JP2018046795A - Method for producing amide compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently producing an amide compound, industrially very important.SOLUTION: The present invention provides a method for producing an amide compound using bacterial cells containing nitrile-hydratase and/or processed bacterial cell products thereof. The method uses bacterial cells suspended in water with an electric conductivity of 100 μS/cm or less, and/or processed bacterial cells thereof.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing an amide compound that uses a microbial cell and / or a treated product thereof as a catalyst and hydrates a nitrile compound to obtain an amide compound.

  As one of the main production methods of amide compounds, a hydration method using a nitrile compound as a raw material is used in many cases, and as an industrial production method of an amide compound such as acrylamide, for example, a metal such as Raney copper A method of hydrating a nitrile compound such as acrylonitrile using copper as a catalyst, or a method of hydrating a nitrile compound using a microbial cell containing nitrile hydratase and / or a treated product thereof as a catalyst in recent years. Are known.

The method of using bacterial cells and / or treated products thereof as a catalyst has attracted industrial attention because of high conversion and selectivity of nitrile compounds such as acrylonitrile.
The activity of the bacterial cell catalyst is unstable and tends to decrease over time. In addition, since the cell catalyst may cause a decrease in activity at a temperature exceeding the heat resistance of the cell, the reaction vessel is usually operated at a temperature at which the activity is maintained. For example, in Patent Document 1, in a method for producing acrylamide from acrylonitrile using a biocatalyst that is easily deactivated by heat, the reaction temperature and the reaction temperature are efficiently removed while being an industrial reaction scale. A method has been proposed in which the temperature difference from the temperature of the cooling water introduced into the heat exchanger is maintained within a certain range. Patent Document 2 discloses a method for producing acrylamide from acrylonitrile using a biocatalyst having a nitrile hydratase, and a method for producing a high-quality acrylamide having a higher concentration with a smaller amount of catalyst, and storing at less than 30 ° C. A method of using the prepared acrylonitrile has been proposed. In Patent Document 3, as a method for efficiently producing an amide compound without reducing the activity of the bacterial cell catalyst, the temperature of the nitrile compound and water supplied to the reaction tank is set to a liquid temperature in the reaction tank of + 10 ° C. or lower. In addition, in the embodiment, the temperature in the storage tank is controlled to be 15 ° C. by immersing the storage tank of acrylonitrile and pure water in a water bath having a temperature of 15 ° C. A method is disclosed in which the temperature of the storage tank is controlled to be 5 ° C. or less by immersing the storage tank of the body catalyst suspension in a water bath at a temperature of 5 ° C. Patent Document 4 discloses that, in producing an amide compound from a nitrile compound, stirring is performed in a reactor as a method for increasing the conversion efficiency from the nitrile compound to the amide compound by improving the dispersibility while suppressing deterioration of the biocatalyst. In the method for producing an amide compound while performing the reaction, a method is disclosed in which the power required for stirring per unit volume of the reaction fluid is set within a range of 0.08 to 0.7 kW / m ³ . Patent Document 5 discloses a method for producing an amide compound from a nitrile compound by using a positive displacement pump for transporting a liquid containing bacterial cells and the amide compound. . In Patent Document 6, as an efficient method for producing an amide compound, a bacterial cell and / or a treated product thereof is reacted using a bacterial cell storage tank equipped with a temperature controller and a stirrer in a method for producing an amide compound. A method for producing an amide compound, characterized in that it is supplied to a tank, is disclosed.
However, in the methods described in Patent Document 1 and Patent Document 2, temperature control is performed only in the reaction tank and the supplied raw material, which is insufficient for efficient use of the bacterial cell catalyst. It was. Moreover, since the method of cooling the storage tank of the suspension of the bacterial cell catalyst disclosed in Patent Document 3 is cooling only from the surface of the storage tank, the liquid in the storage tank cannot be efficiently cooled. The liquid in the storage tank cannot be cooled uniformly, and temperature distribution occurs in the liquid in the storage tank. Furthermore, it has been studied in a storage tank for a small-scale bacterial cell catalyst, and heat removal efficiency on an industrial scale is not taken into consideration. Considering the industrial scale of production, the volume of the cell catalyst storage tank becomes large, and the larger the storage volume, the smaller the ratio of the heat transfer area used for cooling per volume, making heat removal more difficult. It is not sufficient for efficient use of the bacterial cell catalyst. Further, in the methods described in Patent Document 4 and Patent Document 5, although deterioration of the catalyst supplied into the reactor is suppressed, deterioration of the catalyst stored for supply to the reactor is suppressed. It is not possible. Further, in the method described in Patent Document 6, the deterioration of the catalyst stored for supply to the reactor is not sufficiently suppressed, and there is still room for examination.

