Aldehydes enhance the reactivity of nitrile hydratases.

Abstract

This invention provides a simple method for producing amide compounds that improves the productivity of amide compounds. [Solution] In a method for producing an amide compound from a nitrile compound in the presence of a biocatalyst having nitrile hydratase activity, the present invention provides a method for efficiently producing an amide compound by suppressing the decrease in the activity of the biocatalyst and improving the reaction rate for converting the nitrile compound to an amide compound. The method comprises the step of adding an aldehyde compound to a reaction solution containing a nitrile compound, wherein the nitrile compound is at least one selected from acrylonitrile, acetonitrile, etc., the aldehyde compound is at least one selected from formaldehyde, acetaldehyde, etc., which is either a compound having an aldehyde group or a compound that produces a compound having an aldehyde group in solution, and the biocatalyst is a cell or microbial cell containing nitrile hydratase derived from the genus Rhodococcus or Pseudonocardia.

Description

[Technical Field]

[0001] [Related Application] The present specification incorporates the content described in the specification of Japanese Patent Application No. 2021-19409 (filed on Februar y 10, 2021), which is the basis of the priority right of the present application. [Technical Field] The present invention relates to a method for producing an amide compound using nitrile hydratase. More specifically, the present invention relates to a method for improving the reaction rate of nitrile hydratase, a method for suppressing the decrease in activity of nitrile hydratase, and a method for producing an amide compound with an increased reaction rate . [Background Art]

[0002] Amide compounds are industrially important substances and are widely used in a wide range of fields. Their uses include: for acrylamide, it is used in flocculants for wastewater treatment, paper strength agents, and oil recovery agents; for methacryl amide, it is widely used in paints, adhesives and other products.

[0003] As a method for producing an amide compound, an industrial method has conventionally been used, which uses copper in a reduced state as a catalyst to hydrate a nitrile compo und to produce the corresponding amide compound. .

[0004] In recent years, nitrile hydratase, which is an enzyme possessed by microorganisms and has nitrile hydration activity that hydrates nitrile groups and converts them into amide groups, has been discovered. A method for producing the corresponding amide compound from a nitrile compound using said enzyme or microbial cells containing said enzyme has come into use. has nitrile hydration activity that hydrates nitrile groups and converts them into amide groups, has been discovered, and the method for producing the corresponding amide compound from a nitrile compound by using the enzyme or microbial cells having the enzyme is employed. .

[0005] This production method has milder reaction conditions than conventional methods using metal catalysts, and allows nitrile Due to its high conversion rate and high selectivity from the compound to the corresponding amide compound, it is more suitable for It can be said that this is an industrially superior method because it allows for the implementation of simple processes.

[0006] When industrially producing amide compounds using nitrile hydratase, nitridation is performed. It is important to be able to efficiently produce the corresponding amide compound from the compound. Therefore, Nitori Improvement of the enzyme activity of hydratase (Patent Document 1), suppression of activity decrease due to temperature, amidation Improvements in the production efficiency of amide compounds have been reported through improved compound resistance (Patent Document 2).

[0007] Another approach involves investigating organic compounds in nitrile compounds that affect nitrile hydratase activity. By identifying sexual impurities and suppressing the decrease in nitrile hydratase activity, manufacturing efficiency can be improved. Methods for improving this have been reported (Patent Documents 3-5). For example, those present in nitrile compounds A method for efficiently producing amide compounds by reducing the concentration of benzene (Patent text) Reference 3) has been reported. Also, a method for reducing the concentration of hydrogen cyanide in nitrile compounds (Patent document) References 4, 5) have also been reported. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2005-295815 [Patent Document 2] Japanese Patent Publication No. 2010-172295 [Patent Document 3] International Publication No. 2007-043466 [Patent Document 4] Japanese Patent Application Publication No. 11-123098 [Patent Document 5] Japanese Patent Publication No. 2001-288156 SUMMARY OF THE INVENTION PROBLEMS 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 obtained amide compound quality, and a step of removing the metal compound and the like is required after completion of the reaction, which leads to an increase in cost. On the other hand, when purifying a nitrile compound using an ion exchange resin, the increase in the number of steps leads to an increase in cost.

[0010] Further, in the method of Patent Document 5, an alkali compound is added to a raw material nitrile compound, which may increase the labor for adjusting the pH of the nitrile compound before the enzyme reaction, or affect the quality of the obtained amide compound.

[0011] Therefore, a main object of the present invention is to provide a method for more easily improving the productivity of an amide compound by adding an aldehyde compound. MEANS FOR SOLVING THE PROBLEM

[0012] As a result of intensive studies in view of the problems of the prior art, the inventors have found that in a reaction for producing an amide compound from a nitrile compound using a biocatalyst having nitrile hydratase activity, addition of an aldehyde compound improves the reaction rate for converting a nitrile compound to an amide compound, and also can suppress a decrease in the activity of nitrile hydratase, and have completed the present invention.

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

[10] . ​​​[1] Nitrile compounds in the presence of a biocatalyst having nitrile hydratase activity A method for producing an amide compound from, The reaction from the nitrile compound to the amide compound is carried out in the presence of an aldehyde compound. The nitrile compounds listed are acrylonitrile, acetonitrile, methacrylonitrile, and cyanonitrile. At least one selected from pyridine, glyconitrile, and alaninenitrile. The aforementioned method. [2] Nitrile compounds in the presence of a biocatalyst having nitrile hydratase activity. In the production of amide compounds from, the reaction that converts the nitrile compound into an amide compound. A method to improve speed, The step includes adding an aldehyde compound to the reaction solution containing the nitrile compound, The lyl compounds are acrylonitrile, acetonitrile, methacrylonitrile, and cyanopyridin. The method is at least one selected from nitrile, glycolonitrile, and alaninenitrile. Law. [3] Nitrile compounds in the presence of a biocatalyst having nitrile hydratase activity. In the production of amide compounds from the nitrile hydratase biocatalyst A method for suppressing the decrease in activity, The step includes adding an aldehyde compound to the reaction solution containing the nitrile compound, The lyl compounds are acrylonitrile, acetonitrile, methacrylonitrile, and cyanopyridin. The method is at least one selected from nitrile, glycolonitrile, and alaninenitrile. Law. [4] The molar ratio of the aldehyde compound to the cyanide compound in the nitrile compound The method described in any of [1] to [3], where the result is between 0.9 and 15. [5] Biocatalysts with nitrile hydratase activity include those belonging to the genus Rhodococcus and Pseudonoca. Nitrile hydratase derived from the genus Rudia, or cells containing the nitrile hydratase, The method according to any one of [1] to [5], wherein the microbial cells are or processed products thereof. [6] Aldehyde compounds include formaldehyde, acetaldehyde, and propionaldehyde. Hydrate, butyraldehyde, isobutyraldehyde, dialdehyde oxalate, malondial Dehyde, pentanal, isovaleraldehyde, acrolein, crotonaldehyde, thi Glyceraldehyde, Glyceraldehyde, Glycolaldehyde, Furfural, Pig Ndial, trans-2-hexenal, glutaraldehyde, hexanal, hep Tanal, Octanal, Nonanal, Decanal, Paraaldehyde, Benzaldehyde , cinnamaldehyde, perillaldehyde, vanillin, 1-naphthaldehyde, phthalamine Ludehyde, methional, (Z)-7-hexadecenal, glyoxal (oxalate di Aldehydes), paraformaldehyde, acetaldehyde, ammonia, and hexamethylene The method according to any one of [1] to [4], wherein at least one is selected from tetramine. . [7] Aldehyde compounds and, Acrylonitrile, acetonitrile, methacrylonitrile, cyanopyridine, glyconitrile A nitrile compound comprising at least one nitrile compound selected from tolyl and lactonitrile, Composition for the manufacture of compound. [8] A biocatalyst comprising an aldehyde compound and a nitrile hydratase activity, A catalyst composition for the production of chemical compounds. [9] Amid compounds containing an aldehyde compound and an amide compound containing a cyanide compound A chemical composition, wherein the cyanide compound in the amide compound before the aldehyde compound is mixed in. An amide compound composition having a concentration of 0.2 ppm or higher.

