Process for preparing cadaverine dicarboxylate

Abstract

The present application relates to a method for preparing a cadaverine dicarboxylate (cadaverine sebacate, cadaverine undecanediate or cadaverine dodecanediate), 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 sebacate, cadaverine undecanedioate, or cadaverine dodecanedioate) of high purity can be obtained without ion resin exchange, decarburization, and distillation by implementing the method according to the above procedure.

Description

Technical Field

[0001] This disclosure relates to a technique for preparing high-purity cadaverine dicarboxylate (cadaverine sebacate, cadaverine undecanedioate, or cadaverine dodecanedioate) using a microbial fermentation and purification process. Background Technology

[0002] Cadaverine is a foul-smelling, toxic diamine compound produced from the putrefaction of animal tissue. Unlike hexamethylenediamine, cadaverine exists as a liquid at room temperature and more readily absorbs acidic gases such as carbon dioxide from the air, making its transportation and storage difficult. Therefore, salt crystallization not only provides high-quality monomers but also facilitates transportation and storage.

[0003] Meanwhile, cadaverine sebacate, cadaverine undecanoate, and cadaverine dodecanoate are precursors for the polymerization of bio-polyamides, which are engineering plastics widely used in motor vehicles, electrical and electronic components, etc.

[0004] The preparation method of cadaverine dicarboxylate typically includes the following steps: Synthesis of cadaverine: The synthesis of cadaverine involves fermentation by suitable microbial strains capable of producing lysine decarboxylase, an enzyme that catalyzes the decarboxylation of lysine to produce cadaverine.

[0005] Formation of cadaverine dicarboxylate: The formation of cadaverine dicarboxylate involves the reaction of cadaverine with a dicarboxylic acid in the presence of a suitable catalyst. This reaction typically involves mixing the two monomers at an appropriate temperature and stirring for a certain period of time to ensure complete reaction.

[0006] Purification of cadaverine dicarboxylate: After the reaction is complete, various separation and purification techniques such as solvent extraction, chromatography and crystallization are used to purify the crude cadaverine dicarboxylate.

[0007] According to existing technologies, sebacate, undecanediate, and dodecanediate have low solubility, and during the crystallization step of the aforementioned methods, the dicarboxylate (sebacate, undecanediate, or dodecanediate) and cadaverine dicarboxylate (cadaverine sebacate, cadaverine undecanediate, or cadaverine dodecanediate) precipitate together as crystals. Therefore, to produce high-purity cadaverine dicarboxylate, the dicarboxylate needs to be removed before crystallization, resulting in cost burdens due to the additional dicarboxylate removal process, reduced process efficiency, and environmental pollution due to the use of chemical solvents. Summary of the Invention

[0008] Technical issues The problem to be solved by this disclosure is to provide a method for preparing cadaverine dicarboxylate (cadaverine sebacic acid, cadaverine undecanediate, or cadaverine dodecanediate).

[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 C10, C11, or C12 cadaverine dicarboxylate can be prepared in high yields because the dicarboxylic acid crystals do not precipitate as a byproduct. 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 lysine dicarboxylate into cadaverine dicarboxylate.

[0014] 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.

[0015] Specifically, the dicarboxylic acid can be an aliphatic dicarboxylic acid having 10, 11, or 12 carbon atoms. In other words, in this disclosure, a dicarboxylic acid refers to an aliphatic dicarboxylic acid selected from sebacic acid, undecanoic acid, or dodecanoic acid. When the cadaverine dicarboxylate produced by the preparation method of this disclosure is cadaverine sebacic acid, it should be understood that all added or introduced dicarboxylic acids are sebacic acid. When the cadaverine dicarboxylate produced by the preparation method of this disclosure is cadaverine undecanoic acid, it should be understood that all added or introduced dicarboxylic acids are undecanoic acid. When the cadaverine dicarboxylate produced by the preparation method of this disclosure is cadaverine dodecanoic acid, it should be understood that all added or introduced dicarboxylic acids are dodecanoic 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.

[0016] In this disclosure, the term "sebacic acid" is a dicarboxylic acid having 10 carbon atoms and the chemical formula C2. 10 H 18 O4, and is usually found as a white powdery solid. Sebacic acid is also known as decanediol, etc., and is used as a precursor for a variety of industrial products, including polymers and plasticizers.

[0017] In this disclosure, the term "undecanoic acid" refers to a dicarboxylic acid having 11 carbon atoms and the chemical formula C1. 11 H 20 O4. Undecanoic acid is used as a precursor for a variety of industrial products, including polymers and plasticizers.

[0018] In this disclosure, the term "dodecanoic acid" is a carboxylic acid having 12 carbon atoms and the chemical formula C1. 12 H 22 O4. Dodecanoic acid is also known as dodecanoic acid.

[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 sebacate, cadaverine undecanedicate, or cadaverine dodecanedicate.

[0021] Figure 1 and Figure 2This is a flowchart illustrating a method for preparing cadaverine dicarboxylate according to one aspect of this disclosure.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] In the first step, the dicarboxylate 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.

[0029] In this disclosure, the term "diammonium dicarboxylate" refers to a form in which two hydrogen atoms of a dicarboxylic acid are replaced by ammonium, and is used as a primary nitrogen source in culture media. Diammonium dicarboxylate can be prepared by, but is not limited to, gently mixing a dicarboxylic acid with ammonia to achieve a neutral pH, and then concentrating it for use in crystalline or liquid form.

[0030] In the preparation method disclosed herein, by introducing 81 mol% or more of the dicarboxylate supplied throughout the process into the culture medium in the form of diammonium dicarboxylate in the first step, high-purity cadaverine dicarboxylate can be prepared, the process is simplified, and the side effect of dicarboxylate precipitation along with cadaverine dicarboxylate can be reduced.

[0031] Specifically, the diammonium dicarboxylate in the first step may be included in the culture medium at a high ratio, specifically 41 mol% or more of the dicarboxylate supplied throughout the preparation process of this disclosure.

