Cadavering dicarboxylate production process

Abstract

This application relates to a method for producing cadaverine dicarboxylate (cadaverine verphosphate or cadaverine azelaate), comprising a first step of culturing L-lysine-producing microorganisms in a medium supplemented with dicarboxylic acid in the form of diammonium dicarboxylate to obtain lysine dicarboxylate, and a second step of converting lysine dicarboxylate to cadaverine dicarboxylate. According to this application, by carrying out the process according to the above procedure, high-purity cadaverine dicarboxylate (cadaverine verphosphate or cadaverine azelaate) can be obtained without ion resin exchange, decarboxylation, and distillation steps.

Description

Technical Field

[0001] This application relates to a technology for producing high-purity cadaverine dicarboxylate (cadaverine sebacate or cadaverine azelate) using microbial fermentation and purification processes.

Background Art

[0002] Cadaverine is a toxic diamine compound that emits a foul odor generated by the decay of animal tissues. Also, cadaverine dicarboxylate, for example, cadaverine sebacate or cadaverine azelate, is a polymerization precursor of bio-polyamide, and bio-polyamide is one of the engineering plastics widely used in automobiles, electrical and electronic parts, etc.

[0003] The preparation process of cadaverine sebacate or cadaverine azelate generally includes the following steps:

[0004] Synthesis of cadaverine: Cadaverine synthesis involves the fermentation of a suitable microbial strain that can produce lysine decarboxylase, an enzyme that catalyzes the decarboxylation of lysine to cadaverine.

[0005] Production of sebacic acid or azelaic acid: Sebacic acid, which is another monomer required for the production of cadaverine sebacate, can be synthesized by various methods such as the oxidation dimerization of hexane, carbodiimidazole polymerization, and the oxidation of stearic acid. Also, azelaic acid, which is another monomer required for the production of cadaverine azelate, can be synthesized by various methods such as the ozonolysis of oleic acid.

[0006] Formation of cadaverine veraphosphate or cadaverine azelaate: The formation of cadaverine veraphosphate or cadaverine azelaate involves a 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 specific period of time to ensure a complete reaction.

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

[0008] According to conventional technology, suberate or azelaate have low solubility, and during the crystallization process described above, suberate or azelaate precipitate as crystals along with cadaverin suberate or cadaverin azelaate, respectively. Therefore, in order to produce high-purity cadaverin suberate or cadaverin azelaate, suberate or azelaate must be removed before crystallization, which leads to problems such as increased costs due to the additional steps required for suberate or azelaate removal, decreased process efficiency, and environmental pollution due to the use of chemical solvents. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] U.S. Published Patent US 2018-0030430 A1 [Patent Document 2] U.S. Patent Publication Number US 2021-0355514 A1 [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] The problem that this application aims to solve is to provide a method for producing cadaverine dicarboxylate (cadaverine verophosphate or cadaverine azelaate). [Means for solving the problem]

[0011] One object of this application is to provide a method for producing cadaverine dicarboxylate, comprising a first step of culturing L-lysine-producing microorganisms in a medium supplemented with dicarboxylic acid in the form of diammonium dicarboxylate to obtain lysine dicarboxylate, and a second step of converting lysine dicarboxylate to cadaverine dicarboxylate. [Effects of the Invention]

[0012] According to one aspect of this application, L-lysine-producing microorganisms are cultured in a medium containing diammonium dicarboxylate to obtain lysine dicarboxylate, and the lysine dicarboxylate is enzymatically converted to cadaverine dicarboxylate. This eliminates the need for a separate step to remove the dicarboxylate, thus simplifying the process while preventing dicarboxylic acid crystals from precipitating as by-products, and enabling the production of high-purity cadaverine dicarboxylate in high yield. Specifically, the cadaverine dicarboxylate refers to cadaverine verphosphate or cadaverine azelaate. [Brief explanation of the drawing]

[0013] [Figure 1] This is a flowchart illustrating the manufacturing process of cadavering dicarboxylate according to this application. [Figure 2] This is a flowchart illustrating the manufacturing process of a cadavering dicarboxylate according to one aspect of this application. [Modes for carrying out the invention]

[0014] This can be explained in more detail as follows: On the other hand, each description and embodiment disclosed in this application applies to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the categories of this application are not considered to be limited by the specific descriptions described below.

[0015] One aspect of this application provides a method for producing cadaverine dicarboxylate, comprising: a first step of culturing an L-lysine-producing microorganism in a medium to which a dicarboxylic acid has been added in the form of a diammonium dicarboxylate to obtain a lysine dicarboxylate; and a second step of converting the lysine dicarboxylate to a cadaverine dicarboxylate, wherein the dicarboxylate and dicarboxylic acid have 8 or 9 carbon atoms. When the dicarboxylate and dicarboxylic acid have 8 carbon atoms, the dicarboxylate and dicarboxylic acid mean suberate or suberic acid. When the dicarboxylate and dicarboxylic acid have 9 carbon atoms, the dicarboxylate and dicarboxylic acid mean azelaate or azelaic acid.

[0016] In this application, the term "dicarboxylic acid" refers to a dibasic acid containing two carboxyl groups, and is of high importance as a metabolic intermediate in living organisms.

[0017] 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 means one aliphatic dicarboxylic acid selected from suberic acid and azelaic acid. When the cadaverine dicarboxylate to be produced by the production method of the present application is cadaverine suberate, it must be understood that all of the added or input dicarboxylic acid is suberic acid. Also, when the cadaverine dicarboxylate to be produced by the production method of the present application is cadaverine azelate, it must be understood that all of the added or input dicarboxylic acid is azelaic acid. Thus, it is understood that in the present application, the dicarboxylic acid refers to one kind of aliphatic dicarboxylic acid, not a mixture of different kinds of dicarboxylic acids.

[0018] In the present application, the term "Suberic acid" is a dicarboxylic acid having 8 carbon atoms, and has the chemical formula C8H 14 O4, and generally exists as a colorless crystalline solid. The suberic acid is also named octanedioic acid and is utilized as a precursor for various industrial products including polymers and plasticizers.

[0019] In the present application, the term "Azelaic acid" is a dicarboxylic acid having 9 carbon atoms, and has the chemical formula C9H 16 O4, and generally exists in the form of a white powder. The azelaic acid is also named azelaic acid and is utilized as a precursor for various industrial products including polymers and plasticizers.

[0020] In the present application, the term "Cadaverine (CAD)" is a toxic diamine compound that emits a foul odor, and its chemical formula is represented by NH2(CH2)5NH2. Cadaverine is also named 1,5-pentanediamine or pentamethylenediamine. In the present application, it should be understood that cadaverine includes the form of cadaverine dicarboxylate.

