Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/88—Lyases (4.)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y402/00—Carbon-oxygen lyases (4.2)
  • C12Y402/01—Hydro-lyases (4.2.1)
  • C12Y402/01084—Nitrile hydratase (4.2.1.84)

Definitions

• the present specification includes the contents described in the specification of Japanese Patent Application No. 2021-19409 (filed on February 10, 2021), which is the basis of priority of the present application.
• 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 a decrease in activity of nitrile hydratase, a method for producing an amide compound while increasing the reaction rate, and the like.
• Amide compounds are widely used as industrially important substances.
• Acrylamide is widely used as a coagulant for wastewater treatment, a paper strength agent, and an oil recovery agent, and methacrylamide is widely used as paints and adhesives.
• this production method has milder reaction conditions, a higher conversion rate of the nitrile compound to the corresponding amide compound, and a higher selectivity, making it easier to implement a simpler process. can be said to be an industrially excellent technique.
• Patent Document 1 the enzymatic activity of nitrile hydratase is improved (Patent Document 1), the activity is suppressed from decreasing due to temperature, and the production efficiency of amide compounds is improved by improving the resistance to amide compounds (Patent Document 2).
• Patent Document 3-5 a method for improving production efficiency by identifying organic impurities in nitrile compounds that affect the activity of nitrile hydratase and suppressing the decrease in nitrile hydratase activity has been reported.
• Patent Document 3 a method of efficiently producing an amide compound by reducing the concentration of benzene present in a nitrile compound has been reported.
• Patent Documents 4 and 5 techniques for reducing the concentration of hydrocyanic acid in nitrile compounds have also been reported.
• the main object of the present invention is to provide a method for improving the productivity of amide compounds more simply by adding an aldehyde compound.
• the inventors have made intensive studies in view of the problems of the prior art, and found that by adding an aldehyde compound in the reaction for producing an amide compound from a nitrile compound using a biocatalyst having nitrile hydratase activity, The inventors have found that the reaction rate of converting a nitrile compound into an amide compound is improved and that the decrease in activity of nitrile hydratase can be suppressed, thereby completing the present invention.
• a method for producing an amide compound from a nitrile compound in the presence of a biocatalyst having nitrile hydratase activity comprising: 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 acrylonitrile, acetonitrile, methacrylonitrile, cyanopyridine, glycolonitrile and alanine nitrile.
• a method for improving the reaction rate of converting a nitrile compound to an amide compound in the production of an amide compound from a nitrile compound in the presence of a biocatalyst having nitrile hydratase activity comprising: The method, comprising adding an aldehyde compound to a reaction solution containing the nitrile compound, wherein the nitrile compound is at least one selected from acrylonitrile, acetonitrile, methacrylonitrile, cyanopyridine, glycolonitrile and alanine nitrile. .
• a method for producing an amide compound from a nitrile compound in the presence of a biocatalyst having nitrile hydratase activity comprising: The method, comprising adding an aldehyde compound to a reaction solution containing the nitrile compound, wherein the nitrile compound is at least one selected from acrylonitrile, acetonitrile, methacrylonitrile, cyanopyridine, glycolonitrile and alanine nitrile. .
• the biocatalyst having nitrile hydratase activity is a nitrile hydratase derived from the genus Rhodococcus or Pseudonocardia, or a cell containing the nitrile hydratase, a microbial cell, or a processed product thereof, [1]-[ 5].
• the aldehyde compound is formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde, dialdehyde oxalate, malondialdehyde, pentanal, isovaleraldehyde, acrolein, crotonaldehyde, tiglic acid aldehyde, glyceraldehyde, glycolaldehyde , furfural, butandial, trans-2-hexenal, glutaraldehyde, hexanal, heptanal, octanal, nonanal, decanal, paraldehyde, benzaldehyde, cinnamaldehyde, perillaldehyde, vanillin, 1-naphthaldehyde, phthalaldehyde, methional, (Z) - The method according to any one of [1] to [4],
• an aldehyde compound A composition for producing an amide compound, comprising at least one nitrile compound selected from acrylonitrile, acetonitrile, methacrylonitrile, cyanopyridine, glycolonitrile and lactonitrile.
