JP2012031126A - Method for purification of amide compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently removing impurities contained in a liquid containing an amide compound, especially the liquid containing the amide compound which is produced by hydration reaction of a nitrile compound using microorganism cells containing nitrile hydratase or the treated product of the microorganism cells.SOLUTION: The method for purification of an amide compound is characterized by making the liquid containing the amide compound contact with an activated carbon having a pore volume of 0.5 ml/g. The method can be suitably used for acrylamide or methacrylamide.

Description

  The present invention relates to a method for purifying an amide compound. More specifically, the present invention relates to a method for efficiently obtaining a highly pure amide compound by treating an amide compound-containing liquid with activated carbon.

  The amide compound, especially the amide compound product obtained by hydrating a nitrile compound, although the type varies depending on the production method, usually contains polymer substances, surfactants, colored components, eluates, or other impurities. Exists. In order to remove these, for example, Japanese Patent Application Laid-Open No. 61-115495 and Japanese Patent Application Laid-Open No. 61-122253 disclose a purification method using activated carbon, and Japanese Patent Application Laid-Open No. 61-115058 discloses a purification treatment using an ion exchange membrane. JP-A 61-122227 describes a purification method using a porous hollow fiber membrane.

  However, the purification method using the ion exchange membrane or the porous hollow fiber membrane requires a special purification device. For example, a disadvantage in terms of economy cannot be avoided. Moreover, in the purification method using activated carbon, although no special equipment is usually required, impurities are still present in the obtained product, and the effect is still insufficient. In particular, when an amide compound is produced by directly hydrating a nitrile compound using nitrile hydratase or the like, which is an enzyme having nitrile hydration ability, the above-described purification method using activated carbon is a microorganism mixed in the reaction solution. As a result, if the amount is too small, the reaction solution tends to foam, and if the amount is too much, the reaction solution becomes cloudy, which adversely affects product quality. .

  In addition, activated carbons having various pore diameters and specific surface areas are known depending on the raw materials and preparation methods. However, according to the study of the present invention, when the pore volume of activated carbon is small, microorganisms or the like It was found that the protein derived from can not be removed sufficiently.

Japanese Patent Laid-Open No. 61-115495 Japanese Patent Laid-Open No. 61-122253

  Accordingly, an object of the present invention is to provide a method for efficiently removing impurities contained in an amide compound-containing liquid. In particular, the present invention provides a purification method for efficiently removing a protein derived from a microorganism from an amide compound-containing liquid obtained when a corresponding amide compound is produced from a nitrile compound using a microbial cell or a processed product thereof. The task is to do.

  The inventors of the present invention have intensively studied the purification method using activated carbon of the amide compound-containing liquid described above. By contacting the amide compound-containing liquid with activated carbon having a specific pore volume, It has been found that impurities, particularly proteins, contained in the contained liquid can be removed extremely efficiently.

That is, the gist of the present invention is as follows:
[1] A method for purifying an amide compound, comprising contacting an amide compound-containing liquid with activated carbon having a pore volume of 0.5 ml / g or more.
[2] The purification method according to [1], wherein the amide compound-containing liquid is brought into contact with activated carbon under acidic conditions.
[3] The purification method according to [1] or [2], wherein the amide compound-containing liquid is a product liquid obtained by a hydration reaction of a corresponding nitrile compound.
[4] The purification method according to any one of [1] to [3], wherein the amide compound has 2 to 20 carbon atoms.
[5] The purification method according to any one of [1] to [4], wherein the amide compound has a carbon-carbon double bond.
[6] The purification method according to [5], wherein the amide compound is acrylamide or methacrylamide.
[7] The purification method according to any one of [1] to [6], wherein the amide compound is synthesized using a microbial cell containing nitrile hydratase or a processed product of the microbial cell. .
[8] The purification method according to [7], wherein the microbial cell is a transformant in which the nitrile hydratase gene cloned from the microorganism is expressed in an arbitrary host.
[9] The purification method according to any one of [1] to [7], wherein the pH of the amide compound-containing liquid upon contact with activated carbon is 3.5 to 6.5.
[10] The organic acid having an acid dissociation index of 3.5 to 5.5, or an amide compound-containing liquid prepared using the organic acid and a base, is acidic, [2] to [8] Purification method.
[11] The purification method according to [10], wherein the organic acid is acrylic acid or methacrylic acid.
[12] The purification method according to any one of [1] to [11], wherein the activated carbon is activated carbon made of wood or coconut shell.
[13] The purification method according to any one of [1] to [12], wherein the temperature at the time of contact with the activated carbon is 10 to 50 ° C.
[14] The amide compound-containing liquid is brought into contact with activated carbon, and then the activated carbon is separated, and crystals are precipitated with the remaining liquid at a saturation temperature or lower, according to any one of [1] to [13] Purification method.

