JP2016204279A - Purification method for amide compound and purification apparatus for amide compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently removing impurities contained in an amide compound produced by a hydration reaction of a nitrile compound by use of a bacterial cell containing nitrile hydratase and/or a bacterial cell-treated product thereof as a catalyst and to provide a purification apparatus for an amide compound that is suitably used in the purification method.SOLUTION: The purification method for an amide compound comprises mixing a pH regulator such as NaOH or the like with a reaction liquid containing an amide compound such as acrylonitrile or the like produced by use of a bacterial cell containing nitrile hydratase and/or a bacterial cell-treated product thereof so as to regulate the pH to 7.5 to 8.5 and after that, supplying the mixture to a purification treatment tank 1, bringing the mixture into contact with active carbon and then filtering the mixture. The purification apparatus for an amide compound comprises an amide compound-containing reaction liquid supply pipe 2, a pH regulator supply pipe 3, a supply line 4 for supplying a mixture of the reaction liquid and the pH regulator and a purification treatment tank 1 for purifying an amide compound.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for purifying an amide compound containing impurities produced by a hydration reaction of a nitrile compound using a amide compound-containing liquid, in particular, a bacterial cell and / or a treated product thereof as a catalyst, and purification of the amide compound It relates to the device.

  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, Patent Document 1 and Patent Document 2 use a purification method using activated carbon, Patent Document 3 uses a purification method using an ion exchange membrane, and Patent Document 4 uses a porous hollow fiber membrane. A purification method 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. 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.

  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, in particular, when producing an amide compound by directly hydrating a nitrile compound using nitrile hydratase or the like, which is an enzyme having a nitrile hydrating ability, the purification method using activated carbon of the above prior art does not provide the amide compound obtained. The quality is not high enough and there is still room for consideration.

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

  In the purification treatment of activated carbon of an amide compound containing impurities produced by hydration of a nitrile compound using the bacterial cells and / or treated cells thereof, with a suitable pH for efficiently removing impurities A method is used in which a pH regulator is supplied to a purification treatment tank so that there is a range, and the pH of the purification treatment solution is usually in a suitable range. For example, when the pH suitable for the purification treatment is smaller than 7, a method of supplying an acid as a pH regulator to the purification treatment tank, or when the pH suitable for the purification treatment is larger than 7, the base is a pH regulator. The method of supplying to a refinement | purification processing tank is used.

  In the purification process using activated carbon, the purification efficiency decreases when the pH value is outside the range suitable for maintaining good purification efficiency. Therefore, the purification tank is usually maintained at a suitable pH for maintaining good purification efficiency. A purification process is performed. In order to maintain the pH of the purification treatment tank, a pH regulator is supplied to the purification treatment tank and is usually dispersed by mixing and the pH of the purification treatment tank is adjusted.

  However, according to the study by the present inventors, when supplying the pH regulator alone to the purification tank, even if the pH regulator is dispersed after being supplied to the purification tank, the pH is appropriately controlled. It was found that the purification efficiency was lowered. Even if the pH of the purification tank is in the preferred range, the vicinity of the pH regulator supply port is temporarily deviated from the preferred pH range due to the high concentration of the pH regulator, and the amide compound is altered by reaction, polymerization, etc. It is presumed that impurities are generated by this, and the purification efficiency is lowered.

  An object of the present invention is to provide a method for efficiently purifying an amide compound and an apparatus for purifying an amide compound suitably used in the method.

  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, a pH regulator is mixed with the amide compound-containing solution and then supplied to the purification tank A method for purifying an amide compound, wherein the amide compound is acrylamide or methacrylamide, and [3] a microbial cell containing nitrile hydratase and / or Alternatively, an apparatus for purifying an amide compound comprising a purification treatment tank for purifying an amide compound produced using the treated microbial cell product and a supply line for supplying a mixture of the amide compound-containing liquid and the pH regulator.

  In the present invention, by mixing with the amide compound-containing liquid before the pH regulator is supplied to the purification treatment tank, local fluctuations in pH are reduced in the purification treatment tank, and the purification efficiency is lowered. The amide compound can be efficiently purified without any trouble.

