JPH07163387A - Production of b12 - Google Patents

Abstract

PURPOSE:To obtain the subject compound useful as a therapeutic agent for malignant anemia, nervous system disease and methylmalonic aciduria industrially and advantageously by culturing Rhizobium cobalaminogenum capable of producing vitamin B12 in a medium, accumulating and collecting a product. CONSTITUTION:Rhizobium cobalaminogenum 27B47 strain (FERM-BP-4429) capable of producing vitamin B12 is inoculated into a medium containing a cobalt compound (e.g. cobalt chloride) and 5,6-dimethylbenzimidazole compound, cultured at 30 deg.C for 6 days on a rotary shaker and vitamin B12 is formed, accumulated and centrifuged to collect cells. These cells are suspended in an acetic acid buffer, adjusted to pH 5.0, mixed with KCN, heated at 100 deg.C for 15 minutes and centrifuged and the precipitate is removed to give an extracted solution of vitamin B12. Then the extracted solution is treated by an active carbon column and an adsorption resin column and purified to give the objective vitamin B12 useful as a therapeutic agent for malignant anemia, nervous system disease and methylmalonic aciduria.

Description

Detailed Description of the Invention

[0001]

The present invention relates to pernicious anemia, neurological diseases,
The present invention relates to a method for producing vitamin B ₁₂ , which is widely used as a feed additive for poultry and / or livestock, in addition to being used as a therapeutic agent for methylmalonic aciduria.

[0002]

2. Description of the Related Art Regarding the method for producing vitamin B ₁₂ , it is industrially difficult to use the chemical synthesis method due to the complexity of its structure, and the fermentation production method using microorganisms is currently used. . With respect to the production of vitamin B ₁₂ by microorganisms, Streptomyces (Streptomyces) genus Nocardia (Nocardia) sp., Micromonospora (Micromonos
pora ), Aerobacter genus, Agrobacterium genus, Alcaligenes ( Alc
aligenes) genus, Azotobacter (Azotobacter) genus Bacillus (Bacillus) sp., Clostridium (Clostridiu
m) the genus Corynebacterium (Corynebacterium) genus Escherichia (Escherichia) sp., Flavobacterium (F
genus lavobacterium , Mycobacteriu
m) the genus Pseudomonas (Pseudomonas) genus Propionibacterium (Propionibacterium) genus, Proteus (P
roteus) genus Serratia (Serratia) genus Streptococcus (Streptococcus) genus Xanthomonas (Xanthomon
as ) genus, Protaminobacter genus,
Metanobachirusu (Methanobacillus) genus [E Jay Bandame (EJ Vandamme), biotechnology
Biotechnology of Vitamins, Pigments andG
rowth Factors, Elsevier Science Publish
er LTD), 1989, pp. 261-263], Arthrobacter genus (JP-A-52-9449).
8), Klebsiella (Klebsiella) belonging to the genus (JP-A-50-1
32186), Rhodopseudomonu
s) belonging to the genus (JP-A-60-16597), blanking Chile Agrobacterium (Butyribacterium) belonging to the genus (JP-A-62-4419
7), Pseudonocardia genus (Japanese Patent Laid-Open No. 55-96091), Methanosarci ( Methanosarci )
na) genus (JP 1-257490), Yu Mycobacterium (Eubacterium) genus (JP 62-44172), aceto Mycobacterium (Acetobacterium) genus (Japanese 62-1
22593) has been reported. Rhizobium meliloti , Rhizobium
Fazeori (Rhizobium phaseoli), Rhizobium japonicum (Rhizobium japonicum), Rhizobium Torifori (Rhizobium trifolli), Rhizobium leguminosarum (Rhizobium leguminosarum) Various have been reported to accumulate vitamin B ₁₂ [plant Fijii Oh Biology (Plant Physiology ), 38 , 99-1
04 (1963)]. Among the Rhizobium spp., Rhizobium phaseoli and Rhizobium trifolii have been renamed Rhizobium reguminosalum [Fermentation Research Institute, List of Cultures (List).
of Culutures) 1992 Microorganisms (M
icroorganisms), 9th edition, p. 169]. In addition, a part of Rhizobium japonicum was renamed Rhizobium leguminosalum, and the rest was reclassified to Bradyrhizobium japonicum [Fermentation Research Institute, List of Cultures]. 1992, Microorganisms, 9th Edition, p. 169].

