Cadavering dicarboxylate production process

Abstract

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

Description

Technical Field

[0006] , ,

[0007] ,

[0001] This application relates to a technology for producing high - purity cadaverine dicarboxylate (cadaverine succinate or cadaverine glutarate) 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 hexamethylenediamine, 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 is not only for obtaining high - quality monomers but also easy for transportation and storage.

[0003] ​​​​​​​​​​​​​​​​​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, cadaverine succinate or cadaverine glutarate has high solubility in pure water, and this solubility increases significantly with increasing temperature. However, this high solubility leads to high viscosity of the solution, resulting in low recovery rates and making crystallization of cadaverine succinate difficult in aqueous systems. Furthermore, unreacted succinic acid or unreacted glutaric acid crystallizes together, reducing the purity of the final product, cadaverine succinate or cadaverine glutarate. This requires removal before crystallization, leading to additional costs, decreased process efficiency, and environmental pollution due to the use of chemical solvents. [Prior art documents] [Patent Documents]

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

[0010] The problem that this application aims to solve is to provide a method for producing cadaverine dicarboxylate (cadaverine succinate or cadaverine glutarate). [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 C4 or C5 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" refers to a dibasic acid containing two carboxyl groups, which is of high importance as a metabolic intermediate in living organisms. Specifically, the dicarboxylic acid may be an aliphatic dicarboxylic acid having four or five carbon atoms. That is, in this application, the dicarboxylic acid refers to one aliphatic dicarboxylic acid selected from succinic acid and glutaric acid. If the cadaverine dicarboxylic acid salt to be produced by the production method of this application is cadaverine succinate, it must be understood that all the dicarboxylic acid added or introduced is succinic acid. Similarly, if the cadaverine dicarboxylic acid salt to be produced by the production method of this application is cadaverine glutarate, it must be understood that all the dicarboxylic acid added or introduced is glutaric acid. Thus, in this application, it is understood that dicarboxylic acid refers to one type of aliphatic dicarboxylic acid, not a mixture of different types of dicarboxylic acids.

[0017] In this application, the term "succinic acid" refers to a four-carbon dicarboxylic acid with the chemical formula C4H6O4, generally existing as a sour, odorless, white solid. Succinic acid is also known as amber acid, butanedioic acid, and succinic acid, and is used as a precursor in a variety of industrial products, including polymers and plasticizers.

[0018] In this application, the term "Glutaric acid" is a dicarboxylic acid with five carbon atoms, having the chemical formula C5H8O4, and generally exists in the form of colorless crystals. The said glutaric acid is also named 1,3-propanedicarboxylic acid, n-Pyrotartaric acid, etc., and is utilized as a precursor for various industrial products including polymers and plasticizers.

[0019] In this application, the term "Cadaverine, CAD" is a toxic diamine compound with 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.

[0020] In this application, the term "Cadaverine Dicarboxylate" is a substance produced by reacting the diamine form of cadaverine with the component of a dicarboxylic acid, and it has a high industrial utilization degree as a production precursor of polyamide resins. Specifically, in this application, cadaverine dicarboxylate means one cadaverine dicarboxylate selected from cadaverine succinate and cadaverine glutarate.

[0021] Figures 1 and 2 are flowcharts showing the production process of cadaverine dicarboxylate according to an aspect of this application.

[0022] In this application, the term "L-lysine-producing microorganism" includes all wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modifications, and due to reasons such as the insertion of foreign genes, or the enhancement or inactivation of the activity of endogenous genes, etc., a microorganism in which a specific mechanism is weakened or enhanced, and may also be a microorganism that includes genetic modification for the production of L-lysine.

[0023] In one example, the microorganism producing L-lysine of the present application may be, but is not limited to, a microorganism that naturally has the ability to produce L-lysine, a microorganism to which the ability to produce L-lysine is imparted to a microorganism that does not have the ability to produce L-lysine, or a microorganism in which the ability to produce L-lysine is enhanced in a microorganism that has a significantly low ability to produce L-lysine. Specifically, the microorganism producing L-lysine or having the ability to produce L-lysine in the present application may be a microorganism in which a part of the gene in the L-lysine biosynthesis pathway is enhanced or weakened, or a part of the gene in the L-lysine degradation pathway is enhanced or weakened. That the ability to produce L-lysine is "enhanced" or "increased" means that the ability to produce L-lysine is improved as compared with the parent strain or non-transformed microorganism.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] Specifically, the diammonium dicarboxylate salt in the first step may be included in the culture medium in a high proportion relative to the dicarboxylic acid supplied throughout the entire process of the manufacturing method of this invention, specifically in an amount of 61 mol% or more.