[0021] In this application, the term "Cadaverine Dicarboxylate" is a substance produced by reacting the diamine form of cadaverine with a dicarboxylic acid component, and it has a high industrial utilization rate as a precursor for the production of polyamide resins. Specifically, in this application, Cadaverine Dicarboxylate means one Cadaverine Dicarboxylate selected from Cadaverine suberate and Cadaverine azelate.

[0022] In this application, the term "Cadaverine suberate" is a substance produced by reacting the diamine form of cadaverine with suberic acid, which is a dicarboxylic acid component.

[0023] In this application, the term "Cadaverine azelate" is a substance produced by reacting the diamine form of cadaverine with azelaic acid, which is a dicarboxylic acid component.

[0024] Figures 1 and 2 are flowcharts showing the production process of Cadaverine Dicarboxylate according to one aspect of this application.

[0025] In this application, the term "L-lysine-producing microorganism" includes all wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modifications, such as insertion of foreign genes, or enhancement or inactivation of the activity of endogenous genes, resulting in weakened or enhanced specific mechanisms. It may also be a microorganism that includes genetic modifications for the production of L-lysine.

[0026] For example, the L-lysine-producing microorganisms of this application may be, but are not limited to, microorganisms that naturally possess L-lysine-producing ability, microorganisms that lack L-lysine-producing ability but have been conferred with L-lysine-producing ability, or microorganisms that have significantly low L-lysine-producing ability but have been enhanced with L-lysine-producing ability. Specifically, the L-lysine-producing microorganisms or microorganisms that possess L-lysine-producing ability in this application may be microorganisms in which a portion of the genes in the L-lysine biosynthesis pathway is strengthened or weakened, or a portion of the genes in the L-lysine degradation pathway is strengthened or weakened. "Strengthened" or "increased" L-lysine-producing ability means that the L-lysine-producing ability has improved compared to the parent strain or non-mutant microorganisms.

[0027] As a specific example, the microorganism may be a microorganism of the genus Corynebacterium or a microorganism of the genus Escherichia. Microorganisms of the genus Corynebacterium can 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 This may include *Corynebacterium ammoniagenes*, *Corynebacterium pollutisoli*, *Corynebacterium imitans*, *Corynebacterium testudinoris*, or *Corynebacterium flavescens*, and more specifically, *Corynebacterium glutamicum*, but is not limited thereto. Microorganisms of the genus *Escherichia* can include all microorganisms belonging to the genus *Escherichia*.Specifically, these may be Escherichia coli, Escherichia albertii, Escherichia blattae, Escherichia fergusonii, Escherichia hermannii, or Escherichia vulneris, and more specifically, the Escherichia strain may be Escherichia coli, but is not limited to these.

[0028] The culture of the L-lysine-producing microorganism can be carried out according to suitable culture media and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art depending on the selected microorganism. Specifically, the culture may be batch, continuous, or fed-batch, but is not limited thereto.

[0029] In this application, the term "medium" means a substance mixed mainly with nutrients necessary for culturing the microorganisms, supplying nutrients and growth factors, including water which is indispensable for survival and growth. Specifically, the medium used for culturing the L-lysine-producing microorganisms in this application can be any medium used for culturing ordinary microorganisms without particular limitations, except that it contains diammonium dicarboxylate as part or all of the nitrogen source. The microorganisms in this application can be cultured in an ordinary medium containing a nitrogen source including diammonium dicarboxylate, a suitable carbon source, a phosphorus source, inorganic compounds, amino acids and / or vitamins, under aerobic conditions while adjusting the temperature, pH, etc.

[0030] The first step of the method for producing cadaverine dicarboxylate according to this application is to culture L-lysine-producing microorganisms in a medium to which dicarboxylic acid has been added in the form of diammonium dicarboxylate to obtain lysine dicarboxylate. This is carried out through a fermentation process of the lysine-producing microorganisms.

[0031] In the first step, the dicarboxylic acid having eight or nine carbon atoms is supplied to the culture medium in the form of a diammonium dicarboxylate salt, so that it should be understood that in this application, the dicarboxylic acid includes the form of a diammonium dicarboxylate salt.

[0032] In this application, the term "diammonium suberate" refers to a form in which two hydrogen atoms of suberic acid are replaced by ammonium, and is used as the primary nitrogen source for the culture medium. The diammonium suberate may be used in crystalline or liquid form after being prepared by gently mixing suberic acid with aqueous ammonia to a neutral pH and then concentrating it.

[0033] In this application, the term "diammonium azelaite" refers to a form in which two hydrogen atoms of azelaic acid are replaced with ammonium atoms, and is used as the primary nitrogen source for the culture medium. The diammonium azelaite can be used in crystalline or liquid form after being prepared by gently mixing azelaic acid with aqueous ammonia to have a neutral pH and then concentrating it.

[0034] The manufacturing method described in this application simplifies the process while producing high-purity cadaverine dicarboxylate by adding 64 mol% or more of the dicarboxylic acid supplied throughout the entire process to the culture medium in the form of diammonium dicarboxylate in the first step, thereby reducing the side effect of dicarboxylate precipitation together with cadaverine dicarboxylate.

[0035] Specifically, the diammonium dicarboxylate salt in the first step may be included in the culture medium in a high proportion, specifically 64 mol% or more, relative to the dicarboxylic acid supplied throughout the entire process of the manufacturing method of this invention.

[0036] More specifically, the diammonium dicarboxylate in the first step may be present 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 all steps of the manufacturing method of this application.

[0037] The manufacturing method described in this application, by including a large amount of diammonium dicarboxylate in the culture medium, offers the advantage of being able to produce high-purity cadaverine dicarboxylate without performing separate steps to remove the dicarboxylate, such as ion resin exchange, decarboxylation, or distillation, unlike conventional processes that add ammonium sulfate or the like. Furthermore, by using diammonium dicarboxylate, it is possible to easily adjust the molar ratio of dicarboxylic acid added throughout the entire process to cadaverine in the process solution after the second step.

[0038] Specifically, the diammonium dicarboxylate in the first step may be included in such a molar ratio (diammonium dicarboxylate / lysine) with respect to the lysine to be produced that is 0.60 to 1. More specifically, the diammonium dicarboxylate in the first step may be included in such a molar ratio (diammonium dicarboxylate / lysine) with respect to the lysine to be produced that 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, more specifically, 0.79 to 0.99. In this case, the amount of lysine to be produced should be understood to refer to the total amount including all forms of lysine dicarboxylate.

[0039] For example, the diammonium dicarboxylate in the first step may be included in a molar ratio (diammonium dicarboxylate / lysine) within a range defined by one 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 one upper limit selected from 1, 0.99, 0.98, 0.97, 0.96, 0.95, 0.94, 0.93, and 0.92, with respect to the lysine to be produced.

[0040] In one specific example, the dicarboxylate and dicarboxylic acid are suberate and suberic acid, and the diammonium dicarboxylate in the first step may be included in such a way that its molar ratio (diammonium dicarboxylate / lysine) with the lysine to be produced is 0.81 to 1.0.