International Publication No. 2010/038832 Pamphlet International Publication No. 2011/138966 Pamphlet International Publication No. 2012/039407 Pamphlet International Publication No. 2009/113654 Pamphlet JP 2006/187257 pamphlet JP 2015/73490 pamphlet

  An object of the present invention is to provide a method for efficiently producing an amide compound in a hydration reaction of a nitrile compound using a bacterial cell and / or a treated product thereof.

The present inventors have conducted intensive research to solve the above problems. As a result, in a method for producing an amide compound using a bacterial cell containing nitrile hydratase and / or a treated product thereof, the bacterial cell suspended in water having an electric conductivity of 100 μS / cm or less and / or It was found that by using the treated cell product, an amide compound can be efficiently produced without reducing the activity of the cell catalyst, and the present invention has been completed.

That is, the present invention is as follows.
[1] In a method for producing an amide compound using a bacterial cell containing nitrile hydratase and / or a treated product thereof, the bacterial cell suspended in water having an electric conductivity of 100 μS / cm or less and / or A process for producing an amide compound characterized by using the treated cell product [2] The process for producing a nitrile hydratase according to [1], wherein the nitrile hydratase is derived from Pseudonocardia or Rhodococcus The method according to [1] or [2], wherein the amide compound is acrylamide or methacrylamide.

In the present invention, an amide compound can be produced efficiently without reducing the activity of the bacterial cell catalyst.

Hereinafter, the manufacturing method of the amide compound of this invention is demonstrated.
[Method for producing amide compound]
The method for producing an amide compound according to the present invention is a method for producing an amide compound using a microbial cell containing nitrile hydratase and / or a treated product thereof, which is suspended in water having an electric conductivity of 100 μS / cm or less. A step of supplying the turbid cells and / or the processed cells thereof to the reaction tank.
<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. Examples of the cells producing nitrile hydratase include, for example, Nocardia genus, Corynebacterium genus, Bacillus genus, thermophilic Bacillus genus, Pseudomonas genus, Micrococcus (Micrococcus ), Rhodococcus species represented by Rhodochrous species, Acinetobacter genus, Xanthobacter genus, Streptomyces genus, Rhizobium genus, Klebsiella (Klebella) Genus, Enterobacter genus, Erwinia genus, Aeromonas genus, Citrobacter genus, Achromobacter genus, Agrobacterium genus or thermophila species Pseudonocardia, Bacteridium, Bibakuteriumu (Brevibacterium) bacteria belonging to the 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, 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 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 (currently 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 Biotechnology Center of the National Institute of Technology and Evaluation 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 commercially available expression plasmid represented by pET-21a (+), pKK223-3, pUC19, pBluescriptKS (+), pBR322 and the like encodes nitrile hydratase. By incorporating a gene, an expression plasmid for the 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, as long as the strain can grow and 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 microbial cell to be crushed when producing the microbial cell disrupted product is not particularly limited as long as the microbial cell producing nitrile hydratase is included. For example, the culture solution containing the microbial cell itself, the culture solution thereof The collected cells separated and collected by centrifugation, and those collected by washing with physiological saline or the like.

  In the present invention, the bacterial cells and / or the treated cells thereof are supplied to the reaction tank in a state suspended in water having an electric conductivity of 100 μS / cm or less.

  Generally, salt components such as calcium and sodium are dissolved in a small amount in natural water and tap water, and these components are mainly dissolved in an ionic state, so water in which salt components are dissolved is electrically conductive. The degree becomes higher. In the present invention, the electric conductivity of water used for suspending cells and / or treated cells is 100 μS / cm or less, preferably 50 μS / cm or less, more preferably 10 μS / cm or less. When the electric conductivity of water is 100 μS / cm or less, it can be used for suspension as it is. If the electrical conductivity of the water is greater than 100 μS / cm, the water is purified and the electrical conductivity is lowered. The method for purifying water is not particularly limited, and a known method can be used. Examples thereof include distillation, treatment with an ion exchange resin, and filtration with a reverse osmosis membrane. The electrical conductivity of water can be measured by, for example, an alternating current two-electrode method or an electromagnetic induction method.

