Improved nitrile hydratase reactivity using aldehyde

Abstract

To provide a method for producing an amide compound from a nitrile compound, capable of improving the productivity of the amide compound more simply.SOLUTION: The present invention relates to a method for producing an amide compound from a nitrile compound in the presence of a biocatalyst having nitrile hydratase activity. The method includes adding an aldehyde compound into a reaction solution containing the nitrile compound. The biocatalyst having nitrile hydratase activity is a cell or a bacterial cell containing nitrile hydratase derived from the genus Rhodococcus or Pseudonocardia.SELECTED DRAWING: None

Description

[Technical field]

[0001] [Related Applications] This specification includes the contents described in the specification of Patent Application No. 2021-19409 (filed February 10, 2021), which is the priority basis of this application. [Technical field] The present invention relates to a method for producing an amide compound using a nitrile hydratase. More specifically, the present invention relates to a method for improving the reaction rate of nitrile hydratase, a method for suppressing a decrease in the activity of nitrile hydratase, a method for producing an amide compound under conditions of an increased reaction rate, etc. [Background technology]

[0002] Amide compounds are industrially important substances and are used in a wide range of applications, such as for acrylamide as a flocculant for wastewater treatment, a paper strength enhancer, and an oil recovery agent, and for methacrylamide as a paint, adhesive, etc.

[0003] Conventionally, an industrial method for producing an amide compound has been used in which a nitrile compound is hydrated using reduced copper as a catalyst to produce the corresponding amide compound.

[0004] In recent years, nitrile hydratases, which have a nitrile hydration activity that hydrates a nitrile group to convert it into an amide group, have been discovered among enzymes possessed by microorganisms, and a method for producing a corresponding amide compound from a nitrile compound using the enzyme or a microbial cell or the like having the enzyme has been used.

[0005] This production method requires milder reaction conditions than conventional methods that use metal catalysts, and has a high conversion rate and high selectivity from nitrile compounds to the corresponding amide compounds. This makes it easier to set up a simpler process, making it an industrially superior method.

[0006] When amide compounds are industrially produced using nitrile hydratase, it is important to be able to efficiently produce the corresponding amide compounds from the nitrile compounds.Therefore, it has been reported that the efficiency of amide compound production can be improved by improving the enzyme activity of nitrile hydratase (Patent Document 1), suppressing the activity decrease due to temperature, and improving the resistance to amide compounds (Patent Document 2).

[0007] As another method, a method has been reported in which organic impurities in a nitrile compound that affect the activity of nitrile hydratase are identified, and the decrease in the activity of nitrile hydratase is suppressed to improve the production efficiency (Patent Documents 3 to 5). For example, a method has been reported in which an amide compound is efficiently produced by reducing the concentration of benzene present in a nitrile compound (Patent Document 3). In addition, a method has been reported in which the concentration of hydrocyanic acid in a nitrile compound is reduced (Patent Documents 4 and 5). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] JP 2005-295815 A [Patent Document 2] JP 2010-172295 A [Patent Document 3] International Publication No. 2007-043466 Brochure [Patent Document 4] Japanese Patent Application Publication No. 11-123098 [Patent Document 5] JP 2001-288156 A Summary of the Invention [Problem to be solved by the invention]

[0009] However, in the method of Patent Document 4, when a metal compound is added, it may affect the enzyme reaction and the quality of the obtained amide compound, and a step of removing the metal compound etc. is required after the reaction is completed, which leads to an increase in costs. On the other hand, when the nitrile compound is purified using an ion exchange resin, the additional steps lead to an increase in costs.

[0010] In addition, in the method of Patent Document 5, an alkaline compound is added to the raw material nitrile compound, which may increase the effort of adjusting the pH of the nitrile compound before the enzyme reaction and may affect the quality of the obtained amide compound.

[0011] Therefore, a main object of the present invention is to provide a method for improving the productivity of an amide compound more simply by adding an aldehyde compound. [Means for solving the problem]

[0012] Means for Solving the Problems of the Prior Art The inventors have conducted intensive research in view of the problems of the prior art and have found that, in a reaction for producing an amide compound from a nitrile compound using a biocatalyst having nitrile hydratase activity, the addition of an aldehyde compound improves the reaction rate for converting a nitrile compound into an amide compound and also suppresses a decrease in the activity of nitrile hydratase, thereby completing the present invention.

[0013] That is, the present invention provides the following [1] to

[10] . [1] A method for producing an amide compound from a nitrile compound in the presence of a biocatalyst having nitrile hydratase activity, comprising: The above process, wherein the reaction from the nitrile compound to the amide compound is carried out in the presence of an aldehyde compound, and the nitrile compound is at least one selected from the group consisting of acrylonitrile, acetonitrile, methacrylonitrile, cyanopyridine, glycolonitrile and alanine nitrile. [2] A method for improving a reaction rate of converting a nitrile compound into an amide compound in the presence of a biocatalyst having nitrile hydratase activity, comprising: The above method, which comprises a step of adding an aldehyde compound to a reaction liquid containing the nitrile compound, wherein the nitrile compound is at least one selected from the group consisting of acrylonitrile, acetonitrile, methacrylonitrile, cyanopyridine, glycolonitrile and alanine nitrile. [3] A method for suppressing a decrease in activity of a biocatalyst having nitrile hydratase activity in a production of an amide compound from a nitrile compound in the presence of the biocatalyst having nitrile hydratase activity, comprising: The above method, which comprises a step of adding an aldehyde compound to a reaction liquid containing the nitrile compound, wherein the nitrile compound is at least one selected from the group consisting of acrylonitrile, acetonitrile, methacrylonitrile, cyanopyridine, glycolonitrile and alanine nitrile. [4] The method according to any one of [1] to [3], wherein the concentration of the aldehyde compound in the nitrile compound is 0.9 to 15 in terms of molar ratio relative to the concentration of the cyanide compound. [5] The method according to any one of [1] to [5], wherein the biocatalyst having nitrile hydratase activity is a nitrile hydratase derived from the genus Rhodococcus or Pseudonocardia, or a cell, a microbial cell, or a processed product thereof containing the nitrile hydratase. [6] The aldehyde compounds are formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde, oxalic dialdehyde, malondialdehyde, pentanal, isovaleraldehyde, acrolein, crotonaldehyde, tiglic aldehyde, glyceraldehyde, glycolaldehyde, furfural, butanedial, trans-2-hexenal, glutaraldehyde, hexanal, heptane, etc. The method according to any one of [1] to [4], wherein the at least one selected from the group consisting of aldehyde, octanal, nonanal, decanal, paraldehyde, benzaldehyde, cinnamaldehyde, perillaaldehyde, vanillin, 1-naphthaldehyde, phthalaldehyde, methional, (Z)-7-hexadecenal, glyoxal (oxalic acid dialdehyde), paraformaldehyde, acetaldehyde ammonia, and hexamethylenetetramine. [7] An aldehyde compound, and at least one nitrile compound selected from acrylonitrile, acetonitrile, methacrylonitrile, cyanopyridine, glycolonitrile and lactonitrile. [8] A catalyst composition for producing an amide compound, comprising an aldehyde compound and a biocatalyst having nitrile hydratase activity. [9] An amide compound composition containing an aldehyde compound and an amide compound containing a cyanide compound, wherein the concentration of the cyanide compound in the amide compound before being mixed with the aldehyde compound is 0.2 ppm or more.

