Process for preparing cadaverine dicarboxylate

Abstract

The present application relates to a method for preparing a cadaverine dicarboxylate (cadaverine suberate or cadaverine azelate), the method comprising: a first step of obtaining a lysine dicarboxylate by culturing an L-lysine producing microorganism in a culture medium to which a dicarboxylic acid in the form of a diammonium dicarboxylate is added; and a second step of converting the lysine dicarboxylate into a cadaverine dicarboxylate. According to the present application, a cadaverine dicarboxylate (cadaverine suberate or cadaverine azelate) of high purity can be obtained without ion resin exchange, a decarboxylation process, and a distillation process by implementing the method according to the above procedure.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to a technology for preparing high-purity cadaverine dicarboxylate (cadaverine suberate or cadaverine azelate) using microbial fermentation and purification processes. BACKGROUND

[0002] Cadaverine is a foul-smelling, toxic diamine compound produced by the decay of animal tissues. In addition, cadaverine dicarboxylate, such as cadaverine suberate or cadaverine azelate, is a precursor of biopolyamide polymerization, which is one of engineering plastics widely used in automotive vehicles, electrical and electronic components, etc.

[0003] The method of preparing cadaverine suberate or cadaverine azelate generally includes the following steps: Synthesizing cadaverine: synthesizing cadaverine includes fermentation of a suitable microbial strain capable of producing lysine decarboxylase, which is an enzyme that catalyzes the decarboxylation of lysine to produce cadaverine.

[0004] Preparing suberic acid or azelaic acid: suberic acid is another monomer required to produce cadaverine suberate and can be synthesized by various methods such as oxidation of adipamide, polymerization of carbonyl diimidazole, and oxidation of stearic acid. In addition, azelaic acid is another monomer required to produce cadaverine azelate and can be synthesized by various methods such as ozonolysis of oleic acid.

[0005] Forming cadaverine suberate or cadaverine azelate: forming cadaverine suberate or cadaverine azelate involves the reaction of cadaverine with suberic acid or azelaic acid in the presence of a suitable catalyst such as sulfuric acid or phosphoric acid. The reaction generally includes mixing the two monomers at a suitable temperature and stirring for a certain period of time to ensure complete reaction.

[0006] Purifying cadaverine suberate or cadaverine azelate: after the reaction is complete, various separation and purification techniques such as solvent extraction, chromatography, and crystallization are used to purify the crude cadaverine suberate or cadaverine azelate.

[0007] According to the prior art, suberate or azelate has low solubility, and thus there is a problem in that, in the crystallization step of the above method, suberate or azelate is precipitated as crystals together with cadaverine suberate or cadaverine azelate, respectively. Therefore, in order to produce high-purity cadaverine suberate or cadaverine azelate, it is necessary to remove suberate or azelate before crystallization; there are problems such as cost burden due to an additional suberate or azelate removal process, reduced process efficiency, and environmental pollution due to the use of chemical solvents, etc. SUMMARY

[0008] Technical issues The problem to be solved by this disclosure is to provide a method for preparing cadaverine dicarboxylate (cadaverine octanoate or cadaverine nonanoate).

[0009] Technical solution One object of this disclosure is to provide a method for preparing cadaverine dicarboxylate, comprising: a first step of obtaining lysine dicarboxylate by culturing L-lysine producing microorganisms in a culture medium containing dicarboxylate in the form of diammonium dicarboxylate; and a second step of converting lysine dicarboxylate into cadaverine dicarboxylate.

[0010] Beneficial effects According to one aspect of this disclosure, lysine dicarboxylate is obtained by culturing L-lysine-producing microorganisms in a culture medium containing diammonium dicarboxylate, and the lysine dicarboxylate is enzymatically converted to cadaverine dicarboxylate. This simplifies the process because a separate process for removing the dicarboxylate is not required, and high-purity cadaverine dicarboxylate can be prepared in high yields because the dicarboxylic acid crystals do not precipitate as byproducts. Specifically, cadaverine dicarboxylate refers to cadaverine octanoate or cadaverine azelate. Attached Figure Description

[0011] Figure 1 This is a flowchart illustrating the method for preparing cadaverine dicarboxylate according to this disclosure; and Figure 2 This is a flowchart illustrating a method for preparing cadaverine dicarboxylate according to one aspect of this disclosure. Detailed Implementation

[0012] The configuration and effects of this disclosure will be described in detail below. Furthermore, each description and embodiment disclosed herein can also be applied to other descriptions and other embodiments. In other words, all combinations of the various elements disclosed herein fall within the scope of this disclosure. Moreover, the scope of this disclosure is not limited to the specific descriptions below.

[0013] One aspect of this disclosure provides a method for preparing cadaverine dicarboxylate, comprising: a first step of obtaining lysine dicarboxylate by culturing L-lysine-producing microorganisms in a culture medium containing diammonium dicarboxylate in the form of diammonium dicarboxylate; and a second step of converting the lysine dicarboxylate into cadaverine dicarboxylate, wherein the dicarboxylate and the dicarboxylic acid have 8 or 9 carbon atoms.

[0014] When both dicarboxylate and dicarboxylic acid have 8 carbon atoms, they refer to octanoate and octanoic acid, respectively. When both dicarboxylate and dicarboxylic acid have 9 carbon atoms, they refer to azelaate and azelaic acid, respectively.

[0015] In this disclosure, the term "dicarboxylic acid" refers to a dicarboxylic acid containing two carboxyl groups and is very important as a metabolic intermediate in living organisms.

[0016] Specifically, the dicarboxylic acid can be an aliphatic dicarboxylic acid having 8 or 9 carbon atoms. In other words, in this disclosure, a dicarboxylic acid refers to an aliphatic dicarboxylic acid selected from octanoic acid or azelaic acid. When the cadaverine dicarboxylate produced by the preparation method of this disclosure is cadaverine octanoic acid salt, it should be understood that all added or introduced dicarboxylic acids are octanoic acid. Furthermore, when the cadaverine dicarboxylate produced by the preparation method of this disclosure is cadaverine azelaic acid salt, it should be understood that all added or introduced dicarboxylic acids are azelaic acid. Therefore, in this disclosure, a dicarboxylic acid is understood to refer to one type of aliphatic dicarboxylic acid, rather than a mixture of different types of dicarboxylic acids.

[0017] In this disclosure, the term "octanedioic acid" is a dicarboxylic acid having 8 carbon atoms and the chemical formula C8H12H2O. 14 O4, and is usually found as a colorless crystalline solid. Also known as octanedioic acid, it is used as a precursor for a variety of industrial products, including polymers and plasticizers.

[0018] In this disclosure, the term "azelaic acid" is a dicarboxylic acid having 9 carbon atoms and the chemical formula C9H2O. 16 O4, and is usually present as a white powder. Azelaic acid is also known as azelaic acid, etc., and is used as a precursor for a variety of industrial products, including polymers and plasticizers.

[0019] In this disclosure, the term "cadaverine (CAD)" refers to a foul-smelling, toxic diamine compound represented by the chemical formula NH2(CH2)5NH2. Cadaverine can also be called 1,5-pentanediamine or pentamethylenediamine. In this disclosure, cadaverine should be understood to include the cadaverine dicarboxylate form.

