Process for preparing cadaverine dicarboxylate

Abstract

The present application relates to a method for preparing cadaverine dicarboxylate (cadaverine suberate or cadaverine azelaic acid), the method comprising: a first step of obtaining lysine dicarboxylate by culturing a microorganism producing L-lysine in a medium to which dicarboxylic acid is added in the form of diammonium dicarboxylate; and a second step of converting the lysine dicarboxylate into cadaverine dicarboxylate. According to the present application, a high-purity cadaverine dicarboxylate (cadaverine suberate or cadaverine azelaic acid) can be obtained without ion resin exchange, a decarboxylation process, and a distillation process by performing the process according to the above-described procedure.

Description

Cadaverine dicarboxylate manufacturing process

[0001] The present application relates to a technology for producing high-purity cadaverine dicarboxylate (cadaverine suberate or cadaverine azelaate) using a microbial fermentation and purification process.

[0002] Cadaverine is a toxic, foul-smelling diamine compound produced by the decomposition of animal tissue. Furthermore, cadaverine dicarboxylates, such as cadaverine suberate or cadaverine azelaate, serve as polymerization precursors for bio-polyamides, a type of engineering plastic widely used in automotive, electrical, and electronic components.

[0003] The process for preparing cadaverine suberate or cadaverine azelaate generally includes the following steps:

[0004] Synthesis of Cadaverine: Cadaverine synthesis involves the fermentation of suitable microbial strains capable of producing lysine decarboxylase, an enzyme that catalyzes the decarboxylation of lysine to cadaverine.

[0005] Production of Suberic Acid or Azelaic Acid: Suberic acid, another monomer required for the production of cadaverine suberate, can be synthesized by various methods, such as oxydimidation of hexane, polymerization of carbodiimidazole, and oxidation of stearic acid. Azelaic acid, another monomer required for the production of cadaverine azelaate, can also be synthesized by various methods, such as ozonolysis of oleic acid.

[0006] Formation of cadaverine suberate or cadaverine azelaic acid: The formation of cadaverine suberate or cadaverine azelaic acid involves the reaction between cadaverine and suberic acid, or cadaverine and azelaic acid, in the presence of a suitable catalyst, such as sulfuric acid or phosphoric acid. The reaction typically involves mixing the two monomers at a suitable temperature and stirring for a specified period of time to ensure complete reaction.

[0007] Purification of cadaverine suberate or cadaverine azelaate: Once the reaction is complete, the raw material cadaverine suberate or cadaverine azelaate is purified using various separation and purification techniques such as solvent extraction, chromatography, and crystallization.

[0008] According to the prior art, since suberate or azelaite has low solubility, there was a problem that suberate or azelaite precipitated as crystals together with cadaverine suberate or cadaverine azelaite, respectively, during the crystallization process in the above-described process. Therefore, in order to produce high-purity cadaverine suberate or cadaverine azelaite, suberate or azelaite had to be removed before crystallization, and there were problems such as cost burden due to performing an additional process to remove suberate or azelaite, decreased process efficiency, and environmental pollution due to the use of chemical solvents.

[0009] The problem to be solved by the present application is to provide a method for producing cadaverine dicarboxylate (cadaverine suberate or cadaverine azelaate).

[0010] One object of the present application is to provide a method for producing cadaverine dicarboxylate, comprising the first step of culturing an L-lysine producing microorganism in a medium to which dicarboxylic acid is added in the form of diammonium dicarboxylate to obtain lysine dicarboxylate, and the second step of converting the lysine dicarboxylate into cadaverine dicarboxylate.

[0011] According to one embodiment of the present application, by culturing an L-lysine-producing microorganism in a medium containing diammonium dicarboxylate to obtain lysine dicarboxylate, and enzymatically converting the lysine dicarboxylate into cadaverine dicarboxylate, a separate process for removing the dicarboxylate is not required, thereby simplifying the process, and at the same time, crystals of the dicarboxylic acid are not precipitated together as a byproduct, so that a high-purity cadaverine dicarboxylate can be produced in a high yield. Specifically, the cadaverine dicarboxylate refers to cadaverine suberate or cadaverine azelaate.

[0012]

[0013] Figure 1 is a flowchart showing a process for manufacturing cadaverine dicarboxylate according to the present application.

[0014] Figure 2 is a flowchart showing a process for manufacturing cadaverine dicarboxylate according to one embodiment of the present application.

[0015]

[0016] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below.

[0017]

[0018] One aspect of the present application provides a method for producing cadaverine dicarboxylate, comprising: a first step of culturing an L-lysine-producing microorganism in a medium to which dicarboxylic acid is added in the form of diammonium dicarboxylate to obtain lysine dicarboxylate; and a second step of converting the lysine dicarboxylate into cadaverine dicarboxylate, wherein the dicarboxylate and the dicarboxylic acid have 8 or 9 carbon atoms. When the dicarboxylate and the dicarboxylic acid have 8 carbon atoms, the dicarboxylate and the dicarboxylic acid refer to suberic acid or suberic acid. When the dicarboxylate and the dicarboxylic acid have 9 carbon atoms, the dicarboxylate and the dicarboxylic acid refer to azelaic acid or azelaic acid.

[0019]

[0020] In this application, the term "dicarboxylic acid" means a dibasic acid containing two carboxyl groups, and is of high importance as a metabolic intermediate in a living body.

[0021] Specifically, the dicarboxylic acid may be an aliphatic dicarboxylic acid having 8 or 9 carbon atoms. That is, in the present application, the dicarboxylic acid refers to one aliphatic dicarboxylic acid selected from suberic acid and azelaic acid. When the cadaverine dicarboxylate to be produced in the manufacturing method of the present application is cadaverine suberic acid, it should be understood that all of the added or introduced dicarboxylic acids are suberic acid, and further, when the cadaverine dicarboxylate to be produced in the manufacturing method of the present application is cadaverine azelaic acid, it should be understood that all of the added or introduced dicarboxylic acids are azelaic acid. In this way, the dicarboxylic acid in the present application is understood to refer to one type of aliphatic dicarboxylic acid, not a mixture of different types of dicarboxylic acids.

[0022]

[0023] In this application, the term "Suberic acid" refers to a dicarboxylic acid having 8 carbon atoms, C8H 14 It has the chemical formula O4 and generally exists as a colorless crystalline solid. The above-mentioned suberic acid is also called octanedioic acid and is utilized as a precursor for various industrial products, including polymers and plasticizers.

[0024]

[0025] In this application, the term "Azelaic acid" is a dicarboxylic acid having 9 carbon atoms, C9H 16 It has the chemical formula O4 and is generally found in the form of a white powder. Azelaic acid is also called azelaic acid, and is used as a precursor for various industrial products, including polymers and plasticizers.

[0026]

[0027] The term "cadaverine (CAD)" in this application refers to a foul-smelling, toxic diamine compound with the chemical formula NH2(CH2)5NH2. Cadaverine may also be referred to as 1,5-pentanediamine or pentamethylenediamine. It should be understood that the term "cadaverine" in this application includes the cadaverine dicarboxylate salt form.

[0028]

[0029] The term "cadaverine dicarboxylate" in this application refers to a substance manufactured by reacting a dicarboxylic acid component with the diamine-type cadaverine, and has high industrial utility as a precursor for manufacturing polyamide resins. Specifically, cadaverine dicarboxylate in this application refers to one cadaverine dicarboxylate selected from cadaverine suberate and cadaverine azelaate.

[0030] In this application, the term "cadaverine suberate" refers to a substance prepared by reacting cadaverine in the form of the diamine with suberic acid, a dicarboxylic acid component.

[0031] In this application, the term "cadaverine azelate" refers to a substance prepared by reacting azelaic acid, a dicarboxylic acid component, with cadaverine in the form of the above diamine.

[0032]

[0033] Figures 1 and 2 are flowcharts showing a process for manufacturing cadaverine dicarboxylate according to one embodiment of the present application.

[0034]

[0035] In this application, the term "L-lysine producing microorganism" includes both wild type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and may be a microorganism that includes genetic modification for the production of L-lysine, as a microorganism whose specific mechanism has been weakened or strengthened due to causes such as insertion of an external gene or enhancement or inactivation of the activity of an endogenous gene.

