Aspartase mutant and application thereof

Abstract

The invention discloses an aspartase mutant and application thereof. The amino acid sequence of the aspartase mutant has any one or more amino acid mutation sites as shown in a table 1 and corresponding mutation forms relative to the amino acid sequence as shown in SEQ ID NO. 1. Compared with wild-type aspartase (SEQ ID NO: 1), the aspartase mutant disclosed by the invention has the advantages that the enzyme activity, the thermal stability and the capability of catalytically producing beta-alanine are obviously improved, the conversion rate in an acrylic acid ammonification reaction is greater than 99%, and the enzyme activity can still be kept above 85% when the aspartase mutant is used in multiple batches (gt and 10 batches).

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological enzyme catalysis, and particularly relates to an aspartase mutant and application thereof. BACKGROUND

[0002] β-alanine is the only β-type amino acid existing in nature. Although it is a non-protein amino acid, it is widely used in the fields of medicine, chemical industry, food, environment, etc. It can be used not only for preparing high-value-added pharmaceutical intermediates, but also as a precipitating agent in the preparation of medicaments, a flocculating agent for water purification, an electroplating corrosion inhibitor, a detoxifying agent for lead poisoning, a flavoring agent in food, etc.

[0003] The synthesis methods of β-alanine include chemical synthesis and biological synthesis. The chemical method is a high-temperature and high-pressure reaction, which has harsh process conditions, requires high equipment, is difficult to purify the product, and has many by-products in the reaction process. The biological synthesis method is to use pure enzymes or microorganisms capable of producing certain specific enzymes to catalyze the substrate to prepare β-alanine. At present, the biological synthesis method using propylene acid and ammonia has been widely used in industrial production. Many scholars at home and abroad have begun to study the method of producing β-alanine by biological catalysis, and gradually developed from the initial crude natural screening to the more purposeful new technologies such as directional screening, computer modeling, gene editing, protein recombinant expression, enzyme directional evolution and expression (such as Wu, Computational redesign of enzymes for regio-and enantioselective hydroamination; Chinese patent CN109385415A). However, although the existing technology has made some progress in some aspects of β-alanine production, as a whole, there are still many problems to be solved in the aspects of aspartase performance improvement, reaction process optimization, product separation and cost control, etc. For example, such reactions usually occur at 50 degrees, and propylene acid will inevitably undergo some polymerization reactions at this temperature, which will affect the reaction system. In addition, the polymerization inhibitor (such as p-benzenediol) in the propylene acid raw material will have a toxic effect on the enzyme, which will seriously affect the activity and stability of the enzyme in the enzyme catalytic reaction. The existing cells (or enzymes) will be inactivated after single batch use, causing great waste of environment and cost, and having a great influence on the subsequent extraction yield.

[0004] Therefore, it is urgent to develop an aspartase with better performance and the corresponding production process to promote the development of green industrial production of β-alanine by biological enzyme method. SUMMARY

[0005] In response to the shortcomings of the existing technology, the purpose of the present invention is to screen an enzyme and modify it. Starting from the needs of industrial enzymes, enzyme engineering technology is used to improve the enzyme's temperature range, thermal stability and tolerance to inhibitor substances, thereby improving its catalytic properties. On this basis, an immobilized method is adopted to utilize enzyme catalysis to achieve the one-step production of β-alanine using acrylic acid as a substrate.

[0006] To achieve the above object, the present invention provides the following aspartase mutant:

[0007] The amino acid sequence of the aspartase mutant has any one or more amino acid mutation sites and corresponding mutation forms as shown in the following table relative to the amino acid sequence shown in SEQ ID NO.1:

[0008] Amino acid mutation site Location of amino acid mutation site Mutant form N 142 R T 187 C or I N 226 I M 321 I K 324 M or I N 326 T, A, or C L 358 Y .

