WO2021010268A1 - Ｎ－置換アミド化合物／アミド化合物共重合体の製造方法 - Google Patents

Ｎ－置換アミド化合物／アミド化合物共重合体の製造方法 Download PDF

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WO2021010268A1
WO2021010268A1 PCT/JP2020/026794 JP2020026794W WO2021010268A1 WO 2021010268 A1 WO2021010268 A1 WO 2021010268A1 JP 2020026794 W JP2020026794 W JP 2020026794W WO 2021010268 A1 WO2021010268 A1 WO 2021010268A1
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amide compound
acrylamide
substituted
reaction
reaction solution
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French (fr)
Japanese (ja)
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美知子 高橋
章裕 島元
隆文 山口
卓理 秋山
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Mitsubishi Chemical Corp
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Mitsubishi Chemical Corp
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F220/56—Acrylamide; Methacrylamide
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • 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

Definitions

  • the present invention relates to a method for producing a copolymer of acrylamide (amide compound) and N-substituted acrylamide (N-substituted amide compound) (hereinafter, referred to as "N-substituted amide compound / amide compound copolymer"). More specifically, the present invention relates to a method for producing N-substituted acrylamide from acrylamide and amine by a biocatalytic reaction, and obtaining an acrylamide / N-substituted acrylamide copolymer by a polymerization reaction between the produced N-substituted acrylamide and unreacted acrylamide.
  • Copolymers of acrylamide / N-substituted acrylamide are used for paper chemicals such as paints, adhesives, adhesives, coating agents, and paper strength enhancers, polymer flocculants used for treating industrial wastewater and domestic wastewater, and polymer modification. It is a functional polymer used in an extremely diverse field such as pledges, dispersants, thickeners, raw materials for contact lenses and biogels, and reactive diluents for UV curable resins.
  • the conventional method for producing a copolymer of acrylamide / N-substituted acrylamide requires a multi-step process including a step under high temperature conditions for the synthesis of N-substituted acrylamide, and is not an inexpensive method.
  • an amide exchange reaction between (meth) acrylamide and an amine and a Michael addition reaction are carried out at 100 to 250 ° C. using an acrylate of amine as a catalyst, and further liquid phase heat is performed at 160 to 350 ° C.
  • a method of removing an amine by decomposition to obtain N-substituted (meth) acrylamide is disclosed.
  • a main object of the present invention is to provide a method for producing an N-substituted amide compound / amide compound copolymer using a biocatalyst.
  • the present invention provides the following [1]-[7].
  • [1] A step of reacting an amide compound with an amine in the presence of an enzyme that catalyzes an amide exchange reaction.
  • (B) A step of subjecting the obtained reactant to polymerization conditions and polymerizing the N-substituted amide compound and the amide compound to obtain a copolymer.
  • a method for producing an N-substituted amide compound / amide compound copolymer which comprises. [2] The method for producing [1], wherein the enzyme is amidase. [3] The method for producing [1] or [2], wherein the amide compound is acrylamide or methacrylamide.
  • the amine is selected from the group consisting of N, N-dimethyl-1,3-propanediamine, isopropylamine, 2-hydroxyethylamine, tert-butylamine, dimethylamine and 4-amino-4-methyl-2-pentanone.
  • the production method according to any one of [1] to [3], which is one or more of the above.
  • N-substituted acrylamide consists of N- [3- (dimethylamino) propyl] acrylamide, isopropylacrylamide, N- [2-hydroxyethyl] acrylamide, tert-butylacrylamide, NN-dimethylacrylamide and diacetoneacrylamide.
  • the present invention also provides the following [1]-[10].
  • [1] A method for producing a copolymer of acrylamide and N-substituted acrylamide. (1) A step of providing a first reaction solution containing acrylamide, amine and amidase, and (2) N-substituted acrylamide is generated in the first reaction solution by an amide exchange reaction between acrylamide and amine catalyzed by amidase to obtain a second reaction solution containing acrylamide and N-substituted acrylamide. Process and (3) A step of subjecting the second reaction solution to polymerization conditions and polymerizing acrylamide and N-substituted acrylamide to obtain the copolymer. Manufacturing method, including.
  • N-substituted acrylamide is selected from N- [3- (dimethylamino) propyl] acrylamide, isopropylacrylamide, N- [2-hydroxyethyl] acrylamide, tert-butylacrylamide, NN-dimethylacrylamide and diacetoneacrylamide.
  • the production method of [1] or [2] which is one or more.
  • Process and (3) A step of subjecting the second reaction solution to polymerization conditions and polymerizing acrylamide and N-substituted acrylamide to obtain the copolymer.
  • a method for producing a reaction solution containing acrylamide and N-substituted acrylamide (1) A step of providing a first reaction solution containing acrylamide, amine and amidase, and (2) N-substituted acrylamide is generated in the first reaction solution by an amide exchange reaction between acrylamide and amine catalyzed by amidase to obtain a second reaction solution containing acrylamide and N-substituted acrylamide. Process and Manufacturing method, including.