FIG. 1 is a schematic view showing an embodiment of the production apparatus of the present invention. FIG. 2 is a schematic view showing an embodiment of the production apparatus of the present invention.

  The amide compound purification method and purification apparatus of the present invention will be described below.

[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, and a pH regulator is mixed with the liquid containing the amide compound. After that, it has the process of supplying to a refinement processing tank and making it contact with activated carbon.

<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 Such as cells 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 expressing a mutant nitrile hydratase with further improved amide compound resistance, nitrile compound resistance, and temperature resistance 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, and may belong to the genus Bacillus such as Bacillus subtilis. Other strains such as fungi, yeasts and actinomycetes are also included. As an example of such a case, MT-10822 [This strain is the Institute of Biotechnology, Institute of Industrial Technology, Ministry of International Trade and Industry, 1-3 1-3 Higashi, Tsukuba, Ibaraki, on February 7, 1996 (currently Tsukuba, Ibaraki) City East 1-1-1 Tsukuba Center Chuo No. 6 (National Institute of Technology and Evaluation, Patent Biological Deposit Center) under the accession number FERMBP-5785, based on the Budapest Treaty on the international recognition of deposits of bacterial cells under patent procedures It has 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.

  The microbial cells producing nitrile hydratase as described above may be appropriately cultured and grown by a known method 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, as long as the strain can grow and produce nitrile hydratase.

  In the present invention, in order to react the microbial cells producing the above nitrile hydratase with the nitrile compound, various treatments such as collection of cells by centrifugation or the like, and crushing to produce crushed bacterial cells, 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, benzyl cyanide.
Among the nitrile compounds, acrylonitrile and methacrylonitrile are preferable.

<Activated carbon>
In the present invention, purification is carried out by bringing amide compounds produced using nitrile hydratase-containing microbial cells and / or processed microbial cells thereof into contact with activated carbon.

  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, but there is no particular limitation as long as it has adsorption ability, and any of them may be used. It is possible to use. 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 powder form, the activated carbon may be directly added directly to the amide compound-containing liquid, 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.

  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.

<PH regulator>
The pH adjusting agent is used to adjust the pH of the purification treatment solution to a suitable range for maintaining good purification efficiency in the purification treatment.
When the pH suitable for the purification treatment is smaller than 7, an acid can be used as a pH regulator.

As the acid used as the pH regulator, either an inorganic acid or an organic acid can be used. Examples of the inorganic acid include hydrohalic acid such as hydrogen chloride, hydrogen bromide, hydrogen iodide, hypochlorous acid, chlorous acid, chloric acid, perchloric acid, hypobromite, bromite, bromine Examples thereof include halogenated oxo acids such as acid, perbromic acid, hypoiodic acid, iodic acid, iodic acid, and periodic acid, sulfuric acid, nitric acid, phosphoric acid, boric acid, and the like. Examples of the organic acid include carboxylic acid such as 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, 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 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 pH adjustment becomes difficult when an aqueous solution with a high concentration 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 larger than 7, a base can be used as a pH regulator.

  As the base used as the pH regulator, 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. Alkali metal carbonates such as potassium carbonate, lithium hydrogen carbonate, sodium hydrogen carbonate, alkali metal hydrogen carbonates such as potassium hydrogen carbonate, ammonia and the like. Examples of the organic base include trimethylamine, triethylamine, aniline, pyridine and the like. The base used as the pH regulator can be used in any state of gas, solid, and liquid, but considering the ease of supply to the purification treatment tank, it is preferable to use the base in the liquid state. The base in the state is more preferably used as an aqueous solution. Considering the controllability of the pH adjustment of the purification 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 an aqueous solution with a high concentration 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.

<Purification treatment tank>
As the purification treatment tank, a single-stage purification treatment tank composed of one purification treatment apparatus may be used, or a multistage purification treatment tank composed of a plurality of purification treatment apparatuses may be used. As the purification processor, a tank-type purification processor or a tube-type purification processor may be used. As the tank-type purification processor, a purification processor equipped with a stirrer is preferable.