[10] An amide compound composition comprising an amide compound and a compound in which a cyanide compound and an aldehyde compound are bonded together. Effect of the Invention

[0014] According to the present invention, by adding an aldehyde compound to a reaction solution containing a nitrile compound, the reaction rate of the reaction of converting a nitrile compound to an amide compound by a biocatalyst having nitrile hydratase activity can be increased. Furthermore, according to the present invention, the decrease in activity of the biocatalyst having nitrile hydratase activity can be suppressed. Therefore, according to the present invention, an amide compound can be efficiently produced. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] An embodiment of the invention will be described below. (1) Biocatalysts with nitrile hydratase activity In the present invention, nitrile hydratase refers to an enzyme capable of hydrolyzing a nitrile compound to produce the corresponding amide compound. A biocatalyst having nitrile hydratase activity may be a nitrile hydratase protein itself, or may be an animal cell, a plant cell, a cell organelle, or a microbial cell containing nitrile hydratase, or a processed product thereof.

[0016] Examples of the processed material include disrupted animal cells, plant cells, cell organelles, or microbial cells, or enzymes extracted from microbial cells (raw enzymes or purified enzymes); animal cells, plant cells, cell organelles, microbial cells, or enzymes themselves immobilized on a carrier; and the like.

[0017] The treated material also includes animal cells, plant cells, cell organelles, or microbial cells whose proliferation ability has been lost by treatment with a drug.

[0018] Immobilization methods include the entrapment method, the cross-linking method, and the carrier binding method. The entrapment method is a method in which the enzyme is coated with a polymer film. The cross-linking method is a method in which the enzyme is cross-linked with a reagent having two or more functional groups (a multifunctional cross-linking agent). The carrier binding method is a method in which the enzyme is bound to a water-insoluble carrier.

[0019] Examples of the carrier used for immobilization (immobilization carrier) include gas beads, silica gel, polyurethane, polyacrylamide, polyvinyl alcohol, carrageenan, alginic acid, agar, and gelatin.

[0020] Representative examples of such microorganisms include those having nitrile hydratase activity, such as those belonging to the genera Rhodococcus, Gordona, Pseudomonas, Pseudonocardia, Geobacillus, Bacillus, Bacteridium, Micrococcus, Brevibacterium, Corynebacterium, Nocardia, Microbacterium, Fusari, and the like. Examples of microorganisms that may be used include those belonging to the genera Fusarium, Agrobacterium, Acinetobacter, Xanthobacter, Streptomyces, Rhizobium, Klebsiella, Enterobacter, Erwinia, Pantoea, Candida, Aeromonas, Citrobacter, and Achromobacter.

[0021] More specifically, Nocardia sp. N-775 described in JP-B-56-17918, Rhodococcus rhodochrous J-1 described in JP-B-06-55148, Rhodococcus rhodochrous NCIMB41164 described in International Publication WO2005 / 054456, Klebsiella sp. MCI2609 described in JP-A-05-30982, Aeromonas sp. MCI2614 described in JP-A-05-30983, Citrobacter freundii MCI2615 described in JP-A-05-30984, Agrobacterium rhizogenes described in JP-A-05-103681, rhizogenes IAM13570 and Agrobacterium tumefaciens, Xanthobacter flavas JCM1204 described in JP-A-05-161495, Erwinia nigrifluens MAFF03-01435, Enterobacter sp. MCI2707 described in JP-A-05-236975, Streptomyces sp. MCI2691 described in JP-A-05-236976, Rhizobium sp. MCI2610 described in JP-A-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 JP-A-05-15384, Agrobacterium radiobacter described in JP-A-06-14786, radiobacter SC-C15-1, Bacillus smithii SC-J05-1 described in JP-A-07-25494, Pseudonocardia thermophila ATCC19285 described in JP-A-08-56684, Pseudonocardia thermophila JCM3095 described in JP-A-09-275978, and the like.

[0022] The Rhodococcus rhodochrous J-1 strain described in JP-B-06-55148 was deposited on September 18, 1987 under the accession number "FERM BP-1478" at the Patent Organism Depositary of the National Institute of Technology and Evaluation (Chuo 6, 1-1-1 Higashi, Tsukuba, Ibaraki Prefecture (hereinafter the same in this specification)).

[0023] The Rhodococcus rhodochrous strain NCIMB41164 described in International Publication WO2005 / 054456 has been 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.

[0024] Pseudonocardia thermophila JCM3095 described in JP-A-09-275978 was deposited on February 7, 1996 at the National Institute of Technology and Evaluation, Patent Organism Depositary (1-1-1 Central 6, Higashi, Tsukuba, Ibaraki Prefecture) under the accession number "FERM BP-5785".

[0025] In the present invention, one type selected from the above microorganisms can be used alone, or two or more types can be used in combination.

[0026] Furthermore, the gene encoding the nitrile hydratase can be introduced and expressed in microbial cells by ordinary molecular biology techniques (for these molecular techniques, see Sambrook, Fritscj and Maniatis, "Molecular Cloning: A Laboratory Manual" 2nd Edition (1989), Cold Spring Harbor Laboratory Press). That is, in the present invention, an enzyme obtained by expressing a nucleic acid encoding a natural nitrile hydratase (wild type) or a mutant (improved type) thereof in microbial cells can also be used. In the present invention, one type selected from the above enzymes can be used alone, or two or more types can be used in combination.

[0027] The amino acid sequence of wild-type nitrile hydratase is published in NCBI databases such as GenBank (http: / / www.ncbi.nlm.nih.gov / ).

[0028] For example, the accession numbers of the α subunit and β subunit from Rhodococcus rhodochrous J1 (FERM BP-1478) are "P21219" and "P21220", respectively. The accession numbers of the α subunit and β subunit from Rhodococcus rhodochrous M8 (SU1731814) are "ATT79340" and "AAT79339", respectively. The accession numbers of the α subunit and β subunit from Pseudomonas thermophila JCM3095 are "1IRE A" and "1IREB", respectively.

[0029] Examples of the transformant into which the wild-type nitrile hydratase gene has been introduced include, but are not limited to, Escherichia coli MT10770 (FERM P-14756) transformed with nitrile hydratase from the genus Achromobacter (JP Patent Publication No. 8-266277), Escherichia coli MT10822 (FERM BP-5785) transformed with nitrile hydratase from the genus Pseudonocardia (JP Patent Publication No. 9-275978), or a microorganism transformed with nitrile hydratase from the species Rhodococcus rhodochrous (JP Patent Publication No. 4-211379).