  As is clear from the above description, particularly the results of the above examples and comparative examples, according to the method of the present invention, by contacting with activated carbon having a specific pore volume, compared with the conventional purification method using activated carbon. The amide compound can be purified much more effectively. In particular, according to the method of the present invention, when a corresponding amide compound is produced from a nitrile compound using a microbial cell or its treated product, proteins derived from the cell contained in the amide compound are removed very efficiently. Yes.

  The amide compound-containing liquid in the present invention is not particularly limited, and specific examples include a amide compound-containing product liquid obtained by hydrating a nitrile compound having about 2 to 20 carbon atoms. The nitrile compound subjected to the hydration reaction includes a wide range of nitriles such as aliphatic nitriles and aromatic nitriles. As aliphatic nitriles, saturated or unsaturated nitriles having 2 to 6 carbon atoms, for example, aliphatic saturated mononitriles such as acetonitrile, propionitrile, butyronitrile, isobutyronitrile, barrel nitrile, isovaleronitrile, capronitrile, etc .; Aliphatic saturated dinitriles such as malononitrile, succinonitrile, and adiponitrile; aliphatic unsaturated nitriles such as acrylonitrile, methacrylonitrile, and crotononitrile. Aromatic nitriles include benzonitrile, o-, m-, and p-chlorobenzonitrile, o-, m-, and p-fluorobenzonitrile, o-, m-, and p-nitrobenzonitrile, o- Acrylamide, methacrylamide obtained by hydrating a nitrile compound having an unsaturated bond, such as acrylonitrile or methacrylonitrile, and the like. The purification method of the present invention is suitable for the aqueous solution containing it.

  In the present invention, the amide compound-containing liquid to be treated may be obtained by any known hydration method. Namely, a hydration method using sulfuric acid, a catalytic hydration method using a catalyst containing metallic copper such as a Raney copper catalyst, an enzyme (nitrile hydratase) having the ability to hydrate a nitrile compound, and a microbial cell containing the enzyme, Any of those hydrated using the above-mentioned enzyme and microbial cell processed product may be used.

  Among these, nitrile hydratase, which is an enzyme capable of hydrating a nitrile compound, a processed product of nitrile hydratase, a microbial cell containing nitrile hydratase, or a processed product of the microbial cell can be obtained. The amide compound-containing liquid is suitable for the purification method of the present invention. The nitrile hydratase in the above refers to an enzyme having the ability to hydrolyze a nitrile compound to produce a corresponding amide compound.

  Here, as a microorganism containing nitrile hydratase, nitrile hydratase having the ability to hydrolyze a nitrile compound to produce a corresponding amide compound is produced, and nitrile hydratase is produced in 30% by weight acrylamide aqueous solution. The microorganism is not particularly limited as long as it retains the activity. Specifically, the genus Nocardia, the genus Corynebacterium, the genus Bacillus, the thermophilic Bacillus, the Pseudomonas genus, the Micrococcus genus, the Rhodochrous species Rhodococcus genus, Acinetobacter genus, Xanthobacter genus, Streptomyces genus, Rhizobium genus, Klebsiella genus, Enterobacter genus represented by Pseudonocardia represented by the genus Erwinia, Aeromonas, Citrobacter, Achromobacter, Agrobacterium or thermophila A preferred example is a microorganism belonging to the genus Pseudonocardia).