  A supply line for supplying a mixture of the amide compound-containing liquid and the pH regulator is installed in the purification treatment tank. One supply line or a plurality of supply lines may be installed in one purification processor.

  As the supply line of the mixture of the amide compound mixture and the pH adjuster provided in the purification treatment tank, for example, the reaction mixture obtained in the reaction step is used as the amide compound-containing solution, and the mixture of the reaction mixture and the pH adjuster is used. Supply line. Alternatively, an external circulation line equipped with a pump for returning a part of the purification treatment liquid to the purification treatment tank is installed in the purification treatment tank, and a part of the purification treatment liquid taken out from the purification treatment tank as an amide compound-containing liquid And a supply line for a mixture of a part of the purification treatment liquid and the pH adjuster.

The pump provided in the external circulation line in the case of installing the external circulation line equipped with the pump in the refining treatment tank is not particularly limited. For example, a turbo type such as a centrifugal pump, a tilt pump or an axial pump Examples thereof include a pump, and a positive displacement pump such as a reciprocating pump and a rotary pump.
When installing an external circulation line equipped with a pump in the purification tank, a part of the purification solution is taken out from the purification tank using the pump provided in the external circulation line and passes through the external circulation line. And returned to the purification tank.

  The purification treatment tank may or may not have a temperature control device, but it is desirable to have a temperature control device from the viewpoint of controlling the temperature of the purification treatment liquid.

  In the case where the refining treatment tank has a temperature control device, the temperature control apparatus may be provided in the refining treatment tank itself or in an external circulation line installed in the refining treatment tank. Examples of the temperature control device in the purification treatment tank include a heat exchanger. Examples of heat exchangers include those installed directly in purification tanks such as jacket heat exchangers and coil heat exchangers, or multitubular cylindrical exchangers, spiral tube exchangers, and spiral plate exchangers. , Plate-type exchangers, double-pipe-type exchangers and the like installed in the external circulation line of the purification treatment tank.

  Examples of the purification treatment method include: (1) a method in which a mixture of an amide compound-containing solution and a pH regulator and activated carbon are charged in a purification treatment tank all at once (batch method), and (2) an amide compound is contained. The mixture of the liquid and the pH regulator and a part of the activated carbon are charged into the purification tank, and then the reaction is carried out by supplying the mixture of the remaining amide compound-containing liquid and the pH regulator and the activated carbon continuously or intermittently. Method (semi-batch method), (3) Continuous or intermittent supply of a mixture of amide compound-containing liquid and pH regulator and activated carbon, and purification treatment liquid (including amide compound-containing liquid, pH regulator and activated carbon) .)), A method (continuous method) in which the purification treatment is continuously carried out without taking out the entire amount of the purification treatment liquid in the purification treatment tank. 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 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.

Volume of purification treatment tank is not particularly limited, considering the industrial production, usually 0.1 m ³ or more, preferably from 1 to 100 m ^3, more preferably 5 to 50 m ^3. In the case where the purification tank is composed of a plurality of purification processors, the volume refers to the volume of each purification processor.

  The pH in the purification treatment tank is not particularly limited as long as it is in a suitable range for maintaining good purification efficiency, but is preferably in the range of pH 4 to pH 10. When the pH is in the above range, it is preferable in that the purification efficiency by activated carbon can be maintained satisfactorily.

  The pH of the purification tank refers to the pH in the purification processor when the purification tank is composed of only one purification processor, and when the purification tank is composed of a plurality of purification processors. , Refers to the pH within each purification processor. The pH of the purification treatment tank can be measured by, for example, an indicator method, a hydrogen electrode method, a quinhydrone electrode method, an antimony electrode method, or a glass electrode method. The pH of the purification treatment tank can be measured at any location in the purification treatment tank, and specifically, can be measured at the purification treatment tank outlet (purification treatment liquid outlet).

