Process for preparing cadaverine dicarboxylic acid salt

Abstract

The present application relates to a method for preparing cadaverine dicarboxylate (cadaverine sebacate, cadaverine undecanedioate, or cadaverine dodecanedioate), 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, high-purity cadaverine dicarboxylate (cadaverine sebacate, cadaverine undecanedioate, or cadaverine dodecanedioate) can be obtained without ion resin exchange, decarbonization, and distillation 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 sebacate, cadaverine undecanedioic acid salt, or cadaverine dodecanedioic acid salt) using a microbial fermentation and purification process.

[0002] Cadaverine is a toxic, foul-smelling diamine compound produced by the decomposition of animal tissue. Unlike hexanediamine, cadaverine exists in a liquid state at room temperature and readily absorbs carbon dioxide, an acidic gas in the air, making transportation and storage difficult. Therefore, salt-forming crystallization not only yields a high-quality monomer but also facilitates transportation and storage.

[0003] Meanwhile, cadaverine sebacate, cadaverine undecanedioic acid salt, and cadaverine dodecanedioic acid salt are polymerization precursors of bio-polyamide, which is one of the engineering plastics widely used in automobiles, electrical and electronic parts, etc.

[0004] The process for preparing cadaverine dicarboxylate generally includes the following steps:

[0005] 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.

[0006] Formation of cadaverine dicarboxylate: The formation of cadaverine dicarboxylate involves the reaction between cadaverine and a dicarboxylic acid in the presence of a suitable catalyst. 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 dicarboxylate: Once the reaction is complete, the raw material, cadaverine dicarboxylate, is purified using various separation and purification techniques such as solvent extraction, chromatography, and crystallization.

[0008] According to the prior art, since sebacate, undecanedioic acid salt, and dodecanedioic acid salt have low solubility, there was a problem that the dicarboxylate salt (sebacate, undecanedioic acid salt, or dodecanedioic acid salt) was precipitated into crystals together with the cadaverine dicarboxylate salt (cadaverine sebacate, cadaverine undecanedioic acid salt, or cadaverine dodecanedioic acid salt) during the crystallization process in the above-described process. Therefore, in order to produce high-purity cadaverine dicarboxylate salt, the dicarboxylate salt had to be removed before crystallization, and there were problems such as the cost burden due to performing an additional process for removing the dicarboxylate salt, a decrease in 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 dicarboxylic acid salt (cadaverine sebacate, cadaverine undecanedioic acid salt, or cadaverine dodecanedioic acid salt).

[0010]

[0011] 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.

[0012]

[0013] According to one aspect 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, high-purity C10, C11 or C12 cadaverine dicarboxylate can be produced in high yield without dicarboxylic acid crystals being precipitated together as a byproduct.

[0014]

[0015] Figure 1 is a flowchart showing a process for manufacturing cadaverine dicarboxylate according to the present application.

[0016] Figure 2 is a flowchart showing a process for manufacturing cadaverine dicarboxylate according to one embodiment of the present application.

[0017]

[0018] 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.

[0019]

[0020] 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.

[0021]

[0022] The term "dicarboxylic acid" in this application refers to a dibasic acid containing two carboxyl groups, and is highly important as a metabolic intermediate in the body. Specifically, the dicarboxylic acid may be an aliphatic dicarboxylic acid having 10, 11, or 12 carbon atoms. That is, in this application, the dicarboxylic acid refers to an aliphatic dicarboxylic acid selected from sebacic acid, undecanedioic acid, and dodecanedioic acid. If the cadaverine dicarboxylate salt to be produced in the manufacturing method of the present application is cadaverine sebacate, it should be understood that all of the added or introduced dicarboxylic acids are sebacic acid. In addition, if the cadaverine dicarboxylate salt to be produced in the manufacturing method of the present application is cadaverine undecanedioic acid, it should be understood that all of the added or introduced dicarboxylic acids are undecanedioic acid. In addition, if the cadaverine dicarboxylate salt to be produced in the manufacturing method of the present application is cadaverine dodecanedioic acid, it should be understood that all of the added or introduced dicarboxylic acids are dodecanedioic acid. In this way, it is understood that the dicarboxylic acid in the present application refers to one type of aliphatic dicarboxylic acid, not a mixture of different types of dicarboxylic acids.

[0023]

[0024] In this application, the term "sebacic acid" refers to a dicarboxylic acid having 10 carbon atoms and C 10 H 18 It has the chemical formula O4 and generally exists as a white powdery solid. Sebacic acid is also called sebacic acid, sebacic acid, etc., and is used as a precursor for various industrial products, including polymers and plasticizers.

[0025] In this application, the term "undecanedioic acid" refers to a dicarboxylic acid having 11 carbon atoms and C 11 H 20It has the chemical formula O4. The above undecanedioic acid is utilized as a precursor for various industrial products, including polymers and plasticizers.

[0026] In this application, the term "dodecanedioic acid" refers to a carboxylic acid having 12 carbon atoms, C 12 H 22 It has the chemical formula O4. The dodecanedioic acid is also called dodecanedioic acid.

[0027]

[0028] 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.

[0029] The term "cadaverine dicarboxylate" in this application refers to a substance manufactured by reacting a dicarboxylic acid component with the diamine-type cadaverine, which 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 sebacate, cadaverine undecanedioic acid, and cadaverine dodecanedioic acid.

[0030]

[0031] Figures 1 and 2 are flowcharts showing a process for manufacturing cadaverine dicarboxylate according to one embodiment of the present application.

[0032]

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038]

[0039] 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.

[0040]

[0041] In the first step, the dicarboxylic acid 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.

[0042] The term "diammonium dicarboxylate" in this application refers to a form in which two hydrogen atoms of a dicarboxylic acid are replaced with ammonium, and is used as the main nitrogen source of the medium. The diammonium dicarboxylate may be prepared by gently mixing dicarboxylic acid with ammonia water to have a neutral pH, and then concentrated to form crystals or in liquid form, but is not limited thereto.

[0043]

[0044] The manufacturing method of the present application can manufacture cadaverine dicarboxylate with high purity while simplifying the process by adding 81 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.

[0045] Specifically, the diammonium dicarboxylate of the first step can be included in the medium at a high ratio, specifically 41 mol% or more, relative to the dicarboxylic acid supplied throughout the entire process of the manufacturing method of the present application.

[0046] More specifically, the diammonium dicarboxylate of the first step may be included in an amount of 43 mol% or more, 45 mol% or more, 53 mol% or more, 78 mol% or more, 80 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.

[0047]

[0048] 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.

[0049]

[0050] 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 0.7 to 1.11. 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 0.74 to 1.06, 0.76 to 1.04, 0.78 to 1.02, or 0.80 to 1.00, or 0.82 to 0.98, and more specifically 0.84 to 0.96. 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.

[0051] 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 0.7, 0.72, 0.74, 0.78, 0.79, 0.80, 0.82, 0.84, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, and 0.96 and / or an upper limit selected from 1.11, 1.1, 1.08, 1.07, 1.06, 1.05, 1.04, 1.03, 1.02, 1.01, and 1.0.

[0052] In one specific example, the dicarboxylic acid and the dicarboxylic acid are sebacic acid and sebacic 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 0.84-0.86.

[0053] In one specific example, the dicarboxylic acid and dicarboxylic acid are undecanedioic acid and undecanedioic 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 0.84-0.90.

[0054] In one specific example, the dicarboxylic acid and dicarboxylic acid are dodecanedioic acid and dodecanedioic 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 0.86-0.96.

