Cadavering dicarboxylate production process

Abstract

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

Description

Technical Field

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

Background Art

[0002] Cadaverine is a toxic diamine compound that emits a foul odor generated by the decay of animal tissues. Unlike hexanediamine, cadaverine exists in a liquid state at room temperature and more readily absorbs carbon dioxide, an acidic gas in the air, causing many difficulties in transportation and storage. Therefore, salt-forming crystallization not only obtains high-quality monomers but is also easy for transportation and storage.

[0003] On the other hand, cadaverine sebacate, cadaverine undecanedioate, and cadaverine dodecanedioate are polymerization precursors of bio-polyamide (polyamide), and bio-polyamide is one of the engineering plastics widely used in automobiles, electrical and electronic components, etc.

[0004] The preparation process of cadaverine dicarboxylate generally includes the following steps:

[0005] Synthesis of cadaverine: Cadaverine synthesis involves the fermentation of an appropriate microbial strain that can produce 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 dicarboxylic acid in the presence of an appropriate catalyst. The reaction typically includes mixing both monomers at an appropriate temperature and stirring for a specific period to ensure a complete reaction.

[0007] Purification of cadavering dicarboxylate: Once the reaction is complete, the cadavering dicarboxylate, the starting material, is purified using various separation and purification techniques such as solvent extraction, chromatography, and crystallization.

[0008] According to conventional technology, sebacate, undecanediate, and dodecanediate have low solubility, and during the crystallization process described above, there was a problem in that the dicarboxylate salts (sebacate, undecanediate, or dodecanediate) precipitated as crystals along with cadaverine dicarboxylate (cadaverine sebacate, cadaverine undecanediate, or cadaverine dodecanediate). Therefore, in order to produce high-purity cadaverine dicarboxylate, the dicarboxylate salts had to be removed before crystallization, which resulted in problems such as increased costs due to the additional steps required to remove the dicarboxylate salts, decreased process efficiency, and environmental pollution due to the use of chemical solvents. [Prior art documents] [Patent Documents]

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

[0010] The problem that this application aims to solve is to provide a method for producing cadaverine dicarboxylates (cadaverine sebacinate, cadaverine undecane diate, or cadaverine dodecane diate). [Means for solving the problem]

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

[0012] According to one aspect of this application, L-lysine-producing microorganisms are cultured in a medium containing diammonium dicarboxylate to obtain lysine dicarboxylate, and the lysine dicarboxylate is enzymatically converted to cadaverine dicarboxylate. This eliminates the need for a separate step to remove the dicarboxylate, thus simplifying the process while preventing dicarboxylic acid crystals from precipitating as by-products, and enabling the production of high-purity C10, C11, or C12 cadaverine dicarboxylate in high yield. [Brief explanation of the drawing]

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

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

[0015] One aspect of this application provides a method for producing cadaverine dicarboxylate, comprising a first step of culturing an L-lysine-producing microorganism in a medium supplemented with a dicarboxylic acid in the form of a diammonium dicarboxylate to obtain a lysine dicarboxylate, and a second step of converting the lysine dicarboxylate to a cadaverine dicarboxylate.

[0016] In this application, the term "dicarboxylic acid" means a dibasic acid containing two carboxyl groups, and is of high importance as a metabolic intermediate in living organisms. 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 means one aliphatic dicarboxylic acid selected from sebacic acid, undecanedioic acid, and dodecanedioic acid. If the cadaverine dicarboxylate salt to be produced by the manufacturing method of this application is cadaverine sebacate, the added or introduced dicarboxylic acid must be understood to be entirely sebacic acid. If the cadaverine dicarboxylate salt to be produced by the manufacturing method of this application is cadaverine undecanedioic acid, the added or introduced dicarboxylic acid must be understood to be entirely undecanedioic acid. If the cadaverine dicarboxylate salt to be produced by the manufacturing method of this application is cadaverine dodecanedioic acid, the added or introduced dicarboxylic acid must be understood to be entirely dodecanedioic acid. Thus, in this application, it is understood that "dicarboxylic acid" refers to a single aliphatic dicarboxylic acid, not a mixture of different types of dicarboxylic acids.

[0017] In this application, the term "sebacic acid" refers to a dicarboxylic acid having 10 carbon atoms, C 10 H 18 It has the chemical formula O4 and generally exists as a white powder solid. Sebacic acid, also known as sebaic acid or sebasic acid, is used as a precursor in a variety of industrial products, including polymers and plasticizers.

[0018] In this application, the term "undecanedioic acid" refers to a dicarboxylic acid with 11 carbon atoms and has a chemical formula of C 11 H 20 O4. The undecanedioic acid is utilized as a precursor for various industrial products including polymers and plasticizers.

[0019] In this application, the term "Dodecanedioic acid" refers to a carboxylic acid with 12 carbon atoms and has a chemical formula of C 12 H 22 O4. The dodecanedioic acid is also named dodecane diacid.

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

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

[0022] Figures 1 and 2 are flowcharts showing the manufacturing process of cadaverine dicarboxylate according to an aspect of this application.

[0023] In this application, the term "L-lysine-producing microorganism" includes all wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and refers to microorganisms in which a specific mechanism has been weakened or strengthened due to causes such as the insertion of external genes or the enhancement or inactivation of the activity of endogenous genes, and may also refer to microorganisms that have undergone genetic modification for the production of L-lysine.

[0024] For example, the L-lysine-producing microorganisms of this application may be, but are not limited to, microorganisms that naturally possess L-lysine-producing ability, microorganisms that lack L-lysine-producing ability but have been conferred with L-lysine-producing ability, or microorganisms that have significantly low L-lysine-producing ability but have been enhanced with L-lysine-producing ability. Specifically, the L-lysine-producing microorganisms or microorganisms that possess L-lysine-producing ability in this application may be microorganisms in which a portion of the genes in the L-lysine biosynthesis pathway is strengthened or weakened, or a portion of the genes in the L-lysine degradation pathway is strengthened or weakened. "Strengthened" or "increased" L-lysine-producing ability means that the L-lysine-producing ability has improved compared to the parent strain or non-mutant microorganisms.

[0025] As a specific example, the microorganism may be a microorganism of the genus Corynebacterium or a microorganism of the genus Escherichia. Microorganisms of the genus Corynebacterium can include all microorganisms belonging to the genus Corynebacterium. Specifically, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes This may include *Corynebacterium ammoniagenes*, *Corynebacterium pollutisoli*, *Corynebacterium imitans*, *Corynebacterium testudinoris*, or *Corynebacterium flavescens*, and more specifically, *Corynebacterium glutamicum*, but is not limited thereto. Microorganisms of the genus *Escherichia* can include all microorganisms belonging to the genus *Escherichia*.Specifically, these may be Escherichia coli, Escherichia albertii, Escherichia blattae, Escherichia fergusonii, Escherichia hermannii, or Escherichia vulneris, and more specifically, the Escherichia strain may be Escherichia coli, but is not limited to these.

[0026] The culture of the L-lysine-producing microorganism can be carried out according to suitable culture media and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art depending on the selected microorganism. Specifically, the culture may be batch, continuous, or fed-batch, but is not limited thereto.

[0027] In this application, the term "medium" means a substance mixed mainly with nutrients necessary for culturing the microorganisms, supplying nutrients and growth factors, including water which is indispensable for survival and growth. Specifically, the medium used for culturing the L-lysine-producing microorganisms in this application can be any medium used for culturing ordinary microorganisms without particular limitations, except that it contains diammonium dicarboxylate as part or all of the nitrogen source. The microorganisms in this application can be cultured in an ordinary medium containing a nitrogen source including diammonium dicarboxylate, a suitable carbon source, a phosphorus source, inorganic compounds, amino acids and / or vitamins, under aerobic conditions while adjusting the temperature, pH, etc.

[0028] The first step of the method for producing cadaverine dicarboxylate according to this application is to culture L-lysine-producing microorganisms in a medium to which dicarboxylic acid has been added in the form of diammonium dicarboxylate to obtain lysine dicarboxylate. This is carried out through a fermentation process of the lysine-producing microorganisms.

