WO2013151139A1 - Method for producing 1,5-pentamethylenediamine, method for producing 1,5-pentamethylenediisocyanate, method for producing polyisocyanate composition, and method for storing catalyst cell - Google Patents

Method for producing 1,5-pentamethylenediamine, method for producing 1,5-pentamethylenediisocyanate, method for producing polyisocyanate composition, and method for storing catalyst cell Download PDF

Info

Publication number
WO2013151139A1
WO2013151139A1 PCT/JP2013/060355 JP2013060355W WO2013151139A1 WO 2013151139 A1 WO2013151139 A1 WO 2013151139A1 JP 2013060355 W JP2013060355 W JP 2013060355W WO 2013151139 A1 WO2013151139 A1 WO 2013151139A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
base sequence
sequence encoding
changed
reaction
Prior art date
Application number
PCT/JP2013/060355
Other languages
French (fr)
Japanese (ja)
Inventor
大資 望月
正 安楽城
明子 夏地
智美 酒井
友則 秀崎
山崎　聡
俊彦 中川
Original Assignee
三井化学株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 三井化学株式会社 filed Critical 三井化学株式会社
Priority to JP2014509210A priority Critical patent/JP5899309B2/en
Publication of WO2013151139A1 publication Critical patent/WO2013151139A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C263/00—Preparation of derivatives of isocyanic acid
    • C07C263/10—Preparation of derivatives of isocyanic acid by reaction of amines with carbonyl halides, e.g. with phosgene
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • 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
    • 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
    • C12P13/001—Amines; Imines
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01018—Lysine decarboxylase (4.1.1.18)

