WO2006123778A1

WO2006123778A1 - Method for producing cadaverine carbonate, and method for producing polyamide using the same

Info

Publication number: WO2006123778A1
Application number: PCT/JP2006/310037
Authority: WO
Inventors: Masakazu Sato
Original assignee: Ajinomoto Co., Inc.
Priority date: 2005-05-19
Filing date: 2006-05-19
Publication date: 2006-11-23

Abstract

A cadaverine carbonate is produced by using lysine carbonate as a substrate, conducting a pH adjustment by the addition of carbon dioxide, and then carrying out an enzymatic decarboxylation of lysine. A dicarboxylic acid salt is added to the resultant cadaverine carbonate, and a cadaverine dicarboxylic acid salt is produced through an isolation step after a salt exchange reaction with carbonic acid. Cadaverine is produced by concentrating a solution of the cadaverine carbonate, to thereby release carbon dioxide out of the system. A polyamide is produced by using the resultant cadaverine dicarboxylic acid salt or cadaverine.

Description

Specification

Method for producing cadaverine carbonate and method for producing polyamide using the same

[0001] The present invention relates to a method for producing cadaverine carbonate. The present invention also relates to a process for producing cadaverine dicarbonate or cadaverine. The invention further relates to a process for producing polyamide.

Background art

[0002] Petroleum fuels such as naphtha are often used as raw materials for so-called plastic production.

In the case of recycling plastics, the disposal of plastics due to smoldering combustion has become a social problem in recent years because it causes the release of carbon dioxide gas. Therefore, it is encouraging to replace non-petroleum raw materials with the aim of preventing global warming and creating a recycling society.

Non-petroleum raw material power Various types of plastics, including polylactic acid, are being studied. These plastics have different physical properties such as heat resistance depending on their raw materials. Of these, the development of plastics derived from non-petroleum materials with particularly high heat resistance is suitable for the use of polylactic acid under high temperature conditions! /, Point power.

One example of a high heat-resistant plastic is polyamide rosin. Of these, V ヽ is used in a large amount of hexamethylene diamine, which is a 6-carbon diamine, and adipic acid, which is a 6-carbon dicarboxylic acid. Nylon 66 which is a polymer having a molar ratio of 1: 1. However, hexamethyldiamine is produced from benzene, propylene, or butanegen, which also provides naphtha power, and production methods from non-petroleum materials are known!

[0003] On the other hand, pentamethylenediamine having 5 carbon atoms is also called cadaverine and is known to be produced from lysine, one of the amino acids, by lysine decarbonase (LDC) (Non-Patent Document 1). ). Therefore, it can be used under high temperature conditions using non-petroleum raw materials by producing polyamide resin using cadaverine having 5 carbon atoms as raw material instead of hexamethylenediamine having 6 carbon atoms. It is possible to supply plastic materials. Although cadaverine is expected to be used for pharmaceutical intermediates in addition to polyamide sallow, its price is high. Therefore, development of an inexpensive manufacturing method is indispensable for further spread.

[0004] When cadaverine is produced by the action of LDC on lysine, carbon dioxide is generated by decarboxylation of lysine, and cadaverine, a divalent cation, is generated from lysine, a monovalent cation. During the reaction, the pH increases. Therefore, in order to prevent an increase in pH and maintain the enzyme reaction at the optimum pH, it is necessary to add a reaction force or acid in a high concentration buffer solution to the sequential reaction system (Patent Documents 1 and 2). There are also reports that neutralize with adipic acid, which is a dicarboxylic acid, which is often an inorganic or organic acid such as hydrochloric acid, sulfuric acid or phosphoric acid as a neutralizing agent for pH adjustment (Patent Documents 3 and 4). When neutralizers are used, the ability of cadaverine salt to be generated The cadaverine salt power that is produced also produces a by-product of the acid-derived salt added as a neutralizing agent when purifying cadaverine, giving it a significant environmental impact. In order to improve this, dicarboxylic acids such as adipic acid are added when cultivating lysine-fermenting microorganisms, and the resulting lysine / dicarboxylic acid solution is neutralized by acting lysine decarboxylase. There is a method for producing cadaverine dicarboxylic acid (Patent Document 3). By using this method, it is possible to reduce by-product salts in the purification process, but an organic solvent crystallization process is required in the purification process, and an environmental burden is created by the organic solvent. It becomes.

[0005] On the other hand, in the case of producing nylon by polymerizing cadaverine with a dicarboxylic acid, a method is known in which polycondensation is carried out by heating cadaverine dicarboxylate under melting conditions (Non-Patent Document 2). In order to obtain cadaverine dicarboxylate, it may be possible to produce cadaverine dicarbonate by cadaverine salt exchange obtained by using inorganic acid or organic acid as neutralizing agent. In this case, a by-product salt derived from the neutralizing agent is generated, which becomes an environmental burden. In addition, when this by-product salt is recovered and reused, a large amount of recovery equipment is required. On the other hand, when dicarboxylate is used as a neutralizing agent, cadaverine dicarboxylate can be obtained directly. However, organic solvent crystallization is required for the purification process. It is necessary to reinforce the equipment when collecting the waste.

