JPS5995299A - 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/styrene copolymer resin, eluting vitamin B12 with an elutant consisting of an aqueous solution of a lower ketone, etc. CONSTITUTION:A product containing vitamin B12 obtaind 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 with water at 80 deg.C from the mold, 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, it is eluted with an elutant consisting of an aqueous solution of a compound such as a lower ketone, lower ester, lower ether, etc., and an active eluation fraction is collected to give the desired high-purity compound.

Description

[Detailed Description of the Invention] The present invention provides vitamin B1 containing extraneous ingredients! Containing liquid, e.g. fermentation method vitamin B, containing liquid, cell extraction method vitamin 13t
Vitamin B1 containing impurities such as T-containing liquids, etc.
From a ff1-containing solution, a significantly high recovery rate, for example, exceeding about 98% to 100%, can be obtained using simple and easy adsorption and elution operations and equipment 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 13+ in high yield and high purity with an excellent recovery rate, and a method after adsorption operation suitable for implementing this method. This invention relates to a purification separation method using an eluent.

Vitamin BI containing extraneous ingredients! Vitamin Bl from the containing liquid
As one of the methods for separating vitamin B and ', there is a known method to separate vitamin B by adsorption and elution using divinylbenzene/styrene copolymer resin as an adsorbent.
ing. According to that method, the resin is brought into contact with a vitamin Biffi-containing liquid containing undesired components, and vitamin B■ is adsorbed onto the resin, and the adsorbed vitamin B, ! Y
By elution with an eluent to obtain an active eluate fraction, it is possible to obtain purified and separated vitamin B1ff1 in a high yield. When performing elution treatment with hydrous alcohols with an alcohol content of about 50% or less, such as ethanol and 20-fiber isopropanol, the elution rate of vitamin B12 from the resin is slow, so a large amount of the above-mentioned eluent is required. .

Also, is the concentration of vitamin B11 in the eluted active fraction diluted? When recrystallizing if desired, an additional operation of concentration is required, which also has the unavoidable disadvantage of requiring concentration equipment and a heat source.

In particular, in order to obtain a high recovery rate, for example, exceeding 98φ and reaching 100cm, it is necessary to collect vitamin B1 in the active elution fraction.
* It was found that the dilution of the concentration is even more serious, and that the eluent in the elution treatment on an industrial scale is a problem that cannot be expected to yield high yields with simple and easy operations and equipment.

The present inventors have developed vitamin B□ containing extraneous components.

Purified vitamin B is extracted from the containing solution by adsorption-elution method.
l! By overcoming the above-mentioned technical problems in separating and obtaining the crude vitamin B with excellent purity and yield,
Research has been carried out to develop a method that can industrially advantageously purify and separate vitamin B from a containing liquid.

Consequently, as eluents lower ketones such as acetone, methyl ethyl ketone, diethyl ketone, cyclohexanone and methyl isobutyl ketone, lower esters such as ethyl acetate, isopropyl acetate and n-propyl acetate.

An aqueous solution containing an eluent selected from R'6 consisting of lower ethers such as tetrahydrofuran, diethyl ether, 1,4-dioxane, diisopropyl ether, dimethyl cellosolve and diethyl ether is extracted from vitamin B, , '& resin. It has been found that rapid elution is possible and the amount of eluent used can be reduced to 115 to 1715 ml of the eluent in the conventional method using the bathing agent exemplified above.

In addition, the concentration of vitamin B, 2 in the eluted active fraction is 5 to 15 times higher than that of conventional eluents when using the above-mentioned eluent, and when the same fraction is recrystallized if desired, concentration is added. Operation is no longer required.

In particular, even if a high recovery rate of 98% or more than 100 pieces is obtained, vitamin B in the active elution fraction,
In the case of the eluent exemplified above, the concentration is increased 3 to 10 times that of conventional eluents, eliminating the need for a concentration operation, and without the need for additional additional and/or preliminary S production means. With simple and easy elution operation and equipment, 98
Purified vitamin B1 with a high recovery rate of over 100%! It makes it possible to separate and collect vitamin B, which contains extraneous components, and vitamin B1 in the liquid! It has been discovered that the industrial implementation of the purification separation of can be improved very advantageously.

It is therefore an object of the present invention to provide improved vitamin BIff
A purification and separation method for i is provided.