Definitions

  • the present invention relates to a method for producing 1,5-pentamethylenediamine, a method for producing 1,5-pentamethylene diisocyanate, a method for producing a polyisocyanate composition, and a method for preserving catalyst cells.
  • 1,5-pentamethylenediamine is attracting attention as a biomass-derived polymer material, for example, a polyurethane material or a polyamide material.
  • 1,5-pentamethylenediamine and / or 1,5-pentamethylenediamine salt 1,5-pentamethylenediamine
  • lysine and / or a solution of lysine salt (lysine) is used as a raw material.
  • a method of obtaining a solution of 1,5-pentamethylenediamine by allowing a lysine decarboxylase (LDC) derived from microorganisms to act thereon see, for example, Patent Document 1 below.
  • 1,5-pentamethylenediamine or 1,5-pentamethylenediamine salt is industrially produced from lysine or a lysine salt using lysine decarboxylase
  • the lysine decarboxylase used is expensive. Therefore, it is desired to reduce the amount of use as much as possible.
  • proteins or peptides derived from bacterial cells (microorganisms) present in the 1,5-pentamethylenediamine solution are: This is one of the causes of defects in the surface appearance of polyamide resin films and the like.
  • the ratio of the weight of the microbial cells to be used with respect to the total weight of lysine during the reaction is suppressed to 0.002 or less, the aeration rate to the reaction solution is limited to 0.3 vvm or less, and organic It has been proposed to adjust the pH by neutralization with an acid.
  • the reaction rate of the catalyst can be improved, the amount of catalyst used can be reduced, and proteins and peptides can be reduced to prevent surface appearance defects such as fish eyes (for example, (See Patent Documents 2 and 3 below.)
  • lysine decarboxylase has a reaction optimum pH of 5 to 6, and it is known that a decrease in reaction activity occurs on the alkali side (for example, see Non-patent Document 1 below).
  • Non-Patent Document 2 it is known that lysine decarboxylase is subject to reaction inhibition by the produced 1,5-pentamethylenediamine (see, for example, Non-Patent Document 2 below).
  • Patent Document 1 discloses that 1M lysine hydrochloride at 50 mg / L of purified enzyme. Although it is disclosed that 0.97M 1,5-pentamethylenediamine can be produced by decarboxylation, the purification of the enzyme has a disadvantage that it takes a great deal of cost.
  • Patent Documents 2 and 3 disclose that the amount of catalyst used can be reduced (for example, in Example 1 of Patent Document 3, the ratio of the weight of the microbial cells used to the total weight of lysine is 0.0017).
  • the lysine concentration of the reaction solution is about 10%, and it is not an excellent method because of its poor volumetric efficiency for industrial production.
  • Non-Patent Document 2 lysine decarboxylase is inhibited by the generated 1,5-pentamethylenediamine. Enzyme requirement increases. In this respect, if the lysine concentration is lowered as described above, the amount of 1,5-pentamethylenediamine derived from the lysine is reduced, so that the inhibitory effect is reduced and the amount of enzyme required for lysine can be reduced.
  • the production equipment becomes large. Therefore, in industrial production, it is important that the lysine concentration is as high as possible and the amount of enzyme is as small as possible.
  • neutralization with organic acids described in Patent Documents 2 and 3 above is preferable because it becomes a raw material when producing polyamide, but 1,5-pentamethylenediamine is converted as it is or into isocyanate or the like.
  • an impurity When used as an impurity, it becomes an impurity, which is not preferable.
  • a part of polyamide is produced by heating in the purification process, it becomes an impurity to the product and the quality is deteriorated. Therefore, it is important in industrial production that neutralization during the reaction is not performed as much as possible.
  • lysine decarboxylase can be produced in a biological fungus body, and such a fungus body (catalyst fungus body) is collectively produced in a large amount from the viewpoint of cost reduction, Used in line with production plans for 1,5-pentamethylenediamine.
  • the activity of the produced lysine decarbonase may be reduced.
  • An object of the present invention is to provide a method for producing 1,5-pentamethylenediamine at a low cost and in a good yield without adjusting the pH of the reaction solution, and the 1,5-pentamethylene diamine obtained by the method. It is intended to provide a method for producing 1,5-pentamethylene diisocyanate from pentamethylene diami, and further a method for producing a polyisocyanate composition from 1,5-pentamethylene diisocyanate obtained by the method.
  • Another object of the present invention is to provide a method capable of stably storing lysine decarboxylase for a long period of time.
  • the inventors of the present invention have reduced or stopped the reaction rate of lysine decarboxylase due to the oxidant present in the reaction solution, and the enzyme cannot be reduced below a predetermined amount. I found out.
  • the oxidation of the enzyme by the oxidant present in the reaction system, it is possible to prevent a decrease in the enzyme activity. Even if the amount of the enzyme is reduced more than the conventional technology without purifying the enzyme, the yield is 1 It was found that 5-pentamethylenediamine can be produced. Further, since the lysine concentration in the reaction solution is increased and the enzyme activity can be prevented from decreasing even when the reaction time is prolonged, the reaction can be completed. Furthermore, the inventors have found that the reaction can be completed without neutralizing the reaction solution, and completed the present invention.
  • the present invention [1] In a reaction system in which the dissolved oxygen concentration is a saturated dissolved oxygen concentration within 1 hour, L-lysine and / or a salt thereof is converted by lysine decarboxylase and / or mutant lysine decarboxylase.
  • the amount of lysine decarboxylase and / or mutant lysine decarboxylase is 0.0003 parts by mass or more and 0.0015 parts by mass or less in terms of dry cell weight per 1 part by mass of L-lysine and / or its salt.
  • a process for producing 1,5-pentamethylenediamine [7] The method for producing 1,5-pentamethylenediamine according to [6], wherein the step of removing oxygen in the reaction system is a step of replacing dissolved oxygen with an inert gas, [8] The method for producing 1,5-pentamethylenediamine according to [6], wherein the redox potential of the reducing agent is lower than that of physiological saline, [9]
  • the reducing agent is a mercapto compound, sulfide, hydrosulfide, reductive sulfur oxyacid salt, thiourea and its derivatives, a cyclic compound having a hydroxyl group and / or a carboxyl group, a flavonoid compound, a nitrogen-containing complex.
  • 1,5-pentamethylenediamine which is at least one selected from the group consisting of a ring compound, a hydrazyl group compound, and a mucopolysaccharide having a uronic acid group Method
  • the mutant lysine decarboxylase is 137, 138, 286, 290, 295, 303, 317, 335, 352, 353, 386, 443, 466, in the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing.
  • any one of [1] to [9], wherein at least one of the amino acid residues at 475, 553, 710 and 711 is a mutant lysine decarboxylase substituted with another amino acid residue A process for producing 1,5-pentamethylenediamine according to claim 1, [11] A mutant type in which the mutant lysine decarboxylase is substituted with other amino acid residues at the 290th, 335th, 475th, and 711st amino acid residues in the amino acid sequence set forth in SEQ ID NO: 4 in the Sequence Listing The method for producing 1,5-pentamethylenediamine according to [10], which is lysine decarboxylase, [12] The mutant lysine decarboxylase is substituted with other amino acid residues at the 286th, 290th, 335th, 475th and 711th amino acid residues in the amino acid sequence set forth in SEQ ID NO: 4 in the Sequence Listing.
  • the method for producing 1,5-pentamethylenediamine according to [10], which is a mutant lysine decarboxylase, [13] A process for producing 1,5-pentamethylene diisocyanate, characterized in that 1,5-pentamethylenediamine or a salt thereof obtained by the method described in [1] above is isocyanated, [14]
  • the 1,5-pentamethylene diisocyanate obtained by the method described in [13] above is modified so as to contain at least one of the following functional groups (a) to (e):
  • Reducing lysine decarboxylase and / or mutant lysine decarboxylase A method for preserving catalytic cells, characterized by storing in the presence of an agent; [16] The method for preserving a catalytic cell according to
  • the method for producing 1,5-pentamethylenediamine of the present invention since the time during which the dissolved oxygen concentration in the reaction system is the saturated dissolved oxygen concentration is within one hour, lysine decarboxylase or / and mutant lysine decarboxylation Inactivation of the enzyme activity of the enzyme can be suppressed, and even with a high concentration lysine reaction solution, a low enzyme amount and a good yield are obtained, and 1,5-pentamethylenediamine is obtained without adjusting the pH of the reaction solution. be able to. As a result, high-quality 1,5-pentamethylenediamine can be produced at low cost.
  • high-quality 1,5-pentamethylene diisocyanate and polyisocyanate composition can be obtained at a low cost and in a high yield. You can get things.
  • FIG. 1 schematically plots the relationship between the dissolved oxygen concentration and time in the lysine decarboxylation reaction, with the Y axis as the ratio (%) of the dissolved oxygen concentration to the saturated dissolved oxygen concentration and the X axis as the time (minutes). It is an example of the schematic correlation diagram which shows a correlation line.
  • FIG. 2 schematically plots the relationship between the dissolved oxygen concentration and time in the lysine decarboxylation reaction, with the Y axis as the ratio (%) of the dissolved oxygen concentration to the saturated dissolved oxygen concentration and the X axis as the time (minutes). It is another example of the schematic correlation diagram which shows a correlation line.
  • 1,5-pentamethylenediamine refers to 1,5-pentanediamine (H 2 N (CH 2 ) 5 NH 2 ). 1,5-pentamethylenediamine is a useful compound as a polymer raw material or a raw material for synthesizing pharmaceutical intermediates.
  • Lysine decarboxylase The lysine decarboxylase in the present invention is classified into enzyme number EC 4.1.1.18 based on the report of the International Biochemical Union (IUB) Enzyme Committee, and pyridoxal.
  • Phosphoric acid is required as a coenzyme, and L-lysine (also referred to as lysine) to 1,5-pentamethylenediamine (also referred to as pentane 1,5-diamine, 1,5-pentamethylenediamine, PDA) And an enzyme that catalyzes a reaction that produces carbonic acid, a microbial cell that produces this enzyme in a high amount by a technique such as genetic recombination, and a processed product thereof.
  • L-lysine also referred to as lysine
  • 1,5-pentamethylenediamine also referred to as pentane 1,5-diamine, 1,5-pentamethylenediamine, PDA
  • an enzyme that catalyzes a reaction that produces carbonic acid, a microbial cell that produces this enzyme in a high amount by a technique such as genetic recombination, and a processed product thereof.
  • the origin of the lysine decarboxylase of the present invention is not particularly
  • lysine decarboxylase examples include, for example, Bacillus halodurans, Bacillus subtilis, Escherichia coli, Vibrio cholera, and Vibrio cholera.
  • Salmonella typhimurium Hafnia albei
  • Neisseria meningitidis Thermoplasma acid pi
  • examples thereof include those derived from microorganisms such as Corynebacterium glutamicum. From the viewpoint of safety, preferably, those derived from Escherichia coli are used.
  • lysine decarboxylase activity means an activity that catalyzes a reaction in which lysine is decarboxylated and converted to 1,5-pentamethylenediamine. In the present invention, it can be calculated by measuring the amount of 1,5-pentamethylenediamine produced from lysine by high performance liquid chromatography (HPLC).
  • the unit of activity is 1 unit (U) for producing 1 ⁇ mol of 1,5-pentamethylenediamine per minute, and the cell activity is the enzyme activity (U / mg dry cells) per 1 mg of dry cell equivalent weight.
  • the dry cell equivalent weight means the weight that is dried and does not contain moisture.
  • the cell body is separated from the liquid containing the cell body (bacterial body liquid) by a method such as centrifugation or filtration, and the weight becomes constant. Dried, and the weight in terms of dry cells can be determined by measuring the weight.
  • Bacteria The bacteria in the present invention are classified into a plurality of types. In order to avoid misunderstandings, the present invention defines as follows.
  • Catalyst cells Cells that produce lysine decarboxylase at a high level and have higher lysine decarboxylation activity than wild-type strains are referred to as “catalyst cells”. Furthermore, live catalytic cells are defined as “catalytic live cells”, catalytic cells that have stopped growing as “catalytic resting cells”, and catalytic cells that have lost their growth ability as “catalytically dead cells” And (4) Mutant lysine decarboxylase The mutant lysine decarboxylase used in the present invention is mainly a genetic recombination technique in which at least one amino acid residue in the amino acid sequence of wild-type lysine decarboxylase is other.
  • the lysine decarboxylase is characterized by having a mutation substituted in the amino acid residue of lysine and having improved enzyme activity of lysine decarboxylase itself.
  • the amino acids in the amino acid sequence correspond to amino acid residues in lysine decarboxylase, and they are in a corresponding relationship with each other.
  • an amino acid an amino acid represented as an amino acid sequence is indicated, and when referred to as an amino acid residue, an amino acid residue contained in lysine decarboxylase is indicated.
  • the method for preparing a mutant lysine decarboxylase gene may be any known method for introducing a mutation, and can generally be performed by a known method.
  • site-directed mutagenesis (Kramer, W. and frita, HJ, Methods in Enzymology, 1987, 154, 350), recombinant PCR (PCR Technology, Stockton Press, 1989)
  • Examples include a method of chemically synthesizing a specific portion of nucleic acid, a method of treating a gene with hydroxyamine, and a method of treating a strain having the gene with ultraviolet irradiation or a chemical agent such as nitrosoguanidine or nitrous acid.
  • a site-specific mutation method is preferable. Specifically, it is a method of causing site-specific substitution using a commercially available kit based on the wild-type lysine decarboxylase gene.
  • the position of the insertion, deletion or substitution may be any position as long as the lysine decarboxylation activity is not lost.
  • the number of inserted, deleted or substituted amino acid residues include 1 amino acid residue or 2 amino acid residues or more, for example, 1 amino acid residue to 10 amino acid residues, preferably 1 amino acid residue to 5 amino acid residues. Amino acid residues are mentioned.
  • the mutant lysine decarboxylase in the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing, at least one amino acid in the amino acid sequence is substituted with another amino acid whose activity is increased. Has been.
  • amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing is an amino acid sequence of a protein generated from the DNA sequence set forth in SEQ ID NO: 3 in the sequence listing, wherein the N-terminal methionine is the first amino acid,
  • the 129th amino acid is a wing domain
  • the 130th to 183rd amino acids are linker domains
  • these 1st to 183rd amino acids form a 10-mer forming domain.
  • amino acids 184 to 417 are pyridoxal phosphate (PLP enzyme) common domains
  • amino acids 418 to 715 are substrate entrances
  • these amino acids 184 to 715 form an active region domain. .
  • the amino acid present in the 10-mer forming domain and / or the active region domain in the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing is substituted with another amino acid.
  • amino acids present in the wing domain and / or linker domain in the demerization domain, and / or amino acids present in the pyridoxal phosphate common domain and / or substrate entry / exit in the active region domain It is substituted with another amino acid.
  • the amino acid residue corresponding to the amino acid in the amino acid sequence is substituted with another amino acid residue.
  • the mutant lysine decarboxylase is preferably at least 137, 138, 286, 290, 295, 303, 317, 335, 352, 353, 386, 443, 466, 475, 553, the 711th amino acid residue and 14 28, 39, 64, 67, 70, 75, 79, 83, 84, 85, 88, 89, 94, 95, 98, 99, 104, 112, 119, 139, 143, 145, 148, 182, 184 Mutations in which one or more amino acid residues at 253, 262, 430, 446, 460, 471, 506, 524, 539, 544, 546, 623, 626, 636, 646, 648 are substituted with other amino acid residues Type enzymes.
  • the methionine at the N-terminal of the amino acid sequence of the protein generated from the DNA sequence set forth in SEQ ID NO: 3 (SEQ ID NO: 4 in the sequence listing) is number 1 As amino acids 137, 138, 286, 290, 295, 303, 317,
  • the amino acid sequence after the change is not particularly limited as long as it has better properties than before the change, for example, improved specific activity, properties resistant to pH change during the reaction, resistance to reaction products, relaxation of inhibition, etc.
  • the amino acid sequence produced by the sequences described in -6 is particularly preferred.
  • the 14th amino acid in the amino acid present in the 10-mer forming domain was changed from Phe to Gln.
  • the 14th amino acid in the wing domain is changed from Phe to Gln, and the 28th amino acid is changed from Arg to Ile.
  • 39th amino acid changed from Arg to Ile 39th amino acid changed from Arg to Val
  • 64th amino acid changed from Leu to Lys 64th amino acid changed from Leu to Lys
  • 67th amino acid changed from Cys to Thr The 67th amino acid changed from Cys to Leu, the 70th amino acid changed from Ile to Leu, the 70th amino acid changed from Ile to Pro, the 75th amino acid changed to Glu Changed from Pro to Pro, 75th amino acid changed from Glu to His, 79th amino acid changed from Leu to Ile, 83rd amino acid changed from Ala to Leu, 84th amino acid From Asn Changed to Asp, changed the 84th amino acid from Asn to Thr, changed the 85th amino acid from Thr to Pro, changed the 88th amino acid from Thr to Lys, changed
  • Preferred mutant lysine decarboxylase includes an active region domain, specifically, 290th, 335, 475, 711th amino acid residues, 286, 290, 335, 475, 711th amino acid residues, 148 , 646th amino acid residue, 471, 626th amino acid residue, and mutated enzyme in which the 626th and 646th amino acid residues are substituted with other amino acid residues.
  • the mutant enzyme in which the 290th, 335th, 475th, and 711st amino acid residues are substituted with other amino acid residues is N of the amino acid sequence of the protein generated from the DNA sequence described in SEQ ID NO: 3 in the Sequence Listing. This refers to a mutant enzyme having a sequence in which the amino acid at the 4th position of 290, 335, 475, and 711 is replaced with an amino acid different from the original amino acid, with the terminal methionine as the first amino acid.
  • the mutated enzyme in which the 286th, 290th, 335th, 475th, and 711th amino acid residues in the present invention are substituted with other amino acid residues is the amino acid sequence of a protein generated from the DNA sequence shown in SEQ ID NO: 3 in the Sequence Listing.
  • the N-terminal methionine is the first amino acid, and the 286, 290, 335, 475, and 711th amino acids are substituted with amino acids different from the original amino acids.
  • the mutated enzyme in which the 148th and 646th amino acid residues are substituted with other amino acid residues is the N-terminal methionine of the amino acid sequence of the protein generated from the DNA sequence described in SEQ ID NO: 3 in the Sequence Listing. It refers to a mutant enzyme having a sequence in which two amino acids at the 148th and 646th positions are substituted with amino acids different from the original amino acid as the first amino acid.
  • the mutated enzyme in which the 471st and 626th amino acid residues are substituted with other amino acid residues is the N-terminal methionine of the amino acid sequence of the protein generated from the DNA sequence described in SEQ ID NO: 3 in the Sequence Listing.
  • the first amino acid refers to a mutant enzyme having a sequence in which the amino acids at two positions, 471 and 626, are substituted with amino acids different from the original amino acid.
  • the mutated enzyme in which the 626th and 646th amino acid residues are substituted with other amino acid residues is the N-terminal methionine of the amino acid sequence of the protein produced from the DNA sequence described in SEQ ID NO: 3 in the Sequence Listing. This refers to a mutant enzyme having a sequence in which two amino acids at the 626th and 646th positions are substituted with amino acids different from the original amino acid as the first amino acid.
  • amino acid sequence after the change is not particularly limited as long as it has better properties than the enzyme of the original sequence, but the amino acid sequences generated by the sequences shown in Tables 1 to 6 are particularly preferred.
  • the modified mutant lysine decarboxylase is shown as a modified amino acid of the amino acid sequence.
  • the modified mutant lysine decarboxylase may be represented as a modified amino acid sequence. it can.
  • a base sequence encoding Phe which is the 14th amino acid among the amino acid residues present in the 10-mer forming domain Changed from TTT to CAA, the base sequence encoding Gln, the base sequence encoding Leu, the 22nd amino acid, from TTT, the base sequence encoding Leu, the 28th amino acid
  • the base sequence encoding Arg is changed from CGT to ATT, which is the base sequence encoding Ile, and the base sequence encoding Arg, the 39th amino acid, is changed from CGT to Ile, the base sequence encoding Ile.
  • the base sequence encoding Cys is changed from TGC to ACC, which is the base sequence encoding Thr, and the base sequence encoding Cys, the 67th amino acid, is changed to TTA, the base sequence encoding TGC to Leu.
  • the base sequence to be loaded is changed from GAG to CCC, which is the base sequence encoding Pro
  • the base sequence encoding Glu, which is the 75th amino acid is changed from GAG to CAC, which is the base sequence encoding His.
  • the base sequence encoding Leu, the 79th amino acid, has been changed from TTG to ATA, the base sequence encoding Ile, and the base sequence encoding the 83rd amino acid, Ala is a base that encodes Leu from GCT
  • the sequence changed to CTG which is the sequence, the base sequence encoding Ala which is the 83rd amino acid is changed to CTA which is the base sequence encoding Leu from GCT, the base sequence encoding Ala which is the 83rd amino acid Is changed from GCT to CTT, which is the base sequence encoding Leu, the base sequence encoding Ala, which is the 83rd amino acid, is changed to ATA, which is the base sequence encoding Ala, from the GCT, the 83rd amino acid
  • the base sequence encoding the acid Ala is changed from GCT to GCC, which is the base sequence encoding Ala, and the base sequence encoding Asn, the 84th amino acid, is the base sequence en
  • the base sequence encoding Asn which is the 84th amino acid, is changed from AAT to ACA, which is the base sequence encoding Thr, and the base sequence encoding Thr, the 85th amino acid, is changed from ACG to Pro.
  • Changed to CCA which is the base sequence that encodes A
  • the base sequence encoding Thr which is the 88th amino acid
  • AAA which is the base sequence that encodes Lys from ACT
  • Thr which is the 88th amino acid
  • the base sequence to be encoded is changed from ACT to AAG, which is the base sequence encoding Lys, and the base sequence encoding Thr, the 88th amino acid, is changed from ACT to AGA, which is the base sequence encoding Arg.
  • the base sequence encoding Gln, which is changed from CAG to ACT, which is the base sequence encoding Thr, the base sequence encoding Glu, the 104th amino acid, is the base sequence encoding GAA to Asn
  • the base sequence encoding Glu, which is the 104th amino acid has been changed from AAA to GA, which is the base sequence encoding Lys, and the base sequence encoding Asp, which is the 112th amino acid, from GAT.
  • GAG which is the base sequence encoding Glu
  • Gln which is the 119th amino acid
  • AAG which is the base sequence encoding Asn
  • GAG GAG
  • Gln which is the 119th amino acid
  • the base sequence coding for CAG is changed from AAG, which is the base sequence encoding Asn
  • the base sequence encoding Gln, the 119th amino acid is changed from CAG to ATT, the base sequence encoding Ile.
  • the nucleotide sequence encoding Gln, the 119th amino acid was changed from CAG to ACC, the nucleotide sequence encoding Thr, and the nucleotide sequence encoding the G119, the 119th amino acid, encoded Ser from CAG.
  • AGT Changed to AGT, the base sequence that encodes Phe, which is the 137th amino acid, and GTC, which is the base sequence that encodes Val, from TTT, and encodes Lys, the 138th amino acid
  • the base sequence is changed from AAA to ATC which is the base sequence encoding Ile
  • the base sequence encoding Tyr which is the 139th amino acid is changed from TAT to GTA which is the base sequence encoding Val
  • the base sequence encoding Tyr, which is the amino acid of is changed from TAT to GTG, which is the base sequence encoding Val
  • the base sequence encoding Tyr, the 139th amino acid is the base sequence encoding TAT to Cys.
  • the base sequence encoding Tyr, the 145th amino acid, has been changed from CAT to CGT, the base sequence encoding Trg from TAT, and the base sequence encoding Tyr, the 145th amino acid, has changed from TAT to Arg Changed to AGA, which is the base sequence that encodes A, the base sequence encoding Cys, which is the 148th amino acid, changed from AGT, which is the base sequence that encodes Ser, to C, which is the 148th amino acid, C
  • the base sequence encoding ys was changed from TGT to TCT, which is the base sequence encoding Ser, and the base sequence encoding Cys, the 148th amino acid, was changed from TGT to TCC, the base sequence encoding Ser.
  • the base sequence encoding Cys, the 148th amino acid has been changed from TGT to TCA, the base sequence encoding Ser, the base sequence encoding the Cys, the 148th amino acid, encodes Ala from TGT
  • the base sequence was changed to GCG
  • the base sequence encoding Cys, the 148th amino acid was changed to GCA
  • the base sequence encoding Tla to Ala the base encoding Ile
  • the 182th amino acid The sequence is changed from ATT to ATG, which is the base sequence encoding Met
  • the base sequence encoding Val the 184th amino acid among the amino acid residues present in the active region domain, is changed from GTA to Ala.
  • GCC which is the base sequence to be loaded
  • the base sequence encoding Val which is the 184th amino acid
  • GTA to GCA which is the base sequence encoding Ala
  • Met which is the 253rd amino acid.
  • the coding base sequence is changed from ATG to CTA, which is the base sequence encoding Leu
  • the base sequence encoding Phe, the 262nd amino acid is changed from TTC to TAT, which is the base sequence encoding Tyr
  • TTC which is the base sequence encoding Tyr
  • the base sequence encoding Ala has been changed from GCT to GAC, the base sequence encoding Asp, and the base sequence encoding the Lys, 290th amino acid, encodes AAA to His.
  • the base sequence encoding Ser which is the 446th amino acid, has been changed from CAT, which is the base sequence encoding Gln, from TCT, and the base sequence encoding Asp, which is the 460th amino acid, encodes Ile from GAT.
  • the base sequence is changed from GAT to TGT, which is the base sequence encoding Cys
  • the base sequence encoding Asp, the 460th amino acid is changed from CAT, which is the base sequence encoding Gln to Gln.
  • the base sequence encoding Asp, which is the 460th amino acid is changed from CAT, which is the base sequence encoding GAT to Pro, and the base sequence encoding the Asp, which is the 460th amino acid, base sequence encoding GAT to Pro.
  • the base sequence encoding Asp, which is the 460th amino acid is changed to CCG, which is the base sequence encoding GAT to Pro, and the base sequence encoding Asp, which is the 460th amino acid.
  • the base sequence encoding Val is changed from GTT to TTA, which is the base sequence encoding Leu
  • the base sequence encoding Val, the 524th amino acid is changed from GTT to CTG, which is the base sequence encoding Leu.
  • the base sequence encoding Ile, the 539th amino acid was changed from ATC to TGC, the base sequence encoding Cys, and the base sequence encoding the 539th amino acid, Ile, copied Lec from ATC.
  • the coding base sequence is changed from ACC to GCG which is the base sequence encoding Ala
  • the base sequence encoding Thr which is the 544th amino acid is changed from ACC to GCT which is the base sequence encoding Ala
  • the base sequence encoding Thr, the 544th amino acid has been changed from ACC to TCT, the base sequence encoding Ser, the base sequence encoding the 544th amino acid, Thr, the base sequence encoding ACC to Ser Changed to TCC, Thr that encodes Thr, the 544th amino acid, changed to CCT, which is the base sequence encoding Pro from ACC, Salt that encodes Thr, the 544th amino acid
  • the sequence was changed from ACC to CCG which is a base sequence encoding Pro
  • the base sequence encoding Ala which is the 546th amino acid was changed from ACA which is the base sequence encoding Ser to Ger
  • the base sequence encoding the amino acid Leu was changed from CTG to GTA, which is the base
  • the base sequence encoding Ala, which is the 623rd amino acid, is changed from TCA, which is the base sequence encoding Phe, from GCA, and the base sequence encoding Ala, the 623th amino acid, is changed from GCA to Phe.
  • TTC which is the base sequence that encodes A
  • CAG which is the base sequence that encodes Gln from GCA
  • the base sequence encoding Lys is changed from AAA to GTG, which is the base sequence encoding Val
  • the base sequence encoding Tyr, the 636th amino acid is changed from TAC to TGT, which is the base sequence encoding Cys.
  • the base sequence encoding Tyr, the 636th amino acid, has been changed from TAC to CCC, the base sequence encoding Pro, the base sequence encoding the 646th amino acid, Ala, encodes Leu from GCC Changed to TTG, which is the base sequence to be encoded, the base sequence encoding Ala, which is the 646th amino acid, changed from ACC which is the base sequence encoding Ile to GCC, and Met, which is the 648th amino acid, is encoded
  • the base sequence is changed from ATG to TCT which is a base sequence encoding Ser
  • the base sequence encoding Met which is the 648th amino acid is changed from ATG to TCC which is a base sequence encoding Ser
  • the base sequence encoding Lys, the 710th amino acid has been changed from AAA to ACG, the base sequence encoding Thr, the base sequence encoding the 711st amino acid, Glu, is the base sequence encoding GAA to Asp
  • the base sequence encoding Phe the 14th amino acid among the amino acid residues present in the wing domain, encodes a base sequence encoding GTT from TTT
  • the base sequence encoding Leu which is the 22nd amino acid, has been changed from CTT to TTG, which is the base sequence encoding Leu, and the base sequence encoding Arg, the 28th amino acid.
  • CGT to ATT which is the base sequence encoding Ile
  • change base sequence encoding Arg, the 39th amino acid, to ATA the base sequence encoding Ile, from the 39th amino acid
  • the base sequence encoding a certain Arg is changed from CGT to ATC which is the base sequence encoding Ile
  • the base sequence encoding the 39th amino acid Arg is the base sequence encoding CGT to Val
  • the base sequence encoding Leu which is the 64th amino acid, has been changed to AAA, which is the base sequence encoding Lys from CTC, and the base sequence encoding Cys, the 67th amino acid.
  • TGC to ACC which is the base sequence encoding Thr
  • Change from 67th amino acid to Cys which is the base sequence encoding Cys from TGC to TTA, which is the base sequence encoding Leu, 70th amino acid
  • the base sequence encoding a certain Ile is changed from ATT to TTG, which is the base sequence encoding Leu
  • the base sequence encoding Ile, the 70th amino acid is changed from ATT to CTG, the base sequence encoding Leu.
  • the base sequence encoding Ile, the 70th amino acid has been changed from ATT to CCG, which is the base sequence encoding Pro
  • the base sequence encoding the Glu the 75th amino acid, copied GAG to Pro.
  • CCC which is the base sequence to be loaded
  • the base sequence encoding Glu which is the 75th amino acid
  • CAG to CAC which is the base sequence encoding His
  • Leu which is the 79th amino acid.
  • the coding base sequence is changed from TTG to ATA which is the base sequence encoding Ile
  • the base sequence encoding Ala which is the 83rd amino acid is changed from CCT to CTG which is the base sequence encoding Leu
  • the nucleotide sequence encoding Ala, the 83rd amino acid has been changed from CCT, which is the nucleotide sequence encoding Leu, from GCT
  • the nucleotide sequence encoding the Ala, the 83rd amino acid encoding Leu
  • the base sequence encoding Ala, the 83rd amino acid, the base sequence encoding Ala, the 83rd amino acid has been changed from GCT to ATA, the base sequence encoding Ala.
  • GCT Changed from GCT to GCC, which is the base sequence encoding Ala, base sequence encoding Asn, which is the 84th amino acid, changed from GAT, which is the base sequence encoding Asp, to the 84th amino acid, 84th amino acid
  • the base sequence encoding Asn is changed from AAT to ACA, which is the base sequence encoding Thr, and the base sequence encoding Thr, the 85th amino acid, is changed from ACG to CCA, which is the base sequence encoding Pro.
  • the base sequence encoding Leu which is the 98th amino acid, was changed from TTA to ATA, which is the base sequence encoding Ile, and the base sequence encoding Gln, the 99th amino acid.
  • Change from CAG to ACT which is the base sequence encoding Thr
  • Change from base sequence encoding Glu, the 104th amino acid, to AAT the base sequence encoding Asn, 104th amino acid
  • the base sequence encoding Glu which is an acid is changed from GAA to AAA which is a base sequence encoding Lys
  • the base sequence encoding Asp which is the 112th amino acid is a base sequence encoding GAT to Glu
  • the base sequence encoding Gln which is the 119th amino acid, is changed from CAG to AAC, which is the base sequence encoding Asn, and the base sequence encoding the G119, which is the 119th amino acid, is changed from CAG to Asn.
  • a base sequence that encodes AAT a base sequence that encodes Gln, which is the 119th amino acid, has been changed from CAG to ATT, which is a base sequence encoding Ile, and Gln, which is the 119th amino acid.
  • the coding base sequence was changed from CAG to ACC, which is the base sequence encoding Thr, and the base sequence encoding Gln, the 119th amino acid, was changed from CAG to AGT, the base sequence encoding Ser.
  • the base sequence encoding Phe which is the 137th amino acid
  • TTT to GTC
  • GTC which is the base sequence encoding Val
  • Lys the 138th amino acid
  • the base sequence encoding Tyr, the second amino acid has been changed from TAT to GTG, the base sequence encoding Val, and the base sequence encoding Tyr, the 139th amino acid, is a base sequence encoding TAT to Cys.
  • Changed to a certain TGC changed the base sequence encoding Tyr, the 139th amino acid from TAT to ACA, the base sequence encoding Thr, copied the 139th amino acid, Tyr.
  • the base sequence to be changed from TAT to TCT which is the base sequence encoding Ser, the base sequence encoding Tyr which is the 139th amino acid is changed from TAT to AGT which is the base sequence encoding Ser
  • the base sequence encoding Tyr, the 139th amino acid has been changed from TAT to AAC, the base sequence encoding Asn, the base sequence encoding Gly, the 143rd amino acid, is a base encoding GGT to Glu Sequence changed to GAA, base sequence encoding Tyr, the 145th amino acid changed from TAT to CGT, the base sequence encoding Arg, base sequence encoding Tyr, the 145th amino acid Changed from TAT to AGA, which is the base sequence encoding Arg, base sequence encoding Cys, which is the 148th amino acid, changed from AGT, which is the base sequence encoding Ser, to the 148th amino acid
  • the base sequence encoding Cys, which is a mino acid is changed from TGT to TCT,
  • the base sequence encoding Met which is the 253rd amino acid, has been changed from ATG to CTA, which is the base sequence encoding Leu, and the base sequence encoding the P262, which is the 262nd amino acid, encodes Tyr from TTC.
  • the base sequence encoding the first amino acid Ile has been changed from ATT to ACA, the base sequence encoding Thr, and the base sequence encoding the 317th amino acid Phe is the base sequence encoding TTC to Gln.
  • a base sequence encoding Glu which is the 430th amino acid of the amino acid residues present at the substrate entrance / exit, in which the base sequence encoding Glu is changed from GAA to TCC, which is the base sequence encoding Ser.
  • the base sequence encoding Asp which is the 460th amino acid, has been changed from TAT, which is the base sequence encoding Ser, to the 466th amino acid, and the base sequence encoding Pro, the 466th amino acid.
  • Change from CCG to AAC, which is the base sequence encoding Asn change base sequence encoding Pro, the 466th amino acid, from CCG to GGC, which is the base sequence encoding Gly, No. 466
  • the base sequence encoding Pro which is the amino acid of CCG, is changed from CCG to TCT, which is the base sequence encoding Ser, and the base sequence encoding Ser, the 471st amino acid, is the base sequence encoding AGC to Tyr.
  • the base sequence encoding r was changed from ACC to TCT, which is the base sequence encoding Ser, and the base sequence encoding Thr, the 544th amino acid, was changed from ACC to TCC, the base sequence encoding Ser.
  • the base sequence encoding Thr, the 544th amino acid has been changed to CCT, the base sequence encoding Pro from ACC, and the base sequence encoding the Thr, the 544th amino acid, encodes Pro from ACC
  • the base sequence was changed to CCG, the base sequence encoding Ala, the 546th amino acid, was changed to AGC, the base sequence encoding Ser from GCA, and the base encoding Leu, the 553th amino acid.
  • the sequence is changed from CTG to GTA which is the base sequence encoding Val, the base sequence encoding Ala which is the 623rd amino acid is changed from GCA to TGT which is the base sequence encoding Cys, No.
  • the base sequence encoding the first amino acid, Ala is changed from GCA to TTT, the base sequence encoding Phe, and the base sequence encoding the 623th amino acid, Ala, is the base sequence encoding GCA to Phe.
  • TTC Changed to a certain TTC, changed the base sequence encoding Ala, the 623th amino acid, from CAA to CAG, the base sequence encoding Gln, and the base sequence encoding the Lys, the 626th amino acid, AAA Changed from GTG to GTG, the base sequence encoding Val, from TAC to TGT, the base sequence encoding Cys from the 636th amino acid, Tyr, the 636th amino acid, the 636th amino acid
  • the modified base sequence is not particularly limited as long as it has better properties than the enzyme of the original sequence, but base sequences generated by the sequences shown in Tables 1 to 6 are particularly preferred.
  • the method for producing the mutant lysine decarboxylase according to the present invention (hereinafter also simply referred to as “manufacturing method”) is the transformation of the mutant lysine decarboxylase.
  • the body is cultured, and the mutant lysine decarboxylase is recovered from at least one of the transformed transformant of the mutant lysine decarboxylase and the cultured product of the transformant.
  • the transformant of the above-mentioned mutant lysine decarboxylase refers to one transformed with an expression vector containing a nucleic acid represented by a base sequence encoding the amino acid sequence of the above-mentioned mutant lysine decarboxylase.
  • a transformant transformed with an expression vector containing a nucleic acid represented by a base sequence encoding the amino acid sequence of the mutant lysine decarboxylase is cultured.
  • the above mutant lysine decarboxylase is produced.
  • the mutation described above shows stable activity even under severe conditions in which the enzyme is easily inactivated, and does not significantly reduce the initial rate of reaction even when compared with the corresponding wild-type lysine decarboxylase.
  • Type lysine decarboxylase can be produced at low cost.
  • the production method of the mutant lysine decarboxylase according to the present invention is represented by a base sequence encoding the amino acid sequence of the mutant lysine decarboxylase. From the step of culturing a transformant transformed with an expression vector containing a nucleic acid (host cell culturing step), and at least one of the cultured transformant and the culture of the transformant, the mutation It only needs to include a step of recovering the type lysine decarboxylase (mutant lysine decarboxylase recovery step), and may further include other steps as necessary.
  • Transformant culture process is performed by transforming with an expression vector containing a nucleic acid represented by a base sequence encoding the amino acid sequence of the wild-type and / or the mutant lysine decarboxylase. It is a step of culturing the body.
  • the transformant is a transformant transformed with an expression vector containing a nucleic acid represented by a base sequence encoding the amino acid sequence of the wild-type and / or the mutant lysine decarboxylase. There is no particular limitation.
  • the transformant examples include those using cells derived from bacteria, yeast, actinomycetes, filamentous fungi and the like as host cells, and those using cells derived from Escherichia coli and Corynebacterium bacteria as host cells are preferred.
  • the nucleic acid is represented by a base sequence encoding the amino acid sequence of the wild type and the mutant lysine decarboxylase.
  • the base sequence encoding the amino acid sequence of the mutant lysine decarboxylase can be synthesized by a method of introducing a mutation point into the base sequence encoding the corresponding wild-type lysine decarboxylase.
  • the expression vector is not particularly limited as long as it contains a nucleic acid represented by a base sequence encoding the amino acid sequence of the wild type and / or the mutant lysine decarboxylase.
  • transformation efficiency and translation are not limited. From the viewpoint of improving the efficiency, a plasmid vector or a phage vector having the following configuration is more preferable.
  • the expression vector is not particularly limited as long as it contains a wild-type and / or base sequence encoding the mutant lysine decarboxylase and can transform the host cell. If necessary, in addition to the base sequence, a base sequence constituting another region (hereinafter, also simply referred to as “other region”) may be included.
  • regions necessary for the transformant to produce wild type and mutant lysine decarboxylase examples include control regions necessary for the transformant to produce wild type and mutant lysine decarboxylase, regions necessary for autonomous replication, and the like.
  • it may further include a base sequence encoding a selection gene that can be a selection marker.
  • control region necessary for producing the wild type and the mutant lysine decarboxylase examples include, for example, a promoter sequence (including an operator sequence that controls transcription), a ribosome binding sequence (SD sequence), and a transcription termination sequence. And so on.
  • a promoter sequence including an operator sequence that controls transcription
  • SD sequence ribosome binding sequence
  • a transcription termination sequence e.g., a transcription termination sequence.
  • the expression vector is used in addition to the base sequence encoding the wild type and / or the mutant lysine decarboxylase, from the viewpoint of the production efficiency of the wild type and the mutant lysine decarboxylase. It preferably contains a promoter sequence. Further, in addition to the promoter sequence, a ribosome binding sequence, a transcription termination sequence and the like may be included.
  • promoter sequences include, for example, trp promoter of tryptophan operon derived from E. coli and lac promoter of lactose operon, PL promoter and PR promoter derived from lambda phage, gluconate synthase promoter (gnt) derived from Bacillus subtilis, alkaline protease, Examples include a promoter (apr), a neutral protease promoter (npr), and an ⁇ -amylase promoter (amy).
  • a promoter sequence that is uniquely modified or designed such as a tac promoter, can be used.
  • ribosome binding sequence examples include sequences derived from E. coli or Bacillus subtilis, but are not particularly limited as long as the sequence functions in a desired host cell such as Escherichia coli or Bacillus subtilis.
  • ribosome-binding sequence examples include a sequence prepared by DNA synthesis of a consensus sequence that is continuous for 4 bases or more among sequences complementary to the 3 'end region of 16S ribosomal RNA.
  • a transcription termination sequence is not necessarily required, but, for example, a ⁇ -factor-independent one such as a lipoprotein terminator, a trp operon terminator, etc. can be used.
  • sequence order of these control regions on the expression vector is not particularly limited, but considering transcription efficiency, a promoter sequence, a ribosome binding sequence, a gene encoding the target protein, a transcription termination sequence from the 5 ′ end upstream. It is desirable to arrange in order.
  • expression vectors herein include, for example, pBR322, pUC18, Bluescript II SK (+), pKK223-3, pSC101, etc., which have a region capable of autonomous replication in E. coli, such as in Bacillus subtilis. PUB110, pTZ4, pC194, ⁇ 11, ⁇ 1, ⁇ 105 and the like having a region capable of autonomous replication can be used as an expression vector.
  • an expression vector capable of autonomous replication in two or more types of hosts for example, pHV14, TRp7, YEp7, pBS7 and the like can be used as an expression vector.
  • the transformant according to the present invention can be prepared by a known method.
  • the expression vector containing the base sequences encoding the wild type and mutant lysine decarboxylase according to the present invention and the other region as necessary is constructed, and the expression vector is transformed into a desired host cell.
  • the method of conversion etc. are mentioned. Specifically, for example, Sambrook, J. et al. , Et. al. , “Molecular Cloning A Laboratory Manual, 3rd Edition”, Cold Spring Harbor Laboratory Press, (2001), and the like, can be used general methods known in the fields of molecular biology, biotechnology and genetic engineering. it can.
  • a method for introducing into a chromosome using homologous recombination can be used.
  • the transformant according to the present invention not only incorporates the expression vector into the host cell, but also silently converts a low-use codon in the host cell to a high-use codon as necessary. It can also be produced by introducing mutations.
  • the method for introducing a silent mutation is not particularly limited as long as it matches the codon usage in the host cell with the codon of the expression vector, the method of mutation, the type of base to be changed, and the like.
  • the medium for culturing the transformant of the present invention contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the host, and can be a natural medium as long as the transformant can be cultured efficiently. Any of synthetic media may be used.
  • Examples of the carbon source include sugars such as glucose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose, ribose, and starch hydrolysate, for example, alcohols such as glycerol, mannitol, and sorbitol, such as gluconic acid. , Organic acids such as fumaric acid, citric acid, and succinic acid.
  • Such carbon sources may be used alone or in combination.
  • the nitrogen source examples include inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, and organic nitrogen such as soybean hydrolysate, such as ammonia gas and aqueous ammonia.
  • inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate
  • organic nitrogen such as soybean hydrolysate, such as ammonia gas and aqueous ammonia.
  • Such nitrogen sources may be used alone or in combination.
  • inorganic ions include sodium ions, magnesium ions, potassium ions, calcium ions, chlorine ions, manganese ions, iron ions, phosphate ions, and sulfate ions.
  • Such inorganic ions may be used alone or in combination.
  • organic components organic micronutrients
  • organic micronutrients include various amino acids, for example, vitamins such as vitamin B1, required substances such as nucleic acids such as RNA, and yeast extract and the like.
  • Examples of such a medium include LB medium, YT medium, M9 medium, and the like. *
  • LB medium is preferable.
  • the culture conditions for the transformant may be appropriately selected depending on the type of the transformant, the medium, and the culture method.
  • the transformant grows, and the wild type lysine decarboxylase and mutant lysine decarboxylase according to the present invention are used.
  • the culture temperature is, for example, 20 to 45 ° C., preferably 25 to 40 ° C. under aerobic conditions.
  • the culture pH is, for example, 5.0 to 8.5, preferably 6.5 to 8.0, and has a desired mutated lysine decarboxylase activity within a culture period of half to 7 days. Culture may be performed until the protein content is maximized.
  • the culture period of the transformant is preferably 12 to 72 hours, more preferably 14 to 48 hours.
  • the culture pH can be adjusted using, for example, an inorganic or organic acidic or alkaline substance, ammonia gas, or the like.
  • Cultivation can be performed, for example, in a liquid medium containing the above medium by using a normal culture method such as shaking culture, aeration and agitation culture, continuous culture, or fed-batch culture.
  • a normal culture method such as shaking culture, aeration and agitation culture, continuous culture, or fed-batch culture.
  • a transformant can be obtained as a catalyst viable cell.
  • Recovery step of lysine decarboxylase The recovery step of lysine decarboxylase is performed by using wild-type lysine decarboxylase and / or from at least one of the cultured transformant and the culture of the transformant. It is a step of recovering the mutant lysine decarboxylase.
  • a method commonly used in this field can be used as a method for recovering the wild type and / or the above-mentioned mutant lysine decarboxylase according to the present invention after culturing the transformed transformant.
  • the culture of the transformant is, for example, centrifuged or filtered. Etc., a crude enzyme solution can be easily obtained.
  • the cultured transformant is recovered by means such as centrifugation, and the recovered The crude enzyme solution may be recovered by suspending the transformant in water or a buffer and destroying the cell membrane of the transformant according to a known method such as lysozyme treatment, freeze-thawing, or ultrasonic disruption. .
  • the above crude enzyme solution can be concentrated by, for example, an ultrafiltration method and used as a concentrated enzyme by adding, for example, a preservative.
  • a powder enzyme of the wild type and / or the above-mentioned mutant lysine decarboxylase can be obtained by, for example, spray drying.
  • the recovered crude enzyme solution having lysine decarboxylase activity requires separation and purification, for example, salting out with ammonium sulfate or the like, for example, organic solvent precipitation with alcohol or the like, such as dialysis and ultrafiltration, etc.
  • Membrane separation methods by the above for example, known chromatographic separation methods such as ion exchanger chromatography, reverse phase high performance chromatography, affinity chromatography, and gel filtration chromatography can be appropriately combined.
  • the wild type and / or mutant lysine decarboxylase obtained as described above are included in the scope of the present invention.
  • (8) Method for producing catalyst killed cell A cell that has expressed lysine decarboxylase and / or mutant lysine decarboxylase can be killed by heating to obtain a catalyst killed cell.
  • the catalyst viable cells obtained as described above are suspended in a solvent such as water to prepare a cell suspension.
  • concentration of the live catalyst cell in the cell suspension is usually 0.1 to 20% by mass, preferably 1 to 15% by mass in terms of dry cell equivalent.
  • this cell suspension is killed by heating in a warm bath or the like to obtain catalyst killed cells.
  • the heating temperature is not particularly limited as long as the viable bacterial cells can be killed, but is usually 45 to 80 ° C., preferably 50 to 70 ° C.
  • the heating time is usually 5 minutes to 1 hour, preferably 10 to 30 minutes.
  • the catalyst cell may be any of a live catalyst cell, a resting catalyst cell, or a dead catalyst cell.
  • reducing agent a known reducing agent is used, but a reducing agent that can remove dissolved oxygen and does not inhibit the reaction of lysine decarboxylase is preferable.
  • Examples of the reducing agent include those having a low oxidation-reduction potential, and more preferable examples include a reducing agent having a potential lower than the oxidation-reduction potential (+160 mV to +180 mV) at 20 ° C. in physiological saline.
  • the redox potential is measured with a redox potential meter.
  • reducing agent examples include mercapto compounds, sulfides, hydrosulfides, reducing sulfur oxyacid salts, thiourea and derivatives thereof, cyclic compounds having hydroxyl groups and / or carboxyl groups, and flavonoids. Examples thereof include compounds, nitrogen-containing heterocyclic compounds, hydrazyl group compounds, and mucopolysaccharides having uronic acid groups.
  • mercapto compounds include cysteine, N-acetylcysteine, 2-mercaptoethanol, dithioerythritol, dithiothreitol (also known as dithiothreitol), glutathione, and S-acetylmercaptosuccinic anhydride.
  • Examples of the sulfide include sodium sulfide.
  • sulfur oxyacid salts having reducibility include, for example, sodium sulfate, potassium salt, and other physiologically safe salts such as sulfurous acid, bisulfite, thiosulfuric acid, metabisulfite, dithionite, etc. Is mentioned. These salts may be acidic sulfites (bisulfites).
  • thiourea and derivatives thereof examples include thiourea and dimethylthiourea.
  • Examples of the cyclic compound having a hydroxyl group and / or a carboxyl group include physiologically safe salts such as acetylsalicylic acid, ascorbic acid or a sodium salt thereof.
  • Examples of the flavonoid compound include compounds having two or more hydroxyl groups in the ring compound. Specifically, quercetin dihydrate, catechin, epicatechin, Or the hydrate etc. are mentioned.
  • nitrogen-containing heterocyclic compound examples include compounds having a thiazole ring, a thiazoline ring, a thiazolidine ring, a triazole ring, a tetrazole ring, an indole ring, an imidazole ring, a pyridine ring, or a pyrimidin ring.
  • the compound having a thiazole ring include N- (2-thiazolyl) sulfanilamide (N- (2-Thiazolyl) sulfanamide), N-phenacylthiazole bromide and the like.
  • Specific examples of the compound having a thiazoline ring include 2-mercaptothiazoline.
  • Specific examples of the compound having a thiazolidine ring include 2-oxo-4-thiazolidinecarboxylic acid.
  • Specific examples of the compound having a triazole ring include 4- (1,2,3,4-thiatriazo-5-lylamino) phenol hydrate.
  • Specific examples of the compound having an indole ring include N-acetyltryptophan.
  • Examples of the hydrazyl group compound include aminoguanidine hydrochloride.
  • mucopolysaccharide having a uronic acid group examples include heparin.
  • a mercapto compound and a sulfur oxyacid salt having reducibility are mentioned, and more preferably, dithiothreitol (also called dithiothreitol), sodium sulfite (sodium sulfite), sulfite Examples include sodium salt of dithionic acid (hydrosulfite).
  • reducing agents can be used alone or in combination of two or more.
  • the concentration of the reducing agent is not particularly limited as long as it is a concentration that can sufficiently remove dissolved oxygen in the lysine reaction solution and does not deactivate lysine decarbonase. Usually, it is 0.1 to 10 g / L, preferably 0.5 to 5 g / L. If the concentration of the reducing agent is less than the lower limit, lysine decarboxylase and / or mutant lysine decarboxylase may not be stored stably for a long period of time.
  • Such a catalyst cell can be stored for a long period (for example, 80 days or more, preferably 30 days or more) by freezing or refrigeration, for example.
  • the storage temperature of the bacterial cell suspension is, for example, 20 ° C. or lower, preferably 4 ° C. or lower.
  • (10) Method for Producing 1,5-Pentamethylenediamine in the method for producing 1,5-pentamethylenediamine of the present invention, in the reaction system in which the dissolved oxygen concentration is the saturated dissolved oxygen concentration within 1 hour, L -Lysine and / or its salt is subjected to lysine decarboxylation with lysine decarboxylase and / or mutant lysine decarboxylase.
  • the time during which the dissolved oxygen concentration in the reaction system is the saturated dissolved oxygen concentration is set within 1 hour, and the above-mentioned wild type and / or mutant lysine decarboxylase is contacted with lysine, 1,5-pentamethylenediamine is produced.
  • lysine a transformant that expresses wild-type and / or mutant lysine decarboxylase, wild-type and / or mutant lysine decarboxylase (for example, catalytic viable cell), processed product of transformant (for example, a reaction solution is prepared by mixing at least one selected from the group consisting of a dead catalyst cell), a transformant and an immobilized product thereof, and a reaction solvent. And / or mutant lysine decarboxylase is brought into contact with lysine to produce 1,5-pentamethylenediamine by decarboxylase reaction of lysine.
  • the time when the wild-type and / or mutant lysine decarboxylase and lysine first contact each other is defined as the reaction start point. Further, the point of time when the contact between the wild-type and / or mutant lysine decarboxylase and lysine is cut off, or when the amount of 1,5-pentamethylenediamine produced is saturated, is regarded as the reaction end point.
  • the time during which the dissolved oxygen concentration is the saturated dissolved oxygen concentration is limited to within one hour from the reaction start point to the reaction end point.
  • the time during which the dissolved oxygen concentration in the reaction system is the saturated dissolved oxygen concentration is set within one hour, for example, a step of removing oxygen in the reaction system, A step of adding a reducing agent into the reaction system can be included.
  • step of removing oxygen in the reaction system a known method is performed that can remove oxygen dissolved in the reaction system, that is, in the reaction solution.
  • step of removing oxygen in the reaction solution include a step of replacing dissolved oxygen with an inert gas.
  • an inert gas is passed through the reaction solution to exchange dissolved oxygen and inert gas.
  • the aeration amount of the inert gas is, for example, 0.05 to 10 L / hr, preferably 0.1 to 5 L / hr with respect to 100 parts by mass of the reaction solution.
  • the inert gas ventilation time is, for example, 0.5 to 5 hours, preferably 0.1 to 1 hour.
  • the aeration method is not particularly limited and can be bubbled.
  • an inert gas can be passed through the reaction solution before the start of the reaction, the reaction solution after the start of the reaction, and both.
  • the reaction solution before the start of the reaction for example, the catalyst cell body.
  • An inert gas is passed through the lysine solution before the is added.
  • the dissolved oxygen concentration before the start of the reaction is, for example, 90% or less of the saturated dissolved oxygen concentration, preferably 70% or less, more preferably 65% or less, and still more preferably 50% or less.
  • the reducing agent is added to the reaction system, that is, to the reaction solution.
  • reducing agent a known reducing agent is used, but a reducing agent that can reduce the dissolved oxygen concentration and does not inhibit lysine decarboxylase is used. Preferred are those having a low redox potential, and more preferred are reducing agents having a lower potential than the redox potential (+160 mV to +180 mV) at 20 ° C. of physiological saline.
  • such a reducing agent include the above-described mercapto compounds, the above-described sulfides, the above-described hydrosulfides, the above-described sulfur oxyacid salts having reducibility, the above-described thiourea and derivatives thereof, and the above-described hydroxyl groups.
  • sulfur oxyacid salt having reducibility is preferable, and sodium sulfite (sodium sulfite) is more preferable.
  • reducing agents can be used alone or in combination of two or more.
  • the concentration of the reducing agent in the reaction solution may be any concentration that does not deactivate lysine decarboxylase, but is usually about 0.1 to 10 g / L, preferably about 0.1 to 4 g / L.
  • a reducing agent can be added to the reaction solution before the start of the reaction, the reaction solution after the start of the reaction, and both.
  • the reaction solution before the start of the reaction for example, catalyst cells
  • the reducing agent is added to the lysine solution before the is added.
  • the dissolved oxygen concentration before the start of the reaction is, for example, 90% or less of the saturated dissolved oxygen concentration, preferably 70% or less, more preferably 65% or less, and still more preferably 50% or less.
  • the saturated dissolved oxygen concentration in the reaction system is dissolved oxygen in a state where the dissolved oxygen in the reaction system is saturated with oxygen in the air, and can be measured as follows.
  • An oxygen electrode for fermentation (manufactured by CSL-1 Able) is immersed in a solution in which copper sulfate hexahydrate is added to a sodium sulfite solution in advance to make the dissolved oxygen concentration zero, and a dissolved oxygen indicator (MODEL M-1032 Able) Make adjustments so that the indication of “manufactured” is zero. Next, air is passed through the reaction solution, and when the value of the dissolved oxygen indicator is stabilized, the saturated dissolved oxygen concentration is obtained.
  • a dissolved oxygen indicator MODEL M-1032 Able
  • the ratio (%) of the dissolved oxygen concentration to the saturated dissolved oxygen concentration in the reaction system can be measured by the dissolved oxygen indicator adjusted as described above.
  • oxygen in the reaction system may be reduced as the lysine decarboxylation reaction proceeds.
  • the dissolved oxygen concentration in the reaction solution is preferably 65% or less of the saturated dissolved oxygen concentration from the reaction start point to the reaction end point, and the dissolved oxygen concentration is saturated dissolved. It is preferable that the saturated dissolved oxygen concentration is 1% or less within 20 minutes from the point where the oxygen concentration is 65% or less (that is, the reaction start point).
  • the dissolved oxygen concentration is preferably reduced to 65% or less of the saturated dissolved oxygen concentration at the start of the reaction, and the dissolved oxygen concentration increases or decreases during the reaction.
  • the dissolved oxygen concentration increases or decreases in a range not exceeding 65% of the saturated dissolved oxygen concentration, and decreases to 1% or less of the saturated dissolved oxygen concentration as the reaction proceeds, and the required time is Within 20 minutes.
  • the relationship between the dissolved oxygen concentration and the elapsed time in the lysine decarboxylation reaction can be represented as a correlation line.
  • the relationship between the dissolved oxygen concentration and the elapsed time is, for example, the ratio of the dissolved oxygen concentration to the saturated dissolved oxygen concentration (%) on the Y axis and the time ( For example, it is shown as a linear function-like correlation line (see FIG. 1).
  • the correlation indicating the correlation line in which the relationship between the dissolved oxygen concentration and time in the lysine decarboxylation reaction is plotted with the Y axis as the ratio (%) of the dissolved oxygen concentration to the saturated dissolved oxygen concentration and the X axis as the time (min).
  • the area of the portion surrounded by the correlation line, the Y axis and the X axis is preferably less than 1000, more preferably 650 or less.
  • the area unit is, for example, (% ⁇ min).
  • the correlation line between the dissolved oxygen concentration and the elapsed time is shown as a linear function as a schematic conceptual diagram, but the actual correlation line varies depending on the reaction system, and has various curves.
  • the correlation line may be an irregular curve, and the portion surrounded by the correlation line, the Y axis, and the X axis may be discontinuous.
  • the area of the portion surrounded by the correlation line, the Y axis, and the X axis is calculated as the total area of the respective portions.
  • the lysine used as a raw material in the present invention may be a salt thereof.
  • lysine salts include hydrochloride, acetate, carbonate, bicarbonate, sulfate, nitrate, and the like.
  • carbonates and bicarbonates use a large amount of carbon dioxide in the production process and generate a lot of greenhouse gases, from the viewpoint of environmental protection, preferably, hydrochloride, acetate, sulfate, nitrate Etc.
  • lysine hydrochloride is preferable.
  • lysine or a salt thereof purified by adding a reducing agent in the step of purifying lysine can also be used as lysine.
  • the concentration of lysine in the reaction solvent is not particularly limited, but is, for example, 1 to 70% by mass, preferably 10 to 70% by mass, and more preferably 20 to 50% by mass.
  • the required amount of wild-type and mutant lysine decarboxylase in the present invention is a transformant (eg, catalyst) that expresses wild-type and / or mutant lysine decarboxylase, wild-type and / or mutant lysine decarboxylase.
  • a transformant eg, catalyst
  • a processed product of the transformant for example, dead catalyst cell
  • an immobilized product of the processed product and L-lysine and / or With respect to 1 part by mass of the salt
  • lysine decarboxylase and / or mutant lysine decarboxylase is 0.0003 parts by mass or more and 0.0015 parts by mass or less in terms of dry cell weight.
  • reaction solvent examples include water, an aqueous medium, an organic solvent, water, or a mixed liquid of an aqueous medium and an organic solvent.
  • aqueous medium examples include a buffer solution such as a phosphate buffer solution.
  • Any organic solvent may be used as long as it does not inhibit the reaction.
  • the temperature is, for example, 28 to 55 ° C., preferably 35 to 45 ° C.
  • the time is, for example, 0.1 to 72 hours, preferably 1 to 72 hours, Preferably, it is 12 to 36 hours.
  • the reaction pH is, for example, 5.0 to 9.0, preferably 5.5 to 8.5.
  • the reaction can be carried out under shaking, stirring or standing conditions.
  • lysine is decarboxylated and converted into 1,5-pentamethylenediamine, and 1,5-pentamethylenediamine is produced.
  • the 1,5-pentamethylenediamine produced in the present invention may be a salt thereof.
  • Examples of the salt of 1,5-pentamethylenediamine include hydrochloride, acetate, carbonate, bicarbonate, sulfate, nitrate and the like of 1,5-pentamethylenediamine.
  • hydrochloride is preferable.
  • the pH of the reaction solution may increase as lysine is converted to 1,5-pentamethylenediamine.
  • an acidic substance for example, an organic acid, for example, an inorganic acid such as hydrochloric acid
  • an acidic substance can be added to adjust the pH.
  • vitamin B6 and / or a derivative thereof can be added to the reaction solution as necessary.
  • vitamin B6 and / or derivatives thereof examples include pyridoxine, pyridoxamine, pyridoxal, pyridoxal phosphate, and the like.
  • Such vitamin B6 and / or its derivatives may be used alone or in combination.
  • pyridoxal phosphate is preferable.
  • aqueous pentamethylenediamine solution at a pressure of 0.1 kPa to normal pressure using a distillation apparatus equipped with a continuous multistage distillation column, a batch multistage distillation column, etc.
  • a pentamethylenediamine aqueous solution in which a part of water is distilled off can be obtained.
  • the heating temperature is, for example, 25 ° C. or more and less than 90 ° C., preferably 25 ° C. or more and 85 ° C. or less, more preferably 25 ° C. or more and less than 80 ° C., particularly preferably 30 ° C. or more and 70 ° C. or less. is there.
  • the extraction rate of pentamethylenediamine (or a salt thereof) may decrease.
  • the dissolved oxygen concentration in the reaction system is a saturated dissolved oxygen concentration within 1 hour
  • lysine decarboxylase and / or mutant lysine decarboxylation A decrease in the activity of the enzyme can be reduced. Therefore, lysine decarbonization can be performed with excellent reaction efficiency without purifying the enzyme, and the reaction can be completed without adjusting the pH of the reaction solution.
  • an extraction solvent (described later) is brought into contact with an aqueous solution of pentamethylenediamine batchwise, semi-continuously or continuously, and mixed and stirred, so that pentamethylenediamine (or a solution thereof) is obtained.
  • Salt is extracted (distributed) into an extraction solvent (described later), and pentamethylenediamine (or a salt thereof) is separated from the extraction solvent (described later);
  • a tower for example, a spray tower
  • nozzles, orifice plates, baffles, injectors and / or static mixers countercurrent differential type extraction towers, non-stirring type stage extraction towers: revised fifth edition, chemical engineering Handbook, pages 566 to 569, edited by Chemical Engineering Society, Maruzen (1988)
  • an aqueous solution of pentamethylenediamine and an extraction solvent are continuously supplied in a countercurrent flow.
  • liquid-liquid extraction methods can be used alone or in combination of two or more.
  • liquid-liquid extraction method from the viewpoint of production efficiency, a method of continuously extracting (distributing) pentamethylenediamine (or a salt thereof) into an extraction solvent (described later), more specifically, for example, And the above methods (1) to (3).
  • the blending ratio of the pentamethylenediamine aqueous solution and the extraction solvent (described later) in the liquid-liquid extraction is 100 parts by mass of the pentamethylenediamine aqueous solution (if the extraction is continuous, the supply amount per unit time.
  • the extraction solvent (described later) is, for example, 30 to 300 parts by mass, and is preferably 50 to 200 parts by mass, more preferably 50 to 150 parts by mass, particularly preferably from the viewpoint of economy and productivity. Is 80 to 120 parts by mass.
  • pentamethylene diisocyanate (described later) is produced using the pentamethylenediamine obtained from the aqueous solution, and further, an isocyanate-modified product from the pentamethylene diisocyanate.
  • the reaction rate may be low, or the storage stability of the resulting isocyanate-modified product (described later) may be low.
  • the pentamethylenediamine aqueous solution is preferably heated (heat treatment) at 90 ° C. or higher, more preferably 80 ° C. or higher, and particularly preferably the pentamethylenediamine aqueous solution is heated (heat treatment). Without extraction, pentamethylenediamine (or a salt thereof) is directly extracted from the aqueous solution.
  • an aqueous pentamethylenediamine solution and an extraction solvent are, for example, under normal pressure (atmospheric pressure), for example, 5 to 60 ° C., preferably 10 to 60 ° C.
  • normal pressure atmospheric pressure
  • mixing is performed at 15 to 50 ° C., particularly preferably 15 to 40 ° C., for example, by a stirring blade, for example, for 1 to 120 minutes, preferably 5 to 90 minutes, and more preferably 5 to 60 minutes. .
  • the agitating blade is not particularly limited. An anchor etc. are mentioned.
  • the rotation speed in mixing is, for example, 5 to 3000 rpm, preferably 10 to 2000 rpm, and more preferably 20 to 1000 rpm.
  • pentamethylenediamine (or a salt thereof) is extracted into an extraction solvent (described later).
  • a mixture of pentamethylenediamine (or a salt thereof) and an extraction solvent (described later) is allowed to stand, for example, for 5 to 300 minutes, preferably 10 to 240 minutes, and more preferably 20 to 180 minutes.
  • an extraction solvent from which pentamethylenediamine (or a salt thereof) is extracted is used in a known method. Take out.
  • liquid-liquid extraction can be repeated several times (for example, 2 to 5 times).
  • pentamethylenediamine (or a salt thereof) in the aqueous solution of pentamethylenediamine can be extracted into an extraction solvent (described later).
  • the concentration of pentamethylenediamine (or a salt thereof) is, for example, 0.2 to 40% by mass.
  • the amount is 0.3 to 35% by mass, more preferably 0.4 to 30% by mass, and particularly preferably 0.8 to 25% by mass.
  • the yield (extraction rate) of pentamethylenediamine (or a salt thereof) after extraction is, for example, 65 to 100 mol%, preferably 70 to 100 mol%, based on lysine (or a salt thereof). Preferably, it is 80 to 100 mol%, particularly preferably 90 to 100 mol%.
  • pentamethylenediamine (or a salt thereof) can be isolated from a mixture of the obtained extraction solvent (described later) and pentamethylenediamine (or a salt thereof).
  • the isolation of pentamethylenediamine (or a salt thereof) is not particularly limited, but for example, by a distillation apparatus equipped with a continuous multistage distillation column, a batch multistage distillation column, etc., for example, 50 to 182 ° C., 0.1 kPa to ordinary Under pressure, a mixture of the extraction solvent (described later) and pentamethylenediamine (or a salt thereof) is distilled to remove the extraction solvent (described later).
  • examples of the extraction solvent include non-halogen organic solvents.
  • Non-halogen organic solvents are organic solvents that do not contain halogen atoms (fluorine, chlorine, bromine, iodine, etc.) in their molecules, such as non-halogen aliphatic organic solvents, non-halogen alicyclic organic solvents, Non-halogen aromatic organic solvents are exemplified.
  • non-halogen aliphatic organic solvents examples include linear non-halogen aliphatic organic solvents and branched non-halogen aliphatic organic solvents.
  • linear non-halogen aliphatic organic solvents examples include linear non-halogen aliphatic hydrocarbons, linear non-halogen aliphatic ethers, and linear non-halogen aliphatic alcohols. Is mentioned.
  • linear non-halogen aliphatic hydrocarbons examples include n-hexane, n-heptane, n-nonane, n-decane, and n-dodecane.
  • linear non-halogen aliphatic ethers examples include diethyl ether, dibutyl ether, and dihexyl ether.
  • linear non-halogen aliphatic alcohols examples include linear monohydric alcohols having 1 to 3 carbon atoms (eg, methanol, ethanol, n-propanol, isopropanol, etc.), linear carbon atoms of 4 To 7 monohydric alcohols (eg, n-butanol, n-pentanol, n-hexanol, n-heptanol), linear monohydric alcohols having 8 or more carbon atoms (eg, n-octanol, n-nonanol) N-decanol, n-undecanol, n-dodecanol, etc.).
  • monohydric alcohols having 1 to 3 carbon atoms eg, methanol, ethanol, n-propanol, isopropanol, etc.
  • linear carbon atoms of 4 To 7 monohydric alcohols eg, n-butanol, n-pentanol
  • branched non-halogen aliphatic organic solvents include branched non-halogen aliphatic hydrocarbons, branched non-halogen aliphatic ethers, branched non-halogen aliphatic monohydric alcohols, branched Non-halogen aliphatic polyhydric alcohols.
  • branched non-halogen aliphatic hydrocarbons examples include 2-methylpentane, 2,2-dimethylbutane, 2,3-dimethylbutane, 2-methylhexane, 3-methylhexane, and 2,3-dimethylpentane.
  • branched non-halogen aliphatic ethers examples include diisopropyl ether and diisobutyl ether.
  • branched non-halogen aliphatic monohydric alcohols include branched monohydric alcohols having 4 to 7 carbon atoms (for example, 2-butanol, isobutanol, tert-butanol, 2-pentanol, 3-pentane).
  • Tanol isopentanol, 2-methyl-1-butanol, 2-methyl-3-butanol, 2,2-dimethyl-1-propanol, tert-pentanol, 2-hexanol, 3-hexanol, isohexanol, 2- Methyl-2-pentanol, 2-methyl-1-pentanol, 3-methyl-1-pentanol, 2-ethyl-1-butanol, 3,3-dimethyl-1-butanol, 2-heptanol, 3-heptanol 4-heptanol, 5-methyl-1-hexanol, 4-methyl-1-hexanol, 3-methyl-1- Xanol, 2-ethyl-2-methyl-1-butanol, etc., branched monohydric alcohols having 8 or more carbon atoms (for example, isooctanol, isononanol, isodecanol, 5-ethyl-2-nonanol,
  • branched non-halogen aliphatic polyhydric alcohols examples include 2-ethyl-1,3-hexanediol.
  • non-halogen aliphatic organic solvents can be used alone or in combination of two or more.
  • the non-halogen aliphatic organic solvent is preferably a linear non-halogen aliphatic organic solvent, and more preferably a linear non-halogen aliphatic alcohol.
  • pentamethylene diamine can be extracted with high yield.
  • the non-halogen aliphatic organic solvent is preferably a monohydric alcohol having 4 to 7 carbon atoms (a linear monohydric alcohol having 4 to 7 carbon atoms or a branched monohydric alcohol having 4 to 7 carbon atoms). ).
  • pentamethylenediamine or a salt thereof can be efficiently extracted, and further, the content ratio of impurities of pentamethylenediamine or a salt thereof can be reduced.
  • non-halogen alicyclic organic solvent examples include non-halogen alicyclic hydrocarbons (eg, cyclopentane, methylcyclopentane, cyclohexane, methylcyclohexane, ethylcyclohexane, p-menthane, bicyclohexyl, etc.).
  • non-halogen alicyclic hydrocarbons eg, cyclopentane, methylcyclopentane, cyclohexane, methylcyclohexane, ethylcyclohexane, p-menthane, bicyclohexyl, etc.
  • non-halogen alicyclic organic solvents can be used alone or in combination of two or more.
  • Non-halogen aromatic organic solvents include, for example, non-halogen aromatic hydrocarbons (for example, benzene, toluene, xylene, ethylbenzene, isopropylbenzene, 1,3,5-trimethylbenzene, 1,2,3,4- Tetrahydronaphthalene, n-butylbenzene, sec-butylbenzene, tert-butylbenzene, ethylbenzene, etc.) and phenols (eg, phenol, cresol, etc.).
  • non-halogen aromatic hydrocarbons for example, benzene, toluene, xylene, ethylbenzene, isopropylbenzene, 1,3,5-trimethylbenzene, 1,2,3,4- Tetrahydronaphthalene, n-butylbenzene, sec-butylbenzene, tert-butylbenzene, ethylbenzene, etc
  • non-halogen aromatic organic solvents can be used alone or in combination of two or more.
  • non-halogen organic solvent examples include a mixture of aliphatic hydrocarbons and aromatic hydrocarbons, and examples of such a mixture include petroleum ether and petroleum benzine.
  • non-halogen organic solvents can be used alone or in combination of two or more.
  • a halogen-based organic solvent an organic solvent containing a halogen atom in the molecule
  • a halogen-based organic solvent an organic solvent containing a halogen atom in the molecule
  • halogen-based organic solvent examples include halogen-based aliphatic hydrocarbons (for example, chloroform, dichloromethane, carbon tetrachloride, tetrachloroethylene), halogen-based aromatic hydrocarbons (for example, chlorobenzene, dichlorobenzene, chlorotoluene, etc.) Etc.
  • halogen-based aliphatic hydrocarbons for example, chloroform, dichloromethane, carbon tetrachloride, tetrachloroethylene
  • halogen-based aromatic hydrocarbons for example, chlorobenzene, dichlorobenzene, chlorotoluene, etc.
  • halogenated organic solvents can be used alone or in combination of two or more.
  • pentamethylene diisocyanate (described later) is produced using the obtained pentamethylene diamine or a salt thereof, and the pentamethylene diisocyanate (described later) is further reacted to produce isocyanate.
  • the productivity and physical properties for example, yellowing resistance
  • the isocyanate-modified product may be inferior.
  • a polyurethane resin is produced by reacting such a pentamethylene diisocyanate (described later) or an isocyanate-modified product (described later) with an active hydrogen compound (described later), the physical properties of the resulting polyurethane resin (for example, , Mechanical strength, chemical resistance, etc.).
  • the extraction solvent is preferably a non-halogen organic solvent, more preferably a non-halogen aliphatic organic solvent.
  • the boiling point of the extraction solvent is, for example, 60 to 250 ° C., preferably 80 to 200 ° C., and more preferably 90 to 150 ° C.
  • the boiling point of the extraction solvent is less than the lower limit, it may be difficult to separate the extraction solvent from the extraction solvent when pentamethylenediamine or a salt thereof is obtained by extraction from an aqueous pentamethylenediamine solution.
  • the method for obtaining pentamethylenediamine or a salt thereof from an aqueous pentamethylenediamine solution is not limited to the above extraction, and a known isolation and purification method such as distillation can also be employed.
  • (12) Method for Producing 1,5-Pentamethylene Diisocyanate The present invention also relates to 1,5-pentamethylene diisocyanate (hereinafter simply referred to as “1,5-pentamethylene diisocyanate”) from 1,5-pentamethylenediamine (or a salt thereof) thus obtained.
  • a process for producing pentamethylene diisocyanate sometimes referred to as PDI).
  • Examples of the method for synthesizing 1,5-pentamethylene diisocyanate include a method of phosgenating 1,5-pentamethylenediamine (or a salt thereof) (hereinafter sometimes referred to as a phosgenation method), 1, Examples thereof include a method of carbamateizing 5-pentamethylenediamine (or a salt thereof) and then thermally decomposing (hereinafter sometimes referred to as a carbamate method).
  • phosgenation method for example, a method in which pentamethylenediamine is directly reacted with phosgene (hereinafter sometimes referred to as a cold two-stage phosgenation method), or a hydrochloride of pentamethylenediamine is inactive.
  • a cold two-stage phosgenation method a method in which pentamethylenediamine is directly reacted with phosgene
  • a hydrochloride of pentamethylenediamine is inactive.
  • examples thereof include a method of suspending in a solvent (described later) and reacting with phosgene (hereinafter sometimes referred to as a phosgenation method of amine hydrochloride).
  • an inert solvent is charged into a reactor that can be stirred and provided with a phosgene introduction tube, and the pressure in the reaction system is, for example, from normal pressure to 1
  • the pressure is 0.0 MPa, preferably normal pressure to 0.5 MPa
  • the temperature is, for example, 0 to 80 ° C., preferably 0 to 60 ° C.
  • the inert solvent examples include aromatic hydrocarbons such as benzene, toluene, xylene, and ethylbenzene, and fatty acid esters such as ethyl acetate, butyl acetate, and amyl acetate, such as methyl salicylate, dimethyl phthalate, and phthalic acid.
  • Aromatic carboxylic acid esters such as dibutyl and methyl benzoate, for example, chlorinated aromatic hydrocarbons such as monodichlorobenzene, orthodichlorobenzene, and trichlorobenzene, for example, chlorinated hydrocarbons such as chloroform and carbon tetrachloride Etc.
  • inert solvents can be used alone or in combination of two or more.
  • the compounding amount (total amount) of the inert solvent is, for example, 400 to 3000 parts by mass, and preferably 500 to 2000 parts by mass with respect to 100 parts by mass of pentamethylenediamine as a raw material.
  • phosgene is introduced, for example, 1 to 10-fold mol, preferably 1 to 6-fold mol, with respect to one amino group of pentamethylenediamine, and pentamethylene dissolved in the above inert solvent.
  • Add the diamine is added.
  • the reaction solution is maintained at, for example, 0 to 80 ° C., preferably 0 to 60 ° C., and the generated hydrogen chloride is discharged out of the reaction system through a reflux condenser (cold phosgenation reaction). Thereby, the contents of the reactor are made into a slurry.
  • the pressure in the reaction system is, for example, normal pressure to 1.0 MPa, preferably 0.05 to 0.5 MPa, for example, 30 minutes to 5 hours, for example, 80 to 180 ° C.
  • the temperature is raised to a temperature range. After the temperature rise, for example, the reaction is continued for 30 minutes to 8 hours to completely dissolve the slurry (thermal phosgenation reaction).
  • an inert gas such as nitrogen gas is introduced into the reaction system at, for example, 80 to 180 ° C., preferably 90 to 160 ° C. to dissolve excess phosgene and chloride. Purge hydrogen.
  • pentamethylene dicarbamoyl chloride produced by the cold phosgenation reaction is thermally decomposed to produce pentamethylene diisocyanate, and the amine hydrochloride of pentamethylenediamine is further phosgenated to produce pentamethylene diisocyanate. Is done.
  • the hydrochloride of pentamethylenediamine is sufficiently dried and finely pulverized, and then the hydrochloride of pentamethylenediamine is used in the same reactor as in the above-described cold and two-stage phosgenation method. Is stirred in the above inert solvent and dispersed into a slurry.
  • the reaction temperature is maintained at, for example, 80 to 180 ° C., preferably 90 to 160 ° C.
  • the reaction pressure is maintained at, for example, normal pressure to 1.0 MPa, preferably 0.05 to 0.5 MPa.
  • phosgene is introduced over 1 to 10 hours so that the total amount of phosgene is, for example, 1 to 10 times the stoichiometric amount.
  • pentamethylene diisocyanate can be synthesized.
  • the progress of the reaction can be estimated from the amount of hydrogen chloride gas generated and the slurry insoluble in the inert solvent disappearing, and the reaction solution becomes clear and uniform. Further, the generated hydrogen chloride is released out of the reaction system through, for example, a reflux condenser. At the end of the reaction, excess phosgene and hydrogen chloride dissolved by the above method are purged. Thereafter, the mixture is cooled and the inert solvent is distilled off under reduced pressure.
  • pentamethylene diisocyanate tends to increase the concentration (HC) of hydrolyzable chlorine, when adopting the phosgenation method, when it is necessary to reduce HC, for example, a phosgenation reaction is performed.
  • the pentamethylene diisocyanate distilled off after removing the solvent is, for example, 150 to 200 ° C., preferably 160 to 190 ° C., for example, 1 to 8 while passing an inert gas such as nitrogen. Heat treatment is performed for a time, preferably 3 to 6 hours. Thereafter, HC of pentamethylene diisocyanate can be remarkably reduced by performing rectification treatment.
  • the concentration of hydrolyzable chlorine in pentamethylene diisocyanate is, for example, 100 ppm or less, preferably 80 ppm or less, more preferably 60 ppm or less, and particularly preferably 50 ppm or less.
  • the concentration of hydrolyzable chlorine can be measured in accordance with, for example, the test method for hydrolyzable chlorine described in Annex 3 of JIS K-1556 (2000).
  • the concentration of hydrolyzable chlorine exceeds 100 ppm, the reaction rate of trimerization (described later) decreases, and a large amount of trimerization catalyst (described later) may be required, and a large amount of trimerization catalyst (described later) is used.
  • the resulting polyisocyanate composition (described later) has a high degree of yellowing, the number average molecular weight is high, and the viscosity is high.
  • the viscosity and hue may change greatly in the storage process of the polyisocyanate composition (described later) and the manufacturing process of the polyurethane resin (described later).
  • Examples of the carbamate method include a urea method.
  • pentamethylenediamine is first carbamateized to produce pentamethylene dicarbamate (PDC).
  • PDC pentamethylene dicarbamate
  • N-unsubstituted carbamic acid esters examples include N-unsubstituted carbamic acid aliphatic esters (for example, methyl carbamate, ethyl carbamate, propyl carbamate, iso-propyl carbamate, butyl carbamate, isocarbamate).
  • N-unsubstituted carbamic acid esters can be used alone or in combination of two or more.
  • N-unsubstituted carbamic acid esters include N-unsubstituted carbamic acid aliphatic esters.
  • Examples of the alcohol include primary to tertiary monohydric alcohols, and more specific examples include aliphatic alcohols and aromatic alcohols.
  • aliphatic alcohols examples include linear aliphatic alcohols (eg, methanol, ethanol, n-propanol, n-butanol (1-butanol), n-pentanol, n-hexanol, n-heptanol, n-octanol (1-octanol), n-nonanol, n-decanol, n-dodecanol, n-tetradecanol, n-hexadecanol, etc.), branched aliphatic alcohols (eg, iso-propanol, iso) -Butanol, sec-butanol, tert-butanol, iso-pentanol, sec-pentanol, 2-ethylhexanol, iso-decanol, etc.).
  • linear aliphatic alcohols eg, methanol, ethanol, n-prop
  • aromatic alcohols examples include phenol, hydroxytoluene, hydroxyxylene, biphenyl alcohol, naphthalenol, anthracenol, phenanthrenol and the like.
  • These alcohols can be used alone or in combination of two or more.
  • aliphatic alcohols are preferable, and linear aliphatic alcohols are more preferable.
  • the above-described monovalent alcohol having 4 to 7 carbon atoms (linear monovalent alcohol having 4 to 7 carbon atoms, branched monovalent alcohol having 4 to 7 carbon atoms) can be used. .
  • the alcohol when an alcohol (such as a monohydric alcohol having 4 to 7 carbon atoms) is used as an extraction solvent, the alcohol is preferably used as a reaction raw material alcohol.
  • pentamethylene diamine, urea and / or N-unsubstituted carbamic acid ester, and alcohol are blended and reacted preferably in a liquid phase.
  • the blending ratio of pentamethylenediamine, urea and / or N-unsubstituted carbamic acid ester, and alcohol is not particularly limited and can be appropriately selected within a relatively wide range.
  • the blending amount of urea and N-unsubstituted carbamic acid ester and the blending amount of alcohol should be equimolar or more with respect to the amino group of pentamethylenediamine. Therefore, urea and / or N- An unsubstituted carbamate or alcohol itself can also be used as a reaction solvent in this reaction.
  • an alcohol such as a monohydric alcohol having 4 to 7 carbon atoms
  • the alcohol is preferably used as it is as a reaction raw material and a reaction solvent.
  • the amount of urea and / or the above-mentioned N-unsubstituted carbamic acid ester is, for example, 0.5 to 20 times that of one amino group of pentamethylenediamine from the viewpoint of improving the yield of carbamate.
  • Moles preferably 1 to 10 times moles, more preferably 1 to 5 times moles
  • the amount of alcohol blended is 0.5 to 100 times moles with respect to one amino group of pentamethylenediamine, preferably Is 1 to 20 moles, more preferably 1 to 10 moles.
  • a catalyst can also be used.
  • Metal compound for example, lithium methanolate, lithium ethanolate, Lithium propanolate, lithium butanolate, sodium methanolate, potassium tert-butanolate, etc., Group 2 metal compounds (eg magnesium methanolate, calcium methanolate etc.), Group 3 metal compounds (eg cerium oxide) (IV), uranyl acetate, etc.), Group 4 metal compounds (for example, titanium tetraisopropanolate, titanium tetrabutanolate, titanium tetrachloride, titanium tetraphenolate, naphthenic acid Group 5 metal compounds (eg, vanadium (III) chloride, vanadium acetylacetonate, etc.), Group 6 metal compounds (eg, chromium (III) chloride, molybdenum (VI), molyb
  • Zn (OSO 2 CF 3 ) 2 another notation: Zn (OTf) 2 , zinc trifluoromethanesulfonate
  • Zn (OSO 2 C 2 F 5 ) 2 Zn (OSO 2 C 3 F 7 ) 2
  • Zn (OSO 2 C 4 F 9 ) 2 Zn (OSO 2 C 6 H 4 CH 3 ) 2 (p-toluenesulfonic acid zinc)
  • Zn (OSO 2 C 6 H 5 ) 2 Zn ( BF 4 ) 2
  • Zn (PF 6 ) 2 , Hf (OTf) 4 hafnium trifluoromethanesulfonate
  • Sn (OTf) 2 Al (OTf) 3 , Cu (OTf) 2 and the like are also included.
  • These catalysts can be used alone or in combination of two or more.
  • the amount of the catalyst is, for example, 0.000001 to 0.1 mol, preferably 0.00005 to 0.05 mol, per 1 mol of pentamethylenediamine. Even if the amount of the catalyst is larger than this, no further significant reaction promoting effect is observed, but the cost may increase due to an increase in the amount of the catalyst. On the other hand, if the blending amount is less than this, the reaction promoting effect may not be obtained.
  • the addition method of the catalyst does not affect the reaction activity and is not particularly limited by any addition method of batch addition, continuous addition, and plural intermittent additions.
  • reaction solvent is not necessarily required.
  • the operability can be improved by blending the solvent.
  • Solvents that are inactive or poorly reactive with pentamethylenediamine, urea and / or N-unsubstituted carbamic acid esters as reaction raw materials, and alcohol and urethane compounds as reaction products If it is, it will not restrict
  • reaction solvent examples include the extraction solvent in the above-described extraction.
  • reaction solvents aliphatic hydrocarbons and aromatic hydrocarbons are preferably used in view of economy and operability.
  • reaction solvent is preferably the extraction solvent in the above-described extraction.
  • the extracted pentamethylene diisocyanate can be used for the carbamation reaction as it is, and the operability can be improved.
  • reaction solvents can be used alone or in combination of two or more.
  • the amount of the reaction solvent is not particularly limited as long as the target product pentamethylene dicarbamate is dissolved, but industrially, the reaction solvent needs to be recovered from the reaction solution. If the energy consumed for recovery is reduced as much as possible and the amount is large, the reaction substrate concentration decreases and the reaction rate slows down. More specifically, it is usually used in the range of 0.1 to 500 parts by weight, preferably 1 to 100 parts by weight with respect to 1 part by weight of pentamethylenediamine.
  • the reaction temperature is appropriately selected, for example, in the range of 100 to 350 ° C., preferably 150 to 300 ° C.
  • the reaction rate may decrease.
  • the reaction temperature is higher than this, side reaction may increase and the yield of the target product, pentamethylene dicarbamate may decrease.
  • the reaction pressure is usually atmospheric pressure, but may be increased when the boiling point of the component in the reaction solution is lower than the reaction temperature, and further reduced as necessary.
  • the reaction time is, for example, 0.1 to 20 hours, preferably 0.5 to 10 hours. If the reaction time is shorter than this, the yield of the target product, pentamethylene dicarbamate, may decrease. On the other hand, if it is longer than this, it is unsuitable for industrial production.
  • pentamethylenediamine, urea and / or N-unsubstituted carbamic acid ester, alcohol, and, if necessary, a catalyst and a reaction solvent are charged into the reaction vessel under the above-described conditions, and stirred or mixed. do it. Then, pentamethylene dicarbamate is produced in a short time, at a low cost and in a high yield under mild conditions.
  • the obtained pentamethylene dicarbamate usually corresponds to the above pentamethylene diamine used as a raw material component, and more specifically 1,5-pentamethylene dicarbamate is obtained.
  • reaction type either a batch type or a continuous type can be adopted as a reaction type.
  • this reaction is preferably performed while the by-produced ammonia flows out of the system. Furthermore, when N-unsubstituted carbamic acid ester is blended, the reaction is carried out while distilling off the by-produced alcohol out of the system.
  • pentamethylene dicarbamate When the obtained pentamethylene dicarbamate is isolated, for example, excess (unreacted) urea and / or N-unsubstituted carbamic acid ester, excess (unreacted) alcohol, catalyst, pentamethylene dicarbamate.
  • the pentamethylene dicarbamate may be separated from the reaction solution containing carbamate, reaction solvent, by-produced ammonia, and optionally by-produced alcohol by a known separation and purification method.
  • the obtained pentamethylene dicarbamate is thermally decomposed to produce pentamethylene diisocyanate.
  • the pentamethylene dicarbamate obtained as described above is thermally decomposed to produce pentamethylene diisocyanate and alcohol as a by-product.
  • the obtained pentamethylene diisocyanate usually corresponds to the above pentamethylene diamine used as a raw material component, and more specifically 1,5-pentamethylene diisocyanate is obtained.
  • alcohol usually the same kind of alcohol as that used as a raw material component is by-produced.
  • This thermal decomposition is not particularly limited, and for example, a known decomposition method such as a liquid phase method or a gas phase method can be used.
  • pentamethylene diisocyanate and alcohol produced by thermal decomposition can be separated from the gaseous product mixture by fractional condensation.
  • pentamethylene diisocyanate and alcohol produced by thermal decomposition can be separated by, for example, distillation or using a solvent and / or an inert gas as a support material.
  • a liquid phase method is preferably used from the viewpoint of workability.
  • the thermal decomposition reaction of pentamethylene dicarbamate in the liquid phase method is a reversible reaction, it is preferable to suppress the reverse reaction of the thermal decomposition reaction (urethanization reaction of pentamethylene diisocyanate and alcohol).
  • the pentamethylene diisocyanate and / or by-product alcohol is extracted from the reaction mixture as a gas, for example, and separated from the reaction mixture.
  • pentamethylene dicarbamate can be thermally decomposed satisfactorily, and pentamethylene diisocyanate and alcohol generated in the thermal decomposition evaporate, whereby the equilibrium of pentamethylene dicarbamate and pentamethylene diisocyanate is achieved.
  • side reactions such as polymerization of pentamethylene diisocyanate are suppressed.
  • the thermal decomposition temperature is usually 350 ° C. or lower, preferably 80 to 350 ° C., more preferably 100 to 300 ° C. If it is lower than 80 ° C., a practical reaction rate may not be obtained, and if it exceeds 350 ° C., undesirable side reactions such as polymerization of pentamethylene diisocyanate may occur.
  • the pressure at the time of the pyrolysis reaction is preferably a pressure at which the generated alcohol can be vaporized with respect to the above-mentioned pyrolysis reaction temperature. It is preferably 90 kPa.
  • the pentamethylene dicarbamate used for the thermal decomposition may be purified, but the above reaction (that is, reaction of pentamethylenediamine with urea and / or N-unsubstituted carbamic acid ester with alcohol). After completion, excess (unreacted) urea and / or N-unsubstituted carbamic acid ester, excess (unreacted) alcohol, catalyst, reaction solvent, by-product ammonia, and optionally by-product alcohol are recovered and separated. The resulting raw material of pentamethylene dicarbamate may be subsequently pyrolyzed.
  • a catalyst and an inert solvent may be added.
  • these catalysts and inert solvents differ depending on their types, they may be added to the above reaction either before or after distillation separation after the reaction or before or after separation of pentamethylene dicarbamate.
  • the catalyst used for the thermal decomposition is selected from, for example, Sn, Sb, Fe, Co, Ni, Cu, Zn, Cr, Ti, Pb, Mo, Mn, etc. used in the urethanization reaction between isocyanate and hydroxyl group.
  • One or more kinds of simple metals or their oxides, halides, carboxylates, phosphates, metal compounds such as organometallic compounds, and the like are used.
  • Fe, Sn, Co, Sb, and Mn are preferably used because they exhibit the effect of making it difficult to generate by-products.
  • Sn metal catalyst examples include tin oxide, tin chloride, tin bromide, tin iodide, tin formate, tin acetate, tin oxalate, tin octylate, tin stearate, tin oleate, tin phosphate, Examples include dibutyltin chloride, dibutyltin dilaurate, 1,1,3,3-tetrabutyl-1,3-dilauryloxydistanoxane.
  • Examples of the metal catalyst of Fe, Co, Sb, and Mn include acetates, benzoates, naphthenates, and acetylacetonates.
  • the blending amount of the catalyst is, for example, in the range of 0.0001 to 5% by mass, preferably in the range of 0.001 to 1% by mass with respect to the reaction solution as a single metal or a compound thereof.
  • the inert solvent is not particularly limited as long as it dissolves at least pentamethylene dicarbamate, is inert to pentamethylene dicarbamate and isocyanate, and is stable at the temperature in thermal decomposition.
  • the boiling point is preferably higher than that of the isocyanate to be produced.
  • examples of such inert solvents include esters such as dioctyl phthalate, didecyl phthalate, and didodecyl phthalate, such as dibenzyltoluene, triphenylmethane, phenylnaphthalene, biphenyl, diethylbiphenyl, and triethylbiphenyl.
  • Aromatic hydrocarbons and aliphatic hydrocarbons that are commonly used as media.
  • the inert solvent is also available as a commercial product.
  • barrel process oil B-01 aromatic hydrocarbons, boiling point: 176 ° C.
  • barrel process oil B-03 aromatic hydrocarbons, Boiling point: 280 ° C.
  • barrel process oil B-04AB aromatic hydrocarbons, boiling point: 294 ° C.
  • barrel process oil B-05 aromatic hydrocarbons, boiling point: 302 ° C.
  • barrel process oil B-27 Aromatic hydrocarbons, boiling point: 380 ° C
  • barrel process oil B-28AN aromatic hydrocarbons, boiling point: 430 ° C
  • barrel process oil B-30 aromatic hydrocarbons, boiling point: 380 ° C
  • Barrel therm 200 aromatic hydrocarbons, boiling point: 382 ° C.
  • barrel therm 300 aromatic hydrocarbons, boiling point: 344 ° C.
  • barrel therm 400 aromatic hydrocarbons
  • the amount of the inert solvent is, for example, in the range of 0.001 to 100 parts by weight, preferably 0.01 to 80 parts by weight, and more preferably 0.1 to 100 parts by weight with respect to 1 part by weight of pentamethylene dicarbamate.
  • the range is 50 parts by mass.
  • This thermal decomposition reaction is a batch reaction in which pentamethylene dicarbamate, a catalyst and an inert solvent are charged all at once, or a continuous reaction in which pentamethylene dicarbamate is charged in an inert solvent containing a catalyst under reduced pressure. Either can be implemented.
  • pentamethylene diisocyanate and alcohol are generated, and side reactions may generate, for example, allophanate, amines, urea, carbonate, carbamate, carbon dioxide, etc.
  • the obtained pentamethylene diisocyanate is purified by a known method.
  • the carbamate method is not described in detail, but besides the urea method described above, a known carbonate method, that is, pentamethylene dicarbamate is synthesized from pentamethylenediamine and dialkyl carbonate or diaryl carbonate, and the pentamethylene is synthesized.
  • a method of obtaining pentamethylene diisocyanate by thermally decomposing dicarbamate in the same manner as described above can also be employed.
  • the purity of the pentamethylene diisocyanate of the present invention thus obtained is, for example, 95 to 100% by mass, preferably 97 to 100% by mass, more preferably 98 to 100% by mass, and particularly preferably 99 to 100% by mass. %, Most preferably 99.5 to 100% by mass.
  • a stabilizer or the like can be added to pentamethylene diisocyanate.
  • the stabilizer examples include an antioxidant, an acidic compound, a compound containing a sulfonamide group, and an organic phosphite.
  • antioxidants examples include hindered phenol antioxidants, and specific examples include 2,6-di (t-butyl) -4-methylphenol, 2,4,6-triphenol.
  • -T-butylphenol 2,2'-methylenebis- (4-methyl-6-t-butylphenol), 2,2'-thio-bis- (4-methyl-6-t-butylphenol), 4,4'- Thio-bis (3-methyl-6-t-butylphenol), 4,4'-butylidene-bis- (6-t-butyl-3-methylphenol), 4,4'-methylidene-bis- (2,6 -Di-t-butylphenol), 2,2'-methylene-bis- [4-methyl-6- (1-methylcyclohexyl) -phenol], tetrakis- [methylene-3- (3,5-di-t- Butyl-4-hydro Cyphenyl) -propionyl] -methane, 1,3,5-trimethyl-2,4,6-tris- (3,5
  • antioxidants can be used alone or in combination of two or more.
  • the acidic compound examples include organic acidic compounds. Specifically, for example, phosphate ester, phosphite ester, hypophosphite ester, formic acid, acetic acid, propionic acid, hydroxyacetic acid, oxalic acid, lactic acid Citric acid, malic acid, sulfonic acid, sulfonic acid ester, phenol, enol, imide, oxime and the like.
  • organic acidic compounds Specifically, for example, phosphate ester, phosphite ester, hypophosphite ester, formic acid, acetic acid, propionic acid, hydroxyacetic acid, oxalic acid, lactic acid Citric acid, malic acid, sulfonic acid, sulfonic acid ester, phenol, enol, imide, oxime and the like.
  • These acidic compounds can be used alone or in combination of two or more.
  • Examples of the compound containing a sulfonamide group include aromatic sulfonamides and aliphatic sulfonamides.
  • aromatic sulfonamides include benzenesulfonamide, dimethylbenzenesulfonamide, sulfanilamide, o- and p-toluenesulfonamide, hydroxynaphthalenesulfonamide, naphthalene-1-sulfonamide, naphthalene-2-sulfonamide, Examples thereof include m-nitrobenzenesulfonamide and p-chlorobenzenesulfonamide.
  • Examples of the aliphatic sulfonamides include methanesulfonamide, N, N-dimethylmethanesulfonamide, N, N-dimethylethanesulfonamide, N, N-diethylmethanesulfonamide, N-methoxymethanesulfonamide, N- Examples include dodecylmethanesulfonamide, N-cyclohexyl-1-butanesulfonamide, and 2-aminoethanesulfonamide.
  • These compounds containing a sulfonamide group can be used alone or in combination of two or more.
  • organic phosphites include organic phosphite diesters and organic phosphite triesters, and more specifically, for example, triethyl phosphite, tributyl phosphite, tris (2-ethylhexyl) phosphine.
  • tridecyl phosphite trilauryl phosphite, tris (tridecyl) phosphite, tristearyl phosphite, triphenyl phosphite, tris (nonylphenyl) phosphite, tris (2,4-di-t-butylphenyl) Monophosphites such as phosphite, diphenyldecyl phosphite, diphenyl (tridecyl) phosphite, such as distearyl pentaerythrityl diphosphite, di-dodecyl pentaerythritol diphosphite, di-tridecyl From polyhydric alcohols such as pentaerythritol diphosphite, dinonylphenyl pentaerythritol diphosphite, tetraphenyl tetratetrade
  • organic phosphites can be used alone or in combination of two or more.
  • the stabilizer preferably includes an antioxidant, an acidic compound, and a compound containing a sulfonamide group. More preferably, pentamethylene diisocyanate is mixed with an antioxidant and an acidic compound and / or a compound containing a sulfonamide group.
  • the mixing ratio of the stabilizer is not particularly limited, and is appropriately set according to necessity and application.
  • the blending ratio of the antioxidant is, for example, 0.0005 to 0.05 parts by mass with respect to 100 parts by mass of pentamethylene diisocyanate.
  • the compounding ratio of the acidic compound and / or the compound containing a sulfone and group is, for example, 0.0005 to 0.02 with respect to 100 parts by mass of pentamethylene diisocyanate. Part by mass.
  • the present invention further includes a method for producing a polyisocyanate composition.
  • the polyisocyanate composition is obtained by modifying pentamethylene diisocyanate, and contains at least one of the following functional groups (a) to (e).
  • the polyisocyanate composition containing the functional group (isocyanurate group) of (a) is pentamethylene diisocyanate.
  • the trimer (trimer) can be obtained by, for example, reacting pentamethylene diisocyanate in the presence of a known isocyanuration catalyst and trimerization.
  • the polyisocyanate composition containing the functional group (allophanate group) of the above (b) is an allophanate modified product of pentamethylene diisocyanate, and, for example, after reacting pentamethylene diisocyanate with a monoalcohol, a known allophanate is formed. It can be obtained by further reaction in the presence of a catalyst.
  • the polyisocyanate composition containing the functional group (biuret group) of the above (c) is a biuret-modified product of pentamethylene diisocyanate, for example, pentamethylene diisocyanate, for example, water, tertiary alcohol (for example, t -Butyl alcohol, etc.), secondary amines (eg, dimethylamine, diethylamine, etc.) and the like, and then further reacted in the presence of a known biuretization catalyst.
  • pentamethylene diisocyanate for example, pentamethylene diisocyanate
  • tertiary alcohol for example, t -Butyl alcohol, etc.
  • secondary amines eg, dimethylamine, diethylamine, etc.
  • the polyisocyanate composition containing the functional group (urethane group) of (b) is a polyol-modified product of pentamethylene diisocyanate, for example, pentamethylene diisocyanate and a polyol component (for example, trimethylolpropane, etc. ).
  • the polyisocyanate composition containing the functional group (urea group) of (e) is a polyamine-modified product of pentamethylene diisocyanate, and for example, by reaction of pentamethylene diisocyanate with water, a polyamine component (described later), Obtainable.
  • the polyisocyanate composition only needs to contain at least one of the functional groups (a) to (e) described above, and may contain two or more. Such a polyisocyanate composition is produced by appropriately combining the above reactions.
  • polyisocyanate composition examples include pentamethylene diisocyanate trimer (polyisocyanate composition containing isocyanurate group).
  • the trimer of pentamethylene diisocyanate contains polyisocyanate having an iminooxadiazinedione group in addition to the isocyanurate group.
  • a polyurethane resin can be obtained by reacting the above pentamethylene diisocyanate and / or the above polyisocyanate composition with an active hydrogen compound.
  • Examples of the active hydrogen compound include a polyol component (a component mainly containing a polyol having two or more hydroxyl groups), a polyamine component (a compound mainly containing a polyamine having two or more amino groups), and the like.
  • examples of the polyol component include low molecular weight polyols and high molecular weight polyols.
  • the low molecular weight polyol is a compound having two or more hydroxyl groups and a number average molecular weight of less than 400, such as ethylene glycol, propylene glycol, 1,3-propanediol, 1,4-butylene glycol, 1,3-butylene glycol.
  • These low molecular weight polyols can be used alone or in combination of two or more.
  • the high molecular weight polyol is a compound having two or more hydroxyl groups and a number average molecular weight of 400 or more.
  • polyether polyol polyester polyol, polycarbonate polyol, polyurethane polyol, epoxy polyol, vegetable oil polyol, polyolefin polyol, acrylic polyol, vinyl A monomer modified polyol etc. are mentioned.
  • polyether polyol examples include polypropylene glycol and polytetramethylene ether glycol.
  • polypropylene glycol examples include addition polymers of alkylene oxides such as ethylene oxide and propylene oxide (the random and / or two or more types of alkylene oxides) using the above-described low molecular weight polyols or aromatic / aliphatic polyamines as initiators. Including a block copolymer).
  • polytetramethylene ether glycol examples include a ring-opening polymer obtained by cationic polymerization of tetrahydrofuran, and amorphous polytetramethylene ether glycol obtained by copolymerizing the above-described dihydric alcohol with a polymerization unit of tetrahydrofuran.
  • polyester polyol examples include polycondensates obtained by reacting the above-described low molecular weight polyol and polybasic acid under known conditions.
  • polybasic acid examples include oxalic acid, malonic acid, succinic acid, methyl succinic acid, glutaric acid, adipic acid, 1,1-dimethyl-1,3-dicarboxypropane, 3-methyl-3-ethylglutaric acid , Azelaic acid, sebacic acid, other saturated aliphatic dicarboxylic acids (C11-13) such as maleic acid, fumaric acid, itaconic acid, other unsaturated aliphatic dicarboxylic acids such as orthophthalic acid, isophthalic acid, terephthalic acid , Toluene dicarboxylic acid, naphthalene dicarboxylic acid, other aromatic dicarboxylic acids such as hexahydrophthalic acid, other alicyclic dicarboxylic acids such as dimer acid, hydrogenated dimer acid, het acid and other carboxylic acids, And acid anhydrides derived from these carboxylic acids, such as oxalic an
  • polyester polyol for example, a plant-derived polyester polyol, specifically, a hydroxyl group-containing vegetable oil fatty acid (for example, castor oil fatty acid containing ricinoleic acid, 12-hydroxystearic acid, using the above-described low molecular weight polyol as an initiator, And vegetable oil-based polyester polyols obtained by subjecting a hydroxycarboxylic acid such as hydrogenated castor oil fatty acid and the like to a condensation reaction under known conditions.
  • a hydroxycarboxylic acid such as hydrogenated castor oil fatty acid and the like
  • the polyester polyol for example, the above-described low molecular weight polyol (preferably dihydric alcohol) is used as an initiator, for example, lactones such as ⁇ -caprolactone and ⁇ -valerolactone, for example, L-lactide, D- Examples thereof include polycaprolactone polyol, polyvalerolactone polyol obtained by ring-opening polymerization of lactides such as lactide, and lactone polyester polyol obtained by copolymerizing the above-described dihydric alcohol.
  • lactones such as ⁇ -caprolactone and ⁇ -valerolactone
  • L-lactide L-lactide
  • D- Examples thereof include polycaprolactone polyol, polyvalerolactone polyol obtained by ring-opening polymerization of lactides such as lactide, and lactone polyester polyol obtained by copolymerizing the above-described dihydric alcohol.
  • polycarbonate polyol examples include a ring-opening polymer of ethylene carbonate using the above-described low molecular weight polyol (preferably a dihydric alcohol) as an initiator, for example, 1,4-butanediol, 1,5-pentanediol, Examples thereof include amorphous polycarbonate polyols obtained by copolymerizing a dihydric alcohol such as 3-methyl-1,5-pentanediol and 1,6-hexanediol with a ring-opening polymer.
  • the polyurethane polyol is, for example, a ratio in which the equivalent ratio (OH / NCO) of the hydroxyl group (OH) to the isocyanate group (NCO) of the polyester polyol, polyether polyol and / or polycarbonate polyol obtained as described above exceeds 1.
  • polyester polyurethane polyol, polyether polyurethane polyol, polycarbonate polyurethane polyol, or polyester polyether polyurethane polyol can be obtained.
  • epoxy polyol examples include the low molecular weight polyols described above, for example, epoxy polyols obtained by reaction with polyfunctional halohydrins such as epichlorohydrin and ⁇ -methylepichlorohydrin.
  • Examples of the vegetable oil polyol include hydroxyl group-containing vegetable oils such as castor oil and palm oil.
  • castor oil polyol, or ester-modified castor oil polyol obtained by reaction of castor oil fatty acid and polypropylene polyol can be used.
  • polystyrene resin examples include polybutadiene polyol, partially saponified ethylene-vinyl acetate copolymer, and the like.
  • acrylic polyol examples include a copolymer obtained by copolymerizing a hydroxyl group-containing acrylate and a copolymerizable vinyl monomer copolymerizable with the hydroxyl group-containing acrylate.
  • hydroxyl group-containing acrylates examples include 2-hydroxyethyl (meth) acrylate, hydroxypropyl (meth) acrylate, hydroxybutyl (meth) acrylate, 2,2-dihydroxymethylbutyl (meth) acrylate, polyhydroxyalkyl maleate, Examples thereof include polyhydroxyalkyl fumarate.
  • Preferable examples include 2-hydroxyethyl (meth) acrylate.
  • Examples of the copolymerizable vinyl monomer include methyl (meth) acrylate, ethyl (meth) acrylate, propyl (meth) acrylate, isopropyl (meth) acrylate, butyl (meth) acrylate, isobutyl (meth) acrylate, s-butyl ( Alkyl (meth) acrylate, t-butyl (meth) acrylate, pentyl (meth) acrylate, isopentyl (meth) acrylate, hexyl (meth) acrylate, isononyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, cyclohexyl acrylate, etc.
  • (Meth) acrylate (having 1 to 12 carbon atoms), for example, aromatic vinyl such as styrene, vinyltoluene and ⁇ -methylstyrene, vinyl cyanide such as (meth) acrylonitrile, Vinyl monomers containing carboxyl groups such as (meth) acrylic acid, fumaric acid, maleic acid, itaconic acid, or alkyl esters thereof such as ethylene glycol di (meth) acrylate, butylene glycol di (meth) acrylate, hexanediol di ( Alkane polyol poly (meth) acrylates such as meth) acrylate, oligoethylene glycol di (meth) acrylate, trimethylolpropane di (meth) acrylate, trimethylolpropane tri (meth) acrylate, for example 3- (2-isocyanate-2 And vinyl monomers containing an isocyanate group such as -propyl) - ⁇ -methylstyren
  • the acrylic polyol can be obtained by copolymerizing these hydroxyl group-containing acrylate and copolymerizable vinyl monomer in the presence of a suitable solvent and a polymerization initiator.
  • silicone polyols and fluorine polyols.
  • silicone polyol examples include an acrylic polyol in which a silicone compound containing a vinyl group such as ⁇ -methacryloxypropyltrimethoxysilane is blended as the copolymerizable vinyl monomer in the copolymerization of the acrylic polyol described above. .
  • the fluorine polyol for example, in the copolymerization of the acrylic polyol described above, as the copolymerizable vinyl monomer, for example, an acrylic polyol in which a fluorine compound containing a vinyl group such as tetrafluoroethylene or chlorotrifluoroethylene is blended may be mentioned. .
  • the vinyl monomer-modified polyol can be obtained by a reaction between the above-described high molecular weight polyol and a vinyl monomer.
  • the high molecular weight polyol is preferably a high molecular weight polyol selected from polyether polyol, polyester polyol and polycarbonate polyol.
  • examples of the vinyl monomer include the above-described alkyl (meth) acrylate, vinyl cyanide, vinylidene cyanide, and the like. These vinyl monomers can be used alone or in combination of two or more. Of these, alkyl (meth) acrylate is preferable.
  • the vinyl monomer-modified polyol is obtained by reacting these high molecular weight polyol and vinyl monomer in the presence of a radical polymerization initiator (for example, persulfate, organic peroxide, azo compound, etc.), for example. Can be obtained.
  • a radical polymerization initiator for example, persulfate, organic peroxide, azo compound, etc.
  • These high molecular weight polyols can be used alone or in combination of two or more.
  • the high molecular weight polyol is preferably a polyester polyol or an acrylic polyol, more preferably a polyester polyol, and particularly preferably a plant-derived polyester polyol.
  • polyol components can be used alone or in combination of two or more.
  • polyamine component examples include an aromatic polyamine, an araliphatic polyamine, an alicyclic polyamine, an aliphatic polyamine, an amino alcohol, a primary amino group, or an alkoxy having a primary amino group and a secondary amino group.
  • polyamine component examples include silyl compounds and polyoxyethylene group-containing polyamines.
  • aromatic polyamines examples include 4,4'-diphenylmethanediamine and tolylenediamine.
  • Examples of the araliphatic polyamine include 1,3- or 1,4-xylylenediamine or a mixture thereof.
  • alicyclic polyamine examples include 3-aminomethyl-3,5,5-trimethylcyclohexylamine (also known as isophoronediamine), 4,4′-dicyclohexylmethanediamine, 2,5 (2,6) -bis ( Aminomethyl) bicyclo [2.2.1] heptane, 1,4-cyclohexanediamine, 1-amino-3-aminomethyl-3,5,5-trimethylcyclohexane, bis- (4-aminocyclohexyl) methane, diaminocyclohexane 3,9-bis (3-aminopropyl) -2,4,8,10-tetraoxaspiro [5.5] undecane, 1,3- and 1,4-bis (aminomethyl) cyclohexane and mixtures thereof Etc.
  • 3-aminomethyl-3,5,5-trimethylcyclohexylamine also known as isophoronediamine
  • aliphatic polyamine examples include ethylenediamine, propylenediamine, 1,3-propanediamine, 1,4-butanediamine, 1,5-pentanediamine, 1,6-hexamethylenediamine, and hydrazine (including hydrates). ), Diethylenetriamine, triethylenetetramine, tetraethylenepentamine, 1,2-diaminoethane, 1,2-diaminopropane, 1,3-diaminopentane and the like.
  • amino alcohol examples include N- (2-aminoethyl) ethanolamine.
  • alkoxysilyl compound having a primary amino group or a primary amino group and a secondary amino group examples include ⁇ -aminopropyltriethoxysilane and N-phenyl- ⁇ -aminopropyltrimethoxysilane.
  • alkoxysilyl group-containing monoamines such as N- ⁇ (aminoethyl) ⁇ -aminopropyltrimethoxysilane, such as N- ⁇ (aminoethyl) ⁇ -aminopropylmethyldimethoxysilane.
  • polyoxyethylene group-containing polyamine examples include polyoxyalkylene ether diamines such as polyoxyethylene ether diamine. More specifically, for example, PEG # 1000 diamine manufactured by NOF Corporation, Jeffamine ED-2003, EDR-148, XTJ-512 manufactured by Huntsman, etc.
  • polyamine components can be used alone or in combination of two or more.
  • a known additive for example, a plasticizer, an anti-blocking agent, a heat stabilizer, a light stabilizer, an antioxidant, a release agent, a catalyst, a pigment, a dye, Lubricants, fillers, hydrolysis inhibitors and the like can be added.
  • a plasticizer for example, a plasticizer, an anti-blocking agent, a heat stabilizer, a light stabilizer, an antioxidant, a release agent, a catalyst, a pigment, a dye, Lubricants, fillers, hydrolysis inhibitors and the like
  • additives may be added at the time of synthesis of each component, or may be added at the time of mixing / dissolving each component, and may be added after the synthesis.
  • the polyurethane resin can be produced by a polymerization method such as bulk polymerization or solution polymerization.
  • an active hydrogen compound is added to the reaction temperature, for example, 50 to 250 ° C., more preferably, The reaction is carried out at 50 to 200 ° C., for example, for about 0.5 to 15 hours.
  • a pentamethylene diisocyanate and / or polyisocyanate composition and an active hydrogen compound are added to an organic solvent, and the reaction temperature is, for example, 50 to 120 ° C., more preferably 50 to 100 ° C. The reaction is carried out for about 5 to 15 hours.
  • organic solvent examples include ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone and cyclohexanone, nitriles such as acetonitrile, alkyl esters such as methyl acetate, ethyl acetate, butyl acetate and isobutyl acetate, such as n- Aliphatic hydrocarbons such as hexane, n-heptane and octane, for example, alicyclic hydrocarbons such as cyclohexane and methylcyclohexane, for example, aromatic hydrocarbons such as toluene, xylene and ethylbenzene, such as methyl cellosolve acetate , Ethyl cellosolve acetate, methyl carbitol acetate, ethyl carbitol acetate, ethylene glycol ethyl ether acetate, propylene glyco
  • examples of the organic solvent include nonpolar solvents (nonpolar organic solvents).
  • nonpolar organic solvents include aliphatic and naphthenic hydrocarbon organic solvents having an aniline point of, for example, 10 to Examples include non-polar organic solvents having low toxicity and weak dissolving power at 70 ° C., preferably 12 to 65 ° C., and vegetable oils represented by terpene oil.
  • Such a nonpolar organic solvent is available as a commercial product.
  • Examples of such a commercial product include House (manufactured by Shell Chemical Co., Ltd., aniline point 15 ° C.), Swazol 310 (manufactured by Maruzen Petroleum Corporation, aniline point 16 ° C. ), Essonaphtha No. 6 (manufactured by Exxon Chemical Co., Ltd., aniline point 43 ° C.), wax (manufactured by Shell Chemical Co., Ltd., aniline point 43 ° C.), Essonaphtha No.
  • a urethanization catalyst can be added as necessary.
  • amines include tertiary amines such as triethylamine, triethylenediamine, bis- (2-dimethylaminoethyl) ether, N-methylmorpholine, and quaternary ammonium salts such as tetraethylhydroxylammonium, such as imidazole, And imidazoles such as 2-ethyl-4-methylimidazole.
  • tertiary amines such as triethylamine, triethylenediamine, bis- (2-dimethylaminoethyl) ether, N-methylmorpholine
  • quaternary ammonium salts such as tetraethylhydroxylammonium, such as imidazole, And imidazoles such as 2-ethyl-4-methylimidazole.
  • organometallic compounds include tin acetate, tin octylate, tin oleate, tin laurate, dibutyltin diacetate, dimethyltin dilaurate, dibutyltin dilaurate, dibutyltin dimercaptide, dibutyltin maleate, dibutyltin dilaurate, dibutyltin Organic tin compounds such as dineodecanoate, dioctyltin dimercaptide, dioctyltin dilaurate, dibutyltin dichloride, for example, organic lead compounds such as lead octoate and lead naphthenate, for example, organic nickel compounds such as nickel naphthenate, Examples thereof include organic cobalt compounds such as cobalt naphthenate, organic copper compounds such as copper octenoate, and organic bismuth compounds such as bismuth octylate and bismuth
  • examples of the urethanization catalyst include potassium salts such as potassium carbonate, potassium acetate, and potassium octylate.
  • urethanization catalysts can be used alone or in combination of two or more.
  • the (unreacted) pentamethylene diisocyanate and / or polyisocyanate composition can be removed by a known removal means such as distillation or extraction.
  • pentamethylene diisocyanate and / or polyisocyanate composition and an active hydrogen compound are converted into an active hydrogen group (hydroxyl group, amino group) in the active hydrogen compound and pentamethylene diisocyanate and / or polyisocyanate. It is blended so that the equivalent ratio of isocyanate groups (NCO / active hydrogen group) in the composition is, for example, 0.75 to 1.3, preferably 0.9 to 1.1.
  • the polyurethane resin can be obtained by a known method such as a one-shot method or a prepolymer method, depending on the application.
  • a polyurethane resin can also be obtained as an aqueous dispersion (PUD) etc. by another method.
  • a pentamethylene diisocyanate and / or polyisocyanate composition and an active hydrogen compound are mixed with an active hydrogen group (hydroxyl group, amino group) in the active hydrogen compound in the pentamethylene diisocyanate and / or polyisocyanate composition.
  • an active hydrogen group hydroxyl group, amino group
  • the curing reaction is performed at 250 ° C., preferably at room temperature to 200 ° C., for example, for 5 minutes to 72 hours, preferably for 4 to 24 hours.
  • the curing temperature may be a constant temperature, or may be raised or cooled stepwise.
  • a pentamethylene diisocyanate and / or polyisocyanate composition is first reacted with a part of an active hydrogen compound (preferably, a high molecular weight polyol) to form an isocyanate having an isocyanate group at the molecular end.
  • an active hydrogen compound preferably, a high molecular weight polyol
  • a base end prepolymer is synthesized.
  • the obtained isocyanate group-terminated prepolymer and the remainder of the active hydrogen compound preferably, a low molecular weight polyol and / or polyamine component
  • the remainder of the active hydrogen compound is used as a chain extender.
  • a pentamethylene diisocyanate and / or polyisocyanate composition and a portion of the active hydrogen compound may be combined with pentamethylene diisocyanate and / or polyisocyanate for the active hydrogen group in a portion of the active hydrogen compound.
  • Formulated so that the equivalent ratio of isocyanate groups (NCO / active hydrogen group) in the isocyanate composition is, for example, 1.1 to 20, preferably 1.3 to 10, and more preferably 1.3 to 6. (Mixing) and reacting in a reaction vessel at room temperature to 150 ° C., preferably 50 to 120 ° C., for example, for 0.5 to 18 hours, preferably 2 to 10 hours.
  • the urethanization catalyst described above may be added, and after the reaction, if necessary, an unreacted pentamethylene diisocyanate and / or polyisocyanate composition may be added. For example, it can also be removed by a known removal means such as distillation or extraction.
  • the isocyanate group-terminated prepolymer and the remainder of the active hydrogen compound are reacted with the active hydrogen group in the remainder of the active hydrogen compound.
  • the equivalent ratio of isocyanate groups (NCO / active hydrogen groups) in the isocyanate group-terminated prepolymer is, for example, 0.75 to 1.3, preferably 0.9 to 1.1.
  • the curing reaction is performed at room temperature to 250 ° C., preferably at room temperature to 200 ° C., for example, for 5 minutes to 72 hours, preferably for 1 to 24 hours.
  • a polyurethane resin as an aqueous dispersion, for example, first, a pentamethylene diisocyanate and / or polyisocyanate composition and an active hydrogen compound containing a hydrophilic group described below (hereinafter abbreviated as a hydrophilic group-containing active hydrogen compound). To obtain an isocyanate group-terminated prepolymer.
  • an aqueous polyurethane resin in which the isocyanate group-terminated prepolymer is chain-extended with a chain extender can be obtained as an internal emulsion type aqueous dispersion.
  • the isocyanate group-terminated prepolymer is added to water to disperse the isocyanate group-terminated prepolymer.
  • a chain extender is added thereto to chain extend the isocyanate group-terminated prepolymer.
  • the hydrophilic group-containing active hydrogen compound is a compound having both a hydrophilic group and an active hydrogen group.
  • the hydrophilic group include an anionic group (for example, carboxyl group), a cationic group, and a nonionic group (for example, And polyoxyethylene groups). More specifically, examples of the hydrophilic group-containing active hydrogen compound include a carboxylic acid group-containing active hydrogen compound, a polyoxyethylene group-containing active hydrogen compound, and the like.
  • carboxylic acid group-containing active hydrogen compound examples include 2,2-dimethylolacetic acid, 2,2-dimethylollactic acid, 2,2-dimethylolpropionic acid, 2,2-dimethylolbutanoic acid, and 2,2-dimethylolbutyric acid.
  • dihydroxycarboxylic acids such as 2,2-dimethylolvaleric acid, diaminocarboxylic acids such as lysine and arginine, or metal salts and ammonium salts thereof.
  • the polyoxyethylene group-containing active hydrogen compound is a compound having a polyoxyethylene group in the main chain or side chain and having two or more active hydrogen groups, for example, polyethylene glycol, polyoxyethylene side chain-containing polyol ( And compounds having a polyoxyethylene group in the side chain and having two or more active hydrogen groups).
  • hydrophilic group-containing active hydrogen compounds can be used alone or in combination of two or more.
  • chain extender for example, low molecular weight polyols such as the above-described dihydric alcohols and trihydric alcohols described above, for example, diamines such as alicyclic diamines and aliphatic diamines can be used.
  • chain extenders can be used alone or in combination of two or more.
  • the hydrophilic group is neutralized with a known neutralizing agent as necessary.
  • hydrophilic group-containing active hydrogen compound when a hydrophilic group-containing active hydrogen compound is not used as the active hydrogen compound, it can be obtained as an external emulsion type aqueous dispersion by, for example, emulsification using a known surfactant.
  • 1,5-pentamethylene diisocyanate, polyisocyanate composition and polyurethane resin using 1,5-pentamethylenediamine obtained with high production rate, high reaction yield and high quality as raw materials are, for example, polycarbonate, ABS Also suitable for various plastic coating materials for polyethylene terephthalate, nylon, polyolefin, etc., coating raw materials for solar cell backsheet members, exteriors for automobiles and motorcycles, coating materials for metals, binders for ink, etc. it can.
  • the present invention can be applied to a laminate method, an industrial material, a housing / building adhesive material, and a sealing material.
  • thermoplastic or thermosetting polyurethane elastomer it can also be derived into films, sheets, tubes, hoses, powders or flexible gels, and various industrial uses such as medical, clothing, industrial parts, electronic / electrical parts, It can also be deployed in the health care field such as cosmetics. Moreover, it can apply also to capsule materials, such as various fragrance
  • L-lysine and 1,5-pentamethylenediamine were quantified by high performance liquid chromatography (HPLC). These analytical conditions and methods for measuring lysine decarboxylase activity are as follows. ⁇ Analysis conditions for 1,5-pentamethylenediamine> Column: Asahipak ODP-50 4E (manufactured by Showa Denko) Column temperature: 40 ° C Eluent: 0.2 M sodium phosphate (pH 7.7) +2.3 mM sodium 1-octanesulfonate Eluent flow rate: 0.5 mL / min For detection, a post-column derivatization method using orthophthalaldehyde [J. Chromatogr. 83, 353-355 (1973)].
  • the unit of activity was defined as 1 unit for the activity of producing 1 ⁇ mol of 1,5-pentamethylenediamine per minute.
  • the conversion ratio of isocyanate group is the ratio of the area of the peak on the high molecular weight side of the peak of pentamethylene diisocyanate to the total peak area, based on the chromatogram obtained under the following GPC measurement conditions, as the conversion ratio of isocyanate group. .
  • HLC-8020 (manufactured by Tosoh Corporation) Column: G1000HXL, G2000HXL and G3000HXL (above, trade name, manufactured by Tosoh Corporation) connected in series Column temperature: 40 ° C Eluent: Tetrahydrofuran Flow rate: 0.8 mL / min Detection method: Differential refractive index Standard material: Polyethylene oxide (manufactured by Tosoh Corporation, trade name: TSK standard polyethylene oxide) ⁇ Isocyanate trimer concentration (unit: mass%)> The measurement similar to the above (conversion rate of isocyanate group) was performed, and a peak area ratio corresponding to a molecular weight three times that of pentamethylene diisocyanate was defined as an isocyanate trimer concentration.
  • ⁇ Isocyanate group concentration (unit: mass%)> The isocyanate group concentration of the polyisocyanate composition was measured by an n-dibutylamine method according to JIS K-1556 using a potentiometric titrator.
  • ⁇ Viscosity (unit: mPa ⁇ s)> The viscosity at 25 ° C. of the polyisocyanate composition was measured using an E-type viscometer TV-30 manufactured by Toki Sangyo Co., Ltd.
  • ⁇ Hue (unit: APHA)> The hue of the polyisocyanate composition was measured by a method according to JIS K-0071.
  • oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 1 and 2 designed based on the nucleotide sequence of lysine decarboxylase gene (cadA) (GenBank Accession No. AP009048) (consigned to Invitrogen) Synthesized). These primers have restriction enzyme recognition sequences for KpnI and XbaI, respectively, near the 5 'end.
  • the PCR reaction product and the plasmid pUC18 were digested with KpnI and XbaI and ligated using Ligation High (Toyobo), and then the resulting recombinant plasmid was used to use Escherichia coli DH5 ⁇ (Toyobo).
  • the product was transformed.
  • the transformant was cultured on an LB agar medium containing 100 ⁇ g / mL of ampicillin (Am) and X-Gal (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside), and was resistant to Am and white colonies. The resulting transformant was obtained.
  • a plasmid was extracted from the transformant thus obtained.
  • the base sequence of the DNA fragment introduced into the plasmid was the base sequence shown in SEQ ID NO: 3 in the sequence listing.
  • the obtained plasmid having DNA encoding lysine decarboxylase was named pCADA1.
  • SEQ ID NO: 4 A sequence obtained by translating the DNA sequence shown in SEQ ID NO: 3 into an amino acid sequence is shown in SEQ ID NO: 4 in the sequence list.
  • Escherichia coli W3110 strain was transformed with pCADA1 by a conventional method, and the resulting transformant was named W / pCADA1.
  • the transformant was inoculated into 500 ml of LB medium containing 100 ⁇ g / mL of Am in a 2 L baffled Erlenmeyer flask and cultured with shaking at 30 ° C. for 26.5 hours. Thereafter, the culture broth was centrifuged at 8000 rpm for 10 minutes to collect the cells (the dry cell equivalent concentration was 31% (w / w)).
  • This cell suspension was kept in a 58 ° C. water bath for 30 minutes, subjected to heat treatment, and stored frozen at ⁇ 20 ° C. until use.
  • the recovered cells of the transformant W / pCADA1 were suspended in water to prepare a cell suspension with a dry cell equivalent concentration of 12.5% by mass.
  • sodium sulfite was added at 1.0 g / L, kept in a 58 ° C. water bath for 30 minutes, subjected to heat treatment, and stored frozen at ⁇ 20 ° C. until use.
  • the purified enzyme was cultured by the method described above, and the recovered cells were purified by the method of Sabo et al. (Biochemistry 13 (1974) pp. 662-670.). When the enzyme activity of the purified enzyme was measured, 1000 unit / mg of purified enzyme was obtained.
  • reaction cycle comprising denaturation: 96 ° C., 30 seconds, annealing: 55 ° C., 30 seconds, extension reaction: 68 ° C., 5 minutes, 20 seconds using pCADA1 as a template and SEQ ID NO: 5 and SEQ ID NO: 6 PCR was performed under conditions of 16 cycles.
  • the obtained amplified fragment was treated with DpnI and ligated using ligation high, and then the obtained recombinant plasmid was used or the amplified fragment treated with DpnI was directly added to competent cell DH5 ⁇ to obtain Escherichia coli DH5 ⁇ strain.
  • a plasmid was prepared from the prepared strain, the base sequence was determined, and it was confirmed that the target base was substituted.
  • the resulting plasmid was named pCAD2.
  • plasmids from pCAD3 to pCAD20 and from pCAD23 to pCAD119 were constructed.
  • the oligonucleotide sequences used are shown in Tables 1-6.
  • Escherichia coli W3110 strain was transformed with pCADA2 to pCADA20 by a conventional method, and the resulting transformants were named W / pCADA2 to W / pCADA20.
  • Escherichia coli W3110 strain was transformed with pCAD23 to pCAD119 by a conventional method, and the resulting transformants were named W / pCADA23 to W / pCADA119.
  • the transformant was inoculated into 500 ml of LB medium containing 100 ⁇ g / mL of Am in a 2 L baffled Erlenmeyer flask and cultured with shaking at 30 ° C. for 26.5 hours. Thereafter, the culture solution was centrifuged at 8000 rpm for 10 minutes to collect the cells (the dry cell equivalent concentration was 31% by mass).
  • Tables 7 to 9 show the correspondence between the mutation enzyme before mutation and after mutation.
  • PCR was performed under the conditions of 16 cycles of denaturation: 96 ° C., 30 seconds, annealing: 55 ° C., 30 seconds, extension reaction: 68 ° C., 5 minutes 20 seconds.
  • the obtained amplified fragment was treated with DpnI and ligated using ligation high, and then the obtained recombinant plasmid was used to transform E. coli DH5 ⁇ strain.
  • a plasmid was prepared from the prepared strain, the base sequence was determined, and it was confirmed that the target base was substituted.
  • a plasmid is similarly prepared using the nucleotide sequences shown in SEQ ID NOs: 35 and 36.
  • a plasmid is prepared by the above method using the nucleotide sequences shown in SEQ ID NOs: 41 and 42 in this plasmid.
  • plasmid pCADA21 having the quadruple mutant sequence was prepared.
  • the DNA sequence of the obtained quadruple mutant is shown in SEQ ID NO: 43 of the Sequence Listing.
  • the amino acid sequence is shown in SEQ ID NO: 44 in the sequence listing.
  • a plasmid pCADA22 having a quintuple mutant sequence was prepared by preparing a plasmid by the above-described method using the nucleotide sequences shown in SEQ ID NOs: 9 and 10 in this plasmid.
  • This plasmid was transformed into Escherichia coli W3110 strain by a usual method by a usual method, and the resulting transformants were named W / pCADA21 and W / pCADA22.
  • the DNA sequence of the obtained 5-fold mutant is shown in SEQ ID NO: 45 of the sequence listing.
  • the amino acid sequence is shown in SEQ ID NO: 46 in the sequence listing.
  • PCR was performed using pCADA73 as a template and an oligonucleotide having the nucleotide sequences shown in SEQ ID NOs: 235 and 236. Then, PCR was performed under the conditions of 16 cycles of denaturation: 96 ° C., 30 seconds, annealing: 55 ° C., 30 seconds, extension reaction: 68 ° C., 5 minutes, 20 seconds. The obtained amplified fragment was treated with DpnI, and this fragment was used to transform E. coli DH5 ⁇ strain. A plasmid was prepared from the prepared strain, the base sequence was determined, and it was confirmed that the target base was substituted. The resulting plasmid was named pCADA120.
  • PCR was carried out using pCADA95 as a template and an oligonucleotide having the nucleotide sequences shown in SEQ ID NOs: 227 and 228. Then, PCR was performed under the conditions of 16 cycles of denaturation: 96 ° C., 30 seconds, annealing: 55 ° C., 30 seconds, extension reaction: 68 ° C., 5 minutes, 20 seconds. The obtained amplified fragment was treated with DpnI, and this fragment was used to transform E. coli DH5 ⁇ strain. A plasmid was prepared from the prepared strain, the base sequence was determined, and it was confirmed that the target base was substituted. The resulting plasmid was named pCADA121.
  • PCR was performed using pCADA113 as a template and an oligonucleotide having the nucleotide sequences shown in SEQ ID NOs: 235 and 236. Then, PCR was performed under the conditions of 16 cycles of denaturation: 96 ° C., 30 seconds, annealing: 55 ° C., 30 seconds, extension reaction: 68 ° C., 5 minutes, 20 seconds. The obtained amplified fragment was treated with DpnI, and this fragment was used to transform E. coli DH5 ⁇ strain. A plasmid was prepared from the prepared strain, the base sequence was determined, and it was confirmed that the target base was substituted. The resulting plasmid was named pCADA122.
  • the transformant was inoculated into 500 ml of LB medium containing 100 ⁇ g / mL of ampicillin in a 2 L baffled Erlenmeyer flask and cultured with shaking at 30 ° C. for 26.5 hours. Thereafter, the culture solution was centrifuged at 8000 rpm for 10 minutes to collect the cells (the dry cell equivalent concentration was 31% by mass).
  • Example 4 [Example of nitrogen substitution reaction: wild-type enzyme] 120 g of a substrate solution prepared so that the final concentration of L-lysine hydrochloride is 45% by mass and the final concentration of pyridoxal phosphate is 0.15 mM is added to a 300 mL flask, and the reaction solution is added. Prepared.
  • reaction yield when not replacing with nitrogen gas was 95%.
  • Example 5 [Example of nitrogen substitution reaction: quadruple mutant enzyme] 120 g of a substrate solution prepared so that the final concentration of L-lysine hydrochloride is 45% by mass and the final concentration of pyridoxal phosphate is 0.15 mM is added to a 300 mL flask, and the reaction solution is added. Prepared. Next, nitrogen gas was passed through the reaction solution (0.3 L / hr), and the dissolved oxygen sensor was stirred (1 hour) until the dissolved oxygen concentration reached 0 ppm. The pH in the reaction solution at this time was 5.6.
  • W / pCADA21 catalyst dead cells prepared in Example 3 (0.0648 g in terms of dry cell weight, 0.0015 g of catalyst dead cells to 1 g of lysine) were added, and the temperature was 42 ° C. and 200 rpm.
  • the reaction was carried out with care not to allow oxygen to enter the reactor for 24 hours.
  • the oxygen concentration during the reaction period was 0 ppm, which was 0% of the saturated dissolved oxygen concentration (7.4 ppm) at 42 ° C.
  • finish of reaction was 8.0.
  • reaction yield when not replacing with nitrogen gas was 96%.
  • Example 6 [Example of nitrogen substitution reaction: 5-fold mutant enzyme] 120 g of a substrate solution prepared so that the final concentration of L-lysine hydrochloride is 45% by mass and the final concentration of pyridoxal phosphate is 0.15 mM is added to a 300 mL flask, and the reaction solution is added. Prepared. Next, nitrogen gas was passed through the reaction solution (0.3 L / hr), and the dissolved oxygen sensor was stirred (1 hour) until the dissolved oxygen concentration reached 0 ppm. The pH in the reaction solution at this time was 5.6.
  • W / pCADA22 catalyst dead cells prepared in Example 3 (0.0648 g in terms of dry cell weight, 0.0015 g ratio of catalyst dead cells to 1 g of lysine) were added, and at 42 ° C. and 200 rpm.
  • the reaction was carried out with care not to allow oxygen to enter the reactor for 24 hours.
  • the oxygen concentration during the reaction period was 0 ppm, which was 0% of the saturated dissolved oxygen concentration (7.4 ppm) at 42 ° C.
  • finish of reaction was 8.0.
  • Example reaction of reducing agent addition: wild-type enzyme 120 g of a substrate solution prepared so that the final concentration of L-lysine hydrochloride is 10% by mass and the final concentration of pyridoxal phosphate is 0.15 mM is added to a 300 mL flask, and the reaction solution is added. Prepared. Next, sodium sulfite, dithiothreitol, and sodium hydrosulfite were added so that each became 1.0 g / L, and it stirred until the dissolved oxygen concentration was set to 0 ppm with the dissolved oxygen sensor. The pH in the reaction solution at this time was 5.6.
  • catalyst dead cells of W / pCADA1 prepared in Reference Example 1 (0.0036 g in terms of dry cell weight, 0.0003 g of catalyst dead cells relative to 1 g of lysine) were added, and the temperature was 42 ° C. and 200 rpm.
  • the reaction was carried out with care not to allow oxygen to enter the reactor for 24 hours.
  • the dissolved oxygen concentration during the reaction period was 0 ppm, which was 0% of the saturated dissolved oxygen concentration (7.4 ppm) at 42 ° C.
  • the reaction yield when no sodium sulfite was added was 90%.
  • Example 8 [Example of partial substitution with nitrogen: wild-type enzyme] 120 g of a substrate solution prepared so that the final concentration of L-lysine hydrochloride is 45% by mass and the final concentration of pyridoxal phosphate is 0.15 mM is added to a 300 mL flask, and the reaction solution is added. Prepared. Next, nitrogen gas was passed through the reaction solution (0.3 L / hr), and the mixture was stirred until the dissolved oxygen concentration by the dissolved oxygen sensor (fermentation oxygen electrode (CSL-1, manufactured by Able)) reached 5 ppm. The pH in the reaction solution was 5.6.
  • catalyst dead cells of W / pCADA1 prepared in Reference Example 1 (0.0648 g in terms of dry cell weight, 0.0015 g of catalyst dead cells relative to 1 g of lysine) were added, and at 42 ° C. and 200 rpm.
  • the reaction was carried out with care not to allow oxygen to enter the reactor for 24 hours.
  • the dissolved oxygen concentration during the reaction period was 0 ppm, which was 0% of the saturated dissolved oxygen concentration (7.4 ppm) at 42 ° C.
  • finish of reaction was 7.9.
  • Example 9 [Example reaction of sodium sulfite addition: wild-type enzyme] 120 g of a substrate solution prepared so that the final concentration of L-lysine hydrochloride is 45% by mass and the final concentration of pyridoxal phosphate is 0.15 mM is added to a 300 mL flask, and the reaction solution is added. Prepared. Next, sodium sulfite was added so that it might become 1.7 g / L, and it stirred until the dissolved oxygen concentration was set to 0 ppm with the dissolved oxygen sensor. The pH in the reaction solution at this time was 5.6.
  • catalyst dead cells of W / pCADA1 prepared in Reference Example 1 (0.0648 g in terms of dry cell weight, 0.0015 g of catalyst dead cells relative to 1 g of lysine) were added, and at 42 ° C. and 200 rpm.
  • the reaction was carried out with care not to allow oxygen to enter the reactor for 24 hours.
  • the dissolved oxygen concentration during the reaction period was 0 ppm, which was 0% of the saturated dissolved oxygen concentration (7.4 ppm) at 42 ° C.
  • finish of reaction was 8.0.
  • Dissolve lysine hydrochloride (Wako) to 10% by mass take 5 ml each of sodium sulfite added to 1.7 g / L, put them in a PP container with a 15 ml screw cap, and add 0.4% 50 ⁇ l of PLP aqueous solution and 200 ⁇ l of well-stirred culture solution were added after dissolving the above frozen culture solution.
  • the reaction was carried out at 200 rpm, 45 ° C. for 2 hours with the container placed horizontally and parallel to the shaking direction.
  • the reaction was stopped by adding 1 mL of 2M hydrochloric acid to the reaction solution.
  • This reaction stop solution was appropriately diluted with water, and the produced 1,5-pentamethylenediamine was quantified by HPLC.
  • reaction yield of each mutant enzyme was 1.3 times higher than the reaction in which sodium sulfite was not added.
  • Example 11 [Example of reaction with sodium sulfite added: quadruple mutant enzyme] 120 g of a substrate solution prepared so that the final concentration of L-lysine hydrochloride is 45% by mass and the final concentration of pyridoxal phosphate is 0.15 mM is added to a 300 mL flask, and the reaction solution is added. Prepared. Next, sodium sulfite was added so that it might become 1.7 g / L.
  • W / pCADA21 catalyst dead cells prepared in Example 3 (0.0648 g in terms of dry cell weight, 0.0015 g of catalyst dead cells to 1 g of lysine) were added, and the temperature was 42 ° C. and 200 rpm.
  • the reaction was carried out with care not to allow oxygen to enter the reactor for 24 hours.
  • the dissolved oxygen concentration during the reaction period was 0 ppm, which was 0% of the saturated dissolved oxygen concentration (7.4 ppm) at 42 ° C.
  • finish of reaction was 8.0.
  • Example 12 [Reaction example of adding sodium sulfite: 5-fold mutant enzyme] 120 g of a substrate solution prepared so that the final concentration of L-lysine hydrochloride is 45% by mass and the final concentration of pyridoxal phosphate is 0.15 mM is added to a 300 mL flask, and the reaction solution is added. Prepared. Next, sodium sulfite was added so that it might become 1.7 g / L. Next, W / pCADA22 catalyst dead cells prepared in Example 3 (0.0648 g in terms of dry cell weight, 0.0015 g ratio of catalyst dead cells to 1 g of lysine) were added, and at 42 ° C.
  • reaction was carried out with care not to allow oxygen to enter the reactor for 24 hours.
  • the dissolved oxygen concentration during the reaction period was 0 ppm, which was 0% of the saturated dissolved oxygen concentration (7.4 ppm) at 42 ° C.
  • finish of reaction was 8.0.
  • Example 13 [Example reaction of adding sodium sulfite: quadruple mutant enzyme (minimum catalyst addition reaction)] 120 g of a substrate solution prepared so that the final concentration of L-lysine hydrochloride is 45% by mass and the final concentration of pyridoxal phosphate is 0.15 mM is added to a 300 mL flask, and the reaction solution is added. Prepared. Next, sodium sulfite was added so that it might become 1.7 g / L.
  • the catalyst dead cells of W / pCADA21 prepared in Example 3 (0.0324 g in terms of dry cell weight, 0.00075 g of catalyst dead cells relative to 1 g of lysine) were added, and at 42 ° C. and 200 rpm.
  • the reaction was carried out for 48 hours taking care not to allow oxygen to enter the reactor.
  • the dissolved oxygen concentration during the reaction period was 0 ppm, which was 0% of the saturated dissolved oxygen concentration (7.4 ppm) at 42 ° C.
  • finish of reaction was 8.20.
  • Example 14 [Example reaction of adding sodium sulfite: Five-fold mutation enzyme (minimum catalyst addition reaction)] 120 g of a substrate solution prepared so that the final concentration of L-lysine hydrochloride is 45% by mass and the final concentration of pyridoxal phosphate is 0.15 mM is added to a 300 mL flask, and the reaction solution is added. Prepared. Next, sodium sulfite was added so that it might become 1.7 g / L.
  • W / pCADA22 catalyst dead cells prepared in Example 3 (0.0324 g in terms of dry cell weight, 0.00075 g ratio of catalyst dead cells to 1 g of lysine) were added, and the temperature was 42 ° C. and 200 rpm.
  • the reaction was carried out for 48 hours taking care not to allow oxygen to enter the reactor.
  • the dissolved oxygen concentration during the reaction period was 0 ppm, which was 0% of the saturated dissolved oxygen concentration (7.4 ppm) at 42 ° C.
  • finish of reaction was 8.0.
  • Example 15 [Example of post-addition reaction of sodium sulfite: wild-type enzyme] 120 g of a substrate solution prepared so that the final concentration of L-lysine hydrochloride is 45% by mass and the final concentration of pyridoxal phosphate is 0.15 mM is added to a 300 mL flask, and the reaction solution is added. Prepared.
  • the dissolved oxygen concentration became 0% of the saturated dissolved oxygen concentration at 42 ° C.
  • the pH at the start of the reaction was 5.6, and the pH in the reaction solution immediately after the completion of the reaction was 8.0.
  • the residual activity of the dead catalyst cells prepared in the presence of sodium sulfite was 80%.
  • Example 17 [Production of 1,5-pentamethylene diisocyanate] (PDA purification from nitrogen-substituted reaction solution)
  • the reaction solution after completion of the reaction prepared in Example 5 was adjusted to pH 6.0 with sulfuric acid and then centrifuged at 8000 rpm for 20 minutes to remove precipitates such as cells and obtain a supernatant. Next, 30% sodium hydroxide solution was added to the supernatant to adjust the pH to 12.
  • an organic layer extract (n-butanol containing 1,5-pentamethylenediamine) was charged into a four-necked flask equipped with a thermometer, a distillation column, a cooling tube, and a nitrogen introduction tube, Under reduced pressure, the temperature of the oil bath was set to 120 ° C., and n-butanol was distilled off to obtain 1,5-pentamethylenediamine having a purity of 99.9% by mass.
  • a solution obtained by dissolving 400 parts by mass of pentamethylenediamine in 2600 parts by mass of orthodichlorobenzene was fed with a feed pump over 60 minutes, and cold phosgenation was started at 30 ° C. or lower and normal pressure. After completion of the feed, the pressure reactor became a pale brown white slurry.
  • the internal solution of the reactor was pressurized to 0.25 MPa while gradually raising the temperature to 160 ° C., and further subjected to thermal phosgenation at a pressure of 0.25 MPa and a reaction temperature of 160 ° C. for 90 minutes.
  • thermal phosgenation 1100 parts by mass of phosgene was further added.
  • the liquid in the pressurized reactor became a light brown clear solution.
  • nitrogen gas was passed at 100 L / hr at 100 to 140 ° C. for degassing.
  • the distilled pentamethylene diisocyanate was charged into a four-necked flask equipped with a stirrer, a thermometer, a reflux tube, and a nitrogen introduction tube, and at 190 ° C. under normal pressure while introducing nitrogen. Heat treatment was performed for a time.
  • the pentamethylene diisocyanate after the heat treatment is charged into a glass flask, using a distillation tube equipped with a packing, a distillation column equipped with a reflux ratio adjustment timer, and a rectification apparatus equipped with a cooler.
  • the rectification was further performed under reflux at 127 to 132 ° C. and 2.7 KPa to obtain 450 parts by mass of pentamethylene diisocyanate having a purity of 99.8% by mass.
  • Example 18 [Production of polyisocyanate composition]
  • TMP trimethylolpropane
  • 0.25 parts by mass of 2,6-di (tert-butyl) -4-methylphenol and 0.25 parts by mass of tris (tridecyl) phosphite were charged at 80 ° C. for 3 hours.
  • This polyisocyanate composition has a pentamethylene diisocyanate concentration of 0.3% by mass, an isocyanate trimer concentration of 29% by mass, an isocyanate group concentration of 21.8% by mass, a viscosity at 25 ° C. of 9850 mPa ⁇ s, and a hue of APHA40. there were.
  • the 1,5-pentamethylenediamine, 1,5-pentamethylene diisocyanate, and polyisocyanate composition obtained by the method for producing 1,5-pentamethylenediamine of the present invention are, for example, coated as a biomass-derived polymer raw material. It can be suitably used in various industrial fields such as adhesives, sealants, elastomers, gels, binders, films, sheets and capsules, agricultural chemicals and pharmaceutical intermediates.
  • the catalyst cell preserved by the method for preserving the catalyst cell of the present invention can stably store lysine decarboxylase for a long period of time, it is suitable for the production of, for example, biomass-derived polymer raw materials. Can be used.