Patent Document 1: JP 2002-223770 A

Patent Document 2: JP 2002-223771

Patent Document 3: Japanese Patent Application Laid-Open No. 2004-208646 Patent Document 4: JP-A-2005-006650

Non-Patent Document 1: Enzyme Handbook First Edition 636 pages Asakura Shoten

Non-Patent Document 2: Introduction to New Polymer Chemistry, page 22, Chemistry Doujin

Disclosure of the invention

[0006] The present invention minimizes by-product salt generated in the production process of cadaverine carbonate, reduces capital investment for recovery of by-product salt and organic solvent, and provides inexpensive and efficient cadaverine carbonate. It is an object to provide a manufacturing method. Another object of the present invention is to provide a method for producing cadaverine carbonate power cadaverine or cadaverine dicarboxylate, and to provide a method for producing polyamides inexpensively and efficiently using these.

[0007] In order to solve the above problems, the present inventor has intensively studied. As a result, it was found that cadaverine carbonate can be produced efficiently by carrying out enzymatic decarboxylation of lysine while supplying carbon dioxide to produce cadaverine carbonate such as lysine carbonate. Furthermore, it has been found that cadaverine dicarboxylate can be efficiently produced by adding dicarboxylic acid to the obtained cadaverine carbonate. It was also found that cadaverine can be efficiently produced by concentrating the obtained cadaverine carbonate and releasing carbonate ions or hydrogen carbonate ions, which are counter ions of cadaverine, as diacid carbon. As a result, the process of adding a neutralizing agent during the decarboxylation reaction was omitted or the amount added was successfully reduced. Furthermore, operations such as ion exchange resin or organic solvent crystallization, which were conventionally required, are unnecessary, reducing the burden on the environment of by-product salts and organic solvents derived from these operations. We succeeded in reducing the capital investment for recovering and reusing the solvent. Furthermore, it has been found that a polyamide can be efficiently produced by using the obtained cadaverine or cadaverine dicarboxylate, and has completed the present invention.

[0008] That is, the present invention is as follows.

(1) Enzymatic decarboxylation of lysine was performed while adding diacid-carbon to an aqueous solution of lysine carbonate so that the pH of the aqueous solution was maintained at a pH suitable for enzymatic decarboxylation of lysine. A method for producing cadaverine carbonate, comprising producing cadaverine carbonate.

(2) The method according to (1), wherein the pH suitable for the enzymatic decarboxylation reaction is pH 9.0 or less.

(3) The method of (1), wherein the carbon dioxide is added as a gas. (4) The method according to (1), wherein a neutralizing agent other than carbon dioxide is not added during the enzymatic decarboxylation reaction.

(5) The method according to (1), wherein the aqueous solution of lysine carbonate is a lysine carbonate fermentation solution.

(6) The method according to (1), wherein the enzymatic decarboxylation reaction is performed using lysine decarboxylase, a cell that produces lysine decarboxylase, or a treatment product of the cell.

(7) The method according to (1), wherein the cell is a cell modified to increase lysine decarboxylase activity.

(8) By increasing the copy number of the gene encoding lysine decarboxylase or modifying the expression regulatory sequence of the gene so that the expression of the gene is enhanced, (7) The method according to (7), wherein the cell is a recombinant cell having an increased carbonase activity.

(9) The method according to (8), wherein the cell is an Escherichia coli cell, and the gene encoding lysine decarboxylase is the DNA described in (a) or (b) below;

(a) DNA having the base sequence set forth in SEQ ID NO: 11,

(b) A DNA that is hybridized with a polynucleotide having a complementary sequence of the nucleotide sequence of SEQ ID NO: 11 under stringent conditions and encodes a protein having lysine decarboxylase activity.

(10) Cadaverine carbonate is produced by any one of the methods (1) to (9), and cadaverine dicarboxylate is formed by adding dicarboxylic acid to the resulting aqueous solution of daverine carbonate. A process for producing cadaverine dicarboxylate, comprising:

(11) The method according to (10), wherein the dicarboxylic acid is a dicarboxylic acid having 4 to 10 carbon atoms.

(12) The method according to (10), wherein the dicarboxylic acid is adipic acid.

(13) A method for producing polyamide, comprising producing cadaverine dicarboxylate by any one of the methods (10) to (12) and polycondensing the obtained cadaverine dicarboxylate.

(14) A method for producing cadaverine, which comprises producing cadaverine carbonate by the method according to any one of (1) to (9), and concentrating an aqueous solution of the obtained force daverine carbonate to produce cadaverine.

(15) A method for producing polyamide, comprising producing cadaverine by the method of (14) and polycondensing the obtained cadaverine with a dicarboxylic acid. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, details of the present invention will be described.

The method for producing cadaverine carbonate of the present invention comprises adding lysine carbon to an aqueous solution of lysine carbonate while adding diacid-carbon so that the pH of the solution is maintained at a pH suitable for enzymatic decarboxylation of lysine. Carrying out an enzymatic decarboxylation reaction of to produce cadaverine carbonate. Here, lysine may be L-lysine or D-lysine as long as it generates cadaverine by enzymatic decarboxylation, but L-lysine is usually good.

