Method for producing amide compound

Abstract

Provided is a method for efficiently producing an amide compound from a nitrile compound using a biocatalyst, and for producing an amide compound aqueous solution having low foamability. In 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, the pH of the reaction liquid is increased during the hydration reaction. In one example, a hydration reaction is carried out by setting the pH of the reaction liquid in a second half of a reaction in which the content of the amide compound is 40 mass% or more higher than the pH of the reaction liquid in a first half of the reaction in which the content of the amide compound is less than 40 mass% with respect to the total mass of the reaction liquid.

Description

Technical Field

[0001] The present invention relates to a method for producing an amide compound from a nitrile compound using a biocatalyst having nitrile hydratase activity.

[0002] This application claims priority based on Japanese Patent Application No. 2023-043453 filed in Japan on March 17, 2023, and incorporates the contents thereof herein. Background Art

[0003] In recent years, the method of using biocatalysts to produce compounds has been used to produce a variety of compounds due to its advantages such as mild reaction conditions, simplified reaction process, and high purity of reaction products due to few by-products.

[0004] In the production of amide compounds, since the discovery of nitrile hydratase, an enzyme that converts nitrile compounds into amide compounds, the use of biocatalysts has been actively studied (Patent Documents 1 to 6, etc.). Furthermore, processes have also been actively studied, with various schemes proposed, including batch reactions, semi-batch reactions, multi-tank continuous reactions, and tubular reactors.

[0005] For example, Patent Document 5 discloses the difference between the cooling water temperature and the reaction temperature for efficiently removing the heat of reaction, and shows a method for producing an amide compound at low cost by reducing energy costs.

[0006] Patent Document 6 discloses stirring power and Froude number that enable efficient mixing of reaction solutions while preventing volatilization of acrylonitrile during the reaction, and shows a method for producing an amide compound from a nitrile compound at low cost.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: Japanese Patent Application Laid-Open No. 11-123098

[0010] Patent Document 2: Japanese Patent Application Laid-Open No. 7-265091

[0011] Patent Document 3: Japanese Patent Publication No. 56-38118

[0012] Patent Document 4: Japanese Patent Application Laid-Open No. 11-89575

[0013] Patent Document 5: International Publication No. 2010 / 038832

[0014] Patent Document 6: International Publication No. 2009 / 113654 Summary of the Invention

[0015] Problems to be solved by the invention

[0016] However, in order to utilize biocatalysts industrially, it is desirable not only to produce amide compounds at low cost but also to ensure that the produced amide compounds meet certain quality standards. Aqueous amide compound solutions produced by biocatalysis contain biocatalyst-derived proteins as impurities, leading to the problem that the resulting aqueous amide compound solutions tend to foam. This makes the transfer, transportation, and storage of aqueous amide compound solutions difficult. Furthermore, when polymerizing amide compounds to produce amide compound-based polymers, the aqueous amide compound solution can overflow from the polymerization reactor due to foaming, resulting in reduced yields of the amide compound-based polymers.

[0017] The methods of Patent Documents 1 to 6 cannot sufficiently suppress the foaming of the aqueous amide compound solution caused by proteins as impurities.

[0018] The main object of the present invention is to provide a method for producing an aqueous solution of an amide compound having low foaming properties.

[0019] Methods for solving problems

[0020] The present inventors have conducted intensive studies in view of the problems of the prior art and have found that by increasing the pH of the reaction solution during the hydration reaction, the foaming of the resulting aqueous solution of an amide compound can be suppressed, thereby completing the present invention. Specifically, the present invention relates to the following [1] to [8].

[0021] [1] A method for producing an amide compound, comprising: 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.

[0022] [2] The method for producing an amide compound according to [1], wherein the pH of the reaction solution is increased during the reaction compared to the pH at the start of the reaction.

[0023] [3] The method for producing an amide compound according to [1] or [2], wherein the pH of the reaction liquid in the second half of the reaction in which the content of the amide compound is 40% by mass or more is set higher than the pH of the reaction liquid in at least a portion of the first half of the reaction in which the content of the amide compound is less than 40% by mass relative to the total mass of the reaction liquid, and a hydration reaction is performed.

[0024] [4] The method for producing an amide compound according to any one of [1] to [3], wherein the pH of the reaction solution is increased by 0.3 to 1.5.

[0025] [5] The method for producing an amide compound according to any one of [1] to [4], wherein the pH of the reaction solution at the start of the reaction is 6.6 to 7.5.

[0026] [6] The method for producing an amide compound according to any one of [3] to [5], 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 second half of the reaction is set to 7.5 or higher and less than 8.5, and a hydration reaction is performed.

[0027] [7] The method for producing an amide compound according to any one of [1] to [6], wherein the nitrile compound is acrylonitrile or methacrylonitrile.

