阅读说明：本技术 β-丙氨酸的制备方法 (Process for producing beta-alanine ) 是由 苏金环 曾聪明 邱贵森 刘文杰 于 2019-03-20 设计创作，主要内容包括：本发明提供了一种β-丙氨酸的制备方法,包括：将含有富马酸和氨水的反应物在催化剂存在下制备得到β-丙氨酸产物,其中所述催化剂包含含有天冬氨酸酶和L-天冬氨酸-α-脱羧酶的催化组合物,并且在反应过程中加入富马酸,而且加入的富马酸的摩尔总量等于所述反应物中的氨水的初始摩尔量减去所述反应物中的富马酸的初始摩尔量。(The invention provides a preparation method of beta-alanine, which comprises the following steps: preparing a beta-alanine product from reactants comprising fumaric acid and ammonia in the presence of a catalyst, wherein the catalyst comprises a catalytic composition comprising aspartase and L-aspartate-alpha-decarboxylase, and fumaric acid is added during the reaction, and the total molar amount of fumaric acid added equals the initial molar amount of ammonia in the reactants minus the initial molar amount of fumaric acid in the reactants.)

1. A method for producing beta-alanine, comprising:

preparing a reactant containing fumaric acid and ammonia water in the presence of a catalyst to obtain a beta-alanine product,

wherein the catalyst comprises a catalytic composition comprising an aspartase and an L-aspartate-alpha-decarboxylase, and

fumaric acid is added during the reaction, and the total molar amount of fumaric acid added is equal to the initial molar amount of ammonia in the reactant minus the initial molar amount of fumaric acid in the reactant.

2. The method of claim 1, wherein the catalytic composition comprises a purified aspartase and a purified L-aspartate- α -decarboxylase.

3. The method according to claim 1, wherein the catalytic composition comprises cells expressing aspartase and L-aspartate- α -decarboxylase.

4. The method according to claim 3, wherein the cells comprise wet cells, immobilized cells, or cell disruption solution.

5. The method according to claim 3, wherein the microbial cell is derived from a recombinant engineered bacterium.

6. The method according to any one of claims 3 to 5, wherein the microbial cells comprise microbial cells that individually express aspartase and microbial cells that individually express L-aspartate- α -decarboxylase.

7. The production method according to claim 6, wherein the weight percentage of the separately aspartase-expressing microbial cells to the initial fumaric acid in the reaction mixture is 0.5 to 4% (w/w); and the weight percentage of the bacterial cells which individually express the L-aspartate-alpha-decarboxylase and the initial fumaric acid in the reactant is 10-30% (w/w).

8. The method according to any one of claims 3 to 5, wherein the microbial cells comprise microbial cells co-expressing aspartase and L-aspartate- α -decarboxylase.

9. The method according to claim 8, wherein the weight percentage of the co-expressed cells of aspartase and L-aspartate- α -decarboxylase to the initial fumaric acid in the reactant is 10% to 40% (w/w).

10. The production method according to any one of claims 1 to 9, wherein the aspartase is derived from Anaxobacillus flavithermus or Geobacillus thermodernicans; and the L-aspartate-alpha-decarboxylase is derived from Bacillus thermolerans, Anaxobacillus flavthermus or Methanobacillus jannaschii.

11. The production method according to any one of claims 1 to 10, wherein the initial molar ratio of the fumaric acid to the aqueous ammonia in the reactants is 1: 2.

12. the production method according to any one of claims 1 to 11, wherein the fumaric acid added during the reaction is added in a fed-batch manner.

13. The production process according to claim 12, wherein the fumaric acid has a concentration of 50 to 400g/L and is fed at such a rate that the pH during the reaction is controlled to be 6.8 to 7.2.

14. The production method according to any one of claims 1 to 13, wherein the reaction temperature is controlled to be 25 to 55 ℃.

15. The production method according to any one of claims 1 to 14, further comprising, after completion of the catalytic reaction: removing residues from the catalytic composition.