[0009] Preferably, the amino acid sequence of the aspartase mutant has any one of the following mutation combinations (1) to (96) relative to SEQ ID NO.1:

[0010] (1)N142R / T187C / M321I / K324M / N326T;

[0011] (2)N142R / T187I / M321I / K324M / N326T;

[0012] (3)N142R / T187C / M321I / K324I / N326T;

[0013] (4)N142R / T187I / M321I / K324I / N326T;

[0014] (5)N142R / T187C / M321I / K324M / N326A;

[0015] (6)N142R / T187I / M321I / K324M / N326A;

[0016] (7)N142R / T187C / M321I / K324I / N326A;

[0017] (8)N142R / T187I / M321I / K324I / N326A;

[0018] (9)N142R / T187C / M321I / K324M / N326C;

[0019] (10)N142R / T187I / M321I / K324M / N326C;

[0020] (11)N142R / T187C / M321I / K324I / N326C;

[0021] (12)N142R / T187I / M321I / K324I / N326C;

[0022] (13)N142R / T187C / N226I / M321I / K324M / N326T;

[0023] (14)N142R / T187I / N226I / M321I / K324M / N326T;

[0024] (15)N142R / T187C / N226I / M321I / K324I / N326T;

[0025] (16)N142R / T187I / N226I / M321I / K324I / N326T;

[0026] (17)N142R / T187C / N226I / M321I / K324M / N326A;

[0027] (18)N142R / T187I / N226I / M321I / K324M / N326A;

[0028] (19)N142R / T187C / N226I / M321I / K324I / N326A;

[0029] (20)N142R / T187I / N226I / M321I / K324I / N326A;

[0030] (21)N142R / T187C / N226I / M321I / K324M / N326C;

[0031] (22)N142R / T187I / N226I / M321I / K324M / N326C;

[0032] (23)N142R / T187C / N226I / M321I / K324I / N326C;

[0033] (24)N142R / T187I / N226I / M321I / K324I / N326C;

[0034] (25)N142R / T187C / N226I / M321I / K324M / N326T / L358Y;

[0035] (26)N142R / T187I / N226I / M321I / K324M / N326T / L358Y;

[0036] (27)N142R / T187C / N226I / M321I / K324I / N326T / L358Y;

[0037] (28)N142R / T187I / N226I / M321I / K324I / N326T / L358Y;

[0038] (29)N142R / T187C / N226I / M321I / K324M / N326A / L358Y;

[0039] (30)N142R / T187I / N226I / M321I / K324M / N326A / L358Y;

[0040] (31)N142R / T187C / N226I / M321I / K324I / N326A / L358Y;

[0041] (32)N142R / T187I / N226I / M321I / K324I / N326A / L358Y;

[0042] (33)N142R / T187C / N226I / M321I / K324M / N326C / L358Y;

[0043] (34)N142R / T187I / N226I / M321I / K324M / N326C / L358Y;

[0044] (35)N142R / T187C / N226I / M321I / K324I / N326C / L358Y;

[0045] (36)N142R / T187I / N226I / M321I / K324I / N326C / L358Y;

[0046] (37)T187C / M321I / K324M / N326T;

[0047] (38)T187I / M321I / K324M / N326T;

[0048] (39)T187C / M321I / K324I / N326T;

[0049] (40)T187I / M321I / K324I / N326T;

[0050] (41)T187C / M321I / K324M / N326A;

[0051] (42)T187I / M321I / K324M / N326A;

[0052] (43)T187C / M321I / K324I / N326A;

[0053] (44)T187I / M321I / K324I / N326A;

[0054] (45)T187C / M321I / K324M / N326C;

[0055] (46)T187I / M321I / K324M / N326C;

[0056] (47)T187C / M321I / K324I / N326C;

[0057] (48)T187I / M321I / K324I / N326C;

[0058] (49)T187C / N226I / M321I / K324M / N326T;

[0059] (50)T187I / N226I / M321I / K324M / N326T;

[0060] (51)T187C / N226I / M321I / K324I / N326T;

[0061] (52)T187I / N226I / M321I / K324I / N326T;

[0062] (53)T187C / N226I / M321I / K324M / N326A;

[0063] (54)T187I / N226I / M321I / K324M / N326A;

[0064] (55)T187C / N226I / M321I / K324I / N326A;

[0065] (56)T187I / N226I / M321I / K324I / N326A;

[0066] (57)T187C / N226I / M321I / K324M / N326C;

[0067] (58)T187I / N226I / M321I / K324M / N326C;

[0068] (59)T187C / N226I / M321I / K324I / N326C;

[0069] (60)T187I / N226I / M321I / K324I / N326C;

[0070] (61)T187C / N226I / M321I / K324M / N326T / L358Y;

[0071] (62)T187I / N226I / M321I / K324M / N326T / L358Y;

[0072] (63)T187C / N226I / M321I / K324I / N326T / L358Y;

[0073] (64)T187I / N226I / M321I / K324I / N326T / L358Y;