[0003]

However, the production of vitamin B ₁₂ by microorganisms is generally extremely low. In addition, some microorganisms have limited available carbon sources (such as methanol-assimilating bacteria) or require special culture conditions such as anaerobic conditions (propionic acid bacteria, acetobacterium, methane, etc.). There are many industrial production methods that can be performed only under limited conditions, such as producing bacteria), which require a long culture time due to a low growth rate (such as methanogenic bacteria and propion bacteria). Development of improved industrial processes is desired.

[0004]

The present invention provides an inexpensive and efficient industrial process for producing vitamin B ₁₂ . The present inventors have used vitamin B ₁₂ which is widely used for fermentation production as a carbon source, and also the aeration-agitation culture method which is a fermentation production condition which is also commonly used. Rhizobium cobalaminogenum 27B74, a new strain with a new high productivity of vitamin B ₁₂ , was isolated from the soil of Hyogo prefecture in order to develop a method for efficient fermentative production of vitamins. The present invention has been completed based on the findings obtained. That is, the present invention provides (1) a Rhizobium Kobaraminogenumu having an ability to produce vitamin B ₁₂ were cultured in a medium, yielding accumulate vitamin B _12, the preparation of vitamin B _12, characterized in that collecting the ( 2) The production method described in (1) above, wherein the medium contains a cobalt compound, (3) The production method described in (2) above, wherein the cobalt compound is cobalt halide, and (4) the medium is 5,6-dimethylbenzimidazole. The production method according to claim 1, which comprises a compound, (5) the production method according to (1) above, wherein the Rhizobium cobalaminogenum is Rhizobium cobalaminogenum 27B74 strain (FERM BP-4429), and (6) the ability to produce vitamin B _12. Rhizobium cobalaminogenum and (7) microorganism Rhizobium cobalaminogenum 27B
74 strains (FERM BP-4429).

In the present invention, vitamin B ₁₂ is coenzyme type vitamin B ₁₂ [eg, adenosyl cobalamin, methyl cobalamin (methyl type cobalamin)], cyano type vitamin B ₁₂ (cyanocobalamin) or hydroxo type vitamin B ₁₂ (hydroxo). Cobalamin). With good vitamin B ₁₂ producing ability isolated from soil, Rhizobium Kobaraminogenumu (Rhizobium cobala
minogenum ) 27B74 (IFO 15543, FER
The morphological properties of MBP-4429), the growth state in each medium, and the physiological properties are shown below. (A) Morphological properties (when grown on broth agar medium at 28 ° C. for 24 hours) Cell morphology: Bacillus 2. Cell size: 0.5-0.8 μm × 1-2.8 μm 3. Presence or absence of spores: None 4. Motility: Yes (observation after broth liquid culture, growth at 28 ° C. for 16 hours) Gram stainability: negative 6. Anti-acidity: None (B) Growth condition in each medium

[Table 1] (C) Physiological properties

[Table 2] (D) Availability of various sugars

[Table 3]

The production of gas and acid from the above sugars was not observed after 14 days of culture. The above-listed properties are described in Bergey's Manual of Systematic Bacteriology.
Bacteriology) 1st edition (1984), the bacteria were classified according to the genus Rhizobium (Genus Rhizo).
bium ) fungus. The GC content of DNA of this bacterium was determined to be 63.4%. Furthermore, as a result of extracting and analyzing quinones, all had coenzyme Q-10. Moreover, the result of the microbial cell fatty acid composition has 3-hydroxy fatty acid 14: 0, 16: 0, 18: 0 (carbon number: double bond number) as a hydroxy fatty acid, and the ratio is 66 :. It was 17: 7. Linear fatty acids had 16: 0, 18: 0, 18: 1, with 18: 1 being the most abundant. It is already known that many of the genus Rhizobium exhibit the above-mentioned bacterial fatty acid composition. However, linear fatty acids 21: 1 and 19: 0
Is different from the conventionally known bacteria of the genus Rhizobium in that it does not have an identified strain and that acid production is not observed from L-arabinose. further,
The method for DNA homology analysis by Ezaki et al. [Ezaki et al., International Journal of Systematic Bactology]
eriology), 39 , 224-229 (1989)] in comparison with a conventionally known preserved strain of Rhizobium. The results are shown below.