• a catalyst composition for producing an amide compound comprising an aldehyde compound and a biocatalyst having nitrile hydratase activity.
• an aldehyde compound by adding an aldehyde compound to a reaction solution containing a nitrile compound, it is possible to increase the reaction rate of the conversion of a nitrile compound to an amide compound by a biocatalyst having nitrile hydratase activity. Moreover, according to the present invention, it is possible to suppress the decrease in activity of a biocatalyst having nitrile hydratase activity. Therefore, according to the present invention, an amide compound can be produced efficiently.
• nitrile hydratase refers to an enzyme having the ability to hydrolyze a nitrile compound to produce a corresponding amide compound.
• the biocatalyst having nitrile hydratase activity may be the nitrile hydratase protein itself, or may be animal cells, plant cells, cell organelles, microbial cells containing nitrile hydratase, or processed products thereof.
• Examples of the processed products include animal cells, plant cells, cell organelles, crushed products of microbial cells, or enzymes extracted from bacterial cells (primary enzymes or purified enzymes); animal cells, plant cells, cells organelles, microbial cells or enzymes immobilized on carriers; and the like.
• the treated products also include animal cells, plant cells, cell organelles, or microbial cells that have lost their ability to proliferate due to chemical treatment.
• Immobilization methods include entrapment method, cross-linking method, carrier binding method, and the like.
• the entrapment method is a method of coating with a polymer coating.
• the cross-linking method is a method of cross-linking an enzyme with a reagent having two or more functional groups (polyfunctional cross-linking agent).
• the carrier-binding method is a method of binding an enzyme to a water-insoluble carrier.
• substances (immobilization carriers) used for immobilization include gas beads, silica gel, polyurethane, polyacrylamide, polyvinyl alcohol, carrageenan, alginic acid, agar, and gelatin.
• microorganisms include, for example, the genus Rhodococcus, the genus Gordona, the genus Pseudomonas, the genus Pseudonocardia, the genus Geobacillus, and the genus Bacillus having nitrile hydratase activity.
• (Bacillus) genus Bacteridium genus, Micrococcus genus, Brevibacterium genus, Corynebacterium genus, Nocardia genus, Microbacterium genus, Fusarium genus, Agrobacterium genus, Acinetobacter genus, Xanthobacter genus, Streptomyces genus, Rhizobium genus, Klebsiella genus, Enterobacter (Enterobacter) genus, Erwinia genus, Pantoea genus, Candida genus, Aeromonas genus, Citrobacter genus, Achromobacter genus, etc. be done.
• MCI2614 Citrobacter freundii MCI2615 described in JP-A-05-30984, Agrobacterium rhizogenes IAM13570 and Agrobacterium tumefaciens described in JP-A-05-103681 (Agrobacterium faciens), Xanthobacter flavas JCM1204 described in JP-A-05-161495, Erwinia nigrifluens MAFF03-01435, entero described in JP-A-236975 Bacter sp. MCI2707, Streptomyces sp. described in JP-A-05-236976. MCI2691, Rhizobium sp.
• Rhodococcus rhodochrous J-1 strain described in Japanese Patent Publication No. 06-55148 was deposited on September 18, 1987 at the National Institute of Technology and Evaluation Patent Organism Depository under the accession number "FERM BP-1478". It has been deposited at Central 6, 1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture (the same shall apply hereinafter).
• NCIMB41164 ⁇ WO2005/054456 ⁇ NCIMB41164 ⁇ 2003 ⁇ 3 ⁇ 5 ⁇ National Collection of Industrial,Food and Marine Bacteria,Ltd.(NCIMB)(NCIMB Ltd Ferguson Building Craibstone Estate Buksburn Aberdeen AB21 9YA) under accession number NCIMB41164.