  The concentration of the cells and / or the treated cells thereof in the suspension of the cells and / or the treated cells thereof is not particularly limited, but if a high concentration suspension is used, the viscosity increases and the reaction vessel Therefore, it is preferably 0.1 wt% or more and 50 wt% or less, more preferably 0.5 wt% or more and 30 wt% or less, and further preferably 1 wt% or more and 10 wt% or less.

<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.
<Water (raw water)>
The raw material water is not particularly limited, and purified water such as distilled water or ion exchange water can be used, and water having an electric conductivity of 100 μS / cm or less is preferable.
<PH regulator>
The pH adjuster is used to adjust the pH of the reaction solution to a suitable range for keeping the activity of the cell catalyst good.
When the pH suitable for the reaction is less 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 reaction vessel, 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 reaction tank, it is more preferable to use a liquid acid 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 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 reaction is higher than 7, a base can be used as a pH regulator.

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 adjusting agent can be used in any state of gas, solid, and liquid, but considering the ease of supply to the reaction vessel, it is preferable to use the base in the liquid state. The base in a state is more preferably used as an aqueous solution. In consideration of controllability of pH adjustment of the reaction tank, it is more preferable to use a liquid base as an 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.
<Reaction tank>
As the reaction tank, a single-stage reaction tank composed of one reactor may be used, or a multi-stage reaction tank composed of a plurality of reactors may be used. As the reactor, a tank reactor or a tube reactor may be used. As the tank reactor, a reactor equipped with a stirrer is preferable. When the tank reactor has a stirrer, the stirrer blade of the stirrer can be of any shape, for example, propeller blade, flat paddle blade, pitched paddle blade, flat turbine blade, pitched turbine blade, ribbon blade, anchor blade , Full zone wings. One stirring blade may be provided, or a plurality of stirring blades may be provided.
An external circulation line equipped with a circulation pump may be installed in the reaction tank. One external circulation line provided with a circulation pump may be installed in the reaction tank, or a plurality of external circulation lines may be installed.

When an external circulation line equipped with a pump is installed in the reaction tank, the pump provided in the external circulation line is not particularly limited. For example, a turbo pump such as a centrifugal pump, a tilt pump or an axial pump And positive displacement pumps such as reciprocating pumps and rotary pumps.
When an external circulation line equipped with a pump is installed in the reaction tank, a part of the reaction solution is taken out from the reaction tank using the pump provided in the external circulation line and reacted via the external circulation line. Returned to the tank. It is preferable that a temperature control device is installed in the external circulation line installed in the reaction tank. As the temperature control device, a heat exchanger is preferably used. Examples of the heat exchanger include multi-tubular cylindrical, spiral tube, spiral plate, plate, and double tube types.
When installing an external circulation line equipped with a pump in a multistage reaction tank composed of a plurality of reactors, all of the external circulation lines equipped with a pump may be installed in each reactor. It may be installed in only one reactor. In the case of multiple reactors, if the external circulation line equipped with a pump is installed only in one of the reactors, it is installed in the first reactor (the most upstream reactor). It is preferred that

  Using a reaction tank composed of a first-stage tank reactor and a second-stage tube reactor, the reaction liquid taken out from the tank reactor is further reacted in the tube reactor, and the tank reaction A reaction tank in which an external circulation line equipped with a pump is installed in the vessel is more preferable because the conversion rate can be improved.

  The tank reactor and the tube reactor may or may not be equipped with a heat exchanger as long as the nitrile hydratase activity of the bacterial cell catalyst is maintained. In order to control, the reactor preferably comprises a heat exchanger. As the heat exchanger, for example, a multi-tubular cylindrical type, a spiral tube type, a spiral plate type, a plate type, a double pipe type or the like is preferably installed on the external circulation line. The thing of the form directly installed in reactors, such as a formula and a coil type, is mentioned. When the reactor is a tubular reactor, the reactor itself can be composed of a multi-tubular cylindrical or double-tube heat exchanger.

