JPH054073B2 - - Google Patents

Description

[Detailed explanation of the invention] [In industrial use field] The present invention is based on the method of manufacturing of vitamin B ₁₂ , and in detail, a microorganism belonging to the genus the genus of the bias anaerobic bacteria is methanol. The present invention relates to a method for producing vitamin _B12 , which is characterized by culturing.

Vitamin _B12 is involved as an essential factor in various biosynthetic reactions in living organisms, and in addition to being used as a feed additive, it has been found to have excellent medicinal effects for pernicious anemia, chronic neurological diseases, etc. Demand has been increasing in recent years.

[Prior art, problems to be solved by the invention]

Vitamin _B12 , due to the complexity of its structure,
Chemical synthesis is not practical because it requires a complex, multi-stage synthesis process.

There is also a method of extracting vitamin _B12 from animal organs such as cow liver, but this method is difficult because the content of vitamin B12 in animal organs is low, and the supply of animal organs is small and unstable. This cannot be said to be a preferable method as an industrial manufacturing method.

On the other hand, there is also a method of using microorganisms belonging to the genus Pseudomonas, Nocardia, Streptomyces, Arthrobacter, and the like. However, since all of these microorganisms are aerobic microorganisms, a large amount of power is required for aeration and agitation during cultivation, and there is a high risk of contamination with other bacteria, and volatile medium components may be evaporated. many. In addition, many of these microorganisms are generally cultured using sugars such as glucose, fructose, and sucrose, or blackstrap molasses containing these sugars as carbon sources, but these sugars and sugar-containing substances are relatively rare. Because it is expensive and its supply is unstable,
The methods using these microorganisms are also not preferred as industrial methods.

Therefore, if an anaerobic microorganism exists that can assimilate methanol and efficiently produce vitamin _B12 , it can use methanol, which is inexpensive and stably supplied in large quantities as an industrial raw material, as a carbon source. Aeration and stirring are essentially unnecessary, the risk of contamination with bacteria is relatively small, and the amount of volatile medium components volatilized is significantly reduced, making it possible to produce vitamin _B12 industrially. This makes it possible to manufacture the product in an advantageous manner.

[Means and actions for solving problems]

The present inventors have conducted various studies on a method for producing vitamin _B12 using microorganisms and culturing them using methanol as a carbon source. As a result, when a microorganism belonging to the genus Ebacterium, an obligate anaerobic bacterium, is cultured using sugars such as glucose and fructose as a carbon source, the content of vitamin _B12 in the bacterial body does not become very high; When methanol is used as
We obtained a new finding that the B ₁₂ content becomes significantly high, and based on this new finding, we arrived at the present invention.

That is, the present invention relates to Eubacterium (Eu-
bacterium), has the ability to assimilate methanol, and can produce vitamin _B12 .
This is a method for producing vitamin B ₁₂ , which is characterized by culturing under anaerobic conditions using methanol as a carbon source to produce and accumulate vitamin B ₁₂ within the cells of the microorganism.

The microorganisms used in the present invention (hereinafter sometimes simply referred to as microorganisms) belong to the genus Eubacterium, have methanol assimilation ability, and have vitamin
There are no particular restrictions as long as the microorganism can produce B ₁₂ , but a typical example is a microorganism belonging to Eubacterium limosum. Furthermore, the microorganism belonging to the genus Eubacterium used in the present invention is generally a microorganism that can assimilate carbon compounds other than methanol, such as sugar, along with methanol as a carbon source.

Eubacterium limosum is a Gram-positive obligate anaerobic bacterium that produces organic acids such as acetic acid and butyric acid from carbohydrates, but does not assimilate sucrose, ethanol, etc. The mycological properties of Eubacterium limosum are, for example,
Described in the 8th edition of the Manual.

Representative strains belonging to Eubacterium limosum used in the present invention include ATCC8486 strain,
Examples include the ATCC10825 strain, the DSM2593 strain, and the DSM2594 strain, but among these, the ATCC8486 strain is preferred because it has a fast growth rate in a methanol-containing medium.

In culturing the microorganism of the present invention, methanol is used as a carbon source, and it is preferable that CO ₂ and/or CO ^-- ₃ be present in the culture solution. In addition to CO ₂ generated by decomposing methanol as CO ₂ and/or CO ^-- ₃ , carbonic acid compounds such as CO ₂ and/or NaHCO ₃ may be further added. Furthermore, the use of carbon sources other than methanol, such as sugars such as glucose and fructose, and sugar-containing substances, which can be assimilated by this microorganism, is not prohibited.