[0032] More specifically, the diammonium dicarboxylate in the first step may be present in amounts of 61 mol% or more, 68 mol% or more, 73 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.

[0033] 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.

[0034] 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 0.63 to 1. More specifically, the diammonium dicarboxylate in the first step may be included in such a molar ratio (diammonium dicarboxylate / lysine) with respect to the lysine to be produced that is 0.6 to 0.98, 0.73 to 0.98, 0.78 to 0.98, or 0.8 to 0.98, or more specifically, 0.85 to 0.98. 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.

[0035] For example, the diammonium dicarboxylate in the first step may be included in a molar ratio (diammonium dicarboxylate / lysine) within a range defined by one lower limit selected from 0.63, 0.65, 0.7, 0.73, 0.78, 0.8, 0.85, 0.86, 0.87, 0.88, and 0.89 and / or one upper limit selected from 1, 0.99, 0.98, 0.97, 0.96, 0.95, 0.94, 0.93, and 0.92, with respect to the lysine to be produced.

[0036] Specifically, the diammonium dicarboxylate in the first step can be included in a concentration of 0.4 mol / L to 0.8 mol / L based on the L-lysine-producing microbial fermentation broth. More specifically, the diammonium dicarboxylate in the first step can be included in a concentration of 0.48 mol / L to 0.7 mol / L, 0.5 mol / L to 0.7 mol / L, 0.55 mol / L to 0.7 mol / L, or 0.55 mol / L to 0.65 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.

[0037] Lysine dicarboxylate can be produced by fermentation of L-lysine-producing microorganisms in a culture medium containing diammonium dicarboxylate. The lysine dicarboxylate can be added to subsequent processes in the form of a fermentation liquid with the microbial cells still present, or in the form of a fermentation liquid from which the microbial cells have been removed or after purification.

[0038] 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 90 g / L to 300 g / L, 92 g / L to 250 g / L, 92 g / L to 160 g / L, or 92 g / L to 100 g / L based on the L-lysine-producing microorganism fermentation liquid.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] The second step is to convert the lysine dicarboxylate to the target product, cadaverine dicarboxylate (cadaverine succinate or cadaverine glutarate).

[0046] 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.

[0047] The dicarboxylic acid added in the second step may be omitted, or added in amounts of 0.45 mol / L or less, 0.31 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, 0.05 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.

[0048] In one specific example, if the dicarboxylic acid is succinic acid, the dicarboxylic acid added in the second step may be omitted or added in a trace amount of 0.15 mol / L or less.

[0049] In one specific example, if the dicarboxylic acid is glutaric acid, the dicarboxylic acid added in the second step may be omitted or added in a trace amount of 0.08 mol / L or less.

[0050] 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.

[0051] 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.

[0052] 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 1.10 compared to the amount of cadaverine in the process solution after the second step.

[0053] More specifically, the amount of dicarboxylic acid added in the manufacturing method of this application may be in a molar ratio of 0.85-1.07, 0.87-1.06, 0.88-1.05, 0.89-1.03, 0.88-1.02, 0.89-1.0, 0.9-1.10, 0.93-1.08, 0.93-1.07, 0.95-1.06, or 0.95-1.05 compared to the amount of cadaverine in the process solution after the second step.

[0054] As an example, the amount of dicarboxylic acid added in the manufacturing method of this application may be in a molar ratio within a range consisting of one lower limit selected from 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, and 0.95 and / or one upper limit selected from 1.10, 1.09, 1.08, 1.07, 1.06, 1.05, 1.04, 1.03, 1.02, 1.01, and 1.0, relative to the amount of cadaverine in the process solution after the second step.

[0055] In one specific example, if the dicarboxylic acid is succinic 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 1.07, 0.94 to 1.06, or 0.95 to 1.05 compared to the amount of cadaverine in the process solution after the second step.