16. The method of claim 15, further comprising crystallizing the beta-alanine product.

17. The production method according to claim 16, wherein a mother liquor is obtained after the crystallization, and the content of inorganic salts in the mother liquor is less than 10 g/L.

18. The production method according to claim 16, wherein a β -alanine crystal is obtained after the crystallization, and the content of inorganic salts in the crystal is less than 20 mg/g.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for preparing beta-alanine through enzyme catalysis.

Background

Beta-alanine, also known as beta-aminopropionic acid or 3-aminopropionic acid, is a beta-type nonprotein amino acid occurring in nature. Beta-alanine is a multipurpose organic synthetic raw material, is mainly used for synthesizing pantothenic acid, calcium pantothenate, carnosine, pamidronate sodium, balsalazide and the like, is widely applied in the fields of medicines, feeds, foods and the like, and has great market demand.

At present, the production methods of beta-alanine are divided into two major types, namely a chemical synthesis method and a biological method, and the chemical method is still the production method mainly adopted at home and abroad because the research on synthesizing the beta-alanine is early and the process is mature.

The chemical method for producing beta-alanine has two main modes, one is that acrylonitrile and ammonia react at high temperature and high pressure to prepare the beta-alanine, the method has low cost, but the product is difficult to separate due to the side reaction; and the beta-aminopropionitrile and the barium hydroxide are hydrolyzed under the high-temperature condition, the yield of the method is high, but the separation problem also exists due to the fact that a large amount of inorganic salt is generated in the reaction process.

The chemical method generally has the problems of harsh reaction conditions, high equipment requirements, environmental pollution and the like, so that the biological method gradually becomes a research hotspot due to the advantages of mild reaction conditions, high efficiency and environmental friendliness along with the continuous increase of the usage amount of the beta-alanine.

The biological method for producing the beta-alanine is mainly to convert a substrate into the beta-alanine by using microorganisms capable of producing specific enzymes, for example, Zhejiang university of industry (CN1285730) synthesizes the beta-alanine by using acrylic acid and ammonia by using an amination enzyme, and the method has high reaction efficiency and low cost, but has no industrial application report at present because raw materials have strong corrosivity and irritation. Chuanliyang et al (JP10-42886) use organic nitrilase to catalyze beta-aminopropionitrile to synthesize beta-alanine, and the method has the advantages of high raw material price, low reaction concentration and high cost, and is difficult to meet the requirements of industrial production.

Another biological method for synthesizing beta-alanine is to remove alpha carboxyl of L-aspartic acid specifically by using L-aspartic acid-alpha-decarboxylase to generate beta-alanine. However, the existing method has the problems of low enzyme activity and poor enzyme stability.

Disclosure of Invention

The invention provides a preparation method of beta-alanine, which comprises the following steps: preparing a beta-alanine product from reactants comprising fumaric acid and ammonia in the presence of a catalyst, wherein the catalyst comprises a catalytic composition comprising aspartase and L-aspartate-alpha-decarboxylase, and fumaric acid is added during the reaction, and the total molar amount of fumaric acid added equals the initial molar amount of ammonia in the reactants minus the initial molar amount of fumaric acid in the reactants.