[0032] More specifically, the content of diammonium dicarboxylate in the first step can be 43 mol% or more, 45 mol% or more, 53 mol% or more, 78 mol% or more, 80 mol% or more, 81 mol% or more, 82 mol% or more, 83 mol% or more, 84 mol% or more, 85 mol% or more, 86 mol% or more, 87 mol% or more, 88 mol% or more, 89 mol% or more, 90 mol% or more, 91 mol% or more, 92 mol% or more, 93 mol% or more, 94 mol% or more, 95 mol% or more, 96 mol% or more, 97 mol% or more, 98 mol% or more, 99 mol% or more, or 100 mol% of the dicarboxylate supplied throughout the preparation method of this disclosure.

[0033] 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.

[0034] 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 0.7 to 1.11. 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 0.74 to 1.06, 0.76 to 1.04, 0.78 to 1.02, 0.80 to 1.00, or 0.82 to 0.98, more specifically 0.84 to 0.96. 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.

[0035] 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 0.7, 0.72, 0.74, 0.78, 0.79, 0.80, 0.82, 0.84, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, or 0.96 and / or an upper limit selected from 1.11, 1.1, 1.08, 1.07, 1.06, 1.05, 1.04, 1.03, 1.02, 1.01, or 1.0.

[0036] In a specific example, the dicarboxylate and dicarboxylic acid can be sebacic acid and sebacic 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) 0.84 to 0.86.

[0037] In a specific example, the dicarboxylate and dicarboxylic acid can be undecanoic acid salt and undecanoic acid, respectively, and the content 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) can be 0.84 to 0.90.

[0038] In a specific example, the dicarboxylate and dicarboxylic acid can be dodecanoic acid salt and dodecanoic 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) 0.86 to 0.96.

[0039] Specifically, the content of diammonium dicarboxylate in the first step can be 0.3 mol / L to 0.5 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 0.3 mol / L to 0.45 mol / L, 0.35 mol / L to 0.45 mol / L, 0.38 mol / L to 0.45 mol / L, or 0.38 mol / L to 0.4 mol / L, based on the fermentation broth of 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.

[0040] 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.

[0041] 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 50 g / L to 200 g / L, 60 g / L to 150 g / L, 60 g / L to 140 g / L, or 61 g / L to 69 g / L, based on the fermentation broth from the L-lysine-producing microorganisms.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] The second step refers to the conversion of lysine dicarboxylate into the desired product, cadaverine dicarboxylate (cadaverine sebacic acid, cadaverine undecanediate, or cadaverine dodecanediate).

[0049] 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.

[0050] In the second step, dicarboxylic acid may be omitted or added in small amounts of 0.57 mol / L or lower, 0.53 mol / L or lower, 0.48 mol / L or lower, 0.4 mol / L or lower, 0.18 mol / L or lower, 0.15 mol / L or lower, 0.12 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.

[0051] In a specific instance, where the dicarboxylic acid is sebacic acid, a small amount of dicarboxylic acid, such as 0.01 mol / L or more and 0.06 mol / L or less, for example 0.04 mol / L to 0.06 mol / L, may be added in the second step.

[0052] In a specific instance, where the dicarboxylic acid is undecanoic acid, a small amount of dicarboxylic acid, at a concentration of 0.01 mol / L or more and 0.06 mol / L or less, such as 0.02 mol / L to 0.04 mol / L, may be added in the second step.

[0053] In a specific instance, where the dicarboxylic acid is dodecanoic acid, a small amount of dicarboxylic acid at 0.02 mol / L or lower, such as 0.005 mol / L to 0.015 mol / L, may be added in the second step.

[0054] 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.

[0055] 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.

[0056] 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 0.85 to 1.12.

[0057] 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 0.86 to 1.11, 0.87 to 1.10, 0.88 to 1.08, 0.89 to 1.06, 0.90 to 1.05, 0.91 to 1.04, 0.92 to 1.03, 0.93 to 1.02, 0.94 to 1.01, or 0.95 to 1.0 in molar ratio.

[0058] 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, 0.95, or 0.96 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.

[0059] In specific examples, when the dicarboxylate is sebacate, 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 0.9 to 1.10, 0.91 to 1.09, 0.92 to 1.08, 0.93 to 1.06, 0.94 to 1.02, 0.95 to 1.01, or 0.96 to 1.00 in molar ratio.

[0060] In the preparation method disclosed herein, when sebacic acid is added in a molar ratio within the range above relative to the amount of cadaverine to be obtained after the calculated second step, a technical significance is that sebacic acid does not precipitate as crystals along with cadaverine sebacic acid, thus high-purity cadaverine sebacic acid can be produced in high yield.

[0061] By adjusting the molar ratio of the added sebacic acid salt to the desired cadaverine, cadaverine sebacic acid salt with a purity of 98% or higher can be maintained up to the fourth cycle when the mother liquor is recycled. Depending on the number of mother liquor cycles, cadaverine sebacic acid salt can be obtained in total yields of 40% or higher, 42% or higher, 53% or higher, 56% or higher, 64% or higher, 66% or higher, 72% or higher, 73% or higher, or 74% or higher.

[0062] In specific examples, when the dicarboxylate is undecanoic acid salt, 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 0.9 to 1.10, 0.91 to 1.09, 0.92 to 1.08, 0.93 to 1.06, 0.94 to 1.02, or 0.95 to 1.00 in molar ratio.

[0063] In the preparation method disclosed herein, when undecanediate 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 undecanediic acid is found not to precipitate as crystals along with cadaverine undecanediate, thus high-purity cadaverine undecanediate can be produced in high yield.

[0064] By adjusting the molar ratio of the added undecanoate to the desired cadaverine, cadaverine undecanoate with a purity of 99% or higher can be maintained up to the fifth cycle when the mother liquor is recycled. Depending on the number of mother liquor cycles, cadaverine undecanoate can be obtained in total yields of 46% or higher, 48% or higher, 55% or higher, 56% or higher, 61% or higher, 63% or higher, 66% or higher, 68% or higher, 70% or higher, 72% or higher, or 73% or higher.

