Cadaverine adipine salt manufacturing process

Abstract

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

Description

Technical Field

[0001] This application relates to a technology for producing high-purity cadaverine adipate using microbial fermentation and purification processes.

Background Art

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

[0003] The preparation process of cadaverine adipate generally includes the following steps:

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

[0005] Production of adipic acid: Adipic acid, which is another monomer required for the production of cadaverine adipate, can be synthesized by various methods such as the oxidation of cyclohexene or cyclohexanol using nitric acid or air.

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

[0008] According to conventional technology, due to the low solubility of adipicates, a problem arose during the crystallization process described above: adipicates precipitated as crystals along with cadaverine adipicates. Therefore, in order to produce high-purity cadaverine adipicates, adipicates had to be removed before crystallization, which resulted in additional costs due to the extra steps required for adipicate removal, decreased process efficiency, and environmental pollution due to the use of chemical solvents. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] U.S. Patent Application Publication No. 2021 / 0355514 [Patent Document 2] U.S. Patent Application Publication No. 2018 / 0030430 [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 adipate. [Means for solving the problem]

[0011] This application provides a method for producing high-purity cadaverine adipate without the need for conventional ion resin exchange, decarboxylation, or distillation steps, by introducing diammonium adipate into lysine-producing microorganisms and culturing them. [Effects of the Invention]

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

[0013] [Figure 1] This is a flowchart illustrating the manufacturing process for cadaverine adipate according to this application. [Figure 2] This is a flowchart illustrating the manufacturing process of cadaverine adipate according to one aspect of this application. [Figure 3] This graph shows the results of converting lysine adipate to cadaverine adipate using a bacterial strain that expresses lysine decarboxylase. [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 adipine, comprising a first step of culturing an L-lysine-producing microorganism in a medium supplemented with adipic acid in the form of diammonium adipine to obtain lysine adipine, and a second step of converting lysine adipine to cadaverine adipine.

[0016] In this application, the term "Cadaverine (CAD)" refers to 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 adipate.

[0017] In this application, the term "Cadaverine adipate" is a substance produced by reacting adipic acid, which is a component of dicarboxylic acid, with cadaverine in the diamine form, and it has a high industrial utilization degree as a precursor for the production of polyamide resin.

[0018] Figures 1 and 2 are flowcharts showing the production process of cadaverine adipate according to an aspect of this application.

[0019] In this application, the term "L-lysine-producing microorganism" includes all wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modifications, such as the insertion of foreign genes, or the enhancement or inactivation of the activity of endogenous genes, resulting in the weakening or strengthening of specific mechanisms. It may also be a microorganism that includes genetic modification for the production of L-lysine.

[0020] In one example, the microorganism that produces L-lysine in this application may be a microorganism that naturally has the ability to produce L-lysine, a microorganism to which the ability to produce L-lysine has been imparted to a microorganism that originally did not have the ability to produce L-lysine, or a microorganism in which the ability to produce L-lysine has been enhanced in a microorganism that originally had a significantly low ability to produce L-lysine, but is not limited thereto. Specifically, the microorganism that produces L-lysine or has the ability to produce L-lysine in this application may be a microorganism in which a part of the genes in the L-lysine biosynthesis pathway has been enhanced or weakened, or a part of the genes in the L-lysine degradation pathway has been enhanced or weakened. The "enhancement" or "increase" in the ability to produce L-lysine means that the ability to produce L-lysine has been improved compared to the parent strain or non-modified microorganism.

[0021] 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, it may be Escherichia coli, Escherichia albertii, Escherichia blattae, Escherichia fergusonii, Escherichia hermannii or Escherichia vulneris. More specifically, the Escherichia genus strain may be Escherichia coli, but is not limited thereto.

[0022] The cultivation of the L-lysine-producing microorganism can be carried out according to appropriate media and cultivation conditions known in the art. Such a cultivation process can be easily adjusted and used by those skilled in the art according to the selected microorganism. Specifically, the cultivation may be batch, continuous or fed-batch, but is not limited thereto.

[0023] In the present application, the term "medium" means a substance mixed mainly with nutrients necessary for culturing the microorganism, and supplies nutrients and growth factors such as water, which is indispensable for survival and growth. Specifically, the medium used for culturing the L-lysine-producing microorganism of the present application can be any medium used for culturing ordinary microorganisms without particular limitation, except that it is a part or all of the nitrogen source and contains diammonium adipate. The microorganism of the present application can be cultured under aerobic conditions while adjusting the temperature, pH, etc. in an ordinary medium containing a nitrogen source containing diammonium adipate, an appropriate carbon source, a phosphorus source, inorganic compounds, amino acids and / or vitamins.

