Aspartase mutant and application thereof in preparation of beta-alanine

Abstract

The invention discloses an aspartase mutant and an application of the aspartase mutant in preparation of beta-alanine. The amino acid sequence of the aspartase mutant has the difference of one or more amino acid residues selected from the group consisting of T188I, M322I, K325L, N327C and L446K relative to SEQ ID NO: 1. Compared with wild type aspartase LtAspB-WT, the aspartase mutant disclosed by the invention has the advantage that the capability of catalyzing acrylic acid to generate beta-alanine is obviously improved. The aspartase mutant genetically engineered bacterium can catalyze an acrylic acid substrate with the concentration of 300 g / L to react to generate beta-alanine, the effect that the conversion rate exceeds 99% after 4 hours of reaction is achieved, and the aspartase mutant genetically engineered bacterium has industrial application prospects.

Description

Technical Field

[0002] The invention belongs to the technical field of enzyme catalysis, and in particular relates to an aspartase mutant and an application thereof in the preparation of beta-alanine. Background Art

[0004] β-Alanine is a natural β-amino acid that exists in nature. It is easily soluble in water and slightly soluble in ethanol. As a natural non-protein amino acid, β-Alanine does not directly participate in the synthesis of proteins in the body, but participates in the synthesis of a variety of functional substances such as carnosine and vitamin B5. It is a functional amino acid and is widely used in medicine, food, chemical industry and other industries.

[0005] At present, there are three methods for preparing β-alanine: chemical method, enzymatic method and fermentation method. Among them, chemical method and enzymatic method are the main production methods now. Chemical method includes acrylonitrile method, β-aminopropionitrile method, succinimide degradation method, etc., but these methods are high temperature and high pressure reactions, with harsh process conditions, more by-products in the reaction process, complex extraction process, high cost, and unfriendly environment. These inevitable shortcomings lead to the competitiveness of chemical synthesis of β-alanine in the market becoming weaker and weaker. Enzymatic synthesis of β-alanine mainly refers to the decarboxylation of L-aspartic acid under the catalysis of L-aspartate-α-decarboxylase (ADC) to generate β-alanine. Although this method has high catalytic efficiency, mild conditions, and simple and environmentally friendly extraction process, it requires the use of relatively expensive substrate aspartic acid, which leads to high process cost and great restrictions on industrial application. The fermentation method uses cheap glucose as the starting material, but there are many fermentation by-products, complex extraction process, high cost and other problems, which leads to this method being limited to the laboratory research stage.

[0006] Chinese patent application CN110791493 A utilizes an Aspartase AspB mutant from Bacillus sp. YM55-1 to catalyze the reaction of acrylic acid to synthesize β-alanine. After 16 hours of reaction, 12% wt acrylic acid can be catalyzed to convert into β-alanine, with a conversion rate of over 95%. Acrylic acid is low in price, and the enzymatic conversion of acrylic acid provides a new idea for the production of β-alanine. However, there are still problems of long reaction time and low concentration of catalytic acrylic acid, so it is necessary to optimize the technology. Summary of the invention

[0008] In order to solve the problems of long reaction time and low acrylic acid concentration when aspartase mutants in the prior art catalyze acrylic acid to synthesize β-alanine, the present invention transforms and screens aspartase LtAspB derived from Lysinibacillus telephonicus, constructs an aspartase mutant that still has high conversion ability for high-concentration acrylic acid, can efficiently catalyze acrylic acid to produce β-alanine, and has industrial application prospects.

[0009] The present invention solves the above technical problems through the following technical solutions.

[0010] In a first aspect, the present invention provides an aspartase mutant, whose amino acid sequence differs from SEQ ID NO: 1 in one or more amino acid residues selected from T188I, M322I, K325L, N327C and L446K.

[0011] In some embodiments, the difference is selected from any of the following groups:

[0012] (1) T188I;

[0013] (2) M322I;

[0014] (3) K325L;

[0015] (4) N327C;

[0016] (5) L446K;

[0017] (6) T188I, M322I, K325L, N327C and L446K.

