Method for producing amide compound

Abstract

The present invention provides a method in which a biocatalyst is used to efficiently produce an amide compound from a nitrile compound, and which produces an amide compound aqueous solution having low foamability. Provided is a method for producing an amide compound by performing a hydration reaction of a nitrile compound in the presence of a biocatalyst having nitrile hydratase activity, wherein the pH of a reaction liquid is raised during the hydration reaction. In one example, the hydration reaction is performed in a manner such that the pH of the reaction liquid in the latter half of the reaction, in which the content of the amide compound with respect to the total amount of the reaction liquid is not less than 40 mass%, is set higher than the pH of the reaction liquid in the first half of the reaction, in which the content of the amide compound is less than 40 mass%.

Description

Method for producing amide compounds

[0001] The present invention relates to a method for producing an amide compound from a nitrile compound using a biocatalyst having nitrile hydratase activity. This application claims priority to Japanese Patent Application No. 2023-043453, filed on March 17, 2023, the contents of which are incorporated herein by reference.

[0002] In recent years, methods for producing compounds using biocatalysts have been used to produce many compounds because of their advantages, such as mild reaction conditions, simplified reaction processes, and high purity of reaction products due to the small amount of by-products. In the production of amide compounds, the use of biocatalysts has also been actively investigated since the discovery of nitrile hydratase, an enzyme that converts nitrile compounds to amide compounds (Patent Documents 1 to 6, etc.). In addition, the process has also been actively studied, and many proposals have been made, such as batch reactions, semi-batch reactions, multi-vessel continuous reactions, and pipe reactors.

[0003] For example, Patent Document 5 discloses a difference between the cooling water temperature and the reaction temperature for efficiently removing the heat of reaction, thereby showing a method for producing an amide compound at low cost by reducing energy costs. Furthermore, Patent Document 6 discloses stirring power and Froude number that enable efficient mixing of the reaction liquid while preventing volatilization of acrylonitrile during the reaction, and shows a method for producing an amide compound from a nitrile-free compound at low cost.

[0004] JP 11-123098, JP 7-265091, JP 56-38118, JP 11-89575, International Publication No. 2010 / 038832, International Publication No. 2009 / 113654

[0005] However, for industrial use of biocatalysts, it is desirable not only to produce amide compounds at low cost, but also for the produced amide compounds to meet a certain level of quality. Aqueous solutions of amide compounds produced by biocatalytic methods contain proteins derived from the biocatalyst as impurities, which poses a problem of the resulting aqueous solution of amide compounds being prone to foaming. This makes the aqueous solution of amide compounds difficult to handle when transferring, transporting, or storing them. Furthermore, when polymerizing an amide compound to obtain an amide compound-based polymer, foaming can cause the aqueous solution of amide compounds to overflow from the polymerization vessel, resulting in a reduced yield of the amide compound-based polymer. The methods of Patent Documents 1 to 6 are unable to sufficiently suppress foaming of the aqueous solution of amide compounds due to impurity proteins.

[0006] A main object of the present invention is to provide a method for producing an aqueous amide compound solution with low foaming properties.

[0007] The present inventors have conducted extensive research in light of the problems of the prior art and have found that foaming of the resulting aqueous amide compound solution can be suppressed by increasing the pH of the reaction solution during the hydration reaction, thereby completing the present invention. That is, the present invention relates to the following [1] to [8].

[0008] [1] A method for producing an amide compound by hydrating a nitrile compound in the presence of a biocatalyst having nitrile hydratase activity, wherein the pH of the reaction solution is increased during the hydration reaction. [2] A method for producing an amide compound according to [1], wherein the pH of the reaction solution is increased during the reaction from the pH at the start of the reaction. [3] A method for producing an amide compound according to [1] or [2], wherein the hydration reaction is carried out by setting the pH of the reaction solution higher in the latter half of the reaction, when the content of the amide compound is 40% by mass or more, than in at least a part of the first half of the reaction, when the content of the amide compound is less than 40% by mass relative to the total mass of the reaction solution. [4] A method for producing an amide compound according to any of [1] to [3], wherein the pH of the reaction solution is increased by 0.3 to 1.5. [5] A method for producing an amide compound according to any of [1] to [4], wherein the pH of the reaction solution at the start of the reaction is 6.6 to 7.5. [6] The method for producing an amide compound according to any one of [3] to [5], wherein the hydration reaction is carried out by setting the pH of the reaction solution in the first half of the reaction to 6.6 to 7.5 and the pH of the reaction solution in the second half of the reaction to 7.5 or more and less than 8.5. [7] The method for producing an amide compound according to any one of [1] to [6], wherein the nitrile compound is acrylonitrile or methacrylonitrile. [8] The method for producing an amide compound according to any one of [1] to [6], wherein the amide compound is acrylamide or methacrylamide.

[0009] According to the method of the present invention, in a method for producing an amide compound from a nitrile compound using a biocatalyst, by increasing the pH of the reaction solution during the hydration reaction, foaming of the resulting reaction solution, i.e., an aqueous solution of the amide compound, can be suppressed, thereby making the reaction solution easier to handle.