  Further, a transformant obtained by expressing a nitrile hydratase gene cloned from the microorganism in an arbitrary host is also included in the microorganism referred to in the present invention. As an arbitrary host mentioned here, Escherichia coli can be mentioned as a representative example as in Examples described later, but is not particularly limited to Escherichia coli, and Bacillus subtilis and other Bacillus subtilis. Other microbial strains such as genera, yeasts and actinomycetes are also included. As an example of such a case, MT-10822 (this strain was assigned to the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, 1-3 1-3 Higashi, Tsukuba, Ibaraki Prefecture on February 7, 1996. -5785, deposited under the Budapest Treaty on the International Approval of Deposits of Microorganisms in Patent Procedures). In addition, by using recombinant DNA technology, one or more of the constituent amino acids of the enzyme may be substituted, deleted, deleted or inserted with other amino acids to further enhance amide compound resistance, nitrile compound resistance, and temperature resistance. A transformant expressing an improved mutant nitrile hydratase is also included in the microorganism referred to in the present invention.

  When an amide compound is produced using the above-described microorganism, the microorganism or a treated product of the microorganism is usually used. The microbial cells may be prepared by using general methods known in the fields of molecular biology, biotechnology, and genetic engineering. For example, after inoculating the microorganism in a normal liquid medium such as LB medium or M9 medium, an appropriate culture temperature (generally 20 ° C. to 50 ° C., but in the case of thermophilic bacteria, it may be 50 ° C. or higher) And the microorganism is then separated and recovered from the culture solution by centrifugation.

  In addition, the microbial cell treated product in the present invention is an extract or ground product of the above microbial cell, a post-separated product obtained by separating and purifying the nitrile hydratase activity fraction of the extract or ground product, This refers to an immobilized product obtained by immobilizing microbial cells, extracts of the cells, ground products, and post-segregated materials using an appropriate carrier, and so on as long as these have nitrile hydratase activity. Corresponds to the treated bacterial cells.

  In the amide compound to be purified by the present invention, a reaction mode in the case of obtaining a amide compound by hydrating a nitrile compound using a microbial cell containing nitrile hydratase or a treated product of the microbial cell, The corresponding nitrile compound may be subjected to a batch reaction or may be a continuous reaction. Also, the reaction format is not particularly limited, and for example, it may be carried out as a suspended bed or a fixed bed. In this case, the concentration of the catalyst, for example, the microbial cell or the treated microbial product in the reaction solution is not particularly limited as long as the mixing of the aqueous medium and the nitrile compound is not hindered.

  In the above, the concentration of the nitrile compound when the nitrile compound is added at the start of the reaction may be not less than the saturation concentration of the nitrile compound. On the other hand, the upper limit of the concentration is not particularly limited, and may be arbitrarily determined depending on the assumed amide compound concentration and nitrile compound concentration at the end of the reaction.

  Further, the unreacted nitrile compound can be removed from the reaction solution by a means such as distillation after the reaction. Therefore, the nitrile compound can also be added so that the nitrile compound becomes excessive even when the expected amide compound concentration at the end of the reaction is reached. Specifically, for example, when the nitrile compound is acrylonitrile, the saturated concentration of the present compound with respect to water is about 7% by weight at 20 ° C., so about 7% by weight or more is preferable. When methacrylonitrile or crotonnitrile is a nitrile compound, the saturation concentration of these compounds with respect to water is about 2% by weight at 20 ° C., so about 2% by weight or more is preferable.

  The amidation reaction in the above is usually carried out under normal pressure, but can also be carried out under pressure in order to increase the solubility of the nitrile compound in the aqueous medium. The reaction temperature is not particularly limited as long as it is not lower than the freezing point of the aqueous medium. However, when the catalyst contains nitrile hydratase, the reaction temperature is preferably 0 to 50 ° C, more preferably 10 to 30 ° C. .