[0018] In some preferred embodiments, the aspartase mutant has an amino acid sequence as shown in SEQ ID NO: 3.

[0019] The second aspect of the present invention provides a polynucleotide encoding the aspartase mutant as described in the first aspect of the present invention.

[0020] In some preferred embodiments, the polynucleotide has a nucleotide sequence as shown in SEQ ID NO: 4.

[0021] The third aspect of the present invention provides a recombinant expression vector, which comprises the polynucleotide as described in the second aspect of the present invention.

[0022] In some preferred embodiments, the backbone of the recombinant expression vector is a plasmid; the plasmid is preferably selected from pUC, pET, pACYCDuet and pETDuet.

[0023] In some more preferred embodiments, the plasmid is pET21a, pET24a or pET28a.

[0024] The fourth aspect of the present invention provides a genetically engineered bacterium, which expresses the aspartase mutant as described in the first aspect of the present invention, or comprises the polynucleotide as described in the second aspect of the present invention, or comprises the recombinant expression vector as described in the fourth aspect of the present invention.

[0025] In some preferred embodiments, the starting bacteria of the genetically engineered bacteria are selected from Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae and Escherichia coli.

[0026] In some more preferred embodiments, the starting bacteria is Escherichia coli, such as Escherichia coli BL21 (DE3).

[0027] The fifth aspect of the present invention provides a method for preparing an aspartase mutant, the method comprising culturing the genetically engineered bacteria as described in the fourth aspect of the present invention, and obtaining the aspartase mutant from the culture.

[0028] In some preferred embodiments, the culture is seed liquid culture and / or induction culture.

[0029] In some more preferred embodiments, the seed liquid culture refers to: culturing the genetically engineered bacteria at 30-40° C. with shaking for 12-20 h, for example, 16 h.

[0030] In some more preferred embodiments, the induction culture refers to: culturing the genetically engineered bacteria at 30-40°C with shaking until OD600 reaches 0.6-0.8, adding IPTG, cooling to 20-30°C, such as 25°C, and culturing for 22-26 h, such as 24 h.

[0031] In some more preferred embodiments, the culture temperature is 37°C.

[0032] In some more preferred embodiments, the culture medium used in the culture is LB medium.

[0033] In some more preferred embodiments, in the induction culture, the genetically engineered bacteria are inoculated into the culture medium at an inoculation ratio of 0.5%-1.5% (v / v), preferably 1% (v / v).

[0034] In some more preferred embodiments, the rotation speed of the shaking culture is 200 rpm.

[0035] The sixth aspect of the present invention provides a method for producing β-alanine, which comprises contacting and reacting the aspartase mutant according to the first aspect of the present invention with a substrate to produce β-alanine.

[0036] In some preferred embodiments, the substrates are acrylic acid and aqueous ammonia.

[0037] In some preferred embodiments, the method further comprises using magnesium acetate.

[0038] In some embodiments, the aspartase mutant is used in the form of free enzyme, liquid enzyme, solid enzyme powder, immobilized enzyme, purified enzyme, crude enzyme, fermentation broth, cell disruptor, bacterial sludge or bacterial powder.

[0039] In some embodiments, the concentration of acrylic acid is 25-35 wt %; preferably 27.5-32.5 wt %; more preferably 30 wt %.

[0040] In some embodiments, the mass fraction of the aqueous ammonia is 20-30 wt %, preferably 25 wt %.

[0041] In some embodiments, the amount of magnesium acetate added is 0.05 g / L-0.2 g / L; preferably 0.1 g / L.

[0042] In some embodiments, the reaction temperature is 40-60°C; preferably 42-50°C; more preferably 45°C.

[0043] In some embodiments, the pH of the reaction is 9.0-10.0; preferably 9.4-9.7; more preferably 9.5-9.6.

[0044] In some embodiments, the reaction time is 2-8 h; preferably 4 h.