[0041] In one specific example, the dicarboxylate and dicarboxylic acid are azelaate and azelaic acid, and the diammonium dicarboxylate in the first step may be included in such a way that its molar ratio (diammonium dicarboxylate / lysine) with the lysine to be produced is 0.79 to 1.0.

[0042] Specifically, the diammonium dicarboxylate in the first step can be included in a concentration of 0.2 mol / L to 0.8 mol / L based on the L-lysine-producing microbial fermentation broth. More specifically, the diammonium dicarboxylate in the first step can be included in a concentration 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 L-lysine-producing microbial fermentation broth. The diammonium dicarboxylate can be added at the above concentrations so that the ammonium ions act as a nitrogen source during fermentation, and the dicarboxylic acid and lysine form a salt, which may be included in the fermentation broth in a dissolved state.

[0043] Lysine dicarboxylate can be produced by fermentation of L-lysine-producing microorganisms in a culture medium containing diammonium dicarboxylate. The lysine dicarboxylate can be added to subsequent processes in the form of a fermentation liquid with the microbial cells still present, or in the form of a fermentation liquid from which the microbial cells have been removed or after purification.

[0044] The concentration of lysine in the fermentation liquid obtained by culturing the lysine-producing microorganisms in the first step of this application 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 L-lysine-producing microorganism fermentation liquid.

[0045] The carbon sources in the culture 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. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn maceration can also be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and other appropriate amounts of various carbon sources can be used without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited to these uses.

[0046] As a specific example, the carbon source that may be included in the culture medium of this application may include glucose, maltose, or maltose isomers. More specifically, it may include, but is not limited to, one or more selected from glucose, maltose, and maltose isomers (isomaltose), or a combination of two or more.

[0047] The phosphorus source in the culture medium may include monopotassium phosphate, dipotassium phosphate, or corresponding sodium-containing salts. Inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate.

[0048] Furthermore, the culture medium may contain metal salts such as magnesium sulfate or iron sulfate necessary for growth. Finally, essential growth substances such as amino acids and vitamins can be used in addition to the aforementioned substances. Appropriate precursors can also be used in the culture medium. The raw materials may be added to the culture during the culture process in an appropriate manner, for example, in batches or continuously, but are not limited thereto.

[0049] The nitrogen source of the culture medium may include diammonium dicarboxylate. Conventional culture media for microbial culture may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; and organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn maceration, casein hydrolysates, fish or their degradation products, defatted soy cake or its degradation products. In one embodiment of this application, diammonium dicarboxylate may be included as a sole nitrogen source or as an additional nitrogen source.

[0050] After the first step of culturing L-lysine-producing microorganisms in a medium containing diammonium dicarboxylate to produce lysine dicarboxylate, a second step may be performed to convert the lysine dicarboxylate to cadaverine dicarboxylate.

[0051] The second step is to convert the lysine dicarboxylate to the target product, cadaverine dicarboxylate (cadaverine phosphate or cadaverine azelaate).

[0052] In one specific example, the second step may include further adding a dicarboxylic acid to the process liquid. The process liquid may be the fermentation process liquid obtained in the first step.

[0053] The dicarboxylic acid added in the second step may be omitted, or added in amounts 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 in trace amounts of 0.01 mol / L or less, based on the volume of the fermentation liquid obtained in the first step.

[0054] In one specific example, if the dicarboxylic acid is suberic acid, the dicarboxylic acid added in the second step may be omitted or added in a trace amount of 0.1 mol / L or less.

[0055] In one specific example, if the dicarboxylic acid is azelaic acid, the dicarboxylic acid added in the second step may be omitted or added in a trace amount of 0.11 mol / L or less.

[0056] If a dicarboxylic acid is further added to the process solution in the second step, the dicarboxylic acid may, but is not limited to, be added before, after, or simultaneously with the conversion reaction.

[0057] If dicarboxylic acid is further added in the second step, the total amount of dicarboxylic acid added throughout the entire process of the present invention is understood to be the sum of the amount of diammonium dicarboxylate added to the culture medium in the first step and the amount of dicarboxylic acid added in the second step. Also, if no further dicarboxylic acid is added to the process solution in the second step, the total amount of dicarboxylic acid added throughout the entire process of the present invention is understood to be equal to the amount of diammonium dicarboxylate added to the culture medium in the first step.

[0058] Specifically, the amount of dicarboxylic acid added in the manufacturing method of this application can be added considering the amount of cadaverine in the process solution obtained after the second step. Specifically, the amount of dicarboxylic acid added in the manufacturing method of this application may be in a molar ratio of 0.80 to 1.10 compared to the amount of cadaverine in the process solution after the second step.

[0059] More specifically, the amount of dicarboxylic acid added in the manufacturing method of this application may be in a molar ratio of 0.85-1.10, 0.87-1.06, 0.88-1.05, 0.89-1.01, 0.88-1.01, 0.89-1.0, 0.90-1.10, 0.91-1.00, 0.93-1.01, 0.93-1.01, 0.95-1.06, or 0.95-1.05 compared to the amount of cadaverine in the process solution after the second step.

[0060] As an example, the amount of dicarboxylic acid added in the manufacturing method of this application may be in a molar ratio within 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.

[0061] In one specific example, if the dicarboxylic acid is suberic acid, the amount of dicarboxylic acid added in the production method of this application may be in 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 compared to the amount of cadaverine in the process solution after the second step.

[0062] One technical significance of this invention lies in the discovery that, in the manufacturing method of this application, when suberic acid is added in a molar ratio within the aforementioned range relative to the amount of cadaverine calculated to be obtained after the second step, suberic acid does not precipitate as crystals together with cadaverine phosphate, making it possible to produce cadaverine phosphate in high purity and high yield.

[0063] Furthermore, by adjusting the molar ratio of the obtained cadaverine to the added suberic acid, it is possible to maintain a purity of 99% of cadaverine phosphate up to the third cycle during mother liquor circulation, while obtaining cadaverine phosphate in total yields 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 mother liquor cycles.

[0064] In one specific example, if the dicarboxylic acid is azelaic acid, the amount of dicarboxylic acid added in the production method of this application may be in 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 compared to the amount of cadaverine in the process solution after the second step.

[0065] One technical significance of this invention lies in the discovery that, in the manufacturing method of this application, when azelaic acid is added in a molar ratio within the aforementioned range relative to the amount of cadaverine calculated to be obtained after the second step, the azelaic acid does not precipitate as crystals together with cadaverine azelaate, and that it is possible to produce cadaverine azelaate with high purity and high yield.

[0066] Furthermore, by adjusting the molar ratio of the obtained cadaverine to the added azelaic acid, it is possible to maintain a purity of 99% of cadaverine azelaate up to the third cycle of mother liquor circulation, while obtaining cadaverine azelaate with 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 mother liquor cycles.