In addition, unless otherwise specified, “carbonate” includes both carbonate and bicarbonate.

[0010] In the production method of the present invention, an aqueous solution of lysine carbonate is used. The lysine salt contained in this aqueous solution need not be 100% free lysine carbonate, and may partially contain other lysine salts such as lysine hydrochloride and lysine sulfate.

An aqueous solution of lysine carbonate can be obtained, for example, by dissolving lysine carbonate in water. A lysine carbonate fermentation solution (see, for example, JP-A-2002-65287) can also be used as an aqueous lysine carbonate solution.

Further, as shown in the Examples described later, a free lysine base (lysine base) may be dissolved in water, and diacid carbon (CO 2) may be added to this aqueous solution to form an aqueous lysine carbonate solution. in this case

2

There are no particular restrictions on the method of adding carbon dioxide, but for example, it can be added as a gas. When the lysine carbon is added as a gas to obtain a lysine carbonate aqueous solution, the diacid carbon is preferably bubbled through the lysine aqueous solution for 12 hours or longer. The free lysine base may be a purified lysine base because of the raw material, or may be liquid lysine for feed (Japanese Patent Laid-Open No. 2000-256290). Preferably, a highly purified raw material with a small amount of compounds other than carbonate ions and a small amount of compounds other than lysine is preferred. An isolated and purified free lysine base is more preferable.

In addition, a lysine fermentation broth obtained by culturing microorganisms (see, for example, WO95 / 016042, WO95 / 023864, WO96 / 040934, WO00 / 056858, WO00 / 077172, WO01 / 002547, or WO01 / 053459) Preferably, after removing the cells, add carbon dioxide A lysine carbonate aqueous solution may also be used.

[0011] The decarboxylation reaction is performed using the lysine carbonate solution prepared as described above.

Lysine decarboxylation is performed by adding lysine decarboxylase (LDC) to the lysine carbonate solution. The LDC is not particularly limited as long as it acts on lysine to produce cadaverine. As LDC, a purified enzyme may be used, or various cells such as microorganisms, plant cells or animal cells that produce LDC may be used. The number of LDCs or cells producing them may be one type or two or more types.

[0012] Examples of the LDC protein include a protein having the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 12. And a protein having an activity of decarboxylating lysine. Here, several means 1 to 50, preferably 1 to 20, more preferably 1 to LO. The activity of LDC can be measured according to a known method.

The above substitution of amino acid residues in LDC is a conservative substitution so that the activity of LDC protein is maintained. A substitution is a change in which at least one residue in the amino acid sequence is removed and another residue is inserted therein. The amino acids that replace the original amino acids of the LDC protein and are considered conservative substitutions are: Ala to Ser or Thr, Arg to Gln, His or Lys, Asn to Glu, Gln, Lys , His or Asp substitution, Asp to Asn, Glu or Gin substitution, Cys to Ser or Ala substitution, Gin to Asn, Glu, Lys, His, Asp or Arg substitution, Glu to Gly, Asn, Gln, Lys or Asp substitution, Gly to Pro substitution, His to Asn, Lys, Gln, Arg or Tyr substitution, lie to Leu, Met, Val or P he substitution, Leu to Ile , Met, Val or Phe, Lys to Asn, Glu, Gln, His or Arg, Met to Ile, Leu, Val or Phe, Phe to Trp, Tyr, Met, He or Leu Substitution, Ser to Thr or Ala, Thr to Ser or Ala, Trp to Phe or Tyr, Tyr to His, Phe or Trp Substituted, and, Met from Val, and substitution of II e or Leu.

Further, as long as the LDC protein has an activity to decarboxylate lysine, it is 80% or more, preferably 90% or more, more preferably 95%, particularly preferably 98, with the amino acid sequence of SEQ ID NO: 12. It may be a protein having a homologous amino acid sequence of more than%. Amino acid sequence homology was determined using, for example, the algorithm BLAST (Pro. Natl. Acad. Sci. US A, 90, 5873 (1993) by Karlin and Altschul and FASTA (Methods EnzymoL, 183, 63 (1990) by Pearson. can do.

[0013] Alternatively, cells that express LDC may be used as they are. Alternatively, a cell-treated product containing LDC may be used. Examples of the cell treatment product include a cell culture solution, a cell disruption solution, and a fraction thereof. When an enzyme reaction is performed using cells such as microorganisms, plant cells, or animal cells, use of cells treated with organic solvents or surfactants may improve the permeability of the substrate and improve the reactivity. It is generally known. In the enzymatic decarboxylation reaction of lysine, the reactivity can be enhanced by treating the cells producing LDC with an organic solvent or a surfactant. Triton X-100, Tween 20, sodium cholate and CHAPS can be used as the surfactant to be treated, and acetone, xylene and toluene can be used as the organic solvent. More specifically, when Triton X-100 is used, a concentration of 0.01% to 1.0% (wZv) is added, and treatment at 0 ° C to 37 ° C for 2 minutes to 1 hour is appropriate. .