[0024] In this application, the term "adipic acid" refers to a six-carbon dicarboxylic acid with the chemical formula (CH2)4(COOH)2, generally existing as a white crystal or crystalline powder. Adipic acid is also known as adipic acid or hexanedioic acid, and is mostly a precursor for the manufacture of nylon, making it a compound with very high industrial applications.

[0025] The first step of the method for producing cadaverine dicarboxylate according to this application is to culture L-lysine-producing microorganisms in a medium supplemented with adipic acid in the form of diammonium adipate to obtain lysine adipate. This is carried out through a fermentation process of the lysine-producing microorganisms.

[0026] In the first step, the adipic acid is supplied to the culture medium in the form of diammonium adipicate, and it should be understood that in this application, adipic acid includes the form of diammonium adipicate.

[0027] In this application, the term "diammonium adipate" refers to a form in which two hydrogen atoms of adipic acid are replaced with ammonium, and is used as the primary nitrogen source for the culture medium. The diammonium adipate may be used in crystalline or liquid form after being prepared by gently mixing adipic acid with aqueous ammonia to a neutral pH and then concentrating it.

[0028] The manufacturing method described in this application simplifies the process while producing high-purity cadaverine adipic acid by adding 56 mol% or more of the adipic acid supplied throughout the entire process to the culture medium in the form of diammonium adipic acid in the first step, thereby reducing the side effect of adipic acid precipitation together with cadaverine adipic acid.

[0029] Specifically, the diammonium adipic acid in the first step may be included in the culture medium in a high proportion relative to the adipic acid supplied throughout the entire process of the manufacturing method of this invention, specifically in an amount of 81 mol% or more.

[0030] More specifically, the diammonium adipicate in the first step may be present in an amount of 56 mol% or more, 66 mol% or more, 74 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 adipic acid supplied in all steps of the manufacturing method of this application.

[0031] The manufacturing method described in this application, by including a large amount of diammonium adipate in the culture medium, offers the advantage of being able to produce high-purity cadaverine adipate without performing separate adipicate removal steps such as ion resin exchange, decarboxylation, or distillation, unlike conventional processes that add ammonium sulfate or the like. Furthermore, by using diammonium adipate, it is possible to easily adjust the molar ratio of adipic acid added throughout the entire process to cadaverine in the process solution after the second step.

[0032] Specifically, the diammonium adipate in the first step may be included in such a molar ratio (diammonium adipate / lysine) with respect to the lysine to be produced that is 0.55 to 1. More specifically, the diammonium adipate in the first step may be included in such a molar ratio (diammonium adipate / lysine) with respect to the lysine to be produced that is 0.65 to 0.98, 0.73 to 0.98, or 0.85 to 0.98, or more specifically, 0.88 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 adipate.

[0033] For example, the diammonium adipate in the first step may be included in a molar ratio (diammonium adipate / lysine) within a range defined by one lower limit selected from 0.55, 0.60, 0.65, 0.7, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, and 0.92 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.

[0034] Specifically, the diammonium adipic acid in the first step can be included in a concentration of 70 g / L to 140 g / L based on the L-lysine-producing microbial fermentation broth. More specifically, the diammonium adipic acid in the first step can be included in a concentration of 80 g / L to 130 g / L, 90 g / L to 130 g / L, 105 g / L to 130 g / L, or 105 g / L to 120 g / L based on the L-lysine-producing microbial fermentation broth. When diammonium adipic acid is added at the above concentrations, the ammonium ions act as a nitrogen source during fermentation, and adipic acid and lysine form a salt, which may be included dissolved in the fermentation broth.

[0035] Lysine adipate can be produced through fermentation by L-lysine-producing microorganisms in a culture medium containing diammonium adipate. The lysine adipate 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.

[0036] 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, 95 g / L to 250 g / L, 97 g / L to 160 g / L, or 97 g / L to 100 g / L based on the L-lysine-producing microorganism fermentation liquid.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] The nitrogen source of the culture medium may include diammonium adipate. 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 adipate may be included as a sole nitrogen source or as an additional nitrogen source.

[0042] After the first step of culturing L-lysine-producing microorganisms in a medium containing diammonium adipate to produce lysine adipate, a second step may be performed to convert the lysine adipate to cadaverine adipate.