Abstract

L-lysine and/or a salt thereof is subjected to lysine decarboxylation by lysine decarboxylase and/or mutant lysine decarboxylase in a reaction system in which the time that the dissolved oxygen concentration is the saturated dissolved oxygen concentration is within one hour.

Description

１，５－ペンタメチレンジアミンの製造方法、１，５－ペンタメチレンジイソシアネートの製造方法、ポリイソシアネート組成物の製造方法、および、触媒菌体の保存方法Method for producing 1,5-pentamethylenediamine, method for producing 1,5-pentamethylene diisocyanate, method for producing polyisocyanate composition, and method for preserving catalyst cells
　本発明は、１，５－ペンタメチレンジアミンの製造方法、１，５－ペンタメチレンジイソシアネートの製造方法、ポリイソシアネート組成物の製造方法、および、触媒菌体の保存方法に関するものである。 The present invention relates to a method for producing 1,5-pentamethylenediamine, a method for producing 1,5-pentamethylene diisocyanate, a method for producing a polyisocyanate composition, and a method for preserving catalyst cells.
　１，５－ペンタメチレンジアミンは、バイオマス由来のポリマー原料、例えば、ポリウレタン原料、ポリアミド原料として注目されている。 1,5-pentamethylenediamine is attracting attention as a biomass-derived polymer material, for example, a polyurethane material or a polyamide material.
　１，５－ペンタメチレンジアミンおよび／または１，５－ペンタメチレンジアミン塩（１，５－ペンタメチレンジアミン類）を製造する方法として、例えば、リジンおよび／またはリジン塩（リジン類）の溶液を原料とし、これに、微生物由来のリジン脱炭酸酵素（Ｌｙｓｉｎｅ　ｄｅｃａｒｂｏｘｙｌａｓｅ：ＬＤＣ）を作用させることにより、１，５－ペンタメチレンジアミン類の溶液を得る方法が開示されている（例えば、下記特許文献１参照。）。 As a method for producing 1,5-pentamethylenediamine and / or 1,5-pentamethylenediamine salt (1,5-pentamethylenediamine), for example, lysine and / or a solution of lysine salt (lysine) is used as a raw material. And a method of obtaining a solution of 1,5-pentamethylenediamine by allowing a lysine decarboxylase (LDC) derived from microorganisms to act thereon (see, for example, Patent Document 1 below) .)
　一方、リジンまたはリジンの塩からリジン脱炭酸酵素を用いて１，５－ペンタメチレンジアミンまたは１，５－ペンタメチレンジアミンの塩を工業的に製造する場合、使用するリジン脱炭酸酵素が高価であるため、その使用量をできるだけ削減することが望まれている。 On the other hand, when 1,5-pentamethylenediamine or 1,5-pentamethylenediamine salt is industrially produced from lysine or a lysine salt using lysine decarboxylase, the lysine decarboxylase used is expensive. Therefore, it is desired to reduce the amount of use as much as possible.
　また、このような１，５－ペンタメチレンジアミン類をポリアミド樹脂フィルム等の原料として用いる場合には、１，５－ペンタメチレンジアミン溶液中に存在する菌体（微生物）由来のタンパク質やペプチドが、ポリアミド樹脂フィルム等の表面外観の欠陥を招く原因の一つとなっている。 Further, when such 1,5-pentamethylenediamine is used as a raw material for a polyamide resin film or the like, proteins or peptides derived from bacterial cells (microorganisms) present in the 1,5-pentamethylenediamine solution are: This is one of the causes of defects in the surface appearance of polyamide resin films and the like.
　そこで、反応時にリジンの総重量に対して使用する菌体の重量の比率を０．００２以下に抑えることや、さらに、反応液への通気速度を０．３ｖｖｍ以下に制限すること、また、有機酸で中和してｐＨ調整することが、提案されている。これにより、触媒の反応速度を向上させることが可能となり、触媒使用量を削減でき、また、タンパク質やペプチドなどを低減して、フィッシュアイ等の表面外観の欠陥を防止することができる（例えば、下記特許文献２、３参照。）。 Therefore, the ratio of the weight of the microbial cells to be used with respect to the total weight of lysine during the reaction is suppressed to 0.002 or less, the aeration rate to the reaction solution is limited to 0.3 vvm or less, and organic It has been proposed to adjust the pH by neutralization with an acid. As a result, the reaction rate of the catalyst can be improved, the amount of catalyst used can be reduced, and proteins and peptides can be reduced to prevent surface appearance defects such as fish eyes (for example, (See Patent Documents 2 and 3 below.)
　また、リジン脱炭酸酵素は、反応至適ｐＨが５～６にあり、アルカリ側では反応活性の低下が起こることが知られている（例えば、下記非特許文献１参照。）。 In addition, lysine decarboxylase has a reaction optimum pH of 5 to 6, and it is known that a decrease in reaction activity occurs on the alkali side (for example, see Non-patent Document 1 below).
　また、リジン脱炭酸酵素は、生成する１，５－ペンタメチレンジアミンにより反応阻害を受けることが知られている（例えば、下記非特許文献２参照。）。 In addition, it is known that lysine decarboxylase is subject to reaction inhibition by the produced 1,5-pentamethylenediamine (see, for example, Non-Patent Document 2 below).
特開２００９－２０７４９５号公報JP 2009-207495 A 特開２００８－１８７９６３号公報JP 2008-187963 A 特開２００８－２２０１９５号公報JP 2008-220195 A
　上記したように、このような方法では、使用するリジン脱炭酸酵素の使用量をできるだけ削減することが望まれているところ、上記特許文献１には、精製酵素５０ｍｇ／Ｌで１Ｍのリジン塩酸塩を脱炭酸し、０．９７Ｍの１，５－ペンタメチレンジアミンが製造できることが開示されているが、酵素の精製には、多大なコストがかかるという不具合がある。 As described above, in such a method, it is desired to reduce the amount of lysine decarboxylase used as much as possible. Patent Document 1 discloses that 1M lysine hydrochloride at 50 mg / L of purified enzyme. Although it is disclosed that 0.97M 1,5-pentamethylenediamine can be produced by decarboxylation, the purification of the enzyme has a disadvantage that it takes a great deal of cost.
　一方、上記特許文献２および３には、触媒使用量を低減できることが開示されている（例えば、特許文献３の実施例１では、リジンの総重量に対して使用する菌体の重量の比率が０．００１７）。しかし、その反応液のリジン濃度は１０％程度であり、工業的に製造するためには容積効率が悪いため優れた方法とはいえない。 On the other hand, Patent Documents 2 and 3 disclose that the amount of catalyst used can be reduced (for example, in Example 1 of Patent Document 3, the ratio of the weight of the microbial cells used to the total weight of lysine is 0.0017). However, the lysine concentration of the reaction solution is about 10%, and it is not an excellent method because of its poor volumetric efficiency for industrial production.
　すなわち、このような方法では、上記非特許文献２に記載されるように、生成する１，５－ペンタメチレンジアミンによりリジン脱炭酸酵素が阻害を受けるため、反応液のリジン濃度が高まるとリジンに対する酵素必要量は多くなる。この点、上記のようにリジン濃度を下げれば、そのリジンに由来する１，５－ペンタメチレンジアミンの量が低減されるため、阻害効果が低減され、リジンに対する酵素必要量を低減できる。しかし、その一方で、生産設備が大きくなるという不具合がある。そのため、工業的な生産では、リジン濃度はできるだけ高く、なおかつ酵素量はできるだけ少なくすることが重要である。 That is, in such a method, as described in Non-Patent Document 2 above, lysine decarboxylase is inhibited by the generated 1,5-pentamethylenediamine. Enzyme requirement increases. In this respect, if the lysine concentration is lowered as described above, the amount of 1,5-pentamethylenediamine derived from the lysine is reduced, so that the inhibitory effect is reduced and the amount of enzyme required for lysine can be reduced. However, on the other hand, there is a problem that the production equipment becomes large. Therefore, in industrial production, it is important that the lysine concentration is as high as possible and the amount of enzyme is as small as possible.
　また、１，５－ペンタメチレンジアミンの塩の水溶液から１，５－ペンタメチレンジアミンを精製するためには、溶液のｐＨをアルカリとし、有機溶媒で抽出する方法が一般的である。この点、非特許文献１に記載されるように、リジン脱炭酸酵素反応におけるｐＨ条件を５～６にすると、必要なアルカリが増大し精製時の負荷が大きくなり、さらに、廃棄物の塩が大量に出るという不具合がある。 In order to purify 1,5-pentamethylenediamine from an aqueous solution of a salt of 1,5-pentamethylenediamine, a method in which the pH of the solution is alkali and extraction with an organic solvent is common. In this regard, as described in Non-Patent Document 1, when the pH condition in the lysine decarboxylase reaction is set to 5 to 6, the required alkali increases and the load during purification increases, and further, the waste salt is increased. There is a problem that it comes out in large quantities.
　この点、上記特許文献２および３に記載される有機酸による中和は、ポリアミドを生産する場合には、原料となるため好ましいが、１，５－ペンタメチレンジアミンをそのまま、またはイソシアネートなどに変換して用いる場合は不純物となるため好ましくない。また、精製工程での加熱などで一部ポリアミドが生成すると製品への不純物となり品質の低下を招くため好ましくない。そのため反応時の中和はできるだけ行なわないことが工業的な生産では重要である。 In this respect, neutralization with organic acids described in Patent Documents 2 and 3 above is preferable because it becomes a raw material when producing polyamide, but 1,5-pentamethylenediamine is converted as it is or into isocyanate or the like. When used as an impurity, it becomes an impurity, which is not preferable. Further, if a part of polyamide is produced by heating in the purification process, it becomes an impurity to the product and the quality is deteriorated. Therefore, it is important in industrial production that neutralization during the reaction is not performed as much as possible.
　さらに、このような反応において、リジン脱炭酸酵素は、生物菌体内において生産することができ、そのような菌体（触媒菌体）は、低コスト化の観点から、まとめて多量に製造され、１，５－ペンタメチレンジアミンの生産計画に合わせて使用される。 Furthermore, in such a reaction, lysine decarboxylase can be produced in a biological fungus body, and such a fungus body (catalyst fungus body) is collectively produced in a large amount from the viewpoint of cost reduction, Used in line with production plans for 1,5-pentamethylenediamine.
　しかしながら、触媒菌体を製造後に長期間保存すると、生産したリジン脱炭素酵素の活性が低下する場合がある。 However, if the catalyst cells are stored for a long time after production, the activity of the produced lysine decarbonase may be reduced.
　本発明の課題は、低コストで、収率よく、さらに、反応液のｐＨ調整を行わずに、１，５－ペンタメチレンジアミンを製造する方法、また、その方法により得られた１，５－ペンタメチレンジアミから１，５－ペンタメチレンジイソシアネートを製造する方法、さらに、その方法により得られた１，５－ペンタメチレンジイソシアネートからポリイソシアネート組成物を製造する方法を提供することである。 An object of the present invention is to provide a method for producing 1,5-pentamethylenediamine at a low cost and in a good yield without adjusting the pH of the reaction solution, and the 1,5-pentamethylene diamine obtained by the method. It is intended to provide a method for producing 1,5-pentamethylene diisocyanate from pentamethylene diami, and further a method for producing a polyisocyanate composition from 1,5-pentamethylene diisocyanate obtained by the method.
　また、本発明の別の課題は、リジン脱炭酸酵素を長期間安定に保存できる方法を提供することである。 Another object of the present invention is to provide a method capable of stably storing lysine decarboxylase for a long period of time.
　本発明者らは、上記課題を解決するために鋭意検討した結果、リジン脱炭酸酵素は、反応液中に存在する酸化物質により反応速度の低下または停止を来たし、所定量以下に酵素を削減できないことを見出した。反応系内に存在する酸化物質による酵素の酸化を防ぐことで酵素活性の低下を防ぐことが可能となり、酵素を精製することなく、従来技術以上に酵素量を削減しても高収率で１，５－ペンタメチレンジアミンを製造できることを見出した。また、反応液のリジン濃度を高め、反応時間が長くなっても酵素活性の低下を防ぐことが可能となるため、反応が完結できる。更に、反応液の中和を行わなくとも反応が完結できることを見出し、本発明を完成させた。 As a result of intensive studies to solve the above problems, the inventors of the present invention have reduced or stopped the reaction rate of lysine decarboxylase due to the oxidant present in the reaction solution, and the enzyme cannot be reduced below a predetermined amount. I found out. By preventing the oxidation of the enzyme by the oxidant present in the reaction system, it is possible to prevent a decrease in the enzyme activity. Even if the amount of the enzyme is reduced more than the conventional technology without purifying the enzyme, the yield is 1 It was found that 5-pentamethylenediamine can be produced. Further, since the lysine concentration in the reaction solution is increased and the enzyme activity can be prevented from decreasing even when the reaction time is prolonged, the reaction can be completed. Furthermore, the inventors have found that the reaction can be completed without neutralizing the reaction solution, and completed the present invention.
　また、触媒菌体の保存において、リジン脱炭酸酵素遺伝子の組換え微生物を還元剤の存在下で保存すれば、長期間の保存によるリジン脱炭酸酵の活性の低下を防ぐことができることを見出し発明を完成させた。 In addition, in the preservation of catalytic cells, it has been found that if a recombinant microorganism of a lysine decarboxylase gene is stored in the presence of a reducing agent, a decrease in the activity of lysine decarboxylation can be prevented by long-term storage. Was completed.
　すなわち本発明は、
［１］　溶存酸素濃度が飽和溶存酸素濃度である時間が１時間以内である反応系内において、Ｌ－リジンおよび／またはその塩を、リジン脱炭酸酵素および／または変異型リジン脱炭酸酵素によって、リジン脱炭酸反応させることを特徴とする、１，５－ペンタメチレンジアミンの製造方法、
［２］　Ｌ－リジンおよび／またはその塩１質量部に対して、リジン脱炭酸酵素および／または変異型リジン脱炭酸酵素が、乾燥菌体重量換算で、0.0003質量部以上0.0015質量部以下であることを特徴とする、［１］に記載の１，５－ペンタメチレンジアミンの製造方法、
［３］　Ｙ軸を飽和溶存酸素濃度に対する溶存酸素濃度の割合（％）、Ｘ軸を時間（分）として、リジン脱炭酸反応における溶存酸素濃度と時間との関係がプロットされた相関線を示す相関図において、相関線とＹ軸とＸ軸とに囲まれる部分の面積が、１０００未満であることを特徴とする、請求項［１］または［２］に記載の１，５－ペンタメチレンジアミンの製造方法、
［４］　前記面積が、６５０以下であることを特徴とする、［３］に記載の１，５－ペンタメチレンジアミンの製造方法、
［５］　前記溶存酸素濃度が、飽和溶存酸素濃度の６５％以下であり、かつ、前記溶存酸素濃度が、飽和溶存酸素濃度の６５％以下の状態から２０分以内に飽和溶存酸素濃度の１％以下になることを特徴とする、［３］または［４］に記載の１，５－ペンタメチレンジアミンの製造方法、
［６］　反応系内の酸素を除去する工程、および／または、反応系内に還元剤を存在させる工程を含むことを特徴とする、［１］～［５］のいずれか一項に記載の１，５－ペンタメチレンジアミンの製造方法、
［７］　反応系内の酸素を除去する工程が、不活性ガスにより溶存酸素を置換する工程であることを特徴とする、［６］に記載の１，５－ペンタメチレンジアミンの製造方法、
［８］　還元剤の酸化還元電位が、生理食塩水より低いことを特徴とする、［６］に記載の１，５－ペンタメチレンジアミンの製造方法、
［９］　還元剤が、メルカプト化合物、硫化物、水硫化物、還元性を有する硫黄の酸素酸塩、チオウレアおよびその誘導体、ヒドロキシル基および／またはカルボキシル基を有する環状化合物、フラボノイド化合物、窒素含有複素環化合物、ヒドラジル基化合物、および、ウロン酸基を有するムコ多糖類からなる群より選択される少なくとも１種であることを特徴とする、［８］に記載の１，５－ペンタメチレンジアミンの製造方法、
［１０］　変異型リジン脱炭酸酵素が、配列表の配列番号４に記載のアミノ酸配列において、１３７、１３８、２８６、２９０、２９５、３０３、３１７、３３５、３５２、３５３、３８６、４４３、４６６、４７５、５５３、７１０および７１１番目のアミノ酸残基の少なくとも１つが、他のアミノ酸残基に置換されている変異型リジン脱炭酸酵素であることを特徴とする、［１］～［９］のいずれか一項に記載の１，５－ペンタメチレンジアミンの製造方法、
［１１］　変異型リジン脱炭酸酵素が、配列表の配列番号４に記載のアミノ酸配列において、２９０、３３５、４７５および７１１番目のアミノ酸残基が、他のアミノ酸残基に置換されている変異型リジン脱炭酸酵素であることを特徴とする、［１０］に記載の１，５－ペンタメチレンジアミンの製造方法、
［１２］　変異型リジン脱炭酸酵素が、配列表の配列番号４に記載のアミノ酸配列において、２８６、２９０、３３５、４７５および７１１番目のアミノ酸残基が、他のアミノ酸残基に置換されている変異型リジン脱炭酸酵素であることを特徴とする、［１０］に記載の１，５－ペンタメチレンジアミンの製造方法、
［１３］　上記［１］に記載の方法により得られた１，５－ペンタメチレンジアミンまたはその塩を、イソシアネート化することを特徴とする、１，５－ペンタメチレンジイソシアネートの製造方法、
［１４］　上記［１３］に記載の方法により得られた１，５－ペンタメチレンジイソシアネートを、下記（ａ）～（ｅ）の官能基を少なくとも１種含有するように変性することを特徴とする、ポリイソシアネート組成物の製造方法、
（ａ）イソシアヌレート基
（ｂ）アロファネート基
（ｃ）ビウレット基
（ｄ）ウレタン基
（ｅ）ウレア基
［１５］　リジン脱炭酸酵素および／または変異型リジン脱炭酸酵素を発現させた細胞を、還元剤の存在下で保存することを特徴とする、触媒菌体の保存方法、
［１６］　還元剤の酸化還元電位が、生理食塩水より低いことを特徴とする、［１５］に記載の触媒菌体の保存方法、
［１７］　還元剤が、メルカプト化合物、硫化物、水硫化物、還元性を有する硫黄の酸素酸塩、チオウレアおよびその誘導体、ヒドロキシル基および／またはカルボキシル基を有する環状化合物、フラボノイド化合物、窒素含有複素環化合物、ヒドラジル基化合物、および、ウロン酸基を有するムコ多糖類からなる群より選択される少なくとも１種であることを特徴とする、［１５］または［１６］に記載の触媒菌体の保存方法
である。 That is, the present invention
[1] In a reaction system in which the dissolved oxygen concentration is a saturated dissolved oxygen concentration within 1 hour, L-lysine and / or a salt thereof is converted by lysine decarboxylase and / or mutant lysine decarboxylase. A process for producing 1,5-pentamethylenediamine, characterized by subjecting to lysine decarboxylation;
[2] The amount of lysine decarboxylase and / or mutant lysine decarboxylase is 0.0003 parts by mass or more and 0.0015 parts by mass or less in terms of dry cell weight per 1 part by mass of L-lysine and / or its salt. A process for producing 1,5-pentamethylenediamine according to [1], characterized in that
[3] A correlation line in which the relationship between dissolved oxygen concentration and time in lysine decarboxylation reaction is plotted with the Y axis as the ratio (%) of the dissolved oxygen concentration to the saturated dissolved oxygen concentration and the X axis as the time (minutes). The 1,5-pentamethylenediamine according to claim [1] or [2], wherein the area of the portion surrounded by the correlation line, the Y axis and the X axis is less than 1000 in the correlation diagram Manufacturing method,
[4] The method for producing 1,5-pentamethylenediamine according to [3], wherein the area is 650 or less,
[5] The dissolved oxygen concentration is 65% or less of the saturated dissolved oxygen concentration, and the dissolved oxygen concentration is 1% of the saturated dissolved oxygen concentration within 20 minutes from the state of 65% or less of the saturated dissolved oxygen concentration. The process for producing 1,5-pentamethylenediamine according to [3] or [4], characterized in that:
[6] The method according to any one of [1] to [5], including a step of removing oxygen in the reaction system and / or a step of allowing a reducing agent to be present in the reaction system. A process for producing 1,5-pentamethylenediamine,
[7] The method for producing 1,5-pentamethylenediamine according to [6], wherein the step of removing oxygen in the reaction system is a step of replacing dissolved oxygen with an inert gas,
[8] The method for producing 1,5-pentamethylenediamine according to [6], wherein the redox potential of the reducing agent is lower than that of physiological saline,
[9] The reducing agent is a mercapto compound, sulfide, hydrosulfide, reductive sulfur oxyacid salt, thiourea and its derivatives, a cyclic compound having a hydroxyl group and / or a carboxyl group, a flavonoid compound, a nitrogen-containing complex. The production of 1,5-pentamethylenediamine according to [8], which is at least one selected from the group consisting of a ring compound, a hydrazyl group compound, and a mucopolysaccharide having a uronic acid group Method,
[10] The mutant lysine decarboxylase is 137, 138, 286, 290, 295, 303, 317, 335, 352, 353, 386, 443, 466, in the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing. Any one of [1] to [9], wherein at least one of the amino acid residues at 475, 553, 710 and 711 is a mutant lysine decarboxylase substituted with another amino acid residue A process for producing 1,5-pentamethylenediamine according to claim 1,
[11] A mutant type in which the mutant lysine decarboxylase is substituted with other amino acid residues at the 290th, 335th, 475th, and 711st amino acid residues in the amino acid sequence set forth in SEQ ID NO: 4 in the Sequence Listing The method for producing 1,5-pentamethylenediamine according to [10], which is lysine decarboxylase,
[12] The mutant lysine decarboxylase is substituted with other amino acid residues at the 286th, 290th, 335th, 475th and 711th amino acid residues in the amino acid sequence set forth in SEQ ID NO: 4 in the Sequence Listing. The method for producing 1,5-pentamethylenediamine according to [10], which is a mutant lysine decarboxylase,
[13] A process for producing 1,5-pentamethylene diisocyanate, characterized in that 1,5-pentamethylenediamine or a salt thereof obtained by the method described in [1] above is isocyanated,
[14] The 1,5-pentamethylene diisocyanate obtained by the method described in [13] above is modified so as to contain at least one of the following functional groups (a) to (e): A method for producing a polyisocyanate composition,
(A) isocyanurate group (b) allophanate group (c) biuret group (d) urethane group (e) urea group [15] Reducing lysine decarboxylase and / or mutant lysine decarboxylase A method for preserving catalytic cells, characterized by storing in the presence of an agent;
[16] The method for preserving a catalytic cell according to [15], wherein the redox potential of the reducing agent is lower than that of physiological saline,
[17] The reducing agent is a mercapto compound, sulfide, hydrosulfide, reducing sulfur oxyacid salt, thiourea and derivatives thereof, cyclic compound having a hydroxyl group and / or carboxyl group, flavonoid compound, nitrogen-containing complex. Preservation of catalytic cell according to [15] or [16], which is at least one selected from the group consisting of a ring compound, a hydrazyl group compound, and a mucopolysaccharide having a uronic acid group Is the method.
　本発明の１，５－ペンタメチレンジアミンの製造方法によれば、反応系内の溶存酸素濃度が飽和溶存酸素濃度である時間が１時間以内なので、リジン脱炭酸酵素または／および変異型リジン脱炭酸酵素の酵素活性の失活を抑制でき、高濃度のリジン反応液であっても低酵素量で収率よく、さらに、反応液のｐＨ調整を行わずに、１，５－ペンタメチレンジアミンを得ることができる。その結果、低コストで高品質な１，５－ペンタメチレンジアミンを製造できる。 According to the method for producing 1,5-pentamethylenediamine of the present invention, since the time during which the dissolved oxygen concentration in the reaction system is the saturated dissolved oxygen concentration is within one hour, lysine decarboxylase or / and mutant lysine decarboxylation Inactivation of the enzyme activity of the enzyme can be suppressed, and even with a high concentration lysine reaction solution, a low enzyme amount and a good yield are obtained, and 1,5-pentamethylenediamine is obtained without adjusting the pH of the reaction solution. be able to. As a result, high-quality 1,5-pentamethylenediamine can be produced at low cost.
　そのため、本発明の１，５－ペンタメチレンジイソシアネートの製造方法、および、ポリイソシアネート組成物の製造方法によれば、低コストで収率よく、高品質な１，５－ペンタメチレンジイソシアネートおよびポリイソシアネート組成物を得ることができる。 Therefore, according to the method for producing 1,5-pentamethylene diisocyanate and the method for producing a polyisocyanate composition of the present invention, high-quality 1,5-pentamethylene diisocyanate and polyisocyanate composition can be obtained at a low cost and in a high yield. You can get things.
　また、本発明の触媒菌体の保存方法によれば、リジン脱炭酸酵素を長期間安定に保存することができる。 Moreover, according to the method for storing a catalytic cell of the present invention, lysine decarboxylase can be stored stably for a long period of time.
図１は、Ｙ軸を飽和溶存酸素濃度に対する溶存酸素濃度の割合（％）、Ｘ軸を時間（分）として、模式的に、リジン脱炭酸反応における溶存酸素濃度と時間との関係をプロットした相関線を示す模式相関図の一例である。FIG. 1 schematically plots the relationship between the dissolved oxygen concentration and time in the lysine decarboxylation reaction, with the Y axis as the ratio (%) of the dissolved oxygen concentration to the saturated dissolved oxygen concentration and the X axis as the time (minutes). It is an example of the schematic correlation diagram which shows a correlation line. 図２は、Ｙ軸を飽和溶存酸素濃度に対する溶存酸素濃度の割合（％）、Ｘ軸を時間（分）として、模式的に、リジン脱炭酸反応における溶存酸素濃度と時間との関係をプロットした相関線を示す模式相関図の他の一例である。FIG. 2 schematically plots the relationship between the dissolved oxygen concentration and time in the lysine decarboxylation reaction, with the Y axis as the ratio (%) of the dissolved oxygen concentration to the saturated dissolved oxygen concentration and the X axis as the time (minutes). It is another example of the schematic correlation diagram which shows a correlation line.
発明の実施形態Embodiment of the Invention
　以下、本発明について実施の形態を挙げて詳細に説明するが、本発明は以下の説明に限定されるものではなく、その要旨を超えない範囲において種々に変更して実施することができる。 Hereinafter, the present invention will be described in detail with reference to embodiments, but the present invention is not limited to the following description, and various modifications can be made without departing from the scope of the present invention.
　本発明において「１，５－ペンタメチレンジアミン」とは、１，５－ペンタンジアミン（Ｈ２Ｎ（ＣＨ２）５ＮＨ２）をいう。１，５－ペンタメチレンジアミンは、ポリマー原料や医薬中間体の合成原料として有用な化合物である。
（１）リジン脱炭酸酵素
　本発明におけるリジン脱炭酸酵素とは、国際生化学連合（Ｉ．Ｕ．Ｂ．）酵素委員会報告に準拠した酵素番号ＥＣ４．１．１．１８に分類され、ピリドキサールリン酸（ＰＬＰ）を補酵素として要求し、Ｌ－リジン（リジンとも記載する）から１，５－ペンタメチレンジアミン（ペンタン１，５―ジアミン、１，５―ペンタメチレンジアミン、ＰＤＡとも記載する）および炭酸を生成する反応を触媒する酵素、この酵素を遺伝子組み換えなどの技術で高生産している菌体、および、その処理物を指す。本発明のリジン脱炭酸酵素は、その由来は特に限定されるものではなく、例えば、公知の生物に由来するものが挙げられる。リジン脱炭酸酵素として、より具体的には、例えば、バシラス・ハロドゥランス（Ｂａｃｉｌｌｕｓ　ｈａｌｏｄｕｒａｎｓ）、バシラス・サブチリス（Ｂａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ）、エシェリシア・コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）、ビブリオ・コレラ（Ｖｉｂｒｉｏ　ｃｈｏｌｅｒａｅ）、ビブリオ・パラヘモリティカス（Ｖｉｂｒｉｏ　ｐａｒａｈａｅｍｏｌｙｔｉｃｕｓ）、ストレプトマイセス・コエリカーラ（Ｓｔｒｅｐｔｏｍｙｃｅｓ　ｃｏｅｌｉｃｏｌｏｒ）、ストレプトマイセス・ピロサス（Ｓｔｒｅｐｔｏｍｙｃｅｓ　ｐｉｌｏｓｕｓ）、エイケネラ・コロデンス（Ｅｉｋｅｎｅｌｌａ　ｃｏｒｒｏｄｅｎｓ）、イユバクテリウム・アシダミノフィルム（Ｅｕｂａｃｔｅｒｉｕｍ　ａｃｉｄａｍｉｎｏｐｈｉｌｕｍ）、サルモネラ・ティフィムリウム（Ｓａｌｍｏｎｅｌｌａ　ｔｙｐｈｉｍｕｒｉｕｍ）、ハフニア・アルベイ（Ｈａｆｎｉａ　ａｌｖｅｉ）、ナイセリア・メニンギチデス（Ｎｅｉｓｓｅｒｉａ　ｍｅｎｉｎｇｉｔｉｄｉｓ）、テルモプラズマ・アシドフィルム（Ｔｈｅｒｍｏｐｌａｓｍａ　ａｃｉｄｏｐｈｉｌｕｍ）、ピロコッカス・アビシ（Ｐｙｒｏｃｏｃｃｕｓ　ａｂｙｓｓｉ）またはコリネバクテリウム・グルタミカス（Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ　ｇｌｕｔａｍｉｃｕｍ）などの微生物に由来するものが挙げられる。安全性の観点から、好ましくは、Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉに由来するものが挙げられる。 In the present invention, “1,5-pentamethylenediamine” refers to 1,5-pentanediamine (H 2 N (CH 2 ) 5 NH 2 ). 1,5-pentamethylenediamine is a useful compound as a polymer raw material or a raw material for synthesizing pharmaceutical intermediates.
(1) Lysine decarboxylase The lysine decarboxylase in the present invention is classified into enzyme number EC 4.1.1.18 based on the report of the International Biochemical Union (IUB) Enzyme Committee, and pyridoxal. Phosphoric acid (PLP) is required as a coenzyme, and L-lysine (also referred to as lysine) to 1,5-pentamethylenediamine (also referred to as pentane 1,5-diamine, 1,5-pentamethylenediamine, PDA) And an enzyme that catalyzes a reaction that produces carbonic acid, a microbial cell that produces this enzyme in a high amount by a technique such as genetic recombination, and a processed product thereof. The origin of the lysine decarboxylase of the present invention is not particularly limited, and examples thereof include those derived from known organisms. More specific examples of lysine decarboxylase include, for example, Bacillus halodurans, Bacillus subtilis, Escherichia coli, Vibrio cholera, and Vibrio cholera. Vibrio parahaemolyticus, Streptomyces coelicolor, Streptomyces pilosus, Eikenella eucoridae ), Salmonella typhimurium, Hafnia albei, Neisseria meningitidis, Thermoplasma acid pi Examples thereof include those derived from microorganisms such as Corynebacterium glutamicum. From the viewpoint of safety, preferably, those derived from Escherichia coli are used.
　発現する遺伝子は、同様な効果を示せば特に制限はないが、大腸菌由来のｃａｄＡ（ＧｅｎＢａｎｋ　Ａｃｃｅｓｓｉｏｎ　Ｎｏ．ＡＰ００９０４８）が好適である。
（２）リジン脱炭酸酵素活性
　本発明において、リジン脱炭酸酵素活性とは、リジンを脱炭酸して１，５－ペンタメチレンジアミンへと変換する反応を触媒する活性を意味する。本発明においては、リジンからの１，５－ペンタメチレンジアミンの生成量を高速液体クロマトグラフィー（ＨＰＬＣ）で測定することにより、算出することができる。 The gene to be expressed is not particularly limited as long as it exhibits the same effect, but cadA derived from E. coli (GenBank Accession No. AP009048) is preferable.
(2) Lysine decarboxylase activity In the present invention, lysine decarboxylase activity means an activity that catalyzes a reaction in which lysine is decarboxylated and converted to 1,5-pentamethylenediamine. In the present invention, it can be calculated by measuring the amount of 1,5-pentamethylenediamine produced from lysine by high performance liquid chromatography (HPLC).
　活性の単位は、１分間に１μｍｏｌの１，５－ペンタメチレンジアミンを製造する活性を１ｕｎｉｔ（Ｕ）とし、菌体活性は、乾燥菌体換算重量１ｍｇ当たりの酵素活性（Ｕ／ｍｇ　ｄｒｙ　ｃｅｌｌｓ）で表示する。乾燥菌体換算重量とは、乾燥して水分を含まない重量を表わし、例えば、菌体を含む液（菌体液）から遠心分離やろ過などの方法で菌体を分離し、重量が一定になるまで乾燥し、その重量を測定することで乾燥菌体換算重量を求めることができる。
（３）菌体
　本発明における菌体とは、複数の種類に分けられる。誤解を避けるため、本発明においては、以下のように定める。リジン脱炭酸酵素を高生産し、野生株より高いリジン脱炭酸活性を有する菌体を「触媒菌体」とする。さらに、生きている触媒菌体を「触媒生菌体」、生育を休止している触媒菌体を「触媒休止菌体」、増殖能を消失している触媒菌体を「触媒死菌体」とする。
（４）変異型リジン脱炭酸酵素
　本発明における変異型リジン脱炭酸酵素とは、主に遺伝子組換え技術を利用して、野生型リジン脱炭酸酵素のアミノ酸配列において少なくとも１つのアミノ酸残基が他のアミノ酸残基に置換された変異を有し、かつ、リジン脱炭酸酵素自体の酵素活性が向上したことを特徴とするリジン脱炭酸酵素と定義される。なお、アミノ酸配列中のアミノ酸は、リジン脱炭酸酵素中のアミノ酸残基に対応しており、それらは互いに対応関係にある。以下において、アミノ酸と称する場合には、アミノ酸配列として表記されるアミノ酸を示し、アミノ酸残基と称する場合には、リジン脱炭酸酵素中に含まれるアミノ酸残基を示す。 The unit of activity is 1 unit (U) for producing 1 μmol of 1,5-pentamethylenediamine per minute, and the cell activity is the enzyme activity (U / mg dry cells) per 1 mg of dry cell equivalent weight. Is displayed. The dry cell equivalent weight means the weight that is dried and does not contain moisture. For example, the cell body is separated from the liquid containing the cell body (bacterial body liquid) by a method such as centrifugation or filtration, and the weight becomes constant. Dried, and the weight in terms of dry cells can be determined by measuring the weight.
(3) Bacteria The bacteria in the present invention are classified into a plurality of types. In order to avoid misunderstandings, the present invention defines as follows. Cells that produce lysine decarboxylase at a high level and have higher lysine decarboxylation activity than wild-type strains are referred to as “catalyst cells”. Furthermore, live catalytic cells are defined as “catalytic live cells”, catalytic cells that have stopped growing as “catalytic resting cells”, and catalytic cells that have lost their growth ability as “catalytically dead cells” And
(4) Mutant lysine decarboxylase The mutant lysine decarboxylase used in the present invention is mainly a genetic recombination technique in which at least one amino acid residue in the amino acid sequence of wild-type lysine decarboxylase is other. The lysine decarboxylase is characterized by having a mutation substituted in the amino acid residue of lysine and having improved enzyme activity of lysine decarboxylase itself. The amino acids in the amino acid sequence correspond to amino acid residues in lysine decarboxylase, and they are in a corresponding relationship with each other. Hereinafter, when referred to as an amino acid, an amino acid represented as an amino acid sequence is indicated, and when referred to as an amino acid residue, an amino acid residue contained in lysine decarboxylase is indicated.
　本発明において、変異型リジン脱炭酸酵素遺伝子の調製を行う方法は、変異を導入する既知の如何なる方法でもよく、通常は公知の方法で行うことができる。例えば、部位特異的変異法（Ｋｒａｍｅｒ，Ｗ．　ａｎｄ　ｆｒｉｔａ，Ｈ．Ｊ．、　Ｍｅｔｈｏｄｓ　ｉｎ　Ｅｎｚｙｍｏｌｏｇｙ，１９８７年、第１５４巻、第３５０頁）、リコンビナントＰＣＲ法（ＰＣＲ　Ｔｅｃｈｎｏｌｏｇｙ、Ｓｔｏｃｋｔｏｎ　Ｐｒｅｓｓ、１９８９年）、特定の部分の核酸を化学合成する方法、遺伝子をヒドロキシアミン処理する方法、遺伝子を保有する菌株を紫外線照射処理またはニトロソグアニジンや亜硝酸などの化学薬剤で処理する方法などが挙げられる。 In the present invention, the method for preparing a mutant lysine decarboxylase gene may be any known method for introducing a mutation, and can generally be performed by a known method. For example, site-directed mutagenesis (Kramer, W. and frita, HJ, Methods in Enzymology, 1987, 154, 350), recombinant PCR (PCR Technology, Stockton Press, 1989), Examples include a method of chemically synthesizing a specific portion of nucleic acid, a method of treating a gene with hydroxyamine, and a method of treating a strain having the gene with ultraviolet irradiation or a chemical agent such as nitrosoguanidine or nitrous acid.
　このような変異を導入する方法のなかでは、好ましくは、部位特異的変異法が挙げられる。具体的には、野生型リジン脱炭酸酵素遺伝子を基に、市販のキットを利用して、部位特異的な置換を生じさせる方法である。 Among the methods for introducing such mutations, a site-specific mutation method is preferable. Specifically, it is a method of causing site-specific substitution using a commercially available kit based on the wild-type lysine decarboxylase gene.
　アミノ酸残基が挿入、欠失または置換されている場合、その挿入、欠失または置換の位置は、リジン脱炭酸活性を消失させなければどの様な位置であっても構わない。挿入、欠失または置換したアミノ酸残基の数としては、１アミノ酸残基または２アミノ酸残基以上が挙げられ、例えば、１アミノ酸残基～１０アミノ酸残基、好ましくは、１アミノ酸残基～５アミノ酸残基が挙げられる。 When the amino acid residue is inserted, deleted or substituted, the position of the insertion, deletion or substitution may be any position as long as the lysine decarboxylation activity is not lost. Examples of the number of inserted, deleted or substituted amino acid residues include 1 amino acid residue or 2 amino acid residues or more, for example, 1 amino acid residue to 10 amino acid residues, preferably 1 amino acid residue to 5 amino acid residues. Amino acid residues are mentioned.
　本発明において、変異型リジン脱炭酸酵素をコードするアミノ酸配列および塩基配列、または、プライマーの個々の配列に関して、これら互いの相補的な関係に基づいて記述された事項は、特に断らない限り、それぞれの配列と、各配列に対して相補的な配列とについても適用される。各配列に対して相補的な当該配列について本発明の事項を適用する際には、当該相補的な配列が認識する配列について、当業者にとっての技術常識の範囲内で、対応する本明細書に記載された配列に相補的な配列として、明細書全体を読み替えるものとする。 In the present invention, with respect to the amino acid sequence and base sequence encoding the mutant lysine decarboxylase, or the individual sequences of the primers, the matters described based on their complementary relationship are as follows unless otherwise specified. And the sequence complementary to each sequence is also applied. When applying the matter of the present invention to the sequence complementary to each sequence, the sequence recognized by the complementary sequence is described in the corresponding specification within the scope of common technical knowledge for those skilled in the art. The entire specification shall be read as a sequence complementary to the described sequence.
　具体的には、本発明において、変異型リジン脱炭酸酵素は、配列表の配列番号４に記載のアミノ酸配列において、そのアミノ酸配列中のアミノ酸の少なくとも１つが、活性が上昇する他のアミノ酸に置換されている。 Specifically, in the present invention, in the mutant lysine decarboxylase, in the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing, at least one amino acid in the amino acid sequence is substituted with another amino acid whose activity is increased. Has been.
　配列表の配列番号４に記載のアミノ酸配列は、配列表の配列番号３に記載のＤＮＡ配列より生成されるタンパク質のアミノ酸配列であって、そのＮ末端のメチオニンを１番目のアミノ酸として、１～１２９番目のアミノ酸がウィングドメインであり、１３０～１８３番目のアミノ酸がリンカードメインであり、これら１～１８３番目のアミノ酸が１０量体形成ドメインを形成している。また、１８４～４１７番目のアミノ酸がピリドキサールリン酸酵素（ＰＬＰ酵素）共通ドメインであり、４１８～７１５番目のアミノ酸が基質出入口であり、これら１８４～７１５番目のアミノ酸が活性領域ドメインを形成している。 The amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing is an amino acid sequence of a protein generated from the DNA sequence set forth in SEQ ID NO: 3 in the sequence listing, wherein the N-terminal methionine is the first amino acid, The 129th amino acid is a wing domain, the 130th to 183rd amino acids are linker domains, and these 1st to 183rd amino acids form a 10-mer forming domain. In addition, amino acids 184 to 417 are pyridoxal phosphate (PLP enzyme) common domains, amino acids 418 to 715 are substrate entrances, and these amino acids 184 to 715 form an active region domain. .
　そして、本発明における変異型リジン脱炭酸酵素では、配列表の配列番号４に記載のアミノ酸配列において、１０量体形成ドメインおよび／または活性領域ドメインに存在するアミノ酸が、他のアミノ酸に置換されており、詳しくは、１０量体形成ドメイン中のウィングドメインおよび／またはリンカードメインに存在するアミノ酸、および／または、活性領域ドメイン中のピリドキサールリン酸酵素共通ドメインおよび／または基質出入口に存在するアミノ酸が、他のアミノ酸に置換されている。 In the mutant lysine decarboxylase in the present invention, the amino acid present in the 10-mer forming domain and / or the active region domain in the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing is substituted with another amino acid. Specifically, amino acids present in the wing domain and / or linker domain in the demerization domain, and / or amino acids present in the pyridoxal phosphate common domain and / or substrate entry / exit in the active region domain, It is substituted with another amino acid.
　つまり、変異型リジン脱炭酸酵素において、上記のアミノ酸配列中のアミノ酸に対応するアミノ酸残基が、他のアミノ酸残基に置換されている。 That is, in the mutant lysine decarboxylase, the amino acid residue corresponding to the amino acid in the amino acid sequence is substituted with another amino acid residue.
　変異型リジン脱炭酸酵素として、好ましくは、少なくとも１３７、１３８、２８６、２９０、２９５、３０３、３１７、３３５、３５２、３５３、３８６、４４３、４６６、４７５、５５３、７１１番目のアミノ酸残基及び１４、２８、３９、６４、６７、７０、７５、７９、８３、８４、８５、８８、８９、９４、９５、９８、９９、１０４、１１２、１１９、１３９、１４３、１４５、１４８、１８２、１８４、２５３、２６２、４３０、４４６、４６０、４７１、５０６、５２４、５３９、５４４、５４６、６２３、６２６、６３６、６４６、６４８番目のアミノ酸残基を他のアミノ酸残基に１つ以上置換した変異型酵素が挙げられる。 The mutant lysine decarboxylase is preferably at least 137, 138, 286, 290, 295, 303, 317, 335, 352, 353, 386, 443, 466, 475, 553, the 711th amino acid residue and 14 28, 39, 64, 67, 70, 75, 79, 83, 84, 85, 88, 89, 94, 95, 98, 99, 104, 112, 119, 139, 143, 145, 148, 182, 184 Mutations in which one or more amino acid residues at 253, 262, 430, 446, 460, 471, 506, 524, 539, 544, 546, 623, 626, 636, 646, 648 are substituted with other amino acid residues Type enzymes.
　本発明における少なくとも１３７、１３８、２８６、２９０、２９５、３０３、３１７、３３５、３５２、３５３、３８６、４４３、４６６、４７５、５５３、７１１番目のアミノ酸残基及び１４、２８、３９、６４、６７、７０、７５、７９、８３、８４、８５、８８、８９、９４、９５、９８、９９、１０４、１１２、１１９、１３９、１４３、１４５、１４８、１８２、１８４、２５３、２６２、４３０、４４６、４６０、４７１、５０６、５２４、５３９、５４４、５４６、６２３、６２６、６３６、６４６、６４８番目のアミノ酸残基を他のアミノ酸残基に１つ以上置換した変異型酵素とは、配列表の配列番号３に記載のＤＮＡ配列より生成されるタンパク質のアミノ酸配列（配列表の配列番号４）のＮ末端のメチオニンを１番目のアミノ酸として１３７、１３８、２８６、２９０、２９５、３０３、３１７、３３５、３５２、３５３、３８６、４４３、４６６、４７５、５５３、７１１番目のアミノ酸及び１４、２８、３９、６４、６７、７０、７５、７９、８３、８４、８５、８８、８９、９４、９５、９８、９９、１０４、１１２、１１９、１３９、１４３、１４５、１４８、１８２、１８４、２５３、２６２、４３０、４４６、４６０、４７１、５０６、５２４、５３９、５４４、５４６、６２３、６２６、６３６、６４６、６４８番目のアミノ酸を少なくとも１つ以上、元のアミノ酸とは異なるアミノ酸に置換した配列の変異型酵素を指す。ただし８９番目のアミノ酸をアルギニンに変更したものは除く。変更後のアミノ酸配列は変更前よりも良い性質、例えば比活性の向上、反応中のｐＨ変化に強い性質、反応生成物に対する耐性、阻害の緩和等、があれば特に制限はないが、表１～６に記載の配列により生成されるアミノ酸配列は特に好ましい。 In the present invention, at least the 137th, 138, 286, 290, 295, 303, 317, 335, 352, 353, 386, 443, 466, 475, 553, 711th amino acid residues and the 14, 28, 39, 64, 67 70, 75, 79, 83, 84, 85, 88, 89, 94, 95, 98, 99, 104, 112, 119, 139, 143, 145, 148, 182, 184, 253, 262, 430, 446 460, 471, 506, 524, 539, 544, 546, 623, 626, 636, 646, mutated enzyme in which one or more amino acid residues are substituted with other amino acid residues, The methionine at the N-terminal of the amino acid sequence of the protein generated from the DNA sequence set forth in SEQ ID NO: 3 (SEQ ID NO: 4 in the sequence listing) is number 1 As amino acids 137, 138, 286, 290, 295, 303, 317, 335, 352, 353, 386, 443, 466, 475, 553, the 711th amino acid and 14, 28, 39, 64, 67, 70, 75, 79, 83, 84, 85, 88, 89, 94, 95, 98, 99, 104, 112, 119, 139, 143, 145, 148, 182, 184, 253, 262, 430, 446, 460, 471, 506, 524, 539, 544, 546, 623, 626, 636, 646, refers to a mutant enzyme having a sequence in which at least one amino acid is substituted with an amino acid different from the original amino acid. However, the 89th amino acid changed to arginine is excluded. The amino acid sequence after the change is not particularly limited as long as it has better properties than before the change, for example, improved specific activity, properties resistant to pH change during the reaction, resistance to reaction products, relaxation of inhibition, etc. The amino acid sequence produced by the sequences described in -6 is particularly preferred.
　好ましい変異型リジン脱炭酸酵素として、より具体的には、配列表の配列番号４に記載のアミノ酸配列において、１０量体形成ドメインに存在するアミノ酸のうち14番目のアミノ酸をPheからGlnに変更したもの、28番目のアミノ酸をArgからIleに変更したもの、39番目のアミノ酸をArgからIleに変更したもの、39番目のアミノ酸をArgからValに変更したもの、64番目のアミノ酸をLeuからLysに変更したもの、67番目のアミノ酸をCysからThrに変更したもの、67番目のアミノ酸をCysからLeuに変更したもの、70番目のアミノ酸をIleからLeuに変更したもの、70番目のアミノ酸をIleからProに変更したもの、75番目のアミノ酸をGluからProに変更したもの、75番目のアミノ酸をGluからHisに変更したもの、79番目のアミノ酸をLeuからIleに変更したもの、83番目のアミノ酸をAlaからLeuに変更したもの、84番目のアミノ酸をAsnからAspに変更したもの、84番目のアミノ酸をAsnからThrに変更したもの、85番目のアミノ酸をThrからProに変更したもの、88番目のアミノ酸をThrからLysに変更したもの、88番目のアミノ酸をThrからArgに変更したもの、88番目のアミノ酸をThrからAsnに変更したもの、89番目のアミノ酸をLeuからPheに変更したもの、94番目のアミノ酸をAsnからIleに変更したもの、95番目のアミノ酸をAspからProに変更したもの、98番目のアミノ酸をLeuからIleに変更したもの、99番目のアミノ酸をGlnからThrに変更したもの、104番目のアミノ酸をGluからAsnに変更したもの、104番目のアミノ酸をGluからLysに変更したもの、112番目のアミノ酸をAspからGluに変更したもの、119番目のアミノ酸をGlnからAsnに変更したもの、119番目のアミノ酸をGlnからIleに変更したもの、119番目のアミノ酸をGlnからThrに変更したもの、119番目のアミノ酸をGlnからSerに変更したもの、137番目のアミノ酸をPheからValに変更したもの、138番目のアミノ酸をLysからIleに変更したもの、139番目のアミノ酸をTyrからValに変更したもの、139番目のアミノ酸をTyrからCysに変更したもの、139番目のアミノ酸をTyrからThrに変更したもの、139番目のアミノ酸をTyrからSerに変更したもの、139番目のアミノ酸をTyrからAsnに変更したもの、143番目のアミノ酸をGlyからGluに変更したもの、145番目のアミノ酸をTyrからArgに変更したもの、148番目のアミノ酸をCysからSerに変更したもの、148番目のアミノ酸をCysからAlaに変更したもの、182番目のアミノ酸をIleからMetに変更したもの、活性領域ドメインに存在するアミノ酸のうち184番目のアミノ酸をValからAlaに変更したもの、253番目のアミノ酸をMetからLeuに変更したもの、262番目のアミノ酸をPheからTyrに変更したもの、286番目のアミノ酸をAlaからAspに変更したもの、290番目のアミノ酸をLysからHisに変更したもの、295番目のアミノ酸をAlaからSerに変更したもの、303番目のアミノ酸をIleからThrに変更したもの、317番目のアミノ酸をPheからGlnに変更したもの、335番目のアミノ酸をProからAlaに変更したもの、352番目のアミノ酸をGlyからAlaに変更したもの、353番目のアミノ酸をArgからHisに変更したもの、386番目のアミノ酸をGluからSerに変更したもの、430番目のアミノ酸をGluからPheに変更したもの、443番目のアミノ酸をArgからMetに変更したもの、446番目のアミノ酸をSerからTyrに変更したもの、446番目のアミノ酸をSerからGlnに変更したもの、460番目のアミノ酸をAspからIleに変更したもの、460番目のアミノ酸をAspからAsnに変更したもの、460番目のアミノ酸をAspからCysに変更したもの、460番目のアミノ酸をAspからGlnに変更したもの、460番目のアミノ酸をAspからProに変更したもの、460番目のアミノ酸をAspからSerに変更したもの、466番目のアミノ酸をProからAsnに変更したもの、466番目のアミノ酸をProからGlyに変更したもの、466番目のアミノ酸をProからSerに変更したもの、471番目のアミノ酸をSerからTyrに変更したもの、475番目のアミノ酸をGlyからAsnに変更したもの、506番目のアミノ酸をAspからProに変更したもの、524番目のアミノ酸をValからLeuに変更したもの、524番目のアミノ酸をValからLeuに変更したもの、539番目のアミノ酸をIleからCysに変更したもの、539番目のアミノ酸をIleからLeuに変更したもの、544番目のアミノ酸をThrからAlaに変更したもの、544番目のアミノ酸をThrからSerに変更したもの、544番目のアミノ酸をThrからProに変更したもの、546番目のアミノ酸をAlaからSerに変更したもの、553番目のアミノ酸をLeuからValに変更したもの、623番目のアミノ酸をAlaからCysに変更したもの、623番目のアミノ酸をAlaからPheに変更したもの、623番目のアミノ酸をAlaからGlnに変更したもの、626番目のアミノ酸をLysからValに変更したもの、636番目のアミノ酸をTyrからCysに変更したもの、636番目のアミノ酸をTyrからProに変更したもの、646番目のアミノ酸をAlaからLeuに変更したもの、646番目のアミノ酸をAlaからIleに変更したもの、648番目のアミノ酸をMetからSerに変更したもの、710番目のアミノ酸をLysからThrに変更したもの、711番目のアミノ酸をGluからAspに変更したものに少なくとも１箇所以上置換されている変異型リジン脱炭酸酵素が挙げられる。 As a preferred mutant lysine decarboxylase, more specifically, in the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing, the 14th amino acid in the amino acid present in the 10-mer forming domain was changed from Phe to Gln. , 28th amino acid changed from Arg to Ile, 39th amino acid changed from Arg to Ile, 39th amino acid changed from Arg to Val, 64th amino acid changed from Leu to Lys Changed, 67th amino acid changed from Cys to Thr, 67th amino acid changed from Cys to Leu, 70th amino acid changed from Ile to Leu, 70th amino acid changed from Ile Pro changed, 75th amino acid changed from Glu to Pro, 75th amino acid changed from Glu to His, 79th amino acid changed from Leu to Ile, 83rd amino acid changed Ala or Changed to Leu, 84th amino acid changed from Asn to Asp, 84th amino acid changed from Asn to Thr, 85th amino acid changed from Thr to Pro, 88th amino acid changed Thr changed to Lys, 88th amino acid changed from Thr to Arg, 88th amino acid changed from Thr to Asn, 89th amino acid changed from Leu to Phe, 94th Amino acid changed from Asn to Ile, 95th amino acid changed from Asp to Pro, 98th amino acid changed from Leu to Ile, 99th amino acid changed from Gln to Thr, 104 The first amino acid is changed from Glu to Asn, the 104th amino acid is changed from Glu to Lys, the 112th amino acid is changed from Asp to Glu, and the 119th amino acid is changed from Gln to Asn , Amino acid 119 from Gln to Il Changed to e, 119th amino acid changed from Gln to Thr, 119th amino acid changed from Gln to Ser, 137th amino acid changed from Phe to Val, 138th amino acid changed Change from Lys to Ile, change 139th amino acid from Tyr to Val, change 139th amino acid from Tyr to Cys, change 139th amino acid from Tyr to Thr, 139th Amino acid changed from Tyr to Ser, 139th amino acid changed from Tyr to Asn, 143rd amino acid changed from Gly to Glu, 145th amino acid changed from Tyr to Arg, 148 The amino acid changed from Cys to Ser, the 148th amino acid changed from Cys to Ala, the 182nd amino acid changed from Ile to Met, and the 184th amino acid present in the active region domain Is amino acid Val? Changed from Ala to Ala, 253rd amino acid changed from Met to Leu, 262th amino acid changed from Phe to Tyr, 286th amino acid changed from Ala to Asp, 290th amino acid Changed from Lys to His, 295th amino acid changed from Ala to Ser, 303rd amino acid changed from Ile to Thr, 317th amino acid changed from Phe to Gln, 335th The amino acid of was changed from Pro to Ala, the 352nd amino acid was changed from Gly to Ala, the 353rd amino acid was changed from Arg to His, the 386th amino acid was changed from Glu to Ser, 430th amino acid changed from Glu to Phe, 443rd amino acid changed from Arg to Met, 446th amino acid changed from Ser to Tyr, 446th amino acid changed from Ser to Gln 460th amino acid Asp changed from Ile, 460th amino acid changed from Asp to Asn, 460th amino acid changed from Asp to Cys, 460th amino acid changed from Asp to Gln, 460th amino acid Amino acid changed from Asp to Pro, 460th amino acid changed from Asp to Ser, 466th amino acid changed from Pro to Asn, 466th amino acid changed from Pro to Gly, 466 The first amino acid is changed from Pro to Ser, the 471st amino acid is changed from Ser to Tyr, the 475th amino acid is changed from Gly to Asn, and the 506th amino acid is changed from Asp to Pro , 524th amino acid changed from Val to Leu, 524th amino acid changed from Val to Leu, 539th amino acid changed from Ile to Cys, 539th amino acid changed from Ile to Leu 544th No acid changed from Thr to Ala, 544th amino acid changed from Thr to Ser, 544th amino acid changed from Thr to Pro, 546th amino acid changed from Ala to Ser, 553th amino acid changed from Leu to Val, 623rd amino acid changed from Ala to Cys, 623rd amino acid changed from Ala to Phe, 623rd amino acid changed from Ala to Gln , 626th amino acid changed from Lys to Val, 636th amino acid changed from Tyr to Cys, 636th amino acid changed from Tyr to Pro, 646th amino acid changed from Ala to Leu Changed, 646th amino acid changed from Ala to Ile, 648th amino acid changed from Met to Ser, 710th amino acid changed from Lys to Thr, 711th amino acid changed from Glu Less to those changed to Asp Both include mutant lysine decarboxylase in which one or more sites are substituted.
　より具体的には、配列表の配列番号４に記載のアミノ酸配列において、ウィングドメインに存在するアミノ酸のうち14番目のアミノ酸をPheからGlnに変更したもの、28番目のアミノ酸をArgからIleに変更したもの、39番目のアミノ酸をArgからIleに変更したもの、39番目のアミノ酸をArgからValに変更したもの、64番目のアミノ酸をLeuからLysに変更したもの、67番目のアミノ酸をCysからThrに変更したもの、67番目のアミノ酸をCysからLeuに変更したもの、70番目のアミノ酸をIleからLeuに変更したもの、70番目のアミノ酸をIleからProに変更したもの、75番目のアミノ酸をGluからProに変更したもの、75番目のアミノ酸をGluからHisに変更したもの、79番目のアミノ酸をLeuからIleに変更したもの、83番目のアミノ酸をAlaからLeuに変更したもの、84番目のアミノ酸をAsnからAspに変更したもの、84番目のアミノ酸をAsnからThrに変更したもの、85番目のアミノ酸をThrからProに変更したもの、88番目のアミノ酸をThrからLysに変更したもの、88番目のアミノ酸をThrからArgに変更したもの、88番目のアミノ酸をThrからAsnに変更したもの、89番目のアミノ酸をLeuからPheに変更したもの、94番目のアミノ酸をAsnからIleに変更したもの、95番目のアミノ酸をAspからProに変更したもの、98番目のアミノ酸をLeuからIleに変更したもの、99番目のアミノ酸をGlnからThrに変更したもの、104番目のアミノ酸をGluからAsnに変更したもの、104番目のアミノ酸をGluからLysに変更したもの、112番目のアミノ酸をAspからGluに変更したもの、119番目のアミノ酸をGlnからAsnに変更したもの、119番目のアミノ酸をGlnからIleに変更したもの、119番目のアミノ酸をGlnからThrに変更したもの、119番目のアミノ酸をGlnからSerに変更したもの、リンカードメインに存在するアミノ酸のうち137番目のアミノ酸をPheからValに変更したもの、138番目のアミノ酸をLysからIleに変更したもの、139番目のアミノ酸をTyrからValに変更したもの、139番目のアミノ酸をTyrからCysに変更したもの、139番目のアミノ酸をTyrからThrに変更したもの、139番目のアミノ酸をTyrからSerに変更したもの、139番目のアミノ酸をTyrからAsnに変更したもの、143番目のアミノ酸をGlyからGluに変更したもの、145番目のアミノ酸をTyrからArgに変更したもの、148番目のアミノ酸をCysからSerに変更したもの、148番目のアミノ酸をCysからAlaに変更したもの、182番目のアミノ酸をIleからMetに変更したもの、ピリドキサールリン酸酵素共通ドメインに存在するアミノ酸のうち184番目のアミノ酸をValからAlaに変更したもの、253番目のアミノ酸をMetからLeuに変更したもの、262番目のアミノ酸をPheからTyrに変更したもの、286番目のアミノ酸をAlaからAspに変更したもの、290番目のアミノ酸をLysからHisに変更したもの、295番目のアミノ酸をAlaからSerに変更したもの、303番目のアミノ酸をIleからThrに変更したもの、317番目のアミノ酸をPheからGlnに変更したもの、335番目のアミノ酸をProからAlaに変更したもの、352番目のアミノ酸をGlyからAlaに変更したもの、353番目のアミノ酸をArgからHisに変更したもの、386番目のアミノ酸をGluからSerに変更したもの、基質出入口に存在するアミノ酸のうち430番目のアミノ酸をGluからPheに変更したもの、443番目のアミノ酸をArgからMetに変更したもの、446番目のアミノ酸をSerからTyrに変更したもの、446番目のアミノ酸をSerからGlnに変更したもの、460番目のアミノ酸をAspからIleに変更したもの、460番目のアミノ酸をAspからAsnに変更したもの、460番目のアミノ酸をAspからCysに変更したもの、460番目のアミノ酸をAspからGlnに変更したもの、460番目のアミノ酸をAspからProに変更したもの、460番目のアミノ酸をAspからSerに変更したもの、466番目のアミノ酸をProからAsnに変更したもの、466番目のアミノ酸をProからGlyに変更したもの、466番目のアミノ酸をProからSerに変更したもの、471番目のアミノ酸をSerからTyrに変更したもの、475番目のアミノ酸をGlyからAsnに変更したもの、506番目のアミノ酸をAspからProに変更したもの、524番目のアミノ酸をValからLeuに変更したもの、524番目のアミノ酸をValからLeuに変更したもの、539番目のアミノ酸をIleからCysに変更したもの、539番目のアミノ酸をIleからLeuに変更したもの、544番目のアミノ酸をThrからAlaに変更したもの、544番目のアミノ酸をThrからSerに変更したもの、544番目のアミノ酸をThrからProに変更したもの、546番目のアミノ酸をAlaからSerに変更したもの、553番目のアミノ酸をLeuからValに変更したもの、623番目のアミノ酸をAlaからCysに変更したもの、623番目のアミノ酸をAlaからPheに変更したもの、623番目のアミノ酸をAlaからGlnに変更したもの、626番目のアミノ酸をLysからValに変更したもの、636番目のアミノ酸をTyrからCysに変更したもの、636番目のアミノ酸をTyrからProに変更したもの、646番目のアミノ酸をAlaからLeuに変更したもの、646番目のアミノ酸をAlaからIleに変更したもの、648番目のアミノ酸をMetからSerに変更したもの、710番目のアミノ酸をLysからThrに変更したもの、711番目のアミノ酸をGluからAspに変更したものに少なくとも１箇所以上置換されている変異型リジン脱炭酸酵素が挙げられる。 More specifically, in the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing, the 14th amino acid in the wing domain is changed from Phe to Gln, and the 28th amino acid is changed from Arg to Ile. , 39th amino acid changed from Arg to Ile, 39th amino acid changed from Arg to Val, 64th amino acid changed from Leu to Lys, 67th amino acid changed from Cys to Thr , The 67th amino acid changed from Cys to Leu, the 70th amino acid changed from Ile to Leu, the 70th amino acid changed from Ile to Pro, the 75th amino acid changed to Glu Changed from Pro to Pro, 75th amino acid changed from Glu to His, 79th amino acid changed from Leu to Ile, 83rd amino acid changed from Ala to Leu, 84th amino acid From Asn Changed to Asp, changed the 84th amino acid from Asn to Thr, changed the 85th amino acid from Thr to Pro, changed the 88th amino acid from Thr to Lys, changed the 88th amino acid Thr changed to Arg, 88th amino acid changed from Thr to Asn, 89th amino acid changed from Leu to Phe, 94th amino acid changed from Asn to Ile, 95th amino acid Amino acid changed from Asp to Pro, 98th amino acid changed from Leu to Ile, 99th amino acid changed from Gln to Thr, 104th amino acid changed from Glu to Asn, 104 The first amino acid is changed from Glu to Lys, the 112th amino acid is changed from Asp to Glu, the 119th amino acid is changed from Gln to Asn, and the 119th amino acid is changed from Gln to Ile , Amino acid 119 from Gln to Th Changed to r, 119th amino acid changed from Gln to Ser, 137th amino acid in the linker domain changed from Phe to Val, 138th amino acid changed from Lys to Ile 139th amino acid changed from Tyr to Val, 139th amino acid changed from Tyr to Cys, 139th amino acid changed from Tyr to Thr, 139th amino acid changed from Tyr to Ser 139th amino acid changed from Tyr to Asn, 143rd amino acid changed from Gly to Glu, 145th amino acid changed from Tyr to Arg, 148th amino acid changed to Cys Is changed from Ser to Ser, 148th amino acid is changed from Cys to Ala, 182nd amino acid is changed from Ile to Met, and 184 of the amino acids present in the common domain of pyridoxal phosphate The amino acid of the eye was changed from Val to Ala, the 253rd amino acid was changed from Met to Leu, the 262nd amino acid was changed from Phe to Tyr, and the 286th amino acid was changed from Ala to Asp 290th amino acid changed from Lys to His, 295th amino acid changed from Ala to Ser, 303rd amino acid changed from Ile to Thr, 317th amino acid changed from Phe to Gln 335th amino acid changed from Pro to Ala, 352nd amino acid changed from Gly to Ala, 353rd amino acid changed from Arg to His, 386th amino acid changed from Glu to Ser 430th amino acid changed from Glu to Phe, 443rd amino acid changed from Arg to Met, 446th amino acid changed from Ser to Tyr 446th Amino acid changed from Ser to Gln, 460th amino acid changed from Asp to Ile, 460th amino acid changed from Asp to Asn, 460th amino acid changed from Asp to Cys, 460 The first amino acid is changed from Asp to Gln, the 460th amino acid is changed from Asp to Pro, the 460th amino acid is changed from Asp to Ser, and the 466th amino acid is changed from Pro to Asn 466th amino acid changed from Pro to Gly, 466th amino acid changed from Pro to Ser, 471st amino acid changed from Ser to Tyr, 475th amino acid changed from Gly to Asn 506, amino acid changed from Asp to Pro, 524th amino acid changed from Val to Leu, 524th amino acid changed from Val to Leu, 539th amino acid changed from Ile to Cys Changed to 53 9th amino acid changed from Ile to Leu, 544th amino acid changed from Thr to Ala, 544th amino acid changed from Thr to Ser, 544th amino acid changed from Thr to Pro , 546th amino acid changed from Ala to Ser, 553th amino acid changed from Leu to Val, 623rd amino acid changed from Ala to Cys, 623rd amino acid changed from Ala to Phe Changed, 623th amino acid changed from Ala to Gln, 626th amino acid changed from Lys to Val, 636th amino acid changed from Tyr to Cys, 636th amino acid changed from Tyr Pro changed, 646th amino acid changed from Ala to Leu, 646th amino acid changed from Ala to Ile, 648th amino acid changed from Met to Ser, 710th amino acid changed Also changed from Lys to Thr And a mutated lysine decarboxylase in which at least one of the 711st amino acid is changed from Glu to Asp.
　また、好ましい変異型リジン脱炭酸酵素としては、活性領域ドメイン、具体的には、２９０、３３５、４７５、７１１番目のアミノ酸残基、２８６、２９０、３３５、４７５、７１１番目のアミノ酸残基、１４８、６４６番目のアミノ酸残基、４７１、６２６番目のアミノ酸残基、６２６、６４６番目のアミノ酸残基を他のアミノ酸残基に置換した変異型酵素が挙げられる。 Preferred mutant lysine decarboxylase includes an active region domain, specifically, 290th, 335, 475, 711th amino acid residues, 286, 290, 335, 475, 711th amino acid residues, 148 , 646th amino acid residue, 471, 626th amino acid residue, and mutated enzyme in which the 626th and 646th amino acid residues are substituted with other amino acid residues.
　本発明における２９０、３３５、４７５、７１１番目のアミノ酸残基を他のアミノ酸残基に置換した変異型酵素とは配列表の配列番号３に記載のＤＮＡ配列より生成されるタンパク質のアミノ酸配列のＮ末端のメチオニンを１番目のアミノ酸として２９０、３３５、４７５、７１１番目の4箇所のアミノ酸を元のアミノ酸とは異なるアミノ酸に置換した配列の変異型酵素を指す。 In the present invention, the mutant enzyme in which the 290th, 335th, 475th, and 711st amino acid residues are substituted with other amino acid residues is N of the amino acid sequence of the protein generated from the DNA sequence described in SEQ ID NO: 3 in the Sequence Listing. This refers to a mutant enzyme having a sequence in which the amino acid at the 4th position of 290, 335, 475, and 711 is replaced with an amino acid different from the original amino acid, with the terminal methionine as the first amino acid.
　本発明における２８６、２９０、３３５、４７５、７１１番目のアミノ酸残基を他のアミノ酸残基に置換した変異型酵素とは配列表の配列番号３に記載のＤＮＡ配列より生成されるタンパク質のアミノ酸配列のＮ末端のメチオニンを１番目のアミノ酸として２８６、２９０、３３５、４７５、７１１番目の５箇所のアミノ酸を元のアミノ酸とは異なるアミノ酸に置換した配列の変異型酵素を指す。 The mutated enzyme in which the 286th, 290th, 335th, 475th, and 711th amino acid residues in the present invention are substituted with other amino acid residues is the amino acid sequence of a protein generated from the DNA sequence shown in SEQ ID NO: 3 in the Sequence Listing. The N-terminal methionine is the first amino acid, and the 286, 290, 335, 475, and 711th amino acids are substituted with amino acids different from the original amino acids.
　本発明における１４８、６４６番目のアミノ酸残基を他のアミノ酸残基に置換した変異型酵素とは配列表の配列番号３に記載のＤＮＡ配列より生成されるタンパク質のアミノ酸配列のＮ末端のメチオニンを１番目のアミノ酸として１４８、６４６番目の２箇所のアミノ酸を元のアミノ酸とは異なるアミノ酸に置換した配列の変異型酵素を指す。 In the present invention, the mutated enzyme in which the 148th and 646th amino acid residues are substituted with other amino acid residues is the N-terminal methionine of the amino acid sequence of the protein generated from the DNA sequence described in SEQ ID NO: 3 in the Sequence Listing. It refers to a mutant enzyme having a sequence in which two amino acids at the 148th and 646th positions are substituted with amino acids different from the original amino acid as the first amino acid.
　本発明における４７１、６２６番目のアミノ酸残基を他のアミノ酸残基に置換した変異型酵素とは配列表の配列番号３に記載のＤＮＡ配列より生成されるタンパク質のアミノ酸配列のＮ末端のメチオニンを１番目のアミノ酸として４７１、６２６番目の２箇所のアミノ酸を元のアミノ酸とは異なるアミノ酸に置換した配列の変異型酵素を指す。 In the present invention, the mutated enzyme in which the 471st and 626th amino acid residues are substituted with other amino acid residues is the N-terminal methionine of the amino acid sequence of the protein generated from the DNA sequence described in SEQ ID NO: 3 in the Sequence Listing. The first amino acid refers to a mutant enzyme having a sequence in which the amino acids at two positions, 471 and 626, are substituted with amino acids different from the original amino acid.
　本発明における６２６、６４６番目のアミノ酸残基を他のアミノ酸残基に置換した変異型酵素とは配列表の配列番号３に記載のＤＮＡ配列より生成されるタンパク質のアミノ酸配列のＮ末端のメチオニンを１番目のアミノ酸として６２６、６４６番目の２箇所のアミノ酸を元のアミノ酸とは異なるアミノ酸に置換した配列の変異型酵素を指す。 In the present invention, the mutated enzyme in which the 626th and 646th amino acid residues are substituted with other amino acid residues is the N-terminal methionine of the amino acid sequence of the protein produced from the DNA sequence described in SEQ ID NO: 3 in the Sequence Listing. This refers to a mutant enzyme having a sequence in which two amino acids at the 626th and 646th positions are substituted with amino acids different from the original amino acid as the first amino acid.
　変更後のアミノ酸配列は元の配列の酵素よりも良い性質があれば特に制限はないが、それぞれ表１～６に記載の配列により生成されるアミノ酸配列は特に好ましい。 The amino acid sequence after the change is not particularly limited as long as it has better properties than the enzyme of the original sequence, but the amino acid sequences generated by the sequences shown in Tables 1 to 6 are particularly preferred.
　また、上記では、変更変異型リジン脱炭酸酵素が、アミノ酸配列のアミノ酸を変更したものとして示したが、変更変異型リジン脱炭酸酵素は、アミノ酸をコードする塩基配列を変更したものとして示すこともできる。 In the above description, the modified mutant lysine decarboxylase is shown as a modified amino acid of the amino acid sequence. However, the modified mutant lysine decarboxylase may be represented as a modified amino acid sequence. it can.
　そのような変異型リジン脱炭酸酵素としては、配列表の配列番号３に記載のアミノ酸配列において、１０量体形成ドメインに存在するアミノ酸残基のうち14番目のアミノ酸であるPheをコードする塩基配列がTTTからGlnをコードする塩基配列であるCAAに変更したもの、22番目のアミノ酸であるLeuをコードする塩基配列がCTTからLeuをコードする塩基配列であるTTGに変更したもの、28番目のアミノ酸であるArgをコードする塩基配列がCGTからIleをコードする塩基配列であるATTに変更したもの、39番目のアミノ酸であるArgをコードする塩基配列がCGTからIleをコードする塩基配列であるATAに変更したもの、39番目のアミノ酸であるArgをコードする塩基配列がCGTからIleをコードする塩基配列であるATCに変更したもの、39番目のアミノ酸であるArgをコードする塩基配列がCGTからValをコードする塩基配列であるGTGに変更したもの、64番目のアミノ酸であるLeuをコードする塩基配列がCTCからLysをコードする塩基配列であるAAAに変更したもの、67番目のアミノ酸であるCysをコードする塩基配列がTGCからThrをコードする塩基配列であるACCに変更したもの、67番目のアミノ酸であるCysをコードする塩基配列がTGCからLeuをコードする塩基配列であるTTAに変更したもの、70番目のアミノ酸であるIleをコードする塩基配列がATTからLeuをコードする塩基配列であるTTGに変更したもの、70番目のアミノ酸であるIleをコードする塩基配列がATTからLeuをコードする塩基配列であるCTGに変更したもの、70番目のアミノ酸であるIleをコードする塩基配列がATTからProをコードする塩基配列であるCCGに変更したもの、75番目のアミノ酸であるGluをコードする塩基配列がGAGからProをコードする塩基配列であるCCCに変更したもの、75番目のアミノ酸であるGluをコードする塩基配列がGAGからHisをコードする塩基配列であるCACに変更したもの、79番目のアミノ酸であるLeuをコードする塩基配列がTTGからIleをコードする塩基配列であるATAに変更したもの、83番目のアミノ酸であるAlaをコードする塩基配列がGCTからLeuをコードする塩基配列であるCTGに変更したもの、83番目のアミノ酸であるAlaをコードする塩基配列がGCTからLeuをコードする塩基配列であるCTAに変更したもの、83番目のアミノ酸であるAlaをコードする塩基配列がGCTからLeuをコードする塩基配列であるCTTに変更したもの、83番目のアミノ酸であるAlaをコードする塩基配列がGCTからAlaをコードする塩基配列であるATAに変更したもの、83番目のアミノ酸であるAlaをコードする塩基配列がGCTからAlaをコードする塩基配列であるGCCに変更したもの、84番目のアミノ酸であるAsnをコードする塩基配列がAATからAspをコードする塩基配列であるGACに変更したもの、84番目のアミノ酸であるAsnをコードする塩基配列がAATからThrをコードする塩基配列であるACAに変更したもの、85番目のアミノ酸であるThrをコードする塩基配列がACGからProをコードする塩基配列であるCCAに変更したもの、88番目のアミノ酸であるThrをコードする塩基配列がACTからLysをコードする塩基配列であるAAAに変更したもの、88番目のアミノ酸であるThrをコードする塩基配列がACTからLysをコードする塩基配列であるAAGに変更したもの、88番目のアミノ酸であるThrをコードする塩基配列がACTからArgをコードする塩基配列であるAGAに変更したもの、88番目のアミノ酸であるThrをコードする塩基配列がACTからAsnをコードする塩基配列であるAATに変更したもの、89番目のアミノ酸であるLeuをコードする塩基配列がCTCからPheをコードする塩基配列であるTTTに変更したもの、94番目のアミノ酸であるAsnをコードする塩基配列がAATからIleをコードする塩基配列であるATCに変更したもの、95番目のアミノ酸であるAspをコードする塩基配列がGACからProをコードする塩基配列であるCCGに変更したもの、98番目のアミノ酸であるLeuをコードする塩基配列がTTAからIleをコードする塩基配列であるATAに変更したもの、99番目のアミノ酸であるGlnをコードする塩基配列がCAGからThrをコードする塩基配列であるACTに変更したもの、104番目のアミノ酸であるGluをコードする塩基配列がGAAからAsnをコードする塩基配列であるAATに変更したもの、104番目のアミノ酸であるGluをコードする塩基配列がGAAからLysをコードする塩基配列であるAAAに変更したもの、112番目のアミノ酸であるAspをコードする塩基配列がGATからGluをコードする塩基配列であるGAGに変更したもの、119番目のアミノ酸であるGlnをコードする塩基配列がCAGからAsnをコードする塩基配列であるAACに変更したもの、119番目のアミノ酸であるGlnをコードする塩基配列がCAGからAsnをコードする塩基配列であるAATに変更したもの、119番目のアミノ酸であるGlnをコードする塩基配列がCAGからIleをコードする塩基配列であるATTに変更したもの、119番目のアミノ酸であるGlnをコードする塩基配列がCAGからThrをコードする塩基配列であるACCに変更したもの、119番目のアミノ酸であるGlnをコードする塩基配列がCAGからSerをコードする塩基配列であるAGTに変更したもの、137番目のアミノ酸であるPheをコードする塩基配列がTTTからValをコードする塩基配列であるGTCに変更したもの、138番目のアミノ酸であるLysをコードする塩基配列がAAAからIleをコードする塩基配列であるATCに変更したもの、139番目のアミノ酸であるTyrをコードする塩基配列がTATからValをコードする塩基配列であるGTAに変更したもの、139番目のアミノ酸であるTyrをコードする塩基配列がTATからValをコードする塩基配列であるGTGに変更したもの、139番目のアミノ酸であるTyrをコードする塩基配列がTATからCysをコードする塩基配列であるTGCに変更したもの、139番目のアミノ酸であるTyrをコードする塩基配列がTATからThrをコードする塩基配列であるACAに変更したもの、139番目のアミノ酸であるTyrをコードする塩基配列がTATからSerをコードする塩基配列であるTCTに変更したもの、139番目のアミノ酸であるTyrをコードする塩基配列がTATからSerをコードする塩基配列であるAGTに変更したもの、139番目のアミノ酸であるTyrをコードする塩基配列がTATからAsnをコードする塩基配列であるAACに変更したもの、143番目のアミノ酸であるGlyをコードする塩基配列がGGTからGluをコードする塩基配列であるGAAに変更したもの、145番目のアミノ酸であるTyrをコードする塩基配列がTATからArgをコードする塩基配列であるCGTに変更したもの、145番目のアミノ酸であるTyrをコードする塩基配列がTATからArgをコードする塩基配列であるAGAに変更したもの、148番目のアミノ酸であるCysをコードする塩基配列がTGTからSerをコードする塩基配列であるAGTに変更したもの、148番目のアミノ酸であるCysをコードする塩基配列がTGTからSerをコードする塩基配列であるTCTに変更したもの、148番目のアミノ酸であるCysをコードする塩基配列がTGTからSerをコードする塩基配列であるTCCに変更したもの、148番目のアミノ酸であるCysをコードする塩基配列がTGTからSerをコードする塩基配列であるTCAに変更したもの、148番目のアミノ酸であるCysをコードする塩基配列がTGTからAlaをコードする塩基配列であるGCGに変更したもの、148番目のアミノ酸であるCysをコードする塩基配列がTGTからAlaをコードする塩基配列であるGCAに変更したもの、182番目のアミノ酸であるIleをコードする塩基配列がATTからMetをコードする塩基配列であるATGに変更したもの、活性領域ドメインに存在するアミノ酸残基のうち184番目のアミノ酸であるValをコードする塩基配列がGTAからAlaをコードする塩基配列であるGCCに変更したもの、184番目のアミノ酸であるValをコードする塩基配列がGTAからAlaをコードする塩基配列であるGCAに変更したもの、253番目のアミノ酸であるMetをコードする塩基配列がATGからLeuをコードする塩基配列であるCTAに変更したもの、262番目のアミノ酸であるPheをコードする塩基配列がTTCからTyrをコードする塩基配列であるTATに変更したもの、286番目のアミノ酸であるAlaをコードする塩基配列がGCTからAspをコードする塩基配列であるGACに変更したもの、290番目のアミノ酸であるLysをコードする塩基配列がAAAからHisをコードする塩基配列であるCACに変更したもの、295番目のアミノ酸であるAlaをコードする塩基配列がGCA からSerをコードする塩基配列であるTCAに変更したもの、303番目のアミノ酸であるIleをコードする塩基配列がATTからThrをコードする塩基配列であるACAに変更したもの、317番目のアミノ酸であるPheをコードする塩基配列がTTCからGlnをコードする塩基配列であるCAGに変更したもの、335番目のアミノ酸であるProをコードする塩基配列がCCTからAlaをコードする塩基配列であるGCTに変更したもの、352番目のアミノ酸であるGlyをコードする塩基配列がGGCからAlaをコードする塩基配列であるGCAに変更したもの、353番目のアミノ酸であるArgをコードする塩基配列がCGTからHisをコードする塩基配列であるCATに変更したもの、386番目のアミノ酸であるGluをコードする塩基配列がGAAからSerをコードする塩基配列であるTCCに変更したもの、430番目のアミノ酸であるGluをコードする塩基配列がGAAからPheをコードする塩基配列であるTTCに変更したもの、443番目のアミノ酸であるArgをコードする塩基配列がAGAからMetをコードする塩基配列であるATGに変更したもの、446番目のアミノ酸であるSerをコードする塩基配列がTCTからTyrをコードする塩基配列であるTACに変更したもの、446番目のアミノ酸であるSerをコードする塩基配列がTCTからGlnをコードする塩基配列であるCAAに変更したもの、460番目のアミノ酸であるAspをコードする塩基配列がGATからIleをコードする塩基配列であるATTに変更したもの、460番目のアミノ酸であるAspをコードする塩基配列がGATからAsnをコードする塩基配列であるAATに変更したもの、460番目のアミノ酸であるAspをコードする塩基配列がGATからCysをコードする塩基配列であるTGTに変更したもの、460番目のアミノ酸であるAspをコードする塩基配列がGATからGlnをコードする塩基配列であるCAGに変更したもの、460番目のアミノ酸であるAspをコードする塩基配列がGATからProをコードする塩基配列であるCCCに変更したもの、460番目のアミノ酸であるAspをコードする塩基配列がGATからProをコードする塩基配列であるCCTに変更したもの、460番目のアミノ酸であるAspをコードする塩基配列がGATからProをコードする塩基配列であるCCGに変更したもの、460番目のアミノ酸であるAspをコードする塩基配列がGATからSerをコードする塩基配列であるTCAに変更したもの、466番目のアミノ酸であるProをコードする塩基配列がCCGからAsnをコードする塩基配列であるAACに変更したもの、466番目のアミノ酸であるProをコードする塩基配列がCCGからGlyをコードする塩基配列であるGGCに変更したもの、466番目のアミノ酸であるProをコードする塩基配列がCCGからSerをコードする塩基配列であるTCTに変更したもの、471番目のアミノ酸であるSerをコードする塩基配列がAGCからTyrをコードする塩基配列であるTATに変更したもの、475番目のアミノ酸であるGlyをコードする塩基配列がGGCからAsnをコードする塩基配列であるAATに変更したもの、506番目のアミノ酸であるAspをコードする塩基配列がGACからProをコードする塩基配列であるCCAに変更したもの、524番目のアミノ酸であるValをコードする塩基配列がGTTからLeuをコードする塩基配列であるTTAに変更したもの、524番目のアミノ酸であるValをコードする塩基配列がGTTからLeuをコードする塩基配列であるCTGに変更したもの、539番目のアミノ酸であるIleをコードする塩基配列がATCからCysをコードする塩基配列であるTGCに変更したもの、539番目のアミノ酸であるIleをコードする塩基配列がATCからLeuをコードする塩基配列であるCTTに変更したもの、539番目のアミノ酸であるIleをコードする塩基配列がATCからLeuをコードする塩基配列であるCTAに変更したもの、544番目のアミノ酸であるThrをコードする塩基配列がACCからAlaをコードする塩基配列であるGCGに変更したもの、544番目のアミノ酸であるThrをコードする塩基配列がACCからAlaをコードする塩基配列であるGCTに変更したもの、544番目のアミノ酸であるThrをコードする塩基配列がACCからSerをコードする塩基配列であるTCTに変更した
もの、544番目のアミノ酸であるThrをコードする塩基配列がACCからSerをコードする塩基配列であるTCCに変更したもの、544番目のアミノ酸であるThrをコードする塩基配列がACCからProをコードする塩基配列であるCCTに変更したもの、544番目のアミノ酸であるThrをコードする塩基配列がACCからProをコードする塩基配列であるCCGに変更したもの、546番目のアミノ酸であるAlaをコードする塩基配列がGCAからSerをコードする塩基配列であるAGCに変更したもの、553番目のアミノ酸であるLeuをコードする塩基配列がCTGからValをコードする塩基配列であるGTAに変更したもの、623番目のアミノ酸であるAlaをコードする塩基配列がGCAからCysをコードする塩基配列であるTGTに変更したもの、623番目のアミノ酸であるAlaをコードする塩基配列がGCAからPheをコードする塩基配列であるTTTに変更したもの、623番目のアミノ酸であるAlaをコードする塩基配列がGCAからPheをコードする塩基配列であるTTCに変更したもの、623番目のアミノ酸であるAlaをコードする塩基配列がGCAからGlnをコードする塩基配列であるCAGに変更したもの、626番目のアミノ酸であるLysをコードする塩基配列がAAAからValをコードする塩基配列であるGTGに変更したもの、636番目のアミノ酸であるTyrをコードする塩基配列がTACからCysをコードする塩基配列であるTGTに変更したもの、636番目のアミノ酸であるTyrをコードする塩基配列がTACからProをコードする塩基配列であるCCCに変更したもの、646番目のアミノ酸であるAlaをコードする塩基配列がGCCからLeuをコードする塩基配列であるTTGに変更したもの、646番目のアミノ酸であるAlaをコードする塩基配列がGCCからIleをコードする塩基配列であるATCに変更したもの、648番目のアミノ酸であるMetをコードする塩基配列がATGからSerをコードする塩基配列であるTCTに変更したもの、648番目のアミノ酸であるMetをコードする塩基配列がATGからSerをコードする塩基配列であるTCCに変更したもの、710番目のアミノ酸であるLysをコードする塩基配列がAAAからThrをコードする塩基配列であるACGに変更したもの、711番目のアミノ酸であるGluをコードする塩基配列がGAAからAspをコードする塩基配列であるGACに変更したものに少なくとも１箇所以上置換されている変異型リジン脱炭酸酵素が挙げられる。 As such a mutant lysine decarboxylase, in the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing, a base sequence encoding Phe which is the 14th amino acid among the amino acid residues present in the 10-mer forming domain Changed from TTT to CAA, the base sequence encoding Gln, the base sequence encoding Leu, the 22nd amino acid, from TTT, the base sequence encoding Leu, the 28th amino acid The base sequence encoding Arg is changed from CGT to ATT, which is the base sequence encoding Ile, and the base sequence encoding Arg, the 39th amino acid, is changed from CGT to Ile, the base sequence encoding Ile. Modified, base sequence encoding Arg, 39th amino acid changed from CGT to ATC, base sequence encoding Ile, base sequence encoding Arg, 39th amino acid Changed from CGT to GTG, which is the base sequence encoding Val, the base sequence encoding Leu, which is the 64th amino acid, changed from AAA, which is the base sequence encoding Lys, from the CTC, the 67th amino acid The base sequence encoding Cys is changed from TGC to ACC, which is the base sequence encoding Thr, and the base sequence encoding Cys, the 67th amino acid, is changed to TTA, the base sequence encoding TGC to Leu. Changed, the base sequence encoding Ile, the 70th amino acid, changed from ATT to TTG, the base sequence encoding Leu, the base sequence encoding the 70th amino acid, Ile, changed from ATT to Leu Changed to CTG, which is the coding base sequence, changed base sequence encoding Ile, which is the 70th amino acid, to CCG, which is the base sequence encoding ATT to Pro, and copied Glu, which is the 75th amino acid. The base sequence to be loaded is changed from GAG to CCC, which is the base sequence encoding Pro, and the base sequence encoding Glu, which is the 75th amino acid, is changed from GAG to CAC, which is the base sequence encoding His. The base sequence encoding Leu, the 79th amino acid, has been changed from TTG to ATA, the base sequence encoding Ile, and the base sequence encoding the 83rd amino acid, Ala, is a base that encodes Leu from GCT The sequence changed to CTG which is the sequence, the base sequence encoding Ala which is the 83rd amino acid is changed to CTA which is the base sequence encoding Leu from GCT, the base sequence encoding Ala which is the 83rd amino acid Is changed from GCT to CTT, which is the base sequence encoding Leu, the base sequence encoding Ala, which is the 83rd amino acid, is changed to ATA, which is the base sequence encoding Ala, from the GCT, the 83rd amino acid The base sequence encoding the acid Ala is changed from GCT to GCC, which is the base sequence encoding Ala, and the base sequence encoding Asn, the 84th amino acid, is the base sequence encoding AAT to Asp. The base sequence encoding Asn, which is the 84th amino acid, is changed from AAT to ACA, which is the base sequence encoding Thr, and the base sequence encoding Thr, the 85th amino acid, is changed from ACG to Pro. Changed to CCA, which is the base sequence that encodes A, the base sequence encoding Thr, which is the 88th amino acid, from AAA, which is the base sequence that encodes Lys from ACT, and Thr, which is the 88th amino acid The base sequence to be encoded is changed from ACT to AAG, which is the base sequence encoding Lys, and the base sequence encoding Thr, the 88th amino acid, is changed from ACT to AGA, which is the base sequence encoding Arg. The nucleotide sequence encoding Thr, the 88th amino acid, has been changed from ACT to AAT, the nucleotide sequence encoding Asn, and the nucleotide sequence encoding the Leu, the 89th amino acid, is a base encoding CTC to Phe. The sequence changed to TTT, the base sequence encoding Asn, the 94th amino acid, changed from AAT to ATC, the base sequence encoding Ile, the base sequence encoding Asp, the 95th amino acid Changed from GAC to CCG, which is the base sequence encoding Pro, the base sequence encoding Leu, the 98th amino acid, changed from TTA to ATA, the base sequence encoding Ile, the 99th amino acid The base sequence encoding Gln, which is changed from CAG to ACT, which is the base sequence encoding Thr, the base sequence encoding Glu, the 104th amino acid, is the base sequence encoding GAA to Asn The base sequence encoding Glu, which is the 104th amino acid, has been changed from AAA to GA, which is the base sequence encoding Lys, and the base sequence encoding Asp, which is the 112th amino acid, from GAT. Changed to GAG, which is the base sequence encoding Glu, changed base sequence encoding Gln, which is the 119th amino acid, from AAG, which is the base sequence encoding Asn, from GAG, Gln, which is the 119th amino acid The base sequence coding for CAG is changed from AAG, which is the base sequence encoding Asn, and the base sequence encoding Gln, the 119th amino acid, is changed from CAG to ATT, the base sequence encoding Ile. The nucleotide sequence encoding Gln, the 119th amino acid, was changed from CAG to ACC, the nucleotide sequence encoding Thr, and the nucleotide sequence encoding the G119, the 119th amino acid, encoded Ser from CAG. Changed to AGT, the base sequence that encodes Phe, which is the 137th amino acid, and GTC, which is the base sequence that encodes Val, from TTT, and encodes Lys, the 138th amino acid The base sequence is changed from AAA to ATC which is the base sequence encoding Ile, the base sequence encoding Tyr which is the 139th amino acid is changed from TAT to GTA which is the base sequence encoding Val, the 139th position The base sequence encoding Tyr, which is the amino acid of, is changed from TAT to GTG, which is the base sequence encoding Val, and the base sequence encoding Tyr, the 139th amino acid, is the base sequence encoding TAT to Cys. Changed to TGC, base sequence encoding Tyr, the 139th amino acid changed from TAT to ACA, the base sequence encoding Thr, base sequence encoding Tyr, the 139th amino acid The column is changed from TAT to TCT, which is the base sequence encoding Ser, the base sequence encoding Tyr, which is the 139th amino acid, is changed from TAT to AGT, which is the base sequence encoding Ser, the 139th The base sequence encoding the amino acid Tyr is changed from TAT to AAC, which is the base sequence encoding Asn, and the base sequence encoding Gly, the 143rd amino acid, is the base sequence encoding GGT to Glu. The base sequence encoding Tyr, the 145th amino acid, has been changed from CAT to CGT, the base sequence encoding Trg from TAT, and the base sequence encoding Tyr, the 145th amino acid, has changed from TAT to Arg Changed to AGA, which is the base sequence that encodes A, the base sequence encoding Cys, which is the 148th amino acid, changed from AGT, which is the base sequence that encodes Ser, to C, which is the 148th amino acid, C The base sequence encoding ys was changed from TGT to TCT, which is the base sequence encoding Ser, and the base sequence encoding Cys, the 148th amino acid, was changed from TGT to TCC, the base sequence encoding Ser. The base sequence encoding Cys, the 148th amino acid, has been changed from TGT to TCA, the base sequence encoding Ser, the base sequence encoding the Cys, the 148th amino acid, encodes Ala from TGT The base sequence was changed to GCG, the base sequence encoding Cys, the 148th amino acid, was changed to GCA, the base sequence encoding Tla to Ala, the base encoding Ile, the 182th amino acid The sequence is changed from ATT to ATG, which is the base sequence encoding Met, and the base sequence encoding Val, the 184th amino acid among the amino acid residues present in the active region domain, is changed from GTA to Ala. Changed to GCC, which is the base sequence to be loaded, the base sequence encoding Val, which is the 184th amino acid, from GTA to GCA, which is the base sequence encoding Ala, and Met, which is the 253rd amino acid. The coding base sequence is changed from ATG to CTA, which is the base sequence encoding Leu, the base sequence encoding Phe, the 262nd amino acid, is changed from TTC to TAT, which is the base sequence encoding Tyr, The base sequence encoding Ala, the 286th amino acid, has been changed from GCT to GAC, the base sequence encoding Asp, and the base sequence encoding the Lys, 290th amino acid, encodes AAA to His. Changed to CAC, the base sequence encoding Ala, the 295th amino acid, changed from GCA to TCA, the base sequence encoding Ser, and Ile, the 303th amino acid. Base sequence changed from ATT to ACA, which is a base sequence encoding Thr, base sequence encoding Phe, the 317th amino acid, changed from TTC to CAG, a base sequence encoding Gln, 335th The base sequence encoding Pro, which is the amino acid of, is changed from CCT to GCT, which is the base sequence encoding Ala, and the base sequence encoding Gly, the 352nd amino acid, is the base sequence encoding GGC to Ala. Changed to GCA, changed the base sequence encoding Arg, the 353rd amino acid from CGT to CAT, the base sequence encoding His, and the base sequence encoding Glu, the 386th amino acid, from GAA Changed to TCC, which is the base sequence encoding Ser, changed base sequence encoding Glu, which is the 430th amino acid, to TTC, which is the base sequence encoding Phe, from the 430th amino acid, and 443th amino acid The base sequence encoding Arg is changed from AGA to ATG, which is the base sequence encoding Met, and the base sequence encoding Ser, the 446th amino acid, is changed from TCT to TAC, the base sequence encoding Tyr. The base sequence encoding Ser, which is the 446th amino acid, has been changed from CAT, which is the base sequence encoding Gln, from TCT, and the base sequence encoding Asp, which is the 460th amino acid, encodes Ile from GAT. The base sequence encoding Asp, the 460th amino acid, and AAT, the base sequence encoding Asn, from the GAT, and the Asp, the 460th amino acid. The base sequence is changed from GAT to TGT, which is the base sequence encoding Cys, and the base sequence encoding Asp, the 460th amino acid, is changed from CAT, which is the base sequence encoding Gln to Gln. The base sequence encoding Asp, which is the 460th amino acid, is changed from CAT, which is the base sequence encoding GAT to Pro, and the base sequence encoding the Asp, which is the 460th amino acid, base sequence encoding GAT to Pro. The base sequence encoding Asp, which is the 460th amino acid, is changed to CCG, which is the base sequence encoding GAT to Pro, and the base sequence encoding Asp, which is the 460th amino acid. Change from GAT to TCA, which is the base sequence encoding Ser, Change base sequence encoding Pro, which is the 466th amino acid, to AAC, which is the base sequence encoding Asn, from the 466th amino acid, The base sequence encoding Pro is changed from CCG to GGC, which is the base sequence encoding Gly, and the base sequence encoding Pro, the 466th amino acid, is the base sequence encoding CCG to Ser. The base sequence encoding Ser, the 471st amino acid, has been changed from AGC to TAT, the base sequence encoding Tyr, and the base sequence encoding Gly, the 475th amino acid, is GGC. Is changed to AAT, which is the base sequence encoding Asn, the base sequence encoding Asp, which is the 506th amino acid, is changed to CCA, which is the base sequence encoding GAC to Pro, and is the 524th amino acid. The base sequence encoding Val is changed from GTT to TTA, which is the base sequence encoding Leu, and the base sequence encoding Val, the 524th amino acid, is changed from GTT to CTG, which is the base sequence encoding Leu. The base sequence encoding Ile, the 539th amino acid, was changed from ATC to TGC, the base sequence encoding Cys, and the base sequence encoding the 539th amino acid, Ile, copied Lec from ATC. Changed to CTT, which is the base sequence to be loaded, the base sequence encoding Ile, which is the 539th amino acid, changed from CTC, which is the base sequence encoding Leu to ATC, and Thr, which is the 544th amino acid. The coding base sequence is changed from ACC to GCG which is the base sequence encoding Ala, the base sequence encoding Thr which is the 544th amino acid is changed from ACC to GCT which is the base sequence encoding Ala, The base sequence encoding Thr, the 544th amino acid, has been changed from ACC to TCT, the base sequence encoding Ser, the base sequence encoding the 544th amino acid, Thr, the base sequence encoding ACC to Ser Changed to TCC, Thr that encodes Thr, the 544th amino acid, changed to CCT, which is the base sequence encoding Pro from ACC, Salt that encodes Thr, the 544th amino acid The sequence was changed from ACC to CCG which is a base sequence encoding Pro, the base sequence encoding Ala which is the 546th amino acid was changed from ACA which is the base sequence encoding Ser to Ger, and the 553rd The base sequence encoding the amino acid Leu was changed from CTG to GTA, which is the base sequence encoding Val, and the base sequence encoding Ala, the 623rd amino acid, was the base sequence encoding GCA to Cys. The base sequence encoding Ala, which is the 623rd amino acid, is changed from TCA, which is the base sequence encoding Phe, from GCA, and the base sequence encoding Ala, the 623th amino acid, is changed from GCA to Phe. Changed to TTC, which is the base sequence that encodes A, and the base sequence encoding Ala, which is the 623rd amino acid, to CAG, which is the base sequence that encodes Gln from GCA, and the 626th amino acid The base sequence encoding Lys is changed from AAA to GTG, which is the base sequence encoding Val, and the base sequence encoding Tyr, the 636th amino acid, is changed from TAC to TGT, which is the base sequence encoding Cys. The base sequence encoding Tyr, the 636th amino acid, has been changed from TAC to CCC, the base sequence encoding Pro, the base sequence encoding the 646th amino acid, Ala, encodes Leu from GCC Changed to TTG, which is the base sequence to be encoded, the base sequence encoding Ala, which is the 646th amino acid, changed from ACC which is the base sequence encoding Ile to GCC, and Met, which is the 648th amino acid, is encoded The base sequence is changed from ATG to TCT which is a base sequence encoding Ser, the base sequence encoding Met which is the 648th amino acid is changed from ATG to TCC which is a base sequence encoding Ser, The base sequence encoding Lys, the 710th amino acid, has been changed from AAA to ACG, the base sequence encoding Thr, the base sequence encoding the 711st amino acid, Glu, is the base sequence encoding GAA to Asp The mutated lysine decarboxylase in which at least one or more sites are replaced with the GAC that has been changed.