[0056] One technical significance of this invention lies in the discovery that, in the manufacturing method of this application, when succinic 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, succinic acid does not precipitate as crystals together with cadaverine succinate, making it possible to produce cadaverine succinate with high purity and high yield.

[0057] Furthermore, by adjusting the molar ratio of the obtained cadaverine to the added succinic acid, it is possible to maintain a purity of 99% of cadaverine succinate up to the fourth circulation of the mother liquor, while obtaining cadaverine succinate with a total yield of 30% or more, 32% or more, 51% or more, 53% or more, 65% or more, 68% or more, 75% or more, 78% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, or 85% or more, depending on the number of mother liquor circulations.

[0058] In one specific example, if the dicarboxylic acid is glutaric acid, the amount of dicarboxylic acid added in the production method of this application may be in a molar ratio of 0.8 to 1.05, 0.83 to 1.04, 0.85 to 1.03, 0.87 to 1.02, 0.88 to 1.01, or 0.89 to 1.0 compared to the amount of cadaverine in the process solution after the second step.

[0059] One technical significance of this invention lies in the discovery that, in the manufacturing method of this application, when glutaric 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 glutaric acid does not precipitate as crystals together with the cadaverine glutarate, making it possible to produce cadaverine succinate in high purity and high yield.

[0060] Furthermore, by adjusting the molar ratio of the obtained cadaverine to the added glutaric acid, it is possible to maintain a purity of 99% of cadaverine glutarate up to the third cycle of mother liquor circulation, while obtaining cadaverine glutarate in total yields of 50% or more, 49% or more, 50% or more, 52% or more, 74% or more, 75% or more, 77% or more, 86% or more, 87% or more, 88% or more, 92% or more, 93% or more, or 94% depending on the number of mother liquor circulations.

[0061] In the second step, the reaction to convert the lysine dicarboxylate to cadaverine dicarboxylate may be an enzymatic conversion reaction.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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 carbonation-removing enzyme.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] The conversion reaction in the second step may be carried out at 30-60°C, more specifically at 40-50°C, or, for example, at 45°C, but is not limited thereto.

[0071] 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.

[0072] 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.

[0073] The recovery may involve collecting the cadavering dicarboxylate using appropriate methods known in the art.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] The aforementioned concentration step refers to a step of concentrating the converted liquid so that the solid content ratio is high, and may be performed after the filtration step. For example, the concentration may be performed by vacuum concentration using a rotary evaporator.

[0078] Specifically, the concentration step may involve concentrating the solids content to 70-85% (w / w), more specifically to 73-85% (w / w), 75-82% (w / w), 70-80% (w / w), 75-85% (w / w), 74-83% (w / w), 78-82% (w / w), or 73-77% (w / w), and as an example, it may involve concentrating to 75% (w / w). When the solids content is concentrated within the above range in the concentration step, the recovery rate and purity of the cadavering dicarboxylate can be maintained well.

[0079] The concentration step may be carried out at 45-65°C, but is not limited thereto.

[0080] 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.

[0081] Specifically, the cooling may involve cooling the conversion solution to 20-30°C, more specifically to 23-27°C, or, as an example, to 25°C, but is not limited thereto.

[0082] Specifically, the cooling rate may be 5-20°C / hour, more specifically 5-15°C / hour, or, for example, 10°C / hour, but is not limited to these.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] In one embodiment, the recovery step may further include washing, separating, and drying the crystallized process liquid.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] In one embodiment, when the dicarboxylic acid is succinic acid, the method of this application is characterized by obtaining cadaverine succinate with a purity of 99% or more in 0 to 4 cycles of mother liquor circulation. In one embodiment, when the dicarboxylic acid is succinic acid, the cumulative total yield of cadaverine succinate may be more than 80% based on 4 cycles of mother liquor circulation, and more specifically, it may be 81% or more, 82% or more, 83% or more, or 84% or more.

[0093] In one embodiment, when the dicarboxylic acid is glutaric acid, the method of this application is characterized by obtaining cadavering glutarate with a purity of 99% or more in 0 to 3 cycles of mother liquor. In one embodiment, when the dicarboxylic acid is glutaric acid, the cumulative total yield of cadavering glutarate may exceed 90% based on 3 cycles of mother liquor, and more specifically, it may be 92% or more, 93% or more, or 94% or more.

[0094] 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.

[0095] 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.

