JP2009195202A - Method for manufacturing purified pentamethylene diamine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide pentamethylene diamine which is suitable as a material of a polyamide resin. <P>SOLUTION: Pentamethylene diamine is obtained by liberation from a pentamethylene diamine salt solution by alkaline. The anion concentration in the pentamethylene diamine is 2.0 weight ppm or less. As for pentamethylene diamine, one using at least one type selected from a group of lysine decarboxylases, recombinant microorganisms whose lysine decarboxylase activity is improved, cells producing lysine decarboxylases or processed materials of the cells and is produced from lysine is used. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to pentamethylenediamine (common name: cadaverine, abbreviation: PMDA), and more particularly to pentamethylenediamine suitable as a raw material for polyamide resin.

  Most plastic materials use so-called fossil materials. Plastics are discarded except when recycled. In that case, disposal due to combustion or the like is becoming a problem in recent years because it causes the release of carbon dioxide. Therefore, in order to prevent global warming and form a recycling society, it is desired to replace the raw materials for plastic production with raw materials derived from biomass. Such needs range from injection moldings such as films, automobile parts, electrical / electronic parts, mechanical parts, fibers, monofilaments, and the like.

  Polyamide resins are excellent in mechanical strength, heat resistance, chemical resistance, etc., and are used in many fields as one of so-called engineering plastics. Among them, 56 nylon and 56/66 nylon obtained from pentamethylenediamine obtained from lysine as a raw material are highly expected as plant-derived polymers.

The above pentamethylenediamine is obtained by liberation from an aqueous solution of pentamethylenediamine with an alkali. For example, a method is known in which an alkali is added to and mixed with an aqueous pentamethylenediamine salt solution to liberate pentamethylenediamine, followed by extraction with a solvent, followed by distillation (Patent Document 1). In addition, the inventors of the present application first added an alkali to a pentamethylenediamine salt aqueous solution, mixed and separated into a pentamethylenediamine phase and an aqueous phase, and pentamethylenediamine was simply separated from the separated pentamethylenediamine phase. A method for producing pentamethylenediamine, which is characterized in that it is released, has been proposed (Japanese Patent Application No. 2007-247065).
JP 2004-114 A

  The above pentamethylenediamine contains anions derived from pentamethylenediamine salts (for example, chloride ions, sulfate ions, etc.). If there are many such anions, the resulting polyamide resin is used as food. May be restricted when used for film. In the production of polyamide resin, stainless steel is often used as a material for reaction vessels and peripheral equipment. In this case, there is a possibility that the reaction vessel and the like may be corroded and deteriorated.

  This invention is made | formed in view of the said situation, The objective is to provide a pentamethylenediamine suitable as a raw material of a polyamide resin.

  That is, the first gist of the present invention is pentamethylenediamine obtained by liberation with an alkali from a pentamethylenediamine salt aqueous solution, wherein the concentration of anions in the pentamethylenediamine is 2.0 ppm by weight or less. The second gist lies in a polyamide resin obtained by using pentamethylenediamine.

  According to the pentamethylenediamine of the present invention, a polyamide resin having excellent quality can be obtained while suppressing the corrosion deterioration of the reaction vessel. Moreover, since the polyamide resin of this invention has little content of impurity ion, it is suitable for using it as a film for food and drink.

  Hereinafter, the present invention will be described in detail. However, the description of the constituent elements described below is a representative example of embodiments of the present invention, and the present invention is not limited to these contents.

  For example, the pentamethylenediamine in the present invention is subjected to an LDC reaction while adding an acid to a lysine solution so that the pH of the solution is maintained at a pH suitable for enzymatic decarboxylation of lysine (LDC reaction). Can be manufactured. Examples of the acid used here include inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid, and organic acids such as acetic acid. From the obtained reaction product liquid, free pentamethylenediamine can be collected according to the present invention. Furthermore, it is also possible to collect pentamethylenediamine dicarboxylate by using dicarboxylic acid as the acid. The use of sulfuric acid as the acid has the following advantages over the use of hydrochloric acid. That is, in the operation of adding an alkali after the LDC reaction described later to isolate pentamethylenediamine from an aqueous solution of pentamethylenediamine, it is possible to reduce the amount of alkali added and improve phase separation. This increases the purity of the crude pentamethylenediamine.

  Hereinafter, a method for producing pentamethylenediamine dihydrochloride by an LDC reaction using hydrochloric acid as an acid will be described in detail.