[0045] In some embodiments, when the aspartase mutant is used in the form of bacterial sludge, the added mass ratio of the acrylic acid to the bacterial sludge in the reaction is (5-7):1; preferably (5-6):1; more preferably 5.5:1; the added molar ratio of the ammonia water to the acrylic acid in the reaction is 1:1.2-1:2.

[0046] The bacterial mud is prepared by the following method:

[0047] The genetically engineered bacteria expressing the aspartase mutant were centrifuged at 4°C and 10,000 rpm for 5-10 min to collect the bacteria to obtain bacterial sludge.

[0048] The seventh aspect of the present invention provides a reaction end product system for preparing β-alanine by catalysis using acrylic acid as a substrate, the reaction end product system comprising:

[0049] β-Alanine;

[0050] acrylic acid;

[0051] ammonia;

[0052] The aspartase mutant as described in the first aspect of the present invention or the genetically engineered bacteria as described in the fourth aspect of the present invention.

[0053] In some embodiments, the acrylic acid content in the final product system is less than 5%.

[0054] In some embodiments, the acrylic acid content in the final product system is less than 0.5%.

[0055] The eighth aspect of the present invention provides the use of the aspartase mutant as described in the first aspect of the present invention, the polynucleotide as described in the second aspect of the present invention, the recombinant expression vector as described in the third aspect of the present invention, or the genetically engineered bacteria as described in the fourth aspect of the present invention in the preparation of β-alanine.

[0056] In the process of producing β-alanine, acrylic acid is used as a substrate, and the above-mentioned purified aspartase mutant or a microorganism capable of expressing a mutant aspartase is used as a catalyst to catalyze the reaction. In one embodiment, the above-mentioned microorganism can be a whole cell fermentation broth or a whole cell collected by centrifugation or a cell fragment thereof as a catalyst for the reaction.

[0057] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0058] The reagents and raw materials used in the present invention are commercially available.

[0059] The positive and progressive effects of the present invention are:

[0060] Compared with the wild-type aspartase (SEQ ID NO: 1), the aspartase mutant (SEQID NO: 3) constructed by the present invention has significantly improved substrate conversion ability when catalyzing acrylic acid reaction; when the aspartase mutant of the present invention is used to catalyze the substrate acrylic acid to prepare β-alanine, the substrate concentration can be as high as 300 g / L, and the β-alanine conversion rate can be higher than 99% after 4 hours of reaction, and the substrate acrylic acid can be efficiently catalyzed to produce β-alanine, and has industrial application prospects. DETAILED DESCRIPTION

[0062] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0063] The aspartase mutant constructed by the present invention is a mutant of the wild-type aspartase SEQ ID NO: 1 from Lysinibacillus telephonicus, and is a new protein formed after one or more substitutions (T188I, M322I, K325L, N327C, L446K) occur in five amino acids in the sequence of SEQ ID NO: 1. The GenBank accession number of SEQ ID NO: 1 is WP_126295910.1, and the encoding gene is SEQ ID NO: 2 in the sequence table, which is optimized according to the codon preference of Escherichia coli.

[0064] In order to obtain aspartase with higher enzyme performance, the present invention performs point mutation on the gene sequence SEQ ID NO: 2 of SEQ ID NO: 1 through rational design, and obtains a mutant SEQ ID NO: 3 formed by replacing the threonine at position 188 with isoleucine (T188I), replacing the methionine at position 322 with isoleucine (M322I), replacing the lysine at position 325 with leucine (K325L), mutating the asparagine at position 327 to cysteine ​​(N327C), and mutating the leucine at position 446 to lysine (N446K), and the acrylic acid conversion ability of the mutant SEQ ID NO: 3 is significantly improved compared with the wild-type enzyme SEQ ID NO: 1.

[0065] The coding genes involved in the present invention, the expression cassettes and plasmids containing these genes, and the transformants containing the plasmids can all be obtained by genetic engineering construction methods well known to those skilled in the art.

[0066] The above transformant can be any microorganism suitable for expressing aspartase mutants, including bacteria and fungi. Preferably, the microorganism is Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae or Escherichia coli, preferably Escherichia coli, more preferably Escherichia coli BL21 (DE3).