[0055]

[0056] Specifically, the diammonium dicarboxylate of the first step may be included in an amount of 0.3 mol / L to 0.5 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 0.45 mol / L, 0.35 mol / L to 0.45 mol / L, 0.38 mol / L to 0.45 mol / L, or 0.38 mol / L to 0.4 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.

[0057]

[0058] Lysine dicarboxylate can be produced through fermentation of L-lysine-producing microorganisms in a medium containing diammonium dicarboxylate. Lysine dicarboxylate can be introduced into subsequent processes as a fermentation broth containing the cells, in the form of a fermentation broth with the cells removed, or after concentration or purification.

[0059]

[0060] The concentration of lysine in the fermentation broth in which the first-stage lysine-producing microorganism of the present application is cultured is not limited, but may be 50 g / L to 200 g / L, 60 g / L to 150 g / L, 60 g / L to 140 g / L, or 61 g / L to 69 g / L based on the fermentation broth of the L-lysine-producing microorganism.

[0061]

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067]

[0068] 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.

[0069]

[0070] The second step refers to the step of converting lysine dicarboxylate into the target product cadaverine dicarboxylate (cadaverine sebacate, cadaverine undecanedioic acid salt or cadaverine dodecanedioic acid salt).

[0071]

[0072] 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.

[0073] The dicarboxylic acid added in the second step may not be added or may be added in a trace amount of 0.57 mol / L or less, 0.53 mol / L or less, 0.48 mol / L or less, 0.4 mol / L or less, 0.18 mol / L or less, 0.15 mol / L or less, 0.12 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.

[0074] In one specific example, when the dicarboxylic acid is sebacic acid, the dicarboxylic acid added in the second step may be added in a trace amount of 0.01 mol / L or more and 0.06 mol / L or less, for example, 0.04 mol / L to 0.06 mol / L.

[0075] In one specific example, when the dicarboxylic acid is undecanedioic acid, the dicarboxylic acid added in the second step may be added in a trace amount of 0.01 mol / L or more and 0.06 mol / L or less, for example, 0.02 mol / L to 0.04 mol / L.

[0076] In one specific example, when the dicarboxylic acid is dodecanedioic acid, the dicarboxylic acid added in the second step may be added in a trace amount of 0.02 mol / L or less, for example, 0.005 mol / L to 0.015 mol / L.

[0077]

[0078] 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.

[0079] 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.

[0080]

[0081] 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 solution 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 0.85 to 1.12 relative to the amount of cadaverine in the process solution obtained after the second step.

[0082] More specifically, the amount of dicarboxylic acid added in the present application manufacturing method may be a molar ratio of 0.86 to 1.11, 0.87 to 1.10, 0.88 to 1.08, 0.89 to 1.06, 0.90 to 1.05, 0.91 to 1.04, 0.92 to 1.03, 0.93 to 1.02, 0.94 to 1.01, or 0.95 to 1.0 relative to the amount of cadaverine in the process solution after the second step.

[0083] 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, 0.95 and 0.96 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.

[0084]

[0085] In one specific example, when the dicarboxylic acid is sebacic acid, the amount of the dicarboxylic acid added in the manufacturing method of the present application may be a molar ratio of 0.9 to 1.10, 0.91 to 1.09, 0.92 to 1.08, 0.93 to 1.06, 0.94 to 1.02, 0.95 to 1.01, or 0.96 to 1.00 relative to the amount of cadaverine in the process solution after the second step.

[0086] One technical significance of the present invention lies in the discovery that when sebacic acid is added in a molar ratio within the above range in comparison with the amount of cadaverine calculated to be obtained after the second step in the manufacturing method of the present application, sebacic acid does not crystallize together with cadaverine sebacate, thereby enabling the production of high-purity and high-yield cadaverine sebacate.

[0087] In addition, by adjusting the molar ratio of the cadaverine to be obtained and the sebacic acid to be added, it is possible to maintain the purity of cadaverine sebacate at 98% or more up to the fourth cycle during the circulation of the mother liquor, and cadaverine sebacate can be obtained at a total yield of 40% or more, 42% or more, 53% or more, 56% or more, 64% or more, 66% or more, 72% or more, 73% or more, or 74% or more depending on the number of cycles of the mother liquor.

[0088]

[0089] In one specific example, when the dicarboxylic acid is undecanedioic acid, the amount of the dicarboxylic acid added in the manufacturing method of the present application may be a molar ratio of 0.9 to 1.10, 0.91 to 1.09, 0.92 to 1.08, 0.93 to 1.06, 0.94 to 1.02, or 0.95 to 1.00 relative to the amount of cadaverine in the process solution after the second step.

[0090] In the manufacturing method of the present application, one technical significance lies in the fact that when undecanedioic 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, undecanedioic acid does not crystallize together with cadaverine undecanedioic acid salt, thereby enabling the production of cadaverine undecanedioic acid salt with high purity and high yield.

[0091] In addition, by adjusting the molar ratio of the cadaverine to be obtained and the undecanedioic acid to be added, it is possible to maintain the purity of cadaverine undecanedioic acid at 99% or more up to the fifth cycle during the circulation of the mother liquor, and cadaverine undecanedioic acid can be obtained at a total yield of 46% or more, 48% or more, 55% or more, 56% or more, 61% or more, 63% or more, 66% or more, 68% or more, 70% or more, 72% or more, or 73% or more depending on the number of cycles of the mother liquor.

[0092]

[0093] In one specific example, when the dicarboxylic acid is dodecanedioic acid, the amount of dodecanedioic acid added in the manufacturing method of the present application may be a molar ratio of 0.9 to 1.10, 0.91 to 1.09, 0.92 to 1.08, 0.93 to 1.06, 0.94 to 1.02, or 0.95 to 1.00 relative to the amount of cadaverine in the process solution after the second step.

[0094] In the manufacturing method of the present application, one technical significance lies in the fact that when dodecanedioic 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, dodecanedioic acid does not crystallize together with cadaverine dodecanedioic acid salt, thereby enabling the production of cadaverine dodecanedioic acid salt with high purity and high yield.

[0095] In addition, by adjusting the molar ratio of the cadaverine to be obtained and the dodecanedioic acid to be added, it is possible to maintain the purity of cadaverine dodecanedioic acid salt at 98% or more up to the fourth cycle during the circulation of the mother liquor, and cadaverine sebacate salt can be obtained at a total yield of 40% or more, 42% or more, 53% or more, 56% or more, 64% or more, 66% or more, 72% or more, 73% or more, or 74% or more depending on the number of cycles of the mother liquor.

[0096]

[0097] In the second step, the conversion reaction of the lysine dicarboxylate to cadaverine dicarboxylate may be an enzymatic conversion reaction.

[0098] 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.

[0099]

[0100] 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.

[0101] 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.

[0102]

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107]

[0108] 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.

[0109] The conversion reaction of the second step may be performed at 30 to 60°C, more specifically at 40 to 55°C, and for example at 45 to 50°C, but is not limited thereto.

[0110]

[0111] The conversion reaction of the second step may be performed at a pH of 7.5 to 9, more specifically at a pH of 7.8 to 8.7, and even more specifically at a pH of 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.

[0112]

[0113] 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.

[0114]

[0115] The above recovery may be by collecting cadaverine dicarboxylate using a suitable method known in the art.

[0116] 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.

[0117]

[0118] 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.

[0119]

[0120] 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.

[0121] 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.