[0067] In the second step, the reaction to convert the lysine dicarboxylate to cadaverine dicarboxylate may be an enzymatic conversion reaction.

[0068] As one specific example, the lysine dicarboxylate salt may include the lysine dicarboxylate salt obtained in the first step, and, if a dicarboxylic acid is further added in the second step, all of the lysine dicarboxylate salt produced in the second step.

[0069] Specifically, the enzymatic conversion reaction in the second step may be carried out using a protein having lysine decarboxylase activity or a microorganism expressing such a protein.

[0070] As one specific example, a protein having lysine decarboxylase activity can be added to the fermentation liquid in the first step, or a seed culture of a microorganism expressing the said active protein can be added, but the method is not limited to this.

[0071] In this application, the term "decarboxylase" refers to an enzyme that catalyzes the formation of carbon dioxide by removing the carboxyl group, which is an organic acid, and is also known as a decarboxylase, decarboxylase, or carbonic acid release enzyme.

[0072] The aforementioned protein is not particularly limited as long as it exhibits lysine decarboxylase activity, but for example, it may be Pseudomonas thermotolerans PtLDC protein or CadA protein derived from Escherichia coli. However, it is not limited to obtaining the protein sequence using the known database GenBank, expressing it using the aforementioned microorganism, or purchasing and using commercially available enzymes.

[0073] The microorganism expressing the protein may be a microorganism that has been transformed to express the protein. The transformed microorganism is not limited to prokaryotic or eukaryotic microorganisms, as long as it has been transformed to express a protein having decarboxylase activity. The microorganism transformed to express a protein having decarboxylase activity can excrete the protein having the enzyme activity into the seed culture medium and convert the lysine dicarboxylate in the process solution into cadaverine dicarboxylate.

[0074] Specific examples may include strains of microorganisms belonging to the genera Escherichia, Erwinia, Serratia, Providencia, and Corynebacterium. Specifically, these microorganisms may be microorganisms belonging to the genera Escherichia or Corynebacterium, and more specifically, they may be Escherichia coli or Corynebacterium glutamicum, but are not limited to these.

[0075] The conversion reaction in the second step may take place for 20 minutes to 3 hours, more specifically for 0.5 to 1.5 hours, or for example, for 1 hour, but is not limited to these durations.

[0076] The conversion reaction in the second step may be carried out at 30-60°C, more specifically at 40-50°C, or, for example, at 45°C, but is not limited thereto.

[0077] The conversion reaction in the second step may be carried out at a pH of 7.5 to 9, more specifically at 7.8 to 8.7, or more specifically at 8 to 8.5. In one embodiment of the second step, the pH can be adjusted to the above range by selectively adding dicarboxylic acid, and in another embodiment, the pH can be adjusted to the above range by adding CO2. When the pH range of the conversion reaction in the second step is adjusted to the above range, the conversion rate of lysine dicarboxylate to cadaverine dicarboxylate can be significantly increased.

[0078] The method for producing cadavering dicarboxylate according to this application may further include a recovery step of recovering the obtained cadavering dicarboxylate after carrying out the second step.

[0079] The recovery may involve collecting the cadavering dicarboxylate using appropriate methods known in the art.

[0080] For example, cadaverine dicarboxylates can be collected by various chromatography methods such as centrifugation, filtration, concentration, crystallization, extraction, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination of these methods.

[0081] In one specific example of this application, the recovery step may include one or more of the following steps: a filtration step, a concentration step, and a crystallization step.

[0082] The filtration step refers to the step of removing impurities from the conversion solution containing cadavering dicarboxylate obtained in the second step. As an example, the filtration may include removing microbial cells in the cadavering dicarboxylate process solution by membrane separation or by activated carbon filtration.

[0083] The aforementioned concentration step refers to a step in which the solid content ratio in the conversion solution is increased, and may be performed after the filtration step. For example, the concentration may be performed by vacuum concentration using a rotary evaporator.

[0084] Specifically, the concentration step may involve concentrating the solids content to 40-75% (w / w), more specifically, to 40-75% (w / w), 45-70% (w / w), 42-50% (w / w), 50-65% (w / w), 40-50% (w / w), 55-75% (w / w), or 60-70% (w / w), and as an example, to 45% (w / w) or 65% (w / w). When the solids content is concentrated within the above ranges in the concentration step, the recovery rate and purity of the cadavering dicarboxylate can be well maintained.

[0085] The concentration step may be carried out at 45-75°C, but is not limited thereto.

[0086] The crystallization step may also be a cooling crystallization step, which means cooling the conversion solution to precipitate it as crystals and obtain crystals of the final target product, cadaverine dicarboxylate.

[0087] Specifically, the cooling may involve cooling the conversion solution to 20-30°C, more specifically to 20-27°C, and, as an example, to 25°C or 20°C, but is not limited thereto.

[0088] Specifically, the cooling rate may be 3 to 20°C / hour, or more specifically, 3 to 15°C / hour, and for example, it may be 10°C / hour or 3°C / hour, but is not limited to these.

[0089] In one embodiment, the recovery step may further include a stirring step of stirring the process liquid. The stirring step may be performed simultaneously with the cooling crystallization step, in the middle of the cooling crystallization step, before the cooling crystallization step, or after the cooling crystallization step. Such a stirring step allows sufficient time to stir the process liquid, making crystal precipitation easier.

[0090] In one embodiment, the recovery step may further include a mother liquor circulation step in which the mother liquor containing the uncrystallized cadavering dicarboxylate after the crystallization step is recirculated with the feed liquid of the concentration step.

[0091] Specifically, in the mother liquor circulation step, the number of times the mother liquor is circulated 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 0 means that the mother liquor circulation step is not performed.

[0092] Conventionally, in the step of crystallizing cadavering dicarboxylate, a relatively large amount of cadavering dicarboxylate remains in the mother liquor after crystallization, resulting in a low yield of less than 50%. In this application, as a method to improve the yield of cadavering dicarboxylate, the total yield of cadavering dicarboxylate can be increased by recirculating the residual mother liquor of cadavering dicarboxylate one to three times, one to four times, one to five times, one to ten times or more in the feed solution of the concentration step.

[0093] In one embodiment, the recovery step may further include washing, separating, and drying the crystallized process liquid.

[0094] In the drying step described above, moisture contained in the crystals is removed through drying, and a highly pure cadavering dicarboxylate can be produced as a product. After drying, the cadavering dicarboxylate crystals can be provided in powder form, but the dosage form can be varied as needed.

[0095] In one embodiment, the recovery step may further include a decolorization step. The decolorization can be performed using activated carbon, anionic resin, etc., but is not limited thereto.

[0096] Furthermore, the method for producing cadavering dicarboxylate according to this application may include an additional purification step. The purification can be carried out using a suitable method known in the art. For example, if the method for producing cadavering dicarboxylate according to this application includes both a recovery step and a purification step, the recovery step and the purification step may be carried out sequentially or discontinuously, simultaneously or in a single step, regardless of the procedure, but are not limited thereto.