[0029] In the first step, the dicarboxylic acid is supplied to the culture medium in the form of a diammonium dicarboxylate salt, and it should be understood that in this application, the dicarboxylic acid includes the form of a diammonium dicarboxylate salt.

[0030] In this application, the term "diammonium dicarboxylate" refers to a form in which two hydrogen atoms of a dicarboxylic acid are replaced by ammonium, and is used as the primary nitrogen source for the culture medium. The diammonium dicarboxylate can be used in crystalline or liquid form after being gently mixed with aqueous ammonia to prepare a neutral pH, followed by concentration.

[0031] The manufacturing method described in this application simplifies the process while producing high-purity cadaverine dicarboxylate by adding 81 mol% or more of the dicarboxylic acid supplied throughout the entire process to the culture medium in the form of diammonium dicarboxylate in the first step, thereby reducing the side effect of dicarboxylate precipitation together with cadaverine dicarboxylate.

[0032] Specifically, the diammonium dicarboxylate salt in the first step may be included in the culture medium in a high proportion, specifically 41 mol% or more, relative to the dicarboxylic acid supplied throughout the entire process of the manufacturing method of this invention.

[0033] More specifically, the diammonium dicarboxylate in the first step may be present in amounts 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 all steps of the manufacturing method of this application.

[0034] The manufacturing method described in this application, by including a large amount of diammonium dicarboxylate in the culture medium, offers the advantage of being able to produce high-purity cadaverine dicarboxylate without performing separate steps to remove the dicarboxylate, such as ion resin exchange, decarboxylation, or distillation, unlike conventional processes that add ammonium sulfate or the like. Furthermore, by using diammonium dicarboxylate, it is possible to easily adjust the molar ratio of dicarboxylic acid added throughout the entire process to cadaverine in the process solution after the second step.

[0035] Specifically, the diammonium dicarboxylate in the first step may be included in such a molar ratio (diammonium dicarboxylate / lysine) with respect to the lysine to be produced that is 0.7 to 1.11. More specifically, the diammonium dicarboxylate in the first step may be included in such a molar ratio (diammonium dicarboxylate / lysine) with respect to the lysine to be produced that is 0.74 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, or more specifically, 0.84 to 0.96. In this case, the amount of lysine to be produced should be understood to refer to the total amount including all forms of lysine dicarboxylate.

[0036] For example, the diammonium dicarboxylate in the first step may be included in a molar ratio (diammonium dicarboxylate / lysine) with respect to the lysine to be produced, within a range defined by one 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 one 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.

[0037] In one specific example, the dicarboxylate and dicarboxylic acid are sebacate and sebacate, and the diammonium dicarboxylate in the first step may be included in such a way that its molar ratio (diammonium dicarboxylate / lysine) with the lysine to be produced is 0.84 to 0.86.

[0038] In one specific example, the dicarboxylate and dicarboxylic acid are undecane diate and undecane diic acid, and the diammonium dicarboxylate in the first step may be included in such a way that its molar ratio (diammonium dicarboxylate / lysine) with the lysine to be produced is 0.84 to 0.90.

[0039] In one specific example, the dicarboxylate and dicarboxylic acid are dodecane diate and dodecane diic acid, and the diammonium dicarboxylate in the first step may be included in such a way that its molar ratio (diammonium dicarboxylate / lysine) with the lysine to be produced is 0.86 to 0.96.

[0040] Specifically, the diammonium dicarboxylate in the first step can be included in a concentration of 0.3 mol / L to 0.5 mol / L based on the L-lysine-producing microbial fermentation broth. More specifically, the diammonium dicarboxylate in the first step can be included in a concentration of 0.3 mol / L to 0.45 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 L-lysine-producing microbial fermentation broth. The diammonium dicarboxylate can be added at the above concentrations so that the ammonium ions act as a nitrogen source during fermentation, and the dicarboxylic acid and lysine form a salt, which may be included in the fermentation broth in a dissolved state.

[0041] Lysine dicarboxylate can be produced through fermentation by L-lysine-producing microorganisms in a culture medium containing diammonium dicarboxylate. The lysine dicarboxylate can be added to subsequent processes in the form of a fermentation liquid containing microbial cells, a fermentation liquid from which the microbial cells have been removed, or after concentration or purification.

[0042] The concentration of lysine in the fermentation liquid obtained by culturing the lysine-producing microorganisms in the first step of this application is not limited, but may be 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 L-lysine-producing microorganism fermentation liquid.

[0043] The carbon sources in the culture medium may include carbohydrates such as glucose, fructose, sucrose, maltose, and their isomers; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn maceration can also be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and other appropriate amounts of various carbon sources can be used without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited to these uses.

[0044] As a specific example, the carbon source that may be included in the culture medium of this application may include glucose, maltose, or maltose isomers. More specifically, it may include, but is not limited to, one or more selected from glucose, maltose, and maltose isomers (isomaltose), or a combination of two or more.

[0045] The phosphorus source in the culture medium may include monopotassium phosphate, dipotassium phosphate, or corresponding sodium-containing salts. Inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate.

[0046] Furthermore, the culture medium may contain metal salts such as magnesium sulfate or iron sulfate necessary for growth. Finally, essential growth substances such as amino acids and vitamins can be used in addition to the aforementioned substances. Appropriate precursors can also be used in the culture medium. The raw materials may be added to the culture during the culture process in an appropriate manner, for example, in batches or continuously, but are not limited thereto.

[0047] The nitrogen source of the culture medium may include diammonium dicarboxylate. Conventional culture media for microbial culture may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; and organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn maceration, casein hydrolysates, fish or their degradation products, defatted soy cake or its degradation products. In one embodiment of this application, diammonium dicarboxylate may be included as a sole nitrogen source or as an additional nitrogen source.

[0048] After the first step of culturing L-lysine-producing microorganisms in a medium containing diammonium dicarboxylate to produce lysine dicarboxylate, a second step may be performed to convert the lysine dicarboxylate to cadaverine dicarboxylate.

[0049] The second step is to convert the lysine dicarboxylate into the target product, cadaverine dicarboxylate (cadaverine sebacate, cadaverine undecane diate, or cadaverine dodecane diate).

[0050] In one specific example, the second step may include further adding a dicarboxylic acid to the process liquid. The process liquid may be the fermentation process liquid obtained in the first step.

[0051] The dicarboxylic acid added in the second step may be omitted, or added in amounts 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 in trace amounts of 0.01 mol / L or less, based on the volume of the fermentation liquid obtained in the first step.

[0052] In one specific example, if 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.

[0053] In one specific example, if the dicarboxylic acid is undecanediic acid, the dicarboxylic acid added in the second step may be added in a trace amount of 0.01 mol / L to 0.06 mol / L, for example, 0.02 mol / L to 0.04 mol / L.

[0054] In one specific example, if 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.

[0055] If a dicarboxylic acid is further added to the process solution in the second step, the dicarboxylic acid may, but is not limited to, be added before, after, or simultaneously with the conversion reaction.

[0056] If dicarboxylic acid is further added in the second step, the total amount of dicarboxylic acid added throughout the entire process of the present invention is understood to be the sum of the amount of diammonium dicarboxylate added to the culture medium in the first step and the amount of dicarboxylic acid added in the second step. Also, if no further dicarboxylic acid is added to the process solution in the second step, the total amount of dicarboxylic acid added throughout the entire process of the present invention is understood to be equal to the amount of diammonium dicarboxylate added to the culture medium in the first step.

[0057] Specifically, the amount of dicarboxylic acid added in the manufacturing method of this application can be added considering the amount of cadaverine in the process solution obtained after the second step. Specifically, the amount of dicarboxylic acid added in the manufacturing method of this application may be in a molar ratio of 0.85 to 1.12 compared to the amount of cadaverine in the process solution after the second step.