[0065] In specific examples, when the dicarboxylate is dodecanoic acid salt, 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 0.9 to 1.10, 0.91 to 1.09, 0.92 to 1.08, 0.93 to 1.06, 0.94 to 1.02, or 0.95 to 1.00 in molar ratio.

[0066] In the preparation method disclosed herein, when dodecanoic 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 it has been found that dodecanoic acid does not precipitate as crystals along with cadaverine dodecanoic acid salt, thus high-purity cadaverine dodecanoic acid salt can be produced in high yield.

[0067] By adjusting the molar ratio of the added dodecanoate to the desired cadaverine, cadaverine dodecanoate with a purity of 98% or higher can be maintained up to the fourth cycle when the mother liquor is recycled. Depending on the number of mother liquor cycles, cadaverine sebacate can be obtained in total yields of 40% or higher, 42% or higher, 53% or higher, 56% or higher, 64% or higher, 66% or higher, 72% or higher, 73% or higher, or 74% or higher.

[0068] In the second step, the conversion of lysine dicarboxylate to cadaverine dicarboxylate can be an enzymatic conversion reaction.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] There are no particular restrictions on the protein, as long as it exhibits lysine decarboxylase activity. It can be, for example, the PtLDC protein derived from *Pseudomonas thermotolerans* or the CadA protein derived from *Escherichia coli*. The protein sequence can be obtained from the known database GenBank; the protein can be expressed using microorganisms, or a commercially available enzyme can be purchased and used. However, the protein is not limited to these examples.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] The conversion reaction in the second step can be carried out at 30°C to 60°C, more specifically at 40°C to 55°C, for example at 45°C to 50°C, but is not limited thereto.

[0078] 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.

[0079] The method for preparing cadaverine dicarboxylate disclosed herein may further include a recovery step for recovering the obtained cadaverine dicarboxylate after the second step.

[0080] Recovery can be achieved by collecting cadaverine dicarboxylate using suitable methods known in the art.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] The concentration step refers to the process of concentrating the liquid to increase the solids ratio, and it can be performed after the filtration step. For example, concentration can be carried out under reduced pressure using a rotary evaporator.

[0085] Specifically, the concentration step can be performed to concentrate the solids content to 45% to 70% (w / w), more specifically 46% to 68% (w / w), 48% to 67% (w / w), 45% to 65% (w / w), or 50% to 70% (w / w). For example, concentration can be performed to concentrate the solids content to 45% to 55% (w / w). For example, concentration can be performed to concentrate the solids content to 60% to 70% (w / w).

[0086] When the concentration step is carried out to bring the solid content within the above range, the recovery rate and purity of cadaverine dicarboxylate can be maintained very well.

[0087] The concentration step can be carried out at 45°C to 80°C, for example, 55°C to 70°C, but is not limited to this.

[0088] 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.

[0089] Specifically, cooling may involve cooling the conversion liquid to 30°C or lower, more specifically to 27°C or lower, 25°C or lower, 23°C or lower, or 20°C or lower, such as cooling to 10°C, but is not limited thereto.

[0090] Specifically, the cooling rate can be from 0.1°C to 20°C / hour, more specifically from 0.5°C to 1.5°C / hour, for example 1°C / hour, but is not limited thereto.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] In one embodiment, the recovery step may further include washing, separating, and drying the process liquid from the crystallization process.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] In one embodiment, when the dicarboxylate is sebacate, the method of this disclosure can provide cadaverine sebacate with a purity of 99% or higher in 0 to 4 mother liquor cycles. In one embodiment, when the dicarboxylate is sebacate, the cumulative gross yield of cadaverine sebacate can be 70% or higher, more specifically greater than 70%, 71% or higher, 72% or higher, 73% or higher, or 74% or more, based on four mother liquor cycles.

[0101] In one embodiment, when the dicarboxylate is undecanoate, the method of this disclosure can provide cadaverine undecanoate with a purity of 99% or higher in 0 to 5 mother liquor cycles. In one embodiment, when the dicarboxylate is undecanoate, the cumulative gross yield of cadaverine undecanoate can be greater than 68%, for example 70% or higher, based on five mother liquor cycles, and the purity can be 99% or higher.

[0102] In one embodiment, when the dicarboxylate is dodecanoic acid diacidate, the method of this disclosure can provide cadaverine dodecanoic acid diacidate with a purity of 98% or higher in 0 to 4 mother liquor cycles. For example, cadaverine dodecanoic acid diacidate with a purity of 99% or higher can be obtained in 2 to 4 mother liquor cycles. In one embodiment, when the dicarboxylate is dodecanoic acid diacidate, the cumulative gross yield of cadaverine dodecanoic acid diacidate can be 70% or higher, more specifically greater than 70%, 71% or higher, or 72% or higher, based on four mother liquor cycles.

[0103] In one embodiment, the method of this disclosure can provide cadaverine dicarboxylate with a purity of 98% or higher in an overall yield of 22% or higher in 0 to 4 mother liquor cycles.

[0104] The method disclosed herein can produce high-purity cadaverine dicarboxylate without the decarbonization and distillation processes required in conventional liquid cadaverine production.

[0105] 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.

[0106] The preparation method of cadaverine dicarboxylate (cadaverine decanediarate, cadaverine undecanediate, or cadaverine dodecanediate) 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 decanediarate, cadaverine undecanediate, and cadaverine dodecanediate.

[0107] 1. Cadaverine sebacic acid salt Preparation Example 1-1. Production and fermentation process of lysine sebacate 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.

[0108] To produce lysine sebacate using Corynebacterium, in each example, diammonium sebacate was supplied at a level of 92 to 94 g / L instead of the existing ammonium sulfate, and repeated fed-batch fermentations were performed, wherein the carbon source concentration in the culture medium was reduced to 1.2 mol to maintain carbon / nitrogen source balance, to obtain lysine sebacate at a level of 136 to 139 g / L and lysine at a level of 68 g / L. In this example, the molar ratio of sebacate to lysine in the fermentation liquid was adjusted to a level of 0.83 to 0.85.