[0096] The above describes a manufacturing process for cadaverine dicarboxylate (cadaverine succinate or cadaverine glutarate) according to one aspect of this application. Below, the advantageous effects mentioned in this application will be described through experimental results of examples and comparative examples of cadaverine succinate and cadaverine glutarate, respectively.

[0097] Manufacturing Example 1: Production of Cadaverine Succinate Production Example 1-1. Fermentation Process for Lysine Succinate 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.

[0098] For the production of lysine succinate using Corynebacterium, diammonium succinate was supplied at a level of 76-99 g / L to replace the existing ammonium sulfate, and fermentation was carried out, resulting in lysine succinate at a level of 132-172 g / L and lysine at a level of 99 g / L. At that time, in the example, the molar ratio of succinic acid / lysine in the fermentation liquid was adjusted to a level of 0.74-0.96.

[0099] Manufacturing Example 1-2. Conversion reaction of lysine succinate to cadaverine succinate. 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.

[0100] The fermentation liquid of lysine succinate 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 succinate at a concentration of 10% by mass. The conversion reaction temperature was maintained at 45-50°C for approximately 1 hour. The pH was adjusted to 8.0-8.5. For this reason, in Examples 1-1 to 1-3 below, CO2 was added to neutralize the pH. In Examples 1-1 and 1-3, dicarboxylic acid was added at a concentration of 0.03 mol / L (based on the volume of the fermentation liquid of lysine succinate). As a result, it was confirmed that the cadaverine succinate conversion rate was at a level of 97-98%. Specifically, after the conversion reaction, cadaverine succinate was obtained at a level of 117-143 g / L (based on the volume of the fermentation liquid of lysine succinate), and cadaverine was obtained at a level of 68 g / L (based on the volume of the fermentation liquid of lysine succinate). Subsequently, in Examples 1-4 to 1-7, succinic acid was added at a level of 0.02-0.15 mol / L to produce the process liquid.

[0101] Production Example 1-3. Subsequent steps after obtaining cadaverine succinate The following subsequent steps were performed on the process solution containing cadaverine succinate obtained in Production Example 1-2 to obtain crystalline cadaverine succinate.

[0102] [Bacterial cell removal step] Bacterial cells in the cadaverine succinate process solution were removed by membrane filtration using a 0.1 μm membrane.

[0103] [Steps to remove impurities using activated carbon] Activated carbon was added to the filtrate containing cadaverine succinate at a level of 10% based on its weight. 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.

[0104] [Step to concentrate the process solution of cadaverine succinate from which impurities have been removed] The filtered liquid was concentrated in a rotary evaporator under reduced pressure at approximately 45-65°C and 40-70 torr until the solid content of the filtrate was 75-82% by weight.

[0105] [Steps to cool, crystallize, separate, and dry the concentrated cadaverine succinate solution] The concentrated solution was cooled from 45-35°C to 25°C at a rate of 5°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 succinate. After drying the separated crystals for one day, their purity was measured by HPLC.

[0106] [Step of recirculating the mother liquor in the crystallization step] The mother liquor separated above was recycled in the step of [concentrating the process solution of cadaverine succinate from which impurities have been removed].

[0107] Experimental Example 1. Confirmation of changes in purity and yield of cadaverine succinate according to the molar ratio of succinic acid to cadaverine. In Experimental Example 1, the methods described in Production Examples 1-1 to 1-3 were used, and the purity and yield of cadaverine succinate were examined to see how they changed depending on the molar ratio of succinic acid added throughout the entire process to cadaverine in the process solution after the second step.

[0108] Example 1-1: When the molar ratio of succinic acid added to the process solution after the second step is 0.8 Microorganisms were removed by passing 1,000 ml of a cadaverine succinate conversion solution with a succinic acid / cadaverine molar ratio of 0.8 through a 0.1 μm-sized membrane. The cadaverine succinate conversion solution with a succinic acid / cadaverine molar ratio of 0.8 was prepared according to the manufacturing methods of Production Example 1-1 and Production Example 1-2, but the sum of the amount of diammonium succinate added in the fermentation step for producing lysine in Production Example 1-1 and the amount of succinic acid added in the cadaverine succinate conversion reaction in Production Example 1-2 (limited to examples where succinic acid was further added in the second step) was controlled so that it equaled 0.8 of the number of moles of converted cadaverine.