[10] Includes amide compounds and compounds in which cyanide compounds and aldehyde compounds are bonded. A composition of amide compounds. [Effects of the Invention]

[0014] According to the present invention, by adding an aldehyde compound to a reaction solution containing a nitrile compound... Furthermore, amidation of nitrile compounds using a biocatalyst with nitrile hydratase activity The reaction rate of the reaction that converts into a substance can be increased. Furthermore, according to the present invention, Nitori This invention can suppress the decrease in activity of biocatalysts that possess hydratase activity. According to Akira, amide compounds can be produced efficiently. [Modes for carrying out the invention]

[0015] Embodiments of the invention will be described below. (1) Biocatalysts having nitrile hydratase activity In this invention, nitrile hydratase is defined as an enzyme that hydrolyzes nitrile compounds to produce the corresponding enzyme. This refers to an enzyme that has the ability to produce amide compounds. The somatic catalyst can be the nitrile hydratase protein itself, but the nitrile hydratase can be Animal cells, plant cells, organelles, or microbial cells, or processed products thereof, may also be included. stomach.

[0016] The aforementioned processed material may be animal cells, plant cells, organelles, or microbial cells that have been crushed. Enzymes extracted from crushed material or bacterial cells (raw enzymes or purified enzymes); animal cells, plant cells, cells Examples include organelles, microbial cells, or enzymes themselves immobilized on a carrier; and so on.

[0017] Furthermore, the treated material includes animal cells, plant cells, and other cells whose ability to proliferate has been lost through chemical treatment. This includes organelles or even the cells of microorganisms.

[0018] Immobilization methods include the inclusion method, crosslinking method, and carrier bonding method. The inclusion method is a high This method involves coating with a molecular film. The cross-linking method involves using two or more functional groups of an enzyme. This method involves crosslinking with a reagent (multifunctional crosslinking agent). The carrier binding method involves a water-insoluble carrier. This is a method of binding an enzyme to it.

[0019] Examples of materials used for immobilization (immobilization carriers) include gas beads, silica gel, and poly Urethane, polyacrylamide, polyvinyl alcohol, colored ginate, alginic acid, cold Examples include sugars and gelatin.

[0020] A typical example of such a microorganism is, for instance, rhodococ, which possesses nitrile hydratase activity. Rhodococus, Gordona, Pseudomonas Genus seudomonas, Genus Pseudonocardia Geobacillus genus, Bacillus genus, Bacillus Bacteridium genus, Micrococcus s) genus, Brevibacterium genus, Corynebacterium (Corynebacterium) genus, Nocardia genus, Micro Bacterium (Microbacterium) genus, Fusarium (Fusarium) genus Agrobacterium genus, Acinetobacter (Acin etobacter genus, Xanthobacter genus, Strep Streptomyces genus, Rhizobium genus, Klebsiella genus, Enterobacter ) genus, Erwinia genus, Pantoea genus, Candela Candida genus, Aeromonas genus, Citrobacter ( Includes genera such as Citrobacter and Achromobacter. The microorganisms belonging to this group are listed below.

[0021] For more details, see Japanese Patent Publication No. 56-17918, Nocardia sp.N-775 , as described in Japanese Patent Publication No. 06-55148, Rhodococcus rhodoklaus (Rhodoco ccus rhodochrous) J-1, International Open Pamphlet WO2005 / 05 Rhodococcus rhodoclous NCIMB41164 strain, described in Patent No. 4456, Japanese Patent Publication No. 0 Klebsiella sp.MCI2609 as described in Japanese Patent Publication No. 5-30982, Japanese Patent Application Publication No. 05-309 Aeromonas sp.MCI2614 as described in Patent Publication No. 83, Japanese Patent Publication No. 05-30984 Citrobacter freundii M CI2615, Agrobacterium rhisogene described in Japanese Patent Publication No. 05-103681 (Agrobacterium rhizogenes) IAM13570 and Agro Bacterium tumefaciens (Agrobacterium faciens) Xanthobacter flava (Xanthob) as described in Japanese Patent Publication No. 05-161495 actor flavas) JCM1204, Erwinia nigrifluens (Erw inia nigrifluens)MAFF03-01435, JP-A-05-2369 Enterobacter sp. MCI2707 as described in Patent Publication No. 75, Japanese Patent Application Publication No. 05-236976 Streptomyces sp.MCI2691 as described in the publication, Japanese Patent Publication No. 05-236977 Rhizobium sp.MCI2610, Rhizobium sp.MCI2643, and Rhizobium as described in the publication. Rhizobium loti IAM13588, Rhizobium regu Rhizobium legminosarum IAM12609 and Rhizobium merioti IAM12611, special Public Gazette No. 05-15384 describes Candida Guillemondi guilliermondii)NH-2, Pantoea agglomerans (Panto ea agglomerans)NH-3 and Klebsiella neomoniae subspirata Klebsiella pneumoniae NH-26T 2. Agrobacterium radiobacter (Ag) described in Japanese Patent Publication No. 06-14786 robacterium radiobacter)SC-C15-1, JP-A-07-2 Bacillus smithii SC as described in Publication No. 5494 -J05-1, Pseudonocardia thermoph described in Japanese Patent Publication No. 08-56684 Pseudonocardia thermophila ATCC19285, Japanese Patent Publication No. 09-275978 describes Pseudonocardia thermophila (Pse Examples include *Udonocardia thermophila* (JCM3095). Cut.

[0022] The Rhodococcus rhodocrous J-1 strain described in Japanese Patent Publication No. 06-55148 is, Under the accession number "FERM BP-1478", manufactured by an independent administrative agency on September 18, 1987. National Institute of Technology and Evaluation Patent Organism Deposit Center (1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture, Central No. 6 (hereinafter) (as used herein)) is deposited with the same institution.

[0023] Rhodococcus rhodo The Krows NCIMB41164 strain was introduced on March 5, 2003, at National Coll. ection of Industrial,Food and Marine Bac teria,Ltd.(NCIMB)(NCIMB Ltd Ferguson Bui lding Craibstone Estate Buksburn Aberdee It is deposited with NCIMB41164 (n AB21 9YA).

[0024] Pseudonocardia thermophylla as described in Japanese Patent Publication No. 09-275978 Pseudonocardia thermophila (JCM3095) is the recipient number It was registered as "FERM BP-5785" on February 7, 1996, by the Japan Institute of Technology for Product Evaluation (JITES). It has been deposited at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary Center (1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture, Central No. 6). Yes, they are.

[0025] In the present invention, one microorganism selected from the above is used alone or in combination of two or more types. It can be used in this way.

[0026] Furthermore, the gene encoding nitrile hydratase can be obtained by conventional molecular biological methods. It can be introduced and expressed within microbial cells (see below for details on these molecular techniques). :Sambrook, Fritscj and Maniatis, “Molercu lar Cloning:A Laboratory Manual”2nd Editi on(1989),Cold Spring Harbor Laboratory P (ress). In other words, in the present invention, natural nitrile hydratase (wild type) or Using the enzyme obtained by expressing the nucleic acid encoding the mutant (improved) in microbial cells It is also possible to do so. In the present invention, one enzyme selected from the above enzymes may be used alone or two or more. The above can be used in combination.