  Examples of the reaction method include (1) a method in which a reaction is carried out after all of the cell catalyst, reaction raw material (nitrile compound and raw water) and pH regulator are charged in a reaction tank at once (batch reaction), (2) fungus After a part of the body catalyst, reaction raw material (nitrile compound and raw water) and pH regulator are charged into the reaction tank, the remaining cell catalyst, reaction raw material (nitrile compound and raw water) and pH are continuously or intermittently added. A method of performing a reaction by supplying a regulator (semi-batch reaction), (3) a continuous or intermittent supply of a cell catalyst, a reaction raw material (nitrile compound and raw water) and a pH regulator, and a reaction solution (bacteria) Including continuous catalyst, unreacted raw material, pH adjuster and generated amide compound, etc.), while continuously removing the reaction solution in the reaction vessel without taking out the whole reaction solution. Continuous reaction) and the like. Among these, a continuous reaction is preferable because it is easy to industrially produce an amide compound in large quantities and efficiently.

  The reaction is performed in the presence of a cell catalyst. The use form of the cell catalyst is preferably a suspension bed. For example, in the case of continuous reaction, a suspension of the cell catalyst may be prepared and the suspension supplied to the reaction vessel.

  In addition, when using the multistage reaction tank comprised from several reactors as a reaction tank, as the structure, (a) Cell body catalyst, reaction raw material (nitrile compound and raw material water), and pH regulator are the upper reaction. A serial type mode in which a reaction liquid (including a bacterial cell catalyst, an unreacted raw material, a pH regulator, and a generated amide compound) is supplied to the lower reactor and is taken out from the upper reactor, (B) A parallel mode in which a cell catalyst, a reaction raw material (nitrile compound and raw water) and a pH adjuster are directly supplied to two or more reactors (without going through other reactors) can be mentioned.

  For example, in the case of performing a continuous reaction using a multistage reaction tank, the supply destination of the cell catalyst, the reaction raw material (nitrile compound and raw water) and the pH adjuster is the first-stage reactor (the most upstream reaction). It is not limited only to the reactor), but may be a reactor after the second stage (reactor located downstream).

  Although the reaction tank temperature which is the liquid temperature in a reaction tank is based also on the heat resistance of a microbial cell catalyst, it is normally set to 0-50 degreeC, Preferably it is set to 10-40 degreeC. When the reaction vessel temperature is in the above range, it is preferable in that the nitrile hydratase activity of the bacterial cell catalyst can be maintained well.

  The reaction tank temperature refers to the liquid temperature in the reactor when the reaction tank is composed of only one reactor, and in the case where the reaction tank is composed of a plurality of reactors. Refers to the liquid temperature. The reaction vessel temperature can be measured, for example, by a thermocouple method (eg, K type). The reaction vessel temperature can be measured at an arbitrary location in the reaction vessel, and specifically can be measured at the reaction vessel outlet (reaction liquid outlet).

Volume of the reaction vessel 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. When the reaction vessel is composed of a plurality of reactors, the volume refers to the volume of each reactor.

The reaction is generally carried out under normal pressure, but can also be carried out under pressure in order to increase the solubility of the nitrile compound.
The pH in the reaction vessel is not particularly limited as long as it is a suitable range for maintaining the activity of the bacterial cell catalyst, but is preferably in the range of pH 5 to pH 10. When the pH is within the above range, it is preferable in that the nitrile hydratase activity can be favorably maintained.