[0036] In one example, the microorganism producing L-lysine of the present application may be, but is not limited to, a microorganism that naturally has L-lysine production ability, a microorganism that has L-lysine production ability conferred on a microorganism that does not have L-lysine production ability, or a microorganism that has significantly low L-lysine production ability enhanced with L-lysine production ability. Specifically, the microorganism producing L-lysine or the microorganism having L-lysine production ability of the present application may be a microorganism in which some of the genes in the L-lysine biosynthesis pathway are enhanced or weakened, or some of the genes in the L-lysine degradation pathway are enhanced or weakened. "Enhanced" or "increased" L-lysine production ability means that the L-lysine production ability is improved compared to a parent strain or an unmodified microorganism.

[0037] As a specific example, the microorganism may be a microorganism of the genus Corynebacterium or a microorganism of the genus Escherichia. The microorganism of the genus Corynebacterium may include all microorganisms belonging to the genus Corynebacterium. Specifically, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli pollutisoli), Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens, and more specifically, but not limited to, Corynebacterium glutamicum. Microorganisms of the genus Escheria may include all microorganisms belonging to the genus Escheria.Specifically, it may be Escherichia coli, Escherichia albertii, Escherichia blattae, Escherichia fergusonii, Escherichia hermannii or Escherichia vulneris, and more specifically, the strain of the genus Escherichia may be Escherichia coli, but is not limited thereto.

[0038] The above L-lysine-producing microorganism can be cultivated using suitable media and culture conditions known in the art. This cultivation process can be easily adjusted and used by those skilled in the art depending on the selected microorganism. Specifically, the cultivation may be batch, continuous, or fed-batch, but is not limited thereto.

[0039] In this application, the term "medium" refers to a material containing nutrients necessary for culturing the microorganism as a main component, and supplies nutrients and growth factors, including water, which is essential for survival and growth. Specifically, the medium used for culturing the L-lysine-producing microorganism of the present application may be any medium used for culturing general microorganisms without particular limitation, except that it contains diammonium dicarboxylate as part or all of the nitrogen source. The microorganism of the present application can be cultured under aerobic conditions while controlling temperature, pH, etc. in a general medium containing a nitrogen source including diammonium dicarboxylate, an appropriate carbon source, phosphorus, inorganic compounds, amino acids, and / or vitamins.

[0040]

[0041] The first step of the method for producing cadaverine dicarboxylate of the present application is a step of culturing an L-lysine-producing microorganism in a medium to which dicarboxylic acid is added in the form of diammonium dicarboxylate to obtain lysine dicarboxylate. This can be achieved through a fermentation process of the lysine-producing microorganism.

[0042]

[0043] In the first step, the dicarboxylic acid having 8 or 9 carbon atoms may be supplied to the medium in the form of diammonium dicarboxylate, and accordingly, it should be understood that the dicarboxylic acid in the present application includes the form of diammonium dicarboxylate.

[0044] The term "diammonium suberate" in this application refers to a form of suberic acid in which two hydrogen atoms are replaced by ammonium, and is used as the main nitrogen source of the medium. Diammonium suberate may be prepared by gently mixing suberic acid with ammonia water to have a neutral pH, and then concentrated to form crystals or liquids, but is not limited thereto.

[0045] The term "diammonium azelate" in this application refers to a form of azelaic acid in which two hydrogen atoms are replaced by ammonium, and is used as the main nitrogen source of the medium. The diammonium azelate may be prepared by gently mixing azelaic acid with ammonia water to have a neutral pH, and then concentrated to form crystals or in liquid form, but is not limited thereto.

[0046]

[0047] The manufacturing method of the present application can manufacture cadaverine dicarboxylate with high purity while simplifying the process by adding 64 mol% or more of the dicarboxylic acid supplied throughout the process to the medium in the form of diammonium dicarboxylate in the first step, and can reduce the side effect of the dicarboxylate being precipitated together with cadaverine dicarboxylate.

[0048] Specifically, the diammonium dicarboxylate of the first step can be included in the medium at a high ratio, specifically 64 mol% or more, with respect to the dicarboxylic acid supplied throughout the entire process of the present application manufacturing method.

[0049] More specifically, the diammonium dicarboxylate of the first step may be included in an amount of 64 mol% or more, 76 mol% or more, 81 mol% or more, 82 mol% or more, 83 mol% or more, 84 mol% or more, 85 mol% or more, 86 mol% or more, 87 mol% or more, 88 mol% or more, 89 mol% or more, 90 mol% or more, 91 mol% or more, 92 mol% or more, 93 mol% or more, 94 mol% or more, 95 mol% or more, 96 mol% or more, 97 mol% or more, 98 mol% or more, 99 mol% or more, or 100 mol% of the dicarboxylic acid supplied in the entire process of the manufacturing method of the present application.

[0050]

[0051] The manufacturing method of the present application can provide the advantage of manufacturing high-purity cadaverine dicarboxylate by including a large amount of diammonium dicarboxylate in the medium, thereby eliminating the need for a separate dicarboxylate removal step, such as an ion resin exchange, decarboxylation process, or distillation process, unlike conventional processes that involve adding ammonium sulfate, etc. In addition, the use of diammonium dicarboxylate can facilitate the adjustment of the molar ratio of dicarboxylic acid added throughout the entire process / cadaverine in the process solution after the second step.

[0052]

[0053] Specifically, the diammonium dicarboxylate of the first step may be included so that the molar ratio (diammonium dicarboxylate / lysine) with respect to the lysine to be produced is 0.60 to 1. More specifically, the diammonium dicarboxylate of the first step may be included so that the molar ratio (diammonium dicarboxylate / lysine) with respect to the lysine to be produced is 0.6 to 1.0, 0.65 to 0.99, 0.74 to 0.99, or 0.78 to 0.98, 0.8 to 0.98, or 0.83 to 0.98, or 0.08 to 0.96, and even more specifically 0.79 to 0.99. At this time, it should be understood that the amount of lysine to be produced refers to the total amount including all forms of lysine dicarboxylate.

[0054] For example, the diammonium dicarboxylate of the first step may be included in a molar ratio (diammonium dicarboxylate / lysine) with the lysine to be produced in a range consisting of a lower limit selected from 0.60, 0.65, 0.70, 0.73, 0.74, 0.8, 0.83, 0.85, 0.86, 0.87, 0.88, and 0.89 and / or an upper limit selected from 1, 0.99, 0.98, 0.97, 0.96, 0.95, 0.94, 0.93, and 0.92.

[0055] In one specific example, the dicarboxylic acid and dicarboxylic acid are suberic acid and suberic acid, and the diammonium dicarboxylic acid of the first step may be included so that the molar ratio (diammonium dicarboxylic acid / lysine) with the lysine to be produced is 0.81 to 1.0.

[0056] In one specific example, the dicarboxylic acid and dicarboxylic acid are azelaic acid and azelaic acid, and the diammonium dicarboxylic acid of the first step may be included so that the molar ratio (diammonium dicarboxylic acid / lysine) with the lysine to be produced is 0.79 to 1.0.

[0057]

[0058] Specifically, the diammonium dicarboxylate of the first step may be included in an amount of 0.2 mol / L to 0.8 mol / L based on the fermentation broth of the L-lysine-producing microorganism. More specifically, the diammonium dicarboxylate of the first step may be included in an amount of 0.3 mol / L to 0.7 mol / L, 0.4 mol / L to 0.7 mol / L, 0.45 mol / L to 0.7 mol / L, or 0.45 mol / L to 0.53 mol / L based on the fermentation broth of the L-lysine-producing microorganism. By adding the diammonium dicarboxylate at the above concentration, ammonium ions may act as a nitrogen source during fermentation, and dicarboxylic acid and lysine may form a salt and be included in a dissolved state in the fermentation broth.

[0059]

[0060] Lysine dicarboxylate can be produced through fermentation of L-lysine-producing microorganisms in a medium containing diammonium dicarboxylate. Lysine dicarboxylate can be introduced into a subsequent process as a fermentation broth containing the cells, in the form of a fermentation broth with the cells removed, or after purification.