  • [8] (1) A step of providing a first reaction solution containing acrylamide, an amine and an amidase, and (2) N-substituted acrylamide is generated in the first reaction solution by an amide exchange reaction between acrylamide and amine catalyzed by amidase to obtain a second reaction solution containing acrylamide and N-substituted acrylamide. Process and A reaction solution containing acrylamide and N-substituted acrylamide, which is obtained by a production method containing. [9] The reaction solution of [8] for polymerizing acrylamide and N-substituted acrylamide to obtain a copolymer of acrylamide and N-substituted acrylamide. [10] The reaction solution of [8] or [9] containing a recombinant bacterium expressing amidase.
  • the present invention provides a method for producing an N-substituted amide compound / amide compound copolymer using a biocatalyst.
  • the method for producing an N-substituted amide compound / amide compound copolymer according to the present invention includes the following steps (1), (2) and (3), and optionally includes steps (2a) and / or (2b). ..
  • Step (1) A step of providing a first reaction solution containing acrylamide, amine and amidase.
  • Step (2) N-substituted acrylamide is produced in the first reaction solution by an amide exchange reaction between acrylamide and amine catalyzed by amidase, and a second reaction solution containing acrylamide and N-substituted acrylamide is produced. The process of obtaining.
  • Step (2a) A step of removing ammonia produced by the amide transesterification reaction of acrylamide and amine from the second reaction solution.
  • Step (2b) A step of removing the recombinant bacterium from the second reaction solution when the amidase is provided by the recombinant bacterium.
  • Step (3) A step of subjecting the second reaction solution to polymerization conditions and polymerizing acrylamide and N-substituted acrylamide to obtain an acrylamide / N-substituted acrylamide copolymer.
  • the step (2) corresponds to the step (A) of reacting the amide compound with the amine in the presence of an enzyme that catalyzes the amide exchange reaction.
  • step (3) corresponds to the step (B) in which the reaction product obtained in the step (A) is subjected to polymerization conditions and the N-substituted amide compound and the amide compound are polymerized to obtain a copolymer. ..
  • the amide compound is acrylamide or methacrylamide.
  • the amine is not particularly limited as long as it is a compound capable of an amide exchange reaction with an amide compound.
  • Preferred compounds as amines are selected from N, N-dimethyl-1,3-propanediamine, isopropylamine, 2-hydroxyethylamine, tert-butylamine, dimethylamine and 4-amino-4-methyl-2-pentanone. Or more than one, more preferably N, N-dimethyl-1,3-propanediamine.
  • the enzyme that catalyzes the amide exchange reaction is not limited in type and origin as long as it can catalyze the amide exchange reaction. Further, as the enzyme, a wild type can be used, or a mutant type in which the catalytic ability of the amide exchange reaction is imparted by modifying the amino acid sequence can also be used. As the enzyme, a microorganism expressing the enzyme can be used, or one extracted from the microorganism can be used.
  • the enzyme that catalyzes the amide exchange reaction is preferably amidase.
  • an enzyme classified other than amidase can be used as long as it is an enzyme to which amidase activity (catalytic ability of amide exchange reaction) is imparted by modifying the amino acid sequence.
  • the amidase a commercially available product can be obtained and used.
  • an amino acid sequence or a nucleic acid sequence obtained from a database available to the public and synthesized as a recombinant enzyme may be used.
  • the amidase for example, one derived from bacteria can be used.
  • the bacterium is preferably a bacterium belonging to the genus Rhodococcus, Gordonia, Earthlobactor, or Pseudonocardia, and more preferably a bacterium belonging to the genus Rhodococcus.
  • the amidase an amidase derived from the Rhodococcus erythropolis TA37 strain (amino acid sequence is shown in SEQ ID NO: 1 and nucleic acid sequence is shown in SEQ ID NO: 11) is preferably used.
  • amidase a recombinant enzyme synthesized from the amino acid sequence (or nucleic acid sequence) obtained from the database by sequence identity search with the amino acid sequence of SEQ ID NO: 1 (or the nucleic acid sequence of SEQ ID NO: 11) can also be used.
  • an amidase a polypeptide consisting of the amino acid sequences of SEQ ID NOs: 2 to 10 (nucleic acid sequences are shown in SEQ ID NOs: 12 to 20) can be preferably used.
  • the amidase for example, a protein consisting of an amino acid sequence in which one or several amino acids are deleted, inserted, substituted and / or added in the amino acid sequences of SEQ ID NOs: 1 to 10 can be used.
  • the protein maintains the amidase activity that catalyzes the amide exchange reaction with amines.
  • the "several pieces” are, for example, 1 to 40 pieces, 1 to 20 pieces, preferably 1 to 20 pieces, 1 to 10 pieces, more preferably 1 to 5 pieces, 1 to 4 pieces, and particularly preferably 3. One, two or less.
  • a mutation introduction kit using a site-specific mutagenesis method by a known method such as the Kunkel method or the Gapped duplex method, for example, QuikChange TM System-Directed Mutagenesis Kit (Stratagene) , GeneTaylor TM Site-Directed Mutagenesis System (Invitrogen), TakaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc .), etc .: Takara.