[0058] More specifically, the amount of dicarboxylic acid added in the manufacturing method of this application may be in 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 compared to the amount of cadaverine in the process solution after the second step.

[0059] As an example, the amount of dicarboxylic acid added in the manufacturing method of this application may be in a molar ratio within a range consisting of one lower limit selected from 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 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.

[0060] In one specific example, if the dicarboxylic acid is sebacic acid, the amount of dicarboxylic acid added in the production method of this application may be in 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 compared to the amount of cadaverine in the process solution after the second step.

[0061] One technical significance of this invention lies in the discovery that, in the manufacturing method of this application, when sebacic acid is added in a molar ratio within the aforementioned range relative to the amount of cadaverine calculated to be obtained after the second step, the sebacic acid does not precipitate as crystals together with cadaverine sebacate, making it possible to produce cadaverine sebacate with high purity and high yield.

[0062] Furthermore, by adjusting the molar ratio of the obtained cadaverine to the added sebacic acid, it is possible to maintain a purity of 98% or more of cadaverine sebacate up to the fourth cycle of mother liquor circulation, while obtaining cadaverine sebacate with 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 mother liquor circulations.

[0063] In one specific example, if the dicarboxylic acid is undecanediic acid, the amount of dicarboxylic acid added in the production method of this application may be in 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 compared to the amount of cadaverine in the process solution after the second step.

[0064] One technical significance of this invention lies in the discovery that, in the manufacturing method of this application, when undecane dioic acid is added in a molar ratio within the aforementioned range relative to the amount of cadaverine calculated to be obtained after the second step, the undecane dioic acid does not precipitate as crystals together with cadaverine undecane dioate, making it possible to produce cadaverine undecane dioate in high purity and high yield.

[0065] Furthermore, by adjusting the molar ratio of the obtained cadaverine to the added undecanediic acid, it is possible to maintain a purity of 99% or more of cadaverine undecanediate up to the 5th cycle of mother liquor circulation, while obtaining cadaverine undecanediate with 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 mother liquor cycles.

[0066] In one specific example, if the dicarboxylic acid is dodecanedioic acid, the amount of dodecanedioic acid added in the production method of this application may be in 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 compared to the amount of cadaverine in the process solution after the second step.

[0067] One technical significance of this invention lies in the discovery that, in the manufacturing method of this application, when dodecane dioic acid is added in a molar ratio within the aforementioned range relative to the amount of cadaverine calculated to be obtained after the second step, the dodecane dioic acid does not precipitate as crystals together with cadaverin dodecane dioate, making it possible to produce cadaverin dodecane dioate in high purity and high yield.

[0068] Furthermore, by adjusting the molar ratio of the obtained cadaverine to the added dodecanediic acid, it is possible to maintain a purity of 98% or more of cadaverine dodecanediic acid up to the fourth circulation of the mother liquor, while obtaining cadaverine sebacate with 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 mother liquor circulations.

[0069] In the second step, the reaction to convert the lysine dicarboxylate to cadaverine dicarboxylate may be an enzymatic conversion reaction.

[0070] As one specific example, the lysine dicarboxylate salt may include the lysine dicarboxylate salt obtained in the first step, and, if a dicarboxylic acid is further added in the second step, all of the lysine dicarboxylate salt produced in the second step.

[0071] Specifically, the enzymatic conversion reaction in the second step may be carried out using a protein having lysine decarboxylase activity or a microorganism expressing such a protein.

[0072] As one specific example, a protein having lysine decarboxylase activity can be added to the fermentation liquid in the first step, or a seed culture of a microorganism expressing the said active protein can be added, but the method is not limited to this.

[0073] In this application, the term "decarboxylase" refers to an enzyme that catalyzes the formation of carbon dioxide by removing the carboxyl group, which is an organic acid, and is also known as a decarboxylase, decarboxylase, or carbonic acid release enzyme.

[0074] The aforementioned protein is not particularly limited as long as it exhibits lysine decarboxylase activity, but for example, it may be Pseudomonas thermotolerans PtLDC protein or CadA protein derived from Escherichia coli. However, it is not limited to obtaining the protein sequence using the known database GenBank, expressing it using the aforementioned microorganism, or purchasing and using commercially available enzymes.

[0075] The microorganism expressing the protein may be a microorganism that has been transformed to express the protein. The transformed microorganism is not limited to prokaryotic or eukaryotic microorganisms, as long as it has been transformed to express a protein having decarboxylase activity. The microorganism transformed to express a protein having decarboxylase activity can excrete the protein having the enzyme activity into the seed culture medium and convert the lysine dicarboxylate in the process solution into cadaverine dicarboxylate.

[0076] Specific examples may include strains of microorganisms belonging to the genera Escherichia, Erwinia, Serratia, Providencia, and Corynebacterium. Specifically, these microorganisms may be microorganisms belonging to the genera Escherichia or Corynebacterium, and more specifically, they may be Escherichia coli or Corynebacterium glutamicum, but are not limited to these.

[0077] The conversion reaction in the second step may take place for 20 minutes to 3 hours, more specifically, for 0.5 to 1.5 hours, or for example, for 1 hour, but is not limited to these durations.

[0078] The conversion reaction in the second step may be carried out at 30-60°C, more specifically at 40-55°C, or, for example, at 45-50°C, but is not limited thereto.

[0079] The conversion reaction in the second step may be carried out at a pH of 7.5 to 9, more specifically at 7.8 to 8.7, and more specifically at 8 to 8.5. In one embodiment of the second step, the pH can be adjusted to the above range by selectively adding dicarboxylic acid, and in another embodiment, the pH can be adjusted to the above range by adding CO2. When the pH range of the conversion reaction in the second step is adjusted to the above range, the conversion rate of lysine dicarboxylate to cadaverine dicarboxylate can be significantly increased.

[0080] The method for producing cadavering dicarboxylate according to this application may further include a recovery step of recovering the obtained cadavering dicarboxylate after carrying out the second step.

[0081] The recovery may involve collecting the cadavering dicarboxylate using appropriate methods known in the art.

[0082] For example, cadaverine dicarboxylates can be collected by various chromatography methods such as centrifugation, filtration, concentration, crystallization, extraction, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination of these methods.

[0083] In one specific example of this application, the recovery step may include one or more of the following steps: a filtration step, a concentration step, and a crystallization step.

[0084] The filtration step refers to the step of removing impurities from the conversion solution containing cadavering dicarboxylate obtained in the second step. As an example, the filtration may include removing microbial cells in the cadavering dicarboxylate process solution by membrane separation or by activated carbon filtration.

[0085] The aforementioned concentration step refers to a step in which the solid content ratio in the conversion solution is increased, and may be performed after the filtration step. For example, the concentration may be performed by vacuum concentration using a rotary evaporator.

[0086] Specifically, the concentration step may involve concentrating the solids content to 45-70% (w / w), and more specifically, concentrating to 46-68% (w / w), 48-67% (w / w), 45-65% (w / w), or 50-70% (w / w). For example, it may involve concentrating to 45-55% (w / w). For example, it may involve concentrating to 60-70% (w / w).

[0087] When the solid content is concentrated within the range described above in the concentration step, the recovery rate and purity of the cadavering dicarboxylate can be well maintained.

[0088] The concentration step may be carried out at 45-80°C, for example, 55-70°C, but is not limited thereto.

[0089] The crystallization step may also be a cooling crystallization step, which means cooling the conversion solution to precipitate it as crystals and obtain crystals of the final target product, cadaverine dicarboxylate.

[0090] Specifically, the cooling may involve cooling the conversion solution to 30°C or below, and more specifically, it may involve cooling to 27°C or below, 25°C or below, 23°C or below, 20°C or below, or even down to 10°C, but is not limited thereto.

[0091] Specifically, the cooling rate may be 0.1 to 20°C / hour, more specifically, 0.5 to 1.5°C / hour, or, as an example, 1°C / hour, but is not limited to these.

