Method for preparing N-guanidino amino acids

Abstract

The present invention relates to a process for the preparation of N-guanidino amino acids (e.g., GAA) comprising the steps of a) providing a liquid and / or solid mixture comprising N-guanidino amino acids (e.g., GAA), biomass and / or DNA, b) treating the mixture of step a) with an acidic solution and / or an acidic reagent, and c) isolating N-guanidino amino acids (e.g., GAA) from the mixture obtained in step b), characterized in that step b) is carried out at a pH lower than the pKa of the N-guanidino amino acid acetic acid of the mixture provided in step a), said pKa being measured by a pH electrode.

Description

[0001] The present invention relates to a method for preparing N-guanyamino acids, such as guanidinoacetic acid, N-guanidinoamino acid crystals with trapped guanine, and a method for supplementing an animal's diet, wherein the diet is supplemented with N-guanidinoamino acids, such as guanidinoacetic acid, obtained by the method of the present invention, and / or with N-guanidinoamino acids, such as guanidinoacetic acid crystals according to the present invention.

[0002] N-guanidino amino acids are derivatives of amino acids with a guanidine group, which can be obtained by adding cyanamide to the amino acid in question. The most important N-guanidino amino acid is guanidinoacetic acid (GAA), also known as N-guanidinoglycine. It is an endogenous substance in animals and humans and plays a central role in the biosynthesis of creatine. Creatine can be ingested through the diet and / or formed endogenously. Therefore, it is also used as a feed additive in animal nutrition (US2011 / 257075A1). Since it is a direct natural precursor of creatine, supplementation with GAA ensures an optimal supply of creatine in the body, which positively impacts energy transfer in muscle cells. Its biosynthesis occurs from glycine and L-arginine. In mammals, guanidinoacetic acid is formed primarily in the kidneys by transferring the guanidino group of L-arginine to the amino acid glycine using the enzyme L-Arg:Gly-guanyltransferase (AGAT). Starting from L-arginine, L-ornithine is produced, which is then metabolized in the urea cycle via carbamylation to L-citrulline. In a further step, guanidinoacetic acid is methylated with S-adenosylmethionine to creatine by the enzyme guanidinoacetic acid N-methyltransferase (GAMT). Guanidinoacetic acid was first synthesized by Adolph Strecker in 1861 by adding cyanamide to glycine in an aqueous solution, particularly a weak ammonia solution (M. Strecker, compt. Rend. 1861, 52, 1212; cited in Ber. Chem. Ges. (now Eur. J. Inorg. Chem.) 1908, 41, 4385). In later publications, guanidinoacetic acid is prepared from cyanamide and glycine in isopropanol as solvent, using sodium hydroxide as a base (CN102329250A) or sodium carbonate as a base (CN101462983A).

[0003] Published application CN113651726A discloses a process for preparing guanidinoacetic acid by reacting cyanamide with glycine in an alkaline medium, then treating the mixture thus obtained with an acidic solution and / or an acidic reagent, and isolating guanidinoacetic acid from the mixture.

[0004] Published application US2022 / 0388948A1 discloses a method for preparing guanidinoacetic acid containing guanidinoacetic acid in a thermodynamically metastable crystalline modification by crystallization from an aqueous solution in the presence of at least one guanidine compound. The document also discloses the use of the GAA crystals thus obtained as a feed supplement.

[0005] However, these processes have the significant disadvantage of forming by-products melamine and dicyanamide, both of which have negative effects on living organisms.Therefore, alternative processes for the preparation of N-guanidino amino acids such as GAA are considered.

[0006] One alternative method for producing N-guanidino amino acids (e.g., GAA) is to produce N-guanidino amino acids (e.g., GAA) by culturing microbial organisms, such as genetically modified microorganisms or microorganisms modified in other ways, such as by genome editing. This offers advantages over chemical routes, including the absence of problematic byproducts such as melamine and dicyanamide (EP3839051A, CN111748506A, and CN113481139A). For example, EP3839051A discloses a microorganism having an increased ability to produce L-arginine compared to a wild-type microorganism, and / or having an increased enzymatic activity having a carbamoyl-phosphate synthetase function compared to the corresponding enzyme activity in a wild-type microorganism, and comprising at least one gene encoding a protein having an L-arginine:glycine amidinotransferase function. The document also discloses a method for fermentative production of guanidinoacetic acid, comprising the steps of: a) culturing the microorganism in a suitable culture medium under suitable conditions, and b) accumulating guanidinoacetic acid in the culture medium to form a fermentation broth containing guanidinoacetic acid.