[0028] [8] The method for producing an amide compound according to any one of [1] to [6], wherein the amide compound is acrylamide or methacrylamide.

[0029] Effects of the Invention

[0030] According to the method of the present invention, in a method for producing an amide compound from a nitrile compound using a biocatalyst, by raising the pH of the reaction solution during the hydration reaction, foaming of the resulting reaction solution, i.e., the aqueous solution of the amide compound, can be suppressed, thereby facilitating handling of the reaction solution. DETAILED DESCRIPTION

[0031] In this specification, the numerical range represented by “to” means a numerical range including the numerical values ​​before and after “to” as the lower limit and the upper limit.

[0032] The numerical ranges of the contents, various physical property values, and property values ​​disclosed in this specification can be formed by arbitrarily combining the lower limits and upper limits thereof to form new numerical ranges.

[0033] The method for producing an amide compound according to the embodiment is a method for producing an amide compound by hydrating a nitrile compound in the presence of a biocatalyst having nitrile hydratase activity. In one embodiment of this embodiment, the pH of the reaction solution is increased during the hydration reaction.

[0034] Hereinafter, an example of implementation of this aspect will be described.

[0035] (1) Biocatalyst with nitrile hydratase activity

[0036] In this 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 the nitrile hydratase protein itself, or may be animal cells, plant cells, organelles, or microbial cells containing nitrile hydratase, or processed products thereof.

[0037] Examples of the above-mentioned processed products include crushed materials obtained by crushing animal cells, plant cells, organelles or microbial cells, or enzymes (crude enzymes or purified enzymes) extracted from cells; substances obtained by fixing animal cells, plant cells, organelles, microbial cells or enzymes themselves on a carrier, etc.

[0038] Furthermore, the treated substances also include animal cells, plant cells, organelles, or microbial cells that have lost their ability to proliferate due to treatment with a chemical. Microbial cells that have lost their ability to proliferate due to treatment with a chemical are sometimes referred to as "inactivated cells."

[0039] Examples of immobilization methods include embedding, crosslinking, and carrier binding. The embedding method involves coating the enzyme with a polymer membrane. The crosslinking method involves crosslinking the enzyme using a reagent with two or more functional groups, i.e., a multifunctional crosslinking agent. The carrier binding method involves binding the enzyme to a water-insoluble carrier.

[0040] Examples of the carrier used for immobilization include glass beads, silica gel, polyurethane, polyacrylamide, polyvinyl alcohol, carrageenan, alginic acid, agar, and gelatin.

[0041] Representative examples of such microorganisms include Rhodococus, Gordona, Pseudomonas, Pseudonocardia, Geobacillus, Bacillus, Bacteridium, Micrococcus, Brevibacterium, Corynebacterium, Nocardia, and Microbacterium, which have nitrile hydratase activity. microorganisms such as the genus Fusarium, Agrobacterium, Acinetobacter, Xanthobacter, Streptomyces, Rhizobium, Klebsiella, Enterobacter, Erwinia, Pantoea, Candida, Aeromonas, Citrobacter, and Achromobacter.

[0042] 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 strain described in International Publication No. 2005 / 054456, Klebsiella sp. MCI2609 described in Japanese Patent Application Laid-Open No. 05-30982, Aeromonas sp. MCI2614 described in Japanese Patent Application Laid-Open No. 05-30983, Citrobacter freundii MCI2615 described in Japanese Patent Application Laid-Open No. 05-30984, Agrobacterium rhizogenes described in Japanese Patent Application Laid-Open No. 05-103681. rhizogenes) IAM13570 and Agrobacterium faciens, Xanthobacter flavus JCM1204 described in Japanese Patent Application Laid-Open No. 05-161495, Erwinia nigrifluens MAFF03-01435, Enterobacter sp. MCI2707 described in Japanese Patent Application Laid-Open No. 05-236975, Streptomyces sp. MCI2691 described in Japanese Patent Application Laid-Open No. 05-236976, Rhizobium sp. MCI2610 described in Japanese Patent Application Laid-Open No. 05-236977, Rhizobium sp.MCI2643, Rhizobium loti IAM13588, Rhizobium legminosarum IAM12609 and Rhizobium merioti IAM12611, Candida guilliermondii NH-2, Pantoea agglomerans NH-3 and Klebsiella pneumoniae NH-26T2 described in Japanese Patent Application Laid-Open No. 05-15384, Agrobacterium radiobacter SC-C15-1 described in Japanese Patent Application Laid-Open No. 06-14786, Bacillus smithii described in Japanese Patent Application Laid-Open No. 07-25494, smithii) SC-J05-1, Pseudonocardia thermophila ATCC19285 described in Japanese Patent Application Laid-Open No. 08-56684, and Pseudonocardia thermophila JCM3095 described in Japanese Patent Application Laid-Open No. 09-275978.

