Process for the preparation of beta-alanine, beta-alanine salts and pantothenate salts

Abstract

The present application provides a method for preparing beta-alanine, comprising: preparing beta-alanine product by reacting reactants containing fumaric acid and ammonia in the presence of a catalyst, wherein the catalyst comprises a catalytic composition containing aspartase and L-aspartate-alpha-decarboxylase, and fumaric acid is added during the reaction, and the total amount of fumaric acid added is equal to the initial molar amount of ammonia in the reactants minus the initial molar amount of fumaric acid in the reactants. The present application also provides a method for preparing beta-alanine salt (especially calcium beta-alanine, sodium beta-alanine, potassium beta-alanine) and pantothenic acid salt (especially calcium pantothenate, sodium pantothenate, potassium pantothenate).

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an enzyme-catalyzed method for preparing β-alanine, β-alanine salts (especially calcium β-alanine, sodium β-alanine, and potassium β-alanine) and pantothenates (especially calcium pantothenate, sodium pantothenate, and potassium pantothenate). Background Technology

[0002] β-Alanine, also known as β-aminopropionic acid or 3-aminopropionic acid, is a naturally occurring β-type non-protein amino acid. β-Alanine is a versatile organic synthetic raw material, mainly used in the synthesis of pantothenic acid and calcium pantothenate, carnosine, sodium pamidronate, balsalazine, etc. It has wide applications in the pharmaceutical, feed, and food industries, and enjoys a very large market demand.

[0003] Currently, the production methods of β-alanine are divided into two main categories: chemical synthesis and biological methods. Because the chemical synthesis of β-alanine was studied earlier and the process is mature, it is still the main production method used both domestically and internationally.

[0004] There are two main chemical methods for producing β-alanine. One is to react acrylic acid with ammonia under high temperature and pressure. This method is low in cost, but the strong acidity causes great corrosion to the equipment and has high safety requirements. The other method is to first react acrylonitrile with ammonia under high temperature and pressure to prepare β-aminopropionitrile, and then hydrolyze it with sodium hydroxide under high temperature conditions. This method has a low yield and generates a large amount of inorganic salts during the reaction, which presents separation problems.

[0005] Chemical methods generally suffer from problems such as harsh reaction conditions, high equipment requirements, and environmental pollution. Therefore, with the increasing use of β-alanine, biological methods have gradually become a research hotspot due to their advantages of mild reaction conditions, high efficiency, and environmental friendliness.

[0006] Most biological methods for producing β-alanine utilize microorganisms capable of producing specific enzymes to transform substrates into β-alanine. For example, Zhejiang University of Technology (CN1285730) used an aminase to synthesize β-alanine from acrylic acid and ammonia. This method has high reaction efficiency and low cost, but due to the strong corrosiveness and irritation of the raw materials, there are currently no reports of industrial applications. Chuan Liyang et al. (JP10-42886) used an organic nitrile hydrolase to catalyze the synthesis of β-alanine from β-aminopropionitrile. This method has high raw material costs, low reaction concentrations, and high costs, making it difficult to meet the requirements for industrial production.

[0007] Another biological method for synthesizing β-alanine involves using L-aspartate-α-decarboxylase to specifically remove the α-carboxyl group from L-aspartate to generate β-alanine. However, existing methods suffer from low enzyme activity and poor enzyme stability. Summary of the Invention

[0008] On one hand, the present invention provides a method for preparing β-alanine, comprising: preparing a β-alanine product by reacting a mixture containing fumaric acid and ammonia in the presence of a catalyst, wherein the catalyst comprises a catalytic composition containing aspartate enzyme and L-aspartate-α-decarboxylase, and fumaric acid is added during the reaction, wherein the total molar amount of fumaric acid added is equal to the initial molar amount of ammonia in the reactants minus the initial molar amount of fumaric acid in the reactants.

[0009] In some embodiments, the catalytic composition contains purified aspartate aminotransferase and purified L-aspartate-α-decarboxylase. In some embodiments, the catalytic composition contains bacterial cells expressing both aspartate aminotransferase and L-aspartate-α-decarboxylase. In some embodiments, the bacterial cells comprise wet bacterial cells, immobilized bacterial cells, or bacterial cell lysate. In some embodiments, the bacterial cells are derived from recombinant engineered bacteria. In some embodiments, the bacterial cells comprise bacterial cells expressing aspartate aminotransferase alone and bacterial cells expressing L-aspartate-α-decarboxylase alone. In some embodiments, the bacterial cells expressing aspartate aminotransferase alone constitute 0.5% to 4% (w / w) of the initial fumarate in the reactants; and the bacterial cells expressing L-aspartate-α-decarboxylase alone constitute 10% to 30% (w / w) of the initial fumarate in the reactants. In some embodiments, the bacterial cells comprise bacterial cells co-expressing aspartate aminotransferase and L-aspartate-α-decarboxylase. In some embodiments, the bacterial cells co-expressing aspartate and L-aspartate-α-decarboxylase constitute 10% to 40% (w / w) of the initial fumarate in the reactants. In some embodiments, the aspartate is derived from... Anoxybacillus flavithermus or Geobacillus thermodenitrificans The L-aspartate-α-decarboxylase is derived from... Bacillus thermotolerans , Anoxybacillus flavithermus or Methanocaldococcus jannaschii In some embodiments, the aspartate enzyme is derived from... Anoxybacillus flavithermus WK1 or Geobacillus thermodenitrificans NG80-2; the L-aspartate-α-decarboxylase is derived from Quasibacillus thermotolerans , Anoxybacillus flavithermus AK1 or Methanocaldococcus jannaschii DSM 2661.

[0010] In some embodiments, the initial molar ratio of fumaric acid to ammonia in the reactants is 1:2. In some embodiments, the fumaric acid is added during the reaction by a feedstock. In some embodiments, the concentration of the fumaric acid is 50-400 g / L, and the feed rate is such that the pH value is controlled between 6.8 and 7.2 during the reaction. In some embodiments, the reaction temperature is controlled between 25 and 55°C.

[0011] In some embodiments, the method for preparing β-alanine according to the present invention further includes, after the catalytic reaction is completed, removing residues from the catalytic composition. In some embodiments, the method for preparing β-alanine according to the present invention further includes crystallizing the β-alanine product. In some embodiments, a mother liquor is obtained after crystallization, and the content of inorganic salts in the mother liquor is less than 10 g / L. In some embodiments, the mother liquor obtained after crystallization can be recycled. In some embodiments, β-alanine crystals are obtained after crystallization, and the content of inorganic salts in the crystals is less than 20 mg / g.

[0012] On the other hand, the present invention provides a method for preparing β-alanine salt, comprising the following steps: (a) β-alanine is prepared according to the method of the present invention; and (b) The β-alanine obtained in step (a) is reacted with an alkaline solution.

[0013] In some embodiments, the β-alanine salt is an alkali metal salt or an alkaline earth metal salt of β-alanine, and the alkaline solution is an alkaline solution containing alkali metal or alkaline earth metal cations. In some embodiments, the alkaline solution contains KOH, NaOH, Ca(OH)₂, Mg(OH)₂, Al(OH)₃, or combinations thereof. In some embodiments, the Ca(OH)₂ is obtained by reacting calcium oxide with water. In some embodiments, the β-alanine salt is calcium β-alanine, potassium β-alanine, or sodium β-alanine. In some embodiments, the β-alanine salt is calcium β-alanine. In some embodiments, the method for preparing the β-alanine salt of the present invention further includes crystallizing the β-alanine salt.

[0014] On the other hand, the present invention provides a method for preparing pantothenate, comprising the following steps: (a) β-alanine is prepared according to the method of the present invention; (b) The β-alanine obtained in step (a) is reacted with an alkaline solution to prepare β-alanine salt; and (c) React pantothenic acid or pantothenic acid with the β-alanine salt prepared in step (b).

[0015] In some embodiments, the pantothenic acid is D-pantothenic acid. In some embodiments, the pantothenic acid is D-pantothenic acid. In some embodiments, the pantothenic acid (e.g., D-pantothenic acid) or pantothenic acid (e.g., D-pantothenic acid) is dissolved in a solvent before reacting with the β-alanine salt prepared in step (b). In some embodiments, the solvent is methanol or ethanol. In some embodiments, the pantothenate is calcium pantothenate, sodium pantothenate, or potassium pantothenate. In some embodiments, the pantothenate is calcium pantothenate. In some embodiments, the method for preparing the pantothenate according to the present invention further includes crystallizing the pantothenate.

[0016] On the other hand, the present invention provides calcium pantothenate wherein the chloride ion content (by weight) is not higher than 190 ppm, and / or the sodium ion content (by weight) is not higher than 2200 ppm. In some embodiments, the calcium pantothenate is prepared by the method for preparing calcium pantothenate according to the present invention. Brief description of the attached diagram Figure 1 : Showed Anoxybacillus flavithermus The gene sequence encoding aspartate enzyme in the WK1 genome sequence is SEQ ID NO: 1.

[0017] Figure 2 : Showed Geobacillus thermodenitrificans The gene sequence encoding aspartate enzyme in the genomic sequence of NG80-2 is SEQ ID NO: 2.

[0018] Figure 3 : Showed Bacillus thermotolerans middle Quasibacillus thermotolerans strain SGZ-8 Contig4 The gene sequence encoding L-aspartate-α-decarboxylase in the genome sequence is SEQ ID NO:3.

[0019] Figure 4 : Showed Anoxybacillus flavithermus The genome sequence of AK1 encodes the gene sequence SEQ ID NO: 4 for L-aspartate-α-decarboxylase.

[0020] Figure 5 : Showed Methanocaldococcus jannaschii The gene sequence encoding L-aspartate-α-decarboxylase in the genome sequence is SEQ ID NO: 5.

[0021] Figure 6The figure shows the HPLC chromatogram for investigating the linearity of the assay method in the detection of chloride and sodium ions in calcium pantothenate. The figure shows that the retention time of chloride ions in the column is 4.024 min, and the retention time of sodium ions is 4.351 min.

[0022] Figure 7 The standard curve is shown, plotted using the logarithm of the chloride ion concentration (LgC) versus the logarithm of the peak area (LgA), in the experiment for detecting chloride and sodium ion content in calcium pantothenate. Figure 7 A), and a standard curve plotted using the logarithm of sodium ion injection concentration (LgC) and the logarithm of peak area (LgA). Figure 7 B).

[0023] Figure 8 The image shows an HPLC chromatogram of the detection limit of chloride ions in the experiment for detecting chloride and sodium ions in calcium pantothenate.

[0024] Figure 9 The image shows an HPLC chromatogram of the limit of quantitation (LOQ) for chloride ions in the detection of chloride and sodium ions in calcium pantothenate.