[0067] When used as a biocatalyst for producing β-alanine, the aspartase mutant of the present invention can be a free enzyme, an immobilized enzyme, including a purified enzyme, a crude enzyme, a fermentation broth, a cell disruptor, etc.; it can also be a bacterial cell capable of expressing the aspartase mutant, wherein the bacterial cell includes a surviving bacterial cell and a dead bacterial cell that no longer proliferates.

[0068] In one embodiment, microbial cells expressing the aspartic acid mutant SEQ ID NO: 3 can be used as biocatalysts for enzyme catalysis reactions. The forms of the cells include living cells and dead cells that no longer proliferate.

[0069] The present invention is further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0070] This article involves the addition amount, content and concentration of various substances, and the percentages mentioned are all mass percentages unless otherwise specified.

[0071] The molecular biology experiments involved in the examples include plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., and are performed with reference to Molecular Cloning Experiment Guide (3rd edition), edited by J. Sambrook and DW. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002).

[0072] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2 (LB solid medium plus 20 g / L agar powder).

[0073] TB medium: 24 g / L yeast extract, 12 g / L tryptone, 16.43 g / L K2HPO4·3H2O, 2.31 g / L KH2PO4, 5 g / L glycerol, pH 7.0-7.5.

[0074] HPLC determination conditions of substrate acrylic acid and product β-alanine:

[0075] Column: SUPLEX PKB-100, 5 μm, 4.6 × 250 mm;

[0076] Mobile phase: weigh 3.2 g of sodium dihydrogen phosphate into a beaker, add 1000 mL of purified water, adjust the pH to 5.5 with 1 mol / L NaOH, then add 20 mL of acetonitrile, mix well, filter with a 0.45 μm water filter membrane, and degas by ultrasonication for 20 min before use.

[0077] HPLC detection conditions: detection time: 30 min; detection wavelength: 210 nm; column oven temperature: 35°C; pump speed: 0.5 ml / min; injection volume: 20 μl

[0078] β-alanine conversion rate % = molar amount of β-alanine generated by conversion / molar amount of acrylic acid initially added to the reaction × 100%.

[0079] Example 1 Construction of wild-type aspartase gene recombinant Escherichia coli

[0080] For the aspartase LtAspB from Lysinibacillus telephonicus, the GenBank accession number is WP_126295910.1, and the amino acid sequence is SEQ ID NO: 1. Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to perform full gene synthesis and optimized according to the codon preference of Escherichia coli. The optimized gene sequence is SEQ ID NO: 2. After gene synthesis, it was cloned between the Nde I and Hind III restriction sites of the vector pET28a (purchased from Biowind) to obtain the recombinant plasmid pET28a-LtAspB.

[0081] The recombinant plasmid pET28a-LtAspB was transformed into the expression host Escherichia coli BL21 (DE3) (purchased from Hangzhou Baosai Biotechnology Co., Ltd.) by chemical transformation to obtain the recombinant Escherichia coli LtAspB-WT capable of expressing wild-type aspartase.

[0082] SEQ ID NO: 1 Wild-type aspartase from Lysinibacillus telephonicus, GenBank accession number WP_126295910.1

[0083] MVMTEFRIEKDFLGEKEIPKDAYYGVQTMRAVENFPITGYRIHPELIKSLGIVKKAAALANMEADMLKKEIGQFILQACDEVITGKWNDQFIVDPIQGGAGTSINMNANEVIANRALELMGEEKGNYQVISPNSHVNMSQSTNDAFPTATHIAVLSLMNQLLESAKKMQNAFVLKAKEFAGVIKMGR T HLQDAVPILLGQEFEAYARVISRDIDRMSLSIQHLYEVNMGATAVGTGLNADPTYIDAVVQNLNELSGLPLSRAEHLVDATQNTDCYTEVSATLKVCMINMSKIANDLRLMASGPRDGLFEIMLPARQPGSSI M PG K V N PVMAEVVNQVAFQVMGNDLTISQASEAGQFELNVMEPVLFYNLIQSISIMNNVFTVFTEKCVLGIKANAEHLKQYVEQSVGIITAVNPHIGYELAAQIAKEAIATGASVRELCIKSGA LTAEQLDKILDPYEMTHPGIAGGRSLVNK