[0010] In this specification, a numerical range expressed as "to" means a numerical range that includes the numerical values ​​before and after "to" as the lower and upper limits. The numerical ranges of the content, various physical property values, and property values ​​disclosed in this specification can be arbitrarily combined with the lower and upper limits to form new numerical ranges.

[0011] The method for producing an amide compound according to the embodiment is a method for producing an amide compound by subjecting a nitrile compound to a hydration reaction in the presence of a biocatalyst having nitrile hydratase activity. In one embodiment of this aspect, the pH of the reaction solution is increased during the hydration reaction. One embodiment of this aspect is described below.

[0012] (1) Biocatalyst Having Nitrile Hydratase Activity In the present embodiment, nitrile hydratase refers to an enzyme capable of hydrolyzing a nitrile compound to produce the corresponding amide compound. The biocatalyst having nitrile hydratase activity may be a nitrile hydratase protein itself, or may be an animal cell, plant cell, organelle, or microbial cell containing nitrile hydratase, or a processed product thereof.

[0013] Examples of the processed material include disrupted animal cells, plant cells, organelles, or microbial cells, or enzymes extracted from cells (raw enzymes or purified enzymes); animal cells, plant cells, organelles, microbial cells, or enzymes themselves immobilized on a carrier; and the like. The processed material also includes animal cells, plant cells, organelles, or microbial cells that have been treated with a chemical to lose their ability to grow. Microbial cells that have been treated with a chemical to lose their ability to grow are sometimes called "killed cells."

[0014] Immobilization methods include entrapment, cross-linking, and carrier binding. Entrapment is a method of coating with a polymer film. Cross-linking is a method of cross-linking an enzyme with a reagent having two or more functional groups, i.e., a multifunctional cross-linking agent. Carrier binding is a method of binding an enzyme to a water-insoluble carrier.

[0015] Examples of carriers used for immobilization include glass beads, silica gel, polyurethane, polyacrylamide, polyvinyl alcohol, carrageenan, alginic acid, agar, and gelatin.

[0016] Representative examples of such microorganisms include, for example, the genera Rhodococcus, Gordona, Pseudomonas, Pseudonocardia, Geobacillus, Bacillus, Bacteridium, Micrococcus, Brevibacterium, Corynebacterium, Nocardia, Microbacterium, Fusari, and the like, which have nitrile hydratase activity. Examples of microorganisms that can be used include those belonging to the genus Fusarium, Agrobacterium, Acinetobacter, Xanthobacter, Streptomyces, Rhizobium, Klebsiella, Enterobacter, Erwinia, Pantoea, Candida, Aeromonas, Citrobacter, and Achromobacter.

[0017] More specifically, Nocardia sp. N-775 described in Japanese Patent Publication No. 56-17918, Rhodococcus rhodochrous J-1 described in Japanese Patent Publication No. 06-55148, Rhodococcus rhodochrous NCIMB41164 described in International Publication No. 2005 / 054456, Klebsiella sp. MCI2609 described in Japanese Patent Publication No. 05-30982, Aeromonas sp. described in Japanese Patent Publication No. 05-30983, and the like. MCI2614, Citrobacter freundii MCI2615 described in JP-A-05-30984, Agrobacterium rhizogenes IAM13570 and Agrobacterium tumefaciens described in JP-A-05-103681, Xanthobacter flavas JCM1204 described in JP-A-05-161495, Erwinia nigrifluens nigrifluens) MAFF03-01435, Enterobacter sp. MCI2707 described in JP-A-05-236975, Streptomyces sp. MCI2691 described in JP-A-05-236976, Rhizobium sp. MCI2610 and Rhizobium sp. described in JP-A-05-236977. MCI2643, Rhizobium loti IAM13588, Rhizobium legminosarum IAM12609 and Rhizobium melioti IAM12611, Candida guilliermondii NH-2, Pantoea agglomerans NH-3 and Klebsiella pneumoniae subspecies pneumoniae described in JP-A-05-15384. Agrobacterium radiobacter SC-C15-1 described in JP-A-06-14786;Examples of such strains include Bacillus smithii SC-J05-1 described in JP-A No. 07-25494, Pseudonocardia thermophila ATCC19285 described in JP-A No. 08-56684, and Pseudonocardia thermophila JCM3095 described in JP-A No. 09-275978.

[0018] The Rhodococcus rhodochrous J-1 strain described in Japanese Patent Publication No. 06-55148 was deposited on September 18, 1987, under the accession number "FERM BP-1478" at the Patent Organism Depositary, National Institute of Technology and Evaluation (Chuo No. 6, 1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture, Japan (hereinafter the same in this specification)).

[0019] The Rhodococcus rhodochrous NCIMB41164 strain described in WO 2005 / 054456 was deposited on March 5, 2003 at the National Collection of Industrial, Food and Marine Bacteria, Ltd. (NCIMB) (NCIMB Ltd Ferguson Building Craibstone Estate Buksburn Aberdeen AB21 9YA) under accession number NCIMB41164.