[0025] Figure 10 The image shows the HPLC chromatograms of calcium pantothenate samples 1, 2, and 3 in the test for chloride and sodium ion content of calcium pantothenate. Detailed Implementation

[0026] This invention overcomes the shortcomings of existing β-alanine preparation processes and provides a green, efficient, low-cost production process for β-alanine, β-alanine salts, and pantothenate suitable for industrial production.

[0027] On one hand, the present invention provides a method for preparing β-alanine, comprising: preparing a β-alanine product by reacting a mixture containing fumaric acid and ammonia in the presence of a catalyst, wherein the catalyst comprises a catalytic composition containing aspartate enzyme and L-aspartate-α-decarboxylase, and fumaric acid is added during the reaction, wherein the total molar amount of fumaric acid added is equal to the initial molar amount of ammonia in the reactants minus the initial molar amount of fumaric acid in the reactants.

[0028] The “initial molar amount” of fumaric acid or ammonia refers to the initial molar amount of fumaric acid or ammonia before the catalytic reaction begins. The “initial weight of fumaric acid” in the reactants refers to the initial weight of fumaric acid added to the reactants before the catalytic reaction begins. In some embodiments, the initial molar ratio of ammonia to fumaric acid in the reactants is 2:1, or fluctuates within a range of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, and 1% around this value.

[0029] In some embodiments, the reactants containing fumaric acid and ammonia may include ammonium fumarate. Ammonium fumarate is the product obtained by acid-base neutralization of fumaric acid and ammonia. While not theoretically limited, it is considered that in reactants containing fumaric acid and ammonia, at least a portion of the fumaric acid and ammonia will spontaneously form ammonium fumarate. In this application, 1 mole of ammonium fumarate is considered equivalent to 2 moles of ammonia and 1 mole of fumaric acid. Therefore, when the reactants contain only 1 mole of ammonium fumarate, the initial molar ratio of ammonia to fumaric acid is considered to be 2:1.

[0030] In the method provided in this application, the catalyst comprises a catalytic composition containing aspartate enzyme and L-aspartate-α-decarboxylase. Any known aspartate enzyme and L-aspartate-α-decarboxylase can be used. It is known in the art that aspartate enzyme and L-aspartate-α-decarboxylase are naturally expressed in a variety of microorganisms and both possess corresponding catalytic activities.

[0031] In some embodiments, the aspartase and L-aspartate-α-decarboxylase are derived from bacteria. In some embodiments, the aspartase is derived from... Anoxybacillus flavithermus or Geobacillus thermodenitrificans In some embodiments, the aspartate enzyme is derived from... Anoxybacillus flavithermus WK1 strain or Geobacillus thermodenitrificans NG80-2 strain. In some embodiments, the amino acid sequence of the aspartic acid enzyme is identical to, or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with, the amino acid sequence encoded by SEQ ID NO:1 or SEQ ID NO:2.

[0032] In some embodiments, the L-aspartate-α-decarboxylase is derived from... Bacillus thermotolerans, Anoxybacillus flavithermus or Methanocaldococcus jannaschii In some embodiments, the L-aspartate-α-decarboxylase is derived from... Quasibacillus thermotolerans , Anoxybacillus flavithermus AK1 or Methanocaldococcus jannaschiiDSM 2661. In some embodiments, the amino acid sequence of the L-aspartate-α-decarboxylase is identical to, or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with, the amino acid sequence encoded by SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

[0033] In this application, "homology" refers to, for amino acid sequences, comparing a candidate amino acid sequence with a reference amino acid sequence, introducing gaps where necessary, to maximize the number of identical amino acids, and then calculating the percentage of identical amino acids between the two amino acid sequences; for nucleic acid sequences, comparing a candidate nucleic acid sequence with a reference nucleic acid sequence, introducing gaps where necessary, to maximize the number of identical nucleotides, and then calculating the percentage of identical nucleotides between the two nucleic acid sequences. In other words, the percentage of homology between two amino acid sequences (or nucleotide sequences) can be calculated as follows: the number of amino acids (or nucleotides) identical to those in the compared reference sequence divided by the total number of amino acids (or nucleotides) in the candidate sequence or the reference sequence (whichever is shorter). The percentage of homology can be determined through various comparisons known in the art. For example, sequence alignment can be performed using publicly available tools such as BLASTp (National Center for Biotechnology Information (NCBI): http: / / blast.ncbi.nlm.nih.gov / Blast.cgi, see also Altschul SF et al., J. Mol. Biol., 215:403–410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389–3402 (1997)) and ClustalW2 (European Institute for Bioinformatics: http: / / www.ebi.ac.uk / Tools / msa / clustalw2 / , see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23(21):2947-8 (2007)). When using software for sequence alignment, the default parameters provided by the software can be used, or the parameters can be adjusted appropriately according to the needs of the comparison. These are all within the knowledge of those skilled in the art.

[0034] In this application, the aspartate enzyme and L-aspartate-α-decarboxylase in the catalytic composition may be present in any suitable active form, such as, but not limited to, isolated or purified active enzyme proteins, cells expressing the enzyme naturally or recombinantly, or their lysates.

[0035] In some embodiments, the catalytic composition contains purified aspartate enzyme and purified L-aspartate-α-decarboxylase. The purified enzyme may be obtained from microorganisms that naturally express the enzyme, or it may be obtained through recombinant expression followed by purification. Those skilled in the art can prepare purified aspartate enzyme and L-aspartate-α-decarboxylase using conventional techniques. For example, ammonium sulfate precipitation followed by ion exchange chromatography, and then gel chromatography.

[0036] In some embodiments, the catalytic composition contains bacterial cells expressing aspartate aminotransferase and L-aspartate-α-decarboxylase. In some embodiments, the bacterial cells include wet bacterial cells, immobilized bacterial cells, or bacterial cell lysate. In some embodiments, the wet bacterial cells are bacterial cells obtained after solid-liquid separation of bacterial culture fermentation broth, such as bacterial cells collected by centrifugation; the immobilized bacterial cells are immobilized bacterial cells obtained by conventional immobilization methods, such as bacterial cells embedded in sodium alginate; the bacterial cell lysate is a solution obtained by conventionally crushing bacterial cells, such as a high-pressure homogenization solution. The bacterial cell lysate contains the desired enzymes.

[0037] In some embodiments, the bacterial cells expressing aspartate and L-aspartate-α-decarboxylase are derived from wild-type bacteria. For example, wild-type bacteria naturally expressing aspartate, wild-type bacteria naturally expressing L-aspartate-α-decarboxylase, or wild-type bacteria naturally expressing both aspartate and L-aspartate-α-decarboxylase can be used. The wild-type bacteria include, for example, Anoxybacillus flavithermus , Geobacillus thermodenitrificans, Bacillus thermotolerans, Methanocaldococcus jannaschii wait.

[0038] In some embodiments, the bacterial cells expressing aspartate enzyme and L-aspartate-α-decarboxylase are derived from recombinant engineered bacteria. Recombinant engineered bacteria refer to engineered bacteria in which foreign genes have been introduced into a host engineered bacterium via recombinant DNA. Recombinant engineered bacteria can recombinantly express the introduced foreign genes. Those skilled in the art can select a suitable host to express aspartate enzyme and L-aspartate-α-decarboxylase according to their actual needs. In some embodiments, the host is selected from the group consisting of: *Escherichia coli*, ... Escherichia fergusonii , Anoxybacillus flavithermus WK1, Geobacillus thermodenitrificans NG80-2 Bacillus thermotolerans , Anoxybacillus flavithermus AK1 Methanocaldococcus jannaschii, Bacillus cereus, Corynebacterium glutamicum .

[0039] Those skilled in the art can prepare recombinant engineered bacteria using techniques known in the art, as needed. See *Molecular Cloning: A Laboratory Manual (3rd Edition)* (Science Press). The vectors, plasmids, and hosts used in the experiments are all conventional bacterial (e.g., *E. coli*) expression vectors, plasmids, and host series, such as the PET series vectors and plasmids, and the BL21 series host bacteria. The culture media used are conventional bacterial (e.g., *E. coli*) engineered bacteria culture media, such as LB medium. The culture methods used are conventional bacterial (e.g., *E. coli*) engineered bacteria culture methods.

[0040] In some embodiments, the bacterial cells expressing aspartate and L-aspartate-α-decarboxylase include bacterial cells expressing aspartate alone and bacterial cells expressing L-aspartate-α-decarboxylase alone. In some embodiments, the bacterial cells are derived from wild-type bacteria or recombinant engineered bacteria.

[0041] In some embodiments, the weight percentage of the bacterial cells expressing aspartate aminotransferase alone to the initial fumarate in the reactants is 0.5% to 4% (w / w), for example, any value between any two of the following ranges: 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.5%, 3%, 3.5%, 4%, or more, preferably 1% to 2% (w / w); and the weight percentage of the bacterial cells expressing L-aspartate-α-decarboxylase alone to the initial fumarate in the reactants is 10% to 30%. (w / w), for example, any value between any two numerical ranges of 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% or more, preferably 15% to 20% (w / w).

[0042] In some embodiments, the weight of the bacterial cells is calculated as wet weight when determining the weight percentage of the bacterial cells to the initial fumaric acid. In other embodiments, the weight of the bacterial cells is calculated as dry weight when determining the weight percentage of the bacterial cells to the initial fumaric acid. Those skilled in the art can choose according to their actual needs. Those skilled in the art can also convert between dry and wet weight using conventional methods in the prior art, for example, see https: / / bionumbers.hms.harvard.edu / bionumber.aspx?id=109836.

[0043] In some embodiments, the activities of aspartic acid oxidase and L-aspartate-α-decarboxylase produced by the recombinant engineered bacteria used in this invention are at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, or more, higher than those produced by wild-type bacteria. Those skilled in the art can use conventional techniques to determine the activities of aspartic acid oxidase and L-aspartate-α-decarboxylase, for example, by measuring the substrate conversion rate per unit time using fumaric acid and aspartic acid as substrates, respectively.

[0044] While not limited by theory, it is believed that the enzymes or enzyme-expressing cells derived from recombinant engineered bacteria used in this invention have unexpected advantages compared to wild-type bacteria. For example, compared to wild-type bacteria, the recombinant engineered bacteria used in this invention can significantly increase the fumarate reaction concentration, for example, from 100 g / L to 200 g / L, while simultaneously maintaining a high yield and high purity of the β-alanine product.

[0045] In some embodiments, the bacterial cells expressing aspartate and L-aspartate-α-decarboxylase include bacterial cells that co-express aspartate and L-aspartate-α-decarboxylase. In some embodiments, the bacterial cells are derived from recombinant engineered bacteria.

[0046] In some embodiments, the bacterial cells co-expressing aspartate and L-aspartate-α-decarboxylase have a weight percentage of 10% to 40% (w / w) of the initial fumaric acid in the reactants, for example, any value between any two of the following ranges: 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, or more, preferably 20% to 30% (w / w).