  • the entire gene having a sequence containing a deletion or the like may be artificially synthesized.
  • the amidase contains, for example, 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 99.5% or more, and particularly preferably 99.9% or more of the amino acid sequence of SEQ ID NO: 1-10. It is also possible to use a polypeptide consisting of an amino acid sequence having a sequence identity of% or more. Here, the polypeptide maintains an amidase activity that catalyzes an amide exchange reaction with an amine.
  • sequence identity means that both sequences are aligned so that the residues of two amino acid sequences to be compared match as much as possible, and the number of matched residues is divided by the total number of residues. It is expressed as a percentage.
  • a gap is appropriately inserted in one or both of the two sequences to be compared.
  • sequence alignment can be performed using well-known programs such as BLAST, FASTA, and CLUSTALW.
  • the total number of residues is the number of residues counted with one gap as one residue. If the total number of residues counted in this way differs between the two sequences to be compared, the identity (%) is calculated by dividing the total number of residues in the longer sequence by the number of matching residues. ..
  • amidase for example, a protein consisting of an amino acid sequence encoded by a DNA that hybridizes to the complementary strand of the base sequence of SEQ ID NO: 11-20 under stringent conditions can also be used.
  • the protein maintains the amidase activity that catalyzes the amide exchange reaction with amines.
  • stringent conditions for example, a nylon film on which DNA is immobilized is 6 ⁇ SSC (1 ⁇ SSC is 8.76 g of sodium chloride and 4.41 g of sodium citrate dissolved in 1 liter of water), 1%.
  • Conditions for hybridization in a solution containing SDS, 100 ⁇ g / ml salmon sperm DNA, 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, and 0.1% ficol at 65 ° C. for 20 hours with a probe It can be mentioned, but it is not limited to this. Those skilled in the art will set the hybridization conditions in consideration of other conditions such as the probe concentration, the probe length, and the reaction time in addition to the conditions such as the salt concentration and temperature of the buffer. Can be done.
  • the washing conditions after hybridization include, for example, "2 x SSC, 0.1% SDS, 42 ° C.”, "1 x SSC, 0.1% SDS, 37 ° C.”, and the more stringent conditions include, for example, Conditions such as “1 x SSC, 0.1% SDS, 65 ° C.” and "0.5 x SSC, 0.1% SDS, 50 ° C.” can be mentioned.
  • Conditions such as “1 x SSC, 0.1% SDS, 65 ° C.” and "0.5 x SSC, 0.1% SDS, 50 ° C.” can be mentioned.
  • For detailed procedures of hybridization method please refer to Molecular Cloning, A Laboratory Manual 2nd ed. (Cold Spring Harbor Laboratory Press (1989))), Current Protocols in Molecular Biology (John Wiley & Sons (1987-1997)) and the like can be referred to.
  • the synthesis of enzymes such as amidase can be performed using general-purpose molecular biological techniques such as PCR.
  • the enzyme is used as a purified enzyme or a crude enzyme.
  • the recombinant bacterium expressing the enzyme may be used as it is, or the bacterium may be frozen, dried, pulverized or the like.
  • the culture solution obtained by culturing the recombinant bacteria can be used as it is, or the cells obtained from the culture solution by a bacterial collection operation such as centrifugation or a processed product thereof can be used.
  • the cell-treated product include cells treated with acetone, toluene and the like, freeze-dried cells, crushed cells, cell extracts and the like.
  • an enzyme-immobilized enzyme can also be used in the reaction.
  • the method of immobilization is not particularly limited, and can be appropriately selected depending on the type of enzyme, reaction conditions, and the like.
  • a method of immobilization a carrier binding method in which an enzyme is bound to and immobilized on various insoluble carriers such as cellulose, dextrin, resin beads, activated charcoal, silica gel, magnetic particles and polymer film; functional groups in proteins and the functional groups thereof.
  • a cross-linking method that utilizes a chemical bond with a compound that reacts; a comprehensive method that immobilizes the enzyme in the gel without directly chemically modifying the enzyme by incorporating the enzyme into the polymer gel can be mentioned.
  • microorganisms expressing the enzyme can be immobilized and used in the reaction.
  • the method of immobilization is not particularly limited, and a known method can be used.
  • microorganisms immobilized on a polyacrylamide gel can be used as a catalyst.
  • the enzyme is not particularly limited, but can be purified by, for example, the following method.
  • a method for removing low molecular weight substances by (1) fractionation by precipitation, (2) various chromatography, (3) dialysis, ultrafiltration, etc. in a crude enzyme solution obtained by crushing recombinant bacteria and suspending them in a buffer solution. Etc. are applied alone or in combination as appropriate.
  • Recombinant bacteria are prepared by transforming a host microorganism with an expression vector incorporating a gene encoding an enzyme such as amidase.
  • host microorganisms include Escherichia coli, Rhodococcus, Pseudomonas, Corynebacterium, Bacillus, Streptomyces, and Streptomyces in bacteria, and Saccharomyces, Candida, Schizosaccharomyces, and Pichia in yeast.