When the reaction tank is composed of only one reactor, the pH of the reaction tank refers to the pH in the reactor, and when the reaction tank is composed of a plurality of reactors, Of pH. The pH of the reaction vessel 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 reaction vessel can be measured at any location in the reaction vessel, and specifically can be measured at the reaction vessel outlet (reaction liquid outlet).
<Cell catalyst storage tank>
The cell catalyst storage tank has a catalyst supply pipe that holds a cell catalyst suspension and supplies the cell catalyst suspension to the reaction tank. The cell catalyst storage tank may or may not have a temperature control device, but it has a temperature control device from the viewpoint of controlling the liquid temperature of the cell catalyst suspension. It is preferable.
When the cell catalyst storage tank has a temperature control device, the temperature control device may be provided in the cell catalyst storage tank itself, and when the cell catalyst storage tank has an external circulation line, It may be provided. Examples of the temperature control device in the cell catalyst storage tank include a heat exchanger. Examples of the heat exchanger include those installed directly in a cell catalyst storage tank such as a jacket-type heat exchanger and a coil-type heat exchanger, or a multi-tubular cylindrical heat exchanger, a spiral tube heat exchanger, a spiral The thing of the form installed in the external circulation line of a microbial cell catalyst storage tank, such as a plate type heat exchanger, a plate type heat exchanger, and a double tube type heat exchange, is mentioned.
The bacterial cell catalyst storage tank may have a stirrer or may not have a stirrer, but it is preferable to have a stirrer from the viewpoint of controlling the liquid temperature of the bacterial cell catalyst. When the cell catalyst storage tank has a stirrer, the stirring blade of the stirrer can be of any shape, for example, propeller blade, flat paddle blade, pitched paddle blade, flat turbine blade, pitched turbine blade, ribbon blade, anchor blade , Full zone wings. One stirring blade may be provided, or a plurality of stirring blades may be provided.
<Supply of reaction raw materials>
The raw water supplied to the reaction tank may be supplied alone to the reaction tank, or may be supplied to the reaction tank after being mixed with the nitrile compound. When supplying raw material water to the reaction tank alone, there is no particular restriction on the position of the supply port to the reaction liquid in the reaction tank of the raw water supply pipe, and even if it is installed above the reaction liquid, May be installed.
The method for supplying raw water when supplying raw water alone to the reaction vessel is not particularly limited, and a known method can be used. As a method of supplying the raw water to the reaction tank, a device having a liquid transport function can be used. For example, a centrifugal pump, a tilting pump, an axial pump, or a turbo pump, a reciprocating pump, a rotary pump, etc. Pumps such as positive displacement pumps and conveyors such as screw conveyors can be used. As a method for supplying the raw water to the reaction tank, the raw water can be supplied without using the equipment having the liquid transport function. When equipment with a liquid transport function is not used, for example, a raw water storage tank for storing raw water is installed at the top of the reaction tank, the raw water storage tank and the reaction tank are connected by a raw water supply pipe, and gravity is used. There is a method of supplying by dropping. Or the method of supplying using the pressure difference of the raw material water storage tank and reaction tank which arise by pressurizing a raw material water storage tank is mentioned.

There may be one raw water supply pipe for supplying raw water to the reaction tank, or a plurality of raw water supply pipes may be provided for one reaction tank. There may be one supply port for the raw water supply pipe per supply pipe, or a plurality of supply ports may be provided. There is no restriction | limiting in particular in the shape of the supply port of a raw material water supply pipe, Any can be used suitably if it is a shape normally used.
As a method of mixing the raw water and the nitrile compound in the case of supplying the raw water and the nitrile compound to the reaction tank, a known method can be used, for example, a method of stirring and mixing using a mixing tank, The method of mixing raw material water and a nitrile compound in piping is mentioned. In the case of using a mixing tank, the shape of the mixing tank is not particularly limited, but a cylindrical mixing tank is generally used, and it can be used in either a vertical cylindrical shape or a horizontal cylindrical shape. it can. The mixing tank may be provided with a baffle plate or may not be provided with a baffle plate.

In the case of using a mixing tank, a stirring blade having an arbitrary shape can be selected, and examples thereof include a propeller blade, a flat paddle blade, a pitched paddle blade, a flat turbine blade, a pitched turbine blade, a ribbon blade, an anchor blade, and a full zone blade. It is done. One stirring blade may be provided, or a plurality of stirring blades may be provided.
To mix the raw water and the nitrile compound in the pipe, the raw water and the nitrile compound can be directly mixed by combining the raw water supply pipe and the nitrile compound supply pipe. A method of actively mixing by installing a line mixer can be mentioned.

  When the raw water and nitrile compound are mixed and then supplied to the reaction vessel, the nitrile compound may be completely dissolved in water after mixing with water, but it is not necessarily required to be completely dissolved in water. What is necessary is just to mix by arbitrary ratios. Preferably, the ratio of water: nitrile compound is 100: 1 to 1: 100, more preferably 50: 1 to 1:50, and even more preferably 10: 1 to 1:10 by volume.

  When the raw water and the nitrile compound are mixed and then supplied to the reaction vessel, a known method can be used for supplying the raw water and the nitrile compound mixture. Specifically, the above raw water is used alone. The method illustrated by the supply method of raw material water in the case of supplying to a reaction tank can be used.