[0109] Preparation Examples 1-2. Conversion reaction of lysine sebacate to cadaverine sebacate 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.

[0110] The enzyme conversion broth obtained by seed culture with the enzyme strain was supplied at a level of 10% by mass to the fermentation broth for preparing lysine sebacate in Examples 1-1 for conversion reaction. The temperature of the conversion reaction was maintained at 45°C to 50°C, and the pH was adjusted to 8.0 to 8.5. For this purpose, CO2 was introduced for neutralization in Examples 1-1 to 1-4 below. Simultaneously, sebacate was additionally introduced at a level of 0.01 to 0.06 mol / L (based on the volume of the fermentation broth for lysine sebacate) before the conversion reaction in Examples 1-2 to 1-6. As a result, the conversion rate to cadaverine sebacate was found to be at a level of 97% to 98%. Specifically, after the conversion reaction, cadaverine sebacate was obtained at a level of 119 to 140 g / L (based on the volume of the fermentation broth for lysine sebacate), and cadaverine was obtained at a level of 47 g / L (based on the volume of the fermentation broth for lysine sebacate). Subsequently, in Examples 1-5 and 1-6, sebacic acid was additionally introduced at a level of 0.02 to 0.05 mol / L (based on the volume of the fermentation liquid for lysine sebacic acid).

[0111] Preparation Examples 1-3. Subsequent processes after obtaining cadaverine sebacate The process liquid containing cadaverine sebacate obtained in Examples 1-2 was subjected to the following subsequent treatment to obtain cadaverine sebacate in crystalline form.

[0112] [Bacterial Removal Steps] Bacteria in the liquid from the cadaverine sebacate process were removed by membrane filtration using a 0.1 μm membrane.

[0113] [Steps for removing impurities using activated carbon] Activated charcoal was added at a level of 10%, based on the weight of cadaverine sebacate 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.

[0114] [Steps for concentrating cadaverine sebacate 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 50% by weight.

[0115] [Steps for cooling and crystallization, separation, and then drying of the cadaverine sebacate concentrate] The concentrate was cooled from 50°C to 25°C at a rate of 1°C / hr. The precipitated crystals were separated from the mother liquor using a centrifuge. The crystals were washed with 10% water based on the weight of cadaverine sebacate. The separated crystals were dried for one day, and their purity was then determined by HPLC.

[0116] [The step of recycling the mother liquor to the crystallization step] The separated mother liquor is recycled to the step of concentrating the cadaverine sebacate process liquid after impurities have been removed.

[0117] Experimental Example 1-1. The changes in purity and yield of cadaverine sebacate were investigated based on the molar ratio of sebacate to cadaverine in the liquid used for conversion to cadaverine sebacate. 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 sebacate were investigated as varying with the molar ratio of sebacate to cadaverine in the liquid used for conversion to cadaverine sebacate.

[0118] Example 1-1: Case where the molar ratio of added sebacic acid salt to cadaverine in the post-second-step process liquid is 0.85. 1,000 ml of a liquid for the conversion of cadaverine sebacate to cadaverine sebacate with a sebacate / cadaverine molar ratio of 0.85 was supplied and passed through a 0.1 μm membrane to remove microorganisms. The liquid for the conversion of cadaverine sebacate to cadaverine sebacate with a sebacate / cadaverine molar ratio of 0.85 was prepared according to the preparation methods of Preparation Examples 1-1 and 1-2, except that the sum of the amount of diammonium sebacate introduced during the fermentation process for lysine production in Preparation Example 1-1 and the amount of sebacate introduced during the reaction for the conversion to cadaverine sebacate in Preparation Example 1-2 was controlled to be 0.85 of the molar number of cadaverine already converted.

[0119] The filtrate was decolorized with activated carbon, filtered through filter paper, and then concentrated under reduced pressure to a solid content of 50% by weight. The concentrate was cooled from 50°C to 25°C and crystallized. The crystals and mother liquor were separated from each other by centrifugation. The separated crystals were dried for one day, and their purity was measured by HPLC. The mother liquor containing residual sebacic acid was recycled to a cadaverine sebacic acid feed solution with a sebacic acid / cadaverine molar ratio of 0.85, and the crystallization step was repeated. The mother liquor was recycled sequentially.

[0120] Examples 1-2: Case where the molar ratio of added sebacic acid salt to cadaverine in the post-second-step process liquid is 0.89. A liquid for conversion to cadaverine octanoate with a sebacate / cadaverine molar ratio of 0.89 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.

[0121] Examples 1-3: Cases where the molar ratio of added sebacic acid salt to cadaverine in the post-second-step process liquid is 0.96. A liquid for conversion to cadaverine octanoate with a sebacate / cadaverine molar ratio of 0.96 was supplied, and the purification process was carried out in the same manner as in Examples 1-1 while the mother liquor was sequentially recycled.

[0122] Examples 1-4: Cases where the molar ratio of added sebacic acid salt to cadaverine in the post-second-step process liquid is 1.0. A liquid for conversion to cadaverine octanoate with a sebacate / cadaverine molar ratio of 1.0 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.

[0123] In Examples 1-3 and 1-4, the molar ratio of sebacic acid / lysine in the lysine fermentation step was adjusted to 0.85.

[0124] Examples 1-5: Cases where the molar ratio of added sebacic acid salt to cadaverine in the post-second-step process liquid is 1.04. A liquid for conversion to cadaverine octanoate with a sebacate / cadaverine molar ratio of 1.04 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.

[0125] Examples 1-6: Cases where the molar ratio of added sebacic acid salt to cadaverine in the post-second-step process liquid is 1.11. A liquid for conversion to cadaverine octanoate with a sebacate / cadaverine molar ratio of 1.11 was supplied, and the purification process was carried out in the same manner as in Examples 1-1 while the mother liquor was sequentially recycled.

[0126] The measured physical properties of the process liquid and cadaverine sebacate prepared according to Examples 1-1 to 1-6 above are shown in Tables 1 to 5 below.