[0043] The second step involves converting lysine adipine to the target product, cadaverine adipine.

[0044] In one specific example, the second step may include further adding adipic acid to the process liquid. The process liquid may be the fermentation process liquid obtained in the first step.

[0045] The adipic acid added in the second step may be omitted, or it may be added in trace amounts of 0.6 mol / L or less, 0.35 mol / L or less, 0.18 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, or 0.02 mol / L or less, based on the volume of the fermentation liquid obtained in the first step.

[0046] If adipic acid is further added to the process solution in the second step, the adipic acid may, but is not limited to, be added before, after, or simultaneously with the conversion reaction.

[0047] If adipic acid is further added in the second step, the total amount of adipic acid added throughout the entire process of the present invention is understood to be the sum of the amount of diammonium adipicate added to the culture medium in the first step and the amount of adipic acid added in the second step. Also, if adipic acid is not further added to the process solution in the second step, the total amount of adipic acid added throughout the entire process of the present invention is understood to be equal to the amount of diammonium adipicate added to the culture medium in the first step.

[0048] Specifically, the amount of adipic 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 adipic acid added in the manufacturing method of this application may be in a molar ratio of 0.85 to 1.05 compared to the amount of cadaverine in the process solution after the second step.

[0049] More specifically, the amount of adipic acid added in the manufacturing method of this application may be in a molar ratio of 0.87 to 1.04, 0.87 to 1.03, 0.88 to 1.03, 0.88 to 1.02, 0.89 to 1.03, 0.89 to 1.02, 0.89 to 1.01, or 0.9 to 1.01 compared to the amount of cadaverine in the process solution after the second step.

[0050] As an example, the amount of adipic 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 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, and 0.92 and / or one upper limit selected from 1.05, 1.04, 1.03, 1.02, and 1.01, relative to the amount of cadaverine in the process solution after the second step.

[0051] One technical significance of this invention lies in the discovery that, in the manufacturing method described herein, when adipic 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 adipic acid does not precipitate as crystals together with cadaverine adipicate, making it possible to produce cadaverine adipicate in high purity and high yield.

[0052] Furthermore, by adjusting the molar ratio of the obtained cadaverine to the added adipic acid, it is possible to maintain a purity of 99% of cadaverine adipine salt up to the third cycle during mother liquor circulation, while obtaining total yields of 45% or more, 47% or more, 57% or more, 59% or more, 64% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, or 74% or more of cadaverine adipine salt depending on the number of mother liquor circulations.

[0053] In the second step, the reaction to convert lysine adipate to cadaverine adipate may be an enzymatic conversion reaction.

[0054] As one specific example, the lysine adipate may include the lysine adipate obtained in the first step, and, if adipic acid is further added in the second step, all of the lysine adipate produced in the second step.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] The microorganism expressing the protein may be a microorganism that has been transformed to express the protein. The transformed microorganism is not limited to prokaryotes or eukaryotes, 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 lysine adipate in the process solution to cadaverine adipate.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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 adipic 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 adipine to cadaverine adipine can be significantly increased.

[0064] The method for producing cadaverine adipine salt according to this application may further include a recovery step of recovering the obtained cadaverine adipine salt after carrying out the second step.

[0065] The aforementioned recovery may involve collecting cadaverine adipate using appropriate methods known in the art.

[0066] For example, cadaverine adipate 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.

[0067] 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.

[0068] The filtration step refers to the step of removing impurities from the conversion solution containing cadaverine adipate obtained in the second step. As an example, the filtration may include removing microbial cells in the cadaverine adipate process solution by membrane separation or by activated carbon filtration.

[0069] 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.

[0070] Specifically, the concentration step may involve concentrating the solids content to 65-85% (w / w), more specifically to 70-85% (w / w), 72-83% (w / w), 65-80% (w / w), 65-75% (w / w), 75-85% (w / w), 70-80% (w / w), 72-78% (w / w), or 73-77% (w / w), and as an example, it may involve concentrating to 75% (w / w).

[0071] The concentration step may be carried out at 40-60°C, more specifically at 45-55°C, or, for example, at 50°C, but is not limited thereto.

[0072] 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 adipate.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] In one embodiment, the recovery step may further include a mother liquor circulation step in which the mother liquor containing uncrystallized cadaverine adipate after the crystallization step is recirculated with the feed liquid of the concentration step.

[0077] 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.

