JP2009131239A - Method for producing pentamethylenediamine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an improved method for producing pentamethylenediamine in higher yield, which includes separating pentamethylenediamine with an alkali. <P>SOLUTION: The method for producing pentamethylenediamine by adding an alkali to and mixing with a pentamethylenediamine salt solution, separating pentamethylenediamine and precipitating an alkali salt, subsequently treating the solution to isolate pentamethylenediamine, is provided. In the method, pentamethylenediamine adhered to the alkali salt is recovered by treating the alkali salt, precipitated after separating free pentamethylenediamine, with a washing liquid, and the recovered pentamethylenediamine and the pentamethylenediamine are mixed and treated to be isolated. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for producing pentamethylenediamine (also known as 1,5-diaminopentane, common name: cadaverine, abbreviation: PMDA), and more specifically, production of pentamethylenediamine by a simple method from a pentamethylenediamine salt aqueous solution. Regarding the method.

  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 these, 56 nylon, 56/66 nylon and the like made from pentamethylenediamine obtained from lysine have high expectations as plant-derived polymers.

  Conventionally, as a method for producing pentamethylenediamine, a method using a crystallization method during purification is known (Patent Documents 1 and 2). However, the crystallization rate is 40 to 45%, and a high yield cannot be expected by the crystallization method.

  On the other hand, 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 3). Such a method can obtain pentamethylenediamine in a high yield.

  On the other hand, the inventors of the present application first added an alkali to an aqueous solution of pentamethylenediamine, 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). This method differs from the above method in that it separates into a pentamethylenediamine phase and an aqueous phase. Therefore, a distillation method is adopted as an isolation method of pentamethylenediamine from the pentamethylenediamine phase without going through an extraction method. There are benefits that can be done.

JP 2005-6650 A JP 2004-208646 A JP 2004-114 A

  By the way, in each of the above methods for liberating pentamethylenediamine with an alkali, a salt (for example, sodium chloride when sodium hydroxide is added to an aqueous solution of pentamethylenediamine dihydrochloride) is precipitated by the addition of an alkali.

  According to the knowledge of the present inventor, the above-mentioned salt is deposited when pentamethylenediamine is liberated, and hence a non-negligible amount of pentamethylenediamine is adhered. Nevertheless, the above prior art makes no mention of recovery of pentamethylenediamine adhering to the salt.

  This invention is made | formed in view of the said situation, The objective is the manufacturing method of pentamethylenediamine including operation which liberates pentamethylenediamine with an alkali, Comprising: Pentamethylenediamine is obtained with a higher yield. It is an object of the present invention to provide an improved manufacturing method that can be used.

  That is, the first gist of the present invention is to add an alkali to an aqueous solution of pentamethylenediamine and mix it to release the pentamethylenediamine and to precipitate the alkali salt, and then to isolate the pentamethylenediamine. In the method for producing methylene diamine, the separated alkali salt deposited after separating the liberated pentamethylene diamine is treated with a washing solution to recover the pentamethylene diamine adhering to the alkali salt, and the recovered pentamethylene diamine and the pentamethylene are collected. The present invention resides in a process for producing pentamethylenediamine characterized in that it is combined with a diamine and subjected to isolation treatment, and the second aspect is characterized in that it uses pentamethylenediamine obtained by the above method as a raw material. It exists in the polyamide resin.

  According to the present invention, pentamethylenediamine can be obtained with a higher yield.

  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, ie free lysine), but may be a lysine hydrochloride. 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. . Hereinafter, the adjustment of the pH of the reaction solution to a pH suitable for the LDC reaction may be referred to as “neutralization”.

  In the LDC reaction, it is preferable to add at least one vitamin B6 selected from the group of pyridoxine, pyridoxamine, pyridoxal and pyridoxal phosphate for improving the production rate and reaction yield, and pyridoxal phosphate is particularly preferable. The method for adding vitamin B6 is not particularly limited, and may be appropriately added during the reaction.

  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 marcescens such as Serratia marcescens, 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. Plant cells or animal cells can also be used recombinant cells modified to increase LDC activity. The recombinant cell will be described later.

  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 Is 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.

  When the LDC reaction is carried out batchwise, hydrochloric acid can be easily added. The reaction can also be carried out by moving bed column chromatography using a carrier immobilizing LDC, LDC-producing cells, or treatments thereof. In that case, lysine and hydrochloric acid may be injected into an appropriate part of the column so that the reaction proceeds while the pH of the reaction system is maintained within a predetermined range.