Examples of the microorganism include Escherichia bacteria such as E. coli, coryneform bacteria such as Brevibacterium lactofermentum, Bacillus bacteria such as Bacillus subtilis, Serratia marcescens (Serratia marcescens) of Serratia bacteria such as bacteria include eukaryotic cells such as Saccharomyces' cerevisiae (Saccharomyces cervis i _ae). Of these, bacteria, particularly E.coK, are preferred.

The microorganism may be a wild strain or a mutant strain as long as it produces LDC.

It is also possible to use a recombinant modified so as to increase LDC activity (see, for example, JP-A-2002-223770). Microorganisms, plant cells, or animal cells that have been modified to increase LDC activity can be used.

[0015] Recombinant cells modified to increase LDC activity include, for example, increasing the copy number of the gene encoding LDC or regulating the expression of the gene so that the expression of the gene is enhanced. Examples include recombinant cells that have been modified so that LDC activity is increased by modifying the sequence.

As a gene encoding LDC, for example, it has the base sequence of SEQ ID NO: 11 of E. coli DNA can be used. As long as it encodes a protein having LDC activity, a DNA that hybridizes with a polynucleotide having a complementary sequence of the nucleotide sequence of SEQ ID NO: 11 under stringent conditions can also be used. Here, “stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Specifically, it is a condition for washing normal Southerno and Hybridisation60. C, 1 X SSC, 0.1% SDS, preferably 60. C, 0.1 X SSC, 0.1% SDS, more preferably 68. The conditions include washing at a salt concentration and temperature corresponding to C, 0.1 X SSC, 0.1% SDS, more preferably 2 to 3 times.

A gene encoding LDC can be obtained by PCR using a nucleotide sequence-specific primer or probe of the LDC gene or a hybridization method.

Increasing the number of copies of the gene encoding LDC can be achieved by, for example, transforming a cell with a plasmid containing the gene encoding LDC, or integrating the gene encoding LDC into the host cell chromosome by homologous recombination. This can be done by dragging. The plasmid for introducing the gene encoding LDC is not particularly limited as long as it has replication ability in the host cell. For example, in the case of Escherichia coli, for example, pSTV 29 (manufactured by Takara Bio Inc.). ), RSF1010 (Gene vol. 75 (2), p271-288, 1989), pUC19, pBR322, pMW119, and the like, and phage DNA vectors can also be used. Examples of vectors that function in coryneform bacteria include pAM330 (Japanese Patent Laid-Open No. 58-067699), pHM1519 (Japanese Patent Laid-Open No. 58-77895), and pSFK6 (Japanese Patent Laid-Open No. 2000-262288).

Transformation using a plasmid and homologous recombination can be performed according to known methods. When performing transformation or homologous recombination using a plasmid, the expression regulatory region of the LDC gene to be introduced may be modified. Examples of expression control regions include promoters, and strong promoters include, for example, lac promoter, trp promoter, tr c promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, amyE promoter, Examples include a spac promoter and an acid phosphatase promoter.

In addition, mutant strains with enhanced LDC activity may be used for the decarboxylation reaction. For example, such a mutant strain is obtained by subjecting a parent strain or a wild strain to normal mutation treatment, that is, irradiation with X-rays or ultraviolet rays, Or N-methyl Ν 'nitro Ν nitrosoguanidine, etc., treated with a mutagen, etc., and obtained from the obtained mutant strain by selecting a strain with enhanced LDC activity be able to.

Culture for obtaining LDC protein or microorganisms or cells with enhanced LDC activity may be performed by a method suitable for LDC production depending on the microorganisms or cells used. For example, the medium used for the culture may be a normal medium containing a carbon source, a nitrogen source, inorganic ions, and other organic components as required. Carbon sources include sucrose, dulcose, latatoose, galactose, fructose, arabinose, maltose, xylose, trehalose, sugars such as ribose and starch-calyzed hydrolyzate, alcohols such as glycerol, mannitol and sorbitol, darcon Organic acids such as acid, fumaric acid, succinic acid and succinic acid can be used. Nitrogen sources include inorganic ammonium salts such as ammonium sulfate, salt ammonia, and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, and aqueous ammonia. Etc. can be used. As organic micronutrients, it is desirable to contain an appropriate amount of required substances such as vitamins such as vitamin B1, nucleic acids such as adenine and RNA, or yeast sex. In addition to these, a small amount of calcium phosphate, magnesium sulfate, iron ions, manganese ions, etc. is added as necessary.

For example, in the case of E. coli, the culture temperature is controlled to 20-45 ° C and the culture pH is controlled to 5.0-8.0. In addition, inorganic or organic acidic or alkaline substances, ammonia gas, etc. can be used for pH adjustment.

If expression is regulated by a promoter capable of inducing the LDC gene, an inducing agent is added to the medium.

After culturing, the cells can be collected from the culture medium using a centrifuge or a separation membrane. The cells may be used as they are, but when those treatments containing LDC are used, the cells can be disrupted by ultrasonication, French press or enzymatic treatment to extract the enzyme and used as an enzyme extract. .

Further, when purifying the LDC, LDC can be purified by using ammonium sulfate salting-out and various chromatographies according to conventional methods. Purified LDC can be obtained using a carrier. It can also be used in a state where it can be fixed or contacted with the reaction solution through a membrane or the like.