[0078] Conventionally, in the step of crystallizing cadaverine adipate, a relatively large amount of cadaverine adipate (more than 50%) remains in the mother liquor after crystallization, resulting in a problem of low yields of less than 50%. In this application, as a method to improve the yield of cadaverine adipate, the total yield of cadaverine adipate can be increased to 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, or higher by recirculating the residual mother liquor of cadaverine adipate with the feed solution of the concentration step one to three times, one to four times, one to five times, one to ten times or more.

[0079] In one embodiment, the recovery step may further include washing, separating, and drying the crystallized process liquid.

[0080] In the drying step described above, moisture contained in the crystals is removed through drying, allowing for the production of a highly pure cadaverine adipate product. After drying, the cadaverine adipate crystals can be provided in powder form, but the dosage form can vary as needed.

[0081] 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.

[0082] Furthermore, the method for producing cadaverine adipine salt according to this application may include an additional purification step. Such purification can be carried out using a suitable method known in the art. For example, if the method for producing cadaverine adipine salt 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, regardless of the procedure, simultaneously or integrated into a single step, but are not limited thereto.

[0083] The manufacturing method of this application is characterized by not including a separate step for removing adipicates in the recovery step. In the conventional production process of cadaverine adipicates, adipicates precipitate as crystals during the crystallization process, reducing purity. To prevent this, a separate step was required to remove the adipicates before crystallization. However, the manufacturing method of this application offers the technical advantage of being able to produce high-purity cadaverine adipicates without performing a separate step for removing adipicates.

[0084] In one embodiment, the method of this application is characterized by obtaining cadaverine adipate with a purity of 99% or more in 0 to 3 cycles of mother liquor circulation.

[0085] In one embodiment, based on three circulations of the mother liquor, the cumulative total yield of cadaverine adipate may be 65% or more, more specifically, 69%, 70%, 71%, 72%, 73%, 74%, or higher.

[0086] Through the method of this application, high-purity cadaverine adipate can be produced without the decarboxylation and distillation steps that are normally required in the production of cadaverine liquid phase. Examples

[0087] 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.

[0088] The above describes a cadaverine adipate manufacturing process according to one aspect of this application. Below, the advantageous effects mentioned in this application will be described through the experimental results of the examples.

[0089] Production Example 1-1. Fermentation process for lysine adipate Corynebacterium glutamicum strain (KCCM12154P, U.S. Public Registry No. US 2021-0355514 A1) capable of lysine production was obtained through solid-phase culture and flask culture, and after seed culture in a fermenter, the main production fermentation was carried out. The main production fermentation was performed through fermenter culture at 36°C for 30 hours at 900 rpm.

[0090] In each example, diammonium adipate was supplied at a level of 72 to 117 g / L instead of ammonium sulfate, and fermentation was carried out. As a result, lysine adipate was obtained at a level of 116 to 190 g / L, and lysine at a level of 99 g / L. At that time, the molar ratio of adipic acid / lysine on the fermentation liquid was at a level of 0.59 to 0.96 in each example, and in particular, in Examples 4 to 7, the molar ratio of adipic acid / lysine on the fermentation liquid was adjusted to a range of 0.88 to 0.96.

[0091] Manufacturing Example 1-2. Conversion reaction of lysine adipate to cadaverine adipate. 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.

[0092] The conversion reaction was carried out by supplying the seed culture solution of the enzyme strain to the fermentation process liquid of lysine adipate prepared from Production Example 1-1 at a level of 10% by mass percentage. 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 1-4 below, CO2 was added to neutralize the pH, and in Examples 2 and 3, adipic acid was added at a level of 0.02-0.03 mol / L (based on the volume of the fermentation process liquid of lysine adipate). Figure 3 is a graph of the results of the conversion reaction, and it was confirmed that the conversion rate was at a level of 97-98%. After the conversion reaction, cadaverine adipic acid at a level of 99-161 g / L (based on the volume of the fermentation liquid of lysine adipic acid) was obtained, and cadaverine at a level of 68 g / L (based on the volume of the fermentation liquid of lysine adipic acid) was obtained. Subsequently, in the case of Examples 5-7, adipic acid was added at a level of 0.02-0.15 mol / L to produce the process liquid.

[0093] Manufacturing Example 1-3. Subsequent steps after obtaining cadaverine adipine salt. After the conversion reaction according to Production Example 1-2, the following subsequent steps were performed on the process solution containing cadaverine adipine to obtain cadaverine adipine in crystalline form.