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

[0028] For example, α-subu derived from Rhodococcus rhodoklaus J1 (FERM BP-1478) The accession number for the knit is "P21219", and the accession of the β subunit The serial number is "P21220". Also, Rhodococcus rhodoklaus M8 (SU173 The accession number of the α subunit derived from 1814 is "ATT79340". The accession number for the β subunit is "AAT79339". Furthermore, the shoe Pseudomonas thermophila (JCM3) The accession number of the α subunit derived from 095 is "1IRE A", and the β subunit The accessory number for the knit is "1IREB".

[0029] As a transformant into which the wild-type nitrile hydratase gene has been introduced, Achromobac Escherichia coli M transformed with nitrile hydratase from the genus Achromobacter T10770 (FERM P-14756) (Japanese Patent Publication No. Hei 8-266277), Shoe Transformation with nitrile hydratase from the genus Pseudonocardia E. coli MT10822 (FERM BP-5785) (Japanese Patent Publication No. 9-275978) (Report), or Rhodococcus rhodochro Microorganisms transformed with nitrile hydratase of the US species (Japanese Patent Publication No. 4-211379) Examples of objects are given, but the examples are not limited to these.

[0030] A modified (mutant) nitrile hydratase created by amino acid substitutions in wild-type nitrile hydratase. Tarze is known (Japanese Patent Publication No. 2010-172295, Japanese Patent Publication No. 2007-14340) Japanese Patent Publication No. 9, Japanese Patent Publication No. 2007-043910, Japanese Patent Publication No. 2008-253182, Japanese Patent Publication No. 2019-088326, Japanese Patent Publication No. 2019-088327, WO05 / 11 Pamphlet No. 6206, Pamphlet No. WO12 / 164933, WO12 / 1692 (Pamphlet No. 03, Pamphlet WO15 / 186298, etc.) Furthermore, it is also possible to use microorganisms into which these improved nitrile hydratases have been introduced. ru.

[0031] These microorganisms possessing nitrile hydratase activity, or their processed products, immediately after cell preparation. It can be used in amide synthesis reactions, of course, but it can also be stored after cell preparation and used in amide synthesis reactions as needed. It can also be used for this purpose. The method for culturing microorganisms to prepare bacterial cells depends on the type of microorganism. This can be selected as appropriate. Seed culture may be performed before the main culture.

[0032] Microbial cells or processed products thereof possessing nitrile hydratase activity are used in batch reactions. It can also be used in continuous reactions. Furthermore, the reaction format can be a fluidized bed, a fixed bed, or... An appropriate format can be selected, such as a suspension bed. The catalyst temperature in the reaction solution in this case is determined by the aqueous medium. The type of nitrile compound is not particularly limited, as long as it does not interfere with the mixing of the nitrile compounds.

[0033] (2) Nitrile compounds The nitrile compound used as a raw material in the manufacturing method of the present invention is nitrile hydra. Any compound that can be converted to an amide compound by a catalyst having tase activity is not particularly limited. For example, acetonitrile, propionitrile, succinonitrile, adiponitrile, Aliphatic saturated nitriles such as ricolonitrile and lactonitrile, acrylonitrile, meta Aliphatic unsaturated nitriles such as crironitrile, benzonitrile, and phthalodinitrile Examples include aromatic nitriles and heterocyclic nitriles such as cyanopyridine. The nitrile compound in this is preferably acetonitrile, propionitrile, or acryloni Tolyl, methacrylonitrile, n-butyronitrile, isobutyronitrile, cyanopyridin Nitrile compounds such as nitrile, glucolonitrile, and lactonitrile are particularly preferred. Acrylonitrile, methacrylonitrile, acetonitrile, cyanopyridine, glycol These are nitriles and lactonitriles.

[0034] Nitrile compounds generally undergo a purification process before becoming commercially available products. For example, acrylonite Lyl is produced industrially by the ammoxidation of propylene, and cyanide compounds such as hydrogen cyanide are produced. The substance, along with other by-products, is removed by distillation and purification after the reaction.

[0035] However, this process does not completely remove all cyanide compounds from the product. The cyanide compounds contained in the nitrile compounds have nitrile hydratase activity. It is thought that damage to the somatic catalyst leads to decreased activity and a reduction in reaction rate.

[0036] In this invention, an aldehyde compound is added to a reaction solution containing a nitrile compound (to allow it to be present). By doing so, the amount of cyanide compounds contained in the nitrile compound is reduced, and the amount of cyanide compounds This prevents damage to biocatalysts that have nitrile hydratase activity. In other words, nitrile compounds can be converted to amides in the presence of a biocatalyst having nitrile hydratase activity. The reaction rate for conversion to compounds is improved. Also, contact with nitrile compounds improves the nitrile reaction. It is thought that this can suppress the decrease in activity of biocatalysts that possess hydratase activity. .

[0037] (3) Production of amide compounds In this invention, a biocatalyst having nitrile hydratase activity is used to convert nitrile compounds from To produce amide compounds.

[0038] The type of amide compound produced in this invention is not particularly limited, and any amide compound can be used depending on the application. It is possible to manufacture a product. The raw material is a nitrile compound corresponding to the amide compound. You can use it.

[0039] Examples of amide compounds include acrylamide, nicotinamide, and methacrylamide. It can be used. Preferably, it is acrylamide.

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

[0041] A method for producing amide compounds using a biocatalyst having nitrile hydratase activity is particularly... The method is not limited to this, and may, for example, be carried out by a series of reactions that continuously produce amide compounds. Alternatively, this may be carried out by a batch reaction that generates amide compounds discontinuously.

[0042] When carrying out a continuous reaction, the reaction materials, including the biocatalyst, water, and nitrile compound, are supplied to the reactor. Continuous or intermittent introduction and continuous removal of the reaction mixture containing the generated amide compound from the reactor. Alternatively, the reaction mixture can be continuously removed from the reactor without completely removing the reaction mixture, while performing intermittent removals. Amide compounds can be manufactured in a specific manner.

[0043] When performing a batch reaction, the entire amount of reaction materials is charged into the reactor at once before the reaction begins. After adding some of the reaction materials to the reactor, the remaining reaction materials are added continuously or intermittently. Amide compounds can be produced by supplying and reacting them.

[0044] Reactor types include stirred tank type, fixed bed type, fluidized bed type, moving bed type, tubular type, or tower type, etc. Various types of reactors can be used. One reactor may be used, or multiple reactors may be used together. It may be used. When multiple reactors are used, the reaction mixture extracted from the downstream reactors is greater. The concentration of amide compounds in the material increases. Therefore, the final amount obtained depends on the number of reactors. The concentration of the mid compound can be adjusted.

[0045] When carrying out a reaction continuously using multiple reactors, a bio-active nitrile hydratase is used. Reactors that introduce somatic catalysts and nitrile compounds should be used within a range that does not excessively degrade the reaction efficiency, etc. If available, it would not be limited to being introduced only into the reactor located furthest upstream, but also downstream. It can also be introduced into reactors.

[0046] Among the reaction raw materials, the water undergoes a hydration reaction with the nitrile compound when producing the amide compound. It is used for this purpose.