[0020] In this disclosure, the term "cadaverine dicarboxylate" refers to a substance prepared by reacting cadaverine in the form of a diamine with a dicarboxylic acid component, and has high industrial applicability as a precursor for the preparation of polyamide resins. Specifically, in this disclosure, cadaverine dicarboxylate refers to a cadaverine dicarboxylate selected from cadaverine octanoate or cadaverine azelate.

[0021] In this disclosure, the term "cadaverine octanoate" refers to a substance prepared by reacting cadaverine in the form of a diamine with the dicarboxylic acid component octanoic acid.

[0022] In this disclosure, the term "cadaverine azelate" refers to a substance prepared by reacting cadaverine in the form of a diamine with azelate, a dicarboxylic acid component.

[0023] Figure 1 and Figure 2 This is a flowchart illustrating a method for preparing cadaverine dicarboxylate according to one aspect of this disclosure.

[0024] In this disclosure, the term "L-lysine-producing microorganism" includes wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modifications, which are microorganisms whose specific mechanisms are weakened or enhanced due to causes such as the insertion of exogenous genes or the enhancement or inactivation of intrinsic genes, and may be microorganisms containing genetic modifications for the production of L-lysine.

[0025] In one instance, the L-lysine-producing microorganism of this disclosure may be a microorganism naturally capable of producing L-lysine, a microorganism constructed by conferring the ability to produce L-lysine upon a microorganism lacking the ability to produce L-lysine, or a microorganism constructed by enhancing the L-lysine-producing ability of a microorganism with a significantly low ability to produce L-lysine, but is not limited thereto. Specifically, in this disclosure, the L-lysine-producing microorganism or the microorganism capable of producing L-lysine may be a microorganism in which some genes in the L-lysine biosynthesis pathway are enhanced or weakened, or some genes in the L-lysine degradation pathway are enhanced or weakened. An "enhancement" or "increase" in the ability to produce L-lysine means an improvement in the ability to produce L-lysine compared to the parental strain or the unmodified microorganism.

[0026] As a specific example, the microorganism can be a Corynebacterium or an Escherichia. Corynebacterium microorganisms can include all microorganisms belonging to the genus Corynebacterium. Corynebacterium species specifically include *Corynebacterium glutamicum*, *Corynebacterium crudilactis*, *Corynebacterium deserti*, *Corynebacterium efficiens*, *Corynebacterium callunae*, *Corynebacterium stationis*, *Corynebacterium singulare*, *Corynebacterium halotolerans*, *Corynebacterium striatum*, *Corynebacterium ammoniagenes*, *Corynebacterium pollutisoli*, *Corynebacterium imitans*, and *Corynebacterium testis*. *Escherichia coli* or *Corynebacterium flavescens*, and more specifically, *Corynebacterium glutamicum*, but not limited thereto. *Escherichia* microorganisms can include all microorganisms belonging to the genus *Escherichia*. *Escherichia* microorganisms can specifically include *Escherichia coli*, *Escherichia albertii*, *Escherichia blattae*, *Escherichia fergusonii*, *Escherichia hermannii*, or *Escherichia vulneris*, and more specifically, *Escherichia coli*, but not limited thereto.

[0027] The cultivation of L-lysine-producing microorganisms can be carried out using suitable culture media and conditions known in the art. This cultivation process can be readily adapted and used by those skilled in the art based on the selected microorganisms. Specifically, the cultivation can be batch, continuous, or fed-batch, but is not limited to these methods.

[0028] In this disclosure, the term "culture medium" refers to a material in which nutrients required for the cultivation of microorganisms are mixed as the main components, and water, nutrients, and growth factors necessary for their survival and development are provided. Specifically, as the culture medium for cultivating the L-lysine-producing microorganisms of this disclosure, any culture medium used for cultivating common microorganisms can be used without particular limitation, as long as it contains diammonium dicarboxylate as part or all of the nitrogen source. The microorganisms of this disclosure can be cultured under aerobic conditions in a common culture medium containing a nitrogen source (including diammonium dicarboxylate), a suitable carbon source, a phosphorus source, inorganic compounds, amino acids, vitamins, and / or the like, while controlling temperature, pH, etc.

[0029] The first step in the disclosed method for preparing cadaverine dicarboxylate is to obtain lysine dicarboxylate by culturing L-lysine-producing microorganisms in a culture medium containing diammonium dicarboxylate. This can be achieved through a fermentation process of the lysine-producing microorganisms.

[0030] In the first step, a dicarboxylate having 8 or 9 carbon atoms can be supplied to the culture medium in the form of diammonium dicarboxylate, therefore the dicarboxylate of this disclosure should be understood to include the form of diammonium dicarboxylate.

[0031] In this disclosure, the term "diammonium octanoate" refers to a form of octanoic acid in which two hydrogen atoms are replaced by ammonium, and is used as a primary nitrogen source in culture media. Diammonium octanoate can be prepared by, but is not limited to, gently mixing octanoic acid with ammonia to achieve a neutral pH, and then concentrating it for use in crystalline or liquid form.

[0032] In this disclosure, the term "diammonium azelaate" refers to a form of azelaic acid in which two hydrogen atoms are replaced by ammonium, and is used as a primary nitrogen source in culture media. Diammonium azelaate can be prepared by, but is not limited to, gently mixing azelaic acid with ammonia to achieve a neutral pH, and then concentrating it for use in crystalline or liquid form.

[0033] In the preparation method disclosed herein, high-purity cadaverine dicarboxylate can be prepared and the process can be simplified by introducing 64 mol% or more of the dicarboxylate supplied throughout the process into the culture medium in the form of diammonium dicarboxylate in the first step, and the side effect of dicarboxylate precipitation along with cadaverine dicarboxylate can be reduced.

[0034] Specifically, the diammonium dicarboxylate in the first step may be included in the culture medium at a high ratio, specifically 64 mol% or more of the dicarboxylate supplied throughout the preparation process of this disclosure.

[0035] More specifically, the content of diammonium dicarboxylate in the first step can be 64 mol% or more, 76 mol% or more, 81 mol% or more, 82 mol% or more, 83 mol% or more, 84 mol% or more, 85 mol% or more, 86 mol% or more, 87 mol% or more, 88 mol% or more, 89 mol% or more, 90 mol% or more, 91 mol% or more, 92 mol% or more, 93 mol% or more, 94 mol% or more, 95 mol% or more, 96 mol% or more, 97 mol% or more, 98 mol% or more, 99 mol% or more, or 100 mol% of the dicarboxylate supplied throughout the preparation method of this disclosure.

[0036] In the preparation method disclosed herein, by including a large amount of diammonium dicarboxylate in the culture medium, it is possible to prepare high-purity cadaverine dicarboxylate without requiring separate dicarboxylate removal steps, such as ion exchange, decarbonization, or distillation processes. This differs from conventional methods that introduce ammonium sulfate, etc. By using diammonium dicarboxylate, the molar ratio of dicarboxylate added throughout the process to cadaverine in the post-second-step process liquid can be easily adjusted.