　より具体的には、配列表の配列番号３に記載のアミノ酸配列において、ウィングドメインに存在するアミノ酸残基のうち14番目のアミノ酸であるPheをコードする塩基配列がTTTからGlnをコードする塩基配列であるCAAに変更したもの、22番目のアミノ酸であるLeuをコードする塩基配列がCTTからLeuをコードする塩基配列であるTTGに変更したもの、28番目のアミノ酸であるArgをコードする塩基配列がCGTからIleをコードする塩基配列であるATTに変更したもの、39番目のアミノ酸であるArgをコードする塩基配列がCGTからIleをコードする塩基配列であるATAに変更したもの、39番目のアミノ酸であるArgをコードする塩基配列がCGTからIleをコードする塩基配列であるATCに変更したもの、39番目のアミノ酸であるArgをコードする塩基配列がCGTからValをコードする塩基配列であるGTGに変更したもの、64番目のアミノ酸であるLeuをコードする塩基配列がCTCからLysをコードする塩基配列であるAAAに変更したもの、67番目のアミノ酸であるCysをコードする塩基配列がTGCからThrをコードする塩基配列であるACCに変更したもの、67番目のアミノ酸であるCysをコードする塩基配列がTGCからLeuをコードする塩基配列であるTTAに変更したもの、70番目のアミノ酸であるIleをコードする塩基配列がATTからLeuをコードする塩基配列であるTTGに変更したもの、70番目のアミノ酸であるIleをコードする塩基配列がATTからLeuをコードする塩基配列であるCTGに変更したもの、70番目のアミノ酸であるIleをコードする塩基配列がATTからProをコードする塩基配列であるCCGに変更したもの、75番目のアミノ酸であるGluをコードする塩基配列がGAGからProをコードする塩基配列であるCCCに変更したもの、75番目のアミノ酸であるGluをコードする塩基配列がGAGからHisをコードする塩基配列であるCACに変更したもの、79番目のアミノ酸であるLeuをコードする塩基配列がTTGからIleをコードする塩基配列であるATAに変更したもの、83番目のアミノ酸であるAlaをコードする塩基配列がGCTからLeuをコードする塩基配列であるCTGに変更したもの、83番目のアミノ酸であるAlaをコードする塩基配列がGCTからLeuをコードする塩基配列であるCTAに変更したもの、83番目のアミノ酸であるAlaをコードする塩基配列がGCTからLeuをコードする塩基配列であるCTTに変更したもの、83番目のアミノ酸であるAlaをコードする塩基配列がGCTからAlaをコードする塩基配列であるATAに変更したもの、83番目のアミノ酸であるAlaをコードする塩基配列がGCTからAlaをコードする塩基配列であるGCCに変更したもの、84番目のアミノ酸であるAsnをコードする塩基配列がAATからAspをコードする塩基配列であるGACに変更したもの、84番目のアミノ酸であるAsnをコードする塩基配列がAATからThrをコードする塩基配列であるACAに変更したもの、85番目のアミノ酸であるThrをコードする塩基配列がACGからProをコードする塩基配列であるCCAに変更したもの、88番目のアミノ酸であるThrをコードする塩基配列がACTからLysをコードする塩基配列であるAAAに変更したもの、88番目のアミノ酸であるThrをコードする塩基配列がACTからLysをコードする塩基配列であるAAGに変更したもの、88番目のアミノ酸であるThrをコードする塩基配列がACTからArgをコードする塩基配列であるAGAに変更したもの、88番目のアミノ酸であるThrをコードする塩基配列がACTからAsnをコードする塩基配列であるAATに変更したもの、89番目のアミノ酸であるLeuをコードする塩基配列がCTCからPheをコードする塩基配列であるTTTに変更したもの、94番目のアミノ酸であるAsnをコードする塩基配列がAATからIleをコードする塩基配列であるATCに変更したもの、95番目のアミノ酸であるAspをコードする塩基配列がGACからProをコードする塩基配列であるCCGに変更したもの、98番目のアミノ酸であるLeuをコードする塩基配列がTTAからIleをコードする塩基配列であるATAに変更したもの、99番目のアミノ酸であるGlnをコードする塩基配列がCAGからThrをコードする塩基配列であるACTに変更したもの、104番目のアミノ酸であるGluをコードする塩基配列がGAAからAsnをコードする塩基配列であるAATに変更したもの、104番目のアミノ酸であるGluをコードする塩基配列がGAAからLysをコードする塩基配列であるAAAに変更したもの、112番目のアミノ酸であるAspをコードする塩基配列がGATからGluをコードする塩基配列であるGAGに変更したもの、119番目のアミノ酸であるGlnをコードする塩基配列がCAGからAsnをコードする塩基配列であるAACに変更したもの、119番目のアミノ酸であるGlnをコードする塩基配列がCAGからAsnをコードする塩基配列であるAATに変更したもの、119番目のアミノ酸であるGlnをコードする塩基配列がCAGからIleをコードする塩基配列であるATTに変更したもの、119番目のアミノ酸であるGlnをコードする塩基配列がCAGからThrをコードする塩基配列であるACCに変更したもの、119番目のアミノ酸であるGlnをコードする塩基配列がCAGからSerをコードする塩基配列であるAGTに変更したもの、リンカードメインに存在するアミノ酸残基のうち137番目のアミノ酸であるPheをコードする塩基配列がTTTからValをコードする塩基配列であるGTCに変更したもの、138番目のアミノ酸であるLysをコードする塩基配列がAAAからIleをコードする塩基配列であるATCに変更したもの、139番目のアミノ酸であるTyrをコードする塩基配列がTATからValをコードする塩基配列であるGTAに変更したもの、139番目のアミノ酸であるTyrをコードする塩基配列がTATからValをコードする塩基配列であるGTGに変更したもの、139番目のアミノ酸であるTyrをコードする塩基配列がTATからCysをコードする塩基配列であるTGCに変更したもの、139番目のアミノ酸であるTyrをコードする塩基配列がTATからThrをコードする塩基配列であるACAに変更したもの、139番目のアミノ酸であるTyrをコードする塩基配列がTATからSerをコードする塩基配列であるTCTに変更したもの、139番目のアミノ酸であるTyrをコードする塩基配列がTATからSerをコードする塩基配列であるAGTに変更したもの、139番目のアミノ酸であるTyrをコードする塩基配列がTATからAsnをコードする塩基配列であるAACに変更したもの、143番目のアミノ酸であるGlyをコードする塩基配列がGGTからGluをコードする塩基配列であるGAAに変更したもの、145番目のアミノ酸であるTyrをコードする塩基配列がTATからArgをコードする塩基配列であるCGTに変更したもの、145番目のアミノ酸であるTyrをコードする塩基配列がTATからArgをコードする塩基配列であるAGAに変更したもの、148番目のアミノ酸であるCysをコードする塩基配列がTGTからSerをコードする塩基配列であるAGTに変更したもの、148番目のアミノ酸であるCysをコードする塩基配列がTGTからSerをコードする塩基配列であるTCTに変更したもの、148番目のアミノ酸であるCysをコードする塩基配列がTGTからSerをコードする塩基配列であるTCCに変更したもの、148番目のアミノ酸であるCysをコードする塩基配列がTGTからSerをコードする塩基配列であるTCAに変更したもの、148番目のアミノ酸であるCysをコードする塩基配列がTGTからAlaをコードする塩基配列であるGCGに変更したもの、148番目のアミノ酸であるCysをコードする塩基配列がTGTからAlaをコードする塩基配列であるGCAに変更したもの、182番目のアミノ酸であるIleをコードする塩基配列がATTからMetをコードする塩基配列であるATGに変更したもの、ピリドキサールリン酸酵素共通ドメインに存在するアミノ酸残基のうち184番目のアミノ酸であるValをコードする塩基配列がGTAからAlaをコードする塩基配列であるGCCに変更したもの、184番目のアミノ酸であるValをコードする塩基配列がGTAからAlaをコードする塩基配列であるGCAに変更したもの、253番目のアミノ酸であるMetをコードする塩基配列がATGからLeuをコードする塩基配列であるCTAに変更したもの、262番目のアミノ酸であるPheをコードする塩基配列がTTCからTyrをコードする塩基配列であるTATに変更したもの、286番目のアミノ酸であるAlaをコードする塩基配列がGCTからAspをコードする塩基配列であるGACに変更したもの、290番目のアミノ酸であるLysをコードする塩基配列がAAAからHisをコードする塩基配列であるCACに変更したもの、295番目のアミノ酸であるAlaをコードする塩基配列がGCA からSerをコードする塩基配列であるTCAに変更したもの、303番目のアミノ酸であるIleをコードする塩基配列がATTからThrをコードする塩基配列であるACAに変更したもの、317番目のアミノ酸であるPheをコードする塩基配列がTTCからGlnをコードする塩基配列であるCAGに変更したもの、335番目のアミノ酸であるProをコードする塩基配列がCCTからAlaをコードする塩基配列であるGCTに変更したもの、352番目のアミノ酸であるGlyをコードする塩基配列がGGCからAlaをコードする塩基配列であるGCAに変更したもの、353番目のアミノ酸であるArgをコードする塩基配列がCGTからHisをコードする塩基配列であるCATに変更したもの、386番目のアミノ酸であるGluをコードする塩基配列がGAAからSerをコードする塩基配列であるTCCに変更したもの、基質出入口に存在するアミノ酸残基のうち430番目のアミノ酸であるGluをコードする塩基配列がGAAからPheをコードする塩基配列であるTTCに変更したもの、443番目のアミノ酸であるArgをコードする塩基配列がAGAからMetをコードする塩基配列であるATGに変更したもの、446番目のアミノ酸であるSerをコードする塩基配列がTCTからTyrをコードする塩基配列であるTACに変更したもの、446番目のアミノ酸であるSerをコードする塩基配列がTCTからGlnをコードする塩基配列であるCAAに変更したもの、460番目のアミノ酸であるAspをコードする塩基配列がGATからIleをコードする塩基配列であるATTに変更したもの、460番目のアミノ酸であるAspをコードする塩基配列がGATからAsnをコードする塩基配列であるAATに変更したもの、460番目のアミノ酸であるAspをコードする塩基配列がGATからCysをコードする塩基配列であるTGTに変更したもの、460番目のアミノ酸であるAspをコードする塩基配列がGATからGlnをコードする塩基配列であるCAGに変更したもの、460番目のアミノ酸であるAspをコードする塩基配列がGATからProをコードする塩基配列であるCCCに変更したもの、460番目のアミノ酸であるAspをコードする塩基配列がGATからProをコードする塩基配列であるCCTに変更したもの、460番目のアミノ酸であるAspをコードする塩基配列がGATからProをコードする塩基配列であるCCGに変更したもの、460番目のアミノ酸であるAspをコードする塩基配列がGATからSerをコードする塩基配列であるTCAに変更したもの、466番目のアミノ酸であるProをコードする塩基配列がCCGからAsnをコードする塩基配列であるAACに変更したもの、466番目のアミノ酸であるProをコードする塩基配列がCCGからGlyをコードする塩基配列であるGGCに変更したもの、466番目のアミノ酸であるProをコードする塩基配列がCCGからSerをコードする塩基配列であるTCTに変更したもの、471番目のアミノ酸であるSerをコードする塩基配列がAGCからTyrをコードする塩基配列であるTATに変更したもの、475番目のアミノ酸であるGlyをコードする塩基配列がGGCからAsnをコードする塩基配列であるAATに変更したもの、506番目のアミノ酸であるAspをコードする塩基配列がGACからProをコードする塩基配列であるCCAに変更したもの、524番目のアミノ酸であるValをコードする塩基配列がGTTからLeuをコードする塩基配列であるTTAに変更したもの、524番目のアミノ酸であるValをコードする塩基配列がGTTからLeuをコードする塩基配列であるCTGに変更したもの、539番目のアミノ酸であるIleをコードする塩基配列がATCからCysをコードする塩基配列であるTGCに変更したもの、539番目のアミノ酸であるIleをコードする塩基配列がATCからLeuをコードする塩基配列であるCTTに変更したもの、539番目のアミノ酸であるIleをコードする塩基配列がATCからLeuをコードする塩基配列であるCTAに変更したもの、544番目のアミノ酸であるThrをコードする塩基配列がACCからAlaをコードする塩基配列であるGCGに変更したもの、544番目のアミノ酸であるThrをコードする塩基配列がACCからAlaをコードする塩基配列であるGCTに変更したもの、544番目のアミノ酸であるTh
rをコードする塩基配列がACCからSerをコードする塩基配列であるTCTに変更したもの、544番目のアミノ酸であるThrをコードする塩基配列がACCからSerをコードする塩基配列であるTCCに変更したもの、544番目のアミノ酸であるThrをコードする塩基配列がACCからProをコードする塩基配列であるCCTに変更したもの、544番目のアミノ酸であるThrをコードする塩基配列がACCからProをコードする塩基配列であるCCGに変更したもの、546番目のアミノ酸であるAlaをコードする塩基配列がGCAからSerをコードする塩基配列であるAGCに変更したもの、553番目のアミノ酸であるLeuをコードする塩基配列がCTGからValをコードする塩基配列であるGTAに変更したもの、623番目のアミノ酸であるAlaをコードする塩基配列がGCAからCysをコードする塩基配列であるTGTに変更したもの、623番目のアミノ酸であるAlaをコードする塩基配列がGCAからPheをコードする塩基配列であるTTTに変更したもの、623番目のアミノ酸であるAlaをコードする塩基配列がGCAからPheをコードする塩基配列であるTTCに変更したもの、623番目のアミノ酸であるAlaをコードする塩基配列がGCAからGlnをコードする塩基配列であるCAGに変更したもの、626番目のアミノ酸であるLysをコードする塩基配列がAAAからValをコードする塩基配列であるGTGに変更したもの、636番目のアミノ酸であるTyrをコードする塩基配列がTACからCysをコードする塩基配列であるTGTに変更したもの、636番目のアミノ酸であるTyrをコードする塩基配列がTACからProをコードする塩基配列であるCCCに変更したもの、646番目のアミノ酸であるAlaをコードする塩基配列がGCCからLeuをコードする塩基配列であるTTGに変更したもの、646番目のアミノ酸であるAlaをコードする塩基配列がGCCからIleをコードする塩基配列であるATCに変更したもの、648番目のアミノ酸であるMetをコードする塩基配列がATGからSerをコードする塩基配列であるTCTに変更したもの、648番目のアミノ酸であるMetをコードする塩基配列がATGからSerをコードする塩基配列であるTCCに変更したもの、710番目のアミノ酸であるLysをコードする塩基配列がAAAからThrをコードする塩基配列であるACGに変更したもの、711番目のアミノ酸であるGluをコードする塩基配列がGAAからAspをコードする塩基配列であるGACに変更したものに少なくとも１箇所以上置換されている変異型リジン脱炭酸酵素が挙げられる。 More specifically, in the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing, the base sequence encoding Phe, the 14th amino acid among the amino acid residues present in the wing domain, encodes a base sequence encoding GTT from TTT The base sequence encoding Leu, which is the 22nd amino acid, has been changed from CTT to TTG, which is the base sequence encoding Leu, and the base sequence encoding Arg, the 28th amino acid. Change from CGT to ATT, which is the base sequence encoding Ile, change base sequence encoding Arg, the 39th amino acid, to ATA, the base sequence encoding Ile, from the 39th amino acid, The base sequence encoding a certain Arg is changed from CGT to ATC which is the base sequence encoding Ile, the base sequence encoding the 39th amino acid Arg is the base sequence encoding CGT to Val The base sequence encoding Leu, which is the 64th amino acid, has been changed to AAA, which is the base sequence encoding Lys from CTC, and the base sequence encoding Cys, the 67th amino acid. Change from TGC to ACC, which is the base sequence encoding Thr, change from 67th amino acid to Cys, which is the base sequence encoding Cys from TGC to TTA, which is the base sequence encoding Leu, 70th amino acid The base sequence encoding a certain Ile is changed from ATT to TTG, which is the base sequence encoding Leu, and the base sequence encoding Ile, the 70th amino acid, is changed from ATT to CTG, the base sequence encoding Leu. The base sequence encoding Ile, the 70th amino acid, has been changed from ATT to CCG, which is the base sequence encoding Pro, and the base sequence encoding the Glu, the 75th amino acid, copied GAG to Pro. Changed to CCC, which is the base sequence to be loaded, the base sequence encoding Glu, which is the 75th amino acid, changed from CAG to CAC, which is the base sequence encoding His, and Leu, which is the 79th amino acid. The coding base sequence is changed from TTG to ATA which is the base sequence encoding Ile, the base sequence encoding Ala which is the 83rd amino acid is changed from CCT to CTG which is the base sequence encoding Leu, The nucleotide sequence encoding Ala, the 83rd amino acid, has been changed from CCT, which is the nucleotide sequence encoding Leu, from GCT, the nucleotide sequence encoding the Ala, the 83rd amino acid, encoding Leu The base sequence encoding Ala, the 83rd amino acid, the base sequence encoding Ala, the 83rd amino acid, has been changed from GCT to ATA, the base sequence encoding Ala. Changed from GCT to GCC, which is the base sequence encoding Ala, base sequence encoding Asn, which is the 84th amino acid, changed from GAT, which is the base sequence encoding Asp, to the 84th amino acid, 84th amino acid The base sequence encoding Asn is changed from AAT to ACA, which is the base sequence encoding Thr, and the base sequence encoding Thr, the 85th amino acid, is changed from ACG to CCA, which is the base sequence encoding Pro. Modified, base sequence encoding Thr, the 88th amino acid changed from AAA to base sequence encoding ACT to Lys, base sequence encoding Thr, the 88th amino acid changed from ACT to Lys Coding base sequence changed to AAG, 88th amino acid Thr coding base sequence changed from ACT to Arg coding base sequence AGA, 88th amino acid Thr coding The base sequence to be changed from ACT to AAT which is the base sequence encoding Asn, the base sequence encoding Leu which is the 89th amino acid is changed from CTC to TTT which is the base sequence encoding Phe, The base sequence encoding the 94th amino acid Asn is changed from AAT to ATC, which is the base sequence encoding Ile, and the base sequence encoding the 95th amino acid Asp is the base sequence encoding GAC to Pro. The base sequence encoding Leu, which is the 98th amino acid, was changed from TTA to ATA, which is the base sequence encoding Ile, and the base sequence encoding Gln, the 99th amino acid. Change from CAG to ACT, which is the base sequence encoding Thr, Change from base sequence encoding Glu, the 104th amino acid, to AAT, the base sequence encoding Asn, 104th amino acid The base sequence encoding Glu which is an acid is changed from GAA to AAA which is a base sequence encoding Lys, and the base sequence encoding Asp which is the 112th amino acid is a base sequence encoding GAT to Glu The base sequence encoding Gln, which is the 119th amino acid, is changed from CAG to AAC, which is the base sequence encoding Asn, and the base sequence encoding the G119, which is the 119th amino acid, is changed from CAG to Asn. A base sequence that encodes AAT, a base sequence that encodes Gln, which is the 119th amino acid, has been changed from CAG to ATT, which is a base sequence encoding Ile, and Gln, which is the 119th amino acid. The coding base sequence was changed from CAG to ACC, which is the base sequence encoding Thr, and the base sequence encoding Gln, the 119th amino acid, was changed from CAG to AGT, the base sequence encoding Ser. Of the amino acid residues present in the linker domain, the base sequence encoding Phe, which is the 137th amino acid, has been changed from TTT to GTC, which is the base sequence encoding Val, and the 138th amino acid, Lys, is encoded. The base sequence to be changed from AAA to ATC which is a base sequence encoding Ile, the base sequence encoding Tyr which is the 139th amino acid is changed from TAT to GTA which is a base sequence encoding Val, 139 The base sequence encoding Tyr, the second amino acid, has been changed from TAT to GTG, the base sequence encoding Val, and the base sequence encoding Tyr, the 139th amino acid, is a base sequence encoding TAT to Cys. Changed to a certain TGC, changed the base sequence encoding Tyr, the 139th amino acid from TAT to ACA, the base sequence encoding Thr, copied the 139th amino acid, Tyr. The base sequence to be changed from TAT to TCT which is the base sequence encoding Ser, the base sequence encoding Tyr which is the 139th amino acid is changed from TAT to AGT which is the base sequence encoding Ser The base sequence encoding Tyr, the 139th amino acid, has been changed from TAT to AAC, the base sequence encoding Asn, the base sequence encoding Gly, the 143rd amino acid, is a base encoding GGT to Glu Sequence changed to GAA, base sequence encoding Tyr, the 145th amino acid changed from TAT to CGT, the base sequence encoding Arg, base sequence encoding Tyr, the 145th amino acid Changed from TAT to AGA, which is the base sequence encoding Arg, base sequence encoding Cys, which is the 148th amino acid, changed from AGT, which is the base sequence encoding Ser, to the 148th amino acid The base sequence encoding Cys, which is a mino acid, is changed from TGT to TCT, which is the base sequence encoding Ser, and the base sequence encoding Cys, the 148th amino acid, is a base sequence encoding TGT to Ser. Changed to TCC, base sequence encoding Cys, 148th amino acid changed from TGT to TCA, the base sequence encoding Ser, base sequence encoding Cys, 148th amino acid from TGT Changed to GCG, which is the base sequence encoding Ala, changed base sequence encoding Cys, which is the 148th amino acid, from GGT to GCA, which is the base sequence encoding Ala, Ile, which is the 182nd amino acid The nucleotide sequence coding for ATT is changed from ATG to ATG, which is the nucleotide sequence coding for Met, Va which is the 184th amino acid residue among the amino acid residues present in the common domain of pyridoxal phosphate The base sequence encoding l was changed from GTA to GCC, which is the base sequence encoding Ala, and the base sequence encoding Val, the 184th amino acid, was changed from GTA to GCA, the base sequence encoding Ala. The base sequence encoding Met, which is the 253rd amino acid, has been changed from ATG to CTA, which is the base sequence encoding Leu, and the base sequence encoding the P262, which is the 262nd amino acid, encodes Tyr from TTC. The base sequence changed to TAT, the base sequence encoding Ala, the 286th amino acid, changed from GCT to GAC, the base sequence encoding Asp, the base encoding the Lys, the 290th amino acid The sequence was changed from AAA to CAC, which is the base sequence encoding His, the base sequence encoding Ala, the 295th amino acid, was changed from GCA to TCA, the base sequence encoding Ser, 303 The base sequence encoding the first amino acid Ile has been changed from ATT to ACA, the base sequence encoding Thr, and the base sequence encoding the 317th amino acid Phe is the base sequence encoding TTC to Gln. Changed to a CAG, changed the base sequence encoding Pro, the 335th amino acid, from CCT to GCT, the base sequence encoding Ala, the base sequence encoding Gly, the 352nd amino acid Is changed to GCA which is the base sequence encoding Ala, the base sequence encoding Arg which is the 353rd amino acid is changed to CAT which is the base sequence encoding His from CGT, and the 386th amino acid. A base sequence encoding Glu, which is the 430th amino acid of the amino acid residues present at the substrate entrance / exit, in which the base sequence encoding Glu is changed from GAA to TCC, which is the base sequence encoding Ser. Changed from GAA to TTC which is the base sequence encoding Phe, base sequence encoding Arg which is the 443rd amino acid changed from AGA to ATG which is the base sequence encoding Met, the 446th amino acid The nucleotide sequence that encodes Ser is changed from TCT to TAC, which is the nucleotide sequence encoding Tyr, and the nucleotide sequence encoding Ser, the 446th amino acid, is changed to CAA, which is the nucleotide sequence encoding TCT to Gln. Changed, the base sequence encoding Asp, the 460th amino acid, changed from GAT to ATT, the base sequence encoding Ile, the base sequence encoding Asp, the 460th amino acid, changed from GAT to Asn. Changed to AAT, which is the coding base sequence, changed base sequence encoding Asp, which is the 460th amino acid, from GAT to TGT, which is the base sequence encoding Cys, Asp, which is the 460th amino acid The coding base sequence is changed from GAT to CAG, which is the base sequence encoding Gln, the base sequence encoding Asp, the 460th amino acid, is changed from GAT to CCC, which is the base sequence encoding Pro, The base sequence encoding Asp, which is the 460th amino acid, is changed from CAT, which is the base sequence encoding GAT to Pro, and the base sequence encoding the Asp, which is the 460th amino acid, encodes GAT to Pro. The base sequence encoding Asp, which is the 460th amino acid, has been changed from TAT, which is the base sequence encoding Ser, to the 466th amino acid, and the base sequence encoding Pro, the 466th amino acid. Change from CCG to AAC, which is the base sequence encoding Asn, change base sequence encoding Pro, the 466th amino acid, from CCG to GGC, which is the base sequence encoding Gly, No. 466 The base sequence encoding Pro, which is the amino acid of CCG, is changed from CCG to TCT, which is the base sequence encoding Ser, and the base sequence encoding Ser, the 471st amino acid, is the base sequence encoding AGC to Tyr. Changed to TAT, changed base sequence encoding Gly, the 475th amino acid from AGC to base sequence encoding Asn from GGC, base sequence encoding Asp, the 506th amino acid from GAC Changed to CCA, the base sequence encoding Pro, changed base sequence encoding Val, the 524th amino acid, from TTT, the base sequence encoding Leu, Val, the 524th amino acid The base sequence encoding GTT is changed to CTG, which is the base sequence encoding Leu, and the base sequence encoding Ile, the 539th amino acid, is changed from ATC to TGC, which is the base sequence encoding Cys. Modified, the base sequence encoding Ile, the 539th amino acid, changed from ATC to CTT, the base sequence encoding Leu, the base sequence encoding the 539th amino acid, Ile, changed from ATC to Leu Changed to CTA, which is the coding base sequence, changed the base sequence encoding Thr, which is the 544th amino acid, to GCG, which is the base sequence encoding ACC to Ala, and encodes Thr, which is the 544th amino acid The base sequence to be changed from ACC to GCT, which is the base sequence encoding Ala, Th 544th amino acid
The base sequence encoding r was changed from ACC to TCT, which is the base sequence encoding Ser, and the base sequence encoding Thr, the 544th amino acid, was changed from ACC to TCC, the base sequence encoding Ser. The base sequence encoding Thr, the 544th amino acid, has been changed to CCT, the base sequence encoding Pro from ACC, and the base sequence encoding the Thr, the 544th amino acid, encodes Pro from ACC The base sequence was changed to CCG, the base sequence encoding Ala, the 546th amino acid, was changed to AGC, the base sequence encoding Ser from GCA, and the base encoding Leu, the 553th amino acid. The sequence is changed from CTG to GTA which is the base sequence encoding Val, the base sequence encoding Ala which is the 623rd amino acid is changed from GCA to TGT which is the base sequence encoding Cys, No. 623 The base sequence encoding the first amino acid, Ala, is changed from GCA to TTT, the base sequence encoding Phe, and the base sequence encoding the 623th amino acid, Ala, is the base sequence encoding GCA to Phe. Changed to a certain TTC, changed the base sequence encoding Ala, the 623th amino acid, from CAA to CAG, the base sequence encoding Gln, and the base sequence encoding the Lys, the 626th amino acid, AAA Changed from GTG to GTG, the base sequence encoding Val, from TAC to TGT, the base sequence encoding Cys from the 636th amino acid, Tyr, the 636th amino acid, the 636th amino acid A TTG coding sequence changed from TAC to CCC, which is a coding sequence for Pro, and a TG amino acid sequence, Ala, which is the 646th amino acid sequence, is a coding sequence from GCC to Leu. Changed, the base sequence encoding Ala, the 646th amino acid, changed from GCC to ATC, the base sequence encoding Ile, the base sequence encoding Met, the 648th amino acid, from Ser to Ser Changed to TCT, which is the base sequence encoding, changed base sequence encoding Met, which is the 648th amino acid, to TCC, which is the base sequence encoding Ser from ATG, and encodes Lys, which is the 710th amino acid The base sequence to be changed from AAA to ACG which is a base sequence encoding Thr, the base sequence encoding Glu which is the 711st amino acid is changed from GAA to GAC which is a base sequence encoding Asp at least A mutated lysine decarboxylase substituted at one or more sites is mentioned.
　変更後の塩基配列は元の配列の酵素よりも良い性質があれば特に制限はないが、それぞれ表１～６に記載の配列により生成される塩基配列は特に好ましい。
（５）変異型リジン脱炭酸酵素の製造方法
　本発明にかかる上記変異型リジン脱炭酸酵素の製造方法（以下、単に「製造方法」とも言う。）は、上記変異型リジン脱炭酸酵素の形質転換体を培養し、培養された上記変異型リジン脱炭酸酵素の形質転換体および該形質転換体の培養物のうち少なくともいずれか一方から、上記変異型リジン脱炭酸酵素を回収するものである。 The modified base sequence is not particularly limited as long as it has better properties than the enzyme of the original sequence, but base sequences generated by the sequences shown in Tables 1 to 6 are particularly preferred.
(5) Method for Producing Mutant Lysine Decarboxylase The method for producing the mutant lysine decarboxylase according to the present invention (hereinafter also simply referred to as “manufacturing method”) is the transformation of the mutant lysine decarboxylase. The body is cultured, and the mutant lysine decarboxylase is recovered from at least one of the transformed transformant of the mutant lysine decarboxylase and the cultured product of the transformant.
　ここで上記変異型リジン脱炭酸酵素の形質転換体とは、上記変異型リジン脱炭酸酵素のアミノ酸配列をコードする塩基配列で示される核酸を含む発現ベクターで形質転換されたものを示す。 Here, the transformant of the above-mentioned mutant lysine decarboxylase refers to one transformed with an expression vector containing a nucleic acid represented by a base sequence encoding the amino acid sequence of the above-mentioned mutant lysine decarboxylase.
　本発明にかかる上記変異型リジン脱炭酸酵素の製造方法は、上記変異型リジン脱炭酸酵素のアミノ酸配列をコードする塩基配列で示される核酸を含む発現ベクターで形質転換された形質転換体を培養することにより、上記変異型リジン脱炭酸酵素を製造するものである。当該製造方法により、酵素が失活しやすい厳しい条件下においても、安定した活性を示すとともに、対応する野生型リジン脱炭酸酵素と比較しても反応の初速度が大きく低下することのない上記変異型リジン脱炭酸酵素を、低コストで製造することができる。 In the method for producing the mutant lysine decarboxylase according to the present invention, a transformant transformed with an expression vector containing a nucleic acid represented by a base sequence encoding the amino acid sequence of the mutant lysine decarboxylase is cultured. Thus, the above mutant lysine decarboxylase is produced. According to the production method, the mutation described above shows stable activity even under severe conditions in which the enzyme is easily inactivated, and does not significantly reduce the initial rate of reaction even when compared with the corresponding wild-type lysine decarboxylase. Type lysine decarboxylase can be produced at low cost.
　以下に、製造方法に含まれうる各工程を説明するが、本発明にかかる上記変異型リジン脱炭酸酵素の製造方法は、上記変異型リジン脱炭酸酵素のアミノ酸配列をコードする塩基配列で示される核酸を含む発現ベクターで形質転換された形質転換体を培養する工程（宿主細胞培養工程）、および、培養された形質転換体および該形質転換体の培養物のうち少なくともいずれか一方から、上記変異型リジン脱炭酸酵素を回収する工程（変異型リジン脱炭酸酵素回収工程）を含んでいればよく、必要に応じてさらに他の工程を含んでいてもよい。
（６）形質転換体培養工程
　形質転換体培養工程は、野生型および／または上記変異型リジン脱炭酸酵素のアミノ酸配列をコードする塩基配列で示される核酸を含む発現ベクターで形質転換された形質転換体を培養する工程である。
〔形質転換体〕
　本発明にかかる製造方法において、形質転換体とは、野生型および／または上記変異型リジン脱炭酸酵素のアミノ酸配列をコードする塩基配列で示される核酸を含む発現ベクターで形質転換されたものであれば、特に限定されない。 Hereinafter, each step that can be included in the production method will be described. The production method of the mutant lysine decarboxylase according to the present invention is represented by a base sequence encoding the amino acid sequence of the mutant lysine decarboxylase. From the step of culturing a transformant transformed with an expression vector containing a nucleic acid (host cell culturing step), and at least one of the cultured transformant and the culture of the transformant, the mutation It only needs to include a step of recovering the type lysine decarboxylase (mutant lysine decarboxylase recovery step), and may further include other steps as necessary.
(6) Transformant culture process The transformant culture process is performed by transforming with an expression vector containing a nucleic acid represented by a base sequence encoding the amino acid sequence of the wild-type and / or the mutant lysine decarboxylase. It is a step of culturing the body.
[Transformant]
In the production method according to the present invention, the transformant is a transformant transformed with an expression vector containing a nucleic acid represented by a base sequence encoding the amino acid sequence of the wild-type and / or the mutant lysine decarboxylase. There is no particular limitation.
　上記形質転換体は、例えば、細菌、酵母、放線菌、糸状菌など由来の細胞を宿主細胞とするものが挙げられ、大腸菌、コリネバクテリウム属細菌由来の細胞を宿主細胞とするものが好ましい。
〔核酸〕
　上記核酸は、野生型および上記変異型リジン脱炭酸酵素のアミノ酸配列をコードする塩基配列で示される。 Examples of the transformant include those using cells derived from bacteria, yeast, actinomycetes, filamentous fungi and the like as host cells, and those using cells derived from Escherichia coli and Corynebacterium bacteria as host cells are preferred.
[Nucleic acid]
The nucleic acid is represented by a base sequence encoding the amino acid sequence of the wild type and the mutant lysine decarboxylase.
　上記変異型リジン脱炭酸酵素のアミノ酸配列をコードする塩基配列は、対応する野生型リジン脱炭酸酵素をコードする塩基配列に変異点を導入する方法などにより、合成することができる。
〔発現ベクター〕
　上記発現ベクターは、野生型および／または上記変異型リジン脱炭酸酵素のアミノ酸配列をコードする塩基配列で示される核酸を含むものであれば、特に限定されるものではないが、形質転換効率や翻訳効率を向上させるなどの観点より、以下に示すような構成を示すプラスミドベクターや、ファージベクターであることがより好ましい。
〔発現ベクターの基本構成〕
　発現ベクターは、野生型および／または上記変異型リジン脱炭酸酵素をコードする塩基配列を含み、上記宿主細胞を形質転換しうるものであれば、特に限定されない。必要に応じて、該塩基配列の他に、他の領域を構成する塩基配列（以下、単に「他の領域」とも言う。）を含んでいてもよい。 The base sequence encoding the amino acid sequence of the mutant lysine decarboxylase can be synthesized by a method of introducing a mutation point into the base sequence encoding the corresponding wild-type lysine decarboxylase.
[Expression vector]
The expression vector is not particularly limited as long as it contains a nucleic acid represented by a base sequence encoding the amino acid sequence of the wild type and / or the mutant lysine decarboxylase. However, transformation efficiency and translation are not limited. From the viewpoint of improving the efficiency, a plasmid vector or a phage vector having the following configuration is more preferable.
[Basic structure of expression vector]
The expression vector is not particularly limited as long as it contains a wild-type and / or base sequence encoding the mutant lysine decarboxylase and can transform the host cell. If necessary, in addition to the base sequence, a base sequence constituting another region (hereinafter, also simply referred to as “other region”) may be included.
　他の領域としては、例えば、上記形質転換体が、野生型および上記変異型リジン脱炭酸酵素を産生するために必要とする制御領域や、自律複製に必要な領域などが挙げられる。 Examples of other regions include control regions necessary for the transformant to produce wild type and mutant lysine decarboxylase, regions necessary for autonomous replication, and the like.
　また、上記形質転換体の選択を容易にするという観点より、選択マーカーとなりうる選択遺伝子をコードする塩基配列をさらに含んでいてもよい。 In addition, from the viewpoint of facilitating selection of the transformant, it may further include a base sequence encoding a selection gene that can be a selection marker.
　野生型および上記変異型リジン脱炭酸酵素を産生するために必要となる制御領域としては、例えば、プロモーター配列（転写を制御するオペレーター配列を含む。）、リボゾーム結合配列（ＳＤ配列）、転写終結配列などを挙げることができる。
〔原核生物を宿主細胞とした場合の発現ベクター〕
　原核生物を宿主細胞とする場合、発現ベクターは、野生型および／または上記変異型リジン脱炭酸酵素をコードする塩基配列の他に、野生型および上記変異型リジン脱炭酸酵素の産生効率の観点より、プロモーター配列を含んでいることが好ましい。また、プロモーター配列の他に、リボゾーム結合配列や転写終結配列などを含んでいてもよい。 Examples of the control region necessary for producing the wild type and the mutant lysine decarboxylase include, for example, a promoter sequence (including an operator sequence that controls transcription), a ribosome binding sequence (SD sequence), and a transcription termination sequence. And so on.
[Expression vectors when prokaryotes are used as host cells]
When a prokaryote is used as a host cell, the expression vector is used in addition to the base sequence encoding the wild type and / or the mutant lysine decarboxylase, from the viewpoint of the production efficiency of the wild type and the mutant lysine decarboxylase. It preferably contains a promoter sequence. Further, in addition to the promoter sequence, a ribosome binding sequence, a transcription termination sequence and the like may be included.
　プロモーター配列の例としては、例えば、大腸菌由来のトリプトファンオペロンのｔｒｐプロモーターおよびラクトースオペロンのｌａｃプロモーター、ラムダファージ由来のＰＬプロモーターおよびＰＲプロモーターや、枯草菌由来のグルコン酸合成酵素プロモーター（ｇｎｔ）、アルカリプロテアーゼプロモーター（ａｐｒ）、中性プロテアーゼプロモーター（ｎｐｒ）およびα－アミラーゼプロモーター（ａｍｙ）などが挙げられる。 Examples of promoter sequences include, for example, trp promoter of tryptophan operon derived from E. coli and lac promoter of lactose operon, PL promoter and PR promoter derived from lambda phage, gluconate synthase promoter (gnt) derived from Bacillus subtilis, alkaline protease, Examples include a promoter (apr), a neutral protease promoter (npr), and an α-amylase promoter (amy).
　また、ｔａｃプロモーターのように、独自に改変または設計されたプロモーター配列も利用できる。 Also, a promoter sequence that is uniquely modified or designed, such as a tac promoter, can be used.
　リボゾーム結合配列としては、例えば、大腸菌由来または枯草菌由来の配列が挙げられるが、大腸菌や枯草菌などの所望の宿主細胞内で機能する配列であれば特に限定されるものではない。 Examples of the ribosome binding sequence include sequences derived from E. coli or Bacillus subtilis, but are not particularly limited as long as the sequence functions in a desired host cell such as Escherichia coli or Bacillus subtilis.
　上記リボゾーム結合配列としては、例えば、１６ＳリボゾームＲＮＡの３’末端領域に相補的な配列のうち、４塩基以上連続したコンセンサス配列をＤＮＡ合成により作成した配列などが挙げられる。 Examples of the ribosome-binding sequence include a sequence prepared by DNA synthesis of a consensus sequence that is continuous for 4 bases or more among sequences complementary to the 3 'end region of 16S ribosomal RNA.
　転写終結配列は、必ずしも必要ではないが、例えば、ρ因子非依存性のもの、例えば、リポプロテインターミネーター、ｔｒｐオペロンターミネーターなどが利用できる。 A transcription termination sequence is not necessarily required, but, for example, a ρ-factor-independent one such as a lipoprotein terminator, a trp operon terminator, etc. can be used.
　これら制御領域の発現ベクター上での配列順序は、特に制限されるものではないが、転写効率を考慮すると５’末端側上流からプロモーター配列、リボゾーム結合配列、目的蛋白質をコードする遺伝子、転写終結配列の順に並ぶことが望ましい。 The sequence order of these control regions on the expression vector is not particularly limited, but considering transcription efficiency, a promoter sequence, a ribosome binding sequence, a gene encoding the target protein, a transcription termination sequence from the 5 ′ end upstream. It is desirable to arrange in order.
　ここでいう発現ベクターの具体例として、例えば、大腸菌中での自律複製可能な領域を有しているｐＢＲ３２２、ｐＵＣ１８、Ｂｌｕｅｓｃｒｉｐｔ　ＩＩ　ＳＫ（＋）、ｐＫＫ２２３－３、ｐＳＣ１０１など、例えば、枯草菌中での自律複製可能な領域を有しているｐＵＢ１１０、ｐＴＺ４、ｐＣ１９４、ρ１１、φ１、φ１０５などを発現ベクターとして利用することができる。 Specific examples of expression vectors herein include, for example, pBR322, pUC18, Bluescript II SK (+), pKK223-3, pSC101, etc., which have a region capable of autonomous replication in E. coli, such as in Bacillus subtilis. PUB110, pTZ4, pC194, ρ11, φ1, φ105 and the like having a region capable of autonomous replication can be used as an expression vector.
　また、２種類以上の宿主内での自律複製が可能な発現ベクターとして、例えば、ｐＨＶ１４、ＴＲｐ７、ＹＥｐ７、ｐＢＳ７などを発現ベクターとして利用することができる。
〔形質転換体の作製方法〕
　本発明にかかる形質転換体は、公知の方法により作製することができる。例えば、本発明にかかる野生型および変異型リジン脱炭酸酵素をコードする塩基配列と、必要に応じて上記他の領域とを含む上記発現ベクターを構築し、該発現ベクターを所望の宿主細胞に形質転換する方法などが挙げられる。具体的には、例えば、Ｓａｍｂｒｏｏｋ，Ｊ．，ｅｔ．ａｌ．，“Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ　Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，　３ｒｄ　Ｅｄｉｔｉｏｎ”，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ，（２００１）などに記載されている分子生物学、生物工学および遺伝子工学の分野において公知の一般的な方法を利用することができる。 Moreover, as an expression vector capable of autonomous replication in two or more types of hosts, for example, pHV14, TRp7, YEp7, pBS7 and the like can be used as an expression vector.
[Method for producing transformant]
The transformant according to the present invention can be prepared by a known method. For example, the expression vector containing the base sequences encoding the wild type and mutant lysine decarboxylase according to the present invention and the other region as necessary is constructed, and the expression vector is transformed into a desired host cell. The method of conversion etc. are mentioned. Specifically, for example, Sambrook, J. et al. , Et. al. , “Molecular Cloning A Laboratory Manual, 3rd Edition”, Cold Spring Harbor Laboratory Press, (2001), and the like, can be used general methods known in the fields of molecular biology, biotechnology and genetic engineering. it can.
　また、相同組換えを利用した染色体への導入方法を用いることもできる。 Also, a method for introducing into a chromosome using homologous recombination can be used.
　本発明にかかる形質転換体は、上記宿主細胞に上記発現ベクターを組み込むだけではなく、必要に応じて、上記宿主細胞での使用頻度の低いコドンを、使用頻度の高いコドンにするように、サイレント変異を導入することなどを併せて行い作製することもできる。 The transformant according to the present invention not only incorporates the expression vector into the host cell, but also silently converts a low-use codon in the host cell to a high-use codon as necessary. It can also be produced by introducing mutations.
　これにより、発現ベクターに組み込んだ野生型および上記変異型リジン脱炭酸酵素由来のタンパク質の生産量を増加させることができる可能性がある。 This may increase the production amount of the protein derived from the wild type and the mutant lysine decarboxylase incorporated into the expression vector.
　サイレント変異の導入方法は、宿主細胞でのコドン使用頻度に発現ベクターのコドンを合わせるものであれば、その手法、変異点、変更する塩基の種類などは特に制限されない。
〔形質転換体の培養方法〕
　本発明の形質転換体を培養する培地は、宿主が資化し得る炭素源、窒素源、無機塩類などを含有し、形質転換体の培養を効率的に行うことができる培地であれば、天然培地、合成培地のいずれを用いてもよい。 The method for introducing a silent mutation is not particularly limited as long as it matches the codon usage in the host cell with the codon of the expression vector, the method of mutation, the type of base to be changed, and the like.
[Culture method of transformant]
The medium for culturing the transformant of the present invention contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the host, and can be a natural medium as long as the transformant can be cultured efficiently. Any of synthetic media may be used.
　炭素源としては、例えば、グルコース、ラクトース、ガラクトース、フラクトース、アラビノース、マルトース、キシロース、トレハロース、リボース、澱粉の加水分解物などの糖類、例えば、グリセロール、マンニトール、ソルビトールなどのアルコール類、例えば、グルコン酸、フマル酸、クエン酸、コハク酸などの有機酸類などが挙げられる。 Examples of the carbon source include sugars such as glucose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose, ribose, and starch hydrolysate, for example, alcohols such as glycerol, mannitol, and sorbitol, such as gluconic acid. , Organic acids such as fumaric acid, citric acid, and succinic acid.
　このような炭素源は、単独で使用してもよく、あるいは、併用することもできる。 Such carbon sources may be used alone or in combination.
　窒素源としては、例えば、硫酸アンモニウム、塩化アンモニウム、リン酸アンモニウムなどの無機アンモニウム塩、例えば、大豆加水分解物などの有機窒素、例えば、アンモニアガス、アンモニア水などが挙げられる。 Examples of the nitrogen source include inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, and organic nitrogen such as soybean hydrolysate, such as ammonia gas and aqueous ammonia.
　このような窒素源は、単独で使用してもよく、あるいは、併用することもできる。 Such nitrogen sources may be used alone or in combination.
　無機イオンとしては、例えば、ナトリウムイオン、マグネシウムイオン、カリウムイオン、カルシウムイオン、塩素イオン、マンガンイオン、鉄イオン、リン酸イオン、硫酸イオンなどが挙げられる。 Examples of inorganic ions include sodium ions, magnesium ions, potassium ions, calcium ions, chlorine ions, manganese ions, iron ions, phosphate ions, and sulfate ions.
　このような無機イオンは、単独で使用してもよく、あるいは、併用することもできる。　　　 Such inorganic ions may be used alone or in combination. *
　また、培地には、必要に応じて、その他の有機成分（有機微量栄養素）を添加することもできる。そのような有機成分としては、例えば、各種アミノ酸、例えば、ビタミンＢ１などのビタミン類、例えば、ＲＮＡなどの核酸類などの要求物質、さらには、例えば、酵母エキスなどが挙げられる。 Also, other organic components (organic micronutrients) can be added to the medium as necessary. Examples of such organic components include various amino acids, for example, vitamins such as vitamin B1, required substances such as nucleic acids such as RNA, and yeast extract and the like.
　このような培地としては、例えば、ＬＢ培地、ＹＴ培地、Ｍ９培地などが挙げられる。　　　 Examples of such a medium include LB medium, YT medium, M9 medium, and the like. *
　このような培地のなかでは、好ましくは、ＬＢ培地が挙げられる。 Among these mediums, LB medium is preferable.
　形質転換体の培養条件は、上記形質転換体、培地、培養方法の種類により適宜選択すればよく、形質転換体が生育し、本発明にかかる野生型リジン脱炭酸酵素および変異型リジン脱炭酸酵素を産生できる条件であれば特に制限はないが、例えば、大腸菌を培養する場合には、好気条件下において、培養温度が、例えば、２０～４５℃、好ましくは、２５～４０℃であり、培養ｐＨが、例えば、５．０～８．５、好ましくは、６．５～８．０であり、培養期間は半日間～７日間の範囲で、目的の変異型リジン脱炭酸酵素活性を有する蛋白質の含量が最大になるまで培養すればよい。 The culture conditions for the transformant may be appropriately selected depending on the type of the transformant, the medium, and the culture method. The transformant grows, and the wild type lysine decarboxylase and mutant lysine decarboxylase according to the present invention are used. For example, when culturing Escherichia coli, the culture temperature is, for example, 20 to 45 ° C., preferably 25 to 40 ° C. under aerobic conditions. The culture pH is, for example, 5.0 to 8.5, preferably 6.5 to 8.0, and has a desired mutated lysine decarboxylase activity within a culture period of half to 7 days. Culture may be performed until the protein content is maximized.
　形質転換体の培養期間として、好ましくは、１２～７２時間、より好ましくは、１４～４８時間である。なお、培養ｐＨの調整には、例えば、無機または有機の酸性またはアルカリ性物質や、アンモニアガスなどを用いることができる。 The culture period of the transformant is preferably 12 to 72 hours, more preferably 14 to 48 hours. The culture pH can be adjusted using, for example, an inorganic or organic acidic or alkaline substance, ammonia gas, or the like.
　培養は、例えば、上記培地を含有する液体培地中で、上記形質転換体を振とう培養、通気攪拌培養、連続培養、流加培養などの通常の培養方法を用いて行なうことが出来る。 Cultivation can be performed, for example, in a liquid medium containing the above medium by using a normal culture method such as shaking culture, aeration and agitation culture, continuous culture, or fed-batch culture.
　このようにして、形質転換体を、触媒生菌体として得ることができる。
（７）リジン脱炭酸酵素の回収工程
　リジン脱炭酸酵素の回収工程は、培養された形質転換体および該形質転換体の培養物のうち少なくともいずれか一方から、野生型リジン脱炭酸酵素および／または上記変異型リジン脱炭酸酵素を回収する工程である。 Thus, a transformant can be obtained as a catalyst viable cell.
(7) Recovery step of lysine decarboxylase The recovery step of lysine decarboxylase is performed by using wild-type lysine decarboxylase and / or from at least one of the cultured transformant and the culture of the transformant. It is a step of recovering the mutant lysine decarboxylase.
　形質転換した形質転換体を培養した後、本発明にかかる野生型および／または上記変異型リジン脱炭酸酵素を回収する方法は、この分野で慣用されている方法を使用することができる。 A method commonly used in this field can be used as a method for recovering the wild type and / or the above-mentioned mutant lysine decarboxylase according to the present invention after culturing the transformed transformant.
　本発明にかかる野生型リジン脱炭酸酵素および／または上記変異型リジン脱炭酸酵素が、形質転換した形質転換体外に分泌される場合は、該形質転換体の培養物を、例えば、遠心分離、ろ過などを行うことで、粗酵素液を容易に得ることができる。また、本発明にかかる野生型および上記変異型リジン脱炭酸酵素が、形質転換した形質転換体内に蓄積される場合は、培養した該形質転換体を遠心分離などの手段により回収し、回収した該形質転換体を水、もしくは緩衝液に懸濁し、例えば、リゾチーム処理、凍結融解、超音波破砕などの公知の方法に従い該形質転換体の細胞膜を破壊することにより、粗酵素液を回収すればよい。 When the wild-type lysine decarboxylase according to the present invention and / or the mutant lysine decarboxylase is secreted outside the transformed transformant, the culture of the transformant is, for example, centrifuged or filtered. Etc., a crude enzyme solution can be easily obtained. In addition, when the wild type and the mutant lysine decarboxylase according to the present invention are accumulated in the transformed transformant, the cultured transformant is recovered by means such as centrifugation, and the recovered The crude enzyme solution may be recovered by suspending the transformant in water or a buffer and destroying the cell membrane of the transformant according to a known method such as lysozyme treatment, freeze-thawing, or ultrasonic disruption. .
　上記粗酵素液を、例えば、限外ろ過法などにより濃縮し、例えば、防腐剤などを加えて濃縮酵素として利用することが可能である。また、濃縮した後、例えば、スプレードライ法などによって、野生型および／または上記変異型リジン脱炭酸酵素の粉末酵素を得ることもできる。 The above crude enzyme solution can be concentrated by, for example, an ultrafiltration method and used as a concentrated enzyme by adding, for example, a preservative. In addition, after concentration, a powder enzyme of the wild type and / or the above-mentioned mutant lysine decarboxylase can be obtained by, for example, spray drying.
　回収されたリジン脱炭酸酵素活性を有する粗酵素液について、分離精製を必要とする場合は、例えば、硫酸アンモニウムなどによる塩析、例えば、アルコールなどによる有機溶媒沈殿法、例えば、透析および限外ろ過などによる膜分離法、例えば、イオン交換体クロマトグラフィー、逆相高速クロマトグラフィー、アフィニティークロマトグラフィー、ゲルろ過クロマトグラフィーなどの公知のクロマト分離法を適宜組み合わせて行うことができる。 When the recovered crude enzyme solution having lysine decarboxylase activity requires separation and purification, for example, salting out with ammonium sulfate or the like, for example, organic solvent precipitation with alcohol or the like, such as dialysis and ultrafiltration, etc. Membrane separation methods by the above, for example, known chromatographic separation methods such as ion exchanger chromatography, reverse phase high performance chromatography, affinity chromatography, and gel filtration chromatography can be appropriately combined.
　以上のようにして得られた野生型および／または上記変異型リジン脱炭酸酵素は、本発明の範囲に含まれる。
（８）触媒死菌体の製造方法
　リジン脱炭酸酵素および／または変異型リジン脱炭酸酵素を発現させた細胞を、加熱により死滅させることで、触媒死菌体を得ることができる。 The wild type and / or mutant lysine decarboxylase obtained as described above are included in the scope of the present invention.
(8) Method for producing catalyst killed cell A cell that has expressed lysine decarboxylase and / or mutant lysine decarboxylase can be killed by heating to obtain a catalyst killed cell.
　具体的には、まず、上述のように得られた触媒生菌体を、例えば、水などの溶剤に懸濁して、菌体懸濁液を調製する。菌体懸濁液中の触媒生菌体の濃度は、乾燥菌体換算濃度で、通常、０．１～２０質量％、好ましくは、１～１５質量％である。 Specifically, first, the catalyst viable cells obtained as described above are suspended in a solvent such as water to prepare a cell suspension. The concentration of the live catalyst cell in the cell suspension is usually 0.1 to 20% by mass, preferably 1 to 15% by mass in terms of dry cell equivalent.
　次いで、この菌体懸濁液を、温浴などで加熱することにより死滅させ、触媒死菌体を得る。加熱の温度としては、触媒生菌体を死滅できれば構わないが、通常、４５～８０℃、好ましくは、５０～７０℃である。加熱時間は、通常、５分～１時間、好ましくは、１０～３０分間である。 Next, this cell suspension is killed by heating in a warm bath or the like to obtain catalyst killed cells. The heating temperature is not particularly limited as long as the viable bacterial cells can be killed, but is usually 45 to 80 ° C., preferably 50 to 70 ° C. The heating time is usually 5 minutes to 1 hour, preferably 10 to 30 minutes.
　（９）触媒菌体の保存方法
　本発明の触媒菌体の保存方法では、リジン脱炭酸酵素および／または変異型リジン脱炭酸酵素を発現させた細胞を、還元剤の存在下で保存する。具体的には、触媒菌体を含有する上記菌体懸濁液に還元剤を添加する。 (9) Method for Preserving Catalytic Cell In the method for preserving a catalytic cell of the present invention, cells expressing lysine decarboxylase and / or mutant lysine decarboxylase are stored in the presence of a reducing agent. Specifically, a reducing agent is added to the cell suspension containing catalyst cells.
　触媒菌体としては、触媒生菌体、触媒休止菌体または触媒死菌体のいずれであってもよい。 The catalyst cell may be any of a live catalyst cell, a resting catalyst cell, or a dead catalyst cell.
　還元剤としては、公知の還元剤が用いられるが、好ましくは、溶存酸素を除くことができ、リジン脱炭酸酵素の反応を阻害しない還元剤である。 As the reducing agent, a known reducing agent is used, but a reducing agent that can remove dissolved oxygen and does not inhibit the reaction of lysine decarboxylase is preferable.
　還元剤としては酸化還元電位が低いものが挙げられ、さらに好ましくは、生理食塩水の、２０℃における酸化還元電位（＋１６０ｍＶから＋１８０ｍＶ）より低い電位を有する還元剤が挙げられる。 Examples of the reducing agent include those having a low oxidation-reduction potential, and more preferable examples include a reducing agent having a potential lower than the oxidation-reduction potential (+160 mV to +180 mV) at 20 ° C. in physiological saline.
　なお、酸化還元電位は、酸化還元電位計により測定される。 The redox potential is measured with a redox potential meter.
　このような還元剤として、具体的には、メルカプト化合物、硫化物、水硫化物、還元性を有する硫黄の酸素酸塩、チオウレアおよびその誘導体、ヒドロキシル基および／またはカルボキシル基を有する環状化合物、フラボノイド化合物、窒素含有複素環化合物、ヒドラジル基化合物、ウロン酸基を有するムコ多糖類などが挙げられる。 Specific examples of such a reducing agent include mercapto compounds, sulfides, hydrosulfides, reducing sulfur oxyacid salts, thiourea and derivatives thereof, cyclic compounds having hydroxyl groups and / or carboxyl groups, and flavonoids. Examples thereof include compounds, nitrogen-containing heterocyclic compounds, hydrazyl group compounds, and mucopolysaccharides having uronic acid groups.
　メルカプト化合物としては、例えば、システイン、Ｎ―アセチルシステイン、２－メルカプトエタノール、ジチオエリスリトール、ジチオスレイトール（別名ディチオスレイトール）、グルタチオン、Ｓ－アセチルメルカプト無水琥珀酸などが挙げられる。 Examples of mercapto compounds include cysteine, N-acetylcysteine, 2-mercaptoethanol, dithioerythritol, dithiothreitol (also known as dithiothreitol), glutathione, and S-acetylmercaptosuccinic anhydride.
　硫化物としては、例えば、硫化ナトリウムなどが挙げられる。 Examples of the sulfide include sodium sulfide.
　水硫化物としては、例えば、水硫化ナトリウムなどが挙げられる。 Examples of hydrosulfides include sodium hydrosulfide.
　還元性を有する硫黄の酸素酸塩としては、例えば、亜硫酸、重亜硫酸、チオ硫酸、メタ重亜硫酸、亜二チオン酸などの、例えば、ナトリウム塩、カリウム塩、他の生理的に安全な塩などが挙げられる。これらの塩は、酸性亜硫酸塩（重亜硫酸塩）であってもよい。 Examples of sulfur oxyacid salts having reducibility include, for example, sodium sulfate, potassium salt, and other physiologically safe salts such as sulfurous acid, bisulfite, thiosulfuric acid, metabisulfite, dithionite, etc. Is mentioned. These salts may be acidic sulfites (bisulfites).
　チオウレアおよびその誘導体としては、例えば、チオウレア、ジメチルチオウレアなどが挙げられる。 Examples of thiourea and derivatives thereof include thiourea and dimethylthiourea.
　ヒドロキシル基および／またはカルボキシル基を有する環状化合物としては、例えば、アセチルサリチル酸、アスコルビン酸またはそのナトリウム塩などの生理的に安全な塩などが挙げられる。 Examples of the cyclic compound having a hydroxyl group and / or a carboxyl group include physiologically safe salts such as acetylsalicylic acid, ascorbic acid or a sodium salt thereof.
　フラボノイド化合物としては、例えば、素環化合物に２個以上のヒドロキシル基を有する化合物が挙げられ、具体的には、ケルセチン二水和物（Ｑｕｅｒｃｅｔｉｎ　ｄｉｈｙｄｒａｔｅ）、カテキン（Ｃａｔｅｃｈｉｎ）、エピカテキン（Ｅｐｉｃａｔｅｃｈｉｎ）、または、それらの水和物などが挙げられる。 Examples of the flavonoid compound include compounds having two or more hydroxyl groups in the ring compound. Specifically, quercetin dihydrate, catechin, epicatechin, Or the hydrate etc. are mentioned.
　窒素含有複素環化合物としては、例えば、チアゾール環、チアゾリン環、チアゾリジン環、トリアゾール環、テトラゾール環、インドール環、イミダゾール環、ピリヂン環またはピリミヂン環を有する化合物などが挙げられる。 Examples of the nitrogen-containing heterocyclic compound include compounds having a thiazole ring, a thiazoline ring, a thiazolidine ring, a triazole ring, a tetrazole ring, an indole ring, an imidazole ring, a pyridine ring, or a pyrimidin ring.
　チアゾール環を有する化合物として、具体的には、Ｎ－（２－チアゾリル）スルファニルアミド（Ｎ－（２－Ｔｈｉａｚｏｌｙｌ）ｓｕｌｆａｎｉｌａｍｉｄｅ）、Ｎ－フェナシルチアゾールブロマイドなどが挙げられる。 Specific examples of the compound having a thiazole ring include N- (2-thiazolyl) sulfanilamide (N- (2-Thiazolyl) sulfanamide), N-phenacylthiazole bromide and the like.
　チアゾリン環を有する化合物として、具体的には、２－メルカプトチアゾリンなどが挙げられる。 Specific examples of the compound having a thiazoline ring include 2-mercaptothiazoline.
　チアゾリジン環を有する化合物として、具体的には、２－オクソー４－チアゾリジンカルボン酸などが挙げられる。 Specific examples of the compound having a thiazolidine ring include 2-oxo-4-thiazolidinecarboxylic acid.
　トリアゾール環を有する化合物として、具体的には、４－（１，２，３，４－チアトリアゾ－５－リルアミノ）フェノール水和物などが挙げられる。 Specific examples of the compound having a triazole ring include 4- (1,2,3,4-thiatriazo-5-lylamino) phenol hydrate.
　インドール環を有する化合物として、具体的には、Ｎ－アセチルトリプトファンなどを挙げることができる。 Specific examples of the compound having an indole ring include N-acetyltryptophan.
　ヒドラジル基化合物としては、例えば、アミノグアニジン塩酸塩などが挙げられる。 Examples of the hydrazyl group compound include aminoguanidine hydrochloride.
　ウロン酸基を有するムコ多糖類としては、例えば、ヘパリンなどが挙げられる。 Examples of the mucopolysaccharide having a uronic acid group include heparin.
　これら還元剤のうち、好ましくは、メルカプト化合物、還元性を有する硫黄の酸素酸塩が挙げられ、さらに好ましくは、ジチオスレイトール（別名ディチオスレイトール）、亜硫酸のナトリウム塩（亜硫酸ナトリウム）、亜二チオン酸のナトリウム塩（ハイドロサルファイト）が挙げられる。 Among these reducing agents, preferably, a mercapto compound and a sulfur oxyacid salt having reducibility are mentioned, and more preferably, dithiothreitol (also called dithiothreitol), sodium sulfite (sodium sulfite), sulfite Examples include sodium salt of dithionic acid (hydrosulfite).
　さらに、このような還元剤を、後述するビタミンＢ６および／またはその誘導体（リジン脱炭酸酵素の補酵素、具体的には、ピリドキサールリン酸など）と共存させることで、高い触媒安定化効果を発揮することができる。 In addition, when such a reducing agent coexists with vitamin B6 and / or its derivatives (lysine decarboxylase coenzyme, specifically pyridoxal phosphate, etc.) described later, a high catalyst stabilizing effect is exhibited. can do.
　これら還元剤は、単独使用または２種類以上併用することができる。 These reducing agents can be used alone or in combination of two or more.
　還元剤の濃度は、リジン反応液中の溶存酸素を十分に除去できる濃度であって、リジン脱炭素酵素を失活させない濃度であれば、特に限定されないが、菌体懸濁液に対して、通常、０．１～１０ｇ／Ｌ、好ましくは、０．５～５ｇ／Ｌである。還元剤の濃度が上記した下限に満たないと、リジン脱炭酸酵素および／または変異型リジン脱炭酸酵素を長期間安定に保存できない場合がある。 The concentration of the reducing agent is not particularly limited as long as it is a concentration that can sufficiently remove dissolved oxygen in the lysine reaction solution and does not deactivate lysine decarbonase. Usually, it is 0.1 to 10 g / L, preferably 0.5 to 5 g / L. If the concentration of the reducing agent is less than the lower limit, lysine decarboxylase and / or mutant lysine decarboxylase may not be stored stably for a long period of time.
　このような触媒菌体は、例えば、凍結や冷蔵で長期（例えば、８０日以上、好ましくは、３０日以上）に保存することができる。 Such a catalyst cell can be stored for a long period (for example, 80 days or more, preferably 30 days or more) by freezing or refrigeration, for example.
　菌体懸濁液の保存の温度は、例えば、２０℃以下、好ましくは、４℃以下である。
（１０）１，５－ペンタメチレンジアミンの製造方法
　本発明の１，５－ペンタメチレンジアミンの製造方法では、溶存酸素濃度が飽和溶存酸素濃度である時間が１時間以内の反応系内において、Ｌ－リジンおよび／またはその塩を、リジン脱炭酸酵素および／または変異型リジン脱炭酸酵素によって、リジン脱炭酸反応させる。 The storage temperature of the bacterial cell suspension is, for example, 20 ° C. or lower, preferably 4 ° C. or lower.
(10) Method for Producing 1,5-Pentamethylenediamine In the method for producing 1,5-pentamethylenediamine of the present invention, in the reaction system in which the dissolved oxygen concentration is the saturated dissolved oxygen concentration within 1 hour, L -Lysine and / or its salt is subjected to lysine decarboxylation with lysine decarboxylase and / or mutant lysine decarboxylase.
　より具体的には、例えば、反応系内の溶存酸素濃度が飽和溶存酸素濃度である時間を１時間以内にし、リジンに上述の野生型および／または変異型リジン脱炭酸酵素を接触させることにより、１，５－ペンタメチレンジアミンを製造する。 More specifically, for example, the time during which the dissolved oxygen concentration in the reaction system is the saturated dissolved oxygen concentration is set within 1 hour, and the above-mentioned wild type and / or mutant lysine decarboxylase is contacted with lysine, 1,5-pentamethylenediamine is produced.
　すなわち、リジンと、野生型および／または変異型リジン脱炭酸酵素、野生型および／または変異型リジン脱炭酸酵素を発現する形質転換体（例えば、触媒生菌体）、形質転換体の処理物（例えば、触媒死菌体）、形質転換体およびその処理物の固定化物からなる群から選択された少なくとも１種と、反応溶媒とを混合して反応液を調製し、反応液中で、野生型および／または変異型リジン脱炭酸酵素と、リジンとを接触させて、リジンの脱炭酸酵素反応により、１，５－ペンタメチレンジアミンを製造する。 That is, lysine, a transformant that expresses wild-type and / or mutant lysine decarboxylase, wild-type and / or mutant lysine decarboxylase (for example, catalytic viable cell), processed product of transformant ( For example, a reaction solution is prepared by mixing at least one selected from the group consisting of a dead catalyst cell), a transformant and an immobilized product thereof, and a reaction solvent. And / or mutant lysine decarboxylase is brought into contact with lysine to produce 1,5-pentamethylenediamine by decarboxylase reaction of lysine.
　なお、このリジン脱炭酸反応では、野生型および／または変異型リジン脱炭酸酵素とリジンとが最初に接触した時点を、反応開始点とする。また、野生型および／または変異型リジン脱炭酸酵素とリジンとの接触が断たれた時点、または、１，５－ペンタメチレンジアミンの生成量が飽和した時点を、反応終了点とする。 In this lysine decarboxylation reaction, the time when the wild-type and / or mutant lysine decarboxylase and lysine first contact each other is defined as the reaction start point. Further, the point of time when the contact between the wild-type and / or mutant lysine decarboxylase and lysine is cut off, or when the amount of 1,5-pentamethylenediamine produced is saturated, is regarded as the reaction end point.
　そして、このリジン脱炭酸反応では、反応開始点から反応終了点までの間において、溶存酸素濃度が飽和溶存酸素濃度である時間が１時間以内に制限される。 In this lysine decarboxylation reaction, the time during which the dissolved oxygen concentration is the saturated dissolved oxygen concentration is limited to within one hour from the reaction start point to the reaction end point.
　このような１，５－ペンタメチレンジアミンの製造方法は、反応系内の溶存酸素濃度が飽和溶存酸素濃度である時間を１時間以内にするため、例えば、反応系内の酸素を除去する工程、反応系内に還元剤を添加する工程などを含むことができる。 In such a method for producing 1,5-pentamethylenediamine, the time during which the dissolved oxygen concentration in the reaction system is the saturated dissolved oxygen concentration is set within one hour, for example, a step of removing oxygen in the reaction system, A step of adding a reducing agent into the reaction system can be included.
　反応系内の酸素を除去する工程では、反応系内、すなわち、反応液に溶解している酸素を除くことができる公知の方法が実施される。反応液中の酸素を除去する工程として、具体的には、例えば、不活性ガスにより溶存酸素を置換する工程などが挙げられる。 In the step of removing oxygen in the reaction system, a known method is performed that can remove oxygen dissolved in the reaction system, that is, in the reaction solution. Specific examples of the step of removing oxygen in the reaction solution include a step of replacing dissolved oxygen with an inert gas.
　この工程では、具体的には、反応液に不活性ガスを通気して、溶解している酸素と不活性ガスとを交換する。 In this step, specifically, an inert gas is passed through the reaction solution to exchange dissolved oxygen and inert gas.
　不活性ガスとしては、例えば、窒素、アルゴン、ヘリウムなどを用いることができる。反応開始前に溶存酸素を置換するとともに、反応中も酸素が溶け込まないように外部から酸素が入り込まないようにすることが好ましい。 As the inert gas, for example, nitrogen, argon, helium or the like can be used. It is preferable to replace the dissolved oxygen before starting the reaction and to prevent oxygen from entering from the outside so that oxygen does not dissolve during the reaction.
　不活性ガスの通気量は、反応液１００質量部に対して、例えば、０．０５～１０Ｌ／ｈｒ、好ましくは、０．１～５Ｌ／ｈｒである。また、不活性ガスの通気時間は、例えば、０．５～５時間、好ましくは、０．１～１時間である。 The aeration amount of the inert gas is, for example, 0.05 to 10 L / hr, preferably 0.1 to 5 L / hr with respect to 100 parts by mass of the reaction solution. The inert gas ventilation time is, for example, 0.5 to 5 hours, preferably 0.1 to 1 hour.
　また、通気法としては、特に限定されず、バブリングさせることもできる。 Further, the aeration method is not particularly limited and can be bubbled.
　不活性ガスに置換した後、不活性ガスの通気を止めることで、反応液中の炭酸ガスの放出を抑制でき、反応液のｐＨ上昇を抑制できるためより好ましい。 After substituting with an inert gas, it is more preferable to stop the aeration of the inert gas, so that the release of carbon dioxide gas in the reaction solution can be suppressed and the increase in pH of the reaction solution can be suppressed.
　また、この方法では、反応開始前の反応液、反応開始後の反応液、および、その両方に不活性ガスを通じることができるが、好ましくは、反応開始前の反応液（例えば、触媒菌体が添加される前のリジン溶液）に不活性ガスを通じる。 In this method, an inert gas can be passed through the reaction solution before the start of the reaction, the reaction solution after the start of the reaction, and both. Preferably, the reaction solution before the start of the reaction (for example, the catalyst cell body). An inert gas is passed through the lysine solution before the is added.
　このような場合、反応開始前における溶存酸素濃度は、例えば、飽和溶存酸素濃度の９０％以下、好ましくは、７０％以下、より好ましくは、６５％以下、さらに好ましくは、５０％以下である。 In such a case, the dissolved oxygen concentration before the start of the reaction is, for example, 90% or less of the saturated dissolved oxygen concentration, preferably 70% or less, more preferably 65% or less, and still more preferably 50% or less.
　一方、反応系内に還元剤を添加する工程では、反応系内、すなわち、反応液に還元剤を添加する。 On the other hand, in the step of adding a reducing agent to the reaction system, the reducing agent is added to the reaction system, that is, to the reaction solution.
　還元剤としては、公知の還元剤が用いられるが、溶存酸素濃度を低減できる還元剤であり、リジン脱炭酸酵素を阻害しない還元剤が用いられる。好ましくは酸化還元電位が低いものが挙げられ、さらに好ましくは、生理食塩水の、２０℃における酸化還元電位（＋１６０ｍＶから＋１８０ｍＶ）より低い電位を有する還元剤が挙げられる。 As the reducing agent, a known reducing agent is used, but a reducing agent that can reduce the dissolved oxygen concentration and does not inhibit lysine decarboxylase is used. Preferred are those having a low redox potential, and more preferred are reducing agents having a lower potential than the redox potential (+160 mV to +180 mV) at 20 ° C. of physiological saline.
　このような還元剤として、具体的には、上記したメルカプト化合物、上記した硫化物、上記した水硫化物、上記した還元性を有する硫黄の酸素酸塩、上記したチオウレアおよびその誘導体、上記したヒドロキシル基および／またはカルボキシル基を有する環状化合物、上記したフラボノイド化合物、上記した窒素含有複素環化合物、上記したヒドラジル基化合物、上記したウロン酸基を有するムコ多糖類などが挙げられる。 Specific examples of such a reducing agent include the above-described mercapto compounds, the above-described sulfides, the above-described hydrosulfides, the above-described sulfur oxyacid salts having reducibility, the above-described thiourea and derivatives thereof, and the above-described hydroxyl groups. And a cyclic compound having a group and / or a carboxyl group, the above flavonoid compound, the above nitrogen-containing heterocyclic compound, the above hydrazyl group compound, and the above mucopolysaccharide having a uronic acid group.
　これら還元剤のうち、好ましくは、還元性を有する硫黄の酸素酸塩、さらに好ましくは、亜硫酸のナトリウム塩（亜硫酸ナトリウム）が挙げられる。 Among these reducing agents, sulfur oxyacid salt having reducibility is preferable, and sodium sulfite (sodium sulfite) is more preferable.
　これら還元剤は、単独使用または２種類以上併用することができる。 These reducing agents can be used alone or in combination of two or more.
　還元剤の反応液中の濃度は、リジン脱炭酸酵素を失活させない濃度であれば構わないが、通常、０．１～１０ｇ／Ｌ、好ましくは、０．１～４ｇ／Ｌ程度である。 The concentration of the reducing agent in the reaction solution may be any concentration that does not deactivate lysine decarboxylase, but is usually about 0.1 to 10 g / L, preferably about 0.1 to 4 g / L.
　また、この方法では、反応開始前の反応液、反応開始後の反応液、および、その両方に還元剤を添加することができるが、好ましくは、反応開始前の反応液（例えば、触媒菌体が添加される前のリジン溶液）に還元剤を添加する。 In this method, a reducing agent can be added to the reaction solution before the start of the reaction, the reaction solution after the start of the reaction, and both. Preferably, the reaction solution before the start of the reaction (for example, catalyst cells) The reducing agent is added to the lysine solution before the is added.
　このような場合、反応開始前における溶存酸素濃度は、例えば、飽和溶存酸素濃度の９０％以下、好ましくは、７０％以下、より好ましくは、６５％以下、さらに好ましくは、５０％以下である。 In such a case, the dissolved oxygen concentration before the start of the reaction is, for example, 90% or less of the saturated dissolved oxygen concentration, preferably 70% or less, more preferably 65% or less, and still more preferably 50% or less.
　反応系内の溶存酸素濃度が、飽和溶存酸素濃度に対して上記した上限を超えると、十分な収率が得られない場合がある。 If the dissolved oxygen concentration in the reaction system exceeds the upper limit described above with respect to the saturated dissolved oxygen concentration, a sufficient yield may not be obtained.
　なお、反応系内の飽和溶存酸素濃度は、反応系内の溶存酸素が、空気中の酸素で飽和している状態の溶存酸素であり、下記のようにして測定することができる。 The saturated dissolved oxygen concentration in the reaction system is dissolved oxygen in a state where the dissolved oxygen in the reaction system is saturated with oxygen in the air, and can be measured as follows.
　予め亜硫酸ナトリウム溶液に硫酸銅６水和物を添加し溶存酸素濃度をゼロにした溶液に、発酵用酸素電極（ＣＳＬ－１　エイブル社製）を浸漬して、溶存酸素インジケーター（ＭＯＤＥＬ　Ｍ－１０３２　エイブル社製）の指示がゼロになるように調整する。次いで、反応液に空気を通気し、溶存酸素インジケーターの値が安定したところで、飽和溶存酸素濃度とする。 An oxygen electrode for fermentation (manufactured by CSL-1 Able) is immersed in a solution in which copper sulfate hexahydrate is added to a sodium sulfite solution in advance to make the dissolved oxygen concentration zero, and a dissolved oxygen indicator (MODEL M-1032 Able) Make adjustments so that the indication of “manufactured” is zero. Next, air is passed through the reaction solution, and when the value of the dissolved oxygen indicator is stabilized, the saturated dissolved oxygen concentration is obtained.
　また、反応系内の飽和溶存酸素濃度に対する溶存酸素濃度の割合（％）は、上述のように調整した溶存酸素インジケーターにより測定することができる。 Also, the ratio (%) of the dissolved oxygen concentration to the saturated dissolved oxygen concentration in the reaction system can be measured by the dissolved oxygen indicator adjusted as described above.
　また、この方法では、リジン脱炭酸反応の進行とともに、反応系内の酸素が低減される場合がある。 In this method, oxygen in the reaction system may be reduced as the lysine decarboxylation reaction proceeds.
　具体的には、例えば、リジンの脱炭酸反応により生じた炭酸ガスが、反応系内の酸素を追い出す場合、さらには、反応開始後に不活性ガスを通じる場合や、還元剤を添加する場合などには、反応の進行とともに、反応系内の酸素が低減される。 Specifically, for example, when carbon dioxide generated by the decarboxylation reaction of lysine drives off oxygen in the reaction system, and further when passing through an inert gas after starting the reaction, or when adding a reducing agent, etc. As the reaction proceeds, oxygen in the reaction system is reduced.
　このようなリジン脱炭酸反応において、好ましくは、反応液中の溶存酸素濃度が、反応開始点から反応終了点までを通じて、飽和溶存酸素濃度の６５％以下であり、かつ、溶存酸素濃度が飽和溶存酸素濃度の６５％以下である点（すなわち、反応開始点）から２０分以内に、飽和溶存酸素濃度の１％以下になることが好ましい。 In such a lysine decarboxylation reaction, the dissolved oxygen concentration in the reaction solution is preferably 65% or less of the saturated dissolved oxygen concentration from the reaction start point to the reaction end point, and the dissolved oxygen concentration is saturated dissolved. It is preferable that the saturated dissolved oxygen concentration is 1% or less within 20 minutes from the point where the oxygen concentration is 65% or less (that is, the reaction start point).
　すなわち、このリジン脱炭酸反応において、好ましくは、溶存酸素濃度が、反応開始時において、飽和溶存酸素濃度の６５％以下に低減されており、また、反応中に溶存酸素濃度が増減する場合には、その溶存酸素濃度が飽和溶存酸素濃度の６５％を超過することがない範囲で増減し、また、反応の進行に伴って飽和溶存酸素濃度の１％以下にまで減少し、その所要時間が、２０分以内である。 That is, in this lysine decarboxylation reaction, the dissolved oxygen concentration is preferably reduced to 65% or less of the saturated dissolved oxygen concentration at the start of the reaction, and the dissolved oxygen concentration increases or decreases during the reaction. , The dissolved oxygen concentration increases or decreases in a range not exceeding 65% of the saturated dissolved oxygen concentration, and decreases to 1% or less of the saturated dissolved oxygen concentration as the reaction proceeds, and the required time is Within 20 minutes.
　このような条件で反応させることにより、１，５－ペンタメチレンジアミンの収率の向上を図ることができる。 By reacting under such conditions, the yield of 1,5-pentamethylenediamine can be improved.
　また、このように、リジン脱炭酸反応の反応中に溶存酸素濃度が増減する場合、そのリジン脱炭酸反応における溶存酸素濃度と経過時間との関係を相関線として表すことができる。例えば、反応中に溶存酸素濃度が減少する場合には、溶存酸素濃度と経過時間との関係は、例えば、Ｙ軸を飽和溶存酸素濃度に対する溶存酸素濃度の割合（％）、Ｘ軸を時間（分）として、例えば、一次関数的な相関線として示される（図１参照。）。 In this way, when the dissolved oxygen concentration increases or decreases during the lysine decarboxylation reaction, the relationship between the dissolved oxygen concentration and the elapsed time in the lysine decarboxylation reaction can be represented as a correlation line. For example, when the dissolved oxygen concentration decreases during the reaction, the relationship between the dissolved oxygen concentration and the elapsed time is, for example, the ratio of the dissolved oxygen concentration to the saturated dissolved oxygen concentration (%) on the Y axis and the time ( For example, it is shown as a linear function-like correlation line (see FIG. 1).
　そして、Ｙ軸を飽和溶存酸素濃度に対する溶存酸素濃度の割合（％）、Ｘ軸を時間（分）として、リジン脱炭酸反応における溶存酸素濃度と時間との関係がプロットされた相関線を示す相関図において、相関線とＹ軸とＸ軸とに囲まれる部分の面積が、好ましくは、１０００未満、より好ましくは、６５０以下である。なお、面積単位は、例えば、（％・ｍｉｎ）である。 Then, the correlation indicating the correlation line in which the relationship between the dissolved oxygen concentration and time in the lysine decarboxylation reaction is plotted with the Y axis as the ratio (%) of the dissolved oxygen concentration to the saturated dissolved oxygen concentration and the X axis as the time (min). In the figure, the area of the portion surrounded by the correlation line, the Y axis and the X axis is preferably less than 1000, more preferably 650 or less. The area unit is, for example, (% · min).
　このような条件で反応させることにより、１，５－ペンタメチレンジアミンの収率の向上を図ることができる。 By reacting under such conditions, the yield of 1,5-pentamethylenediamine can be improved.
　なお、図１には、模式概念図として溶存酸素濃度と経過時間との相関線を一次関数的に示しているが、実際の相関線は反応系によって異なり、種々の曲線状とされる。 In FIG. 1, the correlation line between the dissolved oxygen concentration and the elapsed time is shown as a linear function as a schematic conceptual diagram, but the actual correlation line varies depending on the reaction system, and has various curves.
　例えば、図２に示すように、相関線が不規則的な曲線状であってもよく、また、相関線とＹ軸とＸ軸とに囲まれる部分が、不連続であってもよい。そのような場合、相関線とＹ軸とＸ軸とに囲まれる部分の面積は、各部分の面積の総面積として算出される。 For example, as shown in FIG. 2, the correlation line may be an irregular curve, and the portion surrounded by the correlation line, the Y axis, and the X axis may be discontinuous. In such a case, the area of the portion surrounded by the correlation line, the Y axis, and the X axis is calculated as the total area of the respective portions.
　本発明で原料として用いられるリジンは、その塩であってもよい。リジンの塩としては、例えば、塩酸塩、酢酸塩、炭酸塩、炭酸水素塩、硫酸塩、硝酸塩などが挙げられる。 The lysine used as a raw material in the present invention may be a salt thereof. Examples of lysine salts include hydrochloride, acetate, carbonate, bicarbonate, sulfate, nitrate, and the like.
　ただし、炭酸塩および炭酸水素塩は、その製造工程において二酸化炭素を多量に使用し、温暖化ガスの発生が多いので、環境保護の観点から、好ましくは、塩酸塩、酢酸塩、硫酸塩、硝酸塩などが挙げられる。 However, since carbonates and bicarbonates use a large amount of carbon dioxide in the production process and generate a lot of greenhouse gases, from the viewpoint of environmental protection, preferably, hydrochloride, acetate, sulfate, nitrate Etc.
　このようなリジンの塩の中で、好ましくは、リジンの塩酸塩が挙げられる。 Among such lysine salts, lysine hydrochloride is preferable.
　さらに、リジンとして、リジンを精製する工程で、還元剤を添加して精製したリジンまたはその塩を用いることもできる。 Furthermore, lysine or a salt thereof purified by adding a reducing agent in the step of purifying lysine can also be used as lysine.
　反応溶媒中におけるリジンの濃度は、特に制限されないが、例えば、１～７０質量％、好ましくは、１０～７０質量％、より好ましくは、２０～５０質量％である。 The concentration of lysine in the reaction solvent is not particularly limited, but is, for example, 1 to 70% by mass, preferably 10 to 70% by mass, and more preferably 20 to 50% by mass.
　本発明における野生型および変異型リジン脱炭酸酵素の必要量は、野生型および／または変異型リジン脱炭酸酵素、野生型および／または変異型リジン脱炭酸酵素を発現する形質転換体（例えば、触媒生菌体）、形質転換体の処理物（例えば、触媒死菌体）、形質転換体およびその処理物の固定化物からなる群から選択された少なくとも１種を用いる場合、Ｌ－リジンおよび／またはその塩１質量部に対して、リジン脱炭酸酵素および／または変異型リジン脱炭酸酵素が、乾燥菌体重量換算で、０．０００３質量部以上０．００１５質量部以下である。 The required amount of wild-type and mutant lysine decarboxylase in the present invention is a transformant (eg, catalyst) that expresses wild-type and / or mutant lysine decarboxylase, wild-type and / or mutant lysine decarboxylase. In the case of using at least one selected from the group consisting of a living cell), a processed product of the transformant (for example, dead catalyst cell), an immobilized product of the processed product, and L-lysine and / or With respect to 1 part by mass of the salt, lysine decarboxylase and / or mutant lysine decarboxylase is 0.0003 parts by mass or more and 0.0015 parts by mass or less in terms of dry cell weight.
　野生型および変異型リジン脱炭酸酵素の使用量が上記範囲であれば、１，５－ペンタメチレンジアミンを効率よく製造することができる。 If the amount of wild-type and mutant lysine decarboxylase used is in the above range, 1,5-pentamethylenediamine can be produced efficiently.
　反応溶媒としては、例えば、水、水性媒体、有機溶媒、水もしくは水性媒体と有機溶媒との混合液が挙げられる。 Examples of the reaction solvent include water, an aqueous medium, an organic solvent, water, or a mixed liquid of an aqueous medium and an organic solvent.
　水性媒体としては、例えば、リン酸緩衝液などの緩衝液などが挙げられる。 Examples of the aqueous medium include a buffer solution such as a phosphate buffer solution.
　有機溶媒としては、反応を阻害しないものであればいずれでもよい。 Any organic solvent may be used as long as it does not inhibit the reaction.
　リジンの脱炭酸酵素反応の条件としては、温度が、例えば、２８～５５℃、好ましくは、３５～４５℃、時間が、例えば、０．１～７２時間、好ましくは、１～７２時間、さらに好ましくは、１２～３６時間である。また、反応ｐＨが、例えば、５．０～９．０、好ましくは、５．５～８．５である。 As conditions for lysine decarboxylase reaction, the temperature is, for example, 28 to 55 ° C., preferably 35 to 45 ° C., the time is, for example, 0.1 to 72 hours, preferably 1 to 72 hours, Preferably, it is 12 to 36 hours. The reaction pH is, for example, 5.0 to 9.0, preferably 5.5 to 8.5.
　反応は、振盪、攪拌または静置条件下で行なうことができる。 The reaction can be carried out under shaking, stirring or standing conditions.
　これにより、リジンが脱炭酸され、１，５－ペンタメチレンジアミンへと変換し、１，５－ペンタメチレンジアミンが製造される。 Thereby, lysine is decarboxylated and converted into 1,5-pentamethylenediamine, and 1,5-pentamethylenediamine is produced.
　本発明で製造される１，５－ペンタメチレンジアミンは、その塩であってもよい。 The 1,5-pentamethylenediamine produced in the present invention may be a salt thereof.
　１，５－ペンタメチレンジアミンの塩としては、例えば、１，５－ペンタメチレンジアミンの塩酸塩、酢酸塩、炭酸塩、炭酸水素塩、硫酸塩、硝酸塩などが挙げられる。 Examples of the salt of 1,5-pentamethylenediamine include hydrochloride, acetate, carbonate, bicarbonate, sulfate, nitrate and the like of 1,5-pentamethylenediamine.
　このような１，５－ペンタメチレンジアミンの塩のなかでは、好ましくは、塩酸塩が挙げられる。 Among such 1,5-pentamethylenediamine salts, hydrochloride is preferable.
　なお、この反応では、得られる１，５－ペンタメチレンジアミンがアルカリ性であるため、リジンが１，５－ペンタメチレンジアミンに変換されるに伴って、反応液のｐＨが増加する場合がある。このような場合には、必要により、酸性物質（例えば、有機酸、例えば、塩酸などの無機酸など）などを添加し、ｐＨを調整することができる。 In this reaction, since the obtained 1,5-pentamethylenediamine is alkaline, the pH of the reaction solution may increase as lysine is converted to 1,5-pentamethylenediamine. In such a case, if necessary, an acidic substance (for example, an organic acid, for example, an inorganic acid such as hydrochloric acid) can be added to adjust the pH.
　また、この反応では、必要により、例えば、ビタミンＢ６および／またはその誘導体を反応液中に添加することもできる。 In this reaction, for example, vitamin B6 and / or a derivative thereof can be added to the reaction solution as necessary.
　ビタミンＢ６および／またはその誘導体としては、例えば、ピリドキシン、ピリドキサミン、ピリドキサール、ピリドキサールリン酸などが挙げられる。 Examples of vitamin B6 and / or derivatives thereof include pyridoxine, pyridoxamine, pyridoxal, pyridoxal phosphate, and the like.
　このようなビタミンＢ６および／またはその誘導体は、単独で使用してもよく、あるいは、併用することもできる。 Such vitamin B6 and / or its derivatives may be used alone or in combination.
　ビタミンＢ６および／またはその誘導体のなかでは、好ましくは、ピリドキサールリン酸が挙げられる。 Among vitamin B6 and / or its derivatives, pyridoxal phosphate is preferable.
　ビタミンＢ６および／またはその誘導体を添加することにより、１，５－ペンタメチレンジアミンの生産速度および反応収率を向上することができる。 By adding vitamin B6 and / or derivatives thereof, the production rate and reaction yield of 1,5-pentamethylenediamine can be improved.
　また、この方法では、得られたペンタメチレンジアミン水溶液から、必要により、水の一部を留去させることができる。 Further, in this method, a part of water can be distilled off from the obtained pentamethylenediamine aqueous solution as necessary.
　より具体的には、例えば、連続多段蒸留塔、回分多段蒸留塔などを備えた蒸留装置などにより、例えば、０．１ｋＰａ～常圧下、ペンタメチレンジアミン水溶液を加熱（熱処理）し、蒸留することにより、水の一部が留去されたペンタメチレンジアミン水溶液を得ることができる。 More specifically, for example, by heating (heat treatment) an aqueous pentamethylenediamine solution at a pressure of 0.1 kPa to normal pressure using a distillation apparatus equipped with a continuous multistage distillation column, a batch multistage distillation column, etc. A pentamethylenediamine aqueous solution in which a part of water is distilled off can be obtained.
　加熱温度としては、例えば、２５℃以上、９０℃未満、好ましくは、２５℃以上、８５℃以下、さらに好ましくは、２５℃以上、８０℃未満、とりわけ好ましくは、３０℃以上、７０℃以下である。 The heating temperature is, for example, 25 ° C. or more and less than 90 ° C., preferably 25 ° C. or more and 85 ° C. or less, more preferably 25 ° C. or more and less than 80 ° C., particularly preferably 30 ° C. or more and 70 ° C. or less. is there.
　ペンタメチレンジアミン水溶液を、９０℃以上で加熱（熱処理）すると、ペンタメチレンジアミン（またはその塩）の抽出率が低下する場合がある。 When the pentamethylenediamine aqueous solution is heated (heat treated) at 90 ° C. or higher, the extraction rate of pentamethylenediamine (or a salt thereof) may decrease.
　本発明の１，５－ペンタメチレンジアミンの製造方法によれば、反応系内の溶存酸素濃度が飽和溶存酸素濃度である時間が１時間以内なので、リジン脱炭酸酵素および／または変異型リジン脱炭酸酵素の活性の低下を低減することができる。そのため、酵素を精製しなくとも、優れた反応効率でリジン脱炭素反応をさせることができ、さらに、反応液のｐＨを調製することなく、反応を完結させることができる。 According to the method for producing 1,5-pentamethylenediamine of the present invention, since the dissolved oxygen concentration in the reaction system is a saturated dissolved oxygen concentration within 1 hour, lysine decarboxylase and / or mutant lysine decarboxylation A decrease in the activity of the enzyme can be reduced. Therefore, lysine decarbonization can be performed with excellent reaction efficiency without purifying the enzyme, and the reaction can be completed without adjusting the pH of the reaction solution.
　つまり、この１，５－ペンタメチレンジアミンの製造方法によれば、低コストで収率よく、さらに、反応液のｐＨ調整を行わずに１，５－ペンタメチレンジアミンを製造することができる。
（１１）１，５－ペンタメチレンジアミンの精製
　そして、この方法では、好ましくは、上記により得られたペンタメチレンジアミン水溶液から、ペンタメチレンジアミン（またはその塩）を抽出する。抽出では、例えば、液－液抽出法が採用される。 That is, according to this method for producing 1,5-pentamethylenediamine, it is possible to produce 1,5-pentamethylenediamine at a low cost with a good yield and without adjusting the pH of the reaction solution.
(11) Purification of 1,5-pentamethylenediamine In this method, preferably, pentamethylenediamine (or a salt thereof) is extracted from the aqueous solution of pentamethylenediamine obtained as described above. In the extraction, for example, a liquid-liquid extraction method is employed.
　液－液抽出法では、例えば、（１）回分的、半連続的または連続的にペンタメチレンジアミン水溶液に、抽出溶媒（後述）を接触させ、混合および撹拌することにより、ペンタメチレンジアミン（またはその塩）を抽出溶媒（後述）へと抽出（分配）し、その抽出溶媒（後述）からペンタメチレンジアミン（またはその塩）を分離する方法、（２）多孔板を備えた塔（例えば、スプレー塔、段型抽出塔など）や、充填物、ノズル、オリフィス板、バッフル、インジェクターおよび／またはスタティックミキサーを備えた塔（向流微分型抽出塔、非撹拌式段型抽出塔：改訂五版　化学工学便覧、ｐ５６６から５６９、化学工学会編、丸善（１９８８））に、ペンタメチレンジアミン水溶液と抽出溶媒（後述）とを、向流で連続的に供給し、ペンタメチレンジアミン（またはその塩）を、抽出溶媒（後述）へと抽出（分配）した後、抽出溶媒（後述）を連続的に流出させ、その抽出溶媒（後述）から、ペンタメチレンジアミン（またはその塩）を分離する方法、（３）邪魔板および撹拌羽根を備えた塔（撹拌式段型抽出塔：改訂五版　化学工学便覧　ｐ５６９から５７４、化学工学会編、丸善（１９８８））に、ペンタメチレンジアミン水溶液と抽出溶媒（後述）とを、向流で連続的に供給し、ペンタメチレンジアミン（またはその塩）を、抽出溶媒（後述）へと抽出（分配）した後、抽出溶媒（後述）を連続的に流出させ、その抽出溶媒（後述）から、ペンタメチレンジアミン（またはその塩）を分離する方法、（４）ミキサーセトラー抽出器、または、遠心式抽出機（改訂五版　化学工学便覧　ｐ５６３から５６６、ｐ５７４、化学工学会編、丸善（１９８８））を用いて、ペンタメチレンジアミン水溶液に、抽出溶媒（後述）を接触させ、ペンタメチレンジアミン（またはその塩）を抽出溶媒（後述）へと抽出（分配）し、その抽出溶媒（後述）からペンタメチレンジアミン（またはその塩）を分離する方法などが採用される。 In the liquid-liquid extraction method, for example, (1) an extraction solvent (described later) is brought into contact with an aqueous solution of pentamethylenediamine batchwise, semi-continuously or continuously, and mixed and stirred, so that pentamethylenediamine (or a solution thereof) is obtained. Salt) is extracted (distributed) into an extraction solvent (described later), and pentamethylenediamine (or a salt thereof) is separated from the extraction solvent (described later); (2) a tower (for example, a spray tower) provided with a perforated plate , Stage type extraction towers, etc.) and columns equipped with packings, nozzles, orifice plates, baffles, injectors and / or static mixers (countercurrent differential type extraction towers, non-stirring type stage extraction towers: revised fifth edition, chemical engineering Handbook, pages 566 to 569, edited by Chemical Engineering Society, Maruzen (1988)), an aqueous solution of pentamethylenediamine and an extraction solvent (described later) are continuously supplied in a countercurrent flow. After extracting (distributing) tamethylenediamine (or a salt thereof) into an extraction solvent (described later), the extraction solvent (described later) is continuously flowed out, and pentamethylenediamine (or its (3) A tower equipped with baffle plates and stirring blades (stirring type extraction tower: revised fifth edition, Chemical Engineering Handbook, p569-574, edited by Chemical Engineering Society, Maruzen (1988)) A methylenediamine aqueous solution and an extraction solvent (described later) are continuously supplied in countercurrent, and pentamethylenediamine (or a salt thereof) is extracted (distributed) into the extraction solvent (described later), and then the extraction solvent (described later). , And a method of separating pentamethylenediamine (or a salt thereof) from the extraction solvent (described later), (4) mixer settler extractor or centrifugal extractor (revised fifth edition) Academic Engineering Handbook, p563 to 566, p574, edited by Chemical Engineering Society, Maruzen (1988)), an extraction solvent (described later) is brought into contact with an aqueous solution of pentamethylenediamine, and pentamethylenediamine (or a salt thereof) is extracted into the extraction solvent For example, a method of extracting (partitioning) into (described later) and separating pentamethylenediamine (or a salt thereof) from the extraction solvent (described later) is employed.
　これら液－液抽出法としては、単独使用または２種類以上併用することができる。 These liquid-liquid extraction methods can be used alone or in combination of two or more.
　液－液抽出法として、生産効率の観点から、好ましくは、ペンタメチレンジアミン（またはその塩）を、抽出溶媒（後述）へと連続的に抽出（分配）する方法、より具体的には、例えば、上記（１）～（３）の方法が挙げられる。 As the liquid-liquid extraction method, from the viewpoint of production efficiency, a method of continuously extracting (distributing) pentamethylenediamine (or a salt thereof) into an extraction solvent (described later), more specifically, for example, And the above methods (1) to (3).
　液－液抽出におけるペンタメチレンジアミン水溶液と抽出溶媒（後述）との配合割合は、ペンタメチレンジアミン水溶液（抽出が連続的である場合は、単位時間あたりの供給量。以下同様。）１００質量部に対して、抽出溶媒（後述）が、例えば、３０～３００質量部であり、経済性および生産性の観点から、好ましくは、５０～２００質量部、さらに好ましくは、５０～１５０質量部、とりわけ好ましくは、８０～１２０質量部である。 The blending ratio of the pentamethylenediamine aqueous solution and the extraction solvent (described later) in the liquid-liquid extraction is 100 parts by mass of the pentamethylenediamine aqueous solution (if the extraction is continuous, the supply amount per unit time. The same applies hereinafter). On the other hand, the extraction solvent (described later) is, for example, 30 to 300 parts by mass, and is preferably 50 to 200 parts by mass, more preferably 50 to 150 parts by mass, particularly preferably from the viewpoint of economy and productivity. Is 80 to 120 parts by mass.
　また、ペンタメチレンジアミン水溶液を、９０℃以上で加熱（熱処理）すると、その水溶液から得られたペンタメチレンジアミンを用いてペンタメチレンジイソシアネート（後述）を製造し、さらに、そのペンタメチレンジイソシアネートからイソシアネート変性体を製造する場合に、反応速度が低い場合や、得られるイソシアネート変性体（後述）の貯蔵安定性が低い場合がある。 Further, when an aqueous pentamethylenediamine solution is heated (heat treatment) at 90 ° C. or higher, pentamethylene diisocyanate (described later) is produced using the pentamethylenediamine obtained from the aqueous solution, and further, an isocyanate-modified product from the pentamethylene diisocyanate. In the case of producing the product, the reaction rate may be low, or the storage stability of the resulting isocyanate-modified product (described later) may be low.
　そのため、好ましくは、ペンタメチレンジアミン水溶液を、９０℃以上で加熱（熱処理）することなく、さらに好ましくは、８０℃以上で加熱することなく、とりわけ好ましくは、ペンタメチレンジアミン水溶液を加熱（熱処理）することなく、その水溶液からそのままペンタメチレンジアミン（またはその塩）を抽出する。 Therefore, the pentamethylenediamine aqueous solution is preferably heated (heat treatment) at 90 ° C. or higher, more preferably 80 ° C. or higher, and particularly preferably the pentamethylenediamine aqueous solution is heated (heat treatment). Without extraction, pentamethylenediamine (or a salt thereof) is directly extracted from the aqueous solution.
　具体的には、液－液抽出では、ペンタメチレンジアミン水溶液と抽出溶媒（後述）とを、例えば、常圧（大気圧）下、例えば、５～６０℃、好ましくは、１０～６０℃、さらに好ましくは、１５～５０℃、とりわけ好ましくは、１５～４０℃において、例えば、撹拌羽根などにより、例えば、１～１２０分間、好ましくは、５～９０分間、さらに好ましくは、５～６０分間混合する。 Specifically, in liquid-liquid extraction, an aqueous pentamethylenediamine solution and an extraction solvent (described later) are, for example, under normal pressure (atmospheric pressure), for example, 5 to 60 ° C., preferably 10 to 60 ° C. Preferably, mixing is performed at 15 to 50 ° C., particularly preferably 15 to 40 ° C., for example, by a stirring blade, for example, for 1 to 120 minutes, preferably 5 to 90 minutes, and more preferably 5 to 60 minutes. .
　撹拌羽根としては、特に限定されないが、例えば、プロペラ、平羽根、角度付平羽根、ピッチ付平羽根、平羽根ディスクタービン、傾斜付羽根ディスクタービン、湾曲羽根、ファウドラー型、ブルーマージン型、ディゾルバー、アンカーなどが挙げられる。 The agitating blade is not particularly limited. An anchor etc. are mentioned.
　また、混合における回転数としては、例えば、５～３０００ｒｐｍ、好ましくは、１０～２０００ｒｐｍ、さらに好ましくは、２０～１０００ｒｐｍである。 Further, the rotation speed in mixing is, for example, 5 to 3000 rpm, preferably 10 to 2000 rpm, and more preferably 20 to 1000 rpm.
　これにより、ペンタメチレンジアミン（またはその塩）を、抽出溶媒（後述）中へと抽出する。 Thereby, pentamethylenediamine (or a salt thereof) is extracted into an extraction solvent (described later).