[0097] The manufacturing method of this application is characterized by not including a separate step for removing the dicarboxylate in the recovery step. Conventionally, in the production process of cadaverine dicarboxylate, the dicarboxylate precipitates as crystals together with the crystal during the crystallization process, reducing purity. To prevent this, a separate step was required to remove the dicarboxylate before crystallization. However, the manufacturing method of this application offers the technical advantage of being able to produce high-purity cadaverine dicarboxylate without performing a separate step for removing the dicarboxylate.

[0098] In one embodiment, when the dicarboxylic acid is suberic acid, the method of this application is characterized by obtaining cadaverin verphosphate with a purity of 99% or more in 0 to 3 cycles of mother liquor. In one embodiment, when the dicarboxylic acid is suberic acid, the cumulative total yield of cadaverin verphosphate may be 65% or more based on 3 cycles of mother liquor, and more specifically, it may be 66%, 67%, 68%, 69%, 70%, or higher.

[0099] In one embodiment, when the dicarboxylic acid is azelaic acid, the method of this application is characterized by obtaining cadaverine azelaate with a purity of 99% or more in 0 to 3 cycles of the mother liquor. In one embodiment, when the dicarboxylic acid is azelaic acid, the cumulative total yield of cadaverine azelaate may be 25% or more, based on 3 cycles of the mother liquor, and more specifically, it may be 26%, 27%, or higher.

[0100] Through the method of this application, high-purity cadaverine dicarboxylate can be produced without the decarboxylation and distillation steps that are normally required in the production of cadaverine liquid phase.

[0101] Examples The present application will be described in more detail below with reference to experimental examples. However, the following embodiments are merely preferred embodiments for illustrative purposes of the present application and are not intended to limit the scope of the rights of this application. On the other hand, technical matters not described herein can be fully understood and easily implemented by a person of ordinary skill who is skilled in the art of this application or a similar art.

[0102] The above describes a manufacturing process for cadaverine dicarboxylate (cadaverine verophosphate or cadaverine azelaate) according to one aspect of this application. Below, the advantageous effects referred to in this application will be described through experimental results of examples and comparative examples of cadaverine verophosphate and cadaverine azelaate, respectively.

[0103] 1. Cadaverin verate Production Example 1-1. Fermentation process for lysine suberate Corynebacterium glutamicum strain (KCCM12154P, U.S. Public Registry No. US 2021-0355514 A1) capable of lysine production was obtained through solid-phase culture and flask culture, and after seed culture in a fermenter, the main production fermentation was carried out. Fermentation was performed at 36°C for 63 hours at 900 rpm via fermenter culture.

[0104] For the production of lysine suberate using Corynebacterium, ammonium suberate was supplied at levels of 67 to 110 g / L in each example to replace the existing ammonium sulfate, and fermentation was carried out. As a result, lysine suberate was obtained at levels of 103 to 170 g / L, and lysine at a level of 79 g / L. At that time, the molar ratio of suberic acid / lysine in the fermentation liquid was adjusted to levels of 0.59 to 0.98 in each example.

[0105] Manufacturing Example 1-2. Conversion reaction of lysine suberate to cadaverin suberate. For the conversion reaction, we used Escherichia coli (US 2018-0030430 A1) in which the PtLDC enzyme, a lysine decarboxylase gene derived from Pseudomonas thermotolerans, was overexpressed using the pET-Deut1 vector.

[0106] The fermentation liquid of lysine suberate produced from Production Example 1-1 was converted using an enzyme-converted solution cultured with the enzyme strain, at a concentration of 10% by mass. The conversion reaction was carried out by supplying this solution to the fermentation liquid of lysine suberate at a concentration of 10% by mass. The conversion reaction temperature was maintained at 45-50°C for approximately 1 hour. The pH was adjusted to 8.0-8.5. For this reason, in Examples 1-1 to 1-5, CO2 was added to neutralize the pH, while in Examples 1-2 to 1-4, suberic acid was added at a concentration of 0.02-0.03 mol / L (based on the volume of the fermentation liquid of lysine suberate) before the conversion reaction. As a result, it was confirmed that the conversion rate of cadaverine suberate was 97-98%. Specifically, after the conversion reaction, cadaverin veraphosphate was obtained at a level of 88-146 g / L (based on the volume of the fermentation liquid of lysine suberaphosphate), and cadaverine was obtained at a level of 54 g / L (based on the volume of the fermentation liquid of lysine succinate). Subsequently, in Examples 1-6 and 1-7, suberic acid was added at a level of 0.05-0.1 mol / L (based on the volume of the fermentation liquid of lysine suberaphosphate).

[0107] Manufacturing Example 1-3. Subsequent steps after obtaining cadaverin verphosphate The following subsequent steps were performed on the process solution containing cadaverin verphosphate obtained in Production Example 1-2 to obtain crystalline cadaverin verphosphate.

[0108] [Bacterial cell removal step] Bacterial cells in the cadaverin phosphate process solution were removed by membrane filtration using a 0.1 μm membrane.

[0109] [Steps to remove impurities using activated carbon] Activated carbon was added to the filtrate after bacterial cell isolation at a level of 10% based on the weight of cadaverins perphosphate. The process solution with the added activated carbon was heated to 60°C and stirred for 1 hour to decolorize it, after which the activated carbon was filtered out through filter paper.

[0110] [Step to concentrate the process solution of cadaverin verphosphate from which impurities have been removed] The filtered liquid was concentrated in a rotary evaporator under reduced pressure at approximately 60°C and 120 Torr until the solid content of the filtrate was 65% by weight.

[0111] [Steps to cool, crystallize, separate, and dry a concentrated solution of cadaverin verphosphate] The 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 10% water based on the weight of cadaverine verate. After drying the separated crystals for one day, their purity was measured by HPLC.

[0112] [Step of recirculating the mother liquor in the crystallization step] The mother liquor separated as described above was recycled in a step to concentrate the process solution of cadaverine verphosphate from which impurities had been removed.

[0113] Experimental Example 1-1. Confirmation of changes in the purity and yield of cadaverin verate according to the molar ratio of suberate to cadaverine in the conversion solution for cadaverin verate. In Experimental Example 1-1, the methods described in Production Examples 1-1 to 1-3 were used, and the purity and yield of cadaverin verphosphate were examined to see how they changed depending on the molar ratio of suberate to cadaverine in the conversion solution.