• one selected from the above microorganisms can be used alone or in combination of two or more.
• the gene encoding nitrile hydratase can be introduced and expressed in microbial cells by conventional molecular biological techniques (for these molecular techniques, see Sambrook, Fritsch 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 kind selected from the above enzymes can be used singly or two or more kinds can be used in combination.
• amino acid sequence of wild-type nitrile hydratase is published in NCBI databases such as GenBank (http://www.ncbi.nlm.nih.gov/).
• accession number of the ⁇ subunit derived from Rhodococcus rhodochrous J1 is "P21219”
• the accession number of the ⁇ subunit is "P21220”
• the accession number of the ⁇ subunit from Rhodococcus rhodochrous M8 is "ATT79340”
• the accession number of the ⁇ subunit is "AAT79339”.
• the accession number for the ⁇ subunit from Pseudomonas thermophila JCM3095 is “1IRE A” and the accession number for the ⁇ subunit is “1IREB”.
• transformants introduced with a wild-type nitrile hydratase gene include Escherichia coli MT10770 (FERM P-14756) transformed with a nitrile hydratase belonging to the genus Achromobacter (JP-A-8-266277); Escherichia coli MT10822 (FERM BP-5785) (JP-A-9-275978) transformed with nitrile hydratase of the genus Pseudonocardia, or Rhodococcus rhodochrous nitrile hydratase (JP-A-4 -211379) are exemplified, but not limited to these.
• An improved (mutant) nitrile hydratase obtained by subjecting a wild-type nitrile hydratase to an amino acid substitution is known (JP-A-2010-172295, JP-A-2007-143409, JP-A-2007-043910, JP 2008-253182, JP 2019-088326, JP 2019-088327, WO05/116206, WO12/164933, WO12/169203, WO15/186298, etc.), Microorganisms into which these improved nitrile hydratases have been introduced can also be used in the method of the present invention.
• microbes having nitrile hydratase activity or their processed products can be used for amide synthesis reaction immediately after preparation of the cells, or can be stored after preparation of the cells and used for amide synthesis reaction as needed.
• a method for culturing microorganisms for preparing cells can be appropriately selected according to the type of microorganism.
• a seed culture may be performed before the main culture.
• the cells of microorganisms having nitrile hydratase activity or their processed products can be used for batch reactions or continuous reactions.
• a suitable reaction form such as a fluidized bed, fixed bed, or suspended bed can be selected.
• the catalyst temperature in the reaction solution at that time is not particularly limited as long as it does not interfere with the mixing of the aqueous medium and the nitrile compound.
• 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 into an amide compound by a catalyst having nitrile hydratase activity.
• aliphatic saturated nitriles such as acetonitrile, propionitrile, succinonitrile, adiponitrile, glycolonitrile, lactonitrile, aliphatic unsaturated nitriles such as acrylonitrile, methacrylonitrile, benzonitrile, phthalodinitrile
• Aromatic nitriles and heterocyclic nitriles such as cyanopyridines are included.
• the nitrile compound in the present invention is preferably a nitrile compound such as acetonitrile, propionitrile, acrylonitrile, methacrylonitrile, n-butyronitrile, isobutyronitrile, cyanopyridine, glucoronitrile, lactonitrile, and particularly more preferably , acrylonitrile, methacrylonitrile, acetonitrile, cyanopyridine, glycolonitrile, lactonitrile.
• a nitrile compound such as acetonitrile, propionitrile, acrylonitrile, methacrylonitrile, n-butyronitrile, isobutyronitrile, cyanopyridine, glucoronitrile, lactonitrile, and particularly more preferably , acrylonitrile, methacrylonitrile, acetonitrile, cyanopyridine, glycolonitrile, lactonitrile.
• Nitrile compounds generally become commercial products after going through a purification process.
