JPS63240792A - Production of menaquinone-4 by fermentation - Google Patents

Abstract

PURPOSE:To produce menaquinone-4 participating in the coagulation of blood and control of calcium in living body, in high efficiency, by culturing a menaquinone-4-producing microorganism in a medium containing lactose as a carbon source. CONSTITUTION:Corynebacterium liquefaciens ATCC 14929, etc., are cultured in a medium containing 5-200g/l, preferably 20-180g/l of glucose as a carbon source in addition to nitrogen source, inorganic salts, minor element, etc., at 20-40 deg.C, preferably 25-35 deg.C and pH 5-9, preferably about 7 for 3-7 days under aerobic condition and the objective menaquinone-4 is separated from the cultured product using a solvent, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention provides a novel industrial method for producing menaquinone-4, which is an important physiologically active substance involved in blood coagulation and calcium regulation in living organisms.

Conventional technology As a method for producing menaquinone-4 using microorganisms, there is a method for producing menaquinone-4 using Flavobacterium sp.
It is known to produce menaquinone-4) [vitamin 58
(7), 278 (1984>, JP-A-61-2)
16696).

In addition, Corynebacterium spp., Brevibacterium spp.
Menaquinone-4 producing bacteria belonging to the genus Microbacterium, Tartobacterium, Aureobacterium or Durham-positive Flavobacterium (JP-A-6L-17
3792), a carotenoid pigment-producing mutant strain of a menaquinone-4 producing bacterium belonging to the genus Corynebacterium (JP-A-6L
-265097) and the like are known.

Problems to be Solved by the Invention There is a need for the development of a method for industrially producing menaquinone-4, which is involved in blood coagulation and calcium regulation in living organisms, at low cost.

Means for Solving the Problems The present inventor conducted research on a method for industrially producing menaquinone-4 at low cost, and found that microorganisms capable of producing menaquinone-4 are used in a medium containing lactose as a carbon source.
The present invention was completed based on the discovery that it shows superior menaquinone-4 productivity.

The present invention will be explained in detail below.

The present invention is characterized by culturing a microorganism capable of producing menaquinone-4 in a medium containing lactose as a carbon source, producing and accumulating menaquinone-4 in the culture, and collecting menaquinone-4 from the culture. Provided is a method for producing menaquinone-4 using a fermentation method.

As the microorganism used in the present invention, any strain can be used as long as it has the ability to produce menaquinone-4.

Specifically, menaquinone-4 producing bacteria belonging to the genus Coliobacterium, Brevibacterium, Microbacterium, Tartobacterium, Aureobacterium, Flavobacterium and the like can be mentioned. As an example of a suitable microorganism, Corynebacterium liquefaciens (Corynebacterium l1quefa
ciens) ATCC14929.

As the medium used in the present invention, either a synthetic medium or a natural medium can be used as long as it contains lactose as a carbon source and appropriately contains a nitrogen source, minerals, and other nutrients.

Specifically, lactose is used as a carbon source at 5 to 200 g/I.
! , preferably a medium containing 20 to 180 g/β. Furthermore, glucose, sucrose, maltose, glycerin, sorbitol, mannitol, molasses, organic acids, fatty acids, etc. can be used in combination with lactose. Nitrogen sources include peptone, polypeptone,
Yeast extract, meat extract, casein hydrolyzate, corn steep liquor, soybean meal, inorganic and organic ammonium salts, and the like can be used alone or in combination.

As the inorganic substance, inorganic salts such as phosphate, magnesium salt, calcium salt, iron salt, manganese salt, sodium salt, potassium salt, etc. are used. Various vitamins such as thiamine, nicotinic acid, biotin, and pantothenic acid are used as other microelements.

Furthermore, by adding a menaquinone-4 biosynthetic precursor and its related substances to the culture medium, the amount of nonaquinone-4 production may be increased.

Cultivation is performed under aerobic conditions such as shaking culture and aerated agitation culture.

The appropriate culture temperature is 20-40°C, preferably 25-35°C. During cultivation, the pH of the culture solution is preferably maintained at 5 to 9, preferably around 7.

As a pH adjuster, ammonia water, water Ml-)lium,
Potassium hydroxide, calcium carbonate, magnesium phosphate, urea, etc. are used.

The culture period is usually 3 to 7 days. Menaquinone-4 accumulates both in the culture solution and in the bacterial cells, but most of it accumulates in the bacterial cells.