[0127] Table 1 shows the process liquids for which mother liquor recycling was not performed.

[0128] [Table 1] Table 2 shows the process liquid for one cycle of the mother liquor.

[0129] [Table 2] Table 3 shows the process liquid for mother liquor circulated twice.

[0130] [Table 3] Table 4 shows the process liquid for three cycles of mother liquor.

[0131] [Table 4] Table 5 shows the process liquid after the mother liquor is circulated four times.

[0132] [Table 5] Based on the experimental results in Tables 1 to 5 above, it has been found that the preparation method disclosed herein can prepare cadaverine sebacate with an overall improved crystallization rate, high purity, and high yield. The method contains a large amount of diammonium sebacate in the first step, reaching 78 to 100 mol% of the total sebacate supplied.

[0133] More specifically, in Example 1-1, no crystals precipitated during the crystallization process. This is because the molar ratio of sebacic acid salt was not high enough to precipitate cadaverine sebacic acid salt crystals.

[0134] In Examples 1-2, crystallization and precipitation continued until the mother liquor was circulated three times, but no crystals were produced when the mother liquor was circulated four times. This is because the sequential circulation of the mother liquor reduced the molar ratio of sebacic acid salt in the feed liquid used for crystallization, and therefore the concentration of sebacic acid salt was not high enough to precipitate cadaverine sebacic acid salt crystals.

[0135] In Examples 1-5, the purity of the crystals was slightly lower than 86% when the mother liquor was not recycled, but the purity increased to 92% when the mother liquor was recycled four times.

[0136] In Examples 1-6, the purity of the crystals was as low as 73% when the mother liquor was not recycled, but the purity increased to 81% when the mother liquor was recycled four times.

[0137] On the other hand, in Examples 1-3, after the mother liquor was recycled four times, 99% high-purity cadaverine sebacate was obtained, and a high total yield of about 72% was achieved.

[0138] In Examples 1-4, after four cycles of mother liquor recycling, 99% high-purity cadaverine sebacate was obtained, and a high total yield of approximately 74% was achieved.

[0139] As described above, a method for preparing high-purity cadaverine sebacate has been discovered based on the molar ratio of sebacate to cadaverine in the conversion liquid (process liquid) supplied after the cadaverine conversion process. Specifically, it has been found that when the amount of sebacate added is adjusted to approximately 0.84 to 0.86 molarly relative to the amount of cadaverine in the process liquid after the second step, an excellent overall yield of 53% or higher can be achieved while maintaining 99% purity of the cadaverine sebacate, after two or more cycles of mother liquor recycling.

[0140] 2. Cadaverine undecanediate Preparation Example 2-1. Production and fermentation process of lysine undecanediate. 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.

[0141] Figure 5 is a graph showing the results of producing lysine undecanoic acid salt by microbial fermentation using ammonium undecanoate according to the preparation example described above.

[0142] To produce lysine undecanoic acid using Corynebacterium, in each example, diammonium undecanoic acid was supplied at a level of 88 to 100 g / L instead of the existing ammonium sulfate, and repeated fed-batch fermentations were performed, wherein the carbon source concentration in the culture medium was reduced to 1.2 mol to maintain carbon / nitrogen source balance, to obtain lysine undecanoic acid at a level of 127 to 145 g / L and lysine at a level of 66 g / L. In this example, the molar ratio of undecanoic acid to lysine in the fermentation liquid was adjusted to a level of 0.78 to 0.89.

[0143] Preparation Example 2-2. Conversion reaction of lysine undecanoic acid salt to cadaverine undecanoic acid salt 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.

[0144] The enzyme conversion broth obtained by seed culture with the enzyme strain was supplied at a level of 10% by mass to the fermentation broth for preparing lysine undecanoic acid salt prepared in Example 2-1 for conversion reaction. The temperature of the conversion reaction was maintained at 45°C to 50°C, and the pH was adjusted to 8.0 to 8.5. For this purpose, CO2 was introduced for neutralization in Examples 2-1 to 2-4 below. Simultaneously, undecanoic acid was additionally introduced at a level of 0.02 to 0.04 mol / L (based on the volume of the fermentation broth for lysine undecanoic acid salt) before the conversion reaction in Examples 2-2 to 2-4. As a result, the conversion rate to cadaverine undecanoic acid salt was found to be at a level of 97% to 98%. After the conversion reaction, cadaverine undecanoic acid salt was obtained at a level of 111 to 140 g / L (based on the volume of the fermentation broth for lysine undecanoic acid salt), and cadaverine was obtained at a level of 45 g / L (based on the volume of the fermentation broth for lysine undecanoic acid salt). Subsequently, in Examples 2-5 and 2-6, undecanoic acid was additionally introduced at a level of 0.06 to 0.08 mol / L to prepare the process liquid.

[0145] Preparation Examples 2-3. Subsequent processes after obtaining cadaverine undecanediate. The process liquid containing cadaverine undecanediate obtained in Preparation Example 2-2 was subjected to the following subsequent treatment to obtain cadaverine undecanediate in crystalline form.

[0146] [Bacterial Removal Steps] Bacteria in the liquid of the cadaverine undecanoate process were removed by membrane filtration using a 0.1 μm membrane.

[0147] [Steps for removing impurities using activated carbon] Activated charcoal was added at a level of 10%, based on the weight of cadaverine undecanoate 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.

[0148] [Steps for concentrating impurity-removed cadaverine undecanediate liquid] 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 65% by weight.

[0149] [Steps for cooling and crystallization, separation, and then drying of the cadaverine undecanediate concentrate] The concentrate was cooled from 50°C to 10°C at a rate of 1°C / hr. The precipitated crystals were separated from the mother liquor using a centrifuge. The crystals were washed with 10% water based on the weight of cadaverine undecanoate. The separated crystals were dried for one day, and their purity was then determined by HPLC.

[0150] [The step of recycling the mother liquor to the crystallization step] The separated mother liquor is recycled to the step of concentrating the cadaverine undecanoate process liquid after impurities have been removed.

