WO2013151139A1 - Procédé pour produire la 1,5-pentaméthylènediamine, procédé pour produire le 1,5-pentaméthylène diisocyanate, procédé pour produire une composition de polyisocyanate, et procédé pour stocker une cellule de catalyseur - Google Patents

Procédé pour produire la 1,5-pentaméthylènediamine, procédé pour produire le 1,5-pentaméthylène diisocyanate, procédé pour produire une composition de polyisocyanate, et procédé pour stocker une cellule de catalyseur 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
English (en)
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/ja
Publication of WO2013151139A1 publication Critical patent/WO2013151139A1/fr

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.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

Selon la présente invention, de la L-lysine et/ou un sel de celle-ci sont soumis à une décarboxylation de lysine par la lysine décarboxylase et/ou une lysine décarboxylase mutante dans un système de réaction dans lequel le temps durant lequel la concentration d'oxygène dissous est la concentration d'oxygène dissous saturée est au plus d'une heure.
PCT/JP2013/060355 2012-04-05 2013-04-04 Procédé pour produire la 1,5-pentaméthylènediamine, procédé pour produire le 1,5-pentaméthylène diisocyanate, procédé pour produire une composition de polyisocyanate, et procédé pour stocker une cellule de catalyseur WO2013151139A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2014509210A JP5899309B2 (ja) 2012-04-05 2013-04-04 １，５−ペンタメチレンジアミンの製造方法、および、触媒菌体の保存方法

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 (fr) 2013-10-10

Family

ID=49300617

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2013/060355 WO2013151139A1 (fr) 2012-04-05 2013-04-04 Procédé pour produire la 1,5-pentaméthylènediamine, procédé pour produire le 1,5-pentaméthylène diisocyanate, procédé pour produire une composition de polyisocyanate, et procédé pour stocker une cellule de catalyseur

Country Status (2)

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

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015137418A1 (fr) * 2014-03-11 2015-09-17 味の素株式会社 Procédé de fabrication de 1,5-pentadiamine mettant en œuvre un mutant de lysine décarboxylase de stabilité thermique améliorée
JP2017527597A (ja) * 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 気相中で１，５−ペンタンジイソシアネートを製造するための方法
CN110157750A (zh) * 2018-02-13 2019-08-23 上海凯赛生物技术研发中心有限公司 一种改良赖氨酸脱羧酶、生产方法及其应用
CN113881719A (zh) * 2020-07-02 2022-01-04 中国科学院过程工程研究所 一种全细胞催化合成1,5-戊二胺的方法
CN115611748A (zh) * 2021-07-14 2023-01-17 上海凯赛生物技术股份有限公司 一种1,5-戊二胺的分离方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08332092A (ja) * 1995-04-04 1996-12-17 Mitsubishi Chem Corp フマル酸の製造法
JPH10108688A (ja) * 1996-08-09 1998-04-28 Nippon Shokubai Co Ltd Ｌ−アスパラギン酸の製造方法
JP2011201863A (ja) * 2010-03-01 2011-10-13 Mitsui Chemicals Inc 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、ペンタメチレンジイソシアネートの製造方法、および、ポリウレタン樹脂

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08332092A (ja) * 1995-04-04 1996-12-17 Mitsubishi Chem Corp フマル酸の製造法
JPH10108688A (ja) * 1996-08-09 1998-04-28 Nippon Shokubai Co Ltd Ｌ−アスパラギン酸の製造方法
JP2011201863A (ja) * 2010-03-01 2011-10-13 Mitsui Chemicals Inc 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、ペンタメチレンジイソシアネートの製造方法、および、ポリウレタン樹脂

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 (fr) * 2014-03-11 2015-09-17 味の素株式会社 Procédé de fabrication de 1,5-pentadiamine mettant en œuvre un mutant de lysine décarboxylase de stabilité thermique améliorée
JPWO2015137418A1 (ja) * 2014-03-11 2017-04-06 味の素株式会社 熱安定性向上リジン脱炭酸酵素変異体を用いる１，５−ペンタジアミンの製造方法
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 (ja) * 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 気相中で１，５−ペンタンジイソシアネートを製造するための方法
CN110157750A (zh) * 2018-02-13 2019-08-23 上海凯赛生物技术研发中心有限公司 一种改良赖氨酸脱羧酶、生产方法及其应用
CN110157750B (zh) * 2018-02-13 2023-09-22 上海凯赛生物技术股份有限公司 一种改良赖氨酸脱羧酶、生产方法及其应用
CN113881719A (zh) * 2020-07-02 2022-01-04 中国科学院过程工程研究所 一种全细胞催化合成1,5-戊二胺的方法
CN115611748A (zh) * 2021-07-14 2023-01-17 上海凯赛生物技术股份有限公司 一种1,5-戊二胺的分离方法

Also Published As

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

Similar Documents

Publication Publication Date Title
JP5961635B2 (ja) １、５−ペンタメチレンジアミンの製造方法
JP5700575B2 (ja) １，５−ペンタメチレンジイソシアネートの製造方法
JP5849088B2 (ja) ペンタメチレンジイソシアネート組成物、ポリイソシアネート変性体組成物、ポリウレタン樹脂およびポリウレア樹脂
JP5899309B2 (ja) １，５−ペンタメチレンジアミンの製造方法、および、触媒菌体の保存方法
JP2011201863A (ja) 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、ペンタメチレンジイソシアネートの製造方法、および、ポリウレタン樹脂
JP6482657B2 (ja) ポリイソシアネート組成物、ポリウレタン樹脂および塗料
JP5711940B2 (ja) ペンタメチレンジアミンまたはその塩の保存方法
JP5623310B2 (ja) ペンタメチレンジアミンまたはその塩、および、その製造方法
JP2019154313A (ja) リジン脱炭酸酵素、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
CN106810677B (zh) 高分子量聚乳酸的制备方法、聚合级丙交酯的制备方法

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