In certain embodiments, the catalytic composition comprises a purified aspartase and a purified L-aspartate- α -decarboxylase. In certain embodiments, the catalytic composition comprises bacterial cells expressing aspartase and L-aspartate- α -decarboxylase. In certain embodiments, the bacterial cells comprise wet bacterial cells, immobilized bacterial cells, or a cell disruption solution. In certain embodiments, the bacterial cells are derived from recombinant engineered bacteria. In some embodiments, the bacterial cells comprise bacterial cells that express aspartase alone and bacterial cells that express L-aspartate- α -decarboxylase alone. In some embodiments, the weight percentage of the cells expressing aspartase alone to the initial fumaric acid in the reaction mixture is 0.5% to 4% (w/w); and the weight percentage of the thallus which independently expresses the L-aspartic acid-alpha-decarboxylase to the initial fumaric acid in the reactant is 10-30% (w/w). In some embodiments, the bacterial cells comprise bacterial cells co-expressing aspartase and L-aspartate- α -decarboxylase. In some embodiments, the weight percentage of the co-expressed cells of aspartase and L-aspartate- α -decarboxylase to the initial fumaric acid in the reactant is 10% to 40% (w/w). In certain embodiments, the aspartase is derived from Anoxybacillus flavithermus or Geobacillus humicola; the L-aspartate-alpha-decarboxylase is derived from Bacillus thermolerans, Anaxobacillus flavthermus or Methanobacillus jannaschii. In certain embodiments, the aspartase is derived from Anoxybacillus flavithermus WK1 or Geobacillus thermophiloidicans NG 80-2; the L-aspartate-alpha-decarboxylase is derived from Quasibacillusthermotolerans, Anaxybacillus flavthermus AK1 or Methanobacillus jannaschiDSM 2661.

In certain embodiments, the initial molar ratio of the fumaric acid to the aqueous ammonia in the reactants is 1: 2. in certain embodiments, the fumaric acid added during the course of the reaction is added in a fed-batch manner. In certain embodiments, the fumaric acid is present in a concentration of 50 to 400g/L at a rate such that the pH during the reaction is controlled between 6.8 and 7.2. In some embodiments, the reaction temperature is controlled between 25 ℃ and 55 ℃.

In certain embodiments, the methods of the present invention further comprise, after the catalytic reaction is complete: removing residues from the catalytic composition. In certain embodiments, the methods of the invention further comprise crystallizing the beta-alanine product. In certain embodiments, a mother liquor is obtained after the 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 the crystallization can be recycled. In certain embodiments, crystals of beta-alanine are obtained after said crystallization, and the content of inorganic salts in said crystals is below 20 mg/g.

Brief Description of Drawings

FIG. 1: the gene sequence of the genome sequence of Anaxybacillus flavithermus WK1 encoding aspartase is shown as SEQ ID NO 1.

FIG. 2: the gene sequence of the gene encoding aspartase of the genomic sequence of Geobacillus thermodernificans NG80-2 is shown as SEQ ID NO 2.

FIG. 3: shows the gene sequence SEQ ID NO 3 of the L-aspartic acid-alpha-decarboxylase in the genome sequence of Quasibacillus thermolerastrain SGZ-8Contig4 in Bacillus thermolerans.

FIG. 4: the gene sequence of the gene sequence encoding L-aspartate-alpha-decarboxylase of Anaxybacillus flavithermus AK1 is shown in SEQ ID NO 4.

FIG. 5: the gene sequence of the gene coding for L-aspartate-alpha-decarboxylase in the genomic sequence of Methanococcus jannaschii is shown as SEQ ID NO 5.

Detailed Description

The invention overcomes the defects of the existing beta-alanine preparation process, and provides a green, high-efficiency and low-cost beta-alanine production process suitable for industrial production.

The invention provides a preparation method of beta-alanine, which comprises the following steps: preparing a beta-alanine product from reactants comprising fumaric acid and ammonia in the presence of a catalyst, wherein the catalyst comprises a catalytic composition comprising aspartase and L-aspartate-alpha-decarboxylase, and fumaric acid is added during the reaction, and the total molar amount of fumaric acid added equals the initial molar amount of ammonia in the reactants minus the initial molar amount of fumaric acid in the reactants.

The "initial molar amount" of fumaric acid or aqueous ammonia means the initial molar amount of fumaric acid or aqueous ammonia before the start of the catalytic reaction. The "initial fumaric acid" weight in the reactants refers to the starting weight of fumaric acid added to the reactants before the catalytic reaction begins. In certain embodiments, the initial molar ratio of the aqueous ammonia to the fumaric acid in the reactants is 2: 1, or within a range that fluctuates by 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% above and below this value.