　次いで、この方法では、ペンタメチレンジアミン（またはその塩）と抽出溶媒（後述）との混合物を、例えば、５～３００分間、好ましくは、１０～２４０分間、さらに好ましくは、２０～１８０分間静置し、その後、ペンタメチレンジアミン（またはその塩）が抽出された抽出溶媒（ペンタメチレンジアミン抽出液、すなわち、抽出溶媒（後述）とペンタメチレンジアミン（またはその塩）との混合物）を、公知の方法により取り出す。 Next, in this method, a mixture of pentamethylenediamine (or a salt thereof) and an extraction solvent (described later) is allowed to stand, for example, for 5 to 300 minutes, preferably 10 to 240 minutes, and more preferably 20 to 180 minutes. Then, an extraction solvent from which pentamethylenediamine (or a salt thereof) is extracted (pentamethylenediamine extract, ie, a mixture of an extraction solvent (described later) and pentamethylenediamine (or a salt thereof)) is used in a known method. Take out.
　なお、１回の液－液抽出によりペンタメチレンジアミン（またはその塩）を十分に抽出できない場合には、複数回（例えば、２～５回）繰り返し液－液抽出することもできる。 If pentamethylenediamine (or a salt thereof) cannot be sufficiently extracted by one liquid-liquid extraction, liquid-liquid extraction can be repeated several times (for example, 2 to 5 times).
　これにより、ペンタメチレンジアミン水溶液中のペンタメチレンジアミン（またはその塩）を、抽出溶媒（後述）に抽出することができる。 Thereby, pentamethylenediamine (or a salt thereof) in the aqueous solution of pentamethylenediamine can be extracted into an extraction solvent (described later).
　このようにして得られる抽出溶媒（抽出溶媒（後述）とペンタメチレンジアミン（またはその塩）との混合物）において、ペンタメチレンジアミン（またはその塩）の濃度は、例えば、０．２～４０質量％、好ましくは、０．３～３５質量％、さらに好ましくは、０．４～３０質量％、とりわけ好ましくは、０．８～２５質量％である。 In the extraction solvent thus obtained (a mixture of the extraction solvent (described later) and pentamethylenediamine (or a salt thereof)), the concentration of pentamethylenediamine (or a salt thereof) is, for example, 0.2 to 40% by mass. Preferably, the amount is 0.3 to 35% by mass, more preferably 0.4 to 30% by mass, and particularly preferably 0.8 to 25% by mass.
　また、抽出後におけるペンタメチレンジアミン（またはその塩）の収率（抽出率）は、リジン（またはその塩）を基準として、例えば、６５～１００モル％、好ましくは、７０～１００モル％、さらに好ましくは、８０～１００モル％、とりわけ好ましくは、９０～１００モル％である。 Further, the yield (extraction rate) of pentamethylenediamine (or a salt thereof) after extraction is, for example, 65 to 100 mol%, preferably 70 to 100 mol%, based on lysine (or a salt thereof). Preferably, it is 80 to 100 mol%, particularly preferably 90 to 100 mol%.
　なお、この方法では、必要により、得られた抽出溶媒（後述）とペンタメチレンジアミン（またはその塩）との混合物から、例えば、ペンタメチレンジアミン（またはその塩）を単離することもできる。ペンタメチレンジアミン（またはその塩）の単離では、特に制限されないが、例えば、連続多段蒸留塔、回分多段蒸留塔などを備えた蒸留装置などにより、例えば、５０～１８２℃、０．１ｋＰａ～常圧下、抽出溶媒（後述）とペンタメチレンジアミン（またはその塩）との混合物を蒸留し、抽出溶媒（後述）を除去する。 In this method, if necessary, for example, pentamethylenediamine (or a salt thereof) can be isolated from a mixture of the obtained extraction solvent (described later) and pentamethylenediamine (or a salt thereof). The isolation of pentamethylenediamine (or a salt thereof) is not particularly limited, but for example, by a distillation apparatus equipped with a continuous multistage distillation column, a batch multistage distillation column, etc., for example, 50 to 182 ° C., 0.1 kPa to ordinary Under pressure, a mixture of the extraction solvent (described later) and pentamethylenediamine (or a salt thereof) is distilled to remove the extraction solvent (described later).
　そして、このような抽出において、抽出溶媒としては、例えば、非ハロゲン系有機溶剤などが挙げられる。 In such extraction, examples of the extraction solvent include non-halogen organic solvents.
　非ハロゲン系有機溶剤は、ハロゲン原子（フッ素、塩素、臭素、ヨウ素など）を分子中に含有しない有機溶剤であって、例えば、非ハロゲン脂肪族系有機溶剤、非ハロゲン脂環族系有機溶剤、非ハロゲン芳香族系有機溶剤などが挙げられる。 Non-halogen organic solvents are organic solvents that do not contain halogen atoms (fluorine, chlorine, bromine, iodine, etc.) in their molecules, such as non-halogen aliphatic organic solvents, non-halogen alicyclic organic solvents, Non-halogen aromatic organic solvents are exemplified.
　非ハロゲン脂肪族系有機溶剤としては、例えば、直鎖状の非ハロゲン脂肪族系有機溶剤、分岐状の非ハロゲン脂肪族系有機溶剤などが挙げられる。 Examples of non-halogen aliphatic organic solvents include linear non-halogen aliphatic organic solvents and branched non-halogen aliphatic organic solvents.
　直鎖状の非ハロゲン脂肪族系有機溶剤としては、例えば、直鎖状の非ハロゲン脂肪族炭化水素類、直鎖状の非ハロゲン脂肪族エーテル類、直鎖状の非ハロゲン脂肪族アルコール類などが挙げられる。 Examples of linear non-halogen aliphatic organic solvents include linear non-halogen aliphatic hydrocarbons, linear non-halogen aliphatic ethers, and linear non-halogen aliphatic alcohols. Is mentioned.
　直鎖状の非ハロゲン脂肪族炭化水素類としては、例えば、ｎ－ヘキサン、ｎ－ヘプタン、ｎ－ノナン、ｎ－デカン、ｎ－ドデカンなどが挙げられる。 Examples of linear non-halogen aliphatic hydrocarbons include n-hexane, n-heptane, n-nonane, n-decane, and n-dodecane.
　直鎖状の非ハロゲン脂肪族エーテル類としては、例えば、ジエチルエーテル、ジブチルエーテル、ジヘキシルエーテルなどが挙げられる。 Examples of linear non-halogen aliphatic ethers include diethyl ether, dibutyl ether, and dihexyl ether.
　直鎖状の非ハロゲン脂肪族アルコール類としては、例えば、直鎖状の炭素数１～３の１価アルコール（例えば、メタノール、エタノール、ｎ－プロパノール、イソプロパノールなど）、直鎖状の炭素数４～７の１価アルコール（例えば、ｎ－ブタノール、ｎ－ペンタノール、ｎ－ヘキサノール、ｎ－ヘプタノールなど）、直鎖状の炭素数８以上の１価アルコール（例えば、ｎ－オクタノール、ｎ－ノナノール、ｎ－デカノール、ｎ－ウンデカノール、ｎ－ドデカノールなど）などが挙げられる。 Examples of linear non-halogen aliphatic alcohols include linear monohydric alcohols having 1 to 3 carbon atoms (eg, methanol, ethanol, n-propanol, isopropanol, etc.), linear carbon atoms of 4 To 7 monohydric alcohols (eg, n-butanol, n-pentanol, n-hexanol, n-heptanol), linear monohydric alcohols having 8 or more carbon atoms (eg, n-octanol, n-nonanol) N-decanol, n-undecanol, n-dodecanol, etc.).
　分岐状の非ハロゲン脂肪族系有機溶剤としては、例えば、分岐状の非ハロゲン脂肪族炭化水素類、分岐状の非ハロゲン脂肪族エーテル類、分岐状の非ハロゲン脂肪族１価アルコール類、分岐状の非ハロゲン脂肪族多価アルコール類などが挙げられる。 Examples of branched non-halogen aliphatic organic solvents include branched non-halogen aliphatic hydrocarbons, branched non-halogen aliphatic ethers, branched non-halogen aliphatic monohydric alcohols, branched Non-halogen aliphatic polyhydric alcohols.
　分岐状の非ハロゲン脂肪族炭化水素類としては、例えば、２－メチルペンタン、２，２－ジメチルブタン、２，３－ジメチルブタン、２－メチルヘキサン、３－メチルヘキサン、２，３－ジメチルペンタン、２，４－ジメチルペンタン、ｎ－オクタン、２－メチルヘプタン、３－メチルヘプタン、４－メチルヘプタン、３－エチルへキサン、２，２－ジメチルへキサン、２，３－ジメチルへキサン、２，４－ジメチルへキサン、２，５－ジメチルへキサン、３，３－ジメチルへキサン、３，４－ジメチルへキサン、２－メチル－３－エチルペンタン、３－メチル－３－エチルペンタン、２，３，３－トリメチルペンタン、２，３，４－トリメチルペンタン、２，２，３，３－テトラメチルブタン、２，２，５－トリメチルヘキサンなどが挙げられる。 Examples of branched non-halogen aliphatic hydrocarbons include 2-methylpentane, 2,2-dimethylbutane, 2,3-dimethylbutane, 2-methylhexane, 3-methylhexane, and 2,3-dimethylpentane. 2,4-dimethylpentane, n-octane, 2-methylheptane, 3-methylheptane, 4-methylheptane, 3-ethylhexane, 2,2-dimethylhexane, 2,3-dimethylhexane, , 4-dimethylhexane, 2,5-dimethylhexane, 3,3-dimethylhexane, 3,4-dimethylhexane, 2-methyl-3-ethylpentane, 3-methyl-3-ethylpentane, 2 , 3,3-trimethylpentane, 2,3,4-trimethylpentane, 2,2,3,3-tetramethylbutane, 2,2,5-trimethylhexane, etc. It is.
　分岐状の非ハロゲン脂肪族エーテル類としては、例えば、ジイソプロピルエーテル、ジイソブチルエーテルなどが挙げられる。 Examples of branched non-halogen aliphatic ethers include diisopropyl ether and diisobutyl ether.
　分岐状の非ハロゲン脂肪族１価アルコール類としては、例えば、分岐状の炭素数４～７の１価アルコール（例えば、２－ブタノール、イソブタノール、ｔｅｒｔ－ブタノール、２－ペンタノール、３－ペンタノール、イソペンタノール、２－メチル－１－ブタノール、２－メチル－３－ブタノール、２，２－ジメチル－１－プロパノール、ｔｅｒｔ－ペンタノール、２－ヘキサノール、３－ヘキサノール、イソヘキサノール、２－メチル－２－ペンタノール、２－メチル－１－ペンタノール、３－メチル－１－ペンタノール、２－エチル－１－ブタノール、３，３－ジメチル－１－ブタノール、２－ヘプタノール、３－ヘプタノール、４－ヘプタノール、５－メチル－１－ヘキサノール、４－メチル－１－ヘキサノール、３－メチル－１－ヘキサノール、２－エチル－２－メチル－１－ブタノールなど）、分岐状の炭素数８以上の１価アルコール（例えば、イソオクタノール、イソノナノール、イソデカノール、５－エチル－２－ノナノール、トリメチルノニルアルコール、２－ヘキシルデカノール、３，９－ジエチル－６－トリデカノール、２－イソヘプチルイソウンデカノール、２－オクチルドデカノールなど）が挙げられる。 Examples of branched non-halogen aliphatic monohydric alcohols include branched monohydric alcohols having 4 to 7 carbon atoms (for example, 2-butanol, isobutanol, tert-butanol, 2-pentanol, 3-pentane). Tanol, isopentanol, 2-methyl-1-butanol, 2-methyl-3-butanol, 2,2-dimethyl-1-propanol, tert-pentanol, 2-hexanol, 3-hexanol, isohexanol, 2- Methyl-2-pentanol, 2-methyl-1-pentanol, 3-methyl-1-pentanol, 2-ethyl-1-butanol, 3,3-dimethyl-1-butanol, 2-heptanol, 3-heptanol 4-heptanol, 5-methyl-1-hexanol, 4-methyl-1-hexanol, 3-methyl-1- Xanol, 2-ethyl-2-methyl-1-butanol, etc., branched monohydric alcohols having 8 or more carbon atoms (for example, isooctanol, isononanol, isodecanol, 5-ethyl-2-nonanol, trimethylnonyl alcohol, 2 -Hexyldecanol, 3,9-diethyl-6-tridecanol, 2-isoheptylisoundecanol, 2-octyldodecanol, etc.).
　分岐状の非ハロゲン脂肪族多価アルコール類としては、例えば、２－エチル－１，３－ヘキサンジオールなどが挙げられる。 Examples of branched non-halogen aliphatic polyhydric alcohols include 2-ethyl-1,3-hexanediol.
　これら非ハロゲン脂肪族系有機溶剤は、単独使用または２種類以上併用することができる。 These non-halogen aliphatic organic solvents can be used alone or in combination of two or more.
　非ハロゲン脂肪族系有機溶剤として、好ましくは、直鎖状の非ハロゲン脂肪族系有機溶剤、さらに好ましくは、直鎖状の非ハロゲン脂肪族アルコール類が挙げられる。 The non-halogen aliphatic organic solvent is preferably a linear non-halogen aliphatic organic solvent, and more preferably a linear non-halogen aliphatic alcohol.
　直鎖状の非ハロゲン脂肪族アルコール類を用いると、ペンタメチレンジアミンを、高収率で抽出することができる。 When linear non-halogen aliphatic alcohols are used, pentamethylene diamine can be extracted with high yield.
　また、非ハロゲン脂肪族系有機溶剤として、好ましくは、炭素数４～７の１価アルコール（直鎖状の炭素数４～７の１価アルコール、分岐状の炭素数４～７の１価アルコール）が挙げられる。 The non-halogen aliphatic organic solvent is preferably a monohydric alcohol having 4 to 7 carbon atoms (a linear monohydric alcohol having 4 to 7 carbon atoms or a branched monohydric alcohol having 4 to 7 carbon atoms). ).
　炭素数４～７の１価アルコールを用いると、ペンタメチレンジアミンまたはその塩を効率良く抽出することができ、さらには、ペンタメチレンジアミンまたはその塩の不純物の含有割合を、低減することができる。 When a monohydric alcohol having 4 to 7 carbon atoms is used, pentamethylenediamine or a salt thereof can be efficiently extracted, and further, the content ratio of impurities of pentamethylenediamine or a salt thereof can be reduced.
　非ハロゲン脂環族系有機溶剤としては、例えば、非ハロゲン脂環族炭化水素類（例えば、シクロペンタン、メチルシクロペンタン、シクロヘキサン、メチルシクロヘキサン、エチルシクロヘキサン、ｐ－メンタン、ビシクロヘキシルなど）が挙げられる。 Examples of the non-halogen alicyclic organic solvent include non-halogen alicyclic hydrocarbons (eg, cyclopentane, methylcyclopentane, cyclohexane, methylcyclohexane, ethylcyclohexane, p-menthane, bicyclohexyl, etc.). .
　これら非ハロゲン脂環族系有機溶剤は、単独使用または２種類以上併用することができる。 These non-halogen alicyclic organic solvents can be used alone or in combination of two or more.
　非ハロゲン芳香族系有機溶剤としては、例えば、非ハロゲン芳香族炭化水素類（例えば、ベンゼン、トルエン、キシレン、エチルベンゼン、イソプロピルベンゼン、１，３，５－トリメチルベンゼン、１，２，３，４－テトラヒドロナフタレン、ｎ－ブチルベンゼン、ｓｅｃ－ブチルベンゼン、ｔｅｒｔ－ブチルベンゼン、エチルベンゼンなど）、フェノール類（例えば、フェノール、クレゾールなど）などが挙げられる。 Non-halogen aromatic organic solvents include, for example, non-halogen aromatic hydrocarbons (for example, benzene, toluene, xylene, ethylbenzene, isopropylbenzene, 1,3,5-trimethylbenzene, 1,2,3,4- Tetrahydronaphthalene, n-butylbenzene, sec-butylbenzene, tert-butylbenzene, ethylbenzene, etc.) and phenols (eg, phenol, cresol, etc.).
　これら非ハロゲン芳香族系有機溶剤は、単独使用または２種類以上併用することができる。 These non-halogen aromatic organic solvents can be used alone or in combination of two or more.
　また、非ハロゲン系有機溶剤としては、例えば、脂肪族炭化水素類と芳香族炭化水素類との混合物なども挙げられ、そのような混合物としては、例えば、石油エーテル、石油ベンジンなどが挙げられる。 In addition, examples of the non-halogen organic solvent include a mixture of aliphatic hydrocarbons and aromatic hydrocarbons, and examples of such a mixture include petroleum ether and petroleum benzine.
　これら非ハロゲン系有機溶剤は、単独使用または２種類以上併用することができる。 These non-halogen organic solvents can be used alone or in combination of two or more.
　なお、抽出溶媒としては、本発明の優れた効果を阻害しない範囲において、例えば、ハロゲン系有機溶剤（ハロゲン原子を分子中に含有する有機溶剤）を用いることもできる。　　　 As the extraction solvent, for example, a halogen-based organic solvent (an organic solvent containing a halogen atom in the molecule) can be used as long as the excellent effects of the present invention are not impaired. *
　ハロゲン系有機溶剤としては、例えば、ハロゲン系脂肪族炭化水素類（例えば、クロロホルム、ジクロロメタン、四塩化炭素、テトラクロロエチレンなど）、ハロゲン系芳香族炭化水素類（例えば、クロロベンゼン、ジクロロベンゼン、クロロトルエンなど）などが挙げられる。 Examples of the halogen-based organic solvent include halogen-based aliphatic hydrocarbons (for example, chloroform, dichloromethane, carbon tetrachloride, tetrachloroethylene), halogen-based aromatic hydrocarbons (for example, chlorobenzene, dichlorobenzene, chlorotoluene, etc.) Etc.
　これらハロゲン系有機溶剤は、単独使用または２種類以上併用することができる。 These halogenated organic solvents can be used alone or in combination of two or more.
　一方、抽出溶媒として、ハロゲン系有機溶剤を用いると、得られるペンタメチレンジアミンまたはその塩を用いてペンタメチレンジイソシアネート（後述）を製造し、さらに、そのペンタメチレンジイソシアネート（後述）を反応させて、イソシアネート変性体（後述）や、ポリウレタン樹脂（後述）を製造する場合において、イソシアネート変性体（後述）の生産性や物性（例えば、耐黄変性など）に劣る場合がある。 On the other hand, when a halogen-based organic solvent is used as an extraction solvent, pentamethylene diisocyanate (described later) is produced using the obtained pentamethylene diamine or a salt thereof, and the pentamethylene diisocyanate (described later) is further reacted to produce isocyanate. In the case of producing a modified product (described later) or a polyurethane resin (described later), the productivity and physical properties (for example, yellowing resistance) of the isocyanate-modified product (described later) may be inferior.
　また、そのようなペンタメチレンジイソシアネート（後述）やイソシアネート変性体（後述）と、活性水素化合物（後述）とを反応させ、ポリウレタン樹脂を製造する場合にも、やはり、得られるポリウレタン樹脂の物性（例えば、機械強度、耐薬品性など）に劣る場合がある。 Also, when a polyurethane resin is produced by reacting such a pentamethylene diisocyanate (described later) or an isocyanate-modified product (described later) with an active hydrogen compound (described later), the physical properties of the resulting polyurethane resin (for example, , Mechanical strength, chemical resistance, etc.).
　そのため、抽出溶媒として、好ましくは、非ハロゲン系有機溶剤、さらに好ましくは、非ハロゲン脂肪族系有機溶剤が挙げられる。 Therefore, the extraction solvent is preferably a non-halogen organic solvent, more preferably a non-halogen aliphatic organic solvent.
　ペンタメチレンジアミンまたはその塩を、非ハロゲン脂肪族系有機溶剤により抽出する場合には、得られるペンタメチレンジアミンまたはその塩を用いて、ペンタメチレンジイソシアネートを製造する場合に、優れた性質を備えるイソシアネート変性体や、優れた性質を備えるポリウレタン樹脂を効率良く製造することができるペンタメチレンジイソシアネートを、製造することができる。 When pentamethylenediamine or a salt thereof is extracted with a non-halogen aliphatic organic solvent, an isocyanate modification having excellent properties is produced when pentamethylene diisocyanate is produced using the obtained pentamethylenediamine or a salt thereof. The pentamethylene diisocyanate which can manufacture efficiently the body and the polyurethane resin provided with the outstanding property can be manufactured.
　また、本発明において、抽出溶媒の沸点は、例えば、６０～２５０℃、好ましくは、８０～２００℃、さらに好ましくは、９０～１５０℃である。 In the present invention, the boiling point of the extraction solvent is, for example, 60 to 250 ° C., preferably 80 to 200 ° C., and more preferably 90 to 150 ° C.
　抽出溶媒の沸点が、上記下限未満であると、ペンタメチレンジアミン水溶液から抽出により、ペンタメチレンジアミンまたはその塩を得る際に、抽出溶媒との分離が困難となる場合がある。 If the boiling point of the extraction solvent is less than the lower limit, it may be difficult to separate the extraction solvent from the extraction solvent when pentamethylenediamine or a salt thereof is obtained by extraction from an aqueous pentamethylenediamine solution.
　一方、抽出溶媒の沸点が、上記上限を超過すると、抽出溶媒とペンタメチレンジアミンまたはその塩との混合物からペンタメチレンジアミンまたはその塩を得る際に、分離工程での消費エネルギーが増大する場合がある。 On the other hand, when the boiling point of the extraction solvent exceeds the above upper limit, when obtaining pentamethylenediamine or a salt thereof from a mixture of the extraction solvent and pentamethylenediamine or a salt thereof, energy consumption in the separation step may increase. .
　また、ペンタメチレンジアミン水溶液からペンタメチレンジアミンまたはその塩を得る方法としては、上記の抽出に限定されず、例えば、蒸留など、公知の単離精製方法を採用することもできる。
（１２）１，５－ペンタメチレンジイソシアネートの製造方法
　また、本発明は、このようにして得られた１，５－ペンタメチレンジアミン（またはその塩）から１，５－ペンタメチレンジイソシアネート（以下、単にペンタメチレンジイソシアネート、ＰＤＩと称する場合がある。）を製造する方法を含んでいる。 Further, the method for obtaining pentamethylenediamine or a salt thereof from an aqueous pentamethylenediamine solution is not limited to the above extraction, and a known isolation and purification method such as distillation can also be employed.
(12) Method for Producing 1,5-Pentamethylene Diisocyanate The present invention also relates to 1,5-pentamethylene diisocyanate (hereinafter simply referred to as “1,5-pentamethylene diisocyanate”) from 1,5-pentamethylenediamine (or a salt thereof) thus obtained. A process for producing pentamethylene diisocyanate, sometimes referred to as PDI).
　１，５－ペンタメチレンジイソシアネートを合成する方法としては、例えば、１，５－ペンタメチレンジアミン（またはその塩）をホスゲン化する方法（以下、ホスゲン化法と称する場合がある。）や、１，５－ペンタメチレンジアミン（（またはその塩））をカルバメート化し、その後、熱分解する方法（以下、カルバメート化法と称する場合がある。）などが挙げられる。 Examples of the method for synthesizing 1,5-pentamethylene diisocyanate include a method of phosgenating 1,5-pentamethylenediamine (or a salt thereof) (hereinafter sometimes referred to as a phosgenation method), 1, Examples thereof include a method of carbamateizing 5-pentamethylenediamine (or a salt thereof) and then thermally decomposing (hereinafter sometimes referred to as a carbamate method).
　ホスゲン化法として、より具体的には、例えば、ペンタメチレンジアミンを直接ホスゲンと反応させる方法（以下、冷熱二段ホスゲン化法と称する場合がある。）や、ペンタメチレンジアミンの塩酸塩を不活性溶媒（後述）中に懸濁させてホスゲンと反応させる方法（以下、アミン塩酸塩のホスゲン化法と称する場合がある。）などが挙げられる。 More specifically, as the phosgenation method, for example, a method in which pentamethylenediamine is directly reacted with phosgene (hereinafter sometimes referred to as a cold two-stage phosgenation method), or a hydrochloride of pentamethylenediamine is inactive. Examples thereof include a method of suspending in a solvent (described later) and reacting with phosgene (hereinafter sometimes referred to as a phosgenation method of amine hydrochloride).
　冷熱二段ホスゲン化法では、例えば、まず、撹拌可能とされ、かつ、ホスゲン導入管を備えた反応器に、不活性溶媒を装入し、反応系内の圧力を、例えば、常圧～１．０ＭＰａ、好ましくは、常圧～０．５ＭＰａとし、また、温度を、例えば、０～８０℃、好ましくは、０～６０℃とする。 In the cold and hot two-stage phosgenation method, for example, first, an inert solvent is charged into a reactor that can be stirred and provided with a phosgene introduction tube, and the pressure in the reaction system is, for example, from normal pressure to 1 The pressure is 0.0 MPa, preferably normal pressure to 0.5 MPa, and the temperature is, for example, 0 to 80 ° C., preferably 0 to 60 ° C.
　不活性溶媒としては、例えば、ベンゼン、トルエン、キシレン、エチルベンゼンなどの芳香族炭化水素類、例えば、酢酸エチル、酢酸ブチル、酢酸アミルなどの脂肪酸エステル類、例えば、サリチル酸メチル、フタル酸ジメチル、フタル酸ジブチル、安息香酸メチルなどの芳香族カルボン酸エステル類、例えば、モノジクロロベンゼン、オルトジクロロベンゼン、トリクロロベンゼンなどの塩素化芳香族炭化水素類、例えば、クロロホルム、四塩化炭素などの塩素化炭化水素類などが挙げられる。 Examples of the inert solvent include aromatic hydrocarbons such as benzene, toluene, xylene, and ethylbenzene, and fatty acid esters such as ethyl acetate, butyl acetate, and amyl acetate, such as methyl salicylate, dimethyl phthalate, and phthalic acid. Aromatic carboxylic acid esters such as dibutyl and methyl benzoate, for example, chlorinated aromatic hydrocarbons such as monodichlorobenzene, orthodichlorobenzene, and trichlorobenzene, for example, chlorinated hydrocarbons such as chloroform and carbon tetrachloride Etc.
　これら不活性溶媒は、単独使用または２種類以上併用することができる。 These inert solvents can be used alone or in combination of two or more.
　不活性溶媒の配合量（総量）は、原料であるペンタメチレンジアミン１００質量部に対して、例えば、４００～３０００質量部、好ましくは、５００～２０００質量部である。　　　 The compounding amount (total amount) of the inert solvent is, for example, 400 to 3000 parts by mass, and preferably 500 to 2000 parts by mass with respect to 100 parts by mass of pentamethylenediamine as a raw material. *
　次いで、この方法では、ホスゲンを、ペンタメチレンジアミンのアミノ基１つに対して、例えば、１～１０倍モル、好ましくは、１～６倍モル導入し、上記の不活性溶媒に溶解したペンタメチレンジアミンを添加する。また、この間、反応液を、例えば、０～８０℃、好ましくは、０～６０℃に維持するとともに、発生する塩化水素を、還流冷却器を通じて反応系外に放出する（冷ホスゲン化反応）。これにより、反応器の内容物をスラリー状とする。 Then, in this method, phosgene is introduced, for example, 1 to 10-fold mol, preferably 1 to 6-fold mol, with respect to one amino group of pentamethylenediamine, and pentamethylene dissolved in the above inert solvent. Add the diamine. During this time, the reaction solution is maintained at, for example, 0 to 80 ° C., preferably 0 to 60 ° C., and the generated hydrogen chloride is discharged out of the reaction system through a reflux condenser (cold phosgenation reaction). Thereby, the contents of the reactor are made into a slurry.
　そして、この冷ホスゲン化反応では、ペンタメチレンジカルバモイルクロリドおよびアミン塩酸塩が生成される。 In this cold phosgenation reaction, pentamethylene dicarbamoyl chloride and amine hydrochloride are produced.
　次いで、この方法では、反応系内の圧力を、例えば、常圧～１．０ＭＰａ、好ましくは、０．０５～０．５ＭＰａとし、例えば、３０分～５時間で、例えば、８０～１８０℃の温度範囲に昇温する。昇温後、例えば、３０分～８時間反応を継続して、スラリー液を完全に溶解させる（熱ホスゲン化反応）。 Next, in this method, the pressure in the reaction system is, for example, normal pressure to 1.0 MPa, preferably 0.05 to 0.5 MPa, for example, 30 minutes to 5 hours, for example, 80 to 180 ° C. The temperature is raised to a temperature range. After the temperature rise, for example, the reaction is continued for 30 minutes to 8 hours to completely dissolve the slurry (thermal phosgenation reaction).
　なお、熱ホスゲン化反応において、昇温時および高温反応時には、溶解ホスゲンが気化して還流冷却器を通じて反応系外に逃げるため、還流冷却器からの還流量が確認できるまでホスゲンを適宜導入する。 In the thermal phosgenation reaction, dissolved phosgene vaporizes and escapes out of the reaction system through the reflux condenser during the temperature rise and the high temperature reaction, so phosgene is appropriately introduced until the reflux amount from the reflux condenser can be confirmed.
　なお、熱ホスゲン化反応終了後、反応系内を、例えば、８０～１８０℃、好ましくは、９０～１６０℃において、窒素ガスなどの不活性ガスを導入し、溶解している過剰のホスゲンおよび塩化水素をパージする。 After the completion of the thermal phosgenation reaction, an inert gas such as nitrogen gas is introduced into the reaction system at, for example, 80 to 180 ° C., preferably 90 to 160 ° C. to dissolve excess phosgene and chloride. Purge hydrogen.
　この熱ホスゲン化反応では、冷ホスゲン化反応で生成したペンタメチレンジカルバモイルクロリドが熱分解され、ペンタメチレンジイソシアネートが生成され、さらに、ペンタメチレンジアミンのアミン塩酸塩がホスゲン化され、ペンタメチレンジイソシアネートが生成される。 In this thermal phosgenation reaction, pentamethylene dicarbamoyl chloride produced by the cold phosgenation reaction is thermally decomposed to produce pentamethylene diisocyanate, and the amine hydrochloride of pentamethylenediamine is further phosgenated to produce pentamethylene diisocyanate. Is done.
　一方、アミン塩酸塩のホスゲン化法では、ペンタメチレンジアミンの塩酸塩を十分に乾燥し、微粉砕した後、上記の冷熱二段ホスゲン化法と同様の反応器内で、ペンタメチレンジアミンの塩酸塩を、上記の不活性溶媒中で撹拌し、分散させて、スラリーとする。 On the other hand, in the phosgenation method of amine hydrochloride, the hydrochloride of pentamethylenediamine is sufficiently dried and finely pulverized, and then the hydrochloride of pentamethylenediamine is used in the same reactor as in the above-described cold and two-stage phosgenation method. Is stirred in the above inert solvent and dispersed into a slurry.
　次いで、この方法では、反応温度を、例えば、８０～１８０℃、好ましくは、９０～１６０℃、反応圧力を、例えば、常圧～１．０ＭＰａ、好ましくは、０．０５～０．５ＭＰａに維持し、ホスゲンを、例えば、１～１０時間かけて、ホスゲン総量が、例えば、化学量論の１～１０倍になるように導入する。 Next, in this method, the reaction temperature is maintained at, for example, 80 to 180 ° C., preferably 90 to 160 ° C., and the reaction pressure is maintained at, for example, normal pressure to 1.0 MPa, preferably 0.05 to 0.5 MPa. Then, for example, phosgene is introduced over 1 to 10 hours so that the total amount of phosgene is, for example, 1 to 10 times the stoichiometric amount.
　これにより、ペンタメチレンジイソシアネートを合成することができる。 Thereby, pentamethylene diisocyanate can be synthesized.
　なお、反応の進行は、発生する塩化水素ガスの量と、上記の不活性溶媒に不溶のスラリーが消失し、反応液が澄明均一になることより推測できる。また、発生する塩化水素は、例えば、還流冷却器を通じて反応系外に放出する。また、反応の終了時には、上記の方法で溶解している過剰のホスゲンおよび塩化水素をパージする。その後、冷却し、減圧下において、不活性溶媒を留去する。 The progress of the reaction can be estimated from the amount of hydrogen chloride gas generated and the slurry insoluble in the inert solvent disappearing, and the reaction solution becomes clear and uniform. Further, the generated hydrogen chloride is released out of the reaction system through, for example, a reflux condenser. At the end of the reaction, excess phosgene and hydrogen chloride dissolved by the above method are purged. Thereafter, the mixture is cooled and the inert solvent is distilled off under reduced pressure.
　ペンタメチレンジイソシアネートは、加水分解性塩素の濃度（ＨＣ）が上昇しやすい傾向にあるため、ホスゲン化法を採用する場合において、ＨＣを低減する必要がある場合には、例えば、ホスゲン化反応させ、脱溶剤させた後、留去させたペンタメチレンジイソシアネートを、例えば、窒素などの不活性ガスを通気しながら、例えば、１５０℃～２００℃、好ましくは、１６０～１９０℃で、例えば、１～８時間、好ましくは、３～６時間加熱処理する。その後、精留処理することによって、ペンタメチレンジイソシアネートのＨＣを著しく低減することができる。 Since pentamethylene diisocyanate tends to increase the concentration (HC) of hydrolyzable chlorine, when adopting the phosgenation method, when it is necessary to reduce HC, for example, a phosgenation reaction is performed. The pentamethylene diisocyanate distilled off after removing the solvent is, for example, 150 to 200 ° C., preferably 160 to 190 ° C., for example, 1 to 8 while passing an inert gas such as nitrogen. Heat treatment is performed for a time, preferably 3 to 6 hours. Thereafter, HC of pentamethylene diisocyanate can be remarkably reduced by performing rectification treatment.
　本発明において、ペンタメチレンジイソシアネートの加水分解性塩素の濃度は、例えば、１００ｐｐｍ以下、好ましくは、８０ｐｐｍ以下、さらに好ましくは、６０ｐｐｍ以下、とりわけ好ましくは、５０ｐｐｍ以下である。 In the present invention, the concentration of hydrolyzable chlorine in pentamethylene diisocyanate is, for example, 100 ppm or less, preferably 80 ppm or less, more preferably 60 ppm or less, and particularly preferably 50 ppm or less.
　なお、加水分解性塩素の濃度は、例えば、ＪＩＳ　Ｋ－１５５６（２０００）の附属書３に記載されている加水分解性塩素の試験方法に準拠して測定することができる。 The concentration of hydrolyzable chlorine can be measured in accordance with, for example, the test method for hydrolyzable chlorine described in Annex 3 of JIS K-1556 (2000).
　加水分解性塩素の濃度が１００ｐｐｍを超過すると、トリマー化（後述）の反応速度が低下し、多量のトリマー化触媒（後述）を必要とする場合があり、トリマー化触媒（後述）を多量に用いると、得られるポリイソシアネート組成物（後述）の黄変度が高くなる場合や、数平均分子量が高くなり、粘度が高くなる場合がある。 When the concentration of hydrolyzable chlorine exceeds 100 ppm, the reaction rate of trimerization (described later) decreases, and a large amount of trimerization catalyst (described later) may be required, and a large amount of trimerization catalyst (described later) is used. In some cases, the resulting polyisocyanate composition (described later) has a high degree of yellowing, the number average molecular weight is high, and the viscosity is high.
　また、加水分解性塩素の濃度が１００ｐｐｍを超過すると、ポリイソシアネート組成物（後述）の貯蔵工程、および、ポリウレタン樹脂（後述）の製造工程において、粘度、色相が大きく変化する場合がある。 In addition, when the concentration of hydrolyzable chlorine exceeds 100 ppm, the viscosity and hue may change greatly in the storage process of the polyisocyanate composition (described later) and the manufacturing process of the polyurethane resin (described later).
　カルバメート化法としては、例えば、尿素法などが挙げられる。 Examples of the carbamate method include a urea method.
　尿素法では、例えば、まず、ペンタメチレンジアミンをカルバメート化し、ペンタメチレンジカルバメート（ＰＤＣ）を生成させる。 In the urea method, for example, pentamethylenediamine is first carbamateized to produce pentamethylene dicarbamate (PDC).
　より具体的には、反応原料として、ペンタメチレンジアミンと、尿素および／またはＮ－無置換カルバミン酸エステルと、アルコールとを反応させる。 More specifically, pentamethylenediamine, urea and / or N-unsubstituted carbamic acid ester, and alcohol are reacted as reaction raw materials.
　Ｎ－無置換カルバミン酸エステルとしては、例えば、Ｎ－無置換カルバミン酸脂肪族エステル類（例えば、カルバミン酸メチル、カルバミン酸エチル、カルバミン酸プロピル、カルバミン酸ｉｓｏ－プロピル、カルバミン酸ブチル、カルバミン酸ｉｓｏ－ブチル、カルバミン酸ｓｅｃ－ブチル、カルバミン酸ｔｅｒｔ－ブチル、カルバミン酸ペンチル、カルバミン酸ｉｓｏ－ペンチル、カルバミン酸ｓｅｃ－ペンチル、カルバミン酸ヘキシル、カルバミン酸ヘプチル、カルバミン酸オクチル、カルバミン酸２－エチルヘキシル、カルバミン酸ノニル、カルバミン酸デシル、カルバミン酸イソデシル、カルバミン酸ドデシル、カルバミン酸テトラデシル、カルバミン酸ヘキサデシルなど）、Ｎ－無置換カルバミン酸芳香族エステル類（例えば、カルバミン酸フェニル、カルバミン酸トリル、カルバミン酸キシリル、カルバミン酸ビフェニル、カルバミン酸ナフチル、カルバミン酸アントリル、カルバミン酸フェナントリルなど）などが挙げられる。 Examples of N-unsubstituted carbamic acid esters include N-unsubstituted carbamic acid aliphatic esters (for example, methyl carbamate, ethyl carbamate, propyl carbamate, iso-propyl carbamate, butyl carbamate, isocarbamate). -Butyl, sec-butyl carbamate, tert-butyl carbamate, pentyl carbamate, iso-pentyl carbamate, sec-pentyl carbamate, hexyl carbamate, heptyl carbamate, octyl carbamate, 2-ethylhexyl carbamate, carbamine Nonyl, decyl carbamate, isodecyl carbamate, dodecyl carbamate, tetradecyl carbamate, hexadecyl carbamate, etc.), N-unsubstituted carbamic acid aromatic esters (for example, Phenyl carbamate, tolyl carbamate, xylyl carbamate, carbamic acid biphenyl, naphthyl carbamate, anthryl carbamate, such as carbamic acid phenanthryl), and the like.
　これらＮ－無置換カルバミン酸エステルは、単独使用または２種類以上併用することができる。 These N-unsubstituted carbamic acid esters can be used alone or in combination of two or more.
　Ｎ－無置換カルバミン酸エステルとして、好ましくは、Ｎ－無置換カルバミン酸脂肪族エステル類が挙げられる。 Preferred examples of N-unsubstituted carbamic acid esters include N-unsubstituted carbamic acid aliphatic esters.
　アルコールとしては、例えば、１～３級の１価のアルコールが挙げられ、より具体的には、例えば、脂肪族アルコール類、芳香族アルコール類などが挙げられる。 Examples of the alcohol include primary to tertiary monohydric alcohols, and more specific examples include aliphatic alcohols and aromatic alcohols.
　脂肪族アルコール類としては、例えば、直鎖状の脂肪族アルコール類（例えば、メタノール、エタノール、ｎ－プロパノール、ｎ－ブタノール（１－ブタノール）、ｎ－ペンタノール、ｎ－ヘキサノール、ｎ－ヘプタノール、ｎ－オクタノール（１－オクタノール）、ｎ－ノナノール、ｎ－デカノール、ｎ－ドデカノール、ｎ－テトラデカノール、ｎ－ヘキサデカノールなど）、分岐状の脂肪族アルコール類（例えば、ｉｓｏ－プロパノール、ｉｓｏ－ブタノール、ｓｅｃ－ブタノール、ｔｅｒｔ－ブタノール、ｉｓｏ－ペンタノール、ｓｅｃ－ペンタノール、２－エチルヘキサノール、ｉｓｏ－デカノールなど）などが挙げられる。 Examples of the aliphatic alcohols include linear aliphatic alcohols (eg, methanol, ethanol, n-propanol, n-butanol (1-butanol), n-pentanol, n-hexanol, n-heptanol, n-octanol (1-octanol), n-nonanol, n-decanol, n-dodecanol, n-tetradecanol, n-hexadecanol, etc.), branched aliphatic alcohols (eg, iso-propanol, iso) -Butanol, sec-butanol, tert-butanol, iso-pentanol, sec-pentanol, 2-ethylhexanol, iso-decanol, etc.).
　芳香族アルコール類としては、例えば、フェノール、ヒドロキシトルエン、ヒドロキシキシレン、ビフェニルアルコール、ナフタレノール、アントラセノール、フェナントレノールなどが挙げられる。 Examples of aromatic alcohols include phenol, hydroxytoluene, hydroxyxylene, biphenyl alcohol, naphthalenol, anthracenol, phenanthrenol and the like.
　これらアルコールは、単独使用または２種類以上併用することができる。 These alcohols can be used alone or in combination of two or more.
　アルコールとして、好ましくは、脂肪族アルコール類、さらに好ましくは、直鎖状の脂肪族アルコール類が挙げられる。 As the alcohol, aliphatic alcohols are preferable, and linear aliphatic alcohols are more preferable.
　また、アルコールとして、好ましくは、上記した炭素数４～７の１価アルコール（直鎖状の炭素数４～７の１価アルコール、分岐状の炭素数４～７の１価アルコール）が挙げられる。 Further, as the alcohol, preferably, the above-described monovalent alcohol having 4 to 7 carbon atoms (linear monovalent alcohol having 4 to 7 carbon atoms, branched monovalent alcohol having 4 to 7 carbon atoms) can be used. .
　さらには、上記した抽出において、抽出溶媒としてアルコール（炭素数４～７の１価アルコールなど）が用いられる場合には、好ましくは、そのアルコールを、反応原料アルコールとして用いる。 Furthermore, in the above-described extraction, when an alcohol (such as a monohydric alcohol having 4 to 7 carbon atoms) is used as an extraction solvent, the alcohol is preferably used as a reaction raw material alcohol.
　そして、この方法では、ペンタメチレンジアミンと、尿素および／またはＮ－無置換カルバミン酸エステルと、アルコールとを配合し、好ましくは、液相で反応させる。 In this method, pentamethylene diamine, urea and / or N-unsubstituted carbamic acid ester, and alcohol are blended and reacted preferably in a liquid phase.
　ペンタメチレンジアミンと、尿素および／またはＮ－無置換カルバミン酸エステルと、アルコールとの配合割合は、特に制限はなく、比較的広範囲において適宜選択することができる。 The blending ratio of pentamethylenediamine, urea and / or N-unsubstituted carbamic acid ester, and alcohol is not particularly limited and can be appropriately selected within a relatively wide range.
　通常は、尿素およびＮ－無置換カルバミン酸エステルの配合量、および、アルコールの配合量が、ペンタメチレンジアミンのアミノ基に対して等モル以上あればよく、そのため、尿素および／または上記したＮ－無置換カルバミン酸エステルや、アルコールそのものを、この反応における反応溶媒として用いることもできる。 Usually, the blending amount of urea and N-unsubstituted carbamic acid ester and the blending amount of alcohol should be equimolar or more with respect to the amino group of pentamethylenediamine. Therefore, urea and / or N- An unsubstituted carbamate or alcohol itself can also be used as a reaction solvent in this reaction.
　また、上記した抽出において抽出溶媒としてアルコール（炭素数４～７の１価アルコールなど）が用いられる場合には、好ましくは、そのアルコールをそのまま、反応原料および反応溶媒として用いる。 In addition, when an alcohol (such as a monohydric alcohol having 4 to 7 carbon atoms) is used as an extraction solvent in the above extraction, the alcohol is preferably used as it is as a reaction raw material and a reaction solvent.
　なお、尿素および／または上記したＮ－無置換カルバミン酸エステルや、アルコールを反応溶媒として兼用する場合には、必要に応じて過剰量の尿素および／または上記したＮ－無置換カルバミン酸エステルやアルコールが用いられるが、過剰量が多いと、反応後の分離工程での消費エネルギーが増大するので、工業生産上、不適となる。 When urea and / or the above-mentioned N-unsubstituted carbamic acid ester or alcohol is also used as a reaction solvent, an excess amount of urea and / or the above-mentioned N-unsubstituted carbamic acid ester or alcohol is used as necessary. However, if the excess amount is large, the energy consumption in the separation step after the reaction increases, which is not suitable for industrial production.
　そのため、尿素および／または上記したＮ－無置換カルバミン酸エステルの配合量は、カルバメートの収率を向上させる観点から、ペンタメチレンジアミンのアミノ基１つに対して、例えば、０．５～２０倍モル、好ましくは、１～１０倍モル、さらに好ましくは、１～５倍モルであり、アルコールの配合量は、ペンタメチレンジアミンのアミノ基１つに対して、０．５～１００倍モル、好ましくは、１～２０倍モル、さらに好ましくは、１～１０倍モルである。 Therefore, the amount of urea and / or the above-mentioned N-unsubstituted carbamic acid ester is, for example, 0.5 to 20 times that of one amino group of pentamethylenediamine from the viewpoint of improving the yield of carbamate. Moles, preferably 1 to 10 times moles, more preferably 1 to 5 times moles, and the amount of alcohol blended is 0.5 to 100 times moles with respect to one amino group of pentamethylenediamine, preferably Is 1 to 20 moles, more preferably 1 to 10 moles.
　また、この方法においては、触媒を用いることもできる。 In this method, a catalyst can also be used.
　触媒としては、特に制限されないが、例えば、周期律表第１族（ＩＵＰＡＣ　Ｐｅｒｉｏｄｉｃ　Ｔａｂｌｅ　ｏｆ　ｔｈｅ　Ｅｌｅｍｅｎｔｓ（ｖｅｒｓｉｏｎ　ｄａｔｅ　２２　Ｊｕｎｅ　２００７）に従う。以下同じ。）金属化合物（例えば、リチウムメタノラート、リチウムエタノラート、リチウムプロパノラート、リチウムブタノラート、ナトリウムメタノラート、カリウム－ｔｅｒｔ－ブタノラートなど）、第２族金属化合物（例えば、マグネシウムメタノラート、カルシウムメタノラートなど）、第３族金属化合物（例えば、酸化セリウム（ＩＶ）、酢酸ウラニルなど）、第４族金属化合物（例えば、チタンテトライソプロパノラート、チタンテトラブタノラート、四塩化チタン、チタンテトラフェノラート、ナフテン酸チタンなど）、第５族金属化合物（例えば、塩化バナジウム（ＩＩＩ）、バナジウムアセチルアセトナートなど）、第６族金属化合物（例えば、塩化クロム（ＩＩＩ）、酸化モリブデン（ＶＩ）、モリブデンアセチルアセトナート、酸化タングステン（ＶＩ）など）、第７族金属化合物（例えば、塩化マンガン（ＩＩ）、酢酸マンガン（ＩＩ）、酢酸マンガン（ＩＩＩ）など）、第８族金属化合物（例えば、酢酸鉄（ＩＩ）、酢酸鉄（ＩＩＩ）、リン酸鉄、シュウ酸鉄、塩化鉄（ＩＩＩ）、臭化鉄（ＩＩＩ）など）、第９族金属化合物（例えば、酢酸コバルト、塩化コバルト、硫酸コバルト、ナフテン酸コバルトなど）、第１０族金属化合物（例えば、塩化ニッケル、酢酸ニッケル、ナフテン酸ニッケルなど）、第１１族金属化合物（例えば、酢酸銅（ＩＩ）、硫酸銅（ＩＩ）、硝酸銅（ＩＩ）、ビス－（トリフェニル－ホスフィンオキシド）－塩化銅（ＩＩ）、モリブデン酸銅、酢酸銀、酢酸金など）、第１２族金属化合物（例えば、酸化亜鉛、塩化亜鉛、酢酸亜鉛、亜鉛アセトニルアセタート、オクタン酸亜鉛、シュウ酸亜鉛、ヘキシル酸亜鉛、安息香酸亜鉛、ウンデシル酸亜鉛など）、第１３族金属化合物（例えば、アルミニウムアセチルアセトナート、アルミニウム－イソブチラート、三塩化アルミニウムなど）、第１４族金属化合物（例えば、塩化スズ（ＩＩ）、塩化スズ（ＩＶ）、酢酸鉛、リン酸鉛など）、第１５族金属化合物（例えば、塩化アンチモン（ＩＩＩ）、塩化アンチモン（Ｖ）、塩化ビスマス（ＩＩＩ）など）などが挙げられる。 Although it does not restrict | limit especially as a catalyst, For example, it follows a periodic table group 1 (IUPAC Periodic Table of the Elements (version date 22 June 2007). The same hereafter) Metal compound (For example, lithium methanolate, lithium ethanolate, Lithium propanolate, lithium butanolate, sodium methanolate, potassium tert-butanolate, etc., Group 2 metal compounds (eg magnesium methanolate, calcium methanolate etc.), Group 3 metal compounds (eg cerium oxide) (IV), uranyl acetate, etc.), Group 4 metal compounds (for example, titanium tetraisopropanolate, titanium tetrabutanolate, titanium tetrachloride, titanium tetraphenolate, naphthenic acid Group 5 metal compounds (eg, vanadium (III) chloride, vanadium acetylacetonate, etc.), Group 6 metal compounds (eg, chromium (III) chloride, molybdenum (VI), molybdenum acetylacetonate, Tungsten oxide (VI), etc.), Group 7 metal compounds (eg, manganese (II) chloride, manganese acetate (II), manganese acetate (III), etc.), Group 8 metal compounds (eg, iron (II) acetate, Iron (III) acetate, iron phosphate, iron oxalate, iron (III) chloride, iron (III) bromide, etc.), Group 9 metal compounds (for example, cobalt acetate, cobalt chloride, cobalt sulfate, cobalt naphthenate, etc.) ), Group 10 metal compounds (eg, nickel chloride, nickel acetate, nickel naphthenate, etc.), Group 11 metal compounds (eg, Copper (II) acid, copper (II) sulfate, copper (II) nitrate, bis- (triphenyl-phosphine oxide) -copper chloride (II), copper molybdate, silver acetate, gold acetate, etc.), Group 12 metals Compounds (eg, zinc oxide, zinc chloride, zinc acetate, zinc acetonyl acetate, zinc octoate, zinc oxalate, zinc hexylate, zinc benzoate, zinc undecylate, etc.), Group 13 metal compounds (eg, aluminum Acetylacetonate, aluminum-isobutyrate, aluminum trichloride, etc.), Group 14 metal compounds (eg, tin (II) chloride, tin (IV) chloride, lead acetate, lead phosphate, etc.), Group 15 metal compounds (eg, , Antimony (III) chloride, antimony (V) chloride, bismuth (III) chloride and the like.
　さらに、触媒としては、例えば、Ｚｎ（ＯＳＯ２ＣＦ３）２（別表記：Ｚｎ（ＯＴｆ）２、トリフルオロメタンスルホン酸亜鉛）、Ｚｎ（ＯＳＯ２Ｃ２Ｆ５）２、Ｚｎ（ＯＳＯ２Ｃ３Ｆ７）２、Ｚｎ（ＯＳＯ２Ｃ４Ｆ９）２、Ｚｎ（ＯＳＯ２Ｃ６Ｈ４ＣＨ３）２（ｐ－トルエンスルホン酸亜鉛）、Ｚｎ（ＯＳＯ２Ｃ６Ｈ５）２、Ｚｎ（ＢＦ４）２、Ｚｎ（ＰＦ６）２、Ｈｆ（ＯＴｆ）４（トリフルオロメタンスルホン酸ハフニウム）、Ｓｎ（ＯＴｆ）２、Ａｌ（ＯＴｆ）３、Ｃｕ（ＯＴｆ）２なども挙げられる。 Furthermore, as a catalyst, for example, Zn (OSO 2 CF 3 ) 2 (another notation: Zn (OTf) 2 , zinc trifluoromethanesulfonate), Zn (OSO 2 C 2 F 5 ) 2 , Zn (OSO 2 C 3 F 7 ) 2 , Zn (OSO 2 C 4 F 9 ) 2 , Zn (OSO 2 C 6 H 4 CH 3 ) 2 (p-toluenesulfonic acid zinc), Zn (OSO 2 C 6 H 5 ) 2 , Zn ( BF 4 ) 2 , Zn (PF 6 ) 2 , Hf (OTf) 4 (hafnium trifluoromethanesulfonate), Sn (OTf) 2 , Al (OTf) 3 , Cu (OTf) 2 and the like are also included.
　これら触媒は、単独使用または２種類以上併用することができる。 These catalysts can be used alone or in combination of two or more.
　また、触媒の配合量は、ペンタメチレンジアミン１モルに対して、例えば、０．０００００１～０．１モル、好ましくは、０．００００５～０．０５モルである。触媒の配合量がこれより多くても、それ以上の顕著な反応促進効果が見られない反面、配合量の増大によりコストが上昇する場合がある。一方、配合量がこれより少ないと、反応促進効果が得られない場合がある。 The amount of the catalyst is, for example, 0.000001 to 0.1 mol, preferably 0.00005 to 0.05 mol, per 1 mol of pentamethylenediamine. Even if the amount of the catalyst is larger than this, no further significant reaction promoting effect is observed, but the cost may increase due to an increase in the amount of the catalyst. On the other hand, if the blending amount is less than this, the reaction promoting effect may not be obtained.
　なお、触媒の添加方法は、一括添加、連続添加および複数回の断続分割添加のいずれの添加方法でも、反応活性に影響を与えることがなく、特に制限されることはない。 In addition, the addition method of the catalyst does not affect the reaction activity and is not particularly limited by any addition method of batch addition, continuous addition, and plural intermittent additions.
　また、この反応において、反応溶媒は必ずしも必要ではないが、例えば、反応原料が固体の場合や反応生成物が析出する場合には、溶媒を配合することにより操作性を向上させることができる。 In this reaction, a reaction solvent is not necessarily required. For example, when the reaction raw material is solid or a reaction product is precipitated, the operability can be improved by blending the solvent.
　溶媒としては、反応原料であるペンタメチレンジアミン、尿素および／またはＮ－無置換カルバミン酸エステル、および、アルコールと、反応生成物であるウレタン化合物などに対して不活性であるか反応性に乏しいものであれば、特に制限されるものではなく、例えば、脂肪族炭化水素類（例えば、ヘキサン、ペンタン、石油エーテル、リグロイン、シクロドデカン、デカリン類など）、芳香族炭化水素類（例えば、ベンゼン、トルエン、キシレン、エチルベンゼン、イソプロピルベンゼン、ブチルベンゼン、シクロヘキシルベンゼン、テトラリン、クロロベンゼン、ｏ－ジクロロベンゼン、メチルナフタレン、クロロナフタレン、ジベンジルトルエン、トリフェニルメタン、フェニルナフタレン、ビフェニル、ジエチルビフェニル、トリエチルビフェニルなど）、エーテル類（例えば、ジエチルエーテル、ジイソプロピルエーテル、ジブチルエーテル、アニソール、ジフェニルエーテル、テトラヒドロフラン、エチレングリコールジメチルエーテル、エチレングリコールジエチルエーテル、エチレングリコールジブチルエーテル、ジエチレングリコールジメチルエーテル、ジエチレングリコールジエチルエーテルなど）、カーボネート類（例えば、ジメチルカーボネート、ジエチルカーボネート、ジプロピルカーボネート、ジブチルカーボネートなど）、ニトリル類（例えば、アセトニトリル、プロピオニトリル、アジポニトリル、ベンゾニトリルなど）、脂肪族ハロゲン化炭化水素類（例えば、塩化メチレン、クロロホルム、１，２－ジクロロエタン、１，２－ジクロロプロパン、１，４－ジクロロブタンなど）、アミド類（例えば、ジメチルホルムアミド、ジメチルアセトアミドなど）、ニトロ化合物類（例えば、ニトロメタン、ニトロベンゼンなど）や、Ｎ－メチルピロリジノン、Ｎ，Ｎ－ジメチルイミダゾリジノン、ジメチルスルホキシドなどが挙げられる。 Solvents that are inactive or poorly reactive with pentamethylenediamine, urea and / or N-unsubstituted carbamic acid esters as reaction raw materials, and alcohol and urethane compounds as reaction products If it is, it will not restrict | limit in particular, For example, aliphatic hydrocarbons (for example, hexane, pentane, petroleum ether, ligroin, cyclododecane, decalins etc.), aromatic hydrocarbons (for example, benzene, toluene) , Xylene, ethylbenzene, isopropylbenzene, butylbenzene, cyclohexylbenzene, tetralin, chlorobenzene, o-dichlorobenzene, methylnaphthalene, chloronaphthalene, dibenzyltoluene, triphenylmethane, phenylnaphthalene, biphenyl, diethylbiphenyl, Ethyl biphenyl, etc.), ethers (eg, diethyl ether, diisopropyl ether, dibutyl ether, anisole, diphenyl ether, tetrahydrofuran, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, ethylene glycol dibutyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether), carbonates (Eg, dimethyl carbonate, diethyl carbonate, dipropyl carbonate, dibutyl carbonate, etc.), nitriles (eg, acetonitrile, propionitrile, adiponitrile, benzonitrile, etc.), aliphatic halogenated hydrocarbons (eg, methylene chloride, chloroform) 1,2-dichloroethane, 1,2-dichloromethane Bread, 1,4-dichlorobutane, etc.), amides (eg, dimethylformamide, dimethylacetamide, etc.), nitro compounds (eg, nitromethane, nitrobenzene, etc.), N-methylpyrrolidinone, N, N-dimethylimidazolidinone And dimethyl sulfoxide.
　さらに、反応溶媒として、例えば、上記した抽出における抽出溶媒も挙げられる。 Furthermore, examples of the reaction solvent include the extraction solvent in the above-described extraction.
　これら反応溶媒のなかでは、経済性、操作性などを考慮すると、脂肪族炭化水素類、芳香族炭化水素類が好ましく用いられる。 Of these reaction solvents, aliphatic hydrocarbons and aromatic hydrocarbons are preferably used in view of economy and operability.
　また、反応溶媒として、好ましくは、上記した抽出における抽出溶媒が挙げられる。 Further, the reaction solvent is preferably the extraction solvent in the above-described extraction.
　抽出溶媒を反応溶媒として用いることにより、抽出されたペンタメチレンジイソシアネートをそのままカルバメート化反応に供することができ、操作性の向上を図ることができる。 By using the extraction solvent as a reaction solvent, the extracted pentamethylene diisocyanate can be used for the carbamation reaction as it is, and the operability can be improved.
　また、このような反応溶媒は、単独もしくは２種以上を組み合わせて用いることができる。 Such reaction solvents can be used alone or in combination of two or more.
　また、反応溶媒の配合量は、目的生成物のペンタメチレンジカルバメートが溶解する程度の量であれば特に制限されないが、工業的には、反応液から反応溶媒を回収する必要があるため、その回収に消費されるエネルギーをできる限り低減し、かつ、配合量が多いと、反応基質濃度が低下して反応速度が遅くなるため、できるだけ少ない方が好ましい。より具体的には、ペンタメチレンジアミン１質量部に対して、通常、０．１～５００質量部、好ましくは、１～１００質量部の範囲で用いられる。 The amount of the reaction solvent is not particularly limited as long as the target product pentamethylene dicarbamate is dissolved, but industrially, the reaction solvent needs to be recovered from the reaction solution. If the energy consumed for recovery is reduced as much as possible and the amount is large, the reaction substrate concentration decreases and the reaction rate slows down. More specifically, it is usually used in the range of 0.1 to 500 parts by weight, preferably 1 to 100 parts by weight with respect to 1 part by weight of pentamethylenediamine.
　また、この反応においては、反応温度は、例えば、１００～３５０℃、好ましくは、１５０～３００℃の範囲において適宜選択される。反応温度がこれより低いと、反応速度が低下する場合があり、一方、これより高いと、副反応が増大して目的生成物であるペンタメチレンジカルバメートの収率が低下する場合がある。 In this reaction, the reaction temperature is appropriately selected, for example, in the range of 100 to 350 ° C., preferably 150 to 300 ° C. When the reaction temperature is lower than this, the reaction rate may decrease. On the other hand, when the reaction temperature is higher than this, side reaction may increase and the yield of the target product, pentamethylene dicarbamate may decrease.
　また、反応圧力は、通常、大気圧であるが、反応液中の成分の沸点が反応温度よりも低い場合には加圧してもよく、さらには、必要により減圧してもよい。 The reaction pressure is usually atmospheric pressure, but may be increased when the boiling point of the component in the reaction solution is lower than the reaction temperature, and further reduced as necessary.
　また、反応時間は、例えば、０．１～２０時間、好ましくは、０．５～１０時間である。反応時間がこれより短いと、目的生成物であるペンタメチレンジカルバメートの収率が低下する場合がある。一方、これより長いと、工業生産上、不適となる。 The reaction time is, for example, 0.1 to 20 hours, preferably 0.5 to 10 hours. If the reaction time is shorter than this, the yield of the target product, pentamethylene dicarbamate, may decrease. On the other hand, if it is longer than this, it is unsuitable for industrial production.
　そして、この反応は、上記した条件で、例えば、反応容器内に、ペンタメチレンジアミン、尿素および／またはＮ－無置換カルバミン酸エステル、アルコール、および、必要により触媒、反応溶媒を仕込み、攪拌あるいは混合すればよい。そうすると、温和な条件下において、短時間、低コストかつ高収率で、ペンタメチレンジカルバメートが生成する。 In this reaction, for example, pentamethylenediamine, urea and / or N-unsubstituted carbamic acid ester, alcohol, and, if necessary, a catalyst and a reaction solvent are charged into the reaction vessel under the above-described conditions, and stirred or mixed. do it. Then, pentamethylene dicarbamate is produced in a short time, at a low cost and in a high yield under mild conditions.
　なお、得られるペンタメチレンジカルバメートは、通常、原料成分として用いられる上記のペンタメチレンジアミンに対応し、より具体的には、１，５－ペンタメチレンジカルバメートが得られる。 The obtained pentamethylene dicarbamate usually corresponds to the above pentamethylene diamine used as a raw material component, and more specifically 1,5-pentamethylene dicarbamate is obtained.
　また、この反応においては、アンモニアが副生される。 In this reaction, ammonia is by-produced.
　また、この反応において、Ｎ－無置換カルバミン酸エステルを配合する場合には、そのエステルに対応するアルコールが副生される。 In this reaction, when N-unsubstituted carbamic acid ester is blended, an alcohol corresponding to the ester is by-produced.
　なお、この反応において、反応型式としては、回分式、連続式いずれの型式も採用することができる。 In this reaction, either a batch type or a continuous type can be adopted as a reaction type.
　また、この反応は、好ましくは、副生するアンモニアを系外に流出させながら反応させる。さらには、Ｎ－無置換カルバミン酸エステルを配合する場合には、副生するアルコールを系外に留出させながら反応させる。 In addition, this reaction is preferably performed while the by-produced ammonia flows out of the system. Furthermore, when N-unsubstituted carbamic acid ester is blended, the reaction is carried out while distilling off the by-produced alcohol out of the system.
　これにより、目的生成物であるペンタメチレンジカルバメートの生成を促進し、その収率を、より一層向上することができる。 Thereby, the production of pentamethylene dicarbamate which is the target product can be promoted, and the yield can be further improved.
　また、得られたペンタメチレンジカルバメートを単離する場合には、例えば、過剰（未反応）の尿素および／またはＮ－無置換カルバミン酸エステル、過剰（未反応）のアルコール、触媒、ペンタメチレンジカルバメート、反応溶媒、副生するアンモニア、場合により副生するアルコールなどを含む反応液から、公知の分離精製方法によって、ペンタメチレンジカルバメートを分離すればよい。 When the obtained pentamethylene dicarbamate is isolated, for example, excess (unreacted) urea and / or N-unsubstituted carbamic acid ester, excess (unreacted) alcohol, catalyst, pentamethylene dicarbamate. The pentamethylene dicarbamate may be separated from the reaction solution containing carbamate, reaction solvent, by-produced ammonia, and optionally by-produced alcohol by a known separation and purification method.
　次いで、このペンタメチレンジイソシアネートの製造方法では、得られたペンタメチレンジカルバメートを熱分解して、ペンタメチレンジイソシアネートを製造する。 Next, in this method for producing pentamethylene diisocyanate, the obtained pentamethylene dicarbamate is thermally decomposed to produce pentamethylene diisocyanate.
　すなわち、このようなイソシアネートの製造方法では、上記によって得られたペンタメチレンジカルバメートを熱分解し、ペンタメチレンジイソシアネート、および、副生物であるアルコールを生成させる。 That is, in such an isocyanate production method, the pentamethylene dicarbamate obtained as described above is thermally decomposed to produce pentamethylene diisocyanate and alcohol as a by-product.
　なお、得られるペンタメチレンジイソシアネートは、通常、原料成分として用いられる上記のペンタメチレンジアミンに対応し、より具体的には、１，５－ペンタメチレンジイソシアネートが得られる。 The obtained pentamethylene diisocyanate usually corresponds to the above pentamethylene diamine used as a raw material component, and more specifically 1,5-pentamethylene diisocyanate is obtained.
　また、アルコールとしては、通常、原料成分として用いられるアルコールと同種のアルコールが、副生する。 In addition, as alcohol, usually the same kind of alcohol as that used as a raw material component is by-produced.
　この熱分解は、特に限定されず、例えば、液相法、気相法などの公知の分解法を用いることができる。 This thermal decomposition is not particularly limited, and for example, a known decomposition method such as a liquid phase method or a gas phase method can be used.
　気相法では、熱分解により生成するペンタメチレンジイソシアネートおよびアルコールは、気体状の生成混合物から、分別凝縮によって分離することができる。また、液相法では、熱分解により生成するペンタメチレンジイソシアネートおよびアルコールは、例えば、蒸留や、担持物質としての溶剤および／または不活性ガスを用いて、分離することができる。 In the vapor phase method, pentamethylene diisocyanate and alcohol produced by thermal decomposition can be separated from the gaseous product mixture by fractional condensation. In the liquid phase method, pentamethylene diisocyanate and alcohol produced by thermal decomposition can be separated by, for example, distillation or using a solvent and / or an inert gas as a support material.
　熱分解として、好ましくは、作業性の観点から、液相法が挙げられる。 As the thermal decomposition, a liquid phase method is preferably used from the viewpoint of workability.
　液相法におけるペンタメチレンジカルバメートの熱分解反応は、可逆反応であるため、好ましくは、熱分解反応の逆反応（ペンタメチレンジイソシアネートとアルコールとのウレタン化反応）を抑制するため、ペンタメチレンジカルバメートを熱分解するとともに、反応混合物からペンタメチレンジイソシアネート、および／または、副生するアルコールを、例えば、気体として抜き出し、それらを分離する。 Since the thermal decomposition reaction of pentamethylene dicarbamate in the liquid phase method is a reversible reaction, it is preferable to suppress the reverse reaction of the thermal decomposition reaction (urethanization reaction of pentamethylene diisocyanate and alcohol). The pentamethylene diisocyanate and / or by-product alcohol is extracted from the reaction mixture as a gas, for example, and separated from the reaction mixture.
　熱分解反応の反応条件として、好ましくは、ペンタメチレンジカルバメートを良好に熱分解できるとともに、熱分解において生成したペンタメチレンジイソシアネートおよびアルコールが蒸発し、これによりペンタメチレンジカルバメートとペンタメチレンジイソシアネートとが平衡状態とならず、さらには、ペンタメチレンジイソシアネートの重合などの副反応が抑制される条件が挙げられる。 As the reaction conditions for the thermal decomposition reaction, it is preferable that pentamethylene dicarbamate can be thermally decomposed satisfactorily, and pentamethylene diisocyanate and alcohol generated in the thermal decomposition evaporate, whereby the equilibrium of pentamethylene dicarbamate and pentamethylene diisocyanate is achieved. In addition, there may be mentioned conditions where side reactions such as polymerization of pentamethylene diisocyanate are suppressed.
　このような反応条件として、より具体的には、熱分解温度は、通常、３５０℃以下であり、好ましくは、８０～３５０℃、さらに好ましくは、１００～３００℃である。８０℃よりも低いと、実用的な反応速度が得られない場合があり、また、３５０℃を超えると、ペンタメチレンジイソシアネートの重合など、好ましくない副反応を生じる場合がある。また、熱分解反応時の圧力は、上記の熱分解反応温度に対して、生成するアルコールが気化し得る圧力であることが好ましく、設備面および用役面から実用的には、０．１３３～９０ｋＰａであることが好ましい。 As such reaction conditions, more specifically, the thermal decomposition temperature is usually 350 ° C. or lower, preferably 80 to 350 ° C., more preferably 100 to 300 ° C. If it is lower than 80 ° C., a practical reaction rate may not be obtained, and if it exceeds 350 ° C., undesirable side reactions such as polymerization of pentamethylene diisocyanate may occur. The pressure at the time of the pyrolysis reaction is preferably a pressure at which the generated alcohol can be vaporized with respect to the above-mentioned pyrolysis reaction temperature. It is preferably 90 kPa.
　また、この熱分解に用いられるペンタメチレンジカルバメートは、精製したものでもよいが、上記反応（すなわち、ペンタメチレンジアミンと、尿素および／またはＮ－無置換カルバミン酸エステルと、アルコールとの反応）の終了後に、過剰（未反応）の尿素および／またはＮ－無置換カルバミン酸エステル、過剰（未反応）のアルコール、触媒、反応溶媒、副生するアンモニア、場合により副生するアルコールを回収して分離されたペンタメチレンジカルバメートの粗原料を用いて、引き続き熱分解してもよい。 The pentamethylene dicarbamate used for the thermal decomposition may be purified, but the above reaction (that is, reaction of pentamethylenediamine with urea and / or N-unsubstituted carbamic acid ester with alcohol). After completion, excess (unreacted) urea and / or N-unsubstituted carbamic acid ester, excess (unreacted) alcohol, catalyst, reaction solvent, by-product ammonia, and optionally by-product alcohol are recovered and separated. The resulting raw material of pentamethylene dicarbamate may be subsequently pyrolyzed.
　さらに、必要により、触媒および不活性溶媒を添加してもよい。これら触媒および不活性溶媒は、それらの種類により異なるが、上記反応時、反応後の蒸留分離の前後、ペンタメチレンジカルバメートの分離の前後の、いずれかに添加すればよい。 Further, if necessary, a catalyst and an inert solvent may be added. Although these catalysts and inert solvents differ depending on their types, they may be added to the above reaction either before or after distillation separation after the reaction or before or after separation of pentamethylene dicarbamate.
　熱分解に用いられる触媒としては、例えば、イソシアネートと水酸基とのウレタン化反応に用いられる、Ｓｎ、Ｓｂ、Ｆｅ、Ｃｏ、Ｎｉ、Ｃｕ、Ｚｎ、Ｃｒ、Ｔｉ、Ｐｂ、Ｍｏ、Ｍｎなどから選ばれる１種以上の金属単体またはその酸化物、ハロゲン化物、カルボン酸塩、リン酸塩、有機金属化合物などの金属化合物などが用いられる。これらのうち、この熱分解においては、Ｆｅ、Ｓｎ、Ｃｏ、Ｓｂ、Ｍｎが副生成物を生じにくくする効果を発現するため、好ましく用いられる。 The catalyst used for the thermal decomposition is selected from, for example, Sn, Sb, Fe, Co, Ni, Cu, Zn, Cr, Ti, Pb, Mo, Mn, etc. used in the urethanization reaction between isocyanate and hydroxyl group. One or more kinds of simple metals or their oxides, halides, carboxylates, phosphates, metal compounds such as organometallic compounds, and the like are used. Among these, in this thermal decomposition, Fe, Sn, Co, Sb, and Mn are preferably used because they exhibit the effect of making it difficult to generate by-products.
　Ｓｎの金属触媒としては、例えば、酸化スズ、塩化スズ、臭化スズ、ヨウ化スズ、ギ酸スズ、酢酸スズ、シュウ酸スズ、オクチル酸スズ、ステアリン酸スズ、オレイン酸スズ、リン酸スズ、二塩化ジブチルスズ、ジラウリン酸ジブチルスズ、１，１，３，３－テトラブチル－１，３－ジラウリルオキシジスタノキサンなどが挙げられる。 Examples of the Sn metal catalyst include tin oxide, tin chloride, tin bromide, tin iodide, tin formate, tin acetate, tin oxalate, tin octylate, tin stearate, tin oleate, tin phosphate, Examples include dibutyltin chloride, dibutyltin dilaurate, 1,1,3,3-tetrabutyl-1,3-dilauryloxydistanoxane.
　Ｆｅ、Ｃｏ、Ｓｂ、Ｍｎの金属触媒としては、例えば、それらの酢酸塩、安息香酸塩、ナフテン酸塩、アセチルアセトナート塩などが挙げられる。 Examples of the metal catalyst of Fe, Co, Sb, and Mn include acetates, benzoates, naphthenates, and acetylacetonates.
　なお、触媒の配合量は、金属単体またはその化合物として、反応液に対して、例えば、０．０００１～５質量％の範囲、好ましくは、０．００１～１質量％の範囲である。 The blending amount of the catalyst is, for example, in the range of 0.0001 to 5% by mass, preferably in the range of 0.001 to 1% by mass with respect to the reaction solution as a single metal or a compound thereof.
　また、不活性溶媒は、少なくとも、ペンタメチレンジカルバメートを溶解し、ペンタメチレンジカルバメートおよびイソシアネートに対して不活性であり、かつ、熱分解における温度において安定であれば、特に制限されないが、熱分解反応を効率よく実施するには、生成するイソシアネートよりも高沸点であることが好ましい。このような不活性溶媒としては、例えば、フタル酸ジオクチル、フタル酸ジデシル、フタル酸ジドデシルなどのエステル類、例えば、ジベンジルトルエン、トリフェニルメタン、フェニルナフタレン、ビフェニル、ジエチルビフェニル、トリエチルビフェニルなどの熱媒体として常用される芳香族系炭化水素や脂肪族系炭化水素などが挙げられる。 The inert solvent is not particularly limited as long as it dissolves at least pentamethylene dicarbamate, is inert to pentamethylene dicarbamate and isocyanate, and is stable at the temperature in thermal decomposition. In order to carry out the reaction efficiently, the boiling point is preferably higher than that of the isocyanate to be produced. Examples of such inert solvents include esters such as dioctyl phthalate, didecyl phthalate, and didodecyl phthalate, such as dibenzyltoluene, triphenylmethane, phenylnaphthalene, biphenyl, diethylbiphenyl, and triethylbiphenyl. Aromatic hydrocarbons and aliphatic hydrocarbons that are commonly used as media.
　また、不活性溶媒は、市販品としても入手可能であり、例えば、バーレルプロセス油Ｂ－０１（芳香族炭化水素類、沸点：１７６℃）、バーレルプロセス油Ｂ－０３（芳香族炭化水素類、沸点：２８０℃）、バーレルプロセス油Ｂ－０４ＡＢ（芳香族炭化水素類、沸点：２９４℃）、バーレルプロセス油Ｂ－０５（芳香族炭化水素類、沸点：３０２℃）、バーレルプロセス油Ｂ－２７（芳香族炭化水素類、沸点：３８０℃）、バーレルプロセス油Ｂ－２８ＡＮ（芳香族炭化水素類、沸点：４３０℃）、バーレルプロセス油Ｂ－３０（芳香族炭化水素類、沸点：３８０℃）、バーレルサーム２００（芳香族炭化水素類、沸点：３８２℃）、バーレルサーム３００（芳香族炭化水素類、沸点：３４４℃）、バーレルサーム４００（芳香族炭化水素類、沸点：３９０℃）、バーレルサーム１Ｈ（芳香族炭化水素類、沸点：２１５℃）、バーレルサーム２Ｈ（芳香族炭化水素類、沸点：２９４℃）、バーレルサーム３５０（芳香族炭化水素類、沸点：３０２℃）、バーレルサーム４７０（芳香族炭化水素類、沸点：３１０℃）、バーレルサームＰＡ（芳香族炭化水素類、沸点：１７６℃）、バーレルサーム３３０（芳香族炭化水素類、沸点：２５７℃）、バーレルサーム４３０（芳香族炭化水素類、沸点：２９１℃）、（以上、松村石油社製）、ＮｅｏＳＫ－ＯＩＬ１４００（芳香族炭化水素類、沸点：３９１℃）、ＮｅｏＳＫ－ＯＩＬ１３００（芳香族炭化水素類、沸点：２９１℃）、ＮｅｏＳＫ－ＯＩＬ３３０（芳香族炭化水素類、沸点：３３１℃）、ＮｅｏＳＫ－ＯＩＬ１７０（芳香族炭化水素類、沸点：１７６℃）、ＮｅｏＳＫ－ＯＩＬ２４０（芳香族炭化水素類、沸点：２４４℃）、ＫＳＫ－ＯＩＬ２６０（芳香族炭化水素類、沸点：２６６℃）、ＫＳＫ－ＯＩＬ２８０（芳香族炭化水素類、沸点：３０３℃）、（以上、綜研テクニックス社製）などが挙げられる。 The inert solvent is also available as a commercial product. For example, barrel process oil B-01 (aromatic hydrocarbons, boiling point: 176 ° C.), barrel process oil B-03 (aromatic hydrocarbons, Boiling point: 280 ° C.), barrel process oil B-04AB (aromatic hydrocarbons, boiling point: 294 ° C.), barrel process oil B-05 (aromatic hydrocarbons, boiling point: 302 ° C.), barrel process oil B-27 (Aromatic hydrocarbons, boiling point: 380 ° C), barrel process oil B-28AN (aromatic hydrocarbons, boiling point: 430 ° C), barrel process oil B-30 (aromatic hydrocarbons, boiling point: 380 ° C) , Barrel therm 200 (aromatic hydrocarbons, boiling point: 382 ° C.), barrel therm 300 (aromatic hydrocarbons, boiling point: 344 ° C.), barrel therm 400 (aromatic hydrocarbons) , Boiling point: 390 ° C.), barrel therm 1H (aromatic hydrocarbons, boiling point: 215 ° C.), barrel therm 2H (aromatic hydrocarbons, boiling point: 294 ° C.), barrel therm 350 (aromatic hydrocarbons, Boiling point: 302 ° C., barrel therm 470 (aromatic hydrocarbons, boiling point: 310 ° C.), barrel therm PA (aromatic hydrocarbons, boiling point: 176 ° C.), barrel therm 330 (aromatic hydrocarbons, boiling point: 257 ° C.), Barrel Therm 430 (aromatic hydrocarbons, boiling point: 291 ° C.), (Made by Matsumura Oil Co., Ltd.), NeoSK-OIL1400 (aromatic hydrocarbons, boiling point: 391 ° C.), NeoSK-OIL1300 (aromatic Group hydrocarbons, boiling point: 291 ° C), NeoSK-OIL330 (aromatic hydrocarbons, boiling point: 331 ° C), NeoSK-OIL170 (good) Aromatic hydrocarbons, boiling point: 176 ° C), NeoSK-OIL240 (aromatic hydrocarbons, boiling point: 244 ° C), KSK-OIL260 (aromatic hydrocarbons, boiling point: 266 ° C), KSK-OIL280 (aromatic carbonization) Hydrogen, boiling point: 303 ° C.) (above, manufactured by Soken Technics).
　不活性溶媒の配合量は、ペンタメチレンジカルバメート１質量部に対して、例えば、０．００１～１００質量部の範囲、好ましくは、０．０１～８０質量部、さらに好ましくは、０．１～５０質量部の範囲である。 The amount of the inert solvent is, for example, in the range of 0.001 to 100 parts by weight, preferably 0.01 to 80 parts by weight, and more preferably 0.1 to 100 parts by weight with respect to 1 part by weight of pentamethylene dicarbamate. The range is 50 parts by mass.
　また、この熱分解反応は、ペンタメチレンジカルバメート、触媒および不活性溶媒を一括で仕込む回分反応、また、触媒を含む不活性溶媒中に、減圧下でペンタメチレンジカルバメートを仕込んでいく連続反応のいずれでも実施することができる。 This thermal decomposition reaction is a batch reaction in which pentamethylene dicarbamate, a catalyst and an inert solvent are charged all at once, or a continuous reaction in which pentamethylene dicarbamate is charged in an inert solvent containing a catalyst under reduced pressure. Either can be implemented.
　また、熱分解では、ペンタメチレンジイソシアネートおよびアルコールが生成するとともに、副反応によって、例えば、アロファネート、アミン類、尿素、炭酸塩、カルバミン酸塩、二酸化炭素などが生成する場合があるため、必要により、得られたペンタメチレンジイソシアネートは、公知の方法により精製される。 In thermal decomposition, pentamethylene diisocyanate and alcohol are generated, and side reactions may generate, for example, allophanate, amines, urea, carbonate, carbamate, carbon dioxide, etc. The obtained pentamethylene diisocyanate is purified by a known method.
　また、カルバメート法としては、詳しくは述べないが、上記した尿素法の他、公知のカーボネート法、すなわち、ペンタメチレンジアミンと、炭酸ジアルキルあるいは炭酸ジアリールとからペンタメチレンジカルバメートを合成し、そのペンタメチレンジカルバメートを、上記と同様に熱分解して、ペンタメチレンジイソシアネートを得る方法などを採用することもできる。 The carbamate method is not described in detail, but besides the urea method described above, a known carbonate method, that is, pentamethylene dicarbamate is synthesized from pentamethylenediamine and dialkyl carbonate or diaryl carbonate, and the pentamethylene is synthesized. A method of obtaining pentamethylene diisocyanate by thermally decomposing dicarbamate in the same manner as described above can also be employed.
　このようにして得られる本発明のペンタメチレンジイソシアネートの純度は、例えば、９５～１００質量％、好ましくは、９７～１００質量％、さらに好ましくは９８～１００質量％、とりわけ好ましくは、９９～１００質量％、最も好ましくは、９９．５～１００質量％である。 The purity of the pentamethylene diisocyanate of the present invention thus obtained is, for example, 95 to 100% by mass, preferably 97 to 100% by mass, more preferably 98 to 100% by mass, and particularly preferably 99 to 100% by mass. %, Most preferably 99.5 to 100% by mass.
　また、ペンタメチレンジイソシアネートには、例えば、安定剤などを添加することができる。 Further, for example, a stabilizer or the like can be added to pentamethylene diisocyanate.
　安定剤としては、例えば、酸化防止剤、酸性化合物、スルホンアミド基を含有する化合物、有機亜リン酸エステルなどが挙げられる。 Examples of the stabilizer include an antioxidant, an acidic compound, a compound containing a sulfonamide group, and an organic phosphite.
　酸化防止剤としては、例えば、ヒンダードフェノール系酸化防止剤などが挙げられ、具体的には、例えば、２，６－ジ（ｔ－ブチル）－４－メチルフェノール、２，４，６－トリ－ｔ－ブチルフェノール、２，２’－メチレンビス－（４－メチル－６－ｔ－ブチルフェノール）、２，２’－チオ－ビス－（４－メチル－６－ｔ－ブチルフェノール）、４，４’－チオ－ビス（３－メチル－６－ｔ－ブチルフェノール）、４，４’－ブチリデン－ビス－（６－ｔ－ブチル－３－メチルフェノール）、４，４’－メチリデン－ビス－（２，６－ジ－ｔ－ブチルフェノール）、２，２’－メチレン－ビス－［４－メチル－６－（１－メチルシクロヘキシル）－フェノール］、テトラキス－［メチレン－３－（３，５－ジ－ｔ－ブチル－４－ヒドロキシフェニル）－プロピオニル］－メタン、１，３，５－トリメチル－２，４，６－トリス－（３，５－ジ－ｔ－ブチル－４－ヒドロキシフェニル）－プロピオニル－メタン、１，３，５－トリメチル－２，４，６－トリス－（３，５－ジ－ｔ－ブチル－４－ヒドロキシベンジル）－ベンゼン、Ｎ，Ｎ’－ヘキサメチレン－ビス－（３，５－ジ－ｔ－ブチル－４－ヒドロキシヒドロ桂皮酸アミド、１，３，５－トリス－（３，５－ジ－ｔ－ブチル－４－ヒドロキシベンジル）－イソシアヌレート、１，１，３－トリス－（５－ｔ－ブチル－４－ヒドロキシ－２－メチルフェニル）－ブタン、１，３，５－トリス－（３，５－ジ－ｔ－ブチル－４－ヒドロキシベンジル）－メシチレン、エチレングリコール－ビス－［３，３－ビス－（３’－ｔ－ブチルー４’－ヒドロキシフェニル）－ブチレート、２，２’－チオジエチル－ビス－３－（３，５－ジ－ｔ－ブチル－４－ヒドロキシフェニル）－プロピオネート、ジ－（３－ｔ－ブチル－４’－ヒドロキシ－５－メチルフェニル）－ジシクロペンタジエン、２，２’－メチレン－ビス－（４－メチル－６－シクロヘキシルフェノール）、１，６－ヘキサンジオール－ビス－（３，５－ジ－ｔ－ブチル－４－ヒドロキシフェニル）－プロピオネート、２，４－ビス－（ｎ－オクチルチオ）－６－（４－ヒドロキシ－３，５－ジ－ｔ－ブチルアニリノ）－１，３，５－トリアジン、ジエチル－３，５－ジ－ｔ－ブチル－４－ヒドロキシベジルホスホネート、トリエチレングリコール－ビス－３－（ｔ－ブチル－４－ヒドロキシ－５－メチルフェニル）－プロピオネート、さらには、例えば、ＩＲＧＡＮＯＸ１０１０、ＩＲＧＡＮＯＸ１０７６、ＩＲＧＡＮＯＸ１０９８、ＩＲＧＡＮＯＸ１１３５、ＩＲＧＡＮＯＸ１７２６、ＩＲＧＡＮＯＸ２４５、ＩＲＧＡＮＯＸ３１１４、ＩＲＧＡＮＯＸ３７９０（以上、ＢＡＳＦジャパン社製、商品名）などが挙げられる。 Examples of the antioxidant include hindered phenol antioxidants, and specific examples include 2,6-di (t-butyl) -4-methylphenol, 2,4,6-triphenol. -T-butylphenol, 2,2'-methylenebis- (4-methyl-6-t-butylphenol), 2,2'-thio-bis- (4-methyl-6-t-butylphenol), 4,4'- Thio-bis (3-methyl-6-t-butylphenol), 4,4'-butylidene-bis- (6-t-butyl-3-methylphenol), 4,4'-methylidene-bis- (2,6 -Di-t-butylphenol), 2,2'-methylene-bis- [4-methyl-6- (1-methylcyclohexyl) -phenol], tetrakis- [methylene-3- (3,5-di-t- Butyl-4-hydro Cyphenyl) -propionyl] -methane, 1,3,5-trimethyl-2,4,6-tris- (3,5-di-t-butyl-4-hydroxyphenyl) -propionyl-methane, 1,3,5 -Trimethyl-2,4,6-tris- (3,5-di-t-butyl-4-hydroxybenzyl) -benzene, N, N'-hexamethylene-bis- (3,5-di-t-butyl -4-hydroxyhydrocinnamic amide, 1,3,5-tris- (3,5-di-t-butyl-4-hydroxybenzyl) -isocyanurate, 1,1,3-tris- (5-t- Butyl-4-hydroxy-2-methylphenyl) -butane, 1,3,5-tris- (3,5-di-t-butyl-4-hydroxybenzyl) -mesitylene, ethylene glycol-bis- [3,3 -Bis- (3'-t Butyl-4′-hydroxyphenyl) -butyrate, 2,2′-thiodiethyl-bis-3- (3,5-di-t-butyl-4-hydroxyphenyl) -propionate, di- (3-t-butyl-4 '-Hydroxy-5-methylphenyl) -dicyclopentadiene, 2,2'-methylene-bis- (4-methyl-6-cyclohexylphenol), 1,6-hexanediol-bis- (3,5-di-) t-butyl-4-hydroxyphenyl) -propionate, 2,4-bis- (n-octylthio) -6- (4-hydroxy-3,5-di-t-butylanilino) -1,3,5-triazine, Diethyl-3,5-di-t-butyl-4-hydroxybezylphosphonate, triethylene glycol-bis-3- (t-butyl-4-hydroxy-5-methylphenol Enil) -propionate, and IRGANOX 1010, IRGANOX 1076, IRGANOX 1098, IRGANOX 1135, IRGANOX 1726, IRGANOX 245, IRGANOX 3114, IRGANOX 3790 (trade name, manufactured by BASF Japan Ltd.) and the like.
　これら酸化防止剤は、単独使用または２種類以上併用することができる。 These antioxidants can be used alone or in combination of two or more.
　酸性化合物としては、例えば、有機酸性化合物が挙げられ、具体的には、例えば、リン酸エステル、亜リン酸エステル、次亜リン酸エステル、ギ酸、酢酸、プロピオン酸、ヒドロキシ酢酸、シュウ酸、乳酸、クエン酸、リンゴ酸、スルホン酸、スルホン酸エステル、フェノール、エノール、イミド、オキシムなどが挙げられる。 Examples of the acidic compound include organic acidic compounds. Specifically, for example, phosphate ester, phosphite ester, hypophosphite ester, formic acid, acetic acid, propionic acid, hydroxyacetic acid, oxalic acid, lactic acid Citric acid, malic acid, sulfonic acid, sulfonic acid ester, phenol, enol, imide, oxime and the like.
　これら酸性化合物は、単独使用または２種類以上併用することができる。 These acidic compounds can be used alone or in combination of two or more.
　スルホンアミド基を含有する化合物としては、例えば、芳香族スルホンアミド類、脂肪族スルホンアミド類などが挙げられる。 Examples of the compound containing a sulfonamide group include aromatic sulfonamides and aliphatic sulfonamides.
　芳香族スルホンアミド類としては、例えば、ベンゼンスルホンアミド、ジメチルベンゼンスルホンアミド、スルファニルアミド、ｏ－およびｐ－トルエンスルホンアミド、ヒドロキシナフタレンスルホンアミド、ナフタレン－１－スルホンアミド、ナフタレン－２－スルホンアミド、ｍ－ニトロベンゼンスルホンアミド、ｐ－クロロベンゼンスルホンアミドなどが挙げられる。 Examples of aromatic sulfonamides include benzenesulfonamide, dimethylbenzenesulfonamide, sulfanilamide, o- and p-toluenesulfonamide, hydroxynaphthalenesulfonamide, naphthalene-1-sulfonamide, naphthalene-2-sulfonamide, Examples thereof include m-nitrobenzenesulfonamide and p-chlorobenzenesulfonamide.
　脂肪族スルホンアミド類としては、例えば、メタンスルホンアミド、Ｎ，Ｎ－ジメチルメタンスルホンアミド、Ｎ，Ｎ－ジメチルエタンスルホンアミド、Ｎ，Ｎ－ジエチルメタンスルホンアミド、Ｎ－メトキシメタンスルホンアミド、Ｎ－ドデシルメタンスルホンアミド、Ｎ－シクロヘキシル－１－ブタンスルホンアミド、２－アミノエタンスルホンアミドなどが挙げられる。 Examples of the aliphatic sulfonamides include methanesulfonamide, N, N-dimethylmethanesulfonamide, N, N-dimethylethanesulfonamide, N, N-diethylmethanesulfonamide, N-methoxymethanesulfonamide, N- Examples include dodecylmethanesulfonamide, N-cyclohexyl-1-butanesulfonamide, and 2-aminoethanesulfonamide.
　これらスルホンアミド基を含有する化合物は、単独使用または２種類以上併用することができる。 These compounds containing a sulfonamide group can be used alone or in combination of two or more.
　有機亜リン酸エステルとしては、例えば、有機亜リン酸ジエステル、有機亜リン酸トリエステルなどが挙げられ、より具体的には、例えば、トリエチルホスファイト、トリブチルホスファイト、トリス（２－エチルヘキシル）ホスファイト、トリデシルホスファイト、トリラウリルホスファイト、トリス（トリデシル）ホスファイト、トリステアリルホスファイト、トリフェニルホスファイト、トリス（ノニルフェニル）ホスファイト、トリス（２，４－ジ－ｔ－ブチルフェニル）ホスファイト、ジフェニルデシルホスファイト、ジフェニル（トリデシル）ホスファイトなどのモノフォスファイト類、例えば、ジステアリル・ペンタエリスリチル・ジホスファイト、ジ・ドデシル・ペンタエリスリトール・ジホスファイト、ジ・トリデシル・ペンタエリスリトール・ジホスファイト、ジノニルフェニル・ペンタエリスリトール・ジホスファイト、テトラフェニル・テトラ・トリデシル・ペンタエリスリチル・テトラホスファイト、テトラフェニル・ジプロピレングリコール・ジホスファイト、トリペンタエリスリトール・トリホスファイトなどの多価アルコールから誘導されたジ、トリあるいはテトラホスファイト類、さらに、例えば、炭素数が１～２０のジ・アルキル・ビスフェノールＡ・ジホスファイト、４，４’－ブチリデン－ビス（３－メチル－６－ｔ－ブチルフェニル－ジ・トリデシル）ホスファイトなどのビスフェノール系化合物から誘導されたジホスファイト類、例えば、水添ビスフェノールＡホスファイトポリマー（分子量２４００～３０００）などのポリホスファイト類、例えば、トリス（２，３－ジクロロプロピル）ホスファイトなどが挙げられる。 Examples of organic phosphites include organic phosphite diesters and organic phosphite triesters, and more specifically, for example, triethyl phosphite, tributyl phosphite, tris (2-ethylhexyl) phosphine. Phyto, tridecyl phosphite, trilauryl phosphite, tris (tridecyl) phosphite, tristearyl phosphite, triphenyl phosphite, tris (nonylphenyl) phosphite, tris (2,4-di-t-butylphenyl) Monophosphites such as phosphite, diphenyldecyl phosphite, diphenyl (tridecyl) phosphite, such as distearyl pentaerythrityl diphosphite, di-dodecyl pentaerythritol diphosphite, di-tridecyl From polyhydric alcohols such as pentaerythritol diphosphite, dinonylphenyl pentaerythritol diphosphite, tetraphenyl tetratetradecyl pentaerythrityl tetraphosphite, tetraphenyl dipropylene glycol diphosphite, tripentaerythritol triphosphite Di-, tri- or tetraphosphites derived, for example, dialkyl bisphenol A diphosphite having 1 to 20 carbon atoms, 4,4′-butylidene-bis (3-methyl-6-t-butyl) Diphosphites derived from bisphenolic compounds such as phenyl-di-tridecyl) phosphite, for example polyphosphines such as hydrogenated bisphenol A phosphite polymer (molecular weight 2400-3000) Aito include, for example, tris (2,3-dichloro-propyl) phosphite and the like.
　これら有機亜リン酸エステルは、単独使用または２種類以上併用することができる。 These organic phosphites can be used alone or in combination of two or more.
　安定剤として、好ましくは、酸化防止剤、酸性化合物、スルホンアミド基を含有する化合物が挙げられる。さらに好ましくは、ペンタメチレンジイソシアネートに、酸化防止剤と、酸性化合物および／またはスルホンアミド基を含有する化合物とを配合し、含有させる。 The stabilizer preferably includes an antioxidant, an acidic compound, and a compound containing a sulfonamide group. More preferably, pentamethylene diisocyanate is mixed with an antioxidant and an acidic compound and / or a compound containing a sulfonamide group.
　これら安定剤を添加することにより、そのペンタメチレンジイソシアネートを用いて得られるイソシアネート変性体（後述）の、貯蔵安定性の向上を図ることができる。 By adding these stabilizers, it is possible to improve the storage stability of an isocyanate-modified product (described later) obtained using the pentamethylene diisocyanate.
　なお、安定剤の配合割合は、特に制限されず、必要および用途に応じて、適宜設定される。 In addition, the mixing ratio of the stabilizer is not particularly limited, and is appropriately set according to necessity and application.
　具体的には、酸化防止剤の配合割合は、ペンタメチレンジイソシアネート１００質量部に対して、例えば、０．０００５～０．０５質量部である。 Specifically, the blending ratio of the antioxidant is, for example, 0.0005 to 0.05 parts by mass with respect to 100 parts by mass of pentamethylene diisocyanate.
　また、酸性化合物および／またはスルホンアンド基を含有する化合物の配合割合（併用される場合には、それらの総量）は、ペンタメチレンジイソシアネート１００質量部に対して、例えば、０．０００５～０．０２質量部である。
（１３）ポリイソシアネート組成物の製造方法
　また、本発明は、さらに、ポリイソシアネート組成物の製造方法を含んでいる。 In addition, the compounding ratio of the acidic compound and / or the compound containing a sulfone and group (the total amount thereof when used in combination) is, for example, 0.0005 to 0.02 with respect to 100 parts by mass of pentamethylene diisocyanate. Part by mass.
(13) Method for Producing Polyisocyanate Composition The present invention further includes a method for producing a polyisocyanate composition.
　ポリイソシアネート組成物は、より具体的には、ペンタメチレンジイソシアネートを変性することにより得られ、下記（ａ）～（ｅ）の官能基を少なくとも１種含有している。
（ａ）イソシアヌレート基
（ｂ）アロファネート基
（ｃ）ビウレット基
（ｄ）ウレタン基
（ｅ）ウレア基
　上記（ａ）の官能基（イソシアヌレート基）を含有するポリイソシアネート組成物は、ペンタメチレンジイソシアネートのトリマー（三量体）であって、例えば、ペンタメチレンジイソシアネートを公知のイソシアヌレート化触媒の存在下において反応させ、三量化することにより、得ることができる。 More specifically, the polyisocyanate composition is obtained by modifying pentamethylene diisocyanate, and contains at least one of the following functional groups (a) to (e).
(A) isocyanurate group (b) allophanate group (c) biuret group (d) urethane group (e) urea group The polyisocyanate composition containing the functional group (isocyanurate group) of (a) is pentamethylene diisocyanate. The trimer (trimer) can be obtained by, for example, reacting pentamethylene diisocyanate in the presence of a known isocyanuration catalyst and trimerization.
　上記（ｂ）の官能基（アロファネート基）を含有するポリイソシアネート組成物は、ペンタメチレンジイソシアネートのアロファネート変性体であって、例えば、ペンタメチレンジイソシアネートとモノアルコールとを反応させた後、公知のアロファネート化触媒の存在下でさらに反応させることにより、得ることができる。 The polyisocyanate composition containing the functional group (allophanate group) of the above (b) is an allophanate modified product of pentamethylene diisocyanate, and, for example, after reacting pentamethylene diisocyanate with a monoalcohol, a known allophanate is formed. It can be obtained by further reaction in the presence of a catalyst.
　上記（ｃ）の官能基（ビウレット基）を含有するポリイソシアネート組成物は、ペンタメチレンジイソシアネートのビウレット変性体であって、例えば、ペンタメチレンジイソシアネートと、例えば、水、第三級アルコール（例えば、ｔ－ブチルアルコールなど）、第二級アミン（例えば、ジメチルアミン、ジエチルアミンなど）などとを反応させた後、公知のビウレット化触媒の存在下でさらに反応させることにより、得ることができる。 The polyisocyanate composition containing the functional group (biuret group) of the above (c) is a biuret-modified product of pentamethylene diisocyanate, for example, pentamethylene diisocyanate, for example, water, tertiary alcohol (for example, t -Butyl alcohol, etc.), secondary amines (eg, dimethylamine, diethylamine, etc.) and the like, and then further reacted in the presence of a known biuretization catalyst.
　上記（ｂ）の官能基（ウレタン基）を含有するポリイソシアネート組成物は、ペンタメチレンジイソシアネートのポリオール変性体であって、例えば、ペンタメチレンジイソシアネートとポリオール成分（例えば、トリメチロールプロパンなど。詳しくは後述）との反応により、得ることができる。 The polyisocyanate composition containing the functional group (urethane group) of (b) is a polyol-modified product of pentamethylene diisocyanate, for example, pentamethylene diisocyanate and a polyol component (for example, trimethylolpropane, etc. ).
　上記（ｅ）の官能基（ウレア基）を含有するポリイソシアネート組成物は、ペンタメチレンジイソシアネートのポリアミン変性体であって、例えば、ペンタメチレンジイソシアネートと水、ポリアミン成分（後述）などとの反応により、得ることができる。 The polyisocyanate composition containing the functional group (urea group) of (e) is a polyamine-modified product of pentamethylene diisocyanate, and for example, by reaction of pentamethylene diisocyanate with water, a polyamine component (described later), Obtainable.
　なお、ポリイソシアネート組成物は、上記（ａ）～（ｅ）の官能基を少なくとも１種含有していればよく、２種以上含有することもできる。そのようなポリイソシアネート組成物は、上記の反応を適宜併用することにより、生成される。 The polyisocyanate composition only needs to contain at least one of the functional groups (a) to (e) described above, and may contain two or more. Such a polyisocyanate composition is produced by appropriately combining the above reactions.
　ポリイソシアネート組成物として、好ましくは、ペンタメチレンジイソシアネートのトリマー（イソシアヌレート基を含有するポリイソシアネート組成物）が挙げられる。 Preferred examples of the polyisocyanate composition include pentamethylene diisocyanate trimer (polyisocyanate composition containing isocyanurate group).
　なお、ペンタメチレンジイソシアネートのトリマーは、イソシアヌレート基の他、さらに、イミノオキサジアジンジオン基などを有するポリイソシアネートを、含んでいる。 The trimer of pentamethylene diisocyanate contains polyisocyanate having an iminooxadiazinedione group in addition to the isocyanurate group.
　そして、上記のペンタメチレンジイソシアネート、および／または、上記のポリイソシアネート組成物と、活性水素化合物とを反応させることにより、ポリウレタン樹脂を得ることができる。 A polyurethane resin can be obtained by reacting the above pentamethylene diisocyanate and / or the above polyisocyanate composition with an active hydrogen compound.
　活性水素化合物としては、例えば、ポリオール成分（水酸基を２つ以上有するポリオールを主として含有する成分）、ポリアミン成分（アミノ基を２つ以上有するポリアミンを主として含有する化合物）などが挙げられる。 Examples of the active hydrogen compound include a polyol component (a component mainly containing a polyol having two or more hydroxyl groups), a polyamine component (a compound mainly containing a polyamine having two or more amino groups), and the like.
　本発明において、ポリオール成分としては、低分子量ポリオールおよび高分子量ポリオールが挙げられる。 In the present invention, examples of the polyol component include low molecular weight polyols and high molecular weight polyols.
　低分子量ポリオールは、水酸基を２つ以上有する数平均分子量４００未満の化合物であって、例えば、エチレングリコール、プロピレングリコール、１，３－プロパンジオール、１，４－ブチレングリコール、１，３－ブチレングリコール、１，２－ブチレングリコール、１，５－ペンタンジオール、１，６－ヘキサンジオール、ネオペンチルグリコール、３－メチル－１，５－ペンタンジオール、２，２，２－トリメチルペンタンジオール、３，３－ジメチロールヘプタン、アルカン（炭素数７～２０）ジオール、１，３－または１，４－シクロヘキサンジメタノールおよびそれらの混合物、１，３－または１，４－シクロヘキサンジオールおよびそれらの混合物、水素化ビスフェノールＡ、１，４－ジヒドロキシ－２－ブテン、２，６－ジメチル－１－オクテン－３，８－ジオール、ビスフェノールＡ、ジエチレングリコール、トリエチレングリコール、ジプロピレングリコールなどの２価アルコール、例えば、グリセリン、トリメチロールプロパンなどの３価アルコール、例えば、テトラメチロールメタン（ペンタエリスリトール）、ジグリセリンなどの４価アルコール、例えば、キシリトールなどの５価アルコール、例えば、ソルビトール、マンニトール、アリトール、イジトール、ダルシトール、アルトリトール、イノシトール、ジペンタエリスリトールなどの６価アルコール、例えば、ペルセイトールなどの７価アルコール、例えば、ショ糖などの８価アルコールなどが挙げられる。 The low molecular weight polyol is a compound having two or more hydroxyl groups and a number average molecular weight of less than 400, such as ethylene glycol, propylene glycol, 1,3-propanediol, 1,4-butylene glycol, 1,3-butylene glycol. 1,2-butylene glycol, 1,5-pentanediol, 1,6-hexanediol, neopentyl glycol, 3-methyl-1,5-pentanediol, 2,2,2-trimethylpentanediol, 3,3 Dimethylol heptane, alkane (carbon number 7-20) diol, 1,3- or 1,4-cyclohexanedimethanol and mixtures thereof, 1,3- or 1,4-cyclohexanediol and mixtures thereof, hydrogenation Bisphenol A, 1,4-dihydroxy-2-butene, 2,6 Dihydric alcohols such as dimethyl-1-octene-3,8-diol, bisphenol A, diethylene glycol, triethylene glycol and dipropylene glycol, for example, trihydric alcohols such as glycerin and trimethylolpropane, such as tetramethylolmethane (penta Erythritol), tetrahydric alcohols such as diglycerin, for example, pentahydric alcohols such as xylitol, for example, hexahydric alcohols such as sorbitol, mannitol, allitol, iditol, dulcitol, altritol, inositol, dipentaerythritol, for example, perseitol, etc. For example, octavalent alcohols such as sucrose.
　これら低分子量ポリオールは、単独使用または２種類以上併用することができる。 These low molecular weight polyols can be used alone or in combination of two or more.
　高分子量ポリオールは、水酸基を２つ以上有する数平均分子量４００以上の化合物であって、例えば、ポリエーテルポリオール、ポリエステルポリオール、ポリカーボネートポリオール、ポリウレタンポリオール、エポキシポリオール、植物油ポリオール、ポリオレフィンポリオール、アクリルポリオール、ビニルモノマー変性ポリオールなどが挙げられる。 The high molecular weight polyol is a compound having two or more hydroxyl groups and a number average molecular weight of 400 or more. For example, polyether polyol, polyester polyol, polycarbonate polyol, polyurethane polyol, epoxy polyol, vegetable oil polyol, polyolefin polyol, acrylic polyol, vinyl A monomer modified polyol etc. are mentioned.
　ポリエーテルポリオールとしては、例えば、ポリプロピレングリコール、ポリテトラメチレンエーテルグリコールなどが挙げられる。 Examples of the polyether polyol include polypropylene glycol and polytetramethylene ether glycol.
　ポリプロピレングリコールとしては、例えば、上記した低分子量ポリオールまたは芳香族／脂肪族ポリアミンを開始剤とする、エチレンオキサイド、プロピレンオキサイドなどのアルキレンオキサイドの付加重合物（２種以上のアルキレンオキサイドのランダムおよび／またはブロック共重合体を含む。）などが挙げられる。 Examples of the polypropylene glycol include addition polymers of alkylene oxides such as ethylene oxide and propylene oxide (the random and / or two or more types of alkylene oxides) using the above-described low molecular weight polyols or aromatic / aliphatic polyamines as initiators. Including a block copolymer).
　ポリテトラメチレンエーテルグリコールとしては、例えば、テトラヒドロフランのカチオン重合により得られる開環重合物や、テトラヒドロフランの重合単位に上記した２価アルコールを共重合した非晶性ポリテトラメチレンエーテルグリコールなどが挙げられる。　　　 Examples of the polytetramethylene ether glycol include a ring-opening polymer obtained by cationic polymerization of tetrahydrofuran, and amorphous polytetramethylene ether glycol obtained by copolymerizing the above-described dihydric alcohol with a polymerization unit of tetrahydrofuran. *
　ポリエステルポリオールとしては、例えば、上記した低分子量ポリオールと多塩基酸とを、公知の条件下、反応させて得られる重縮合物が挙げられる。 Examples of the polyester polyol include polycondensates obtained by reacting the above-described low molecular weight polyol and polybasic acid under known conditions.
　多塩基酸としては、例えば、シュウ酸、マロン酸、コハク酸、メチルコハク酸、グルタール酸、アジピン酸、１，１－ジメチル－１，３－ジカルボキシプロパン、３－メチル－３－エチルグルタール酸、アゼライン酸、セバシン酸、その他の飽和脂肪族ジカルボン酸（Ｃ１１～１３）、例えば、マレイン酸、フマル酸、イタコン酸、その他の不飽和脂肪族ジカルボン酸、例えば、オルソフタル酸、イソフタル酸、テレフタル酸、トルエンジカルボン酸、ナフタレンジカルボン酸、その他の芳香族ジカルボン酸、例えば、ヘキサヒドロフタル酸、その他の脂環族ジカルボン酸、例えば、ダイマー酸、水添ダイマー酸、ヘット酸などのその他のカルボン酸、および、それらカルボン酸から誘導される酸無水物、例えば、無水シュウ酸、無水コハク酸、無水マレイン酸、無水フタル酸、無水２－アルキル（Ｃ１２～Ｃ１８）コハク酸、無水テトラヒドロフタル酸、無水トリメリット酸、さらには、これらのカルボン酸などから誘導される酸ハライド、例えば、シュウ酸ジクロライド、アジピン酸ジクロライド、セバシン酸ジクロライドなどが挙げられる。 Examples of the polybasic acid include oxalic acid, malonic acid, succinic acid, methyl succinic acid, glutaric acid, adipic acid, 1,1-dimethyl-1,3-dicarboxypropane, 3-methyl-3-ethylglutaric acid , Azelaic acid, sebacic acid, other saturated aliphatic dicarboxylic acids (C11-13) such as maleic acid, fumaric acid, itaconic acid, other unsaturated aliphatic dicarboxylic acids such as orthophthalic acid, isophthalic acid, terephthalic acid , Toluene dicarboxylic acid, naphthalene dicarboxylic acid, other aromatic dicarboxylic acids such as hexahydrophthalic acid, other alicyclic dicarboxylic acids such as dimer acid, hydrogenated dimer acid, het acid and other carboxylic acids, And acid anhydrides derived from these carboxylic acids, such as oxalic anhydride, succinic anhydride Acid, maleic anhydride, phthalic anhydride, 2-alkyl (C12-C18) succinic anhydride, tetrahydrophthalic anhydride, trimellitic anhydride, and acid halides derived from these carboxylic acids, such as sulphur Acid dichloride, adipic acid dichloride, sebacic acid dichloride and the like can be mentioned.
　また、ポリエステルポリオールとして、例えば、植物由来のポリエステルポリオール、具体的には、上記した低分子量ポリオールを開始剤として、ヒドロキシル基含有植物油脂肪酸（例えば、リシノレイン酸を含有するひまし油脂肪酸、１２－ヒドロキシステアリン酸を含有する水添ひまし油脂肪酸など）などのヒドロキシカルボン酸を、公知の条件下、縮合反応させて得られる植物油系ポリエステルポリオールなどが挙げられる。 Further, as the polyester polyol, for example, a plant-derived polyester polyol, specifically, a hydroxyl group-containing vegetable oil fatty acid (for example, castor oil fatty acid containing ricinoleic acid, 12-hydroxystearic acid, using the above-described low molecular weight polyol as an initiator, And vegetable oil-based polyester polyols obtained by subjecting a hydroxycarboxylic acid such as hydrogenated castor oil fatty acid and the like to a condensation reaction under known conditions.
　また、ポリエステルポリオールとして、例えば、上記した低分子量ポリオール（好ましくは、２価アルコール）を開始剤として、例えば、ε－カプロラクトン、γ－バレロラクトンなどのラクトン類や、例えば、Ｌ－ラクチド、Ｄ－ラクチドなどのラクチド類などを開環重合して得られる、ポリカプロラクトンポリオール、ポリバレロラクトンポリオール、さらには、それらに上記した２価アルコールを共重合したラクトン系ポリエステルポリオールなどが挙げられる。 Further, as the polyester polyol, for example, the above-described low molecular weight polyol (preferably dihydric alcohol) is used as an initiator, for example, lactones such as ε-caprolactone and γ-valerolactone, for example, L-lactide, D- Examples thereof include polycaprolactone polyol, polyvalerolactone polyol obtained by ring-opening polymerization of lactides such as lactide, and lactone polyester polyol obtained by copolymerizing the above-described dihydric alcohol.