[0020] Pseudonocardia thermophila JCM3095 described in Japanese Patent Application Laid-Open No. 09-275978 was deposited on February 7, 1996, with the National Institute of Technology and Evaluation (Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki Prefecture) under the accession number "FERM BP-5785".

[0021] In this embodiment, one type of microorganism having desirable properties selected from the above-mentioned microorganisms can be used alone, or two or more types can be used in combination.

[0022] The gene encoding the nitrile hydratase can be introduced and expressed in microbial cells by conventional molecular biological techniques. For details of these molecular biological techniques, see Sambrook, Fritsch and Maniatis, "Molecular Cloning: A Laboratory Manual," 2nd Edition (1989), Cold Spring Harbor Laboratory Press. That is, in this embodiment, an enzyme obtained by expressing a nucleic acid encoding a natural nitrile hydratase (wild-type) or a mutant (improved) thereof in microbial cells can also be used.

[0023] In this embodiment, one enzyme selected from the above enzymes can be used alone, or two or more enzymes can be used in combination.

[0024] The amino acid sequence of wild-type nitrile hydratase has been published in NCBI databases such as GenBank (http: / / www.ncbi.nlm.nih.gov / ). For example, the accession number of the α subunit derived from Rhodococcus rhodochrous J1 (FERM BP-1478) is "P21219", and the accession number of the β subunit is "P21220". In addition, the accession number of the α subunit derived from Rhodococcus rhodochrous M8 (SU1731814) is "ATT79340", and the accession number of the β subunit is "AAT79339". Furthermore, the accession number of the α subunit derived from Pseudomonas thermophila JCM3095 is "1IREA," and the accession number of the β subunit is "1IREB."

[0025] Examples of the transformant into which a wild-type nitrile hydratase gene has been introduced include, but are not limited to, Escherichia coli MT10770 (FERM P-14756) transformed with a nitrile hydratase from the genus Achromobacter (Japanese Patent Laid-Open No. 8-266277), Escherichia coli MT10822 (FERM BP-5785) transformed with a nitrile hydratase from the genus Pseudonocardia (Japanese Patent Laid-Open No. 9-275978), or a microorganism transformed with a nitrile hydratase from the species Rhodococcus rhodochrous (Japanese Patent Laid-Open No. 4-211379).

[0026] Improved (mutant) nitrile hydratases in which amino acid substitutions have been made in wild-type nitrile hydratases are known (JP 2010-172295 A, JP 2007-143409 A, JP 2007-043910 A, JP 2008-253182 A, JP 2019-088326 A, JP 2019-088327 A, WO 2005 / 116206, WO 2012 / 164933, WO 2012 / 169203, WO 2015 / 186298, etc.). In the method of this embodiment, microorganisms into which these improved nitrile hydratases have been introduced can also be used.

[0027] These microorganisms having nitrile hydratase activity or processed products thereof can be used in the amide synthesis reaction immediately after the preparation of the bacterial cells, or can be stored after the preparation of the bacterial cells and used in the amide synthesis reaction as needed. The method for culturing the microorganisms to prepare the bacterial cells can be appropriately selected depending on the type of microorganism. Seed culture may be performed before the main culture. The bacterial cells of the microorganisms having nitrile hydratase activity or processed products thereof can be used in either a batch reaction or a continuous reaction. In addition, an appropriate reaction format can be selected, such as a fluidized bed, fixed bed, or suspension bed. In this case, the temperature of the biocatalyst in the reaction solution is not particularly limited, as long as it does not interfere with the mixing of the aqueous medium and the nitrile compound.

[0028] (2) Nitrile Compound The nitrile compound used as a raw material in the production method of this embodiment is not particularly limited as long as it is a compound that can be converted to an amide compound by a biocatalyst having nitrile hydratase activity. Examples include saturated aliphatic nitriles such as acetonitrile, propionitrile, succinonitrile, and adiponitrile; unsaturated aliphatic nitriles such as acrylonitrile and methacrylonitrile; aromatic nitriles such as benzonitrile and phthalodinitrile; and heterocyclic nitriles such as nicotinonitrile. The nitrile compound in this embodiment is preferably a nitrile compound having 2 to 4 carbon atoms, such as acetonitrile, propionitrile, acrylonitrile, methacrylonitrile, n-butyronitrile, or isobutyronitrile. This embodiment is particularly effective with acrylonitrile, methacrylonitrile, and acetonitrile.

[0029] (3) Raw Water The water used as a raw material (raw water) is used in the hydration reaction with acrylonitrile when producing acrylamide. Examples of water include pure water; aqueous solutions of acids, salts, etc. dissolved in water; and the like. Examples of acids include phosphoric acid, acetic acid, citric acid, boric acid, acrylic acid, formic acid, etc. Examples of salts include sodium salts, potassium salts, ammonium salts, etc. of the above acids. Specific examples of water include, but are not limited to, pure water, ultrapure water, city water, etc.; and buffer solutions such as Tris buffer, phosphate buffer, acetate buffer, citrate buffer, and borate buffer. The pH of the raw water at 20°C is preferably 5 to 9.

