JPS6240295A - Production of vitamin b12 - Google Patents

Abstract

PURPOSE:To efficiently collect vitamin B12 from microbial cells, by cultivating a microorganism, belonging to the genus Eubaterium and having methanol assimilating property under anaerobic conditions in producing the vitamin B12 by the microorganism. CONSTITUTION:A microorganism, belonging to the genus Eubacterium and having the methanol assimilating property and ability to produce vitamin B12, e.g. Eubacterium limosum, is cultivated in a culture medium containing methanol as a carbon source and a nitrogen source, e.g. peptone or meat extract, and carbon source, e.g. sugar, under anaerobic conditions. Microbial cells recovered by the solid-liquid separation are suspended to form a microbial suspension, which is acidified. A cyanide is added thereto and the aimed vitamin B12 is extracted and separated by heating.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for producing vitamin 812, and more specifically, the present invention relates to a method for producing vitamin 812, and more specifically, a method for culturing a microorganism belonging to the genus Niebacterium, which is a unique anaerobic bacterium, using methanol as the main carbon source. The present invention relates to a method for producing vitamin B12, which is characterized by the following.

Vitamin B12 is involved as an essential factor in various biosynthetic reactions in vivo, and has been found to have excellent medicinal effects for pernicious anemia, chronic neurological diseases, etc., in addition to being used as a feed additive. Demand has been increasing in recent years.

[Prior art, problems to be solved by the invention] Vitamin B12 has a complex structure.

Chemical synthesis is not practical because it requires a complex, multi-stage synthesis process.

Another method is to extract vitamin B12 from animal organs such as cow liver, but this method is not suitable for industrial use because the content of vitamin B12 in animal organs is low, and the supply of animal organs is small and unstable. It cannot be said that this is a preferable manufacturing method.

On the other hand, there is also a method of using microorganisms belonging to the genus Pseudomonas, Nocardia, Streptomyces, and 7-Slobacter. 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 cultured using sugars such as glucose, fructose, and shakerose, or blackstrap molasses containing these sugars, as a general KR source, but these sugars and sugar-containing substances are Since these microorganisms are relatively expensive and their supply is unstable, methods using these microorganisms are also not preferred as industrial methods.

Therefore, it has methanol assimilation ability and vitamin 81
If an anaerobic microorganism exists that can efficiently produce 2, it would use methanol, which is inexpensive and stably supplied in large quantities as an industrial raw material, as a carbon source, essentially eliminating the need for aeration and stirring, and eliminating contamination by bacteria. The risk of being damaged is relatively small, and the amount of volatilization of volatile medium components is significantly reduced, making it possible to industrially advantageously produce vitamin B12.

[Means and actions for solving problems]

The present inventors have conducted various studies on a method for producing vitamin B12 by culturing microorganisms using methanol as a carbon source. As a result, the vitamin B12 content in the microbial cells does not become very high when the anaerobic microorganisms of the genus Niebacterium are cultured using sugars such as glucose and fructose as carbon sources; We obtained new knowledge that when methanol was used as a liquid, the vitamin B content in the bacterial cells increased significantly.
The present invention was achieved based on this new knowledge.

That is, the present invention relates to Eu-bacterium (Eu-bacterium
A microorganism belonging to the genus B. erium, capable of assimilating methanol, and capable of producing vitamin 812 is cultured under exhaust conditions using methanol as a carbon source, and vitamin B is produced and accumulated in the microbial cells of the microorganism. This is a method for producing vitamin B12, which is characterized by the following.

The microorganisms used in the present invention (hereinafter sometimes simply referred to as microorganisms) are not particularly limited as long as they belong to the genus Niebacterium, have methanol assimilation ability, and can produce vitamin B12. However, a typical example is Eubacterium lim
osum) There are microorganisms that belong to K. Furthermore, the microorganism belonging to the genus Niebacterium used in the present invention is generally a microorganism that can assimilate methanol as well as carbon compounds other than methanol such as KW&.

Batatherium lithosum does not assimilate ethanol, etc. The chemical properties of N. limosum are, for example, %Ber-gey's Manua
1 Retail listed in the 8th edition.

Representative strains belonging to Niebacterium limosum used in the present invention include ATCCaa86 strain%ATCC10
825 strain, DSM 2593 strain and DSM 25
Among them, 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 Cot and /l or COs exist in this culture solution. This Co2 and/or force XI' can be produced as CO5-1 by decomposition of methanol.

. 2゜! □. . 2 and u/l,,,yu,8□. . 3 -□□ Any carbonate compound may be further added. Also, glucose and fructose are used as carbon sources.
1. It does not preclude the combined use of carbon sources other than methanol that can be assimilated by the microorganisms, such as any sugars or sugar-containing substances.

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 can be adjusted depending on the type of microorganism, other ordinary culture media, and in addition to carbon sources, nitrogen sources, minerals, vitamins, etc. can also be included.

Mention may be made of hefton, yeast extract, meat extract, cornstap liquor 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, for example, 5,6-dimethylpenzimidazole,
Addition of choline, betaine, etc. is preferable because it improves the production of vitamin B, and these compounds are presumed to act as precursors of vitamin B12. Also.

It is preferable to include a detection agent such as resazurin in the culture solution to determine the reduction state of the culture solution.

Since this microorganism is an 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. When actually culturing the culture 111, 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.

In addition, the culture methods include batch culture and continuous culture. A difference in culture is also possible.

The culture temperature varies depending on the microorganism and cannot be generally specified, but in practice, it is usually 20 to 45°C.

The temperature is preferably 30 to 40°C. The culture pH varies depending on the microorganism and cannot be generally specified, but in practice, it is usually about pH 5 to 8, preferably about pH 6 to 7.