When the raw water and the nitrile compound are mixed and then supplied to the reaction tank, the supply pipe for supplying the raw water and the mixture of the nitrile compound to the reaction tank may be one per reaction tank. May be. The supply port of the supply pipe may be one for each supply pipe, or a plurality of supply ports may be provided. There is no restriction | limiting in particular in the shape of the supply port of a supply pipe | tube, Any can be used suitably if it is a shape normally used.
The nitrile compound supplied to the reaction vessel may be supplied to the reaction vessel after being mixed with the raw water, or may be supplied alone to the reaction vessel. When supplying the nitrile compound alone to the reaction vessel, there is no particular restriction on the position of the supply port to the reaction solution in the reaction vessel of the nitrile compound supply pipe, and even if it is installed above the reaction solution, May be installed.
The supply method of the nitrile compound when supplying the nitrile compound alone to the reaction vessel is not particularly limited, and a known method can be used. Specifically, the raw material water is supplied to the reaction vessel. The method exemplified in the method can be used.
There may be one nitrile compound supply pipe for supplying the nitrile compound to the reaction tank, or a plurality of nitrile compound supply pipes per reaction tank. The supply port of the nitrile compound supply pipe may be one for each supply pipe, or a plurality of supply ports may be provided. The shape of the supply port of the nitrile compound supply pipe is not particularly limited, and any shape that is usually used can be suitably used.
<Supply of pH regulator>
The pH adjuster supplied to the reaction tank may be supplied alone to the reaction tank, or may be supplied to the reaction tank after being mixed with the aqueous medium and / or the mixture containing the aqueous medium supplied to the reaction tank. When supplying the pH regulator alone to the reaction tank, the position of the supply port to the reaction liquid in the reaction tank of the pH regulator supply pipe is not particularly limited. You may install in a reaction liquid.
The supply method of the pH adjuster when supplying the pH adjuster alone to the reaction vessel is not particularly limited, and a known method can be used. Specifically, the above raw water is supplied to the reaction vessel. The method exemplified in the supplying method can be used.
The number of pH adjusting agent supply pipes for supplying the pH adjusting agent to the reaction vessel may be one per reaction vessel or may be plural. The supply port of the supply pipe for the pH adjusting agent may be one for each supply pipe, or a plurality of supply ports may be provided. The shape of the supply port of the pH adjusting agent supply pipe is not particularly limited, and any shape that is usually used can be suitably used.
The aqueous medium in which the pH adjusting agent is mixed when the pH adjusting agent and the aqueous medium and / or the mixture containing the aqueous medium are mixed and then supplied to the reaction vessel is preferably raw water. Examples of the mixture containing an aqueous medium in which the pH adjuster is mixed include a reaction liquid containing a mixture of raw water and a nitrile compound, a cell catalyst, an unreacted raw material, a pH adjuster, and a generated amide compound. .

As a method of mixing the pH regulator and the raw water when the pH regulator is mixed with the raw water and then fed to the reaction vessel, a known method can be used. The method illustrated by the method of mixing a nitrile compound can be used.
A known method can be used as the method for supplying the mixture of the pH regulator and the raw water when the pH regulator is mixed with the raw water and then supplied to the reaction tank. The method illustrated by the supply method of raw material water in the case of supplying to a reaction tank independently can be used.
The pH adjusting agent to be supplied to the reaction tank and the aqueous medium and / or the mixture containing the aqueous medium may be mixed at an arbitrary ratio, and preferably the pH adjusting agent: the aqueous medium and / or the mixture containing the aqueous medium. The ratio is 1: 1 to 1: 100,000,000, more preferably 1:10 to 1: 10,000,000 in volume ratio.

  A known method can be used as a method of mixing the mixture of the pH regulator, the raw water and the nitrile compound when the pH regulator is mixed with the raw water and the nitrile compound and then supplied to the reaction vessel. The method exemplified in the method of mixing the raw material water and the nitrile compound can be used.

pH regulator and cell catalyst, unreacted material, pH when supplying to the reaction vessel after mixing with a reaction solution containing a pH adjuster and cell catalyst, unreacted raw material, pH adjuster and generated amide compound, etc. As a method of mixing the reaction liquid containing the regulator and the generated amide compound, for example, the following method can be used. An external circulation line equipped with a pump for returning a part of the reaction solution to the reaction tank is installed in the reaction tank, and the reaction is performed as a mixture containing an aqueous medium by combining the external circulation line and the pH regulator supply pipe. Examples include a method in which a part of the reaction liquid taken out from the tank is used and a part of the reaction liquid and the pH adjuster are directly mixed in the external circulation line. A part of the reaction liquid and the pH regulator directly mixed in the external circulation line are supplied to the reaction tank as they are using the external circulation line.