[0074] (65)T187C / N226I / M321I / K324M / N326A / L358Y;

[0075] (66)T187I / N226I / M321I / K324M / N326A / L358Y;

[0076] (67)T187C / N226I / M321I / K324I / N326A / L358Y;

[0077] (68)T187I / N226I / M321I / K324I / N326A / L358Y;

[0078] (69)T187C / N226I / M321I / K324M / N326C / L358Y;

[0079] (70)T187I / N226I / M321I / K324M / N326C / L358Y;

[0080] (71)T187C / N226I / M321I / K324I / N326C / L358Y;

[0081] (72)T187I / N226I / M321I / K324I / N326C / L358Y;

[0082] (73)T187C / M321I / K324M / N326T / L358Y;

[0083] (74)T187I / M321I / K324M / N326T / L358Y;

[0084] (75)T187C / M321I / K324I / N326T / L358Y;

[0085] (76)T187I / M321I / K324I / N326T / L358Y;

[0086] (77)T187C / M321I / K324M / N326A / L358Y;

[0087] (78)T187I / M321I / K324M / N326A / L358Y;

[0088] (79)T187C / M321I / K324I / N326A / L358Y;

[0089] (80)T187I / M321I / K324I / N326A / L358Y;

[0090] (81)T187C / M321I / K324M / N326C / L358Y;

[0091] (82)T187I / M321I / K324M / N326C / L358Y;

[0092] (83)T187C / M321I / K324I / N326C / L358Y;

[0093] (84)T187I / M321I / K324I / N326C / L358Y;

[0094] (85)N142R / T187C / M321I / K324M / N326T / L358Y;

[0095] (86)N142R / T187I / M321I / K324M / N326T / L358Y;

[0096] (87)N142R / T187C / M321I / K324I / N326T / L358Y;

[0097] (88)N142R / T187I / M321I / K324I / N326T / L358Y;

[0098] (89)N142R / T187C / M321I / K324M / N326A / L358Y;

[0099] (90)N142R / T187I / M321I / K324M / N326A / L358Y;

[0100] (91)N142R / T187C / M321I / K324I / N326A / L358Y;

[0101] (92)N142R / T187I / M321I / K324I / N326A / L358Y;

[0102] (93)N142R / T187C / M321I / K324M / N326C / L358Y;

[0103] (94)N142R / T187I / M321I / K324M / N326C / L358Y;

[0104] (95)N142R / T187C / M321I / K324I / N326C / L358Y;

[0105] (96)N142R / T187I / M321I / K324I / N326C / L358Y.

[0106] The present invention also provides a polynucleotide encoding the aspartase mutant and a recombinant plasmid comprising the polynucleotide.

[0107] Furthermore, the recombinant plasmid uses pET-28a(+) as an expression vector; when the gene sequence of the aspartase mutant is inserted into the pET-28a(+) expression vector, the selected restriction enzyme sites are NdeⅠ restriction site and EcoRI restriction site.

[0108] The present invention also provides a recombinant genetically engineered bacterium, which comprises the above-mentioned aspartase mutant or the above-mentioned recombinant plasmid.

[0109] Furthermore, the host cell of the recombinant genetically engineered bacteria is Escherichia coli BL21 (DE3).

[0110] The present invention also provides a method for preparing the above-mentioned aspartase mutant, which comprises culturing the above-mentioned recombinant genetically engineered bacteria, obtaining the aspartase mutant wet cells from the culture of the recombinant genetically engineered bacteria, or further preparing the aspartase mutant wet cells into aspartase mutant immobilized cells.

[0111] Furthermore, the recombinant genetically engineered bacteria are cultured using a three-stage fermentation method, firstly through a first-stage seed fermentation, then through a second-stage seed fermentation, and finally through a high-density fermentation.

[0112] The first-stage seed fermentation culture conditions are: temperature 30-40°C, preferably 37°C; rotation speed 200-220 rpm; culture time 8-10 hours;

[0113] The secondary seed fermentation culture conditions are: temperature 30-40°C, preferably 37°C; rotation speed 150-200 rpm; culture time 3-4 hours;

[0114] The ratio of glucose, ammonium sulfate, phosphate buffer, citric acid, and magnesium sulfate in the high-density fermentation medium is 0.8-1.5%: 0.08-0.15%: 1.0-1.5%: 0.15-0.20%: 0.05-0.1% (W / V); culture conditions: culture at 30-40°C in the early stage, preferably at 37°C, pH 6.7-6.9, preferably controlled by ammonia water; ventilation volume 1.0-1.5 vvm, preferably 1.0 vvm; tank pressure 0.03-0.08 MPa, preferably 0.05 MPa; stirring speed 200-800 rpm, preferably 500 rpm; dissolved oxygen not less than 20 vol%; when OD600 reaches 20-25, cooling to the induction temperature of 25-30°C; adding IPTG (isopropyl-β-D-thiogalactopyranoside) to a final concentration of 0.2-0.4 mg / L, preferably 0.3 mg / L.