[0061]

[0062] The concentration of lysine in the fermentation liquid in which the first-stage lysine-producing microorganism of the present application is cultured is not limited, but may be 60 g / L to 200 g / L, 70 g / L to 150 g / L, 72 g / L to 140 g / L, or 72 g / L to 79 g / L based on the fermentation liquid of the L-lysine-producing microorganism.

[0063]

[0064] The carbon source of the above medium may include carbohydrates such as glucose, fructose, sucrose, maltose, and their isomers; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. In addition, natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane bagasse, and corn steep liquor may be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugar) may be used, and other appropriate carbon sources may be used in various ways without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0065] As a specific example, the carbon source that may be included in the culture medium of the present application may include glucose, maltose, or maltose isomers. More specifically, the carbon source may include at least one selected from among glucose, maltose, and maltose isomers (isomaltose), or a combination of two or more thereof, but is not limited thereto.

[0066] The above-mentioned badge may include potassium phosphate monobasic, potassium phosphate dibasic, or their corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and the like.

[0067] Additionally, the culture medium may contain metal salts, such as magnesium sulfate or iron sulfate, necessary for growth. Finally, in addition to the above substances, essential growth substances, such as amino acids and vitamins, may be used. Appropriate precursors may also be used in the culture medium. The above-mentioned raw materials may be added to the culture in an appropriate manner during the cultivation process, for example, batchwise or continuously, but are not limited thereto.

[0068] The nitrogen source of the above medium may include diammonium dicarboxylate. A typical medium for microbial culture may include an inorganic nitrogen source such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc. as a nitrogen source; an organic nitrogen source such as amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. However, in one embodiment of the present application, diammonium dicarboxylate may be included as the sole nitrogen source or as an additional nitrogen source.

[0069]

[0070] After the first step of producing lysine dicarboxylate by culturing an L-lysine producing microorganism in a medium containing diammonium dicarboxylate, a second step of converting the lysine dicarboxylate into cadaverine dicarboxylate can be performed.

[0071]

[0072] The second step refers to the step of converting lysine dicarboxylate into the target product, cadaverine dicarboxylate (cadaverine suberate or cadaverine azelaate).

[0073]

[0074] In one specific embodiment, the second step may further comprise adding a dicarboxylic acid to the process liquid. The process liquid may be the fermentation process liquid obtained in the first step.

[0075] The dicarboxylic acid added in the second step may not be added or may be added in a trace amount of 0.4 mol / L or less, 0.25 mol / L or less, 0.15 mol / L or less, 0.12 mol / L or less, 0.11 mol / L or less, 0.1 mol / L or less, 0.08 mol / L or less, 0.06 mol / L or less, 0.05 mol / L or less, 0.04 mol / L or less, 0.03 mol / L or less, 0.02 mol / L or less, or 0.01 mol / L or less based on the volume of the process solution, specifically based on the volume of the fermentation process solution obtained in the first step.

[0076] In one specific example, when the dicarboxylic acid is suberic acid, the dicarboxylic acid added in the second step may not be added or may be added in a trace amount of 0.1 mol / L or less.

[0077] In one specific example, when the dicarboxylic acid is azelaic acid, the dicarboxylic acid added in the second step may not be added or may be added in a trace amount of 0.11 mol / L or less.

[0078]

[0079] When dicarboxylic acid is additionally added to the process solution in the second step, the dicarboxylic acid may be added before, after, or simultaneously with the conversion reaction, but is not limited thereto.

[0080] If dicarboxylic acid is additionally added in the second step, it is understood that the amount of dicarboxylic acid added throughout the process of the manufacturing method of the present application is the sum of the amount of diammonium dicarboxylate added to the medium in the first step and the amount of dicarboxylic acid added in the second step. In addition, if dicarboxylic acid is not additionally added to the process solution in the second step, it is understood that the amount of dicarboxylic acid added throughout the process of the manufacturing method of the present application is the same as the amount of diammonium dicarboxylate added to the medium in the first step.

[0081]

[0082] Specifically, the amount of dicarboxylic acid added in the present application's manufacturing method may be added taking into account the amount of cadaverine in the process liquid obtained after the second step. Specifically, the amount of dicarboxylic acid added in the present application's manufacturing method may be a molar ratio of 0.80 to 1.10 relative to the amount of cadaverine in the process liquid obtained after the second step.

[0083] More specifically, the amount of dicarboxylic acid added in the present application manufacturing method may be a molar ratio of 0.85 to 1.10, 0.87 to 1.06, 0.88 to 1.05, 0.89 to 1.01, 0.88 to 1.01, 0.89 to 1.0, 0.90 to 1.10, 0.91 to 1.00, 0.93 to 1.01, 0.93 to 1.01, 0.95 to 1.06 or 0.95 to 1.05 relative to the amount of cadaverine in the process solution after the second step.

[0084] For example, the amount of dicarboxylic acid added in the present application manufacturing method may be included in a molar ratio in a range consisting of one lower limit selected from 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, and 0.95 and / or one upper limit selected from 1.10, 1.09, 1.08, 1.07, 1.06, 1.05, 1.04, 1.03, 1.02, 1.01, and 1.0 relative to the amount of cadaverine in the process solution after the second step.

[0085]

[0086] In one specific example, when the dicarboxylic acid is suberic acid, the amount of the dicarboxylic acid added in the manufacturing method of the present application may be a molar ratio of 0.80 to 1.10, 0.85 to 1.05, 0.87 to 1.03, 0.89 to 1.02, 0.91 to 1.00, 0.91 to 1.10, 0.90 to 1.05, or 0.90 to 1.02 relative to the amount of cadaverine in the process solution after the second step.

[0087] In the manufacturing method of the present application, one technical significance lies in the fact that when suberic acid is added in a molar ratio within the above range compared to the amount of cadaverine calculated to be obtained after the second step, suberic acid does not crystallize together with cadaverine suberic acid salt, thereby enabling the production of high-purity and high-yield cadaverine suberic acid salt.

[0088] In addition, by adjusting the molar ratio of the cadaverine to be obtained and the suberic acid to be added, it is possible to maintain the purity of cadaverine suberic acid at 99% up to the third cycle during the circulation of the mother liquor, and cadaverine suberic acid can be obtained at a total yield of 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, or 70% or more depending on the number of cycles of the mother liquor.

[0089]

[0090] In one specific example, when the dicarboxylic acid is azelaic acid, the amount of the dicarboxylic acid added in the manufacturing method of the present application may be a molar ratio of 0.80 to 1.10, 0.85 to 1.05, 0.87 to 1.03, 0.89 to 1.02, 0.91 to 1.00, 0.91 to 1.10, 0.90 to 1.05, 0.90 to 1.02, or 0.90 to 1.0 relative to the amount of cadaverine in the process solution after the second step.

[0091]

[0092] In the manufacturing method of the present application, one technical significance lies in the fact that it was discovered that when azelaic acid is added in a molar ratio within the above range compared to the amount of cadaverine calculated to be obtained after the second step, azelaic acid does not crystallize together with cadaverine azelaite, thereby enabling the production of high-purity and high-yield cadaverine azelaite.

[0093] In addition, by adjusting the molar ratio of the cadaverine to be obtained and the azelaic acid to be added, it is possible to maintain the purity of cadaverine azelaic acid at 99% up to the third cycle during the circulation of the mother liquor, and obtain cadaverine azelaic acid at a total yield of 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, or 30% or more, depending on the number of cycles of the mother liquor.

[0094]

[0095] In the second step, the conversion reaction of the lysine dicarboxylate to cadaverine dicarboxylate may be an enzymatic conversion reaction.

[0096] In one specific example, the lysine dicarboxylate may include both the lysine dicarboxylate obtained in the first step and the lysine dicarboxylate produced in the second step when a dicarboxylic acid is additionally added in the second step.

[0097]

[0098] Specifically, the enzymatic conversion reaction of the second step can be performed using a protein having lysine decarboxylase activity or a microorganism expressing a protein having the activity.

[0099] As a specific example, a protein having lysine decarboxylase activity may be added to the fermentation process liquid of the first stage, or a starter culture of a microorganism expressing a protein having the activity may be added, but is not limited thereto.

[0100]

[0101] The term "decarboxylase" in this application refers to an enzyme that catalyzes the production of carbonic acid by removing a carboxyl group, which is an organic acid, and may also be named decarboxylase, decarboxylase, decarboxylase, decarboxylase, or carbonic acid decarboxylase.