[0043] The Rhodococcus rhodochrous J-1 strain described in Japanese Patent Gazette No. 06-55148 was deposited on September 18, 1987 with the Patent Organism Depository of Japan (Central No. 6, Higashi 1-1-1, Tsukuba City, Ibaraki Prefecture) under the deposit number "FERM BP-1478".

[0044] The Rhodococcus rhodochrous strain NCIMB41164 described in International Publication No. 2005 / 054456 was deposited on March 5, 2003, with the National Collection of Industrial, Food and Marine Bacteria, Ltd. (NCIMB Ltd., Ferguson Building, Craibstone Estate, Buksburn, Aberdeen AB219YA) under the accession number NCIMB41164.

[0045] Pseudonocardiathermophila JCM3095 described in Japanese Patent Application Laid-Open No. 09-275978 was deposited on February 7, 1996, with the Patent Organism Depository of Japan (Central No. 6, Higashi 1-1-1, Tsukuba City, Ibaraki Prefecture) under the deposit number "FERM BP-5785."

[0046] In this embodiment, one kind selected from the above-mentioned microorganisms and having desired characteristics can be used alone or in combination of two or more kinds.

[0047] The gene encoding nitrile hydratase can be introduced into microbial cells and expressed using conventional molecular biology methods. For information on these molecular biology methods, see Sambrook, Fritsch, and Maniatis, "Molecular Cloning: A Laboratory Manual," 2nd ed. (1989), Cold Spring Harbor Laboratory Press. Specifically, in this embodiment, an enzyme obtained by expressing a nucleic acid encoding a natural nitrile hydratase (wild-type) or a mutant (modified) thereof in microbial cells can also be used.

[0048] In the present embodiment, one type selected from the above enzymes can be used alone or two or more types can be used in combination.

[0049] The amino acid sequence of wild-type nitrile hydratase is published in NCBI databases such as GenBank (http: / / www.ncbi.nlm.nih.gov / ).

[0050] For example, the accession number (Accession No.) for the α subunit from Rhodococcus rhodochrous J1 (FERM BP-1478) is "P21219," and the accession number for the β subunit is "P21220." Furthermore, the accession number for the α subunit from Rhodococcus rhodochrous M8 (SU1731814) is "ATT79340," and the accession number for the β subunit is "AAT79339." Furthermore, the accession number for the α subunit from Pseudomonas thermophila JCM3095 is "1IREA," and the accession number for the β subunit is "1IREB."

[0051] Examples of transformants into which a wild-type nitrile hydratase gene has been introduced include Escherichia coli MT10770 (FERM P-14756) transformed with a nitrile hydratase of the genus Achromobacter (Japanese Patent Application Laid-Open No. 8-266277), Escherichia coli MT10822 (FERM BP-5785) transformed with a nitrile hydratase of the genus Pseudonocardia (Japanese Patent Application Laid-Open No. 9-275978), or microorganisms transformed with a nitrile hydratase of the species Rhodococcus rhodochrous (Japanese Patent Application Laid-Open No. 4-211379), but are not limited thereto.

[0052] Improved (mutant) nitrile hydratases in which amino acid substitutions are performed on wild-type nitrile hydratases are known (Japanese Patent Application Publication No. 2010-172295, Japanese Patent Application Publication No. 2007-143409, Japanese Patent Application Publication No. 2007-043910, Japanese Patent Application Publication No. 2008-253182, Japanese Patent Application Publication No. 2019-088326, Japanese Patent Application Publication No. 2019-088327, International Publication No. 2005 / 116206, International Publication No. 2012 / 164933, International Publication No. 2012 / 169203, International Publication No. 2015 / 186298, etc.).

[0053] In the method of the present embodiment, microorganisms into which these improved nitrile hydratases have been introduced can also be used.

[0054] These microorganisms having nitrile hydratase activity or processed products thereof can be used in the amide synthesis reaction immediately after bacterial cell preparation, or they can be stored after bacterial cell preparation and then used in the amide synthesis reaction as needed. The culture method of the microorganism used to prepare the bacterial cell can be appropriately selected depending on the type of microorganism. Seed culture can also be performed before the main culture.

[0055] The cells of the microorganism having nitrile hydratase activity or its processed product can be used for batch reaction or continuous reaction. In addition, the reaction format can be selected from a fluidized bed, fixed bed, suspended bed or other appropriate formats. The temperature of the biocatalyst in the reaction solution at this time is not particularly limited as long as it does not hinder the mixing of the aqueous medium and the nitrile compound.