[0007]

[Table 4] The 27B74 strain did not show homology as the same species as any of the strains. Therefore, it was concluded that this strain is a new strain belonging to the genus Rhizobium, and the strain name is cobalaminogenum ( co
balaminogenum ). In this specification, the IFO number is a deposit number for the Fermentation Research Institute (IFO, 2-1785, 13 Sanhonmachi, Yodogawa-ku, Osaka-shi, Osaka Prefecture), and the FERM BP number is the biotechnology technology of the Agency of Industrial Science and Technology. Research institute (FRI 1-3-3 East, Tsukuba City, Ibaraki Prefecture)
No.) under the Budapest Treaty. Rhizobium cobalaminogenum 27B74 was 1993 9
To IFO on the 2nd of the month Accession number IFO 15543, 199
To FRI on September 29, 3rd, Accession number FERM BP-4
429, respectively.

For culturing the vitamin B ₁₂ producing bacterium obtained as described above, the same method as that for culturing ordinary microorganisms is used. That is, as the medium, in addition to carbon sources, nitrogen sources, inorganic substances, metal salts, or yeast extract, yeast cells, substances such as ribonucleic acid, amino acids, if necessary,
A medium containing a nutrient source such as vitamins is used.
For example, as a carbon source, glucose, choucloth, maltose, sorbitol, starch, saccharified starch solution,
Carbohydrates such as molasses, various organic acids such as pyruvic acid, fumaric acid, malic acid and succinic acid, alcohols such as ethanol and methanol, amines such as betaine, choline and monoethanolamine are used. As the nitrogen source, in addition to organic nitrogen sources such as peptone, corn steep liquor, soybean powder, and urea, ammonium salts such as sulfuric acid, nitric acid, hydrochloric acid, and carbonic acid, and inorganic nitrogen sources such as ammonia gas and ammonia water are used. Used alone or as a mixture. As other nutrient sources, in addition to substances such as yeast extract, yeast cells, ribonucleic acid, etc., inorganic salts such as calcium salts, magnesium salts, potassium salts, phosphates, amino acids, vitamins, etc. are appropriately selected. The above may be used alone or as a mixture. Furthermore, a defoaming agent such as silicone oil and a surfactant such as polyalkylene glycol ether can be added to the medium, if necessary. Further, it is preferable to add a cobalt compound or a 5,6-dimethylbenzimidazole compound as a precursor of vitamin B ₁₂ . These compounds increase the yield of vitamin B _{12 in} the medium. As the cobalt compound, any cobalt source or its precursor can be used. Examples of the cobalt compound include cobalt halides (eg, cobalt chloride, cobalt bromide, etc.), cobalt nitrate, cobalt sulfide, cobalt acetate, ammonium sulfate cobalt, cobalt carbonate, cobalt 4-cyclohexylbutyrate, cobalt 2-ethylhexanoate, and the like.
Examples thereof include cobalt hydroxide, cobalt phosphate, cobalt oxide and cobalt thiocyanate. Also, 5,6-
As the dimethylbenzimidazole compound, any 5,6-dimethylbenzimidazole source or its precursor can be used. Examples of the 5,6-dimethylbenzimidazole compound include 5,6-dimethylbenzimidazole, nicotinamide, nicotinate adenine dinucleotide (NAD), nicotinamide adenine dinucleotide (NaAD), nicotinate mononucleotide (NaMN), and nicotine. Examples thereof include amide adenine dinucleotide phosphate (NADP) and nicotinic acid.