  As described above, the reaction proceeds satisfactorily by neutralizing the pH, which increases with the formation of pentamethylenediamine by the LDC reaction, successively using hydrochloric acid. Since the pentamethylenediamine thus produced is a diamine, the pentamethylenediamine produced by the reaction accumulates in the reaction solution as a divalent hydrochloride.

  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.

Moreover, the pentamethylenediamine phase (crude pentamethylenediamine) after liquid separation is an ion other than water and pentamethylenediammonium ion [(C ₅ H ₁₀ (NH ₃ +) ₂ ] dissolved therein (hereinafter referred to as “impurity ion”). Therefore, the above-mentioned crude pentamethylenediamine cannot be used as it is as a raw material for the polyamide resin. It is necessary to isolate and purify pentamethylenediamine, and the impurity ions include a counter anion of pentamethylene diammonium ion (for example, chloride ion, sulfate ion, etc.) and the added alkali component. .

  By the way, when the distillation method is adopted as a means for isolating pentamethylenediamine, the crude pentamethylenediamine having a large amount of impurity ions precipitates a salt during the distillation. In particular, in the case of continuous distillation, the deposited salt accumulates and heat transfer decreases. Invite etc. Therefore, the water concentration in the crude pentamethylenediamine is usually 70 wt% or less, preferably 50 wt% or less, more preferably 25 wt% or less. A high moisture concentration is not preferable because the concentration of ions dissolved therein increases. The concentration of impurity ions in the crude pentamethylenediamine is usually 10 wt% or less, preferably 4 wt% or less. A high impurity ion concentration is not preferable because the amount of salt deposited after distillation increases. As a material for a distillation column or the like, it is preferable to use a relatively inexpensive stainless steel in view of strength and economy. Considering the corrosion of stainless steel by chloride ions, the chloride ion concentration in the crude pentamethylenediamine is usually 4 wt% or less, preferably 2 wt% or less.

  As the alkali used for the separation of the pentamethylenediamine phase and the aqueous phase, alkalis having a high basicity, specifically, sodium hydroxide, potassium hydroxide, calcium hydroxide and the like are used. When an alkali having a low basicity is used, it may not be separated into a pentamethylenediamine phase and an aqueous phase. The pentamethylenediamine phase is separated from the phase-separated pentamethylenediamine phase and aqueous phase. There is no restriction | limiting in particular in the method to fractionate, A well-known method can be used. A method for isolating pentamethylenediamine from the collected pentamethylenediamine phase is not particularly limited, but the above-described distillation method is preferable because of its high yield.

  A feature of the present invention is that the pentamethylenediamine adhering to the alkali salt is recovered by treating with a washing liquid after separating the released pentamethylenediamine from the alkali salt precipitated when the pentamethylenediamine is liberated by alkali.

  The alkali salt is sodium chloride when sodium hydroxide is added to an aqueous solution of pentamethylenediamine dihydrochloride, and sodium sulfate when sodium hydroxide is added to an aqueous solution of pentamethylenediamine sulfate.

  As a method for separating the released pentamethylenediamine, a known liquid-liquid separation method, for example, a method of extracting one liquid phase separated into two phases from the top of the liquid-liquid interface or from the bottom of the liquid phase can be used. In the case of separating the alkali salt from the liquid, a known solid-liquid separation method such as filtration or centrifugation can be employed as the method.

  The alkali salt cleaning solution is not particularly limited as long as pentamethylenediamine adhering to the alkali salt can be separated and recovered. The liquid does not need to dissolve the alkali salt at all, but a liquid in which the dissolved amount of the alkali salt at the washing treatment temperature is 50 wt% or less of the deposited amount is preferable. Specific examples of the alkali salt cleaning liquid include the following.

(1) An alkali salt aqueous solution having an alkali salt concentration at the washing treatment temperature of 50% or more of the saturation solubility is used. The concentration of the alkali salt with respect to the saturation solubility is preferably 70%, more preferably 90% or more.

(2) An alkaline aqueous solution having an alkali concentration at the washing treatment temperature of 50% or more of the saturation solubility is used. The alkali concentration with respect to the saturation solubility is preferably 70%, more preferably 90% or more.

(3) Use an alkali salt-containing alkaline aqueous solution in which the alkali salt concentration at the washing treatment temperature is 50% or more of the saturation solubility and the alkali concentration at the washing treatment temperature is 50% or more of the saturation solubility. The alkali salt and alkali concentration ranges are as described above.