[0030] (4) Production of an amide compound from a nitrile compound using a biocatalyst Any of the following reactions (i) to (iii) can be applied to the method of producing an amide compound from a nitrile compound using a biocatalyst having nitrile hydratase activity according to this embodiment: (i) A method in which all reaction raw materials including the biocatalyst, acrylonitrile, and raw material water are charged into a reactor at once and then reacted (batch reaction); (ii) A method in which a portion of the reaction raw materials is charged into a reactor, and then the remaining reaction raw materials are continuously or intermittently supplied and reacted (semi-batch reaction); (iii) A method in which the reaction raw materials are continuously or intermittently supplied and the reaction mixture containing the reaction raw materials and the produced acrylamide is continuously or intermittently removed, without completely withdrawing the reaction mixture from the reactor (continuous reaction).

[0031] The type of reactor is not particularly limited, and various types of reactors can be used, such as agitated, fixed bed, fluidized bed, moving bed, tower, and tubular reactors. Among these, agitated reactors are preferred because they can promote dispersion and mixing of raw materials. Reactors of different types can also be combined and connected.

[0032] The apparatus used in the multi-tank continuous reaction comprises two or more reaction vessels connected in series, and produces an amide compound from a nitrile compound and water by continuous reactions using a biocatalyst in each reactor. More specifically, in the continuous reaction apparatus, the raw materials to be reacted are first added to the reactor located most upstream and the reaction vessel connected thereto to initiate the reaction, and the reaction proceeds as the reaction liquid is sequentially transferred to reaction vessels located downstream. The reaction liquid containing the produced amide compound, i.e., the target aqueous amide compound solution, can then be recovered from the reaction vessel located most downstream. The biocatalyst can be separated from the recovered reaction liquid and resupplied to the reaction vessels.

[0033] The number of reactors (reaction tanks) is not particularly limited and can be appropriately selected depending on the reaction conditions, etc. For example, 2 to 20 reactors are preferred, 2 to 12 reactors are more preferred, and 2 to 10 reactors are even more preferred. Some reactors may be connected in parallel as necessary. The reactors may be independent of each other, or a large reactor may be divided into multiple reactors by partition walls. In the case of a reactor divided by partition walls, each space divided by the partition wall is considered to be a single reactor.

[0034] The tank to which the nitrile compound, biocatalyst, raw material water, other auxiliaries, etc. are supplied is not limited to the single most upstream tank, but may be one tank or two or more tanks. The latter (downstream) tanks are used for reaction cut-off and aging, and the reaction liquid containing the product can be extracted from the most downstream tank (final tank) or a tank located upstream thereof. The number of tanks to which raw materials are supplied and the number of tanks for aging, etc. can be appropriately selected depending on the reaction conditions, reaction scale, etc.

[0035] The stirring device is preferably a stirring blade. The shape of the stirring blade is not particularly limited, and examples thereof include a paddle, a disk turbine, a propeller, a helical ribbon, an anchor, and a Pfaudle.

[0036] A water-soluble monocarboxylate having two or more carbon atoms can be added to the reaction liquid. The timing of adding the water-soluble monocarboxylate is not particularly limited, and the water-soluble monocarboxylate can be added to the reactor located most upstream, and the water-soluble monocarboxylate contained in the reaction liquid can move downstream together with the reaction liquid, thereby being contained in the reaction liquid in each reactor. Alternatively, the water-soluble monocarboxylate can be added to each reactor before or after the start of the reaction.

[0037] Adding a water-soluble monocarboxylic acid salt having two or more carbon atoms can improve the stability of acrylamide in the reaction solution. The water-soluble monocarboxylic acid salt may be either a saturated monocarboxylic acid salt or an unsaturated monocarboxylic acid salt. Examples of saturated carboxylic acids include acetic acid, propionic acid, and n-caproic acid. Examples of unsaturated carboxylic acids include acrylic acid and methacrylic acid. Examples of salts include sodium salts, potassium salts, and ammonium salts of the saturated or unsaturated monocarboxylic acids. These water-soluble monocarboxylic acid salts can be used alone or in combination of two or more. The amount of water-soluble monocarboxylic acid salt added is preferably 20 to 5,000 mg / kg of acid relative to the amount of acrylamide produced.

[0038] <pH Control of Reaction Solution> In this embodiment, with regard to the pH of the reaction solution in which acrylonitrile is hydrated to produce acrylamide, the pH of the reaction solution is increased during the hydration reaction. By increasing the pH of the reaction solution during the hydration reaction, foaming of the resulting aqueous amide compound solution can be suppressed and foaming properties can be reduced, making the reaction solution easier to handle. The pH of the reaction solution can be measured by any known method, such as the indicator method, metal electrode method, glass electrode method, semiconductor sensor method, etc. In this embodiment, measurement by the glass electrode method, which is widely used industrially, is preferred.