[0037] Specifically, the amount of diammonium dicarboxylate in the first step can be such that the molar ratio of diammonium dicarboxylate to lysine to be produced (diammonium dicarboxylate / lysine) is 0.60 to 1. More specifically, the amount of diammonium dicarboxylate in the first step can be such that the molar ratio of diammonium dicarboxylate to lysine to be produced (diammonium dicarboxylate / lysine) is 0.6 to 1.0, 0.65 to 0.99, 0.74 to 0.99, or 0.78 to 0.98, 0.8 to 0.98, or 0.83 to 0.98, or 0.8 to 0.96, more specifically 0.79 to 0.99. In this case, the amount of lysine to be produced should be understood as the total amount including lysine in the form of lysine dicarboxylate.

[0038] As an example, the amount of diammonium dicarboxylate in the first step can be such that the molar ratio of diammonium dicarboxylate to lysine to be produced (diammonium dicarboxylate / lysine) is within a range consisting of a lower limit selected from 0.60, 0.65, 0.70, 0.73, 0.74, 0.8, 0.83, 0.85, 0.86, 0.87, 0.88, or 0.89 and / or an upper limit selected from 1, 0.99, 0.98, 0.97, 0.96, 0.95, 0.94, 0.93, or 0.92.

[0039] In a specific example, the dicarboxylate and dicarboxylic acid can be octanoate and octanoic acid, respectively, and the content of diammonium dicarboxylate in the first step can make the molar ratio of diammonium dicarboxylate to lysine to be produced (diammonium dicarboxylate / lysine) 0.81 to 1.0.

[0040] In a specific example, the dicarboxylate and dicarboxylic acid can be azelaate and azelaic acid, respectively, and the content of diammonium dicarboxylate in the first step can make the molar ratio of diammonium dicarboxylate to lysine to be produced (diammonium dicarboxylate / lysine) 0.79 to 1.0.

[0041] Specifically, the content of diammonium dicarboxylate in the first step can be from 0.2 mol / L to 0.8 mol / L, based on the fermentation broth of L-lysine production microorganisms. More specifically, the content of diammonium dicarboxylate in the first step can be from 0.3 mol / L to 0.7 mol / L, 0.4 mol / L to 0.7 mol / L, 0.45 mol / L to 0.7 mol / L, or 0.45 mol / L to 0.53 mol / L, based on the fermentation broth of L-lysine production microorganisms. By adding diammonium dicarboxylate at the above concentrations, ammonium ions can serve as a nitrogen source during fermentation, and the dicarboxylate and lysine can form a salt, thus being contained in the fermentation broth in a dissolved state.

[0042] Lysine dicarboxylate can be produced by fermentation of L-lysine-producing microorganisms in a culture medium containing diammonium dicarboxylate. Lysine dicarboxylate can be introduced into subsequent processes either as is, in the form of fermentation broth containing bacteria, in the form of fermentation broth after bacteria have been removed, or after purification.

[0043] The concentration of lysine in the fermentation broth from which the lysine-producing microorganisms are cultured in the first step of this disclosure is not limited, but may be from 60 g / L to 200 g / L, 70 g / L to 150 g / L, 72 g / L to 140 g / L, or 72 g / L to 79 g / L, based on the fermentation broth from the L-lysine-producing microorganisms.

[0044] 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 hydrolysate, molasses, saccharin paste, rice bran, cassava, bagasse, and corn steep liquor may be used. Specifically, carbohydrates such as glucose and sterilized and pretreated molasses (i.e., molasses converted to reducing sugars) may be used, and other appropriate amounts of carbon sources may be used without restriction in various ways. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0045] As a specific example, carbon sources that may be included in the culture media of this disclosure may include glucose, maltose, or maltose isomers. More specifically, carbon sources may include one or more selected from glucose, maltose, or maltose isomers (isomaltose), or combinations of two or more thereof, but are not limited thereto.

[0046] The phosphorus source in the culture medium may include potassium dihydrogen phosphate and dipotassium hydrogen phosphate or their corresponding sodium-containing salts. As inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc., can be used.

[0047] The culture medium may contain metal salts required for growth, such as magnesium sulfate or ferric sulfate. Finally, in addition to these substances, growth-essential substances such as amino acids and vitamins may also be used. Precursors suitable for the culture medium may be used. The above-mentioned materials may be added to the culture medium in an appropriate manner, for example, in batches or continuously during the culture process, but are not limited thereto.

[0048] The nitrogen source of the culture medium may include diammonium dicarboxylate. As a nitrogen source, a common culture medium for culturing microorganisms may contain 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 extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition products, defatted soybean meal or its decomposition products, but in one aspect of this disclosure, diammonium dicarboxylate may be included as the sole nitrogen source or as an additional nitrogen source.

[0049] After the first step of producing lysine dicarboxylate by culturing L-lysine-producing microorganisms in a culture medium containing diammonium dicarboxylate, a second step of converting lysine dicarboxylate into cadaverine dicarboxylate can be carried out.

[0050] The second step refers to the conversion of lysine dicarboxylate into the desired product, cadaverine dicarboxylate (cadaverine octanoate or cadaverine nonanoate).

[0051] In a specific example, the second step may include adding a dicarboxylic acid to the process liquid. The process liquid may be the fermentation process liquid obtained in the first step.

[0052] In the second step, dicarboxylic acid may be omitted or added in small amounts of 0.4 mol / L or lower, 0.25 mol / L or lower, 0.15 mol / L or lower, 0.12 mol / L or lower, 0.11 mol / L or lower, 0.1 mol / L or lower, 0.08 mol / L or lower, 0.06 mol / L or lower, 0.05 mol / L or lower, 0.04 mol / L or lower, 0.03 mol / L or lower, 0.02 mol / L or lower, or 0.01 mol / L or lower, based on the volume of the process liquid, specifically the volume of the fermentation process liquid obtained in the first step.

[0053] In specific instances, where the dicarboxylic acid is octanoic acid, the dicarboxylic acid may not be added in the second step, or a small amount of 0.1 mol / L or less of the dicarboxylic acid may be added.

[0054] In specific instances, where the dicarboxylic acid is azelaic acid, the dicarboxylic acid may not be added in the second step, or a small amount of 0.11 mol / L or less of the dicarboxylic acid may be added.

[0055] In the second step, if a dicarboxylic acid is added to the process liquid, the dicarboxylic acid may be added before, after, or simultaneously with the conversion reaction, but is not limited to this.

[0056] When a dicarboxylic acid is added in the second step, the amount of dicarboxylic acid added throughout the preparation method of this disclosure is understood to be the sum of the amount of diammonium dicarboxylic acid added to the culture medium in the first step and the amount of dicarboxylic acid added in the second step. When no dicarboxylic acid is added to the process liquid in the second step, the amount of dicarboxylic acid added throughout the preparation method of this disclosure is understood to be the same as the amount of diammonium dicarboxylic acid added to the culture medium in the first step.

[0057] Specifically, the amount of dicarboxylate added in the preparation method of this disclosure can be determined by considering the amount of cadaverine in the process liquid obtained after the second step. Specifically, the molar ratio of the amount of dicarboxylate added in the preparation method of this disclosure to the amount of cadaverine in the process liquid after the second step can be from 0.80 to 1.10.

[0058] More specifically, the amount of dicarboxylate added in the preparation method of this disclosure, relative to the amount of cadaverine in the process liquid after the second step, can be 0.85 to 1.10, 0.87 to 1.06, 0.88 to 1.05, 0.89 to 1.01, 0.88 to 1.01, 0.89 to 1.0, 0.90 to 1.10, 0.91 to 1.00, 0.93 to 1.01, 0.93 to 1.01, 0.95 to 1.06, or 0.95 to 1.05 in molar ratio.