[0056] (2) Nitrile compounds

[0057] The nitrile compound used as a raw material in the production method of this embodiment is not particularly limited, as long as it can be converted into an amide compound by a biocatalyst exhibiting nitrile hydratase activity. Examples include aliphatic saturated nitriles such as acetonitrile, propionitrile, succinonitrile, and adiponitrile; aliphatic unsaturated nitriles such as acrylonitrile and methacrylonitrile; aromatic nitriles such as benzonitrile and phthalonitrile; and heterocyclic nitriles such as nicotinonitrile. Nitrile compounds in this embodiment are preferably nitrile compounds having 2 to 4 carbon atoms, such as acetonitrile, propionitrile, acrylonitrile, methacrylonitrile, n-butyronitrile, and isobutyronitrile. Acrylonitrile, methacrylonitrile, and acetonitrile are particularly effective in this embodiment.

[0058] (3) Raw water

[0059] Water (raw water) used as a raw material is used for the hydration reaction with acrylonitrile when producing acrylamide. Examples of water include: pure water; aqueous solutions obtained by dissolving acids, salts, etc. in water; and the like. Examples of acids include phosphoric acid, acetic acid, citric acid, boric acid, acrylic acid, and formic acid. Examples of salts include sodium salts, potassium salts, and ammonium salts of the above acids. Specific examples of water are not particularly limited, and examples include pure water, ultrapure water, tap water, and other water; and buffer solutions such as Tris buffer, phosphate buffer, acetate buffer, citrate buffer, and borate buffer. The raw water preferably has a pH of 5 to 9 at 20°C.

[0060] (4) Production of amide compounds from nitrile compounds using biocatalysts

[0061] The method of producing an amide compound from a nitrile compound using a biocatalyst having nitrile hydratase activity according to the present embodiment can be applied to any of the following reactions (i) to (iii).

[0062] (i) A method in which all the raw materials including the biocatalyst, acrylonitrile, and raw water are fed into the reactor at once and then reacted (batch reaction)

[0063] (ii) A method in which a portion of the reaction raw materials is fed into the reactor and the remaining reaction raw materials are continuously or intermittently fed to carry out the reaction (semi-batch reaction)

[0064] (iii) A method for continuous production without withdrawing all of the reaction mixture in the reactor while continuously or intermittently supplying the reaction raw materials and continuously or intermittently withdrawing the reaction mixture containing the reaction raw materials and the produced acrylamide (continuous reaction)

[0065] The reactor type is not particularly limited; various types of reactors may be used, including stirred-type, fixed-bed, fluidized-bed, moving-bed, tower, and tubular reactors. Of these, stirred-type reactors are preferred because they facilitate dispersion and mixing of the raw materials. Reactors of different types may also be combined and connected.

[0066] The apparatus used in a multi-tank continuous reaction comprises two or more reaction tanks connected in series. An amide compound is produced from a nitrile compound and water through a continuous reaction using a biocatalyst within each reactor. More specifically, in the continuous reaction apparatus, the initial reaction starting materials are added to the reactor located most upstream and the reaction tank connected thereto to initiate the reaction. The reaction proceeds while the reaction liquid is sequentially moved to the downstream reaction tank. The reaction liquid containing the generated amide compound, i.e., the target amide compound aqueous solution, is then recovered from the downstream reaction tank. Alternatively, the biocatalyst may be separated from the recovered reaction liquid and supplied to the reaction tank again.

[0067] The number of reactors (reaction tanks) is not particularly limited and can be appropriately selected according to reaction conditions. For example, it is preferably 2 to 20, more preferably 2 to 12, and even more preferably 2 to 10. In the reactor, reactors connected in parallel may also be present as needed. The reactors may be independently connected or a large reactor may be separated into multiple reactors by a bulkhead. In the case of a reactor separated by a bulkhead, each space separated by the bulkhead is considered to be one reactor.

[0068] The tank for supplying the nitrile compound, biocatalyst, raw water, and other additives is not limited to the single upstream tank and may be a single tank or multiple tanks. The tanks in the latter half (downstream) are used for reaction termination and maturation, and the reaction liquid containing the product can be withdrawn from the downstream tank (final tank) or a tank upstream thereof.

[0069] The number of tanks for supplying raw materials and the number of tanks for aging can be appropriately selected according to the reaction conditions, reaction scale, and the like.

[0070] 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 spiral ribbon, an anchor, and a Pfaudler.

[0071] A water-soluble monocarboxylate having a carbon number of 2 or more may be added to the reaction solution. The timing of adding the water-soluble monocarboxylate is not particularly limited. The water-soluble monocarboxylate may be added to the reactor located on the uppermost side, so that the water-soluble monocarboxylate contained in the reaction solution moves downstream together with the reaction solution, thereby being included in the reaction solution in each reactor. Alternatively, the water-soluble monocarboxylate may be added to each reactor before or after the reaction is started.

[0072] By adding a water-soluble monocarboxylate having 2 or more carbon atoms, the stability of acrylamide in the reaction solution can be improved.

[0073] The water-soluble monocarboxylate may be any of a saturated monocarboxylate and an unsaturated monocarboxylate. Examples of saturated carboxylic acids include acetic acid, propionic acid, and n-hexanoic 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 monocarboxylates may be used alone or in combination of two or more.