[0007] However, the fermentative synthesis of N-guanidino amino acids, such as GAA, results in product particles containing DNA (deoxyribonucleic acid) from the fermented biomass and / or genetically modified microorganisms or microorganisms modified in other ways, such as by genome editing, which are used for fermentation as impurities. The biomass and / or DNA are trapped in the N-guanidino amino acids (e.g., GAA crystals) formed during the fermentation process. Therefore, classical separation techniques such as filtration cannot remove or at least deplete this impurity.

[0008] Therefore, there remains a need for a process for producing N-guanidino amino acids (eg, guanidinoacetic acid) by fermentation in the absence of biomass and / or DNA as impurities or in the presence of at least reduced amounts of any of these impurities.

[0009] A solution to this problem has been found in that a liquid and / or solid mixture obtained from a fermentation process comprising N-guanidino amino acids, such as GAA, biomass and / or DNA is treated with an acidic solution and / or an acidic agent.

[0010] Therefore, one object of the present invention is a process for preparing an N-guanidino amino acid, such as GAA, comprising the following steps:

[0011] a) providing a liquid and / or solid mixture comprising an N-guanidino amino acid (e.g. GAA), biomass and / or DNA,

[0012] b) treating the mixture of step a) with an acidic solution and / or an acidic agent, and

[0013] c) separating N-guanidino amino acids, such as GAA, from the mixture obtained in step b),

[0014] Characterized in that step b) is carried out at a pH below the pKa of the N-guanidinoglycolic acid of the mixture provided in step a), said pKa being measured by a pH electrode.

[0015] Preferably, the biomass and / or DNA in the mixture provided in step a) is produced by fermentation of N-guanidino amino acids, such as GAA.

[0016] The method according to the present invention allows the removal or at least depletion of biomass and / or DNA from any type of liquid and / or solid mixture containing N-guanidino amino acids, such as GAA, biomass and / or DNA. The solid mixture can be the aforementioned N-guanidino amino acid crystals, such as GAA, in which the DNA of the microorganisms used in the fermentation is trapped, or it can be any other type of solid mixture containing the fermentation product N-guanidino amino acid, such as GAA in a non-crystalline form, biomass and / or DNA. Alternatively, in the case of a liquid mixture, the method according to the present invention allows the removal of DNA of the microorganisms used in the fermentation process from a fermentation broth containing N-guanidino amino acids.

[0017] In one embodiment of the method according to the invention, the liquid mixture of step a) comprises or consists of a fermentation broth, wherein the fermentation broth comprises the N-guanidino amino acid; and the solid mixture of step a) comprises or consists of N-guanidino amino acid crystals, wherein the crystals comprise biomass and / or DNA.

[0018] It has been found to be beneficial to carry out step b) at a pH below the pKa of the N-guanidino amino acid of the mixture provided in step a). This results in protonation of the guanidino amino acid, which facilitates the dissolution of the N-guanidino amino acid in question and the removal or at least depletion of any biomass and / or DNA therefrom. Therefore, the pH at which step b) is carried out depends on the individual N-guanidino amino acid, such as GAA, provided in step a), as well as the medium in which step b) is carried out. Preferably, step b) is carried out in an aqueous medium. For example, when the N-guanidino amino acid provided in step a) is guanidinoacetic acid, step b) is carried out at a pH below the pKa of guanidinoacetic acid, i.e., below about 2.9, as measured by a pH electrode. Depending on the individual composition of the mixture provided in step a), the pH at which step b) is carried out may differ from the specific value of the pKa of the N-guanidino amino acid, i.e., the pH may differ from 2.9 for guanidinoacetic acid. In the context of the present invention, deviations from the explicitly mentioned pH values ​​are still encompassed by the scope of the present invention, provided that they allow the benefits of the present invention to be achieved.