[0030] Improved (mutated) 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, WO05 / 116206 pamphlet, WO12 / 164933 pamphlet, WO12 / 169203 pamphlet, WO15 / 186298 pamphlet, etc.), and in the method of the present invention, microorganisms into which these improved nitrile hydratases have been introduced can also be used.

[0031] These microorganisms having nitrile hydratase activity or processed products thereof can be used for amide synthesis reaction immediately after preparation of the bacterial cells, or can be stored after preparation of the bacterial cells and used for amide synthesis reaction as necessary. The method of culturing the microorganism for preparing the bacterial cells can be appropriately selected depending on the type of the microorganism. Seed culture may be performed before main culture.

[0032] The cells of the microorganism having nitrile hydratase activity or a processed product thereof can be used for a batch reaction or a continuous reaction. The reaction system can be appropriately selected from a fluidized bed, a fixed bed, a suspension bed, and the like. In this case, the catalyst temperature in the reaction liquid is not particularly limited as long as it does not interfere with the mixing of the aqueous medium and the nitrile compound.

[0033] (2) Nitrile compounds The nitrile compound used as a raw material in the production method of the present invention is not particularly limited as long as it is a compound that can be converted to an amide compound by a catalyst having nitrile hydratase activity. For example, aliphatic saturated nitriles such as acetonitrile, propionitrile, succinonitrile, adiponitrile, glycolonitrile, and lactonitrile, aliphatic unsaturated nitriles such as acrylonitrile and methacrylonitrile, aromatic nitriles such as benzonitrile and phthalodinitrile, and heterocyclic nitriles such as cyanopyridine can be mentioned. The nitrile compound in the present invention is preferably a nitrile compound such as acetonitrile, propionitrile, acrylonitrile, methacrylonitrile, n-butyronitrile, isobutyronitrile, cyanopyridine, glucolonitrile, and lactonitrile, and particularly preferably acrylonitrile, methacrylonitrile, acetonitrile, cyanopyridine, glycolonitrile, and lactonitrile.

[0034] Nitrile compounds are generally made into commercial products through a purification process. For example, acrylonitrile is industrially produced by the ammoxidation of propylene, and cyanide compounds such as hydrocyanic acid are removed by distillation purification after the reaction together with other by-products.

[0035] However, the product contains cyanide compounds that cannot be completely removed by this procedure. It is believed that the cyanide compounds contained in the nitrile compounds damage the biocatalysts that have nitrile hydratase activity, leading to a decrease in activity and a decrease in reaction rate.

[0036] In the present invention, by adding (allowing to be present) an aldehyde compound in a reaction solution containing a nitrile compound, the amount of cyanide contained in the nitrile compound can be reduced, and damage to a biocatalyst having nitrile hydratase activity caused by the cyanide can be prevented. That is, the reaction rate of converting a nitrile compound to an amide compound in the presence of a biocatalyst having nitrile hydratase activity is improved. It is also considered that the decrease in activity of a biocatalyst having nitrile hydratase activity due to contact with a nitrile compound can be suppressed.

[0037] (3) Production of amide compounds In the present invention, an amide compound is produced from a nitrile compound using a biocatalyst having nitrile hydratase activity.

[0038] The type of amide compound produced by the present invention is not particularly limited, and an amide compound according to the application can be produced. As a raw material, a nitrile compound corresponding to the amide compound can be used.

[0039] Examples of the amide compound include acrylamide, nicotinamide, methacrylamide, etc., with acrylamide being preferred.

[0040] When acrylonitrile is used as the nitrile compound, acrylamide is obtained, when methacrylonitrile is used as the nitrile compound, methacrylamide is obtained, and when cyanopyridine is used as the nitrile compound, nicotinamide is obtained.

[0041] The method for producing an amide compound using a biocatalyst having nitrile hydratase activity is not particularly limited, and may be, for example, a continuous reaction for continuously producing an amide compound, or a batch reaction for discontinuously producing an amide compound.

[0042] When a continuous reaction is carried out, reaction raw materials including a biocatalyst, water and a nitrile compound are continuously or intermittently introduced into a reactor, and a reaction mixture including a produced amide compound is continuously or intermittently removed from the reactor, thereby enabling the continuous production of an amide compound without withdrawing the entire amount of the reaction mixture in the reactor.

[0043] When a batch reaction is carried out, the amide compound can be produced by charging all of the reaction raw materials into a reactor at once and then reacting them, or by charging a portion of the reaction raw materials into a reactor and then continuously or intermittently supplying the remaining reaction raw materials and reacting them.

[0044] The type of reactor may be various types such as a stirred tank type, fixed bed type, fluidized bed type, moving bed type, tube type, or tower type. One reactor may be used, or multiple reactors may be used in combination. When multiple reactors are used, the concentration of the amide compound in the reaction mixture taken out of the reactor at the downstream side becomes higher. Therefore, the concentration of the amide compound finally obtained can be adjusted by the number of reactors.

[0045] When a plurality of reactors are used to continuously carry out reactions, the reactor into which the biocatalyst having nitrile hydratase activity and the nitrile compound are introduced is not limited to the reactor located at the most upstream, and they may also be introduced into reactors downstream thereof, so long as the efficiency of the reaction is not excessively deteriorated.

[0046] Of the reaction raw materials, raw material water is used in a hydration reaction with a nitrile compound when an amide compound is produced.

[0047] The raw water may be water or an aqueous solution in which an acid or salt is dissolved in water. Examples of acids include phosphoric acid, acetic acid, citric acid, boric acid, etc. Examples of salts include sodium salts, potassium salts, ammonium salts, etc. of the above acids. The type of raw water is not limited, and examples include pure water, city water, Tris buffer, phosphate buffer, acetate buffer, citrate buffer, borate buffer, etc. The pH (25°C) of the raw water used is not particularly limited as long as the biocatalyst can react efficiently. For example, it can be 4 to 10, preferably 5 to 9. By setting the pH to 4 or more, the enzyme activity of the biocatalyst can be sufficiently increased. By setting the pH to 10 or less, the deactivation of the biocatalyst can be suppressed.

[0048] The amount of the biocatalyst used can be appropriately selected depending on the type of the biocatalyst used, the reaction conditions, etc. For example, it is preferable to adjust the activity of the biocatalyst introduced into the reactor to about 50 to 500 U per 1 mg of dry cells at a reaction temperature of 10° C. The unit U means that 1 micromole of an amide compound is produced from a nitrile compound per minute, and is a value measured using the nitrile compound used in the production.

[0049] At least one water-soluble monocarboxylate having 2 or more carbon atoms may be added to the raw material used in the hydration reaction of acrylonitrile or the reaction mixture during or after the hydration reaction for the purpose of assisting stabilization. The water-soluble monocarboxylate may be either a saturated monocarboxylate or an unsaturated monocarboxylate. Examples of the saturated carboxylic acid include acetic acid, propionic acid, and n-caproic acid. Examples of the unsaturated carboxylic acid include acrylic acid, methacrylic acid, and vinylacetic acid. Representative salts include sodium salts, potassium salts, and ammonium salts. The amount of the water-soluble monocarboxylate to be added is preferably 20 to 5000 mg / kg of acid relative to the acrylamide in the finally obtained reaction mixture (aqueous acrylamide solution).

