JP2019196317A - Refining method of amide compound - Google Patents

Abstract

To provide a method for efficiently refining an amide- compound-containing solution, particularly an amide compound produced by hydration reaction of a nitryl compound using a fungus and/or a fungus processed product as a catalyst.SOLUTION: In a method for refining an amide compound produced using a fungus containing nitrile-hydratase and/or a fungus processed product, a coagulant is added to an amide-compound-containing solution to coagulate the fungus and/or the fungus processed product. The obtained mixture is then brought into contact with activated carbon followed by solid liquid separation.SELECTED DRAWING: None

Description

  The present invention relates to a method for purifying an amide compound containing an impurity produced by a hydration reaction of a nitrile compound using a amide compound-containing liquid, in particular, a fungus body and / or a treated product thereof as a catalyst.

  Although amide compounds, especially amide compound-containing liquids obtained by hydrating nitrile compounds, vary in type depending on the production method, they usually contain polymer substances, surfactants, colored substances, eluates, or other impurities. To do. In order to remove these impurities, for example, Patent Document 1 and Patent Document 2 disclose a purification method using activated carbon, Patent Document 3 discloses a purification method using an ion exchange membrane, and Patent Document 4 describes a porous hollow fiber. A membrane purification process is described.

  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. In addition, the purification method using activated carbon usually does not require special equipment, but impurities are still present in the obtained product, and the effect is still insufficient.

  Patent Document 5 proposes a method of bringing an amide compound-containing solution into contact with activated carbon under acidic conditions as a simple and efficient purification method. However, particularly in the case of producing an amide compound by directly hydrating a nitrile compound using nitrile hydratase or the like, which is an enzyme having nitrile hydration ability, the purification method using activated carbon of the above prior art has sufficiently high purification efficiency. It cannot be said that the quality of the obtained amide compound is sufficiently high. Further, according to the conventional knowledge, the amide compound having an unsaturated bond still has room for examination because the compound becomes unstable in the acidic region and the polymerization reaction easily occurs.

Japanese Patent Laid-Open No. 61-115495 Japanese Patent Laid-Open No. 61-122253 JP-A-61-115058 Japanese Patent Application Laid-Open No. 61-122227 JP 2001-270857 A

  An object of the present invention is to provide a method for efficiently purifying an amide compound in a purification treatment of an amide compound containing impurities produced by a hydration reaction of a nitrile compound using a bacterial cell and / or a treated product thereof. It is to provide.

  The present inventors have conducted intensive research to solve the above problems. As a result, the inventors have found that the above problems can be solved by a method for purifying an amide compound having the following steps, and have reached the present invention.

That is, the present invention is as follows.
[1] In a method for purifying an amide compound produced using a nitrile hydratase-containing microbial cell and / or a processed product thereof, an flocculating agent is added to the amide compound-containing solution, and the microbial compound and / or A method for purifying an amide compound, comprising aggregating the cell-treated product and then contacting with activated carbon, followed by solid-liquid separation.
[2] The present invention is characterized in that a flocculant is added to an amide compound-containing solution under basic conditions to aggregate the cells and / or the treated cells thereof, and then contacted with activated carbon, followed by solid-liquid separation. The purification method according to [1] above.
[3] The purification method according to [1] or [2], wherein the amide compound is acrylamide or methacrylamide.
[4] The purification method according to any one of [1] to [3], wherein the flocculant is an inorganic flocculant.
[5] The purification method according to any one of [1] to [3], wherein the flocculant is an organic flocculant.

  In the present invention, the amide compound can be efficiently purified by contact with activated carbon after the addition of the flocculant.

  Hereinafter, the purification method of the amide compound of the present invention will be described.

[Purification method of amide compound]
The method for purifying an amide compound according to the present invention is a method for purifying an amide compound produced using a bacterial cell containing nitrile hydratase and / or a treated product thereof, wherein a flocculant is added to the liquid containing the amide compound. And aggregating the microbial cells and / or treated microbial cells thereof, and then contacting the activated carbon, followed by solid-liquid separation. The “amide compound-containing liquid” to which a flocculant is added in the step of solid-liquid separation is a liquid containing an amide compound produced using a bacterial body containing nitrile hydratase and / or a treated product thereof. Point to.

<Bacteria and / or treated cells containing nitrile hydratase>
In the present invention, a microbial cell containing nitrile hydratase and / or a treated product thereof (hereinafter, also simply referred to as “bacterial cell catalyst”) is used as a hydration catalyst of a nitrile compound to an amide compound.