• acrylonitrile is industrially produced by the ammoxidation method of propylene, and cyanide compounds such as hydrocyanic acid are removed together with other by-products by distillation after the reaction.
• the product contains cyanide compounds that cannot be completely removed by this operation. It is thought that the cyanide contained in the nitrile compound damages the biocatalyst having nitrile hydratase activity, leading to a decrease in activity and reaction rate.
• the amount of cyanide contained in the nitrile compound is reduced, and damage to the biocatalyst having nitrile hydratase activity due to the cyanide. can be prevented. That is, the reaction rate for converting a nitrile compound to an amide compound in the presence of a biocatalyst having nitrile hydratase activity is improved. In addition, it is thought that the decrease in activity of the biocatalyst having nitrile hydratase activity due to contact with the nitrile compound can be suppressed.
• an amide compound is produced from a nitrile compound using a biocatalyst having nitrile hydratase activity.
• the type of amide compound produced in the present invention is not particularly limited, and an amide compound can be produced according to the application.
• a raw material a nitrile compound corresponding to the amide compound can be used.
• Amide compounds include acrylamide, nicotinamide, and methacrylamide. Acrylamide is preferred.
• Acrylamide is obtained when acrylonitrile is used as the nitrile compound, and methacrylamide is obtained when methacrylonitrile is used as the nitrile compound. Nicotinamide is obtained when cyanopyridine is used as the nitrile compound.
• the method for producing an amide compound using a biocatalyst having nitrile hydratase activity is not particularly limited. For example, it may be carried out by a continuous reaction that continuously produces an amide compound, or a discontinuous production of an amide compound. A batch reaction may also be performed.
• the reaction raw materials containing the biocatalyst, water and nitrile compound are continuously or intermittently introduced into the reactor, and the reaction mixture containing the produced amide compound is continuously or intermittently discharged from the reactor.
• An amide compound can be continuously produced while removing the reaction mixture without withdrawing the entire amount of the reaction mixture in the reactor.
• reaction raw materials are charged into the reactor in its entirety at one time and then reacted, or after a part of the reaction raw materials are charged into the reactor, the remaining reaction raw materials are fed continuously or intermittently.
• An amide compound can be produced by reacting with
• reactors can be used, such as a stirred tank type, fixed bed type, fluidized bed type, moving bed type, tubular type, or tower type.
• One reactor may be used, or a plurality of reactors may be used in combination.
• the concentration of the amide compound in the reaction mixture taken out from the downstream reactor is higher. Therefore, the concentration of the finally obtained amide compound can be adjusted by the number of reactors.
• the reactor into which the biocatalyst with nitrile hydratase activity and the nitrile compound are introduced should be the most upstream as long as the efficiency of the reaction is not excessively deteriorated. It is not limited to introducing only into the reactor located at , but can also be introduced into reactors further downstream.
• the raw water is used for the hydration reaction with the nitrile compound when producing the amide compound.
• raw water examples include water; or an aqueous solution in which acids or salts are dissolved in water.
• Acids include phosphoric acid, acetic acid, citric acid, boric acid and the like.
• salts include sodium salts, potassium salts, ammonium salts and the like of the above acids.
• the type of raw water is not limited, but examples include pure water, city water, Tris buffer, phosphate buffer, acetate buffer, citrate buffer, borate buffer, and the like.
• the pH (25° C.) of the raw material water to be used is also not particularly limited as long as the biocatalyst can react efficiently. For example, it can be 4-10, preferably 5-9. By adjusting the pH to 4 or higher, the enzymatic activity of the biocatalyst can be sufficiently enhanced. Deactivation of the biocatalyst can be suppressed by adjusting the pH to 10 or less.
• the amount of biocatalyst used can be appropriately selected depending on the type of biocatalyst used and reaction conditions. For example, it is preferable to adjust the activity of the biocatalyst to be introduced into the reactor to be about 50 to 500 U per 1 mg of dry cells at a reaction temperature of 10°C.
