JPS5995298A - Separation and purification of vitamin b12 - Google Patents

Abstract

PURPOSE:To obtain the titled compound by a simple device by easy operations industrially advantageously, by bringing a solution comprising vitamin B12 containing admixtures into contact with a divinylbenzene/syrene copolymer resin, adsorbing vitamin B12 to the resin, washing it with pure water at a specific temperature, eluting it with an eluent. CONSTITUTION:A product containing vitamin B12 obtained by cultivating a strain of Propionibacterium shermanii IFO12391 capable of producing vitamin B12 is centrifuged, and filtered to give a mold. Vitamin B12 together with admixtures is extracted from the mold with water at 80 deg.C, to give an aqueous solution of crude vitamin B12. The solution comprising vitamin B12 containing admixtures is brought into contact with a divinylbenzene/styrene copolymer resin, vitamin B12 is adsorbed on the resin, the resin is washed with pure water or a solution with low concentration of an acid in pure water at 30-70 deg.C, adsorbed vitamin B12 is eluted with an elutant, and an active eluate fraction is collected to give the desired high-purity compound.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention contains vitamin B1ff1 containing extraneous components.
Liquid, such as fermentation method vitamin BI! Containing liquid, cell extraction method Vitamin Bl! Simple and easy adsorption, washing, and elution operations and equipment for vitamin B□-containing liquids containing impure components, such as vitamin B□-containing liquids, without requiring additional operations such as additional purification or pre-purification. A method for purifying and separating vitamin B□, which can separate and recover purified vitamin B□ in high purity and in a high yield with satisfactory purity, for example, exceeding about 60 cm and reaching as high as 80 cm, and this method This invention relates to a purification separation method using a cleaning treatment solution after an adsorption operation suitable for use in the implementation of the present invention.

Vitamin Bl from vitamin B□-containing liquid containing extraneous ingredients!
One method for separating vitamin B1! is to use divinylbenzene/styrene copolymer resin as an adsorbent and perform an adsorption-washing-elution operation. There are known methods for containing liquid. According to that method, the resin and a vitamin B1ff1-containing solution containing extraneous components are brought into contact at 10 to 60°C,
High purity vitamin B1! is obtained by adsorbing vitamin B□ onto the resin, washing the resin, and eluting the adsorbed vitamin Bst'l with an eluent to obtain an active elution fraction. , with high yield, F? It is possible to obtain I-manufacturing and dispensing.

However, after adsorption treatment, water, low concentration hydrous alcohols such as 20% methanol water, 10% ethanol water, 5% probanol water, and low concentration acid solutions such as 1%
Phosphoric acid aqueous solution, 1-thiacetic acid aqueous solution, 1-thioboric acid aqueous solution, 0
．． When cleaning with 1% hydrochloric acid, etc., depending on the type and amount of impurities, the cleaning effect may be small even if the cleaning solutions are used in combination, and the active fraction may be eluted with an eluent after cleaning. Vitamin BI in classification! Purity was low. Furthermore, even when the washing treatment liquids were combined and used in large quantities, the purity of the active elution fraction was still unsatisfactory. Furthermore, in hydrous alcohol, absorbed vitamin B1! is partially desorbed from the resin, resulting in a yield loss, and on an industrial scale, it also has the unavoidable disadvantage of requiring additional equipment for the recovery of the alcohol. In particular, fermentation method vitamin B□-containing liquid, cell extraction method vitamin B□-containing liquid 1
j Vitamin B1 with a relatively large amount of undesirable components! This is even worse for liquids containing crude vitamins B and 2, and the cleaning liquid used in the cleaning process after the adsorption treatment of these crude vitamin B and 2-containing liquids has high purity and high yield and is purified vitamin B! It has been found that this is a problem that cannot be ignored in a method for purifying and separating vitamin B1t that can separate and recover vitamin B1t.