  Nitrile hydratase refers to an enzyme (protein) having the ability to hydrolyze a nitrile compound to produce a corresponding amide compound (hereinafter also referred to as “nitrile hydratase activity”).

  The microbial cell containing nitrile hydratase is not particularly limited as long as it produces nitrile hydratase and retains nitrile hydratase activity in an aqueous solution of a nitrile compound and an amide compound. The nitrile hydratase-producing cells include Nocardia, Corynebacterium, Bacillus, thermophilic Bacillus, Pseudomonas, and Micrococcus. , Rhodococcus genus represented by Rhodochrous species, Acinetobacter genus, Xanthobacter genus, Streptomyces genus, Rhizobium genus, Klebsiella genus, Klebsiella Representative of the genus Enterobacter, Erwinia, Aeromonas, Citrobacter, Achromobacter, Agrobacterium or thermophila Genus Pseudonocardia, Bacteridium, Brevibak Bacteria such as belonging to the helium (Brevibacterium) genus can be mentioned. These may be used alone or in combination of two or more.

  In addition, a transformant in which a nitrile hydratase gene cloned from these bacterial cells is highly expressed in an arbitrary host, and one or more of the constituent amino acids of the nitrile hydratase using other DNA by using recombinant DNA technology And a transformant in which a mutant nitrile hydratase having further improved amide compound resistance, nitrile compound resistance, and temperature resistance is expressed by substitution, deletion, deletion, or insertion. In addition, as an arbitrary host mentioned here, Escherichia coli is exemplified as a representative example as described later, but is not particularly limited to Escherichia coli, and is not limited to Escherichia coli, for example, Bacillus subtilis. Other strains such as fungi, yeasts and actinomycetes are also included. As an example of such a case, MT-10822 [This strain was founded on February 7, 1996, 1-3-1 Higashi 1-chome, Tsukuba, Ibaraki Prefecture, Institute of Biotechnology, Institute of Industrial Technology, Ministry of International Trade and Industry (current Kisarazu, Chiba Prefecture). 2-5-8 Kazusa-shi Kazusa-shi Based on the Budapest Treaty on International Approval for Deposits of Bacteria in Patent Procedures under the Accession Number FERMBP-5785 to the Japan Biotechnology Center Patent Biological Deposit Center) Have been deposited. ].

  Among these bacterial cells, in terms of having a highly active and highly stable nitrile hydratase, cells belonging to the genus Pseudonocardia and the nitrile hydratase gene cloned from the bacterial cells can be used in any host. A highly expressed transformant and a transformant expressing a mutant nitrile hydratase are preferred. In addition, the said transformant is preferable at the point which raises the stability of nitrile hydratase more and the activity per microbial cell is higher.

  Also preferred are Rhodococcus rhodochrous J-1, which can highly express nitrile hydratase in the cells, and transformants in which the nitrile hydratase gene cloned from the cells is highly expressed in any host. . The microbial cells producing the nitrile hydratase can be prepared by a general method known in the fields of molecular biology, biotechnology, and genetic engineering.

  The recombinant vector according to the present invention contains a gene encoding nitrile hydratase, and can be obtained by linking a gene encoding nitrile hydratase to the vector. The vector is not particularly limited. For example, a gene encoding nitrile hydratase in a commercially available expression plasmid represented by pET-21a (+), pKK223-3, pUC19, pBluescriptKS (+), pBR322, etc. Thus, the expression plasmid of nitrile hydratase can be constructed. The host organism to be used for transformation is not limited as long as the recombinant vector is stable and capable of self-propagation and can express foreign DNA traits. For example, E. coli is a good example. The transformant having the ability to produce nitrile hydratase can be obtained by introducing it into Bacillus subtilis, yeast or the like.

  Bacteria that produce nitrile hydratase as described above may be appropriately cultured and grown by known methods to produce nitrile hydratase. As a medium used in this case, either a synthetic medium or a natural medium can be used as long as it contains a suitable amount of carbon source, nitrogen source, inorganic salts and other nutrients. For example, after inoculating the cells 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, 50 ° C. or higher) However, it can also be prepared by culturing. Culturing can be performed using a conventional culture method such as shaking culture, aeration and agitation culture, continuous culture, or fed-batch culture in a liquid medium containing the culture components. The culture temperature of the transformant is preferably 15 to 37 ° C. The culture conditions are not particularly limited, and may be appropriately selected depending on the type of culture and the culture method. It is sufficient that the strain grows and can produce nitrile hydratase.