[0019] In principle, the process according to the invention is not subject to any restrictions with regard to a specific acidic reagent in step b). However, sulfuric acid was found to be a suitable acid for step b).

[0020] In another embodiment of the process according to the invention, the pKa of the acidic reagent of step b) is from -4 to +4, preferably from -3 to +3.

[0021] In principle, the method according to the invention, in particular step b) of the method, is not subject to any restrictions with regard to a specific temperature. However, it is preferred that step b) is carried out at an elevated temperature, as elevated temperatures have been found to be beneficial to the efficiency of step b), in particular with regard to the extent to which any biomass and / or DNA is removed or at least depleted from the liquid or solid mixture comprising guanidinoamino acids (e.g. GAA) and the time taken for this.

[0022] In another embodiment of the process according to the invention, step b) is carried out at elevated temperature.

[0023] In the context of the present invention, an elevated temperature is in particular any temperature of 25° C. or higher. The upper limit of the elevated temperature is given by various factors, such as the boiling temperature of the solvent and the decomposition temperature of organic substances other than N-guanidino amino acids (e.g. GAA) (which are residues of the fermentation process), such as sugars. For example, step b) is carried out at a temperature of 25 to 90° C., 25 to 85° C., 25 to 80° C., 25 to 75° C., 25 to 70° C., 25 to 65° C., 25 to 60° C., 25 to 55° C. or 25 to 50° C.

[0024] In a preferred embodiment of the process according to the invention, step b) is carried out at a temperature of at least 25°C.

[0025] N-guanidino amino acids are synthesized by fermentation to produce product particles, wherein DNA (deoxyribonucleic acid) from genetically or otherwise modified microorganisms used in the fermentation is captured as an impurity. The DNA is captured in GAA crystals formed during the fermentation process. In step c), the liquid and / or solid mixture comprising N-guanidino amino acids (e.g., GAA), biomass, and DNA is treated with an acidic solution and / or an acidic agent, resulting in hydrolysis or at least partial hydrolysis of the DNA.

[0026] In another embodiment of the method according to the invention, step b) comprises acidic hydrolysis of the DNA.

[0027] After the acidic treatment in step b) of the process according to the invention, the N-guanidino amino acid, for example GAA, is separated from the mixture obtained in step b) in step c). This separation can be carried out by ultrafiltration followed by concentration of the permeate thus obtained, which contains the N-guanidino amino acid, for example GAA.

[0028] In yet another embodiment of the method according to the present invention, step c) comprises the following steps:

[0029] c1) removing biomass from the mixture obtained in step b) by ultrafiltration to obtain a permeate, wherein the permeate comprises N-guanidino amino acids, such as GAA, and

[0030] c2) concentrating the permeate obtained in step c1).

[0031] The permeate obtained in step c1) may still contain any undesirable residues from the fermentation. Therefore, it is preferred that the permeate obtained in step c2) is not concentrated to dryness, for example the solvent is not completely removed at reduced pressure and / or elevated temperature. On the contrary, it is preferred that only a portion of the medium of the permeate is removed in the concentration of step c2) and the permeate from step c2) thus concentrated, comprising N-guanidino amino acids (e.g. GAA), is fed to a crystallization process. Preferably, the concentration is carried out at an elevated temperature, i.e. a temperature of at least 25°C, preferably at least 30°C, 35°C or 40°C, and / or under reduced pressure. Although the reduced pressure is not subject to any restrictions regarding a specific value, the upper limit of the elevated temperature is given by various factors, such as the boiling point of the solvent and the decomposition temperature of organic substances other than N-guanidino amino acids (e.g. GAA) (which are residues from the fermentation process, such as sugars). For example, step c2) is performed at a temperature of 40 to 100°C, 45 to 100°C, 50 to 100°C, 55 to 100°C, 60 to 100°C, 65 to 100°C, 70 to 100°C, 75 to 100°C or 80 to 100°C.

[0032] In yet another embodiment, the method according to the present invention further comprises the following steps:

[0033] d) feeding the isolated N-guanidino amino acid (eg GAA) obtained in step c) to a crystallization process.