The present inventors have discovered that vitamin B1 contains extraneous ingredients! By overcoming the above-mentioned technical problems in separating and obtaining purified vitamin B□ from the containing liquid by adsorption-washing-elution method, we can obtain the crude vitamin B1 with excellent purity and yield! Research has been carried out to develop a method that can industrially advantageously purify and separate vitamin B and gg from the liquid containing it.

As a result, the temperature of the cleaning treatment liquid after the adsorption operation was 30.
℃~70℃, pure water such as ion-exchanged water or distilled water or a low concentration acid bath solution does not desorb vitamins Bl and Y, and can quickly remove impurities attached to the resin surface in small amounts. It has an excellent cleaning ability of rinsing away vitamin B, and has a selective cleaning ability of sufficiently high purification of vitamin B in the active fraction eluted with an eluent after washing treatment. 13 tg of purified vitamins are separated with a satisfactory purity of about 60% to 80% by simple and easy adsorption, washing-elution operations and equipment without requiring any additional and/or preliminary purification means. We discovered that it is possible to collect

Therefore, an object of the present invention is to provide an improved method for purifying and separating vitamin B□.

Another object of the invention is to use a cleaning treatment solution after the adsorption operation which is suitable for use in carrying out such a method.

The cleaning treatment liquid used in the present invention is pure water such as ion-exchanged water or distilled water or a low concentration acid solution of pure water having a temperature of 60 to 70°C, preferably 40 to 6o'C. While water and low concentration acid solutions in conventional methods have poor affinity for impurities, vitamin B□ has a high affinity for impurities attached to the resin surface and is adsorbed on the resin.
Impurities can be selectively and efficiently washed away without being desorbed from the resin.

It is an organic acid. Furthermore, the acid solution referred to in the present invention is 5wt
% or less, preferably 1 wt% or less.

The impurities mentioned here include fatty carboxylates such as sodium acetate, sodium propionate, sodium butyrate, sodium valerate, etc., amino acids such as glutamic acid, aspartic acid, proline, leucine, and nucleobases such as adenine, guanine, cytosine,
Examples include compounds such as thymine and uracil. For example, when the water temperature is 50°C, the solubility of Kochu et al. in water is 25
It is about 1.6 to 5.0 times higher than ℃.

Furthermore, the cleaning treatment liquid used in the present invention is pure water such as ion-exchanged water or distilled water that does not contain a water-soluble organic solvent, and in the conventional method, it is difficult to use water-containing alcohol at a low concentration on an industrial scale. Although the disadvantage of requiring incidental equipment for solvent recovery cannot be avoided, since solvent recovery is not required, it can be carried out industrially advantageously.

The present invention uses a divinylbenzene/styrenic copolymer resin as an adsorbent, which is a copolymer resin obtained from divinylbenzene, styrene, or a functional derivative thereof as the main polymer component, or Divinylbenzene, styrene, or their functional derivatives are used as main components. nK is the following formula, where R is 03 to C having a carbon-carbon double bond.
A copolymer resin derived from an aromatic polycarboxylic acid unsaturated alkyl ester (hereinafter referred to as DST resin), where n is an unsaturated alkyl residue of 1°, and n is 2 or 3. There is).

These resins are generally obtained by polymerizing the above monomers using a known radical polymerization initiator. Here, the content of styrene or its functional derivative is usually 60 to 80 wt%96, preferably 45 to 70 wt%, and when the aromatic polycarboxylic acid unsaturated alkyl ester is used, the content is 0.1 to 30 wt%. Preferably 1 to 10 wt% is 44-11/1-t.

According to the present invention, the above-mentioned resin is brought into contact with a vitamin 13tt-containing liquid containing undesired components, and the resin is mixed with vitamin Bt.
High-purity vitamin Btt 'f is adsorbed, the resin is washed with the washing treatment liquid, and the adsorbed vitamin Btt 'r' is eluted with an eluent to obtain an active elution fraction. B*t Y can be purified and separated in high yield without the need for additional and/or pre-purification means, with simple and easy adsorption and elution operations and equipment RY.