[0047] Examples of raw material water include water, or aqueous solutions of acids or salts dissolved in water. Examples of acids include phosphoric acid, acetic acid, citric acid, and boric acid. Examples of salts include the above acids. Examples include sodium salts, potassium salts, and ammonium salts. The type of raw water is also not limited. However, for example, pure water, tap water, Tris buffer, phosphate buffer, acetate buffer, citrate buffer Examples include borate buffer solutions. The pH of the raw water used (at 25°C) is also important for the biocatalyst to work efficiently. The reaction rate is not particularly limited as long as it is sufficient. For example, it can be 4 to 10, preferably 5 to 9. Yes, it is possible. By raising the pH to 4 or higher, the enzyme activity of the biocatalyst can be sufficiently increased. By lowering the pH to 10 or below, the deactivation of biocatalysts can be suppressed.

[0048] The amount of biocatalyst used can be appropriately selected depending on the type of biocatalyst used, the reaction conditions, etc. Yes, it is possible. For example, the activity of the biocatalyst introduced into the reactor is such that at a reaction temperature of 10°C, the dry cell count is 1 m It is preferable to adjust the amount to approximately 50 to 500 U per gram. (T) means producing 1 micromol of amide compound from a nitrile compound per minute. The taste is measured using the nitrile compounds used in the manufacturing process.

[0049] The reaction raw materials used in the hydration reaction of acrylonitrile described above, or during the hydration reaction To aid in stabilizing the reaction mixture after the reaction, a water-soluble monocarbon with 2 or more carbon atoms is added. At least one type of salt acid may be added. The water-soluble monocarboxylate is saturated monocarb Either a carboxylate or an unsaturated monocarboxylate salt may be used. Examples of saturated carboxylic acids include acetic acid and plutonium. Examples include lopionic acid and n-caproic acid. Unsaturated carboxylic acids include acrylic acid. Examples include methacrylic acid and vinylacetic acid. Salts include sodium salts and potassium salts. Ammonium salts are typical. The amount of the water-soluble monocarboxylate added is the final amount obtained In the reaction mixture (aqueous solution of acrylamide), add 20 to 5000 mg of acid relative to the acrylamide. A quantity of kg is preferable.

[0050] The amount of nitrile compound used depends on the type of biocatalyst used, the reaction conditions, the scale of the reaction, and whether it is a continuous reaction. The appropriate method can be selected depending on whether it is a batch reaction or not.

[0051] The reaction temperature is not particularly limited as long as the biocatalyst efficiently promotes the reaction. For example, 5 to 50°C, preferably 10 to 40°C, and more preferably 15 to 35°C. This can be achieved. By raising the reaction temperature to 10°C or higher, the reaction activity of the biocatalyst can be sufficiently increased. This can be achieved by keeping the reaction temperature below 50°C, thereby suppressing the deactivation of the biocatalyst. It is possible.

[0052] The reaction time is not particularly limited and can be any reaction format, such as batch reaction, post-reaction reaction, or continuous reaction. The duration can be appropriately selected depending on the reaction scale and other factors. For example, 0.1 to 60 hours, preferably. This can be 1 to 50 hours, more preferably 2 to 40 hours.

[0053] When producing amide compounds by a continuous reaction, when removing the reaction mixture from the reactor... The fluid velocity is set so that the reaction mixture in the reactor can be produced continuously without having to remove the entire contents. The rate of introduction of the nitrile compound and biocatalyst should be determined accordingly.

[0054] Furthermore, to stabilize the reaction, additives are added to the nitrile compound or the reaction solution. It is also possible.

[0055] The concentration of the amide compound in the aqueous solution of the amide compound obtained in the present invention is the concentration of the amide compound obtained. The amide compound can be appropriately selected depending on the intended use of the compound. For example, the concentration of the amide compound. This is added in an amount of 25 to 65% by mass, preferably, relative to the total mass of the aqueous solution of the resulting amide compound. The amount can be 30-60% by mass, more preferably 35-55% by mass. Amide compound By keeping the concentration below 65% by mass, the precipitation of amide compound crystals at room temperature is prevented. This can be done. Also, by setting the concentration of the amide compound to 25% by mass or more, storage and This can reduce the volume of tanks used for storage and lower transportation costs. Cut.

[0056] The amide compounds obtained by the present invention can be used directly in polymerization reactions, It can also be stored until use. Furthermore, aldehyde compounds can be removed as needed. It is also possible to store amide compounds, and polymerization inhibitors and polymerization initiators may be added as needed. It can be used with various additives added as needed.

[0057] (4) Aldehyde compounds In this invention, in the presence of a biocatalyst having nitrile hydratase activity, a nitrile compound In a method for producing an amide compound from a nitrile compound, the reaction of the nitrile compound to the amide compound The reaction 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 are obtained. No. For example, formaldehyde, acetaldehyde, propionaldehyde, buty Isobutyraldehyde, malondialdehyde, pentanal, isovale Acrolein, crotonaldehyde, tigrine aldehyde, glyceryl aldehyde Glycolaldehyde, furfural, butanedial, trans-2- Xenal, glutaraldehyde, hexanal, heptanal, octanal, nonana Decanal, paraaldehyde, benzaldehyde, cinnamaldehyde, perillium Ludehyde, vanillin, 1-naphthaldehyde, phthalaldehyde, methional, (Z) Examples include -7-Hexadecenal.

[0059] Aldehyde compounds include not only compounds having an aldehyde group (-CHO), but also compounds that can be found in water or This also includes compounds that produce aldehyde compounds in solution. Examples include glyoxal (dialdehyde oxalate), paraformaldehyde, and acetone. Examples include aldehyde ammonia and hexamethylenetetramine.

[0060] Among these, aldehyde compounds with a molecular weight of about 200 or less are preferred, and the molecular weight is Aldehyde compounds with a concentration of approximately 100 or less are more preferable.

[0061] Particularly preferred are formaldehyde, acetaldehyde, and propyl alcohol. Examples include ionaldehyde and butyraldehyde. In this specification, "adding" to the reaction solution means "to be present" in the reaction solution. This shall include the following.

[0062] In the present invention, the amount of aldehyde compound added to the reaction solution containing the nitrile compound is: Nitrile compounds are converted to amide compounds in the presence of a biocatalyst with nitrile hydratase activity. The reaction rate to convert to can be increased, or when in contact with a nitrile compound. If the decrease in activity of biocatalysts having nitrile hydratase activity can be suppressed, It is not particularly limited.

[0063] The amount of aldehyde compound added to the reaction solution is determined in relation to the amount of cyanide compound in the reaction solution. The molar ratio is 0.9 to 15, preferably 1.5 to 10.0, more preferably 2.0 to 6. It can be set to .0.

[0064] The cyanide compound content in acrylonitrile is determined by extraction with an alkaline solution followed by the use of silver nitrate. It can be determined by titration, or measured by spectrophotometric method.

[0065] The amount of aldehyde present in the reaction solution should be 0 in molar ratio to the cyanide compound content. By setting it to 9 or higher, nitrile hydratase activity is maintained in the presence of a biocatalyst having nitrile hydratase activity. This can increase the reaction rate for converting a compound to an amide compound, or Decreased activity of biocatalysts with nitrile hydratase activity upon contact with tolyl compounds. It can be suppressed.

[0066] On the other hand, the amount of aldehyde present in the reaction solution is determined by the molar content relative to the cyanide compound content. The reason for keeping the ratio below 15 is that adding more aldehyde compounds will not improve the effect. Because it's difficult to deal with.

[0067] In this specification, cyanide compounds include hydrogen cyanide (HCN); cyanide ion (C N - ); containing cyanide compounds such as sodium cyanide and potassium cyanide or reaction conditions The substance shall release cyanide or cyanide ions.

[0068] The timing for adding the aldehyde compound to the reaction solution containing the nitrile compound is as follows: The following are not particularly limited as long as they can exert the effects described above, and have nitrile hydratase activity. Before contacting the biocatalyst with the nitrile compound, or at the time of contact (simultaneously) or after contact It may also be done later (i.e., after the enzymatic reaction of the nitrile compound by the biocatalyst has started).