[0151] Experimental Example 2-1. The changes in purity and yield of cadaverine undecanediate were investigated based on the molar ratio of undecanediate to cadaverine in the liquid used for conversion to cadaverine undecanediate. 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 undecanoic acid salt were investigated as to how they varied depending on the molar ratio of undecanoic acid salt to cadaverine in the liquid used for conversion to cadaverine undecanoic acid salt.

[0152] Example 2-1: Case where the molar ratio of added undecanoate / cadaverine in the post-second step liquid is 0.79. 1,000 ml of a liquid for the conversion of undecanediate to cadaverine undecanediate with a molar ratio of 0.79 was supplied and passed through a 0.1 μm membrane to remove microorganisms. The liquid for the conversion of undecanediate to cadaverine undecanediate with a molar ratio of 0.79 was prepared according to the preparation methods of Preparation Examples 2-1 and 2-2, except that the sum of the amount of undecanediate of diammonium undecanediate introduced during the fermentation process for lysine production in Preparation Example 2-1 and the amount of undecanediic acid introduced during the reaction for the conversion to cadaverine undecanediate in Preparation Example 2-2 was controlled to be 0.79 of the molar number of cadaverine already converted.

[0153] The filtrate was decolorized with activated carbon, filtered through filter paper, and then concentrated under reduced pressure to a solid content of 65% by weight. The concentrate was cooled from 50°C to 10°C and crystallized. The crystals and mother liquor were separated from each other by centrifugation. The separated crystals were dried for one day, and their purity was measured by HPLC. The mother liquor containing residual undecanoic acid salt was recycled to a cadaverine undecanoic acid salt feed solution with a molar ratio of undecanoic acid salt to cadaverine salt of 0.79, and the crystallization step was repeated. The mother liquor was recycled sequentially.

[0154] Example 2-2: Case where the molar ratio of added undecanoate / cadaverine in the post-second step liquid is 0.91. A liquid for conversion to cadaverine undecanoate was supplied with a molar ratio of undecanoate / cadaverine of 0.91, 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 2-3: Case where the molar ratio of added undecanoate / cadaverine in the post-second step liquid is 0.95. A liquid for conversion to cadaverine undecanoate was supplied with a molar ratio of 0.95, and the purification process was carried out in the same manner as in Example 2-1 while the mother liquor was recycled sequentially.

[0156] Examples 2-4: Cases where the molar ratio of added undecanoate / cadaverine in the post-second-step process liquid is 1.0. A liquid for conversion to cadaverine undecanoate was supplied with a molar ratio of 1.0, and the purification process was carried out in the same manner as in Example 2-1 while the mother liquor was recycled sequentially.

[0157] In Examples 2-3 and 2-4, the molar ratio of undecanoate / lysine in the lysine fermentation step was adjusted to 0.89.

[0158] Examples 2-5: Cases where the molar ratio of added undecanoate / cadaverine in the post-second-step process liquid is 1.04. A liquid for conversion to cadaverine undecanoate was supplied with a molar ratio of undecanoate / cadaverine of 1.04, and the purification process was carried out in the same manner as in Example 2-1 while the mother liquor was recycled sequentially.

[0159] Examples 2-6: Cases where the molar ratio of added undecanoate / cadaverine in the post-second-step process liquid is 1.09. A liquid for conversion to cadaverine undecanoate was supplied with a molar ratio of undecanoate / cadaverine of 1.09, and the purification process was carried out in the same manner as in Example 2-1 while the mother liquor was recycled sequentially.

[0160] The measured physical properties of the process liquid and cadaverine undecanediate prepared according to Examples 2-1 to 2-6 above are shown in Tables 6 to 11 below.

[0161] Table 6 shows the process liquids for which mother liquor recycling was not performed.

[0162] [Table 6] Table 7 shows the process liquid for one mother liquor cycle.

[0163] [Table 7] Table 8 shows the process liquid for mother liquor circulated twice.

[0164] [Table 8] Table 9 shows the process liquid for three cycles of mother liquor.

[0165] [Table 9] Table 10 shows the process liquid after the mother liquor has been circulated four times.

[0166] [Table 10] Table 11 shows the process liquid after five cycles of mother liquor circulation.

[0167] [Table 11] Based on the experimental results in Tables 1 to 6 above, it has been found that the preparation method disclosed herein can prepare cadaverine undecanediate with improved overall crystallization rate, high purity and high yield. The method contains a large amount of diammonium undecanediate in the first step, reaching 83 to 100 mol% of the total undecanediate supplied.

[0168] Specifically, in Example 2-1, 99% high-purity cadaverine undecanediate was obtained, but a total yield of 62% was only achieved after five cycles of mother liquor recycling. This was because the molar ratio of undecanediate was not high enough to precipitate cadaverine undecanediate crystals.

[0169] In Example 2-2, 99% high-purity cadaverine undecanediate was obtained, but a low overall yield of 67% was only achieved after five cycles of mother liquor recycling. This was because the molar ratio of undecanediate was not high enough to precipitate cadaverine undecanediate crystals.

[0170] In Examples 2-5, the purity of the crystals was slightly lower than 91% when the mother liquor was not recycled, but the purity increased to 96% when the mother liquor was recycled five times.

[0171] In Examples 2-6, the purity of the crystals was as low as 82% when the mother liquor was not recycled, but the purity increased to 92% when the mother liquor was recycled five times.

[0172] In Examples 2-3, after five cycles of mother liquor recycling, 99% high-purity cadaverine undecanoate was obtained, and a high total yield of approximately 70% was achieved.

[0173] In Examples 2-4, after five cycles of mother liquor recycling, 99% high-purity cadaverine undecanoate was obtained, and a high total yield of approximately 73% was achieved.

[0174] As described above, it has been found that the purity of the prepared cadaverine undecanoate can vary depending on the molar ratio of undecanoic acid to cadaverine in the conversion liquid (process liquid) supplied after the cadaverine conversion process. In particular, it has been found that when the amount of undecanoate added is adjusted to approximately 0.95 to 1.00 molar ratio relative to the amount of cadaverine in the process liquid after the second step, an excellent overall yield of 55% or higher can be achieved while maintaining a purity of 99% for cadaverine undecanoate, after two or more cycles of mother liquor recycling.