Vitamin B1 including the above-mentioned extraneous ingredients! Examples of liquids containing vitamin 1Btt-producing bacteria include Propionibacterium, Streptomyces, and Arthrobacterium.
For example, B11 produced in the culture solution or cultured cells obtained by culturing Corynebacterium genus Corynebacterium can be extracted with water having a temperature of 80°C.

According to the method of the present invention, vitamin B1. Vitamin B□ is adsorbed onto the resin adsorbent by bringing the containing liquid into contact with the resin, and any means that can bring the two into sufficient contact can be adopted as the mode of contact. For example, the adsorbent may be combined with the vitamin Bl! A batch method can be adopted in which the containing liquid is mixed with the liquid and, if desired, the two are brought into contact with each other by stirring.'5! , the adsorbent is packed in a suitable column, and the vitamin Bl!
Column chromatography, which allows the liquid to pass through, can also be employed. In the case of a batch method, the pH' of the vitamin B□-containing solution? Appropriate pH conditions, e.g.
The pH is adjusted to about 5 to about 8, more preferably about pH 7, and an appropriate amount of the adsorbent is added, such as a solution containing about 1 to about 50 vol/vol, for example, about 10 minutes to about 2 vol/vol. The mixing system can be gently stirred for a period of time, usually about 20 minutes to about 1 hour. The temperature for the adsorption operation may be room temperature, but it is preferable to use as low a temperature as possible, for example, a temperature of about 10 to about 60°C. Also, in the case of column fist chromatography,
Under the same pH and temperature conditions as above, the vitamin B1
! This can be carried out by passing the containing liquid through a packed bed of the adsorbent.

According to the present invention, by cleaning the resin on which vitamin B□ has been adsorbed as described above with the cleaning treatment liquid, impurities other than vitamin B□ can be removed without desorption of vitamin Blt from the resin. Fatty carboxylic acid salts such as propionic acid, sodium, sodium butyrate, and sodium valerate; amino acid compounds such as glutamic acid, aspartic acid, proline, leucine, and alanine;
Saccharide compounds such as glucose, fructose, ribose, and galactose, as well as nucleobase compounds such as adenine, guanine, cytosine, thymine, and uracil, are easily washed away from the resin, and after the washing treatment, C) Purity of the active elution fraction. Vitamin BI, which contains impurities such as those exemplified above as well as other sigma), contains 0.
It is possible to purify and separate ρ and vitamin B□.

The cleaning solution may be purified water having a temperature of 60°C to 70°C, preferably about 45°C to 55°C, and a general ion exchange resin such as Anno Kuitoite IR-120B, Anno (
-Using a tower filled with light IRA-410 etc.
Specific resistance value 1 obtained by treating tap water Y 1 t/day
Ion-exchange pure water having a value of 00x 1a' ohm or more can be used, and for example, acetic acid 1. Acids such as phosphoric acid and sulfuric acid Y0.1-1. Ow t% gold content, temperature 5
An aqueous member having a temperature of 0°C to 70°C, preferably about 45 to 55°C can be exemplified. .

The type of cleaning treatment liquid can be appropriately selected depending on the type and amount of interfering components, the type of adsorbent resin, etc.

According to the present invention, a vitamin B11-containing solution containing undesirable components is obtained by elution with a vitamin B1tk eluent adsorbed to the resin washed as described above to obtain an active elution fraction. Vitamin B0 can be purified and separated from

As the eluent, common eluents can be used, such as an aqueous solution containing an eluent selected from the group consisting of lower alcohols, acids, alkalis, and salts.

Specific examples of such eluents include, for example, lower alcohols such as methanol, ethanol, and impropatol; for example, phosphoric acid.

Acids such as acetic acid, boric acid, hydrochloric acid; for example, ammonium water phosphate.