<Supply of pH regulator>
The pH adjuster is mixed with the amide compound-containing liquid supplied to the purification treatment tank and then supplied to the purification treatment tank. There is no particular restriction on the installation position of the supply port to the purification treatment liquid in the purification treatment tank of the supply line for supplying the mixture of the amide compound-containing liquid and the pH adjusting agent to the purification treatment tank. Or you may install in a refinement | purification processing liquid. The shape of the supply port of the mixture of the amide compound-containing liquid and the pH regulator is not particularly limited, and any shape that is usually used can be suitably used.

  As a method of mixing the pH regulator and the amide compound-containing liquid when the pH regulator is mixed with the amide compound-containing liquid and then supplied to the purification treatment tank, a known method can be used. The method etc. which mix a regulator and an amide compound containing liquid in piping are mentioned. In order to mix the pH regulator and the amide compound-containing liquid in the pipe, the pH regulator and the amide compound-containing liquid can be directly mixed by combining the pH regulator supply pipe and the amide compound-containing liquid supply pipe. Furthermore, the method of mixing actively by installing line mixers, such as a static mixer, in piping is mentioned.

  The pH regulator and amide compound-containing liquid supplied to the purification treatment tank may be mixed at an arbitrary ratio. Preferably, the ratio of the pH regulator: amide compound-containing liquid is 1: 1 to 1: 100,000,000, more preferably 1:10 to 1: 10,000,000. A known method can be used for supplying the mixture of the pH adjuster and the amide compound-containing liquid when the pH adjuster is mixed with the amide compound-containing liquid and then supplied to the purification treatment tank.

[Purification equipment for amide compounds]
An apparatus for purifying an amide compound according to the present invention includes a purification treatment tank for purifying an amide compound produced using a nitrile hydratase-containing microbial cell and / or a processed microbial cell product thereof, an amide compound-containing liquid, and a pH regulator. And a supply line for supplying the mixture.
The configuration of the refining treatment tank is as described above.

  Hereinafter, although the specific example of the refiner | purifier of the amide compound of this invention is demonstrated with reference to drawings, this invention is not limited to this.

  The production apparatus of FIG. 1 includes a purification treatment tank 1 provided with a stirrer 7, a reaction mixture supply pipe 2 and a pH adjustment agent supply pipe 3 connected to the purification treatment tank 1 through a supply pipe 4 for a mixture of a reaction mixture and a pH adjustment agent. With.

  The production apparatus of FIG. 2 includes a refining treatment tank 1 provided with a stirrer 7, an external circulation line 8 installed in a reaction tank 1 connected to the reaction tank 1 through a supply pipe 6 of a mixture of a refining treatment liquid and a pH regulator, and a pH. A regulator supply pipe 3 and a reaction mixture supply pipe 2 obtained in the reaction step are provided. A circulation pump 9 and a heat exchanger 10 are provided on the external circulation line.

  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 tank reactor with a SUS jacket heat exchanger (volume: 1 m ³ ) having a tank inner diameter of 1 m and a straight body length of 1.36 m, equipped with a stirrer as the first reactor, and a volume of 0.5 m as the second reactor. ³ SUS double tube reactors were prepared. 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 kg of water in advance. The wet cells obtained by the above culture method were suspended in pure water. This suspension was continuously fed at a rate of 11 kg / h while stirring in the first reactor. Further, acrylonitrile having a purity of 99.8% was continuously fed to the first reactor through an acrylonitrile feed pipe at a rate of 32 kg / h. Pure water was continuously supplied to the first reactor through a pure water supply pipe at a rate of 37 kg / h. The temperature of the reaction solution during the reaction is 20 ° C., and 5 ° C. cooling water is circulated through the double tube of the heat exchanger and the second reactor installed on the external circulation line of the first reactor. Control was performed. 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 continuously to the first reactor via the pH adjuster supply pipe. Supplied. The reaction pH was measured using the glass electrode method at the outlet of the first reactor.

  The reaction solution is continuously withdrawn from the first reactor at a rate of 80 kg / h so that the reaction solution is 1 m above the bottom of the tank, and continuously supplied to the second reactor. The reaction was allowed to proceed further in the second reactor.