  In the present invention, in order to react the bacterial body producing the above-mentioned nitrile hydratase with a nitrile compound, various treatments such as collection of bacteria by centrifugation, etc., and crushing to produce a crushed bacterial body, The microbial cells that have been subjected to any of these treatments are collectively referred to as processed microbial cells.

  The form of the disrupted cell is not particularly limited as long as it contains a nitrile hydratase-producing cell. For example, the culture solution containing the cell itself, the culture solution is centrifuged and separated and recovered. And those obtained by washing the collected cells with physiological saline or the like.

<Nitrile compound>
Examples of the nitrile compound include an aliphatic nitrile compound having 2 to 20 carbon atoms and an aromatic nitrile compound having 6 to 20 carbon atoms, which may be used alone or in combination of two or more.

  Examples of the aliphatic nitrile compound include saturated or unsaturated nitriles having 2 to 6 carbon atoms; specifically, acetonitrile, propionitrile, butyronitrile, isobutyronitrile, valeronitrile, isovaleronitrile, capronitrile Aliphatic saturated mononitriles such as malononitrile, succinonitrile, adiponitrile, and the like; and aliphatic unsaturated nitriles such as acrylonitrile, methacrylonitrile, and crotonnitrile.

  Examples of the aromatic nitrile compound include benzonitrile, o-, m- or p-chlorobenzonitrile, o-, m- or p-fluorobenzonitrile, o-, m- or p-nitrobenzonitrile, o- , M- or p-tolunitrile, o-, m- or p-cyanopyridine, benzyl cyanide.

  Among the nitrile compounds, acrylonitrile and methacrylonitrile are preferable.

<Flocculant>
In the present invention, a step of adding a flocculant to the amide compound-containing liquid produced using a bacterial cell catalyst containing nitrile hydratase to aggregate the bacterial cell catalyst, and then contacting the activated carbon catalyst, followed by solid-liquid separation Have

  As the flocculant, either an inorganic flocculant or an organic flocculant can be used. Examples of inorganic flocculants include aluminum flocculants such as aluminum sulfate, aluminum chloride, and polyaluminum chloride, and iron flocculants such as ferrous sulfate, ferric sulfate, polyferric sulfate, and ferric chloride. Is mentioned. Examples of organic flocculants include cationic polymer flocculants such as acrylic acid esters and methacrylic acid esters, anionic polymer flocculants such as carboxylic acids and sulfonic acids, and nonionic polymer flocculants. And amphoteric polymer flocculants.

  In the present invention, the amount of the flocculant used for the flocculation treatment of the amide compound-containing liquid is difficult to obtain a sufficient flocculating effect if it is too small, and it becomes uneconomical if used too much. The amount used is usually in the range of 0.0001 to 20% by weight, more preferably in the range of 0.0005 to 10% by weight, based on the amide compound-containing liquid. Further, when a powdery agent is used as the flocculant, the flocculant may be directly added directly to the amide compound-containing liquid, or once the flocculant is dissolved in a medium such as water to obtain a solution. You may make it add or supply a thing to an amide compound containing liquid.

<Activated carbon>
In the present invention, the flocculant is added to the amide compound-containing solution to aggregate the cells and / or the treated cells thereof, and then purified by contacting with activated carbon.

  Activated carbon generally has coal, wood, coconut shell, etc. as raw materials, but there is no particular limitation as long as it has adsorption capacity, and any of them should be used. Is possible. 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. Further, the method for contacting the activated carbon with the amide compound-containing liquid is not particularly limited. For example, when activated carbon is used particularly in powder form, the activated carbon is directly added to the amide compound-containing liquid after the addition of the flocculant. Alternatively, the activated carbon may be once dispersed in a medium such as water, and a slurry may be added to or supplied to the amide compound-containing liquid.

  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, and a centrifuge, and further, batch and continuous. Any of the formulas 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.

<PH regulator>
In the present invention, a pH adjusting agent may be used. The pH adjuster is used to adjust the pH of the purification treatment solution to a suitable range for maintaining good purification efficiency in the purification treatment. The addition of the pH adjusting agent may be performed, for example, before the addition of the flocculant, simultaneously with the addition of the flocculant, after the addition of the flocculant, and before contact with the activated carbon.

  When the pH suitable for the purification treatment is smaller than 7, an acid can be used as a pH adjuster.