  • Aspergillus genus is mentioned as a filamentous fungus.
  • recombinant bacteria not only the above-mentioned recombinant bacteria, but also a cell-treated product such as a frozen product, a dried product or a crushed product of the recombinant bacteria may be collectively referred to as "recombinant bacteria”.
  • the concentration of the amide compound in the first reaction solution is preferably 0.1% by mass or more. Further, the amide compound concentration is preferably 50% by mass or less, more preferably 30% by mass or less, and more preferably 15% by mass or less in order to suppress the inactivation of the enzyme by a high concentration substrate. More preferred. The amide compound concentration may be, for example, 2 to 11% by mass.
  • the amine concentration of the first reaction solution is preferably 0.1% by mass or more. Further, the amine concentration is preferably 30% by mass or less, more preferably 20% by mass or less, in order to suppress the inactivation of the enzyme by a high concentration substrate. The amine concentration may be, for example, 2 to 7% by mass.
  • the amidase activity of the enzyme in the first reaction solution is preferably 100 U / L to 100,000 U / L.
  • the solvent of the first reaction solution is preferably an aqueous solvent, and a buffer can be added to the aqueous solvent if necessary.
  • the pH of the first reaction solution can be preferably 8 to 12, more preferably 9 to 11.
  • the buffer is not particularly limited as long as the pH can be maintained, and for example, sodium carbonate and sodium hydrogen carbonate can be used.
  • At least a part of the amide compound is converted to an N-substituted amide compound by amide exchange with an amine. Further, since the conversion efficiency of the amide compound by amidase to the N-substituted amide compound is usually not 100%, the amide compound that has not been subjected to the amide exchange with the amine remains in the reaction solution. As a result, a reaction solution containing an amide compound and an N-substituted amide compound is obtained as the second reaction solution.
  • the second reaction solution may contain an enzyme such as amidase or a recombinant bacterium in addition to the amide compound and the N-substituted amide compound.
  • the N-substituted amide compound produced depends on the selection of the amide compound and amine to be used.
  • the N-substituted amide compounds produced are specifically N- [3- (dimethylamino) propyl] acrylamide, isopropylacrylamide, N- [2-hydroxyethyl] acrylamide, tert-butylacrylamide, NN-dimethylacrylamide and One or more selected from diacetone acrylamide.
  • the N-substituted amide compound is preferably N- [3- (dimethylamino) propyl] acrylamide.
  • the amide compound is acrylamide and the amine is N, N-dimethyl-1,3-propanediamine.
  • reaction temperature is preferably 4 to 90 ° C, more preferably 10 to 40 ° C, and even more preferably 15 to 30 ° C in order to suppress the inactivation of the enzyme due to heat.
  • the reaction time is a time sufficient for producing the N-substituted amide compound at the desired concentration, and is not particularly limited, but is, for example, 7 to 96 hours.
  • the second reaction solution obtained in step (2) is (I) Any of the group selected from the group consisting of a Michael addition reaction product of an amide compound and an amine, a hydrolyzate of an amide compound, ammonia or a salt thereof, a component derived from a microbial cell, and a medium component for culturing the microorganism.
  • a Michael addition reaction product of an amide compound and an amine a hydrolyzate of an amide compound, ammonia or a salt thereof, a component derived from a microbial cell, and a medium component for culturing the microorganism.
  • Amide compound and May include.
  • reaction solution in addition to the (iii) N-substituted amide compound and the (iii) amide compound, ammonia or a salt thereof produced by an amide exchange reaction between the amide compound and the amine; the amide compound and the amine are contained.
  • a Michael addition reaction product produced by a Michael addition reaction separate from the amide exchange reaction; any one or more components selected from a hydrolyzate of an amide compound, a component derived from a microbial cell, and a medium component for culturing the microorganism.
  • This second reaction solution is used for producing an N-substituted amide compound / amide compound copolymer in the subsequent step (3), and has the following features.
  • the start of polymerization may be delayed, but when the second reaction solution obtained in the present invention is subjected to a polymerization reaction, there is no such drawback. It can be used for polymerization as it is without purification.
  • the second reaction solution obtained in the present invention is directly subjected to a polymerization reaction, the molecular weight of the obtained polymer is appropriately adjusted, so that the obtained polymer is less likely to be insolubilized (gelled).
  • the molecular weight can be increased by changing the polymerization conditions, but there is a feature that it is difficult to insolubilize because there are few cross-linking components.
  • the component (i) above does not affect the polymerization and is considered to have a buffering ability, even if the second reaction solution obtained in the present invention is directly subjected to the polymerization reaction, it is possible to carry out the polymerization at the time of polymerization.
  • the change in pH can be controlled to be small, and the reaction is stable.
  • the amide compound concentration of the second reaction solution obtained in the step (2) is preferably 0.1% by mass or more and 50% by mass or less.
  • the concentration of the N-substituted amide compound in the second reaction solution obtained in the step (2) is preferably 0.1% by mass or more and 30% by mass or less.