[0092] In one embodiment, the recovery step may further include a stirring step of stirring the process liquid. The stirring step may be performed simultaneously with the cooling crystallization step, in the middle of the cooling crystallization step, before the cooling crystallization step, or after the cooling crystallization step. Such a stirring step allows sufficient time to stir the process liquid, making crystal precipitation easier.

[0093] In one embodiment, the recovery step may further include a mother liquor circulation step in which the mother liquor containing the uncrystallized cadavering dicarboxylate after the crystallization step is recirculated with the feed liquid of the concentration step.

[0094] Specifically, in the mother liquor circulation step, the number of times the mother liquor is circulated may be 0 to 3 times, 1 to 3 times, 1 to 4 times, 1 to 5 times, 1 to 10 times, or more. In this case, a number of times the mother liquor is 0 means that the mother liquor circulation step is not performed.

[0095] Conventionally, in the step of crystallizing cadavering dicarboxylate, a relatively large amount of cadavering dicarboxylate remains in the mother liquor after crystallization, resulting in a low yield of less than 50%. In this application, as a method to improve the yield of cadavering dicarboxylate, the total yield of cadavering dicarboxylate can be increased by recirculating the residual mother liquor of cadavering dicarboxylate one to three times, one to four times, one to five times, one to ten times or more in the feed solution of the concentration step.

[0096] In one embodiment, the recovery step may further include washing, separating, and drying the crystallized process liquid.

[0097] In the drying step described above, moisture contained in the crystals is removed through drying, and a highly pure cadavering dicarboxylate can be produced as a product. After drying, the cadavering dicarboxylate crystals can be provided in powder form, but the dosage form can be varied as needed.

[0098] In one embodiment, the recovery step may further include a decolorization step. The decolorization can be performed using activated carbon, anionic resin, etc., but is not limited thereto.

[0099] Furthermore, the method for producing cadavering dicarboxylate according to this application may include an additional purification step. The purification can be carried out using a suitable method known in the art. For example, if the method for producing cadavering dicarboxylate according to this application includes both a recovery step and a purification step, the recovery step and the purification step may be carried out sequentially or discontinuously, simultaneously or in a single step, regardless of the procedure, but are not limited thereto.

[0100] The manufacturing method of this application is characterized by not including a separate step for removing the dicarboxylate in the recovery step. Conventionally, in the production process of cadaverine dicarboxylate, the dicarboxylate precipitates as crystals together with the crystal during the crystallization process, reducing purity. To prevent this, a separate step was required to remove the dicarboxylate before crystallization. However, the manufacturing method of this application offers the technical advantage of being able to produce high-purity cadaverine dicarboxylate without performing a separate step for removing the dicarboxylate.

[0101] In one embodiment, when the dicarboxylic acid is sebaic acid, the method of this application is characterized by obtaining cadaverine sebacate with a purity of 99% or more in 0 to 4 cycles of mother liquor circulation. In one embodiment, when the dicarboxylic acid is sebaic acid, the cumulative total yield of cadaverine sebacate may be 70% or more based on 4 cycles of mother liquor circulation, and more specifically, it may be greater than 70%, 71% or more, 72% or more, 73% or more, or 74% or more.

[0102] In one embodiment, when the dicarboxylic acid is undecanediic acid, the method of this application is characterized by obtaining cadaverine undecanediic acid with a purity of 99% or more in the 0th to 5th cycles of the mother liquor. In one embodiment, when the dicarboxylic acid is undecanediic acid, the cumulative total yield of cadaverine undecanediic acid may be greater than 68%, for example 70% or more, based on the 5th cycle of the mother liquor, and the purity may be 99% or more.

[0103] In one embodiment, when the dicarboxylic acid is dodecanedioic acid, the method of this application can be characterized by obtaining cadaverindodecanediate with a purity of 98% or more after 0 to 4 cycles of mother liquor circulation. As an example, it can be characterized by obtaining cadaverindodecanediate with a purity of 99% or more after 2 to 4 cycles of mother liquor circulation. In one embodiment, when the dicarboxylic acid is dodecanedioic acid, the cumulative total yield of cadaverindodecanediate may be 70% or more based on 4 cycles of mother liquor circulation, and more specifically, it may be greater than 70%, 71% or more, or 72% or more.

[0104] In one embodiment, the method of this application is characterized by obtaining a cadavering dicarboxylate with a total yield of 22% or more and a purity of 98% or more in 0 to 4 cycles of the mother liquor.

[0105] Through the method of this application, high-purity cadaverine dicarboxylate can be produced without the decarboxylation and distillation steps that are normally required in the production of cadaverine liquid phase.

[0106] Examples The present application will be described in more detail below with reference to experimental examples. However, the following embodiments are merely preferred embodiments for illustrative purposes of the present application and are not intended to limit the scope of the rights of this application. On the other hand, technical matters not described herein can be fully understood and easily implemented by a person of ordinary skill who is skilled in the art of this application or a similar art.

[0107] The above describes a manufacturing process for cadaverine dicarboxylates (cadaverine sebacate, cadaverine undecane diate, or cadaverine dodecane diate) according to one aspect of this application. Below, the advantageous effects referred to in this application will be described through experimental results of examples and comparative examples of cadaverine sebacate, cadaverine undecane diate, or cadaverine dodecane diate.

[0108] 1. Cadaverine sebacinate Production Example 1-1. Fermentation process for the production of lysine sebacate salt. Corynebacterium glutamicum strain (KCCM12154P, publication number US 2021-0355514 A1) capable of lysine production was obtained through solid-phase culture and flask culture, and after seed culture in a fermenter, the main production fermentation was carried out. Fermentation was performed at 36°C for 63 hours at 900 rpm via fermenter culture.

[0109] For the production of lysine sebacate using Corynebacterium, ammonium sebacate was supplied at a level of 92-94 g / L to replace existing ammonium sulfate in each example. Repeated fed-batch fermentation was performed with the concentration of the carbon source in the culture medium reduced to 1.2 mol to maintain the balance of carbon source / nitrogen source. As a result, lysine sebacate was obtained at a level of 136-139 g / L, and lysine at a level of 68 g / L. At that time, the molar ratio of sebatic acid / lysine in the fermentation liquid was adjusted to a level of 0.83-0.85 in each example.

[0110] Manufacturing Example 1-2. Conversion reaction of lysine sebacate to cadaverine sebacate. For the conversion reaction, we used Escherichia coli (US 2018-0030430 A1) in which the PtLDC enzyme, a lysine decarboxylase gene derived from Pseudomonas thermotolerans, was overexpressed using the pET-Deut1 vector.

[0111] The fermentation liquid of lysine sebacate produced from Production Example 1-1 was converted using an enzyme-converted solution cultured with the enzyme strain, at a concentration of 10% by mass. The conversion reaction was carried out by supplying this solution to the fermentation liquid of lysine sebacate produced from Production Example 1-1 at a concentration of 10% by mass. The conversion reaction temperature was maintained at 45-50°C, and the pH was adjusted to 8.0-8.5. For this reason, in Examples 1-1 to 1-4 below, CO2 was added to neutralize the pH. On the other hand, in Examples 1-2 to 1-6, sebaic acid was added at a concentration of 0.01-0.06 mol / L (based on the volume of the fermentation liquid of lysine sebacate) before the conversion reaction. As a result, it was confirmed that the conversion rate of cadaverine sebacate was at a level of 97-98%. Specifically, after the conversion reaction, cadaverine sebacate was obtained at a level of 119-140 g / L (based on the volume of the fermentation liquid of lysine sebacate), and cadaverine was obtained at a level of 47 g / L (based on the volume of the fermentation liquid of lysine sebacate). Subsequently, in Examples 1-5 and 1-6, sebaic acid was added at a level of 0.02-0.05 mol / L (based on the volume of the fermentation liquid of lysine sebacate).

[0112] Manufacturing Example 1-3. Subsequent steps after obtaining cadaverine sebacate The following subsequent steps were performed on the process solution containing cadaverine sebacate obtained in Production Example 1-2 to obtain cadaverine sebacate in crystalline form.