[0059] As an example, the amount of dicarboxylate added in the preparation method of this disclosure, relative to the amount of cadaverine in the process liquid after the second step, can be in a molar ratio within a range consisting of a 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 or 0.95 and / or an upper limit selected from 1.10, 1.09, 1.08, 1.07, 1.06, 1.05, 1.04, 1.03, 1.02, 1.01 or 1.0.

[0060] In specific examples, when the dicarboxylate is octanoate, the amount of dicarboxylate added in the preparation method of this disclosure, relative to the amount of cadaverine in the process liquid after the second step, can be 0.80 to 1.10, 0.85 to 1.05, 0.87 to 1.03, 0.89 to 1.02, 0.91 to 1.00, 0.91 to 1.10, 0.90 to 1.05, or 0.90 to 1.02 in molar ratio.

[0061] In the preparation method disclosed herein, when octanedia salt is added in a molar ratio within the range above relative to the calculated amount of cadaverine to be obtained after the second step, a technical significance is that octanedia salt does not precipitate as crystals along with cadaverine octanedia salt, thus high-purity cadaverine octanedia salt can be produced in high yield.

[0062] By adjusting the molar ratio of the added octopaate to the desired cadaverine, cadaverine octopaate with a purity of 99% can be maintained until the third cycle when the mother liquor is recycled. Depending on the number of mother liquor cycles, cadaverine octopaate can be obtained in total yields of 62% or higher, 63% or higher, 64% or higher, 65% or higher, 66% or higher, 67% or higher, 68% or higher, 69% or higher, or 70% or higher.

[0063] In specific examples, when the dicarboxylate is azelaate, the amount of dicarboxylate added in the preparation method of this disclosure, relative to the amount of cadaverine in the process liquid after the second step, can be 0.80 to 1.10, 0.85 to 1.05, 0.87 to 1.03, 0.89 to 1.02, 0.91 to 1.00, 0.91 to 1.10, 0.90 to 1.05, 0.90 to 1.02, or 0.90 to 1.0.

[0064] In the preparation method disclosed herein, when azelaic acid salt is added in a molar ratio within the range above relative to the calculated amount of cadaverine to be obtained after the second step, a technical significance is that azelaic acid does not precipitate as crystals along with cadaverine azelaic acid salt, thus enabling the production of high-purity cadaverine azelaic acid salt in high yield.

[0065] By adjusting the molar ratio of the added azelate to the desired cadaverine, cadaverine azelate with a purity of 99% can be maintained until the third cycle when the mother liquor is recycled. Depending on the number of mother liquor cycles, cadaverine azelate can be obtained in total yields of 25% or higher, 26% or higher, 27% or higher, 28% or higher, 29% or higher, or 30% or higher.

[0066] In the second step, the conversion of lysine dicarboxylate to cadaverine dicarboxylate can be an enzymatic conversion reaction.

[0067] As a specific example, if a dicarboxylic acid is introduced in the second step, the lysine dicarboxylate may include both the lysine dicarboxylate obtained in the first step and the lysine dicarboxylate produced in the second step.

[0068] Specifically, the enzymatic conversion reaction in the second step can be carried out using a protein that exhibits lysine decarboxylase activity or a microorganism that expresses a protein that exhibits such activity.

[0069] As a specific example, a protein exhibiting lysine decarboxylase activity or a seed culture of a microorganism expressing a protein exhibiting such activity may be introduced into the fermentation liquid of the first step, but this is not limited to that.

[0070] In this disclosure, the term "decarboxylase" is an enzyme that catalyzes the elimination of the carboxyl group of an organic acid to produce carbon dioxide; it may also be called a carboxyl lyase, carbon-carbon lyase, etc.

[0071] There are no particular restrictions on the protein as long as it exhibits lysine decarboxylase activity, but it can be, for example, a PtLDC protein derived from Pseudomonas thermotolerans or a CadA protein derived from Escherichia coli. However, the protein sequence can be obtained from a known database, GenBank, and the protein can be expressed by microorganisms or can be purchased and used with commercially available enzymes, but the protein is not limited to these.

[0072] The microorganism expressing the protein can be a microorganism transformed to express the protein. The transforming microorganism is not limited to prokaryotic or eukaryotic microorganisms, as long as it is transformed to express a protein exhibiting decarboxylase activity. By releasing the protein exhibiting enzymatic activity into a seed culture medium, the microorganism transformed to express the protein exhibiting decarboxylase activity can convert lysine dicarboxylate in the process liquid to cadaverine dicarboxylate.

[0073] Specific examples of the microorganisms may include strains of the genera *Escherichia*, *Erwinia*, *Serratia*, *Providencia*, and *Corynebacterium*. Specifically, the microorganisms may be those belonging to the genera *Escherichia* or *Corynebacterium*, more specifically, *Escherichia coli* or *Corynebacterium glutamicum*, but are not limited thereto.

[0074] The conversion reaction in the second step can take 20 minutes to 3 hours, more specifically 0.5 to 1.5 hours, such as 1 hour, but is not limited to this.

[0075] The conversion reaction in the second step can be carried out at 30°C to 60°C, more specifically at 40°C to 50°C, for example at 45°C, but is not limited thereto.

[0076] The conversion reaction in the second step can be carried out at a pH of 7.5 to 9, more specifically at a pH of 7.8 to 8.7, and even more specifically at a pH of 8 to 8.5. In one embodiment of the second step, the pH can be adjusted to the above range by selectively and additionally introducing a dicarboxylic acid; 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.

[0077] The method for preparing cadaverine dicarboxylate disclosed herein may further include a recovery step for recovering the obtained cadaverine dicarboxylate after the second step.

[0078] Recovery can be achieved by collecting cadaverine dicarboxylate using suitable methods known in the art.

[0079] For example, cadaverine dicarboxylate can be collected by centrifugation, filtration, concentration, crystallization, extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography and affinity chromatography), HPLC or a combination of these methods.

[0080] In specific examples of this disclosure, the recovery step may include one or more of a filtration step, a concentration step, or a crystallization step.

[0081] The filtration step refers to the step of removing impurities from the conversion liquid containing cadaverine dicarboxylate obtained in the second step. As an example, filtration may include removing bacteria from the cadaverine dicarboxylate process liquid by means of membrane filtration or activated carbon filtration.

[0082] The concentration step refers to the step of concentrating the liquid to increase the solids ratio, and it can be carried out after the filtration step. For example, concentration can be carried out under reduced pressure using a rotary evaporator.

[0083] Specifically, the concentration step can be performed to concentrate the solids content to 40% to 75% (w / w), more specifically 40% to 75% (w / w), 45% to 70% (w / w), 42% to 50% (w / w), 50% to 65% (w / w), 40% to 50% (w / w), 55% to 75% (w / w), or 60% to 70% (w / w), for example 45% (w / w) or 65% (w / w). When the concentration step is performed to bring the solids content within the above ranges, the recovery rate and purity of cadaverine dicarboxylate can be maintained very well.

[0084] The concentration step can be carried out at temperatures ranging from 45°C to 75°C, but is not limited to this.