In certain embodiments, the reactant comprising fumaric acid and aqueous ammonia may comprise ammonium fumarate. The ammonium fumarate refers to a product obtained by neutralizing fumaric acid and ammonia water with acid and alkali. Without being limited by theory, it is believed that at least a portion of the fumaric acid and a portion of the aqueous ammonia spontaneously form ammonium fumarate in the reactant comprising fumaric acid and aqueous ammonia. In the present application, 1 mole of ammonium fumarate is considered to correspond to 2 moles of aqueous ammonia and 1 mole of fumaric acid. Therefore, when only 1 mole of ammonium fumarate was contained in the reaction mixture, it was considered that the ratio of the initial molar amounts of ammonia water and fumaric acid therein was 2: 1.

in the methods provided herein, the catalyst comprises a catalytic composition comprising an aspartase and an L-aspartate- α -decarboxylase. Any known aspartase and L-aspartate- α -decarboxylase can be used. It is well known in the art that aspartase and L-aspartate- α -decarboxylase are known to be naturally expressed in a variety of microorganisms and have corresponding catalytic activities.

In certain embodiments, the aspartase and the L-aspartate- α -decarboxylase are derived from a bacterium. In certain embodiments, the aspartase is derived from Anoxybacillus flavithermus or Geobacillus humicola. In certain embodiments, the aspartase is derived from Anaxobacillus flavhermus WK1 strain or Geobacillus thermodernicans NG80-2 strain. In certain embodiments, the amino acid sequence of the aspartase is the same as, or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to, the amino acid sequence encoded by SEQ ID No. 1 or SEQ ID No. 2.

In certain embodiments, the L-aspartate- α -decarboxylase is derived from bacillus thermophilans, anaerobacterium flavhermus, or methanocadococcus jannaschii. In certain embodiments, the L-aspartate- α -decarboxylase is derived from Quasibacillus thermolerans, Anaxybacillus flavithermus AK1 or Methanobacillus jannaschii DSM 2661. In certain embodiments, the amino acid sequence of the L-aspartate- α -decarboxylase is identical to the amino acid sequence encoded by SEQ ID No. 3, SEQ ID No. 4 or SEQ ID No. 5, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology thereto.

"homology" in the present application means that, with respect to amino acid sequences, a candidate amino acid sequence is aligned with a reference amino acid sequence, and a gap is introduced, if necessary, so as to maximize the number of identical amino acids, and on the basis of this, the percentage of identical amino acids between the two amino acid sequences is calculated; for nucleic acid sequences, the candidate nucleic acid sequences are aligned with the aligned nucleic acid sequences, and intervals are introduced, if necessary, to maximize the number of identical nucleotides, and the percentage of identical nucleotides between the two nucleic acid sequences is calculated on the basis thereof. The comparison to determine the percentage of homology can be performed in a variety of ways known in the art. For example, alignment can be performed using publicly available tools such as BLASTp (national center for biotechnology information (NCBI):http://blast.ncbi.nlm.nih.gov/ Blast.cgisee also, Altschul S.F.et al, J.mol.biol., 215: 403-; stephenf et al, Nucleic Acids res., 25: 3389-3402 (1997)), ClustalW2 (european institute for bioinformatics website:http://www.ebi.ac.uk/Tools/msa/clustalw2/see, e.g., Higgins D.G.et al, Methods in Enzymology, 266: 383-; larkin M.A.et al, Bioinformatics (Oxford, England), 23(21):2947-8 (2007)). When software is used to perform the sequence alignment, default parameters provided by the software can be used, and parameters can be adjusted appropriately according to the needs of alignment, which are within the knowledge of those skilled in the art.

In the present application, the aspartase and the L-aspartate- α -decarboxylase in the catalytic composition may be present in any suitable active form, such as, but not limited to, an isolated or purified active enzyme protein, a cell naturally or recombinantly expressing said enzyme, or a lysate thereof.