[0074] The amount of the water-soluble monocarboxylate added is preferably 20 to 5000 mg / kg in terms of acid relative to the generated acrylamide.

[0075] <pH Control of Reaction Solution>

[0076] In this embodiment, the pH of the reaction solution in which acrylonitrile is hydrated to produce acrylamide is raised during the hydration reaction. Raising the pH of the reaction solution during the hydration reaction suppresses foaming of the resulting aqueous amide compound solution, reducing foaming properties and facilitating handling of the reaction solution.

[0077] The pH of the reaction solution can be measured by a known method, for example, an indicator method, a metal electrode method, a glass electrode method, a semiconductor sensor method, etc. In this embodiment, measurement is preferably performed by the glass electrode method which is widely used industrially.

[0078] In one embodiment, for example, during the hydration reaction, the pH of the reaction solution is made higher than the pH at the start of the reaction.

[0079] The pH of the reaction solution at the start of the reaction can be set to, 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 setting the pH of the reaction solution at the start of the reaction within the above range, an amide compound can be efficiently obtained from the nitrile compound.

[0080] When the pH of the reaction solution is raised, the pH increase is preferably from 0.3 to 1.5, more preferably from 0.4 to 1.4, and even more preferably from 0.5 to 1.2. When the pH increase is within this range, foaming of the resulting aqueous amide compound solution is easily suppressed, and the hydration reaction efficiency tends to be highly effective.

[0081] In a preferred example, the hydration reaction is carried out by setting the pH in at least a portion of the second half of the reaction to be higher than the pH of the reaction solution in the first half of the reaction.

[0082] Here, the term "first half of the reaction" refers to "the period 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 term "second half of the reaction" refers to "the period after the concentration of the amide compound in the reaction solution becomes 40% by mass or more." In other words, 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 is referred to as the first half of the reaction, and the period during which the content of the amide compound in the product relative to the total mass of the reaction solution is 40% by mass or more is referred to as the second half of the reaction. The total mass of the reaction solution refers to the mass of the entire reaction solution contained in the reaction tank of interest, including the mass of the amide compound and the nitrile compound.

[0083] By making the pH of the reaction liquid in at least a portion of the second half of the reaction higher than the pH of the reaction liquid in the first half of the reaction, the reaction can be carried out without reducing the reaction efficiency, and the foaming of the obtained aqueous solution of the amide compound can be suppressed and the foaming property can be reduced, thereby facilitating the handling of the reaction liquid.

[0084] The pH of the reaction liquid in the second half of the reaction may be higher than the pH of the reaction liquid in the first half of the reaction only in a portion of the second half of the reaction, or may be higher than the pH of the reaction liquid in the first half of the reaction throughout the second half of the reaction. However, in order to easily reduce the foaming property of the obtained aqueous solution of the amide compound, it is preferred to set the pH of the reaction liquid in the second half of the reaction higher than the pH of the reaction liquid in the first half of the reaction throughout the second half of the reaction.

[0085] The pH of the reaction solution in the first half of the reaction can be set to, 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 setting 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 the nitrile compound.

[0086] The pH of the reaction solution in the second half of the reaction can be set to, for example, 7.5 or higher 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 setting the pH of the reaction solution in the second half of the reaction to 7.5 or higher, foaming of the reaction solution can be sufficiently suppressed. By setting the pH of the reaction solution in the second half of the reaction to less than 8.5, efficient production of the amide compound can be achieved.

[0087] In this embodiment, if the pH of the reaction liquid in the second half of the reaction is higher than the pH of the reaction liquid in the first half of the reaction, the difference is not limited. For example, the difference between the pH of the reaction liquid in the second half of the reaction and the pH of the reaction liquid in the first half of the reaction can be set to 0.3 to 1.5, preferably to 0.4 to 1.4, and more preferably to 0.5 to 1.2.

[0088] In this embodiment, the method for adjusting the pH of the reaction solution is not limited, and the pH of the reaction solution can be adjusted by appropriately adding an acid or a base to the reaction solution according to the pH of the reaction solution.

[0089] As the acid, either an inorganic acid or an organic acid can be used. Examples of the inorganic acid include hydrohalic acids such as hydrogen chloride, hydrogen bromide, and hydrogen iodide; halogenated oxygen acids 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; and sulfuric acid, nitric acid, phosphoric acid, and boric acid. Examples of the organic acid 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.

[0090] When using these acids, they can be used in any state of gas, solid, or liquid, but considering the ease of supply to the reaction tank, it is preferred to use a substance in a liquid or solid state. The concentration of the acid when using the acid as the liquid is not particularly limited and can be appropriately selected.

[0091] As the base, any inorganic base or 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; 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.