[0085] The crystallization step can be a cooling crystallization step, which refers to the step of cooling the conversion liquid and precipitating the conversion liquid into crystals to obtain the final desired product, cadaverine dicarboxylate crystals.

[0086] Specifically, cooling may involve cooling the conversion liquid to 20°C to 30°C, more specifically to 20°C to 27°C, for example to 25°C to 20°C, but is not limited thereto.

[0087] Specifically, the cooling rate can be from 3°C to 20°C / hour, more specifically from 3°C to 15°C / hour, such as 10°C / hour or 3°C / hour, but is not limited thereto.

[0088] In one embodiment, the recovery step may further include a stirring step of the process liquid. The stirring step may be performed simultaneously with the cooling crystallization step, during the cooling crystallization step, before the cooling crystallization step, or after the cooling crystallization step. This stirring step provides sufficient time for agitation of the process liquid and can promote crystal precipitation.

[0089] In one embodiment, the recovery step may further include a mother liquor recycling step, in which the mother liquor containing uncrystallized cadaverine dicarboxylate is recycled to the feed liquid of the concentration step after the crystallization step.

[0090] Specifically, in the mother liquor recycling step, the number of times the mother liquor is recycled can 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 mother liquor recycling number of 0 means that the mother liquor recycling step is not performed.

[0091] Until now, in the crystallization step of cadaverine dicarboxylate, a relatively large amount of cadaverine dicarboxylate remains in the mother liquor after crystallization, resulting in a yield as low as less than 50%. In this disclosure, as a method to improve the yield of cadaverine dicarboxylate, the mother liquor containing the residual cadaverine dicarboxylate is recycled to the feed liquid of the concentration step 1 to 3 times, 1 to 4 times, 1 to 5 times, 1 to 10 times or more, thereby increasing the overall yield of cadaverine dicarboxylate.

[0092] In one embodiment, the recovery step may further include washing, separating, and drying the process liquid from the crystallization process.

[0093] In the drying step, moisture contained in the crystals can be removed, and high-purity cadaverine dicarboxylate can be commercialized. After drying, cadaverine dicarboxylate crystals can be provided in powder form, but the dosage form can be varied as needed.

[0094] In one implementation, the recycling step may also include a decolorization step. Decolorization can be carried out using activated carbon, anion exchange resin, etc., but is not limited to these.

[0095] The method for preparing cadaverine dicarboxylate disclosed herein may include additional purification steps. Purification can be performed by suitable methods known in the art. In one example, where the method for preparing cadaverine dicarboxylate disclosed herein includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously without regard to the order, or they may be performed simultaneously or by integrating them into a single step, but are not limited thereto.

[0096] The preparation method disclosed herein can omit a separate process for removing the dicarboxylate in the recovery step. In conventional methods for producing cadaverine dicarboxylate, the dicarboxylate precipitates as crystals during crystallization, reducing purity. To prevent this, a separate process for removing the dicarboxylate is required before crystallization. However, the preparation method disclosed herein offers the technical advantage of producing high-purity cadaverine dicarboxylate without a separate dicarboxylate removal process.

[0097] In one embodiment, when the dicarboxylate is octanoate, the method of this disclosure can provide cadaverine octanoate with a purity of 99% or higher in 0 to 3 mother liquor cycles. In one embodiment, when the dicarboxylate is octanoate, the cumulative gross yield of cadaverine octanoate can be 65% or higher, more specifically 66%, 67%, 68%, 69%, 70% or higher, based on three mother liquor cycles.

[0098] In one embodiment, when the dicarboxylate is azelaate, the method of this disclosure can provide cadaverine azelaate with a purity of 99% or higher in 0 to 3 mother liquor cycles. In one embodiment, when the dicarboxylate is azelaate, the cumulative gross yield of cadaverine azelaate can be 25% or higher, more specifically 26%, 27% or higher, based on three mother liquor cycles.

[0099] The method disclosed herein can produce high-purity cadaverine dicarboxylate without the decarbonization and distillation processes required in conventional liquid cadaverine production.

[0100] Methods of implementing the present invention The present disclosure will now be described in more detail with reference to embodiments. However, the following embodiments are merely illustrative of preferred embodiments of the present disclosure and are not intended to limit the scope of the disclosure thereto. Furthermore, those skilled in the art or similar fields will fully understand and readily implement any technical matters not described herein.

[0101] The preparation method of cadaverine dicarboxylate (cadaverine octanoate or cadaverine azelate) according to one aspect of this disclosure has been explained above. The beneficial effects mentioned in this disclosure are explained below through experimental results of examples and comparative examples of cadaverine octanoate and cadaverine azelate.

[0102] 1. Cadaverine octanoate Preparation Example 1-1. Production and fermentation process of lysine octanoate Seeds of a strain of Corynebacterium glutamicum capable of producing lysine (KCCM12154P, US 2021-0355514 A1) were obtained through solid-phase and shake-flask cultures. Seed culture was then carried out in a fermenter, followed by primary production and fermentation. Fermentation was conducted in the fermenter at 36°C and 900 rpm for 63 hours.

[0103] To produce lysine octanoate using Corynebacterium, diammonium octanoate is supplied at a level of 67 to 110 g / L to replace the existing ammonium sulfate, and fermentation is carried out to obtain lysine octanoate at a level of 103 to 170 g / L and lysine at a level of 79 g / L. In this example, the molar ratio of octanoate to lysine in the fermentation liquid is adjusted to a level of 0.59 to 0.98.

[0104] Preparation Examples 1-2. Conversion reaction of lysine octanoate to cadaverine octanoate For the transformation reaction, E. coli (US 2018-0030430 A1) overexpressing PtLDC enzyme using the pET-Deut1 vector was used. PtLDC enzyme is a lysine decarboxylase gene derived from thermostable Pseudomonas.

[0105] The enzyme conversion broth obtained by seed culture of the enzyme strain was supplied at a level of 10% by mass to the fermentation broth for preparing lysine octanoate in Examples 1-1 for conversion reaction. The conversion reaction was carried out for approximately 1 hour while the temperature was maintained at a level of 45°C to 50°C. The pH was adjusted to 8.0 to 8.5, for which CO2 was introduced for neutralization in Examples 1-1 to 1-5 below, and octanoic acid was additionally introduced at a level of 0.02 mol / L to 0.03 mol / L (based on the volume of the fermentation broth for lysine octanoate) before the conversion reaction in Examples 1-2 to 1-4. As a result, the conversion rate to cadaverine octanoate was found to be at a level of 97% to 98%. Specifically, after the conversion reaction, cadaverine octanoate was obtained at a level of 88 to 146 g / L (based on the volume of the fermentation broth for lysine octanoate), and cadaverine was obtained at a level of 54 g / L (based on the volume of the fermentation broth for lysine succinate). Subsequently, in Examples 1-6 and 1-7, octanoic acid was additionally introduced at a level of 0.05 to 0.1 mol / L (based on the volume of the fermentation liquid for lysine octanoate).

[0106] Preparation Examples 1-3. Subsequent processes after obtaining cadaverine octanoate The process liquid containing cadaverine octanoate obtained in Examples 1-2 was subjected to the following subsequent treatment to obtain cadaverine octanoate in crystalline form.

[0107] [Bacterial Removal Steps] Bacteria in the liquid of the cadaverine octanoate process were removed by membrane filtration using a 0.1 μm membrane.