[0018] In the method of the present invention, a decarboxylation reaction is carried out using the cells expressing LDC protein or LDC gene obtained as described above or a treated product thereof. When performing the decarboxylation reaction, lysine carbonate as a substrate may be further added according to the progress of the reaction. In addition, LDC protein and LDC gene-expressing cells and cell treatment solution may be added directly to the reaction solution at the start of the reaction, or may be added in portions according to the progress of the reaction.

[0019] By supplying carbon dioxide to the reaction solution, the pH is adjusted to a pH suitable for the enzymatic decarboxylation reaction of lysine. This pH is adjusted by adjusting the purity, flow rate and pressure of the enzyme reaction system. The pH is usually 9.0 or less, preferably pH 5.0-9.0, more preferably pH 7.0-9.0.

Cadaverine is produced by decarboxylation of lysine. At this time, lysine, which is a monovalent cation, decarboxylation, cadaverine, which is a divalent cation, is converted into cadaverine, and the carbonic acid present in the aqueous solution becomes a counter ion, and cadaverine carbonate is obtained in the reaction solution.

If the reaction pH can be maintained within the above pH range, strict pH adjustment is not necessary during the decarboxylation reaction. However, as the reaction proceeds, the carbon dioxide released from the lysine force is released as the reaction solution and the pH rises. Therefore, carbon dioxide is added to the reaction solution and adjusted so that the pH of the reaction solution falls within the above range. The carbon dioxide to be added may be a gas, a liquid, or a solid (dry ice), but is preferably stored as a gas. The diacid carbon may be a mixed gas containing other gases, but is preferably 100% pure carbon dioxide. The carbon dioxide addition may be continuous or intermittent. A neutralizing agent other than carbon dioxide may be added at the same time. However, in order not to generate a double salt, it is preferable that the neutralizing agent only contains carbon dioxide.

[0020] The reaction temperature of the decarboxylase reaction is preferably within the range where the enzyme reaction is maximized, the release of carbon dioxide from the solution is minimized, and the reaction pH is kept within the above pH range. 20-50 ° C, more preferably 25-45 ° C.

[0021] The enzymatic decarboxylation reaction can be accelerated by adding vitamin B6, which is a coenzyme. There is no limitation on the type of vitamin B6, but one of pyridoxine, pyridoxamine and pyridoxal phosphate may be preferable. More preferably pyridoxal phosphate (PLP) The The addition concentration is not particularly limited, but is preferably a concentration of O.lmM or more.

By adding dicarboxylic acid to the aqueous solution of cadaverine carbonate obtained by the method for producing cadaverine carbonate of the present invention to produce cadaverine dicarboxylate, and recovering the cadaverine dicarbonate, cadaverine is recovered. Dicarboxylates can be produced. That is, cadaverine dicarboxylate is formed in the solution by a salt exchange reaction between carbonic acid and dicarboxylic acid. On the other hand, carbonate ions are released out of the system as carbon dioxide. The dicarboxylic acid added at this time should be equimolar with cadaverine. The dicarboxylic acid to be added is preferably a dicarboxylic acid having 4 to 10 carbon atoms! Examples of the dicarboxylic acid having 4 to 10 carbon atoms include succinic acid, maleic acid, fumaric acid, glutaric acid, adipic acid, sebacic acid, terephthalic acid, and isophthalic acid. More preferred are 6-carbon adipic acid or 8 carbon-terephthalic acid.

[0023] The obtained cadaverine dicarboxylate can be easily separated and recovered by, for example, the following method. First, after sterilization by centrifugation, decolorization is preferably performed by activated carbon treatment to remove PLP and impurities. Next, cadaverine dicarboxylate crystals can be obtained by concentration under reduced pressure.

[0024] Production Method of Kudaverine>

Cadaverine can be produced by concentrating an aqueous solution of cadaverine carbonate obtained by the method for producing cadaverine carbonate of the present invention. Cadaverine can be easily separated and recovered by, for example, the following method.

That is, the aqueous solution of cadaverine carbonate is decolorized by treatment with activated carbon to remove PLP and impurities after removing cells by centrifugation or the like. Next, by performing concentration, carbonate ions and hydrogen carbonate ions are released into the atmosphere as carbon dioxide and carbon dioxide, and cadaverine can be obtained after water evaporation. Concentration is preferably performed under reduced pressure, and can be efficiently concentrated by heating. The heating temperature is preferably 40 to 100 ° C.

Compared with the known ion exchange resin method and organic solvent crystallization method, the method of the present invention does not use a large amount of water in the refining process, does not generate by-product salt in the process of resin regeneration, Melting This is an excellent and simple method that does not use a medium.

Polyamides can be produced using cadaverine dicarboxylate or cadaverine obtained by the above method. Examples of the polyamide production method include a method of performing a polycondensation reaction using cadaverine dicarboxylic acid salt and a method of performing a polycondensation reaction using cadaverine and dicarboxylic acid. The polycondensation reaction can be carried out according to a known method (see, for example, “Plastic Materials Course [16] Polyamide resin” (Nikkan Kogyo Shimbun)). For example, there is a heat polycondensation method in which cadaverine dicarboxylate is mixed with water and the mixture is heated and dehydrated for condensation. Further, cadaverine and dicarboxylic acid may be mixed with water, and the mixture may be polycondensed while being dehydrated by heating.