[0102] The above protein is not particularly limited as long as it exhibits lysine decarboxylase activity, but may be, for example, PtLDC protein from Pseudomonas thermotolerans or CadA protein derived from Escherichia coli. However, the protein sequence may be obtained using GenBank, a known database, or expressed using the above microorganism, or a commercially available enzyme may be purchased and used, and is not limited thereto.

[0103] The microorganism expressing the above protein may be a microorganism transformed to express the protein. The transformed microorganism is not limited to a prokaryotic microorganism or a eukaryotic microorganism, as long as it is transformed to express a protein having decarboxylase activity. The microorganism transformed to express the protein having decarboxylase activity can convert lysine dicarboxylate in the process solution into cadaverine dicarboxylate by secreting the protein having the enzymatic activity into the starter culture solution.

[0104] Specific examples thereof may include strains of Escherichia, Erwinia, Serratia, Providencia, and coryneform microorganisms. The microorganism may specifically be a microorganism belonging to the genus Escherichia or the genus Corynebacterium, and more specifically, may be, but is not limited to, Escherichia coli or Corynebacterium glutamicum.

[0105]

[0106] The conversion reaction of the second step may be performed for 20 minutes to 3 hours, more specifically for 0.5 to 1.5 hours, and for example, for 1 hour, but is not limited thereto.

[0107] The conversion reaction of the second step may be performed at 30 to 60°C, more specifically at 40 to 50°C, and for example, at 45°C, but is not limited thereto.

[0108]

[0109] The conversion reaction of the second step may be performed at a pH of 7.5 to 9, more specifically at 7.8 to 8.7, and even more specifically at 8 to 8.5. In one embodiment of the second step, the pH may be adjusted to the above range by optionally adding a dicarboxylic acid, and in another embodiment, the pH may be adjusted to the above range by adding CO2. When the pH range of the second step conversion reaction is adjusted to the above range, the conversion rate of lysine dicarboxylate to cadaverine dicarboxylate may be significantly increased.

[0110]

[0111] The method for producing cadaverine dicarboxylate of the present application may further include a recovery step of recovering the obtained cadaverine dicarboxylate after performing the second step.

[0112]

[0113] The above recovery may be by collecting cadaverine dicarboxylate using a suitable method known in the art.

[0114] For example, cadaverine dicarboxylate can be collected by various chromatographic methods such as centrifugation, filtration, concentration, crystallization, extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination of these methods.

[0115]

[0116] In one specific embodiment of the present application, the recovery step may include one or more of a filtration step, a concentration step, and a crystallization step.

[0117]

[0118] The above filtration step refers to a step of removing impurities from the conversion solution containing cadaverine dicarboxylate obtained in the second step. As an example, the filtration may include removing cells in the cadaverine dicarboxylate process solution by performing membrane separation, etc., or performing activated carbon filtration, etc.

[0119] The above concentration step refers to a step of concentrating to increase the solid content ratio in the conversion solution, and may be performed after the filtration step. As an example, the concentration may be performed by reducing pressure concentration using a rotary evaporator.

[0120] Specifically, the above concentration step may be to concentrate so that the solid content becomes 40 to 75% (w / w), and more specifically, to concentrate so that it becomes 40 to 75% (w / w), 45 to 70% (w / w), 42 to 50% (w / w), 50 to 65% (w / w), 40 to 50% (w / w), 55 to 75% (w / w), or 60 to 70% (w / w), and for example, to concentrate so that it becomes 45% (w / w) or 65% (w / w). When the solid content is concentrated in the above range in the above concentration step, the recovery rate and purity of cadaverine dicarboxylate can be excellently maintained.

[0121] The above concentration step may be performed at, but is not limited to, 45 to 75°C.

[0122]

[0123] The above crystallization step may be a cooling crystallization step, which means a step of cooling the conversion solution and precipitating it into crystals to obtain the final target product, cadaverine dicarboxylate crystals.

[0124] Specifically, the cooling may be cooling the conversion solution to 20 to 30°C, more specifically to 20 to 27°C, and for example to 25°C or 20°C, but is not limited thereto.

[0125] Specifically, the cooling rate may be 3-20°C / hour, more specifically 3-15°C / hour, and for example, but not limited to, 10°C / hour, or 3°C / hour.

[0126]

[0127] In one embodiment, the recovery step may further include a stirring step for stirring the process solution. The stirring step may be performed simultaneously with the cooling crystallization step, during the cooling crystallization step, before the cooling crystallization step, or after the cooling crystallization step. This stirring step provides sufficient time for the process solution to be stirred, thereby facilitating crystal precipitation.

[0128]

[0129] In one embodiment, the recovery step may further include a mother liquor circulation step of recycling the mother liquor containing non-crystallized cadaverine dicarboxylate after the crystallization step to the feed liquor of the concentration step.

[0130] Specifically, in the mother liquor circulation step, the number of times the mother liquor is cycled may be 0 to 3 times, 1 to 3 times, 1 to 4 times, 1 to 5 times, 1 to 10 times, or more. In this case, a number of times the mother liquor is cycled being 0 means that the mother liquor circulation step is not performed.

[0131]

[0132] In the past, there was a problem that a relatively large amount of cadaverine dicarboxylate still remained in the mother liquor remaining after crystallization in the step of crystallizing cadaverine dicarboxylate, resulting in a low yield of less than 50%. In the present application, as a method for improving the yield of cadaverine dicarboxylate, the total yield of cadaverine dicarboxylate can be increased by recycling the remaining mother liquor of cadaverine dicarboxylate as a feed liquid of the concentration step 1 to 3 times, 1 to 4 times, 1 to 5 times, 1 to 10 times or more.

[0133]

[0134] In one embodiment, the recovery step may further include washing, separating, and drying the crystallized process solution.

[0135] In the above drying step, moisture contained in the crystals is removed through drying, and a high-purity cadaverine dicarboxylate can be commercialized. After drying, the cadaverine dicarboxylate crystals can be provided in powder form, but the formulation can be varied as needed.

[0136]

[0137] In one embodiment, the recovery step may further include a decolorization step. The decolorization may be performed using, but is not limited to, activated carbon, anion resin, or the like.

[0138]

[0139] Additionally, the method for producing cadaverine dicarboxylate of the present application may include an additional purification step. The purification may be performed using any suitable method known in the art. In one example, when the method for producing cadaverine dicarboxylate of the present application includes both a recovery step and a purification step, the recovery process and the purification process may be performed continuously or discontinuously, regardless of the order, or may be performed simultaneously or integrated into a single step, but is not limited thereto.

[0140]

[0141] The manufacturing method of the present application may be characterized by not including a separate process for removing the dicarboxylate salt in the recovery step. In the case of the conventional cadaverine dicarboxylate production process, the dicarboxylate salt precipitates as crystals together during the crystallization process, lowering the purity. To prevent this, a separate process for removing the dicarboxylate salt before crystallization was required. However, the manufacturing method of the present application provides the technical advantage of producing high-purity cadaverine dicarboxylate salt without performing a separate dicarboxylate salt removal process.

[0142]

[0143] In one embodiment, when the dicarboxylic acid is suberic acid, the method of the present application may be characterized in that cadaverine suberic acid having a purity of 99% or higher can be obtained in the 0th to 3rd cycle of the mother liquor. In one embodiment, when the dicarboxylic acid is suberic acid, the cumulative total yield of cadaverine suberic acid may be 65% or higher, and more specifically, 66%, 67%, 68%, 69%, 70% or higher, based on the 3rd cycle of the mother liquor.

[0144] In one embodiment, when the dicarboxylic acid is azelaic acid, the method of the present application may be characterized in that cadaverine azelaic acid having a purity of 99% or higher can be obtained in the 0th to 3rd cycle of the mother liquor. In one embodiment, when the dicarboxylic acid is azelaic acid, the cumulative total yield of cadaverine azelaic acid may be 25% or higher, and more specifically, 26%, 27%, or higher, based on the 3rd cycle of the mother liquor.

[0145]

[0146] The method of the present application can produce high-purity cadaverine dicarboxylate without the decarboxylation and distillation processes required in the conventional liquid cadaverine production.