[0047] In the method for preparing β-alanine provided in this application, the pH of the solution gradually increases as the catalytic reaction proceeds. The inventors of this application add fumaric acid during the reaction to control the pH value of the reaction. While not limited by theory, it is believed that fumaric acid has significant advantages over conventional inorganic acid regulators in the prior art in at least the following two aspects: Firstly, fumaric acid can effectively lower the pH value of the reaction solution to a suitable range; secondly, the added fumaric acid can also serve as a reaction substrate, being consumed through the catalytic reaction to generate β-alanine, thus avoiding the introduction of additional impurities. Conversely, if an inorganic acid regulator is used to adjust the pH, it will form ammonium salts of inorganic acids with ammonia in the reaction system, leading to the generation of impurities, thus requiring additional impurity removal steps.

[0048] In the method for preparing β-alanine provided in this application, fumaric acid and ammonia water are first converted into the intermediate product aspartic acid under the catalysis of aspartic acidase. Then, aspartic acid does not need to be further purified or extracted. It is directly converted into the final product β-alanine under the catalysis of L-aspartate-α-decarboxylase. This method can realize the one-step direct generation of β-alanine without the need to extract the intermediate product aspartic acid.

[0049] Another technical advantage of this invention is that it can significantly reduce the residual ammonia, fumaric acid, and ammonium fumarate in the product. In the method for preparing β-alanine provided in this application, the total molar amount of fumaric acid added is equal to the initial molar amount of ammonia in the reactants minus the initial molar amount of fumaric acid in the reactants, thereby ensuring that the ammonia in the reactants reacts completely while avoiding excess fumaric acid in the product. Furthermore, ammonia and fumaric acid are converted into β-alanine after catalytic reaction, thus significantly reducing the content of uncatalyzed ammonium fumarate.

[0050] In some embodiments of this application, the initial molar ratio of fumaric acid to ammonia is designed to be 1:2. For example, assuming the initial molar amount of fumaric acid is 1 mol and the initial molar amount of ammonia is 2 mol. During the reaction, to avoid pH elevation, the inventors cleverly control the pH value by adding fumaric acid during the reaction process, and precisely control the amount of fumaric acid added, ensuring that the molar amount of added fumaric acid (1 mol) is equal to the initial molar amount of ammonia in the reactants (2 mol) minus the initial molar amount of fumaric acid in the reactants (1 mol). This ensures complete reaction of the ammonia in the reactants while avoiding excess fumaric acid in the product. The reaction process for preparing β-alanine from fumaric acid and ammonia can be summarized as follows: In some embodiments, fumaric acid is added during the reaction by a fed-batch method. In some embodiments, the feeding rate of the fumaric acid is such that the pH value during the reaction is controlled between 6.8 and 7.2. In some embodiments, the concentration of the fumaric acid is 50-400 g / L, and its feeding rate is such that the pH value during the reaction is controlled between 6.8 and 7.2, for example, any value between any two of the ranges of 6.8, 6.9, 7.0, 7.1, 7.2, or higher. In some embodiments, the pH value of the reaction system can be detected during the fed-batch process, thereby adjusting the feeding rate of the fumaric acid during the reaction. For example, in some embodiments, when the concentration is 100 g / L, the feeding rate depends on controlling the pH value during the reaction to be between 6.8 and 7.1. In some embodiments, when the concentration is 200 g / L, the feeding rate depends on controlling the pH value during the reaction to be between 6.9 and 7.2.

[0051] In some embodiments, the reaction temperature of the method for preparing β-alanine according to the present invention is controlled between 25 and 55°C, for example, any value between any two of the following ranges: 25°C, 30°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, and preferably between 35 and 42°C.

[0052] In some embodiments, after the catalytic reaction is completed, the method for preparing β-alanine according to the present invention further includes: removing residues from the catalytic composition.

[0053] In this application, "catalytic reaction" refers to the reaction process in which fumaric acid and ammonia react in the presence of a catalyst to produce β-alanine. Those skilled in the art can use various methods to determine whether the catalytic reaction has ended. In some embodiments, the reaction is monitored (e.g., using HPLC) after the fumaric acid addition is complete. When the fumaric acid content is <0.5% (w / v), for example 0.4%, 0.3%, 0.2%, 0.1% or even lower, and the molar conversion of aspartic acid is >99%, for example 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or even 100%, the reaction is considered complete. Those skilled in the art can use conventional methods to calculate the molar conversion of aspartic acid, for example, by using HPLC to determine the amount of each component in the reaction mixture, thereby determining the molar conversion of aspartic acid.

[0054] In some embodiments, the residues include large particulate impurities, such as cells, bacterial fragments, aggregates, and flocs, as well as small molecule impurities, such as nucleic acids and nucleic acid fragments, proteins, and culture medium components in bacterial culture media. Those skilled in the art can remove the catalyst residues from the mixture using conventional separation methods, such as filtration, centrifugation, microfiltration, and ultrafiltration, as needed.

[0055] In some embodiments, the filtration is achieved using filter paper or filter cloth. The filter paper or filter cloth described in this invention can be commercially available, such as filter paper or filter cloth manufactured by companies like GE Healthcare Life Sciences, Spice, and Asahi Kasei. In some embodiments, the pore size of the filter paper or filter cloth is 10–150 μm, for example, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, or any value between any two of these ranges. Those skilled in the art can select an appropriate pore size for the filter paper or filter cloth to remove impurities based on their size.

[0056] In some embodiments, microfiltration is achieved by passing the reaction solution through a microfiltration membrane. The microfiltration membrane described in this invention can be a commercially available microfiltration membrane, such as the hollow fiber microfiltration membrane series manufactured by GE Healthcare Life Sciences, Spice, Asahi Kasei, etc. In some embodiments, the pore size of the microfiltration membrane is 0.1 μm to 0.6 μm, for example, 0.1 μm, 0.15 μm, 0.2 μm, 0.22 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm, 0.6 μm, or any value between any two of the above ranges. Those skilled in the art can select an appropriate microfiltration membrane pore size to remove impurities based on their size.

[0057] In some embodiments, ultrafiltration is achieved by passing the reaction solution through an ultrafiltration membrane. The ultrafiltration membrane described in this invention can be a commercially available ultrafiltration membrane, such as the ultrafiltration hollow fiber membrane series manufactured by companies like GE Healthcare Life Sciences, Spice, and Asahi Kasei. In some embodiments, the ultrafiltration membrane is a hollow fiber ultrafiltration membrane with a pore size of 5kD to 500kD, for example, a hollow fiber ultrafiltration membrane with a pore size of 5kD, 6kD, 7kD, 8kD, 9kD, 10kD, 20kD, 30kD, 40kD, 50kD, 60kD, 70kD, 80kD, 90kD, 100kD, 150kD, 200kD, 250kD, 300kD, 350kD, 400kD, 450kD, 500kD, or any value between any two of these ranges. Those skilled in the art can select an appropriate ultrafiltration membrane pore size to remove impurities based on their size.

[0058] In some embodiments, the method for preparing β-alanine according to the present invention further includes concentrating the β-alanine product. In some embodiments, the concentration is achieved by reducing pressure, for example, by pumping the reaction solution after filtration, microfiltration, or ultrafiltration into a concentration device for concentration under reduced pressure to 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10 of the original volume, or any value between any two of the above ranges.

[0059] In some embodiments, the method for preparing β-alanine according to the present invention further includes crystallizing the β-alanine product. In some embodiments, the crystallization is achieved by lowering the temperature and adding an organic solvent (e.g., methanol, ethanol, etc.), for example, by adding 1, 2, 3, or 4 times the volume of organic solvent dropwise to a concentrated reaction solution, and crystallizing at low temperature conditions (e.g., 10°C, 5°C, or lower).

[0060] In the method for preparing β-alanine provided in this application, a mother liquor is obtained after crystallization. In this application, "mother liquor" refers to the liquid remaining after β-alanine crystallization following the catalytic reaction. The content of inorganic salts in the mother liquor is less than 10 g / L, for example, less than 9 g / L, 8 g / L, 7 g / L, 6 g / L, 5 g / L, 4 g / L, 3 g / L, 2 g / L, 1 g / L, 0.5 g / L, etc. In some embodiments, the mother liquor obtained during the crystallization process does not contain inorganic salts. Those skilled in the art can use conventional techniques to determine the content of inorganic salts in the mother liquor, for example, using an ammonia nitrogen detector (Shanghai Yidian Scientific Instruments Co., Ltd.) to detect residual ammonium ions in the mother liquor. Not limited by theory, another technical advantage of this invention is that the remaining mother liquor after the reaction is essentially free of impurities such as inorganic salts, allowing for recycling and avoiding the discharge of industrial wastewater, thus making it more environmentally friendly. In some embodiments, β-alanine crystals are obtained after crystallization, and the content of inorganic salts in the crystals is less than 20 mg / g, for example less than 19 mg / g, 18 mg / g, 17 mg / g, 16 mg / g, 15 mg / g, 14 mg / g, 13 mg / g, 12 mg / g, 11 mg / g, 10 mg / g, 9 mg / g, 8 mg / g, 7 mg / g, 6 mg / g, 5 mg / g, 4 mg / g, 3 mg / g, 2 mg / g, 1 mg / g, etc.

[0061] The method for preparing β-alanine described in this invention has the following advantages compared with the prior art: Using inexpensive fumaric acid as the starting substrate, β-alanine can be directly generated in one step without the need for intermediate extraction of aspartic acid, which simplifies the production process, reduces production costs, and is environmentally friendly. The thermostable enzyme was efficiently expressed in engineered bacteria, exhibiting high enzyme activity, good enzyme stability, high substrate concentration, and high conversion efficiency, with molar conversion rates of fumaric acid and aspartic acid both exceeding 99%. The pH value is controlled by adding fumaric acid, and no other inorganic acids (such as phosphoric acid, sulfuric acid, hydrochloric acid, nitric acid, etc.) are introduced during the entire reaction process. The molar amount of fumaric acid, ammonia water and added fumaric acid in the reactants are precisely controlled during the reaction process, so that no by-products and inorganic salts are generated in the reaction process, the product extraction process is simple and the product purity is high.

[0062] The mother liquor obtained after β-alanine crystallization contains almost no inorganic salts or other impurities. Theoretically, the mother liquor can be recycled, thereby reducing production costs.

[0063] On the other hand, the present invention provides a method for preparing β-alanine salt, comprising the following steps: (a) β-alanine is prepared according to the method of the present invention; and (b) The β-alanine obtained in step (a) is reacted with an alkaline solution.

[0064] Specifically, in some embodiments, the method for preparing β-alanine salt according to the present invention includes the following steps: (a) A β-alanine product is prepared from reactants containing fumaric acid and ammonia in the presence of a catalyst, wherein the catalyst comprises a catalytic composition containing aspartate enzyme and L-aspartate-α-decarboxylase, and fumaric acid is added during the reaction, wherein the total molar amount of fumaric acid added is equal to the initial molar amount of ammonia in the reactants minus the initial molar amount of fumaric acid in the reactants; and (b) The β-alanine obtained in step (a) is reacted with an alkaline solution.