In certain embodiments, the catalytic composition comprises a purified aspartase and a purified L-aspartate- α -decarboxylase. The purified enzyme may be obtained by isolation and purification from a microorganism naturally expressing the enzyme, or may be obtained by recombinant expression followed by isolation and purification. Purified aspartase and L-aspartate-alpha-decarboxylase enzymes can be prepared by one skilled in the art using routine techniques in the art. For example, ammonium sulfate precipitation followed by ion exchange chromatography followed by gel chromatography.

In certain embodiments, the catalytic composition comprises bacterial cells expressing aspartase and L-aspartate- α -decarboxylase. In certain embodiments, the bacterial cells comprise wet bacterial cells, immobilized bacterial cells, or a cell disruption solution. In some embodiments, the wet cells are cells obtained by solid-liquid separation of a bacterial culture broth, such as cells collected by centrifugation; the immobilized thallus is obtained by wet thallus through a conventional immobilization means, such as thallus embedded by sodium alginate; the thallus crushing liquid is a solution obtained by crushing thallus conventionally, such as high-pressure homogenate crushing liquid. The cell disruption solution contains a desired enzyme.

In certain embodiments, the bacterial cells expressing aspartase and L-aspartate- α -decarboxylase are derived from wild-type bacteria. For example, wild-type bacteria that naturally express aspartase, wild-type bacteria that naturally express L-aspartate- α -decarboxylase, or wild-type bacteria that naturally express both aspartase and L-aspartate- α -decarboxylase can be used. The wild-type bacteria include, for example, Anaxybacillus flavithermus, Geobacillus hermodeniticans, Bacillus thermophilus, Methanococcus jannaschii and the like.

In some embodiments, the bacterial cells expressing aspartase and L-aspartate- α -decarboxylase are derived from recombinant engineered bacteria. The recombinant engineering bacteria are engineering bacteria introduced with exogenous genes in host engineering bacteria by a recombinant DNA method. The recombinant engineering bacteria can express the introduced exogenous gene in a recombination mode. Those skilled in the art can select appropriate hosts for expressing aspartase and L-aspartate- α -decarboxylase according to their actual needs. In certain embodiments, the host is selected from the group consisting of: escherichia coli, Escherichia coli fergusonii, Anaxybacillus flavhermus WK1, Geobacillus thermodernicans NG80-2, Bacillus thermolerans, Anaxybacillus flavhermus AK1, Methanobacillus jannaschii, Bacillus cereus, Corynebacterium glutamicum.

The skilled in the art can prepare recombinant engineered bacteria by using technical means known in the art according to actual needs, see book of molecular cloning experiments (3 rd edition) (scientific press). The vectors, plasmids and hosts adopted in the experiment are all vectors, plasmids and host series adopted by conventional bacteria (such as escherichia coli) expression, such as PET series vectors and plasmids, BL21 series host bacteria; the culture medium used is a culture medium of engineering bacteria of conventional bacteria (such as Escherichia coli), such as LB culture medium; the adopted culture method is a conventional bacteria (such as Escherichia coli) engineering bacteria culture method.

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

In certain embodiments, the weight percentage of the separately aspartase-expressing bacterial cells to the initial fumaric acid in the reactant is 0.5% to 4% (w/w), e.g., 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 any value between any two of the above numerical ranges, preferably 1% to 2% (w/w); and the weight percentage of the bacterial cells expressing L-aspartate- α -decarboxylase alone to the initial fumaric acid in the reactant is 10% to 30% (w/w), for example, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% or any value between any two of the above numerical ranges, preferably 15% to 20% (w/w).