[0050] The amount of the nitrile compound used can be appropriately selected depending on the type of biocatalyst used, the reaction conditions, the reaction scale, whether the reaction is continuous or batchwise, and the like.

[0051] The reaction temperature is not particularly limited as long as the biocatalyst can efficiently promote the reaction. For example, the reaction temperature can be 5 to 50°C, preferably 10 to 40°C, and more preferably 15 to 35°C. By setting the reaction temperature at 10°C or higher, the reaction activity of the biocatalyst can be sufficiently increased. By setting the reaction temperature at 50°C or lower, the deactivation of the biocatalyst can be suppressed.

[0052] The reaction time is not particularly limited and can be appropriately selected depending on the reaction type (e.g., batch reaction, batch reaction, continuous reaction, etc.) and the reaction scale, etc. For example, the reaction time can be 0.1 to 60 hours, preferably 1 to 50 hours, and more preferably 2 to 40 hours.

[0053] When the amide compound is produced by a continuous reaction, the flow rate at which the reaction mixture is removed from the reactor may be determined in accordance with the introduction rates of the nitrile compound and the biocatalyst so that the amide compound can be produced continuously without withdrawing the entire amount of the reaction mixture in the reactor.

[0054] In order to stabilize the reaction, an additive may be added to the nitrile compound or the reaction liquid.

[0055] The concentration of the amide compound in the aqueous solution of the amide compound obtained in the present invention can be appropriately selected depending on the intended use of the amide compound. For example, the concentration of the amide compound can be 25 to 65% by mass, preferably 30 to 60% by mass, and more preferably 35 to 55% by mass, based on the total mass of the aqueous solution of the amide compound obtained. By setting the concentration of the amide compound to 65% by mass or less, it is possible to prevent crystals of the amide compound from precipitating at room temperature. In addition, by setting the concentration of the amide compound to 25% by mass or more, it is possible to reduce the tank volume used for storage and keeping, and to reduce transportation costs.

[0056] The amide compound obtained by the present invention can be used as it is for polymerization reaction, or can be stored until use. In addition, the aldehyde compound can be removed as necessary. When storing the amide compound, various additives such as a polymerization inhibitor and a polymerization initiator can be added as necessary.

[0057] (4) Aldehyde compounds In the present invention, in the method for producing an amide compound from a nitrile compound in the presence of a biocatalyst having nitrile hydratase activity, the reaction of the nitrile compound to an amide compound is carried out in the presence of an aldehyde compound.

[0058] In the present invention, the aldehyde compound is not particularly limited as long as the above-mentioned effects can be obtained. For example, formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde, malondialdehyde, pentanal, isovaleraldehyde, acrolein, crotonaldehyde, tiglic aldehyde, glyceraldehyde, glycolaldehyde, furfural, butanedial, trans-2-hexenal, glutaraldehyde, hexanal, heptanal, octanal, nonanal, decanal, paraldehyde, benzaldehyde, cinnamaldehyde, perillaaldehyde, vanillin, 1-naphthaldehyde, phthalaldehyde, methional, (Z)-7-hexadecenal, etc. can be mentioned.

[0059] The aldehyde compound includes not only compounds having an aldehyde group (-CHO) but also compounds that generate aldehyde compounds in water or in a solution. Examples of such aldehyde compounds include glyoxal (oxalic acid dialdehyde), paraformaldehyde, acetaldehyde ammonia, and hexamethylenetetramine.

[0060] Among these, aldehyde compounds having a molecular weight of about 200 or less are preferred, and aldehyde compounds having a molecular weight of about 100 or less are more preferred.

[0061] Particularly preferred aldehyde compounds are formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, and the like. In this specification, "adding" to a reaction liquid includes "making present" in the reaction liquid.

[0062] In the present invention, the amount of the aldehyde compound to be added to the reaction solution containing a nitrile compound is not particularly limited as long as it can increase the reaction rate of converting a nitrile compound to an amide compound in the presence of a biocatalyst having nitrile hydratase activity, or can suppress a decrease in the activity of the biocatalyst having nitrile hydratase activity when brought into contact with a nitrile compound.

[0063] The amount of the aldehyde compound added to the reaction liquid can be set to a molar ratio of 0.9 to 15, preferably 1.5 to 10.0, and more preferably 2.0 to 6.0 relative to the content of the cyanide compound in the reaction liquid.

[0064] The content of cyanide compounds in acrylonitrile can be determined by extraction with an alkaline solution, followed by titration using silver nitrate, or by spectrophotometry.

[0065] By adjusting the amount of aldehyde present in the reaction solution to be 0.9 or more in terms of molar ratio relative to the content of the cyanide compound, the reaction rate of converting a nitrile compound to an amide compound in the presence of a biocatalyst having nitrile hydratase activity can be increased, or a decrease in the activity of the biocatalyst having nitrile hydratase activity when contacted with a nitrile compound can be suppressed.

[0066] On the other hand, the amount of aldehyde present in the reaction solution is set to 15 or less in terms of molar ratio relative to the content of the cyanide compound, because adding more than this amount of aldehyde compound makes it difficult to obtain improved effects.

[0067] In this specification, the cyanide compound includes hydrocyanic acid (HCN); cyanide ion (CN - ); contains a cyanide compound such as sodium cyanide or potassium cyanide, or releases cyanide or cyanide ions under reaction conditions.

[0068] The timing of adding the aldehyde compound to the reaction solution containing the nitrile compound is not particularly limited as long as the above-mentioned effect can be exhibited, and the aldehyde compound may be added before, during (simultaneous with) contacting the biocatalyst having nitrile hydratase activity with the nitrile compound, or after contacting (i.e., after initiation of the enzymatic reaction of the nitrile compound by the biocatalyst).

[0069] Preferably, the aldehyde compound is added before contacting the biocatalyst having nitrile hydratase activity with the nitrile compound. When the aldehyde compound is added after the start of the reaction, it is considered that the more soon after the start of the reaction (the earlier the better), the more the above-mentioned effect can be obtained.

[0070] The method of adding the aldehyde compound to the reaction solution is not particularly limited as long as the aldehyde compound is present in the reaction solution at a desired concentration. For example, the aldehyde compound may be added in a solid form, or may be added after dissolving the aldehyde compound in a solvent used in the reaction, water, or the like.

[0071] (5) Improved reaction rate for converting nitrile compounds to amide compounds The present invention provides a method for improving the reaction rate of an enzymatic reaction for converting a nitrile compound into an amide compound in the presence of a biocatalyst having nitrile hydratase activity, the method comprising the step of adding an aldehyde compound to a reaction solution containing the nitrile compound. As the nitrile compound, at least one selected from acrylonitrile, acetonitrile, methacrylonitrile, cyanopyridine, glycolonitrile and alanine nitrile can be used.

