JP2019089727A - Method of purifying amide compound - Google Patents

Abstract

To provide a method of purifying efficiently an amide compound produced by a hydration reaction of a nitrile compound using as a catalyst an amide compound-containing liquid, particularly a cell and/or a processed cell thereof.SOLUTION: The present invention provides a method for purifying an amide compound produced using a cell and/or a processed cell thereof containing nitrile hydratase, where, a flocculant is added to an amide compound-containing liquid, to flocculate the cell and/or the processed cell thereof, and then subject it to solid-liquid separation.SELECTED DRAWING: None

Description

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

  Although an amide compound, particularly an amide compound-containing liquid obtained by hydrating a nitrile compound varies in type depending on its production method, usually, a polymer substance, surfactant, coloring substance, eluate, or other impurities are present Do. In order to remove these impurities, for example, Patent Document 1 and Patent Document 2 perform a purification treatment method with activated carbon, Patent Document 3 a purification treatment method with an ion exchange membrane, and Patent Document 4 a porous hollow fiber A membrane purification process has been described.

  However, the purification method using the above-mentioned ion exchange membrane or porous hollow fiber membrane requires a special purification apparatus, and the demerit in terms of economy can not be avoided in its implementation. Moreover, in the purification method using activated carbon, although special equipment is not usually required, impurities are still present in the obtained product, and the effect is still insufficient.

  In addition, Patent Document 5 proposes a method of bringing an amide compound-containing liquid into contact with activated carbon under acidic conditions as a simple and efficient purification method. However, in particular, when the nitrile compound is directly hydrated using nitrile hydratase or the like having an ability to hydrate nitrile to produce an amide compound, the purification efficiency with activated carbon of the above prior art is sufficiently high in purification efficiency. However, the quality of the resulting amide compound can not be said to be sufficiently high, and there is still room for investigation.

Japanese Patent Application Laid-Open No. 61-115495 Japanese Patent Application 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 a method of efficiently purifying an amide compound in the purification treatment of an amide compound containing an impurity produced by hydration reaction of a nitrile compound using a cell and / or a treated product of the cell. It is to provide.

  The present inventors have intensively studied to solve the above-mentioned problems. As a result, they have found that the above-mentioned problems can be solved by the method for purifying an amide compound having the following steps, and the present invention has been achieved.

  That is, the present invention is as follows.

  [1] A method of purifying a cell containing the nitrile hydratase and / or an amide compound produced using the cell treated product thereof, comprising adding an aggregating agent to the liquid containing an amide compound to thereby produce the cell and / or the cell A method of purifying an amide compound comprising solid-liquid separation of the treated product of the cells after aggregation.

  [2] The purification according to the above-mentioned [1], characterized in that a coagulant is added to the amide compound-containing solution under basic conditions, and the cells and / or the treated product of the cells is subjected to aggregation after solid-liquid separation. Method.

  [3] The purification method according to the above [1] or [2], wherein the amide compound is acrylamide or methacrylamide.

  [4] The purification method according to any one of the above [1] to [3], wherein the coagulant is an inorganic coagulant.

  [5] The purification method according to any one of the above [1] to [3], wherein the coagulant is an organic coagulant.

  In the present invention, the amide compound can be efficiently purified by using a flocculant.

  Hereafter, the purification method of the amide compound of this invention is demonstrated.

[Method for purifying amide compound]
The method for purifying an amide compound of the present invention is a method of purifying a cell containing a nitrile hydratase and / or an amide compound produced using the cell treated product thereof, wherein an aggregating agent is added to the amide compound-containing liquid The method comprises solid-liquid separation after aggregation of the cells and / or a treated product of the cells. The “amide compound-containing liquid” to which a flocculant is added in the step of solid-liquid separation is a liquid containing a nitrile hydratase-containing cell and / or an amide compound produced using the cell-treated product thereof. Point to.