[0069] Preferably, the aldehyde compound is a biocatalyst having nitrile hydratase activity. Add before contact with the lyl compound. If adding after the reaction has started, start the reaction. Add the aldehyde compound as soon as possible (the sooner the better). It is believed that the more you add, the greater the above effects will be obtained.

[0070] One method for adding an aldehyde compound to the reaction solution is to add an aldehyde compound of the desired concentration to the reaction solution. The process is not particularly limited as long as a aldehyde compound is present. For example, adding a solid aldehyde compound. It is also possible to add the aldehyde compound in a solution of the solvent used in the reaction or in water, etc. It can also be added.

[0071] (5) Improving the reaction rate of converting nitrile compounds to amide compounds This invention relates to nitrification in the presence of a biocatalyst having nitrile hydratase activity. In the production of an amide compound from a compound, an aldehyde is added to the reaction solution containing the nitrile compound. A method for converting nitrile compounds to amide compounds, characterized by including a step of adding a compound. The present invention provides a method for improving the reaction rate of an enzymatic reaction. The nitrile compound is a Crilonitrile, acetonitrile, methacrylonitrile, cyanopyridine, glyconitrile At least one selected from lyl and alanine nitrile can be used.

[0072] It has been reported that aldehydes are used to prevent polymerization (stabilize) acrylamide, (WO2011 / 102510), Aldehyde compounds possess nitrile hydratase activity. Preventing damage to the biocatalyst and improving the reaction rate is a first in this invention. It was discovered.

[0073] In this invention, "improving the reaction rate" means in the case where no aldehyde compound is present. This means that the reaction rate is higher compared to [another method].

[0074] The reaction rate is measured by the decrease in the amount of nitrile compound present in the reaction system per unit time. Methods for measuring the amount of compound added or methods for measuring the amount of amide compound added to the reaction system include measuring the amount of compound added or the amount of compound added to the reaction system. Gas chromatography is a method for measuring nitrile compounds or amide compounds. Known methods such as those mentioned above can be used.

[0075] (6) Suppression of activity reduction of biocatalysts having nitrile hydratase activity In the presence of a biocatalyst having nitrile hydratase activity, nitrile compounds are converted to In the production of a mid compound, an aldehyde compound is added to the reaction solution containing the nitrile compound. The biocatalyst having nitrile hydratase activity, characterized by including a step of adding The present invention provides a method for suppressing the decrease in activity. The nitrile compound is acrylonitrile. Acetonitrile, methacrylonitrile, cyanopyridine, glyconitrile, and alanine At least one type selected from nitriles can be used.

[0076] As mentioned above, the activity of nitrile hydratase is affected by impurities in the reaction mixture and reaction conditions. It is easily accepted. According to the method of the present invention, no additional steps are required, and the quality of the amide compound is not affected. This method allows for the simple suppression of the decrease in nitrile hydratase activity without causing any adverse effects.

[0077] (7) Compositions for the production of amide compounds This invention relates to aldehyde compounds and acrylonitrile, acetonitrile, methacrylonite At least one selected from lyl, cyanopyridine, glyconitrile and lactonitrile We also provide compositions for producing amide compounds, which include various nitrile compounds.

[0078] In the amide compound production composition of the present invention, the aldehyde compound is "(4) aldehyde The compounds listed in "Compounds" can be used in accordance with the description in that section. The substance is used in the method for producing amide compounds described in (3), and efficiently amidates It enables the manufacture of goods.

[0079] (8) Catalyst composition for the production of amide compounds The present invention includes an aldehyde compound and a biocatalyst having nitrile hydratase activity. We also provide catalyst compositions for the production of amide compounds.

[0080] In the catalyst composition for producing amide compounds of the present invention, aldehyde compounds and nitrile hydrides Biocatalysts having latex activity are "(4) aldehyde compounds" and "(1) d As described in "Biocatalysts possessing trillhydratase activity".

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

[0082] In the first embodiment, the amide compound composition is an amide compound and an aldehyde compound The substance contains a particular compound, and the concentration of the cyanide compound in the amide compound is 0.2 ppm or higher. The characteristic is that the cyanide compound and the amide compound may be separate compounds or be combined. This is also acceptable. The concentration of the cyanide compound should be the same as the concentration before the addition of the aldehyde compound.

[0083] In the second embodiment, the amide compound composition comprises an amide compound and a cyanide compound It includes compounds to which aldehyde compounds are bonded. [Examples]

[0084] The present invention will be described in more detail below using examples, but the present invention is not limited to these examples. It is not something that should be done. In this specification, "%" refers to "mass%".

[0085] [Example 1] (Preparation of bacterial cells) Rhodococcus rhodochrous J-1 (FERM) BP-1478) Rhodococcus rhodoclous J-1 strain possessing nitrile hydratase activity [Rhod [ococcus rhodochrous J-1 (FERMBP-1478)] Cosine 2.0%, urea 1.0%, peptone 0.5%, yeast extract 0.3%, and cobalt chloride The cells were cultured aerobically at 30°C using a medium containing 0.05% of the active ingredient (pH 7.0). This mixture was then incubated for 50 minutes. The cells were washed with M phosphate buffer (pH 7.0) to obtain a bacterial cell suspension (3% in terms of dry cell content).

[0086] (Preparation of acrylonitrile with a specified hydrogen cyanide concentration) Industrial acrylonitrile (manufactured by Mitsubishi Chemical Corporation) with a hydrogen cyanide concentration of 2.1 ppm by weight. To achieve this, a 1000 ppm cyanide standard solution (manufactured by Hayashi Pure Chemical Industries) was added.

[0087] (Method for measuring cyanide concentration) Add 9.6g of pure water and 0.4g of acrylonitrile (manufactured by Mitsubishi Chemical Corporation) to a test tube and refrigerate for 25 minutes. The mixture was left to stand at °C for 30 minutes. Then, CyaniVer.3 from the HACH analysis kit was added. After vortexing for 30 seconds, let it stand for 30 seconds. Add CyaniVer.4 and 10 I vortexed for a second, then added CyaniVer.5 and vortexed for two minutes.

[0088] After standing at 25°C for 30 minutes, use a spectrophotometer (DR500001) (HACH Corporation) to measure the absorbance. The absorbance was measured using a sample. As a result, the hydrogen cyanide concentration was found to be 2.1 ppm.

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

[0090] (Amide formation reaction) In a glass case with a lid and an internal volume of 13.5 ml, add 3.15 g of pH 7.0 phosphate buffer, as described above. The bacterial cells were added to the reaction solution and diluted with pH 7.0 phosphate buffer to achieve an activity of 1460 U. 3.15 g of the diluted bacterial solution was added and stirred while maintaining a temperature of 20°C. Then the aforementioned 10 ppm 4.8 mL of acrylonitrile containing acetaldehyde was added to start the reaction. After 4 hours... The reaction solution is collected and subjected to gas chromatography (column: PoraPack-PS (Wat (Manufactured by ers, 1m, 210℃, Carrier gas: Helium, Detector: FID) Acrylic The concentration of nitrile was measured.

[0091] [Example 2] Except for the acetaldehyde concentration in acrylonitrile being 6.0 ppm in Example 1, the other difference is that the concentration of acetaldehyde in acrylonitrile in Example 1 was set to 6.0 ppm. The procedure was carried out in the same manner as in Example 1.

[0092] [Example 3] Except for the acetaldehyde concentration in acrylonitrile being 3.0 ppm in Example 1, the rest of the code is the same. The procedure was carried out in the same manner as in Example 1.