　ポリカーボネートポリオールとしては、例えば、上記した低分子量ポリオール（好ましくは、２価アルコール）を開始剤とするエチレンカーボネートの開環重合物や、例えば、１，４－ブタンジオール、１，５－ペンタンジオール、３－メチル－１，５－ペンタンジオールや１，６－ヘキサンジオールなどの２価アルコールと、開環重合物とを共重合した非晶性ポリカーボネートポリオールなどが挙げられる。 Examples of the polycarbonate polyol include a ring-opening polymer of ethylene carbonate using the above-described low molecular weight polyol (preferably a dihydric alcohol) as an initiator, for example, 1,4-butanediol, 1,5-pentanediol, Examples thereof include amorphous polycarbonate polyols obtained by copolymerizing a dihydric alcohol such as 3-methyl-1,5-pentanediol and 1,6-hexanediol with a ring-opening polymer.
　また、ポリウレタンポリオールは、例えば、上記により得られたポリエステルポリオール、ポリエーテルポリオールおよび／またはポリカーボネートポリオールを、イソシアネート基（ＮＣＯ）に対する水酸基（ＯＨ）の当量比（ＯＨ／ＮＣＯ）が１を超過する割合で、ポリイソシアネートと反応させることによって、ポリエステルポリウレタンポリオール、ポリエーテルポリウレタンポリオール、ポリカーボネートポリウレタンポリオール、あるいは、ポリエステルポリエーテルポリウレタンポリオールなどとして得ることができる。 The polyurethane polyol is, for example, a ratio in which the equivalent ratio (OH / NCO) of the hydroxyl group (OH) to the isocyanate group (NCO) of the polyester polyol, polyether polyol and / or polycarbonate polyol obtained as described above exceeds 1. By reacting with polyisocyanate, polyester polyurethane polyol, polyether polyurethane polyol, polycarbonate polyurethane polyol, or polyester polyether polyurethane polyol can be obtained.
　エポキシポリオールとしては、例えば、上記した低分子量ポリオール、例えば、エピクロルヒドリン、β－メチルエピクロルヒドリンなどの多官能ハロヒドリンとの反応により得られるエポキシポリオールなどが挙げられる。 Examples of the epoxy polyol include the low molecular weight polyols described above, for example, epoxy polyols obtained by reaction with polyfunctional halohydrins such as epichlorohydrin and β-methylepichlorohydrin.
　植物油ポリオールとしては、例えば、ひまし油、やし油などのヒドロキシル基含有植物油などが挙げられる。例えば、ひまし油ポリオール、または、ひまし油脂肪酸とポリプロピレンポリオールとの反応により得られるエステル変性ひまし油ポリオールなどが挙げられる。 Examples of the vegetable oil polyol include hydroxyl group-containing vegetable oils such as castor oil and palm oil. For example, castor oil polyol, or ester-modified castor oil polyol obtained by reaction of castor oil fatty acid and polypropylene polyol can be used.
　ポリオレフィンポリオールとしては、例えば、ポリブタジエンポリオール、部分ケン価エチレン－酢酸ビニル共重合体などが挙げられる。 Examples of the polyolefin polyol include polybutadiene polyol, partially saponified ethylene-vinyl acetate copolymer, and the like.
　アクリルポリオールとしては、例えば、ヒドロキシル基含有アクリレートと、ヒドロキシル基含有アクリレートと共重合可能な共重合性ビニルモノマーとを、共重合させることによって得られる共重合体などが挙げられる。 Examples of the acrylic polyol include a copolymer obtained by copolymerizing a hydroxyl group-containing acrylate and a copolymerizable vinyl monomer copolymerizable with the hydroxyl group-containing acrylate.
　ヒドロキシル基含有アクリレートとしては、例えば、２－ヒドロキシエチル（メタ）アクリレート、ヒドロキシプロピル（メタ）アクリレート、ヒドロキシブチル（メタ）アクリレート、２，２－ジヒドロキシメチルブチル（メタ）アクリレート、ポリヒドロキシアルキルマレエート、ポリヒドロキシアルキルフマレートなどが挙げられる。好ましくは、２－ヒドロキシエチル（メタ）アクリレートなどが挙げられる。 Examples of hydroxyl group-containing acrylates include 2-hydroxyethyl (meth) acrylate, hydroxypropyl (meth) acrylate, hydroxybutyl (meth) acrylate, 2,2-dihydroxymethylbutyl (meth) acrylate, polyhydroxyalkyl maleate, Examples thereof include polyhydroxyalkyl fumarate. Preferable examples include 2-hydroxyethyl (meth) acrylate.
　共重合性ビニルモノマーとしては、例えば、メチル（メタ）アクリレート、エチル（メタ）アクリレート、プロピル（メタ）アクリレート、イソプロピル（メタ）アクリレート、ブチル（メタ）アクリレート、イソブチル（メタ）アクリレート、ｓ－ブチル（メタ）アクリレート、ｔ－ブチル（メタ）アクリレート、ペンチル（メタ）アクリレート、イソペンチル（メタ）アクリレート、ヘキシル（メタ）アクリレート、イソノニル（メタ）アクリレート、２－エチルヘキシル（メタ）アクリレート、シクロヘキシルアクリレートなどのアルキル（メタ）アクリレート（炭素数１～１２）、例えば、スチレン、ビニルトルエン、α－メチルスチレンなどの芳香族ビニル、例えば、（メタ）アクリロニトリルなどのシアン化ビニル、例えば、（メタ）アクリル酸、フマル酸、マレイン酸、イタコン酸などのカルボキシル基を含むビニルモノマー、または、そのアルキルエステル、例えば、エチレングリコールジ（メタ）アクリレート、ブチレングリコールジ（メタ）アクリレート、ヘキサンジオールジ（メタ）アクリレート、オリゴエチレングリコールジ（メタ）アクリレート、トリメチロールプロパンジ（メタ）アクリレート、トリメチロールプロパントリ（メタ）アクリレートなどのアルカンポリオールポリ（メタ）アクリレート、例えば、３－（２－イソシアネート－２－プロピル）－α－メチルスチレンなどのイソシアネート基を含むビニルモノマーなどが挙げられる。 Examples of the copolymerizable vinyl monomer include methyl (meth) acrylate, ethyl (meth) acrylate, propyl (meth) acrylate, isopropyl (meth) acrylate, butyl (meth) acrylate, isobutyl (meth) acrylate, s-butyl ( Alkyl (meth) acrylate, t-butyl (meth) acrylate, pentyl (meth) acrylate, isopentyl (meth) acrylate, hexyl (meth) acrylate, isononyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, cyclohexyl acrylate, etc. (Meth) acrylate (having 1 to 12 carbon atoms), for example, aromatic vinyl such as styrene, vinyltoluene and α-methylstyrene, vinyl cyanide such as (meth) acrylonitrile, Vinyl monomers containing carboxyl groups such as (meth) acrylic acid, fumaric acid, maleic acid, itaconic acid, or alkyl esters thereof such as ethylene glycol di (meth) acrylate, butylene glycol di (meth) acrylate, hexanediol di ( Alkane polyol poly (meth) acrylates such as meth) acrylate, oligoethylene glycol di (meth) acrylate, trimethylolpropane di (meth) acrylate, trimethylolpropane tri (meth) acrylate, for example 3- (2-isocyanate-2 And vinyl monomers containing an isocyanate group such as -propyl) -α-methylstyrene.
　そして、アクリルポリオールは、これらヒドロキシル基含有アクリレート、および、共重合性ビニルモノマーを、適当な溶剤および重合開始剤の存在下において共重合させることにより得ることができる。 The acrylic polyol can be obtained by copolymerizing these hydroxyl group-containing acrylate and copolymerizable vinyl monomer in the presence of a suitable solvent and a polymerization initiator.
　また、その他、例えば、シリコーンポリオールやフッ素ポリオールが挙げられる。 In addition, other examples include silicone polyols and fluorine polyols.
　シリコーンポリオールとしては、例えば、上記したアクリルポリオールの共重合において、共重合性ビニルモノマーとして、例えば、γ－メタクリロキシプロピルトリメトキシシランなどのビニル基を含むシリコーン化合物が配合されたアクリルポリオールが挙げられる。 Examples of the silicone polyol include an acrylic polyol in which a silicone compound containing a vinyl group such as γ-methacryloxypropyltrimethoxysilane is blended as the copolymerizable vinyl monomer in the copolymerization of the acrylic polyol described above. .
　フッ素ポリオールとしては、例えば、上記したアクリルポリオールの共重合において、共重合性ビニルモノマーとして、例えば、テトラフルオロエチレン、クロロトリフルオロエチレンなどのビニル基を含むフッ素化合物が配合されたアクリルポリオールが挙げられる。 As the fluorine polyol, for example, in the copolymerization of the acrylic polyol described above, as the copolymerizable vinyl monomer, for example, an acrylic polyol in which a fluorine compound containing a vinyl group such as tetrafluoroethylene or chlorotrifluoroethylene is blended may be mentioned. .
　ビニルモノマー変性ポリオールは、上記した高分子量ポリオールと、ビニルモノマーとの反応により得ることができる。 The vinyl monomer-modified polyol can be obtained by a reaction between the above-described high molecular weight polyol and a vinyl monomer.
　高分子量ポリオールとして、好ましくは、ポリエーテルポリオール、ポリエステルポリオールおよびポリカーボネートポリオールから選択される高分子量ポリオールが挙げられる。 The high molecular weight polyol is preferably a high molecular weight polyol selected from polyether polyol, polyester polyol and polycarbonate polyol.
　また、ビニルモノマーとしては、例えば、上記したアルキル（メタ）アクリレート、シアン化ビニルまたはシアン化ビニリデンなどが挙げられる。これらビニルモノマーは、単独使用または２種類以上併用することができる。また、これらのうち、好ましくは、アルキル（メタ）アクリレートが挙げられる。 Further, examples of the vinyl monomer include the above-described alkyl (meth) acrylate, vinyl cyanide, vinylidene cyanide, and the like. These vinyl monomers can be used alone or in combination of two or more. Of these, alkyl (meth) acrylate is preferable.
　そして、ビニルモノマー変性ポリオールは、これら高分子量ポリオール、および、ビニルモノマーを、例えば、ラジカル重合開始剤（例えば、過硫酸塩、有機過酸化物、アゾ系化合物など）の存在下などにおいて反応させることにより得ることができる。 The vinyl monomer-modified polyol is obtained by reacting these high molecular weight polyol and vinyl monomer in the presence of a radical polymerization initiator (for example, persulfate, organic peroxide, azo compound, etc.), for example. Can be obtained.
　これら高分子量ポリオールは、単独使用または２種類以上併用することができる。 These high molecular weight polyols can be used alone or in combination of two or more.
　高分子量ポリオールとして、好ましくは、ポリエステルポリオール、アクリルポリオールが挙げられ、さらに好ましくは、ポリエステルポリオールが挙げられ、とりわけ好ましくは、植物由来のポリエステルポリオールが挙げられる。 The high molecular weight polyol is preferably a polyester polyol or an acrylic polyol, more preferably a polyester polyol, and particularly preferably a plant-derived polyester polyol.
　これらポリオール成分は、単独使用または２種類以上併用することができる。 These polyol components can be used alone or in combination of two or more.
　ポリアミン成分としては、例えば、芳香族ポリアミン、芳香脂肪族ポリアミン、脂環族ポリアミン、脂肪族ポリアミン、アミノアルコール、第１級アミノ基、または、第１級アミノ基および第２級アミノ基を有するアルコキシシリル化合物、ポリオキシエチレン基含有ポリアミンなどが挙げられる。 Examples of the polyamine component include an aromatic polyamine, an araliphatic polyamine, an alicyclic polyamine, an aliphatic polyamine, an amino alcohol, a primary amino group, or an alkoxy having a primary amino group and a secondary amino group. Examples thereof include silyl compounds and polyoxyethylene group-containing polyamines.
　芳香族ポリアミンとしては、例えば、４，４’－ジフェニルメタンジアミン、トリレンジアミンなどが挙げられる。 Examples of aromatic polyamines include 4,4'-diphenylmethanediamine and tolylenediamine.
　芳香脂肪族ポリアミンとしては、例えば、１，３－もしくは１，４－キシリレンジアミンまたはその混合物などが挙げられる。 Examples of the araliphatic polyamine include 1,3- or 1,4-xylylenediamine or a mixture thereof.
　脂環族ポリアミンとしては、例えば、３－アミノメチル－３，５，５－トリメチルシクロヘキシルアミン（別名：イソホロンジアミン）、４，４’－ジシクロヘキシルメタンジアミン、２，５（２，６）－ビス（アミノメチル）ビシクロ［２．２．１］ヘプタン、１，４－シクロヘキサンジアミン、１－アミノ－３－アミノメチル－３，５，５－トリメチルシクロヘキサン、ビス－（４－アミノシクロヘキシル）メタン、ジアミノシクロヘキサン、３，９－ビス（３－アミノプロピル）－２，４，８，１０－テトラオキサスピロ［５．５］ウンデカン、１，３－および１，４－ビス（アミノメチル）シクロヘキサンおよびそれらの混合物などが挙げられる。 Examples of the alicyclic polyamine include 3-aminomethyl-3,5,5-trimethylcyclohexylamine (also known as isophoronediamine), 4,4′-dicyclohexylmethanediamine, 2,5 (2,6) -bis ( Aminomethyl) bicyclo [2.2.1] heptane, 1,4-cyclohexanediamine, 1-amino-3-aminomethyl-3,5,5-trimethylcyclohexane, bis- (4-aminocyclohexyl) methane, diaminocyclohexane 3,9-bis (3-aminopropyl) -2,4,8,10-tetraoxaspiro [5.5] undecane, 1,3- and 1,4-bis (aminomethyl) cyclohexane and mixtures thereof Etc.
　脂肪族ポリアミンとしては、例えば、エチレンジアミン、プロピレンジアミン、１，３－プロパンジアミン、１，４－ブタンジアミン、１，５－ペンタンジアミン、１，６－ヘキサメチレンジアミン、ヒドラジン（水和物を含む。）、ジエチレントリアミン、トリエチレンテトラミン、テトラエチレンペンタミン、１，２－ジアミノエタン、１，２－ジアミノプロパン、１，３－ジアミノペンタンなどが挙げられる。 Examples of the aliphatic polyamine include ethylenediamine, propylenediamine, 1,3-propanediamine, 1,4-butanediamine, 1,5-pentanediamine, 1,6-hexamethylenediamine, and hydrazine (including hydrates). ), Diethylenetriamine, triethylenetetramine, tetraethylenepentamine, 1,2-diaminoethane, 1,2-diaminopropane, 1,3-diaminopentane and the like.
　アミノアルコールとしては、例えば、Ｎ－（２－アミノエチル）エタノールアミンなどが挙げられる。 Examples of amino alcohol include N- (2-aminoethyl) ethanolamine.
　第１級アミノ基、または、第１級アミノ基および第２級アミノ基を有するアルコキシシリル化合物としては、例えば、γ－アミノプロピルトリエトキシシラン、Ｎ－フェニル－γ－アミノプロピルトリメトキシシランなどのアルコキシシリル基含有モノアミン、例えば、Ｎ－β（アミノエチル）γ－アミノプロピルトリメトキシシラン、例えば、Ｎ－β（アミノエチル）γ－アミノプロピルメチルジメトキシシランなどが挙げられる。 Examples of the alkoxysilyl compound having a primary amino group or a primary amino group and a secondary amino group include γ-aminopropyltriethoxysilane and N-phenyl-γ-aminopropyltrimethoxysilane. Examples include alkoxysilyl group-containing monoamines such as N-β (aminoethyl) γ-aminopropyltrimethoxysilane, such as N-β (aminoethyl) γ-aminopropylmethyldimethoxysilane.
　ポリオキシエチレン基含有ポリアミンとしては、例えば、ポリオキシエチレンエーテルジアミンなどのポリオキシアルキレンエーテルジアミンなどが挙げられる。より具体的には、例えば、日本油脂社製のＰＥＧ＃１０００ジアミンや、ハンツマン社製のジェファーミンＥＤ―２００３、ＥＤＲ－１４８、ＸＴＪ－５１２などが挙げられる。 Examples of the polyoxyethylene group-containing polyamine include polyoxyalkylene ether diamines such as polyoxyethylene ether diamine. More specifically, for example, PEG # 1000 diamine manufactured by NOF Corporation, Jeffamine ED-2003, EDR-148, XTJ-512 manufactured by Huntsman, etc.
　これらポリアミン成分は、単独使用または２種類以上併用することができる。 These polyamine components can be used alone or in combination of two or more.
　なお、本発明では、必要に応じて、公知の添加剤、例えば、可塑剤、ブロッキング防止剤、耐熱安定剤、耐光安定剤、酸化防止剤、離型剤、触媒、さらには、顔料、染料、滑剤、フィラー、加水分解防止剤などを添加することができる。これら添加剤は、各成分の合成時に添加してもよく、あるいは、各成分の混合・溶解時に添加してもよく、さらには、合成後に添加することもできる。 In the present invention, a known additive, for example, a plasticizer, an anti-blocking agent, a heat stabilizer, a light stabilizer, an antioxidant, a release agent, a catalyst, a pigment, a dye, Lubricants, fillers, hydrolysis inhibitors and the like can be added. These additives may be added at the time of synthesis of each component, or may be added at the time of mixing / dissolving each component, and may be added after the synthesis.
　そして、ポリウレタン樹脂は、例えば、バルク重合や溶液重合などの重合方法により製造することができる。 The polyurethane resin can be produced by a polymerization method such as bulk polymerization or solution polymerization.
　バルク重合では、例えば、窒素気流下において、ペンタメチレンジイソシアネートおよび／またはポリイソシアネート組成物を撹拌しつつ、これに、活性水素化合物を加えて、反応温度、例えば、５０～２５０℃、さらに好ましくは、５０～２００℃で、例えば、０．５～１５時間程度反応させる。 In bulk polymerization, for example, while stirring a pentamethylene diisocyanate and / or polyisocyanate composition under a nitrogen stream, an active hydrogen compound is added to the reaction temperature, for example, 50 to 250 ° C., more preferably, The reaction is carried out at 50 to 200 ° C., for example, for about 0.5 to 15 hours.
　溶液重合では、有機溶剤に、ペンタメチレンジイソシアネートおよび／またはポリイソシアネート組成物、活性水素化合物を加えて、反応温度、例えば、５０～１２０℃、さらに好ましくは、５０～１００℃で、例えば、０．５～１５時間程度反応させる。 In solution polymerization, a pentamethylene diisocyanate and / or polyisocyanate composition and an active hydrogen compound are added to an organic solvent, and the reaction temperature is, for example, 50 to 120 ° C., more preferably 50 to 100 ° C. The reaction is carried out for about 5 to 15 hours.
　有機溶剤としては、例えば、アセトン、メチルエチルケトン、メチルイソブチルケトン、シクロヘキサノンなどのケトン類、例えば、アセトニトリルなどのニトリル類、酢酸メチル、酢酸エチル、酢酸ブチル、酢酸イソブチルなどのアルキルエステル類、例えば、ｎ－ヘキサン、ｎ－ヘプタン、オクタンなどの脂肪族炭化水素類、例えば、シクロヘキサン、メチルシクロヘキサンなどの脂環族炭化水素類、例えば、トルエン、キシレン、エチルベンゼンなどの芳香族炭化水素類、例えば、メチルセロソルブアセテート、エチルセロソルブアセテート、メチルカルビトールアセテート、エチルカルビトールアセテート、エチレングリコールエチルエーテルアセテート、プロピレングリコールメチルエーテルアセテート、３－メチル－３－メトキシブチルアセテート、エチル－３－エトキシプロピオネートなどのグリコールエーテルエステル類、例えば、ジエチルエーテル、テトラヒドロフラン、ジオキサンなどのエーテル類、例えば、塩化メチル、塩化メチレン、クロロホルム、四塩化炭素、臭化メチル、ヨウ化メチレン、ジクロロエタンなどのハロゲン化脂肪族炭化水素類、例えば、Ｎ－メチルピロリドン、ジメチルホルムアミド、Ｎ，Ｎ’－ジメチルアセトアミド、ジメチルスルホキシド、ヘキサメチルホスホニルアミドなどの極性非プロトン類などが挙げられる。 Examples of the organic solvent include ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone and cyclohexanone, nitriles such as acetonitrile, alkyl esters such as methyl acetate, ethyl acetate, butyl acetate and isobutyl acetate, such as n- Aliphatic hydrocarbons such as hexane, n-heptane and octane, for example, alicyclic hydrocarbons such as cyclohexane and methylcyclohexane, for example, aromatic hydrocarbons such as toluene, xylene and ethylbenzene, such as methyl cellosolve acetate , Ethyl cellosolve acetate, methyl carbitol acetate, ethyl carbitol acetate, ethylene glycol ethyl ether acetate, propylene glycol methyl ether acetate, 3-methyl-3-meth Glycol ether esters such as sibutyl acetate and ethyl-3-ethoxypropionate, for example, ethers such as diethyl ether, tetrahydrofuran and dioxane, such as methyl chloride, methylene chloride, chloroform, carbon tetrachloride, methyl bromide, Halogenated aliphatic hydrocarbons such as methylene iodide and dichloroethane, and polar aprotics such as N-methylpyrrolidone, dimethylformamide, N, N′-dimethylacetamide, dimethyl sulfoxide, hexamethylphosphonylamide, etc. It is done.
　さらに、有機溶剤としては、例えば、非極性溶剤（非極性有機溶剤）が挙げられ、これら非極性溶剤としては、脂肪族、ナフテン系炭化水素系有機溶剤を含む、アニリン点が、例えば、１０～７０℃、好ましくは、１２～６５℃の、低毒性で溶解力の弱い非極性有機溶剤や、ターペン油に代表される植物性油などが挙げられる。 Further, examples of the organic solvent include nonpolar solvents (nonpolar organic solvents). Examples of these nonpolar solvents include aliphatic and naphthenic hydrocarbon organic solvents having an aniline point of, for example, 10 to Examples include non-polar organic solvents having low toxicity and weak dissolving power at 70 ° C., preferably 12 to 65 ° C., and vegetable oils represented by terpene oil.
　かかる非極性有機溶剤は、市販品として入手可能であり、そのような市販品としては、例えば、ハウス（シェル化学社製、アニリン点１５℃）、スワゾール３１０（丸善石油社製、アニリン点１６℃）、エッソナフサＮｏ．６（エクソン化学社製、アニリン点４３℃）、ロウス（シェル化学社製、アニリン点４３℃）、エッソナフサＮｏ．５（エクソン社製、アニリン点５５℃）、ペガゾール３０４０（モービル石油社製、アニリン点５５℃）などの石油炭化水素系有機溶剤、その他、メチルシクロヘキサン（アニリン点４０℃）、エチルシクロヘキサン（アニリン点４４℃）、ガムテレピンＮ（安原油脂社製、アニリン点２７℃）などのターペン油類などが挙げられる。 Such a nonpolar organic solvent is available as a commercial product. Examples of such a commercial product include House (manufactured by Shell Chemical Co., Ltd., aniline point 15 ° C.), Swazol 310 (manufactured by Maruzen Petroleum Corporation, aniline point 16 ° C. ), Essonaphtha No. 6 (manufactured by Exxon Chemical Co., Ltd., aniline point 43 ° C.), wax (manufactured by Shell Chemical Co., Ltd., aniline point 43 ° C.), Essonaphtha No. 5 (Exxon, aniline point 55 ° C), Pegasol 3040 (Mobil Petroleum, aniline point 55 ° C) and other hydrocarbon organic solvents, methylcyclohexane (aniline point 40 ° C), ethylcyclohexane (aniline point) 44 ° C.), and turpentine oils such as gum turpentine N (manufactured by Yasuru Seiyaku Co., Ltd., aniline point 27 ° C.).
　さらに、上記重合反応においては、必要に応じて、例えば、ウレタン化触媒を添加することができる。 Furthermore, in the above polymerization reaction, for example, a urethanization catalyst can be added as necessary.
　アミン類としては、例えば、トリエチルアミン、トリエチレンジアミン、ビス－（２－ジメチルアミノエチル）エーテル、Ｎ－メチルモルホリンなどの３級アミン類、例えば、テトラエチルヒドロキシルアンモニウムなどの４級アンモニウム塩、例えば、イミダゾール、２－エチル－４－メチルイミダゾールなどのイミダゾール類などが挙げられる。 Examples of amines include tertiary amines such as triethylamine, triethylenediamine, bis- (2-dimethylaminoethyl) ether, N-methylmorpholine, and quaternary ammonium salts such as tetraethylhydroxylammonium, such as imidazole, And imidazoles such as 2-ethyl-4-methylimidazole.
　有機金属化合物としては、例えば、酢酸錫、オクチル酸錫、オレイン酸錫、ラウリル酸錫、ジブチル錫ジアセテート、ジメチル錫ジラウレート、ジブチル錫ジラウレート、ジブチル錫ジメルカプチド、ジブチル錫マレエート、ジブチル錫ジラウレート、ジブチル錫ジネオデカノエート、ジオクチル錫ジメルカプチド、ジオクチル錫ジラウリレート、ジブチル錫ジクロリドなどの有機錫系化合物、例えば、オクタン酸鉛、ナフテン酸鉛などの有機鉛化合物、例えば、ナフテン酸ニッケルなどの有機ニッケル化合物、例えば、ナフテン酸コバルトなどの有機コバルト化合物、例えば、オクテン酸銅などの有機銅化合物、例えば、オクチル酸ビスマス、ネオデカン酸ビスマスなどの有機ビスマス化合物などが挙げられる。 Examples of organometallic compounds include tin acetate, tin octylate, tin oleate, tin laurate, dibutyltin diacetate, dimethyltin dilaurate, dibutyltin dilaurate, dibutyltin dimercaptide, dibutyltin maleate, dibutyltin dilaurate, dibutyltin Organic tin compounds such as dineodecanoate, dioctyltin dimercaptide, dioctyltin dilaurate, dibutyltin dichloride, for example, organic lead compounds such as lead octoate and lead naphthenate, for example, organic nickel compounds such as nickel naphthenate, Examples thereof include organic cobalt compounds such as cobalt naphthenate, organic copper compounds such as copper octenoate, and organic bismuth compounds such as bismuth octylate and bismuth neodecanoate.
　さらに、ウレタン化触媒として、例えば、炭酸カリウム、酢酸カリウム、オクチル酸カリウムなどのカリウム塩が挙げられる。 Furthermore, examples of the urethanization catalyst include potassium salts such as potassium carbonate, potassium acetate, and potassium octylate.
　これらウレタン化触媒は、単独使用または２種類以上併用することができる。 These urethanization catalysts can be used alone or in combination of two or more.
　また、上記重合反応においては、（未反応の）ペンタメチレンジイソシアネートおよび／またはポリイソシアネート組成物を、例えば、蒸留や抽出などの公知の除去手段により除去することができる。 In the polymerization reaction, the (unreacted) pentamethylene diisocyanate and / or polyisocyanate composition can be removed by a known removal means such as distillation or extraction.
　バルク重合および溶液重合では、例えば、ペンタメチレンジイソシアネートおよび／またはポリイソシアネート組成物と、活性水素化合物とを、活性水素化合物中の活性水素基（水酸基、アミノ基）に対するペンタメチレンジイソシアネートおよび／またはポリイソシアネート組成物中のイソシアネート基の当量比（ＮＣＯ／活性水素基）が、例えば、０．７５～１．３、好ましくは、０．９～１．１となるように配合する。 In bulk polymerization and solution polymerization, for example, pentamethylene diisocyanate and / or polyisocyanate composition and an active hydrogen compound are converted into an active hydrogen group (hydroxyl group, amino group) in the active hydrogen compound and pentamethylene diisocyanate and / or polyisocyanate. It is blended so that the equivalent ratio of isocyanate groups (NCO / active hydrogen group) in the composition is, for example, 0.75 to 1.3, preferably 0.9 to 1.1.
　また、上記重合反応をより工業的に実施する場合には、ポリウレタン樹脂は、その用途に応じて、例えば、ワンショット法およびプレポリマー法などの公知の方法により、得ることができる。また、その他の方法により、ポリウレタン樹脂を、例えば、水系ディスパージョン（ＰＵＤ）などとして得ることもできる。 Further, when the polymerization reaction is carried out more industrially, the polyurethane resin can be obtained by a known method such as a one-shot method or a prepolymer method, depending on the application. Moreover, a polyurethane resin can also be obtained as an aqueous dispersion (PUD) etc. by another method.
　ワンショット法では、例えば、ペンタメチレンジイソシアネートおよび／またはポリイソシアネート組成物と活性水素化合物とを、活性水素化合物中の活性水素基（水酸基、アミノ基）に対するペンタメチレンジイソシアネートおよび／またはポリイソシアネート組成物中のイソシアネート基の当量比（ＮＣＯ／活性水素基）が、例えば、０．７５～１．３、好ましくは、０．９～１．１となるように処方（混合）した後、例えば、室温～２５０℃、好ましくは、室温～２００℃で、例えば、５分～７２時間、好ましくは、４～２４時間硬化反応させる。なお、硬化温度は、一定温度であってもよく、あるいは、段階的に昇温または冷却することもできる。 In the one-shot method, for example, a pentamethylene diisocyanate and / or polyisocyanate composition and an active hydrogen compound are mixed with an active hydrogen group (hydroxyl group, amino group) in the active hydrogen compound in the pentamethylene diisocyanate and / or polyisocyanate composition. Is formulated (mixed) such that the equivalent ratio of isocyanate groups (NCO / active hydrogen group) is, for example, 0.75 to 1.3, preferably 0.9 to 1.1. The curing reaction is performed at 250 ° C., preferably at room temperature to 200 ° C., for example, for 5 minutes to 72 hours, preferably for 4 to 24 hours. The curing temperature may be a constant temperature, or may be raised or cooled stepwise.
　また、プレポリマー法では、例えば、まず、ペンタメチレンジイソシアネートおよび／またはポリイソシアネート組成物と活性水素化合物の一部（好ましくは、高分子量ポリオール）とを反応させて、分子末端にイソシアネート基を有するイソシアネート基末端プレポリマーを合成する。次いで、得られたイソシアネート基末端プレポリマーと、活性水素化合物の残部（好ましくは、低分子量ポリオールおよび／またはポリアミン成分）とを反応させて、硬化反応させる。なお、プレポリマー法において、活性水素化合物の残部は、鎖伸長剤として用いられる。 In the prepolymer method, for example, a pentamethylene diisocyanate and / or polyisocyanate composition is first reacted with a part of an active hydrogen compound (preferably, a high molecular weight polyol) to form an isocyanate having an isocyanate group at the molecular end. A base end prepolymer is synthesized. Next, the obtained isocyanate group-terminated prepolymer and the remainder of the active hydrogen compound (preferably, a low molecular weight polyol and / or polyamine component) are reacted to cause a curing reaction. In the prepolymer method, the remainder of the active hydrogen compound is used as a chain extender.
　イソシアネート基末端プレポリマーを合成するには、ペンタメチレンジイソシアネートおよび／またはポリイソシアネート組成物と活性水素化合物の一部とを、活性水素化合物の一部中の活性水素基に対するペンタメチレンジイソシアネートおよび／またはポリイソシアネート組成物中のイソシアネート基の当量比（ＮＣＯ／活性水素基）が、例えば、１．１～２０、好ましくは、１．３～１０、さらに好ましくは、１．３～６となるように処方（混合）し、反応容器中にて、例えば、室温～１５０℃、好ましくは、５０～１２０℃で、例えば、０．５～１８時間、好ましくは、２～１０時間反応させる。なお、この反応においては、必要に応じて、上記したウレタン化触媒を添加してもよく、また、反応終了後には、必要に応じて、未反応のペンタメチレンジイソシアネートおよび／またはポリイソシアネート組成物を、例えば、蒸留や抽出などの公知の除去手段により、除去することもできる。 To synthesize an isocyanate group-terminated prepolymer, a pentamethylene diisocyanate and / or polyisocyanate composition and a portion of the active hydrogen compound may be combined with pentamethylene diisocyanate and / or polyisocyanate for the active hydrogen group in a portion of the active hydrogen compound. Formulated so that the equivalent ratio of isocyanate groups (NCO / active hydrogen group) in the isocyanate composition is, for example, 1.1 to 20, preferably 1.3 to 10, and more preferably 1.3 to 6. (Mixing) and reacting in a reaction vessel at room temperature to 150 ° C., preferably 50 to 120 ° C., for example, for 0.5 to 18 hours, preferably 2 to 10 hours. In this reaction, if necessary, the urethanization catalyst described above may be added, and after the reaction, if necessary, an unreacted pentamethylene diisocyanate and / or polyisocyanate composition may be added. For example, it can also be removed by a known removal means such as distillation or extraction.
　次いで、得られたイソシアネート基末端プレポリマーと、活性水素化合物の残部とを反応させるには、イソシアネート基末端プレポリマーと、活性水素化合物の残部とを、活性水素化合物の残部中の活性水素基に対するイソシアネート基末端プレポリマー中のイソシアネート基の当量比（ＮＣＯ／活性水素基）が、例えば、０．７５～１．３、好ましくは、０．９～１．１となるように処方（混合）し、例えば、室温～２５０℃、好ましくは、室温～２００℃で、例えば、５分～７２時間、好ましくは、１～２４時間硬化反応させる。 Next, in order to react the obtained isocyanate group-terminated prepolymer with the remainder of the active hydrogen compound, the isocyanate group-terminated prepolymer and the remainder of the active hydrogen compound are reacted with the active hydrogen group in the remainder of the active hydrogen compound. It is formulated (mixed) so that the equivalent ratio of isocyanate groups (NCO / active hydrogen groups) in the isocyanate group-terminated prepolymer is, for example, 0.75 to 1.3, preferably 0.9 to 1.1. For example, the curing reaction is performed at room temperature to 250 ° C., preferably at room temperature to 200 ° C., for example, for 5 minutes to 72 hours, preferably for 1 to 24 hours.
　また、ポリウレタン樹脂を水系ディスパージョンとして得るには、例えば、まず、ペンタメチレンジイソシアネートおよび／またはポリイソシアネート組成物と、後述する親水基を含有する活性水素化合物（以下、親水基含有活性水素化合物と略する。）を含む活性水素化合物とを反応させることにより、イソシアネート基末端プレポリマーを得る。 In order to obtain a polyurethane resin as an aqueous dispersion, for example, first, a pentamethylene diisocyanate and / or polyisocyanate composition and an active hydrogen compound containing a hydrophilic group described below (hereinafter abbreviated as a hydrophilic group-containing active hydrogen compound). To obtain an isocyanate group-terminated prepolymer.
　次いで、得られたイソシアネート基末端プレポリマーと鎖伸長剤とを水中で反応させて分散させる。これによって、イソシアネート基末端プレポリマーが鎖伸長剤によって鎖伸長された水性ポリウレタン樹脂を、内部乳化型の水系ディスパージョンとして得ることができる。 Next, the obtained isocyanate group-terminated prepolymer and the chain extender are reacted and dispersed in water. Thus, an aqueous polyurethane resin in which the isocyanate group-terminated prepolymer is chain-extended with a chain extender can be obtained as an internal emulsion type aqueous dispersion.
　イソシアネート基末端プレポリマーと鎖伸長剤とを水中で反応させるには、例えば、まず、イソシアネート基末端プレポリマーを水に添加して、イソシアネート基末端プレポリマーを分散させる。次いで、これに鎖伸長剤を添加して、イソシアネート基末端プレポリマーを鎖伸長する。 In order to react the isocyanate group-terminated prepolymer and the chain extender in water, for example, first, the isocyanate group-terminated prepolymer is added to water to disperse the isocyanate group-terminated prepolymer. Next, a chain extender is added thereto to chain extend the isocyanate group-terminated prepolymer.
　親水基含有活性水素化合物は、親水基と活性水素基とを併有する化合物であって、親水基としては、例えば、アニオン性基（例えば、カルボキシル基など）、カチオン性基、ノニオン性基（例えば、ポリオキシエチレン基など）が挙げられる。親水基含有活性水素化合物として、より具体的には、カルボン酸基含有活性水素化合物、ポリオキシエチレン基含有活性水素化合物などが挙げられる。 The hydrophilic group-containing active hydrogen compound is a compound having both a hydrophilic group and an active hydrogen group. Examples of the hydrophilic group include an anionic group (for example, carboxyl group), a cationic group, and a nonionic group (for example, And polyoxyethylene groups). More specifically, examples of the hydrophilic group-containing active hydrogen compound include a carboxylic acid group-containing active hydrogen compound, a polyoxyethylene group-containing active hydrogen compound, and the like.
　カルボン酸基含有活性水素化合物としては、例えば、２，２－ジメチロール酢酸、２，２－ジメチロール乳酸、２，２－ジメチロールプロピオン酸、２，２－ジメチロールブタン酸、２，２－ジメチロール酪酸、２，２－ジメチロール吉草酸などのジヒドロキシルカルボン酸、例えば、リジン、アルギニンなどのジアミノカルボン酸、または、それらの金属塩類やアンモニウム塩類などが挙げられる。 Examples of the carboxylic acid group-containing active hydrogen compound include 2,2-dimethylolacetic acid, 2,2-dimethylollactic acid, 2,2-dimethylolpropionic acid, 2,2-dimethylolbutanoic acid, and 2,2-dimethylolbutyric acid. And dihydroxycarboxylic acids such as 2,2-dimethylolvaleric acid, diaminocarboxylic acids such as lysine and arginine, or metal salts and ammonium salts thereof.
　ポリオキシエチレン基含有活性水素化合物は、主鎖または側鎖にポリオキシエチレン基を含み、２つ以上の活性水素基を有する化合物であって、例えば、ポリエチレングリコール、ポリオキシエチレン側鎖含有ポリオール（側鎖にポリオキシエチレン基を含み、２つ以上の活性水素基を有する化合物）などが挙げられる。 The polyoxyethylene group-containing active hydrogen compound is a compound having a polyoxyethylene group in the main chain or side chain and having two or more active hydrogen groups, for example, polyethylene glycol, polyoxyethylene side chain-containing polyol ( And compounds having a polyoxyethylene group in the side chain and having two or more active hydrogen groups).
　これら親水基含有活性水素化合物は、単独使用または２種類以上併用することができる。 These hydrophilic group-containing active hydrogen compounds can be used alone or in combination of two or more.
　鎖伸長剤としては、例えば、上記した２価アルコール、上記した３価アルコールなどの低分子量ポリオール、例えば、脂環族ジアミン、脂肪族ジアミンなどのジアミンなどを使用することができる。 As the chain extender, for example, low molecular weight polyols such as the above-described dihydric alcohols and trihydric alcohols described above, for example, diamines such as alicyclic diamines and aliphatic diamines can be used.
　これら鎖伸長剤は、単独使用または２種類以上併用することができる。 These chain extenders can be used alone or in combination of two or more.
　このように、親水基含有活性水素化合物を含む活性水素化合物を使用する場合には、必要により、親水基を公知の中和剤で中和する。 Thus, when an active hydrogen compound containing a hydrophilic group-containing active hydrogen compound is used, the hydrophilic group is neutralized with a known neutralizing agent as necessary.
　また、活性水素化合物として、親水基含有活性水素化合物を使用しない場合には、例えば、公知の界面活性剤を用いて乳化することにより、外部乳化型の水系ディスパージョンとして得ることができる。 Further, when a hydrophilic group-containing active hydrogen compound is not used as the active hydrogen compound, it can be obtained as an external emulsion type aqueous dispersion by, for example, emulsification using a known surfactant.
　そして、高生産速度、高反応収率および高品質で得られる１，５－ペンタメチレンジアミンを原料とした、１,５－ペンタメチレンジイソシアネート、ポリイソシアネート組成物およびポリウレタン樹脂は、例えば、ポリカーボネート、ＡＢＳ、ポリエチレンテレフタレート、ナイロン、ポリオレフィンなどへの各種プラスチックコート材料、太陽電池のバックシート部材へのコーティング原材料や自動車、二輪車などへの外装、金属などへのコート材料、インキなどのバインダーにも好適に使用できる。また、イソシアネート基あるいは水酸基末端のポリウレタンプレポリマーに誘導することにより、ラミネート工法や工業用、住宅・建築用接着材料やシーリング材料にも適用できる。さらには、熱可塑性あるいは熱硬化性ポリウレタンエラストマーとして、フィルム、シート、チューブ、ホース、パウダーあるいは柔軟なゲルなどにも誘導でき、各種産業用途、例えば、医療、衣料、工業部材、電子・電気部材、化粧品などのヘルスケア分野にも展開できる。また、アミノ基を有した化合物と本イソシアネートおよびポリイソシアネート組成物を反応させることにより、各種香料、薬剤などのカプセル材にも適用できる。 1,5-pentamethylene diisocyanate, polyisocyanate composition and polyurethane resin using 1,5-pentamethylenediamine obtained with high production rate, high reaction yield and high quality as raw materials are, for example, polycarbonate, ABS Also suitable for various plastic coating materials for polyethylene terephthalate, nylon, polyolefin, etc., coating raw materials for solar cell backsheet members, exteriors for automobiles and motorcycles, coating materials for metals, binders for ink, etc. it can. Moreover, by introducing into a polyurethane prepolymer having an isocyanate group or a hydroxyl group terminal, the present invention can be applied to a laminate method, an industrial material, a housing / building adhesive material, and a sealing material. Furthermore, as a thermoplastic or thermosetting polyurethane elastomer, it can also be derived into films, sheets, tubes, hoses, powders or flexible gels, and various industrial uses such as medical, clothing, industrial parts, electronic / electrical parts, It can also be deployed in the health care field such as cosmetics. Moreover, it can apply also to capsule materials, such as various fragrance | flavor and a chemical | medical agent, by making this isocyanate and polyisocyanate composition react with the compound which has an amino group.
　以下、本発明を実施例にて詳細に説明する。しかしながら、本発明はそれらに何ら限定されるものではない。 Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited to them.
　なお、Ｌ－リジンおよび１，５－ペンタメチレンジアミンは高速液体クロマトグラフ（ＨＰＬＣ）により定量した。これらの分析条件およびリジン脱炭酸酵素活性の測定方法は次の通りである。
＜１，５－ペンタメチレンジアミンの分析条件＞
カラム：Ａｓａｈｉｐａｋ　ＯＤＰ－５０　４Ｅ（昭和電工社製）
カラム温度：４０℃
溶離液：０．２Ｍ　リン酸ナトリウム（ｐＨ７．７）＋２．３ｍＭ　１－オクタンスルホン酸ナトリウム
溶離液の流量：０．５ｍＬ／ｍｉｎ
　検出には、オルトフタルアルデヒドを用いたポストカラム誘導体化法〔Ｊ．Ｃｈｒｏｍａｔｏｇｒ．，８３，３５３－３５５（１９７３）〕を用いた。
＜リジン脱炭酸酵素活性の測定方法＞
　２００ｍＭ　Ｌ－リジン一塩酸塩および０．１５ｍＭ　ピリドキサールリン酸（広島和光工業社製）を含む２００ｍＭ　リン酸ナトリウム緩衝液（ｐＨ７．０）に、菌体懸濁液またはその処理物を添加して、合計０．２ｍＬとし、３７℃で６分間反応した。反応液に、０．２Ｍ　塩酸を１ｍＬ添加して、反応を停止した。この反応停止液を水で適当に希釈し、生成した１，５－ペンタメチレンジアミンを、ＨＰＬＣにより定量した。 L-lysine and 1,5-pentamethylenediamine were quantified by high performance liquid chromatography (HPLC). These analytical conditions and methods for measuring lysine decarboxylase activity are as follows.
<Analysis conditions for 1,5-pentamethylenediamine>
Column: Asahipak ODP-50 4E (manufactured by Showa Denko)
Column temperature: 40 ° C
Eluent: 0.2 M sodium phosphate (pH 7.7) +2.3 mM sodium 1-octanesulfonate Eluent flow rate: 0.5 mL / min
For detection, a post-column derivatization method using orthophthalaldehyde [J. Chromatogr. 83, 353-355 (1973)].
<Method for measuring lysine decarboxylase activity>
To a 200 mM sodium phosphate buffer (pH 7.0) containing 200 mM L-lysine monohydrochloride and 0.15 mM pyridoxal phosphate (manufactured by Hiroshima Wako Kogyo Co., Ltd.), the cell suspension or a treated product thereof was added, The total was 0.2 mL, and the reaction was performed at 37 ° C. for 6 minutes. 1 mL of 0.2M hydrochloric acid was added to the reaction solution to stop the reaction. The reaction stop solution was appropriately diluted with water, and the produced 1,5-pentamethylenediamine was quantified by HPLC.
　活性の単位は、１分間に１μｍｏｌの１，５－ペンタメチレンジアミンを生成する活性を、１ｕｎｉｔとした。
＜ペンタメチレンジイソシアネート濃度（単位：質量％）＞
　後述する実施例１３で得られたペンタメチレンジイソシアネートを用い、以下のＨＰＬＣ分析条件下で得られたクロマトグラムの面積値から作成した検量線により、ポリイソシアネート組成物中のペンタメチレンジイソシアネートの濃度を算出した。 The unit of activity was defined as 1 unit for the activity of producing 1 μmol of 1,5-pentamethylenediamine per minute.
<Pentamethylene diisocyanate concentration (unit: mass%)>
Using the pentamethylene diisocyanate obtained in Example 13 described later, the concentration of pentamethylene diisocyanate in the polyisocyanate composition was calculated from a calibration curve created from the area values of the chromatogram obtained under the following HPLC analysis conditions. did.
　　装置：Ｐｒｏｍｉｎｅｎｃｅ（島津製作所社製）
　　１）　ポンプ　ＬＣ－２０ＡＴ
　　２）　デガッサ　ＤＧＵ－２０Ａ３
　　３）　オートサンプラ　ＳＩＬ－２０Ａ
　　４）　カラム恒温槽　ＣＯＴ－２０Ａ
　　５）　検出器　ＳＰＤ－２０Ａ
　　カラム：ＳＨＩＳＥＩＤＯ　ＳＩＬＩＣＡ　ＳＧ－１２０
　　カラム温度：４０℃
　　溶離液：ｎ－ヘキサン／メタノール／１，２－ジクロロエタン＝９０／５／５（体積比）
　　流量：０．２ｍＬ／ｍｉｎ
　　検出方法：ＵＶ　２２５ｎｍ
＜イソシアネート基の転化率（単位：％）＞
　イソシアネート基の転化率は、以下のＧＰＣ測定条件において得られたクロマトグラムにより、全ピーク面積に対するペンタメチレンジイソシアネートのピークよりも高分子量側にあるピークの面積の割合を、イソシアネート基の転化率とした。 Device: Prominence (manufactured by Shimadzu Corporation)
1) Pump LC-20AT
2) Degasser DGU-20A3
3) Autosampler SIL-20A
4) Column thermostat COT-20A
5) Detector SPD-20A
Column: SHISEIDO SILICA SG-120
Column temperature: 40 ° C
Eluent: n-hexane / methanol / 1,2-dichloroethane = 90/5/5 (volume ratio)
Flow rate: 0.2mL / min
Detection method: UV 225 nm
<Conversion rate of isocyanate group (unit:%)>
The conversion ratio of isocyanate group is the ratio of the area of the peak on the high molecular weight side of the peak of pentamethylene diisocyanate to the total peak area, based on the chromatogram obtained under the following GPC measurement conditions, as the conversion ratio of isocyanate group. .
　　装置：ＨＬＣ－８０２０（東ソー社製）
　　カラム：Ｇ１０００ＨＸＬ、Ｇ２０００ＨＸＬおよびＧ３０００ＨＸＬ（以上、商品名、東ソー社製）を直列に連結
　　カラム温度：４０℃
　　溶離液：テトラヒドロフラン
　　流量：０．８ｍＬ／ｍｉｎ
　　検出方法：示差屈折率
　　標準物質：ポリエチレンオキシド（東ソー社製、商品名：ＴＳＫ標準ポリエチレンオキシド）
＜イソシアネート３量体濃度（単位：質量％）＞
　上記した（イソシアネート基の転化率）と同様の測定を行い、ペンタメチレンジイソシアネートの３倍の分子量に相当するピーク面積比率を、イソシアネート３量体濃度とした。
＜イソシアネート基濃度（単位：質量％）＞
　ポリイソシアネート組成物のイソシアネート基濃度は、電位差滴定装置を用いて、ＪＩＳ　Ｋ－１５５６に準拠したｎ－ジブチルアミン法により、測定した。
＜粘度（単位：ｍＰａ・ｓ）＞
　東機産業社製のＥ型粘度計ＴＶ－３０を用いて、ポリイソシアネート組成物の２５℃における粘度を測定した。
＜色相（単位：ＡＰＨＡ）＞
　ＪＩＳ　Ｋ－００７１に準拠した方法により、ポリイソシアネート組成物の色相を測定した。
（参考例１）
［リジン脱炭酸酵素遺伝子（ｃａｄＡ）のクローニング］
　Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　Ｗ３１１０株（ＡＴＣＣ２７３２５）から常法に従い調製したゲノムＤＮＡをＰＣＲの鋳型に用いた。 Apparatus: HLC-8020 (manufactured by Tosoh Corporation)
Column: G1000HXL, G2000HXL and G3000HXL (above, trade name, manufactured by Tosoh Corporation) connected in series Column temperature: 40 ° C
Eluent: Tetrahydrofuran Flow rate: 0.8 mL / min
Detection method: Differential refractive index Standard material: Polyethylene oxide (manufactured by Tosoh Corporation, trade name: TSK standard polyethylene oxide)
<Isocyanate trimer concentration (unit: mass%)>
The measurement similar to the above (conversion rate of isocyanate group) was performed, and a peak area ratio corresponding to a molecular weight three times that of pentamethylene diisocyanate was defined as an isocyanate trimer concentration.
<Isocyanate group concentration (unit: mass%)>
The isocyanate group concentration of the polyisocyanate composition was measured by an n-dibutylamine method according to JIS K-1556 using a potentiometric titrator.
<Viscosity (unit: mPa · s)>
The viscosity at 25 ° C. of the polyisocyanate composition was measured using an E-type viscometer TV-30 manufactured by Toki Sangyo Co., Ltd.
<Hue (unit: APHA)>
The hue of the polyisocyanate composition was measured by a method according to JIS K-0071.
(Reference Example 1)
[Cloning of lysine decarboxylase gene (cadA)]
Genomic DNA prepared from Escherichia coli W3110 strain (ATCC 27325) according to a conventional method was used as a PCR template.
　ＰＣＲ用のプライマーには、リジン脱炭酸酵素遺伝子（ｃａｄＡ）（ＧｅｎＢａｎｋ　Ａｃｃｅｓｓｉｏｎ　Ｎｏ．ＡＰ００９０４８）の塩基配列に基づいて設計した配列番号１および２に示す塩基配列を有するオリゴヌクレオチド（インビトロジェン社に委託して合成した）を用いた。これらのプライマーは、５’末端付近にそれぞれＫｐｎＩおよびＸｂａＩの制限酵素認識配列を有する。 As primers for PCR, oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 1 and 2 designed based on the nucleotide sequence of lysine decarboxylase gene (cadA) (GenBank Accession No. AP009048) (consigned to Invitrogen) Synthesized). These primers have restriction enzyme recognition sequences for KpnI and XbaI, respectively, near the 5 'end.
　上記のゲノムＤＮＡ１ｎｇ／μＬおよび各プライマー０．５ｐｍｏｌ／μＬを含む２５μＬのＰＣＲ反応液を用いて、変性：９４℃、３０秒間、アニーリング：５５℃、３０秒間、伸長反応：６８℃、２分間からなる反応サイクルを３０サイクルの条件でＰＣＲを行った。 Using 25 μL of a PCR reaction solution containing 1 ng / μL of genomic DNA and 0.5 pmol / μL of each primer, denaturation: 94 ° C., 30 seconds, annealing: 55 ° C., 30 seconds, extension reaction: 68 ° C., from 2 minutes PCR was performed under the following reaction cycle of 30 cycles.
　ＰＣＲ反応産物およびプラスミドｐＵＣ１８（タカラバイオ社製）をＫｐｎＩおよびＸｂａＩで消化し、ライゲーション・ハイ（東洋紡社製）を用いて連結した後、得られた組換えプラスミドを用いて、Ｅｓｃｈｒｉｃｈｉａ　ｃｏｌｉ　ＤＨ５α（東洋紡社製）を形質転換した。形質転換体を、アンピシリン（Ａｍ）１００μｇ／ｍＬおよびＸ－Ｇａｌ（５－ブロモ－４－クロロ－３－インドリル－β－Ｄ－ガラクトシド）を含むＬＢ寒天培地で培養し、Ａｍ耐性でかつ白色コロニーとなった形質転換体を得た。このようにして得られた形質転換体よりプラスミドを抽出した。 The PCR reaction product and the plasmid pUC18 (Takara Bio) were digested with KpnI and XbaI and ligated using Ligation High (Toyobo), and then the resulting recombinant plasmid was used to use Escherichia coli DH5α (Toyobo). The product was transformed. The transformant was cultured on an LB agar medium containing 100 μg / mL of ampicillin (Am) and X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside), and was resistant to Am and white colonies. The resulting transformant was obtained. A plasmid was extracted from the transformant thus obtained.
　通常の塩基配列の決定法に従い、プラスミドに導入されたＤＮＡ断片の塩基配列が配列表の配列番号３に示す塩基配列であることを確認した。 In accordance with the usual method for determining the base sequence, it was confirmed that the base sequence of the DNA fragment introduced into the plasmid was the base sequence shown in SEQ ID NO: 3 in the sequence listing.
　得られたリジン脱炭酸酵素をコードするＤＮＡを持つプラスミドをｐＣＡＤＡ１と命名した。 The obtained plasmid having DNA encoding lysine decarboxylase was named pCADA1.
　また、配列表の配列番号３に記載のＤＮＡ配列をアミノ酸配列に翻訳した配列を、配列表の配列番号４に示した。
［形質転換体の調製］
　ｐＣＡＤＡ１を用いてＥｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　Ｗ３１１０株を通常の方法で形質転換し、得られた形質転換体をＷ／ｐＣＡＤＡ１と命名した。 A sequence obtained by translating the DNA sequence shown in SEQ ID NO: 3 into an amino acid sequence is shown in SEQ ID NO: 4 in the sequence list.
[Preparation of transformant]
Escherichia coli W3110 strain was transformed with pCADA1 by a conventional method, and the resulting transformant was named W / pCADA1.
　該形質転換体を２Ｌのバッフル付き三角フラスコ中のＡｍ１００μｇ／ｍＬを含むＬＢ培地５００ｍｌに接種し、３０℃にて２６．５時間振盪培養した。その後、培養液を８０００ｒｐｍで１０分間遠心分離し、菌体を回収した（乾燥菌体換算濃度は３１％（ｗ／ｗ））。
［触媒菌体の超音波破砕物の調製］
　得られた形質転換体Ｗ／ｐＣＡＤＡ１の回収菌体を希釈液（０．１５ｍＭ　ピリドキサールリン酸および５ｇ／Ｌ　ウシアルブミン（ＳＩＧＭＡ社製）を含む１０ｍＭ　リン酸ナトリウム緩衝液（ｐＨ７．０））に懸濁し、菌体懸濁液を調製した。そして、菌体懸濁液をそれぞれバイオラピュター（オリンパス社製）により氷水中で１５分間破砕した。
［触媒死菌体の調製］
　形質転換体Ｗ／ｐＣＡＤＡ１の回収菌体を水に懸濁し、乾燥菌体換算濃度１２．５質量％の菌体懸濁液を調製した。この菌体懸濁液を５８℃の湯浴で３０分間保温し、熱処理を施し、使用するまで－２０℃で凍結保存した。
［還元剤添加による触媒死菌体の調製］
　形質転換体Ｗ／ｐＣＡＤＡ１の回収菌体を水に懸濁し、乾燥菌体換算濃度１２．５質量％の菌体懸濁液を調製した。この菌体懸濁液に亜硫酸ナトリウムを１．０ｇ／Ｌとなるように加え５８℃の湯浴で３０分間保温し、熱処理を施し、使用するまで－２０℃で凍結保存した。
［酵素活性の測定］
　精製酵素は上記の方法で形質転換体を培養し、回収した菌体をＳａｂｏら（Ｂｉｏｃｈｅｍｉｓｔｒｙ　１３（１９７４）ｐｐ．６６２－６７０．）の方法により精製した。精製した酵素の酵素活性を測定したところ１０００ｕｎｉｔ／ｍｇの精製酵素が得られた。　　　 The transformant was inoculated into 500 ml of LB medium containing 100 μg / mL of Am in a 2 L baffled Erlenmeyer flask and cultured with shaking at 30 ° C. for 26.5 hours. Thereafter, the culture broth was centrifuged at 8000 rpm for 10 minutes to collect the cells (the dry cell equivalent concentration was 31% (w / w)).
[Preparation of ultrasonically crushed catalyst cells]
The recovered cells of the obtained transformant W / pCADA1 were suspended in a diluent (10 mM sodium phosphate buffer (pH 7.0) containing 0.15 mM pyridoxal phosphate and 5 g / L bovine albumin (manufactured by SIGMA)). It became cloudy and a cell suspension was prepared. The bacterial cell suspensions were each crushed for 15 minutes in ice water using a bioraptor (manufactured by Olympus).
[Preparation of dead catalyst cells]
The recovered cells of the transformant W / pCADA1 were suspended in water to prepare a cell suspension having a dry cell equivalent concentration of 12.5% by mass. This cell suspension was kept in a 58 ° C. water bath for 30 minutes, subjected to heat treatment, and stored frozen at −20 ° C. until use.
[Preparation of dead catalyst cells by adding a reducing agent]
The recovered cells of the transformant W / pCADA1 were suspended in water to prepare a cell suspension with a dry cell equivalent concentration of 12.5% by mass. To this cell suspension, sodium sulfite was added at 1.0 g / L, kept in a 58 ° C. water bath for 30 minutes, subjected to heat treatment, and stored frozen at −20 ° C. until use.
[Measurement of enzyme activity]
The purified enzyme was cultured by the method described above, and the recovered cells were purified by the method of Sabo et al. (Biochemistry 13 (1974) pp. 662-670.). When the enzyme activity of the purified enzyme was measured, 1000 unit / mg of purified enzyme was obtained.
　また、触媒死菌体の活性を測定したところ、１００ｕｎｉｔ／ｍｇ－乾燥菌体（Ｄｒｙ　Ｃｅｌｌ）であった。
（参考例２）
［変異酵素の作製］
　ｐＣＡＤＡ１を鋳型として表１～６に示す塩基配列を有するオリゴヌクレオチド（インビトロジェン社に委託して合成した）を用いて、ＰＣＲを行った。 Further, when the activity of the dead catalyst cell was measured, it was 100 unit / mg-dry cell.
(Reference Example 2)
[Production of mutant enzyme]
PCR was performed using pCADA1 as a template and oligonucleotides having base sequences shown in Tables 1 to 6 (synthesized by commissioning Invitrogen).
　即ち、ｐＣＡＤＡ１を鋳型として配列表の配列番号５と配列番号６を用いて変性：９６℃、３０秒間、アニーリング：５５℃、３０秒間、伸長反応：６８℃、５分間２０秒からなる反応サイクルを１６サイクルの条件でＰＣＲを行った。 That is, a reaction cycle comprising denaturation: 96 ° C., 30 seconds, annealing: 55 ° C., 30 seconds, extension reaction: 68 ° C., 5 minutes, 20 seconds using pCADA1 as a template and SEQ ID NO: 5 and SEQ ID NO: 6 PCR was performed under conditions of 16 cycles.
　得られた増幅断片をＤｐｎＩ処理し、ライゲーション・ハイを用いて連結した後、得られた組換えプラスミドを用いて、または、ＤｐｎＩ処理した増幅断片を直接コンピテントセルＤＨ５αに添加し、大腸菌ＤＨ５α株を形質転換した。作成した株よりプラスミドを調製して塩基配列を決定し目的の塩基が置換されていることを確認した。得られたプラスミドをｐＣＡＤ２と命名した。 The obtained amplified fragment was treated with DpnI and ligated using ligation high, and then the obtained recombinant plasmid was used or the amplified fragment treated with DpnI was directly added to competent cell DH5α to obtain Escherichia coli DH5α strain. Was transformed. A plasmid was prepared from the prepared strain, the base sequence was determined, and it was confirmed that the target base was substituted. The resulting plasmid was named pCAD2.
　同様にｐＣＡＤ３からｐＣＡＤ２０およびｐＣＡＤ２３からｐＣＡＤ１１９のプラスミドを構築した。使用したオリゴヌクレオチドの配列を表１～６に示す。 Similarly, plasmids from pCAD3 to pCAD20 and from pCAD23 to pCAD119 were constructed. The oligonucleotide sequences used are shown in Tables 1-6.
　ｐＣＡＤＡ２からｐＣＡＤＡ２０を用いてＥｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　Ｗ３１１０株を通常の方法で形質転換し、得られた形質転換体をＷ／ｐＣＡＤＡ２～Ｗ／ｐＣＡＤＡ２０と命名した。同様にｐＣＡＤ２３からｐＣＡＤ１１９を用いてＥｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　Ｗ３１１０株を通常の方法で形質転換し、得られた形質転換体をＷ／ｐＣＡＤＡ２３～Ｗ／ｐＣＡＤＡ１１９と命名した。 Escherichia coli W3110 strain was transformed with pCADA2 to pCADA20 by a conventional method, and the resulting transformants were named W / pCADA2 to W / pCADA20. Similarly, Escherichia coli W3110 strain was transformed with pCAD23 to pCAD119 by a conventional method, and the resulting transformants were named W / pCADA23 to W / pCADA119.
　該形質転換体を２Ｌのバッフル付き三角フラスコ中のＡｍ１００μｇ／ｍＬを含むＬＢ培地５００ｍｌに接種し、３０℃にて２６．５時間振盪培養した。その後、培養液を８０００ｒｐｍで１０分間遠心分離し、菌体を回収した（乾燥菌体換算濃度は３１質量％）。 The transformant was inoculated into 500 ml of LB medium containing 100 μg / mL of Am in a 2 L baffled Erlenmeyer flask and cultured with shaking at 30 ° C. for 26.5 hours. Thereafter, the culture solution was centrifuged at 8000 rpm for 10 minutes to collect the cells (the dry cell equivalent concentration was 31% by mass).
　触媒菌体の超音波破砕物の調製及び触媒死菌体の調製は参考例１の方法に従った。 Preparation of ultrasonic disruption product of catalyst cells and preparation of dead catalyst cells were carried out according to the method of Reference Example 1.
　また、変異酵素の変異前と変異後との対応関係を、表７～９に示す。 In addition, Tables 7 to 9 show the correspondence between the mutation enzyme before mutation and after mutation.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
（参考例３）
［多重変異株の作成］
　ｐＣＡＤＡ５を鋳型として配列番号１９および２０に示す塩基配列を有するオリゴヌクレオチド（インビトロジェン社に委託して合成した）を用いて、ＰＣＲを行った。 (Reference Example 3)
[Create multiple mutants]
PCR was performed using pCADA5 as a template and oligonucleotides having the nucleotide sequences shown in SEQ ID NOS: 19 and 20 (synthesized by commissioning Invitrogen).
　そして、変性：９６℃、３０秒間、アニーリング：５５℃、３０秒間、伸長反応：６８℃、５分間２０秒からなる反応サイクルを１６サイクルの条件でＰＣＲを行った。 Then, PCR was performed under the conditions of 16 cycles of denaturation: 96 ° C., 30 seconds, annealing: 55 ° C., 30 seconds, extension reaction: 68 ° C., 5 minutes 20 seconds.
　得られた増幅断片をＤｐｎＩ処理し、ライゲーション・ハイを用いて連結した後、得られた組換えプラスミドを用いて、大腸菌ＤＨ５α株を形質転換した。作成した株よりプラスミドを調製して塩基配列を決定し目的の塩基が置換されていることを確認した。このプラスミドを鋳型として同様に配列番号３５および３６に示す塩基配列を用いて同様にプラスミドを作製し、さらにこのプラスミドに配列番号４１および４２に示す塩基配列を用いて上記の方法でプラスミドを作製することにより４重変異体の配列をもつプラスミドｐＣＡＤＡ２１を作製した。得られた４重変異体のＤＮＡ配列を配列表の配列番号４３に示す。アミノ酸配列を配列表の配列番号４４に示す。 The obtained amplified fragment was treated with DpnI and ligated using ligation high, and then the obtained recombinant plasmid was used to transform E. coli DH5α strain. A plasmid was prepared from the prepared strain, the base sequence was determined, and it was confirmed that the target base was substituted. Similarly, using this plasmid as a template, a plasmid is similarly prepared using the nucleotide sequences shown in SEQ ID NOs: 35 and 36. Further, a plasmid is prepared by the above method using the nucleotide sequences shown in SEQ ID NOs: 41 and 42 in this plasmid. Thus, plasmid pCADA21 having the quadruple mutant sequence was prepared. The DNA sequence of the obtained quadruple mutant is shown in SEQ ID NO: 43 of the Sequence Listing. The amino acid sequence is shown in SEQ ID NO: 44 in the sequence listing.
　さらにこのプラスミドに配列番号９および１０に示す塩基配列を用いて上記の方法でプラスミドを作製することにより５重変異体の配列をもつプラスミドｐＣＡＤＡ２２を作製した。このプラスミドを通常の方法でＥｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　Ｗ３１１０株を通常の方法で形質転換し、得られた形質転換体をＷ／ｐＣＡＤＡ２１およびＷ／ｐＣＡＤＡ２２と命名した。得られた５重変異体のＤＮＡ配列を配列表の配列番号４５に示す。アミノ酸配列を配列表の配列番号４６に示す。 Furthermore, a plasmid pCADA22 having a quintuple mutant sequence was prepared by preparing a plasmid by the above-described method using the nucleotide sequences shown in SEQ ID NOs: 9 and 10 in this plasmid. This plasmid was transformed into Escherichia coli W3110 strain by a usual method by a usual method, and the resulting transformants were named W / pCADA21 and W / pCADA22. The DNA sequence of the obtained 5-fold mutant is shown in SEQ ID NO: 45 of the sequence listing. The amino acid sequence is shown in SEQ ID NO: 46 in the sequence listing.
　次にｐＣＡＤＡ７３を鋳型として配列番号２３５および２３６に示す塩基配列を有するオリゴヌクレオチドを用いて、ＰＣＲを行った。そして、変性：９６℃、３０秒間、アニーリング：５５℃、３０秒間、伸長反応：６８℃、５分間２０秒からなる反応サイクルを１６サイクルの条件でＰＣＲを行った。得られた増幅断片をＤｐｎＩ処理し、この断片を用いて、大腸菌ＤＨ５α株を形質転換した。作成した株よりプラスミドを調製して塩基配列を決定し目的の塩基が置換されていることを確認した。得られたプラスミドをｐＣＡＤＡ１２０と命名した。 Next, PCR was performed using pCADA73 as a template and an oligonucleotide having the nucleotide sequences shown in SEQ ID NOs: 235 and 236. Then, PCR was performed under the conditions of 16 cycles of denaturation: 96 ° C., 30 seconds, annealing: 55 ° C., 30 seconds, extension reaction: 68 ° C., 5 minutes, 20 seconds. The obtained amplified fragment was treated with DpnI, and this fragment was used to transform E. coli DH5α strain. A plasmid was prepared from the prepared strain, the base sequence was determined, and it was confirmed that the target base was substituted. The resulting plasmid was named pCADA120.
　次にｐＣＡＤＡ９５を鋳型として配列番号２２７および２２８に示す塩基配列を有するオリゴヌクレオチドを用いて、ＰＣＲを行った。そして、変性：９６℃、３０秒間、アニーリング：５５℃、３０秒間、伸長反応：６８℃、５分間２０秒からなる反応サイクルを１６サイクルの条件でＰＣＲを行った。得られた増幅断片をＤｐｎＩ処理し、この断片を用いて、大腸菌ＤＨ５α株を形質転換した。作成した株よりプラスミドを調製して塩基配列を決定し目的の塩基が置換されていることを確認した。得られたプラスミドをｐＣＡＤＡ１２１と命名した。 Next, PCR was carried out using pCADA95 as a template and an oligonucleotide having the nucleotide sequences shown in SEQ ID NOs: 227 and 228. Then, PCR was performed under the conditions of 16 cycles of denaturation: 96 ° C., 30 seconds, annealing: 55 ° C., 30 seconds, extension reaction: 68 ° C., 5 minutes, 20 seconds. The obtained amplified fragment was treated with DpnI, and this fragment was used to transform E. coli DH5α strain. A plasmid was prepared from the prepared strain, the base sequence was determined, and it was confirmed that the target base was substituted. The resulting plasmid was named pCADA121.
　次にｐＣＡＤＡ１１３を鋳型として配列番号２３５および２３６に示す塩基配列を有するオリゴヌクレオチドを用いて、ＰＣＲを行った。そして、変性：９６℃、３０秒間、アニーリング：５５℃、３０秒間、伸長反応：６８℃、５分間２０秒からなる反応サイクルを１６サイクルの条件でＰＣＲを行った。得られた増幅断片をＤｐｎＩ処理し、この断片を用いて、大腸菌ＤＨ５α株を形質転換した。作成した株よりプラスミドを調製して塩基配列を決定し目的の塩基が置換されていることを確認した。得られたプラスミドをｐＣＡＤＡ１２２と命名した。 Next, PCR was performed using pCADA113 as a template and an oligonucleotide having the nucleotide sequences shown in SEQ ID NOs: 235 and 236. Then, PCR was performed under the conditions of 16 cycles of denaturation: 96 ° C., 30 seconds, annealing: 55 ° C., 30 seconds, extension reaction: 68 ° C., 5 minutes, 20 seconds. The obtained amplified fragment was treated with DpnI, and this fragment was used to transform E. coli DH5α strain. A plasmid was prepared from the prepared strain, the base sequence was determined, and it was confirmed that the target base was substituted. The resulting plasmid was named pCADA122.
　これらのプラスミド、ｐＣＡＤＡ１２０、ｐＣＡＤＡ１２１、ｐＣＡＤＡ１２２を通常の方法でＥｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　Ｗ３１１０株を形質転換し、得られた形質転換体をＷ／ｐＣＡＤＡ１２０、Ｗ／ｐＣＡＤＡ１２１、およびＷ／ｐＣＡＤＡ１２２と命名した。 These plasmids, pCADA120, pCADA121, and pCADA122 were transformed into Escherichia coli W3110 strain by a conventional method, and the obtained transformants were named W / pCADA120, W / pCADA121, and W / pCADA122.
　該形質転換体を２Ｌのバッフル付き三角フラスコ中のアンピシリン１００μｇ／ｍＬを含むＬＢ培地５００ｍｌに接種し、３０℃にて２６．５時間振盪培養した。その後、培養液を８０００ｒｐｍで１０分間遠心分離し、菌体を回収した（乾燥菌体換算濃度は３１質量％）。 The transformant was inoculated into 500 ml of LB medium containing 100 μg / mL of ampicillin in a 2 L baffled Erlenmeyer flask and cultured with shaking at 30 ° C. for 26.5 hours. Thereafter, the culture solution was centrifuged at 8000 rpm for 10 minutes to collect the cells (the dry cell equivalent concentration was 31% by mass).
　触媒菌体の超音波破砕物の調製および触媒死菌体の調製は参考例１の方法に従った。
（実施例４）
［窒素置換の反応例：野生型酵素］
　３００ｍＬのフラスコに、Ｌ－リジン塩酸塩の終濃度が４５質量％となるように、かつ、ピリドキサールリン酸の終濃度が０．１５ｍＭとなるように調製した基質溶液１２０ｇを加えて、反応液を調製した。次に、窒素ガスを反応液に通気（０．３Ｌ／ｈｒ）し、溶存酸素センサー（発酵用酸素電極（ＣＳＬ－１、エイブル社製）による溶存酸素濃度が０ｐｐｍとなるまで攪拌（１時間）した。このときの反応液中のｐＨは、５．６であった。次に、参考例１で調製したＷ／ｐＣＡＤＡ１の触媒死菌体（乾燥菌体重量換算０．０６４８ｇ、リジン１ｇに対する触媒死菌体の比率０．００１５ｇ）を添加して、４２℃、２００ｒｐｍで、２４時間、反応器に酸素が入らない様に注意して反応を行った。反応期間中の溶存酸素濃度は、０ｐｐｍであり、４２℃における飽和溶存酸素濃度（７．４ｐｐｍ）の０％であった。また、反応終了直後の反応液中のｐＨは、７．９であった。 Preparation of ultrasonic crushed material of catalyst cells and preparation of dead catalyst cells were carried out according to the method of Reference Example 1.
(Example 4)
[Example of nitrogen substitution reaction: wild-type enzyme]
120 g of a substrate solution prepared so that the final concentration of L-lysine hydrochloride is 45% by mass and the final concentration of pyridoxal phosphate is 0.15 mM is added to a 300 mL flask, and the reaction solution is added. Prepared. Next, nitrogen gas was passed through the reaction solution (0.3 L / hr) and stirred until the dissolved oxygen concentration by the dissolved oxygen sensor (fermentation oxygen electrode (CSL-1, Able)) was 0 ppm (1 hour) The pH in the reaction solution at this time was 5.6 Next, W / pCADA1 catalyst dead cells prepared in Reference Example 1 (0.0648 g in terms of dry cell weight, catalyst for 1 g of lysine) The dead cells were added at a rate of 0.0015 g), and the reaction was carried out at 42 ° C. and 200 rpm for 24 hours taking care not to allow oxygen to enter the reactor.The dissolved oxygen concentration during the reaction period was 0 ppm. It was 0% of the saturated dissolved oxygen concentration (7.4 ppm) at 42 ° C. The pH in the reaction solution immediately after the completion of the reaction was 7.9.
　このときの反応収率は、９９％であった。 The reaction yield at this time was 99%.
　なお、窒素ガスで置換しない場合の反応収率は９５％であった。
（実施例５）
［窒素置換の反応例：４重変異酵素］
　３００ｍＬのフラスコに、Ｌ－リジン塩酸塩の終濃度が４５質量％となるように、かつ、ピリドキサールリン酸の終濃度が０．１５ｍＭとなるように調製した基質溶液１２０ｇを加えて、反応液を調製した。次に、窒素ガスを反応液に通気（０．３Ｌ／ｈｒ）し、溶存酸素センサーにより溶存酸素濃度が０ｐｐｍとなるまで攪拌（１時間）した。このときの反応液中のｐＨは、５．６であった。次に、実施例３で調製したＷ／ｐＣＡＤＡ２１の触媒死菌体（乾燥菌体重量換算０．０６４８ｇ、リジン１ｇに対する触媒死菌体の比率０．００１５ｇ）を添加して、４２℃、２００ｒｐｍで、２４時間、反応器に酸素が入らない様に注意して反応を行った。反応期間中の存酸素濃度は、０ｐｐｍであり、４２℃における飽和溶存酸素濃度（７．４ｐｐｍ）の０％であった。また、反応終了直後の反応液中のｐＨは、８．０であった。 In addition, the reaction yield when not replacing with nitrogen gas was 95%.
(Example 5)
[Example of nitrogen substitution reaction: quadruple mutant enzyme]
120 g of a substrate solution prepared so that the final concentration of L-lysine hydrochloride is 45% by mass and the final concentration of pyridoxal phosphate is 0.15 mM is added to a 300 mL flask, and the reaction solution is added. Prepared. Next, nitrogen gas was passed through the reaction solution (0.3 L / hr), and the dissolved oxygen sensor was stirred (1 hour) until the dissolved oxygen concentration reached 0 ppm. The pH in the reaction solution at this time was 5.6. Next, W / pCADA21 catalyst dead cells prepared in Example 3 (0.0648 g in terms of dry cell weight, 0.0015 g of catalyst dead cells to 1 g of lysine) were added, and the temperature was 42 ° C. and 200 rpm. The reaction was carried out with care not to allow oxygen to enter the reactor for 24 hours. The oxygen concentration during the reaction period was 0 ppm, which was 0% of the saturated dissolved oxygen concentration (7.4 ppm) at 42 ° C. Moreover, pH in the reaction liquid immediately after completion | finish of reaction was 8.0.
　このときの反応収率は、９９％であった。 The reaction yield at this time was 99%.
　なお、窒素ガスで置換しない場合の反応収率は、９６％であった。
（実施例６）
［窒素置換の反応例：５重変異酵素］
　３００ｍＬのフラスコに、Ｌ－リジン塩酸塩の終濃度が４５質量％となるように、かつ、ピリドキサールリン酸の終濃度が０．１５ｍＭとなるように調製した基質溶液１２０ｇを加えて、反応液を調製した。次に、窒素ガスを反応液に通気（０．３Ｌ／ｈｒ）し、溶存酸素センサーにより溶存酸素濃度が０ｐｐｍとなるまで攪拌（１時間）した。このときの反応液中のｐＨは、５．６であった。次に、実施例３で調製したＷ／ｐＣＡＤＡ２２の触媒死菌体（乾燥菌体重量換算０．０６４８ｇ、リジン１ｇに対する触媒死菌体の比率０．００１５ｇ）を添加して、４２℃、２００ｒｐｍで、２４時間、反応器に酸素が入らない様に注意して反応を行った。反応期間中の存酸素濃度は、０ｐｐｍであり、４２℃における飽和溶存酸素濃度（７．４ｐｐｍ）の０％であった。また、反応終了直後の反応液中のｐＨは、８．０であった。 In addition, the reaction yield when not replacing with nitrogen gas was 96%.
(Example 6)
[Example of nitrogen substitution reaction: 5-fold mutant enzyme]
120 g of a substrate solution prepared so that the final concentration of L-lysine hydrochloride is 45% by mass and the final concentration of pyridoxal phosphate is 0.15 mM is added to a 300 mL flask, and the reaction solution is added. Prepared. Next, nitrogen gas was passed through the reaction solution (0.3 L / hr), and the dissolved oxygen sensor was stirred (1 hour) until the dissolved oxygen concentration reached 0 ppm. The pH in the reaction solution at this time was 5.6. Next, W / pCADA22 catalyst dead cells prepared in Example 3 (0.0648 g in terms of dry cell weight, 0.0015 g ratio of catalyst dead cells to 1 g of lysine) were added, and at 42 ° C. and 200 rpm. The reaction was carried out with care not to allow oxygen to enter the reactor for 24 hours. The oxygen concentration during the reaction period was 0 ppm, which was 0% of the saturated dissolved oxygen concentration (7.4 ppm) at 42 ° C. Moreover, pH in the reaction liquid immediately after completion | finish of reaction was 8.0.
　このときの反応収率は、９９％であった。 The reaction yield at this time was 99%.
　なお、窒素ガスで置換しない場合の反応収率は、９６％であった。
（実施例７）
［還元剤添加の反応例：野生型酵素］
　３００ｍＬのフラスコに、Ｌ－リジン塩酸塩の終濃度が１０質量％となるように、かつ、ピリドキサールリン酸の終濃度が０．１５ｍＭとなるように調製した基質溶液１２０ｇを加えて、反応液を調製した。次に、亜硫酸ナトリウム、ディチオスレイトール、ハイドロサルファイトナトリウムをそれぞれが１．０ｇ／Ｌとなるように添加し、溶存酸素センサーにより溶存酸素濃度が０ｐｐｍとなるまで攪拌した。このときの反応液中のｐＨは、５．６であった。次に、参考例１で調製したＷ／ｐＣＡＤＡ１の触媒死菌体（乾燥菌体重量換算０．００３６ｇ、リジン１ｇに対する触媒死菌体の比率０．０００３ｇ）を添加して、４２℃、２００ｒｐｍで、２４時間、反応器に酸素が入らない様に注意して反応を行った。反応期間中の溶存酸素濃度は、０ｐｐｍであり、４２℃における飽和溶存酸素濃度（７．４ｐｐｍ）の０％であった。 In addition, the reaction yield when not replacing with nitrogen gas was 96%.
(Example 7)
[Example reaction of reducing agent addition: wild-type enzyme]
120 g of a substrate solution prepared so that the final concentration of L-lysine hydrochloride is 10% by mass and the final concentration of pyridoxal phosphate is 0.15 mM is added to a 300 mL flask, and the reaction solution is added. Prepared. Next, sodium sulfite, dithiothreitol, and sodium hydrosulfite were added so that each became 1.0 g / L, and it stirred until the dissolved oxygen concentration was set to 0 ppm with the dissolved oxygen sensor. The pH in the reaction solution at this time was 5.6. Next, catalyst dead cells of W / pCADA1 prepared in Reference Example 1 (0.0036 g in terms of dry cell weight, 0.0003 g of catalyst dead cells relative to 1 g of lysine) were added, and the temperature was 42 ° C. and 200 rpm. The reaction was carried out with care not to allow oxygen to enter the reactor for 24 hours. The dissolved oxygen concentration during the reaction period was 0 ppm, which was 0% of the saturated dissolved oxygen concentration (7.4 ppm) at 42 ° C.
　このときの反応収率は、９９％であった。 The reaction yield at this time was 99%.
　なお、亜硫酸ナトリウムを添加しない場合の反応収率は、９０％であった。
（実施例８）
［窒素による部分置換の反応例：野生型酵素］
　３００ｍＬのフラスコに、Ｌ－リジン塩酸塩の終濃度が４５質量％となるように、かつ、ピリドキサールリン酸の終濃度が０．１５ｍＭとなるように調製した基質溶液１２０ｇを加えて、反応液を調製した。次に、窒素ガスを反応液に通気（０．３Ｌ／ｈｒ）し、溶存酸素センサー（発酵用酸素電極（ＣＳＬ－１、エイブル社製）による溶存酸素濃度が５ｐｐｍとなるまで攪拌した。このときの反応液中のｐＨは、５．６であった。 The reaction yield when no sodium sulfite was added was 90%.
(Example 8)
[Example of partial substitution with nitrogen: wild-type enzyme]
120 g of a substrate solution prepared so that the final concentration of L-lysine hydrochloride is 45% by mass and the final concentration of pyridoxal phosphate is 0.15 mM is added to a 300 mL flask, and the reaction solution is added. Prepared. Next, nitrogen gas was passed through the reaction solution (0.3 L / hr), and the mixture was stirred until the dissolved oxygen concentration by the dissolved oxygen sensor (fermentation oxygen electrode (CSL-1, manufactured by Able)) reached 5 ppm. The pH in the reaction solution was 5.6.
　次に、参考例１で調製したＷ／ｐＣＡＤＡ１の触媒死菌体（乾燥菌体重量換算０．０６４８ｇ、リジン１ｇに対する触媒死菌体の比率０．００１５ｇ）を添加して、４２℃、２００ｒｐｍで、２４時間、反応器に酸素が入らない様に注意して反応を行った。反応期間中の溶存酸素濃度は、０ｐｐｍであり、４２℃における飽和溶存酸素濃度（７．４ｐｐｍ）の０％であった。また、反応終了直後の反応液中のｐＨは、７．９であった。 Next, catalyst dead cells of W / pCADA1 prepared in Reference Example 1 (0.0648 g in terms of dry cell weight, 0.0015 g of catalyst dead cells relative to 1 g of lysine) were added, and at 42 ° C. and 200 rpm. The reaction was carried out with care not to allow oxygen to enter the reactor for 24 hours. The dissolved oxygen concentration during the reaction period was 0 ppm, which was 0% of the saturated dissolved oxygen concentration (7.4 ppm) at 42 ° C. Moreover, pH in the reaction liquid immediately after completion | finish of reaction was 7.9.
　このときの反応収率は、９８％であった。 The reaction yield at this time was 98%.
　なお、窒素ガスで置換しない場合の反応収率は９５％であった。
（実施例９）
［亜硫酸ナトリウム添加の反応例：野生型酵素］
　３００ｍＬのフラスコに、Ｌ－リジン塩酸塩の終濃度が４５質量％となるように、かつ、ピリドキサールリン酸の終濃度が０．１５ｍＭとなるように調製した基質溶液１２０ｇを加えて、反応液を調製した。次に、亜硫酸ナトリウムを１．７ｇ／Ｌとなるように添加し、溶存酸素センサーにより溶存酸素濃度が０ｐｐｍとなるまで攪拌した。このときの反応液中のｐＨは、５．６であった。次に、参考例１で調製したＷ／ｐＣＡＤＡ１の触媒死菌体（乾燥菌体重量換算０．０６４８ｇ、リジン１ｇに対する触媒死菌体の比率０．００１５ｇ）を添加して、４２℃、２００ｒｐｍで、２４時間、反応器に酸素が入らない様に注意して反応を行った。反応期間中の溶存酸素濃度は、０ｐｐｍであり、４２℃における飽和溶存酸素濃度（７．４ｐｐｍ）の０％であった。また、反応終了直後の反応液中のｐＨは、８．０であった。 In addition, the reaction yield when not replacing with nitrogen gas was 95%.
Example 9
[Example reaction of sodium sulfite addition: wild-type enzyme]
120 g of a substrate solution prepared so that the final concentration of L-lysine hydrochloride is 45% by mass and the final concentration of pyridoxal phosphate is 0.15 mM is added to a 300 mL flask, and the reaction solution is added. Prepared. Next, sodium sulfite was added so that it might become 1.7 g / L, and it stirred until the dissolved oxygen concentration was set to 0 ppm with the dissolved oxygen sensor. The pH in the reaction solution at this time was 5.6. Next, catalyst dead cells of W / pCADA1 prepared in Reference Example 1 (0.0648 g in terms of dry cell weight, 0.0015 g of catalyst dead cells relative to 1 g of lysine) were added, and at 42 ° C. and 200 rpm. The reaction was carried out with care not to allow oxygen to enter the reactor for 24 hours. The dissolved oxygen concentration during the reaction period was 0 ppm, which was 0% of the saturated dissolved oxygen concentration (7.4 ppm) at 42 ° C. Moreover, pH in the reaction liquid immediately after completion | finish of reaction was 8.0.
　このときの反応収率は、９９％であった。 The reaction yield at this time was 99%.
　なお、亜硫酸ナトリウムを添加しない場合の反応収率は、９５％であった。
（実施例１０）
［亜硫酸ナトリウム添加の反応例：単独変異型酵素］
　Ｗ／ｐＣＡＤＡ１、および、Ｗ／ｐＣＡＤＡ２３～１１９を３ｍｌのＬＢ培地（Ｄｉｆｃｏ　Ｃａｔ．２４４６２０）を入れた試験管に植菌し、ＩＰＴＧ（イソプロピルーβーチオガラクトピラノシド）が０．１ｍＭとなるように添加し、３３℃、２００ｒｐｍで２４時間培養した。得られた培養液１ｍｌを１．５ｍｌチューブに入れ、使用するまで－２０℃で保存した。１０質量％となるようにリジン塩酸塩（Ｗａｋｏ）を溶解し、亜硫酸ナトリウムを１．７ｇ／Ｌとなるように加えた液をそれぞれ５ｍｌとり、１５ｍｌスクリューキャップつきのＰＰ容器に入れ、０．４％ＰＬＰ水溶液を５０μｌ、及び上記で凍らせた培養液を溶解した後、良く攪拌したものを２００μｌ添加した。 The reaction yield when no sodium sulfite was added was 95%.
(Example 10)
[Example of reaction with sodium sulfite added: Single mutant enzyme]
W / pCADA1 and W / pCADA23 to 119 are inoculated into a test tube containing 3 ml of LB medium (Difco Cat. 244620), and IPTG (isopropyl-β-thiogalactopyranoside) becomes 0.1 mM. And cultured at 33 ° C. and 200 rpm for 24 hours. 1 ml of the obtained culture broth was placed in a 1.5 ml tube and stored at −20 ° C. until use. Dissolve lysine hydrochloride (Wako) to 10% by mass, take 5 ml each of sodium sulfite added to 1.7 g / L, put them in a PP container with a 15 ml screw cap, and add 0.4% 50 μl of PLP aqueous solution and 200 μl of well-stirred culture solution were added after dissolving the above frozen culture solution.
　反応は容器を振盪方向に並行、水平に設置し、２００ｒｐｍ、４５℃、２時間反応を行った。反応液に２Ｍ塩酸を１ｍＬ添加して反応を停止した。この反応停止液を水で適当に希釈し、生成した１、５－ペンタメチレンジアミンをＨＰＬＣにより定量した。 The reaction was carried out at 200 rpm, 45 ° C. for 2 hours with the container placed horizontally and parallel to the shaking direction. The reaction was stopped by adding 1 mL of 2M hydrochloric acid to the reaction solution. This reaction stop solution was appropriately diluted with water, and the produced 1,5-pentamethylenediamine was quantified by HPLC.
　反応収率は、何れの変異型酵素も亜硫酸ナトリウムを添加しなかった反応に比べて１．３倍以上高い収率を示した。
（実施例１１）
［亜硫酸ナトリウム添加の反応例：４重変異酵素］
　３００ｍＬのフラスコに、Ｌ－リジン塩酸塩の終濃度が４５質量％となるように、かつ、ピリドキサールリン酸の終濃度が０．１５ｍＭとなるように調製した基質溶液１２０ｇを加えて、反応液を調製した。次に、亜硫酸ナトリウムを１．７ｇ／Ｌとなるように添加した。次に、実施例３で調製したＷ／ｐＣＡＤＡ２１の触媒死菌体（乾燥菌体重量換算０．０６４８ｇ、リジン１ｇに対する触媒死菌体の比率０．００１５ｇ）を添加して、４２℃、２００ｒｐｍで、２４時間、反応器に酸素が入らない様に注意して反応を行った。反応期間中の溶存酸素濃度は、０ｐｐｍであり、４２℃における飽和溶存酸素濃度（７．４ｐｐｍ）の０％であった。また、反応終了直後の反応液中のｐＨは、８．０であった。 The reaction yield of each mutant enzyme was 1.3 times higher than the reaction in which sodium sulfite was not added.
(Example 11)
[Example of reaction with sodium sulfite added: quadruple mutant enzyme]
120 g of a substrate solution prepared so that the final concentration of L-lysine hydrochloride is 45% by mass and the final concentration of pyridoxal phosphate is 0.15 mM is added to a 300 mL flask, and the reaction solution is added. Prepared. Next, sodium sulfite was added so that it might become 1.7 g / L. Next, W / pCADA21 catalyst dead cells prepared in Example 3 (0.0648 g in terms of dry cell weight, 0.0015 g of catalyst dead cells to 1 g of lysine) were added, and the temperature was 42 ° C. and 200 rpm. The reaction was carried out with care not to allow oxygen to enter the reactor for 24 hours. The dissolved oxygen concentration during the reaction period was 0 ppm, which was 0% of the saturated dissolved oxygen concentration (7.4 ppm) at 42 ° C. Moreover, pH in the reaction liquid immediately after completion | finish of reaction was 8.0.
　このときの反応収率は、９９％であった。 The reaction yield at this time was 99%.
　なお、亜硫酸ナトリウムを添加しない場合の反応収率は、９６％であった。
（実施例１２）
［亜硫酸ナトリウム添加の反応例：５重変異酵素］
　３００ｍＬのフラスコに、Ｌ－リジン塩酸塩の終濃度が４５質量％となるように、かつ、ピリドキサールリン酸の終濃度が０．１５ｍＭとなるように調製した基質溶液１２０ｇを加えて、反応液を調製した。次に、亜硫酸ナトリウムを１．７ｇ／Ｌとなるように添加した。次に、実施例３で調製したＷ／ｐＣＡＤＡ２２の触媒死菌体（乾燥菌体重量換算０．０６４８ｇ、リジン１ｇに対する触媒死菌体の比率０．００１５ｇ）を添加して、４２℃、２００ｒｐｍで、２４時間、反応器に酸素が入らない様に注意して反応を行った。反応期間中の溶存酸素濃度は、０ｐｐｍであり、４２℃における飽和溶存酸素濃度（７．４ｐｐｍ）の０％であった。また、反応終了直後の反応液中のｐＨは、８．０であった。 The reaction yield when no sodium sulfite was added was 96%.
Example 12
[Reaction example of adding sodium sulfite: 5-fold mutant enzyme]
120 g of a substrate solution prepared so that the final concentration of L-lysine hydrochloride is 45% by mass and the final concentration of pyridoxal phosphate is 0.15 mM is added to a 300 mL flask, and the reaction solution is added. Prepared. Next, sodium sulfite was added so that it might become 1.7 g / L. Next, W / pCADA22 catalyst dead cells prepared in Example 3 (0.0648 g in terms of dry cell weight, 0.0015 g ratio of catalyst dead cells to 1 g of lysine) were added, and at 42 ° C. and 200 rpm. The reaction was carried out with care not to allow oxygen to enter the reactor for 24 hours. The dissolved oxygen concentration during the reaction period was 0 ppm, which was 0% of the saturated dissolved oxygen concentration (7.4 ppm) at 42 ° C. Moreover, pH in the reaction liquid immediately after completion | finish of reaction was 8.0.
　このときの反応収率は、９９％であった。 The reaction yield at this time was 99%.
　なお、亜硫酸ナトリウムを添加しない場合の反応収率は、９６％であった。
（実施例１３）
［亜硫酸ナトリウム添加の反応例：４重変異酵素（最小触媒添加反応）］
　３００ｍＬのフラスコに、Ｌ－リジン塩酸塩の終濃度が４５質量％となるように、かつ、ピリドキサールリン酸の終濃度が０．１５ｍＭとなるように調製した基質溶液１２０ｇを加えて、反応液を調製した。次に、亜硫酸ナトリウムを１．７ｇ／Ｌとなるように添加した。次に、実施例３で調製したＷ／ｐＣＡＤＡ２１の触媒死菌体（乾燥菌体重量換算０．０３２４ｇ、リジン１ｇに対する触媒死菌体の比率０．０００７５ｇ）を添加して、４２℃、２００ｒｐｍで、４８時間、反応器に酸素が入らない様に注意して反応を行った。反応期間中の溶存酸素濃度は、０ｐｐｍであり、４２℃における飽和溶存酸素濃度（７．４ｐｐｍ）の０％であった。また、反応終了直後の反応液中のｐＨは、８．２０であった。 The reaction yield when no sodium sulfite was added was 96%.
(Example 13)
[Example reaction of adding sodium sulfite: quadruple mutant enzyme (minimum catalyst addition reaction)]
120 g of a substrate solution prepared so that the final concentration of L-lysine hydrochloride is 45% by mass and the final concentration of pyridoxal phosphate is 0.15 mM is added to a 300 mL flask, and the reaction solution is added. Prepared. Next, sodium sulfite was added so that it might become 1.7 g / L. Next, the catalyst dead cells of W / pCADA21 prepared in Example 3 (0.0324 g in terms of dry cell weight, 0.00075 g of catalyst dead cells relative to 1 g of lysine) were added, and at 42 ° C. and 200 rpm. The reaction was carried out for 48 hours taking care not to allow oxygen to enter the reactor. The dissolved oxygen concentration during the reaction period was 0 ppm, which was 0% of the saturated dissolved oxygen concentration (7.4 ppm) at 42 ° C. Moreover, pH in the reaction liquid immediately after completion | finish of reaction was 8.20.
　このときの反応収率は、９９％であった。 The reaction yield at this time was 99%.
　なお、亜硫酸ナトリウムを添加しない場合の反応収率は、９１％であった。
（実施例１４）
［亜硫酸ナトリウム添加の反応例：５重変異酵素（最小触媒添加反応）］
　３００ｍＬのフラスコに、Ｌ－リジン塩酸塩の終濃度が４５質量％となるように、かつ、ピリドキサールリン酸の終濃度が０．１５ｍＭとなるように調製した基質溶液１２０ｇを加えて、反応液を調製した。次に、亜硫酸ナトリウムを１．７ｇ／Ｌとなるように添加した。次に、実施例３で調製したＷ／ｐＣＡＤＡ２２の触媒死菌体（乾燥菌体重量換算０．０３２４ｇ、リジン１ｇに対する触媒死菌体の比率０．０００７５ｇ）を添加して、４２℃、２００ｒｐｍで、４８時間、反応器に酸素が入らない様に注意して反応を行った。反応期間中の溶存酸素濃度は、０ｐｐｍであり、４２℃における飽和溶存酸素濃度（７．４ｐｐｍ）の０％であった。また、反応終了直後の反応液中のｐＨは、８．０であった。 The reaction yield when no sodium sulfite was added was 91%.
(Example 14)
[Example reaction of adding sodium sulfite: Five-fold mutation enzyme (minimum catalyst addition reaction)]
120 g of a substrate solution prepared so that the final concentration of L-lysine hydrochloride is 45% by mass and the final concentration of pyridoxal phosphate is 0.15 mM is added to a 300 mL flask, and the reaction solution is added. Prepared. Next, sodium sulfite was added so that it might become 1.7 g / L. Next, W / pCADA22 catalyst dead cells prepared in Example 3 (0.0324 g in terms of dry cell weight, 0.00075 g ratio of catalyst dead cells to 1 g of lysine) were added, and the temperature was 42 ° C. and 200 rpm. The reaction was carried out for 48 hours taking care not to allow oxygen to enter the reactor. The dissolved oxygen concentration during the reaction period was 0 ppm, which was 0% of the saturated dissolved oxygen concentration (7.4 ppm) at 42 ° C. Moreover, pH in the reaction liquid immediately after completion | finish of reaction was 8.0.
　このときの反応収率は、９９％であった。 The reaction yield at this time was 99%.
　なお、亜硫酸ナトリウムを添加しない場合の反応収率は、９１％であった。
（実施例１５）
［亜硫酸ナトリウムの後添加の反応例：野生型酵素］
　３００ｍＬのフラスコに、Ｌ－リジン塩酸塩の終濃度が４５質量％となるように、かつ、ピリドキサールリン酸の終濃度が０．１５ｍＭとなるように調製した基質溶液１２０ｇを加えて、反応液を調製した。次いで、参考例１で調製したＷ／ｐＣＡＤＡ１の触媒死菌体（乾燥菌体重量換算０．０６４８ｇ、リジン１ｇに対する触媒死菌体の比率０．００１５ｇ）を添加した後、さらに、亜硫酸ナトリウムを１．７ｇ／Ｌとなるようにを添加して、４２℃、２００ｒｐｍで、２４時間、反応器に酸素が入らない様に注意して反応を開始した。 The reaction yield when no sodium sulfite was added was 91%.
(Example 15)
[Example of post-addition reaction of sodium sulfite: wild-type enzyme]
120 g of a substrate solution prepared so that the final concentration of L-lysine hydrochloride is 45% by mass and the final concentration of pyridoxal phosphate is 0.15 mM is added to a 300 mL flask, and the reaction solution is added. Prepared. Next, after adding the dead catalyst cell of W / pCADA1 prepared in Reference Example 1 (0.0648 g in terms of dry cell weight, 0.0015 g of the dead catalyst cell to 1 g of lysine), sodium sulfite was further added to 1 Then, the reaction was started at 42 ° C. and 200 rpm for 24 hours so as not to allow oxygen to enter the reactor.
　反応開始３０分後には、溶存酸素濃度は、４２℃における飽和溶存酸素濃度の０％となった。反応開始時のｐＨは５．６であり、反応終了直後の反応液中のｐＨは、８．０であった。 30 minutes after the start of the reaction, the dissolved oxygen concentration became 0% of the saturated dissolved oxygen concentration at 42 ° C. The pH at the start of the reaction was 5.6, and the pH in the reaction solution immediately after the completion of the reaction was 8.0.
　このときの反応収率は、９９％であった。
（実施例１６）
［還元剤存在下での触媒死菌体の保存安定性］
　参考例１で調製したＷ／ｐＣＡＤＡ１の触媒死菌体を、４℃で８０日間保存した。 The reaction yield at this time was 99%.
(Example 16)
[Storage stability of dead catalyst cells in the presence of reducing agent]
The dead W / pCADA1 catalyst prepared in Reference Example 1 was stored at 4 ° C. for 80 days.
　亜硫酸ナトリウム存在下で調製した触媒死菌体の残存活性は、８０％であった。 The residual activity of the dead catalyst cells prepared in the presence of sodium sulfite was 80%.
　無添加で調製した触媒死菌体の残存活性は、４０％であった。
（実施例１７）
［１，５－ペンタメチレンジイソシアネートの製造］
（窒素置換した反応液からのＰＤＡ精製）
　実施例５で調製した反応終了後の反応液を硫酸でｐＨを６．０に調整した後、８０００ｒｐｍ、２０分の遠心分離により、菌体などの沈殿物を除去し、上清を得た。次に、この上清に、３０％水酸化ナトリウム溶液を加えて、ｐＨを１２に調整した。
（精製ＰＤＡの濃縮）
　分液ロートに１，５－ペンタメチレンジアミン水溶液１００質量部をｎ－ブタノール（抽出溶媒）１００質量部と仕込み、１０分間混合し、その後、３０分間静置した。次いで、有機層（１，５－ペンタメチレンジアミンを含むｎ－ブタノール）を抜き出した。 The residual activity of the dead catalyst cells prepared without addition was 40%.
(Example 17)
[Production of 1,5-pentamethylene diisocyanate]
(PDA purification from nitrogen-substituted reaction solution)
The reaction solution after completion of the reaction prepared in Example 5 was adjusted to pH 6.0 with sulfuric acid and then centrifuged at 8000 rpm for 20 minutes to remove precipitates such as cells and obtain a supernatant. Next, 30% sodium hydroxide solution was added to the supernatant to adjust the pH to 12.
(Concentration of purified PDA)
In a separatory funnel, 100 parts by mass of a 1,5-pentamethylenediamine aqueous solution was charged with 100 parts by mass of n-butanol (extraction solvent), mixed for 10 minutes, and then allowed to stand for 30 minutes. Next, the organic layer (n-butanol containing 1,5-pentamethylenediamine) was extracted.
　次いで、温度計、蒸留塔、冷却管および窒素導入管を備えた４つ口フラスコに、有機層の抽出液（１，５－ペンタメチレンジアミンを含むｎ－ブタノール）１００質量部を仕込み、１００ｋＰａの減圧下、オイルバスの温度を１２０℃として、ｎ－ブタノールを留去させ、純度９９．９質量％の１，５－ペンタメチレンジアミンを得た。
（ＰＤＩの合成）
　電磁誘導撹拌機、自動圧力調整弁、温度計、窒素導入ライン、ホスゲン導入ライン、凝縮器および原料フィードポンプを備え付けたジャケット付き加圧反応器に、オルトジクロロベンゼン２０００質量部を仕込んだ。次いで、ホスゲン２３００質量部をホスゲン導入ラインから加え、撹拌を開始した。反応器のジャケットには冷水を通し、内温を約１０℃に保った。そこへ、ペンタメチレンジアミン４００質量部をオルトジクロロベンゼン２６００質量部に溶解した溶液を、フィードポンプにて６０分かけてフィードし、３０℃以下、常圧下で冷ホスゲン化を開始した。フィード終了後、加圧反応器内は淡褐白色スラリー状液となった。 Subsequently, 100 parts by mass of an organic layer extract (n-butanol containing 1,5-pentamethylenediamine) was charged into a four-necked flask equipped with a thermometer, a distillation column, a cooling tube, and a nitrogen introduction tube, Under reduced pressure, the temperature of the oil bath was set to 120 ° C., and n-butanol was distilled off to obtain 1,5-pentamethylenediamine having a purity of 99.9% by mass.
(Synthesis of PDI)
2000 parts by mass of orthodichlorobenzene was charged into a jacketed pressurized reactor equipped with an electromagnetic induction stirrer, an automatic pressure control valve, a thermometer, a nitrogen introduction line, a phosgene introduction line, a condenser and a raw material feed pump. Next, 2300 parts by mass of phosgene was added from the phosgene introduction line, and stirring was started. Cold water was passed through the reactor jacket to keep the internal temperature at about 10 ° C. A solution obtained by dissolving 400 parts by mass of pentamethylenediamine in 2600 parts by mass of orthodichlorobenzene was fed with a feed pump over 60 minutes, and cold phosgenation was started at 30 ° C. or lower and normal pressure. After completion of the feed, the pressure reactor became a pale brown white slurry.
　次いで、反応器の内液を、徐々に１６０℃まで昇温しながら、０．２５ＭＰａに加圧し、さらに圧力０．２５ＭＰａ、反応温度１６０℃で９０分間、熱ホスゲン化した。なお、熱ホスゲン化の途中で、ホスゲン１１００質量部を、さらに添加した。熱ホスゲン化の過程で、加圧反応器内液は、淡褐色澄明溶液となった。熱ホスゲン化終了後、１００～１４０℃において、窒素ガスを１００Ｌ／ｈｒで通気し、脱ガスした。 Next, the internal solution of the reactor was pressurized to 0.25 MPa while gradually raising the temperature to 160 ° C., and further subjected to thermal phosgenation at a pressure of 0.25 MPa and a reaction temperature of 160 ° C. for 90 minutes. During the thermal phosgenation, 1100 parts by mass of phosgene was further added. During the thermal phosgenation, the liquid in the pressurized reactor became a light brown clear solution. After completion of the thermal phosgenation, nitrogen gas was passed at 100 L / hr at 100 to 140 ° C. for degassing.
　次いで、減圧下でオルトジクロルベンゼンを留去した後、同じく減圧下でペンタメチレンジイソシアネートを留去させた。 Subsequently, orthodichlorobenzene was distilled off under reduced pressure, and pentamethylene diisocyanate was then distilled off under reduced pressure.
　次いで、留去させたペンタメチレンジイソシアネートを、攪拌機、温度計、還流管、および、窒素導入管を備えた４つ口フラスコに装入し、窒素を導入しながら、常圧下で、１９０℃、３時間加熱処理を行った。 Next, the distilled pentamethylene diisocyanate was charged into a four-necked flask equipped with a stirrer, a thermometer, a reflux tube, and a nitrogen introduction tube, and at 190 ° C. under normal pressure while introducing nitrogen. Heat treatment was performed for a time.
　次いで、加熱処理後のペンタメチレンジイソシアネートを、ガラス製フラスコに装入し、充填物を充填した蒸留管、還流比調節タイマーを装着した蒸留塔、および、冷却器を装備する精留装置を用いて、１２７～１３２℃、２．７ＫＰａの条件下、さらに還流しながら精留し、純度９９．８質量％のペンタメチレンジイソシアネートを４５０質量部得た。
（実施例１８）
［ポリイソシアネート組成物の製造］
　攪拌機、温度計、還流管、および、窒素導入管を備えた４つ口フラスコに、ペンタメチレンジイソシアネートを５００質量部、トリメチロールプロパン（以下、ＴＭＰと略する場合がある。）を５．８質量部、２，６－ジ（ｔｅｒｔ－ブチル）－４－メチルフェノールを０．２５質量部、トリス（トリデシル）ホスファイトを０．２５質量部装入し、８０℃で３時間反応させた。この溶液を６０℃に降温した後、トリマー化触媒としてＮ－（２－ヒドロキシプロピル）－Ｎ，Ｎ，Ｎ－トリメチルアンモニウム－２－エチルヘキサノエートを０．１質量部添加した。１時間半反応させた後、ｏ－トルエンスルホンアミドを０．１２質量部添加した（イソシアネート基の転化率：２０質量％）。得られた反応液を薄膜蒸留装置（真空度０．０９３ＫＰａ、温度１５０℃）に通液して未反応のペンタメチレンジイソシアネートを除去し、さらに、得られた組成物１００質量部に対し、ｏ－トルエンスルホンアミドを０．０２質量部添加し、ポリイソシアネート組成物を得た。 Next, the pentamethylene diisocyanate after the heat treatment is charged into a glass flask, using a distillation tube equipped with a packing, a distillation column equipped with a reflux ratio adjustment timer, and a rectification apparatus equipped with a cooler. The rectification was further performed under reflux at 127 to 132 ° C. and 2.7 KPa to obtain 450 parts by mass of pentamethylene diisocyanate having a purity of 99.8% by mass.
(Example 18)
[Production of polyisocyanate composition]
In a four-necked flask equipped with a stirrer, a thermometer, a reflux tube, and a nitrogen introduction tube, 500 parts by mass of pentamethylene diisocyanate and 5.8 masses of trimethylolpropane (hereinafter sometimes abbreviated as TMP). 0.25 parts by mass of 2,6-di (tert-butyl) -4-methylphenol and 0.25 parts by mass of tris (tridecyl) phosphite were charged at 80 ° C. for 3 hours. After the temperature of this solution was lowered to 60 ° C., 0.1 part by mass of N- (2-hydroxypropyl) -N, N, N-trimethylammonium-2-ethylhexanoate was added as a trimerization catalyst. After reacting for 1.5 hours, 0.12 parts by mass of o-toluenesulfonamide was added (conversion rate of isocyanate group: 20% by mass). The obtained reaction solution was passed through a thin film distillation apparatus (vacuum degree: 0.093 KPa, temperature: 150 ° C.) to remove unreacted pentamethylene diisocyanate. Further, with respect to 100 parts by mass of the obtained composition, o- 0.02 part by mass of toluenesulfonamide was added to obtain a polyisocyanate composition.
　このポリイソシアネート組成物のペンタメチレンジイソシアネート濃度は０．３質量％、イソシアネート３量体濃度は２９質量％、イソシアネート基濃度は２１．８質量％、２５℃における粘度は９８５０ｍＰａ・ｓ、色相はＡＰＨＡ４０であった。 This polyisocyanate composition has a pentamethylene diisocyanate concentration of 0.3% by mass, an isocyanate trimer concentration of 29% by mass, an isocyanate group concentration of 21.8% by mass, a viscosity at 25 ° C. of 9850 mPa · s, and a hue of APHA40. there were.
　本発明の１，５－ペンタメチレンジアミンの製造方法により得られる１，５－ペンタメチレンジアミン、１，５－ペンタメチレンジイソシアネート、および、ポリイソシアネート組成物は、例えば、バイオマス由来のポリマー原料として、コーティング、接着剤、シーラント、エラストマー、ゲル、バインダー、フィルム、シートおよびカプセルなどの材料、農薬、医薬の中間体などの種々の産業分野において、好適に用いることができる。 The 1,5-pentamethylenediamine, 1,5-pentamethylene diisocyanate, and polyisocyanate composition obtained by the method for producing 1,5-pentamethylenediamine of the present invention are, for example, coated as a biomass-derived polymer raw material. It can be suitably used in various industrial fields such as adhesives, sealants, elastomers, gels, binders, films, sheets and capsules, agricultural chemicals and pharmaceutical intermediates.
　また、本発明の触媒菌体の保存方法により保存される触媒菌体は、リジン脱炭酸酵素を長期間安定に保存することができるため、例えば、バイオマス由来のポリマー原料などの製造において、好適に用いることができる。 Further, since the catalyst cell preserved by the method for preserving the catalyst cell of the present invention can stably store lysine decarboxylase for a long period of time, it is suitable for the production of, for example, biomass-derived polymer raw materials. Can be used.