<A cell containing nitrile hydratase and/or a treated product of the cell>
In the present invention, a cell containing nitrile hydratase and / or a cell treated product of the cell (hereinafter, these are simply referred to as "cell body catalyst") is used as a hydration catalyst for the amide compound of the nitrile compound.

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

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

  Also, transformants in which the nitrile hydratase gene cloned from these cells is highly expressed in any host, and one or two or more of the constituent amino acids of the nitrile hydratase as other amino acids using recombinant DNA technology Also included are transformants in which a mutant-type nitrile hydratase is expressed, in which amide compound resistance, nitrile compound resistance and temperature resistance are further improved by substitution, deletion, deletion or insertion. In addition, although E. coli (Escherichia coli) is mentioned as a representative example as an arbitrary host mentioned here as in the examples described later, it is not particularly limited to E. coli, and Bacillus genus such as Bacillus subtilis. Other strains such as fungi, yeasts and actinomycetes are also included. As an example of such a thing, MT-10 822 (this strain is a life engineering industrial research institute of the industrial technology institute of the Ministry of International Trade and Industry of the Ministry of International Trade and Industry of the east 1-chome, Tsukuba, Ibaraki prefecture on February 7, 1996 (now Kisarazu, Chiba prefecture) Based on the Budapest Treaty on the International Recognition of the Deposit of Bacterial Cells under Patent Procedure as Accession No. FERMBP-5785 to the City Administration Team 2-5-8, National Institute of Technology and Evaluation, National Institute of Technology and Evaluation, Biotechnology Technology Center Has been deposited. ] Is mentioned.

  Among these cells, cells belonging to the genus Pseudonocardia and having a nitrile hydratase gene cloned from the cells can be used as an arbitrary host in that they have high activity and high stability nitrile hydratase. It is preferable to use a transformant that is highly expressed and a transformant that expresses a mutant form of nitrile hydratase. The above transformant is preferable in that it improves the stability of nitrile hydratase and the activity per cell is higher.

  In addition, Rhodococcus rhodochrous J-1 capable of highly expressing nitrile hydratase in the cell body, and a transformant in which the nitrile hydratase gene cloned from the cell is highly expressed in any host are also preferable. . Bacterial cells producing the above nitrile hydratase can be prepared by general methods 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, and for example, a gene encoding nitrile hydratase in a commercially available expression plasmid typified by pET-21a (+), pKK223-3, pUC19, pBluescript KS (+) and pBR322 etc. An expression plasmid of the nitrile hydratase can be constructed by Further, as a host organism used for transformation, any recombinant organism can be used as long as it is stable and can be self-propagated and can express foreign DNA traits, for example, E. coli is a preferred example, but only E. coli is preferred. By introducing into Bacillus subtilis, yeast or the like, transformants having the ability to produce nitrile hydratase can be obtained.

  Bacterial cells producing 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, any medium such as a synthetic medium or a natural medium may be used as long as it is a medium containing an appropriate amount of carbon source, nitrogen source, inorganic salts and other nutrients. For example, after inoculating cells in a normal liquid medium such as LB medium or M9 medium, appropriate culture temperature (generally 20 ° C. to 50 ° C., but in the case of thermophilic bacteria, 50 ° C. or higher) It may be prepared by culturing it in The culture can be carried out using a common culture method such as shaking culture, aeration and stirring culture, continuous culture, or fed-batch culture in a liquid medium containing the above-mentioned culture components. As a culture | cultivation temperature of a transformant, 15-37 degreeC is preferable. The culture conditions are not particularly limited, and may be appropriately selected according to 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 above-mentioned nitrile hydratase-producing cells with a nitrile compound, various treatments may be performed, such as collecting bacteria by centrifugation or the like, or crushing to prepare crushed cells. The cells treated with any of these treatments are collectively referred to as treated cells.