[0122]

[0123] Specifically, the above concentration step may be to concentrate so that the solid content becomes 45-70% (w / w), and more specifically, to concentrate so that it becomes 46-68% (w / w), 48-67% (w / w), 45-65% (w / w), 50-70% (w / w). For example, it may be to concentrate so that it becomes 45-55% (w / w). For example, it may be to concentrate so that it becomes 60-70% (w / w).

[0124] When the solid content is concentrated to the above range in the above concentration step, the recovery rate and purity of cadaverine dicarboxylate can be maintained excellently.

[0125] The above concentration step may be performed at a temperature of 45 to 80°C, for example, 55°C to 70°C, but is not limited thereto.

[0126]

[0127] 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.

[0128] Specifically, the cooling may be cooling the conversion liquid to 30°C or lower, and more specifically, cooling may be cooling to 27°C or lower, 25°C or lower, 23°C or lower, 20°C or lower, for example, 10°C, but is not limited thereto.

[0129] Specifically, the cooling rate may be 0.1-20°C / hour, more specifically 0.5-1.5°C / hour, and for example, 1°C / hour, but is not limited thereto.

[0130]

[0131] 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.

[0132]

[0133] 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.

[0134] 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.

[0135]

[0136] 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.

[0137]

[0138] In one embodiment, the recovery step may further include washing, separating, and drying the crystallized process solution.

[0139] 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.

[0140]

[0141] 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.

[0142]

[0143] 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.

[0144]

[0145] 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.

[0146]

[0147] In one embodiment, when the dicarboxylic acid is sebacic acid, the method of the present application may be characterized in that cadaverine sebacate having a purity of 99% or higher can be obtained in the 0th to 4th cycle of the mother liquor. In one embodiment, when the dicarboxylic acid is sebacic acid, based on the 4th cycle of the mother liquor, the accumulated total yield of cadaverine sebacate may be 70% or higher, and more specifically, may be more than 70%, 71% or higher, 72% or higher, 73% or higher, or 74% or higher.

[0148] In one embodiment, when the dicarboxylic acid is undecanedioic acid, the method of the present application may be characterized in that cadaverine undecanedioic acid salt having a purity of 99% or higher can be obtained in the 0th to 5th cycle of the mother liquor. In one embodiment, when the dicarboxylic acid is undecanedioic acid, based on the 5th cycle of the mother liquor, the cumulative total yield of cadaverine undecanedioic acid salt may be greater than 68%, for example, greater than 70%, and the purity may be greater than 99%.

[0149] In one embodiment, when the dicarboxylic acid is dodecanedioic acid, the method of the present application may be characterized in that cadaverine dodecanedioic acid salt having a purity of 98% or higher can be obtained in the 0th to 4th circulation of the mother liquor. For example, the method may be characterized in that cadaverine dodecanedioic acid salt having a purity of 99% or higher can be obtained in the 2nd to 4th circulation of the mother liquor. In one embodiment, when the dicarboxylic acid is dodecanedioic acid, the cumulative total yield of cadaverine dodecanedioic acid salt may be 70% or higher, and more specifically, may be more than 70%, 71% or higher, or 72% or higher, based on the 4th circulation of the mother liquor.

[0150] In one embodiment, the method of the present application may be characterized in that it can obtain cadaverine dicarboxylate having a purity of 98% or more and a total yield of 22% or more in the 0th to 4th cycle of the mother liquor.

[0151]

[0152] 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.

[0153] 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.

[0154]

[0155] In the above, the manufacturing process of cadaverine dicarboxylic acid salt (cadaverine sebacate, cadaverine undecanedioic acid salt, or cadaverine dodecanedioic acid salt) according to one embodiment of the present application has been described. In the following, the advantageous effects mentioned in the present application will be examined through experimental results of examples and comparative examples of cadaverine sebacate, cadaverine undecanedioic acid salt, or cadaverine dodecanedioic acid salt, respectively.

[0156]

[0157] 1. Cadaverine sebacate

[0158] Manufacturing Example 1-1. Fermentation process for producing lysine sebacate

[0159] A Corynebacterium glutamicum strain (KCCM12154P, 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.

[0160] In order to produce lysine sebacate using Corynebacterium, ammonium sebacate to replace the existing ammonium sulfate was supplied at a level of 92-94 g / L for each example, and the concentration of the carbon source included in the culture medium was lowered to 1.2 mol to maintain the carbon source / nitrogen source balance. As a result of performing repeated fed-batch fermentation, lysine sebacate was obtained at a level of 136-139 g / L and lysine was obtained at a level of 68 g / L. At this time, the molar ratio of sebacic acid / lysine in the fermentation process liquid in the examples was adjusted to a level of 0.83 to 0.85.

[0161]

[0162] Manufacturing Example 1-2. Conversion reaction of lysine sebacate to cadaverine sebacate

[0163] 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.

[0164] The enzyme conversion solution obtained by culturing the enzyme strain in the fermentation process solution of lysine sebacate prepared in 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, and the pH was adjusted to 8.0 to 8.5. For this purpose, CO2 was added for neutralization in the following Examples 1-1 to 1-4. Meanwhile, in Examples 1-2 to 1-6, sebacic acid was additionally added at a level of 0.01 to 0.06 mol / L (based on the volume of the fermentation process solution of lysine sebacate) before the conversion reaction. As a result, it was confirmed that the conversion rate of cadaverine sebacate was at the level of 97 to 98%. Specifically, after the conversion reaction, cadaverine sebacate was obtained at a level of 119 to 140 g / L (based on the volume of the fermentation process liquid of lysine sebacate), and cadaverine was obtained at a level of 47 g / L (based on the volume of the fermentation process liquid of lysine sebacate). In Examples 1-5 and 1-6, sebacic acid was additionally added at a level of 0.02 to 0.05 mol / L (based on the volume of the fermentation process liquid of lysine sebacate).

[0165]

[0166] Manufacturing Example 1-3. Subsequent processes after obtaining cadaverine sebacate

[0167] The following subsequent process was performed on the process solution containing cadaverine sebacate obtained in Manufacturing Example 1-2 to obtain cadaverine sebacate in crystal form.

[0168]

[0169] [Bacterial removal step]

[0170] The cells in the cadaverine sebacate process solution were removed by performing membrane filtration with a size of 0.1 μm.

[0171] [Step to remove impurities using activated carbon]

[0172] Add activated carbon at a level of 10% based on the weight of cadaverine sebacate 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.

[0173] [Step of concentrating the cadaverine sebacate process solution from which impurities have been removed]

[0174] 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 50% by weight.

[0175] [Step of cooling, crystallizing, and separating the cadaverine sebacate concentrate and then drying it]

[0176] The above concentrate was cooled from 50°C to 25°C at a rate of 1°C / hr. The precipitated crystals were separated from the mother liquor using a centrifuge. The crystals were washed with water containing 10% cadaverine sebacate by weight. The separated crystals were dried for one day, and their purity was measured by HPLC.

[0177]

[0178] [Step of recycling the mother liquor to the crystallization stage]

[0179] The mother liquor separated above was recycled to the step of concentrating the cadaverine sebacate process solution from which impurities were removed.

[0180]

[0181] Experimental Example 1-1. Confirmation of changes in cadaverine sebacate purity and yield according to the molar ratio of sebacate and cadaverine in the cadaverine sebacate conversion solution.

[0182] 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 sebacate changed depending on the molar ratio of sebacate and cadaverine in the cadaverine sebacate conversion solution.