[0072] Although it has been reported that aldehydes are used to prevent (stabilize) the polymerization of acrylamide (WO2011 / 102510), it was discovered for the first time in the present invention that an aldehyde compound prevents damage to a biocatalyst having nitrile hydratase activity and improves the reaction rate.

[0073] In the present invention, "improving the reaction rate" means that the reaction rate is higher than in the absence of the aldehyde compound.

[0074] The reaction rate can be measured by measuring the amount of the nitrile compound present in the reaction system that decreases per hour or by measuring the amount of the amide compound that increases in the reaction system. The amount of the nitrile compound or the amide compound can be measured by a known method such as gas chromatography analysis.

[0075] (6) Suppression of the decrease in activity of biocatalysts with nitrile hydratase activity The present invention provides a method for suppressing a decrease in activity of a biocatalyst having nitrile hydratase activity in the production of an amide compound from a nitrile compound in the presence of the biocatalyst having nitrile hydratase activity, the method comprising the step of adding an aldehyde compound to a reaction solution containing the nitrile compound. As the nitrile compound, at least one selected from the group consisting of acrylonitrile, acetonitrile, methacrylonitrile, cyanopyridine, glycolonitrile and alanine nitrile can be used.

[0076] As described above, the activity of nitrile hydratase is easily affected by impurities in the reaction solution and reaction conditions. According to the method of the present invention, it is possible to easily suppress the decrease in the activity of nitrile hydratase without requiring an additional step and without affecting the quality of the amide compound.

[0077] (7) Composition for producing amide compound The present invention also provides a composition for producing an amide compound, which comprises an aldehyde compound and at least one nitrile compound selected from acrylonitrile, acetonitrile, methacrylonitrile, cyanopyridine, glycolonitrile and lactonitrile.

[0078] In the composition for producing an amide compound of the present invention, the aldehyde compound may be any of the compounds described in "(4) Aldehyde Compound" in accordance with the description in the same section. The composition is used in the method for producing an amide compound described in (3), and enables efficient production of the amide compound.

[0079] (8) Catalyst composition for producing amide compounds The present invention also provides a catalyst composition for producing an amide compound, comprising an aldehyde compound and a biocatalyst having nitrile hydratase activity.

[0080] In the catalyst composition for producing an amide compound of the present invention, the aldehyde compound and the biocatalyst having nitrile hydratase activity are as described in "(4) Aldehyde compound" and "(1) Biocatalyst having nitrile hydratase activity", respectively.

[0081] (9) Amide compound composition The present invention also provides an amide compound composition comprising an aldehyde compound and a cyanide compound.

[0082] In a first embodiment, the amide compound composition contains an amide compound and an aldehyde compound, and the concentration of the cyanide compound in the amide compound is 0.2 ppm or more. The cyanide compound and the amide compound may be separate compounds or may be combined. The concentration of the cyanide compound is the concentration before the addition of the aldehyde compound.

[0083] In a second embodiment, the amide compound composition includes an amide compound and a compound in which a cyanide compound and an aldehyde compound are combined. EXAMPLES

[0084] The present invention will be described in more detail below using examples, but the present invention is not limited to these examples. In this specification, "%" indicates "% by mass".

[0085] [Example 1] (Preparation of Bacterial Cells) Rhodococcus rhodochrous J-1 (FERMBP-1478) Rhodococcus rhodochrous J-1 (FERMBP-1478) having nitrile hydratase activity was aerobically cultured at 30° C. in a medium (pH 7.0) containing 2.0% glucose, 1.0% urea, 0.5% peptone, 0.3% yeast extract, and 0.05% cobalt chloride. The medium was washed with 50 mM phosphate buffer (pH 7.0) to prepare a bacterial cell suspension (3% in terms of dry cells).

[0086] (Adjusting acrylonitrile to a given concentration of hydrocyanic acid) A 1000 ppm cyanide standard solution (manufactured by Hayashi Pure Chemical Industries) was added to industrial acrylonitrile (manufactured by Mitsubishi Chemical Corporation) so that the hydrocyanic acid concentration was 2.1 ppm by weight.

[0087] (Method for measuring hydrogen cyanide concentration) 9.6g of pure water and 0.4g of acrylonitrile (Mitsubishi Chemical) were placed in a test tube and left to stand at 25°C for 30 minutes. Then, CyaniVer.3 from the HACH analysis kit was added, vortexed for 30 seconds, and left to stand for 30 seconds. CyaniVer.4 was added and vortexed for 10 seconds, and CyaniVer.5 was added and vortexed for 2 minutes.

[0088] After leaving the mixture at 25°C for 30 minutes, the absorbance was measured using a spectrophotometer (DR500001) (HACH Co., Ltd.) The hydrogen cyanide concentration was found to be 2.1 ppm.

[0089] (Preparation of acrylonitrile containing acetaldehyde) Acetaldehyde reagent (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to industrial acrylonitrile (manufactured by Mitsubishi Chemical Corporation) to a concentration of 10 ppm by weight.

[0090] (Amide formation reaction) 3.15 g of pH 7.0 phosphate buffer and 3.15 g of diluted bacterial solution prepared by diluting the aforementioned bacterial cells with pH 7.0 phosphate buffer so that the activity of the reaction solution was 1460 U were added to a glass case with a lid having an internal volume of 13.5 ml, and the mixture was stirred while controlling the temperature at 20°C. 4.8 mL of acrylonitrile containing 10 ppm acetaldehyde was added to the mixture to start the reaction. After 4 hours, the reaction solution was sampled and the concentration of acrylonitrile was measured by gas chromatography (column: PoraPack-PS (Waters), 1 m, 210°C, carrier gas: helium, detector: FID).

[0091] [Example 2] The same procedure as in Example 1 was carried out except that the acetaldehyde concentration in the acrylonitrile was 6.0 ppm.

[0092] [Example 3] The same procedure as in Example 1 was repeated except that the acetaldehyde concentration in the acrylonitrile was 3.0 ppm.

[0093] [Example 4] The same procedure as in Example 1 was carried out, except that the hydrogen cyanide concentration in the acrylonitrile was changed to 1.1 ppm.

[0094] [Comparative Example 1] The experiment was carried out in the same manner as in Example 1, except that no acetaldehyde was added to the acrylonitrile.

[0095] [Comparative Example 2] The same procedure as in Example 1 was carried out except that the hydrogen cyanide concentration in the acrylonitrile was 1.1 ppm and no acetaldehyde was added to the acrylonitrile.

[0096] Table 1 shows the concentration ratio of acrylonitrile 4 hours after the start of the reaction, compared to the case where no aldehyde compound was added. This concentration ratio can be calculated using the following formula. Concentration ratio [%] = (Acrylonitrile concentration when aldehyde is added / Acrylonitrile concentration when aldehyde is not added) x 100 For compounds that generate aldehyde compounds in water or solution, the aldehyde / cyanide ratio was calculated based on the number of moles of the aldehyde compound generated in water or solution.

[0097] [Table 1]

[0098] In the examples in which acetaldehyde was added, it was confirmed that the concentration of acrylonitrile was lower and the reaction rate of conversion to an amide compound was higher, regardless of whether the hydrocyanic acid concentration was 2.1 ppm or 1.1 ppm, compared to the comparative examples in which acetaldehyde was not added.