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

<Nitrile compound>
As a nitrile compound, a C2-C20 aliphatic nitrile compound and a C6-C20 aromatic nitrile compound are mentioned, for example, You may use by 1 type and may use 2 or more types together.

  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 and adiponitrile, and aliphatic unsaturated nitriles such as acrylonitrile, methacrylonitrile and crotononitrile.

  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-toluene nitrile, o-, m- or p-cyanopyridine, benzyl cyanide.

  Among the nitrile compounds, acrylonitrile and methacrylonitrile are preferable.

Flocculant
In the present invention, there is the step of adding a flocculant to an amide compound-containing solution produced using a microbial cell catalyst containing nitrile hydratase, and subjecting the microbial cell catalyst to solid-liquid separation after aggregation.

  As the coagulant, either an inorganic coagulant or an organic coagulant can be used. Examples of inorganic coagulants include aluminum coagulants such as aluminum sulfate, aluminum chloride and polyaluminum chloride, and iron coagulants such as ferrous sulfate, ferric sulfate, polyferric sulfate and ferric chloride Can be mentioned. As an organic type coagulant | flocculant, for example, cation type polymer coagulant | flocculants, such as acrylic acid ester type and a methacrylate ester type | system | group, anionic polymer coagulant | flocculants, such as carboxylic acid type and sulfonic acid type, nonionic polymer coagulant | flocculant And amphoteric polymer flocculants.

  In the present invention, it is difficult to obtain a sufficient aggregation effect if the amount of aggregating agent used when aggregating the amide compound-containing liquid is too small, and it is uneconomical to use too much. The amount thereof 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. When a powdery agent is used as the aggregating agent, the aggregating agent may be added directly to the amide compound-containing solution as it is, or the aggregating agent is once dissolved in a medium such as water to prepare a solution. The substance may be added to or supplied to the amide compound-containing liquid.

  Next, in the solid-liquid separation step of the present invention, the aggregate of the microbial cell catalyst is separated from the aggregation-treated amide compound-containing solution to obtain a purified solution of the amide compound-containing solution. There is no particular limitation on the method of separating the aggregates of the microbial cell catalyst, as long as it is a method using a generally used solid-liquid separation device, and examples thereof include pressure filters, vacuum filters, and centrifuges. May be either a batch system or a continuous system. Further, in the present invention, the solution containing the amide compound after separation of the aggregates of the above-mentioned microbial cell catalyst is cooled, and further purification is carried out by adopting a method of crystallizing the desired amide compound from the solution. It is also possible to obtain amide compounds.

<PH regulator>
The pH adjuster is used to adjust the pH of the purification treatment solution to a suitable range for keeping the purification efficiency in the purification treatment good.

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

  As an acid used as a pH regulator, any of an inorganic acid and an organic acid can be used. As an inorganic acid, for example, hydrohalic acid such as hydrogen chloride, hydrogen bromide, hydrogen iodide, etc., hypochlorous acid, chlorous acid, chloric acid, perchloric acid, hypobromous acid, brominated acid, bromine Examples thereof include halogenated oxo acids such as acid, perbromic acid, hypoiodite, hypoiodite, iodic acid and periodic acid, sulfuric acid, nitric acid, phosphoric acid and oxalic acid. Examples of organic acids include carboxylic acids such as formic acid, acetic acid, propionic acid, acrylic acid, methacrylic acid, crotonic acid, oxalic acid, malonic acid, fumaric acid, maleic acid, citric acid, lactic acid, benzoic acid and the like, and methanesulfonic acid, Sulfonic acids such as ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like can be mentioned. The acid used as a pH regulator can be used in any state of gas, solid or liquid, but considering the ease of supply to the purification treatment tank, it is preferable to use the liquid state, and it is preferable to use gas or solid It is more preferable to use the acid in the form of an aqueous solution. In consideration of the controllability of pH control of the purification treatment tank, it is more preferable to use the acid in the liquid state as an 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 using a high concentration aqueous solution, preferably 0.1 wt% or more and 99 wt% or less, more preferably 1 wt% or more and 90 wt% The content is more preferably 1% by weight or more and 50% by weight or less.