[0175] 3. Cadaverine dodecanediate Preparation Example 3-1. Production and fermentation process of lysine dodecanoate. 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.

[0176] To produce lysine using Corynebacterium, in each example, diammonium dodecanoate was supplied at a level of 93 to 106 g / L instead of the existing ammonium sulfate, and repeated fed-batch fermentations were performed, wherein the carbon source concentration in the culture medium was reduced to 1.2 mol to maintain carbon / nitrogen source balance, to obtain lysine dodecanoate at a level of 132 to 151 g / L and lysine at a level of 61 g / L. In this example, the molar ratio of dodecanoate to lysine in the fermentation liquid was adjusted to a level of 0.83 to 0.95.

[0177] Preparation Example 3-2. Conversion reaction of lysine dodecanoic acid salt to cadaverine dodecanoic acid salt 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.

[0178] The enzyme conversion broth obtained by seed culture with the enzyme strain was supplied at a level of 10% by mass to the fermentation process broth for preparing lysine dodecanoate in Example 3-1 for conversion reaction. The temperature of the conversion reaction was maintained at 45°C to 50°C, and the pH was adjusted to 8.0 to 8.5. For this purpose, CO2 was introduced for neutralization in Examples 3-1 to 3-4 below. Simultaneously, dodecanoic acid was additionally introduced at a level of 0.01 mol / L (based on the volume of the fermentation process broth for lysine dodecanoate) before the conversion reaction in Examples 3-2 to 3-4. As a result, the conversion rate to cadaverine dodecanoate was found to be at a level of 97% to 98%. Specifically, after the conversion reaction, cadaverine dodecanoate was obtained at a level of 116 to 136 g / L (based on the volume of the fermentation process broth for lysine dodecanoate), and cadaverine was obtained at a level of 42 g / L (based on the volume of the fermentation process broth for lysine dodecanoate). Subsequently, in Examples 3-5 and 3-6, dodecanoic acid was additionally introduced at a level of 0.03 to 0.05 mol / L (based on the volume of the fermentation process liquid of lysine dodecanoic acid) to prepare the process liquid.

[0179] Preparation Example 3-3. Subsequent processes after obtaining cadaverine dodecanediate. The process liquid containing cadaverine dodecanoate obtained in Preparation Example 3-2 was subjected to the following subsequent treatment to obtain cadaverine dodecanoate in crystalline form.

[0180] [Bacterial Removal Steps] Bacteria in the liquid of the cadaverine dodecanoate process were removed by membrane filtration using a 0.1 μm membrane.

[0181] [Steps for removing impurities using activated carbon] Activated charcoal was added at a level of 10%, based on the weight of cadaverine dodecanoate 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.

[0182] [Steps for concentrating impurity-removed cadaverine dodecanediate liquid] 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 50% by weight.

[0183] [Steps for cooling and crystallizing, separating and then drying the cadaverine dodecanediate concentrate] The concentrate was cooled from 50°C to 10°C at a rate of 1°C / hr. The precipitated crystals were separated from the mother liquor using a centrifuge. The crystals were washed with 10% water based on the weight of cadaverine dodecanoate. The separated crystals were dried for one day, and their purity was then determined by HPLC.

[0184] [The step of recycling the mother liquor to the crystallization step] The separated mother liquor is recycled to the step of concentrating the cadaverine dodecanoate process liquid after impurities have been removed.

[0185] Experimental Example 3-1. The changes in purity and yield of cadaverine dodecanoate were investigated based on the molar ratio of dodecanoate to cadaverine in the liquid used for conversion to cadaverine dodecanoate. In Experimental Example 3-1, the method according to Preparation Examples 3-1 to 3-3 was used, except that the purity and yield of cadaverine dodecanoate were investigated as a function of the molar ratio of dodecanoate to cadaverine in the liquid used for conversion to cadaverine dodecanoate.

[0186] Example 3-1: Case where the molar ratio of added dodecanoate / cadaverine in the post-second step process liquid is 0.85. 1,000 ml of a liquid for the conversion of cadaverine to cadaverine dodecanoic acid with a dodecanoic acid / cadaverine molar ratio of 0.85 was supplied and passed through a 0.1 μm membrane to remove microorganisms. The liquid for the conversion of cadaverine to cadaverine dodecanoic acid with a dodecanoic acid / cadaverine molar ratio of 0.85 was prepared according to the preparation methods of Preparation Examples 3-1 and 3-2, except that the sum of the amount of dodecanoic acid introduced into the fermentation process of lysine production in Preparation Example 3-1 and the amount of dodecanoic acid introduced into the reaction for the conversion of cadaverine to cadaverine dodecanoic acid in Preparation Example 3-2 was controlled to be 0.85 of the molar number of cadaverine already converted.

[0187] The filtrate was decolorized with activated carbon, filtered through filter paper, and then concentrated under reduced pressure to a solid content of 50% by weight. The concentrate was cooled from 50°C to 10°C and crystallized. The crystals and mother liquor were separated from each other by centrifugation. The separated crystals were dried for one day, and their purity was measured by HPLC. The mother liquor containing residual dodecanoic acid was recycled to a cadaverine dodecanoic acid feed solution with a dodecanoic acid / cadaverine molar ratio of 0.85, and the crystallization step was repeated. The mother liquor was recycled sequentially.

[0188] Example 3-2: Case where the molar ratio of added dodecanoic acid salt to cadaverine in the post-second step liquid is 0.9. A liquid for conversion to cadaverine dodecanoate was supplied with a dodecanoate / cadaverine molar ratio of 0.9, and the purification process was carried out in the same manner as in Example 3-1 while the mother liquor was recycled sequentially.