[0113] [Bacterial cell removal step] Bacterial cells in the cadaverine sebacate process solution were removed by membrane filtration using a 0.1 μm membrane.

[0114] [Steps to remove impurities using activated carbon] Activated carbon was added to the filtrate after bacterial cell isolation at a level of 10% based on the weight of cadaverine sebacate. The process solution with the added activated carbon was heated to 60°C and stirred for 1 hour to decolorize it, after which the activated carbon was filtered through filter paper.

[0115] [Step to concentrate the process solution of cadaverine sebacate from which impurities have been removed] The filtered liquid was concentrated in a rotary evaporator under reduced pressure at approximately 55-70°C and 120 Torr until the solid content of the filtrate was 50% by weight.

[0116] [Steps to cool, crystallize, separate, and dry a concentrated cadaverine sebacate solution] The 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 10% water based on the weight of cadaverine sebacate. After drying the separated crystals for one day, their purity was measured by HPLC.

[0117] [Step of recirculating the mother liquor in the crystallization step] The mother liquor separated as described above was recycled in a step to concentrate the process solution of cadaverine sebacate from which impurities had been removed.

[0118] Experimental Example 1-1. Confirmation of changes in the purity and yield of cadaverine sebacate in the conversion solution of cadaverine sebacate according to the molar ratio of sebacate to cadaverine. In Experimental Example 1-1, the methods described in Production Examples 1-1 to 1-3 were used, and the purity and yield of cadaverine sebacate were examined to see how they changed depending on the molar ratio of sebacate to cadaverine in the conversion solution.

[0119] Example 1-1: When the molar ratio of added sebacic acid / cadaverine in the process solution after the second step is 0.85 Microorganisms were removed by passing 1,000 ml of a cadaverine sebacate conversion solution, in which the sebacate / cadaverine molar ratio was 0.85, through a 0.1 μm-sized membrane. The cadaverine sebacate conversion solution, in which the sebacate / cadaverine molar ratio was 0.85, was prepared according to the manufacturing methods of Production Example 1-1 and Production Example 1-2, but in Production Example 1-1, the amount of sebacate added to the ammonium sebacate in the fermentation process for producing lysine and the amount of sebacate added in Production Example 1-2, the cadaverine sebacate conversion reaction, were controlled so that the total amount of sebacate added was 0.85 times the number of moles of converted cadaverine.

[0120] The filtrate was decolorized using activated carbon, filtered through filter paper, and then concentrated under reduced pressure to a level of 50% based on solid content weight. The concentrate was cooled and crystallized from 50°C to 25°C. The crystals and mother liquor were separated by centrifugation, and the separated crystals were dried for one day, after which their purity was measured by HPLC. The mother liquor, which still contained sebacate, was recirculated with a feed solution of cadaverine sebacate with a sebacate / cadaverine molar ratio of 0.85, and the crystallization step was repeated. The recirculation of the mother liquor was carried out sequentially.

[0121] Examples 1-2: When the molar ratio of added sebacic acid / cadaverine in the process solution after step 2 is 0.89 Upon receiving a conversion solution of cadaverine sebacate with a sebacate / cadaverine molar ratio of 0.89, the mother liquor was sequentially recycled and the purification process proceeded, as in Example 1-1.

[0122] Examples 1-3: When the molar ratio of added sebacic acid / cadaverine in the process solution after step 2 is 0.96 Upon receiving a conversion solution of cadaverine sebacate with a sebacate / cadaverine molar ratio of 0.96, the mother liquor was sequentially recycled and the purification process proceeded, as in Example 1-1.

[0123] Examples 1-4: When the molar ratio of added sebacic acid / cadaverine in the process solution after step 2 is 1.0 Upon receiving a conversion solution of cadaverine sebacate with a sebacate / cadaverine molar ratio of 1.0, the mother liquor was sequentially recycled and the purification process proceeded, as in Example 1-1.

[0124] In Examples 1-3 to 1-4, the molar ratio of sebacic acid to lysine in the lysine fermentation step was adjusted to 0.85.

[0125] Examples 1-5: When the molar ratio of added sebacic acid / cadaverine in the process solution after step 2 is 1.04 The mother liquor was sequentially recycled in the same manner as in Example 1, after receiving a conversion solution of cadaverine sebacate with a sebacate / cadaverine molar ratio of 1.04, and the purification process was carried out.

[0126] Examples 1-6: When the molar ratio of added sebacic acid / cadaverine in the process solution after step 2 is 1.11 Upon receiving a conversion solution of cadaverine sebacate with a sebacate / cadaverine molar ratio of 1.11, the mother liquor was sequentially recycled and the purification process proceeded, as in Example 1-1.

[0127] The physical properties measured for the process solutions and cadaverine sebacate produced by the above-described Examples 1-1 to 1-6 are shown in Tables 1 to 5.

[0128] Table 1 relates to process fluids that do not circulate the mother liquor.

[0129] [Table 1]

[0130] Table 2 shows the process fluid after the mother liquor has been circulated once.

[0131] [Table 2]

[0132] Table 3 shows the process fluid after the mother liquor has been circulated twice.

[0133] [Table 3]

[0134] Table 4 shows the process fluid after the mother liquor has been circulated three times.

[0135] [Table 4]

[0136] Table 5 shows the process fluid after the mother liquor has been circulated four times.

[0137] [Table 5]

[0138] Based on the experimental results shown in Tables 1 to 5, it was confirmed that the manufacturing method of this invention, which incorporates a large amount of diammonium sebacate from the first step at a level of 78-100 mol% of the total supplied sebacate, enables the production of cadaverine sebacate with generally improved crystallinity, high purity, and high yield.

[0139] More specifically, in Example 1-1, no crystals precipitated even after the crystallization process was carried out. This is because the molar ratio of sebacate was low and insufficient for cadaverine sebacate crystals to precipitate.

[0140] In Examples 1-2, crystals precipitated up to the third mother liquor circulation, but no crystals were formed during the fourth circulation. This is because the sequentially circulated mother liquor lowered the molar ratio of sebacate in the crystallization supply solution, resulting in a sebacate concentration that was insufficient to precipitate cadaverine sebacate crystals.

[0141] In Examples 1-5, the purity of the crystals was somewhat low at 86% when the mother liquor was not circulated, but it increased to 92% when the mother liquor circulation progressed for the fourth time.

[0142] In Examples 1-6, the purity of the crystals was low at 73% when the mother liquor was not circulated, but it increased to 81% when the mother liquor circulation progressed for the fourth time.

[0143] On the other hand, in Examples 1-3, high-purity cadaverine sebacate with a purity of 99% was obtained up to the fourth mother liquor circulation, achieving a high total yield of approximately 72%.

[0144] Examples 1-4 yielded cadaverine sebacate with 99% high purity up to the fourth mother liquor circulation, achieving a high total yield of approximately 74%.

[0145] Thus, we confirmed that a method for producing high-purity cadaverine sebacate can be provided depending on the molar ratio of sebacate to cadaverine in the conversion solution (process solution) supplied after the cadaverine conversion step. In particular, we confirmed that when the amount of sebacate added is adjusted to a molar ratio of approximately 0.84 to 0.86 compared to the amount of cadaverine in the process solution after the second step, it is possible to maintain a purity of 99% of cadaverine sebacate even after two or more mother liquor cycles while showing an excellent total yield of 53% or more.

[0146] 2. Cadaverine undecane diate Production Example 2-1. Fermentation process for lysine undecane diate Corynebacterium glutamicum strain (KCCM12154P, U.S. Patent Publication No. US 2021-0355514 A1) capable of lysine production was obtained through solid-phase culture and flask culture, and after seed culture in a fermenter, the main production fermentation was carried out. Fermentation was performed at 36°C for 30 hours at 900 rpm through fermenter culture.