Menaquinone-4 is isolated from the culture by using a single or mixed solvent such as methanol, ethanol, acetone, or chloroform to obtain a menaquinone-4-containing extract, which is then subjected to partition extraction with an organic solvent, silica gel, Sephadex, etc. Purification can be carried out by combining column chromatography, thin layer chromatography, etc.

Quantification of menaquinone-4 in samples was performed using Shimpack 0.
0S (manufactured by Shimabara Seisakusho), Zorbax ODS,
Reversed phase partition type high performance liquid chromatography using Nisil NQC-18 (both manufactured by Gas Chrom Industries Co., Ltd.) is used.

The present invention will be specifically illustrated by giving Examples below.

Example 1 Peptone 1 g/dfl, meat extract Ig/J, NaCA
'0.3g, /j! A seed medium consisting of the composition (pH 7, 2
) 30 Qml was placed in a 2β Erlenmeyer flask and sterilized.

In this, Corynebacterium liquefaciens ATCC
14929 was inoculated and cultured with shaking at 28°C for 24 hours.

30 Qml of the culture solution was mixed with yeast extract 2g/di, potassium phosphate 0.1g/d1, dipotassium phosphate 0.
05g/d1, magnesium sulfate heptahydrate 0.1g/a
Various carbon sources shown in Table 1 were added at 30 g/A each to a basic medium (pH 7.2) consisting of ammonium sulfate 0.25 g/dl, calcium carbonate 0.5 g/d1, and ferrous sulfate 0.1 mg/J. A 5β volume jar fermenter containing production medium 3β was inoculated, and the rotation speed was 400 rpm.
The cells were cultured for 5 days under the culture conditions of 31 aeration per minute and a temperature of 28°C. Menaquinone at this time
4 production J was as shown in Table 1.

Menaquinone-
The production amount of 4 was 18.7 mg/β, which was about 3 times or more compared to other carbon sources, and the production amount improved over time.

Table 1 (mg/β) Glucose 3.6 Glycerin 2
．． 6 manninto 1.9 sorbitol 2.4 ri tosu l
8 Bathyucrose 5.9 Maltose 5.8 Starch 4.0 Next, the culture solution 21 cells obtained as described above were obtained. 45 Qml of methanol was added to this and extracted three times at 55°C. The extract was concentrated, and the resulting oil was mixed with 30 Q
ml of hexane was added and insoluble matter was filtered. 8g in p liquid
of silica gel was added and stirred to adsorb the target product onto the silica gel.

After washing out non-adsorbed substances, the target substance was eluted using 5 Qml of ethyl acetate. The eluate was concentrated under reduced pressure to form an oil], 3
0 mg was obtained.

Next, this oily substance was dissolved in Qml of acetone, and this acetone solution was spotted on 8 sheets of 7Liligel thin layer 60F2S (manufactured by Merck & Co., Ltd.), and toluene:ethyl acetate-9:1
It was developed using the following developing solvent. Rfj! After scraping off the part showing ultraviolet absorption of 0.8, extracting with acetone and concentrating,
It was again dissolved in 4 ml of acetone.

This is silica gel 60 soaked in paraffin in advance.
Spotted on 8 sheets of F254 (manufactured by Merck & Co., Ltd.), developed with a developing solvent of acetone:water-95:5, scraped off the part with an Rf value of 0.62, which corresponds to the standard product of menaquinone-4, extracted with acetone, and awl. 18 mg of menaquinone-4 was obtained. This was confirmed to be menaquinone-4 through reverse phase thin layer chromatography and high performance liquid chromatography.

Example 2 Corynebacterium liquefaciens ATCC 14929 was cultured in the same manner as in Example 1 using a production medium to which lactose was added to the concentrations shown in Table 2. The amount of menaquinone-4 produced is as shown in Table 2.

Table 2 Effects of the Invention According to the method of the present invention, the fermentation production of menaquinone-4 can be significantly improved, so menaquinone-4 can be supplied in large quantities at low cost.

Procedural correction layer (voluntary) September 12, 1988

Claims (1)

[Claims] A fermentation method characterized by culturing a microorganism capable of producing menaquinone-4 in a medium containing lactose as a carbon source, producing and accumulating menaquinone-4 in the culture, and collecting menaquinone-4 from the culture. A method for producing menaquinone-4 by.