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

  As a base used as a pH regulator, any of an inorganic base and an organic base can be used. As the inorganic base, for example, hydroxides of alkali metals such as lithium hydroxide, sodium hydroxide and potassium hydroxide, hydroxides of alkaline earth metals such as magnesium hydroxide and calcium hydroxide, lithium carbonate and sodium carbonate And carbonates of alkali metals such as potassium carbonate, lithium hydrogen carbonate, hydrogen carbonates of alkali metals such as sodium hydrogen carbonate and potassium hydrogen carbonate, and ammonia. Examples of the organic base include trimethylamine, triethylamine, aniline and pyridine. Although the base used as a pH regulator can be used in any state of gas, solid or liquid, it is preferable to use the one in liquid state, considering ease of supply to the purification treatment tank, and it is preferable to use gas or solid It is more preferable to use the base in the state of as an aqueous solution. In consideration of the controllability of pH control of the purification treatment tank, it is more preferable to use the base in the liquid state as an aqueous solution. The concentration of the base when used as an aqueous solution is not particularly limited, but it is difficult to adjust the pH when using a high concentration aqueous solution, preferably 0.1 wt% to 99 wt%, more preferably 1 wt% to 90 wt%. The content is more preferably 1% by weight or more and 50% by weight or less.

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

  As a method of carrying out the aggregation in the purification treatment method in the present invention, for example, (1) A method (batch method) in which the aggregation step is carried out after all the amide compound-containing liquid and the coagulant are charged into the purification treatment tank at once. A method of carrying out the aggregation step by feeding the remaining amide compound-containing liquid and the coagulant continuously or intermittently after charging the amide compound-containing liquid and a part of the coagulant into the purification treatment tank (semi-batch method), 3) Purification in the purification processing tank while continuously or intermittently supplying the amide compound-containing liquid and the flocculant, and continuously or intermittently taking out the purified treatment liquid containing the amide compound-containing liquid and the flocculant and the like The method (continuous method) which performs an aggregation process continuously, without taking out a processing liquid whole quantity is mentioned. Among these, the continuous method is preferable in that it is easy to industrially produce a large amount of amide compounds efficiently.

  In the present invention, the temperature at which the amide compound-containing liquid is purified and treated with a coagulant is not particularly limited as long as crystals of the amide compound do not precipitate and the stability thereof is not affected. It is the range of 0-80 ° C. In particular, the purification treatment temperature in the case of purifying an amide compound-containing liquid having an unsaturated bond such as an acrylamide or methacrylamide-containing liquid is 60 ° C. or less, further 10 to 50 ° C. to prevent generation of a polymerization reaction. Is preferred.

  EXAMPLES The present invention will next be described in detail by way of examples, which should not be construed as limiting the invention thereto.

Example 1
[Preparation of cells containing nitrile hydratase]
According to the method described in Example 1 of Japanese Patent Application Laid-Open No. 2001-340091, no. 3 Clonal bacterial cells were obtained, and cultured in the same manner as in Example 1 to obtain wet bacterial cells containing nitrile hydratase.

[Production of Acrylamide]
The reaction was carried out under the following conditions in order to obtain a product having an acrylamide concentration of 50% by weight in an aqueous solution as a final product.

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

  In the first reactor, 400 g of water was charged 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. Also, acrylonitrile was continuously supplied at a rate of 32 g / h and pure water as raw material water at a rate of 38 g / h. The temperature control was performed by immersing both the first reactor and the second reactor in a water bath at a temperature of 10 to 20 ° C. so that the temperature of the reaction solution during the reaction was 15 ° C. The amount of feed was adjusted so that the reaction pH was 7.5 to 8.5 using a 0.1 M aqueous solution of NaOH as a pH adjuster, and then fed to the first reactor.