The methanol concentration in the culture solution is not particularly limited as long as it allows microorganisms to propagate suitably. The methanol concentration in the culture solution varies depending on the type of microorganism, other medium components, culture temperature, etc., and cannot be determined unconditionally, but in practice it is usually 5% by volume or less, especially 1.5% by volume or less. preferable. Furthermore, like a normal culture medium, it is also possible to contain a nitrogen source, minerals, vitamins, etc. in addition to a carbon source.

Examples of nitrogen sources include ammonium salts, peptones, yeast extracts, meat extracts, cornstarch and casamino acids. Examples of minerals include phosphates, magnesium salts, potassium salts,
Calcium salts, manganese salts, cobalt salts,
Iron salts, zinc salts, etc. can be used. Furthermore, addition of 5,6-dimethylbenzimidazole, choline, betaine, etc. is preferable since it improves the production amount of vitamin _B12 , and these compounds are presumed to act as precursors of vitamin _B12 . Further, it is preferable to include a detecting agent such as resazurin in the culture solution to determine the reduction state of the culture solution.

Since this microorganism is an obligate anaerobic bacterium, it is necessary to culture it under anaerobic conditions. Aerobic culture requires energy such as stirring power and oxygen supply power, whereas anaerobic culture essentially does not require these energies. In actual culture, static culture is employed, but either aeration agitation culture using nitrogen gas or carbon dioxide gas or power agitation culture is also possible. However, the power required at this time is extremely small compared to aerobic culture.

Moreover, as a culture method, both batch culture and continuous culture are possible.

Although the culture temperature varies depending on the microorganism and cannot be specified unconditionally, it is generally set at 20 to 45°C, preferably 30 to 40°C for practical purposes. The culture pH differs depending on the microorganism and cannot be unambiguously determined, but in practice it is usually about
An example is pH 5-8, preferably about PH 6-7.
In actual culture, it is preferable to keep the pH of the culture solution constant, and an alkali such as caustic soda, soda carbonate, or aqueous ammonia is used to adjust the pH.

Vitamin B ₁₂ can be recovered from the culture solution by a method known per se. That is, since vitamin B ₁₂ is essentially produced and accumulated within the bacterial cells, it can be separated from the bacterial cells recovered from the culture solution by solid-liquid separation means such as centrifugation. When separating and recovering vitamin B ₁₂ as cyano vitamin B ₁₂ , the pH of a suspension of bacterial cells in water (a bacterial suspension) is acidified, preferably with an acid such as sulfuric acid.
Extractive separation is possible by adjusting the pH to 4 to 5, adding cyanide such as potassium cyanide to about 50 to 200 ppm, and then heating to 85 to 120°C. In addition, when obtaining vitamin _B12 in the form of methyl or coenzyme, the bacterial cells are extracted using a solvent such as methanol, ethanol, acetone, etc. in the dark using a conventional method.
Vitamin _B12 extracted and separated from bacterial cells is further purified as necessary. This purification is performed by phenol extraction, column chromatography using an ion exchange resin or cellulose, or a combination of these methods.

〔Example〕

The present invention will be explained in more detail with reference to Examples.

In Comparative Examples and Examples, vitamin B ₁₂ was quantified by a microbial method using Lactolus reichmanii (IFO-3376), a vitamin B ₁₂ -requiring mutant strain. In addition, resazurin, a detection agent, was added to the culture solution, and the culture solution was kept in a reduced state so that resazurin remained colorless.

Comparative Example 1 A medium having the composition shown in Table 1 was used for culturing Eubacterium limosum (ATCC8486).