(4) Use an organic solvent. Examples of the organic solvent include methanol, ethanol, propanol, hexanol, isopropanol, acetonitrile, acetone, and the like, in addition to the same organic solvent used for the extraction solvent.

  The aqueous alkali solution does not need to be prepared for the purpose of washing, and is recovered from the pentamethylenediamine phase by phase separation in a method of separating the pentamethylenediamine phase and the aqueous phase by adding an alkali. It can be prepared using an aqueous alkaline solution. In the case of an aqueous alkali solution prepared for washing purposes, the pentamethylenediamine phase can be phase-separated after washing and used as an alkali added to the aqueous pentamethylenediamine salt solution. When using the recovered aqueous alkali solution, when adding alkali to the pentamethylenediamine salt aqueous solution and performing phase separation, (1) First, the precipitated alkali salt is recovered, and then the liquid phase part is separated into two phases. Then, the pentamethylenediamine phase is recovered, and then the separated aqueous phase and the previously recovered alkali salt are stirred to wash the alkali salt, or (2) the liquid phase is phase separated. Then, after the pentamethylenediamine phase is recovered, the alkali salt can be continuously washed by stirring the aqueous alkali solution as the aqueous phase containing the precipitated alkali salt as it is. In addition, said organic solvent is preferable for washing | cleaning of the alkali salt in the method of extracting the pentamethylenediamine liberated with the alkali with the solvent, and the same kind of solvent as the extraction solvent is usually used.

  As the washing method, methods such as sprinkling washing and suspension washing can be employed. The washing temperature is usually 0 to 60 ° C, preferably 15 to 50 ° C. The amount of the cleaning liquid used varies depending on the cleaning method and cannot be determined unconditionally. However, the ratio to the alkali salt to be cleaned is usually about 0.1 to 5 times by weight, preferably about 1 to 3 times by weight. is there.

  Next, a method for modifying microorganisms so that LDC activity is increased will be exemplified. For other cells, the LDC activity can be similarly increased by appropriately modifying the following method so as to be suitable for it. I can do it.

  The LDC activity is increased by, for example, enhancing the expression of a gene encoding LDC (LDC gene). Enhanced expression of the LDC gene is achieved by increasing the copy number of the LDC gene. For example, an LDC gene fragment may be ligated with a vector that functions in a microorganism, preferably a multicopy vector, to produce a recombinant DNA, which is introduced into a suitable host and transformed.

  Increasing the LDC gene copy number can also be achieved by having multiple copies of the LDC gene on the chromosomal DNA of the microorganism. In order to introduce a gene in multiple copies on the chromosomal DNA of a microorganism, homologous recombination is performed using a sequence present in multiple copies on the chromosomal DNA as a target. As a sequence present in multiple copies on chromosomal DNA, repetitive DNA and inverted repeat present at the end of a transposable element can be used. Alternatively, as disclosed in JP-A-2-109985, a target gene can be mounted on a transposon and transferred to introduce multiple copies onto chromosomal DNA.

  In addition to the gene amplification described above, the increase in LDC activity can also be achieved by replacing an expression regulatory sequence such as a promoter of LDC gene on chromosomal DNA or plasmid with a strong one. For example, lac promoter, trp promoter, trc promoter and the like are known as strong promoters. Further, as disclosed in International Publication No. 00/18935 pamphlet, it is possible to introduce a base substitution of several bases into the promoter region of a gene and modify it to be more powerful. These promoter substitutions or modifications enhance LDC gene expression and increase LDC activity. Modification of these expression regulatory sequences may be combined with increasing the copy number of the gene.

  The replacement of the expression regulatory sequence can be performed, for example, in the same manner as the gene replacement using a temperature sensitive plasmid. E. Examples of the vector having a temperature-sensitive replication origin of E. coli include plasmid pMAN997 described in International Publication No. 99/03988 pamphlet. The expression regulatory sequence can also be replaced by a method using red recombinase of λ phage (Datsenko, KA, Proc. Natl. Acad. Sci. USA (2000) 97 (12), 6640-6645). Can be done.

  The LDC gene is not particularly limited as long as the encoded LDC can be effectively used for the LDC reaction. Examples thereof include bacteria such as E. coli, plants such as glass beans, and the LDC gene of microorganisms described in JP-A-2002-223770.