[0115] Furthermore, the preparation method of the aspartase mutant immobilized cells is as follows:

[0116] adding an adsorption carrier to a bacterial suspension of the aspartase mutant, stirring and adsorbing the suspension, and then adding polyethyleneimine and glutaraldehyde for cross-linking to obtain immobilized cells of the aspartase mutant;

[0117] Wherein, the adsorption carrier is selected from diatomaceous earth, bentonite, sodium alginate or activated carbon.

[0118] The present invention also provides use of the aspartase mutant in catalyzing the production of β-alanine.

[0119] Furthermore, the application uses acrylic acid and ammonia as substrate raw materials.

[0120] Furthermore, when catalyzing the production of β-alanine, the pH is set to 8.0-9.0, the reaction temperature is 35-50° C., and the reaction time is 6-12 h.

[0121] Preferably, when catalyzing the production of β-alanine, the reaction temperature is 35-37° C.; the catalyst is immobilized cells of an aspartase mutant; wherein the mass ratio of the aspartase mutant immobilized cells: acrylic acid: ammonia water is 3-8%: 30-40%: 30-48%.

[0122] The beneficial effects of the present invention are:

[0123] Compared with wild-type aspartase (Bacillus sp. YM55-1, SEQ ID NO. 1), the aspartase mutant constructed in the present invention has significantly improved enzyme activity and thermal stability as well as the ability to catalyze the production of β-alanine.

[0124] When used as a biological enzyme system to catalyze the production of β-alanine, the aspartase mutant of the present invention has a wide reaction temperature range (30°C to 50°C), and its activity can be maintained at more than 85% when used in multiple batches (>10 batches). During the catalytic reaction, the substrate concentration can be as high as 450g / L, and the β-alanine conversion rate can be higher than 80% after 4 hours of reaction. The reaction can be basically completed in 8 to 10 hours, with a conversion rate of 99%. In particular, when enzyme-immobilized cells are used for the conversion reaction, after the conversion is completed, the immobilized cells of the aspartase mutant of the present invention can be directly separated from the conversion liquid by suction filtration and reused for the next batch of conversion; the obtained conversion liquid can be simply extracted and crystallized for the preparation of β-alanine. The whole process is easy to operate, product separation is simpler, the extraction yield is significantly improved, and the immobilized cells have a high reuse rate, which significantly reduces production costs and lays the foundation for industrial green production upgrades. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] Figure 1 The figure is a schematic diagram of the reaction system in which immobilized cells of the aspartase mutant of the present invention catalyze the addition of ammonia to acrylic acid to prepare β-alanine.

[0126] Figure 2 This is a graph showing the progress of the reaction of preparing β-alanine by adding ammonia to acrylic acid catalyzed by immobilized cells of the aspartase mutant of the present invention in Example 4.

[0127] Figure 3 This is a graph showing the relative enzyme activity after the aspartase mutant of the present invention was repeatedly used to immobilize cells 10 times in Example 5. DETAILED DESCRIPTION

[0128] The present invention will be further described below with reference to the embodiments.

[0129] Unless otherwise specified, the abbreviated symbols of amino acids and their corresponding codons in the present invention are conventional in the art.

[0130] Example 1 Preparation of aspartase mutants

[0131] The method for preparing an aspartase mutant of the present invention comprises constructing a recombinant expression vector and transfecting it into competent host cells to obtain a genetically engineered strain expressing aspartase; the strain is cultured, collected, and then immobilized. The specific steps are as follows:

[0132] (1) Construction of aspartase strain

[0133] The gene sequence of aspartase from Bacillus sp. YM55-1 (WT, GenBank: AB028242.1, amino acid sequence shown in SEQ ID NO. 1) was codon-optimized based on the codon preference of E. coli, and the optimized sequence, SEQ ID NO. 2, was synthesized. Site-directed mutagenesis was performed on the optimized sequence, and the specific amino acid mutation sites and mutation patterns are shown in Table 1:

[0134] Table 1

[0135] Amino acid mutation site Location of amino acid mutation site Mutant form N 142 R T 187 C or I N 226 I M 321 I K 324 M or I N 326 T, A, or C L 358 Y .