Another object of the invention is to provide vitamin BI! suitable for use in carrying out such a method. The purpose is to use an eluent for purification and separation.

The present invention uses a divinylbenzene/styrene copolymer resin as an adsorbent, which is a copolymer obtained by polymerizing divinylbenzene, styrene, or a functional derivative thereof as the main polymer component. The main component is resin, or divinylbenzene, styrene or a functional derivative thereof.
A copolymer resin derived from an unsaturated alkyl ester of an aromatic polycarboxylic acid, represented by 03 to C8o unsaturated alkyl residue having a double bond between carbons, and n being 2 or 6. Hereinafter, it may be abbreviated as DST resin).

These resins are generally obtained by polymerizing the above-mentioned polymers using a known radical polymerization initiator. Here, the content of styrene or its functional derivative is usually 60 to 80 wt%, preferably 45 to 70 wt%, and when the aromatic polyvalent carbon or unsaturated alkyl ester is used, the content is 0.1 ~50 wt%, preferably 1~10 wt%.

According to the present invention, the resin described above is brought into contact with a liquid containing vitamin B containing undesired components, and the resin is injected with vitamin B,
High-purity vitamin B1. Y can be obtained in high yield and purified and separated using simple and easy adsorption and elution operations and equipment without the need for additional and/or pre-purification means.

Vitamin B1 including the above-mentioned extraneous ingredients! For example, the solution containing vitamin 13tt producing bacteria such as Propionibacterium, Streptomyces, Arthrobacterium, Corynebacterium, Quadripseudomonas, Mycobacterium, Pseudomonas, etc. Bl produced in the culture solution or cultured bacterial cells obtained by culturing
t 'al' extract extracted with water having a temperature of 80 °C,
Vitamin B, such as a mixed solution in which the cell membranes of bacterial cells are physically or ultrasonically destroyed.

Examples include liquid containing liquid.

According to the method of the present invention, vitamin 13tt is adsorbed onto the resin adsorbent by bringing the vitamin 13tt-containing solution containing the above-mentioned miscellaneous components into contact with the resin. Any means that can bring the material into contact with the material can be used. For example, the adsorbent requires the vitamin 13
A batch method can be adopted in which the adsorbent is mixed with the tt-containing liquid and brought into contact with the two by stirring if desired, or the adsorbent is packed in a suitable column and the vitamin 1as is mixed in the packed bed.
It is also possible to employ a column chromatography method in which a t-containing liquid is passed through. In the case of a batch method, the pH of the vitamin B1m-containing solution is adjusted to an appropriate pH condition, for example, about pH 5 to about 8, more preferably about pH 7,
A suitable amount of the adsorbent, such as from about 1 to about 50 v o 1 /
The adsorbent may be added in an amount such as vol. of the liquid containing the adsorbent, and the process may be carried out with gentle mixing and stirring, for example, for about 10 minutes to about 2 hours, 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 can be exemplified. Also, in the case of one-sided column chromatography, under the same pH and temperature conditions as above,
This can be carried out by passing the vitamin B-containing liquid through a packed bed of adhesive.

After adsorption treatment, water, low concentration hydrous alcohols e.g.
0% methanol water, 10% ethanol water, 5% isopropanol water, low concentration acid solution such as 1% phosphoric acid aqueous solution, 1
It is preferable that the sample be washed with an aqueous solution of thiacetic acid, an aqueous solution of 1-thioboric acid, 0.1-dihydrochloric acid, etc., and then subjected to an elution treatment with an eluent.

According to the present invention, vitamin Bl! adsorbed on the resin as described above! 'f By elution with an eluent and obtaining an active elution fraction, vitamin B containing impurities can be purified and separated from the containing solution.

The eluent used in the present invention is an aqueous solution containing an eluent selected from the group consisting of lower ketones, lower esters, and lower ethers. Here, the number of carbon atoms in the compound is 2 to q, preferably 6 to 7, and lower ketones such as cyclohexanone and methyl isobutyl ketone; Esters: Examples include lower ethers such as tetrahydrofuran, diethyl ether, 1,4-dioxane, diisopropyl ether, dimethyl cellosolve, and diethyl cellosolve. The type and concentration of such eluents are
It can be selected as appropriate depending on the type and amount of interfering components, the type of adsorbent resin, etc.