[0084] SEQ ID NO: 2 The encoding gene of wild-type aspartase from Lysinibacillus telephonicus, which is optimized according to the codon preference of Escherichia coli

[0085]

[0086] Example 2 Construction of mutation point library by semi-rational design

[0087] The binding site of the substrate α-carboxyl group in the pocket of LtAspB protein was predicted by Discovery Studio software. Using pET28a-LtAspB as the starting plasmid, single-point saturation mutation and iterative saturation mutation were performed at positions T188, M322, K325, N327 and L446, respectively. The screening method of mutant proteins was as follows: the recombinant plasmid (pET28a as a vector) containing the point-mutated LtAspB protein was transformed into Escherichia coli BL21 (DE3) competent cells to obtain mutant transformants. The recombinant Escherichia coli LtAspB-WT expressing wild-type aspartase was used as a control and inoculated into 5 mL LB medium (containing kanamycin at a final concentration of 50 μg / ml), and cultured at 37°C in a shaker at 200 rpm for 16 hours to obtain seed solution. Each sub-liquid was inoculated into fresh TB medium (containing kanamycin at a final concentration of 50 μg / ml) at a ratio of 1%, and cultured in a shaker at 37°C at 200 rpm until OD 600 0.6-0.8, add IPTG with a final concentration of 0.1 mM, cool to 25℃ and induce culture for 24 hours, centrifuge at 4℃, 10000 rpm for 5 min, collect the bacteria, which are the corresponding enzymes.

[0088] Example 3 Screening of aspartase mutants

[0089] The aspartase mutants obtained in Example 2 were used to perform the acrylic acid enzyme conversion reaction. First, acrylic acid substrate (25% acrylic acid, concentrated ammonia solution corrected to pH 9.5) was prepared. 60 mL of acrylic acid substrate was added to 3.0 g of bacterial sludge of each aspartase mutant. After mixing evenly, the mixture was reacted at 45°C for 30 minutes. Then, the sample was centrifuged at 4°C and 12,000 rpm for 10 minutes. The supernatant was taken and the conversion rate of β-alanine was determined by HPLC. Table 1 lists the single-point mutants and five-point mutants with the highest conversion rates among the screened mutants.

[0090] Table 1 Screening of aspartase mutants

[0091] Wild type / mutant number Wild type / mutant mutation point β-Alanine conversion rate % LtAspB-WT No mutation 1.2% LtAspB-m1-17 T188I 23.3% LtAspB-m2-14 M322I 34.5% LtAspB-m3-11 K325L 14.2% LtAspB-m4-16 N327C 17.8% LtAspB-m5-8 L446K 22.6% LtAspB-m6-77 T188I / M322I / K325L / M327C / L446K 53.9%

[0092] As can be seen from the table, we obtained the mutant protein LtAspB-m6-77, the amino acid sequence of which is shown in SEQ ID NO: 3, which also contains five point mutations, namely T188I, M322I, K325L, N327C and L446K, and its coding sequence is shown in SEQ ID NO: 4. Compared with the wild-type enzyme SEQ ID NO: 1, the mutant protein LtAspB-m6-77 has a 44-fold increase in β-alanine conversion rate in the same reaction time.