[0114] Example 1-1: When the molar ratio of suberic acid added to the process solution after the second step is 0.6 Microorganisms were removed by passing 1,000 ml of a cadaverin-verphosphate conversion solution with a suberic acid / cadaverine molar ratio of 0.6 through a 0.1 μm-sized membrane. The cadaverin-verphosphate conversion solution with a suberic acid / cadaverine molar ratio of 0.61 was produced according to the production methods of Production Example 1-1 and Production Example 1-2, but the sum of the amount of suberate added in the ammonium suberate in the fermentation step for producing lysine in Production Example 1-1 and the amount of suberate added in the cadaverin-verphosphate conversion reaction in Production Example 1-2 was controlled so that it equaled 0.6 moles of converted cadaverine.

[0115] The filtrate was decolorized using activated carbon, filtered through filter paper, and then concentrated under reduced pressure to a solid content of 65%. The concentrate was cooled and crystallized from 50°C to 25°C. The crystals and mother liquor were separated by centrifugation, and the separated crystals were dried for one day, after which their purity was measured by HPLC. The mother liquor, which still contained suberate, was recirculated with a feed solution of cadaverin-suberate with a suberic acid / cadaverine molar ratio of 0.6, and the crystallization step was repeated. The recirculation of the mother liquor was carried out sequentially.

[0116] Examples 1-2: When the molar ratio of suberic acid added to the process solution after the second step is 0.70 Upon receiving a conversion solution of cadaverine veraphosphate with a suberic acid / cadaverine molar ratio of 0.70, the mother liquor was sequentially recycled and the purification process proceeded as in Example 1-1.

[0117] Examples 1-3: When the molar ratio of suberic acid added to the process solution after the second step is 0.79 Upon receiving a conversion solution of cadaverine veraphosphate with a suberic acid / cadaverine molar ratio of 0.79, the mother liquor was sequentially recycled and the purification process proceeded, as in Example 1-1.

[0118] Examples 1-4: When the molar ratio of added suberic acid / cadaverine is 0.91 Upon receiving a conversion solution of cadaverine veraphosphate with a suberic acid / cadaverine molar ratio of 0.91, the mother liquor was sequentially recycled and the purification process proceeded as in Example 1-1.

[0119] Examples 1-5: When the molar ratio of added suberic acid / cadaverine is 1.00 Upon receiving a conversion solution of cadaverine veraphosphate with a suberic acid / cadaverine molar ratio of 1.00, the mother liquor was sequentially recycled and the purification process proceeded as in Example 1-1.

[0120] In Examples 1-4 to 1-5, the molar ratio of suberic acid to lysine in the lysine fermentation step was adjusted to 0.83 to 0.98.

[0121] Examples 1-6: When the molar ratio of added suberic acid / cadaverine is 1.10 Upon receiving a conversion solution of cadaverine veraphosphate with a suberic acid / cadaverine molar ratio of 1.10, the mother liquor was sequentially recycled and the purification process proceeded, as in Example 1-1.

[0122] Examples 1-7: When the molar ratio of added suberic acid / cadaverine is 1.19 Upon receiving a conversion solution of cadaverine veraphosphate with a suberic acid / cadaverine molar ratio of 1.19, the mother liquor was sequentially recycled and the purification process proceeded, as in Example 1-1.

[0123] The physical properties measured for the process solutions and cadaverines verphosphate produced by the above-described Examples 1-1 to 1-7 are shown in Tables 1 to 4.

[0124] Table 1 relates to process fluids that do not circulate the mother liquor.

[0125] [Table 1]

[0126] Table 2 shows the process fluid after the mother liquor has been circulated once.

[0127] [Table 2]

[0128] Table 3 shows the process fluid after the mother liquor has been circulated twice.

[0129] [Table 3]

[0130] Table 4 shows the process fluid after the mother liquor has been circulated three times.

[0131] [Table 4]

[0132] Based on the experimental results shown in Tables 1 to 4, it was confirmed that the manufacturing method of this invention, which incorporates a large amount of diammonium suberate from the first step at a level of 84-100 mol% of the total supplied suberic acid, enables the production of cadaverin suberate with generally improved crystallinity, high purity, and high yield.

[0133] More specifically, in Example 1-1, no crystals precipitated even after the crystallization process was carried out. This is because the molar ratio of suberate was low and insufficient for cadaverin suberate crystals to precipitate.

[0134] In Examples 1-2, crystals precipitated during the first mother liquor circulation, but no crystals were formed during the second circulation. This is because, after the first circulation of the mother liquor, the molar ratio of suberate in the crystallization feed solution became too low to adequately precipitate cadaverin suberate crystals.

[0135] In Examples 1-3, crystals precipitated up to the second mother liquor circulation, but no crystals were formed during the third circulation. This is because the sequentially circulated mother liquor lowered the molar ratio of suberate in the crystallization supply solution, resulting in a suberate concentration that was insufficient to precipitate cadaverin suberate crystals.

[0136] In Examples 1-6, the purity remained slightly lower at 85% even during the third mother liquor circulation. This is because, as the mother liquor circulated sequentially, suberic acid accumulated in the crystallization feed solution precipitated as an impurity.

[0137] In Examples 1-7, the purity decreased slightly to 74% even during the third mother liquor circulation. This is because, as the mother liquor circulated sequentially, suberic acid accumulated in the crystallization feed solution precipitated as an impurity.

[0138] On the other hand, in Examples 1-4, high-purity cadaverinsverate with a purity of 99% was obtained up to the third mother liquor circulation, and a high overall yield of approximately 65% ​​was achieved.

[0139] Examples 1-5 yielded cadaverin verphosphate with a purity of 99% up to the third mother liquor circulation, achieving a high overall yield of approximately 70%.

[0140] Thus, we have discovered that a method for producing high-purity cadaverin phosphate can be provided depending on the molar ratio of suberic acid added throughout the entire process to the amount of cadaverine in the process solution after the second step. In particular, we confirmed that when the amount of suberic acid added is adjusted to a molar ratio of approximately 0.91 to 1.00 compared to the amount of cadaverine in the process solution after the second step, it is possible to maintain a purity of 99% of cadaverin phosphate while achieving an excellent total yield of 65% or more, even after three or more mother liquor cycles.

[0141] 2. Cadaverine azelaine Production Example 2-1. Fermentation process for lysine azelaite Corynebacterium glutamicum strain (KCCM12154P, U.S. Patent Publication No. US 2021-0355514 A1) capable of lysine production was obtained through solid-phase culture and flask culture, and after seed culture in a fermenter, the main production fermentation was carried out. Fermentation was performed at 36°C for 30 hours at 900 rpm through fermenter culture.

[0142] For the production of lysine azelaate using Corynebacterium, ammonium azelaate was supplied at levels of 67 to 107 g / L in each example to replace the existing ammonium sulfate, and fermentation was carried out. As a result, lysine azelaate was obtained at levels of 100 to 161 g / L, and lysine at a level of 73 g / L. At that time, the molar ratio of azelaic acid / lysine in the fermentation liquid was adjusted to levels of 0.6 to 0.96 in each example.