  • the solvent of the second reaction solution obtained in the step (2) is the same as that of the first reaction solution.
  • step (2) at least a part of the amide compound is converted to an N-substituted amide compound by amide exchange with an amine, but the residue remains unreacted in the reaction solution.
  • an N-substituted amide compound / amide compound copolymer is produced by a polymerization reaction of the unreacted amide compound and the N-substituted amide compound.
  • the amide compound and the N-substituted amide compound in the second reaction solution may be subjected to a polymerization reaction without purification and separation. Further, if necessary, the amide compound can be added to the reaction solution.
  • the polymerization method is not particularly limited as long as it is a method of polymerizing in an aqueous solvent, and an aqueous solution polymerization method, an adiabatic polymerization method, a continuous polymerization method and the like can be adopted. Furthermore, a suspension polymerization method or an emulsion polymerization method using a non-aqueous solvent can also be adopted.
  • the polymerization is started by adding a polymerization initiator to the second reaction solution and generating free radicals by heat, light, a redox reaction or the like. It is common to add a solution of the polymerization initiator to the second reaction solution, but the second reaction solution may be added dropwise to the solution of the polymerization initiator.
  • a solution of the polymerization initiator may be added to a suspension prepared by suspending or emulsifying the second reaction solution in a non-aqueous solvent, and the non-aqueous solvent or non-aqueous solvent may be added.
  • the polymerization initiator solution and the second reaction solution may be added to the aqueous solvent and the suspension or emulsion of water.
  • the concentration of the amide compound in the second reaction solution in the step (3) is preferably 5% by mass or more.
  • the amide compound concentration is preferably 50% by mass or less, more preferably 30% by mass or less, and further preferably 20% by mass or less from the viewpoint of suppressing the heat of polymerization.
  • the amide compound concentration may be, for example, about 10% by mass.
  • the concentration of the N-substituted amide compound in the second reaction solution in the step (3) is preferably 0.1% by mass or more. Further, the concentration of the N-substituted amide compound is preferably 30% by mass or less, more preferably 20% by mass or less, and further preferably 15% by mass or less from the viewpoint of suppressing the heat of polymerization.
  • the second reaction solution in the step (3) may contain other monomers, if necessary.
  • examples of other monomers include acrylic acid for amphoterizing the polymer, a polyfunctional monomer for branching or cross-linking the polymer, and a dimethylacrylamide monomer for modifying the polymer.
  • the concentration of the other monomer varies depending on the purpose and type thereof, but is preferably 0 to 30% by mass.
  • Any solvent may be used as the solvent for the second reaction solution, but an aqueous solvent is preferable. In the case of an aqueous solvent, the preferable pH of the second reaction solution is 2 to 10.
  • Conventionally general-purpose polymerization initiators can be used, for example, peroxides such as potassium persulfate, ammonium persulfate, benzoyl peroxide, hydrogen peroxide, and t-butyl hydroperoxide; azobisisobutyro. Azo compounds such as nitriles; photo-decomposition-type polymerization initiators such as benzoin ethyl ale; and sodium hydrogen peroxide, sodium sulfite, hydrosulfite sodium, triethanolamine, sulfate that form initiators by redox reaction with the above peroxides.
  • a reducing agent such as ferrous iron; can be used.
  • One of these polymerization initiators can be used alone or in combination of two or more.
  • the concentration of the polymerization initiator varies depending on the type and temperature, but is preferably 10 to 50,000 ppm, more preferably 20 to 40,000 ppm.
  • the temperature of the polymerization reaction more accurately than the decomposition temperature of the catalyst, varies depending on the half-life of 10 hours, but is preferably 0 ° C. to 90 ° C.
  • the time of the polymerization reaction is, for example, 30 minutes to 10 hours.
  • the copolymer composition obtained in the step (3) is (I) Any of the group selected from the group consisting of a Michael addition reaction product of an amide compound and an amine, a hydrolyzate of an amide compound, ammonia or a salt thereof, a component derived from a microbial cell, and a medium component for culturing the microorganism.
  • the copolymer composition obtained in the present invention contains the component (i) above, but since these components do not post-crosslink the polymer, the obtained polymer is insolubilized (gelled) even during storage. It is difficult to do and the copolymerization is stable. Further, since the component (i) exerts a buffering effect, it has an effect that the copolymer can be stably stored even against changes in the external environment such as pH.
  • the conversion rate of the amide compound to the N-substituted amide compound / amide compound copolymer by the polymerization reaction is, for example, 98% or more.
  • the conversion rate of the N-substituted amide compound to the copolymer by the polymerization reaction is, for example, 99.8% or more.
  • an N-substituted amide compound is produced from an amide compound and an amine by a biocatalytic reaction (step (2)), and the produced N-substituted amide compound is polymerized with an unreacted amide compound.
  • the production process can be simplified by a continuous process of producing an N-substituted amide compound / amide compound copolymer by the reaction (step (3)).
  • the production method according to the present invention does not require a multi-step step requiring high temperature conditions for the synthesis of the N-substituted amide compound, which can contribute to cost reduction.