[0093] [Example 4] Except for setting the hydrogen cyanide concentration in acrylonitrile to 1.1 ppm in Example 1, the results are the same as in Example 1. The same procedure was followed.

[0094] [Comparative Example 1] The experiment was conducted in the same manner as in Example 1, except that acetaldehyde was not added to the acrylonitrile. They did that.

[0095] [Comparative Example 2] The hydrogen cyanide concentration in acrylonitrile is 1.1 ppm, and acetaldehyde is added to acrylonitrile. The procedure was the same as in Example 1, except that the additive was omitted.

[0096] Table 1 shows the results after 4 hours from the start of the reaction, compared to the case without the addition of the aldehyde compound. The concentration ratio of acrylonitrile is shown. This concentration ratio can be calculated using the following formula. Concentration ratio [%] = (Acrylonitrile concentration when aldehyde is added / amount of aldehyde added) (Acrylonitrile concentration without addition) × 100 Furthermore, compounds that produce aldehyde compounds in water or solution are generated in water or solution. The aldehyde / cyanide ratio was calculated based on the number of moles of aldehyde compounds.

[0097] [Table 1]

[0098] In the example where acetaldehyde was added, compared to the comparative example where acetaldehyde was not added... And, whether the hydrogen cyanide concentration is 2.1 ppm or 1.1 ppm, the concentration of acrylonitrile is It was confirmed that the reaction rate for conversion to amide compounds was low and high.

[0099] [Test Example 1] The effect of the following compounds on the amide compound formation reaction was evaluated according to Example 1. No.1: Formaldehyde (30.03), No.2: Acetaldehyde (44.05), No.3: Propylene Glycol No. 4: Butyraldehyde (58.08), No. 5: Hexanal (10 0.16), No.6: Heptanal (114.18), No.7: Octanal (128.21), No.8: Nonana No. 9: Decanal (142.24), No. 10: Formic acid (46.03), No. 11: Shu Acid dialdehyde (58.04), No. 12: meso-erythritol (122.12), No. 13: paraaldehyde Hydrate (132.16), No. 14: Paraformaldehyde (30.03 × n), No. 15: Benzaldehyde (106.12), No.16: Cinnamaldehyde (132.16), No.17: Perillaldehyde (150.22) ), No.18: Vanillin (152.15), No.19: Acetaldehyde Ammonia (183.25), No.2 0: Hexamethylenetetramine (140.19) The values ​​in parentheses are molecular weight (g / mol). No. 11, 14, 19, 20: Forms aldehyde compounds in water.

[0100] Acrylonitrile was prepared to have a hydrogen cyanide concentration of 2.0 ppm.

[0101] The aforementioned bacterial cells were placed in a lidded glass case with an internal volume of 13.5 ml, and the activity level was 184. Add 1.00 g of diluted bacterial solution, diluted with pH 7.0 phosphate buffer to a concentration of 0 U, and each aluminum Add the dehyde compound to the reaction solution at a pH of 7.0 phosphate buffer to achieve a concentration of 5 ppm or 10 ppm. Diluted solution was added, and finally pH 7.0 phosphate buffer was added to bring the total volume to 6.2. The amount was set to g. The resulting solution was stirred while maintaining a controlled temperature of 25°C. Acrylonitrile 4 was added to it. 8 mL (equivalent to 50% acrylamide, 38% by weight) was added, and the reaction was started. 3.5 After 4.5, 5.0, and 6.0 hours, the reaction solution was collected and subjected to gas chromatography (color). Material: PoraPack-PS (Waters), 1m, 210℃, Carrier gas: H The concentration of acrylonitrile was measured using a luminescent detector (FID).

[0102] Table 2 shows the results at 3.5 hours and 6 hours after the start of the reaction, without the addition of the aldehyde compound. The concentration ratio of acrylonitrile compared to the case without it is shown. This concentration ratio can be calculated using the following formula. . Concentration ratio [%] = (Acrylonitrile concentration when aldehyde is added / amount of aldehyde added) (Acrylonitrile concentration without addition) × 100 Furthermore, compounds that produce aldehyde compounds in water or solution are generated in water or solution. The aldehyde / cyanide ratio was calculated based on the number of moles of aldehyde compounds.

[0103] [Table 2]

[0104] For aldehyde compounds other than formic acid, meso-erythritol, and para-aldehydes The addition of aldehyde compounds, compared to the case without addition, the concentration of acrylonitrile It was confirmed that the reaction rate for converting nitrile compounds to amide compounds is improved, as the reaction rate is lower. It was done.

[0105] [Example 5] (Preparation of transformants containing nitrile hydratase derived from Rhodococcus rhodoclous M8 strain) ) (1) Chromosomal DNA preparation from Rhodococcus rhodoclous M8 strain (hereinafter referred to as M8 strain) Made The M8 strain (SU1731814) can be obtained from the Russian Strain Center IBFM (VKPM S-926). ru.

[0106] M8 strain in 100 mL of MYK (0.5% polypeptone, 0.3% bactoyst extract, 0.3% bactoyst extract) Tomoruto extract, 0.2% K2HPO4, 0.2% KH2PO4) medium (pH 7.0), incubated with shaking at 30°C for 72 hours. The culture medium was centrifuged, and the collected cells were placed in a Saline-EDTA solution (0.1M EDTA, 0.15M NaCl). The suspension was resuspended in 4 mL of (pH 8.0). 8 mg of lysozyme was added to the suspension and shaken at 37°C for 1-2 hours, It froze at -20°C.

[0107] Next, add 10 mL of Tris-SDS solution (1% SDS, 0.1 M NaCl, 0.1 M Tris-HCl (pH 9.0)) to the suspension. The following was added while gently shaking. Furthermore, proteinase K (Merck) was added to the suspension. Add the final concentration (0.1 mg) and shake at 37°C for 1 hour. Next, add an equal volume of TE-saturated phenol and stir. After mixing (TE: 10mM Tris-HCl, 1mM EDTA (pH 8.0)), the mixture was centrifuged. The upper layer was collected and twice the amount of ethanol was used. After adding the enzyme, the DNA was wrapped around a glass rod. Then, this was sequentially divided into 90%, 80%, and 70% enzymes. The phenol was removed by centrifugation with tanol.

[0108] Next, the DNA is dissolved in 3 mL of TE buffer and then incubated with ribonuclease A solution (100°C for 15 minutes). (Heat-treated) was added to a total concentration of 10 μg / mL and shaken at 37°C for 30 minutes. Furthermore, proteinase K (Merck) was added and shaken at 37°C for 30 minutes. An equal volume of TE-saturated phenol was then added. After centrifugation, the mixture was separated into upper and lower layers.

[0109] After adding an equal amount of TE-saturated phenol to the upper layer and centrifuging it, the upper and lower layers were separated. This procedure was repeated again. Then, the same amount of chloroform (4% isoamyl alcohol) was added to the upper layer. (containing ethanol) was added and the mixture was centrifuged, and the upper layer was collected. Then, twice the amount of ethanol was added to the upper layer. Furthermore, the DNA was wound up and recovered using a glass rod to obtain chromosomal DNA.

[0110] (2) Production of nitrile hydratase expression plasmid derived from strain M8 The nitrile hydratase gene derived from strain M8 (SEQ ID NO: 5) is described in Non-Patent Literature (Veiko, VP et al., Cloning,nucleotide sequence of nitrile hydratase gene from Rhodococcus rhodochr It is described in ous M8, Biotekhnologiia (Mosc.), 5, 3-5 (1995), and the β subunit The amino acid sequences of the α subunit and activator are listed in order in Sequence ID No. 6, sequence number 6. This is shown in sequence number 7 and sequence number 8. Based on this sequence information, the following primers (sequence number 8) 1) and 2) were synthesized, and the prepared M8 strain genomic DNA was used as a template for PCR under the following reaction conditions. .