<Supply of bacterial cell catalyst>
The bacterial cell catalyst supplied to the reaction tank may be supplied alone to the reaction tank, or may be supplied to the reaction tank after being mixed with the aqueous medium and / or the mixture containing the aqueous medium supplied to the reaction tank. . When supplying the cell catalyst alone to the reaction vessel, the installation position of the supply port to the reaction solution in the reaction vessel of the cell catalyst supply pipe is not particularly limited. You may install in a reaction liquid.
The supply method of the bacterial cell catalyst when supplying the bacterial cell catalyst alone to the reaction vessel is not particularly limited, and a known method can be used. Specifically, the above raw water is supplied to the reaction vessel. The method exemplified in the supplying method can be used.
There may be one or a plurality of cell catalyst supply pipes for supplying the cell catalyst to the reaction tank. There may be one supply port of the supply pipe for the bacterial cell catalyst, or a plurality of supply ports may be provided for each supply pipe. The shape of the supply port of the fungus body catalyst supply pipe is not particularly limited, and any shape having a shape normally used can be used.
The aqueous medium in which the bacterial cell catalyst is mixed when the bacterial cell catalyst and the aqueous medium and / or the mixture containing the aqueous medium are mixed and then supplied to the reaction vessel is preferably raw water. Examples of the mixture containing an aqueous medium in which the bacterial cell catalyst is mixed include a reaction liquid containing a mixture of raw water and a nitrile compound, a bacterial cell catalyst, an unreacted raw material, a pH regulator, and a generated amide compound. .

As a method of mixing the bacterial cell catalyst and the raw water when mixing the bacterial cell catalyst with the raw material water and supplying it to the reaction tank, a known method can be used. The method illustrated by the method of mixing a nitrile compound can be used.
A known method can be used as a method for supplying the mixture of the bacterial cell catalyst and the raw material water when the bacterial cell catalyst is mixed with the raw material water and then supplied to the reaction vessel. The method illustrated with the supply method of the raw material water in the case of supplying to a reaction tank independently can be used.

  The bacterial cell catalyst to be supplied to the reaction tank and the aqueous medium and / or the mixture containing the aqueous medium may be mixed at an arbitrary ratio. Preferably, the bacterial cell catalyst: the aqueous medium and / or the mixture containing the aqueous medium The ratio is 1: 1 to 1: 100,000,000, more preferably 1:10 to 1: 10,000,000 in volume ratio.

  As a method of mixing the cell catalyst, the raw material water and the mixture of the nitrile compound when the cell catalyst is mixed with the mixture of the raw material water and the nitrile compound and then supplied to the reaction vessel, a known method can be used, Specifically, the method exemplified in the method of mixing the raw material water and the nitrile compound can be used.

As a method for mixing the cell catalyst with a reaction solution containing the cell catalyst, unreacted raw material, pH adjuster, generated amide compound, and the like, for example, the following method can be used. An external circulation line equipped with a pump for returning a part of the reaction solution to the reaction tank is installed in the reaction tank, and the reaction is performed as a mixture containing an aqueous medium by combining the external circulation line and the cell catalyst supply pipe. Examples include a method in which a part of the reaction liquid taken out from the tank is used and a part of the reaction liquid and the bacterial cell catalyst are directly mixed in the external circulation line. Part of the reaction solution and the bacterial cell catalyst directly mixed in the external circulation line are supplied to the reaction tank as they are using the external circulation line.

The amount of the bacterial cell catalyst used varies depending on the reaction conditions, the type of catalyst and its form, but is usually 10 to 50,000 ppm by weight, preferably 10 to 50,000 ppm by weight, preferably in terms of the dry cell weight of the cells. 50 to 30,000 ppm by weight.

The reaction time (retention time of the reaction solution) is usually 0.5 to 50 hours, preferably 2 to 25 hours. When a multistage reaction vessel is used, the reaction time refers to the total reaction time (retention time of the reaction liquid) in all reactors.
In the method for producing an amide compound of the present invention, the obtained amide compound can be recovered and purified by, for example, a concentration operation (eg, evaporation and concentration), activated carbon treatment, ion exchange treatment, filtration treatment, and crystallization operation. .