[0092] When using these alkalis, they can be used in any state of gas, solid, or liquid, but considering the ease of supply to the reaction tank, it is preferred to use an alkali in a liquid or solid state. The concentration of the alkali when using the alkali as the liquid is not particularly limited and can be appropriately selected.

[0093] 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. Setting the reaction temperature at 10°C or higher can sufficiently enhance the reactivity of the biocatalyst. Setting the reaction temperature at 50°C or lower can prevent deactivation of the biocatalyst. Furthermore, 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.

[0094] The amount of catalyst is actually compared in units of catalyst rather than actual weight. In the nitrile hydratase activity, 1 U is defined as the amount of enzyme that purifies 1 micromole of acrylamide in 1 minute. The specific activity of the nitrile hydratase of this embodiment can be, for example, 50 U / mg or greater, preferably 80 U / mg or greater, and more preferably 100 U / mg or greater, based on dry bacterial cells.

[0095] Example

[0096] Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to the following Examples. In the Examples, "%" means "mass %".

[0097] [Preparation of biocatalyst]

[0098] <Production of a bacterial catalyst derived from Rhodococcus rhodochrous J1>

[0099] Pre-culture conditions:

[0100] (Culture medium composition)

[0101] Fructose: 2%, polypeptone: 5% (Nippon Pharmaceutical Co., Ltd.), yeast extract: 0.3% (Oriental Yeast Industry Co., Ltd.), KH2PO4: 0.1%, K2HPO4: 0.1%, MgSO4H2O: 0.1%, pH 7.

[0102] (Cultivation Method)

[0103] 100 mL of the culture medium was dispensed into 500 mL Erlenmeyer flasks, 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.

[0104] Main culture conditions:

[0105] (Culture medium composition)

[0106] Initial culture medium: yeast extract: 0.2%, KH2PO4: 0.1%, K2HPO4: 0.1%, MgSO4·7H2O: 0.1%, CoCl2·6H2O: 0.002%, ammonium sulfate: 0.025%, fructose: 2%, urea: 2%, ethanol: 0.4%, Pluronic L61: 0.1% (Asahi Denka Kogyo Co., Ltd.), pH 7. Note: "Pluronic" is a registered trademark.

[0107] Then, culture medium was added; fructose: 20%, ethanol: 5%, ammonium sulfate: 6%, pH 6.5.

[0108] (Cultivation Method)

[0109] 2 L of the initial culture medium was dispensed into a 3 L mini-fermenter and sterilized in an autoclave at 121°C for 20 minutes. Fructose, ethanol, and urea were sterile filtered separately before addition to the culture medium. 0.45 μm filter paper manufactured by Advantec Toyo Co., Ltd. was used for filtration.

[0110] After culturing for 43 hours at a tank pressure of 0.098 MPa, a stirring speed of 600 rpm, a ventilation volume of 1 vvm, pH 7, and a temperature of 30°C, the cells were washed with 50 mM phosphate buffer (pH 7.7) to obtain a bacterial suspension with a dry bacterial weight of 15%.

[0111] <Example 1>

[0112] Seven reaction tanks (internal volume: 1.3 L) equipped with jacketed coolers were connected in series so that the reaction liquid flowed sequentially from the first tank to the seventh tank.

[0113] 50 mM phosphate buffer (pH 7.0) was continuously supplied to the first tank at 525 mL / hr, acrylonitrile was continuously supplied at 150 mL / hr, and a diluted bacterial cell suspension prepared by diluting a bacterial cell suspension containing 15% of the dry cell weight 40-fold with 50 mM phosphate buffer was continuously supplied at 100 mL / hr. Acrylonitrile alone was continuously supplied to the second tank at 120 mL / hr, acrylonitrile alone was continuously supplied to the third tank at 120 mL / hr, and acrylonitrile alone was continuously supplied to the fourth tank at 80 mL / hr to initiate the reaction.

[0114] The height of the overflow pipe from each tank was adjusted so that the reaction liquid volume in each tank was 1 L, and the reaction liquid was transferred to the next tank by overflow. The temperature of the reaction liquid in tanks 1 to 7 was controlled using jacket cooling water (10°C) so that the temperature of each tank was 20°C.

[0115] Two paddle blades (blade diameter: 350 mm, blade width: 100 mm) were used to adjust the stirring power per unit reaction liquid flow in all the reactors from tank 1 to tank 7 to 0.08 kW / m 3 (Froud number: 0.057) Here, the power required for stirring per unit reaction liquid fluid is calculated by dividing the power required for stirring in each reactor by the liquid volume (1 L = 0.001 ml 3 ) and calculated.

[0116] The pH of the reaction solution in each tank was measured with a KCl supply type pH detector, and a 0.06 N sodium hydroxide aqueous solution was automatically added to adjust the pH to a set value.