[0183]

[0184] Example 1-1: When the molar ratio of added sebacic acid / cadaverine in the process solution after the second step is 0.85

[0185] 1,000 ml of a cadaverine sebacate conversion solution having a sebacate / cadaverine molar ratio of 0.85 was supplied and passed through a 0.1 μm membrane to remove microorganisms. The cadaverine sebacate conversion solution having a sebacate / cadaverine molar ratio of 0.85 was manufactured by following the manufacturing methods of Manufacturing Examples 1-1 and 1-2, except that the sum of the amount of sebacate added among the ammonium sebacate added in the fermentation process for producing lysine in Manufacturing Example 1-1 and the amount of sebacate added in the cadaverine sebacate conversion reaction in Manufacturing Example 1-2 was controlled to be 0.85 of the mole number of cadaverine converted.

[0186] The filtrate was decolorized using activated carbon, filtered through a filter paper, and concentrated under reduced pressure to a solid content of 50% by weight. 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 sebacate was recycled to the cadaverine sebacate feed solution having a sebacate / cadaverine molar ratio of 0.85 and then subjected to another crystallization step. The recycling of the mother liquor was performed sequentially.

[0187]

[0188] Example 1-2: When the molar ratio of added sebacic acid / cadaverine in the process solution after the second step is 0.89

[0189] A cadaverine sebacate conversion solution having a sebacate / cadaverine molar ratio of 0.89 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 1-3: When the molar ratio of added sebacic acid / cadaverine in the process solution after the second step is 0.96

[0192] A cadaverine sebacate conversion solution having a sebacate / cadaverine molar ratio of 0.96 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]

[0194] Example 1-4: When the molar ratio of added sebacic acid / cadaverine in the process solution after the second step is 1.0

[0195] A cadaverine sebacate conversion solution having a sebacate / 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 1-1.

[0196] In Examples 1-3 to 1-4, the molar ratio of sebacic acid / lysine in the lysine fermentation step was adjusted to 0.85.

[0197]

[0198] Example 1-5: When the molar ratio of added sebacic acid / cadaverine in the process solution after the second step is 1.04

[0199] A cadaverine sebacate conversion solution having a sebacate / cadaverine molar ratio of 1.04 was supplied, and the mother liquor was sequentially recycled and a purification process was performed in the same manner as in Example 1.

[0200]

[0201] Example 1-6: When the molar ratio of added sebacic acid / cadaverine in the process solution after the second step is 1.11

[0202] A cadaverine sebacate conversion solution having a sebacate / cadaverine molar ratio of 1.11 was supplied, and the mother liquor was sequentially recycled and a purification process was performed in the same manner as in Example 1-1.

[0203] The physical property measurements for the process solutions and cadaverine sebacate prepared according to the above-described Examples 1-1 to 1-6 are as shown in Tables 1 to 5 below.

[0204]

[0205] Table 1 shows the process solution that did not circulate the mother liquor.

[0206] No mother liquor circulation Example 1-1 Example 1-2 Example 1-3 Example 1-4 Example 1-5 Example 1-6 Sebacic acid / cadaverine molar ratio 0.85 0.89 0.96 11.04 1.11 Crystallization feed New feed, ml 1000 1000 1000 1000 1000 1000 Amount of cadaverine in crystallization solution, g 166 166 166 166 166 Amount of cadaverine in crystals produced, g No crystals produced 353740 3531 Amount of cadaverine in mother liquor, g - 13 11 29 127 13 11 35 Result Purity, % - 9899998673 Yield, % - 2122242119 Total yield, % - 2122242119

[0207] Table 2 shows the process solution that has been circulated once.

[0208] 1 cycle of mother liquor circulation Example 1-1 Example 1-2 Example 1-3 Example 1-4 Example 1-5 Example 1-6 Sebacic acid / cadaverine molar ratio 0.85 0.89 0.96 11.04 1.11 Crystallization feed New feed, ml - 1000 1000 1000 1000 1000 Amount of cadaverine in crystallization solution, g - 297 295 293 297 301 Cadaverine in produced crystals, g - 6 266 706 356 Cadaverine in mother liquor, g - 235 229 223 234 245 Result Purity, % - 989 899 8878 Yield, % - 21 222 421 19 Total yield, % - 38 40 423 834

[0209] Table 3 shows the process solution that was cycled twice.

[0210] Mother liquor circulation 2 times Example 1-1 Example 1-2 Example 1-3 Example 1-4 Example 1-5 Example 1-6 Sebacic acid / cadaverine molar ratio 0.85 0.89 0.96 11.04 1.11 Crystallization feed New feed, ml - 1000 1000 1000 1000 1000 Amount of cadaverine in crystallization solution, g - 40 1395389 400 4 11 Amount of cadaverine in produced crystals, g - 8489938577 Amount of cadaverine in mother liquor, g - 317306297315334 Result Purity, % - 98999990 8 1 Yield, % - 2 122242 119 Total yield, % - 5 153565 146

[0211] Table 4 shows the process solution after circulating the mother liquor three times.

[0212] Mother liquor circulation 3 times Example 1-1 Example 1-2 Example 1-3 Example 1-4 Example 1-5 Example 1-6 Sebacic acid / cadaverine molar ratio 0.85 0.89 0.96 11.04 1.11 Crystallization feed New feed, ml - 1000 1000 1000 1000 1000 Amount of cadaverine in crystallization solution, g - 48347246348 1500 Amount of cadaverine in produced crystals, g - 101 106 110 10294 Amount of cadaverine in mother liquor, g - 382366353379406 Result Purity, % - 9899999 184 Yield, % - 2122242119 Total yield, % - 6164666256

[0213] Table 5 shows the process solution after circulating the mother liquor four times.

[0214] Mother liquor circulation 4 times Example 1-1 Example 1-2 Example 1-3 Example 1-4 Example 1-5 Example 1-6 Sebacic acid / cadaverine molar ratio 0.85 0.89 0.96 11.04 1.11 Crystallization feed New feed, ml - 1000 1000 1000 1000 1000 Amount of cadaverine in crystallization solution, g - 548 532 519 545 572 Amount of cadaverine in crystals produced, g - No crystals produced 120 123 116 107 Amount of cadaverine in mother liquor, g - 413395429465 Result Purity, % - 99999 285 Yield, % - 22242119 Total yield, % - 72747065

[0215] By summarizing the experimental results according to Tables 1 to 5 above, it was confirmed that the presently applied manufacturing method, in which the first-stage diammonium sebacate is included in a large amount at 78-100 mol% of the total supplied sebacic acid, enables the production of cadaverine sebacate with an overall improved crystallization rate, high purity, and high yield.

[0216]

[0217] 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 sebacate salt was low, which was not sufficient for crystals of cadaverine sebacate salt to precipitate.

[0218] In Example 1-2, crystals were precipitated up to the third cycle of mother liquor circulation, but no crystals were formed in the fourth cycle. This is because the sequentially circulated mother liquor lowered the molar ratio of sebacate to the crystallization feed solution, and thus the concentration of sebacate was not sufficient to precipitate crystals of cadaverine sebacate.

[0219] In Example 1-5, the purity of the crystal was somewhat low at 86% when the mother liquor was not circulated, but the purity increased to 92% when the mother liquor was circulated 4 times.

[0220] In Example 1-6, the purity of the crystal was low at 73% when the mother liquor was not circulated, but the purity increased to 81% when the mother liquor was circulated 4 times.