[0147] The present application will be described in more detail below through examples. However, the following examples are merely preferred embodiments intended to illustrate the present application and are therefore not intended to limit the scope of the present application. Furthermore, technical details not described herein can be readily understood and implemented by those skilled in the technical field of the present application or similar fields.

[0148]

[0149] The above has described a process for manufacturing cadaverine dicarboxylate (cadaverine suberate or cadaverine azelaic acid salt) according to one embodiment of the present application. Below, the advantageous effects mentioned in the present application will be examined through experimental results of examples and comparative examples of cadaverine suberate or cadaverine azelaic acid salt, respectively.

[0150]

[0151] 1. Cadaverine suberate

[0152]

[0153] Manufacturing Example 1-1. Fermentation process for producing lysine suberate

[0154] A Corynebacterium glutamicum strain (KCCM12154P, US Publication No. US 2021-0355514 A1) capable of producing lysine was cultured on solid surfaces and in flasks to obtain seed cultures. After seed culture in a fermenter, the main production fermentation was performed. Fermentation was performed at 36°C for 63 hours, with the fermenter cultured at 900 rpm.

[0155] In order to produce lysine suberate using Corynebacterium, ammonium suberate was supplied at a level of 67 to 110 g / L for each example to replace the existing ammonium sulfate, and as a result of fermentation, lysine suberate was obtained at a level of 103 to 170 g / L and lysine was obtained at a level of 79 g / L. At this time, the molar ratio of suberate / lysine in the fermentation process solution in the examples was adjusted to a level of 0.59 to 0.98.

[0156]

[0157] Manufacturing Example 1-2. Conversion reaction of lysine suberate to cadaverine suberate

[0158] For the conversion reaction, E. coli (US Patent Publication No. US 2018-0030430 A1) overexpressing the PtLDC enzyme, a lysine decarboxylase gene derived from Pseudomonas thermotolerans, using the pET-Deut1 vector was used.

[0159] The enzyme conversion solution obtained by culturing the above enzyme strain in the fermentation process solution of lysine suberate prepared from the above Manufacturing Example 1-1 was supplied at a level of 10% by mass percentage to the process solution to perform the conversion reaction. The conversion reaction temperature was maintained at 45 to 50°C for about 1 hour. The pH was adjusted to 8.0 to 8.5, and for this purpose, in the cases of the following Examples 1-1 to 1-5, CO2 was added for neutralization, and in the cases of Examples 1-2 to 1-4, suberate was additionally added at a level of 0.02 to 0.03 mol / L (based on the volume of the fermentation process solution of lysine suberate) before the conversion reaction. As a result, it was confirmed that the conversion rate of cadaverine suberate was at the level of 97 to 98%. Specifically, after the conversion reaction, cadaverine suberate was obtained at a level of 88 to 146 g / L (based on the volume of the fermentation process liquid of lysine suberate), and cadaverine was obtained at a level of 54 g / L (based on the volume of the fermentation process liquid of lysine succinate). Subsequently, in Examples 1-6 and 1-7, suberate was additionally added at a level of 0.05 to 0.1 mol / L (based on the volume of the fermentation process liquid of lysine suberate).

[0160]

[0161] Manufacturing Example 1-3. Subsequent processes after obtaining cadaverine suberate

[0162] The following subsequent process was performed on the process solution containing cadaverine suberate obtained in Manufacturing Example 1-2 to obtain cadaverine suberate in crystal form.

[0163] [Bacterial removal step]

[0164] The cells in the cadaverine suberate process solution were removed by performing membrane filtration with a size of 0.1 μm.

[0165] [Step to remove impurities using activated carbon]

[0166] Add activated carbon at a level of 10% based on the weight of cadaverine suberate to the filtrate from which the cells were separated. Heat the process solution with activated carbon added to 60℃, stir for 1 hour to decolorize, and then filter the activated carbon through a filter paper.

[0167] [Step of concentrating the cadaverine suberate process solution from which impurities have been removed]

[0168] The filtered liquid above was concentrated under reduced pressure at about 60°C and 120 Torr in a rotary evaporator until the solid content of the filtrate became 65% by weight.

[0169] [Step of cooling, crystallizing, and separating the cadaverine suberate concentrate and then drying it]

[0170] The above concentrate was cooled from 50°C to 25°C at a rate of 10°C / hr. The precipitated crystals were separated from the mother liquor using a centrifuge. The crystals were washed with water containing 10% of the weight of cadaverine suberate. The separated crystals were dried for one day, and their purity was measured by HPLC.

[0171] [Step of recycling the mother liquor to the crystallization stage]

[0172] The mother liquor separated above was recycled to the step of concentrating the cadaverine suberate process solution from which impurities were removed.

[0173]

[0174] Experimental Example 1-1. Confirmation of changes in cadaverine suberate purity and yield according to the molar ratio of suberate and cadaverine in the cadaverine suberate conversion solution.

[0175] In Experimental Example 1-1, the method according to Manufacturing Examples 1-1 to 1-3 was used to confirm how the purity and yield of cadaverine suberate change depending on the molar ratio of suberate and cadaverine in the cadaverine suberate conversion solution.

[0176]

[0177] Example 1-1: When the molar ratio of added suberic acid / cadaverine in the process solution after the second step is 0.6

[0178] 1,000 ml of a cadaverine suberate conversion solution having a suberic acid / cadaverine molar ratio of 0.6 was supplied and passed through a 0.1 μm membrane to remove microorganisms. A cadaverine suberate conversion solution having a suberic acid / cadaverine molar ratio of 0.61 was produced by following the production methods of Manufacturing Examples 1-1 and 1-2, except that the sum of the amount of suberic acid added among the ammonium suberate added in the fermentation process for producing lysine in Manufacturing Example 1-1 and the amount of suberic acid added in the cadaverine suberate conversion reaction in Manufacturing Example 1-2 was controlled to be 0.6 of the mole number of cadaverine that was converted.

[0179]

[0180] The filtrate was decolorized using activated carbon, filtered through a filter paper, and concentrated under reduced pressure to a solid content of 65%. The concentrate was cooled from 50°C to 25°C and crystallized. The crystals and mother liquor were separated by centrifugation, and the separated crystals were dried for one day and their purity was measured by HPLC. The mother liquor containing the remaining suberic acid was recycled to the cadaverine suberic acid feed solution having a suberic acid / cadaverine molar ratio of 0.6, and then subjected to another crystallization step. The recycling of the mother liquor was performed sequentially.

[0181]

[0182] Example 1-2: When the molar ratio of added suberic acid / cadaverine in the process solution after the second step is 0.70

[0183] A cadaverine suberate conversion solution having a suberic acid / cadaverine molar ratio of 0.70 was supplied, and the mother liquor was sequentially recycled and a purification process was performed in the same manner as in Example 1-1.

[0184]

[0185] Example 1-3: When the molar ratio of added suberic acid / cadaverine in the process solution after the second step is 0.79

[0186] A cadaverine suberate conversion solution having a suberic acid / cadaverine molar ratio of 0.79 was supplied, and the mother liquor was sequentially recycled and a purification process was performed in the same manner as in Example 1-1.

[0187]

[0188] Example 1-4: When the molar ratio of added suberic acid / cadaverine is 0.91

[0189] A cadaverine suberate conversion solution having a suberic acid / cadaverine molar ratio of 0.91 was supplied, and the mother liquor was sequentially recycled and a purification process was performed in the same manner as in Example 1-1.

[0190]

[0191] Example 1-5: When the molar ratio of added suberic acid / cadaverine is 1.00

[0192] A cadaverine suberate conversion solution having a suberic acid / cadaverine molar ratio of 1.00 was supplied, and the mother liquor was sequentially recycled and a purification process was performed in the same manner as in Example 1-1.

[0193] For Examples 1-4 to 1-5, the molar ratio of suberic acid / lysine in the lysine fermentation step was adjusted to 0.83-0.98.

[0194]

[0195] Example 1-6: When the molar ratio of added suberic acid / cadaverine is 1.10

[0196] A cadaverine suberate conversion solution having a suberic acid / cadaverine molar ratio of 1.10 was supplied, and the mother liquor was sequentially recycled and a purification process was performed in the same manner as in Example 1-1.