Claims (17)

  1. 　溶存酸素濃度が飽和溶存酸素濃度である時間が１時間以内である反応系内において、Ｌ－リジンおよび／またはその塩を、リジン脱炭酸酵素および／または変異型リジン脱炭酸酵素によって、リジン脱炭酸反応させることを特徴とする、１，５－ペンタメチレンジアミンの製造方法。 In a reaction system in which the dissolved oxygen concentration is the saturated dissolved oxygen concentration within 1 hour, L-lysine and / or a salt thereof is lysine decarboxylated by lysine decarboxylase and / or mutant lysine decarboxylase. A process for producing 1,5-pentamethylenediamine, characterized by reacting.
  2. 　Ｌ－リジンおよび／またはその塩１質量部に対して、リジン脱炭酸酵素および／または変異型リジン脱炭酸酵素が、乾燥菌体重量換算で、０．０００３質量部以上０．００１５質量部以下であることを特徴とする、請求項１に記載の１，５－ペンタメチレンジアミンの製造方法。 With respect to 1 part by mass of L-lysine and / or a salt thereof, lysine decarboxylase and / or mutant lysine decarboxylase is 0.0003 parts by mass or more and 0.0015 parts by mass or less in terms of dry cell weight. The method for producing 1,5-pentamethylenediamine according to claim 1, wherein:
  3. 　Ｙ軸を飽和溶存酸素濃度に対する溶存酸素濃度の割合（％）、Ｘ軸を時間（分）として、リジン脱炭酸反応における溶存酸素濃度と時間との関係がプロットされた相関線を示す相関図において、
    　相関線とＹ軸とＸ軸とに囲まれる部分の面積が、１０００未満であることを特徴とする、請求項１に記載の１，５－ペンタメチレンジアミンの製造方法。     In the correlation diagram showing the correlation line in which the relationship between the dissolved oxygen concentration and time in the lysine decarboxylation reaction is plotted, where the Y axis is the ratio (%) of the dissolved oxygen concentration to the saturated dissolved oxygen concentration and the X axis is the time (minutes) ,
    2. The method for producing 1,5-pentamethylenediamine according to claim 1, wherein the area surrounded by the correlation line, the Y axis, and the X axis is less than 1000.
  4. 　前記面積が、６５０以下であることを特徴とする、請求項３に記載の１，５－ペンタメチレンジアミンの製造方法。 The method for producing 1,5-pentamethylenediamine according to claim 3, wherein the area is 650 or less.
  5. 　前記溶存酸素濃度が、飽和溶存酸素濃度の６５％以下であり、かつ、
    　前記溶存酸素濃度が、飽和溶存酸素濃度の６５％以下の状態から２０分以内に飽和溶存酸素濃度の１％以下になる
    ことを特徴とする、請求項３に記載の１，５－ペンタメチレンジアミンの製造方法。     The dissolved oxygen concentration is 65% or less of the saturated dissolved oxygen concentration, and
    The 1,5-pentamethylenediamine according to claim 3, wherein the dissolved oxygen concentration becomes 1% or less of the saturated dissolved oxygen concentration within 20 minutes from the state of 65% or less of the saturated dissolved oxygen concentration. Manufacturing method.
  6. 　反応系内の酸素を除去する工程、および／または、反応系内に還元剤を存在させる工程を含むことを特徴とする、請求項１に記載の１，５－ペンタメチレンジアミンの製造方法。 The method for producing 1,5-pentamethylenediamine according to claim 1, comprising a step of removing oxygen in the reaction system and / or a step of allowing a reducing agent to be present in the reaction system.
  7. 　反応系内の酸素を除去する工程が、不活性ガスにより溶存酸素を置換する工程であることを特徴とする、請求項６に記載の１，５－ペンタメチレンジアミンの製造方法。 The method for producing 1,5-pentamethylenediamine according to claim 6, wherein the step of removing oxygen in the reaction system is a step of replacing dissolved oxygen with an inert gas.
  8. 　還元剤の酸化還元電位が、生理食塩水より低いことを特徴とする、請求項６に記載の１，５－ペンタメチレンジアミンの製造方法。 The method for producing 1,5-pentamethylenediamine according to claim 6, wherein the redox potential of the reducing agent is lower than that of physiological saline.
  9. 　還元剤が、メルカプト化合物、硫化物、水硫化物、還元性を有する硫黄の酸素酸塩、チオウレアおよびその誘導体、ヒドロキシル基および／またはカルボキシル基を有する環状化合物、フラボノイド化合物、窒素含有複素環化合物、ヒドラジル基化合物、および、ウロン酸基を有するムコ多糖類からなる群より選択される少なくとも１種であることを特徴とする、請求項８に記載の１，５－ペンタメチレンジアミンの製造方法。 The reducing agent is a mercapto compound, sulfide, hydrosulfide, reducing oxygen oxyacid salt, thiourea and its derivatives, cyclic compounds having a hydroxyl group and / or carboxyl group, flavonoid compounds, nitrogen-containing heterocyclic compounds, The method for producing 1,5-pentamethylenediamine according to claim 8, wherein the method is at least one selected from the group consisting of a hydrazyl group compound and a mucopolysaccharide having a uronic acid group.
  10. 　変異型リジン脱炭酸酵素が、配列表の配列番号４に記載のアミノ酸配列において、１３７、１３８、２８６、２９０、２９５、３０３、３１７、３３５、３５２、３５３、３８６、４４３、４６６、４７５、５５３、７１０および７１１番目のアミノ酸残基の少なくとも１つが、他のアミノ酸残基に置換されている変異型リジン脱炭酸酵素であることを特徴とする、請求項１に記載の１，５－ペンタメチレンジアミンの製造方法。 Mutant lysine decarboxylase is 137, 138, 286, 290, 295, 303, 317, 335, 352, 353, 386, 443, 466, 475, 553 in the amino acid sequence set forth in SEQ ID NO: 4 in the Sequence Listing. The 1,5-pentamethylene according to claim 1, wherein at least one of the amino acid residues 710 and 711 is a mutant lysine decarboxylase substituted with another amino acid residue A method for producing diamine.
  11. 　変異型リジン脱炭酸酵素が、配列表の配列番号４に記載のアミノ酸配列において、２９０、３３５、４７５および７１１番目のアミノ酸残基が、他のアミノ酸残基に置換されている変異型リジン脱炭酸酵素であることを特徴とする、請求項１０に記載の１，５－ペンタメチレンジアミンの製造方法。 The mutant lysine decarboxylase is a mutant lysine decarboxylase in which the amino acid residues at positions 290, 335, 475 and 711 are substituted with other amino acid residues in the amino acid sequence set forth in SEQ ID NO: 4 in the Sequence Listing. The method for producing 1,5-pentamethylenediamine according to claim 10, wherein the method is an enzyme.
  12. 　変異型リジン脱炭酸酵素が、配列表の配列番号４に記載のアミノ酸配列において、２８６、２９０、３３５、４７５および７１１番目のアミノ酸残基が、他のアミノ酸残基に置換されている変異型リジン脱炭酸酵素であることを特徴とする、請求項１０に記載の１，５－ペンタメチレンジアミンの製造方法。 Mutant lysine decarboxylase is a mutant lysine in which the 286th, 290th, 335th, 475th and 711st amino acid residues are substituted with other amino acid residues in the amino acid sequence set forth in SEQ ID NO: 4 in the Sequence Listing The method for producing 1,5-pentamethylenediamine according to claim 10, wherein the method is decarboxylase.
  13. 　溶存酸素濃度が飽和溶存酸素濃度である時間が１時間以内である反応系内において、Ｌ－リジンおよび／またはその塩を、リジン脱炭酸酵素および／または変異型リジン脱炭酸酵素によって、リジン脱炭酸反応させることにより得られた１，５－ペンタメチレンジアミンまたはその塩を、イソシアネート化することを特徴とする、１，５－ペンタメチレンジイソシアネートの製造方法。 In a reaction system in which the dissolved oxygen concentration is the saturated dissolved oxygen concentration within 1 hour, L-lysine and / or a salt thereof is lysine decarboxylated by lysine decarboxylase and / or mutant lysine decarboxylase. A process for producing 1,5-pentamethylene diisocyanate, characterized in that 1,5-pentamethylenediamine or a salt thereof obtained by reacting is isocyanated.
  14. 　溶存酸素濃度が飽和溶存酸素濃度である時間が１時間以内である反応系内において、Ｌ－リジンおよび／またはその塩を、リジン脱炭酸酵素および／または変異型リジン脱炭酸酵素によって、リジン脱炭酸反応させることにより得られた１，５－ペンタメチレンジアミンまたはその塩を、イソシアネート化することにより得られた１，５－ペンタメチレンジイソシアネートを、
    　下記（ａ）～（ｅ）の官能基を少なくとも１種含有するように変性することを特徴とする、ポリイソシアネート組成物の製造方法。
    （ａ）イソシアヌレート基
    （ｂ）アロファネート基
    （ｃ）ビウレット基
    （ｄ）ウレタン基
    （ｅ）ウレア基     In a reaction system in which the dissolved oxygen concentration is the saturated dissolved oxygen concentration within 1 hour, L-lysine and / or a salt thereof is lysine decarboxylated by lysine decarboxylase and / or mutant lysine decarboxylase. 1,5-pentamethylene diisocyanate obtained by subjecting 1,5-pentamethylenediamine or a salt thereof obtained by reacting to isocyanate to
    A method for producing a polyisocyanate composition, which is modified so as to contain at least one of the following functional groups (a) to (e):
    (A) isocyanurate group (b) allophanate group (c) biuret group (d) urethane group (e) urea group
  15. 　リジン脱炭酸酵素および／または変異型リジン脱炭酸酵素を発現させた細胞を、還元剤の存在下で保存することを特徴とする、触媒菌体の保存方法。 A method for preserving a catalytic cell, comprising storing cells expressing lysine decarboxylase and / or mutant lysine decarboxylase in the presence of a reducing agent.
  16. 　還元剤の酸化還元電位が、生理食塩水より低いことを特徴とする、請求項１５に記載の触媒菌体の保存方法。 The method for preserving catalytic cells according to claim 15, wherein the redox potential of the reducing agent is lower than that of physiological saline.
  17. 　還元剤が、メルカプト化合物、硫化物、水硫化物、還元性を有する硫黄の酸素酸塩、チオウレアおよびその誘導体、ヒドロキシル基および／またはカルボキシル基を有する環状化合物、フラボノイド化合物、窒素含有複素環化合物、ヒドラジル基化合物、および、ウロン酸基を有するムコ多糖類からなる群より選択される少なくとも１種であることを特徴とする、請求項１５に記載の触媒菌体の保存方法。 The reducing agent is a mercapto compound, sulfide, hydrosulfide, reducing oxygen oxyacid salt, thiourea and its derivatives, cyclic compounds having a hydroxyl group and / or carboxyl group, flavonoid compounds, nitrogen-containing heterocyclic compounds, The method for preserving a catalytic cell according to claim 15, wherein the method is at least one selected from the group consisting of a hydrazyl group compound and a mucopolysaccharide having a uronic acid group.
PCT/JP2013/060355 2012-04-05 2013-04-04 Method for producing 1,5-pentamethylenediamine, method for producing 1,5-pentamethylenediisocyanate, method for producing polyisocyanate composition, and method for storing catalyst cell WO2013151139A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2014509210A JP5899309B2 (en) 2012-04-05 2013-04-04 Method for producing 1,5-pentamethylenediamine and method for preserving catalyst cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2012086667 2012-04-05
JP2012-086667 2012-04-05