[0221]

[0222] On the other hand, examples 1-3 were able to obtain 99% high-purity cadaverine sebacate up to the fourth cycle of mother liquor circulation, and achieve a high total yield of approximately 72%.

[0223] Example 1-4 was able to obtain 99% high-purity cadaverine sebacate up to the fourth cycle of mother liquor circulation, and achieve a high total yield of approximately 74%.

[0224]

[0225] In this way, it was confirmed that a method for producing high-purity cadaverine sebacate can be provided depending on the molar ratio of sebacic acid and cadaverine in the conversion solution (process solution) supplied after the cadaverine conversion process. In particular, it was confirmed that when the amount of added sebacic acid was adjusted to a molar ratio of about 0.84 to 0.86 with respect to the amount of cadaverine in the process solution after the second step, an excellent total yield of 53% or more can be exhibited while maintaining a purity of 99% of cadaverine sebacate even after two or more cycles of mother liquor circulation.

[0226]

[0227] 2. Cadaverine undecanedioic acid salt

[0228] Manufacturing Example 2-1. Fermentation process for producing lysine undecanedioic acid salt

[0229] 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.

[0230] FIG. 5 is a graph showing the results of producing lysine undecanedioic acid salt through microbial fermentation using ammonium undecanedioic acid salt according to the manufacturing example method described above. For the production of lysine undecanedioic acid salt using Corynebacterium, ammonium undecanedioic acid salt to replace the existing ammonium sulfate was supplied at a level of 88 to 100 g / L for each example, and the concentration of the carbon source included in the culture medium was lowered to 1.2 mol to maintain the carbon source / nitrogen source balance. As a result of repeated fed-batch fermentation, lysine undecanedioic acid salt was obtained at a level of 127 to 145 g / L, and lysine was obtained at a level of 66 g / L. At this time, the molar ratio of undecanedioic acid / lysine in the fermentation process solution in the examples was adjusted to a level of 0.78 to 0.89.

[0231]

[0232] Manufacturing Example 2-2. Conversion reaction of lysine undecanedioic acid salt to cadaverine undecanedioic acid salt

[0233] 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.

[0234] The enzyme conversion solution obtained by culturing the above enzyme strain in the fermentation process solution of lysine undecanedioic acid salt prepared in Manufacturing Example 2-1 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, and the pH was adjusted to 8.0 to 8.5. For this purpose, CO2 was added for neutralization in the following Examples 2-1 to 2-4. Meanwhile, in Examples 2-2 to 2-4, undecanedioic acid was additionally added at a level of 0.02 to 0.04 mol / L (based on the volume of the fermentation process solution of lysine undecanedioic acid salt) before the conversion reaction. As a result, it was confirmed that the conversion rate of cadaverine undecanedioic acid salt was at the level of 97 to 98%. After the conversion reaction, cadaverine undecanedioic acid was obtained at a level of 111 to 140 g / L (based on the volume of the fermentation process liquid of lysine undecanedioic acid), and cadaverine was obtained at a level of 45 g / L (based on the volume of the fermentation process liquid of lysine undecanedioic acid). In the cases of Examples 2-5 and 2-6, undecanedioic acid was additionally added at a level of 0.06 to 0.08 mol / L to prepare a process liquid.

[0235]

[0236] Manufacturing Example 2-3. Subsequent processes after obtaining cadaverine undecanedioic acid salt

[0237] The following subsequent process was performed on the process solution containing cadaverine undecanedioic acid salt obtained in Manufacturing Example 2-2 to obtain cadaverine undecanedioic acid salt in crystal form.

[0238] [Bacterial removal step]

[0239] The cells in the cadaverine undecanedioic acid solution were removed by performing membrane filtration with a size of 0.1 μm.

[0240] [Step to remove impurities using activated carbon]

[0241] Add activated carbon at a level of 10% based on the weight of cadaverine undecanedioic acid salt 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.

[0242] [Step of concentrating the cadaverine undecanedioic acid solution from which impurities have been removed]

[0243] 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 65% by weight.

[0244] [Step of cooling, crystallizing, and separating the cadaverine undecanedioic acid salt concentrate and then drying it]

[0245] The above concentrate was cooled from 50°C to 10°C at a rate of 1°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 undecanedioic acid salt. The separated crystals were dried for one day, and their purity was measured by HPLC.

[0246] [Step of recycling the mother liquor to the crystallization stage]

[0247] The mother liquor separated above was recycled to the step of concentrating the cadaverine undecanedioic acid salt process solution from which impurities were removed.

[0248]

[0249] Experimental Example 2-1. Changes in the purity and yield of cadaverine undecanedioic acid salt according to the molar ratio of undecanedioic acid salt and cadaverine in the cadaverine undecanedioic acid salt conversion solution.

[0250] 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 undecanedioic acid salt change depending on the molar ratio of undecanedioic acid salt and cadaverine in the cadaverine undecanedioic acid salt conversion solution.

[0251] Example 2-1: When the molar ratio of added undecanedioic acid / cadaverine in the process solution after the second step is 0.79

[0252] 1,000 ml of a cadaverine undecanedioic acid conversion solution having an undecanedioic acid / cadaverine molar ratio of 0.79 was supplied and passed through a 0.1 μm membrane to remove microorganisms. The cadaverine undecanedioic acid conversion solution having an undecanedioic acid / cadaverine molar ratio of 0.79 was manufactured by following the manufacturing methods of Manufacturing Examples 2-1 and 2-2, but controlling the sum of the amount of undecanedioic acid added among the ammonium undecanedioic acid added in the fermentation process for producing lysine in Manufacturing Example 2-1 and the amount of undecanedioic acid added in the cadaverine undecanedioic acid conversion reaction in Manufacturing Example 2-2 so that it could be 0.79 of the mole number of cadaverine that was converted.

[0253] 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 10°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 undecanedioic acid was recycled to the cadaverine undecanedioic acid feed solution having an undecanedioic acid / cadaverine molar ratio of 0.79, and then subjected to another crystallization step. The recycling of the mother liquor was performed sequentially.

[0254]

[0255] Example 2-2: When the molar ratio of added undecanedioic acid / cadaverine in the process solution after the second step is 0.91

[0256] A cadaverine undecanedioic acid conversion solution having an undecanedioic 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 2-1.

[0257] Example 2-3: When the molar ratio of added undecanedioic acid / cadaverine in the process solution after the second step is 0.95

[0258] A cadaverine undecanedioic acid conversion solution having an undecanedioic acid / cadaverine molar ratio of 0.95 was supplied, and the mother liquor was sequentially recycled and a purification process was performed in the same manner as in Example 2-1.

[0259] Example 2-4: When the molar ratio of added undecanedioic acid / cadaverine in the process solution after the second step is 1.0

[0260] A cadaverine undecanedioic acid conversion solution having an undecanedioic 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.

[0261] In the case of Examples 2-3 to 2-4, the molar ratio of undecanedioic acid / lysine in the lysine fermentation step was adjusted to 0.89.

[0262] Example 2-5: When the molar ratio of added undecanedioic acid / cadaverine in the process solution after the second step is 1.04

[0263] A cadaverine undecanedioic acid conversion solution having an undecanedioic acid / cadaverine molar ratio of 1.04 was supplied, and the mother liquor was sequentially recycled and a purification process was performed in the same manner as in Example 2-1.

[0264] Example 2-6: When the molar ratio of added undecanedioic acid / cadaverine in the process solution after the second step is 1.09

[0265] A cadaverine undecanedioic acid conversion solution having an undecanedioic acid / cadaverine molar ratio of 1.09 was supplied, and the mother liquor was sequentially recycled and a purification process was performed in the same manner as in Example 2-1.