[0189] Example 3-3: Case where the molar ratio of added dodecanoate / cadaverine in the post-second step process liquid is 0.95. A liquid for conversion to cadaverine dodecanoate was supplied with a dodecanoate / cadaverine molar ratio of 0.95, and the purification process was carried out in the same manner as in Example 3-1 while the mother liquor was recycled sequentially.

[0190] Examples 3-4: Cases where the molar ratio of added dodecanoate / cadaverine in the post-second-step process liquid is 1.0. A liquid for conversion to cadaverine dodecanoate was supplied with a dodecanoate / cadaverine molar ratio of 1.0, and the purification process was carried out in the same manner as in Example 3-1 while the mother liquor was recycled sequentially.

[0191] In Examples 3-3 and 3-4, the molar ratio of dodecanoate / lysine in the lysine fermentation step was adjusted to 0.9 to 0.95.

[0192] Examples 3-5: Cases where the molar ratio of added dodecanoate / cadaverine in the post-second-step process liquid is 1.04. A liquid for conversion to cadaverine dodecanoate was supplied with a dodecanoate / cadaverine molar ratio of 1.04, and the purification process was carried out in the same manner as in Example 3-1 while the mother liquor was recycled sequentially.

[0193] Examples 3-6: Cases where the molar ratio of added dodecanoate / cadaverine in the post-second-step process liquid is 1.09. A liquid for conversion to cadaverine dodecanoate was supplied with a dodecanoate / cadaverine molar ratio of 1.09, and the purification process was carried out in the same manner as in Example 3-1 while the mother liquor was recycled sequentially.

[0194] The measured physical properties of the process liquid and cadaverine dodecanoate prepared according to Examples 3-1 to 3-6 above are shown in Tables 12 to 16 below.

[0195] Table 12 shows the process liquids for which mother liquor recycling was not performed.

[0196] [Table 12] Table 13 shows the process liquid for one mother liquor cycle.

[0197] [Table 13] Table 14 shows the process liquid for mother liquor circulated twice.

[0198] [Table 14] Table 15 shows the process liquid for three cycles of mother liquor.

[0199] [Table 15] Table 16 shows the process liquid after four cycles of mother liquor circulation.

[0200] [Table 16] Based on the experimental results in Tables 12 to 16 above, it has been found that the preparation method disclosed herein can prepare cadaverine dodecanoic acid tar ...

[0201] More specifically, in Example 3-1, no crystals precipitated during the crystallization process. This is because the molar ratio of dodecanoate was not high enough to precipitate cadaverine dodecanoate crystals.

[0202] In Example 3-2, crystallization and precipitation continued until the mother liquor was circulated three times, but no crystals were produced when the mother liquor was circulated four times. This is because the sequential circulation of the mother liquor reduced the molar ratio of dodecanoate in the feed liquid used for crystallization, and therefore the concentration of dodecanoate was not high enough to precipitate cadaverine dodecanoate crystals.

[0203] In Examples 3-5, the purity of the crystals was slightly lower than 86% when the mother liquor was not recycled, but the purity increased to 92% when the mother liquor was recycled four times.

[0204] In Examples 3-6, the purity of the crystals was as low as 73% when the mother liquor was not recycled, but the purity increased to 81% when the mother liquor was recycled four times.

[0205] In Examples 3-3, after four cycles of mother liquor recycling, 99% high-purity cadaverine dodecanoate was obtained, and a high total yield of approximately 72% was achieved.

[0206] In Examples 3-4, after four cycles of mother liquor recycling, 99% high-purity cadaverine dodecanoate was obtained, and a high total yield of approximately 74% was achieved.

[0207] As described above, it has been found that the purity of the prepared cadaverine dodecanoate can vary depending on the molar ratio of dodecanoate to cadaverine in the conversion liquid (process liquid) supplied after the cadaverine conversion process. In particular, it has been found that when the amount of added dodecanoate is adjusted to approximately 0.95 to 1.00 molar ratio relative to the amount of cadaverine in the process liquid after the second step, an excellent overall yield of 53% or higher can be achieved while maintaining a purity of 99% for cadaverine succinate, after two or more cycles of mother liquor recycling.

[0208] 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 is to obtain lysine dicarboxylate by culturing L-lysine-producing microorganisms in a culture medium containing diammonium dicarboxylate. and 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 0.90 to 1.05, and The dicarboxylate is an aliphatic dicarboxylate having 10 to 12 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 0.84 to 0.

96.

4. The method according to claim 1, wherein the dicarboxylic acid salt and the dicarboxylic acid are sebacic acid salt and sebacic acid, respectively. 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 0.84 to 0.

86.

5. The method according to claim 1, wherein the dicarboxylate and the dicarboxylic acid are undecanoic acid salt and undecanoic acid, respectively. 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 0.84 to 0.

90.

6. The method according to claim 1, wherein the dicarboxylate and the dicarboxylic acid are dodecanoic acid salt and dodecanoic acid, respectively, 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 0.86 to 0.

96.

7. 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.

8. The method according to claim 1, wherein the dicarboxylate and the dicarboxylic acid are sebacic acid salt and sebacic acid, respectively. The amount of dicarboxylate added is 0.96 to 1.00 molar ratio relative to the amount of cadaverine in the process liquid after the second step.

9. The method according to claim 1, wherein the dicarboxylic acid salt and the dicarboxylic acid are undecanoic acid salt and undecanoic acid, respectively. The amount of dicarboxylate added is 0.95 to 1.00 molar ratio relative to the amount of cadaverine in the process liquid after the second step.

10. The method according to claim 1, wherein the dicarboxylate and the dicarboxylic acid are dodecanoic acid salt and dodecanoic acid, respectively, and The amount of dicarboxylate added is 0.95 to 1.00 molar ratio relative to the amount of cadaverine in the process liquid after the second step.

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

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

13. The method of claim 12, 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.

14. The method of claim 12, 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.

15. The method of claim 13, wherein the concentration step is to concentrate the solids content to 45% to 70% (w / w).

16. 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.

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

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

19. The method of claim 13, wherein a cadaverine dicarboxylate with a purity of 98% or higher is obtained within 0 to 4 cycles of mother liquor recycling.

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