  • the production method according to the present invention is an enzymatic reaction at a low temperature (for example, 100 ° C.
  • the impurities required for purification are extremely limited, and even if these impurities are present, polymerization delay, cross-linking, gelation, etc. are not caused.
  • the currently used method by reaction at high temperature is extremely likely to generate other functional monomers that cause cross-linking due to condensation of raw materials, chain transfers, condensates that lead to polymerization delay, and aromatic compounds. It was expensive and a purification process was essential. Therefore, the second reaction solution of the present invention, which does not cause polymerization delay and is extremely unlikely to cause reactions such as cross-linking and gelation, can omit the purification step. Therefore, in the present invention, the N-substituted amide compound / The amide compound copolymer can be produced very efficiently.
  • the step (2) and the step (3) can be a continuous process as described above, but if necessary, a second reaction is performed between the step (2) and the step (3). It may include a step of adding an amide compound and / or other monomer to the liquid. Further, in the step (2), an amide compound and / or another monomer may be added to the second reaction solution.
  • the manufacturing method according to the present invention may include the following steps (2a) and / or steps (2b) between the steps (2) and the steps (3), if necessary.
  • Step (2a) ammonia produced by the amide transesterification reaction between the amide compound and the amine is removed from the second reaction solution.
  • this step is not essential, it is preferably performed to obtain an N-substituted amide compound / amide compound copolymer that does not contain ammonia as a contaminant.
  • Ammonia may be removed by means such as aeration, or if there is no practical problem in the presence of the ammonia salt, it may be neutralized with an acid such as hydrochloric acid.
  • Step (2b) In this step, recombinant bacteria expressing an enzyme such as amidase are removed from the second reaction solution. Although this step is not essential, it is preferably performed to obtain an N-substituted amide compound / amide compound copolymer that does not contain recombinant bacteria as a contaminant. Conventional general-purpose methods such as centrifugation, membrane filtration, cake filtration, agglomeration separation, and activated carbon treatment can be applied alone or in combination for removing recombinant bacteria.
  • a recombinant bacterium expressing amidase was prepared by transforming a host microorganism with the expression vector of 2 above.
  • DN1 strain see Patent Document 4
  • DN1 strain competent cell solution was ice-cooled for 30 minutes. The mixed solution was placed in a cuvette and subjected to electric pulse treatment at 20 kV / cm and 200 OHMS by a gene transfer device (Gene Pulser, BIO RAD).
  • the mixed solution was allowed to stand for 10 minutes under ice-cooling, and heat-shocked at 37 ° C. for 10 minutes.
  • MYK medium 0.5% polypeptone, 0.3% bactoyeast extract, 0.3% bactomolt extract, 0.2% K 2 HPO 4 , 0.2% KH 2 PO 4
  • 10 ⁇ g / ml was applied to MYK agar medium containing canamycin and cultured at 30 ° C. for 3 days. Colonies with confirmed plasmid introduction were isolated as amidase-expressing recombinant bacteria.
  • amidase activity of the recombinant bacteria in 3 above was measured by the following method.
  • a culture solution containing a recombinant was added to a reaction solution (100 mM acrylamide, 50 mM N, N-dimethyl-1,3-propanediamine, 50 mM sodium hydrogen carbonate buffer (pH 9)) and reacted at 37 ° C. overnight. ..
  • the reaction solution obtained by filtering the cells using a 0.45 ⁇ m filter was subjected to gas chromatography analysis, and N- [3- [3-] produced by an amide exchange reaction between acrylamide and N, N-dimethyl-1,3-propanediamine.
  • the concentration of (dimethylamino) propyl] acrylamide was measured.
  • Analytical conditions Analytical equipment: Gas chromatograph GC-2014S (manufactured by Shimadzu Corporation) Detector: FID (detected at 250 ° C) Column: Rtx-5Amine (30 mm x 0.25 mm I.D. x 1.0 ⁇ m df Restek) Column temperature: 50 °C, 2min ⁇ (20 °C / min) ⁇ 160 °C, 0min ⁇ (30 °C / min) ⁇ 240 °C, 5min
  • Table 3 shows the amidase activity of the recombinant bacteria by the amount of N- [3- (dimethylamino) propyl] acrylamide produced (relative value with the amount produced in the recombinant bacteria by the expression vector pSJTA37 as 1). The production of N- [3- (dimethylamino) propyl] acrylamide was confirmed in all recombinant bacteria.
  • Example 1 Production of acrylamide / N-substituted acrylamide copolymer
  • 1. Process (1) Prepared in Example 1 with 3.2% (v / v) of 50% acrylamide aqueous solution (Mitsubishi Chemical Corporation) and 2.3% of N, N-dimethyl-1,3-propanediamine (Tokyo Chemical Industry Co., Ltd.). A first reaction solution containing the recombinant bacteria (1%) and water was prepared in a reaction vessel. A jacketed stirring tank having an internal volume of 500 mL was used as the reaction tank. The reaction solution was adjusted to pH 10 by adding a 6M HCl solution. The amidase activity in the first reaction solution was 1000 U / L.