[0039] In one embodiment, for example, during the hydration reaction, the pH of the reaction solution is increased from the pH at the start of the reaction. The pH of the reaction solution at the start of the reaction can be, for example, 6.6 to 7.5, preferably 6.8 to 7.5, more preferably 6.9 to 7.4, and even more preferably 7.0 to 7.3. By keeping the pH of the reaction solution at the start of the reaction within the above range, an amide compound can be efficiently obtained from a nitrile compound. The increase in pH when increasing the pH of the reaction solution is preferably 0.3 to 1.5, more preferably 0.4 to 1.4, and even more preferably 0.5 to 1.2. If the increase in pH is within the above range, foaming of the resulting amide compound aqueous solution is easily suppressed and the effect of improving the efficiency of the hydration reaction tends to be high.

[0040] In a preferred example, the hydration reaction is carried out by setting the pH of the reaction solution in at least a portion of the second half of the reaction higher than the pH of the reaction solution in the first half of the reaction. Here, the "first half of the reaction" refers to "from the start of the reaction until the concentration of the amide compound in the reaction solution becomes less than 40% by mass," and the "second half of the reaction" refers to "from the point at which the concentration of the amide compound in the reaction solution becomes equal to or greater than 40% by mass." In other words, the first half of the reaction refers to the period during which the content of the amide compound in the product relative to the total mass of the reaction solution is less than 40% by mass, and the second half of the reaction refers to the period during which the content of the amide compound in the product relative to the total mass of the reaction solution is equal to or greater than 40% by mass. The total mass of the reaction solution refers to the mass of the entire reaction solution contained in the reaction vessel of interest, including the mass of the amide compound and the nitrile compound.

[0041] By increasing the pH of the reaction solution in at least a part of the second half of the reaction to a value higher than the pH of the reaction solution in the first half of the reaction, the reaction can be carried out without decreasing the reaction efficiency, and further, foaming of the resulting aqueous amide compound solution can be suppressed, reducing foamability, making the reaction solution easier to handle. The pH of the reaction solution in the second half of the reaction may be increased only in a part of the second half of the reaction to a value higher than the pH of the reaction solution in the first half of the reaction, or may be increased throughout the entire second half of the reaction to a value higher than the pH of the reaction solution in the first half of the reaction. In particular, it is preferable to set the pH of the reaction solution in the second half of the reaction to a value higher than the pH of the reaction solution in the first half of the reaction throughout the entire second half of the reaction, as this tends to reduce foamability of the resulting aqueous amide compound solution.

[0042] The pH of the reaction solution in the first half of the reaction can be, for example, 6.6 to 7.5, preferably 6.8 to 7.5, more preferably 6.9 to 7.4, and even more preferably 7.0 to 7.3. By adjusting the pH of the reaction solution in the first half of the reaction to 6.6 to 7.5, an amide compound can be efficiently obtained from a nitrile compound. The pH of the reaction solution in the second half of the reaction can be, for example, 7.5 or more and less than 8.5, preferably 7.5 to 8.4, more preferably 7.6 to 8.3, and even more preferably 7.8 to 8.3. By adjusting the pH of the reaction solution in the second half of the reaction to 7.5 or more, foaming of the reaction solution can be sufficiently suppressed. By adjusting the pH of the reaction solution in the second half of the reaction to less than 8.5, an amide compound can be efficiently produced.

[0043] In this embodiment, the difference in pH between the reaction solution in the latter half of the reaction and the reaction solution in the first half of the reaction is not limited as long as the pH is higher than the pH of the reaction solution in the latter half of the reaction. For example, the difference in pH between the reaction solution in the latter half of the reaction and the reaction solution in the first half of the reaction can be 0.3 to 1.5, preferably 0.4 to 1.4, and more preferably 0.5 to 1.2.

[0044] In this embodiment, the method for adjusting the pH of the reaction solution is not limited. The pH of the reaction solution can be adjusted by adding an acid or a base to the reaction solution as appropriate depending on the pH of the reaction solution.

[0045] The acid may be either an inorganic acid or an organic acid. Examples of inorganic acids include halogenated hydroacids such as hydrogen chloride, hydrogen bromide, and hydrogen iodide, halogenated oxoacids such as hypochlorous acid, chlorous acid, chloric acid, perchloric acid, hypobromous acid, bromous acid, bromic acid, perbromic acid, hypoiodous acid, iodous acid, iodic acid, and periodic acid, sulfuric acid, nitric acid, phosphoric acid, and boric 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, and benzoic acid, and sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0046] When these acids are used, they can be used in any state of gas, solid, or liquid, but in consideration of ease of supply to the reaction vessel, it is preferable to use them in a liquid or solid state. When the acid is used as a liquid, the concentration of the acid is not particularly limited and can be selected appropriately.

[0047] The base may be either an inorganic base or an organic base. 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, alkali metal carbonates such as lithium carbonate, sodium carbonate, and potassium carbonate, alkali metal bicarbonates such as lithium bicarbonate, sodium bicarbonate, and potassium bicarbonate, and ammonia. Examples of the organic base include trimethylamine, triethylamine, aniline, and pyridine.

[0048] When using these bases, they can be used in any state of gas, solid, or liquid, but in consideration of ease of supply to the reaction vessel, it is preferable to use them in a liquid or solid state. When using the base as a liquid, the concentration of the base is not particularly limited and can be selected appropriately.