In certain embodiments, the weight of the biomass is calculated as the wet weight when calculating the weight percentage of the biomass to initial fumaric acid. In certain embodiments, the weight of the biomass is calculated as a dry weight percentage of the biomass to initial fumaric acid. The skilled person can select it according to their actual needs. The person skilled in the art can also carry out the conversion between dry weight and wet weight according to conventional means in the prior art, for example with reference tohttps:// bionumbers.hms.harvard.edu/bionumber.aspx？id＝109836。

In certain embodiments, the recombinantly engineered bacteria used in the present invention produce aspartase and L-aspartate- α -decarboxylase with activities 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% and above, greater than the activities of aspartase and L-aspartate- α -decarboxylase produced by the wild-type bacterium. The activity of aspartase and L-aspartate- α -decarboxylase can be determined by one skilled in the art using conventional techniques, e.g., determining the conversion of substrate per unit time using fumarate and aspartate, respectively, as substrates.

Without being limited by theory, it is believed that enzymes derived from recombinant engineered bacteria or bacteria expressing enzymes used in the present invention have unexpected advantages over wild-type bacteria. For example, the recombinant engineered bacteria used in the present invention are capable of greatly increasing the fumaric acid reaction concentration, for example, from 100g/L to 200g/L, compared to the wild-type bacteria, while also maintaining high yield and high purity of the beta-alanine product.

In certain embodiments, the cells expressing aspartase and L-aspartate- α -decarboxylase comprise cells co-expressing aspartase and L-aspartate- α -decarboxylase. In certain embodiments, the bacterial cells are derived from recombinant engineered bacteria.

In certain embodiments, the weight percentage of the co-expressing cells of aspartase and L-aspartate- α -decarboxylase to the initial fumaric acid in the reactant is 10% to 40% (w/w), e.g., 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% or any value between any two of the above ranges, preferably 20% to 30% (w/w).

In the method provided by the present application, as the catalytic reaction proceeds, the pH of the solution gradually increases, and the present inventors added fumaric acid during the reaction to control the pH of the reaction. Without being limited by theory, it is believed that fumaric acid has distinct advantages over the inorganic acid modifiers conventionally used in the prior art in at least two respects: on one hand, the fumaric acid can effectively reduce the pH value of the reaction solution to a proper range, and on the other hand, the added fumaric acid can be used as a reaction substrate and is consumed through a catalytic reaction to generate beta-alanine, so that the introduction of additional impurities can be avoided; on the contrary, if the inorganic acid regulator is used to adjust the pH, an ammonium salt of the inorganic acid is formed with aqueous ammonia in the reaction system, resulting in the generation of impurities, and thus an additional impurity removal step is required.

In the method provided by the application, under the catalytic action of aspartase, fumaric acid and ammonia water firstly generate intermediate product aspartic acid, then the aspartic acid does not need to be further purified or extracted, and the final product beta-alanine is further generated directly under the catalytic action of L-aspartic acid-alpha-decarboxylase, so that the one-step method can be realized for directly generating the beta-alanine without extracting the intermediate product aspartic acid.

Another technical advantage of the present invention is that residual ammonia, fumaric acid, and ammonium fumarate in the product can be significantly reduced. In the method provided by the application, the total molar amount of the added fumaric acid is equal to the initial molar amount of the ammonia water in the reactant minus the initial molar amount of the fumaric acid in the reactant, so that the ammonia water in the reactant is ensured to react completely, and excessive fumaric acid in the product is avoided. And the ammonia water and the fumaric acid are converted into beta-alanine after catalytic reaction, so that the content of uncatalyzed ammonium fumarate is also obviously reduced.

In certain embodiments of the present application, the initial molar amount ratio of fumaric acid to aqueous ammonia is designed to be 1:2, for example, assuming that the initial molar amount of fumaric acid is 1mol and the initial molar amount of aqueous ammonia is 2 mol. In order to avoid the pH increase during the reaction, the applicant's inventors skillfully controlled the pH by adding fumaric acid during the reaction, and precisely controlled the amount of fumaric acid added during the reaction, so that the molar amount of fumaric acid added (1mol) is equal to the initial molar amount of aqueous ammonia in the reactant (2mol) minus the initial molar amount of fumaric acid in the reactant (1mol), thereby ensuring the completion of the reaction of aqueous ammonia in the reactant while avoiding an excessive amount of fumaric acid in the product. The reaction process for preparing beta-alanine from fumaric acid and ammonia can be summarized as follows:

in certain embodiments, the fumaric acid added during the course of the reaction is added in a fed-batch manner. In some embodiments, the fumaric acid is fed at a rate such that the pH during the reaction is controlled between 6.8 and 7.2. In certain embodiments, the fumaric acid is present in a concentration of 50 to 400g/L at a ramp rate such that the pH during the reaction is controlled between 6.8 and 7.2, for example, a pH of 6.8, 6.9, 7.0, 7.1, 7.2, or any value between any two of the above ranges. In certain embodiments, the pH of the reaction system may be measured during the feeding, thereby adjusting the feeding rate of fumaric acid during the reaction. For example, in some embodiments, the flow rate is controlled to be in the range of 6.8 to 7.1 at a concentration of 100g/L, depending on the pH during the reaction. In certain embodiments, the flow rate is controlled to be in the range of 6.9 to 7.2 depending on the pH during the reaction, when the concentration is 200 g/L.

In certain embodiments, the reaction temperature of the process of the invention is controlled between 25 ℃ and 55 ℃, for example, between 25 ℃, 30 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, 45 ℃, 46 ℃, 47 ℃, 48 ℃, 49 ℃, 50 ℃, 51 ℃, 52 ℃, 53 ℃, 54 ℃, 55 ℃ or any number between any two of the above ranges, preferably between 35 ℃ and 42 ℃.

In some embodiments, after the catalytic reaction is finished, the preparation method of the invention further comprises: removing residues from the catalytic composition.

In the present application, "catalytic reaction" refers to a reaction process in which fumaric acid and aqueous ammonia are reacted in the presence of a catalyst to produce a β -alanine product. One skilled in the art can employ various means to determine whether the catalytic reaction is complete. In certain embodiments, the reaction is monitored (e.g., by HPLC) after the fumaric acid stream is completed, and the end of the reaction is indicated when the fumaric acid content is < 0.5% (w/v), e.g., 0.4%, 0.3%, 0.2%, 0.1% or even less, and the molar conversion of aspartic acid is > 99%, e.g., 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or even 100%. The molar conversion of aspartic acid can be calculated by one skilled in the art using conventional means in the art, for example, by determining the amount of each component in the reaction mixture using HPLC to determine the molar conversion of aspartic acid.

In certain embodiments, the residue comprises large particle impurities, such as cells, bacterial debris, aggregates, flocs, and the like, as well as small molecule impurities, such as nucleic acids and nucleic acid fragments in bacterial culture media, proteins, media components, and the like. The skilled person can remove the catalyst residues from the mixture using conventional separation means, e.g. one or more of filtration, centrifugation, microfiltration, ultrafiltration, etc., depending on their actual needs.

In certain embodiments, the filtration is achieved by using filter paper or filter cloth. The filter paper or filter cloth in the present invention may be commercially available filter paper or filter cloth, such as those manufactured by GE Healthcare Life Sciences, Shibi pure, Asahi chemical Co. In certain embodiments, the filter paper or filter cloth has a pore size of 10 to 150 μm, such as 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 number between any two of the above numerical ranges. The skilled person can select a suitable filter paper or filter cloth pore size to remove impurities according to the size of the impurities.

In certain embodiments, the microfiltration is achieved by passing the reaction solution through a microfiltration membrane. The microfiltration membrane according to the present invention may be a commercially available microfiltration membrane, such as a microfiltration hollow fiber membrane series produced by GE Healthcare Life Sciences, Shibi pure, Asahi chemical Co. In certain embodiments, the pore size of the microfiltration membrane is between 0.1 μm and 0.6 μm, such as 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 numerical ranges. The skilled person can select the appropriate pore size of the microfiltration membrane to remove impurities according to the size of the impurities.