[0094] [Bacterial cell removal step] Bacterial cells in the cadaverine adipate process solution were removed by membrane filtration with a pore size of 0.1 μm.

[0095] [Steps to remove impurities using activated carbon] Activated carbon was added to the filtrate from which the bacterial cells had been removed, at a level of 10% based on the weight of cadaverine adipate. The mixture was heated to 60°C and stirred for 1 hour to decolorize it, and then the activated carbon was filtered through filter paper.

[0096] [Step to concentrate the process solution of cadaverine adipine salt from which impurities have been removed] The filtrate from which the activated carbon had been removed was concentrated in a rotary evaporator under reduced pressure at approximately 50°C and 120 Torr until the solid content was 75% by weight.

[0097] [Steps to cool, crystallize, separate, and dry a concentrated solution of cadaverine adipinate] The concentrate was cooled from 50°C to 25°C at a rate of 10°C / hr. The precipitated crystals were separated from the mother liquor using a centrifuge. The crystals were washed with 10% water based on the weight of cadaverine adipate. After drying the separated crystals for one day, their purity was measured by HPLC.

[0098] [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 adipate from which impurities have been removed].

[0099] Experimental Example 1. Confirmation of changes in purity and yield of cadaverine adipate according to the molar ratio of adipate 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 adipate were examined to see how they changed depending on the molar ratio of adipic acid added throughout the entire process to cadaverine in the process solution after the second step.

[0100] Example 1: When the molar ratio of adipic acid added to the process solution after the second step is 0.6 1,000 ml of a cadaverine adipate conversion solution with an adipic acid / cadaverine molar ratio of 0.6 was passed through a 0.1 μm pore size membrane to remove bacterial cells. The cadaverine adipate conversion solution with an adipic acid / cadaverine molar ratio of 0.6 was produced by controlling the total number of moles of adipic acid added in the conversion reaction between diammonium adipate and cadaverine adipate, which were added in the fermentation process for producing lysine adipate, so that it was 0.6 times the number of moles of converted cadaverine (see Equation 1 below).

[0101]

number

[0102] The filtrate from which the bacterial cells were removed was decolorized using activated carbon and filtered through filter paper, then concentrated under reduced pressure to a solid content of 75%. The concentrate was cooled and crystallized from 60°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 adipic acid, was recirculated with a feed solution of cadaverine adipic acid with an adipic acid / cadaverine molar ratio of 0.6, and the crystallization step was repeated. The recirculation of the mother liquor was carried out sequentially.

[0103] Example 2: When the molar ratio of adipic acid added to the process solution after the second step is 0.71 Following the supply of a conversion solution of cadaverine adipic acid with an adipic acid / cadaverine molar ratio of 0.71, the mother liquor was sequentially recycled and the purification process proceeded as in Example 1.

[0104] Example 3: When the molar ratio of adipic acid added to the process solution after the second step is 0.8 A conversion solution of cadaverine adipinate, with an adipic acid / cadaverine molar ratio of 0.8, was supplied, and the mother liquor was sequentially recycled and the purification process proceeded as in Example 1.

[0105] Example 4: When the molar ratio of adipic acid added to the process solution after the second step is 0.9 Following the supply of a conversion solution of cadaverine adipinate with an adipic acid / cadaverine molar ratio of 0.9, the mother liquor was sequentially recycled and the purification process proceeded as in Example 1.

[0106] Example 5: When the molar ratio of adipic acid added to the process solution after the second step is 1.01 The mother liquor was sequentially recycled and the purification process proceeded as in Example 1, after receiving a conversion solution of cadaverine adipic acid with a molar ratio of adipic acid / cadaverine of 1.01.

[0107] In Examples 4 and 5, the molar ratio of adipic acid to lysine in the lysine fermentation step was adjusted to 0.85-0.98.

[0108] Example 6: When the molar ratio of adipic acid added to the process solution after the second step is 1.1 The mother liquor was sequentially recycled and the purification process proceeded as in Example 1, after receiving a conversion solution of cadaverine adipic acid with a molar ratio of adipic acid / cadaverine of 1.1.

[0109] Example 7: When the molar ratio of adipic acid added to the process solution after the second step is 1.2 Following the supply of a conversion solution of cadaverine adipic acid with an adipic acid / cadaverine molar ratio of 1.2, the mother liquor was sequentially recycled and the purification process proceeded as in Example 1.

[0110] The physical properties measured for the process solutions and cadaverine adipate produced by the above-described Examples 1 to 7 are shown in Tables 1 to 4.