[0197]

[0198] Example 1-7: When the molar ratio of added suberic acid / cadaverine is 1.19

[0199] A cadaverine suberate conversion solution having a suberic acid / cadaverine molar ratio of 1.19 was supplied, and the mother liquor was sequentially recycled and a purification process was performed in the same manner as in Example 1-1.

[0200]

[0201] The physical property measurements for the process solutions and cadaverine suberate prepared according to the above-described Examples 1-1 to 1-7 are as shown in Tables 1 to 4 below.

[0202]

[0203] Table 1 shows the process solution that did not circulate the mother liquor.

[0204]

[0205] No mother liquor circulation Example 1-1 Example 1-2 Example 1-3 Example 1-4 Example 1-5 Example 1-6 Example 1-7 Suberic acid / cadaverine molar ratio 0.60 0.70 0.79 0.91 1.00 1.10 1.19 Crystallization feed New feed, ml 1000 1000 1000 1000 1000 1000 1000 Amount of cadaverine in crystallization solution, g 2 3 4 2 3 4 2 3 4 2 3 4 2 3 4 2 3 4 2 3 4 Amount of cadaverine in crystals produced, g No crystals produced 3 3 4 5 5 3 6 1 5 0 3 9 Amount of cadaverine in mother liquor, g - 2 0 0 1 8 9 1 8 0 1 7 1 8 4 1 9 5 Result Purity, % - 999999997656 Yield, %-141923262117Total yield, %-141923262117

[0206] Table 2 shows the process solution that has been circulated once.

[0207] Mother liquor circulation 1 timeExample 1-1Example 1-2Example 1-3Example 1-4Example 1-5Example 1-6Example 1-7Suberic acid / cadaverine mole ratio 0.600.700.790.911.001.101.19Crystallization feed solutionNew feed solution, ml-100010001000100010001000Amount of cadaverine in crystallization solution, g-434422414406417428Amount of cadaverine in produced crystals, g-Crystals not produced81951058971Amount of cadaverine in mother liquor, g-342319301329357Result Purity, %--9999998165Yield, %--1923262117Total yield, %--3541453830

[0208] Table 3 shows the process solution that was cycled twice.

[0209] Mother liquor circulation 2 times Example 1-1 Example 1-2 Example 1-3 Example 1-4 Example 1-5 Example 1-6 Example 1-7 Suberic acid / cadaverine mole ratio 0.60 0.70 0.79 0.91 1.00 1.10 1.19 Crystallization feed New feed, ml--1000 1000 1000 1000 1000 Amount of cadaverine in crystallization solution, g--575552535562591 Amount of cadaverine in produced crystals, g--110 126139 12098 Amount of cadaverine in mother liquor, g--465426396443493 Result Purity, %--9999998 370 Yield, %--19 232621 17 Total yield, %--4754595142

[0210] Table 4 shows the process solution after circulating the mother liquor three times.

[0211] Mother liquor circulation 3 times Example 1-1 Example 1-2 Example 1-3 Example 1-4 Example 1-5 Example 1-6 Example 1-7 Suberic acid / cadaverine molar ratio 0.6 0.7 0.7 9 0.9 1 1.0 1.1 1.1 9 Crystallization feed New feed, ml--1000 1000 1000 1000 1000 Amount of cadaverine in crystallization solution, g--699659629676726 Amount of cadaverine in crystals produced, g--No crystals produced 151163144121 Amount of cadaverine in mother liquor, g---508466532606 Result Purity, %---99998574 Yield, %---23262117 Total yield, %---65706252

[0212] By summarizing the experimental results according to Tables 1 to 4 above, it was confirmed that the presently applied manufacturing method, which includes a large amount of diammonium suberate in the first step at 84-100 mol% of the total supplied suberic acid, enables the production of cadaverine suberate with an overall improved crystallization rate, high purity, and high yield.

[0213]

[0214] More specifically, in the case of Example 1-1, no crystals were precipitated even when the crystallization process was performed. This is because the molar ratio of suberic acid salt was low, which was not sufficient for crystals of cadaverine suberic acid salt to precipitate.

[0215] In Example 1-2, crystals were formed up to the first cycle of mother liquor circulation, but no crystals were formed in the second cycle. This is because the molar ratio of suberic acid salt in the crystallization feed solution was low after the first cycle of mother liquor circulation, making it insufficient for cadaverine suberic acid salt crystals to be formed.

[0216] In Examples 1-3, crystals were formed up to the second cycle of mother liquor circulation, but no crystals were formed in the third cycle. This is because the sequentially circulated mother liquor lowered the molar ratio of suberic acid salt to the crystallization feed solution, and thus the concentration of suberic acid salt was not sufficient to precipitate crystals of cadaverine suberic acid salt.

[0217] In Example 1-6, the purity dropped slightly to 85% even after the third cycle of mother liquor circulation. This is because the suberic acid accumulated in the crystallization feed liquid was precipitated as an impurity as the mother liquor was sequentially circulated.

[0218] In Example 1-7, the purity dropped slightly to 74% even after the third cycle of mother liquor circulation. This is because suberic acid accumulated in the crystallization feed liquid was precipitated as an impurity as the mother liquor was sequentially circulated.

[0219]

[0220] On the other hand, Example 1-4 was able to obtain 99% high-purity cadaverine suberate up to the third cycle of mother liquor circulation, and achieve a high total yield of approximately 65%.

[0221] Example 1-5 was able to obtain 99% high-purity cadaverine suberate up to the third cycle of mother liquor circulation, and achieve a high total yield of approximately 70%.

[0222]

[0223] In this way, it was determined that a method for producing high-purity cadaverine suberate can be provided depending on the molar ratio of suberic acid added throughout the entire process relative to the amount of cadaverine in the process solution after the second step. In particular, it was confirmed that when the amount of suberic acid added was adjusted to a molar ratio of about 0.91 to 1.00 relative to the amount of cadaverine in the process solution after the second step, an excellent total yield of 65% or more could be achieved while maintaining a purity of 99% of cadaverine suberate even after three or more cycles of mother liquor circulation.

[0224]

[0225] 2. Cadaverine azelaate

[0226]

[0227] Manufacturing Example 2-1. Fermentation process for producing lysine azelaic acid

[0228] A Corynebacterium glutamicum strain (KCCM12154P, US Patent Publication No. US 2021-0355514 A1) capable of producing lysine was cultured on solid surfaces and in flasks to obtain seed cultures. After seed culture in a fermenter, the main production fermentation was performed. Fermentation was performed at 36°C for 30 hours, with the fermenter cultured at 900 rpm.

[0229] In order to produce lysine azelaic acid using Corynebacterium, ammonium azelaic acid was supplied at a level of 67 to 107 g / L for each example to replace the existing ammonium sulfate, and as a result of fermentation, lysine azelaic acid at a level of 100 to 161 g / L and lysine at a level of 73 g / L were obtained. At this time, the azelaic acid / lysine molar ratio in the fermentation process solution in the examples was adjusted to a level of 0.6 to 0.96.

[0230]

[0231] Manufacturing Example 2-2. Conversion reaction of lysine azelaate to cadaverine azelaate

[0232] For the conversion reaction, E. coli (US Patent Publication No. US 2018-0030430 A1) overexpressing the PtLDC enzyme, a lysine decarboxylase gene derived from Pseudomonas thermotolerans, using the pET-Deut1 vector was used.

[0233] The enzyme conversion solution obtained by culturing the enzyme strain above was supplied to the fermentation process solution of lysine azelaic acid prepared from the above Manufacturing Example 2-1 at a mass percentage of 10% to the process solution to perform the conversion reaction. The conversion reaction temperature was maintained at 45°C and performed for approximately 1 hour. The pH was adjusted to 8.0 to 8.5, and for this purpose, in the cases of the following Examples 2-1 to 2-5, CO2 was added for neutralization, and in the cases of Examples 2-3 to 2-5, azelaic acid was additionally added at a level of 0.01 to 0.04 mol / L (based on the volume of the fermentation process solution of lysine azelaic acid). As a result, it was confirmed that the conversion rate of cadaverine azelaic acid was at the level of 97 to 98%. Specifically, after the conversion reaction, cadaverine suberate was obtained at a level of 87 to 142 g / L (based on the volume of the fermentation process liquid of lysine suberate), and cadaverine was obtained at a level of 50 g / L (based on the volume of the fermentation process liquid of lysine succinate). In Examples 2-6 and 2-7, azelaic acid was additionally added at a level of 0.06 to 0.11 mol / L (based on the volume of the fermentation process liquid of lysine azelaate) after the conversion reaction.