  The lysine used as a raw material is usually preferably a free base (lysine base), that is, a free lysine, but may be a hydrochloride of lysine. The lysine may be either L-lysine or D-lysine as long as it produces pentamethylenediamine by the LDC reaction, but L-lysine is usually preferred because it is easily available. The lysine may be a purified lysine, or a fermentation liquid containing lysine as long as the pentamethylenediamine produced by the LDC reaction can form a salt with hydrochloric acid.

  As a solvent for preparing the lysine solution, water is preferably used. Since the pH of the reaction solution is adjusted with hydrochloric acid, it is not necessary to use other pH adjusting agents or buffers, but a buffer solution may be used as the solvent. Examples of such a buffer include sodium acetate buffer. However, from the viewpoint of forming a salt of pentamethylenediamine and hydrochloric acid, it is preferable not to use a buffering agent or to suppress the concentration to a low level even if it is used.

  When free lysine is used as lysine, hydrochloric acid is added to the lysine solution to adjust the pH to be suitable for the LDC reaction. The lower limit of specific pH is usually 4.0, preferably 5.0, more preferably 5.5, and the upper limit is usually 8.0, preferably 7.0, more preferably 6.5. is there. Hereinafter, the adjustment of the pH of the reaction solution to a pH suitable for the LDC reaction may be referred to as “neutralization”.

  The LDC reaction can be performed, for example, by adding lysine decarboxylase (LDC) to the lysine solution neutralized as described above. The LDC is not particularly limited as long as it acts on lysine to produce pentamethylenediamine. As LDC, a purified enzyme may be used, and cells such as plant cells and animal cells may be used in addition to microorganisms that produce LDC. Two or more types of LDCs or cells producing them may be used in combination. Further, the cells may be used as they are, or a cell treatment product containing LDC may be used. Examples of the cell treatment product include a cell disruption solution and a fraction thereof.

  Examples of the microorganism include Escherichia bacteria such as E. coli, coryneform bacteria such as Brevibacterium lactofermentum, Bacillus bacteria such as Bacillus subtilis, Serratia -Bacteria such as Serratia spp. Such as Serratiamarcescens, and eukaryotic cells such as Saccharomyces cerevisiae. Among these, bacteria, especially E. coli. E. coli is preferred.

  The microorganism may be a wild strain or a mutant strain as long as it produces LDC. Moreover, the recombinant strain modified so that LDC activity may increase may be sufficient.

  After starting the reaction by adding LDC to the lysine solution, as the reaction proceeds, carbon dioxide released from the lysine is released from the reaction solution, and the pH rises. Therefore, hydrochloric acid is added to the reaction solution so that the pH of the reaction solution is in the above range. Hydrochloric acid may be added continuously or as long as the pH is maintained in the above range. The reaction temperature is not particularly limited as long as LDC acts on lysine to produce pentamethylenediamine, but the lower limit is usually 20 ° C., preferably 30 ° C., and the upper limit is usually 60 ° C., preferably 40 ° C.

  The raw material lysine or lysine hydrochloride may be added to the reaction solution in its entirety at the start of the reaction, or may be added in portions as the LDC reaction proceeds.

  The pentamethylenediamine dihydrochloride obtained by the LDC reaction is isolated and purified from the reaction solution according to the present invention. The pentamethylenediamine dihydrochloride may be in a solution or a crystal depending on the use mode.

  Next, a method for separating and isolating pentamethylenediamine from an aqueous solution of pentamethylenediamine will be described. As this method, (1) a method in which an alkali is added to a pentamethylenediamine salt aqueous solution and mixed to release pentamethylenediamine and extracted with a solvent and then distilled, (2) an alkali is added to the pentamethylenediamine salt aqueous solution, Any method of mixing and separating the solution into a pentamethylenediamine phase and an aqueous phase and isolating pentamethylenediamine from the separated pentamethylenediamine phase by distillation or the like may be used.

  First, the method (1) will be described.

  As the method (1), the method described in JP-A-2004-114 described above can be employed. Specifically, an alkali is added to and mixed with an aqueous pentamethylenediamine salt solution, the pH is adjusted to 12 to 14, and extraction is performed with a polar organic solvent. As a solvent used for extraction, polar organic solvents such as aniline, cyclohexanone, 1-octanol, isobutyl alcohol, cyclohexanol, and chloroform are preferable. Isolation of pentamethylenediamine from the extraction solvent can be performed by a known distillation means such as vacuum distillation.

  Next, the method (2) will be described.