[0109] The filtrate was decolorized using activated carbon, filtered through filter paper, and then concentrated under reduced pressure for 40 torr to a solids content of 78% by weight. The concentrate was cooled and crystallized from 35°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 succinate, was recirculated with a feed solution of cadaverine succinate with a succinic acid / cadaverine molar ratio of 0.8, and the concentration step was repeated. The recirculation of the mother liquor was carried out sequentially.

[0110] Examples 1-2: When the molar ratio of succinic acid added to the process solution after the second step is 0.9 A conversion solution of cadaverine succinate, with a succinic acid / cadaverine molar ratio of 0.9, was supplied, and the mother liquor was sequentially recycled and the purification process proceeded as in Example 1-1.

[0111] Examples 1-3: When the molar ratio of succinic acid added to the process solution after the second step is 0.95 Upon receiving a conversion solution of cadaverine succinate with a succinic acid / cadaverine molar ratio of 0.95, the mother liquor was sequentially recycled and the purification process proceeded, as in Example 1-1.

[0112] Examples 1-4: When the molar ratio of succinic acid added to the process solution after the second step is 1.01 Upon receiving a conversion solution of cadaverine succinate with a succinic acid / cadaverine molar ratio of 1.01, the mother liquor was sequentially recycled and the purification process proceeded, as in Example 1-1.

[0113] Examples 1-5: When the molar ratio of succinic acid added to the process solution after the second step is 1.05 Upon receiving a conversion solution of cadaverine succinate with a succinic acid / cadaverine molar ratio of 1.05, the mother liquor was sequentially recycled and the purification process proceeded, as in Example 1-1.

[0114] In Examples 1-3 to 1-5, the molar ratio of succinic acid to lysine in the lysine fermentation step was adjusted to 0.88-0.96.

[0115] Examples 1-6: When the molar ratio of succinic acid added to the process solution after the second step is 1.10 Upon receiving a conversion solution of cadaverine succinate with a succinic acid / cadaverine molar ratio of 1.10, the mother liquor was sequentially recycled and the purification process proceeded, as in Example 1-1.

[0116] Examples 1-7: When the molar ratio of succinic acid added to the process solution after the second step is 1.20 Upon receiving a conversion solution of cadaverine succinate with a succinic acid / cadaverine molar ratio of 1.20, the mother liquor was sequentially recycled and the purification process proceeded, as in Example 1-1.

[0117] The physical properties measured for the process solutions and cadaverine succinate produced by the above-described Examples 1-1 to 1-7 are shown in Tables 1 to 5.

[0118] Table 1 relates to process fluids that do not circulate the mother liquor.

[0119] [Table 1]

[0120] Table 2 shows the process fluid after the mother liquor has been circulated once.

[0121] [Table 2]

[0122] Table 3 relates to the process fluid after the mother liquor has been circulated twice.

[0123] [Table 3]

[0124] Table 4 relates to the process fluid after the mother liquor has been circulated three times.

[0125] [Table 4]

[0126] Table 5 relates to the process fluid after the mother liquor has been circulated four times.

[0127] [Table 5]

[0128] Based on the experimental results shown in Tables 1 to 5, it was confirmed that the manufacturing method of this invention, which includes a large amount of diammonium succinate in the first step at a level of 81 to 100 mol% of the total supplied succinic acid, enables the production of cadaverine succinate with generally improved crystallinity, high purity, and high yield.

[0129] More specifically, Example 1-1 had a high purity of 99% after the fourth mother liquor cycle, but a low yield of 70.8%. This was because the sequentially circulated mother liquor lowered the molar ratio of succinate in the crystallization feed solution, and the concentration of succinate was insufficient compared to the concentration of cadaverine succinate.

[0130] Examples 1-2 showed a high purity of 99% after the fourth cycle of the mother liquor, but had a slightly lower yield of 79.5%. This was because the sequentially circulated mother liquor lowered the molar ratio of succinate in the crystallization feed solution, resulting in a succinate concentration that was insufficient compared to the cadaverine succinate concentration.

[0131] On the other hand, Examples 1-3 exhibited a high purity of 99% after the fourth mother liquor step and a high total yield of 82.8%.

[0132] Examples 1-4 exhibited a high purity of 99% after the fourth mother liquor step and a high total yield of 85.5%.