After the polycondensation, the molecular weight can be increased by solid phase polymerization. Solid-phase polymerization proceeds by heating in a vacuum or in an inert gas in the temperature range from 100 ° C to the melting point, and polyamides with insufficient molecular weight can be converted to high molecular weight by heating polycondensation. .

Various types of polyamides can be produced depending on the type of dicarboxylic acid used for polycondensation. The dicarboxylic acid that can be used for polycondensation is not particularly limited as long as it can be used for the production of polyamide. For example, succinic acid, maleic acid, fumaric acid, glutaric acid, adipic acid, sebacic acid, terephthalic acid And isophthalic acid. For example, when adipic acid is used for polycondensation, 5,6-nylon can be obtained, and when terephthalic acid is used for polycondensation, 5, T-nylon can be obtained.

Example

[0026] [Example; pH adjustment of lysine carbonate solution by addition of carbon dioxide]

100% carbon dioxide gas is bubbled into the solution at 5, 10, 20, 50 mL / min for 200 mL of 100 g / L L-lysine solution, and L-lysine carbonate is formed while stirring equivalent to lOOrpm. I went. At this time, the pH during the reaction was measured. The results are shown in Table 1.

[0027] [Table 1] Table 1 Relationship between CO ₂ addition flow rate and pH of L-lysine solution

[0028] The pH of the L-lysine solution depends on the CO flow rate at the beginning of the reaction. The larger the addition flow rate, the lower the pH.

2

After 18 hours, the lower speed is equilibrated at a pH that depends on the CO addition flow rate.

2

The pH reached a constant value. After that, when CO addition is interrupted, pH tends to be alkaline

2

The solution should have enough CO dissolved in the solution to neutralize L-lysine.

2

confirmed.

From this result, when CO is added to the L-lysine solution to produce L-lysine carbonate, CO

2 2 By increasing the amount added, more CO is dissolved in the solution, and the pH of the reaction solution is further lowered.

2

It was confirmed that it was possible to lower

If the pH of the L-lysine carbonate solution of the reaction substrate is different from the optimum reaction pH of the enzyme (LDC), it is possible to adjust the pH of the reaction solution to the optimum pH of the enzyme reaction by the above method. Was confirmed.

[0029] [Example 1: Construction of plasmid cadA220]

(Construction of LDC expression plasmid derived from Escherichia coli)

E.coli-derived LDC gene (cadA) (N. Watson et al., Journal of bacteriolog y, (1992) vol. 174, 530-540; SY Neng and GN Bennet, Journal of bacteriology (19 92) vol 174, 2659-2669), and designed a PCR primer having the base sequence shown in 5, -gtcgacactgcacacggctggcgg-3, (SEQ ID NO: 1) and 5, -gttagcggcacgtacacctgcctgg-3, (SEQ ID NO: 2). A DNA fragment containing cadA was amplified by PCR using the E. coli W3110 (ATCC39936) chromosome as a saddle.

The amplified DNA fragment was cleaved with Kpnl and Sphl, and the obtained fragment (2,468 bp) was inserted into the Kpnl and Sphl cleavage sites of pUC18 (Tacarano) to prepare plasmid pcadA.

[0030] (Construction of Enterobacter acid phosphatase gene expression plasmid)

As described in Journal of Bioscience and Bioengineering (2001) Vol. 92, No.l, 50-54 ( Alternatively, it is excised from the chromosomal DNA derived from Enterobacter aerogenes IFO 12010 strain with restriction enzyme Sail and restriction enzyme Kpnl from Enterobacter aerogenes IFO 12010 strain. A 1.6 kbp DNA fragment was isolated and a plasmid pEAM330 linked to PUC118 was prepared.

(Construction of LDC high expression plasmid)

The above plasmid pcadA as a cage type, 5'-ggggtacctgtgagggtgttttcatg tg ttctc-3 (Tatsumi column number 3) and 5-tattgcaataacgttcatcgcgaaagcgttaacgg-3 '(the first column number 4) each oligonucleotide 0.4mM and buffer for KOD plus ( TOYOBO), dATP, dC TP, dGTP, dTTP 0.2 mM each, MgSO ImM and KOD plus polymerase (TOYOBO)

Four

50 μL reaction solution containing 1 unit at 94 ° C for 30 seconds, then 94 ° C for 15 seconds, 55 ° C for 30 seconds, 68 ° C for 2 minutes 30 seconds 25 Repeated PCR was performed to amplify the cadA gene portion.

Further, PCR was performed under the same conditions using the above plasmid pEAM330 as the cage type, 5′-gctctagaattttttcaatg tgattt-3 (酉歹酉歹 ¾ · No. 5) and a-gtgattcaatattgcaataacgttcatctacatttccttacggtgtta-. (SEQ ID NO: 6) oligonucleotide as primers. The promoter sequence portion of acid phosphatase was amplified. The reaction solution was subjected to agarose gel electrophoresis, and each amplified DNA fragment was recovered using a Microspin column (manufactured by Amersham's Pharmacia Biotech).