  Further, the pH of the reaction solution at the time of the amidation reaction is not particularly limited as long as the nitrile hydratase activity is maintained, but it is preferably in the range of pH 6 to 10, more preferably pH 7 to 9. It is a range.

  In the purification of the amide compound in the present invention, the pH of the liquid containing the amide compound is not particularly limited, but is preferably acidic, more preferably pH 2 or more and less than 7, and still more preferably pH 3.5 to 6.5. It is performed by contacting with activated carbon in the range of.

  In the present invention, in order to make an amide compound-containing solution acidic, it is usually necessary to add an acid to the amide compound-containing solution, but the acid type must be one that does not affect the stability of the amide compound. A wide range can be used, for example, mineral acids such as sulfuric acid and nitric acid, or organic acids such as acetic acid and acrylic acid, and two or more kinds may be used. Among these, in the present invention, the use of a weak acid is effective in that the pH adjustment is easier and more stable, and a higher purification effect and stability of the amide compound are obtained. It is very preferable to adjust to the above pH range using both a weak acid and a base.

  The weak acid preferably used is preferably one having an acid dissociation index (pKa; 25 ° C., in water) of 2.0 to 6.0, more preferably 3.5 to 5.5 in consideration of the pH control range. Is more preferable. Representative of these acids are aliphatic saturated monocarboxylic acids such as acetic acid, propionic acid, octanoic acid and valeric acid, aliphatic unsaturated monocarboxylic acids such as acrylic acid, crotonic acid and methacrylic acid, oxalic acid and adipine. Examples thereof include aliphatic polycarboxylic acids such as acid, succinic acid and maleic acid, and aromatic carboxylic acids such as benzoic acid. The base is preferably a strong base such as sodium hydroxide or potassium hydroxide.

  In the present invention, when the amide compound to be treated is particularly one having an unsaturated bond, when it is subjected to a polymerization reaction after purification, an acid is contained in the obtained polymer. Inconvenience of remaining or freeing will be caused. Therefore, when an amide compound having an unsaturated bond as described above is to be purified, the acid has an unsaturated bond and can be copolymerized with the amide compound. Specifically, it is preferable to use acrylic acid, methacrylic acid, or crotonic acid.

  In the present invention, the concentration of the acid or the acid and the base depends on the property of the amide compound-containing liquid and the pKa of the acid used, but is usually 10 ppm to 5% by weight with respect to the amide compound-containing liquid in terms of acid. Range.

  Further, in the present invention, the amide compound-containing liquid is brought into contact with activated carbon under the above-described acidic conditions. There is no particular limitation, and any method may be used in which the amide compound-containing solution is prepared under acidity simultaneously with the addition of activated carbon.

The activated carbon used in the present invention has a pore volume of 0.5 ml / g or more, preferably 0.5 ml / g to 10 ml / g, more preferably 0.6 ml / g to 5 ml / g.
When the pore volume is 0.5 ml / g or more, when producing a corresponding amide compound from a nitrile compound using a microorganism containing nitrile hydratase or a processed product thereof, from the amide compound-containing liquid as a product, Since it becomes possible to remove efficiently the protein derived from microorganisms, it is preferable.

The pore volume of the activated carbon can be measured by a gas adsorption method or a mercury intrusion method.
Activated carbon having a pore volume of 0.5 ml / g or more can be appropriately selected from commercially available activated carbon. For example, product names PM-SX of Kuraray Co., Ltd. and 6MW-K and SW50 sold by Calgon Carbon Japan Co., Ltd. can be mentioned.

The activated carbon can be used in any of powdery and granular form. Moreover, what is necessary is just to use the apparatus suitable for the particle size of the used activated carbon also in the apparatus which performs a refinement | purification process. For example, when powdered activated carbon is used, it can be carried out either batchwise or continuously in a tank in which the liquid can be stirred. Moreover, when using granular activated carbon, the continuous process by a packed tower format other than the said format is also possible.
In addition, activated carbon generally includes coal, wood, coconut shell, and the like as raw materials. However, there is a special limitation as long as the pore volume is in the above range and has adsorption ability. Any of them can be used. However, when the amide compound to be treated has an unsaturated bond in particular, considering the storage stability and ease of polymerization of the amide compound, the activated carbon should have a low metal content. It is preferable to use, and it is more preferable to use a raw material made of wood or a coconut shell.