[0234]

[0235] Manufacturing Example 2-3. Subsequent processes after obtaining cadaverine azelaic acid salt

[0236] The following subsequent process was performed on the process solution containing cadaverine azelaic acid salt obtained in Manufacturing Example 2-2 to obtain cadaverine azelaic acid salt in crystal form.

[0237] [Bacterial removal step]

[0238] The cells in the cadaverine azelaic acid solution were removed by performing membrane filtration with a size of 0.1 μm.

[0239] [Step to remove impurities using activated carbon]

[0240] Add activated carbon at a level of 10% based on the weight of cadaverine azelaic acid to the filtrate from which the cells were separated. Heat the process solution with added activated carbon to 60℃, stir for 1 hour to decolorize, and then filter the activated carbon through a filter paper.

[0241] [Step of concentrating the cadaverine azelaic acid solution from which impurities have been removed]

[0242] The filtered liquid above was concentrated under reduced pressure at approximately 55 to 70°C and 120 Torr in a rotary evaporator until the solid content of the filtrate became 45% by weight.

[0243] [Step of cooling, crystallizing, and separating the cadaverine azelaic acid concentrate and then drying it]

[0244] The above concentrate was cooled from 60°C to 25°C at a rate of 10°C / hr. Thereafter, it was cooled from 25°C to 20°C at a rate of 3°C / hr. The mixture was then stirred at 20°C for 24 hours. The precipitated crystals were separated from the mother liquor using a centrifuge. The crystals were washed with water containing 10% of the weight of cadaverine azelaic acid. The separated crystals were dried at low temperature (20°C) for one to two days, and their purity was measured using HPLC.

[0245] [Step of recycling the mother liquor to the crystallization stage]

[0246] The mother liquor separated above was recycled to the [step of concentrating the cadaverine azelaic acid process solution from which impurities have been removed].

[0247]

[0248] Experimental Example 2-1. Confirmation of changes in cadaverine azelaite purity and yield according to the molar ratio of azelaite and cadaverine in the cadaverine azelaite conversion solution.

[0249] In Experimental Example 2-1, the method according to Manufacturing Examples 2-1 to 2-3 was used to confirm how the purity and yield of cadaverine azelaic acid salt change depending on the molar ratio of azelaic acid salt and cadaverine in the cadaverine azelaic acid conversion solution.

[0250]

[0251] Example 2-1: When the molar ratio of added azelaic acid / cadaverine is 0.61

[0252] 1,000 ml of a cadaverine azelaate conversion solution having an azelaate / cadaverine molar ratio of 0.61 was supplied and passed through a 0.1 μm membrane to remove microorganisms. The cadaverine azelaate conversion solution having an azelaate / cadaverine molar ratio of 0.6 was manufactured by following the manufacturing methods of Manufacturing Examples 2-1 and 2-2, but controlling the sum of the azelaate input amount of the ammonium azelaate input in the fermentation process for producing lysine in Manufacturing Example 2-1 and the azelaate input amount in the cadaverine azelaate conversion reaction in Manufacturing Example 2-2 to be 0.61 of the mole number of cadaverine converted.

[0253] The filtrate was decolorized using activated carbon, filtered through a filter paper, and concentrated under reduced pressure to a solid content of 45%. The concentrate was cooled from 60℃ to 25℃ and then stirred for 24 hours to crystallize. The crystals and mother liquor were separated by centrifugation, and the separated crystals were dried at low temperature for one day or up to two days, and then the purity was measured by HPLC. The mother liquor containing residual azelaite was recycled to the cadaverine azelaite feed solution having an azelaite / cadaverine molar ratio of 0.61, and then the crystallization step was performed again. The recycling of the mother liquor was performed sequentially.

[0254]

[0255] Example 2-2: When the molar ratio of added azelaic acid / cadaverine is 0.71

[0256] A cadaverine azelaic acid conversion solution having an azelaic acid / cadaverine molar ratio of 0.71 was supplied, and the mother liquor was sequentially recycled and a purification process was performed in the same manner as in Example 2-1.

[0257]

[0258] Example 2-3: When the molar ratio of added azelaic acid / cadaverine is 0.8

[0259] A cadaverine azelaic acid conversion solution having an azelaic acid / cadaverine molar ratio of 0.8 was supplied, and the mother liquor was sequentially recycled and a purification process was performed in the same manner as in Example 2-1.

[0260]

[0261] Example 2-4: When the molar ratio of added azelaic acid / cadaverine is 0.9

[0262] A cadaverine azelaic acid conversion solution having an azelaic acid / cadaverine molar ratio of 0.9 was supplied, and the mother liquor was sequentially recycled and a purification process was performed in the same manner as in Example 2-1.

[0263]

[0264] Example 2-5: When the molar ratio of added azelaic acid / cadaverine is 1.0

[0265] A cadaverine azelaic acid conversion solution having an azelaic acid / cadaverine molar ratio of 1.0 was supplied, and the mother liquor was sequentially recycled and a purification process was performed in the same manner as in Example 2-1. In the case of Examples 2-4 and 2-5, the azelaic acid / lysine molar ratio in the lysine fermentation step was adjusted to 0.80-0.96.

[0266]

[0267] Example 2-6: When the molar ratio of added azelaic acid salt / cadaverine is 1.1

[0268] A cadaverine azelaic acid conversion solution having an azelaic acid / cadaverine molar ratio of 1.1 was supplied, and the mother liquor was sequentially recycled and a purification process was performed in the same manner as in Example 2-1.

[0269]

[0270] Example 2-7: When the molar ratio of added azelaic acid / cadaverine is 1.2

[0271] A cadaverine azelaic acid conversion solution having an azelaic acid / cadaverine molar ratio of 1.2 was supplied, and the mother liquor was sequentially recycled and a purification process was performed in the same manner as in Example 2-1.

[0272]

[0273] The physical property measurements for the process solution and cadaverine azelaic acid salt prepared according to Examples 2-1 to 2-7 described above are as shown in Tables 5 to 8 below.

[0274]

[0275] Table 5 is for the process solution that did not circulate the mother liquor.

[0276] No mother liquor circulation Example 2-1 Example 2-2 Example 2-3 Example 2-4 Example 2-5 Example 2-6 Example 2-7 Azelaic acid / cadaverine molar ratio 0.6 10.7 10.8 00.9 01.0 1.1 01.2 0 Crystallization feed New feed ml 1000 1000 1000 1000 1000 1000 1000 Amount of cadaverine in crystallization solution, g 154.2 154.2 154.2 154.2 154.2 154.2 Amount of cadaverine in produced crystals, g No crystals produced 20.9 23.1 24.8 26.2 24.5 22.7 Amount of cadaverine in mother liquor, g-133.3131.1129.4128.0129.7131.5Result Purity, %-98%98%98%99%97%92%Yield, %-13.6%15.0%16.1%17.0%15.9%14.7%Total Yield, %-13.6%15.0%16.1%17.0%15.9%14.7%

[0277] Table 6 shows the process solution that has been circulated once.

[0278] Mother liquor circulation 1 timeExample 2-1Example 2-2Example 2-3Example 2-4Example 2-5Example 2-6Example 2-7Azelaic acid / cadaverine mole ratio0.610.710.800.901.001.101.20Crystallization feed solutionNew feed solution ml100010001000100010001000Amount of cadaverine in crystallization solution, g170.9166.8163.9161.3164.6167.6Amount of cadaverine in produced crystals, g-24.826.026.927.426.825.7Amount of cadaverine in mother liquor, g-146.1140.8137.0133.9137.9141.9Result purity, %98%99%99%99%93%84%Yield, %15.4%16.1%16.7%17.0%16.6%15.9%Total Yield, %19.8%21.2%21.1%21.3%21.1%20.6%

[0279] Table 7 shows the process solution that was circulated twice.