Table 1 KH ₂ PO ₄ 3g Na ₂ HPO ₄ 2g MgSO ₄・7H ₂ O 0.4g Na ₂ CO ₃ 1.0g (NH ₄ ) ₂ SO ₄ 0.5g CH ₃ COONa・3H ₂ O 1.0g Cysteine/hydrochloride 0.5 g CaCl ₂・2H ₂ O 0.1g Yeast extract 10g Resazurin (2% solution) 0.1ml Iron citrate・xH ₂ O 60mg ZnSO ₄・7H ₂ O 10mg MnCl ₂・4H ₂ O 10mg CuSO ₄・5H ₂ O 0.5mg KI 1mg (NH ₄ ) ₆ Mo ₇ O ₂₄・4H ₂ O 1mg CoCl ₂・6H ₂ O 10mg H ₃ BO ₃ 1mg NiSO ₄・6H ₂ O 1mg Biotin 50μg Folic acid 10μg Thiamine/hydrochloride 10mg Riboflavin 100μg Niacin 100μg Pantothene Calcium acid 5 mg p-aminobenzoic acid 100 μg Thioctic acid 100 μg Water 1 750 ml of a medium with the composition shown in Table 1 and an additional 30 g/glucose was added to a culture tank and sterilized under high pressure at 120°C for 20 minutes. Thereafter, it was cooled under a nitrogen stream. Eubacte-rium limosum was prepared in advance by static culture in a 100 ml Erlenmeyer flask using a medium similar to this medium.
(ATCC8486) was inoculated with about 30 ml of seed culture solution, and the culture temperature was 35°C with gentle stirring at 150 rpm.
Anaerobic culture was carried out under nitrogen flow while controlling the temperature to 7.3 with aqueous ammonia. At 15 hours and 25 hours after the start of culture, 25 g of glucose was added to the culture tank. The production amount of vitamin _B12 was approximately 6 mg/30 hours after the start of culture.
The content per gram of dried bacterial cells was approximately 0.30 mg. Even if the culture was continued, no bacterial growth was observed, and the production of vitamin _B12 did not increase.

Comparative Example 2 The same procedure as Comparative Example 1 was conducted except that a medium to which fructose was added instead of glucose as a carbon source was used, and fructose was also used as the carbon in the seed culture solution.

After about 20 hours and about 35 hours had passed after the start of culture, 25 g of fructose was added to the culture tank. The amount of vitamin B ₁₂ produced in 45 hours after the start of culture was approximately 8 mg/g of dry bacterial cells.
The B ₁₂ content was 0.26 mg, and the production of vitamin B ₁₂ did not increase even if the culture was continued beyond this point.

Example 1 750 ml of a medium to which 10 ml/g of methanol was added instead of glucose as a carbon source was placed in a 1-volume culture tank and sterilized at 120° C. for 20 minutes. After adding NaHCO ₃ , which was sterilized by filtration separately, at a concentration of 2 g/
The same procedure as in Comparative Example 1 was conducted except that the pH was adjusted to 7.3 with carbon dioxide gas and methanol was used instead of glucose in the seed culture. Methanol was added at appropriate times so that the methanol concentration in the culture solution was 0.1 to 0.8% by volume. The amount of vitamin B ₁₂ produced was measured approximately 40 hours after the start of culture, and it was approximately 27 mg/g, and the vitamin B ₁₂ content per 1 g of dry bacterial cells was approximately 4 mg.
Therefore, the amount of vitamin B ₁₂ produced was significantly increased compared to each of Comparative Example 1 and Comparative Example 2.

Example 2 750 ml of a medium containing 10 ml of methanol and 2 g of choline chloride instead of glucose in the culture composition was placed in a 1-volume culture tank and sterilized at 120° C. for 20 minutes. 2g/separately filter-sterilized NaHCO ₃
After adding so that the pH becomes 7.3 with carbon dioxide gas,
The same procedure as in Comparative Example 1 was conducted except that methanol was used instead of glucose in the seed culture. Methanol concentration in culture solution is 0.1-0.8% by volume
5,6-Dimethylbenzimidazole was added at appropriate times to achieve a concentration of 5,6-dimethylbenzimidazole at 20 hours after the start of the culture. About 40 hours after the start of culture, dried bacterial cells 1
The vitamin B ₁₂ content per g was 4.8 mg, and the vitamin B ₁₂ production was 33 mg/g.

〔Effect of the invention〕

By culturing microorganisms belonging to the genus Eubacterium, which are anaerobic microorganisms, using methanol, which is inexpensive and stably supplied, as a carbon source, we can produce vitamins that are highly valuable as pharmaceuticals and feed additives.
It has become possible to produce B ₁₂ with less energy, less risk of contamination with bacteria, and less emission of volatile culture medium components, making it extremely advantageous industrially.

Claims (1)

[Claims] 1 Belongs to the genus Eubacterium, has methanol assimilation ability, and has vitamin
1. A method for producing vitamin B ₁₂ , which comprises culturing a microorganism capable of producing B _{12 under anaerobic conditions using methanol as a carbon source, and producing and accumulating vitamin B 12 in} the cells of the microorganism.