[0099] [Test Example 1] According to Example 1, the effect of the following compounds on the amide compound production reaction was evaluated. No.1: Formaldehyde (30.03), No.2: Acetaldehyde (44.05), No.3: Propionaldehyde (58.08), No.4: Butyraldehyde (72.11), No.5: Hexanal (100.16), No.6: Heptanal (114.18), No.7: Octanal (128.21), No.8: Nonanal (142.24), No.9: Decanal (156.27), No.10: Formic acid (46.03), No.11: Oxalic acid dialdehyde (58.0 4), No.12: meso-erythritol (122.12), No.13: paraldehyde (132.16), No.14: paraformaldehyde (30.03×n), No.15: benzaldehyde (106.12), No.16: cinnamaldehyde (132.16), No.17: perilla aldehyde (150.22), No.18: vanillin (152.15), No.19: acetaldehyde ammonia (183.25), No.20: hexamethylenetetramine (140.19) The numbers in parentheses are molecular weights (g / mol). No.11, 14, 19, 20: Produces aldehyde compounds in water.

[0100] The acrylonitrile was adjusted so that the hydrocyanic acid concentration was 2.0 ppm.

[0101] In a glass case with a lid having an internal volume of 13.5 ml, 1.00 g of the diluted bacterial solution obtained by diluting the aforementioned bacterial cells with a pH 7.0 phosphate buffer so that the charged activity in the reaction solution was 1840 U was added, and a diluted solution obtained by diluting each aldehyde compound with a pH 7.0 phosphate buffer so that the concentration in the reaction solution was 5 ppm or 10 ppm was added, and finally, a pH 7.0 phosphate buffer was added to make the total amount 6.2 g. The obtained solution was stirred while controlling the temperature at 25 ° C. 4.8 mL of acrylonitrile (equivalent to 50% acrylamide, 38 wt%) was added to this to start the reaction. After 3.5, 4.5, 5.0, and 6.0 hours, the reaction solution was sampled, and the concentration of acrylonitrile was measured by gas chromatography (column: PoraPack-PS (Waters), 1 m, 210 ° C., carrier gas: helium, detector: FID).

[0102] Table 2 shows the concentration ratio of acrylonitrile at 3.5 hours and 6 hours after the start of the reaction, compared with the case where no aldehyde compound was added. This concentration ratio is calculated by the following formula. Concentration ratio [%] = (Acrylonitrile concentration when aldehyde is added / Acrylonitrile concentration when aldehyde is not added) x 100 For compounds that generate aldehyde compounds in water or solution, the aldehyde / cyanide ratio was calculated based on the number of moles of the aldehyde compound generated in water or solution.

[0103] [Table 2]

[0104] For aldehyde compounds other than formic acid, meso-erythritol, and paraldehyde, it was confirmed that the addition of an aldehyde compound reduced the concentration of acrylonitrile and increased the reaction rate of conversion of nitrile compounds to amide compounds compared to the case where the aldehyde compound was not added.

[0105] [Example 5] (Preparation of transformants containing nitrile hydratase derived from Rhodococcus rhodochrous M8 strain) (1) Preparation of chromosomal DNA from Rhodococcus rhodochrous M8 strain (hereinafter referred to as M8 strain) The M8 strain (SU1731814) is available from the Russian Strain Center IBFM (VKPM S-926).

[0106] The M8 strain was cultured in 100 mL of MYK (0.5% polypeptone, 0.3% Bacto yeast extract, 0.3% Bacto malt extract, 0.2% K2HPO4, 0.2% KH2PO4) medium (pH 7.0) with shaking at 30°C for 72 hours. The culture was centrifuged, and the collected cells were suspended in 4 mL of saline-EDTA solution (0.1 M EDTA, 0.15 M NaCl (pH 8.0)). 8 mg of lysozyme was added to the suspension, which was then shaken at 37°C for 1 to 2 hours and then frozen at -20°C.

[0107] Next, 10 mL of Tris-SDS solution (1% SDS, 0.1 M NaCl, 0.1 M Tris-HCl (pH 9.0)) was added to the suspension while gently shaking. Furthermore, proteinase K (Merck) (final concentration 0.1 mg) was added to the suspension and shaken at 37°C for 1 hour. Next, an equal volume of TE-saturated phenol was added, stirred (TE: 10 mM Tris-HCl, 1 mM EDTA (pH 8.0)), and centrifuged. The upper layer was collected, a double volume of ethanol was added, and the DNA was wound around a glass rod. This was then centrifuged in succession with 90%, 80%, and 70% ethanol to remove the phenol.

[0108] Next, DNA was dissolved in 3 mL of TE buffer, and ribonuclease A solution (heat-treated at 100°C for 15 minutes) was added to a concentration of 10 μg / mL, followed by shaking at 37°C for 30 minutes. Proteinase K (Merck) was then added and the mixture was shaken at 37°C for 30 minutes. An equal volume of TE-saturated phenol was added to the mixture, followed by centrifugation to separate the mixture into an upper and lower layer.

[0109] The upper layer was further separated into upper and lower layers after adding an equal volume of TE-saturated phenol and centrifuging. This procedure was repeated once more. The upper layer was then added with the same volume of chloroform (containing 4% isoamyl alcohol) and centrifuged to recover the upper layer. Next, twice the volume of ethanol was added to the upper layer, and the DNA was wound around a glass rod and recovered to obtain chromosomal DNA.

[0110] (2) Construction of an expression plasmid for nitrile hydratase from M8 strain The M8 strain-derived nitrile hydratase gene (SEQ ID NO: 5) is described in a non-patent document (Veiko, VP et al., Cloning, nucleotide sequence of nitrile hydratase gene from Rhodococcus rhodochrous M8, Biotekhnologiia (Mosc.), 5, 3-5 (1995)), and the amino acid sequences of the β subunit, α subunit, and activator are shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. Based on this sequence information, the following primers (SEQ ID NO: 1 and 2) were synthesized, and PCR was performed under the following reaction conditions using the prepared genomic DNA of the M8 strain as a template.

[0111] Primer: M8-1: 5'-GGTCTAGAATGGATGGTATCCACGACACAGGC-3' (SEQ ID NO: 1) M8-2: 5'-cccctgcaggtcagtcgatgatggccatcgattc-3' (SEQ ID NO: 2)

[0112] Reaction mixture composition: Template DNA (M8 strain genomic DNA) 1 μl Primer M8-1 0.5 μl Primer M8-2 0.5 μl Sterile water 8μl PrimeSTAR (Takara Bio) 10μl Total volume: 20 μl

[0113] Temperature cycle: 30 cycles of 98℃ 10 seconds, 55℃ 5 seconds, and 72℃ 30 seconds

[0114] Next, the obtained plasmid DNA was cleaved with restriction enzymes XbaI and Sse8387I, and then electrophoresed on a 0.7% agarose gel to recover a 1.6 kb nitrile hydratase gene fragment (SEQ ID NO: 5), which was then introduced into the XbaI-Sse8387I site of the plasmid pSJ042. The obtained plasmid was named pSJ-N01A. Note that pSJ042 was prepared by the method shown in JP 2008-154552 A as a plasmid expressing J1 strain-derived nitrile hydratase in Rhodococcus, and the plasmid pSJ023 used for the preparation of pSJ042 was deposited as a transformant ATCC12674 / pSJ023 (FERM BP-6232) at the National Institute of Advanced Industrial Science and Technology (6 Central, 1-1-1 Higashi, Tsukuba, Ibaraki Prefecture) on March 4, 1997.