  As the acid used as the pH adjuster, either an inorganic acid or an organic acid can be used. Examples of inorganic acids include hydrohalic acids such as hydrogen chloride, hydrogen bromide, and hydrogen iodide, hypochlorous acid, chlorous acid, chloric acid, perchloric acid, hypobromite, bromite, bromine Examples thereof include halogenated oxoacids such as acid, perbromic acid, hypoiodic acid, iodic acid, iodic acid, and periodic acid, sulfuric acid, nitric acid, phosphoric acid, and boric acid. Examples of organic acids include formic acid, acetic acid, propionic acid, acrylic acid, methacrylic acid, crotonic acid, succinic acid, malonic acid, fumaric acid, maleic acid, citric acid, lactic acid, benzoic acid and other carboxylic acids such as methanesulfonic acid, Examples include sulfonic acids such as ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. The acid used as the pH adjusting agent can be used in any state of gas, solid and liquid, but considering the ease of supply to the purification tank, it is preferable to use the acid in the liquid state. The acid in the state is more preferably used as an aqueous solution. In consideration of controllability of pH adjustment of the purification treatment tank, it is more preferable to use a liquid acid as the aqueous solution. The concentration of the acid when used as an aqueous solution is not particularly limited, but it is difficult to adjust the pH when a high concentration aqueous solution is used. Therefore, it is preferably 0.1 wt% or more and 99 wt% or less, more preferably 1 wt% or more and 90 wt%. Hereinafter, it is more preferably 1 wt% or more and 50 wt% or less.

  When the pH suitable for the purification treatment is higher than 7, a base can be used as a pH adjuster.

  As the base used as the pH adjuster, either an inorganic base or an organic base can be used. Examples of the inorganic base include alkali metal hydroxides such as lithium hydroxide, sodium hydroxide, and potassium hydroxide, alkaline earth metal hydroxides such as magnesium hydroxide and calcium hydroxide, lithium carbonate, and sodium carbonate. And alkali metal carbonates such as potassium carbonate, alkali metal hydrogen carbonates such as lithium hydrogen carbonate, sodium hydrogen carbonate and potassium hydrogen carbonate, and ammonia. Examples of the organic base include trimethylamine, triethylamine, aniline, and pyridine. The base used as the pH adjuster can be used in any state of gas, solid, and liquid, but considering the ease of supply to the purification tank, it is preferable to use the liquid state, gas or solid The base in the state is more preferably used as an aqueous solution. In consideration of controllability of pH adjustment of the purification treatment tank, it is more preferable to use a liquid base as the aqueous solution. The concentration of the base when used as an aqueous solution is not particularly limited, but pH adjustment becomes difficult when a high-concentration aqueous solution is used. Therefore, it is preferably 0.1 wt% or more and 99 wt% or less, more preferably 1 wt% or more and 90 wt%. Hereinafter, it is more preferably 1 wt% or more and 50 wt% or less.

  The pH in the purification treatment is not particularly limited as long as it is in a suitable range for maintaining good purification efficiency. However, when aggregation is performed, preferably basic conditions, more preferably pH 7.5 to pH 10.5. Is in range. When the pH is in the above range, it is preferable in that the aggregation efficiency by the aggregating agent can be maintained well.

  Examples of the method for aggregating in the purification treatment method of the present invention include: (1) a method (batch method) in which the amide compound-containing liquid and the aggregating agent are charged in the purification treatment tank all at once and then the aggregation step is performed (batch method); A method in which a part of the amide compound-containing liquid and the flocculant is charged into the purification treatment tank, and then the remaining amide compound-containing liquid and the flocculant are supplied continuously or intermittently to perform the agglomeration process (half-batch method), ( 3) Purification in the purification tank while continuously or intermittently supplying the amide compound-containing liquid and the flocculant and continuously or intermittently removing the purification treatment liquid containing the amide compound-containing liquid and the flocculant. The method (continuous method) which performs an agglomeration process continuously, without taking out the whole quantity of a processing liquid is mentioned. Among these, the continuous method is preferable in that it is easy to produce an amide compound industrially in large quantities and efficiently.

  In the present invention, the temperature at which the amide compound-containing liquid is purified by the flocculant is not particularly limited as long as the amide compound crystals do not precipitate and do not affect the stability. It is in the range of 0-80 ° C. In particular, in the case of purifying an amide compound-containing liquid having an unsaturated bond, such as an acrylamide or methacrylamide-containing liquid, the purification treatment temperature is 60 ° C. or lower, more preferably 10 to 50 ° C. to prevent the occurrence of a polymerization reaction. The range of is preferable.

  Next, examples of the present invention will be specifically described, but the present invention is not limited to these examples.