  In the present invention, when the amount of activated carbon used for purification treatment of the amide compound is too small, it is difficult to obtain a sufficient purification effect. The amount is usually in the range of 0.01 to 20% by weight, more preferably in the range of 0.05 to 10% by weight with respect to the amide compound-containing liquid. When activated carbon is used in particular in the form of powder, the activated carbon may be added directly to the amide compound-containing liquid as it is, or once activated carbon is dispersed in a medium such as water to form a slurry. You may make it add or supply to an amide compound containing liquid.

  In the present invention, the temperature at which the amide compound-containing liquid is purified by activated carbon is not particularly limited as long as the amide compound crystals are not precipitated and the stability thereof is not affected. It is carried out in the range of -80 ° C. In particular, when purifying an amide compound-containing liquid having an unsaturated bond, such as an acrylamide or methacrylamide-containing liquid, in order to prevent gelation due to the occurrence of a polymerization reaction, it is in the range of 60 ° C. or lower, and further in the range of 10-50 ° C. It is preferable to contact with activated carbon. The time required for the contact treatment with activated carbon is usually in the range of 0.5 to 20 hours, although it depends on the type of treatment and the amount of activated carbon.

  Next, in the present invention, the activated carbon is separated from the contact-treated amide compound-containing liquid to obtain a purified liquid of the amide compound-containing liquid. The method for separating activated carbon is not particularly limited as long as it is a method using a commonly used solid-liquid separation device, and examples thereof include a pressure filter, a vacuum filter, a centrifuge, and the like. Any of continuous type may be used. Further, in the present invention, a further purified amide compound is obtained by adopting a method of cooling the amide compound-containing liquid after separating the activated carbon and crystallizing the target amide compound from the liquid. Is also possible.

  In this example, amino acid substitutions that retain nitrile hydratase activity are obtained by site-specific mutation. However, even if a recombinant plasmid is constructed by a method other than site-specific mutation and introduced into a host cell based on the type of base to be replaced with the mutation point disclosed in the examples, Similar results can be obtained. For example, a DNA fragment having a base sequence in which the base sequence of the DNA corresponding to the mutation point disclosed in the Examples becomes the sequence after amino acid substitution is synthesized with a DNA synthesizer or the like, and separated from the obtained fragment separately. By substituting the region corresponding to the fragment of pPT-DB1 previously prepared, the intended recombinant plasmid can be obtained.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated still in detail, this invention is not limited at all by the following examples. In the following, HPLC analysis of the reaction solution is performed using ULTRON 80HG (50 × 8 φmm) as a column, 10 mM phosphoric acid aqueous solution as a developing solution, and acrylamide is detected by absorbance at 220 nm. Moreover, in order to confirm the effect of this invention, the protein contained in the obtained amide compound containing liquid was analyzed. The protein concentration was dialyzed and removed from the amide compound-containing solution using a semipermeable membrane, and then quantified using a protein analysis kit manufactured by Biorat to determine the protein removal rate.

Example 1
100 ml of a medium having the following composition was prepared in a 500 ml baffled Erlenmeyer flask and sterilized by autoclaving at 121 ° C. for 20 minutes. Ampicillin was added to this medium so that the final concentration would be 50 μg / ml, and then MT-10822 strain (FERM BP-5785) was inoculated in the ears and cultured at 37 ° C. and 130 rpm for 20 hours. Only bacterial cells were separated from the culture solution by centrifugation (15000G × 15 minutes), then resuspended in 50 ml of physiological saline, and then centrifuged again to obtain wet cells. .
Medium composition (pH 7.5)
Yeast extract 5.0 g / L
Polypeptone 10.0g / L
NaCl 5.0g / L
Cobalt chloride hexahydrate 10.0mg / L
Ferric sulfate heptahydrate 40.0mg / L