[0065] In some embodiments, the β-alanine obtained in step (a) is dissolved in a solvent before reacting with the alkaline solution. In some embodiments, the β-alanine obtained in step (a) is dissolved in water before reacting with the alkaline solution.

[0066] In some embodiments, the β-alanine salt of the present invention is an alkali metal salt of β-alanine, for example, an alkali metal salt containing a cation of any one of lithium (Li), sodium (Na), potassium (K), rubidium (Rb), and cesium (Cs). In some embodiments, the β-alanine salt is sodium β-alanine or potassium β-alanine. In some embodiments, the β-alanine salt of the present invention is an alkaline earth metal salt of β-alanine, for example, an alkaline earth metal containing a cation of any one of beryllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), and radium (Ra). In some embodiments, the β-alanine salt of the present invention is calcium β-alanine.

[0067] In this application, "alkaline solution" refers to a solution with a pH > 7 at room temperature or a hydroxide ion concentration greater than a hydrogen ion concentration. In some embodiments, the selected alkaline solution is chosen to be capable of reacting with β-alanine under suitable conditions. In some embodiments, the alkaline solution is an alkaline solution containing alkali metal or alkaline earth metal cations. In some embodiments, the alkaline solution comprises NaOH, KOH, Ca(OH)₂, Mg(OH)₂, Al(OH)₃, or combinations thereof.

[0068] Depending on the target β-alanine salt, those skilled in the art can choose a suitable alkaline solution for the reaction. In some embodiments, the alkaline solution can also be prepared fresh for use. For example, in the preparation of β-alanine calcium salt, calcium oxide is added to an aqueous solution of β-alanine. The calcium oxide then reacts with water to generate Ca(OH)₂, which in turn reacts with β-alanine to generate β-alanine calcium salt (the specific reaction process is shown below). In this preparation method, the feed ratio (weight ratio) of β-alanine, calcium oxide, and water is between 3:1:5 and 3:1:17, such as 3:1:6, 3:1:7, 3:1:8, 3:1:9, 3:1:10, 3:1:11, 3:1:12, 3:1:13, 3:1:14, 3:1:15, 3:1:16, etc. In some embodiments, the feed ratio (weight ratio) of β-alanine, calcium oxide and water is between 3:1:6 and 3:2:6, for example 3:1.1:6, 3:1.2:6, 3:1.3:6, 3:1.4:6, 3:1.5:6, 3:1.6:6, 3:1.7:6, 3:1.8:6, 3:1.9:6, 3:2:6, etc. In some embodiments, step (b) of the method for preparing β-alanine salt according to the present invention is carried out under suitable conditions. For example, in some embodiments, the reaction temperature of step (b) is controlled between 40 and 100°C, for example, any value between any two of the following ranges: 40°C, 50°C, 60°C, 70°C, 80°C, 85°C, 90°C, 95°C, 100°C, or preferably between 80 and 100°C, for example, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, 100°C, etc.

[0069] In some embodiments, to improve the yield of β-alanine salt, a temperature control method of heating-cooling-heating is used in step (b) to ensure the reaction proceeds to completion. For example, the reaction solution is first heated to 80-100°C (e.g., 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, 100°C), and then cooled to 50-70°C (e.g., 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C). Keep the solution at 67°C, 68°C, 69°C, or 70°C for a certain period of time (e.g., 30 minutes to 1 hour), filter while hot, and then heat the filtrate to 80-100°C (e.g., 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, or 100°C) and keep it at that temperature for a certain period of time (e.g., 30 minutes to 1 hour), and then filter again until solid precipitates.

[0070] In some embodiments, the method for preparing β-alanine salt according to the present invention further includes concentrating the β-alanine salt product. In some embodiments, the concentration is achieved by reducing pressure, for example, by pumping the reaction solution after filtration, microfiltration, or ultrafiltration into a concentration device for concentration under reduced pressure to any value between any two numerical ranges of 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, or more of the original volume.

[0071] In some embodiments, the method for preparing β-alanine salt according to the present invention further includes crystallizing the β-alanine salt product. In some embodiments, the crystallization is achieved by lowering the temperature, adding an organic solvent (e.g., methanol, ethanol, isopropanol, etc.) or water, for example, by adding 1 to 10 times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, etc.) of the mass of organic solvent or water to a concentrated reaction solution, and crystallizing at a low temperature of 0-10°C (e.g., 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, 4°C, 3°C, 2°C, 1°C, 0°C) or lower.

[0072] In some embodiments, the preparation method of β-alanine salt according to the present invention includes: placing β-alanine prepared according to the method of the present invention and a solvent (e.g., water) in a container, then adding an alkaline solution or calcium oxide, heating to 80-100°C and refluxing for 0.5-5 hours, then cooling to 50-70°C and holding for 0.5-5 hours, filtering, then heating the filtrate to 80-100°C and holding for 0.5-5 hours, filtering, and crystallizing.

[0073] The method for preparing β-alanine salt described in this invention has the following advantages compared with the prior art: (1) It can save costs to a large extent and is suitable for industrial production; (2) In the conventional preparation process of β-alanine calcium, the usual practice is to dissolve β-alanine prepared using an inorganic acid regulator and calcium oxide in methanol for reaction, rather than using water as a solvent, because water would dissolve the salt impurities introduced in the conventional preparation process of β-alanine. Therefore, the preparation method of β-alanine salt described in this invention not only avoids the introduction of additional impurities that would affect the synthesis of pantothenate, but also avoids the use of organic solvents (e.g., methanol), thereby reducing production costs and being environmentally friendly.

[0074] On the other hand, the present invention provides a method for preparing pantothenate, comprising the following steps: (a) β-alanine is prepared according to the method of the present invention; (b) The β-alanine obtained in step (a) is reacted with an alkaline solution to prepare β-alanine salt; and (c) React pantothenic acid or pantothenic acid with the β-alanine salt prepared in step (b).

[0075] Specifically, in some embodiments, the method for preparing pantothenate according to the present invention includes the following steps: (a) A β-alanine product is prepared by reactants containing fumaric acid and ammonia in the presence of a catalyst, wherein the catalyst comprises a catalytic composition containing aspartate enzyme and L-aspartate-α-decarboxylase, and fumaric acid is added during the reaction, wherein the total molar amount of fumaric acid added is equal to the initial molar amount of ammonia in the reactants minus the initial molar amount of fumaric acid in the reactants. (b) The β-alanine obtained in step (a) is reacted with an alkaline solution to prepare β-alanine salt; and (c) React pantothenic acid or pantothenic acid with the β-alanine salt prepared in step (b).

[0076] Pantothenic acid lactone belongs to the class of γ-butyric acid lactones and is an intermediate of pantothenic acid. In some embodiments, the pantothenic acid lactone used in this invention is DL-pantothenic acid lactone (i.e., a pantothenic acid lactone without optical activity). In some embodiments, the pantothenic acid lactone used in this invention is D-pantothenic acid lactone (the specific reaction process is shown below). The pantothenic acid is a ring-opening form of pantothenic lactone. In some embodiments, the pantothenic acid used in this invention is DL-pantothenic acid (i.e., pantothenic acid without optical activity). In some embodiments, the pantothenic acid used in this invention is D-pantothenic acid.

[0077] In some embodiments, before reacting pantothenic acid or pantothenic acid with the β-alanine salt prepared according to the method of the present invention, the pantothenic acid or pantothenic acid is first dissolved in a solvent (e.g., water, methanol, ethanol, isopropanol, n-propanol, butanol, pentanol, ether, benzene, chloroform, etc.). In some embodiments, before reacting pantothenic acid or pantothenic acid with the β-alanine salt prepared according to the method of the present invention, the pantothenic acid or pantothenic acid is first dissolved in methanol or ethanol. In some embodiments, before reacting pantothenic acid or pantothenic acid with the β-alanine salt prepared according to the method of the present invention, the pantothenic acid or pantothenic acid is first dissolved in methanol.

[0078] In some embodiments, the molar ratio of pantothenic acid lactone (e.g., D-pantothenic acid lactone) to β-alanine salt is between 2:1 and 2:1.5, such as 2:1.05, 2:1.1, 2:1.15, 2:1.2, 2:1.25, 2:1.3, 2:1.35, 2:1.4, 2:1.45, 2:1.5, etc. In some embodiments, the feed ratio (mass ratio) of the solvent (e.g., methanol) to panthenolactone (e.g., D-panthenolactone) is between 2:1 and 5:1, for example 2:1, 2.5:1, 3:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, 5:1, etc.

[0079] In some embodiments, the method for preparing pantothenate according to the present invention further includes filtering and / or drying the β-alanine salt obtained in step (b) before step (c). One advantage of this approach is that it removes the water generated during the preparation of β-alanine salt, thereby preventing water from affecting the formation of pantothenate (e.g., calcium pantothenate, sodium pantothenate, potassium pantothenate) in step (c), and greatly improving the quality of the pantothenate (e.g., calcium pantothenate, sodium pantothenate, potassium pantothenate).

[0080] In some embodiments, step (c) of the method for preparing pantothenate according to the present invention is carried out under suitable reaction conditions. In some embodiments, the reaction temperature of step (c) is controlled between 40 and 100°C, for example, any value between any two of the following ranges: 40°C, 50°C, 60°C, 70°C, 80°C, 85°C, 90°C, 95°C, 100°C, preferably between 40 and 60°C.

[0081] In some embodiments, the method for preparing pantothenate according to the present invention further includes concentrating the pantothenate product obtained in step (c). In some embodiments, the concentration is achieved by reducing pressure, for example, by pumping the reaction solution after filtration, microfiltration, or ultrafiltration into a concentration device for concentration under reduced pressure to any value between any two of the following values: 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, or more of the original volume.

[0082] In some embodiments, the method for preparing pantothenate according to the present invention further includes crystallizing the pantothenate product obtained in step (c). In some embodiments, the crystallization is achieved by lowering the temperature, adding an organic solvent (e.g., methanol, ethanol, isopropanol, etc.) or water, for example, by adding a small amount of water of crystallization dropwise to a concentrated reaction solution, and crystallizing at low temperatures (e.g., 0°C, -5°C, -6°C, -7°C, -8°C, -9°C, -10°C, -11°C, -12°C, -13°C, -14°C, -15°C or lower). While not bound by any theoretical constraints, it is generally believed that lower temperatures are more conducive to the crystallization of the pantothenate product, and simultaneously result in a higher yield of pantothenate.

[0083] In some embodiments, the method for preparing pantothenate according to the present invention includes: placing pantothenic acid lactone (e.g., D-pantothenic acid lactone) and a solvent (e.g., methanol) in a container, then adding β-alanine salt (e.g., calcium β-alanine, sodium β-alanine, potassium β-alanine) prepared by the method of the present invention, heating to 40-60°C and reacting for 1-20 hours, filtering, and crystallizing.