[Example 1]
[Preparation of microbial cells containing nitrile hydratase]
In accordance with the method described in Example 1 of JP-A-2001-340091, no. Three clone cells were obtained and similarly cultured by the method of Example 1 to obtain wet cells containing nitrile hydratase.

[Production of acrylamide]
In order to obtain a final product having an acrylamide concentration in the aqueous solution of 50% by weight, the reaction was performed under the following conditions.

  A 1 L glass flask equipped with a stirrer was prepared as the first reactor, and a Teflon (registered trademark) tube 20 m having an inner diameter of 5 mm was prepared as the second reactor. The raw water supply pipe, the pH regulator supply pipe, the acrylonitrile supply pipe, and the cell catalyst supply pipe are each directly connected to the first reactor.

  The first reactor was charged with 400 g of water in advance. The wet cells obtained by the preparation of the cells were suspended in pure water. This suspension was continuously fed at a rate of 10 g / h while stirring in the first reactor. Further, acrylonitrile was continuously supplied at a rate of 32 g / h, and pure water was continuously supplied as raw water at a rate of 38 g / h. The temperature of the reaction solution during the reaction was controlled by immersing both the first reactor and the second reactor in a water bath having a temperature of 10 to 20 ° C. A 0.1 M NaOH aqueous solution was used as a pH adjuster, the supply amount was adjusted so that the reaction pH was 7.5 to 8.5, and the resultant was supplied to the first reactor.

  In order to keep the liquid level of the first reactor constant, the reaction solution is continuously withdrawn from the first reactor at a rate of 80 g / h and continuously supplied to the second reactor. The reaction was allowed to proceed further within.

  Analysis was performed under the following HPLC conditions 50 hours after the start of the reaction. The conversion to acrylamide at the outlet of the first reactor was 95%, and the acrylonitrile concentration at the outlet of the second reactor was below the detection limit (10 ppm). The following).

Here, the analysis conditions were as follows.
・ Acrylamide analysis conditions:
High performance liquid chromatograph: LC-10A system
(Manufactured by Shimadzu Corporation)
(UV detector wavelength 250 nm, column temperature 40 ° C.)
Separation column: SCR-101H (manufactured by Shimadzu Corporation)
Eluent: 0.05% (volume basis) -phosphoric acid aqueous solution / acrylonitrile Analysis conditions:
High performance liquid chromatograph: LC-10A system
(Manufactured by Shimadzu Corporation)
(UV detector wavelength 200 nm, column temperature 40 ° C.)
Separation column: Wakosil-II 5C18HG (manufactured by Wako Pure Chemical Industries)
Eluent: 7% (volume basis) -acetonitrile, 0.1 mM-acetic acid,
An aqueous solution containing 0.2 mM sodium acetate at each concentration.

[Purification of acrylamide]
300 ppm of polyaluminum chloride manufactured by Nippon Chemical Industry Co., Ltd. was added to the reaction solution containing the bacterial cell catalyst obtained by the above production method, adjusted to pH 9 using a 0.1 M NaOH aqueous solution, and then 30 ° C at 25 ° C. Aggregation treatment was performed by stirring for a minute. Next, 2 wt% of powdered activated carbon PM-SX manufactured by Kuraray Chemical Co., Ltd. was added, and the mixture was stirred at 25 ° C. for 5 hours to perform adsorption treatment. Solid-liquid separation was performed by centrifuging the purified treatment solution at 7500 rpm / min for 5 minutes.

  The protein removal rate was determined by comparing the amount of protein contained in each of the reaction solution before the purification treatment and the supernatant solution after the purification treatment.

  The protein concentration was quantified using a protein analysis kit manufactured by Biorat after the amide compound contained in each of the reaction solution and the supernatant was removed by dialysis using a semipermeable membrane. The protein removal rate after the purification treatment was 99.5% or more.

  100 mL of methanol was added to 10 mL of the supernatant after purification, and the transmittance at 360 nm was measured. As a result, it was 99.9% or more, and the presence of a polymer of acrylamide was not observed.

[Example 2]
In the purification of acrylamide in Example 1, instead of adding polyaluminum chloride as a flocculant to 300 ppm, it was the same as Example 1 except that aluminum sulfate manufactured by Nippon Chemical Industry Co., Ltd. was added to 300 ppm. The purification process was performed.

  The protein removal rate was determined by comparing the amount of protein contained in each of the reaction solution before the purification treatment and the supernatant solution after the purification treatment.

  The protein removal rate after the purification treatment was 99.5% or more.