  The reaction liquid is continuously withdrawn from the first reactor at a rate of 80 g / h and continuously supplied to the second reactor so as to keep the liquid level of the first reactor constant, and the second reactor The reaction proceeded further inside.

  The analysis was carried out under the following HPLC conditions 50 hours after the start of the reaction: 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 ppm It became below.

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

Purification of Acrylamide
300 ppm of polyaluminum chloride manufactured by Nippon Chemical Industrial Co., Ltd. is added to the reaction solution containing the bacterial cell catalyst obtained by the above-mentioned production method, and the pH is adjusted to 9 using a 0.1 M aqueous NaOH solution. The aggregation treatment was performed by stirring for a minute. Solid-liquid separation was performed by centrifuging the flocculation treatment liquid 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 purification treatment and the supernatant fluid after 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 liquid and the supernatant liquid was dialyzed off with a semipermeable membrane. The protein removal rate after the purification treatment was 99.5% or more.

Example 2
The purification of acrylamide of Example 1 is the same as Example 1 except that aluminum sulfate made by Nippon Chemical Industry Co., Ltd. is added so as to be 300 ppm instead of adding polyaluminum chloride as the coagulant so as to be 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 purification treatment and the supernatant fluid after purification treatment.

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

[Example 3]
In the purification of acrylamide of Example 1, except that polyaluminum chloride is added so as to be 50 ppm as a coagulant, except that a polymer coagulant Akofloc N-100 manufactured by MT Aqua Polymer Co., Ltd. is added so as to be 100 ppm. The purification treatment was carried out 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 purification treatment and the supernatant fluid after purification treatment.

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

Example 4
In the purification of the acrylamide of Example 1, except that polyaluminum chloride was added so as to be 50 ppm as a coagulant, except that a polymer coagulant Akofloc A-100 manufactured by MT Aqua Polymer Co., Ltd. was added so as to be 100 ppm. The purification treatment was carried out 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 purification treatment and the supernatant fluid after purification treatment.

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

[Example 5]
In the purification of acrylamide of Example 1, except that polyaluminum chloride is added so as to be 50 ppm as a coagulant, except that a polymer coagulant Akofloc C-500N manufactured by MT Aqua Polymer Co., Ltd. is added so as to be 100 ppm. The purification treatment was carried out in the same manner as in Example 1.

Comparative Example 1
In the purification of acrylamide in Example 1, 300 ppm of polyaluminum chloride was added to the reaction solution, and after adjusting to pH 9 with 0.1 M NaOH aqueous solution, instead of performing aggregation treatment, the reaction solution was treated without treatment at 7500 rpm / min. Solid-liquid separation was performed by centrifugation for 5 minutes under the conditions.

  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 purification treatment was 80%.

[Example 6]
In the purification of the acrylamide of Example 1, after adding 300 ppm of polyaluminum chloride to the reaction solution and then adjusting to pH 9 using a 0.1 M aqueous NaOH solution, a 0.5 wt% acrylic acid aqueous solution reaction solution is used. The purification treatment was carried out 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 90%.

Claims (5)

  In the method of purifying an amide compound produced by using a cell containing nitrile hydratase and / or a treated product of the cell, a coagulant is added to the liquid containing an amide compound, and the cell and / or the cell is A method of purifying an amide compound comprising solid-liquid separation of the treated product after aggregation.   The purification method according to claim 1, characterized in that the flocculating agent is added to the amide compound-containing liquid under basic conditions, and the cells and / or the treated product of the cells is subjected to aggregation after solid-liquid separation.   The purification method according to claim 1 or 2, wherein the amide compound is acrylamide or methacrylamide.   The purification method according to any one of claims 1 to 3, wherein the coagulant is an inorganic coagulant.   The purification method according to any one of claims 1 to 3, wherein the coagulant is an organic coagulant.