In actual culture, it is preferable to keep the pH of the culture solution constant, and the pH can be adjusted using caustic soda, sodium carbonate,
An alkali such as Nmoni 7 water is used.

Vitamin 812 can be recovered from the culture solution by a method known per se. That is, since vitamin B is essentially produced and accumulated within the microbial cells, it can be separated from the microbial cells collected from the culture solution by solid-liquid separation means such as centrifugation. Vitamin 812 as cyano vitamin B1
In the case of separating and recovering as 2, the pH of a suspension of bacterial cells in water (M-body suspension) is adjusted to acidic, preferably pH 4 to 5, with an acid such as sulfuric acid, and the pH is adjusted to acidic, preferably pH 4 to 5. After adding about 50-200 ppm of cyanide, 85-12
By heating to 0°C, paper extraction separation is possible. When vitamin 812 is obtained in the form of methyl or coenzyme, it is extracted from the bacterial cells in the dark using a solvent such as methanol, ethanol, 7-setone, etc. by a conventional method. Vitamin B extracted and separated from itself is further purified if 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 B12 was quantified by a microbial method using Lactobacillus reichmanii CIFO-3576)ft, a vitamin B-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 I Eubacterium limosum (ATCC84
86), a medium having the composition shown in Table 1 was used.

Table 1 KH*POa 5. 1ilNazHP
O429.

Mg5Oi・7H200,4g 3. 4) 2504 0.517:.

NaxCOsLogCHsCOON
a ・5HzOl, 09 cydstein hydrochloride
0.5gCaC1*・2HzOO,IJ Yeast extract 10g Resazurin (296 solution (fund) 01td
-Iron citrate・xH2O60daZnSO4・7H2010R9 MnC1z・4HzO101it CuSO4・5H200,519 Kl + ■(
NH4) 6Mo 7o24-aHρ 1~CoC
l2-6HzO10# H3BO31try NiSQ<・(SH201 dapiotin 50 41 leaves
Acid 10 μg Thiamine・
Hydrochloride 10 1n9 Riboflavin 100 μ9 Knife Schiff
100 μ9 calcium pantothenate 5 dap-7 minobenzoic acid
100 μI thioctic acid
100 μI water
11 Add 30 more to the medium with the composition shown in Table 1.
Medium containing g/g glucose 750- to 1! The mixture was placed in a culture tank and autoclaved at 120°C for 20 minutes, followed by cooling under a nitrogen stream. Eubacterium Iimosum (A
Approximately 5 arnl of seed culture of TCC8486 was inoculated,
Slowly stir at 150 rpm at a culture temperature of 35°C,
Anaerobic culture was carried out under a nitrogen stream while controlling the culture pH to 7.3 with 77 Monis water. 15 days after starting culture
After the passage of time and after 25 hours, 25 g of glucose was added to the culture tank. Thirty hours after the start of culture, the production amount of vitamin B was about 6119/l, and the content per 1.9 dry bacterial cells was about 0.3019.

Even if the culture was continued, no bacterial growth was observed, and the amount of vitamin 812 produced did not increase.

Comparative Example 2 Using a medium supplemented with Fruct□ instead of glucose as a carbon source, the seed culture solution was
1□ The same procedure as Comparative Example 11 was carried out except that fructose was also used as Kel carbon.

After about 20 hours and about 35 hours had passed after the start of culture, 25 II of 7lactose was added to the culture tank. 45 hours after the start of culture, the vitamin B12 production amount was approximately 81119/l, and the 812 content per 1 g of dry bacterial cells was 0.2689, so even if the culture was continued beyond this point, the vitamin B12 production value would increase. I didn't.

Example 1 10 m of methanol was added to glucose as a carbon source.
Put 750mj of medium added with 1/9 into an 11 volume culture tank,
It was sterilized at 120°C for 20 minutes.

Separately, filter-sterilized NaHCOi was added to a concentration of 2 f//11, and then the pH was adjusted to 7.3 with carbon dioxide gas. Ta. After starting culture, about 4
When the vitamin B12 production amount was measured at 0 hours, it was approximately 279/l, and the vitamin B content per 1 g of dry bacterial cells was approximately 4 caps. , vitamin B production increased significantly12
I added it.
[Example 2
(□ Medium composition: instead of l-X ・' J
/-t.

"1゛°"／l＞! ＃jJ［か°”72 fl
Add 750 ILl of culture medium to an 11-volume culture tank and sterilize it at 12°C for 20 minutes.
18°HCOs Ir 2 ,! i'/l
K″h!') mhnL, fF-'4, .

The pH of the extract was adjusted to 7.3 with carbon dioxide gas, and methanol was used instead of glucose in the seed culture, and methanol was added at appropriate times.When 20 hours had passed after the start of the culture, the pH was 5.6-
Dimethylpenzimidazole was added at 20W/IK.

At about 40 hours after the start of culture, the vitamin B content per 19 dry bacterial cells was 4.8 μ, and the vitamin B production was 33 da/l.

〔Effect of the invention〕

Microorganisms belonging to the genus Niebacterium, which are anaerobic microorganisms, are available at low prices! By culturing with a stable supply of methanol as a carbon source, Pitamin B, which is highly valuable as a pharmaceutical and feed additive, can be produced using less industrial water, less risk of contamination with bacteria, and a volatile culture medium. The amount of volatilization of the components has been reduced, making it possible to produce it industrially with great advantage.

Patent applicant: Mitsubishi Gas Chemical Co., Ltd. Representative Kazuyoshi Nagano

Claims (1)

[Claims] Belongs to the genus Eubacterium, has methanol assimilation ability, and contains vitamin B_1_2
Vitamin B_1_2 is produced and accumulated in the cells of the microorganism by culturing a microorganism capable of producing it under anaerobic conditions using methanol as a carbon source.
_2 manufacturing method.