[0084] On the other hand, the present invention provides a calcium pantothenate wherein the chloride ion content (by weight) is not higher than 190 ppm, and / or the sodium ion content (by weight) is not higher than 2200 ppm. In some embodiments, the chloride ion content (by weight) in the calcium pantothenate provided by the present invention is not higher than 180 ppm, not higher than 170 ppm, not higher than 160 ppm, not higher than 150 ppm, not higher than 140 ppm, not higher than 130 ppm, not higher than 120 ppm, not higher than 110 ppm, not higher than 100 ppm, not higher than 90 ppm, not higher than 80 ppm, not higher than 70 ppm, not higher than 60 ppm, not higher than 50 ppm, not higher than 40 ppm, not higher than 30 ppm, not higher than 20 ppm, and not higher than 10 ppm. For example, in some embodiments, the chloride ion content (by weight) in the calcium pantothenate provided by the present invention is 1 ppm ~ 190 ppm, 1 ppm ~ 180 ppm, 1 ppm ~ 170 ppm, 1 ppm ~ 160 ppm, 1 ppm ~ 150 ppm, 1 ppm ~ 140 ppm, 1 ppm ~ 130 ppm, 1 ppm ~ 120 ppm, 1 ppm ~ 110 ppm, 1 ppm ~ 100 ppm, 1 ppm ~ 90 ppm, 1 ppm ~ 80 ppm, 1 ppm ~ 70 ppm, 1 ppm ~ 60 ppm, 1 ppm ~ 50 ppm, 1 ppm ~ 40 ppm, 1 ppm ~ 30 ppm, 1 ppm ~ 20 ppm, 1 ppm ~ 10 ppm, or any value between any two of the above ranges.

[0085] In some embodiments, the sodium ion content (by weight) in the calcium pantothenate provided by the present invention is not higher than 2150 ppm, not higher than 2100 ppm, not higher than 2050 ppm, not higher than 2000 ppm, not higher than 1950 ppm, not higher than 1900 ppm, not higher than 1850 ppm, and not higher than 1800 ppm. For example, in some embodiments, the sodium ion content (by weight) in the calcium pantothenate provided by the present invention is 1800 ppm ~ 2200 ppm, 1800 ppm ~ 2150 ppm, 1800 ppm ~ 2100 ppm, 1800 ppm ~ 2050 ppm, 1800 ppm ~ 2000 ppm, 1800 ppm ~ 1950 ppm, 1800 ppm ~ 1900 ppm, 1800 ppm ~ 1850 ppm, or any value between any two of the above ranges.

[0086] In some embodiments, the calcium pantothenate wherein the chloride ion content (by weight) is not higher than 190 ppm, and / or the sodium ion content (by weight) is not higher than 2200 ppm is prepared by the method according to the present invention. The method for preparing calcium pantothenate in some embodiments includes the following steps: (a) β-alanine is prepared according to the method of the present invention; (b) The β-alanine obtained in step (a) is reacted with an alkaline solution containing calcium salt to prepare calcium β-alanine; and (c) React pantothenic acid or pantothenic acid with the calcium β-alanine prepared in step (b).

[0087] Specifically, in some embodiments, the method for preparing calcium pantothenate according to the present invention includes the following steps: (a) A β-alanine product is prepared by reactants containing fumaric acid and ammonia in the presence of a catalyst, wherein the catalyst comprises a catalytic composition containing aspartate enzyme and L-aspartate-α-decarboxylase, and fumaric acid is added during the reaction, wherein the total molar amount of fumaric acid added is equal to the initial molar amount of ammonia in the reactants minus the initial molar amount of fumaric acid in the reactants. (b) The β-alanine obtained in step (a) is reacted with an alkaline solution containing a calcium salt (e.g., calcium hydroxide) to prepare calcium β-alanine; and (c) React pantothenic acid or pantothenic acid with the calcium β-alanine prepared in step (b).

[0088] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0089] Example Example 1: Fermentation culture of engineered Escherichia coli Fermentation culture of engineered *Escherichia coli*, inorganic salt fermentation medium formula: NaCl 1 g / L; MgSO4-7H2O 1 g / L; FeSO4-7H2O 0.02 g / L; ZnSO4-7H2O 0.03 g / L; CuSO4-5H2O 0.005 g / L; (NH4)2SO4 4 g / L; KH2PO4 4 g / L, 121 o Sterilize at 15 minutes for 15 minutes, then cool to 37°C. o C. Vaccination, dose 2%, at 37 oFermentation was carried out under C conditions, with dissolved oxygen maintained at 30%-50%, aeration rate of 1 VVM, and ammonia added to adjust pH and provide nitrogen source, maintaining pH at 7.5. Glucose was added to provide carbon source, with residual sugar maintained at 0.1-0.5 g / L, and cultured until OD. 600 = 30, temperature drops to 30 o C was added to induce fermentation with IPTG, and fermentation was stopped after 30 hours of induction culture. OD 600 The concentration reached approximately 110, and the wet weight of the bacterial cells after centrifugation reached approximately 120 g / L.

[0090] Example 2: Synthesis of β-alanine Example 2.1 Refer to *Molecular Cloning: A Laboratory Manual (3rd Edition)* (Science Press) and Li Y. et al. Appl. Microbiol. Biotechnol. 2017, 101, 6015-6021 Preparations were made containing [materials derived from...] Anoxybacillus flavithermus WK1 aspartate aminotransferase (hereinafter referred to as "enzyme 1") is derived from engineered E. coli and contains... Bacillus thermotolerans The engineered strain of *E. coli* containing L-aspartate-α-decarboxylase (hereinafter referred to as "enzyme 2") was fermented in conventional LB medium or by centrifugation (5000 rpm, 5 min) using the method in Example 1 to obtain wet engineered *E. coli* strains for later use.

[0091] To prepare 3 L of ammonium fumarate solution (100 g fumaric acid / L), the initial molar amount of fumaric acid is 2.586 mol, the initial molar amount of ammonia is 5.172 mol, and 3 g of ammonia solution containing [amount missing] is added. Anoxybacillus flavithermus The wet cells of engineered E. coli containing enzyme 1 of WK1 (at a weight ratio of 1% to the initial fumaric acid), and 60g containing [the enzyme sourced from...]. Bacillus thermotolerans The reaction was initiated with wet cells of engineered Escherichia coli containing enzyme 2 (at a weight ratio of 20% to the initial fumaric acid). 2.586 mol of fumaric acid (100 g fumaric acid / L) was added (totaling 5.172 mol of fumaric acid to the initial amount in the reactants). The pH was controlled at 7.0, and the reaction temperature at 37°C. After the fumaric acid addition was complete, the reaction was monitored by HPLC. The reaction was terminated after 18 h when the fumaric acid content was <0.5% (w / v) and the molar conversion rate of aspartic acid was >99%.

[0092] The reaction solution was passed through a 0.4µm microfiltration membrane to remove large particulate impurities, such as cells, bacterial debris, aggregates, and flocs. Small molecule impurities, such as nucleic acids and nucleic acid fragments, proteins, and culture medium components, were removed through a 10KD ultrafiltration membrane. The solution was concentrated under reduced pressure to 1 / 5 of its original volume. Three times the volume of methanol was added dropwise, and crystallization was carried out at 10°C. The crystals were then filtered and dried to obtain 390.5 g of a white solid with a β-alanine content of 98.7%, yielding a yield of 83.7%. The methanol in the mother liquor was recovered and recycled.

[0093] Example 2.2 The same literature preparation method as Example 2.1 yielded a product containing [source name missing]. Geobacillus thermodenitrificans NG80-2 contains enzyme 1 of engineered E. coli and bacteria derived from Anoxybacillus flavithermus The engineered Escherichia coli strain with enzyme 2 of AK1 was fermented in conventional LB medium or by the method in Example 1, followed by centrifugation (5000 rpm, 5 min) to obtain wet engineered Escherichia coli. 1g of cells was added to 10ml of pH 7.0 phosphate buffer, stirred evenly, and then the cells were homogenized by high pressure to obtain the engineered Escherichia coli cell lysate for later use.

[0094] To prepare 3 L of ammonium fumarate solution (150 g fumaric acid / L), the initial molar amount of fumaric acid is 3.879 mol, the initial molar amount of ammonia is 7.758 mol, and 9 g of solution containing [ammonia source] is added. Geobacillus thermodenitrificans NG80-2 enzyme 1 E. coli engineered bacterial cell lysate (its weight ratio to initial fumaric acid is 2%) and 135g containing enzyme 1 derived from Anoxybacillus flavithermus The reaction was initiated with a lysate of engineered E. coli cells containing enzyme 2 of AK1 (at a weight ratio of 30% to the initial fumaric acid). 3.879 mol of fumaric acid (150 g fumaric acid / L) was added (totaling 7.758 mol of fumaric acid to the initial amount in the reactants). The pH was controlled at 7.0, and the reaction temperature at 37°C. After the fumaric acid addition was complete, the reaction was monitored by HPLC. The reaction was terminated after 24 h when the fumaric acid content was <0.5% (w / v) and the molar conversion rate of aspartic acid was >99%.

[0095] The reaction solution was passed through a 0.4µm microfiltration membrane to remove large particulate impurities, such as cells, bacterial debris, aggregates, and flocs. Small molecule impurities, such as nucleic acids and nucleic acid fragments, proteins, and culture medium components, were removed through a 6KD ultrafiltration membrane. The solution was concentrated under reduced pressure to 1 / 4 of its original volume. Three times the volume of methanol was added dropwise, and crystallization was carried out at 5°C. The crystals were then filtered and dried to obtain 622.5 g of a white solid with a β-alanine content of 99.1%, yielding a yield of 89.3%. The methanol in the mother liquor was recovered and recycled.

[0096] Example 2.3 The same preparation method as in Example 2.1 yielded a product containing [material] derived from [source]. Anoxybacillus flavithermus WK1 enzyme 1 E. coli engineered bacteria and containing derived from Methanocaldococcus jannaschii The engineered strain of *E. coli* containing enzyme 2 was fermented in conventional LB medium or by the method described in Example 1, followed by centrifugation (5000 rpm, 5 min) to obtain wet engineered *E. coli* strains for later use.