[0147] Figure 5 is a graph showing the results of producing lysine undecanediate through microbial fermentation using ammonium undecanediate according to the manufacturing example method described above. For the production of lysine undecanediate using Corynebacterium, ammonium undecanediate was supplied at a level of 88-100 g / L to replace existing ammonium sulfate in each example. To maintain the balance of carbon source / nitrogen source, the concentration of the carbon source in the culture medium was reduced to 1.2 mol. Repeated fed-batch fermentation was performed, resulting in lysine undecanediate at a level of 127-145 g / L and lysine at a level of 66 g / L. At that time, the molar ratio of undecanediate / lysine in the fermentation liquid was adjusted to a level of 0.78-0.89 in the example.

[0148] Manufacturing Example 2-2. Conversion reaction of lysine undecane diate to cadaverine undecane diate For the conversion reaction, we used Escherichia coli (US 2018-0030430 A1) in which the PtLDC enzyme, a lysine decarboxylase gene derived from Pseudomonas thermotolerans, was overexpressed using the pET-Deut1 vector.

[0149] The fermentation liquid of lysine undecane diate produced from the above-mentioned production example 2-1 was converted using an enzyme conversion solution obtained by culturing the enzyme strain in a starter culture, and the conversion reaction was carried out by supplying the solution to the process liquid at a level of 10% by mass percentage. The conversion reaction temperature was maintained at a level of 45-50°C, and the pH was adjusted to 8.0-8.5. For this reason, in the cases of Examples 2-1 to 2-4 below, CO2 was added to neutralize the pH. On the other hand, in Examples 2-2 to 2-4, undecane diate was added at a level of 0.02-0.04 mol / L (based on the volume of the fermentation liquid of lysine undecane diate) before the conversion reaction. As a result, it was confirmed that the conversion rate of cadaverine undecane diate was at a level of 97-98%. After the conversion reaction, cadaverine undecanediate was obtained at a level of 111-140 g / L (based on the volume of the fermentation liquid of lysine undecanediate), and cadaverine was obtained at a level of 45 g / L (based on the volume of the fermentation liquid of lysine undecanediate). Subsequently, in Examples 2-5 and 2-6, undecanediate was added at a level of 0.06-0.08 mol / L to produce the process liquid.

[0150] Manufacturing Example 2-3. Subsequent steps after obtaining cadaverine undecane diate The process solution containing cadaverine undecane diate obtained in Production Example 2-2 was subjected to the following subsequent steps to obtain cadaverine undecane diate in crystalline form.

[0151] [Bacterial cell removal step] Bacterial cells in the cadaverine undecane diate process solution were removed by membrane filtration using a 0.1 μm membrane.

[0152] [Steps to remove impurities using activated carbon] Activated carbon was added to the filtrate after bacterial cell isolation at a level of 10% based on the weight of cadaverine undecane diate. The process solution with the added activated carbon was heated to 60°C and stirred for 1 hour to decolorize it, after which the activated carbon was filtered through filter paper.

[0153] [Step to concentrate the process solution of cadaverine undecane diate from which impurities have been removed] The filtered liquid was concentrated in a rotary evaporator under reduced pressure at approximately 55-70°C and 120 Torr until the solid content of the filtrate was 65% by weight.

[0154] [Steps to cool, crystallize, separate, and dry a concentrated solution of cadaverine undecane diate] The 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 10% water based on the weight of cadaverine undecane diate. After drying the separated crystals for one day, their purity was measured by HPLC.

[0155] [Step of recirculating the mother liquor in the crystallization step] The mother liquor separated as described above was recycled in a step to concentrate the process solution of cadaverine undecane diate from which impurities had been removed.

[0156] Experimental Example 2-1. Confirmation of changes in the purity and yield of cadaverine undecane diate according to the molar ratio of undecane diate to cadaverine in the conversion solution for cadaverine undecane diate. In Experimental Example 2-1, the methods described in Production Examples 2-1 to 2-3 were used, and the purity and yield of cadaverine undecane diate were investigated to see how they changed depending on the molar ratio of undecane diate to cadaverine in the conversion solution.

[0157] Example 2-1: When the molar ratio of undecanedioic acid added to the process solution after the second step is 0.79 Microorganisms were removed by passing 1,000 ml of a cadaverine undecane diate conversion solution, in which the molar ratio of undecane diate / cadaverine was 0.79, through a 0.1 μm-sized membrane. The cadaverine undecane diate conversion solution, in which the molar ratio of undecane diate / cadaverine was 0.79, was prepared according to the manufacturing methods of Production Example 2-1 and Production Example 2-2, but the amount of undecane diate added in the ammonium undecane diate for the fermentation process to produce lysine in Production Example 2-1 and the amount of undecane diate added in the cadaverine undecane diate conversion reaction in Production Example 2-2 were controlled so that the total amount of undecane diate added was 0.79 of the number of moles of converted cadaverine.

[0158] The filtrate was decolorized using activated carbon, filtered through filter paper, and then concentrated under reduced pressure to a solid content of 65%. The concentrate was cooled and crystallized from 50°C to 10°C. The crystals and mother liquor were separated by centrifugation, and the separated crystals were dried for one day, after which their purity was measured by HPLC. The mother liquor, which still contained undecane dioxide, was recirculated with a feed solution of cadaverine undecane dioxide with an undecane dioxide / cadaverine molar ratio of 0.79, and the crystallization step was repeated. The recirculation of the mother liquor was carried out sequentially.

[0159] Example 2-2: When the molar ratio of undecanedioic acid added to the process solution after the second step is 0.91 Following the supply of a conversion solution of cadaverine undecanediate, in which the molar ratio of undecanediate / cadaverine was 0.91, the mother liquor was sequentially recycled and the purification process proceeded, as in Example 2-1.

[0160] Examples 2-3: When the molar ratio of undecanedioic acid added to the process solution after the second step is 0.95 Following the supply of a conversion solution of cadaverine undecanediate, in which the molar ratio of undecanediate / cadaverine was 0.95, the mother liquor was sequentially recycled and the purification process proceeded, as in Example 2-1.

[0161] Example 2-4: When the molar ratio of undecanedioic acid added to the process solution after the second step is 1.0 A conversion solution of cadaverine undecanediate, with a molar ratio of undecanediate / cadaverine of 1.0, was supplied, and the mother liquor was sequentially recycled and the purification process proceeded as in Example 2-1.

[0162] In Examples 2-3 to 2-4, the molar ratio of undecanediic acid to lysine in the lysine fermentation step was adjusted to 0.89.

[0163] Example 2-5: When the molar ratio of undecanedioic acid added to the process solution after the second step is 1.04 Following the supply of a conversion solution of cadaverine undecanediate, in which the molar ratio of undecanediate / cadaverine was 1.04, the mother liquor was sequentially recycled and the purification process proceeded, as in Example 2-1.

[0164] Example 2-6: When the molar ratio of undecanedioic acid added to the process solution after the second step is 1.09 Following the supply of a conversion solution of cadaverine undecanediate, in which the molar ratio of undecanediate / cadaverine was 1.09, the mother liquor was sequentially recycled and the purification process proceeded, as in Example 2-1.

[0165] The physical property measurements for the process solutions and cadaverine undecane diate produced by the above-described Examples 2-1 to 2-6 are shown in Tables 6 to 11.

[0166] Table 6 relates to process fluids that do not circulate the mother liquor.

[0167] [Table 6]

[0168] Table 7 shows the process fluid after the mother liquor has been circulated once.

[0169] [Table 7]

[0170] Table 8 shows the process fluid after the mother liquor has been circulated twice.

[0171] [Table 8]

[0172] Table 9 shows the process fluid after the mother liquor has been circulated three times.

[0173] [Table 9]

[0174] Table 10 shows the process fluid after the mother liquor has been circulated four times.

[0175] [Table 10]

[0176] Table 11 shows the process fluid after the mother liquor has been circulated five times.

[0177] [Table 11]

[0178] Based on the experimental results shown in Tables 1 to 6, it was confirmed that the manufacturing method of this invention, which incorporates a large amount of diammonium undecanediate from the first step at a level of 83 to 100 mol% of the total supplied undecanediate, enables the production of cadaverine undecanediate with generally improved crystallinity, high purity, and high yield.