(sodium oxide)-r1αrhe=i Alkalies such as ammonium and ammonium nitrate; for example, sodium carbonate,
Salts such as sodium acetate, sodium phosphate, potassium phosphate; many examples may be mentioned. The type of eluent can be selected as appropriate depending on the type and amount of interfering components, the type of adsorbent resin, etc., but it is preferable to use an aqueous solution of lower alcohols, such as 50% methanol, 15% cetanol, etc. Examples include the use of hydroalcohols having an alcohol content of about 60% or less, such as 5% isopropanol. The elution operation can also be carried out at room temperature, and although there is no particular need for heating or cooling, it may be carried out if desired. For example, operating temperatures of about 20 to about 60°C can be exemplified.

The active elution fraction thus eluted can be obtained and, if desired, subjected to concentration, recrystallization, etc.

Hereinafter, several examples will be shown in which vitamin B□ is purified and separated by the method of the present invention using the cleaning treatment liquid 7 of the present invention.

Example 1 Vitamin B obtained by culturing the vitamin B□ producing bacterium Propionibacterium shamanii IFO12391 strain
The product containing 11 was centrifuged, and 80
℃Cl: Vitamin 13tt was extracted together with impurities using the water contained in the mixture to prepare a crude vitamin B□ aqueous solution. (Vitamin Bll content I S' Op p m) This solution 25
The column filled with D'8T resin particles of 0 Cc'l 5 CC was allowed to flow at a flow rate of K 0.4 cc for 1 minute using an upward flow method to adsorb vitamin B□. Continuous temperature 50℃
53 cc of ion-exchanged pure water with 16 mL of water was passed through the sink, and the impurities were discharged to the adsorption waste liquid. Next, water containing KCN is passed through to convert it into vitamin BI2, ie, dianocobalamin (CN-B12), which has -C,N groups. Subsequently, CN-B□ was eluted with 30% methanol water, and 40 cc of active fraction was collected and dried to obtain CN@Bi powder. The yield from the aqueous vitamin B0 solution to the eluate was in the range of IC1O. Crude vitamin B □ Crude vitamin BI including impurities in the aqueous solution! Purified vitamin B1 □Powdered vitamin B1. Example 2 Vitamin B1 with a 300-fold improvement in specific activity! Vitamin BI obtained by culturing Propioniva P Thylium shamanii IFO12391 strain! The product containing the product is centrifuged, and the separated bacterial cells are washed with water at 80°C to remove vitamin B1ff along with impurities.
1 was extracted to create a 13tt crude vitamin aqueous solution.

This solution (containing vitamin Bs: 40 ppm) was passed through a column packed with 750 cc of DST resin particles at a flow rate of 3.4 cc per minute using an upward flow method. Let it absorb. Continuously pour 50 cc of a 0.5% acetic acid aqueous solution at a temperature of 65°C and remove the impurities. Drains into adsorption waste liquid. Then, water containing KCN is passed to convert the -CN group to 1-vitamin Bit, ie, dianocobalamin (cN-B).Continued 6
Elute CN@B1ff1 with 0 time methanol water, 40 c
Collect the active fraction of c, dry it and CN@B,! A powder was obtained. Crude vitamin BI! The yield from aqueous solution to □ eluate is 9
Crude vitamin BI was 8%! Crude vitamin B1 including impurities in aqueous solution! Purified vitamin Bl compared to powder
ffi powder vitamins! The specific activity was improved by 500 times.

Patent applicant: Nippon Oil Co., Ltd. Agent: Patent Attorney Shigeru Mura 1-1

Claims (2)

[Claims] (1) After contacting vitamin B containing unaccompanied components, the liquid solution containing it, and a divinylbenzene/styrene copolymer resin, and washing the resin with vitamin 13+tY adsorbed, the adsorbed vitamin B, & A method for purifying and separating vitamin B□ from a vitamin Bit-containing liquid containing impurities, characterized by obtaining an active elution fraction by elution with an eluent. (2) The copolymer resin is divinylbenzene. A copolymer resin derived from styrene or its functional derivatives, or a functional derivative of divinylbenzene, styrene, etc. and the following formula, where R represents a carbon-carbon double bond. C8~C with
The copolymer resin according to claim 1, which is a copolymer resin derived from an aromatic polycarboxylic acid unsaturated alkyl ester represented by Method.