[0034] To promote crystallization in step c), the pH of the N-guanidino amino acid (e.g., GAA) separated in step c) or the concentrated permeate containing the N-guanidino amino acid (e.g., GAA) obtained in step c2) is preferably set to a pH greater than the pKa of the N-guanidino amino acid (e.g., GAA) and less than the pKb of the N-guanidino amino acid (e.g., GAA), each measured using a pH electrode. This results in deprotonation of the guanidino group of the N-guanidino amino acid (e.g., GAA), which promotes crystallization of the N-guanidino amino acid (e.g., GAA) in question. The pH at which step d) is performed depends on the individual N-guanidino amino acid, e.g., GAA, provided in step a), as well as the medium in which step b) is performed. Preferably, step d) is performed in an aqueous medium. For example, when the N-guanidino amino acid provided in step a) is guanidinoacetic acid, step d) is performed at a pH greater than the pKa of guanidinoacetic acid and less than the pKb of guanidinoacetic acid, for example, at a pH greater than about 2.9 and less than about 10.91. Depending on the respective composition of the mixture provided in step a), the pH in step d) may differ from the specific values ​​of pKa and pKb of the N-guanidino amino acid (e.g., GAA). In the context of the present invention, deviations from the explicitly mentioned pH values ​​are still encompassed by the scope of the present invention, as long as they allow the benefits of the present invention to be achieved.

[0035] In a preferred embodiment of the method according to the invention, step d) is performed at a pH greater than the pKa of the N-guanidino amino acid (eg GAA) and lower than the pKb of the N-guanidino amino acid (eg GAA), each measured by a pH electrode.

[0036] After the crystals of the N-guanidino amino acid (eg, GAA) are formed, the crystals can be easily separated from the mother liquor of the crystallization process and dried.

[0037] In another preferred embodiment of the process according to the invention, the crystals obtained in step d) are isolated by filtration and dried.

[0038] In principle, the method is not subject to any restrictions with regard to a specific N-guanidino amino acid, such as GAA. However, the commercially most relevant N-guanidino amino acid is guanidinoacetic acid.

[0039] In one embodiment of the invention, the N-guanidino amino acid is guanidinoacetic acid.

[0040] Amino acids or derivatives thereof produced during the fermentation process, such as N-guanidino amino acids, such as guanidinoacetic acid, can be used for animal nutrition, such as supplementing animal diets. In step c), the liquid and / or solid mixture comprising N-guanidino amino acids, biomass and / or DNA is treated with an acidic solution and / or an acidic reagent to cause the hydrolysis of the DNA. As a result of this treatment, the obtained N-guanidino amino acids (such as GAA) do not contain or at least deplete the DNA of the microorganism used for fermentative production of N-guanidino amino acids (such as GAA). Guanine is a degradation product from acidic hydrolysis, which can be found in N-guanidino amino acids such as guanidinoacetic acid. The obtained N-guanidino amino acids, such as GAA, obtained by the method according to the present invention can be used in animal diet supplements without any restrictions or requirements. On the other hand, the presence of guanine in the N-guanidino amino acids (such as GAA) crystals allows identification of whether the N-guanidino amino acids (such as GAA) are prepared or purified by the method according to the present invention.

[0041] Therefore, another object of the present invention are crystals of N-guanidino amino acids, such as GAA, wherein said crystals contain trapped guanine.

[0042] In one embodiment of the crystal according to the present invention, the N-guanidinoamino acid is guanidinoacetic acid.

[0043] In another embodiment of the crystal according to the present invention, an N-guanidinoamino acid such as guanidinoacetic acid is obtained by the method according to the present invention.

[0044] Another object of the present invention is a method for supplementing an animal's diet, wherein the diet is supplemented with an N-guanidinoamino acid, such as guanidinoacetic acid, obtained by the method of the present invention and / or with N-guanidinoamino acid, such as guanidinoacetic acid crystals according to the present invention. Example:

[0045] After fermentation and inactivation of the fermentation broth, the pH of the fermentation broth was adjusted to 1.5 using 75% w / w H2SO4 at 50°C. The biomass was removed by ultrafiltration, and the temperature of the broth was kept constant at 50°C. The resulting permeate was concentrated at 80°C and used for the crystallization step. The permeate fed to the crystallization process was adjusted to pH 8 using 25% w / w NH4OH. The crystals formed were separated by suction and dried.