[0133] Examples 1-5 exhibited a high purity of 99% after the fourth mother liquor cycle and a high total yield of 82.5%.

[0134] Examples 1-6 had a slightly low purity of 98.1% after the fourth mother liquor step, but also a slightly low yield of 79.3%. This is because succinic acid exceeding its solubility precipitated as crystals, reducing the purity.

[0135] Examples 1-7 showed a low purity of 89% after the fourth mother liquor step, but also a low yield of 76%. This is because succinic acid exceeding its solubility precipitated as crystals, reducing the purity.

[0136] Thus, we have discovered that a method for producing high-purity cadaverine succinate can be provided depending on the molar ratio of succinic acid added throughout the entire process compared to the amount of cadaverine in the process solution after the second step. In particular, we confirmed that when the amount of succinic acid added is adjusted to a molar ratio of approximately 0.95 to 1.05 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 succinate even after four or more mother liquor cycles while showing an excellent total yield of 82% or more.

[0137] Manufacturing Example 2. Manufacturing of Cadaverin Glutarate Production Example 2-1. Fermentation process for the production of lithyringe talate. 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 30 hours at 900 rpm via fermenter culture.

[0138] For the production of lysine thalates using Corynebacterium, diammonium glutarate was supplied at levels of 66-100 g / L to replace existing ammonium sulfate, and fermentation was carried out, resulting in lysine thalates at levels of 111-167 g / L and lysine at levels of 92 g / L. At that time, in the example, the molar ratio of glutaric acid / lysine in the fermentation liquid was adjusted to levels of 0.63-0.95.

[0139] Manufacturing Example 2-2. Conversion reaction of lithyring tarate to cadavering tarate 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.

[0140] The enzyme conversion solution, obtained by culturing the enzyme strain in the fermentation process liquid of the resingle thalate produced in the above-mentioned Production Example 2-1, was supplied to the process liquid at a level of 10% by mass percentage to carry out the conversion reaction. The conversion reaction temperature was maintained at 45-50°C for approximately 1 hour. The pH was adjusted to 8.0-8.5. For this reason, in Examples 2-1 to 2-4 below, CO2 was added to neutralize the pH, and in Examples 2-1 to 2-3 and 2-4, dicarboxylic acid was added at a level of 0.01-0.05 mol / L (based on the volume of the resingle thalate fermentation process liquid). As a result, it was confirmed that the conversion rate of cadaverin thalate was at a level of 97-98%. Specifically, after the conversion reaction, cadaverine succinate was obtained at a level of 101-141 g / L (based on the volume of the fermentation liquid of lithylen thalate), and cadaverine was obtained at a level of 63 g / L (based on the volume of the fermentation liquid of lithylen thalate). Subsequently, in Examples 2-4 to 2-7, glutaric acid was added at a level of 0.02-0.08 mol / L to produce the process liquid.

[0141] Production Example 2-3. Subsequent steps after obtaining cadaverin glutarate The process solution containing cadaverin gluthrate obtained in Production Example 2-2 was subjected to the following subsequent steps to obtain crystalline cadaverin gluthrate.

[0142] [Bacterial cell removal step] Bacterial cells in the cadaveral glutarate process solution were removed by membrane filtration using a 0.1 μm membrane.

[0143] [Steps to remove impurities using activated carbon] Activated carbon was added to the filtrate containing cadaveral glutarate at a level of 10% based on its weight. 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.

[0144] [Step to concentrate the process solution of cadaveral glutarate from which impurities have been removed] The filtered liquid was concentrated in a rotary evaporator under reduced pressure at approximately 45-65°C and 40-70 torr until the solid content of the filtrate was 75-82% by weight.

[0145] [Steps to cool, crystallize, separate, and dry a concentrated cadaveral glutarate solution] The concentrated solution was cooled from 50-35°C to 25°C at a rate of 5°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 cadaverin glutarate. After drying the separated crystals for one day, their purity was measured by HPLC.

[0146] [Step of recirculating the mother liquor in the crystallization step] The mother liquor separated above was recycled in the step of [concentrating the process solution of cadaveral glutarate from which impurities have been removed].