[0108] [Steps for removing impurities using activated carbon] Activated charcoal was added at a level of 10%, based on the weight of cadaverine octanoate in the filtrate from which the bacteria were isolated. The process liquid with added activated charcoal was heated to 60°C and stirred for 1 hour to decolorize, and then the activated charcoal was filtered through filter paper.

[0109] [Steps for concentrating cadaverine octanoate liquid after impurities have been removed] The filtered liquid was concentrated in a rotary evaporator under reduced pressure at approximately 60°C and 120 Torr until the solid content in the filtrate became 65% by weight.

[0110] [Steps for cooling and crystallization, separation, and then drying of the cadaverine octanoate concentrate] 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 octanoate. The separated crystals were dried for one day, and their purity was then determined by HPLC.

[0111] [The step of recycling the mother liquor to the crystallization step] The separated mother liquor is recycled to the step of concentrating the cadaverine octanoate process liquid after impurities have been removed.

[0112] Experimental Example 1-1. The changes in purity and yield of cadaverine octanoate were investigated based on the molar ratio of octanoate to cadaverine in the liquid used for conversion to cadaverine octanoate. In Experimental Example 1-1, the method according to Preparation Examples 1-1 to 1-3 was used, except that the purity and yield of cadaverine octanoate were investigated as varying with the molar ratio of octanoate to cadaverine in the liquid used for conversion to cadaverine octanoate.

[0113] Example 1-1: Case where the molar ratio of added octanoate to cadaverine in the post-second-step process liquid is 0.6. 1,000 ml of a liquid for the conversion of octanoate to cadaverine octanoate with a molar ratio of 0.6 was supplied and passed through a 0.1 μm membrane to remove microorganisms. A liquid for the conversion of octanoate to cadaverine octanoate with a molar ratio of 0.61 was prepared according to the preparation methods of Preparation Examples 1-1 and 1-2, except that the sum of the amount of diammonium octanoate introduced during the fermentation process for lysine production in Preparation Example 1-1 and the amount of octanoate introduced during the conversion reaction in Preparation Example 1-2 was controlled to be 0.6 of the molar number of cadaverine already converted.

[0114] The filtrate was decolorized with activated carbon, filtered through filter paper, and then concentrated under reduced pressure to a solids content of 65%. The concentrate was cooled from 50°C to 25°C for crystallization. The crystals and mother liquor were separated by centrifugation, and the separated crystals were dried for one day, then their purity was measured by HPLC. The mother liquor containing residual octanoate was recycled to a cadaverine octanoate feed solution with an octanoate / cadaverine molar ratio of 0.6, and the crystallization step was repeated. The mother liquor was recycled sequentially.

[0115] Examples 1-2: Case where the molar ratio of added octanoate to cadaverine in the post-second-step process liquid is 0.70. A liquid for conversion to cadaverine octanoate with a molar ratio of 0.70 is supplied, and the purification process is carried out in the same manner as in Examples 1-1 while the mother liquor is recycled sequentially.

[0116] Examples 1-3: Cases where the molar ratio of added octanoate to cadaverine in the post-second-step process liquid is 0.79. A liquid for conversion to cadaverine octanoate with a molar ratio of 0.79 was supplied, and the purification process was carried out in the same manner as in Examples 1-1 while the mother liquor was recycled sequentially.

[0117] Examples 1-4: Cases where the added octanoate / cadaverine molar ratio is 0.91 A liquid for conversion to cadaverine octanoate with a molar ratio of 0.91 was supplied, and the purification process was carried out in the same manner as in Examples 1-1 while the mother liquor was recycled sequentially.

[0118] Examples 1-5: Cases where the molar ratio of added octanoate / cadaverine is 1.00 A liquid for conversion to cadaverine octanoate with a molar ratio of 1.00 is supplied, and the purification process is carried out in the same manner as in Examples 1-1 while the mother liquor is recycled sequentially.

[0119] In Examples 1-4 and 1-5, the molar ratio of octanoate / lysine in the lysine fermentation step was adjusted to 0.83 to 0.98.

[0120] Examples 1-6: Cases where the added octanoate / cadaverine molar ratio is 1.10 A liquid for conversion to cadaverine octanoate with a molar ratio of 1.10 is supplied, and the purification process is carried out in the same manner as in Examples 1-1 while the mother liquor is recycled sequentially.

[0121] Examples 1-7: Cases where the added octanoate / cadaverine molar ratio is 1.19 A liquid for conversion to cadaverine octanoate with a molar ratio of 1.19 was supplied, and the purification process was carried out in the same manner as in Examples 1-1 while the mother liquor was recycled sequentially.

[0122] The measured physical properties of the process liquid and cadaverine octanoate prepared according to Examples 1-1 to 1-7 above are shown in Tables 1 to 4 below.

[0123] Table 1 shows the process liquids for which mother liquor recycling was not performed.

[0124] [Table 1] Table 2 shows the process liquid for one cycle of the mother liquor.

[0125] [Table 2] Table 3 shows the process liquid for mother liquor circulated twice.

[0126] [Table 3] Table 4 shows the process liquid for three cycles of mother liquor.

[0127] [Table 4] Based on the experimental results in Tables 1 to 4 above, it has been found that the preparation method disclosed herein can prepare cadaverine octanoate with improved overall crystallization rate, high purity and high yield. The method contains a large amount of diammonium octanoate in the first step, reaching 84 to 100 mol% of the total octanoate supplied.

[0128] More specifically, in Example 1-1, no crystals precipitated during the crystallization process. This is because the molar ratio of octanoate was not high enough to precipitate cadaverine octanoate crystals.

[0129] In Examples 1-2, crystals precipitated until the mother liquor was circulated once, but no crystals were produced when the mother liquor was circulated twice. This is because after the mother liquor was circulated once, the molar ratio of octanoate in the feed liquid used for crystallization decreased, and therefore the molar ratio was not high enough to precipitate cadaverine octanoate crystals.

[0130] In Examples 1-3, crystallization and precipitation continued until the mother liquor was circulated twice, but no crystals were produced when the mother liquor was circulated three times. This is because the sequential circulation of the mother liquor reduced the molar ratio of octanoate in the feed liquid used for crystallization, and therefore the concentration of octanoate was not high enough to precipitate cadaverine octanoate crystals.

[0131] In Examples 1-6, when the mother liquor was also circulated three times, the purity was slightly lower, at 85%. This is because when the mother liquor was circulated sequentially, the octanediic acid accumulated in the feed liquid used for crystallization precipitated as an impurity.

[0132] In Examples 1-7, when the mother liquor was also circulated three times, the purity was slightly lower, at 74%. This is because when the mother liquor was circulated sequentially, the octanoic acid accumulated in the feed liquid used for crystallization precipitated as an impurity.

[0133] On the other hand, in Examples 1-4, after the mother liquor was recycled three times, 99% high-purity cadaverine octanoate was obtained, and a high total yield of about 65% was achieved.

[0134] In Examples 1-5, after three cycles of mother liquor recycling, 99% high-purity cadaverine octanoate was obtained, and a high total yield of approximately 70% was achieved.