The concentration of the aqueous solution is usually 1 to 50 vol%, preferably 5 to 50 vol.
40vol chi will be adopted. 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, can be recrystallized without being concentrated.

Hereinafter, some examples of separating vitamin B and diluted vitamin B by the method of the present invention using the adsorbent of the present invention will be shown below.

Example 1 Vitamin B obtained by culturing the vitamin Btt producing bacterium Propionibacterium shamanii IF012391 strain
1! The containing product was centrifuged and separated from white wood at 80%
Vitamin Bl 2 Y along with impurities by water with ℃
The crude vitamin B1m was extracted and cultured IV. (Vitamin 1at containing liquid 120 ppm) This bath U2SL
cc was passed through a column packed with 5 cc of DST resin particles at a flow rate of 0.4 cc per minute using an upward flow method to obtain vitamin Bl! Let it be absorbed. Approximately 50 cc of 1% acetic acid water is continuously flowed to drain the impurities into the adsorption waste liquid. Then KCN
Vitamin B1 with 10N group by flowing water containing 'jz
*i.e. dianocobalamin c CN -Bl *)
Convert to

Subsequently, CN-B was eluted with 60% methanol water.
The active fraction of cc was collected and dried to obtain 30 m9 of CN.13tt powder. Yield from culture solution to eluate is 100%
Met. Also, the vitamin Blt concentration in the eluate was 0.7.
Heat at 5 mtg/cc.

On the other hand, following the same procedure until conversion to dianocobalamin, CN11B1. Powder 3oη? I got it.

The yield from the culture solution to the eluate was 1 oos. In addition, the concentration of vitamin BIt in the eluate was 757n9/Ct.
tC-There was.

That is, the concentration of vitamin B1m in the eluate is extremely high when acetone water is used.

Example 2 Vitamin B obtained by culturing the vitamin Blt-producing bacterium Propionibacterium shamanii-IF012391 strain
I! The containing product was centrifuged, and the separated bacterial cells were separated into 8
Vitamin B*t along with impurities by water with 0℃
K was extracted to prepare a crude vitamin B and ffi culture solution. (
Vitamin B1! (Content: 20 ppm)
800 cc of L was passed through a column packed with 5 cc of DST resin particles at a flow rate of 0.4 cc for 1 minute using an upward flow method to obtain vitamin Bl! Let it absorb Y. Approximately 60 cc of 1% acetic acid water is continuously flowed to drain impurities into the adsorption waste liquid. Next, KCNi-containing water was poured to extract vitamin B11 having a -CN group, i.e. dianocobalamin (cN-B1).
Convert to 1). Then add 15% isopropanol in water.
l'J-13BY was eluted and 80 cc of active fraction was collected and dried to obtain CN@B, powder 15.71n9y.

The yield from the culture solution to the eluate was 98 cm. Also, vitamin BI in the eluate! Concentration is 0.201n97 cc
There was a time.

On the other hand, following the same procedure until conversion to dianocobalamin, cN11B1!
The active fraction of 7.5 cc was collected, dried and cN
@B1! 15.7 m9 of powder V was obtained. The yield from the culture solution to the eluate was 98 m. Also, vitamin B1 in the eluate! The concentration was 2.09Tn9/cc.

In other words, vitamin B! ! The concentration of is extremely high when dioxane water is used.

Patent applicant: Nippon Oil Co., Ltd. Agent: Patent attorney Shigeru Nomura Masu :・、-i

Claims (2)

[Claims] (1) Bringing a vitamin B11-containing liquid containing interfering components into contact with a divinylbenzene/styrene copolymer resin,
Vitamin B11 Y is adsorbed onto the resin, and the adsorbed vitamin B11 is eluted with an eluent consisting of an aqueous solution of a compound selected from the group consisting of lower ketones, lower esters, and lower ethers to obtain an active elution fraction. Vitamin B1. Vitamin B1 from the containing liquid! How to purify and separate. (2) The copolymer resin is a copolymer resin derived from divinylbenzene, styrene or a functional derivative thereof, or a copolymer resin derived from divinylbenzene, styrene or a functional derivative thereof and the following formula, where R is 03-C having a carbon-carbon double bond
Claim 1, which is a copolymer resin derived from an aromatic polycarboxylic acid unsaturated alkyl ester having an unsaturated alkyl residue of 8° and n being 2 or 3. Method described.