  • Steps (2) and (2b) The temperature of the reaction solution was controlled to 20 ° C., and the amide exchange reaction was carried out for 7 hours with stirring so that the recombinant bacteria were uniformly dispersed to obtain a second reaction solution.
  • the obtained second reaction solution was membrane-filtered with a filter having a pore size of 0.45 ⁇ m to remove recombinant bacteria.
  • the second reaction solution from which the recombinant bacteria had been removed was subjected to the same gas chromatography analysis as in Example 1 (4), and was produced by an amide exchange reaction between acrylamide and N, N-dimethyl-1,3-propanediamine.
  • the concentration of N- [3- (dimethylamino) propyl] acrylamide was measured.
  • the acrylamide concentration was 0.1% and the N- [3- (dimethylamino) propyl] acrylamide concentration was 1.1%.
  • the temperature of the jacket was raised to 70 ° C., 15,000 ppm of 2,2'-azobis (2-methylpropioamidine) dihydrochloride was added as a pair of monomers under a nitrogen seal, and the mixture was stirred for 3 hours to carry out a polymerization reaction.
  • a part of the reaction solution after the polymerization reaction was taken, and the residual monomer was measured by liquid chromatography (column: ODP2 HP4E 250 mm ⁇ 4.6 mm UV 207 nm detection).
  • the polymerization conversion rate of the monomer was 98% (see Table 4).
  • the polymerization conversion rate of the monomer was calculated by the following formula (the same applies to the following Examples and Comparative Examples).
  • Polymerization conversion of monomer (%) ⁇ [(concentration of amide compound before the start of polymerization reaction) + (concentration of N-substituted amide compound before start of polymerization reaction)]-[(concentration of amide compound after completion of polymerization reaction) ) + (Concentration of N-substituted amide compound after completion of polymerization reaction)] ⁇ / [(Concentration of amide compound before initiation of polymerization reaction) + (Concentration of N-substituted amide compound before initiation of polymerization reaction)] Moreover, the solution temperature at the time of polymerization was measured with a digital thermometer (the same applies to the following Examples and Comparative Examples). The maximum temperature reached of the solution during polymerization was 75.4 ° C (see FIG. 1).
  • Example 1 As a result, the conversion rate of the monomer was 98% (see Table 4), and the maximum temperature reached by the solution during polymerization was 78.6 ° C. (see FIG. 1). From these results, in Example 1, impurities (Michael addition reaction product of amide compound and amine, hydrolyzate of amide compound, ammonia or salt thereof) were added to the second reaction solution obtained by the enzymatic reaction of the present invention. , Recombinant cell-derived components, medium components, etc.), but it can be confirmed that the conversion rate equivalent to that of Comparative Example 1 in which the reaction solution containing no of these impurities was subjected to the polymerization reaction was achieved. It was. Further, it was found that Example 1 had a lower maximum temperature reached during the polymerization reaction than Comparative Example 1, and the polymerization reaction could be safely carried out.
  • Example 2 Production of acrylamide / N-substituted acrylamide copolymer
  • An amide exchange reaction was carried out in the same manner as in Example 1 except that the reaction time was 24 hours and the pH of the reaction solution was 9, except that the concentration of the recombinant bacteria was 3.7%, the reaction time was 24 hours, and a second reaction solution was obtained. It was.
  • the amidase activity in the first reaction solution was 16000 U / L.
  • the acrylamide concentration of the second reaction solution from which the recombinant bacteria had been removed was 4.3%, and the N- [3- (dimethylamino) propyl] acrylamide concentration was 6.5%.
  • Example 2 the polymerization reaction was carried out in the same manner as in the step (3) of Example 2, and the polymerization conversion rate of the monomer and the solution temperature at the time of polymerization were measured.
  • the conversion rate of the monomer was 99% (see Table 4), and the maximum temperature reached by the solution during polymerization was 89.8 ° C. (see FIG. 2).
  • impurities Moichael addition reaction product of amide compound and amine, hydrolyzate of amide compound, ammonia or its like
  • Example 3 Production of acrylamide / N-substituted acrylamide copolymer
  • the second reaction solution was subjected to the amide exchange reaction in the same manner as in Example 1 except that the reaction time was 24 hours and the pH of the reaction solution was 9, except that the concentration of the recombinant bacteria was 7.7% and the concentration of the recombinant bacteria was 5.0%.
  • the amidase activity in the first reaction solution was 12000 U / L.
  • the acrylamide concentration of the second reaction solution from which the recombinant bacteria had been removed was 1.3%, and the N- [3- (dimethylamino) propyl] acrylamide concentration was 8.6%.
  • Example 3 As a result, the conversion rate of the monomer was 99% (see Table 4), and the maximum temperature reached by the solution during polymerization was 77.3 ° C. (see FIG. 3). From these results, also in Example 3, impurities (Michael addition reaction product of amide compound and amine, hydrolyzate of amide compound, ammonia or its like) are contained in the second reaction solution obtained by the enzymatic reaction of the present invention. It was confirmed that even in the presence of salts, recombinant bacterial cell-derived components, medium components, etc.), the same conversion rate as in Comparative Example 3 in which the reaction solution containing no of these impurities was subjected to the polymerization reaction was achieved. did it. Further, it was found that the maximum temperature reached during the polymerization reaction of Example 3 was lower than that of Comparative Example 3, and the polymerization reaction could be safely carried out.