[0097] Prepare 3 L of ammonium fumarate solution (75 g fumaric acid / L), with an initial fumaric acid concentration of 1.939 mol and an initial ammonia concentration of 3.878 mol. Add 20 mM pyridoxal phosphate (PLP) and 2.25 g of ammonia solution containing... Anoxybacillus flavithermus The wet cells of engineered E. coli containing enzyme 1 of WK1 (at a weight ratio of 1% to initial fumaric acid) and 33.75g containing [the enzyme sourced from...]. Methanocaldococcus jannaschii The reaction was initiated with wet cells of engineered *E. coli* bacteria containing enzyme 2 (at a weight ratio of 15% to the initial fumaric acid). 1.939 mol of fumaric acid (75 g fumaric acid / L) was added (totaling 3.878 mol of fumaric acid to the initial amount in the reactants). The pH was controlled at 7.0, and the reaction temperature was 37°C. O C. After the fumaric acid addition is completed, the reaction is monitored by HPLC. The reaction is terminated when the fumaric acid content is <0.5% (w / v) and the molar conversion of aspartic acid is >99% after 24 h of reaction.

[0098] The reaction solution was passed through a 0.4µm microfiltration membrane to remove large particulate impurities, such as cells, bacterial debris, aggregates, and flocs. Small molecule impurities, such as nucleic acids and nucleic acid fragments, proteins, and culture medium components, were removed through a 10 KD ultrafiltration membrane. The solution was concentrated under reduced pressure to 1 / 7 of its original volume and slowly cooled to 4°C for crystallization. The crystals were then filtered and dried to obtain 227.6 g of a white solid with a β-alanine content of 99.4%, yielding a yield of 65.5%. The mother liquor was recycled after methanol was recovered.

[0099] Example 2.4 The co-expression was prepared according to the same literature as in Example 2.1. Anoxybacillus flavithermus WK1 enzyme 1 and derived from Bacillus thermotolerans The engineered strain of *E. coli* containing enzyme 2 was fermented in conventional LB medium or by the method described in Example 1, followed by centrifugation (5000 rpm, 5 min) to obtain wet engineered *E. coli* strains for later use.

[0100] Prepare 3 L of ammonium fumarate solution (100 g fumaric acid / L). The initial molar amount of fumaric acid is 2.586 mol, and the initial molar amount of ammonia is 5.172 mol. Add 90 g of co-expressing... Anoxybacillus flavithermus WK1 enzyme 1 and derived from Bacillus thermotolerans The reaction was initiated with wet cells of engineered Escherichia coli containing enzyme 2 (at a weight ratio of 30% to the initial fumaric acid). 2.586 mol of fumaric acid (100 g fumaric acid / L) was added (totaling 5.172 mol of fumaric acid to the initial amount in the reactants). The pH was controlled at 7.0, and the reaction temperature at 37°C. After the fumaric acid addition was complete, the reaction was monitored by HPLC. The reaction was terminated after 27 h when the fumaric acid content was <0.5% (w / v) and the molar conversion rate of aspartic acid was >99%.

[0101] The reaction solution was passed through a 0.4µm microfiltration membrane to remove large particulate impurities, such as cells, bacterial debris, aggregates, and flocs. Small molecule impurities, such as nucleic acids and nucleic acid fragments, proteins, and culture medium components, were removed through a 10KD ultrafiltration membrane. The solution was concentrated under reduced pressure to 1 / 6 of its original volume and slowly cooled to 5°C for crystallization. The crystals were then filtered and dried to obtain 338.5 g of a white solid with a β-alanine content of 99.3%, yielding a 73% yield. The mother liquor was recycled after methanol was recovered.

[0102] Example 2.5 Referring to the same literature as Example 2.1, co-expressed proteins derived from [the original text] were prepared using conventional LB medium or by fermentation using the method of Example 1. Anoxybacillus flavithermus WK1 enzyme 1 and derived from Bacillus thermotolerans After obtaining enzyme 2 from E. coli cells, prepare a mixed solution of 8% polyvinyl alcohol and 2.5% sodium alginate, add 10% E. coli cells, mix well, and then use a peristaltic pump to drip the solution from a height of 10 cm into a crosslinking agent containing 2% calcium chloride and 3% boric acid. After solidification for 8 hours, filter, and rinse repeatedly with distilled water 3-4 times to immobilize the cells for later use.

[0103] Prepare 3 L of ammonium fumarate solution (75 g fumaric acid / L). The initial molar amount of fumaric acid is 1.939 mol, and the initial molar amount of ammonia is 3.878 mol. Add 67.5 g of co-expressing agent derived from... Anoxybacillus flavithermus WK1 enzyme 1 and derived from Bacillus thermotolerans The reaction was initiated using immobilized E. coli cells containing enzyme 2 (at a weight ratio of 30% to the initial fumaric acid). 1.939 mol of fumaric acid (75 g fumaric acid / L) was added (totaling 3.878 mol of fumaric acid to the initial amount in the reactants). The pH was controlled at 7.0, and the reaction temperature was 37°C. At °C, the reaction was monitored by HPLC after the fumaric acid addition was completed. The reaction was terminated when the fumaric acid content was <0.5% (w / v) and the molar conversion of aspartic acid was >99% after 20 h of reaction.

[0104] Large particulate impurities, such as cells, bacterial debris, aggregates, and flocs, were removed from the reaction solution by passing it through a filter cloth. Small molecule impurities, such as nucleic acids and nucleic acid fragments, proteins, and culture medium components, were removed by passing it through a 10 KD ultrafiltration membrane. The solution was concentrated under reduced pressure to 1 / 7 of the original reaction solution volume. Three times the volume of methanol was added dropwise, and crystallization was carried out at 10°C. The crystals were then filtered and dried to obtain 296.7 g of a white solid with a β-alanine content of 98.9% and a yield of 85%. The methanol in the mother liquor was recovered and recycled.

[0105] Example 3: Comparison of salt-free and salt-containing processes The applicant prepared β-alanine using the preparation method of the present invention (hereinafter referred to as the "salt-free process") and a preparation method using appropriate inorganic acids (hereinafter referred to as the "salt process"), and compared the content of β-alanine, ammonia nitrogen, and ammonia nitrogen content per gram of β-alanine obtained by the two processes.

[0106] Salt-free process: Referring to the same literature as Example 2.1, salt-free materials were prepared by fermentation using conventional LB medium or the method of Example 1, containing... Anoxybacillus flavithermus WK1 enzyme 1 in whole cells of E. coli and containing [derived from] Bacillus thermotolerans Whole cells of E. coli containing enzyme 2, for later use.

[0107] To prepare 3 L of ammonium fumarate solution (100 g fumaric acid / L), the initial molar amount of fumaric acid is 2.586 mol, the initial molar amount of ammonia is 5.172 mol, and 3 g of solution containing [ammonia source] is added. Anoxybacillus flavithermus WK1 enzyme 1 E. coli whole cells (with a weight ratio of 1% to initial fumaric acid) and 60g containing from Bacillus thermotolerans The reaction was initiated using whole cells of *E. coli* containing enzyme 2 (at a weight ratio of 20% to the initial fumaric acid). 2.586 mol of fumaric acid (100 g fumaric acid / L) was added (totaling 5.172 mol of fumaric acid to the initial amount in the reactants). The pH was controlled at 7.0, and the reaction temperature at 37°C. After the fumaric acid addition was complete, the reaction was monitored by HPLC. The reaction was terminated after 18 h when the fumaric acid content was <0.5% (w / v) and the molar conversion of aspartic acid was >99%. The ammonium ion concentration in the reaction solution was measured to be 0.82 g / L using an ammonia nitrogen detector (Shanghai Yidian Scientific Instruments Co., Ltd.).

[0108] Control experiment Salt-containing process: Referring to the same literature as Example 2.1, the salt-containing process was prepared by fermentation using conventional LB medium or the method of Example 1. Anoxybacillus flavithermus WK1 enzyme 1 in whole cells of E. coli and containing [derived from] Bacillus thermotolerans Whole cells of E. coli containing enzyme 2, for later use.

[0109] To prepare 3 L of ammonium fumarate solution (200 g fumaric acid / L), the initial molar amount of fumaric acid is 5.172 mol, the initial molar amount of ammonia is 10.344 mol, and 6 g of solution containing [ammonia source] is added. Anoxybacillus flavithermus WK1 enzyme 1 E. coli whole cells (with a weight ratio of 1% to initial fumaric acid) and 120g containing from Bacillus thermotolerans The reaction was initiated using whole cells of *E. coli* containing enzyme 2 (at a weight ratio of 20% to initial fumaric acid). H₂SO₄ was added to maintain the pH at 7.0, and the reaction temperature was 37°C. The reaction was monitored by HPLC. The reaction was terminated after 18 hours when the fumaric acid content was <0.5% (w / v) and the molar conversion of aspartic acid was >99%. The ammonium ion concentration in the reaction solution was measured to be 23.5 g / L using an ammonia nitrogen detector (Shanghai Yidian Scientific Instruments Co., Ltd.).

[0110] Both the salt-free and salt-containing processes described above employ the same post-treatment process: large particulate impurities, such as cells, bacterial debris, aggregates, and flocs, are removed using a 0.4µm microfiltration membrane; small molecule impurities, such as nucleic acids and nucleic acid fragments, proteins, and culture medium components, are removed using a 10KD ultrafiltration membrane; the mixture is then concentrated under reduced pressure to 1 / 5 of the original reaction volume, crystallized at 10°C, and finally filtered and dried. The results are as follows: (1) In the salt-free process, 207g of β-alanine was obtained, with a content of 98.6% and an ammonia nitrogen content of 13mg / g per gram of β-alanine. The ammonia nitrogen content in the recovered liquid was 2.3g / L.

[0111] (2) In the salt process, 290g of β-alanine was obtained with a content of 55.6%, and the ammonia nitrogen content in each gram of β-alanine was 910mg / g. The ammonia nitrogen content in the recovered liquid was 51.7g / L.

[0112] Example 4: Recycling of Salt-Free Process The applicant uses the preparation method of this invention for repeated use in salt-free processes.

[0113] Three batches of reaction solution were prepared according to the salt-free process in Example 2.5, and were numbered A, B, and C respectively.

[0114] (1) Reaction solution A was concentrated under reduced pressure to 1 / 5 of the original reaction solution volume, crystallized at 10°C, filtered and dried to obtain 212g of β-alanine with a content of 98.7% and an ammonia nitrogen content of 11mg / g per gram of β-alanine. 510ml of recovery solution was obtained, with an ammonia nitrogen content of 2.5g / L.

[0115] (2) The recovered solution of reaction solution A in (1) above was added to reaction solution B, and the mixture was concentrated under reduced pressure to 1 / 5 of the original reaction solution volume. Crystallization was carried out at 10°C, filtered and dried to obtain 393g of β-alanine with a content of 98.3% and an ammonia nitrogen content of 10mg / g per gram of β-alanine. 620ml of the recovered solution was obtained, and the ammonia nitrogen content in the recovered solution was 5.7g / L.

[0116] (3) Add the recovered liquid from (2) above to reaction liquid C, and concentrate it under reduced pressure to 1 / 5 of the original reaction liquid volume. Crystallize at 10°C, filter and dry to obtain 410g of β-alanine with a content of 98.1% and an ammonia nitrogen content of 11mg / g per gram of β-alanine. 650ml of the recovered liquid has an ammonia nitrogen content of 5.8g / L.