[0049] The reaction temperature during hydration of acrylonitrile, i.e., the temperature of the reaction solution, is not particularly limited, but is preferably 10 to 50°C, more preferably 15 to 40°C, and even more preferably 20 to 35°C. By setting the reaction temperature to 10°C or higher, the reaction activity of the biocatalyst can be sufficiently increased. Furthermore, by setting the reaction temperature to 50°C or lower, deactivation of the biocatalyst can be prevented. Furthermore, in order to reduce the heat removal load on the reactor, it is preferable to supply water or acrylonitrile at a temperature at least 5°C lower than the reaction temperature.

[0050] The amount of catalyst is actually compared in units, not in terms of the actual weight of the catalyst. Regarding nitrile hydratase activity, the amount of enzyme that produces 1 micromole of acrylamide per minute is defined as 1 U. The specific activity of the nitrile hydratase of this embodiment is, for example, 50 U / mg or more, preferably 80 U / mg or more, more preferably 100 U / mg or more, based on dry cells.

[0051] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to the following examples. In the examples, "%" indicates "% by mass".

[0052] [Preparation of Biocatalyst] <Preparation of Bacterial Catalyst Derived from Rhodococcus rhodochrous J1> Pre-culture conditions: (Culture medium composition) Fructose: 2%, Polypeptone: 5% (Nihon Pharmaceutical Co., Ltd.), Yeast extract: 0.3% (Oriental Yeast Co., Ltd.), KH ２ P.O. ４ : 0.1%, K ２ HPO ４ :0.1%, MgSO ４ H ２ O: 0.1%, pH 7. (Cultivation method) 100 mL of the medium was dispensed into a 500 mL Erlenmeyer flask, plugged with cotton, and sterilized in an autoclave at 121°C for 20 minutes. Rhodococcus rhodochrous J1 (FERM BP-1478) was inoculated and cultured with shaking at 30°C for 48 hours.

[0053] Main culture conditions: (Culture medium composition) Initial medium: yeast extract: 0.2%, KH ２ P.O.４ : 0.1%, K ２ HPO ４ :0.1%, MgSO ４ ・7H ２ O: 0.1%, CoCl ２ ・6H ２ O: 0.002%, ammonium sulfate: 0.025%, fructose: 2%, urea: 2%, ethanol: 0.4%, Pluronic L61: 0.1% (Asahi Denka Kogyo Co., Ltd.), pH 7. "Pluronic" is a registered trademark. Post-addition medium: fructose: 20%, ethanol: 5%, ammonium sulfate: 6%, pH 6.5. (Cultivation method) 2 L of initial medium was dispensed into a 3 L mini jar fermenter and sterilized by autoclaving at 121°C for 20 minutes. However, fructose, ethanol, and urea were separately aseptically filtered and added to the medium. A 0.45 μm filter paper manufactured by Advantec Toyo Co., Ltd. was used for filtration. After culturing for 43 hours at a tank pressure of 0.098 MPa, stirring speed of 600 rpm, aeration rate of 1 vvm, pH 7, and temperature of 30°C, the culture was washed with 50 mM phosphate buffer (pH 7.7) to obtain a bacterial cell suspension with a dry cell weight of 15%.

[0054] Example 1 Seven reactors (internal volume: 1.3 L) equipped with jacket coolers were connected in series so that the reaction solution flowed sequentially from the first to seventh reactors. 50 mM phosphate buffer (pH 7.0) was continuously supplied to the first reactor at 525 mL / hr, acrylonitrile at 150 mL / hr, and a diluted bacterial cell suspension prepared by diluting a 15% bacterial cell suspension by 40 times with 50 mM phosphate buffer at 100 mL / hr. Acrylonitrile alone was continuously supplied to the second reactor at 120 mL / hr, acrylonitrile alone was continuously supplied to the third reactor at 120 mL / hr, and acrylonitrile alone was continuously supplied to the fourth reactor at 80 mL / hr to initiate the reaction.

[0055] The height of the overflow pipe from which the reaction solution flowed out of each tank was adjusted so that the volume of the reaction solution in each of the first to seventh tanks was 1 L, and the reaction solution was sent to the next tank by overflow. The temperature of the reaction solution in each of the first to seventh tanks was controlled to 20°C using cooling water (10°C) in the jacket.

[0056] Using two paddle blades (blade diameter: 350 mm, blade width: 100 mm), the stirring power per reaction liquid fluid in all reactors from the first to the seventh vessels was set to 0.08 kW / m ３ (Froude number: 0.057). Here, the stirring power required per reaction fluid was calculated by multiplying the stirring power required in each reactor by the liquid volume (1 L = 0.001 m ３ ) was divided by the

[0057] The pH of the reaction solution in each tank was measured with a KCl refill type pH detector and adjusted to the set value by automatically adding 0.06 N sodium hydroxide aqueous solution. The pH of the reaction solutions in tanks 1, 2, 3, 4, 5, 6, and 7 was set to 7.2, 7.2, 7.5, 7.7, 8.0, and 8.0, respectively.