  E. as a host microorganism When using E. coli, E. coli is used. The LDC gene derived from E. coli is preferred. E. As the LDC gene of E. coli, cadA gene and LDC gene are known from US Pat. No. 5,827,698, and among these, cadA gene is preferable. E. The sequence of the E. coli cadA gene is known (N. Watson et al., Journal of bacteriology (1992) vol. 174, p. 530-540; SYMenget al. Journal of bacteriology (1992) vol. 174, p .2659-2668; GenBank accession M76411), by PCR using primers prepared based on the sequence, E. coli. It can be isolated from E. coli chromosomal DNA. Examples of such primers include primers having the base sequences shown in SEQ ID NOs: 1 and 2.

  In order to prepare a recombinant DNA by ligating the obtained LDC gene and the vector, the vector is cleaved with a restriction enzyme suitable for the end of the LDC gene, and the gene and the vector are used using a ligase such as T4 DNA ligase. Can be connected. E. Examples of the vector for E. coli include pUC18, pUC19, pSTV29, pHSG299, pHSG399, pHSG398, RSF1010, pBR322, pACYC184, pMW219 and the like.

  The LDC gene may be a wild type or a mutant type. For example, the cadA gene may encode an LDC containing one or several amino acid substitutions, deletions, insertions or additions at one or more positions, as long as the activity of the encoded LDC is not impaired. Good. Here, “several” is usually 2 to 50, preferably 2 to 30, more preferably 2 to 10, although it varies depending on the position and type of amino acid residues in the three-dimensional structure of the protein.

  The DNA encoding the protein substantially the same as LDC as described above may be substituted, deleted, inserted, added or inverted by, for example, site-directed mutagenesis. It is obtained by modifying the base sequence of the cadA gene. The modified DNA as described above can also be obtained by a conventionally known mutation treatment. As the mutation treatment, a method in which DNA before mutation treatment is treated in vitro with hydroxylamine or the like, a microorganism (eg, Escherichia bacterium) holding the DNA before mutation treatment is treated with ultraviolet light or N-methyl-N′-nitro- A method of treating with a mutagen that is usually used for mutagenesis treatment such as N-nitrosoguanidine (NTG), ethylmethanesulfonic acid (EMS) and the like can be mentioned.

  By expressing the DNA having the mutation as described above in an appropriate cell and examining the activity of the expression product, DNA encoding a protein substantially identical to LDC can be obtained. Further, it hybridizes under stringent conditions with a probe having a sequence (or a part of the sequence) of the coding region of cadA gene (GenBank accession M76411), for example, from DNA encoding LDC having a mutation or a cell carrying the same. A DNA encoding a protein that is soybean and has an activity equivalent to that of LDC is obtained. The “stringent conditions” referred to herein are conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Although it is difficult to quantify this condition clearly, for example, DNAs having high homology, for example, DNAs having a homology of 70% or more, preferably 80% or more, more preferably 90% or more, are present. 60 ° C., 1 × SSC, 0.1% SDS (preferably, 0 ° C., which is a condition in which DNAs are not hybridized with each other and DNAs having low homology do not hybridize with each other or washing conditions of normal Southern hybridization. For example, 1 × SSC, 0.1% SDS).

  A partial sequence of the cadA gene can also be used as a probe. Such a probe can be prepared by PCR using an oligonucleotide prepared based on a known cadA gene base sequence as a primer and a DNA fragment containing the cadA gene as a template. When a DNA fragment having a length of about 300 bp is used as a probe, hybridization washing conditions include 50 ° C., 2 × SSC, and 0.1% SDS.

  Specifically, the DNA encoding a protein substantially the same as LDC is usually 70% or more, preferably 80% or more, more preferably 90% or more homology with the amino acid sequence encoded by a known cadA gene. And a DNA encoding a protein having LDC activity.

  In order to introduce the recombinant DNA into the microorganism, the transformation method reported so far may be performed. For example, E.I. A method for increasing the permeability of DNA by treating recipient cells with calcium chloride as reported for E. coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970 )), And a method of introducing competent cells from proliferating cells and introducing DNA as reported for Bacillus subtilis (Ducan, CH, Wilson, GAand Young, FE, Gene, 1,153 ( 1997)). In addition, as known for Bacillus subtilis, actinomycetes and yeast, the DNA-receptive cells are put into a protoplast or spheroplast state that easily incorporates the recombinant DNA, and the recombinant DNA is introduced into the DNA-receptor. (Chang, S. and Choen, SN, Molec, Gen. Genet., 168, 111 (1979); Bibb, MJ, Ward, JMand Hopwood, OA, Nature, 274, 398 (1978); Hinnen, A., Hicks, JBand Fink, GRProc. Natl. Acad. Sci. USA, 75 1929 (1978)) can also be applied. Furthermore, microorganisms can also be transformed by the electric pulse method (Japanese Patent Laid-Open No. 2-207791).