[0117] (4) The recovered liquid from (3) above was directly concentrated into a solid under reduced pressure to obtain 275g of β-alanine with a content of 90.1% and an ammonia nitrogen content of 11mg / g per gram of β-alanine.

[0118] As can be seen from the results of Examples 3 and 4, the β-alanine obtained by the salt-free process of the present invention is significantly superior to the β-alanine obtained by the salt process in terms of content, ammonia nitrogen, and ammonia nitrogen content per gram of β-alanine. Moreover, the mother liquor obtained after crystallization in the salt-free process is still almost free of inorganic salts and other impurities after multiple cycles, thereby reducing production costs.

[0119] Example 5: Synthesis of β-alanine calcium Example 5.1 17.84 g (0.2 mol) of β-alanine prepared in Example 2 and 100 g of water were added sequentially to a three-necked flask. After stirring and dissolving at room temperature, 6.21 g (0.108 mol) of 98% calcium oxide was slowly added. After the addition was complete, the mixture was heated under reflux for 1 hour, stirring was stopped, and the mixture was allowed to cool naturally to 60°C and held at that temperature for 1 hour. The mixture was filtered while hot, activated carbon was added to the filtrate, and the mixture was heated to 90°C and held at that temperature for 30 minutes. The mixture was then filtered again, and the filtrate was concentrated until a solid precipitated. Isopropanol, in an amount of 4-6 times the mass of β-alanine, was added, and the mixture was stirred and beaten to crystallize. The mixture was cooled to below 10°C, filtered, and dried to obtain 20.5 g of calcium β-alanine, with a yield of 93%.

[0120] Example 5.2 89.3 g (1 mol) of β-alanine prepared in Example 2 and 500 g of water were added sequentially to a three-necked flask. After stirring and dissolving at room temperature, 30.98 g (0.54 mol) of 98% calcium oxide was slowly added. After the addition was complete, the mixture was heated under reflux for 1 hour, stirring was stopped, and the mixture was allowed to cool naturally to 60°C and held at that temperature for 1 hour. The mixture was filtered while hot, activated carbon was added to the filtrate, and the mixture was heated to 90°C and held at that temperature for 30 minutes. The mixture was then filtered again, and the filtrate was concentrated until a solid precipitated. Isopropanol, in an amount of 4-6 times the mass of β-alanine, was added, and the mixture was stirred and slurried to crystallize. The mixture was cooled to below 10°C, filtered, and dried to obtain 102.1 g of calcium β-alanine, with a yield of 94.21%.

[0121] Example 5.3 44.37 g (0.5 mol) of β-alanine prepared in Example 2 and 190 g of water were added sequentially to a three-necked flask. After stirring and dissolving at room temperature, 15.45 g (0.27 mol) of 98% calcium oxide was slowly added. After the addition was complete, the mixture was heated under reflux for 1 hour. Stirring was stopped, and the mixture was allowed to cool naturally to 60°C and held at that temperature for 1 hour. The mixture was filtered while hot, and activated carbon was added to the filtrate. The mixture was then heated to 90°C and held at that temperature for 30 minutes. After filtration, the filtrate was concentrated until a solid precipitated. Ethanol of 4-6 times the mass of β-alanine was added, and the mixture was stirred and slurried to crystallize. The mixture was cooled to below 10°C, filtered, and dried to obtain 43.6 g of calcium β-alanine, with a yield of 80.96%.

[0122] Example 5.4 195.8 g (2.2 mol) of β-alanine prepared in Example 2 and 1000 g of water were added sequentially to a three-necked flask. After stirring and dissolving at room temperature, 67.9 g (1.19 mol) of 98% calcium oxide was slowly added. After the addition was complete, the mixture was heated under reflux for 1 hour. Stirring was stopped, and the mixture was allowed to cool naturally to 60°C and kept at that temperature for 1 hour. The mixture was filtered while hot, and activated carbon was added to the filtrate. The mixture was then heated to 90°C and kept at that temperature for 30 minutes. After filtration, the filtrate was concentrated until a solid precipitated. The mixture was cooled to below 10°C, filtered, and dried to obtain 116 g of calcium β-alanine, with a yield of 75.50%.

[0123] Example 5.5 89.1 g (1 mol) of β-alanine prepared in Example 2 and 160 g of water were added to a three-necked flask. After stirring and dissolving at room temperature, 28.7 g (0.505 mol) of 98% calcium oxide was slowly added. The addition was exothermic, so the temperature was controlled below 60°C. After the addition was complete, the mixture was heated under reflux for 0.5 hours. Stirring was stopped, and the mixture was allowed to cool naturally to 60°C and kept at that temperature for 1 hour. The mixture was filtered while hot, and activated carbon was added to the filtrate. The mixture was then heated to 90°C and kept at that temperature for 30 minutes. After filtration, the mother liquor was concentrated to dryness and dried in an oven to obtain 104.8 g of calcium β-alanine, with a yield of 97.01%.

[0124] Example 5.6 89.1 g (1 mol) of β-alanine prepared in Example 2 and 160 g of water were added to a three-necked flask. After stirring and dissolving at room temperature, 28.7 g (0.505 mol) of 98% calcium oxide was slowly added. The addition was exothermic, so the temperature was controlled below 60°C. After the addition was complete, the mixture was heated under reflux for 1 hour. Stirring was stopped, and the mixture was allowed to cool naturally to 60°C and kept at that temperature for 1 hour. The mixture was filtered while hot, and activated carbon was added to the filtrate. The mixture was then heated to 90°C and kept at that temperature for 30 minutes. After filtration, the mother liquor was concentrated to dryness and dried in an oven to obtain 105.5 g of calcium β-alanine, with a yield of 97.71%.

[0125] Example 5.7 89.1 g (1 mol) of β-alanine prepared in Example 2 and 160 g of water were added to a three-necked flask. After stirring and dissolving at room temperature, 28.7 g (0.505 mol) of 98% calcium oxide was slowly added. The addition was exothermic, so the temperature was controlled below 60°C. After the addition was complete, the mixture was heated under reflux for 3 hours. Stirring was stopped, and the mixture was allowed to cool naturally to 60°C and kept at that temperature for 1 hour. The mixture was filtered while hot, and activated carbon was added to the filtrate. The mixture was then heated to 90°C and kept at that temperature for 30 minutes. After filtration, the mother liquor was concentrated to dryness and dried in an oven to obtain 100.7 g of calcium β-alanine, with a yield of 93.25%.

[0126] Example 5.8 89.1 g (1 mol) of β-alanine prepared in Example 2 and 160 g of water were added to a three-necked flask. After stirring and dissolving at room temperature, 28.2 g (0.5 mol) of 98% calcium oxide was slowly added. The addition was exothermic, so the temperature was controlled below 60°C. After the addition was complete, the mixture was heated to reflux for 1 hour. Stirring was stopped, and the mixture was allowed to cool naturally to 60°C and kept at that temperature for 1 hour. The mixture was filtered while hot, and activated carbon was added to the filtrate. The mixture was then heated to 90°C and kept at that temperature for 30 minutes. After filtration, the mother liquor was concentrated to dryness and dried in an oven to obtain 97.4 g of calcium β-alanine, with a yield of 90.22%.

[0127] The reaction conditions and results of Examples 5.1 to 5.8 are summarized in Table 1 below.

[0128] Table 1: Summary of Synthesis Conditions and Results of β-Alanine Calcium Example 6: Synthesis of Calcium Pantothenate Example 6.1 60.1 g (0.46 mol) of D-pantothenic acid lactone and 225 g of methanol were added to a three-necked flask and stirred at room temperature until dissolved. 49.5 g (0.23 mol) of calcium β-alanine prepared in Example 5 was slowly added while stirring, taking care to prevent clumping during the addition process. After the addition was complete, the temperature was raised to 50°C and reacted for 1 hour. The mixture was filtered, and the filtrate was stirred at 25°C for 2 hours. The mixture was then cooled to 0°C, and a small amount of water of crystallization was added. After the addition was complete, the mixture was cooled to -5°C and crystallized at -5°C for 16 hours. The mixture was filtered, and the filter cake was dried to obtain 57.1 g of white solid with a purity of 99.7%, a yield of 53.1%, and a specific rotation of 26.1°.

[0129] Example 6.2 58.5 g (0.45 mol) of D-pantothenic acid lactone and 225 g of methanol were added to a three-necked flask and stirred at room temperature until dissolved. Then, 49.5 g (0.23 mol) of calcium β-alanine prepared in Example 5 was slowly added while stirring, taking care to prevent clumping during the addition process. After the addition was complete, the temperature was raised to 50°C and reacted for 1 hour. The mixture was filtered, and the filtrate was stirred at 25°C for 2 hours. After cooling to 0°C, a small amount of water of crystallization (without seed crystals) was added. After the addition was complete, the mixture was cooled to -10°C and crystallized for 16 hours. A small amount of solid precipitated, and after filtration, 18.1 g of white solid was obtained, with a purity of 98.0% and a yield of 16.4%. The specific rotation was 25.2°.

[0130] Example 6.3 585 g (4.5 mol) of D-pantothenic acid lactone and 2250 g of methanol were added to a three-necked flask and stirred at room temperature until dissolved. 495 g (2.3 mol) of calcium β-alanine prepared in Example 5 was slowly added while stirring to prevent clumping. After the addition was complete, the temperature was raised to 50°C and reacted for 1 hour. The mixture was filtered, and the filtrate was stirred at 25°C for 2 hours. After cooling to 0°C, a small amount of water of crystallization was added. After the addition was complete, the mixture was cooled to -5°C and crystallized at -5°C for 16 hours. The mixture was filtered, and the filter cake was dried to obtain 566.7 g of white solid with a purity of 99.6%, a yield of 52.8%, and a specific rotation of 26.4°.

[0131] Example 6.4 317 g (2.44 mol) of D-pantothenic acid lactone and 1442 g of methanol were added to a three-necked flask and stirred at room temperature until dissolved. 375 g (1.74 mol) of calcium β-alanine prepared in Example 5 was slowly added while stirring to prevent clumping. After the addition was complete, the temperature was raised to 50°C and reacted for 1 hour. The mixture was filtered, and the filtrate was stirred at 25°C for 2 hours. After cooling to 0°C, a small amount of water of crystallization was added. After the addition was complete, the mixture was cooled to -10°C and crystallized at -10°C for 16 hours. The mixture was filtered, and the filter cake was dried to obtain 529.9 g of white solid with a purity of 99.8%, a yield of 91.1%, and a specific rotation of 26.7°.

[0132] Example 6.5 Add 58.5 g (0.45 mol) of D-pantothenic acid lactone and 225 g of water to a three-necked flask, stir at room temperature until dissolved, and slowly add 49.5 g (0.23 mol) of calcium β-alanine prepared in Example 5 while stirring. Prevent agglomeration during the addition process. After the addition is complete, raise the temperature to 50°C and react for 1 hour. Monitor the reaction progress with liquid phase and calculate the reaction yield as 2%-4% by liquid phase normalization method.