[0136] The target fragment and the pET28a(+) plasmid were then double-digested with NdeI and EcoRI, respectively. The digested product was recovered after completion of the reaction. The digested target fragment and the pET-28a(+) vector were then mixed in a specific ratio and DNA ligase was added to initiate a ligation reaction. The ligation product was then transformed into competent E. coli DH5α cells and plated on a plate containing kanamycin-resistant LB solid medium for screening. Upon verification, an aspartase mutant strain was obtained.

[0137] (2) Construction of genetically engineered bacteria

[0138] The mutated gene is used as the target gene, and the plasmid pET28a(+) is used as the expression vector. The target gene fragment and the expression vector pET-28a(+) are double-digested with Nde I and EcoR I, respectively. After the reaction, the digested product is recovered. The digested target fragment and the pET-28a(+) vector are then mixed in a specific ratio, and DNA ligase is added to perform a ligation reaction to obtain a recombinant plasmid. The recombinant plasmid is then introduced into competent Escherichia coli BL21(DE3) cells for transformation, and then plated on a plate containing LB solid medium containing kanamycin resistance for screening. After verification, a genetically engineered bacterium expressing the aspartase mutant is obtained.

[0139] (3) Fermentation culture: adopt three-stage fermentation, first through primary seed fermentation, then through secondary seed fermentation, and finally through high-density fermentation;

[0140] The first-level seed fermentation formula is LB medium (Luria-Bertani medium), and the culture conditions are: temperature 37° C.; rotation speed 200-220 rpm; and culture time 10 hours.

[0141] The secondary seed formula is LB medium (Luria-Bertani medium), culture conditions: temperature 37°C, rotation speed 200 rpm, culture time 3.5 hours. After the OD600 absorbance value reaches 5 or above, high-density fermentation is carried out;

[0142] The high-density fermentation formula uses glucose as the carbon source (at a ratio of 1% (W / V)) and ammonium sulfate as the nitrogen source (at a ratio of 0.1% (W / V)). It also includes 1.2% (W / V) phosphate buffer, 0.20% (W / V) citric acid, and 0.05% (W / V) magnesium sulfate. Culture conditions include initial incubation at 37°C; pH maintained between 6.7 and 6.9; aeration rate of 1.0 vvm; tank pressure of 0.05 MPa; agitation speed: initially 300 rpm, increasing to 500 rpm after 3 hours; and a minimum dissolved oxygen level of at least 20%.

[0143] When OD600 reaches 20-25, cool down to the induction temperature of 25-26°C, add IPTG (isopropyl-β-D-thiogalactoside) to make the final concentration of 0.3 mg / L, continue fermentation for 10 hours after induction and then release the tank.

[0144] (3) Collecting bacteria

[0145] The fermented bacterial solution was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the precipitate - bacterial cells were collected.

[0146] (4) Diatomaceous earth immobilized cells

[0147] Add 5g of bacterial cells and 0.3g of diatomaceous earth to 50mL of Tris-HCl buffer. Add 1.5mL of 8% polyethyleneimine (PEI) with stirring, and flocculate for 2.0 hours. Then, add 5% glutaraldehyde and cross-link for 1.5 hours. Filter, rinse the filter cake three times with Tris-HCl buffer, squeeze it into long strips with a syringe, air-dry it at room temperature, and grind it into granules.

[0148] The wet bacteria of the aspartase mutant or the diatomaceous earth-immobilized cells of the aspartase mutant obtained by the above preparation process can be used to catalyze the ammonia addition of acrylic acid to produce β-alanine. Figure 1The following is a reaction formula for the use of the aspartase mutant of the present invention in the production of β-alanine. Specifically, a biocatalytic reaction is performed using acrylic acid and ammonia as substrates in an enzyme system primarily composed of immobilized cells of the aspartase mutant. The system comprises 3-8% by mass of the immobilized cells, 30-40% by mass of acrylic acid, and 30-48% by mass of ammonia. After the biocatalytic reaction, β-alanine is obtained.