  Culture for obtaining microorganisms or cells producing LDC may be performed by a method suitable for production of LDC depending on the microorganism or cells used.

  For example, the medium may be a normal medium containing a carbon source, a nitrogen source, inorganic ions, and other organic components as required. Carbon sources include glucose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose, sugars such as ribose and starch hydrolysates, alcohols such as glycerol, mannitol and sorbitol, gluconic acid, fumaric acid, citric acid And organic acids such as succinic acid can be used. As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, aqueous ammonia, and the like can be used. As the organic micronutrients, it is preferable to contain an appropriate amount of vitamins such as vitamin B1, required substances such as nucleic acids such as adenine and RNA, yeast extract and the like. In addition to these, a small amount of calcium phosphate, magnesium sulfate, iron ion, manganese ion or the like is added as necessary.

  The culture is performed in E. coli. In the case of E. coli, it is preferable to carry out the reaction under aerobic conditions for about 16 to 72 hours. In addition, inorganic or organic acidic or alkaline substance, ammonia gas, etc. can be used for pH adjustment. In addition, when the expression of the LDC gene is regulated by an inducible promoter, an inducing agent is added to the medium.

  After culturing, the cells can be recovered from the culture solution by collecting them with a centrifuge or a membrane. The cells may be used as they are, but when those treatments containing LDC are used, the cells are disrupted by ultrasonication, French press or enzymatic treatment, and the enzyme is extracted to obtain a cell-free extract. Furthermore, when purifying LDC, ammonium sulfate or various chromatographies can be used in accordance with conventional methods.

  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. Solid-phase polymerization can be performed by heating in a vacuum or in an inert gas at a temperature of 100 ° C. or higher and a melting point or lower, for example.

  The degree of polymerization of the polyamide resin of the present invention is not particularly limited, and the lower limit of the relative viscosity at 25 ° C. of a 98% sulfuric acid solution having a concentration of 0.01 g / mL is usually 1.5, preferably 2.0. Yes, and 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 particularly preferably from 2.0 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 cyanurate, 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.

  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.

(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) Moisture measurement:
Using a Karl Fischer type moisture meter ("CA-06" manufactured by Mitsubishi Chemical Corporation), the moisture concentration in the pentamethylenediamine phase was measured.

(4) 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).

(5) 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 this lysate and shaking gently at room temperature for 10 minutes, the whole amount was centrifuged (5,000 × g, 20 minutes, 10 to 12 ° C.) to obtain a supernatant fraction. The fraction was collected and sodium acetate was added to a concentration of 0.3 M, and then twice the amount of ethanol was added and mixed. The precipitate collected by centrifugation (15,000 g (g indicates gravitational acceleration), 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 synthesis designed based on the gene sequence of the E. coli K12-MG1655 strain (Genbank Database Accession No. U00096) for which the entire genome sequence has been reported. Performed by PCR using DNA (SEQ ID NO: 1 and SEQ ID NO: 2).

Reaction solution composition:
Template DNA 1 μL, PfxDNA polymerase (manufactured by Invitrogen) 0.2 μL, 1 × concentration attached 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 Kpn I and restriction enzyme Sph I. This DNA preparation was separated by 0.75% agarose (SeaKem GTGagarose: manufactured by FMC BioProducts) gel electrophoresis, and visualized by ethidium bromide staining to detect a fragment of about 2.6 kb containing cadA, and QIAquickGel Extraction The target DNA fragment was recovered using Kit (manufactured by QIAGEN).

  The recovered DNA fragment was mixed with an E. coli plasmid vector pUC18 (Takara Shuzo) cut with restriction enzymes Kpn I and restriction enzyme Sph I and mixed with 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. The obtained plasmid DNA was cleaved with restriction enzyme KpnI and restriction enzyme SphI to confirm that an inserted fragment of about 2.5 kb was confirmed, and the E. coli strains containing pCAD1 and pCAD1 were identified as JM109 / pCAD1, respectively. Named.