[0117] The pH of the reaction solutions in tanks 1, 2, 3, 4, 5, 6, and 7 were set to 7.2, 7.2, 7.2, 7.5, 7.7, 8.0, and 8.0, respectively.

[0118] One day after the start of the reaction, after confirming that the pH in each reaction tank maintained the set value, the acrylamide concentration of the reaction solution in each reaction tank was measured using a refractometer (Atago: RX-5000). The acrylamide concentrations in tanks 1, 2, 3, 4, 5, 6, and 7 were 19, 31, 38, 43, 49, 50, and 50%, respectively. The acrylamide concentration of the reaction solution flowing out of tank 7 reached 50% of the target concentration. Separately, the acrylonitrile concentration in the reaction solution in tank 7 was measured using gas chromatography (column: Waters, PoraPak-PS, 1 m, 180°C; carrier gas: helium; detector: FID). The unreacted acrylonitrile (AN) content in tank 7 was 10 ppm, indicating high reaction efficiency. An unreacted acrylonitrile concentration of 100 ppm or less is preferred for improved quality during acrylamide polymerization.

[0119] Next, 300 mL of the reaction solution from tank 7 was added to a 500 mL graduated cylinder and allowed to stand in a thermostatic bath at 25°C for 10 minutes. A glass ball filter (Kihara glass filter 504G) was placed in the center of the graduated cylinder, 5 mm from the bottom, and the pressure was set to 0.5 kg / cm 3 Air was passed through the reaction solution at a rate of 800cc / min. When the reaction solution began to foam, aeration was stopped when the foam level stabilized, and the time until the foam disappeared was measured. The shorter the time until the foam disappeared, the less protein was carried over from the biocatalyst into the aqueous acrylamide solution. For quality control, the time until the foam disappeared was within 30 seconds. A time until the foam disappeared of 5 seconds met the required quality.

[0120] <Example 2>

[0121] Reactions were carried out in the same manner as in Example 1 except that the pH of the reaction solutions in tanks 1, 2, 3, 4, 5, 6, and 7 was set to 6.8, 7.0, 7.5, 8.3, 8.3, 8.3, and 8.3, respectively.

[0122] One day after the start of the reaction, after confirming that the pH in each reaction tank maintained the set value, the acrylamide concentration of the reaction solution in each reaction tank was measured. The acrylamide concentrations in tanks 1, 2, 3, 4, 5, 6, and 7 were 18, 31, 36, 42, 48, 50, and 50%, respectively. The acrylamide concentration of the reaction solution flowing out of tank 7 reached 50% of the target concentration. Furthermore, the acrylonitrile concentration in the reaction solution in tank 7 was measured, and the unreacted acrylonitrile concentration was 20 ppm, indicating high reaction efficiency.

[0123] The defoaming time was measured using the reaction solution in tank 7 and was found to be 2 seconds, which met the required quality.

[0124] <Example 3>

[0125] Reactions were carried out in the same manner as in Example 1 except that the pH of the reaction solutions in tanks 1, 2, 3, 4, 5, 6, and 7 was set to 6.8, 6.8, 6.8, 8.3, 8.3, 8.3, and 8.3.

[0126] One day after the start of the reaction, after confirming that the pH in each reaction tank maintained the set value, the acrylamide concentration of the reaction solution in each reaction tank was measured. The acrylamide concentrations in tanks 1, 2, 3, 4, 5, 6, and 7 were 18, 30, 35, 41, 47, 49, and 50%, respectively. The acrylamide concentration of the reaction solution flowing out of tank 7 reached 50% of the target concentration. Furthermore, the acrylonitrile concentration in the reaction solution in tank 7 was measured, and the unreacted acrylonitrile concentration was 30 ppm, indicating high reaction efficiency.

[0127] The defoaming time was measured using the reaction solution in tank 7 and was found to be 2 seconds, which met the required quality.

[0128] <Example 4>

[0129] Reactions were carried out in the same manner as in Example 1 except that the pH of the reaction solutions in tanks 1, 2, 3, 4, 5, 6, and 7 was set to 7.5, 7.5, 7.5, 8.5, 8.5, 8.5, and 8.5.

[0130] One day after the start of the reaction, after confirming that the pH in each reaction tank maintained the set value, the acrylamide concentration of the reaction solution in each reaction tank was measured. The acrylamide concentrations in tanks 1, 2, 3, 4, 5, 6, and 7 were 18, 30, 34, 41, 47, 49, and 50%, respectively. The acrylamide concentration of the reaction solution flowing out of tank 7 reached 50% of the target concentration. Furthermore, the acrylonitrile concentration in the reaction solution in tank 7 was measured, and the unreacted acrylonitrile concentration was 300 ppm. Examples 1 to 3, in which the pH in the second half of the reaction was controlled to less than 8.5, exhibited higher reaction efficiency.

[0131] The defoaming time was measured using the reaction solution in tank 7 and was found to be 2 seconds, which met the required quality.