The culture is usually carried out under aerobic conditions. Specifically, it is carried out, for example, by shaking or aeration-agitation deep culture. Usually, the pH of the medium is preferably in the range of about 4-9. If pH fluctuations are observed during culturing, in order to correct this to a preferable range, acid (eg, sulfuric acid, hydrochloric acid, acetic acid, etc.), alkali (eg, calcium carbonate,
Sodium hydroxide, ammonia gas, ammonia water, etc.)
May be added as appropriate. The culture temperature is usually selected from the range of about 20 ° C to 45 ° C, and a temperature suitable for the growth of microorganisms and accumulation of vitamin B ₁₂ is selected. Culturing is carried out until the vitamin B ₁₂ accumulation amount is substantially maximized, but the purpose can usually be reached by culturing for about 2 to 10 days. Vitamin B ₁₂ producing bacteria belonging to Rhizobium cobalaminogenum can be mutated by irradiation with ultraviolet rays, radiation, etc., single cell separation, various mutation treatments, and other conventional means, as in the case of other bacteria.
Even such mutant strains or naturally obtained mutant strains that are substantially different from each other in comparison with the above taxonomical properties and have the ability to produce vitamin B ₁₂ are All can be used in the method of the present invention.

Vitamin B ₁₂ and vitamin B ₁₂ from the culture
In order to separate and collect each component, a commonly known conventional purification means, for example, a solvent extraction method using phenol, butanol, a precipitation method, a chromatography method using an ion exchange resin, silica gel, activated carbon, etc. J. Florent and L. Ninet, Vitamin B ₁₂ Microbial Technology HJ Pepler
And Day Pearlman (HJ Peppler and D. Per
Lman), Academic Press NW (Academ
ic Press, NY), pages 497-519 (1979)
Year)〕. More specifically, for example, by centrifuging a culture to obtain bacterial cells, an inorganic acid (eg, hydrochloric acid, sulfuric acid, etc.) or an organic acid (eg, acetic acid, tartaric acid, etc.) is used to obtain p.
After adjusting H to about 5.0, cyanide is added and heated to extract into water as a cyano type. When vitamin B ₁₂ is obtained in the form of methyl or coenzyme, alcohols (eg, ethanol, propanol, etc.), ketones (eg, acetone,
Extract with (methyl ethyl ketone, etc.). When extracting vitamin B ₁₂ from the bacterial cells, it is also effective to crush the bacterial cells by a method known per se, if desired.

[0011]

The present invention will be described in more detail with reference to the following examples. Unless otherwise specified,% and ratio in the examples indicate% by volume and ratio by volume, respectively. Example 1 5 ml of a broth liquid medium containing 1% (wt%) sucrose was dispensed into a test tube with a cotton plug and then sterilized by heating. Rhizobium cobalaminogenum 27B74 (IFO 1554, which had been grown on this medium in advance on broth agar at 30 ° C. for 3 days.
3, FERM BP-4429), one platinum loop inoculation,
The cells were cultured at 30 ° C. for 24 hours with shaking. 20 ml of the medium having the composition shown in [Table 5] was placed in a 200 ml flask and sterilized by heating. Then, 1.0 ml of the above culture was inoculated into the flask and cultured at 30 ° C. for 6 days on a rotary shaker at 230 rpm. Cells were collected from 1 liter of the culture obtained by the same method as above by centrifugation. The obtained bacterial cells were suspended in an acetate buffer to adjust the pH to 5.0, and KCN was added to the suspension to give a final concentration of 100 μg / ml, followed by heating at 100 ° C. for 15 minutes. After cooling, the precipitate was removed by centrifugation to obtain a vitamin B ₁₂ extract. The amount of vitamin B _{12 in the} extract was quantified by high performance liquid chromatography. The column used was ODP-50 4.6 × 150 mm (manufactured by Asahi Kasei), and 40 mM ammonium acetate: acetonitrile = 8 was used for elution of vitamin B ₁₂ from the column.
8:12 was used. The vitamin B ₁₂ content in the eluate was determined by measuring the absorbance at 361 nm. As a result, it was confirmed that 22 μg / ml of vitamin B ₁₂ was produced in the above culture. Next, the above vitamin B
_{The 12} extract was applied to an activated carbon column to adsorb vitamin B ₁₂ . After washing the column with distilled water,
Elution with 0% acetone. Collect the vitamin B ₁₂ fractions,
After concentration under reduced pressure, it was applied to a column of Dowex 50. After washing the column with water, the adsorbed substance was eluted with a 50 mM sodium acetate buffer (pH 6.5). Vitamin B
_Twelve fractions were collected, neutralized, and reapplied to the activated carbon column. After washing the column with distilled water, remove the adsorbate by 40%
Elute with acetone. Vitamin B ₁₂ fractions were collected and concentrated under reduced pressure, and then acetone was added to this to obtain vitamin B ₁₂
Was crystallized to obtain 16.5 mg of vitamin B ₁₂ (cyano type) crystals.