[0147] Experimental Example 2. Confirmation of changes in purity and yield of cadaverine glutarate according to the molar ratio of glutarate to cadaverine. In Experimental Example 2, the methods described in Production Examples 2-1 to 2-3 were used, and the purity and yield of cadaverine glutarate were examined to see how they changed depending on the molar ratio of glutaric acid added throughout the entire process and cadaverine in the process solution after the second step.

[0148] Example 2-1: When the molar ratio of glutaric acid added / cadaverine in the process solution after the second step is 0.7 Microorganisms were removed by passing 1,000 ml of a cadaverin glutarate conversion solution, in which the molar ratio of glutarate to cadaverine was 0.7, through a 0.1 μm-sized membrane. The cadaverin glutarate conversion solution, in which the molar ratio of glutarate to cadaverine was 0.7, was prepared according to the manufacturing methods of Production Example 2-1 and Production Example 2-2, but the sum of the amount of ammonium glutarate added in the fermentation step for producing lysine in Production Example 2-1 and the amount of glutaric acid added in the cadaverin glutarate conversion reaction in Production Example 2-2 (limited to examples in which glutaric acid was further added in the second step) was controlled so that it equaled 0.7 of the number of moles of converted cadaverine.

[0149] The filtrate was decolorized using activated carbon, filtered through filter paper, and then concentrated under reduced pressure for 40 torr to a solids content of 75% by 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 glutarate, was recirculated with a feed solution of cadaverine glutarate with a glutaric acid / cadaverine molar ratio of 0.7, and the concentration step was repeated. The recirculation of the mother liquor was carried out sequentially.

[0150] Example 2-2: When the molar ratio of glutaric acid added / cadaverine in the process solution after the second step is 0.79 A conversion solution of cadaverine glutarate with a glutaric acid / cadaverine molar ratio of 0.79 was supplied, and the mother liquor was sequentially recycled and the purification process proceeded as in Example 2-1.

[0151] Example 2-3: When the molar ratio of glutaric acid added / cadaverine in the process solution after the second step is 0.89 Following the supply of a cadaverine glutarate conversion solution with a glutaric acid / cadaverine molar ratio of 0.89, the mother liquor was sequentially recycled and the purification process proceeded, as in Example 2-1.

[0152] Example 2-4: When the molar ratio of glutaric acid added / cadaverine in the process solution after the second step is 0.95 A conversion solution of cadaverine glutarate with a glutaric acid / cadaverine molar ratio of 0.95 was supplied, and the mother liquor was sequentially recycled and the purification process proceeded as in Example 2-1.

[0153] Example 2-5: When the molar ratio of glutaric acid added / cadaverine in the process solution after the second step is 1.0 A conversion solution of cadaverine glutarate with a glutarate / cadaverine molar ratio of 1.0 was supplied, and the mother liquor was sequentially recycled and the purification process proceeded as in Example 2-1.

[0154] In Examples 2-3 to 2-5, the molar ratio of glutaric acid to lysine in the lysine fermentation step was adjusted to 0.79-0.95.

[0155] Example 2-6: When the molar ratio of glutaric acid added / cadaverine in the process solution after the second step is 1.05 A conversion solution of cadaverine glutarate with a glutarate / cadaverine molar ratio of 1.05 was supplied, and the mother liquor was sequentially recycled and the purification process proceeded as in Example 2-1.

[0156] Example 2-7: When the molar ratio of glutaric acid added / cadaverine in the process solution after the second step is 1.10 A conversion solution of cadaverine glutarate with a glutarate / cadaverine molar ratio of 1.1 was supplied, and the mother liquor was sequentially recycled and the purification process proceeded as in Example 2-1.

[0157] The physical property measurements for the process solutions and cadaveral glutarates produced by the above-described Examples 2-1 to 2-7 are shown in Tables 6 to 9.

[0158] Table 6 relates to process fluids that do not circulate the mother liquor.

[0159] [Table 6]

[0160] Table 7 shows the process fluid after the mother liquor has been circulated once.

[0161] [Table 7]

[0162] Table 8 relates to the process fluid after the mother liquor has been circulated twice.

[0163] [Table 8]

[0164] Table 9 relates to the process fluid after the mother liquor has been circulated three times.