• the unit U (unit) means that 1 ⁇ mole of an amide compound is produced from a nitrile compound per minute, and is a value measured using a nitrile compound used for production.
• At least one water-soluble monocarboxylic acid salt having 2 or more carbon atoms is added to the reaction 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. may be added.
• the water-soluble monocarboxylic acid salt may be either a saturated monocarboxylic acid salt or an unsaturated monocarboxylic acid salt.
• Saturated carboxylic acids include acetic acid, propionic acid, n-caproic acid and the like.
• Unsaturated carboxylic acids include acrylic acid, methacrylic acid, vinylacetic acid and the like. Salts are typically sodium salts, potassium salts, and ammonium salts.
• the amount of the water-soluble monocarboxylic acid salt to be added is preferably such that the acid is 20 to 5000 mg/kg with respect to acrylamide in the finally obtained reaction mixture (acrylamide aqueous solution).
• the amount of nitrile compound used can be appropriately selected depending on the type of biocatalyst used, reaction conditions, reaction scale, continuous reaction or batch reaction, etc.
• the reaction temperature is not particularly limited as long as the biocatalyst can efficiently promote the reaction.
• it can be 5 to 50°C, preferably 10 to 40°C, more preferably 15 to 35°C.
• the reaction temperature can be 5 to 50°C, preferably 10 to 40°C, more preferably 15 to 35°C.
• the reaction time is not particularly limited, and can be appropriately selected according to the reaction format and reaction scale, such as batch reaction, batch reaction, and continuous reaction. For example, it can be 0.1 to 60 hours, preferably 1 to 50 hours, more preferably 2 to 40 hours.
• the fluid velocity when taking out the reaction mixture from the reactor is adjusted so that the nitrile compound and the biocatalyst can be produced continuously without taking out the entire amount of the reaction mixture in the reactor. It may be determined according to the introduction speed.
• Additives can also be added to the nitrile compound or reaction solution in order to stabilize the reaction.
• the concentration of the amide compound in the aqueous solution of the amide compound obtained in the present invention can be appropriately selected according to the purpose of use of the obtained amide compound.
• the concentration of the amide compound can be 25 to 65% by mass, preferably 30 to 60% by mass, more preferably 35 to 55% by mass, based on the total mass of the resulting aqueous solution of the amide compound.
• concentration of the amide compound By setting the concentration of the amide compound to 65% by mass or less, precipitation of crystals of the amide compound at room temperature can be prevented.
• the concentration of the amide compound to 25% by mass or more, it is possible to reduce the volume of the tank used for storage and storage, and to reduce the transportation cost.
• the amide compound obtained by the present invention can be used for the polymerization reaction as it is, or can be stored until use. Moreover, an aldehyde compound can also be removed as needed. When storing the amide compound, various additives such as a polymerization inhibitor and a polymerization initiator may be added and used as necessary.
• the aldehyde compound is not particularly limited as long as the above effect can be obtained.
• Aldehyde compounds include not only compounds having an aldehyde group (-CHO), but also compounds that generate aldehyde compounds in water or in solution. Examples of such aldehyde compounds include glyoxal (oxalic acid dialdehyde), paraformaldehyde, acetaldehyde ammonia, hexamethylenetetramine and the like.
• aldehyde compounds with a molecular weight of about 200 or less are preferable, and aldehyde compounds with a molecular weight of about 100 or less are more preferable.
• aldehyde compounds are formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde and the like.
• “adding" to the reaction liquid includes “existing" in the reaction liquid.
• the amount of the aldehyde compound added to the reaction solution containing the nitrile compound can increase the reaction rate of converting the nitrile compound to the amide compound in the presence of a biocatalyst having nitrile hydratase activity, or However, it is not particularly limited as long as it can suppress the decrease in activity of the biocatalyst having nitrile hydratase activity when brought into contact with the nitrile compound.