[0266] The physical property measurements for the process solution and cadaverine undecanedioic acid salt prepared according to Examples 2-1 to 2-6 described above are as shown in Tables 6 to 11 below.

[0267]

[0268] Table 6 is for the process solution that did not circulate the mother liquor.

[0269] No mother liquor circulation Example 2-1 Example 2-2 Example 2-3 Example 2-4 Example 2-5 Example 2-6 Undecanedioic acid / cadaverine molar ratio 0.79 0.91 0.95 1.01 04 1.09 Crystallization feed New feed, ml 1000 1000 1000 1000 1000 1000 Amount of cadaverine in crystallization solution, g 113 113 113 113 113 Amount of crystals produced, g 109 119 124 127 132 136 Amount of cadaverine in mother liquor, g 78 757 372 7577 Result Purity, % 999999999 182 Yield, % 313435363432 Total yield, % 313435363432

[0270] Table 7 shows the process solution that has been circulated once.

[0271] Mother liquor circulation 1 time Example 2-1 Example 2-2 Example 2-3 Example 2-4 Example 2-5 Example 2-6 Undecanedioic acid / cadaverine molar ratio 0.79 0.91 0.95 1.01 04 1.09 Crystallization feed solution New feed solution, ml 1000 1000 1000 1000 1000 1000 Amount of cadaverine in crystallization solution, g 19 118 8 187 185 188 190 Amount of crystals produced, g 17 8 19 5 20 32 0 9 22 7 24 5 Amount of cadaverine in mother liquor, g 13 4 12 5 12 2 1 1 8 11 9 11 9 Result Purity, % 99 100 99999 38 7 Yield, % 30 33 35 36 37 37 Total yield, %414546484747

[0272] Table 8 shows the process solution that was circulated twice.

[0273] Mother liquor circulation 2 times Example 2-1 Example 2-2 Example 2-3 Example 2-4 Example 2-5 Example 2-6 Undecanedioic acid / cadaverine molar ratio 0.79 0.91 0.95 1.01 04 1.09 Crystallization feed solution New feed solution, ml 1000 1000 1000 1000 1000 1000 Amount of cadaverine in crystallization solution, g 2 47 239 235 2322 3223 Amount of crystals produced, g 2 19 24 325 326 1277 293 Amount of cadaverine in mother liquor, g 1 77 160 154 148 147 146 Result Purity, % 999999999 489 Yield, % 28 3335 36 37 37 Total yield, %485355565757

[0274] Table 9 shows the process solution after circulating the mother liquor three times.

[0275] Mother liquor circulation 3 times Example 2-1 Example 2-2 Example 2-3 Example 2-4 Example 2-5 Example 2-6 Undecanedioic acid / cadaverine molar ratio 0.79 0.91 0.95 1.01 04 1.09 Crystallization feed solution New feed solution, ml 1000 1000 1000 1000 1000 Amount of cadaverine in crystallization solution, g 290 274267261260 259 Amount of crystals produced, g 243274385 29430 9323 Amount of cadaverine in mother liquor, g 212186176167165 163 Result Purity, % 9999 100 100 9590 Yield, % 2732334363737 Total yield, %535961636464

[0276] Table 10 shows the process solution after circulating the mother liquor four times.

[0277] Mother liquor circulation 4 times Example 2-1 Example 2-2 Example 2-3 Example 2-4 Example 2-5 Example 2-6 Undecanedioic acid / cadaverine molar ratio 0.79 0.91 0.95 1.01 04 1.09 Crystallization feed solution New feed solution, ml 1000 1000 1000 1000 1000 1000 Amount of cadaverine in crystallization solution, g 3 26 299 289 280 278 277 Amount of crystals produced, g 2 5 6 292 30 5 3 15 3 29 34 3 Amount of cadaverine in mother liquor, g 2 4 3 20 5 19 17 9 17 7 174 Result Purity, % 999999 100959 1 Yield, % 2 5 3 13 4 36 37 37 Total yield, %576466686969

[0278] Table 11 shows the process solution after circulating the mother liquor five times.

[0279] Mother liquor circulation 5 times Example 2-1 Example 2-2 Example 2-3 Example 2-4 Example 2-5 Example 2-6 Undecanedioic acid / cadaverine molar ratio 0.79 0.91 0.95 1.01 04 1.09 Crystallization feed solution New feed solution, ml 1000 1000 1000 1000 1000 Amount of cadaverine in crystallization solution, g 357318304292290288 Amount of crystals produced, g 26 2304318329342355 Amount of cadaverine in mother liquor, g 27 3221202186184181 Result Purity, % 99 10099 1009692 Yield, % 24 3134363737 Total yield, %606870737373

[0280] By summarizing the experimental results according to Tables 1 to 6 above, it was confirmed that the presently applied manufacturing method, in which the first-stage diammonium undecanedioic acid salt is included in a large amount at 83-100 mol% of the total supplied undecanedioic acid, enables the production of cadaverine undecanedioic acid salt with an overall improved crystallization rate, high purity, and high yield.

[0281]

[0282] Specifically, in the case of Example 2-1, 99% high-purity cadaverine undecanedioic acid salt was obtained, but a low yield of 62% was achieved up to the fifth cycle of mother liquor circulation. This is because the molar ratio of undecanedioic acid salt was low, which was not sufficient for crystals of cadaverine undecanedioic acid salt to precipitate.

[0283] Example 2-2 obtained 99% high-purity cadaverine undecanedioic acid salt through mother liquor circulation, but achieved a low yield of 67% in total yield up to the fifth mother liquor circulation. This is because the molar ratio of undecanedioic acid salt was low, which was not sufficient for crystal precipitation of cadaverine undecanedioic acid salt.

[0284] In Example 2-5, the purity of the crystal was somewhat low at 91% when the mother liquor was not circulated, but the purity increased to 96% when the mother liquor was circulated 5 times.

[0285] In Example 2-6, the purity of the crystal was low at 82% when the mother liquor was not circulated, but the purity increased to 92% when the mother liquor was circulated 5 times.

[0286] Example 2-3 obtained 99% high-purity cadaverine undecanedioic acid salt up to the fifth cycle of mother liquor circulation, and also achieved a high yield of approximately 70% in total yield.

[0287] Example 2-4 obtained 99% high-purity cadaverine undecanedioic acid salt up to the fifth cycle of mother liquor circulation, and also achieved a high yield of approximately 73% in total yield.

[0288]

[0289] In this way, it was confirmed that the purity of the manufactured cadaverine undecanedioic acid salt can vary depending on the molar ratio of undecanedioic acid and cadaverine in the conversion solution (process solution) supplied after the cadaverine conversion process. In particular, it was confirmed that when the amount of added undecanedioic acid was adjusted to a molar ratio of about 0.95 to 1.00 with respect to the amount of cadaverine in the process solution after the second step, it was possible to maintain a purity of 99% of cadaverine undecanedioic acid salt even after two or more cycles of mother liquor circulation while exhibiting an excellent total yield of 55% or more.

[0290]

[0291] 3. Cadaverine dodecanedioic acid salt

[0292]

[0293] Manufacturing Example 3-1. Fermentation process for producing lysine dodecanedioic acid salt

[0294] 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.