[0149] Example 2 Enzyme activity determination

[0150] Preparation of substrate: 500 mM acrylic acid, adjusted to pH 9.0 with aqueous ammonia.

[0151] Enzyme activity assay steps:

[0152] A certain amount of the fermented aspartase mutant wet bacteria or immobilized cells of the present invention was added to the substrate and reacted at 35° C. for 0.5 h. The reaction solution was diluted with mobile phase and filtered through a 0.22 μm filter membrane for HPLC detection.

[0153] Liquid phase detection conditions:

[0154] Hypersil NH2, 4.6 mm × 250 mm, 5 μm; mobile phase: phosphoric acid aqueous solution: acetonitrile = 3:7 (volume ratio), phosphoric acid aqueous solution preparation method: 1.7011 g potassium dihydrogen phosphate was dissolved in 250 mL of purified water; detection wavelength: 205 nm; flow rate: 1 mL / min; column temperature: 30°C; injection volume: 20 μl.

[0155] The enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the conversion of 1 μmol of β-alanine per minute at 35°C and pH 9.0. The specific activity (U / g) is defined as the number of enzyme units per gram of wet bacteria or immobilized cells.

[0156] The mutation sites of the aspartase mutant protein of the present invention and the corresponding relative activity (mutant / WT) in catalyzing the addition of acrylic acid to ammonia are shown in Table 2:

[0157] Table 2

[0158]

[0159]

[0160]

[0161]

[0162] The results show that the enzyme activity of the aspartase mutant of the present invention is increased by 2 to 45 times compared with the wild type.

[0163] Thermostability of aspartase mutants

[0164] The wild-type aspartase cells and aspartase mutant cells AspB-1 to AspB-96 were incubated at 60°C, respectively. The samples were cooled in ice bath every 1 h. The enzyme activity was determined according to the method of Example 2. The time when the enzyme activity decreased to about 50% of the original enzyme activity was the half-life of the enzyme at the temperature, and the temperature stability of the aspartase was determined.

[0165] The half-life of the wild-type aspartase cells and some aspartase mutant cells of the application was determined, and the results are shown in Table 3. The half-life of the wild-type aspartase was only 3 h at 60°C, while the half-life of the aspartase mutants of the application was more than 6 h, and the half-life of mutant AspB-42 was 12 h at 60°C, which was significantly higher than that of the wild-type aspartase.

[0166] Table 3

[0167]

[0168] Application of aspartase mutants of the application in preparation of β-alanine

[0169] The diatomite-immobilized cells of aspartase mutants AspB-1 to AspB-96 were prepared according to the procedure of Example 1. In a 5 L catalytic system, 250 g of each mutant immobilized cells, 2000 g of acrylic acid, and 2100 g of ammonia were added, and the pH was set at 9.0. The reaction temperature was 37°C, and the reaction time was 10 h. The reaction liquid containing β-alanine was obtained. The immobilized cells and the reaction supernatant were collected by filtration, 0.2% activated carbon was added, and the mixture was stirred at 65°C for 2 h for decolorization treatment. The decolorized liquid was obtained by filtration again. The decolorized liquid was concentrated by rotation to 60%, the concentration temperature was set at 60°C, and the temperature was reduced to 8-10°C. 3-5 BV of ethanol was slowly added for crystallization. The crystals were washed with 1 BV of ethanol, centrifuged, and dried in a drying device at a temperature of 60°C. The β-alanine product was obtained after drying.

[0170] Under the action of the aspartase mutant cells of the application, the β-alanine product (white crystalline solid) was more than 2.2 kg, the conversion rate of acrylic acid reached more than 99% in 10 hours of reaction, and the product yield was more than 89%. The yield of some aspartase mutants and the weight of the obtained β-alanine product are shown in Table 4. The catalytic reaction process curve of aspartase mutant cells AspB-42 is shown in Figure 2 .

[0171] Table 4

[0172]

[0173]

[0174] Example 5 Experiment on the number of times immobilized cells can be reused

[0175] Continuous conversion was carried out in a 1L reaction system, with the acrylic acid concentration in the system controlled at 30%, the pH adjusted to 9.0 with ammonia water, the temperature at 35°C, and 50g of AspB-42 immobilized cells added. After each reaction was completed, the immobilized cells were filtered and used for the next catalytic reaction. The enzyme activity of the immobilized cells in the first use was set as 100%. After the immobilized cells were reused 10 times, the relative enzyme activity was calculated as follows: Figure 3 As shown, after being used 10 times, 85% of the initial enzyme activity was still retained.