[0135] As described above, a method for preparing high-purity cadaverine octopaate has been disclosed, which can be based on the molar ratio of octopaate added throughout the process to cadaverine in the post-second-step process liquid. In particular, it has been found that when the amount of octopaate added is adjusted to approximately 0.91 to 1.00 molar ratio relative to the amount of cadaverine in the post-second-step process liquid, an excellent overall yield of 65% or higher can be achieved while maintaining 99% purity of the cadaverine octopaate, after three or more cycles of mother liquor recycling.

[0136] 2. Cadanilide azelaate Preparation Example 2-1. Production and fermentation process of lysine azelaate Seeds of a strain of Corynebacterium glutamicum capable of producing lysine (KCCM12154P, US 2021-0355514 A1) were obtained through solid-phase and shake-flask cultures. Seed culture was then carried out in a fermenter, followed by primary production and fermentation. Fermentation was conducted in the fermenter at 36°C and 900 rpm for 30 hours.

[0137] To produce lysine azelaate using Corynebacterium, diammonium azelaate is supplied at a level of 67 to 107 g / L to replace the existing ammonium sulfate, and fermentation is carried out to obtain lysine azelaate at a level of 100 to 161 g / L and lysine at a level of 73 g / L. In this example, the molar ratio of azelaate to lysine in the fermentation liquid is adjusted to a level of 0.6 to 0.96.

[0138] Preparation Example 2-2. Conversion reaction of lysine azelaate to cadaverine azelaate For the transformation reaction, E. coli (US 2018-0030430 A1) overexpressing PtLDC enzyme using the pET-Deut1 vector was used. PtLDC enzyme is a lysine decarboxylase gene derived from thermostable Pseudomonas.

[0139] The enzyme conversion broth obtained by seed culture of the enzyme strain was supplied at a level of 10% by mass to the fermentation broth for preparing lysine azelaate in Example 2-1 for conversion reaction. The conversion reaction was carried out for approximately 1 hour while the temperature was maintained at 45°C. The pH was adjusted to 8.0 to 8.5, for which CO2 was introduced for neutralization in Examples 2-1 to 2-5 below, and azelaic acid was additionally introduced at a level of 0.01 to 0.04 mol / L (based on the volume of the fermentation broth for lysine azelaate) in Examples 2-3 to 2-5. As a result, the conversion rate to cadaverine azelaate was found to be at a level of 97% to 98%. Specifically, after the conversion reaction, cadaverine octanoate was obtained at a level of 87 to 142 g / L (based on the volume of the fermentation broth for lysine octanoate), and cadaverine was obtained at a level of 50 g / L (based on the volume of the fermentation broth for lysine succinate). Subsequently, in Examples 2-6 and 2-7, after the conversion reaction, azelaic acid was additionally introduced at a level of 0.06 to 0.11 mol / L (based on the volume of the fermentation liquid for lysine azelaate).

[0140] Preparation Examples 2-3. Subsequent processes after obtaining cadaverine azelate The process liquid containing cadaverine azelate obtained in Preparation Example 2-2 was subjected to the following subsequent treatment to obtain cadaverine azelate in crystalline form.

[0141] [Bacterial Removal Steps] Bacteria in the liquid from the cadaverine azelate process were removed by membrane filtration using a 0.1 μm membrane.

[0142] [Steps for removing impurities using activated carbon] Activated charcoal was added at a level of 10%, based on the weight of cadaverine azelate in the filtrate from which the bacteria were isolated. The process liquid with added activated charcoal was heated to 60°C and stirred for 1 hour to decolorize, and then the activated charcoal was filtered through filter paper.

[0143] [Steps for concentrating cadaverine azelate solution after impurities have been removed] The filtered liquid was concentrated in a rotary evaporator at a reduced pressure of about 55°C to 70°C and 120 Torr until the solid content in the filtrate became 45% by weight.

[0144] [Steps for cooling and crystallization, separation, and then drying of the cadaverine azelate concentrate] The concentrate was cooled from 60°C to 25°C at a rate of 10°C / hr. Then, the concentrate was cooled to 25°C and then further cooled to 20°C at a rate of 3°C / h. The concentrate was then stirred at 20°C for 24 hours. The precipitated crystals were separated from the mother liquor using a centrifuge. The crystals were washed with 10% water based on the weight of cadaverine azelate. The separated crystals were dried at low temperature (20°C) for one to two days, and their purity was then determined by HPLC.

[0145] [The step of recycling the mother liquor to the crystallization step] The separated mother liquor is recycled to the step of concentrating the impurity-removed cadaverine azelate process liquid.

[0146] Experimental Example 2-1. The changes in purity and yield of cadaverine azelate were investigated based on the molar ratio of azelate to cadaverine in the liquid used for conversion to cadaverine azelate. In Experimental Example 2-1, the method according to Preparation Examples 2-1 to 2-3 was used, except that the purity and yield of cadaverine azelate were investigated as a function of the molar ratio of azelate to cadaverine in the liquid used for conversion to cadaverine azelate.

[0147] Example 2-1: The molar ratio of added azelaate / cadaverine is 0.61. 1,000 ml of azelaate / cadaverine molar ratio of 0.61 was supplied as a liquid for the conversion to cadaverine azelaate and passed through a 0.1 μm membrane to remove microorganisms. The liquid for the conversion to cadaverine azelaate with an azelaate / cadaverine molar ratio of 0.61 was prepared according to the preparation methods of Preparation Example 2-1 and Preparation Example 2-2, except that the sum of the amount of azelaate of diammonium azelaate introduced during the fermentation process for lysine production in Preparation Example 2-1 and the amount of azelaate introduced during the reaction for the conversion to cadaverine azelaate in Preparation Example 2-2 was controlled to be 0.61 of the molar number of cadaverine already converted.

[0148] The filtrate was decolorized with activated carbon, filtered through filter paper, and then concentrated under reduced pressure to a solid content of 45%. The concentrate was cooled from 60°C to 25°C and then stirred for 24 hours to crystallize. The crystals and mother liquor were separated by centrifugation, and the separated crystals were dried at low temperature for one to two days, and their purity was measured by HPLC. The mother liquor containing residual azelaate was recycled to a cadaverine azelaate feed solution with a molar ratio of azelaate / cadaverine of 0.61, and the crystallization step was repeated. The mother liquor was recycled sequentially.

[0149] Example 2-2: Case where the added azelaate / cadaverine molar ratio is 0.71 A liquid for conversion to cadaverine azelate was supplied with a molar ratio of azelate / cadaverine of 0.71, and the purification process was carried out in the same manner as in Example 2-1 while the mother liquor was recycled sequentially.

[0150] Examples 2-3: Case where the molar ratio of added azelaate / cadaverine is 0.8 A liquid for conversion to cadaverine azelate was supplied with a molar ratio of azelate / cadaverine of 0.8, and the purification process was carried out in the same manner as in Example 2-1 while the mother liquor was recycled sequentially.

[0151] Examples 2-4: Cases where the added azelaate / cadaverine molar ratio is 0.9 A liquid for conversion to cadaverine azelate was supplied with a molar ratio of azelate / cadaverine of 0.9, and the purification process was carried out in the same manner as in Example 2-1 while the mother liquor was recycled sequentially.

[0152] Examples 2-5: Cases where the molar ratio of added azelaate / cadaverine is 1.0 A liquid for conversion to cadaverine azelate with a molar ratio of 1.0 is supplied, and the purification process is carried out in the same manner as in Example 2-1 while the mother liquor is recycled sequentially.