  By adding a certain amount or more of alkali to the aqueous solution of the pentamethylenediamine salt and mixing, the pentamethylenediamine is liberated and further separated into a pentamethylenediamine phase and an aqueous phase. Most of the salt precipitates on the aqueous phase side.

  When adding an alkali, the amino group equivalent of an alkali changes with kinds of pentamethylenediamine salt. For example, in the case of pentamethylenediamine dihydrochloride, it is usually at least 1.51 equivalents, preferably at least 2.50 equivalents, more preferably at least 3.50 equivalents of the amino group equivalent of pentamethylenediamine. In the case of pentamethylenediamine sulfate, it is usually at least 0.50 equivalents, preferably at least 1.00 equivalents, more preferably at least 1.51 equivalents of the amino group equivalent of pentamethylenediamine. When the amount of alkali added is too small, the pentamethylenediamine phase and the aqueous phase may not be separated. The amount of alkali added can be determined experimentally according to the counter ion (acid-derived content) of the pentamethylenediamine salt.

  The pentamethylenediamine of the present invention is a pentamethylenediamine obtained by liberation from an aqueous solution of a pentamethylenediamine salt with an alkali, and the anion concentration in the pentamethylenediamine is 2.0 ppm by weight or less. Features.

  Examples of the anion include an anion (counter anion) liberated from a pentamethylenediamine salt. When the pentamethylenediamine salt is a hydrochloride, it is a chloride ion, and when it is a sulfate, a sulfate ion. It is. When the concentration of anions exceeds 2.0 ppm by weight, the obtained polyamide resin has a large content of impurity ions, so it may be unsuitable for use as a film or film for food or drink. is there. Further, when the anion is a chloride ion, stainless steel, which is generally used as a material for the reaction vessel, may be corroded. The anion concentration is preferably 1.0 ppm by weight or less, more preferably 0.5 ppm by weight or less, and particularly preferably 0.1 ppm by weight or less.

A method for obtaining the pentamethylenediamine of the present invention, that is, a method for removing anions is not particularly limited, and an ion exchange resin method, a distillation method, or a combination method thereof can be employed. A distillation method employing an ion exchange resin method as a pretreatment is advantageous from the viewpoints of selecting a material such as a distillation column and reducing the load during distillation. In the ion exchange resin method, a commercially available anion exchange resin can be appropriately selected and used.
In addition, although the cation (for example, sodium ion) derived from an alkali may be contained in pentamethylenediamine, such a cation is obtained by, for example, an ion exchange method using a distillation method or a cation exchange resin. It is possible to remove to a concentration similar to the above-described anion concentration.

  The polyamide resin of the present invention comprising the above pentamethylenediamine as a raw material contains a pentamethylenediamine unit and a dicarboxylic acid unit as constituent components, but within the range not impairing the effects of the present invention, other copolymerization components May be contained.

  Examples of the copolymer component include amino acids such as 6-aminocaproic acid, 11-aminoundecanoic acid, 12-aminododecanoic acid and paraaminomethylbenzoic acid, lactams such as ε-caprolactam and ω-laurolactam, oxalic acid and malonic acid Aliphatic dicarboxylic acids such as succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, undecanedioic acid, dodecanedioic acid, brassic acid, tetradecanedioic acid, pentadecanedioic acid, octadecanedioic acid , Cycloaliphatic dicarboxylic acid and other alicyclic dicarboxylic acids, phthalic acid, isophthalic acid, terephthalic acid, naphthalenedicarboxylic acid and other aromatic dicarboxylic acids, ethylenediamine, 1,3-diaminopropane, 1,4-diaminobutane, 1,6 -Diaminohexane, 1,7-diaminohept Tan, 1,8-diaminooctane, 1,9-diaminononane, 1,10-diaminodecane, 1,11-diaminoundecane, 1,12-diaminododecane, 1,13-diaminotridecane, 1,14-diaminotetradecane 1,15-diaminopentadecane, 1,16-diaminohexadecane, 1,17-diaminoheptadecane, 1,18-diaminooctadecane, 1,19-diaminononadecane, 1,20-diaminoeicosane, 2-methyl- Aliphatic diamines such as 1,5-diaminopentane, cyclohexanediamine, alicyclic diamines such as bis- (4-aminohexyl) methane, and aromatic diamines such as xylylenediamine. Two or more of these copolymer components may be used in combination.