[0176] The experiment of repeated use of other mutants was the same as that of AspB-42. The enzyme activity data of some bacteria after repeated use for 10 times are shown in Table 5.

[0177] Table 5

[0178] Bacteria name Reuse 10 times relative to the initial enzyme activity AspB-34 80％ AspB-37 82％ AspB-38 75％ AspB-42 85％ AspB-43 70％ AspB-49 81％ AspB-58 80％ AspB-65 83％ AspB-69 79％ AspB-86 82％ AspB-94 75％

[0179] The above data show that the immobilized cells of the aspartase mutant of the present invention have good stability.

[0180] All technical features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may also be replaced by other features having the same, equivalent, or similar functions. Therefore, unless otherwise specified, each feature disclosed in this specification is merely an example of a series of equivalent or similar features.

[0181] In addition, from the above description, those skilled in the art can easily understand the key features of the present disclosure from this disclosure. Without departing from the spirit and scope of the present disclosure, many modifications can be made to the invention to adapt to various different purposes and conditions of use. Therefore, such modifications are also intended to fall within the scope of the appended claims.

Claims

1. An aspartase mutant, characterized in that: The amino acid sequence thereof has any one or more amino acid mutation sites and corresponding mutation forms as shown in the following table relative to the amino acid sequence shown in SEQ ID NO.1: 。 2. The aspartase mutant according to claim 1, characterized in that The amino acid sequence of the aspartase mutant has any one of the following mutation combinations (1) to (96) relative to SEQ ID NO.1: (1) N142R / T187C / M321I / K324M / N326T; (2) N142R / T187I / M321I / K324M / N326T; (3) N142R / T187C / M321I / K324I / N326T; (4) N142R / T187I / M321I / K324I / N326T; (5) N142R / T187C / M321I / K324M / N326A; (6) N142R / T187I / M321I / K324M / N326A; (7) N142R / T187C / M321I / K324I / N326A; (8) N142R / T187I / M321I / K324I / N326A; (9) N142R / T187C / M321I / K324M / N326C; (10) N142R / T187I / M321I / K324M / N326C; (11) N142R / T187C / M321I / K324I / N326C; (12) N142R / T187I / M321I / K324I / N326C; (13) N142R / T187C / N226I / M321I / K324M / N326T; (14) N142R / T187I / N226I / M321I / K324M / N326T; (15) N142R / T187C / N226I / M321I / K324I / N326T; (16) N142R / T187I / N226I / M321I / K324I / N326T; (17) N142R / T187C / N226I / M321I / K324M / N326A; (18) N142R / T187I / N226I / M321I / K324M / N326A; (19) N142R / T187C / N226I / M321I / K324I / N326A; (20) N142R / T187I / N226I / M321I / K324I / N326A; (21) N142R / T187C / N226I / M321I / K324M / N326C; (22) N142R / T187I / N226I / M321I / K324M / N326C; (23) N142R / T187C / N226I / M321I / K324I / N326C; (24) N142R / T187I / N226I / M321I / K324I / N326C; (25)N142R / T187C / N226I / M321I / K324M / N326T / L358Y; (26)N142R / T187I / N226I / M321I / K324M / N326T / L358Y; (27)N142R / T187C / N226I / M321I / K324I / N326T / L358Y; (28)N142R / T187I / N226I / M321I / K324I / N326T / L358Y; (29)N142R / T187C / N226I / M321I / K324M / N326A / L358Y; (30)N142R / T187I / N226I / M321I / K324M / N326A / L358Y; (31)N142R / T187C / N226I / M321I / K324I / N326A / L358Y; (32)N142R / T187I / N226I / M321I / K324I / N326A / L358Y; (33)N142R / T187C / N226I / M321I / K324M / N326C / L358Y; (34)N142R / T187I / N226I / M321I / K324M / N326C / L358Y; (35)N142R / T187C / N226I / M321I / K324I / N326C / L358Y; (36)N142R / T187I / N226I / M321I / K324I / N326C / L358Y; (37)T187C / M321I / K324M / N326T; (38)T187I / M321I / K324M / N326T; (39)T187C / M321I / K324I / N326T; (40)T187I / M321I / K324I / N326T; (41)T187C / M321I / K324M / N326A; (42)T187I / M321I / K324M / N326A; (43)T187C / M321I / K324I / N326A; (44)T187I / M321I / K324I / N326A; (45)T187C / M321I / K324M / N326C; (46)T187I / M321I / K324M / N326C; (47)T187C / M321I / K324I / N326C; (48)T187I / M321I / K324I / N326C; (49)T187C / N226I / M321I / K324M / N326T; (50)T187I / N226I / M321I / K324M / N326T; (51)T187C / N226I / M321I / K324I / N326T; (52)T187I / N226I / M321I / K324I / N326T; (53)T187C / N226I / M321I / K324M / N326A; (54)T187I / N226I / M321I / K324M / N326A; (55)T187C / N226I / M321I / K324I / N326A; (56)T187I / N226I / M321I / K324I / N326A; (57)T187C / N226I / M321I / K324M / N326C; (58)T187I / N226I / M321I / K324M / N326C; (59)T187C / N226I / M321I / K324I / N326C; (60)T187I / N226I / M321I / K324I / N326C; (61)T187C / N226I / M321I / K324M / N326T / L358Y; (62)T187I / N226I / M321I / K324M / N326T / L358Y; (63)T187C / N226I / M321I / K324I / N326T / L358Y; (64)T187I / N226I / M321I / K324I / N326T / L358Y; (65)T187C / N226I / M321I / K324M / N326A / L358Y; (66)T187I / N226I / M321I / K324M / N326A / L358Y; (67)T187C / N226I / M321I / K324I / N326A / L358Y; (68)T187I / N226I / M321I / K324I / N326A / L358Y; (69)T187C / N226I / M321I / K324M / N326C / L358Y; (70)T187I / N226I / M321I / K324M / N326C / L358Y; (71)T187C / N226I / M321I / K324I / N326C / L358Y; (72)T187I / N226I / M321I / K324I / N326C / L358Y; (73)T187C / M321I / K324M / N326T / L358Y; (74)T187I / M321I / K324M / N326T / L358Y; (75)T187C / M321I / K324I / N326T / L358Y; (76)T187I / M321I / K324I / N326T / L358Y; (77)T187C / M321I / K324M / N326A / L358Y; (78)T187I / M321I / K324M / N326A / L358Y; (79) T187C / M321I / K324I / N326A / L358Y; (80)T187I / M321I / K324I / N326A / L358Y; (81) T187C / M321I / K324M / N326C / L358Y; (82) T187I / M321I / K324M / N326C / L358Y; (83) T187C / M321I / K324I / N326C / L358Y; (84) T187I / M321I / K324I / N326C / L358Y; (85) N142R / T187C / M321I / K324M / N326T / L358Y; (86) N142R / T187I / M321I / K324M / N326T / L358Y; (87) N142R / T187C / M321I / K324I / N326T / L358Y; (88) N142R / T187I / M321I / K324I / N326T / L358Y; (89) N142R / T187C / M321I / K324M / N326A / L358Y; (90) N142R / T187I / M321I / K324M / N326A / L358Y; (91) N142R / T187C / M321I / K324I / N326A / L358Y; (92) N142R / T187I / M321I / K324I / N326A / L358Y; (93) N142R / T187C / M321I / K324M / N326C / L358Y; (94) N142R / T187I / M321I / K324M / N326C / L358Y; (95) N142R / T187C / M321I / K324I / N326C / L358Y; (96) N142R / T187I / M321I / K324I / N326C / L358Y.

3. A polynucleotide, characterized in that The polynucleotide encodes the aspartase mutant according to claim 1 or 2.

4. A recombinant plasmid, characterized in that The recombinant plasmid comprises the polynucleotide according to claim 3.

5. A recombinant genetically engineered bacterium, characterized in that: The recombinant genetically engineered bacteria comprises the aspartase mutant according to claim 1 or 2, or the recombinant plasmid according to claim 4.

6. Use of the aspartase mutant according to claim 1 or 2 in catalytic production of β-alanine.

7. The use according to claim 6, characterized in that Acrylic acid and ammonia are used as substrates.

8. The use according to claim 7, characterized in that The reaction pH is 8.0~9.0, the reaction temperature is 35~50℃, and the reaction time is 6~12h.

9. The use according to claim 7, characterized in that The catalyst is immobilized cells of aspartase mutant.

10. The use according to claim 9, characterized in that The mass ratio of aspartase mutant immobilized cells: acrylic acid: ammonia water is 3-8%: 30-40%: 30-48%.

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