<Preparation of aqueous solution of pentamethylenediamine dihydrochloride>
The reaction solution (pentamethylenediamine dihydrochloride aqueous solution) used in the following Reference Examples 1 and 2 was prepared by the following method using 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).

<Preparation of pentamethylenediamine sulfate aqueous solution>
The reaction solution (pentamethylenediamine sulfate aqueous solution) used in the following Reference Examples 3 and 4 was prepared by the following method using a cadA amplified strain and 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).

Reference example 1:
<Purification and isolation of pentamethylenediamine>
(1) Concentration:
A pentamethylenediamine dihydrochloride aqueous solution was treated with a rotary evaporator (R205V-0) manufactured by Shibata Kagaku Co., Ltd. to prepare a concentrated solution having a pentamethylenediamine concentration of 40 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% hydroxylation prepared by dissolving 118.6 g (pure sodium hydroxide: 117.4 g) of sodium hydroxide (reagent special grade: Hayashi Junyaku Co., Ltd., purity 99%) in 126.0 g of water in advance. 244.6 g of an aqueous sodium solution was placed in a stirring vessel together with 150 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) 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.

(4) 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 3.1 and the melting point was 255 ° C. The results of Reference Example 1 are shown in Table 3.

Reference example 2:
In Reference Example 1, 48 wt% prepared by dissolving 166.4 g (pure sodium hydroxide: 164.7 g) of sodium hydroxide (Hayashi Junyaku Co., Ltd., reagent grade: pearl, purity 99%) in 176.7 g of water Purified and isolated pentamethylenediamine and produced a polyamide resin in the same manner as in Reference Example 1 except that 343.1 g of a sodium hydroxide aqueous solution was used. The obtained polyamide resin had a relative viscosity of 3.1 and a melting point of 255 ° C. The results of Reference Example 2 are shown in Table 3.

Comparative Reference Example 1:
In Reference Example 1, 48 wt% prepared by dissolving 47.6 g (purity sodium hydroxide: 47.1 g) of sodium hydroxide (Hayashi Junyaku Co., Ltd., reagent grade: pearl, purity 99%) in 50.5 g of water The sample was mixed and allowed to stand in the same manner as in Reference Example 1 except that 98.1 g of a sodium hydroxide aqueous solution was used, but the pentamethylenediamine phase was separated without separation into a pentamethylenediamine phase and an aqueous phase. I couldn't. The results of Comparative Reference Example 1 are shown in Table 3.

Comparative Reference Example 2:
In Reference Example 1, 48 wt% prepared by dissolving 71.1 g (purity sodium hydroxide: 70.4 g) of sodium hydroxide (Hayashi Junyaku Co., Ltd., reagent grade: pearl, purity 99%) in 75.6 g of water The mixture was mixed and allowed to stand in the same manner as in Reference Example 1 except that 146.7 g of a sodium hydroxide aqueous solution was used. However, the pentamethylenediamine phase was separated without separation into a pentamethylenediamine phase and an aqueous phase. I couldn't. The results of Comparative Reference Example 2 are shown in Table 3.

Reference Example 3:
<Purification and isolation of pentamethylenediamine>
(1) Concentration:
A pentamethylenediamine sulfate aqueous solution was treated with a rotary evaporator (R205V-0) manufactured by Shibata Kagaku Co., Ltd. to prepare a concentrated solution having a pentamethylenediamine concentration of 16.3 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% hydroxide prepared by dissolving 12.9 g (pure sodium hydroxide: 12.8 g) of sodium hydroxide (Hayashi Junyaku Co., Ltd., reagent grade: pearl, purity 99%) in 13.7 g of water in advance. 26.6 g of an aqueous sodium solution was placed in a stirring container together with 100 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) Isolation of pentamethylenediamine:
Purified pentamethylenediamine was isolated in the same manner as in Reference Example 1.

(4) Concentration measurement and weight calculation:
Concentration measurement and weight calculation were performed in the same manner as in Reference Example 1, and the purification yield of pentamethylenediamine was calculated.

<Manufacture of polyamide resin>
A polyamide resin was produced in the same manner as in Reference Example 1 and analyzed. The relative viscosity was 3.1 and the melting point was 255 ° C. The results of Reference Example 3 are shown in Table 4.