In certain embodiments, the ultrafiltration is achieved by passing the reaction solution through an ultrafiltration membrane. The ultrafiltration membrane of the present invention may be a commercially available ultrafiltration membrane, such as a hollow fiber ultrafiltration membrane series produced by GE Healthcare Life Sciences, Shibi pure, Asahi chemical company, and the like. In certain embodiments, the ultrafiltration membrane is a hollow fiber ultrafiltration membrane having a pore size of 5kD to 500kD, such as a hollow fiber ultrafiltration membrane having 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 in between any two of the above ranges. The skilled person can select the appropriate pore size of the ultrafiltration membrane to remove impurities according to the size of the impurities.

In certain embodiments, the methods of making described herein further comprise concentrating the beta-alanine product. In certain embodiments, the concentration is achieved by reducing pressure, for example, pumping the filtered, microfiltered or ultrafiltered reactant solution 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 or any value between any two of the above numerical ranges.

In certain embodiments, the methods of the invention further comprise crystallizing the beta-alanine product. In some embodiments, the crystallization is achieved by reducing the temperature and adding an organic solvent (e.g., methanol, ethanol, etc.), for example, by adding 1-fold, 2-fold, 3-fold, 4-fold equal volume of organic solvent dropwise to the concentrated reaction solution and crystallizing at low temperature (e.g., 10 ℃, 5 ℃ or lower).

In the process provided herein, a mother liquor is obtained after said crystallization. In this application, "mother liquor" refers to the liquid remaining after crystallization of beta-alanine after the completion of the catalytic reaction. The content of inorganic salts in the mother liquor is less than 10g/L, such as less than 9g/L, 8g/L, 7g/L, 6g/L, 5g/L, 4g/L, 3g/L, 2g/L, 1g/L, 0.5g/L, and the like. In certain embodiments, the mother liquor obtained during the crystallization process is free of inorganic salts. The content of inorganic salts in the mother liquor can be determined by one skilled in the art using conventional techniques, for example, an ammonia nitrogen detector (Shanghai apparatus and electrosciences apparatus Co., Ltd.) is used to detect residual ammonium ions in the mother liquor. Without being limited by theory, another technical advantage of the present invention is that the mother liquor remaining after the reaction is finished contains substantially no impurities such as inorganic salts, and can be recycled, thereby avoiding discharge of industrial wastewater, and thus being more environmentally friendly. In certain embodiments, crystals of beta-alanine are obtained after said crystallization and the content of inorganic salts in said crystals is less than 20mg/g, such as less than 19mg/g, 18mg/g, 17mg/g, 16mg/g, 15mg/g, 14mg/g, 13mg/g, 12mg/g, 11mg/g, 10mg/g, 9mg/g, 8mg/g, 7mg/g, 6mg/g, 5mg/g, 4mg/g, 3mg/g, 2mg/g, 1mg/g, etc.

Compared with the prior art, the invention has the following advantages:

1. cheap fumaric acid is used as an initial substrate, beta-alanine is directly generated by a one-step method, and aspartic acid is not required to be extracted, so that the production process is simplified, the production cost is reduced, and the method is green and environment-friendly;

2. the thermostable enzyme is efficiently expressed in engineering bacteria, the enzyme activity is high, the enzyme stability is good, the reaction substrate concentration is high, the conversion efficiency is high, and the molar conversion rate of fumaric acid and aspartic acid is more than 99%;

3. the pH value is controlled by adding fumaric acid in a flowing manner, other inorganic acids (such as phosphoric acid, sulfuric acid, hydrochloric acid, nitric acid and the like) are not introduced in the whole reaction process, and the molar weight of the fumaric acid, the molar weight of ammonia water and the molar weight of the fumaric acid in the reactants in the reaction process are accurately controlled, so that no by-product and inorganic salt are generated in the reaction process, the product extraction process is simple, and the product purity is high.

4. The mother liquor obtained after the beta-alanine is crystallized hardly contains inorganic salt and other impurities, and theoretically, the mother liquor can be recycled, so that the production cost is reduced.

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.