[0143] Manufacturing Example 2-2. Conversion reaction of lysine azelaate to cadaverine azelaate For the conversion reaction, we used Escherichia coli (US 2018-0030430 A1) in which the PtLDC enzyme, a lysine decarboxylase gene derived from Pseudomonas thermotolerans, was overexpressed using the pET-Deut1 vector.

[0144] The enzyme conversion solution, obtained by culturing the enzyme strain in the fermentation process liquid of lysine azelaate produced from the above-mentioned Production Example 2-1, was supplied to the process liquid at a level of 10% by mass percentage to carry out the conversion reaction. The conversion reaction temperature was maintained at 45°C for approximately 1 hour. The pH was adjusted to 8.0-8.5. Therefore, in Examples 2-1 to 2-5 below, CO2 was added to neutralize the pH, and in Examples 2-3 to 2-5, azelaic acid was added at a level of 0.01-0.04 mol / L (based on the volume of the fermentation process liquid of lysine azelaate). As a result, it was confirmed that the conversion rate of cadaverine azelaate was at a level of 97-98%. Specifically, after the conversion reaction, cadaverin veraphosphate was obtained at a level of 87-142 g / L (based on the volume of the fermentation liquid of lysine suberaphosphate), and cadaverine was obtained at a level of 50 g / L (based on the volume of the fermentation liquid of lysine succinate). Subsequently, in Examples 2-6 and 2-7, azelaic acid was added at a level of 0.06-0.11 mol / L (based on the volume of the fermentation liquid of lysine azelaate) after the conversion reaction.

[0145] Manufacturing Example 2-3. Subsequent steps after obtaining cadaverine azelaate The following subsequent steps were performed on the process solution containing cadaverine azelaate obtained in Production Example 2-2 to obtain cadaverine azelaate in crystalline form.

[0146] [Bacterial cell removal step] Bacterial cells in the cadaverine azelaate process solution were removed by membrane filtration using a 0.1 μm membrane.

[0147] [Steps to remove impurities using activated carbon] Activated carbon was added to the filtrate after bacterial cell isolation at a level of 10% based on the weight of cadaverine azelaate. The process solution with the added activated carbon was heated to 60°C and stirred for 1 hour to decolorize it, after which the activated carbon was filtered through filter paper.

[0148] [Step to concentrate the process solution of cadaverine azelaate from which impurities have been removed] The filtered liquid was concentrated in a rotary evaporator under reduced pressure at approximately 55-70°C and 120 Torr until the solid content of the filtrate was 45% by weight.

[0149] [Steps to cool, crystallize, separate, and dry the concentrated cadaverine azelaate solution] The concentrate was cooled from 60°C to 25°C at a rate of 10°C / hr. It was then cooled to 25°C and then to 20°C at a rate of 3°C / hr. Afterward, it was stirred at 20°C for 24 hours. The precipitated crystals were separated from the mother liquor using a centrifuge. The crystals were washed with 10% water based on the weight of cadaverine azelaate. The separated crystals were dried at low temperature (20°C) for one to two days, and their purity was measured by HPLC.

[0150] [Step of recirculating the mother liquor in the crystallization step] The mother liquor separated above was recycled in the step of [concentrating the process solution of cadaverine azelaate from which impurities have been removed].

[0151] Experimental Example 2-1. Confirmation of changes in the purity and yield of cadaverine azelaate according to the molar ratio of azelaate to cadaverine in the conversion solution for cadaverine azelaate. In Experimental Example 2-1, the methods described in Production Examples 2-1 to 2-3 were used, and the purity and yield of cadaverine azelaate were examined to see how they changed depending on the molar ratio of azelaate to cadaverine in the conversion solution.

[0152] Example 2-1: When the molar ratio of azelaic acid / cadaverine added is 0.61 Microorganisms were removed by passing 1,000 ml of a cadaverine azelaate conversion solution, in which the azelaate / cadaverine molar ratio was 0.61, through a 0.1 μm-sized membrane. The cadaverine azelaate conversion solution, in which the azelaate / cadaverine molar ratio was 0.6, was produced according to the production methods of Production Example 2-1 and Production Example 2-2, but the sum of the amount of azelaate added in the ammonium azelaate introduced in the fermentation step for producing lysine in Production Example 2-1 and the amount of azelaic acid salt introduced in the cadaverine azelaate conversion reaction in Production Example 2-2 was controlled so that it equaled 0.61 of the number of moles of converted cadaverine.

[0153] The filtrate was decolorized using activated carbon, filtered through filter paper, and then concentrated under reduced pressure to a solid content of 45%. The concentrate was cooled from 60°C to 25°C, 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 or more days, and their purity was measured by HPLC. The mother liquor, which still contained azelaate, was recirculated with a feed solution of cadaverine azelaate with an azelaate / cadaverine molar ratio of 0.61, and the crystallization step was repeated. The recirculation of the mother liquor was carried out sequentially.

[0154] Example 2-2: When the molar ratio of azelaic acid / cadaverine added is 0.71 Following the supply of a conversion solution of cadaverine azelaate, in which the molar ratio of azelaic acid to cadaverine was 0.71, the mother liquor was sequentially recycled and the purification process proceeded as in Example 2-1.

[0155] Example 2-3: When the molar ratio of azelaic acid / cadaverine added is 0.8 Following the supply of a conversion solution of cadaverine azelaate, in which the molar ratio of azelaic acid to cadaverine was 0.8, the mother liquor was sequentially recycled and the purification process proceeded as in Example 2-1.

[0156] Example 2-4: When the molar ratio of azelaic acid / cadaverine added is 0.9 Following the supply of a conversion solution of cadaverine azelaate, in which the molar ratio of azelaic acid to cadaverine was 0.9, the mother liquor was sequentially recycled and the purification process proceeded as in Example 2-1.

[0157] Example 2-5: When the molar ratio of azelaic acid / cadaverine added is 1.0 As in Example 2-1, the mother liquor was sequentially recycled after receiving a conversion solution of cadaverine azelaate with an azelaic acid / cadaverine molar ratio of 1.0, and the purification process proceeded. In Examples 2-4 and 2-5, the azelaic acid / lysine molar ratio in the lysine fermentation step was adjusted to 0.80 to 0.96.

[0158] Example 2-6: When the molar ratio of azelaate / cadaverine added is 1.1 Following the supply of a conversion solution of cadaverine azelaate, in which the molar ratio of azelaic acid to cadaverine was 1.1, the mother liquor was sequentially recycled and the purification process proceeded as in Example 2-1.

[0159] Example 2-7: When the molar ratio of azelaic acid / cadaverine added is 1.2 Upon receiving a conversion solution of cadaverine azelaate, in which the molar ratio of azelaic acid to cadaverine was 1.2, the mother liquor was sequentially recycled and the purification process proceeded as in Example 2-1.

[0160] The physical properties measured for the process solutions and cadaverine azelaate produced by the above-described Examples 2-1 to 2-7 are shown in Tables 5 to 8.