[0280] Mother liquor circulation 2 times Example 2-1 Example 2-2 Example 2-3 Example 2-4 Example 2-5 Example 2-6 Example 2-7 Azelaic acid / cadaverine molar ratio 0.6 10.7 10.8 00.9 01.0 1.1 01.2 0 Crystallization feed New feed ml-1000 1000 1000 1000 1000 - Amount of cadaverine in crystallization solution, g-212.0 203.1 196.1 190.9 197.6 - Amount of cadaverine in crystals formed, g- No crystals formed29.4 31.1 32.5 30.7 - Amount of cadaverine in mother liquor, g--173.7 164.9 158.4 166.9 - Purity of result, %--98%98%99%86%-Yield, %--14.5%15.9%17.0%15.5%-Total Yield, %--22.8%23.6%24.6%23.9%-

[0281] Table 8 shows the process solution that was circulated three times.

[0282] Mother liquor circulation 3 times Example 2-1 Example 2-2 Example 2-3 Example 2-4 Example 2-5 Example 2-6 Example 2-7 Azelaic acid / cadaverine molar ratio 0.6 10.7 10.8 00.9 01.0 01.1 01.2 0 Crystallization feed New feed ml--1000 1000 1000--Amount of cadaverine in crystals formed, g--105.4 249.7 23 2.0--Amount of cadaverine in crystals formed, g--No crystals formed 38.3 39.4--Amount of cadaverine in mother liquor, g--- 211.5 19 2.5--Result purity, %---99% 99%--Yield, %---15.3 17.0--Total yield, %---27.1%26.8%--

[0283] By summarizing the experimental results according to Tables 5 to 8 above, it was confirmed that the present application manufacturing method, which includes a large amount of diammonium azelaic acid in the first step at 81-100 mol% of the total supplied azelaic acid, enables the production of cadaverine azelaic acid with an overall improved crystallization rate, high purity, and high yield.

[0284]

[0285] More specifically, in the case of Example 2-1, even when the crystallization process was performed, no crystals were precipitated. This is because the molar ratio of azelaic acid salt was low, which was not sufficient for crystals of cadaverine azelaic acid salt to precipitate.

[0286] In Example 2-2, crystals were precipitated up to the first cycle of mother liquor circulation, but no crystals were formed in the second cycle. This is because the molar ratio of azelaic acid salt in the crystallization feed solution was low after the first cycle of mother liquor circulation, making it insufficient for cadaverine azelaic acid salt crystals to precipitate.

[0287] In Example 2-3, crystals were precipitated up to the second cycle of mother liquor circulation, but no crystals were formed in the third cycle. This is because the sequentially circulated mother liquor lowered the molar ratio of azelaic acid salt to the crystallization feed solution, and thus the concentration of azelaic acid salt was not sufficient to precipitate crystals of cadaverine azelaic acid salt.

[0288] In Example 2-6, crystals were precipitated up to the second cycle of mother liquor circulation, but the purity of the crystals dropped slightly to 86%. This is because azelaic acid accumulated in the crystallization feed liquid as the mother liquor was sequentially circulated, causing azelaic acid to precipitate as an impurity.

[0289]

[0290] On the other hand, Example 2-4 was able to obtain 99% high-purity cadaverine azelaic acid salt up to the third cycle of mother liquor circulation, and achieve a high total yield of approximately 27.1%.

[0291] Example 2-5 obtained 99% high-purity cadaverine azelaic acid salt up to the third cycle of mother liquor circulation, and achieved a high total yield of approximately 26.8%.

[0292] In this way, it was found that a method for producing high-purity cadaverine azelaic acid salt can be provided depending on the molar ratio of azelaic acid added in the entire process to the amount of cadaverine in the process solution after the second step.

[0293]

[0294] In particular, when the amount of added azelaic acid is adjusted to a molar ratio of about 0.9 to 1.00 compared to the amount of cadaverine in the process solution after the second step, it was confirmed that an excellent total yield of 26.8% or more can be exhibited while maintaining a cadaverine azelaic acid purity of 99% even after three or more cycles of mother liquor circulation.

[0295]

[0296] From the above description, those skilled in the art will understand that the present application can be implemented in other specific forms without altering its technical concept or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of this application should be interpreted to include all changes or modifications derived from the meaning and scope of the following claims and their equivalents, rather than the detailed description above.

Claims

1. A first step of culturing an L-lysine producing microorganism in a medium containing dicarboxylic acid in the form of diammonium dicarboxylate to obtain lysine dicarboxylate; and A method for producing cadaverine dicarboxylate, comprising a second step of converting lysine dicarboxylate into cadaverine dicarboxylate, The amount of dicarboxylic acid added is a molar ratio of 0.85 to 1.05 relative to the amount of cadaverine in the process solution after the second step, The above dicarboxylic acid is an aliphatic dicarboxylic acid having 8 or 9 carbon atoms. Method for preparing cadaverine dicarboxylate.

2. In paragraph 1, The diammonium dicarboxylate of the above first step is comprised of 81 mol% or more of the total supplied dicarboxylic acid. Method for preparing cadaverine dicarboxylate.

3. In paragraph 1, A method for producing cadaverine dicarboxylate, wherein the diammonium dicarboxylate of the first step is included so that the molar ratio (diammonium dicarboxylate / lysine) with the lysine to be produced is 0.79 to 0.

99.

4. In paragraph 1, The above dicarboxylic acid salts and dicarboxylic acids are suberic acid salts and suberic acid, A method for producing cadaverine dicarboxylate, wherein the diammonium dicarboxylate of the first step is included so that the molar ratio (diammonium dicarboxylate / lysine) with the lysine to be produced is 0.81 to 1.

0.

5. In paragraph 1, The above dicarboxylic acid salts and dicarboxylic acids are azelaic acid salts and azelaic acid, A method for producing cadaverine dicarboxylate, wherein the diammonium dicarboxylate of the first step is included so that the molar ratio (diammonium dicarboxylate / lysine) with the lysine to be produced is 0.79 to 1.

0.

6. A method for producing cadaverine dicarboxylate, wherein the conversion reaction of the second step in the first paragraph is performed at a pH of 8.0 to 8.

5.

7. In paragraph 1, The above dicarboxylic acid salts and dicarboxylic acids are suberic acid salts and suberic acid, A method for producing cadaverine dicarboxylate, wherein the amount of added dicarboxylic acid is a molar ratio of 0.91 to 1.0 relative to the amount of cadaverine in the process solution after the second step.

8. In paragraph 1, The above dicarboxylic acid salts and dicarboxylic acids are azelaic acid salts and azelaic acid, A method for producing cadaverine dicarboxylate, wherein the amount of added dicarboxylic acid is a molar ratio of 0.90 to 1.0 relative to the amount of cadaverine in the process solution after the second step.

9. In paragraph 1, A method for producing cadaverine dicarboxylate, wherein the second step further comprises adding a dicarboxylic acid to the process solution.

10. In paragraph 1, A method for producing cadaverine dicarboxylate, further comprising a recovery step of recovering cadaverine dicarboxylate after performing the above second step.

11. In paragraph 10, A method for producing cadaverine dicarboxylate, wherein the recovery step comprises at least one step selected from the group consisting of a filtration step, a concentration step, and a cooling crystallization step.

12. In the 10th paragraph, the recovery step A method for producing cadaverine dicarboxylate, further comprising a mother liquor circulation step of recycling the mother liquor containing non-crystallized cadaverine dicarboxylate after the cooling crystallization step as a feed liquid of a concentration step.

13. A method for producing cadaverine dicarboxylate, wherein the concentration step is performed so that the solid content becomes 40-75% (w / w) in the 11th paragraph.

14. In paragraph 1, A method for producing cadaverine dicarboxylate, wherein in the second step, converting lysine dicarboxylate into cadaverine dicarboxylate is performed using a protein having lysine decarboxylase activity or a microorganism expressing a protein having the activity.

15. In paragraph 1, A method for producing cadaverine dicarboxylate, wherein the above L-lysine producing microorganism is Corynebacterium glutamicum.

16. A method for producing cadaverine dicarboxylate, characterized in that the recovery step in the first paragraph does not include a separate process for removing dicarboxylate from the process solution.

17. In paragraph 11, A method for producing cadaverine dicarboxylate, wherein cadaverine dicarboxylate having a purity of 99% or higher can be obtained in the 0th to 3rd cycle of the above-mentioned mother liquor.

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