Reference example 4:
<Purification and isolation of pentamethylenediamine>
(1) Concentration:
The pentamethylenediamine sulfate aqueous solution was treated with a rotary evaporator (R205V-0) manufactured by Shibata Kagakusha to prepare a concentrated solution having a pentamethylenediamine concentration of 38.8 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% hydroxide prepared by previously dissolving 64.8 g of sodium hydroxide (Hayashi Junyaku Co., Ltd., reagent grade: pearl, purity 99%) in 69.0 g of water. 133.8 g of an aqueous sodium solution was placed in a stirring vessel together with 60.2 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) Isolation of pentamethylenediamine:
Purified pentamethylenediamine was isolated in the same manner as in Reference Example 1.

(4) Concentration measurement and weight calculation:
Concentration measurement and weight calculation were performed in the same manner as in Reference Example 1, and the purification yield of pentamethylenediamine was calculated.

<Manufacture of polyamide resin>
A polyamide resin was produced in the same manner as in Reference Example 1 and analyzed. The relative viscosity was 3.1 and the melting point was 255 ° C. The results of Reference Example 4 are shown in Table 4.

  In each of the above examples, “phase separability” was evaluated in the following manner at the time of phase separation by a separatory funnel in the separation process of the pentamethylenediamine phase. That is, the time until complete phase separation was measured, and the case where the required time was within 5 minutes was evaluated as “◎”, and the case where it exceeded 5 minutes and within 30 minutes was evaluated as “◯”. Note that the phase separation was visually determined.

  Comparative Reference Examples 1 and 2 do not phase separate into a pentamethylenediamine phase and an aqueous phase, whereas Reference Examples 1 and 2 and Reference Examples 3 and 4 undergo phase separation. When the pentamethylenediamine dihydrochloride is used (Reference Examples 1 and 2) and the pentamethylenediamine sulfate is used (Reference Examples 3 and 4), it can be seen that there is the following difference. As shown in Reference Example 3, in the case of sulfate, the “amino group equivalent ratio” is small as 1.00, but the phase separation is good. In the case of hydrochloride, phase separation does not occur when “amino group equivalent ratio” is 1.00 (see Comparative Reference Example 1). And, as shown in Reference Example 4, when sulfate was used and “amino group equivalent ratio” was increased, hydrochloride was used and “amino group equivalent ratio” was increased to the same extent as above ( Compared with Reference Example 2), the phase separation is remarkably improved. In this case, the concentration of residual inorganic ions (sulfate ions) is also low, and the quality of the purified pentamethylenediamine is good.

Example 1:
In the same manner as in Reference Example 1, a concentrated solution of pentamethylenediamine dihydrochloride aqueous solution (pentamethylenediamine concentration 40 wt%) was prepared. On the other hand, 166.4 g (pure sodium hydroxide: 164.7 g) of sodium hydroxide (Hayashi Junyaku Co., Ltd. reagent special grade: 99% pure) was dissolved in 176.7 g of water to obtain 48 wt% sodium hydroxide. An aqueous solution was prepared.

  150 g of the above concentrated solution was put in a stirring vessel, and 343.1 g of a 48 wt% aqueous sodium hydroxide solution was added and mixed well to precipitate a solid. The precipitated solid was separated by filtration. The liquid after filtration was put into a separating funnel and mixed well, and then allowed to stand to phase-separate into a pentamethylenediamine phase and an aqueous phase to obtain a pentamethylenediamine phase and an aqueous phase.

  Next, 50.0 g of the above solid and 134.4 g of the above aqueous phase (sodium chloride-saturated sodium hydroxide aqueous solution) as a cleaning liquid are placed in a stirring vessel and stirred, and the pentamethylene diamine adhering to the solid is washed again. The solid was separated by filtration. The liquid after solid separation was separated into an aqueous phase and a small amount of pentamethylenediamine phase. Each item shown in Table 5 before and after washing the solid was measured. The results are shown in Table 5.

  The pentamethylene diamine phase separated during the solid precipitation and the pentamethylene diamine phase separated during the solid washing were combined, and the same procedure as in “Isolation of pentamethylene diamine” in Reference Example 1 was performed. Purified pentamethylenediamine was recovered. Then, using this purified pentamethylenediamine, a polyamide resin was produced in the same manner as in Reference Example 1. The obtained polyamide resin had a relative viscosity of 3.1 and a melting point of 255 ° C.

Example 2:
In Example 1, 65.2 g (pure sodium hydroxide: 64.5 g) of sodium hydroxide (special grade made by Hayashi Junyaku Co., Ltd .: pearl form, purity 99%) was dissolved in 69.2 g of water as a cleaning solution. Purified pentamethylenediamine was recovered by washing the solid and isolating pentamethylenediamine in the same manner as in Example 1 except that the prepared 48 wt% sodium hydroxide aqueous solution was used. In the same manner as in Example 1, each item shown in Table 5 before and after washing the solid was measured. The results are shown in Table 5.