[0111] Table 1 relates to process fluids that do not circulate the mother liquor.

[0112] [Table 1]

[0113] Table 2 shows the process fluid after the mother liquor has been circulated once.

[0114] [Table 2]

[0115] Table 3 relates to the process fluid after the mother liquor has been circulated twice.

[0116] [Table 3]

[0117] Table 4 relates to the process fluid after the mother liquor has been circulated three times.

[0118] [Table 4]

[0119] Based on the experimental results shown in Tables 1 to 4, it was confirmed that the manufacturing method of this invention, which includes a large amount of diammonium adipate from the first step at a level of 81 to 100 mol% of the total supplied adipic acid, enables the production of cadaverine adipate with generally improved crystallinity, high purity, and high yield.

[0120] More specifically, in Example 1, no crystals precipitated even after the crystallization process was carried out. This is because the molar ratio of adipic acid was low and insufficient for cadaverine adipicate crystals to precipitate.

[0121] In Example 2, crystals precipitated during the first mother liquor circulation, but no crystals were formed during the second circulation. This is because, after the first circulation of the mother liquor, the molar ratio of adipic acid in the crystallization feed solution became too low to adequately precipitate cadaverine adipicate crystals.

[0122] In Example 3, crystals precipitated up to the second mother liquor circulation, but no crystals were formed during the third circulation. This is because the sequentially circulated mother liquor lowered the molar ratio of adipic acid in the crystallization supply solution, resulting in an insufficient concentration of adipic acid for the precipitation of cadaverine adipic acid crystals.

[0123] In Example 4, high-purity cadaverine adipine salt with a purity of 99% was obtained up to the third mother liquor circulation, and a high overall yield of approximately 70% was also achieved.

[0124] In Example 5, high-purity cadaverine adipine salt with a purity of 99% was obtained up to the third mother liquor circulation, and a high overall yield of approximately 74% was also achieved.

[0125] In Example 6, crystals precipitated up to the second mother liquor circulation, but the purity of the crystals decreased slightly to 86%. This is because, as the mother liquor circulated sequentially, adipic acid accumulated in the crystallization supply solution and precipitated as an impurity.

[0126] In Example 7, the purity of the crystals decreased slightly to 84% after the first mother liquor circulation. This is because, as the mother liquor circulated sequentially, adipic acid accumulated in the crystallization feed solution and precipitated as an impurity.

[0127] Thus, we have discovered that a method for producing high-purity cadaverine adipic acid can be provided depending on the molar ratio of adipic 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 adipic acid added is adjusted to a molar ratio of approximately 0.9 to 1.01 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 adipic acid while achieving an excellent total yield of 69% or more, even after three or more mother liquor cycles.

[0128] 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 supplemented with adipic acid in the form of diammonium adipate to obtain lysine adipate; and A method for producing cadaverine adipine salt, comprising a second step of converting lysine adipine salt to cadaverine adipine salt, The amount of adipic acid added is present in a molar ratio of 0.85 to 1.05 compared to the amount of cadaverine in the process solution after the second step. Method for manufacturing cadaverine adipine salt.

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

3. The method for producing cadaverine adipate according to claim 1, wherein the diammonium adipate in the first step is included in such a way that the molar ratio (diammonium adipate / lysine) with the lysine to be produced is 0.85 to 0.

98.

4. The method for producing cadaverine adipate 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 method for producing cadaverine adipic acid salt according to claim 1, wherein the amount of adipic acid added is in a molar ratio of 0.88 to 1.02 compared to the amount of cadaverine in the process solution after the second step.

6. The method for producing cadaverine adipate according to claim 1, further comprising a recovery step of recovering cadaverine adipate after carrying out the second step.

7. The method for producing cadaverine adipate according to claim 6, 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.

8. The aforementioned recovery step is, The method for producing cadaverine adipate according to claim 7, further comprising a mother liquor circulation step of recirculating the mother liquor containing uncrystallized cadaverine adipate after the cooling crystallization step with the supply liquid of the concentration step.

9. The method for producing cadaverine adipate according to claim 7, wherein the concentration step involves concentrating the salt so that the solid content is 70-80% (w / w).

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

11. The method for producing cadaverine adipine salt according to claim 1, wherein the L-lysine-producing microorganism is Corynebacterium glutamicum.

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

13. The method for producing cadaverine adipate according to claim 8, wherein cadaverine adipate with a purity of 99% or more can be obtained in the 0 to 3 cycles of the mother liquor.

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