[0295] For lysine production using Corynebacterium, ammonium dodecanedioic acid to replace the existing ammonium sulfate was supplied at a level of 93-106 g / L for each example, and the concentration of the carbon source included in the culture medium was lowered to 1.2 mol to maintain the carbon source / nitrogen source balance. As a result of repeated fed-batch fermentation, lysine dodecanedioic acid was obtained at a level of 132-151 g / L, and lysine was obtained at a level of 61 g / L. At this time, the dodecanedioic acid / lysine molar ratio in the fermentation process liquid in the examples was adjusted to a level of 0.83 to 0.95.

[0296]

[0297] Manufacturing Example 3-2. Conversion reaction of lysine dodecanedioic acid salt to cadaverine dodecanedioic acid salt

[0298] 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.

[0299] The enzyme conversion solution obtained by culturing the enzyme strain above was supplied to the fermentation process solution of lysine dodecanedioic acid salt prepared in Manufacturing Example 3-1 at a mass percentage of 10% to the process solution to perform the conversion reaction. The conversion reaction temperature was maintained at 45 to 50°C, and the pH was adjusted to 8.0 to 8.5. For this purpose, CO2 was added for neutralization in Examples 3-1 to 3-4. Meanwhile, in Examples 3-2 to 3-4, dodecanedioic acid was additionally added at a level of 0.01 mol / L (based on the volume of the fermentation process solution of lysine dodecanedioic acid salt) before the conversion reaction. As a result, it was confirmed that the conversion rate of cadaverine dodecanedioic acid salt was at the level of 97 to 98%. Specifically, after the conversion reaction, cadaverine dodecanedioic acid salt was obtained at a level of 116 to 136 g / L (based on the volume of the fermentation process liquid of lysine dodecanedioic acid salt), and cadaverine was obtained at a level of 42 g / L (based on the volume of the fermentation process liquid of lysine dodecanedioic acid salt). In the cases of Examples 3-5 and 3-6, dodecanedioic acid was additionally added at a level of 0.03 to 0.05 mol / L (based on the volume of the fermentation process liquid of lysine dodecanedioic acid salt) to prepare a process liquid.

[0300]

[0301] Manufacturing Example 3-3. Subsequent processes after obtaining cadaverine dodecanedioic acid salt

[0302] The following subsequent process was performed on the process solution containing cadaverine dodecanedioic acid salt obtained in Manufacturing Example 3-2 to obtain cadaverine dodecanedioic acid salt in crystal form.

[0303] [Bacterial removal step]

[0304] The cells in the cadaverine dodecanedioic acid solution were removed by performing membrane filtration with a size of 0.1 μm.

[0305] [Step to remove impurities using activated carbon]

[0306] Add activated carbon at a level of 10% based on the weight of cadaverine dodecanedioic acid salt 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.

[0307] [Step of concentrating the cadaverine dodecanedioic acid solution from which impurities have been removed]

[0308] The filtered liquid above was concentrated under reduced pressure at approximately 55-70°C and 120 Torr in a rotary evaporator until the solid content of the filtrate became 50% by weight.

[0309] [Step of cooling, crystallizing, and separating the cadaverine dodecanedioic acid salt concentrate and then drying it]

[0310] The above concentrate was cooled from 50°C to 10°C at a rate of 1°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 dodecanedioic acid salt. The separated crystals were dried for one day, and their purity was measured by HPLC.

[0311] [Step of recycling the mother liquor to the crystallization stage]

[0312] The mother liquor separated above was recycled to the step of concentrating the cadaverine dodecanedioic acid salt process solution from which impurities were removed.

[0313]

[0314] Experimental Example 3-1. Changes in the purity and yield of cadaverine dodecanedioic acid salt according to the molar ratio of dodecanedioic acid salt and cadaverine in the cadaverine dodecanedioic acid salt conversion solution.

[0315] In Experimental Example 3-1, the method according to Manufacturing Examples 3-1 to 3-3 was used to confirm how the purity and yield of cadaverine dodecanedioic acid salt change depending on the molar ratio of dodecanedioic acid salt and cadaverine in the cadaverine dodecanedioic acid salt conversion solution.

[0316]

[0317] Example 3-1: When the molar ratio of added dodecanedioic acid / cadaverine in the process solution after the second step is 0.85

[0318] 1,000 ml of a cadaverine dodecanedioic acid salt conversion solution having a dodecanedioic acid salt / cadaverine molar ratio of 0.85 was supplied and passed through a 0.1 μm membrane to remove microorganisms. The cadaverine dodecanedioic acid salt conversion solution having a dodecanedioic acid salt / cadaverine molar ratio of 0.85 was manufactured according to the manufacturing methods of Manufacturing Examples 3-1 and 3-2, except that the sum of the amount of dodecanedioic acid salt added in the fermentation process for producing lysine in Manufacturing Example 3-1 and the amount of dodecanedioic acid salt added in the cadaverine dodecanedioic acid salt conversion reaction in Manufacturing Example 3-2 was controlled to be 0.85 of the mole number of cadaverine that was converted.

[0319] The filtrate was decolorized using activated carbon, filtered through a filter paper, and concentrated under reduced pressure to a solid content of 50%. The concentrate was cooled from 50°C to 10°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 residual dodecanedioic acid salt was recycled to the cadaverine dodecanedioic acid salt feed solution having a dodecanedioic acid salt / cadaverine molar ratio of 0.85, and then subjected to another crystallization step. The recycling of the mother liquor was performed sequentially.

[0320]

[0321] Example 3-2: When the molar ratio of added dodecanedioic acid / cadaverine in the process solution after the second step is 0.9

[0322] A cadaverine dodecanedioic acid conversion solution having a dodecanedioic 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 3-1.

[0323] Example 3-3: When the molar ratio of added dodecanedioic acid / cadaverine in the process solution after the second step is 0.95

[0324] A cadaverine dodecanedioic acid conversion solution having a dodecanedioic acid / cadaverine molar ratio of 0.95 was supplied, and the mother liquor was sequentially recycled and a purification process was performed in the same manner as in Example 3-1.

[0325] Example 3-4: When the molar ratio of added dodecanedioic acid / cadaverine in the process solution after the second step is 1.0

[0326] A cadaverine dodecanedioic acid conversion solution having a dodecanedioic 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 3-1.

[0327] In the case of Examples 3-3 to 3-4, the molar ratio of dodecanedioic acid / lysine in the lysine fermentation step was adjusted to 0.9-0.95.

[0328] Example 3-5: When the molar ratio of added dodecanedioic acid / cadaverine in the process solution after the second step is 1.04

[0329] A cadaverine dodecanedioic acid conversion solution having a dodecanedioic acid / cadaverine molar ratio of 1.04 was supplied, and the mother liquor was sequentially recycled and a purification process was performed in the same manner as in Example 3-1.

[0330] Example 3-6: When the molar ratio of added dodecanedioic acid / cadaverine in the process solution after the second step is 1.09

[0331] A cadaverine dodecanedioic acid conversion solution having a dodecanedioic acid / cadaverine molar ratio of 1.09 was supplied, and the mother liquor was sequentially recycled and a purification process was performed in the same manner as in Example 3-1.

[0332] The physical property measurements for the process solution and cadaverine dodecanedioic acid salt prepared according to Examples 3-1 to 3-6 described above are as shown in Tables 12 to 16 below.

[0333]

[0334] Table 12 is for the process solution that did not circulate the mother liquor.