[0058] One day after the start of the reaction, after confirming that the pH of each reaction tank was maintained at the set value, the acrylamide concentration of the reaction solution in each reaction tank was measured using a refractometer (RX-5000, manufactured by Atago Co., Ltd.). The acrylamide concentrations in tanks 1, 2, 3, 4, 5, 6, and 7 were 19, 31, 38, 43, 49, 50, and 50%, respectively, and the acrylamide concentration of the reaction solution flowing out of tank 7 reached 50% of the target concentration. The acrylonitrile concentration of the reaction solution in tank 7 was also measured by gas chromatography (column: Waters PoraPak-PS, 1 m, 180°C; carrier gas: helium; detector: FID). The unreacted acrylonitrile (AN) concentration in tank 7 was 10 ppm, indicating high reaction efficiency. A concentration of unreacted acrylonitrile of 100 ppm or less is also preferred in terms of improving the quality of acrylamide polymerization.

[0059] Next, 300 mL of the reaction solution in the seventh tank was placed in a 500 mL measuring cylinder and allowed to stand in a thermostatic bath at 25° C. for 10 minutes. A glass ball filter (Kinoshita Glass Filter 504G) was placed in the center of the measuring cylinder, 5 mm from the bottom, and a pressure of 0.5 kg / cm was applied. ３Air was aerated at 800 cc / min. The reaction solution began to foam, and when the foam height stabilized, the aeration was stopped and the time until the foam disappeared was measured. The shorter the time until the foam disappeared, the less protein was carried from the biocatalyst into the aqueous acrylamide solution. Quality requires that the time until the foam disappeared be within 30 seconds. The time until the foam disappeared was 5 seconds, which met the required quality.

[0060] Example 2 The reaction was carried out in the same manner as in Example 1, except that the pH of the reaction solution in the first, second, third, fourth, fifth, sixth, and seventh tanks was set to 6.8, 7.0, 7.5, 8.3, 8.3, 8.3, and 8.3, respectively. One day after the start of the reaction, it was confirmed that the pH of each reaction tank was maintained at the set value. The acrylamide concentration of the reaction solution in each reaction tank was then measured. The acrylamide concentrations in the first, second, third, fourth, fifth, sixth, and seventh tanks were 18, 31, 36, 42, 48, 50, and 50%, respectively. The acrylamide concentration of the reaction solution flowing out of the seventh tank reached 50% of the target concentration. Furthermore, the acrylonitrile concentration of the reaction solution in the seventh tank was measured. The unreacted acrylonitrile concentration was 20 ppm, indicating high reaction efficiency. The defoaming time measured using the reaction solution in the seventh tank was 2 seconds, satisfying the required quality.

[0061] Example 3 The reaction was carried out in the same manner as in Example 1, except that the pH of the reaction solution in the first, second, third, fourth, fifth, sixth, and seventh tanks was set to 6.8, 6.8, 6.8, 8.3, 8.3, 8.3, and 8.3, respectively. One day after the start of the reaction, it was confirmed that the pH of each reaction tank was maintained at the set value. The acrylamide concentration of the reaction solution in each reaction tank was then measured. The acrylamide concentrations in the first, second, third, fourth, fifth, sixth, and seventh tanks were 18, 30, 35, 41, 47, 49, and 50%, respectively. The acrylamide concentration of the reaction solution flowing out of the seventh tank reached 50% of the target concentration. Furthermore, the acrylonitrile concentration of the reaction solution in the seventh tank was measured. The unreacted acrylonitrile concentration was 30 ppm, indicating high reaction efficiency. The defoaming time measured using the reaction solution in the seventh tank was 2 seconds, satisfying the required quality.

[0062] Example 4 The reaction was carried out in the same manner as in Example 1, except that the pH of the reaction solution in the first, second, third, fourth, fifth, sixth, and seventh tanks was set to 7.5, 7.5, 7.5, 8.5, 8.5, 8.5, and 8.5. One day after the start of the reaction, it was confirmed that the pH of each reaction tank was maintained at the set value. Then, the acrylamide concentration of the reaction solution in each reaction tank was measured. The acrylamide concentrations in the first, second, third, fourth, fifth, sixth, and seventh tanks were 18, 30, 34, 41, 47, 49, and 50%, respectively. The acrylamide concentration of the reaction solution flowing out of the seventh tank reached 50% of the target concentration. Furthermore, the acrylonitrile concentration of the reaction solution in the seventh tank was measured. The unreacted acrylonitrile concentration was 300 ppm. The reaction efficiency was higher in Examples 1 to 3, in which the pH in the latter half of the reaction was controlled to less than 8.5. The defoaming time measured using the reaction solution in the seventh tank was 2 seconds, satisfying the required quality.