Example 3:
In Example 1, 26.4 wt prepared by dissolving 35.7 g of sodium chloride (special grade of reagent manufactured by Kishida Chemical Co., Ltd., purity 99.5%) (95.5 g of pure sodium chloride) in 98.7 g of water as a cleaning solution. Purified pentamethylenediamine was recovered by washing the solid and isolating the pentamethylenediamine in the same manner as in Example 1 except that a sodium chloride aqueous solution was used. In the same manner as in Example 1, each item shown in Table 5 before and after washing the solid was measured. The results are shown in Table 5.

Example 4:
In the same manner as in Reference Example 1, a concentrated solution (concentration 40 wt%) of an aqueous pentamethylenediamine dihydrochloride solution was prepared. On the other hand, 47.6 g (purity sodium hydroxide: 47.1 g) of sodium hydroxide (Hayashi Junyaku Co., Ltd. reagent special grade: pearl shape, purity 99%) was dissolved in 50.5 g of water to obtain 48 wt% sodium hydroxide An aqueous solution was prepared.

  150 g of the concentrated solution was put in a stirring vessel, and 98.1 g of a 48 wt% aqueous sodium hydroxide solution was added and mixed well to precipitate a solid. The precipitated solid was separated by filtration. Since the liquid after filtration was not phase-separated into a pentamethylenediamine phase and an aqueous phase, pentamethylenediamine was recovered by extraction with chloroform (Wako Pure Chemical Industries, Ltd.).

  Next, 50.0 g of the above solid and 50.0 g of chloroform (manufactured by Wako Pure Chemical Industries, Ltd.) as a cleaning liquid were placed in a stirring vessel and stirred, the pentamethylenediamine adhering to the solid was washed, and the solid was separated again by filtration. . Chloroform after solid separation contained a small amount of pentamethylenediamine. Each item shown in Table 5 before and after washing the solid was measured in the same manner as in Example 1, and the results are shown in Table 5. The cadaverine was isolated by combining the chloroform after the extraction operation and the above-mentioned chloroform after the solid washing, and performing distillation under reduced pressure (30 mmHg, 80 ° C.). As in Example 1, each item shown in Table 5 before and after washing the solid was measured. The results are shown in Table 5.

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

Claims (10)

  In the method for producing pentamethylenediamine, an alkali is added to and mixed with an aqueous pentamethylenediamine salt solution to liberate pentamethylenediamine and precipitate an alkali salt, and then the pentamethylenediamine is isolated. Treating the alkali salt deposited after separating the pentamethylenediamine with a washing solution to recover the pentamethylenediamine adhering to the alkali salt, and combining the recovered pentamethylenediamine and the above pentamethylenediamine for isolation treatment A process for producing pentamethylenediamine, characterized in that   The manufacturing method of Claim 1 which uses the liquid whose amount of melt | dissolving of the alkali salt in cleaning process temperature is 50 wt% or less of precipitation amount as a washing | cleaning liquid.   The manufacturing method of Claim 1 which uses the alkali salt aqueous solution whose density | concentration of the alkali salt in cleaning process temperature is 50% or more of saturation solubility as a washing | cleaning liquid.   The manufacturing method of Claim 1 which uses the aqueous alkali solution whose density | concentration of the alkali in washing | cleaning processing temperature is 50% or more of saturated solubility as a washing | cleaning liquid.   The manufacturing method of Claim 1 which uses an organic solvent as a washing | cleaning liquid.   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 manufacturing method according to any one of claims 1 to 5, wherein   The production method according to claim 1, wherein the pentamethylenediamine salt is pentamethylenediamine hydrochloride, pentamethylenediamine sulfate, pentamethylenediamine carbonate, pentamethylenediamine acetate or pentamethylenediamine nitrate.   The production method according to any one of claims 1 to 7, wherein the alkali is sodium hydroxide, potassium hydroxide or calcium hydroxide.   The production method according to claim 1, wherein an alkali is added to and mixed with an aqueous pentamethylenediamine salt solution to separate the solution into a pentamethylenediamine phase and an aqueous phase.   A polyamide resin comprising pentamethylenediamine obtained by the method according to any one of claims 1 to 9 as a raw material.

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