  As a method for producing the polyamide resin, known methods can be used, and specifically disclosed in “Polyamide Resin Handbook” (published by Nikkan Kogyo Co., Ltd .: Osamu Fukumoto). For example, as a method for producing polyamide 56, a method (heat polycondensation) in which pentamethylenediamine adipate is mixed in the presence of water and heated to proceed with a dehydration reaction is preferable. Here, the heat polycondensation is a production process in which the maximum temperature of the polymerization reaction product in the production of polyamide resin is increased to 200 ° C. or higher. The upper limit of the maximum reaction temperature is usually 300 ° C. or lower in consideration of the thermal stability during the polymerization reaction. There is no restriction | limiting in particular in a polymerization system, A batch system or a continuous system can be employ | adopted.

  The polyamide resin produced after the above heat polycondensation can be further subjected to solid phase polymerization. Thereby, the molecular weight of the polyamide resin can be increased. The solid phase polymerization can be performed, for example, by heating in a vacuum or an inert gas at a temperature of 100 ° C. or higher and lower than the melting point of the polyamide resin.

  The degree of polymerization of the polyamide resin of the present invention is not particularly limited, and the lower limit is usually 1.5, preferably 2.0, as the relative viscosity at 25 ° C. of a 98% sulfuric acid solution having a concentration of 0.01 g / mL. The upper limit is usually 8.0, preferably 5.5. When the relative viscosity is less than 1.5, the practical strength is insufficient. On the other hand, when the relative viscosity exceeds 8.0, the fluidity is lowered and the moldability is impaired. From the viewpoint of moldability, the relative viscosity is particularly preferably from 3.0 to 5.5 for extrusion molding of films, fibers, monofilaments and the like, and from 2.2 to 3.5 for injection molding.

  In the polyamide resin in the present invention, other components, such as antioxidants and heat stabilizers (hindered phenols, hydroquinones, phosphites and their substitution products, copper halide, Iodine compounds, etc.), weathering agents (resorcinol, salicylate, benzotriazole, benzophenone, hindered amine, etc.), mold release agents and lubricants (aliphatic alcohol, aliphatic amide, aliphatic bisamide, bisurea, polyethylene wax, etc. ), Pigments (phthalocyanine, carbon black, etc.), dyes (nigrosin, aniline black, etc.), plasticizers (octyl p-oxybenzoate, N-butylbenzenesulfonamide, etc.), antistatic agents (alkyl sulfate type anionic antistatic agents) Agent, quaternary ammonium salt type cationic system Antistatic agents, nonionic antistatic agents such as polyoxyethylene sorbitan monostearate, betaine amphoteric antistatic agents, etc.), flame retardants (hydramines such as melamine isocyanurate, magnesium hydroxide, aluminum hydroxide, Ammonium polyphosphate, brominated polystyrene, brominated polyphenylene oxide, brominated polycarbonate, brominated epoxy resins, combinations of these brominated flame retardants and antimony trioxide, etc., other polymers (other polyamides, polyethylene, polypropylene) Polyester, polycarbonate, polyphenylene ether, polyphenylene sulfide, liquid crystal polymer, polysulfone, polyether sulfone, ABS resin, SAN resin, polystyrene, etc.). The compounding can be carried out at any stage from resin polymerization to molding, but a dry blend method or a melt-kneading method using an extruder is preferred.

  The polyamide resin in the present invention can be molded into a desired shape by any molding method such as injection molding, film molding, melt spinning, blow molding, vacuum molding, etc., for example, injection molded products, films, sheets, filaments , Tapered filaments, fibers and the like. The polyamide resin in the present invention can also be used for adhesives, paints and the like.

  Specific examples of applications of the polyamide resin in the present invention include the following parts as automobile / vehicle-related parts. That is, intake manifold, clip with hinge (molded product with hinge), cable tie, resonator, air cleaner, engine cover, rocker cover, cylinder head cover, timing belt cover, gasoline tank, gasoline sub tank, radiator tank, intercooler tank, oil reservoir Tank, oil pan, electric power steering gear, oil strainer, canister, engine mount, junction block, relay block, connector, corrugated tube, protector and other automotive under hood parts, door handle, fender, hood bulge, roof rail leg, door mirror stay, Automotive exterior parts such as bumpers, spoilers, wheel covers, cup holders, console boxes , An accelerator pedal, a clutch pedal, a shift lever pedestals, automobile interior parts such as a shift lever knob and the like.