[0161] Table 5 pertains to process fluids that do not circulate the mother liquor.

[0162] [Table 5]

[0163] Table 6 shows the process fluid after the mother liquor has been circulated once.

[0164] [Table 6]

[0165] Table 7 relates to the process fluid after the mother liquor has been circulated twice.

[0166] [Table 7]

[0167] Table 8 relates to the process fluid after the mother liquor has been circulated three times.

[0168] [Table 8]

[0169] Based on the experimental results shown in Tables 5 to 8, it was confirmed that the manufacturing method of this invention, which includes a large amount of diammonium azelaate from the first step at a level of 81 to 100 mol% of the total supplied azelaic acid, enables the production of cadaverine azelaate with generally improved crystallization rate, high purity, and high yield.

[0170] More specifically, in Example 2-1, no crystals precipitated even after the crystallization process was carried out. This is because the molar ratio of azelaate was low and insufficient for cadaverine azelaate crystals to precipitate.

[0171] In Example 2-2, crystals precipitated during the first mother liquor circulation, but no crystals were formed during the second circulation. This is because, after the first circulation of the mother liquor, the molar ratio of azelaate in the crystallization feed solution became too low to adequately precipitate cadaverine azelaate crystals.

[0172] In Examples 2-3, crystals precipitated during the first two mother liquor cycles, but no crystals were formed during the third cycle. This was because the sequentially circulated mother liquor lowered the molar ratio of azelaate in the crystallization supply solution, resulting in an insufficient concentration of azelaate for the precipitation of cadaverine azelaate crystals.

[0173] In Example 2-6, crystals precipitated up to the second mother liquor circulation, but the purity of the crystals decreased slightly to 86%. This is because azelaic acid accumulated in the crystallization supply solution as the mother liquor circulated sequentially, and the azelaic acid precipitated as an impurity.

[0174] On the other hand, in Examples 2-4, high-purity cadaverine azelaate with a purity of 99% was obtained up to the third mother liquor circulation, achieving a high total yield of approximately 27.1%.

[0175] Examples 2-5 yielded cadaverine azelaate with 99% purity up to the third mother liquor circulation, achieving a high overall yield of approximately 26.8%.

[0176] Thus, we have discovered that a method for producing high-purity cadaverine azelaate can be provided depending on the molar ratio of azelaic acid added throughout the entire process compared to the amount of cadaverine in the process solution after the second step.

[0177] In particular, when the amount of azelaic acid added is adjusted to a molar ratio of approximately 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 achieved while maintaining a purity of 99% of cadaverine azelaate even after three or more mother liquor cycles.

[0178] From the above description, a person skilled in the art to which this application pertains will understand that this application can be implemented in other specific forms without altering its technical idea or essential features. In this regard, it should be understood that the embodiments described above are merely illustrative and not limiting. The scope of this application should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, as described below, and their equivalent concepts, rather than from the above detailed description.

Claims

1. The first step is to culture L-lysine-producing microorganisms in a medium to which dicarboxylic acid has been added in the form of diammonium dicarboxylate to obtain lysine dicarboxylate; and A method for producing cadavering dicarboxylate, comprising a second step of converting lysine dicarboxylate to cadavering dicarboxylate, The amount of dicarboxylic acid added is in a molar ratio of 0.85 to 1.05 compared to the amount of cadaverine in the process solution after the second step. A method for producing a cadaverine dicarboxylate, wherein the dicarboxylic acid is an aliphatic dicarboxylic acid having eight or nine carbon atoms.

2. The method for producing a cadaverine dicarboxylate according to claim 1, wherein the diammonium dicarboxylate of the first step is present in an amount of 81 mol% or more of the total supplied dicarboxylic acid.

3. The method for producing cadaverine dicarboxylate according to claim 1, wherein the diammonium dicarboxylate in the first step is included in such a way that the molar ratio (diammonium dicarboxylate / lysine) with the lysine to be produced is 0.79 to 0.

99.

4. The dicarboxylate salt and dicarboxylic acid are suberate and suberic acid, The method for producing cadaverine dicarboxylate according to claim 1, wherein the diammonium dicarboxylate in the first step is included in such a way that its molar ratio (diammonium dicarboxylate / lysine) with the lysine to be produced is 0.81 to 1.

0.

5. The dicarboxylate salt and dicarboxylic acid are azelaic acid and azelaic acid, The method for producing cadaverine dicarboxylate according to claim 1, wherein the diammonium dicarboxylate in the first step is included in such a way that its molar ratio (diammonium dicarboxylate / lysine) with the lysine to be produced is 0.79 to 1.

0.

6. The method for producing a cadavering dicarboxylate according to claim 1, wherein the conversion reaction in the second step is carried out at a pH of 8.0 to 8.

5.

7. The dicarboxylate salt and dicarboxylic acid are suberate and suberic acid, The method for producing cadaverine dicarboxylate according to claim 1, wherein the amount of dicarboxylic acid added is in a molar ratio of 0.91 to 1.0 compared to the amount of cadaverine in the process solution after the second step.

8. The dicarboxylate salt and dicarboxylic acid are azelaic acid and azelaic acid, The method for producing cadaverine dicarboxylate according to claim 1, wherein the amount of dicarboxylic acid added is in a molar ratio of 0.90 to 1.0 compared to the amount of cadaverine in the process solution after the second step.

9. The method for producing a cadaverine dicarboxylate according to claim 1, wherein the second step further comprises adding a dicarboxylic acid to the process solution.

10. The method for producing a cadavering dicarboxylate according to claim 1, further comprising a recovery step of recovering the cadavering dicarboxylate after performing the second step.

11. The method for producing a cadavering dicarboxylate according to claim 10, 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. The aforementioned recovery step is, The method for producing a cadavering dicarboxylate according to claim 10, further comprising a mother liquor circulation step of recirculating the mother liquor containing the uncrystallized cadavering dicarboxylate after the cooling crystallization step with the feed liquid of the concentration step.

13. The method for producing a cadavering dicarboxylate according to claim 11, wherein the concentration step is to concentrate the salt so that the solid content is 40 to 75% (w / w).

14. The method for producing a cadavering dicarboxylate according to claim 1, wherein in the second step, the conversion of the lysine dicarboxylate to a cadavering dicarboxylate is carried out using a protein having lysine decarboxylase activity or a microorganism expressing such a protein.

15. The method for producing cadavering dicarboxylate according to claim 1, wherein the L-lysine-producing microorganism is Corynebacterium glutamicum.

16. The method for producing cadaverine dicarboxylate according to claim 1, characterized in that the recovery step does not include a separate step for removing the dicarboxylate from the process liquid.

17. The method for producing a cadavering dicarboxylate according to claim 11, wherein a cadavering dicarboxylate with a purity of 99% or more can be obtained with 0 to 3 circulations of the mother liquor.

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