[0111] Primer: M8-1: 5'-GGTCTAGAATGGATGGTATCCACGACACAGGC-3'(Sequence ID 1) M8-2: 5'-cccctgcaggtcagtcgatgatggccatcgattc-3'(Sequence ID 2)

[0112] Reaction solution 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: The reaction consists of 30 cycles of 98°C for 10 seconds, 55°C for 5 seconds, and 72°C for 30 seconds.

[0114] Next, the obtained plasmid DNA was cleaved with restriction enzymes XbaI and Sse8387I, and then 0.7% agarose sedge. Electrophoresis was performed using a nasal electrophoresis tool to recover a 1.6kb nitrile hydratase gene fragment (SEQ ID NO: 5). The plasmid was then introduced into the XbaI-Sse8387I site of plasmid pSJ042. The resulting plasmid was then used to create pSJ-N01 It was named A. Furthermore, pSJ042 is derived from the nitrile hydratase of the J1 strain in Rhodococcus. The plasmid to be expressed was prepared by the method shown in Japanese Patent Publication No. 2008-154552, p The plasmid pSJ023 used to create SJ042 was the transformant ATCC12674 / pSJ023(FERM BP-6232) ) as the Patent Organism Deposit Center of the National Institute of Advanced Industrial Science and Technology (Higashi 1, Tsukuba City, Ibaraki Prefecture) It was deposited at 1-1-1 Chuo 6 on March 4, 1997.

[0115] (3) Preparation of ATCC12674 transformants Logarithmic growth phase of Rhodococcus rhodochrous ATCC strain 12674 The bacterial cells are collected using a centrifuge, washed three times with ice-cold sterile water, suspended in sterile water, and then... Incompetent cells were fabricated.

[0116] Mix 1 μl of the plasmid DNA (pSJ-N01A) prepared above with 10 μl of ATCC12674 competent cells. Combined and chilled on ice for 30 minutes. The DNA and bacterial cell suspension was placed in a cuvette, and the gene transfer apparatus Gene P Electrical pulse treatment was performed using ulser (BIO RAD) at 20kV / cm and 200 OHMS. Let stand on ice for 10 minutes, then heat shock at 37°C for 10 minutes, and then in MYK medium (0.5% polypeptone, 0.3% Bactoyst extract, 0.3% Bactomalt extract, 0.2% K2HPO4, 0.2% KH2PO4) Add 500 μl and let stand at 30°C for 24 hours, then spread onto MYK agar medium containing 10 μg / ml kanamycin. The cells were incubated at 30°C for 3 days.

[0117] The plasmids of the obtained colonies were examined, and a transformant (ATCC12674 / pSJ-N01A) was obtained.

[0118] (4) Culture of recombinant bacteria The transformant (ATCC12674 / pSJ-N01A) obtained in the above process was placed in MYK medium (50 μg / ml Each was inoculated into kanamycin and cultured with shaking at 30°C for 2 days, then incubated in GGPK medium (1.5 % glucose, 1% monosodium glutamate, 0.1% yeast extract, 0.05% K2HP O4, 0.05%KH2PO4, 0.05%Mg2O4·7H2O, 1% CoCl2, 0.1 The cells were inoculated with 1% urea (50 μg / ml kanamycin, pH 7.2) and incubated at 30°C for 3 days. The cells were cultured with intermittent shaking and collected by centrifugation. Then, they were incubated in 100 mM phosphate buffer (pH 7.0). The bacterial cells were washed with ) and a bacterial cell suspension was prepared.

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

[0120] Acrylonitrile was prepared to have a hydrogen cyanide concentration of 2.0 ppm.

[0121] Into a glass case with a lid having an internal volume of 13.5 ml, the bacterial cells described above are charged such that the amount of the bacterial cells charged in the reaction solution is 5.6 mg, 1.00 g of a diluted bacterial solution obtained by dilution with a pH 7.0 phosphate buffer is added, and each aldehy de compound is adjusted to 10 ppm, 25 ppm or 50 ppm in the reaction solution by diluting with p H 7.0 phosphate buffer to obtain a diluted solution, which is then added, and finally pH 7.0 phosphate buffer is added to adjust the total amount to 9.0 g. The resulting solution was stirred while being controlled at 25°C. To this was added acry lonitrile (1.0 g) to initiate the reaction. After 6.0 hours, the reaction solution was collected, the concentration of acrylonitrile was measured by gas chromatography (column: PoraPack-PS (manufactured by 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 to the case where no aldehyde compound is added. This concentration ratio is obtained by the following formula. Concentration ratio [%]=(acrylonitrile concentration when aldehyde is added / acrylonitrile concentration when aldehyde is not adde d)×100 For compounds that generate an aldehyde compound in water or a solution, the aldehyde / cyanide ratio was calculated based on the number of moles of the aldehyde compound generated in water or the solution.

[0123]

Table 3

[0124] In the M8 strain-derived transformant (ATCC12674 / pSJ-N01A), addition of an aldehyde compound results in the addition ​​Compared to cases where this is not done, the concentration of acrylonitrile is lower, and nitrile compounds are converted to amid compounds. It was confirmed that the reaction rate for conversion to matter improved.

[0125] [Example 6] (1) Expresses nitrile hydratase derived from Pseudonocardia thermophylla JCM3095 strain Preparation of DN1 transformants Plasmid pPT-DB1 is derived from Pseudonocardia thermophila obtained in Japanese Patent Publication No. 9-275978. Plasmi containing the nitrile hydratase gene derived from strain JCM3095 (hereinafter referred to as strain JCM3095) It is a transformed strain (MT-10822 strain) introduced into E. coli HB101, and is recognized by the Japan Industrial Safety and Health Agency (JISA). It is deposited at the Technical Research Institute (1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture, Japan, Central No. 6).

[0126] The nitrile hydratase gene (SEQ ID NO: 9) of the JCM3095 strain is described in Japanese Patent Publication No. 9-275978. Therefore, the amino acid sequences of the β subunit, α subunit, and activator are listed in order. This is shown in sequence numbers 10, 11, and 12. Based on this sequence information, the following The primers (SEQ ID NOs: 3, 4) were synthesized, and PCR was performed using pPT-DB1 as a template under the following reaction conditions. The pRT-DB1 used as a template was prepared from the MT-10822 strain according to standard procedures.

[0127] Primer: PSN-1: 5'-GGTCTAGAATGAACGGCGTGTACGACGTCGGC-3'(Sequence ID 3) PSN-2: 5'-ccCCTGCAGGTCAGGACCGCACGGCCGGGTGGAC-3'(Sequence ID 4)

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

[0129] Temperature cycling: 30 cycles of reaction at 98°C for 10 seconds, 55°C for 5 seconds, and 72°C for 30 seconds

[0130] Using the obtained PCR product (SEQ ID NO: 10), a plasmid was prepared in the same manner as in Example 5(2), and named pSJ-N02A. The obtained plasmid was introduced into AT CC12674 by the same method as in Example 5(3), to obtain a transformant (ATCC12674 / pSJ-N02A).

