₃ -15 (FERM No. 81
No. 16) was obtained.

B Collection of menaquinone One 100 ml of peptone-glycerol liquid medium (M31 medium)
The mixture was placed in an Erlenmeyer flask and heat sterilized at 120 ° C. for 20 minutes. Other flasks should have the same media composition as above.
0.1 g / various kinds of surfactants were added, and heat sterilization was performed at 120 ° C. for 20 minutes.

In each flask, the strain obtained in the above A was used for 1 day at 30 ° C. using the above M31 medium containing no surfactant,
Preculture (test tube culture) obtained was Flavobacterium Aqua tile K ₃ 1 volume% -15 cells of the to a medium
The cells were inoculated and subjected to rotary shaking culture at a culture temperature of 30 ° C. On the third day after the start of culture, the culture solution was centrifuged and the content of menaquinone-4 in the culture supernatant was measured. 2 ml of n-butanol was added to 1 ml of the culture supernatant, which was shaken for 10 minutes and then centrifuged at 3000 rpm for 5 minutes, and the supernatant was subjected to high performance liquid chromatography analysis to analyze menaquinone-4.

The results are shown in Table 1.

Example 2 Glycerol 10 g /, polypeptone 15 g /, yeast extract _{1g /, K 2 HPO 4 3g} /, NaCl 2g /, MgSO 4 · 7H 2 O
Emulgen 430 was added to 0.2 g / basic medium.
(Products of Kao Corporation) 0.1 /, 0.5g /,
100 ml of the medium added so that the amount became 1.0 g / was placed in a 1-volume Erlenmeyer flask and sterilized by heating at 120 ° C. for 20 minutes. The strain obtained in the same manner as in Example 1A was obtained by preculture (test tube culture) at 30 ° C. for 1 day using the above basic medium,
Flavobacterium Aqua tile K ₃ was inoculated 1 volume% -15 cells of the to a medium, Natsuta lines cultured with shaking rotation at a culture temperature 30 ° C.. On the third day after the start of the culture, the culture was centrifuged and the content of menaquinone-4 in the culture supernatant was measured. The results are shown in Table 2.

Example 3 The same procedure as in Example 2 was carried out except that Licanone UA5012 (a product of New Japan Rika Co., Ltd.) was used as the surfactant.

Table 3 shows the content of menaquinone-4 in the culture supernatant.

Example 4 The same procedure as in Example 2 was carried out except that Newcol 2614 (a product of Shin Nippon Rika Co., Ltd.) was used as the surfactant.

Table 4 shows the content of menaquinone-4 in the culture supernatant.

EFFECT OF THE INVENTION According to the present invention, menaquinone, which is highly valuable as a pharmaceutical,
4 can be easily and efficiently produced by culturing a menaquinone-4-producing microorganism.

Claims (1)

[Claims] 1. Menaquinone-4, which is discharged from the microbial cells of a microorganism belonging to the genus Flavobacterium and has the ability to produce menaquinone-4, is collected. Manufacturing method.