[0012]

[Table 5]

Example 2 In order to obtain each component of vitamin B _{12 in the} coenzyme form, Example 1
The bacterial cells were collected from 1 liter of the culture obtained in 1. above by centrifugation. 80% ethanol (without CN) concentration in the dark
The resulting cells were heated and extracted at 80 ° C. for 20 minutes. The ethanol of the extract was removed under reduced pressure to obtain an aqueous solution. The obtained aqueous solution was used as Sep-Pak C18.
After desalting with (Waters), the coenzyme-type vitamin B ₁₂ was identified by high performance liquid chromatography. Column is Lichrospher RP
18 (5 × 250 mm) (manufactured by Merck) was used, and the elution from the column was carried out by a gradient method using 85 mM phosphoric acid (pH 3.0) and acetonitrile. The concentration of acetonitrile was adjusted to 0% (at the start of elution) to 50% (25 minutes after the start of elution). The content of coenzyme-type vitamin B ₁₂ in the eluate was determined by measuring the absorbance at 361 nm. As a result, adenosyl cobalamin (Ado-B ₁₂ ) (18 μg / ml), methylcobalamin (CH ₃ -B ₁₂ ) (2.5 μg / ml) and hisosoxocobalamin (OH-B ₁₂ ) were added to the culture. (1.0 μg / m
l) was found to have been produced.

Comparative Example In the same manner as in Example 1, Rhizobium meliloti ( Rh
izobium meliloti ) IFO 14782, Rhizobium
Rhizobium galegae IFO 14965,
Rhizobium Fuakui (Rhizobium huakuii) IFO
15243, Rhizobiu
m leguminosarum ) IFO 13337, Rhizobium
Rhizobium leguminosarum IFO
14784, Rhizobium loti
IFO 13336 and Rhizobium tropisis ( Rh
Izobium tropici ) IFO 15247 was cultured at 30 ° C. for 6 days. The vitamin B ₁₂ content of the extract was quantified from the obtained culture using the method described in Example 1. The results are shown in [Table 6].

[Table 6]

[0015]

Industrial Applicability According to the present invention, vitamin B ₁₂ can be industrially manufactured at low cost, easily and efficiently.

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Claims (7)

[Claims] 1. Rhizobium cobalaminogenum having the ability to produce vitamin B ₁₂ is cultured in a medium to obtain vitamin B _12.
A method for producing vitamin B ₁₂ , which comprises producing and accumulating ₁₂ and collecting this. 2. The medium contains a cobalt compound.
The manufacturing method described. 3. The method according to claim 2, wherein the cobalt compound is cobalt halide. 4. The method according to claim 1, wherein the medium contains a 5,6-dimethylbenzimidazole compound. 5. Rhizobium cobalaminogenum is Rhizobium cobalaminogenum strain 27B74 (FERM BP-
4429). 6. Rhizobium cobalaminogenum having the ability to produce vitamin B ₁₂ . 7. The microorganism Rhizobium cobalaminogenum 2
7B74 strain (FERMBP-4429).