  Further, the polyamide resin in the present invention includes fishing-related materials such as fishing lines, fishing nets, switches, ultra-small slide switches, DIP switches, switch housings, lamp sockets, cable ties, connectors, connector housings, connector shells, ICs. Sockets, coil bobbins, bobbin covers, relays, relay boxes, condenser cases, motor internal parts, small motor cases, gears / cams, dancing pulleys, spacers, insulators, casters, terminal blocks, power tool housings, starter insulation parts , Fuse box, terminal housing, bearing retainer, speaker diaphragm, heat-resistant container, microwave oven part, rice cooker part, printer ribbon guide, etc. Product parts, computer-related parts, facsimile-copier-related parts, can be used in various applications, such as machine-related parts.

  In particular, since the polyamide resin in the present invention has a low content of impurity ions, it is suitable for use as a film or membrane for food and drink.

  Examples Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the description of these examples.

<Physical property measurement method>
(1) Measurement of each concentration of pentamethylenediamine, chloride ion and sulfate ion:
Using ion chromatography, the concentration was determined based on a calibration curve in the concentration range described in Table 1 below prepared for each measurement item. Table 2 shows the ion chromatography conditions.

(2) Measurement of sodium ion concentration:
It measured by atomic absorption analysis using "AAnalyst 100" by PerkinElmer Japan. The sodium ion concentration in the sample was adjusted to the range of a calibration curve prepared in advance (range of 0.1 to 1 ppm).

(3) Measurement of relative viscosity:
The sample was dissolved in 98% concentrated sulfuric acid to obtain a solution having a concentration of 0.01 g / mL, and measurement was performed at 25 ° C. using an Ostwald viscometer. The relative viscosity was defined as (sample solution drop time) / (concentrated sulfuric acid drop time).

(4) DSC (differential scanning calorimetry):
Using a DSC made by Seiko Electronics Industry, about 5 mg of a sample was taken under a nitrogen atmosphere and measured in the following manner. That is, after the polyamide resin is completely melted and held for 3 minutes, the temperature of the exothermic peak (temperature-decreasing crystallization temperature Tc) that appears when the temperature is decreased to 30 ° C. at a temperature-decreasing rate of 20 ° C./min. After maintaining at 30 ° C. for 3 minutes, the endothermic peak temperature (melting point Tm) observed when the temperature was increased from 30 ° C. at a rate of temperature increase of 20 ° C./min was determined. When there were a plurality of endothermic peaks, the highest temperature was defined as the melting point Tm.

<Preparation of LDC gene (cadA) enhanced strain>
(A) E. coli DNA extraction:
Escherichia coli JM109 strain is cultured in 10 mL of LB medium (composition: tryptone 10 g, yeast extract 5 g, NaCl 5 g dissolved in 1 L of distilled water) until the late logarithmic growth phase, and the resulting cells are 10 mg / mL. Of lysozyme in 10 mM: NaCl / 20 mM, Tris buffer (pH 8.0) / 1 mM, EDTA · 2Na solution 0.15 mL.

  Next, proteinase K was added to the above suspension so that the final concentration was 100 μg / mL, and the mixture was incubated at 37 ° C. for 1 hour. Furthermore, sodium dodecyl sulfate was added to a final concentration of 0.5 wt%, and the mixture was incubated at 50 ° C. for 6 hours for lysis. After adding an equal amount of phenol / chloroform solution to the lysate and shaking gently at room temperature for 10 minutes, the whole amount was centrifuged (5,000 g (g indicates gravitational acceleration), 20 minutes, 10-12 ° C. The supernatant fraction was collected, sodium acetate was added to a concentration of 0.3 M, and 2 times the amount of ethanol was added and mixed. The precipitate collected by centrifugation (15,000 × g, 2 minutes) was washed with 70% ethanol and then air-dried. To the obtained DNA, 5 mL of 10 mM Tris buffer (pH 7.5) -1 mM: EDTA · 2Na solution was added and left overnight at 4 ° C., and used as template DNA for subsequent PCR.

(B) Cloning of cadA:
E. coli cadA is obtained by using the DNA prepared in (A) above as a template and a synthetic DNA designed based on the sequence of the gene of E. coli K12-MG1655 strain (Genbank Database Accession No. U00096) for which the entire genome sequence has been reported. Performed by PCR using (SEQ ID NO: 1 and SEQ ID NO: 2).

Reaction solution composition:
Template DNA 1 μL, Pfx DNA polymerase (manufactured by Invitrogen) 0.2 μL, 1-fold concentration buffer, 0.3 μM each primer, 1 mM: MgSO 4, 0.25 μM dNTPs were mixed to make a total volume of 20 μL.