[0179] Specifically, in Example 2-1, cadaverine undecane diate with a high purity of 99% was obtained, but the total yield was low at 62% even after five cycles of mother liquor circulation. This is because the molar ratio of undecane diate was low and insufficient for the precipitation of cadaverine undecane diate crystals.

[0180] Example 2-2 yielded high-purity cadaverine undecane diate with 99% mother liquor circulation, but achieved a low yield of 67% overall after 5 mother liquor circulations. This is because the molar ratio of undecane diate was low and insufficient for cadaverine undecane diate crystals to precipitate.

[0181] In Example 2-5, the purity of the crystals was somewhat low at 91% when the mother liquor was not circulated, but it increased to 96% after the fifth mother liquor circulation.

[0182] In Example 2-6, the purity of the crystals was low at 82% when the mother liquor was not circulated, but it increased to 92% after the fifth mother liquor circulation.

[0183] In Examples 2-3, high-purity cadaverine undecane diate with a purity of 99% was obtained up to the 5th mother liquor circulation, and a high overall yield of approximately 70% was also achieved.

[0184] Examples 2-4 yielded cadaverine undecane diate with a purity of 99% up to the 5th mother liquor circulation, and achieved a high overall yield of approximately 73%.

[0185] Thus, it was confirmed that the purity of the cadaverine undecanediate produced can vary depending on the molar ratio of undecanediic acid to cadaverine in the conversion solution (process solution) supplied after the cadaverine conversion step. In particular, it was confirmed that when the amount of undecanediic acid added is adjusted to a molar ratio of approximately 0.95 to 1.00 compared to the amount of cadaverine in the process solution after the second step, it is possible to maintain a purity of 99% of cadaverine undecanediate even after two or more mother liquor cycles while showing an excellent total yield of 55% or more.

[0186] 3. Cadaverindodecane diate Production Example 3-1. Fermentation process for the production of lysine dodecane diate. Corynebacterium glutamicum strain (KCCM12154P, U.S. Patent Publication No. US 2021-0355514 A1) capable of lysine production was obtained through solid-phase culture and flask culture, and after seed culture in a fermenter, the main production fermentation was carried out. Fermentation was performed at 36°C for 30 hours at 900 rpm through fermenter culture.

[0187] For lysine production using Corynebacterium, ammonium dodecanediate was supplied at levels of 93-106 g / L to replace existing ammonium sulfate in each example. Repeated fed-batch fermentation was performed with the concentration of the carbon source in the culture medium reduced to 1.2 mol to maintain the balance of carbon source / nitrogen source. As a result, lysine dodecanediate was obtained at levels of 132-151 g / L, and lysine at levels of 61 g / L. At that time, the molar ratio of dodecanediate / lysine in the fermentation liquid was adjusted to levels of 0.83-0.95 in each example.

[0188] Manufacturing Example 3-2. Conversion reaction of lysine dodecane diate to cadaverin dodecane diate. For the conversion reaction, we used Escherichia coli (US 2018-0030430 A1) in which the PtLDC enzyme, a lysine decarboxylase gene derived from Pseudomonas thermotolerans, was overexpressed using the pET-Deut1 vector.

[0189] The fermentation liquid of lysine dodecane diate produced from the above-mentioned production example 3-1 was converted by supplying an enzyme conversion solution, in which the enzyme strain was cultured as a starter culture, to the fermentation liquid at a level of 10% by mass percentage. The conversion reaction temperature was maintained at 45-50°C, and the pH was adjusted to 8.0-8.5. For this reason, in Examples 3-1 to 3-4 below, CO2 was added to neutralize the pH. On the other hand, in Examples 3-2 to 3-4, dodecane diate was added at a level of 0.01 mol / L (based on the volume of the fermentation liquid of lysine dodecane diate) before the conversion reaction. As a result, it was confirmed that the conversion rate of cadaverin dodecane diate was at a level of 97-98%. Specifically, after the conversion reaction, cadaverin dodecanediote was obtained at a level of 116-136 g / L (based on the volume of the fermentation liquid of lysine dodecanediote), and cadaverine was obtained at a level of 42 g / L (based on the volume of the fermentation liquid of lysine dodecanediote). Subsequently, in Examples 3-5 and 3-6, dodecanediote was added at a level of 0.03-0.05 mol / L (based on the volume of the fermentation liquid of lysine dodecanediote) to produce the process liquid.

[0190] Manufacturing Example 3-3. Subsequent steps after obtaining cadaverindodecane diate The process solution containing cadaverindodecanediate obtained in Production Example 3-2 was subjected to the following subsequent steps to obtain cadaverindodecanediate in crystalline form.

[0191] [Bacterial cell removal step] Bacterial cells in the cadavelindodecanediate process solution were removed by membrane filtration using a 0.1 μm membrane.

[0192] [Steps to remove impurities using activated carbon] Activated carbon was added to the filtrate after bacterial cell isolation at a level of 10% based on the weight of cadavelindodecane diate. The process solution with the added activated carbon was heated to 60°C and stirred for 1 hour to decolorize it, after which the activated carbon was filtered out through filter paper.

[0193] [Step to concentrate the process solution of cadaverindodecane diate from which impurities have been removed] The filtered liquid was concentrated in a rotary evaporator under reduced pressure at approximately 55-70°C and 120 Torr until the solid content of the filtrate was 50% by weight.

[0194] [Steps to cool, crystallize, separate, and dry a concentrated solution of cadaverindodecane diate] The 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 10% water based on the weight of cadavelindodecanediate. After drying the separated crystals for one day, their purity was measured by HPLC.

[0195] [Step of recirculating the mother liquor in the crystallization step] The mother liquor separated as described above was recycled in a step to concentrate the process solution of cadaverindodecanediate from which impurities had been removed.

[0196] Experimental Example 3-1. Confirmation of changes in the purity and yield of cadaverindodecanediate according to the molar ratio of dodecanediate to cadaverine in the conversion solution for cadaverindodecanediate. Experimental Example 3-1 uses the methods described in Production Examples 3-1 to 3-3, and investigates how the purity and yield of cadaverindodecanediate change depending on the molar ratio of dodecanediate to cadaverine in the conversion solution.

[0197] Example 3-1: When the molar ratio of dodecanediic acid added to the process solution after the second step is 0.85 Microorganisms were removed by passing 1,000 ml of a cadaverin-dodecanediate conversion solution, in which the molar ratio of dodecanediate / cadaverine was 0.85, through a 0.1 μm-sized membrane. The cadaverin-dodecanediate conversion solution, in which the molar ratio of dodecanediate / cadaverine was 0.85, was prepared according to the manufacturing methods of Production Example 3-1 and Production Example 3-2, but in Production Example 3-1, the amount of dodecanediate added in the ammonium dodecanediate fermentation process for producing lysine and the amount of dodecanediate added in Production Example 3-2, the cadaverin-dodecanediate conversion reaction were controlled so that the total amount of dodecanediate added was 0.85 times the number of moles of converted cadaverine.

[0198] The filtrate was decolorized using activated carbon, filtered through filter paper, and then concentrated under reduced pressure to a solid content of 50%. The concentrate was cooled and crystallized from 50°C to 10°C. The crystals and mother liquor were separated by centrifugation, and the separated crystals were dried for one day, after which their purity was measured by HPLC. The mother liquor, which still contained dodecane dioxide, was recirculated with a feed solution of cadaverin-dodecane dioxide with a dodecane dioxide / cadaverin molar ratio of 0.85, and the crystallization step was repeated. The recirculation of the mother liquor was carried out sequentially.

[0199] Example 3-2: When the molar ratio of dodecanediic acid added to the process solution after the second step is 0.9 Upon receiving a conversion solution of cadaverin-dodecanediate with a dodecanediate / cadaverine molar ratio of 0.9, the mother liquor was sequentially recycled and the purification process proceeded, as in Example 3-1.