• the amount of the aldehyde compound added to the reaction solution is 0.9 to 15, preferably 1.5 to 10.0, more preferably 2.5 to 1.5 in terms of molar ratio to the content of the cyanide compound in the reaction solution. It can be from 0 to 6.0.
• the content of cyanide in acrylonitrile can be determined by titration using silver nitrate after extraction with an alkaline solution. Alternatively, it can be measured by absorptiometry.
• Conversion of a nitrile compound to an amide compound in the presence of a biocatalyst having nitrile hydratase activity by adjusting the amount of aldehyde present in the reaction solution to a molar ratio of 0.9 or more with respect to the content of the cyanide compound.
• the reaction rate can be increased, or the decrease in activity of a biocatalyst having nitrile hydratase activity when brought into contact with a nitrile compound can be suppressed.
• the reason why the amount of aldehyde present in the reaction solution is set to 15 or less in terms of molar ratio to the content of the cyanide compound is that even if the aldehyde compound is added in excess, it is difficult to obtain an improvement in the effect. is.
• the cyanide compounds include hydrocyanic acid (HCN); cyanide ions (CN ⁇ ); sodium cyanide, potassium cyanide, and other cyanide compounds, or compounds that release cyanide or cyanide ions under reaction conditions. and
• the timing of adding the aldehyde compound to the reaction solution containing the nitrile compound is not particularly limited as long as the above effects can be exhibited. It may be during (simultaneously) or after contact (that is, after initiation of the enzymatic reaction of the nitrile compound by the biocatalyst).
• the aldehyde compound is added before the biocatalyst having nitrile hydratase activity is brought into contact with the nitrile compound.
• the aldehyde compound is added after the start of the reaction, it is believed that the earlier the aldehyde compound is added (the earlier it is added), the more the above effect can be obtained.
• the method of adding the aldehyde compound to the reaction solution is not particularly limited as long as the desired concentration of the aldehyde compound is present in the reaction solution.
• a solid aldehyde compound can be added, or an aldehyde compound dissolved in a solvent, water, or the like used for the reaction can be added.
• the present invention relates to the production of an amide compound from a nitrile compound in the presence of a biocatalyst having nitrile hydratase activity, in a reaction solution containing the nitrile compound.
• a method for improving the reaction rate of an enzymatic reaction that converts a nitrile compound to an amide compound comprising the step of adding an aldehyde compound.
• At least one selected from acrylonitrile, acetonitrile, methacrylonitrile, cyanopyridine, glycolonitrile and alaninenitrile can be used as the nitrile compound.
• aldehydes to prevent (stabilize) polymerization of acrylamide has been reported (WO2011/102510), but aldehyde compounds prevent damage to biocatalysts having nitrile hydratase activity and improve the reaction rate. was discovered for the first time in the present invention.
• "improving the reaction rate” means that the reaction rate is higher than when the aldehyde compound is not present.
• Methods for measuring the reaction rate include a method of measuring the amount of nitrile compounds present in the reaction system that decreases per time, and a method of measuring the amount of amide compounds that increase in the reaction system.
• a method for measuring the nitrile compound or amide compound a known method such as gas chromatography analysis can be used.
• the present invention provides a method for suppressing a decrease in activity of the biocatalyst having nitrile hydratase activity, comprising the step of: At least one selected from acrylonitrile, acetonitrile, methacrylonitrile, cyanopyridine, glycolonitrile and alaninenitrile can be used as the nitrile compound.
• the activity of nitrile hydratase is easily affected by impurities in the reaction solution and reaction conditions.
• INDUSTRIAL APPLICABILITY According to the method of the present invention, the decrease in nitrile hydratase activity can be easily suppressed without requiring an additional step and without affecting the quality of the amide compound.
• a composition for producing an amide compound is for producing an amide compound, comprising an aldehyde compound and at least one nitrile compound selected from acrylonitrile, acetonitrile, methacrylonitrile, cyanopyridine, glycolonitrile and lactonitrile. Compositions are also provided.