Reaction temperature conditions:
As a DNA thermal cycler, “PTC-200” manufactured by MJ Research was used, and a cycle consisting of 94 ° C. for 20 seconds, 60 ° C. for 20 seconds and 72 ° C. for 2.5 minutes was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute and 20 seconds, and the heat retention at 72 ° C. in the final cycle was 10 minutes.

  After completion of PCR, the amplified product was purified by ethanol precipitation and then cleaved with restriction enzyme KpnI and restriction enzyme SphI. This DNA preparation was separated by 0.75% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis, and visualized by ethidium bromide staining to detect a fragment of about 2.6 kb containing cadA, and QIAquickGelExtraction The target DNA fragment was recovered using Kit (manufactured by QIAGEN).

  The recovered DNA fragment was mixed with a DNA fragment prepared by cleaving E. coli plasmid vector pUC18 (Takara Shuzo) with restriction enzymes KpnI and restriction enzyme SphI, and ligation kit ver. After ligation using 2 (Takara Shuzo), the resulting plasmid DNA was used to transform E. coli (JM109 strain). The recombinant Escherichia coli thus obtained was placed in an LB agar medium containing 50 μg / mL: ampicillin, 0.2 mM: IPTG (isopropyl-β-D-thiogalactopyranoside) and 50 μg / mL: X-Gal. Smeared.

  Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. By cleaving the obtained plasmid DNA with restriction enzymes KpnI and restriction enzyme SphI, it was confirmed that an inserted fragment of about 2.5 kb was observed, and the E. coli strains containing pCAD1 and pCAD1 were named JM109 / pCAD1, respectively. did.

Example 1:
<Preparation of aqueous solution of pentamethylenediamine dihydrochloride>
The pentamethylenediamine dihydrochloride aqueous solution was prepared by the following method using a cadA amplified strain and lysine hydrochloride as a raw material.

(1) CadA amplified strain culture:
E. After pre-culturing E. coli JM109 / pCAD1 in a flask containing LB medium, 3 mL of the culture solution was inoculated into a 1 L flask containing 100 mL of LB medium having a double concentration, and stirred and cultured at 35 ° C. and 250 rpm. At 4 hours from the start of culture, sterilized IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.5 mM, and then the culture was continued for 14 hours.

(2) Separation and storage of bacterial cells:
The culture solution was centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, and the cells were collected. The obtained wet cells were suspended in 50 mM sodium acetate buffer so as to be 1/20 of the culture volume and stored at 4 ° C. until required for the reaction.

(3) Production of pentamethylenediamine dihydrochloride:
Hydrochloric acid is added to a 50% (w / v) lysine base solution (manufactured by Kyowa Hakko Kogyo Co., Ltd.) so that the pH is 6.0, and demineralized water is further added so that the lysine concentration is 140 g / L. A substrate solution (3 L) was made. The entire amount of the substrate solution was put into a 5 L culture tank, and pyridoxal phosphate was added to a concentration of 0.1 mM. E. The reaction was started by adding E. coli JM109 / pCAD1 cells so that the OD660 was 0.5. The reaction conditions were 37 ° C., 0.5 vvm aeration, and 200 rpm. The pH of the solution was controlled to be 6.5 by adding 2.5 M hydrochloric acid, and the reaction was continued for 30 hours. At the end of the reaction, the residual lysine concentration was 0.05 g / L or less. The solution after the reaction was subjected to inactivation treatment of the bacterial cells (121 ° C., 20 minutes).

<Purification and isolation of pentamethylenediamine>
(1) Concentration:
The aqueous solution of pentamethylenediamine dihydrochloride was concentrated with a rotary evaporator (R205V-0) manufactured by Shibata Kagaku Co., Ltd. to prepare a concentrated solution having a pentamethylenediamine concentration of 42.7 wt%. The concentration conditions are an oil bath temperature of 65 ° C., a rotation speed of 100 rpm, and a degree of vacuum of 100 Torr.

(2) Sorting of pentamethylenediamine phase:
48 wt% concentration of water prepared by dissolving 522.1 g (purity sodium hydroxide: 516.9 g) of sodium hydroxide (Hayashi Junyaku Co., Ltd. reagent special grade: pearl shape, purity 99%) in 554.8 g of water in advance. 1076.9 g of an aqueous sodium oxide solution was placed in a stirring vessel together with 441.0 g of the concentrated solution and mixed well to precipitate a solid, which was separated by filtration. And after putting the liquid after filtration into a separatory funnel and mixing sufficiently, it left still and phase-separated into the pentamethylenediamine phase and the water phase. Then, only the light liquid pentamethylenediamine phase was fractionated.