[0131] (4) Culture of recombinant bacteria The transformant (ATCC12674 / pSJ-N02A) obtained in the above step was inoculated into MYK medium (50 μg / ml kanamycin), cultured with shaking at 30°C for 2 days, and then inoculated at 1% into GGPK medium (1.5 % glucose, 1% sodium glutamate, 0.1% yeast extract, 0.05% K2HP O4, 0.05% KH2PO4, 0.05% MgSO4·7H2O, 1% CoCl2, 0.1 % urea, 50 μg / ml kanamycin, pH 7.2). The culture was subjected to shaking culture at 30°C for 3 days, and cells were collected by centrifugation. Thereafter, the cells were washed with 100 mM phosphate buffer (pH 7.0 ) to prepare a cell suspension.

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

[0133] Acrylonitrile was prepared to have a hydrogen cyanide concentration of 2.0 ppm.

[0134] The aforementioned bacterial cells were placed in a glass case with a lid and an internal volume of 13.5 ml, with a total bacterial count of 4.1 Add 1.00 g of diluted bacterial solution, diluted with pH 7.0 phosphate buffer to a concentration of mg, and each of the aluminum The dehyde compound was diluted in pH 7.0 phosphate buffer to a concentration of 7 ppm in the reaction solution. The solution was added, and finally, pH 7.0 phosphate buffer was added to bring the total volume to 8.5 g. The solution was stirred while maintaining a controlled temperature of 25°C. 1.5 g of acrylonitrile was added to this solution, and the reaction was performed. The reaction was initiated. After 6.0 hours, the reaction solution was collected and subjected to gas chromatography (column: P oraPack-PS (Waters Corporation), 1m, 210℃, Carrier gas: Helium The concentration of acrylonitrile was measured using a detector (FID).

[0135] Table 4 shows the results after 6 hours from the start of the reaction, compared to the case without the addition of the aldehyde compound. This shows the concentration ratio of acrylonitrile. This concentration ratio can be calculated using the following formula. Concentration ratio [%] = (Acrylonitrile concentration when aldehyde is added / amount of aldehyde added) (Acrylonitrile concentration without addition) × 100 Furthermore, compounds that produce aldehyde compounds in water or solution are generated in water or solution. The aldehyde / cyanide ratio was calculated based on the number of moles of aldehyde compounds.

[0136] [Table 4]

[0137] The addition of aldehyde compounds increased the concentration of acrylonitrile compared to when they were not added. It was confirmed that the reaction rate for converting nitrile compounds to amide compounds is improved, as the reaction rate is lower. It was done. [Industrial applicability]

[0138] This invention involves combining amide compounds such as acrylamide and methacrylamide with nitrile compounds. It is useful in biological industrial production, which involves manufacturing from materials.

[0139] All publications, patents, and patent applications cited herein are used as direct reference herein. It shall be incorporated into the interior. [Sequence Listing Free Text]

[0140] Sequence ID 1: Primer M8-1 Sequence ID 2: Primer M8-2 Sequence ID 3: Primer PSN-1 Sequence ID 4: Primer PSN-2

Claims

1. In the presence of a biocatalyst having nitrile hydratase activity, nitrile compounds are converted to A A method for producing mid compounds, The step includes adding an aldehyde compound to the reaction solution containing the nitrile compound, The nitrile compound is acrylonitrile, acetonitrile, methacrylonitrile, shea It is at least one selected from nopyridine, glyconitrile, and alaninenitrile. 、 The aldehyde compound is a compound having an aldehyde group, or an aldehyde in water or solution. Compounds that produce compounds having a group, such as formaldehyde, acetaldehyde, pro Pionaldehyde, butyraldehyde, isobutyraldehyde, dialdehyde oxalate, Malondialdehyde, pentanal, isovaleraldehyde, glyceraldehyde, glyc Collaldehyde, furfural, butanedial, glutaraldehyde, hexanal Heptanal, Octanal, Nonanal, Decanal, Paraaldehyde, Benzal Dehyde, cinnamaldehyde, perillaldehyde, vanillin, 1-naphthaldehyde, f Taraldehyde, methional, paraformaldehyde, acetaldehyde, ammonia, and It is at least one selected from hexamethylenetetramine, The biocatalyst having nitrile hydratase activity is a member of the genus Rhodococcus or Pseudono The method is a cell or microbial cell containing nitrile hydratase derived from the genus Cardia. 。

2. From nitrile compounds in the presence of a biocatalyst having nitrile hydratase activity In the production of amide compounds, the reaction rate for converting the nitrile compound to the amide compound is A method to improve, The step includes adding an aldehyde compound to the reaction solution containing the nitrile compound, The nitrile compound is acrylonitrile, acetonitrile, methacrylonitrile, shea It is at least one selected from nopyridine, glyconitrile, and alaninenitrile. 、 The aldehyde compound is a compound having an aldehyde group, or an aldehyde in water or solution. Compounds that produce compounds having a group, such as formaldehyde, acetaldehyde, pro Pionaldehyde, butyraldehyde, isobutyraldehyde, dialdehyde oxalate, Malondialdehyde, pentanal, isovaleraldehyde, glyceraldehyde, glyc Collaldehyde, furfural, butanedial, glutaraldehyde, hexanal Heptanal, Octanal, Nonanal, Decanal, Paraaldehyde, Benzal Dehyde, cinnamaldehyde, perillaldehyde, vanillin, 1-naphthaldehyde, f Taraldehyde, methional, paraformaldehyde, acetaldehyde, ammonia, and It is at least one selected from hexamethylenetetramine, The biocatalyst having nitrile hydratase activity is a member of the genus Rhodococcus or Pseudono The method is a cell or microbial cell containing nitrile hydratase derived from the genus Cardia. 。

3. From nitrile compounds in the presence of a biocatalyst having nitrile hydratase activity In the production of amide compounds, the activity of the biocatalyst having nitrile hydratase activity is reduced. A method of suppressing the lower The step includes adding an aldehyde compound to the reaction solution containing the nitrile compound, The nitrile compound is acrylonitrile, acetonitrile, methacrylonitrile, shea It is at least one selected from nopyridine, glyconitrile, and alaninenitrile. 、 The aldehyde compound is a compound having an aldehyde group, or an aldehyde in water or solution. Compounds that produce compounds having a group, such as formaldehyde, acetaldehyde, pro Pionaldehyde, butyraldehyde, isobutyraldehyde, dialdehyde oxalate, Malondialdehyde, pentanal, isovaleraldehyde, glyceraldehyde, glyc Collaldehyde, furfural, butanedial, glutaraldehyde, hexanal Heptanal, Octanal, Nonanal, Decanal, Paraaldehyde, Benzal Dehyde, cinnamaldehyde, perillaldehyde, vanillin, 1-naphthaldehyde, f Taraldehyde, methional, paraformaldehyde, acetaldehyde, ammonia, and It is at least one selected from hexamethylenetetramine, The biocatalyst having nitrile hydratase activity is a member of the genus Rhodococcus or Pseudono The method is a cell or microbial cell containing nitrile hydratase derived from the genus Cardia. 。

4. The molar ratio of the aldehyde compound to the cyanide compound in the nitrile compound is 0. The method according to any one of claims 1 to 3, wherein the value is 9 to 15.

5. Aldehyde compounds and, Acrylonitrile, acetonitrile, methacrylonitrile, cyanopyridine, glyco A nitrile compound comprising at least one nitrile compound selected from nitriles and alanine nitriles, The method according to any one of claims 1 to 4, which is carried out using a composition for producing amide compounds. Law.

6. Amid compounds comprising aldehyde compounds and biocatalysts having nitrile hydratase activity The method according to any one of claims 1 to 4, wherein a catalyst composition for manufacturing is used.

7. The aforementioned cells or microbial cells are cells or microbial cells that have lost their ability to proliferate due to drug treatment. The method according to any one of claims 1 to 6.

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