  • SEQ ID NO: 1 Amino acid sequence of amidase of Rhodococcus erythropolis strain TA37
  • SEQ ID NO: 2 Amino acid sequence of amidase of Rhodococcus erythropolis SK121
  • SEQ ID NO: 3 Amino acid sequence of amidase of Rhodococcus erythropolis PR4
  • SEQ ID NO: 4 Amino acid sequence of amidase of Rhodococcus qingshengii CW25 No. 5: Gordonia alkanivorans NBRC 16433 amidase amino acid sequence No. 6: Arthrobacter sp. MWB30 amidase amino acid sequence No. 7: Pseudonocardia sp.

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5818346A (ja) 1981-07-20 1983-02-02 ドイチエ・テキサコ・ア−ゲ− Ｎ−置換アクリルアミド及びメタクリルアミドの製造方法
JPH10337185A (ja) 1997-06-06 1998-12-22 Mitsubishi Rayon Co Ltd アスパルターゼ活性を有する新規なタンパク質および該タンパク質をコードする遺伝子ｄｎａ
JP2011200132A (ja) 2010-03-24 2011-10-13 Mitsubishi Rayon Co Ltd ニトリルヒドラターゼ遺伝子を欠失又は不活性化させた微生物
RU2539033C1 (ru) 2013-10-21 2015-01-10 Федеральное государственное унитарное предприятие "Государственный научно-исследовательский институт генетики и селекции промышленных микроорганизмов" (ФГУП "ГосНИИгенетика") РЕКОМБИНАНТНЫЙ ШТАММ БАКТЕРИЙ Rhodococcus rhodochrous, ОБЛАДАЮЩИЙ КОНСТИТУТИВНОЙ АЦИЛИРУЮЩЕЙ АКТИВНОСТЬЮ, И СПОСОБ СИНТЕЗА N-ЗАМЕЩЕННЫХ АКРИЛАМИДОВ С ИСПОЛЬЗОВАНИЕМ ЭТОГО ШТАММА В КАЧЕСТВЕ БИОКАТАЛИЗАТОРА
CN105348130A (zh) * 2015-12-02 2016-02-24 天津久日新材料股份有限公司 一种n、n-二甲基丙基丙烯酰胺的制备方法
CN105439890A (zh) * 2015-12-31 2016-03-30 沈阳化工研究院有限公司 一种催化酰胺化制备n-（3-二甲氨基丙基）丙烯酰胺的方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5818346A (ja) 1981-07-20 1983-02-02 ドイチエ・テキサコ・ア−ゲ− Ｎ−置換アクリルアミド及びメタクリルアミドの製造方法
JPH10337185A (ja) 1997-06-06 1998-12-22 Mitsubishi Rayon Co Ltd アスパルターゼ活性を有する新規なタンパク質および該タンパク質をコードする遺伝子ｄｎａ
JP2011200132A (ja) 2010-03-24 2011-10-13 Mitsubishi Rayon Co Ltd ニトリルヒドラターゼ遺伝子を欠失又は不活性化させた微生物
RU2539033C1 (ru) 2013-10-21 2015-01-10 Федеральное государственное унитарное предприятие "Государственный научно-исследовательский институт генетики и селекции промышленных микроорганизмов" (ФГУП "ГосНИИгенетика") РЕКОМБИНАНТНЫЙ ШТАММ БАКТЕРИЙ Rhodococcus rhodochrous, ОБЛАДАЮЩИЙ КОНСТИТУТИВНОЙ АЦИЛИРУЮЩЕЙ АКТИВНОСТЬЮ, И СПОСОБ СИНТЕЗА N-ЗАМЕЩЕННЫХ АКРИЛАМИДОВ С ИСПОЛЬЗОВАНИЕМ ЭТОГО ШТАММА В КАЧЕСТВЕ БИОКАТАЛИЗАТОРА
CN105348130A (zh) * 2015-12-02 2016-02-24 天津久日新材料股份有限公司 一种n、n-二甲基丙基丙烯酰胺的制备方法
CN105439890A (zh) * 2015-12-31 2016-03-30 沈阳化工研究院有限公司 一种催化酰胺化制备n-（3-二甲氨基丙基）丙烯酰胺的方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
"Current Protocols in Molecular Biology", 1987, JOHN WILEY & SONS
"GenBank", Database accession no. JX894244
"Molecular Cloning, A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
CAS, no. 3845-76-9
TORRE, OLIVER ET AL.: "Study of the Chemoselectivity in the Aminolysis Reaction of Methyl Acrylate Catalysed Lipase B from Candida antarctica", ADV. SYNTH. CATAL., vol. 347, 2005, pages 1007 - 1014, XP055788758 *

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