[0153] In Examples 2-4 and 2-5, the molar ratio of azelate to lysine in the lysine fermentation step was adjusted to 0.80 to 0.96.

[0154] Examples 2-6: Cases where the molar ratio of added azelaate / cadaverine is 1.1 A liquid for conversion to cadaverine azelate was supplied with a molar ratio of azelate / cadaverine of 1.1, and the purification process was carried out in the same manner as in Example 2-1 while the mother liquor was recycled sequentially.

[0155] Examples 2-7: Cases where the added azelaate / cadaverine molar ratio is 1.2 A liquid for conversion to cadaverine azelate with a molar ratio of 1.2 is supplied, and the purification process is carried out in the same manner as in Example 2-1 while the mother liquor is recycled sequentially.

[0156] The measured physical properties of the process liquid and cadaverine azelate prepared according to Examples 2-1 to 2-7 above are shown in Tables 5 to 8 below.

[0157] Table 5 shows the process liquids for which mother liquor recycling was not performed.

[0158] [Table 5] Table 6 shows the process liquid for one mother liquor cycle.

[0159] [Table 6] Table 7 shows the process liquid for mother liquor circulated twice.

[0160] [Table 7] Table 8 shows the process liquid for three cycles of mother liquor.

[0161] [Table 8] Based on the experimental results in Tables 5 to 8 above, it has been found that the preparation method disclosed herein can prepare cadaverine azelate with improved overall crystallization rate, high purity and high yield. The method contains a large amount of diammonium azelate in the first step, reaching 81 to 100 mol% of the total azelate supplied.

[0162] More specifically, in Example 2-1, no crystals precipitated during the crystallization process. This is because the molar ratio of azelaate was not high enough to precipitate cadaverine azelaate crystals.

[0163] In Example 2-2, crystals precipitated until the mother liquor was circulated once, but no crystals were produced when the mother liquor was circulated twice. This is because after the mother liquor was circulated once, the molar ratio of azelaate in the feed liquid used for crystallization decreased, and therefore the molar ratio was not high enough to precipitate cadaverine azelaate crystals.

[0164] In Examples 2-3, crystallization and precipitation continued until the mother liquor was circulated twice, but no crystals were produced when the mother liquor was circulated three times. This is because the sequential circulation of the mother liquor reduced the molar ratio of azelaate in the feed liquid used for crystallization, and therefore the concentration of azelaate was not high enough to precipitate cadaverine azelaate crystals.

[0165] In Examples 2-6, crystals precipitated until the mother liquor was circulated twice, but the purity of the crystals was slightly lower, at 86%. This is because azelaic acid accumulated in the feed liquid used for crystallization precipitates as an impurity when the mother liquor is circulated sequentially.

[0166] On the other hand, in Examples 2-4, after the mother liquor was recycled three times, 99% high-purity cadaverine azelate was obtained, and a high total yield of about 27.1% was achieved.

[0167] In Examples 2-5, after three cycles of mother liquor recycling, 99% high-purity cadaverine azelate was obtained, and a high total yield of approximately 26.8% was achieved.

[0168] As described above, a method for preparing high-purity cadaverine azelate has been revealed, which can be based on the molar ratio of azelate added throughout the process to cadaverine in the process liquid after the second step.

[0169] In particular, it has been found that when the amount of azelate added is adjusted to approximately 0.9 to 1.00 molar ratio relative to the amount of cadaverine in the post-second process liquid, an excellent total yield of 26.8% or higher can be achieved while maintaining 99% purity of cadaverine azelate, after three or more cycles of mother liquor recycling.

[0170] Based on the foregoing description, those skilled in the art will understand that this disclosure can be implemented in other specific forms without altering its technical concept or essential features. Therefore, it should be understood that the above embodiments are not restrictive in any respect, but rather illustrative. The scope of this disclosure is defined by the appended claims and not by the following description, and all changes and modifications derived from the meaning and scope of the claims or their equivalents should be interpreted as including within the scope of this disclosure.

Claims

1. A method for preparing cadaverine dicarboxylate, comprising: The first step involves obtaining lysine dicarboxylate by culturing L-lysine-producing microorganisms in a culture medium containing diammonium dicarboxylate. The second step involves converting lysine dicarboxylate into cadaverine dicarboxylate, in which... The amount of dicarboxylate added relative to the amount of cadaverine in the process liquid after the second step, expressed as a molar ratio, is 0.85 to 1.05, and The dicarboxylic acid salt is an aliphatic dicarboxylic acid salt having 8 or 9 carbon atoms.

2. The method according to claim 1, wherein the diammonium dicarboxylate contained in the first step is 81 mol% or more of the total dicarboxylate supplied.

3. The method according to claim 1, wherein the content of diammonium dicarboxylate in the first step is such that the molar ratio of the diammonium dicarboxylate to the lysine to be produced (diammonium dicarboxylate / lysine) is 0.79 to 0.

99.

4. The method according to claim 1, wherein the dicarboxylate and the dicarboxylic acid are respectively octanoate and octanoic acid, and The content of diammonium dicarboxylate in the first step is such that the molar ratio of the diammonium dicarboxylate to the lysine to be produced (diammonium dicarboxylate / lysine) is 0.81 to 1.

0.

5. The method according to claim 1, wherein the dicarboxylate and the dicarboxylic acid are respectively azelaate and azelaic acid, and The content of diammonium dicarboxylate in the first step is such that the molar ratio of the diammonium dicarboxylate to the lysine to be produced (diammonium dicarboxylate / lysine) is 0.79 to 1.

0.

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

5.

7. The method according to claim 1, wherein the dicarboxylate and the dicarboxylic acid are respectively octanoate and octanoic acid, and The amount of dicarboxylate added relative to the amount of cadaverine in the process liquid after the second step is 0.91 to 1.0 in molar ratio.

8. The method according to claim 1, wherein the dicarboxylate and the dicarboxylic acid are respectively azelaate and azelaic acid, and The amount of dicarboxylate added relative to the amount of cadaverine in the process liquid after the second step is 0.90 to 1.0 in molar ratio.

9. The method of claim 1, wherein the second step comprises adding a dicarboxylic acid to the process liquid.

10. The method of claim 1, further comprising a recovery step of recovering cadaverine dicarboxylate after the second step.

11. The method of claim 10, wherein the recovery step comprises at least one or more steps selected from the group consisting of a filtration step, a concentration step, and a cooling crystallization step.

12. The method of claim 10, wherein the recovery step further comprises a mother liquor recycling step, wherein the mother liquor containing uncrystallized cadaverine dicarboxylate is recycled to the feed liquid of the concentration step after the cooling crystallization step.

13. The method of claim 11, wherein the concentration step is to concentrate the solids content to 40% to 75% (w / w).

14. The method of claim 1, wherein a protein having lysine decarboxylase activity or a microorganism expressing a protein having said activity is used to perform the conversion of lysine dicarboxylate to cadaverine dicarboxylate in the second step.

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

16. The method of claim 1, wherein the recovery step does not include a separate process for removing dicarboxylate from the process liquid.

17. The method of claim 11, wherein a cadaverine dicarboxylate with a purity of 99% or higher is obtained within 0 to 3 cycles of mother liquor recycling.

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