(3) Ion exchange resin treatment:
To a strongly basic ion exchange resin (Mitsubishi Chemical Co., Ltd. product: SA10A OH type) previously washed with deionized water, the fractionated pentamethylenediamine phase was converted to SV = 1 hr-1 (per hour, ion exchange resin capacity. The same amount of liquid was passed through).

(4) Isolation of pentamethylenediamine:
Pentamethylenediamine was isolated by distillation from the pentamethylenediamine aqueous solution subjected to the above treatment. First, water was distilled off at an oil bath temperature of 90 ° C. and a reduced pressure of 50 Torr, and then purified pentamethylenediamine was isolated under the conditions of an oil bath temperature of 110 ° C. and a reduced pressure of 20 Torr.

(5) Concentration measurement and weight calculation:
The weight and pentamethylenediamine concentration of the pentamethylenediamine dihydrochloride aqueous solution after concentration, the weight of the distillate, and the pentamethylenediamine concentration were measured. Thereafter, the weight of pentamethylenediamine in the concentrated liquid (pentamethylenediamine dihydrochloride aqueous solution after concentration) and the weight of pentamethylenediamine in the distillate were calculated. Furthermore, the purification yield of pentamethylenediamine was calculated.

<Manufacture of polyamide resin>
After adding 19.5 g of water to 8.2 g of the purified pentamethylenediamine prepared above, 11.8 g of adipic acid (manufactured by Honshu Chemical Industry) was added to adjust the pH to 8.8 to 8.9, and phosphorous acid 0.5 g of 0.2% phosphorous acid aqueous solution prepared in advance using (Wako Pure Chemical Industries Reagent Wako Special Grade) was added and heated to 70 ° C. to completely dissolve the mixture to obtain a raw material aqueous solution. . 40 g of the prepared aqueous raw material solution was placed in an autoclave and replaced with nitrogen to form a nitrogen atmosphere. The autoclave was immersed in an oil bath at a temperature of 270 ° C. and held at an internal pressure of 1.57 MPa for 2 hours. Next, after gradually releasing the pressure in the autoclave, the pressure was further reduced to 460 Torr and held for 1 hour to complete the reaction. After completion of the reaction, the reaction mixture was allowed to cool in a reduced pressure state, and after cooling, the contents were taken out and analyzed. The relative viscosity was 3.3 and the melting point was 255 ° C. Table 3 summarizes the results of Example 1.

Comparative Example 1:
In Example 1, it carried out like Example 1 except not having performed ion exchange resin treatment in the case of isolation of pentamethylenediamine. Table 3 summarizes the results of Comparative Example 1.

Example 2:
<Preparation of pentamethylenediamine sulfate aqueous solution>
The pentamethylenediamine sulfate aqueous solution was prepared by the following method using a cadA amplified strain, using lysine sulfate as a raw material.

(1) CadA amplified strain culture:
E. After pre-culturing E. coli JM109 / pCAD1 in a flask containing LB medium, 3 mL of the culture solution was inoculated into a 1 L flask containing 100 mL of LB medium having a double concentration, and stirred and cultured at 35 ° C. and 250 rpm. At 4 hours from the start of culture, sterilized IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.5 mM, and then the culture was continued for 14 hours.

(2) Separation and storage of bacterial cells:
The culture solution was centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, and the cells were collected. The obtained wet cells were suspended in 50 mM sodium acetate buffer so as to be 1/20 of the culture volume and stored at 4 ° C. until required for the reaction.

(3) Production of pentamethylenediamine sulfate:
Concentrated sulfuric acid and demineralized water were added to a 50% (w / v) lysine base solution (manufactured by Kyowa Hakko Kogyo Co., Ltd.) so that the pH was 6.0, and a substrate solution (lysine concentration 140 g / L) was obtained. 3L). The entire amount of the substrate solution was put into a 5 L culture tank, and pyridoxal phosphate was added to a concentration of 0.1 mM. E. The reaction was started by adding E. coli JM109 / pCAD1 cells so that the OD660 was 0.5. The reaction conditions were 37 ° C., no ventilation, and 400 rpm. The pH of the solution was controlled to be 6.5 by adding 1 M sulfuric acid, and the reaction was continued for 30 hours. At the end of the reaction, the residual lysine concentration was 0.05 g / L or less. The solution after the reaction was subjected to inactivation treatment of the bacterial cells (121 ° C., 20 minutes).