• composition for producing an amide compound of the present invention the compound described in "(4) Aldehyde compound” can be used according to the description in the same paragraph.
• the composition is used in the method for producing an amide compound described in (3) and enables efficient production of an amide compound.
• the present invention also provides a catalyst composition for producing amide compounds, which contains an aldehyde compound and a biocatalyst having nitrile hydratase activity.
• the aldehyde compound and the biocatalyst having nitrile hydratase activity are described in "(4) Aldehyde compound” and “(1) Biocatalyst having nitrile hydratase activity", respectively. That's right.
• the present invention also provides an amide compound composition containing an aldehyde compound and a cyanide compound.
• the first embodiment is characterized in that 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. Let the density
• the amide compound composition contains an amide compound and a compound in which a cyanide compound and an aldehyde compound are bonded.
• Example 1 Rhodococcus rhodochrous J-1 (FERMBP-1478) 2.0% glucose, 1.0% urea, 0.5% peptone, 0.3% yeast extract, and It was cultured aerobically at 30° C. using a medium (pH 7.0) containing 0.05% cobalt chloride. This was washed with 50 mM phosphate buffer (pH 7.0) to obtain a cell suspension (3% in terms of dry cells).
• Example 2 The procedure of Example 1 was repeated except that the acetaldehyde concentration in acrylonitrile in Example 1 was changed to 6.0 ppm.
• Example 3 The procedure of Example 1 was repeated except that the acetaldehyde concentration in acrylonitrile in Example 1 was changed to 3.0 ppm.
• Example 4 The procedure of Example 1 was repeated except that the concentration of hydrocyanic acid in acrylonitrile in Example 1 was changed to 1.1 ppm.
• Example 1 The experiment was performed as in Example 1, except that no acetaldehyde was added to the acrylonitrile.
• Example 1 was repeated except that the concentration of hydrocyanic acid in acrylonitrile was 1.1 ppm and acetaldehyde was not added to acrylonitrile.
• Example 1 According to Example 1, the effects of the following compounds on the amide compound formation reaction were evaluated.
• 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: Dialdehyde oxalate (58.04), 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 (
• reaction solution was collected and subjected to gas chromatography (column: PoraPack-PS (manufactured by Waters), 1 m, 210° C., carrier gas: helium, Detector: FID) was used to measure the concentration of acrylonitrile.
• PoraPack-PS manufactured by Waters
• carrier gas helium
• aldehyde compounds other than formic acid, meso-erythritol, and paraldehyde the addition of the aldehyde compound lowers the concentration of acrylonitrile and improves the reaction rate of conversion from the nitrile compound to the amide compound, compared to the case where the aldehyde compound is not added. was confirmed.
• Example 5 (Preparation of Transformant Having Nitrile Hydratase Derived from Rhodococcus rhodochrous M8 Strain)
• M8 strain M8 strain
• SU1731814 can be obtained from Russian Strain Center IBFM (VKPM S-926).
• the M8 strain was shake-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) 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, shaken at 37°C for 1-2 hours, and then frozen at -20°C.
• Tris-SDS solution 1% SDS, 0.1 M NaCl, 0.1 M Tris-HCl (pH 9.0)
• proteinase K final concentration: 0.1 mg
• TE-saturated phenol and stirring TE: 10 mM Tris-HCl, 1 mM EDTA (pH 8.0)
• the mixture was centrifuged. The upper layer was collected, 2 volumes of ethanol was added, and the DNA was wound with a glass rod. After that, it was centrifuged with 90%, 80% and 70% ethanol in order to remove phenol.
• the DNA was dissolved in 3 mL of TE buffer, ribonuclease A solution (heated at 100°C for 15 minutes) was added to 10 ⁇ g/mL, and shaken at 37°C for 30 minutes. Further, proteinase K (Merck) was added and shaken at 37°C for 30 minutes. An equal amount of TE-saturated phenol was added thereto, and after centrifugation, the mixture was separated into an upper layer and a lower layer.