  Analysis was conducted 200 hours after the start of the reaction under the following HPLC conditions. The conversion to acrylamide at the first reactor outlet was 95%, and the acrylonitrile concentration at the second reactor outlet was below the detection limit (10 Weight ppm or less). The acrylamide concentration at the outlet of the second reactor was 53.1% by weight.

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, 0.2 mM sodium acetate at each concentration
Aqueous solution

[Purification of acrylamide]
As a purification treatment tank, a tank-type purification treatment tank with a SUS jacket type heat exchanger (volume: 0.5 m ³ ) having a tank inner diameter of 0.8 m and a straight body length of 1 m was prepared. The second reactor outlet pipe was connected to the purification treatment tank. The pH regulator supply pipe is a junction pipe connected to the second reactor outlet pipe, and the reaction mixture and the pH regulator are mixed in the junction pipe and supplied to the purification treatment tank as a mixture. The activated carbon supply pipe is directly connected to the purification treatment tank.

  The reaction mixture containing the bacterial cell catalyst obtained by the above production method was continuously supplied to the purification treatment tank at a rate of 80 kg / h. A 0.5% by weight-acrylic acid aqueous solution was used as a pH adjuster, and the supply amount was adjusted so that the pH of the purified treatment solution was 4.9 to 5.1. The pH regulator was joined to the second reactor outlet pipe via the pH regulator supply pipe, mixed with the reaction mixture, and then continuously supplied to the purification treatment tank. The pH of the purification treatment solution was measured using a glass electrode method at the outlet of the purification treatment tank. Powdered activated carbon PM-SX manufactured by Kuraray Chemical Co., Ltd. was continuously supplied to the purification treatment tank at a rate of 0.5 kg / h. The purification treatment liquid was continuously taken out from the purification treatment tank at a rate of 80 kg / h and filtered.

  The filtrate was collected 100 hours after the start of the purification treatment. The protein removal rate was calculated | required by comparing the quantity of the protein contained in each of the reaction liquid before the refinement | purification process obtained from the 2nd reactor exit, and the filtrate after a refinement | purification process.

  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 filtrate 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 filtrate after the purification treatment, and the transmittance at 360 nm was measured. As a result, it was 99.9% or more, and the presence of an acrylamide polymer was not observed.

[Example 2]
In the purification of acrylonitrile in Example 1, acrylamide was purified in the same manner as in Example 1 except that instead of 0.5 wt% -acrylic acid aqueous solution as a pH regulator, 1 wt% -sulfuric acid aqueous solution was used. Went. The filtrate was collected 100 hours after the start of the purification treatment, and the protein removal rate was determined by the same method as in Example 1. The protein removal rate was 99.5% or more.

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

[Comparative Example 1]
In the purification of acrylonitrile in Example 1, the acrylamide was purified in the same manner as in Example 1 except that the supply pipe for the pH regulator was directly connected to the purification tank and the pH regulator was directly fed to the purification tank. went. The filtrate was collected 100 hours after the start of the purification treatment, and the protein removal rate was determined by the same method as in Example 1. The protein removal rate was 95%.
100 mL of methanol was added to 10 mL of the filtrate after the purification treatment, and the transmittance at 360 nm was measured. As a result, it was 98.5%, and the presence of a polymer of acrylamide was recognized.

1: Purification tank 2: Reaction mixture supply pipe 3: pH regulator supply pipe 4: Feed pipe for mixture of reaction mixture and pH regulator 4 ': Feed pipe for mixture of purification treatment liquid and pH regulator 5: Stirrer 6 : External circulation line 7: Circulation pump 8: Heat exchanger 9: Cooling water inlet 10: Cooling water outlet

Claims (3)

In a method for purifying a microbial compound containing nitrile hydratase and / or an amide compound produced by treating the microbial compound, a pH regulator is mixed in the amide compound-containing solution and then supplied to a purification treatment tank. Method for purifying amide compound characterized by contacting with The production method according to claim 1, wherein the amide compound is acrylamide or methacrylamide. A purification treatment tank for purifying an amide compound produced using a nitrile hydratase-containing microbial cell and / or a processed microbial cell product thereof, and a supply line for supplying a mixture of the amide compound-containing liquid and the pH regulator Amide compound purification equipment