Next, the amplified fragment mixture is made into a bowl shape, and oligonucleotides of SEQ ID NO: 3 and SEQ ID NO: 5 are used as primers, and 94 ° C for 15 seconds, 55 ° C for 30 seconds, 68 ° C in a reaction solution having the same composition. PCR was repeated 25 times in a cycle of 2 minutes and 30 seconds to construct a chimeric enzyme gene. Each amplified DNA fragment was recovered using a Microspin column (Amersham 'Farmasia' Biotech) and digested with Xbal and Pstl. This was ligated to the Xbal-Pstl site of plasmid pUC119. The CadA expression plasmid was constructed by force and named pcadA202.

Next, using the QuickChange 'Site-Directed Mutagenesis Kit (Stratagene was used, mutation was introduced into the expression control region of pcadA202 in accordance with the following procedure in order to increase CadA production.

First, plasmid pcadA202 is used as a cocoon, and as a primer, 5, -ggacatataacaccgtaagg PCR was performed using aggaatgtagatgaacgttattgc-3, (self-sequence number 7) and 5, -gcaataacgttcatctacattcctccttacggt gttatatgtcc-3, (SEQ ID NO: 8) oligonucleotide according to the method described in the manual to construct plasmid pcadA210.

Next, PCR was performed using the plasmid pcadA210 as a saddle type, and using 5, -gaattttttcaatgtgattttg acatttacttccagatgac-ύ (item ti column ¾ · No. 9) and 5-gtcatctggaagtaaatgtcaaaatcacattgaaaaa attc-3 ′ (SEQ ID NO: 10) oligonucleotide as primers. A plasmid was constructed. This CadA high expression plasmid was named pcadA220. pcadA220 is designed to enhance LDC expression by modifying the constitutive expression promoter and ribosome binding site of the acid phosphatase gene of the genus Enterobacter.

All constructed plasmids were sequenced using the 310 Genetic analyzer (ABI) by the Dye Terminator method using the DNA Sequencing Kit Dye Terminator Cycle Sequencing Ready Reaction (PERKIN ELMER). It was confirmed that it was introduced.

Also, E. coli JM109 strain (Takara Bio) was transformed with plasmid pcadA220, and the resulting transformant was named Escherichia coli cadA220. In this strain, the constitutive expression promoter of the Enterobacter genus acid phosphatase gene and the ribosome binding site were modified and succeeded in higher expression of LDC.

[Example 2: Production of cadaverine carbonate]

(Culture of Escherichia coli cadA220 strain)

Escherichia coli cadA220 strain is inoculated on LB medium plate for 1 ase and cultured at 26 ° C for 晚, and then cultured cells are sown for 1 ase and inoculated into 50 mL of liquid LB medium at 28 ° C. C. Shaking culture was performed for 8 hours under conditions of 150 rpm, and a culture solution was obtained in advance.

The pre-culture solution obtained was inoculated into 10 mL of the pre-culture medium shown below, and pre-cultured at a total volume of 300 mL under the conditions of 28 ° C, 700 rpm, pH 7.0, aeration rate of 300 mL / min. . Ammonia was used to adjust the culture pH. When the consumption of sugar in the preculture medium was completed, the culture was terminated to obtain a preculture solution.

Preculture medium composition

Glucose 25g / L ゝ MgSO 7aq 1.0g / L, KH PO 1.4g / L ゝ (NH) SO 5.0g / L, soybean Hydrochloric acid decomposition product 0.45g / L in terms of inorganic nitrogen, FeSO 7aq 20mg / L, MnSO 5H O 20mg / L,

4 4 2

Thiamine HC1 1.0mg / L, Antifoam O.lmL / L

The medium was mixed and adjusted to pH 5.0 with KOH aqueous solution.

[0033] 15 mL of the obtained preculture solution was added to the main culture medium having the same composition as that of the preculture medium, and the total volume was added.

The main culture was performed at 300 ° C., 30 ° C., 700 rpm, pH 7.0, and aeration rate of 300 mL / min. The culture pH was adjusted with ammonia, and when the sugar consumption in the main culture medium was completed, the culture was terminated to obtain Escherichia coli cadA220 cells.

[0034] (Pretreatment of Escherichia coli cadA220 strain)

The obtained Escherichia coli cadA220 strain was subjected to the pretreatment shown below before the enzyme reaction. The bacterial cell culture solution was centrifuged at 6,000 rpm for 10 minutes at 4 ° C to obtain precipitated bacterial cells.

A 1/5 volume of 0.1% Triton X_100, 0.2 M Tris-HCl (pH 7.4) solution of 1/5 of the culture solution used for the centrifugation was added to the precipitated cells to suspend the precipitated cells uniformly. After suspending, the solution was ice-cooled for 10 minutes to obtain an enzyme solution.

[Preparation of lysine carbonate solution]

Carbon dioxide was added to L-lysine (pharmaceutical grade) solution 200 g / L 200 mL at a flow rate of 60 mL / min. Stirring was carried out under conditions of 150 rpm and 25 ° C., and a solution equilibrated at a predetermined pH was used as an L-lysine carbonate solution for the following reaction.