[0063] Comparative Example 1: The reaction was carried out in the same manner as in Example 1, except that the pH of the reaction solution in the first, second, third, fourth, fifth, sixth, and seventh tanks was all set to 7.0. One day after the start of the reaction, it was confirmed that the pH of each reaction tank was maintained at the set value. The acrylamide concentrations of the reaction solution in each reaction tank were then measured. The acrylamide concentrations in the first, second, third, fourth, fifth, sixth, and seventh tanks were 19, 31, 36, 44, 49, and 50%, respectively. The acrylamide concentration of the reaction solution flowing out of the seventh tank reached 50% of the target concentration. Furthermore, the acrylonitrile concentration of the reaction solution in the seventh tank was measured. No unreacted acrylonitrile was detected, satisfying the required quality. The defoaming time measured using the reaction solution in the seventh tank was 150 seconds, which did not satisfy the required quality.

[0064] Comparative Example 2: The reaction was carried out in the same manner as in Example 1, except that the pH of the reaction solution in the first, second, third, fourth, fifth, sixth, and seventh tanks was all set to 7.5. One day after the start of the reaction, it was confirmed that the pH of each reaction tank was maintained at the set value. The acrylamide concentrations of the reaction solution in each reaction tank were then measured. The acrylamide concentrations in the first, second, third, fourth, fifth, sixth, and seventh tanks were 18, 30, 36, 43, 49, 50, and 50%, respectively. The acrylamide concentration of the reaction solution flowing out of the seventh tank reached 50% of the target concentration. Furthermore, the acrylonitrile concentration of the reaction solution in the seventh tank was measured. The unreacted acrylonitrile concentration was 10 ppm, satisfying the required quality. The defoaming time measured using the reaction solution in the seventh tank was 80 seconds, which did not satisfy the required quality.

[0065] Comparative Example 3: The reaction was carried out in the same manner as in Example 1, except that the pH of the reaction solution in the first, second, third, fourth, fifth, sixth, and seventh tanks was all set to 8.0. One day after the start of the reaction, it was confirmed that the pH of each reaction tank was maintained at the set value. The acrylamide concentrations of the reaction solution in each reaction tank were then measured. The acrylamide concentrations in the first, second, third, fourth, fifth, sixth, and seventh tanks were 17, 30, 35, 41, 48, 50, and 50%, respectively. The acrylamide concentration of the reaction solution flowing out of the seventh tank reached 50% of the target concentration. Furthermore, the acrylonitrile concentration of the reaction solution in the seventh tank was measured. The unreacted acrylonitrile concentration was 120 ppm, which slightly did not meet the required quality. The defoaming time measured using the reaction solution in the seventh tank was 45 seconds, which did not meet the required quality.

[0066]

[0067] The results of Examples 1 to 3 are summarized as follows. In the first half of the reaction (first tank, second tank, third tank), where the content of the amide compound relative to the total mass of the reaction solution in each reaction tank was less than 40% by mass, the pH of the reaction solution was set to a first pH, and the hydration reaction of the nitrile compound was carried out. Due to this hydration reaction, the concentration of the amide compound in the reaction solution increased as the reaction solution progressed from the first tank to the third tank. The content of the amide compound relative to the total mass of the reaction solution that entered the third tank into the fourth tank was 40% by mass or more. In the second half of the reaction from the fourth tank onwards, the pH of the reaction solution was set to a second pH, and the hydration reaction continued. Here, the second pH was required to be higher than the first pH. As a result, in all of Examples 1 to 4, the aqueous acrylamide solution obtained in the seventh tank exhibited suppressed foaming and low foaming properties. Furthermore, Examples 1 to 3, in which the second pH in the second half of the reaction was controlled to less than 8.5, had a lower concentration of unreacted acrylonitrile and higher reaction efficiency than Example 4, in which the second pH was 8.5.

[0068] The present invention is useful in the industrial production of amide compounds such as acrylamide and methacrylamide.

Claims

1. A method for producing an amide compound by subjecting a nitrile compound to a hydration reaction in the presence of a biocatalyst having nitrile hydratase activity, wherein the pH of the reaction solution is increased during the hydration reaction. A method for producing an amide compound.

2. The method for producing an amide compound according to claim 1, wherein the pH of the reaction solution during the reaction is increased compared to the pH at the start of the reaction.

3. The pH of the reaction solution in at least a part of the latter half of the reaction, where the content of the amide compound is 40% by mass or more, is set higher than the pH of the reaction solution in the first half of the reaction, where the content of the amide compound is less than 40% by mass with respect to the total mass of the reaction solution, and the hydration reaction is carried out. The method for producing an amide compound according to claim 1 or 2.

4. The method for producing an amide compound according to claim 1 or 2, wherein the pH of the reaction solution is increased by 0.3 to 1.

5.

5. The method for producing an amide compound according to claim 1 or 2, wherein the pH of the reaction solution at the start of the reaction is 6.6 to 7.

5.

6. The method for producing an amide compound according to claim 3, wherein the pH of the reaction solution in the first half of the reaction is set to 6.6 to 7.5, and the pH of the reaction solution in the latter half of the reaction is set to 7.5 or more and less than 8.5, and the hydration reaction is carried out.

7. The method for producing an amide compound according to claim 1 or 2, wherein the nitrile compound is acrylonitrile or methacrylonitrile.

8. The method for producing an amide compound according to claim 1 or 2, wherein the amide compound is acrylamide or methacrylamide.