[0133] Example 6.6 58.5 g (0.45 mol) of D-pantothenic acid lactone and 225 g of methanol were added to a three-necked flask and stirred at room temperature until dissolved. 49.5 g (0.23 mol) of calcium β-alanine prepared in Example 5 was slowly added while stirring to prevent clumping. After the addition was complete, the mixture was stirred at 25°C for 20 hours, cooled to 0°C, and a small amount of water of crystallization was added. After the addition was complete, the mixture was cooled to -10°C and crystallized at -10°C for 16 hours. The crystals were filtered, and the filter cake was dried to obtain 92.3 g of white solid with a purity of 98.1%, a yield of 83.6%, and a specific rotation of 26.3°.

[0134] Example 6.7 Add 58.5 g (0.45 mol) of D-pantothenic acid lactone and 225 g of isopropanol to a three-necked flask and stir at room temperature until dissolved. Slowly add 49.5 g (0.23 mol) of calcium β-alanine prepared in Example 5 while stirring, taking precautions to prevent agglomeration during the addition process. After the addition is complete, raise the temperature to 50°C and react for 1 hour. Monitor the reaction progress with the liquid phase and calculate the reaction yield as 5%-8% using the liquid phase normalization method.

[0135] Example 6.8 317 g (2.44 mol) of D-pantothenic acid lactone and 1568 g of the mother liquor from Example 6.4 were added to a three-necked flask and stirred at room temperature until dissolved. 375 g (1.74 mol) of calcium β-alanine prepared in Example 5 was slowly added while stirring to prevent clumping. After the addition was complete, the temperature was raised to 50°C and reacted for 1 hour. The mixture was filtered, and the filtrate was stirred at 25°C for 2 hours. After cooling to 0°C, a small amount of water of crystallization was added. After the addition was complete, the mixture was cooled to -10°C and crystallized at -10°C for 16 hours. The mixture was then filtered, and the mother liquor and eluent were reused in the next batch of reaction. The filter cake was dried to obtain 541 g of white solid with a purity of 99.6%, a yield of 93.1%, and a specific rotation of 26.3°.

[0136] The reaction conditions and results of Examples 6.1 to 6.8 are summarized in Table 2 below.

[0137] Table 2: Summary of synthesis conditions and results of calcium pantothenate Example 7: Experiment on the detection of chloride and sodium ion content in calcium pantothenate According to the European Pharmacopoeia 10.0, the chloride ion content in calcium pantothenate must not exceed 200 ppm. The applicant tested the chloride and sodium ion content in the calcium pantothenate prepared by the method of this invention (hereinafter referred to as "Test Sample 1") and compared it with the chloride and sodium ion content in two commercially available calcium pantothenate products. The two commercially available calcium pantothenate products were purchased from Xinfeng Pharmaceutical Co., Ltd. (hereinafter referred to as "Test Sample 2") and Zhejiang Hangzhou Xinfu Pharmaceutical Co., Ltd. (hereinafter referred to as "Test Sample 3"), respectively.

[0138] Example 7.1 Examination of Linear Relationship Potassium chloride and sodium sulfate standards were prepared into aqueous solutions with chloride ion concentrations of 4.79 mg / mL and sodium ion concentrations of 3.25 mg / mL. These solutions were then diluted to prepare aqueous solutions containing different concentrations of chloride and sodium ions: chloride ion concentrations of 0.0240 mg / mL, 0.0479 mg / mL, 0.0958 mg / mL, 0.1916 mg / mL, and 0.3832 mg / mL; and sodium ion concentrations of 0.0325 mg / mL, 0.0650 mg / mL, 0.13 mg / mL, 0.26 mg / mL, and 0.65 mg / mL. 10 μL of each solution was injected and analyzed using HPLC. The HPLC parameters are shown below. Column: Acclaim Trinity P2, 3.0*100mm*3μm Mobile phase: 100 mM ammonium formate buffer (pH 3.65), isocratic elution Column flow rate: 0.3 mL / min Column temperature: 30℃ Detector: ELSD HPLC chromatogram as shown Figure 6 As shown, chloride ions eluted at approximately 4.024 minutes. A standard curve was plotted using the logarithm of the chloride ion injection concentration (LgC) and the logarithm of the peak area (LgA). The regression equation was calculated as Y = 1.4528X + 7.5318, R0. 2 =0.9977. See the standard curve. Figure 7 A. The results showed that when the chloride ion injection concentration was in the range of 0.0240 mg / mL to 0.3832 mg / mL, there was a good linear relationship between the concentration and the peak area.

[0139] like Figure 6As shown, sodium ions eluted at approximately 4.351 minutes. A standard curve was plotted using the logarithm of the sodium ion injection concentration (LgC) and the logarithm of the peak area (LgA). The regression equation was calculated as Y = 1.2843X + 7.4906, R0. 2 =0.9974. See the standard curve. Figure 7 B. The results showed that when the sodium ion injection concentration was in the range of 0.0325 mg / mL to 0.6500 mg / mL, there was a good linear relationship between the sodium ion injection concentration and the peak area.

[0140] Example 7.2 Chloride ion recovery rate Weigh 396.3 mg of calcium pantothenate sample 1, dissolve and dilute with water to a final volume of 10 mL, and use this as the sample. Weigh 401.5 mg of calcium pantothenate sample 1, add 0.2 mL of chloride ion standard (4.79 mg / mL), dissolve and dilute with water to a final volume of 10 mL, and use this as the spiked sample. The chloride ion content was calculated using a standard curve, and the chloride ion recovery rate was found to be 105.4%, indicating good recovery.

[0141] Example 7.3 Limits of detection and limits of quantitation for chloride ions Weigh 392.6 mg of calcium pantothenate sample 1, add 0.2 mL of potassium chloride standard with a chloride ion concentration of 0.0479 mg / mL, and then add water to dissolve and dilute to a final volume of 10 mL. HPLC was used for determination.

[0142] Detection limit test results are as follows Figure 8 As shown, the signal-to-noise ratio of chloride ions is greater than 3. The results indicate that the detection limit of chloride ions in calcium pantothenate aqueous solution is 0.0025% (by weight).

[0143] Weigh 400.2 mg of calcium pantothenate sample 1, add 1.0 mL of potassium chloride standard with a chloride ion concentration of 0.0479 mg / mL, and then add water to dissolve and dilute to a final volume of 10 mL. Analyze using HPLC.

[0144] The results of the limit of quantitation experiment are as follows Figure 9 As shown, the signal-to-noise ratio of chloride ions is greater than 10. The results indicate that the limit of quantitation of chloride ions in calcium pantothenate aqueous solution is 0.0125% (by weight).

[0145] Example 7.4 Test of calcium pantothenate sample Calcium pantothenate samples 1, 2, and 3 were each prepared into 40 mg / mL aqueous solutions for HPLC analysis. The HPLC results are shown in Table 3. Figure 10As shown in the figure. The results showed that chloride ions were not detected in test sample 1, while chloride ions were detected in test samples 2 and 3. The sodium ion content (by weight) detected in test sample 1 was 0.18%, which was much lower than the sodium ion content (by weight) in test samples 2 and 3 (0.50% and 0.68%, respectively).

[0146] Table 3: Chloride and Sodium Ion Content in Each Sample

Claims

1. A method for preparing β-alanine, comprising: (1) β-alanine product was prepared by reactants containing fumaric acid and ammonia in the presence of a catalyst, wherein: The catalyst comprises recombinant engineered bacterial cells expressing aspartate enzyme and L-aspartate-α-decarboxylase, wherein the aspartate enzyme is derived from... Anoxybacillus flavithermus WK1 The L-aspartate-α-decarboxylase is derived from Bacillus thermotolerans ; Fumaric acid is added in a fed-batch manner during the reaction, and the total molar amount of fumaric acid added is equal to the initial molar amount of ammonia in the reactants minus the initial molar amount of fumaric acid in the reactants. (2) Monitor the reaction after the fumaric acid addition is completed. When the fumaric acid content is <0.5% (w / v) and the molar conversion of aspartic acid is >99%, the catalytic reaction ends. (3) The β-alanine product is crystallized to obtain a mother liquor, wherein the content of inorganic salts in the mother liquor is less than 10 g / L, and methanol or ethanol is not added during the crystallization; and (4) The mother liquor obtained after crystallization is recycled; The reaction pH was controlled between 6.8 and 7.2, and the reaction temperature was between 35 and 42 degrees Celsius. O Between C; The bacterial cells comprise recombinant engineered cells expressing aspartate aminotransferase alone and recombinant engineered cells expressing L-aspartate-α-decarboxylase alone; the weight percentage of the recombinant engineered cells expressing aspartate aminotransferase alone to the initial fumarate in the reactants is 1% (w / w); and the weight percentage of the recombinant engineered cells expressing L-aspartate-α-decarboxylase alone to the initial fumarate in the reactants is 20% (w / w); or The bacterial cells comprise recombinant engineered cells that co-express aspartate enzyme and L-aspartate-α-decarboxylase; and the weight percentage of the recombinant engineered cells co-expressing aspartate enzyme and L-aspartate-α-decarboxylase to the initial fumarate in the reactants is 30% (w / w).

2. The preparation method according to claim 1, wherein the bacterial cells include wet bacterial cells, immobilized bacterial cells, or bacterial cell disruption liquid.

3. The preparation method according to any one of claims 1-2, wherein the initial molar ratio of fumaric acid to ammonia in the reactants is 1:

2.

4. The preparation method according to any one of claims 1-3, wherein the concentration of fumaric acid is 50-400 g / L, and the flow rate is such that the pH value is controlled at 7.0 during the reaction.

5. The preparation method according to any one of claims 1-4, wherein the reaction temperature is controlled at 37°C. O C.

6. The preparation method according to any one of claims 1-5, wherein after the catalytic reaction is completed, it further comprises: Remove the residues from the recombinant engineered bacteria.

7. The preparation method according to any one of claims 1-6, wherein no organic solvent is added during the crystallization.

8. The preparation method according to any one of claims 1-7, wherein β-alanine crystals are obtained after crystallization, and the content of inorganic salts in the crystals is less than 20 mg / g.

9. A method for preparing β-alanine salt, comprising the following steps: β-alanine was prepared by the method according to any one of claims 1-8; The β-alanine obtained in step (a) is dissolved in water and then reacted with an alkaline solution.

10. A method for preparing pantothenate, comprising the following steps: β-alanine was prepared by the method according to any one of claims 1-8; The β-alanine obtained in step (a) is dissolved in water and then reacted with an alkaline solution to obtain β-alanine salt; and The pantothenic acid or pantothenic acid is reacted with the β-alanine salt prepared in step (b).

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