The wet cells 1.5 g obtained above were suspended in 98.5 g of 0.3 mM NaOH aqueous solution, and 36 g of acrylonitrile was added all at once to this suspension and reacted while stirring at 10 ° C. . The reaction solution was analyzed by HPLC analysis 24 hours after the start of the reaction. As a result, only acrylamide was present in the reaction solution (concentration = 35% by weight), and acrylonitrile was not observed. The pH of this reaction solution was 8.0.
This reaction solution was adjusted to pH 5 with a 10% aqueous sulfuric acid solution, and 2% by weight of powdered activated carbon PM-SX (pore volume: 1.38 ml / g) manufactured by Kuraray Chemical Co., Ltd. was added to the reaction solution at 25 ° C. After stirring for 5 hours, filtration was performed with filter paper. When the protein concentration in the obtained filtrate was measured, the removal rate was 99% or more.

(Example 2)
In Example 1, instead of using powdered activated carbon PM-SX manufactured by Kuraray Chemical Co., Ltd., powdered activated carbon S-W50 (pore volume: 1.08 ml / g) manufactured by Calgon Carbon Japan Co., Ltd. was used. The same operation as in Example 1 was performed to obtain a filtrate. When the protein concentration in the obtained filtrate was measured, the removal rate was 99% or more.

(Example 3)
In Example 1, instead of using powdered activated carbon PM-SX manufactured by Kuraray Chemical Co., Ltd., powdered activated carbon 6 MW-K (pore volume: 0.64 ml / g) manufactured by Calgon Carbon Japan Co., Ltd. was used except 2% by weight. The same operation as in Example 1 was performed to obtain a filtrate. When the protein concentration in the obtained filtrate was measured, the removal rate was 99% or more.

(Comparative example)
In Example 1, instead of using powdered activated carbon PM-SX made by Kuraray Chemical Co., Ltd. The same operation as in Example 1 was performed to obtain a filtrate. When the protein concentration in the obtained filtrate was measured, the removal rate was 50%.

Claims (14)

  A method for purifying an amide compound, comprising contacting an amide compound-containing liquid with activated carbon having a pore volume of 0.5 ml / g or more.   The purification method according to claim 1, wherein the amide compound-containing liquid is contacted with activated carbon under acidic conditions.   The purification method according to claim 1 or 2, wherein the amide compound-containing liquid is a product liquid obtained by a hydration reaction of a corresponding nitrile compound.   The purification method according to any one of claims 1 to 3, wherein the amide compound has 2 to 20 carbon atoms.   The purification method according to any one of claims 1 to 4, wherein the amide compound has a carbon-carbon double bond.   The purification method according to claim 5, wherein the amide compound is acrylamide or methacrylamide.   The purification method according to any one of claims 1 to 6, wherein the amide compound is synthesized using a microbial cell containing nitrile hydratase or a processed product of the microbial cell.   8. The purification method according to claim 7, wherein the microbial cell is a transformant in which the nitrile hydratase gene cloned from the microorganism is expressed in an arbitrary host.   The purification method according to any one of claims 2 to 7, wherein the pH of the amide compound-containing liquid upon contact with activated carbon is 3.5 to 6.5.   The purification according to any one of claims 2 to 8, wherein the amide compound-containing liquid is prepared acidic using an organic acid having an acid dissociation index of 3.5 to 5.5, or the organic acid and a base. Method.   The purification method according to claim 10, wherein the organic acid is acrylic acid or methacrylic acid.   The purification method according to any one of claims 1 to 11, wherein the activated carbon is activated carbon made of wood or coconut shell.   The purification method according to any one of claims 1 to 12, wherein the temperature at the time of contact with the activated carbon is 10 to 50 ° C. The purification method according to any one of claims 1 to 13, wherein after the amide compound-containing liquid is brought into contact with activated carbon, the activated carbon is separated, and the residual liquid is set to a saturation temperature or lower to precipitate crystals.