  As described above, an amide compound corresponding to the nitrile compound as a reaction raw material, for example, (meth) acrylonitrile, (meth) acrylamide can be obtained.

  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.

[Purification of water]
As a distillation apparatus, a glass 5 L round bottom flask equipped with a Dimroth type cooling coagulator and a packed tower corresponding to 8 stages was prepared.

The apparatus was charged with 3 kg of tap water in a round bottom flask and distilled. An oil bath was used for heating, and the fraction until the distillation temperature reached 100 ° C. was defined as the first fraction. 2 kg of a fraction having a distillation temperature of 100 to 101 ° C. was obtained as a main fraction.
The electric conductivity of tap water was measured by an alternating current two power source method and found to be 180 μS / cm. Further, when the main sample was analyzed in the same manner, the electric conductivity was 10 μS / cm. Hereinafter, the water after the distillation treatment is referred to as purified water a.

[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 tap water in advance. 200 g of wet cells obtained by the above culture method were suspended in 800 g of purified water. This suspension was continuously fed at a rate of 10 g / h while stirring in the first reactor. Acrylonitrile was continuously supplied at a rate of 32 g / h, and tap water was continuously supplied 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 solution was continuously 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 various concentrations

[Example 2]
[Purification of water]
As an ion exchange apparatus, a packed column of cation exchange resin and anion exchange resin were prepared, and 500 cc of resin was filled in each packed column. The cation exchange resin tower and the anion exchange resin tower were connected.

3 kg of tap water was passed through the cation exchange resin tower and then the anion exchange resin tower at a flow rate of 300 cc / h to obtain 2.5 kg of ion exchange treated water.
The electric conductivity of tap water was measured by an alternating current two power source method and found to be 180 μS / cm. Further, when the ion exchange treated water was analyzed in the same manner, the electric conductivity was 3 μS / cm. Hereinafter, the ion exchange treated water is referred to as purified water b.

[Production of acrylamide]
In the production of acrylamide of Example 1, acrylamide was produced in the same manner as in Example 1, except that purified water b was used instead of using purified water a for diluting wet microbial cells containing nitrile hydratase. went. When HPLC analysis was performed 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 or less). .

[Example 3]
[Purification of water]
As a reverse osmosis membrane treatment apparatus, a reverse osmosis membrane having an effective area of 200 cm 2 was prepared.

3 kg of tap water was passed through the reverse osmosis membrane at a flow rate of 300 cc / h to obtain 2 kg of reverse osmosis membrane treated water.
The electric conductivity of tap water was measured by an alternating current two power source method and found to be 180 μS / cm. When the reverse osmosis membrane treated water was analyzed in the same manner, the electric conductivity was 8 μS / cm. Hereinafter, the reverse osmosis membrane treated water is referred to as purified water c.

[Production of acrylamide]
In the production of acrylamide of Example 1, acrylamide was produced in the same manner as in Example 1 except that purified water c was used instead of purified water a for diluting wet microbial cells containing nitrile hydratase. went. When HPLC analysis was performed 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 or less). .

[Comparative example]
In the production of acrylamide of Example 1, acrylamide was produced in the same manner as in Example 1 except that tap water was used instead of purified water a. When HPLC analysis was performed 50 hours after the start of the reaction, the conversion to acrylamide at the outlet of the first reactor was 92%, and the acrylonitrile concentration at the outlet of the second reactor was 15 ppm.
Since acrylonitrile was detected at the outlet of the second reactor, the supply rate of the wet cell suspension was changed from 10 g / h to 12 g / h in order to complete the reaction. Acrylamide was produced. When HPLC analysis was performed 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 or less). Completed.

Claims (3)

In a method for producing an amide compound using a bacterial cell containing nitrile hydratase and / or a treated product thereof, the bacterial cell suspended in water and / or the bacterial cell having an electric conductivity of 100 μS / cm or less Process for producing amide compound characterized by using treated product The method according to claim 1, wherein the nitrile hydratase is a nitrile hydratase derived from Pseudonocardia or Rhodococcus. The production method according to claim 1 or 2, wherein the amide compound is acrylamide or methacrylamide.