[0093] SEQ ID NO: 3 Amino acid sequence of mutant aspartase LtAspB-m6-77

[0094] MVMTEFRIEKDFLGEKEIPKDAYYGVQTMRAVENFPITGYRIHPELIKSLGIVKKAAALANMEADMLKKEIGQFILQACDEVITGKWNDQFIVDPIQGGAGTSINMNANEVIANRALELMGEEKGNYQVISPNSHVNMSQSTNDAFPTATHIAVLSLMNQLLESAKKMQNAFVLKAKEFAGVIKMGR I HLQDAVPILLGQEFEAYARVISRDIDRMSLSIQHLYEVNMGATAVGTGLNADPTYIDAVVQNLNELSGLPLSRAEHLVDATQNTDCYTEVSATLKVCMINMSKIANDLRLMASGPRDGLFEIMLPARQPGSSI I PG L V C PVMAEVVNQVAFQVMGNDLTISQASEAGQFELNVMEPVLFYNLIQSISIMNNVFTVFTEKCVLGIKANAEHLKQYVEQSVGIITAVNPHIGYELAAQIAKEAIATGASVRELCIKSGA K TAEQLDKILDPYEMTHPGIAGGRSLVNK

[0095] SEQ ID NO: 4 Gene sequence encoding mutant aspartase LtAspB-m6-77

[0096]

[0097] Example 4 Biocatalytic production of β-alanine from acrylic acid using aspartase mutant LtAspB-m6-77

[0098] Weigh 250 g of acrylic acid into a 2 L three-necked flask, slowly add 25% mass fraction of ammonia water while stirring in an ice bath, adjust the pH of the system to 9.5, add 0.1 g of magnesium acetate, add water to make the total volume of the reaction system 1 L, raise the temperature to 45 ° C, add 50 g of the aspartase mutant LtAspB-m6-77 obtained in Example 2, and stir the reaction at 220 rpm for 4 h. The conversion rate was 99.5%.

[0099] Example 5 Optimal pH for the biocatalytic production of β-alanine from acrylic acid using the aspartase mutant LtAspB-m6-77

[0100] 250 g of acrylic acid was weighed into a 2 L three-necked flask, and 25% ammonia water was slowly added dropwise while stirring in an ice bath, and the pH of the system was adjusted to 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, and 9.9, respectively. 0.1 g of magnesium acetate was added, and water was added to make the total volume of the reaction system 1 L. The temperature was raised to 45 ° C, and 50 g of the bacterial sludge of the aspartase mutant LtAspB-m6-77 obtained in Example 2 was added. After stirring at 220 rpm for 4 h, the conversion rates were 93.2%, 96.4%, 99.5%, 99.5%, 95.1%, 90.1%, and 87.5%, respectively.

[0101] Example 6 Maximum substrate concentration for the biocatalytic production of β-alanine from acrylic acid using the aspartase mutant LtAspB-m6-77

[0102] 250, 275, 300, 325, and 350 g of acrylic acid were weighed into a 2 L three-necked flask, and 25% ammonia water was slowly added dropwise while stirring in an ice bath to adjust the pH of the system to 9.5. 0.1 g of magnesium acetate was added and water was added to make the total volume of the reaction system 1 L. The temperature was raised to 45 ° C, and 50 g of the bacterial sludge of the aspartase mutant LtAspB-m6-77 obtained in Example 2 was added to each of them. After stirring at 220 rpm for 4 h, the conversion rates were 99.5%, 99.6%, 99.5%, 96.2%, and 95.7%, respectively.

[0103] In summary, the aspartase mutant LtAspB-m6-77 (SEQ ID NO: 3) constructed in the present invention can quickly catalyze the reaction of up to 300 g / L of acrylic acid substrate to produce β-alanine when a small amount of bacterial sludge is used, and has prospects for industrial development and application.

Claims

1. An aspartase mutant, characterized in that: Its amino acid sequence differs from SEQ ID NO: 1 in that it has one or more amino acid residues selected from T188I, M322I, K325L, N327C and L446K.

2. The aspartase mutant according to claim 1, characterized in that The difference is selected from any of the following groups: (1) T188I; (2) M322I; (3) K325L; (4) N327C; (5) L446K; (6) T188I, M322I, K325L, N327C and L446K; Preferably, the aspartase mutant has an amino acid sequence as shown in SEQ ID NO:

3.