  100 mL of methanol was added to 10 mL of the supernatant after purification, and the transmittance at 360 nm was measured. As a result, it was 99.9% or more, and the presence of a polymer of acrylamide was not observed.

[Example 3]
In the purification of acrylamide of Example 1, instead of adding polyaluminum chloride as a flocculant to 300 ppm, except that MT Aqua Polymer Co., Ltd. polymer flocculant Acofloc N-100 was added to 100 ppm. Was purified in the same manner as in Example 1.

  The protein removal rate was determined by comparing the amount of protein contained in each of the reaction solution before the purification treatment and the supernatant solution after the purification treatment.

  The protein removal rate after the purification treatment was 99.5% or more.

  100 mL of methanol was added to 10 mL of the supernatant after purification, and the transmittance at 360 nm was measured. As a result, it was 99.9% or more, and the presence of a polymer of acrylamide was not observed.

[Example 4]
In the purification of acrylamide of Example 1, instead of adding polyaluminum chloride as a flocculant to 300 ppm, except that MT Aqua Polymer Co., Ltd. polymer flocculant Acofloc A-100 was added to 100 ppm. Was purified in the same manner as in Example 1.

  The protein removal rate was determined by comparing the amount of protein contained in each of the reaction solution before the purification treatment and the supernatant solution after the purification treatment.

  The protein removal rate after the purification treatment was 99.5% or more.

  100 mL of methanol was added to 10 mL of the supernatant after purification, and the transmittance at 360 nm was measured. As a result, it was 99.9% or more, and the presence of a polymer of acrylamide was not observed.

[Example 5]
In the purification of acrylamide in Example 1, instead of adding polyaluminum chloride as a flocculant to 300 ppm, except that the polymer flocculant Acofloc C-500N manufactured by MT Aqua Polymer Co., Ltd. was added to 100 ppm. Was purified in the same manner as in Example 1.

  The protein removal rate was determined by comparing the amount of protein contained in each of the reaction solution before the purification treatment and the supernatant solution after the purification treatment.

  The protein removal rate after the purification treatment was 99.5% or more.

  100 mL of methanol was added to 10 mL of the supernatant after purification, and the transmittance at 360 nm was measured. As a result, it was 99.9% or more, and the presence of a polymer of acrylamide was not observed.

[Example 6]
In the purification of acrylamide in Example 1, instead of adding 300 ppm of polyaluminum chloride to the reaction solution and adjusting the pH to 9 using a 0.1 M NaOH aqueous solution, a 0.5 wt% -acrylic acid aqueous solution reaction solution was used. Then, the purification treatment was performed in the same manner as in Example 1 except that the pH was adjusted to 5.

  The protein removal rate was determined by comparing the amount of protein contained in each of the reaction solution and the filtrate after centrifugation.

  The protein removal rate after the purification treatment was 99.5% or more.

  100 mL of methanol was added to 10 mL of the supernatant after purification, and the transmittance at 360 nm was measured. As a result, it was 98.3% and the presence of a polymer of acrylamide was recognized.

[Comparative Example 1]
In the purification of acrylamide in Example 1, 300 ppm of polyaluminum chloride was added to the reaction solution, adjusted to pH 9 with 0.1 M NaOH aqueous solution, and then subjected to an agglomeration treatment and then an adsorption treatment with powdered activated carbon. The liquid was subjected to solid-liquid separation by centrifugation for 5 minutes without treatment at 7500 rpm / min.

  The protein removal rate was determined by comparing the amount of protein contained in each of the reaction solution and the filtrate after centrifugation.

  The protein removal rate after the purification treatment was 80%.

  100 mL of methanol was added to 10 mL of the supernatant after purification, and the transmittance at 360 nm was measured. As a result, it was 99.9% or more, and the presence of a polymer of acrylamide was not observed.

Claims (5)

  In a method for purifying an amide compound produced using a bacterial cell containing nitrile hydratase and / or a processed product thereof, the flocculant is added to the amide compound-containing solution and / or the bacterial cell. A method for purifying an amide compound, comprising aggregating a treated product and then contacting with activated carbon, followed by solid-liquid separation.   The flocculant is added to the amide compound-containing liquid under basic conditions to aggregate the cells and / or the treated cells thereof, and then contacted with activated carbon, followed by solid-liquid separation. The purification method according to 1.   The purification method according to claim 1 or 2, wherein the amide compound is acrylamide or methacrylamide.   The purification method according to claim 1, wherein the flocculant is an inorganic flocculant.   The purification method according to claim 1, wherein the flocculant is an organic flocculant.