[0115] (3) Preparation of ATCC12674 transformants Logarithmic growth phase cells of Rhodococcus rhodochrous ATCC 12674 strain were collected by centrifugation, washed three times with ice-cold sterilized water, and suspended in sterilized water to prepare competent cells.

[0116] The plasmid DNA (pSJ-N01A) 1μl prepared above and ATCC12674 competent cells 10μl were mixed and cooled on ice for 30 minutes. The DNA and bacterial cell suspension was placed in a cuvette and subjected to electric pulse treatment at 20kV / cm and 200 OHMS using a gene introduction device Gene Pulser (BIO RAD). The electric pulse treatment solution was left on ice for 10 minutes, heat-shocked at 37℃ for 10 minutes, and 500μl of MYK medium (0.5% polypeptone, 0.3% Bacto yeast extract, 0.3% Bacto malt extract, 0.2% K2HPO4, 0.2% KH2PO4) was added and left at 30℃ for 24 hours, then spread on MYK agar medium containing 10μg / ml kanamycin and cultured at 30℃ for 3 days.

[0117] The plasmid in the resulting colonies was confirmed, and a transformant (ATCC12674 / pSJ-N01A) was obtained.

[0118] (4) Cultivation of recombinant bacteria The transformants (ATCC12674 / pSJ-N01A) obtained in the above process were inoculated into MYK medium (50 μg / ml kanamycin), cultured with shaking at 30°C for 2 days, and then inoculated into GGPK medium (1.5% glucose, 1% sodium glutamate, 0.1% yeast extract, 0.05% K2HPO4, 0.05% KH2PO4, 0.05% Mg2O4·7H2O, 1% CoCl2, 0.1% urea, 50 μg / ml kanamycin, pH 7.2) at 1%. Cultured with shaking at 30°C for 3 days and collected by centrifugation. The cells were then washed with 100 mM phosphate buffer (pH 7.0) to prepare a cell suspension.

[0119] (5) Evaluation of the effect on the amide compound formation reaction According to Example 1, the effect of propionaldehyde on the amide compound production reaction of the M8 strain-derived transformant (ATCC12674 / pSJ-N01A) was evaluated.

[0120] The acrylonitrile was adjusted so that the hydrocyanic acid concentration was 2.0 ppm.

[0121] In a glass case with a lid having an internal volume of 13.5 ml, 1.00 g of the diluted bacterial solution obtained by diluting the aforementioned bacterial cells with a pH 7.0 phosphate buffer so that the amount of bacteria charged in the reaction solution was 5.6 mg was added, and a diluted solution obtained by diluting each aldehyde compound with a pH 7.0 phosphate buffer so that the amount of each aldehyde compound in the reaction solution was 10 ppm, 25 ppm, or 50 ppm was added, and finally, a pH 7.0 phosphate buffer was added to make the total amount 9.0 g. The resulting solution was stirred while controlling the temperature at 25 ° C. 1.0 g of acrylonitrile was added to this to start the reaction. After 6.0 hours, the reaction solution was collected and the concentration of acrylonitrile was measured by gas chromatography (column: PoraPack-PS (Waters), 1 m, 210 ° C., carrier gas: helium, detector: FID).

[0122] Table 3 shows the concentration ratio of acrylonitrile 6 hours after the start of the reaction, compared with the case where no aldehyde compound was added. This concentration ratio is calculated using the following formula: Concentration ratio [%] = (Acrylonitrile concentration when aldehyde is added / Acrylonitrile concentration when aldehyde is not added) x 100 For compounds that generate aldehyde compounds in water or solution, the aldehyde / cyanide ratio was calculated based on the number of moles of the aldehyde compound generated in water or solution.

[0123] [Table 3]

[0124] It was confirmed that the addition of an aldehyde compound to the M8 strain-derived transformant (ATCC12674 / pSJ-N01A) reduced the concentration of acrylonitrile and increased the reaction rate of converting nitrile compounds to amide compounds compared to when no aldehyde compound was added.

[0125] [Example 6] (1) Construction of DN1 transformants expressing nitrile hydratase from Pseudonocardia thermophila JCM3095 strain Plasmid pPT-DB1 is a plasmid containing the nitrile hydratase gene derived from Pseudonocardia thermophila JCM3095 strain (hereinafter referred to as JCM3095 strain) obtained in JP 09-275978 A, and has been deposited at the National Institute of Advanced Industrial Science and Technology (6-1-1 Central, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) as a transformed strain (MT-10822 strain) introduced into Escherichia coli HB101.

[0126] The nitrile hydratase gene (SEQ ID NO: 9) of the JCM3095 strain is described in JP-A-9-275978, and the amino acid sequences of the β subunit, α subunit, and activator are shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively. Based on this sequence information, the following primers (SEQ ID NO: 3 and SEQ ID NO: 4) were synthesized, and PCR was performed using pPT-DB1 as a template under the following reaction conditions. pRT-DB1 used as a template was prepared from the MT-10822 strain by a standard method.

[0127] Primer: PSN-1: 5'-GGTCTAGAATGAACGGCGTGTACGACGTCGGC-3' (SEQ ID NO: 3) PSN-2: 5'-ccCCTGCAGGTCAGGACCGCACGGCCGGGTGGAC-3' (SEQ ID NO: 4)

[0128] Reaction mixture composition: Template DNA (pPT-DB1) 1 μl Primer PSN-1 0.5 μl Primer PSN-2 0.5 μl Sterile water 8μl PrimeSTAR (Takara Bio) 10μl Total volume: 20 μl

[0129] Temperature cycle: 30 cycles of 98℃ 10 seconds, 55℃ 5 seconds, and 72℃ 30 seconds

[0130] A plasmid was prepared using the obtained PCR product (SEQ ID NO: 10) in the same manner as in Example 5(2) and named pSJ-N02A. The obtained plasmid was introduced into ATCC12674 in the same manner as in Example 5(3) to obtain a transformant (ATCC12674 / pSJ-N02A).

[0131] (4) Cultivation of recombinant bacteria The transformants (ATCC12674 / pSJ-N02A) obtained in the above process were inoculated into MYK medium (50 μg / ml kanamycin), cultured with shaking at 30°C for 2 days, and then inoculated into GGPK medium (1.5% glucose, 1% sodium glutamate, 0.1% yeast extract, 0.05% K2HPO4, 0.05% KH2PO4, 0.05% Mg2O4·7H2O, 1% CoCl2, 0.1% urea, 50 μg / ml kanamycin, pH 7.2) at 1%. Cultured with shaking at 30°C for 3 days and collected by centrifugation. The cells were then washed with 100 mM phosphate buffer (pH 7.0) to prepare a cell suspension.