<Purification and isolation of pentamethylenediamine>
(1) Concentration:
The aqueous solution of pentamethylenediamine sulfate was concentrated using a rotary evaporator (R205V-0) manufactured by Shibata Kagaku Co., Ltd. to prepare a concentrated solution having a pentamethylenediamine concentration of 38.2 wt%. The concentration conditions are an oil bath temperature of 65 ° C., a rotation speed of 100 rpm, and a degree of vacuum of 100 Torr.

(2) Sorting of pentamethylenediamine phase:
Water with a concentration of 48 wt% prepared in advance by dissolving 343.0 g (purity sodium hydroxide: 339.6 g) of sodium hydroxide (Hayashi Junyaku Co., Ltd., reagent grade: pearl, purity 99%) in 364.5 g of water. 707.5 g of an aqueous sodium oxide solution was placed in a stirring vessel together with 324.0 g of the concentrated liquid and mixed well to precipitate a solid, which was separated by filtration. And after putting the liquid after filtration into a separatory funnel and mixing sufficiently, it left still and phase-separated into the pentamethylenediamine phase and the water phase. Then, only the light liquid pentamethylenediamine phase was fractionated.

(3) Ion exchange resin treatment:
The separated pentamethylenediamine phase was treated with a strongly basic ion exchange resin (manufactured by Mitsubishi Chemical Corporation: SA10A OH type) previously washed with deionized water at a liquid flow rate of SV = 1 hr-1.

(4) Isolation of pentamethylenediamine:
Pentamethylenediamine was isolated from the separated pentamethylenediamine phase by distillation. First, water was distilled off at an oil bath temperature of 90 ° C. and a reduced pressure of 50 Torr, and then purified pentamethylenediamine was isolated under the conditions of an oil bath temperature of 110 ° C. and a reduced pressure of 20 Torr.

(5) Concentration measurement and weight calculation:
The weight and pentamethylenediamine concentration of the pentamethylenediamine sulfate aqueous solution after concentration, the weight of the distillate, and the pentamethylenediamine concentration were measured. Thereafter, the weight of pentamethylenediamine in the concentrated liquid (pentamethylenediamine sulfate aqueous solution after concentration) and the weight of pentamethylenediamine in the distillate were calculated. Furthermore, the purification yield of pentamethylenediamine was calculated.

<Manufacture of polyamide resin>
After adding 19.5 g of water to 8.2 g of the purified pentamethylenediamine prepared above, 11.8 g of adipic acid (manufactured by Honshu Chemical Industry) was added to adjust the pH to 8.8 to 8.9, and phosphorous acid 0.5 g of 0.2% phosphorous acid aqueous solution prepared in advance using (Wako Pure Chemical Industries Reagent Wako Special Grade) was added and heated to 70 ° C. to completely dissolve the mixture to obtain a raw material aqueous solution. . 40 g of the prepared aqueous raw material solution was placed in an autoclave and replaced with nitrogen to form a nitrogen atmosphere. The autoclave was immersed in an oil bath at a temperature of 270 ° C. and held at an internal pressure of 1.57 MPa for 2 hours. Next, after gradually releasing the pressure in the autoclave, the pressure was further reduced to 460 Torr and held for 1 hour to complete the reaction. After completion of the reaction, the reaction mixture was allowed to cool in a reduced pressure state, and after cooling, the contents were taken out and analyzed. The relative viscosity was 3.3 and the melting point was 255 ° C. Table 3 summarizes the results of Example 2.

Production process diagram of LDC gene (cadA) -enhanced strain

Claims (5)

  A pentamethylenediamine obtained by liberation with an alkali from an aqueous pentamethylenediamine salt solution, wherein the concentration of anions in the pentamethylenediamine is 2.0 ppm by weight or less.   The pentamethylenediamine according to claim 1, wherein the anion is liberated from a pentamethylenediamine salt.   Pentamethylenediamine is produced from lysine using at least one selected from the group of lysine decarboxylase, recombinant microorganisms with improved lysine decarboxylase activity, cells producing lysine decarboxylase, or processed products of the cells. The pentamethylenediamine according to claim 1 or 2, wherein   The pentamethylenediamine according to any one of claims 1 to 3, wherein the pentamethylenediamine salt is at least one selected from the group consisting of hydrochloride, sulfate, carbonate, acetate, and nitrate.   The polyamide resin obtained by using the pentamethylenediamine in any one of Claims 1-4 as a raw material.

Images