[0200] Example 3-3: When the molar ratio of dodecanediic acid added to the process solution after the second step is 0.95 A conversion solution of cadaverin-dodecanediate, with a dodecanediate / cadaverine molar ratio of 0.95, was supplied, and the mother liquor was sequentially recycled and the purification process proceeded as in Example 3-1.

[0201] Examples 3-4: When the molar ratio of dodecanediic acid added to the process solution after the second step is 1.0 Following the supply of a conversion solution of cadaverin-dodecanediate with a dodecanediate / cadaverine molar ratio of 1.0, the mother liquor was sequentially recycled and the purification process proceeded, as in Example 3-1.

[0202] In Examples 3-3 to 3-4, the molar ratio of dodecanediic acid to lysine in the lysine fermentation step was adjusted to 0.9 to 0.95.

[0203] Examples 3-5: When the molar ratio of dodecanediic acid added to the process solution after the second step is 1.04 A conversion solution of cadaverin-dodecanediate, with a dodecanediate / cadaverine molar ratio of 1.04, was supplied, and the mother liquor was sequentially recycled and the purification process proceeded as in Example 3-1.

[0204] Examples 3-6: When the molar ratio of dodecanediic acid added to the process solution after the second step is 1.09 Following the supply of a cadaverin-dodecanediate conversion solution with a dodecanediate / cadaverine molar ratio of 1.09, the mother liquor was sequentially recycled and the purification process proceeded, similar to Example 3-1.

[0205] The physical property measurements for the process solutions and cadaverindodecane diate produced by the above-described Examples 3-1 to 3-6 are shown in Tables 12 to 16.

[0206] Table 12 relates to process fluids that do not circulate the mother liquor.

[0207] [Table 12]

[0208] Table 13 shows the process fluid after the mother liquor has been circulated once.

[0209] [Table 13]

[0210] Table 14 shows the process fluid after the mother liquor has been circulated twice.

[0211] [Table 14]

[0212] Table 15 shows the process fluid after the mother liquor has been circulated three times.

[0213] [Table 15]

[0214] Table 16 shows the process fluid after the mother liquor has been circulated four times.

[0215] [Table 16]

[0216] Based on the experimental results shown in Tables 12 to 16, it was confirmed that the manufacturing method of this invention, which includes a large amount of diammonium dodecanediote in the first step at a level of 89-100 mol% of the total supplied dodecanediote, enables the production of cadaverindodecanediote with generally improved crystallization rate, high purity, and high yield. Specifically, in the case of Example 3-1, no crystals precipitated even after the crystallization process was carried out. This is because the molar ratio of dodecanediote was low and insufficient for cadaverindodecanediote crystals to precipitate.

[0217] In Example 3-2, crystals precipitated up to the third mother liquor circulation, but no crystals were formed during the fourth circulation. This is because the sequentially circulated mother liquor lowered the molar ratio of dodecane diate in the crystallization supply solution, resulting in a concentration of dodecane diate that was insufficient to precipitate cadaverindodecane diate crystals.

[0218] In Examples 3-5, the purity of the crystals was somewhat low at 86% when the mother liquor was not circulated, but it increased to 92% when the mother liquor circulation progressed for the fourth time.

[0219] In Examples 3-6, the purity of the crystals was low at 73% when the mother liquor was not circulated, but it increased to 81% when the mother liquor circulation progressed for the fourth time.

[0220] In Example 3-3, high-purity cadaverindodecane diate with a purity of 99% was obtained up to the fourth mother liquor circulation, and a high overall yield of approximately 72% was also achieved.

[0221] Examples 3-4 yielded cadaverindodecane diate with a purity of 99% up to the fourth mother liquor circulation, and achieved a high overall yield of approximately 74%.

[0222] Thus, it was confirmed that the purity of the cadaverin-dodecanediate produced can vary depending on the molar ratio of dodecanediic acid to cadaverine in the conversion solution (process solution) supplied after the cadaverine conversion step. In particular, it was confirmed that when the amount of dodecanediic acid added is adjusted to a molar ratio of approximately 0.95 to 1.00 compared to the amount of cadaverine in the process solution after the second step, it is possible to maintain a purity of 99% of cadaverin-dodecanediate even after two or more mother liquor cycles while showing an excellent total yield of 53% or more.

[0223] From the above description, a person skilled in the art to which this application pertains will understand that this application can be implemented in other specific forms without altering its technical idea or essential features. In this regard, it should be understood that the embodiments described above are merely illustrative and not limiting. The scope of this application should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, as described below, and their equivalent concepts, rather than from the above detailed description.

Claims

1. The first step is to culture L-lysine-producing microorganisms in a medium to which dicarboxylic acid has been added in the form of diammonium dicarboxylate to obtain lysine dicarboxylate; and A method for producing cadavering dicarboxylate, comprising a second step of converting lysine dicarboxylate to cadavering dicarboxylate, The amount of dicarboxylic acid added is in a molar ratio of 0.90 to 1.05 molars relative to the amount of cadaverine in the process solution after the second step. A method for producing cadaverine dicarboxylate, wherein the dicarboxylic acid is an aliphatic dicarboxylic acid having 10 to 12 carbon atoms.

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

3. The method for producing cadaverine dicarboxylate according to claim 1, wherein the diammonium dicarboxylate in the first step is included in such a way that the molar ratio (diammonium dicarboxylate / lysine) with the lysine to be produced is 0.84 to 0.

96.

4. The dicarboxylate salt and dicarboxylic acid are sebacate salt and sebacate acid, The method for producing cadaverine dicarboxylate according to claim 1, wherein the diammonium dicarboxylate in the first step is included in such a way that the molar ratio (diammonium dicarboxylate / lysine) with the lysine to be produced is 0.84 to 0.

86.

5. The dicarboxylate salt and dicarboxylic acid are undecane diate and undecane diic acid, The method for producing cadaverine dicarboxylate according to claim 1, wherein the diammonium dicarboxylate in the first step is included in such a way that its molar ratio (diammonium dicarboxylate / lysine) with the lysine to be produced is 0.84 to 0.

90.

6. The dicarboxylate salt and dicarboxylic acid are dodecane diate and dodecane diic acid, The method for producing cadaverine dicarboxylate according to claim 1, wherein the diammonium dicarboxylate in the first step is included in such a way that the molar ratio (diammonium dicarboxylate / lysine) with the lysine to be produced is 0.86 to 0.

96.

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

5.

8. The dicarboxylate salt and dicarboxylic acid are sebacate salt and sebacate acid, The method for producing cadaverine dicarboxylate according to claim 1, wherein the amount of dicarboxylic acid added is in a molar ratio of 0.96 to 1.00 compared to the amount of cadaverine in the process solution after the second step.

9. The dicarboxylate salt and dicarboxylic acid are undecane diate and undecane diic acid, The method for producing cadaverine dicarboxylate according to claim 1, wherein the amount of dicarboxylic acid added is in a molar ratio of 0.95 to 1.00 compared to the amount of cadaverine in the process solution after the second step.

10. The dicarboxylate salt and dicarboxylic acid are dodecane diate and dodecane diic acid, The method for producing cadaverine dicarboxylate according to claim 1, wherein the amount of dicarboxylic acid added is in a molar ratio of 0.95 to 1.00 compared to the amount of cadaverine in the process solution after the second step.

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

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

13. The method for producing a cadavering dicarboxylate according to claim 12, 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. The aforementioned recovery step is, The method for producing a cadavering dicarboxylate according to claim 12, further comprising a mother liquor circulation step of recirculating the mother liquor containing the uncrystallized cadavering dicarboxylate after the cooling crystallization step with the feed liquid of the concentration step.

15. The method for producing a cadavering dicarboxylate according to claim 13, wherein the concentration step is to concentrate the salt so that the solid content is 45 to 70% (w / w).

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

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

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

19. The method for producing a cadavering dicarboxylate according to claim 13, wherein a cadavering dicarboxylate with a purity of 98% or more can be obtained with 0 to 4 circulations of the mother liquor.

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