3. A polynucleotide, characterized in that The polynucleotide encodes the aspartase mutant as described in claim 1 or 2; preferably, the polynucleotide has a nucleotide sequence as shown in SEQ ID NO:

4.

4. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the polynucleotide according to claim 3; Preferably, the backbone of the recombinant expression vector is a plasmid; the plasmid is preferably selected from pUC, pET, pACYCDuet and pETDuet; More preferably, the plasmid is pET21a, pET24a or pET28a.

5. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria expresses the aspartase mutant according to claim 1 or 2, or comprises the polynucleotide according to claim 3, or comprises the recombinant expression vector according to claim 4; Preferably, the starting bacteria of the genetically engineered bacteria are selected from Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae and Escherichia coli; More preferably, the starting bacteria is Escherichia coli, such as Escherichia coli BL21 (DE3).

6. A method for preparing an aspartase mutant, characterized in that: The method comprises culturing the genetically engineered bacteria as claimed in claim 5, and obtaining the aspartase mutant from the culture; Preferably, the culture is seed liquid culture and / or induction culture; The seed liquid culture preferably refers to: culturing the genetically engineered bacteria at 30-40° C. with shaking for 12-20 h, for example, 16 h; The induction culture preferably refers to: culturing the genetically engineered bacteria at 30-40°C with shaking until OD600 is 0.6-0.8, adding IPTG, cooling to 20-30°C, such as 25°C, and culturing for 22-26 h, such as 24 h; More preferably, the culture temperature is 37°C; and / or, the culture medium used is LB culture medium; and / or, in the induction culture, the genetically engineered bacteria are inoculated into the culture medium at an inoculation ratio of 0.5%-1.5% (v / v), preferably 1% (v / v); and / or, the rotation speed of the shaking culture is 200 rpm.

7. A method for producing β-alanine, characterized in that The method comprises contacting and reacting the aspartase mutant according to claim 1 or 2 with a substrate to produce β-alanine; Preferably, the substrate is acrylic acid and aqueous ammonia; and / or, the method further comprises using magnesium acetate.

8. The method according to claim 7, characterized in that The aspartase mutant is used in the form of free enzyme, liquid enzyme, solid enzyme powder, immobilized enzyme, purified enzyme, crude enzyme, fermentation broth, cell disruptor, bacterial sludge or bacterial powder; and / or, the concentration of the acrylic acid is 25-35 wt %; preferably 27.5-32.5 wt %; more preferably 30 wt %; And / or, the mass fraction of the aqueous ammonia is 20-30wt%; preferably 25wt%; And / or, the amount of magnesium acetate added is 0.05 g / L-0.2 g / L; preferably 0.1 g / L; And / or, the reaction temperature is 40-60°C; preferably 42-50°C; more preferably 45°C; And / or, the pH of the reaction is 9.0-10.0; preferably 9.4-9.7; more preferably 9.5-9.6; And / or, the reaction time is 2-8 h; preferably 4 h; Preferably, when the aspartase mutant is used in the form of bacterial sludge, the mass ratio of the acrylic acid to the bacterial sludge in the reaction is (5-7):1; preferably (5-6):1; more preferably 5.5:1; the molar ratio of the ammonia water to the acrylic acid in the reaction is 1:1.2-1:2; The bacterial mud is prepared by the following method: The genetically engineered bacteria expressing the aspartase mutant were centrifuged at 4°C and 10,000 rpm for 5-10 min to collect the bacteria to obtain bacterial sludge.

9. A reaction end product system for producing β-alanine by catalysis using acrylic acid as a substrate, characterized in that: The reaction end product system comprises: β-Alanine; acrylic acid; ammonia; The aspartase mutant according to claim 1 or 2 or the genetically engineered bacterium according to claim 5; Preferably, the acrylic acid content in the final product system is less than 5%; More preferably, the acrylic acid content in the final product system is less than 0.5%.

10. Use of the aspartase mutant according to claim 1 or 2, the polynucleotide according to claim 3, the recombinant expression vector according to claim 4 or the genetically engineered bacteria according to claim 5 in the preparation of β-alanine.