• ribonuclease A solution heated at 100°C for 15 minutes
• proteinase K Merck
• M8 strain-derived nitrile hydratase expression plasmid The M8 strain-derived nitrile hydratase gene (SEQ ID NO: 5) was obtained from Biotechnologiia (Mosc.), 5, 3-5 (1995)), and the amino acid sequences of the ⁇ subunit, ⁇ subunit, and activator are shown in sequence as SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. It was shown to. Based on these sequence information, the following primers (SEQ ID NOS: 1 and 2) were synthesized, and PCR was performed using the prepared genomic DNA of the M8 strain as a template under the following reaction conditions.
• M8-1 5'-GGTCTAGAATGGATGGTATCCACCGACACAGGC-3' (SEQ ID NO: 1)
• M8-2 5'-cccctgcaggtcagtcgatgatggccatcgattc-3' (SEQ ID NO: 2)
• Reaction liquid composition Template DNA (M8 strain genomic DNA) 1 ⁇ l Primer M8-1 (SEQ ID NO: 15) 0.5 ⁇ l Primer M8-2 (SEQ ID NO: 16) 0.5 ⁇ l 8 ⁇ l of sterile water PrimeSTAR (Takara Bio) 10 ⁇ l Total volume 20 ⁇ l
• Temperature cycle 30 cycles of 98°C for 10 seconds, 55°C for 5 seconds, and 72°C for 30 seconds
• pSJ-N01A a plasmid expressing nitrile hydratase derived from strain J1 in Rhodococcus spp.
• ATCC12674 transformant Rhodococcus rhodochrous strain ATCC 12674 strain logarithmic growth phase cells were collected with a centrifuge, washed three times with ice-cold sterilized water, and sterilized with sterilized water. Suspended to prepare competent cells.
• the plasmid of the obtained colony was confirmed and a transformant (ATCC12674/pSJ-N01A) was obtained.
• Example 1 Evaluation of Effect on Amide Compound Production 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.
• Example 6 (1) Preparation of DN1 transformant expressing nitrile hydratase derived from Pseudonocardia thermophila JCM3095 strain
• Plasmid pPT-DB1 is Pseudonocardia thermophila JCM3095 strain obtained in JP-A-9-275978 (hereinafter referred to as A plasmid containing the nitrile hydratase gene derived from E. coli HB101 was introduced into Escherichia coli HB101 (MT-10822 strain). It is deposited at No. 1 Central No. 6).
• the nitrile hydratase gene (SEQ ID NO: 9) of strain JCM3095 is described in JP-A-9-275978. and SEQ ID NO: 12. Based on these sequence information, the following primers (SEQ ID NOs: 3 and 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.
• PSN-1 5'-GGTCTAGAATGAACGGCGTGTACGACGTCGGC-3' (SEQ ID NO: 3)
• PSN-2 5'-ccCCTGCAGGTCAGGACCGCACGGCCGGGTGGAC-3' (SEQ ID NO: 4)
• Reaction liquid composition Template DNA (pPT-DB1) 1 ⁇ l Primer PSN-1 (SEQ ID NO: 17) 0.5 ⁇ l Primer PSN-2 (SEQ ID NO: 18) 0.5 ⁇ l 8 ⁇ l of sterile water PrimeSTAR (Takara Bio) 10 ⁇ l Total volume 20 ⁇ l
• Temperature cycle 30 cycles of 98°C for 10 seconds, 55°C for 5 seconds, and 72°C for 30 seconds
• a plasmid was prepared in the same manner as in Example 5 (2) and named pSJ-N02A.
• the resulting plasmid was introduced into ATCC12674 in the same manner as in Example 5 (3) to obtain a transformant (ATCC12674/pSJ-N02A).
• the present invention is useful in industrial biological production of amide compounds such as acrylamide and methacrylamide from nitrile compounds.
• 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

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