[0335] No mother liquor circulation Example 3-1 Example 3-2 Example 3-3 Example 3-4 Example 3-5 Example 3-6 Dodecanedioic acid / cadaverine molar ratio 0.85 0.9 0.95 11.04 1.09 Crystallization feed New feed, ml 1000 1000 1000 1000 1000 1000 Amount of cadaverine in crystallization solution, g 166 166 166 166 166 166 Crystals produced, g No crystals produced 353740 3531 Mother liquor, g - 131129 127 131135 Result Purity, % - 9899998673 Yield, % - 2122242119 Total yield, % - 2122242119

[0336] Table 13 shows the process solution that has been circulated once.

[0337] Mother liquor circulation 1 time Example 3-1 Example 3-2 Example 3-3 Example 3-4 Example 3-5 Example 3-6 Dodecanedioic acid / cadaverine molar ratio 0.85 0.9 0.95 11.04 1.09 Crystallization feed solution New feed solution, ml-1000 1000 1000 1000 1000 Amount of cadaverine in crystallization solution, g-297295 293297301 Produced crystals, g-6266706356 Mother liquor, g-235229223234245 Resulting purity, %-9898998878 Yield, %-2122242119 Total yield, %-3840423834

[0338] Table 14 shows the process solution that was cycled twice.

[0339] Mother liquor circulation 2 times Example 3-1 Example 3-2 Example 3-3 Example 3-4 Example 3-5 Example 3-6 Dodecanedioic acid / cadaverine molar ratio 0.85 0.9 0.95 11.04 1.09 Crystallization feed solution New feed solution, ml - 1000 1000 1000 1000 1000 Amount of cadaverine in crystallization solution, g - 40 1395389 400 411 Produced crystals, g - 8489938577 Mother liquor, g - 317306297315334 Result Purity, % - 989999908 1 Yield, % - 2122242119 Total yield, % - 5153565146

[0340] Table 15 shows the process solution that was circulated three times.

[0341] Mother liquor circulation 3 times Example 3-1 Example 3-2 Example 3-3 Example 3-4 Example 3-5 Example 3-6 Dodecanedioic acid / cadaverine molar ratio 0.85 0.9 0.95 11.04 1.09 Crystallization feed solution New feed solution, ml - 1000 1000 1000 1000 1000 Amount of cadaverine in crystallization solution, g - 48347246348 1500 Produced crystals, g - 101 106 110 10294 Mother liquor, g - 382366353379406 Result Purity, % - 9899999 184 Yield, % - 2122242119 Total yield, % - 6164666 256

[0342] Table 16 shows the process solution after circulating the mother liquor four times.

[0343] Mother liquor circulation 4 times Example 3-1 Example 3-2 Example 3-3 Example 3-4 Example 3-5 Example 3-6 Dodecanedioic acid / cadaverine molar ratio 0.85 0.9 0.95 11.04 1.09 Crystallization feed New feed, ml - 1000 1000 1000 1000 1000 Amount of cadaverine in crystallization solution, g - 548 532 519 545 572 Crystals produced, g - No crystals produced 120 123 116 107 Mother liquor, g - 413395429 465 Result Purity, % - 99999 285 Yield, % - 22242119 Total yield, % - 72747065

[0344] In summary of the experimental results according to Tables 12 to 16 above, it was confirmed that the present invention's manufacturing method, in which the first-stage diammonium dodecanedioic acid salt is included in a large amount at 89-100 mol% of the total supplied dodecanedioic acid, enables the production of cadaverine dodecanedioic acid salt with an overall improved crystallization rate, high purity, and high yield. Specifically, in the case of Example 3-1, crystals were not precipitated even when the crystallization process was performed. This is because the molar ratio of dodecanedioic acid salt was low, which was not sufficient for the precipitation of crystals of cadaverine dodecanedioic acid salt.

[0345]

[0346] In Example 3-2, crystals were precipitated up to the third cycle of mother liquor circulation, but no crystals were formed in the fourth cycle. This is because the sequentially circulated mother liquor lowered the molar ratio of dodecanedioic acid salt to the crystallization feed liquid, and thus the concentration of dodecanedioic acid salt was not sufficient to precipitate crystals of cadaverine dodecanedioic acid salt.

[0347] In Example 3-5, the purity of the crystal was somewhat low at 86% when the mother liquor was not circulated, but the purity increased to 92% when the mother liquor was circulated 4 times.

[0348] In Example 3-6, the purity of the crystal was low at 73% when the mother liquor was not circulated, but the purity increased to 81% when the mother liquor was circulated 4 times.

[0349] Example 3-3 obtained 99% high-purity cadaverine dodecanedioic acid salt up to the fourth cycle of mother liquor circulation, and also achieved a high yield of approximately 72% in total yield.

[0350] In Example 3-4, 99% high-purity cadaverine dodecanedioic acid salt was obtained up to the fourth cycle of mother liquor circulation, and a high total yield of approximately 74% was achieved.

[0351]

[0352] In this way, it was confirmed that the purity of the cadaverine dodecanedioic acid salt produced can vary depending on the molar ratio of dodecanedioic acid and cadaverine in the conversion solution (process solution) supplied after the cadaverine conversion process. In particular, it was confirmed that when the amount of added dodecanedioic acid was adjusted to a molar ratio of about 0.95 to 1.00 with respect to the amount of cadaverine in the process solution after the second step, it was possible to maintain a purity of 99% of cadaverine dodecanedioic acid salt even after two or more cycles of mother liquor circulation while exhibiting an excellent total yield of 53% or more.

[0353]

[0354] 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 0.90 to 1.05 molar ratio to the amount of cadaverine in the process solution after the second step, The above dicarboxylic acid is an aliphatic dicarboxylic acid having 10 to 12 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 0.84 to 0.

96.

4. In paragraph 1, The above dicarboxylic acid salts and dicarboxylic acids are sebacic acid salts and sebacic 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 0.84-0.

86.

5. In paragraph 1, The above dicarboxylic acid salts and dicarboxylic acids are undecanedioic acid salts and undecanedioic 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 0.84-0.

90.

6. In paragraph 1, The above dicarboxylic acid salts and dicarboxylic acids are dodecanedioic acid salts and dodecanedioic 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 0.86-0.

96.

7. 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.

8. In paragraph 1, The above dicarboxylic acid salts and dicarboxylic acids are sebacic acid salts and sebacic acid, A method for producing cadaverine dicarboxylate, wherein the amount of added dicarboxylic acid is a molar ratio of 0.96 to 1.00 relative to the amount of cadaverine in the process solution after the second step.

9. In paragraph 1, The above dicarboxylic acid salts and dicarboxylic acids are undecanedioic acid salts and undecanedioic acid, A method for producing cadaverine dicarboxylate, wherein the amount of added dicarboxylic acid is a molar ratio of 0.95 to 1.00 relative to the amount of cadaverine in the process solution after the second step.

10. In paragraph 1, The above dicarboxylic acid salts and dicarboxylic acids are dodecanedioic acid salts and dodecanedioic acid, A method for producing cadaverine dicarboxylate, wherein the amount of added dicarboxylic acid is a molar ratio of 0.95 to 1.00 relative to the amount of cadaverine in the process solution after the second step.

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

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

13. In paragraph 12, 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.

14. In the 12th 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.

15. A method for producing cadaverine dicarboxylate, wherein the concentration step is performed so that the solid content becomes 45 to 70% (w / w) in the 13th paragraph.

16. 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.

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

18. 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.

19. In paragraph 13, A method for producing cadaverine dicarboxylate, wherein cadaverine dicarboxylate having a purity of 98% or higher can be obtained in the 0th to 4th cycle of the above-mentioned mother liquor.

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