[0046] A portion of the acidic concentrated permeate was stored in a refrigerator over a period of approximately 2 weeks. The GAA content in the permeate was determined regularly by HPLC.

[0047] Description of HPLC method:

[0048] Column: ThermoScientific HyperCarb100x4,6 35007-104630

[0049] Eluent:

[0050] Eluent A:

[0051] 2.3g ammonium dihydrogen phosphate (NH4H2PO4) and

[0052] 2.6g diammonium hydrogen phosphate ((NH4)2HPO4)

[0053] Dissolve in 2L of purified water

[0054] Eluent B:

[0055] 2.3g ammonium dihydrogen phosphate (NH4H2PO4) and

[0056] 2.6g diammonium hydrogen phosphate ((NH4)2HPO4)

[0057] Dissolve in 1 L purified water and mix with 1 L acetonitrile

[0058] The following gradient was used in the HPLC method:

[0059]

[0060] The results of the determination of GAA content are summarized in the following table:

[0061] date GAA content [g / kg] 01.04.2022 120.9 07.04.2022 118.5 12.04.2022 119.9 13.04.2022 119.3

[0062] The results showed that the GAA content was constant over a 2-week period within the accuracy of the measurements, implying that there was no degradation of GAA in the acidic concentrated permeate.

Claims

1. A method for preparing N-guanidino amino acid, comprising the following steps: a) providing a liquid and / or solid mixture comprising N-guanidino amino acids, biomass and / or DNA, b) treating the mixture of step a) with an acidic solution and / or an acidic agent, and c) separating the N-guanidino amino acid from the mixture obtained in step b), Characterized in that step b) is carried out at a pH below the pKa of the N-guanidinoglycolic acid of the mixture provided in step a), said pKa being measured by a pH electrode.

2. The method according to claim 1 , wherein the liquid mixture of step a) comprises or consists of a fermentation broth, wherein the fermentation broth comprises an N-guanidino amino acid; and the solid mixture of step a) comprises or consists of N-guanidino amino acid crystals, wherein the crystals comprise biomass and / or DNA.

3. The method according to any one of claims 1 to 2, wherein the pKa of the acidic reagent of step b) is -4 to +4.

4. The process according to claim 1, wherein step b) is carried out at elevated temperature.

5. The process according to any one of claims 1 to 4, wherein step b) is carried out at a temperature of at least 25°C.

6. The method according to any one of claims 1 to 5, wherein step b) comprises acidic hydrolysis of DNA.

7. The method according to any one of claims 1 to 6, wherein step c) comprises the following steps: c1) removing the biomass from the mixture obtained in step b) by ultrafiltration to obtain a permeate, wherein the permeate comprises an N-guanidino amino acid, and c2) concentrating the permeate obtained in step c1).

8. The method according to any one of claims 1 to 7, further comprising the steps of: d) feeding the isolated N-guanidino amino acid obtained in step c) to a crystallization process.

9. The method according to claim 8, wherein step d) is performed at a pH greater than the pKa of the N-guanidino amino acid and lower than the pKb of the N-guanidino amino acid, each measured by a pH electrode.

10. The process according to claim 8 or 9, wherein the crystals obtained in step d) are separated from the mother liquor of the crystallization process by filtration and dried.

11. The method of any one of claims 1 to 10, wherein the N-guanidinoamino acid is guanidinoacetic acid.

12. N-guanidino amino acid crystal, characterized in that The crystals contain trapped guanine.

13. The N-guanidino amino acid crystal according to claim 12, wherein the N-guanidino amino acid is guanidinoacetic acid.

14. The N-guanidino amino acid crystal according to claim 12 or 13, wherein the N-guanidino amino acid is obtained by the method according to any one of claims 1 to 11.

15. Method for supplementing an animal's diet, wherein the diet is supplemented with an N-guanidino amino acid obtained by the method of any one of claims 1 to 11 and / or with N-guanidino amino acid crystals of any one of claims 12 to 14.

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