[0132] (5) Evaluation of the effect on the amide compound formation reaction According to Example 1, the effect of propionaldehyde on the amide compound production reaction of the transformant (ATCC12674 / pSJ-N02A) was evaluated.

[0133] The acrylonitrile was adjusted so that the hydrocyanic acid concentration was 2.0 ppm.

[0134] In a glass case with a lid having an internal volume of 13.5 ml, 1.00 g of the diluted bacterial solution obtained by diluting the aforementioned bacterial cells with a pH 7.0 phosphate buffer solution so that the amount of bacteria charged in the reaction solution was 4.1 mg was added, and a diluted solution obtained by diluting each aldehyde compound with a pH 7.0 phosphate buffer solution so that the concentration in the reaction solution was 7 ppm was added, and finally, a pH 7.0 phosphate buffer solution was added to make the total amount 8.5 g. The obtained solution was stirred while controlling the temperature at 25 ° C. 1.5 g of acrylonitrile was added to this to start the reaction. After 6.0 hours, the reaction solution was collected and the concentration of acrylonitrile was measured by gas chromatography (column: PoraPack-PS (Waters), 1 m, 210 ° C., carrier gas: helium, detector: FID).

[0135] Table 4 shows the concentration ratio of acrylonitrile 6 hours after the start of the reaction, compared with the case where no aldehyde compound was added. This concentration ratio is calculated using the following formula: Concentration ratio [%] = (Acrylonitrile concentration when aldehyde is added / Acrylonitrile concentration when aldehyde is not added) x 100 For compounds that generate aldehyde compounds in water or solution, the aldehyde / cyanide ratio was calculated based on the number of moles of the aldehyde compound generated in water or solution.

[0136] [Table 4]

[0137] It was confirmed that the addition of an aldehyde compound reduced the concentration of acrylonitrile and increased the reaction rate of conversion of a nitrile compound to an amide compound, compared to the case where an aldehyde compound was not added. [Industrial Applicability]

[0138] INDUSTRIAL APPLICABILITY The present invention is useful in the biological industrial production of amide compounds such as acrylamide and methacrylamide from nitrile compounds.

[0139] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. [Sequence List Free Text]

[0140] SEQ ID NO: 1: Primer M8-1 SEQ ID NO: 2: Primer M8-2 SEQ ID NO: 3: primer PSN-1 SEQ ID NO: 4: Primer PSN-2

Claims

1. A method for producing an amide compound from a nitrile compound in the presence of a biocatalyst having nitrile hydratase activity, comprising the steps of: adding an aldehyde compound to a reaction liquid containing the nitrile compound, the nitrile compound is at least one selected from acrylonitrile, acetonitrile, methacrylonitrile, cyanopyridine, glycolonitrile, and alanine nitrile; The aldehyde compound is a compound having an aldehyde group or a compound that generates a compound having an aldehyde group in water or a solution, and is at least one selected from formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde, oxalic acid dialdehyde, malondialdehyde, pentanal, isovaleraldehyde, glyceraldehyde, glycolaldehyde, furfural, butanedial, glutaraldehyde, hexanal, heptanal, octanal, nonanal, decanal, paraldehyde, benzaldehyde, cinnamaldehyde, perillaldehyde, vanillin, 1-naphthaldehyde, phthalaldehyde, methional, paraformaldehyde, acetaldehyde ammonia, and hexamethylenetetramine; The method as described above, wherein the biocatalyst having nitrile hydratase activity is a cell or a microbial cell containing nitrile hydratase derived from the genus Rhodococcus or Pseudonocardia.

2. A method for improving a reaction rate of converting a nitrile compound into an amide compound in the presence of a biocatalyst having nitrile hydratase activity, comprising: adding an aldehyde compound to a reaction liquid containing the nitrile compound, the nitrile compound is at least one selected from acrylonitrile, acetonitrile, methacrylonitrile, cyanopyridine, glycolonitrile, and alanine nitrile; The aldehyde compound is a compound having an aldehyde group or a compound that generates a compound having an aldehyde group in water or a solution, and is at least one selected from formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde, oxalic acid dialdehyde, malondialdehyde, pentanal, isovaleraldehyde, glyceraldehyde, glycolaldehyde, furfural, butanedial, glutaraldehyde, hexanal, heptanal, octanal, nonanal, decanal, paraldehyde, benzaldehyde, cinnamaldehyde, perillaldehyde, vanillin, 1-naphthaldehyde, phthalaldehyde, methional, paraformaldehyde, acetaldehyde ammonia, and hexamethylenetetramine; The method as described above, wherein the biocatalyst having nitrile hydratase activity is a cell or a microbial cell containing nitrile hydratase derived from the genus Rhodococcus or Pseudonocardia.

3. A method for suppressing a decrease in activity of a biocatalyst having nitrile hydratase activity in a production of an amide compound from a nitrile compound in the presence of the biocatalyst having nitrile hydratase activity, comprising: adding an aldehyde compound to a reaction liquid containing the nitrile compound, the nitrile compound is at least one selected from acrylonitrile, acetonitrile, methacrylonitrile, cyanopyridine, glycolonitrile, and alanine nitrile; The aldehyde compound is a compound having an aldehyde group or a compound that generates a compound having an aldehyde group in water or a solution, and is at least one selected from formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde, oxalic acid dialdehyde, malondialdehyde, pentanal, isovaleraldehyde, glyceraldehyde, glycolaldehyde, furfural, butanedial, glutaraldehyde, hexanal, heptanal, octanal, nonanal, decanal, paraldehyde, benzaldehyde, cinnamaldehyde, perillaldehyde, vanillin, 1-naphthaldehyde, phthalaldehyde, methional, paraformaldehyde, acetaldehyde ammonia, and hexamethylenetetramine; The method as described above, wherein the biocatalyst having nitrile hydratase activity is a cell or a microbial cell containing nitrile hydratase derived from the genus Rhodococcus or Pseudonocardia.

4. The method according to any one of claims 1 to 3, wherein the concentration of the aldehyde compound in the nitrile compound is 0.9 to 15 in terms of molar ratio relative to the concentration of the cyanide compound.

5. An aldehyde compound, The method according to any one of claims 1 to 4, which is carried out using a composition for producing an amide compound, comprising at least one nitrile compound selected from the group consisting of acrylonitrile, acetonitrile, methacrylonitrile, cyanopyridine, glycolonitrile and alanine nitrile.

6. The method according to any one of claims 1 to 4, which is carried out using a catalyst composition for producing an amide compound, comprising an aldehyde compound and a biocatalyst having nitrile hydratase activity.

7. The method according to any one of claims 1 to 6, wherein the cells or microbial cells have been treated with a drug to lose their ability to proliferate.

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