Publications (1)

Publication Number Publication Date
WO2013151139A1 true WO2013151139A1 (en) 2013-10-10

Family

ID=49300617

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2013/060355 WO2013151139A1 (en) 2012-04-05 2013-04-04 Method for producing 1,5-pentamethylenediamine, method for producing 1,5-pentamethylenediisocyanate, method for producing polyisocyanate composition, and method for storing catalyst cell

Country Status (2)

Country Link
JP (1) JP5899309B2 (en)
WO (1) WO2013151139A1 (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015137418A1 (en) * 2014-03-11 2015-09-17 味の素株式会社 Method for producing 1,5-pentadiamine using increased-thermal-stability lysine decarboxylase mutant
JP2017527597A (en) * 2014-09-19 2017-09-21 コベストロ、ドイチュラント、アクチエンゲゼルシャフトＣｏｖｅｓｔｒｏ Ｄｅｕｔｓｃｈｌａｎｄ Ａｇ Process for producing 1,5-pentane diisocyanate in the gas phase
CN110157750A (en) * 2018-02-13 2019-08-23 上海凯赛生物技术研发中心有限公司 A kind of improvement lysine decarboxylase, production method and its application
CN113881719A (en) * 2020-07-02 2022-01-04 中国科学院过程工程研究所 Method for synthesizing 1, 5-pentanediamine through whole-cell catalysis
CN115611748A (en) * 2021-07-14 2023-01-17 上海凯赛生物技术股份有限公司 Separation method of 1, 5-pentanediamine

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08332092A (en) * 1995-04-04 1996-12-17 Mitsubishi Chem Corp Production of fumaric acid
JPH10108688A (en) * 1996-08-09 1998-04-28 Nippon Shokubai Co Ltd Production of l-aspartic acid
JP2011201863A (en) * 2010-03-01 2011-10-13 Mitsui Chemicals Inc Pentamethylene diisocyanate, polyisocyanate composition, method for preparing the pentamethylene diisocyanate and polyurethane resin

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08332092A (en) * 1995-04-04 1996-12-17 Mitsubishi Chem Corp Production of fumaric acid
JPH10108688A (en) * 1996-08-09 1998-04-28 Nippon Shokubai Co Ltd Production of l-aspartic acid
JP2011201863A (en) * 2010-03-01 2011-10-13 Mitsui Chemicals Inc Pentamethylene diisocyanate, polyisocyanate composition, method for preparing the pentamethylene diisocyanate and polyurethane resin

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MENG S.Y. ET AL.: "Nucleotide sequence of the Escherichia coli cad operon: a system for neutralization of low extracellular pH", J. BACTERIOL., vol. 174, no. 8, April 1992 (1992-04-01), pages 2659 - 2669, XP000978358 *
TAKATSUKA Y. ET AL.: "Gene cloning and molecular characterization of lysine decarboxylase from Selenomonas ruminantium delineate its evolutionary relationship to ornithine decarboxylases from eukaryotes", J. BACTERIOL., vol. 182, no. 23, December 2000 (2000-12-01), pages 6732 - 6741, XP055080140, DOI: doi:10.1128/JB.182.23.6732-6741.2000 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015137418A1 (en) * 2014-03-11 2015-09-17 味の素株式会社 Method for producing 1,5-pentadiamine using increased-thermal-stability lysine decarboxylase mutant
JPWO2015137418A1 (en) * 2014-03-11 2017-04-06 味の素株式会社 Method for producing 1,5-pentadiamine using lysine decarboxylase mutant with improved thermal stability
US10358638B2 (en) 2014-03-11 2019-07-23 Ajinomoto Co., Inc. Method of producing 1,5-pentadiamine using lysine decarboxylase mutant having improved thermal stability
JP2017527597A (en) * 2014-09-19 2017-09-21 コベストロ、ドイチュラント、アクチエンゲゼルシャフトＣｏｖｅｓｔｒｏ Ｄｅｕｔｓｃｈｌａｎｄ Ａｇ Process for producing 1,5-pentane diisocyanate in the gas phase
CN110157750A (en) * 2018-02-13 2019-08-23 上海凯赛生物技术研发中心有限公司 A kind of improvement lysine decarboxylase, production method and its application
CN110157750B (en) * 2018-02-13 2023-09-22 上海凯赛生物技术股份有限公司 Improved lysine decarboxylase, production method and application thereof
CN113881719A (en) * 2020-07-02 2022-01-04 中国科学院过程工程研究所 Method for synthesizing 1, 5-pentanediamine through whole-cell catalysis
CN115611748A (en) * 2021-07-14 2023-01-17 上海凯赛生物技术股份有限公司 Separation method of 1, 5-pentanediamine

Also Published As

Publication number Publication date
JP5899309B2 (en) 2016-04-06
JPWO2013151139A1 (en) 2015-12-17

Similar Documents

Publication Publication Date Title
JP5961635B2 (en) Method for producing 1,5-pentamethylenediamine
JP5700575B2 (en) Process for producing 1,5-pentamethylene diisocyanate
JP5849088B2 (en) Pentamethylene diisocyanate composition, polyisocyanate modified composition, polyurethane resin and polyurea resin
JP5899309B2 (en) Method for producing 1,5-pentamethylenediamine and method for preserving catalyst cells
JP2011201863A (en) Pentamethylene diisocyanate, polyisocyanate composition, method for preparing the pentamethylene diisocyanate and polyurethane resin
JP6482657B2 (en) Polyisocyanate composition, polyurethane resin and paint
JP5711940B2 (en) Method for preserving pentamethylenediamine or a salt thereof
JP5623310B2 (en) PENTAMethylenediamine or a salt thereof, and a production method thereof
JP2019154313A (en) Lysine decarboxylases, methods for producing 1,5-pentamethylene diisocyanate, and methods for producing polyisocyanate compositions
CN106810677B (en) Preparation method, the preparation method of polymer grade lactide of poly-lactic acid in high molecular weight

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13771985

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2014509210

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13771985

Country of ref document: EP

Kind code of ref document: A1