[0165] [Table 9]

[0166] Based on the experimental results shown in Tables 6 to 9, it was confirmed that the manufacturing method of this invention, which includes a large amount of diammonium glutarate in the first step at a level of 88-100 mol% of the total supplied glutaric acid, enables the production of cadaveral glutarate with generally improved crystallinity, high purity, and high yield.

[0167] More specifically, Example 2-1 had a high purity of 99% after the third mother liquor cycle, but a low yield of 86.1%. This is because the sequentially circulated mother liquor lowered the molar ratio of glutarate in the crystallization feed solution, resulting in a glutarate concentration that was insufficient compared to the cadaveral glutarate concentration.

[0168] Example 2-2 exhibits a high purity of 99% and a high yield of 90.2% after the third stage of mother liquor processing.

[0169] On the other hand, Examples 2-3 exhibited a high purity of 99% and a high yield of 92.8% after the third stage of mother liquor processing.

[0170] Examples 2-4 exhibited a high purity of 99% and a high total yield of 93.9% after the third stage of mother liquor processing.

[0171] Examples 2-5 exhibited a high purity of 99% and a high total yield of 94.7% after the third stage of mother liquor processing.

[0172] Examples 2-6 exhibited a low purity of 95.5% and a yield of 94.2% during the third stage of the mother liquor reaction. This was because glutaric acid exceeding its solubility precipitated as crystals, resulting in low purity and insufficient concentration compared to that of cadaveral glutarate.

[0173] Examples 2-7 exhibited an even lower purity than Example 3, which had a purity of 91.6% after the third mother liquor cycle, and a slightly lower yield of 93.8%. This was because glutaric acid exceeding its solubility precipitated as crystals, lowering the purity and resulting in a concentration insufficient compared to that of cadaveral glutarate.

[0174] Thus, we have discovered that a method for producing high-purity cadaverin glutarate can be provided depending on the molar ratio of glutaric acid added throughout the entire process to the amount of cadaverine in the process solution after the second step. In particular, we confirmed that when the amount of glutaric acid added is adjusted to a molar ratio of approximately 0.89 to 1.0 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 glutarate while showing an excellent total yield of 92% or more, even after three or more mother liquor cycles.

[0175] From the above description, a person skilled in the art to which this application pertains will understand that this application can be implemented in other specific forms without altering its technical idea or essential features. In this regard, it should be understood that the embodiments described above are merely illustrative and not limiting. The scope of this application should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, as described below, and their equivalent concepts, rather than from the above detailed description.

Claims

1. The first step is to culture L-lysine-producing microorganisms in a medium to which dicarboxylic acid has been added in the form of diammonium dicarboxylate to obtain lysine dicarboxylate; and A method for producing cadavering dicarboxylate, comprising a second step of converting lysine dicarboxylate to cadavering dicarboxylate, The amount of dicarboxylic acid added is in a molar ratio of 0.85 to 1.07 compared to the amount of cadaverine in the process solution after the second step. The dicarboxylic acid is an aliphatic dicarboxylic acid having 4 or 5 carbon atoms. Method for producing cadaverine dicarboxylate.

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 0.78 to 0.

98.

4. 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.

5. The aforementioned dicarboxylic acid is succinic 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 1.05 compared to the amount of cadaverine in the process solution after the second step.

6. The dicarboxylic acid is glutaric acid, The method for producing cadaverine dicarboxylate according to claim 1, wherein the amount of dicarboxylic acid added is in a molar ratio of 0.89 to 1.0 compared to the amount of cadaverine in the process solution after the second step.

7. 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.

8. 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.

9. The method for producing a cadavering dicarboxylate according to claim 7, 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.

10. The aforementioned recovery step is, The method for producing a cadavering dicarboxylate according to claim 7, 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.

11. The method for producing a cadavering dicarboxylate according to claim 8, wherein the concentration step is to concentrate the salt so that the solid content is 75-82% (w / w).

12. 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.

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

14. 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.

15. The aforementioned dicarboxylic acid is succinic acid, The method for producing a cadavering dicarboxylate according to claim 9, wherein a cadavering dicarboxylate with a purity of 99% or more can be obtained in the 0 to 4 cycles of the mother liquor.

16. The dicarboxylic acid is glutaric acid, The method for producing a cadavering dicarboxylate according to claim 9, wherein a cadavering dicarboxylate with a purity of 99% or more can be obtained in the 0 to 3 cycles of the mother liquor.

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