[0132] Comparative Example 1

[0133] Reactions were carried out in the same manner as in Example 1, except that the pH of the reaction solutions in tanks 1, 2, 3, 4, 5, 6, and 7 was all set to 7.0.

[0134] One day after the start of the reaction, after confirming that the pH in each reaction tank maintained the set value, the acrylamide concentration of the reaction solution in each reaction tank was measured. The acrylamide concentrations in tanks 1, 2, 3, 4, 5, 6, and 7 were 19, 31, 36, 44, 49, and 50%, respectively. The acrylamide concentration of the reaction solution flowing out of tank 7 reached 50% of the target concentration. Furthermore, the acrylonitrile concentration in the reaction solution in tank 7 was measured, and no unreacted acrylonitrile was detected, thus meeting the required quality.

[0135] The defoaming time was measured using the reaction solution in the seventh tank and was found to be 150 seconds, which did not meet the required quality.

[0136] Comparative Example 2

[0137] Reactions were carried out in the same manner as in Example 1, except that the pH of the reaction solutions in tanks 1, 2, 3, 4, 5, 6, and 7 was all set to 7.5.

[0138] One day after the start of the reaction, after confirming that the pH in each reaction tank maintained the set value, the acrylamide concentration of the reaction solution in each reaction tank was measured. The acrylamide concentrations in tanks 1, 2, 3, 4, 5, 6, and 7 were 18, 30, 36, 43, 49, 50, and 50%, respectively. The acrylamide concentration of the reaction solution flowing out of tank 7 reached 50% of the target concentration. Furthermore, the acrylonitrile concentration in the reaction solution in tank 7 was measured, and the unreacted acrylonitrile concentration was 10 ppm, meeting the required quality.

[0139] The defoaming time was measured using the reaction solution in tank 7 and was found to be 80 seconds, which did not meet the required quality.

[0140] Comparative Example 3

[0141] Reactions were carried out in the same manner as in Example 1, except that the pH of the reaction solutions in tanks 1, 2, 3, 4, 5, 6, and 7 was all set to 8.0.

[0142] One day after the start of the reaction, after confirming that the pH in each reaction tank maintained the set value, the acrylamide concentration of the reaction solution in each reaction tank was measured. The acrylamide concentrations in tanks 1, 2, 3, 4, 5, 6, and 7 were 17, 30, 35, 41, 48, 50, and 50%, respectively. The acrylamide concentration of the reaction solution flowing out of tank 7 reached 50% of the target concentration. Furthermore, the acrylonitrile concentration in the reaction solution in tank 7 was measured, and the unreacted acrylonitrile concentration was 120 ppm, slightly below the required quality.

[0143] The defoaming time was measured using the reaction solution in tank 7 and was found to be 45 seconds, which did not meet the required quality.

[0144] [Table 1]

[0145]

[0146] The results of Examples 1 to 3 are summarized as follows. In the first half of the reaction (the 1st tank, the 2nd tank, and the 3rd tank) in which the content of the amide compound relative to the total mass of the reaction solution is less than 40% by mass of each reaction tank, the pH of the reaction solution is set to the first pH, and the hydration reaction of the nitrile compound is carried out. Through this hydration reaction, as the reaction solution moves from the 1st tank to the 3rd tank, the concentration of the amide compound in the reaction solution increases. Relative to the total mass of the reaction solution entering the 4th tank from the 3rd tank, the content of the amide compound is 40% by mass or more. In the second half of the reaction after the 4th tank, the pH of the reaction solution is set to the second pH, and the hydration reaction is continued. Here, the second pH is higher than the first pH. As a result, in Examples 1 to 4, the foaming of the acrylamide aqueous solution obtained in the 7th tank was suppressed and the foaming property was low. In addition, compared with Example 4 in which the second pH in the second half of the reaction was controlled to be less than 8.5, the concentration of unreacted acrylonitrile was low and the reaction efficiency was higher.

[0147] Industrial applicability

[0148] 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, comprising 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. 2 . The method for producing an amide compound according to claim 1 , wherein the pH of the reaction solution is made higher during the reaction than the pH at the start of the reaction.

3. The method for producing an amide compound according to claim 1 or 2, wherein the pH of the reaction solution in at least a portion of the second half of the reaction in which 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 in which the content of the amide compound is less than 40% by mass relative to the total mass of the reaction solution, and the hydration reaction is carried out. 4 . The method for producing an amide compound according to claim 1 , 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 , 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 second half of the reaction is set to 7.5 or higher and lower than 8.5, and the hydration reaction is performed. 7 . The method for producing an amide compound according to claim 1 , wherein the nitrile compound is acrylonitrile or methacrylonitrile. 8 . The method for producing an amide compound according to claim 1 , wherein the amide compound is acrylamide or methacrylamide.

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