[0036] (Production reaction of cadaverine carbonate)

To 200 mL of the obtained L-lysine carbonate 200 g / L (converted to L-lysine), add 5 mL of the enzyme solution and pyridoxal-5-phosphate (PLP) so as to correspond to O.lmM, and carry out the enzyme reaction. Started. The reaction was carried out under the conditions of stirring at 150 rpm, 25 ° C. and carbon dioxide gas 60 mL / min, and no other neutralizing agent was added during the reaction.

Enzymatic reaction was carried out, and as a result, 128 g / L (converted to cadaverine) of cadaverine carbonate was confirmed in the reaction solution.

[0037] [Example 3: Purification of cadaverine carboxylate]

An equimolar amount of 80.9 g of adipic acid crystals was added to 400 mL of 141.4 g / L cadaverine carbonate solution, and the mixture was stirred and mixed. Stirring dissolves adipic acid, The reaction was continued until the generation of carbon dioxide and the reaction pH became constant. As a result, a cadaverine adipic acid solution was obtained.

The obtained cadaverine adipic acid solution was centrifuged (12,000 rpm, 10 minutes) to remove the cells and their residues, and a supernatant fraction was obtained.

10% dry weight (vs. cadaverine) of activated carbon was added to each obtained supernatant and reacted at 50 ° C for 30 minutes. Thereafter, the activated carbon was removed by filtration to obtain a cadaverine carbonate solution. By this operation, PLP and impurities in the reaction solution were separated.

200 mL of the obtained cadaverine adipate solution was concentrated under reduced pressure using an evaporator. By this operation, water in the solution was removed and finally 64.0 g of cadaverine adipate was obtained.

[Example 4; Purification of cadaverine]

The cadaverine carbonate solution obtained in Example 2 was first centrifuged at 12,000 rpm for 10 minutes at 4 ° C. The microbial cells and microbial cell residues in the reaction solution were removed, and the supernatant fraction was obtained.

Cadaverine was purified by concentrating 300 mL of the obtained cadaverine carbonate solution under reduced pressure at 40 ° C. using an evaporator. This operation removed carbon dioxide and water from the solution, and 28.7 g of cadaverine was finally obtained.

Industrial applicability

[0039] In the present invention, by using diacid carbon as the counter ion of cadaverine, the production of by-product salts is minimized, the purification process is simplified, and inexpensive cadaverine carbonate, and Using this, it was possible to supply cadaverine or cadaverine dicarboxylate and to produce polyamides using these.

Claims

The scope of the claims

[I] Enzymatic decarboxylation of lysine was carried out while adding diacid-carbon to an aqueous solution of lysine carbonate so that the pH of the aqueous solution was maintained at a pH suitable for enzymatic decarboxylation of lysine. A method for producing cadaverine carbonate, comprising producing daverine carbonate.

[2] The method according to [1], wherein the pH suitable for the enzymatic decarboxylation reaction is pH 9.0 or less.

3. The method according to claim 1, wherein the carbon dioxide is added as a gas.

[4] The method according to [1], wherein a neutralizing agent other than carbon dioxide and carbon dioxide is not added during the enzymatic decarboxylation reaction.

5. The method according to claim 1, wherein the lysine carbonate aqueous solution is a lysine carbonate fermentation solution.

6. The method according to claim 1, wherein the enzymatic decarboxylation reaction is performed using lysine decarboxylase, a cell producing lysine decarboxylase, or a treated product of the same.

7. The method according to claim 6, wherein the cell is a cell modified so that lysine decarboxylase activity is increased.

[8] By increasing the copy number of the gene encoding lysine decarboxylase or modifying the expression regulatory sequence of the gene so that the expression of the gene is enhanced, The method according to claim 7, which is a recombinant cell having an increased activity.

[9] The method according to claim 8, wherein the cell is an Escherichia coli cell, and the gene encoding lysine decarboxylase is the DNA according to (a) or (b) below:

(a) DNA having the base sequence set forth in SEQ ID NO: 11,

[10] Cadaverine carbonate is produced by the method according to any one of claims 1 to 9, and cadaverine dicarboxylate is produced by adding dicarboxylic acid to the obtained aqueous solution of cadaverine carbonate. A method for producing cadaverine dicarboxylate.

[II] The method according to claim 10, wherein the dicarboxylic acid is a dicarboxylic acid having a carbon number of 4 to LO.

12. The method according to claim 10, wherein the dicarboxylic acid is adipic acid.

[13] The cadaverine dicarboxylate is obtained by the method according to any one of claims 10 to 12. A process for producing a polyamide, comprising polycondensing a cadaverine dicarboxylate produced and obtained.

[14] Production of cadaverine, comprising producing cadaverine carbonate by the method according to any one of claims 1 to 9, and concentrating the obtained aqueous solution of cadaverine carbonate to produce cadaverine. Law.

[15] A process for producing a polyamide, comprising producing cadaverine by the method according to claim 14, and polycondensing the obtained cadaverine with dicarboxylic acid.